THE MALARIA VACCINE DILEMMA:
NOVEL APPROCHES TO ADENOVIRAL VECTORED MALARIA VACCINES
By
Nathaniel Jerome Thibodeau Schuldt

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Genetics
2012

THE MALARIA VACCINE DILEMMA:
NOVEL APPROCHES TO ADENOVIRAL VECTORED MALARIA VACCINES
By
Nathaniel Jerome Thibodeau Schuldt
Despite the discovery more than 30 years ago that artificial or “unnatural” protection
against malaria is achievable, a practical protective malaria vaccine has yet to be realized. Recent
developments in sub-unit malaria vaccine platforms are bridging the gap between high levels of
protection and feasibility. However, the current leading sub-unit vaccine, RTS,S, has only
demonstrated the ability to induce protection from malaria infection in up 56% of RTS,S
vaccinees. Though encouraging, these results may fall short of protection levels generally
considered to be required to achieve eradication of malaria. The uses of viral vectored vaccine
platforms have recently been pursued to further improve the efficacy of malaria vaccines.
Adenovirus serotype 5 (Ad5) based vaccine platforms have demonstrated potent anti-malaria
immune responses, although it is clear more potent Ad5-induced immune responses are required
if Ad5-based malaria vaccines are to confer protection. Through explication of Ad interactions
with the innate immune system we have uncovered multiple targets that could be exploited to
improve the immunogenicity of Ad-based malaria vaccines. We have also attempted to
overcome an oft cited difficulty with use of Ad5 in malaria vaccine platforms, namely the high
seroprevalence of Ad5. We sought to improve Ad-induced immunogenicity in Ad5 immune
patients by the use of an alternative serotype of Ad (Adenovirus serotype 4 (Ad4)) in
heterologous prime boost regimens with Ad5. Instead, we uncovered a previously unknown
cross-reactivity between these two Ad serotypes that resulted in severely ablated
immunogenicity. We then tested the utility of various immunomodulators expressed from Ad

vectors to stimulate innate immune system responses and ultimately improve adaptive responses
to Ad-expressed malaria antigens. We found a promising immunomodulator in a SLAM receptor
adaptor protein (EAT-2). Co-injection of an Ad5 vaccine expressing EAT-2 with an Ad5 vaccine
expressing a malaria antigen (Circumsporozoite protein (CSP)) improved CSP specific CD8+ T
cell responses and in vivo cytotoxicity. Currently, we are testing the ability of this
immunomodulator to improve protection against a mouse malaria challenge model. Our research
has unearthed multiple valuable advancements in Ad-based vaccine technology that can be
utilized in malaria and non-malaria vaccine platforms alike.

Copyright by
NATHANIEL JEROME THIBODEAU SCHULDT
2012

I would like to dedicate this dissertation to beloved parents Marcia and Jerry Schuldt. It is
because of them that I am where I am today. With my fulfillment of the requirements to earn a
Doctorate of Philosophy in Genetics my parents will have nurtured the first and second PhDs in
my extended family. It is by no coincidence that my older brother and I have both achieved this
goal. Rather, it is due to the excellent examples of hard work and determination that my parents
have set for us. I am proud to share this honor with my parents and the rest of my family for it is
only thanks to them that I am here.

v

ACKNOWLEDGEMENTS
I would like to make use of the following section to thank those who have contributed to
my development throughout my graduate career. First, I wish to thank my mentor Dr. Andrea
Amalfitano. Dr. Amalfitano was absolutely primary to my accomplishments and his hard work
and perseverance served to motivate me to strive to achieve as he has. Dr. Amalfitano kindly
took me into his lab in 2007 and instantly began guiding me on my path toward becoming a
scientist. His gentle leadership encourages independent thought and rewards hard work. Under
Dr. Amalfitano’s tutelage I believe I have grown tremendously as a scientist in both my
mechanical skill and more importantly my intellectual process. Dr. Amalfitano’s impact will
forever shape my career and I cannot express my gratitude enough for the opportunity and for the
guidance he has given me. Thank you Dr. Amalfitano.
I would also like to thank all the professors who have served on my guidance committee;
Dr. Michelle Fluck, Dr. Sungjin Kim, Dr. Yong-Hui Zheng, Dr. Ian York, Dr. Christina Chan,
and Dr. Norbert Kaminski. My committee members challenged me and helped to prepare me for
the difficulties that accompany a career pursuing answers to difficult scientific questions. They
helped me to be both confident in what I know and to realize what I have yet to learn. All the
while treating me with respect and encouraging me to continue. Possibly most importantly, they
exemplified how to have constructive intelligent open-minded discussions. I am indebted to
every one of my committee members for their guidance and support during my graduate studies.
I would like to also thank the Genetics Program for accepting me into the program and
giving me this amazing opportunity. Particularly, I would like to thank Genetics Program
Director Dr. Barb Sears and Associate Director Dr. John LaPres. Dr. Sears and Dr. LaPres have
both given much of their time to the Genetics Program to ensure that the program continues to be

vi

a source of excellent scientists. It is in large part because of their hard work that graduates from
the Genetics Program proudly say they obtained a degree from the Genetics Program at
Michigan State University. I am grateful for the opportunity to serve as Genetics Student
President where I was mentored by both Dr. Sears and Dr. LaPres and participated in executive
committee meetings where I was able to observe firsthand what goes into running an academic
program at a large university. Additionally, I want to thank Dr. Sears for allowing me to be a
teaching assistant in her undergraduate genetics course and for selecting me as a writing
facilitator for the genetics writing group. Dr. Sears helped me to develop teaching and
organization skills that have improved my ability to convey my message. Participating in these
programs helped me to further understand the field of genetics and improve my scientific
writing. The Genetics Writing Group provides a unique education that is not offered by many
other programs or courses and for that opportunity I am grateful.
I cannot thank the Genetics Program without singling out Genetics Program Secretary
Jeannine Lee. Jeannine has assisted me since the day I first sent my application into the Genetics
Program. Jeannine is absolutely crucial to the program and to graduate students in the program.
Jeannine has helped with everything from getting financial support to organizing graduation. To
list everything she has helped me out with would be exhausting. Whenever I needed assistance
Jeannine helped promptly and with a smile. Thank you Jeannine, for keeping me organized and
on track.
I want to sincerely thank Dr. Dan Appledorn for being a great mentor and friend. Dr.
Appledorn was the Post Doc in the Amalfitano lab when I arrived. His enthusiasm for research
was absolutely infectious. Dr. Appledorn played a direct role in developing my skills as a
scientist by teaching me methods, experiment design, and results interpretation. I could always

vii

trust Dr. Appledorn to give me honest feedback. Dr. Appledorn was an excellent scientific role
model who demonstrated hard work and led by example. I consider Dr. Appledorn to be the
archetypal Post Doc, whom I plan on modeling myself after as I pursue my own Post Doc career.
I want to thank the Amalfitano Lab Tech, Sarah Godbehere-Roosa. Sarah provides the
structure and organization that a science lab requires to function. Without Sarah research in the
Amalfitano lab would likely come to a screeching halt. Additionally, Sarah has been a trusted
friend and confidant throughout my graduate career.
I also want to thank current Post Doc and former Amalfitano Lab graduate student Dr.
Sergey Seregin and past and present Amalfitano Lab graduate students; Yasser Aldhamen, Tyler
Voss, Aaron Mcbride, Joyce Liu, Dion Quiroga, Youssef Kousa, David Rastall. The Amalfitano
Lab has always been a welcoming friendly group. We have all grown together as scientists and
helped each other out considerably along the way in both the laboratory and classroom. Thank
you to all of the Amalfitano Lab undergraduates of past and present as well; Megan Hoban,
Jennifer Zender, Topher Busuito, John David, Brandi Burke, Kristen Kenny, Laura Harding,
Will Nance, Will Depas. I have made some very important and lifelong friendships while
working with them. Thank you to the entire Amalfitano Lab.
I want to thank the many Michigan State University facilities that have made my research
possible; ULAR (animal care), flow cytometry (Dr. Louis King), the Histo core, Microscopy (Dr.
Melinda Frame), and Sequencing. I am so very grateful for their expertise, excellent work, and
flexibility.
Finally, I want to thank my family; my two brothers Adam and Tyler, my sisters Sara and
Megan, my Mom and Dad, and of course my wonderful wife Christina. My beautiful wife
Christina has supported me both emotionally and financially. Christina has endured much during

viii

my graduate career and has never been anything less than completely supportive and generous,
as is her nature. Thankfully, I have the rest of our life together to try to make it up to her. I want
to thank my older brother, Dr. Adam Schuldt, for forging the trail for me. His advice and input
has helped shape me as a scientist and a person. Finally, I wish to thank the two people who have
shaped me more than any other, my parents Marcia and Jerry Schuldt. Thank you Mom and Dad,
for everything, I mean everything. They both have made endless sacrifices for their five children
and we do not thank them enough. They are beyond doubt, the hardest working people I have
ever known. Thank you all so much.

ix

TABLE OF CONTENTS
List of Tables ………...………………………………………………………………………....xii
List of Figures ....…………………………………………………………...………………...…xiii
Key Symbols or Abbreviations

…………………………………………………………..xvi

Chapter 1
Introduction: Malaria Vaccines: Focus on Adenovirus based vectors
….………………….1
1.1
Pathogenesis ....………………………………………………………………......2
1.2
Epidemiology ....………………………………………………………………......3
1.3
Prevention
....………………………………………………………………......3
1.4
Natural infection and hopes for a malaria vaccine ……………………………..4
1.5
Previous examples of putative malaria vaccines
……………………………..4
1.6
RTS,S
……...……………………………………………………………...7
1.7
Viral vectors as malaria vaccines
……….…………………………………….9
1.8
Adenovirus based malaria vaccines ……….…………………………………...10
1.9
Innate/adaptive immune systems
….………………………………………...11
1.10 rAd5 use in malaria vaccines …………...……………………………………….12
1.11 Pre-existing Ad immunity
…………...……………………………………….13
1.12 Alternative rAd serotypes for use in malaria vaccines
……………...….…14
1.13 Summary
……………………………………………………………………17
Chapter 2
Adenovirus induced innate immune responses
……..……………………………………..18
2.1
Introduction ……………………………………………………………………19
2.2
Results
……………………………………………………………………23
2.3
Discussion
……………………………………………………………………44
Chapter 3
Efficacy when utilizing Adenovirus serotype 4 and 5 vaccines expressing Circumsporozoite
protein in naïve and Ad5 immune mice
….……………………………………………...…48
3.1
Introduction ……………………………………………………………………49
3.2
Results
……………………………………………………………………51
3.3
Discussion
…..………………………………………………………………..73
Chapter 4
Vaccine platforms combining Circumsporozoite protein and potent immune modulators, rEA or
EAT-2, paradoxically result in opposing immune responses ……………………………………78
4.1
Introduction …………………………………………………………....………79
4.2
Results
…………………………………………………………………....83
4.3
Discussion
………..…………………………………………………………106
x

Chapter 5
Capability of advanced generation, Adenovirus based malaria vaccines to prevent malaria
infection
…………………………………………..……………………………………....112
5.1
Introduction …………………………………………………………………..113
5.2
Results
…………………………………………………………………..114
5.3
Discussion
……..……………………………………………………………121
Chapter 6
Summary and future directions
..……….……………………………………………...…124
6.1
Ad interactions with the innate immune system
…………………………..125
6.2
Studies of alternative serotypes
……………...…………………………...126
6.3
Immunomodulation …………………………………………………………..128
6.4
Challenge study
..………………………………………………………....130
Chapter 7
Material and methods …………………………………..………………………………………132
Bibliography ………….…………………………………………………………………….....143

xi

LIST OF TABLES
Table 1

Ad5-LacZ-induced gene expression in liver at 6 hpi in complement KO mice (fold
over mock) ..…………….…………………………………………………….24

Table 2

Ad5-LacZ-induced gene expression in a liver in mCR1/2-KO mice (fold over
C57BL/6 WT Mock) ...………………………………………………………….26

Table 3

Ad5 induced gene expression in a liver of C57BL/6 WT mice (fold over mock, 6
hpi) …………….…………………………………………………….………..40

Table 4

Mean spot forming splenocytes in Ad5 immune and Ad5 naïve vaccinated mice
....…………………………………………………………………………...…….66

xii

LIST OF FIGURES
Figure 1

Plasma cytokine and chemokine elevations after intravenous adenovirus (Ad)
injection
....……………………………………...……………………….....27

Figure 2

Murine complement receptor 1/2 (mCR1/2) mitigates Adenovirus (Ad)-mediated
cytokine and chemokine release in C57BL/6 mice ……...….………………....29

Figure 3

Ad neutralizing antibody titers are C3-dependent

Figure 4

Anti-recombinant adenovirus (rAd)-specific antibodies are dependent on both
alternative pathway (AP) and classical pathway (CP) of complement
……33

Figure 5

mCR1/2-KO mice exhibit significantly reduced Adenovirus (Ad) capsid-specific
neutralizing antibodies titer ……………...…………………………………….35

Figure 6

mCR1/2-KO mice exhibit significantly reduced Adenovirus (Ad) vector capsidspecific humoral immune responses ……………………………………………36

Figure 7

Anti transgene (GFP)-specific antibodies are C3-dependent ..…………………..39

Figure 8

Ad5 vectors “capsid-displaying” retro-DAF complement inhibitor significantly
reduce Ad dependent activation of endothelial cells in C57BL/6 mice
…....41

Figure 9

Ad4-CSP/Ad5-CSP heterologous prime boost results in improved quality of T cell
response
………………….………………………………………………...52

Figure 10

Ad5-CSP/Ad5-CSP vaccination resulted in higher percentage of tetramer positive
CD8+ T cells than Ad4-CSP/Ad5-CSP in the spleen ……..……………………..54

Figure 11

Memory responses triggered by vaccination with homologous and heterologous
prime boost regimens utilizing Ad4-CSP and Ad5-CSP in Ad naïve mice…...…56

Figure 12

Ad4-CSP/Ad4-CSP and Ad5-CSP/Ad5-CSP vaccinated animals have no
significant cross stimulation of splenocytes …………………………..………..57

Figure 13

All vaccinations stimulated significantly higher anti-CSP total IgG than
unvaccinated and AD4-CSP/Ad4-CSP vaccination in Ad naïve animals

xiii

……….…………………...32

…....59

Figure 14

Sub-isotype analysis of IgG antibody from plasma of mice vaccinated with
heterologous and homologous prime boost regimens utilizing Ad4-CSP and Ad5CSP ……………..……………………………………………………………..60

Figure 15

Th1 to Th2 ratio (IgG2a/IgG1) of plasma from vaccinated Ad naïve animals ….61

Figure 16

Ad5-CSP/Ad5-CSP and Ad4-CSP/Ad5-CSP both stimulated more percent
specific killing than unvaccinated animals ………..…………………………..63

Figure 17

IFNγ secretion of cells from Ad5 immune mice vaccinated with heterologous and
homologous prime boost regimens utilizing Ad4-CSP and Ad5-CSP
…....64

Figure 18

CD8+ T cell activation in Ad5 immune animals vaccinated with heterologous or
homologous prime boost regimens utilizing Ad4-CSP and Ad5-CSP
……67

Figure 19

All vaccinations in Ad5 immune animals resulted in significantly higher
percentages of CD8+ CSP tetramer positive cells than unvaccinated Ad5 immune
animals
……...…………………………………………………………….68

Figure 20

Memory responses triggered by vaccination with homologous and heterologous
prime boost regimens utilizing Ad4-CSP and Ad5-CSP in Ad5 immune animals
……….……..…………………………………………………………………….69

Figure 21

Homologous prime boost regimens favor a Tcm cell phenotype in the peripheral
blood of Ad5 immmune mice …..………………………………………………..70

Figure 22

All vaccinations stimulated significantly higher anti-CSP total IgG than
unvaccinated and Ad5-CSP/Ad5-CSP vaccination in Ad5 immune animals .…..72

Figure 23

Ad5-CSP construction

Figure 24

Ad5-CSP Stimulates CSP specific T and B cell responses

Figure 25

TLR agonist, rEA, induced innate cytokines 6 hours post injection …………....87

Figure 26

Immuno-modulating proteins conversely affect IFNγ secreting splenocytes ...….90

Figure 27

CSP expression does not interfere with antigen specific immune responses against
other transgenes at low doses ………….………………………………….…….91

……...……………………………………..……...84

xiv

……….……..…….85

Figure 28

7

Ad-GFP/rEA combined with 5x10 vp/mouse of Ad5-CSP begins to display a
6

diminished CSP specific CMI response after a dose of 5x10 vp/mouse
………….………………………………………………………………..……….92
Figure 29

Co-expression of CSP and EAT-2 stimulates more potent CSP specific CMI
responses
…………………….……………………………………..……….94

Figure 30

Expression of GFP does not interfere with CSP specific CMI responses
…………...………………………………………………………………..……...96

Figure 31

Co-expression of CSP and EAT-2 increases the breadth of response against CSP
………………………………………………………………………..…………..97

Figure 32

Improved degranulation of CD8+ T cells in mice co-vaccinated with Ad5-CSP
and Ad-EAT2 …………...……………………………………………………...100

Figure 33

Co-expression of CSP and EAT-2 increases cytolytic activity of CSP specific T
cells ……………………...…………………………………………………...101

Figure 34

Induction of CSP specific antibody responses by Ad5-CSP vaccines augmented
by rEA or EAT-2
………….……………………………………………….103

Figure 35

Sub-isotype analysis of IgG antibody from plasma of mice co-vaccinated with
Ad5-CSP and Ad-EAT2
…………………………………………………..104

Figure 36

CD3+ CD8- IFNγ+ cells respond similarly to both vaccine regimens

Figure 37

Co-vaccination with Ad5-PbCSP and Ad5-EAT2 drastically increases PbCSP
specific CMI responses
…………………..………………..………..……116

Figure 38

Giemsa stain strongly correlates with % FITC+ red blood cells

Figure 39

Parasitemia was similar between treatments at 7 and 14 days post challenge …119

Figure 40

CSP sequence ………………....………………………………………………..135

xv

…..105

….……….118

Key Symbols or Abbreviations
µL

microliters

µM

micromoles

AAALAC

Association for Assessment and Accreditation of Laboratory
Animal Care

AAV

Adeno-associated virus

ABSL-2

Animal Biosafety level 2

Ad

Adenovirus

Ad26

Adenovirus Serotype 26

Ad26CSP

Adenovirus Serotype 26 expressing circumsporozoite protein

Ad35

Adenovirus Serotype 35

Ad35CSP

Adenovirus Serotype 35 expressing circumsporozoite protein

Ad35PyCS

Adenovirus Serotype 35 expressing Plasmodium yoelli
Circumsporozoite protein

Ad4

Adenovirus serotype 4

Ad4-CSP

Adenovirus serotype 4 expressing circumsporozoite protein

Ad5

Adenovirus serotype 5

Ad5-CSP

Adenovirus serotype 5 expressing circumsporozoite protein

Ad5-GFP-IX-dDAF_REO

Adenovirus serotype 5 with protein IX capsid fusion to human
decay accelerating factor in the retro-orientation expressing GFP

Ad5-IX-GFP

Adenovirus serotype 5 with protein IX capsid fusion to green
fluorescent protein

Ad5PfCS

Adenovirus serotype 5 expressing Plasmodium falciparum
circumsporozoite protein

Ad5PyCS

Adenovirus serotype 5 expressing Plasmodium yoelli
circumsporozoite protein

xvi

ADAR

Adenosine deaminase, ribonucleic acid specific

AdC9

Chimpanzee adenovirus serotype 9

Ad-GFP

Adenovirus expressing green fluorescent protein

Ad-GFP/rEA

Adenovirus expressing green fluorescent protein and recombinant
eimeria tenella antigen

AMA-1

Apical membrane antigen 1

ANOVA

Analysis of variance

AP

Alternative pathway

AP-C3

Acute respiratory disease

AS01B

Adjuvant series 01B

AS02A

Adjuvant series 02A

BALB/cJ

Albino laboratory mouse strain

CAR

Coxsackievirue and adenovirus receptor

C1

Complement component 1

C1q-KO

Complement component 1q knock out mouse

C1qrs

Complement complex made up of complement components 1, q, r,
and s

C1r

Complement component 1r

C1s

Complement component C1s

C2

Complement component 2

C3

Complement component 3

C3-C5

Complement component 3/complement component 5 convertase

C3d

Complement component 3d

C4

Complement component 4

xvii

C4-KO

Complement component 4 knock out mouse

C57BL/6

Black laboratory mouse strain

C6

Complement component 6

C7

Complement component 7

C8

Complement component 8

C9

Complement component 9

CAR

Coxsackie adenovirus receptor

CCL7

Chemokine (C-C motif) ligand 7

CCPR

Complement control protein repeats

CD107a

Cluster of differentiation 107a also known as Lysosomalassociated membrane protein

CD127

Cluster of differentiation 127 also known as Interleukin-7 receptor
subunit alpha

CD14

Cluster of differentiation 14

CD19

B lymphocyte antigen CD19 Cluster of differentiation 19

CD2

Cluster of differentiation 2

CD22

Cluster of differentiation 22

CD3

Cluster of differentiation 3

CD4

Cluster of differentiation 4

CD46

Cluster of differentiation also known as membrane cofactor protein

CD62L

Cluster of differentiation 62 ligand also known as L-selectin

CD69

Cluster of differentiation 69

CD8

Cluster of differentiation 8

CFSE

Carboxyfluorescein diacetate

xviii

ChAd63

Chimpanzee adenovirus serotype 63

CMI

Cell mediated immunity

CMV

Cytomegalovirus

CP

Classical pathway of complement

CP-C3

Classical pathway of complement component 3 convertase

CR

Complement receptor

CR1

Complement receptor 1

CR1/2

Complement receptor 1/2

CR2

Complement receptor 2

CRACC

CD2-like receptor activating cytotoxic cells

Crry

Rodent specific complement receptor 1 related gene/protein-y

CS

Circumsporozoite

CSP

Circumsporozoite protein

CTL

Cytotoxic T lymphocyte

CXCL9

Chemokine (C-X-C motif) ligand 9

DAF

Decay accelerating factor

DMEM

Dulbecco/vogt modified eagle’s minimal essential medium

DNA

Deoxyribonucleic acid

Dpi

Days post injection

E1

E1 region of Adenovirus genome encodes proteins trans-acting
transcription factor

E2b

E2b region of Adenovirus genome encodes Adenovirus
polymerase

E3

E3 region of Adenovirus genome encodes multiple immune
defense proteins
xix

E4

E4 region of Adenovirus genome encodes genes for lytic growth

EAT-2

Ewing’s sarcoma related transcript 2

ELISA

Enzyme-linked immunosorbant Assay

ELISpot

Enzyme-linked immunosorbant spot assay

E-selectin

Also known as cluster of differentiation 62 (CD62)

FB

Factor B

FB-KO

Factor B knock-out

FcγRIIB

Fcγ receptor IIB also known as cluster of differentiation 32 (CD32)

Gag

Human immunodeficiency virus viral core protein

GATA-3

Trans-acting T cell specific transcription factor that recognizes the
deoxyribonucleic acid sequence GATA

G-CSF

Granulocyte colony stimulating factor

GFP

Green fluorescent protein

GSK

Glaxo-Smith-Kline

hAd

Human Adenovirus

hAd5

Human Adenovirus serotype 5

HBsAg

Hepatitis B surface antigen

HEK 293

Human embryonic kidney 293 cells

HIV

Human immunodeficiency virus

hpi

Hours post injection

ICAM

Intracellular cell adhesion protein

ICS

Intracellular staining

IFNα

Interferon alpha

xx

IFNβ

Interferon beta

IgA

Immunoglobulin A

IgG

Immunoglobulin G

IgG1

Immunoglobulin G1

IgG2a

Immunoglobulin G2a

IgG2b

Immunoglobulin G2b

IgG2c

Immunoglobulin G2c

IgG3

Immunoglobulin G3

IgM

Immunoglobulin M

IL-12

Interleukin 12

Il-12 (p40)

Interleukin 12 subunit protein 40

IM

Intramuscular

IRF-1

Interferon regulatory protein 1

IRF-7

Interferon regulatory protein 7

IRF-8

Interferon regulatory protein 8

ITSM

immune-receptor tyrosine-based switch motif

IV

Intravenous

JAK-1

Janus kinase 1

JAK-3

Janus kinase 3

kb

kilobase

KC

Also known as chemokine (C-X-C motif) ligand 1

kD

Kilodalton

KO

Knock out

xxi

LAG-3

Lymphocyte-activation gene 3

MASP

Mannose-binding lectin-associated serine protease

MBL

Mannose-binding lectin

MCP

Monocyte chemotactic protein

MCP-1

Monocyte chemotactic protein 1

mCR

Murine complement receptor

mCR1/2

Murine complement receptor 1/2

ME.TRAP

multi-epitope thrombospondin related adhesive protein

MIP-1β

Macrophage inflammatory protein -1β

mL

milliliter

MPL

Monophosphoryl lipid A

MSP-1

Merozoite specific protein 1

MVA

Modified vaccinia Ankara

MyD88

Myeloid differentiation primary response gene 88

NALP

A Nucleotide-binding oligomerization domain-containing protein
(NOD)-like receptor

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B cells

NF-κB-RelA

Nuclear factor kappa-light-chain-enhancer of activated B cells
subunit Rel A also known as protein 65 (p65)

ng

Nanograms

NIH

National institute of health

NK

Natural Killer

NLR

Nucleotide-binding oligomerization domain-containing protein
(NOD)-like receptors

NOD-1

Nucleotide-binding oligomerization domain-containing protein 1
xxii

NOD-2

Nucleotide-binding oligomerization domain-containing protein 2

O.D.

Optical density

OAS1a

2’-5’ oligoadenylate synthetase 1 a

ORF

Open reading frame

P. berhei

Plasmodium berghei

P. falciparum

Plasmodium falciparum

P. yoelli

Plasmodium yoelli

P. vivax

Plasmodium vivax

PAMP

Pathogen-associated molecular pattern

PBMC

Peripheral blood mononuclear cells

PBS

Phosphate buffered saline

PD-1

Programmed death 1

Pfs25

Plasmodium falciparum surface protein 25

pg

picagrams

PHS

Public health services

pIX

Adenovirus surface protein IX

polyA

Polyadenylation

qRT-PCR

Quantitative reverse transcriptase polymerase chain reaction

QS21

Quillaja saponaria extract 21

rAd

Recombinant adenovirus

rAd35

Recombinant adenovirus serotype 35

rAd5

Recombinant adenovirus serotype 5

RANTES

Regulated upon activation, normal T cell expressed, and secreted

xxiii

rEA

Eimeria tenella antigen

RIG

Retinoic acid-inducible gene protein

RLR

Retinoic acid-inducible gene protein (RIG)-like receptor

RNA

Ribonucleic acid

rRNA

Ribosomal ribonucleic acid

RTS,S

Hepatitis B surface antigen-circumsporozoite protein fusion based
malaria vaccine developed by Glaxo-Smith-Kline

RTS,S/AS01B

Hepatitis B surface antigen-circumsporozoite protein fusion based
malaria vaccine developed by Glaxo-Smith-Kline adjuvanted with
adjuvant series 01B

SFC

Spot forming cells

SLAM

Signaling lymphocytic activation molecule

SOCS-1

Suppressor of cytokine signaling1

SOCS-3

Suppressor of cytokine signaling 3

TBK-1

TANK-binding kinase 1

Tcm

Central memory T cells

Tem

Effector memory T cells

Tet

tetramer

Th1

T helper cell 1

Th2

T helper cell 2

TK

Thymidine kinase

TLR

Toll-like receptor

TLR2

Toll-like receptor 2

TLR3

Toll-like receptor 3

TLR6

Toll-like receptor 6
xxiv

TLR9

Toll-like receptor 9

TMB

3,3’,5,5’ tetramethylbenzidine

TNFα

Tumor necrosis factor alpha

TRAF2-bp

Tumor necrosis factor receptor associated factor 2-bp

TRAP

Tumor necrosis factor receptor associated protein

TRIM30

Tripartite-motif protein 30

TSR

Thrombospondin-like type I repeat region

VCAM

Vascular cell adhesion molecule

vp

Viral particles

WT

Wildtype

xxv

Chapter 1:
Introduction: Malaria Vaccines: Focus on Adenovirus based vectors

1

1.1 Pathogenesis:
Five protozoan parasites are known to cause malaria in humans, Plasmodium falciparum,
Plasmodium ovale, Plasmodium malariae, Plasmodium vivax, and Plasmodium knowlesi, with P.
falciparum being the most deadly accounting for 80% of all malaria cases, and 90% of all
malaria deaths.[1] The parasites are transferred from human to human by the bite of the
Anopholes mosquito. As a consequence of the intermediate host, the parasites have both a
complex mosquito, and human, life cycle. The human stage begins when an infected mosquito
takes a blood meal from a human. The parasites traverse the mosquito’s proboscis from the
salivary gland and enter the humans in the form of sporozoites. The sporozoites rapidly travel to
the liver through the blood stream where they infect hepatocytes in what is known as the exoerythrocytic phase. Here the parasites multiply into thousands of merozoites. Merozoites then
leave the liver and enter the blood stream where they infect red blood cells, this begins the
erythrocytic phase. Once inside the erythrocyte the merozoite transforms into a trophozoite, this
stage is sometimes referred to as the feeding stage. The trophozoite nucleus then divides
asexually to produce a multi-nucleated schizont. The schizont divides into many mononucleated
merozoites which are then released back into the blood stream to infect more erythrocytes.
Occasionally, when merozoites enter erythrocytes they transform into male or female
gametocytes and do not rupture the erythrocyte. Gametocyte infected red blood cells can then be
picked up by another mosquito taking a blood meal beginning the mosquito stage of the
lifecycle.
In the mosquito gut the male and female gametocytes merge creating a diploid zygote
that forms an oocyst in the intestinal wall of the mosquito. Inside the oocyst multiple cell
divisions take place resulting in the production of many sporozoites. Sporozoites then travel to

2

the salivary gland of the mosquito reinitiating the human stage of the life cycle when the
mosquito bites another human.
1.2 Epidemiology:
Recently the numbers of malaria cases and malaria deaths have decreased worldwide in
large part due to use of pesticides and bed nets that together kill or prevent mosquitoes from
biting susceptible humans. In 2009, 225 million people were infected with malaria, down from
244 million in 2005.[2] While this is an encouraging trend, there were still 781,000 malaria
deaths worldwide, indicating new preventions must be developed and employed if malaria is to
be eradicated. Malaria has been considered "eliminated" in the United States of America since
1970 and there were no locally acquired cases of P.falciparum reported in the European region in
2009.[2] However, malaria still remains a prominent threat in areas of South America, SubSaharan Africa and Southeast Asia placing roughly one third of the world’s population at risk of
contracting malaria.[2]
1.3 Prevention:
Malaria prevention focuses on three main targets; vector control, prophylactics, and
vaccination. The use of long lasting insecticide treated bed nets and indoor residual spraying
have proven to be successful and relatively economical strategies to reduce the risk of malaria
transmission, provided high coverage is achieved and sustained.[3,4] However, mosquito
pesticide resistance and behavioral adaptations (like biting immediately after sunset and before
sunrise) that allow the mosquito to circumvent these preventions have already been
detected.[5,6]
Chemical prophylactics are used to prevent parasite replication in the human host. They
are often used successfully, however, no prophylactic is 100% effective and most prophylactics

3

are strain specific. The current preferred chemoprophylactics include chloroquine, proguanil,
doxycycline, and mefloquine.[7,8] Unfortunately, uncomfortable gastrointestinal, hematological,
and/or neurological side effects often accompany utilization of these medications.[7,9] In
addition the prophylactics must be taken regularly to be effective, that and the uncomfortable
side effects can make it difficult for some people to remain compliant on the treatment. Overuse
of the prophylactic chloroquine in eastern Africa has lead to chloroquine resistant P. falciparum
parasites in those regions. As a result, close monitoring of parasite resistance is required in order
to rapidly change the prophylactic in use should resistance occur.[10]
1.4 Natural Infection and hopes for a malaria vaccine:
Sadly, the majority of malaria related deaths occur in children, since many adults have
acquired immunity to malaria "naturally" over time as a result of surviving repeated malaria
infections.[11] While it appears that natural immunity to human malaria is largely IgG antibody
mediated, it has proven difficult to pinpoint the specific antigens these antibodies
target.[11,12,13] Antibody responses to malaria antigens are generally short-lived, possibly
because natural malaria infections hinder the development of B cell memory
responses.[11,12,13,14,15] For example, P. falciparum infection can induce expression of a T
cell inhibitory receptor called Programmed Death-1 (PD-1), leading to poor CD4+ T cell
responses. Simultaneous blockade of both PD-1 ligand and Lymphocyte Activation Gene - 3
(LAG-3: a negative regulator of T cell function) together can allow for more rapid clearance of
blood stage infections in mouse models, and has been recently targeted as a strategy to treat
active malaria infection in humans.[16]
1.5 Previous examples of putative malaria vaccines:

4

Alternative malaria prevention methods attempt to proactively vaccinate individuals from
malaria infection (i.e.: “unnatural” immunity). Potent humoral and/or CD8+ T cell responses
against multiple malaria antigens have been identified to be partially responsible for protection
from malaria infection. Based upon these findings, it has been postulated that pre-emptive
induction of adaptive immune responses to malaria derived antigens may be of benefit in
preventing the symptoms of subsequent malaria infection, if not protection from malaria
infections in general.
We know that artificial or “unnatural” inductions of immunity to malaria are achievable.
In 1975 mosquitoes infected with P. vivax or P. falciparum were irradiated (preventing live
parasite transmission) and then used to bite a human volunteer, as a result the volunteer was
protected from natural malaria infections for a short period of time.[17] Although the experiment
was subsequently validated in larger groups of human volunteers, the approach was not practical
as many hundreds of bites were required, and the resulting protection was no-less short
lived.[18,19] Despite this disadvantage, the approach still remains one of the most protective
malaria vaccine platforms to date, as protection rates approached over 90%.
Another non-irradiated mosquito bite based vaccine platform utilizes chloroquine to
control parasite infection. In this platform P. falciparum infected mosquitoes are allowed to bite
volunteers while chloroquine is administered to prevent blood stage infection by the live
parasites. Chloroquine controlled infection has shown high rates of effector memory mediated
protection upon parasite rechallenge that lasted for up to 2 years, a significant increase over the
few months observed with use of irradiated mosquito based vaccine platforms.[20,21]
Chloroquine controlled infection also decreased the amount of mosquito bites required for
protection, from many hundreds of bites to only 10-15.[20] Notably, chloroquine controlled

5

infection also decreases the number of PD-1 expressing CD4+ T cells improving the CD4+ T
cell exhaustion phenotype.[16] However, due to extensive use of chloroquine in East Africa,
some strains of P. falciparum have developed resistance to the prophylactic.[10] Since nonchloroqine resistant strains must be used in this method, further research must be conducted to
ensure that chloroqine controlled vaccination with non-chloroquine resistant parasites can protect
against challenge with chloroquine resistant parasites.
The use of purified, radiation attenuated sporozoite’s as a prophylactic malaria vaccine is
another method attempted for use in humans. In this method sporozoites are harvested from the
salivary glands of irradiated mosquitoes and injected with a needle rather than via bites from the
irradiated mosquitoes. Analysis of immune correlates of protection performed after vaccinations
with irradiated sporozoites suggest that CD8+ T cell responses against the liver stage of the
parasite appear to be more important for achieving protection, relative to antibody
responses.[22,23,24,25] Further supporting this correlation, studies where CD8+ T cells specific
for liver stage malaria antigens were passively transferred into naive mice demonstrated
protection from intravenous (IV) sporozoite challenge.[26]
Unfortunately the method requires higher doses of sporozoites and was demonstrated to
be poorly immunogenic in human trials.[23,27] More recent animal studies confirmed that
protection was improved by IV injection of irradiated sporozoites, and future human trials will
be required to assess the immunogenicity of IV injected irradiated sporozoites.[23] The
complexities of harvesting, storing, and transporting purified irradiated sporozoites is a concern
as well, as sporozoites are very fragile outside of their mosquito host. Attempts to cryopreserve
irradiated sporozoites demonstrated that they do not survive the freeze thaw process well.

6

Inoculations with cryopreserved irradiated sporozoites also required fourfold more sporozoites
than fresh sporozoite preparations to achieve the same effectiveness in animal models.[23]
Use of live (non-irradiated) but genetically attenuated sporozoites to increase the
immunogenicity and therefore decrease the number sporozoites required to achieve protection
has also been recently described. In a mouse model of malaria, attenuated P. yoelli sporozoites
that have been genetically engineered to arrest late in the liver stage were capable of stimulating
broader and more potent CD8+ T cell responses to malaria antigens (including blood stage
antigens) than what was observed using irradiated P. yoelli sporozoites as the vaccinating
antigen.[28] Mice vaccinated with lower numbers of the genetically attenuated sporozoites
displayed a wider range of antigen responses and were also protected against blood stage
challenge as compared to purified radiation attenuated sporozoites injected IV.[28] Whether
these results translate to P. falciparum and human malaria infections remains to be seen.
The use of attenuated sporozoites, whether purified and injected, or administered through
mosquito bites, has shown promising results in the laboratory. However, the necessity for
multiple bites from infected mosquitoes and the inability to mass produce and preserve purified
sporozoites according to regulatory standards for human use, has prompted the development of
alternative, subunit based vaccines targeting specific malaria parasite antigens.
1.6 RTS,S:
Circumsporozoite protein (CSP) is the most abundantly expressed protein during the
sporozoite stage and is found both on the surface of sporozoites and in the plasma membrane and
cytoplasm of infected hepatocytes during early liver infection.[29] CSP has been repeatedly
shown to be an immunodominant protective antigen.[22,26,30] In fact, when transgenic mice
were altered to express and therefore tolerate CSP, the absence of a CSP specific B and T cell

7

response dramatically decreased the ability of irradiated sporozoites to protect the transgenic
mice from malaria challenge.[30] Conversely, mice vaccinated with irradiated sporozoites
engineered to express CSP of a different malaria parasite species were still capable of stimulating
protective immunity when challenged with parasites of the same species as the irradiates
sporozoites, indicating that species specific B and T cell responses to CSP are not required for
protection.[31,32] Despite these conflicting findings CSP remains the most commonly utilized
malaria antigen in malaria vaccines and there is a wealth of evidence demonstrating that CSP, if
not required, is protective on some level.
CSP is a 58 kD protein composed of a C-terminus containing the thrombospondin-like
type I repeat region ((TSR) involved in liver sinusoid attachment), a central region of [NANP]
repeats, and a N-terminal site that when in contact with the liver sinusoid is cleaved exposing the
TSR.[33,34,35] The most successful malaria subunit vaccine candidate to date is composed of a
novel fusion between the immunogenic hepatitis B surface antigen (HBsAg), and amino acids
207-395 of CSP from P. falciparum strain NF54, clone 3D7.[36] Developed by Glaxo-SmithKline (GSK), the fusion protein is thought to spontaneously form a pseudo virion structure that
displays CSP on its surface.
Initial tests proved the fusion to be poorly immunogenic, so potent adjuvants were added
to increase overall immunogenicity.[37,38,39,40,41] These adjuvants include AS02A (squalenein-water emulsion containing monophosphoryl lipid A (MPL)) and AS01B (liposome
preparation of MPL) and a plant extract known as QS21). Inclusion of these adjuvants with the
HBsAg-CSP fusion protein (referred to as RTS,S) has achieved up to 50% protection from
malaria infection in several human trials.[36,37,38,39,42,43] Most recently, a phase 3 study was
conducted with RTS,S/AS01B on 6000 infants aged 5 to 17 months in 7 African countries, and

8

managed to achieve 56% protection against naturally occurring clinical malaria infections.[44]
While encouraging, these results are well below that achieved by most vaccines currently used
for other infectious diseases, higher protection rates are likely required to eradicate malaria from
the endemic regions.
The efficacy of early forms of the fusion vaccine was attributed to induction of high CSP
specific antibody titers.[45] Inclusion of AS01B maintained anti-CSP antibody titers while also
significantly increasing T cell responses to CSP as well, a finding that positively correlated with
improved protection of vaccinated volunteers from experimental malaria challenges via bites
from P. falciparum infected mosquitoes.[46] The result suggests that further improvement of T
cell responses to malaria antigens such as CSP is desirable of a putative malaria vaccine.
Vaccination regimens that utilize a priming vaccination with one type of vaccine platform
followed by boosting vaccinations with a different (heterologous) vaccine platform, each of
which includes the same antigenic target may provide increased T cell immune responses to the
desired antigen relative to sole use of one vaccine platform (homologous prime-boosting).
Heterologous prime-boost vaccinations combining the use of the RTS,S/AS01B platform with
virus vector based malaria vaccine platforms that also express CSP induced more potent and
longer lasting CSP specific CD8+ T cell responses in rhesus macaques, relative to use of either
vaccine platform alone.[47] Such studies suggest that the use of virus based vaccine platforms
may improve malaria antigen specific adaptive immune responses, and/or increase potency when
used in heterologous prime-boost vaccination regimens in malaria targeted vaccine formulations,
as summarized below.
1.7 Viral vectors as malaria vaccines:

9

Viral based vaccines provide a means by which to rapidly activate the host innate
immune system (due to the presence of pathogen associated molecular patterns (PAMPs))
simultaneous with delivery malaria antigen expressing genes without the requirement of
additional adjuvants per se. Viral vectors can also be used to overcome the manufacturing
hurdles that accompany mosquito and/or sporozoite based vaccine formulations, as some viral
vectors are relatively easy to produce to high titer. Many viral vectors have been utilized as
potential malaria vaccines inclusive of alphavirus, flavivirus, morbillivirus, adeno-associated
virus (AAV), modified vaccinia virus Ankara (MVA), and adenovirus (Ad).[48] MVA based
vaccines targeting malaria antigen have been shown to induce both CD4+ and CD8+ T cells
responses, and have even been easily administered via a microneedle array transdermal
patch.[49] However, MVA based malaria vaccines have been more commonly successful as a
boosting vaccination used in heterologous prime-boost vaccination combinations, especially
when paired with Ad based malaria vaccines.[50]
1.8 Adenovirus based vaccines:
The Ad family of viruses have an icosahedral capsid that protects the non-enveloped
linear double stranded DNA genome.[51] There are at least 52 human serotypes divided into
subgroups A-F primarily based on lack of cross-neutralization by antisera. Of these serotypes,
human adenovirus serotype 5 of subgroup C (Ad5) is the most studied and well understood.
Wildtype Ad5 enters cells via interactions with the coxsackievirus and adenovirus receptor
(CAR), as well secondary interactions with integrins. Ad5 has a 36 kb genome that is
functionally divided into early and late genes based on temporal expression relative to the initial
infection event. Early gene transcription is initiated by the E1 gene products which function in
trans to augment expression of the other Ad encoded transcription units. Deletion of the E1

10

region of the Ad genome partially renders the virus replication incompetent and provides space
to incorporate a gene encoding an antigen of interest for expression by the recombinant virus
upon infection.[52,53] E1 deleted hAd5s ([E1-]Ad5s) are cultivated in special HEK 293 cells
that have the E1 region incorporated into their genome and can therefore supply E1 in trans to
initiate replication.[52,53] Recombinant viruses are then purified to high titers by use of cesium
chloride gradients, or when large scale applications are required, column
chromatography.[52,53,54] High titer Ad vectors can then be easily administered intravenously,
intramuscularly, subcutaneously, intranasally, and even orally.[55,56,57,58,59] Further
attenuation by removing different combinations (or all) of the Ad5 E2b, E3, and E4 genes can
provide a cloning capacity of over 33kb have also been described, with some having unique
abilities as a vaccine platform.[51,60,61,62]
1.9 Innate/adaptive immune systems:
The innate immune system heavily influences and augments the development of an
antigen specific adaptive response.[63] Innate immune responses are stimulated through multiple
receptors and sensors specially suited for detecting the aforementioned PAMPs, that include
foreign protein, DNA, RNA, and polysaccharides. These receptors include membrane and
endosome bound receptors called toll-like receptors (TLRs), intracellular receptors like NODlike receptors (NLRs) and RIG-like receptors (RLRs), the inflammasome/NALP pathway, and
the complement pathway. These receptors and sensors recognize multiple pathogen associated
signals and activate innate immunity (which includes multiple cytokine and chemokines) in
response to the detected infection. Without an associated danger signal many antigens would
otherwise be well tolerated by the immune system. Deliberate stimulation of an innate immune
response by adjuvants can provide the danger signals necessary to signify the antigen provided

11

by a vaccine as a pathogen, resulting in a more robust adaptive response against the antigen of
choice. TLRs, NLRs and RLRs, inflamasome/NALP pathway, and complement mediated
induction of innate cytokines have all been shown to greatly augment adaptive responses against
the pathogens from which the PAMPs originate from.[64,65,66,67,68,69,70]
rAds have shown great promise as a vaccine platform for several reasons. In general they
are highly regarded for their ability to stimulate potent cellular (CD8+ T cells) and humoral
adaptive responses against expressed antigens, a feature thought to be due in part by the Ad
capsid and genome simultaneously stimulating several arms of the innate immune response,
including the NLRs, TLRs, complement system, and the inflammasome/NALP
pathways.[65,69,71,72,73] Stimulation of the innate immune system in this multi-faceted fashion
likely promotes the development of robust adaptive responses against a specific antigen,
responses that would otherwise be less immunogenic without these associated "danger
signals".[63] Ad5 based vectors have proven very successful at stimulating adaptive responses
against several antigens, including those targeting rabies, cancer (P815 tumor), and HIV, as well
as malaria.[60,74,75,76] My research has elucidated Ad vector interactions with complement
that could lead to improvements in Ad vector design and immunogenicity.
1.10 rAd5 use in malaria vaccines:
Ad5 based malaria targeted vaccines have repeatedly matched or surpassed the ability of
other vaccine platforms to induce beneficial, malaria antigen specific adaptive immune
responses.[59,75,77,78,79] For example, a rAd5 expressing P. falciparum CSP (Ad5PfCS)
provoked equivalent CSP specific lymphocyte activation and antibody titers as the leading
adjuvanted malaria subunit vaccine, RTS,S/AS01B, without the use of additional adjuvants.[75]
In mouse models of malaria, rAd5s expressing P. yoelii derived CSP (Ad5PyCS) provided CSP

12

specific CD8+ T cell mediated protection from malaria challenge with live, intravenously
injected P. yoelii sporozoites.[59,77] Ad5PyCS induced CSP specific T cell responses greater
than what was observed in mice similarly vaccinated with irradiated sporozoites.[78] While
potent CD8+ T cell responses have been implicated as being important for protection against preerythrocytic antigens, Ad5 based vaccines are also capable of inducing humoral responses
against erythrocytic stage antigens. rAd5s expressing blood stage antigen candidates such as the
apical membrane antigen-1 (AMA-1) or the merozoite surface protein-1 (MSP-1), induced antiantigen antibody titers equivalent to adjuvant enhanced protein based vaccines. The Ad based
malaria vaccines were also able to successfully induce malaria antigen specific IgG in vaccinated
rabbits to levels that inhibited P. falciparum in erythrocytes growth assays.[79]
A rAd5 vaccine formulation combining two Ad vectors, with one expressing the CSP and
the other expressing the erythrocytic stage antigen AMA-1, has recently moved forward to safety
and efficacy studies in humans. The vaccine formulation was found to be well tolerated;
however, prime-boost vaccinations with the vaccine were not able to provide sterile protection
from malaria challenge (vaccinated volunteers exposed to mosquitoes infected with P.
falciparum). Additionally, the second vaccine dose appeared less immunogenic than the first,
suggesting that anti-Ad5 immune responses induced by the priming vaccination may have
prevented boosting of malaria antigen specific immune responses.[80,81,82] This notion has
been supported recently, as the repeated use of DNA based priming vaccinations (encoding CSP
and AMA-1) followed by boosting with rAd based malaria vaccines expressing the same
antigens provided for up to 27% protection from malaria challenge (i.e.: exposure to mosquitoes
infected with P. falciparum).[81]
1.11 Pre-existing Ad immunity:

13

In Africa and Southeast Asia over 50% of the population has significantly elevated antihAd5 neutralizing antibody titers.[83,84] Pre-existing immunity to wildtype hAd5 is common in
the sub-Saharan regions of Africa and correlates with weakened hAd5 vaccine vector induced
immune responses to rAd5 expressed antigens.[85] Pre-existing Ad5 immunity encompasses not
only hAd5 neutralizing antibodies, but cytotoxic T cell responses against hAd5 infected cells as
well.[86,87,88] For example, advanced generation E1 deleted Ad5 based vectors that are
additionally deleted for the Ad polymerase gene can allow for induction of robust antigen
specific immune responses in Ad5 immune animals, including non-human primates, a property
that may be due to avoidance of pre-existing cellular immune responses to the Ad polymerase
protein.[60,61,89]
1.12 Alternative rAd serotypes for use in malaria vaccines:
Although it is important to note that malaria typically affects young children and infants
in Africa, which rarely possess neutralizing antibody against hAd5, the use of alternative Ad
serotypes (other than Ad5) may afford increased levels of efficacy in an Ad5 immune
population.[48] As a result of the seroprevalence of hAd5, alternative serotypes of Ad are being
studied more frequently for use in HIV and malaria vaccines.
Use of subgroup B derived recombinant Adenovirus serotype 35 (rAd35) has become a
very popular alternative to rAd5 in vaccinations designed for use in areas of high Ad5
seroprevalence. Ad35 has a neutralizing antibody seroprevalence of less than 20% in malaria
endemic regions (Africa and Southeast Asia).[83,84] Much like Ad5, Ad35 can be made
replication defective by deletion of the Ad35 E1 genes, and E1 deleted Ad35 based vaccines
have been shown to be capable of stimulating potent malaria antigen specific adaptive immune
responses. For example, an Ad35 vaccine expressing the CSP (Ad35PfCS) stimulates potent CSP

14

specific B and T cell responses in mice equivalent to those induced by either Ad5PfCS or
RTS,S/ASO1B.[75] Comparison of Ad35 vaccines expressing P. yoelii CSP (Ad35PyCS) and
Ad5PyCS showed that both vaccines were capable of stimulating B and T cell responses. These
responses positively correlated with the vaccines reducing parasite infection in the liver of
vaccinated animals after challenge (injection) with purified live P. yoelii sporozoites, in this case
as measured by parasite rRNA levels present in the liver of challenged animals.[90] Furthermore,
Ad35PyCS induced similarly high responses in hAd5 immune mice; whereas Ad5PyCS could
not, as CSP responses were severely ablated in Ad5 immune mice treated with Ad5PyCS.[90]
Ad35 utilizes CD46 to gain entrance into human cells, which is ubiquitously expressed in
humans but is only expressed in the testis of mice. Therefore, results of research performed in
mice may be a poor predictor of efficacy or safety in humans.[91] However, Ad35PfCSP has
also been shown to be effective when used as a priming vaccine in rhesus macaques prior to
boosting with either RTS,S/ASO1B or Ad5PfCS.[47,92] Based upon these promising results,
several clinical trials are currently underway to assess the safety and efficacy of rAd35 based
malaria vaccine platforms in humans, inclusive of an Ad35PfCSP phase I trial also using
Ad26PfCSP in a heterologous prime boost based regimen.Ad serotype 26 (Ad26) is another rare
Ad serotype with seroprevalence below 20% in Africa and Southeast Asia.[83,84] Ad26 is a
member of Ad subgroup D, which uses a combination of CAR, CD46, and sialic acid to gain
entry into cells. Use of malaria targeted rAd26 vaccines in the context of heterologous prime
boost vaccinations with CSP, or Ad35 expressing CSP were able to induce potent, and long
lasting IFNγ+, TNFα+, CD8+ T cells specific for CSP in mice.[93]
In a similar vein to the use of alternative human Ad serotypes, rAd based malaria
vaccines derived from Simian derived Ad serotypes have also been utilized as alternatives to

15

Ad5 malaria vaccines, as there should theoretically be no pre-existing immunity to simian Ads in
the human population. For example, a single dose of a simian Ad (AdC9) expressing an
enhanced form of the P. berghei thrombospondin-related anonymous protein (ME.TRAP) (a
protein found on the surface of sporozoites) provided a potent Tem cell response and protection
from purified P. berghei sporozoite challenge in hAd5 seropositive mice.[94] Simian Ad based
vaccines also can serve as potent priming vaccinations in heterologous prime boost studies with
MVA, much like hAds.[95,96,97,98] For example, following priming vaccination with a Simian
Ad expressing ME.TRAP, boosting with an MVA expressing ME.TRAP, increased the
polyfunctionality of the resultant TRAP specific T cell responses, and increased protection from
purified P. berghei sporozoite challenge in a mouse model.[98] In vaccinations aimed at
inhibiting transmission of malaria parasites from human to mosquito host, simian Ads (ChAd63)
expressing the ookinete surface protein (Pfs25) were used as a priming vaccination, which were
then boosted with an MVA vaccine expressing the same antigen in a P. berghei mouse model of
malaria. Specifically, the ChAd63 and MVA based immunizations were just as effective at
eliciting anti-Pfs25 IgG, and at reducing oocyst intensity in a standard feeding assay as was
similar use of hAd5 and the MVA vaccines expressing the same antigen.[99] These results held
true in rhesus monkey studies where priming vaccinations with a simian Ad vaccine, followed by
boosting vaccinations with an MVA vaccine also expressing AMA-1, resulted in long lasting
multifunctional CD8+ T cell responses to AMA-1.[97]
In general, alternative serotype Ads have not proven particularly more immunogenic or
safer than Ad5 in Ad5 naïve subjects.[75,90] It should also be noted that alternative serotype Ads
can vary dramatically not only in their abilities to infect certain types of cells, but also in their
differential abilities to stimulate the innate immune system.[84,100,101] Therefore, extensive

16

biodistribution and safety studies for each serotype should be undertaken, as baseline data from
widespread use of rAd based on the Ad5 serotype may not be applicable.
1.13 Summary:
It has been over 30 years since the discovery that immunity to malaria can be obtained
prophylactically, yet development of a viable, highly efficacious malaria vaccine has not been
achieved to date. Though some in the malaria field may be disheartened by this drought of
progress, the studies and efforts to date confirm that an effective malaria vaccine can likely be
produced. As it currently stands, sporozoite based vaccine platforms may be an impractical
method by which to vaccinate the many millions of people in need of malaria protection. The
RTS,S platform can overcome some of these shortcomings, and though laudable, contemporary
RTS,S based vaccine regimens have only reached a 56% level of protection.[44]
Viral vector based malaria vaccine platforms provide an alternative means by which to
produce large amounts of potentially efficacious malaria vaccines. This dissertation describes my
contribution to important works performed in our lab focused on elucidation of Ad interactions
with the innate immune system, and then capitalizes upon these insights by development of
advanced, Ad based malaria vaccines that utilize rare Ad serotypes or express potent and novel
immunomodulators. These studies have contributed to improvements in Ad based malaria
vaccines that have the potential to close the gap between protective efficacy and ease of
production and inoculation, which together could one day help to achieve global malaria
eradication.

17

Chapter 2:
Adenovirus induced innate immune responses

Portions of this chapter are derived from the following three research articles that have been
previously published:
Complex interactions with several arms of the complement system dictate innate and humoral
immunity to adenoviral vectors. Gene Therapy (2008); 15: 1606-1617
Authors: Appledorn DM, McBride A, Seregin S, Scott, JM, Schuldt N, Kiang A, Godbehere S,
Amalfitano A: Schuldt NJ – assisted with qRT-PCR measuring liver transcriptome
dysregulation and performed ELISAs measuring Total IgG and subisotypes

CR1/2 is an important suppressor of Adenovirus-induced innate immune responses and is
required for induction of neutralizing antibodies. Gene Therapy (2009); 16: 1245-1259
Authors: Seregin SS, Aldhamen YA, Appledorn DM, Schuldt NJ, McBride AJ, Buhold M,
Godbehere SS, Amalfitano A: Schuldt NJ – assisted with qRT-PCR measuring liver
transcriptome dysregulation and performed ELISAs measuring Total IgG and subisotypes

Adenovirus capsid-display of the retro-oriented human complement inhibitor DAF reduces Advector triggered immune responses in vitro and in vivo. Blood (2010); 116(10): 1669-1677
Authors: Seregin SS, Aldhamen YA, Appledorn DM, Hartman ZC, Schuldt NJ, Scott J,
Godbehere S, Jiang H, Frank M, Amalfitano A: Schuldt NJ – assisted with construction of Ad5GFP-IX-dDAF-REO virus

18

2.1 Introduction:
rAd vectors have been used extensively as both gene transfer and vaccine vectors. While
Ad induction of the innate system is a major obstacle in its efficacy as a gene transfer vector, it
can be exploited to intensify an adaptive immune reaction to a transgene for the purpose of
directing immunity to that transgene. Despite the popularity of Ad based vectors very little is
actually known about specific interaction between Ad and the innate immune system or how Ad
induction of the innate immune systems leads to adaptive responses. Understanding the complex
interaction between Ads and the innate immune system is relevant to both gene transfer and
vaccine functions of Ad vectors. With this knowledge we can develop less immunogenic Ad
based gene transfer vectors and/or Ad based vaccine vectors that induce more immunogenic
responses against transgenes.
We have previously demonstrated the involvement of complement system and uncovered
the importance of TLR intracellular signaling in forming adaptive responses subsequent to Ad
exposures.[67,68,69,102,103] Here we focus on further elucidating interactions between the
complement system and rAds. The complement system serves to recognize various pathogens
through detection of PAMPs and then initiate a cascade of immune responses aimed at clearing
the pathogen. The complement system can be divided into three activation pathways; the
classical pathway (CP), the alternative pathway (AP), and the mannan-binding lectin pathway.
The classical complement pathway begins with the binding of antibody to pathogenic invaders.
Antibody bound to the surface of a pathogen forms a complex that is recognized by the C1qrs
protein complex. The C1r and C1s subunits of the complex become activated by crossproteolysis and recruit C4. C4 is then cleaved by C1s and binds the surface of the pathogen. C2
binds to C4 and is also cleaved by the activated C1s to form CP-C3 convertase. C3 convertases

19

then cleave C3 to assemble the C3-C5 convertase which leads to the cleavage of multiple C3 and
C5 molecules. Cleaved C5 binds with C6 and C7 and begins to form the membrane attack
complex. C8 binds to this complex and inserts itself into the surface of the pathogen and assists
in the assembly of multiple C9 proteins to form a pore on the pathogen surface. The membrane
attack complex disrupts the ionic and osmotic equilibrium leading to destruction of the pathogen.
Conversely, the alternative pathway recognizes pathogen motifs and results in direct binding and
activation of C3 independent of antibody binding. In the alternative pathway, C3 bound to the
pathogen membrane recruits Factor B which is then cleaved by Factor D resulting in the active
AP-C3 convertase. This complex then binds properdin, which stably links the complex to the
pathogen surface. Then, much like the classical pathway, the AP-C3 convertase cleaves multiple
C3 molecules, which can then bind the AP-C3 convertase and form the alternative pathway C5
convertase. From here the alternative pathway functions identically to the classical pathway. The
mannan-binding lectin pathway differs only in that carbohydrate motifs on bacterial pathogens
are recognized by mannose-binding lectin (MBL) and mannose associated serine protease
(MASP) molecules which directly activate C4 and C2 to create the C3 convertase in a similar
manner to the classical pathway. Unchecked complement activation can result in severe tissue
damage. Therefore, the complement system has multiple proteins (complement receptors (CR),
decay accelerating factor (DAF), membrane co-factor protein (MCP)) for the purpose of downregulating the complement system preventing damage and ultimately returning the system to a
monitoring mode.[104]
Activation of the complement system can induce a pathogen specific humoral
response.[64,105,106] Following opsonization of the pathogen by C3, B cells and dendritic cells
can bind to the pathogen through interactions with complement receptors CR1 and CR2. CR1 is

20

also involved in clearance of immune complexes and B cell maturation. In humans, CR2 is found
only on the surface of B cells, follicular dendritic cells, and thymocytes. Human CR2 binding to
C3d-opsonized pathogens has been shown to lower B cell activation 1000 fold.
Murine complement receptors (mCRs) 1 and 2 are products of alternative splicing from
the same gene and are known to be expressed on B cells and dendritic cells similar to human
CR2.[107] Although murine CR1/2 protein’s role in adaptive immunity is well studied, its role in
inhibiting/regulating murine complement has not been described, possibly because the parallel
acting protein, Crry, was suggested to play the predominant role in controlling complement
activation in most mouse models. We feel that the role of murine CR1/2 protein in complement
dependent innate immune responses may be more important than previously considered.
Our previous studies demonstrate that rAds activate the complement system in a C3dependent manner.[67,102] Here we attempt to determine what roles CP and AP of complement
have in creating humoral responses. We also analyze mCR1/2’s role in regulating innate and
humoral responses to rAd and to transgenes. Knowledge of anti-Ad humoral responses could be
used to improve upon current generations of Ad based vectors and may even result in the
development of rAds that can circumvent previous Ad immunity. This knowledge can also be
used to ablate the Ad-induced innate immune response to Ad-based gene therapy vectors making
them less immunogenic and therefore extending expression of the therapeutic transgene.
Our results led us to hypothesize that genetic engineering of the native Ad capsid in a
manner that minimized its capacity to activate the complement system would mitigate Ad capsid
induced, complement dependent, immune responses. To test this hypothesis we engineered a rAd
vector to display complement inhibitor DAF on the surface of the Ad capsid by fusing DAF to
pIX in the retro-oriented form of the human DAF protein (thereby displaying the primary DAF

21

amino acid sequence in a more native conformation relative to the Ad capsid surface). Human
DAF contains four complement control protein repeats (CCPRs) that decreases complement
activation by increasing the rate of decay of the classical and alternative C3 convertases
generated during pathogen mediated complement activation.[108] DAF-fusion to viral
capsid/envelope proteins has been shown to retain functionality of the CCPRs. For example,
baculovirus, retrovirus, and lentivirus have all been shown to successfully evade complement
mediated lysis when DAF was incorporated onto their surface.[109,110,111,112] Additionally it
has been shown that proteins displayed at the C-terminus of Ad5 protein IX can retain their
functionality (GFP, TK).[113,114] Here we created a novel cDNA encoding a retro-form of
human DAF in order to better simulate natural DAF orientation of human cell surfaces. This
“retro-DAF” was incorporated into an adenoviral capsid by fusion to pIX. Multiple studies have
previously demonstrated retention of functionality in retro-oriented proteins.[115,116,117,118]
We confirm that the native Ad capsid can “capsid-display” the natural complement inhibitor
decay accelerating factor DAF as a C-terminal fusion protein with the Ad capsid protein, pIX.
Ad capsid-display of DAF can minimize the induction of the complement system, and
complement dependent innate immune responses.

22

2.2 Results:
Ad-induced transcriptome dysregulation is modulated by factors of both classical and
alternative pathway and is regulated by CR1/2 in murine livers
We have previously described C3-dependent changes to the transcriptome of murine
livers after administration of rAd.[67] Here we further elucidate the role of complement in
transcriptome dysregulation by using CP-deficient mice (C4-KO and C1q-KO) and AP deficient
mice (FB-KO). Additionally, we investigate C3-dependence of other complement regulated Adinduced gene inductions previously untested. Six hours after systemic injection of 7.5x10

10

vp

per mouse of adenovirus, liver lysates were collected and tested by qRT-PCR for transcript
levels of TLR related proteins (MyD88, TRAF2-bp, TRIM30, CD14, and TLRs 2, 3, 6, and 9),
endothelial cell activation markers (intracellular adhesion molecule (ICAM), vascular cell
adhesion molecule (VCAM and E-selectin), interferon response markers (IRF-1 and OAS1a), and
negative regulators of cytokine signaling (SOCS-1 and SOCS-3) (Table 1). All of the above have
previously been shown to be dysregulated by rAd in a complement dependent manner.[67] While
we did find that dysregulation in all genes except E-selectin and IRF-7 was dependent upon
presence of a functional C3 protein, we were unable to determine if these changes were
dependent on AP or CP. Surprisingly, we observed significantly higher levels of ADAR, ICAM,
IRF-7, TLR2, and TRIM30 gene inductions in FB-KO mice and higher levels of CXCL9 and Eselectin in C1q-KO mice (P<0.05). Suggesting that FB and C1q are not only required to activate
transcription of multiple genes, but also negatively regulate genes. Lower levels of TLR2 were
observed in C4-KO mice while there was no change in C1q-KO mice indicating functional C4,
but not C1q, is required for complete induction of TLR2.

23

Table 1: Ad5-LacZ-induced gene expression in liver at 6 hpi (fold over mock). *Student’s t-tests
were completed between C57BL/6 and each respective genotype. Statistically significant
decreases are highlighted in light gray boxes. Statistically significant increases are highlighted in
dark gray boxes with bold numerals.

24

When gene induction was similarly tested using mCR1/2-KO mice in a larger panel of
genes we observed further induction of 13 genes when compared to Ad-injected wildtype mice at
6hpi. Indicating these genes (ADAR, GATA-3, ICAM1, JAK3, MYD88, NOD-1, OAS-1, SOCS-1,
TBK-1, TLR2, TLR3, TRAF2bp, and VCAM1) are Ad-induce, but mCR1/2 suppressed (Table 2).
Taken together with data from C3-KO experiments we can determine mCR1/2 plays a significant
role in down-regulating Ad-induced C3-dependent proinflammatory gene expression.
Ad-induced cytokine and chemokine responses are C3 dependent and are suppressed by
the presence of functional CR1/2
To describe the role of several complement proteins in Ad-induced innate and adaptive
responses we injected AP deficient mice (FB-KO), CP deficient mice (C4-KO and C1q-KO)
CR1/2-KO, and C3-KO mice intravenously with 7.5x10

10

vp per mouse. We then measured the

amounts of cytokines known to be induced by Ad at 6 hpi by multiplex bead-based enzyme
linked immunosorbant assay. Under these conditions only C3-KO mice demonstrated
significantly decreased amounts of Ad-induced RANTES, MCP-1, IL-12p40, G-CSF, and KC,
indicating Ad-induced cytokine and chemokine production are C3-dependent, but may only be
partially dependent on C4, C1q, and FB if at all (Figure 1). Conversely, CR1/2-KO mice
demonstrated increased levels of Ad-induced RANTES, MCP-1, and G-CSF, supporting a role
for CR1/2 in suppressing compliment activation after systemic Ad administration (Figure 2).
Inductions of rAd specific humoral responses are dependent on functioning AP, CP, and
CR1/2.
We assessed the role of complement in shaping neutralizing antibody responses in vivo
by measuring plasma borne anti-Ad neutralizing antibody in C3-KO, FB-KO, and C1q-KO mice
30 dpi after intravenous injection of 7.5x10

10

vp per mouse. We observed dramatically decrease
25

Table 2: Ad5-LacZ-induced gene expression in a liver (fold over C57BL/6_WT_Mock) The
numbers represent mean ± s.d. Statistical analysis was completed using one-way ANOVA with a
Student-Newman-Keuls post hoc test, P<0.05 was deemed a statistically significant difference.
Note, when significant P<0.001 was observed in majority of cases. N=4 for mock-injected
groups, N=6 for virus injected groups was used. Significant differences compared to
C57BL/6_WT_Mock are highlighted in grey color. Significant inductions of transcriptional
activation in CR1/2-KO_Ad5-LacZ group compared to WT_Ad5-LacZ group are indicated in
table with black frame and boldface font. Note that no significant differences were detected
between mock-injected WT and CR1/2-KO mice.

26

27

Figure 1 cont’d: Plasma cytokine and chemokine elevations after intravenous adenovirus (Ad)
injection. C57BL/6, C3-KO, Factor B knockout (FB-KO), C4-KO and C1q-KO mice were
10
injected with 7.5x10 viral particle (vp) per mouse of rAd5-LacZ. Plasma samples were isolated
at 6 h.p.i. and analyzed using a Bio-Plex bead-based enzyme-linked immunosorbent assay
(ELISA) assay (see Materials and methods). Bars represent mean±s.d. Student’s t-tests were
completed between mock-injected samples (†P<0.05 and ††P<0.01), virus-injected samples
(#P<0.05 and ##P<0.01) and between mock and virus-injected samples within the same
genotype (*P<0.05 and **P<0.01). N=3 for all samples tested.

28

29

Figure 2 cont’d: Murine complement receptor 1/2 (mCR1/2) mitigates Adenovirus (Ad)mediated cytokine and chemokine release in C57BL/6 mice. C57BL/6 wild-type (WT) and
11
CR1/2-KO mice were intravenously injected with 0.75x10 vp per mouse of Ad5-LacZ vector.
Plasma samples were collected at 1 and 6 h post-virus injection (hpi). Plasma samples were
analyzed using a multiplexed bead array-based system. Statistical analysis was completed using
two-way analysis of variance (ANOVA) with a Bonferroni post hoc test. The N=4 for mock
(phosphate-based saline; PBS)-injected animals, N=6 for virus-injected mice at 1 hpi and N=12
for virus-injected mice at 6 hpi. The bars represent mean ± s.d. *,**Indicate plasma cytokine
values that are statistically different from those in mock-injected animals of the same treatment
at the same time point (that is, CR1/2-KO_Ad5-LacZ group from CR1/2-KO_Mock group),
P<0.05, P<0.001, respectively. #,##Indicate statistically different values in CR1/2-KO_Ad5LacZ group compared to WT_Ad5-LacZ group at the same time point, P<0.05, P<0.001,
respectively.

30

anti-rAd neutralizing antibody in plasma of C3-KO mice. High variability or lack of assay
sensitivity did not allow us to determine if the observed decrease of rAd-specific neutralizing
antibody in C3-KO was due to AP, CP, or a combined result of both (Figure 3).
To determine if the observed decrease in rAd-specific neutralizing antibody was a result
of overall decreases in titers of IgG, we analyzed plasma for titers of total IgG (Figure 4). We
observed that total anti-Ad IgG was significantly decreased in both C3-KO and FB-KO mice.
We further characterized this response by analyzing the same plasma for sub-isotypes of IgG
(Figure 4). IgG 1 and IgG3 were both significantly lower in C3-KO mice, while IgG2c was
significantly higher in C3-KO mice. C1q-KO had significantly decreased levels of plasma borne
IgG1 and IgG2c. FB-KO mice only showed decreased titers of IgG3. IgG2c/IgG1 ratio is
believed to indicate relative contribution of the Th1/Th2 response. Based on the remarkably
different IgG2c and IgG1 tiers in C3-KO mice we hypothesized there may be a shift in the
Th1/Th2 balance. When we analyzed the IgG2c/IgG1 ratio we found an eight fold increase in
Th1 type antibody compared to Th2 antibody in C3-KO mice as compared to wildtype.
When we similarly analyzed mCR1/2 role in Ad-specific neutralizing antibody we found
mCR1/2-KO mice had significantly diminished capacity to make anti-Ad neutralizing antibody
that was nearly identical to what was observed in C3-KO mice. These data indicate C3dependent induction of neutralizing antibody could be mediated by interactions involving
mCR1/2 (Figure 5). Further analysis of total IgG and sub-isotypes also showed decreased titers
of total anti-Ad IgG, IgG1 and IgG3 similar to C3-KO mice. However, mCR1/2-KO mice also
showed a diminished capacity to induce titers of IgG2a, IgM, IgA, and IgG2b (Figure 6).
When we analyzed the same KO animal’s ability to induce humoral responses against Ad
expressed transgenes we found C3-KO mice actually had increased titers of total anti-transgene

31

Figure 3: Ad neutralizing antibody titers are C3-dependent. C57BL/6, C3-KO, Factor B
10
knockout (FB-KO) and C1q-KO mice were injected with 7.5x10 vp per mouse of rAd5-GFP
(N=3 for recombinant adenovirus (rAd)-naïve animals, N=4 for all immunized animals). Plasma
samples were isolated at 30 d.p.i. and analyzed for neutralizing antibodies using four successive
dilutions of plasma (see Materials and methods). Bars represent mean ± s.d. ‘**’ represents a
statistical difference, as measured by a homoscedastic t-test, in neutralizing antibody titers found
in plasma derived from C57BL/6 animals versus C3-KO animals (P<0.01).

32

33

Figure 4 cont’d: Anti-recombinant adenovirus (rAd)-specific antibodies are dependent on both
alternative pathway (AP) and classical pathway (CP) of complement. C57BL/6 (N=3 naive and
N=7 immunized), C3-KO (N=4 naive and N=9 immunized), Factor B knockout (FB-KO)
(N=4 naive and N=11 immunized) and C1q-KO (N=3 naive and N=5 immunized) mice were
10
injected with 7.5x10 vp per mouse of rAd5-GFP. (a) At 30 d.p.i., plasma was analyzed for
anti-rAd capsid-specific total IgG antibodies at the appropriate dilution (1:800). Bars represent
mean ± s.d. (b) Plasma was also analyzed for various IgG subclasses. Data points represent mean
± s.d. *P<0.05 and **P<0.01 indicate a statistical difference between C57BL/6 and each
respective genotype. (c) The IgG2c/IgG1 ratio, indicative of a Th1/Th2 response, was calculated
based on subclass titering.

34

Figure 5: mCR1/2-KO mice exhibit significantly reduced Adenovirus (Ad) capsid-specific
neutralizing antibodies titer. Three groups of mice were treated as described in Materials and
methods: Wild-type (WT)_mock (N=4), WT_Ad5-LacZ (N=5), CR1/2-KO_Ad5-LacZ (N=5).
Plasma samples were collected at 28 dpi and assayed for neutralizing antibodies using successive
dilutions (see Materials and methods). The error bars represent ± s.d. Statistical analysis was
completed using one-way analysis of variance (ANOVA) with a Student-Newman-Keuls post
hoc test, P<0.05 was deemed a statistically significant difference. *,**Indicate values,
statistically different from those in WT_mock-injected animals, P<0.05, P<0.001, respectively.
#,## Indicate statistically different values in CR1/2-KO_Ad5-LacZ group compared to
WT_Ad5-LacZ group, P<0.05, P<0.001, respectively.

35

36

Figure 6 cont’d: mCR1/2-KO mice exhibit significantly reduced Adenovirus (Ad) vector
capsid-specific humoral immune responses. Three groups of mice were treated as described in
Materials and methods: Wild-type (WT)_mock (N=4), WT_Ad5-LacZ (N=5), CR1/2-KO_Ad5LacZ (N=5). Plasma samples, collected at 14 dpi and 28 dpi, were analyzed for anti Ad capsidspecific total immunoglobulin M (IgM), IgA and IgG antibodies and various IgG subclasses. The
error bars represent±s.d. Statistical analysis was completed using two-tailed Student’s t-test to
compare two groups of virus-injected animals. ##Indicate statistically different values in CR1/2KO_Ad5-LacZ group compared to WT_Ad5-LacZ group, P<0.001. *,**Indicate values,
statistically different from animals of the same group at different time point, P<0.05, P<0.001,
respectively.

37

IgG with a strong bias of the Th1/Th2 response toward Th1 (Figure 7). mCR1/2 mice levels of
anti-transgene IgG were relatively identical to wildtype mice (data not shown).
Capsid display of DAF minimizes Ad dependent complement activation in vivo
Ad capsid mediated complement activation has been indirectly associated with toxicity,
as many of these toxicities can be avoided when Ad vectors are injected into C3-KO
mice.[66,67,101] Based on our results we hypothesized that genetic engineering of a rAd to
display a natural complement inhibitor (DAF) as a C-terminal fusion on an Ad-capsid protein
(pIX) would mitigate Ad capsid induced complement.
We constructed Ad viruses displaying DAF in a native (N terminus exposed to the
extracellular milieu) conformation by producing a synthetic cDNA encoding the primary amino
acid sequence of DAF in reverse order relative to the native DAF gene sequence. This was then
sub-cloned into the C-terminus of the pIX open reading frame resulting in a re-orientation of the
human DAF relative to the Ad capsid (Ad5-GFP-IX-dDAF_REO).
Animals were either mock injected or intravenously injected with 7.5x10

10

vp per mouse

with Ad5 expressing GFP (Ad5-GFP), Ad displaying GFP on pIX (Ad5-IX-GFP), or Ad5-GFPIX-dDAF_REO. 6hpi liver dysregulation was measured as performed previously. Multiple
transcripts were found to be significantly decreased in Ad5-GFP-IX-dDAF_REO treated
wildtype mice as compared to Ad5-GFP treated wildtype mice (CXCL-9, ICAM, IRF-7, IRF-8,
JAK-1, JAK-3, MyD88, NF-κB-RelA, OAS-1a, TBK-1, TLR-6, and TRAF2bp) (Table 3).
When we analyzed levels of cytokines in the plasma at 6hpi we found that mice treated
with Ad5-GFP-IX-dDAF_REO had significantly less induction of IL-12(p40) and MCP-1 (at
low dose) and MIP-1β (at high dose) as compared to conventional Ad5 vectors (Figure 8).

38

Figure 7: Anti transgene (GFP)-specific antibodies are C3-dependent. C57BL/6 (N=3 naive and
N=7 immunized), C3-KO (N=4 naive and N=9 immunized), Factor B knockout (FB-KO)
(N=4 naive and N=11 immunized) and C1q-KO (N=3 naïve and N=5 immunized) mice were
10
injected with 7.5x10 vp per mouse of rAd5-GFP. (a) At 30 d.p.i., plasma was analyzed for
anti-GFP-specific total IgG antibodies at the appropriate dilution (1:800). Bars represent mean ±
s.d. (b) Plasma was also analyzed for various IgG subclasses. Data points represent mean ± s.d.
*P<0.05 and **P<0.01 indicate a statistical difference between C57BL/6 and each respective
genotype. (c) The IgG2c/ IgG1 ratio, indicative of a Th1/Th2 response, was calculated based on
subclass titering.

39

11

Table 3: Medium dose of virus used for injection: 0.75x10 vp/mouse. The numbers represent
Mean ± SD. Statistical analysis was completed using One Way ANOVA with a StudentNewman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. N=4 for
mock injected groups, N=5 for virus injected groups. Significant differences compared to
WT_Mock are highlighted in light grey color. Significant reductions of transcriptional activation
compared to WT_Ad5-GFP group are indicated in table with black frame and boldface font;
significant induction of transcriptional activation compared to WT_Ad5-GFP group are indicated
by dark grey color. Gene names highlighted in bold were shown to be induced in C3 dependent
manner after systemic Ad injection.

40

41

Figure 8 cont’d: Ad5 vectors “capsid-displaying” retro-DAF complement inhibitor significantly
reduce Ad dependent activation of endothelial cells in C57BL/6 mice. C57BL/6 WT and C3-KO
11
11
mice were intravenously injected with (A) 0.75x10 vp/mouse (medium dose) or (B) 2x10
vp/mouse (high dose) of Ad5 based control and experimental vectors. Plasma samples, collected
at 6 hpi (N=6 for virus treated groups, N=4 for Mock injected groups) were analyzed using a
multiplexed bead array based quantitative system. The bars represent Mean ± SD. Statistical
analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test. *,
** - Indicate values, statistically different from those in Mock injected animals of both
genotypes, 30 p<0.05, p<0.001 respectively. # - Indicate significant reduction of EC activation as
compared to both WT_Ad5-GFP and WT_Ad5-IX-dGFP injected mice, p<0.05. Note that levels
of EC activation triggered by Ad5-GFP-IX-dDAF_REO novel Ad closely parallel the levels
observed in C3-KO mice treated with conventional Ad5-GFP vector (shaded bars).

42

However, multiple cytokines and chemokines known to be induced by rAds were not reduced by
display of DAF on the Ad capsid (G-CSF, RANTES, KC, and IL-6) (Figure 8).

43

2.3 Discussion:
In these experiments we focused upon elucidating Ad interactions with the complement
system, and how these interactions shape important Ad-induced immune responses. These
discoveries have implications in gene transfer protocols as well as Ad vaccines, as evidenced by
our successful display of DAF on the surface of an Ad capsid resulting in reduced Ad-induced
immune responses. We and others have demonstrated Ad opsonization by complement.[66,119]
Here we attempted to tease out which complement pathways are involved in multiple aspects of
Ad-induced innate immune responses.
We show that intravenous injection of rAd induces the transcription of multiple genes in
the liver in a C3-dependent manner inclusing MyD88, many TLRs, and endothelial activation
markers like VCAM and ICAM. However, we were unable to delineate the specific roles of CP
and AP in these responses. We did however detect a negative role for FB in regulation of ADAR,
IRF-7, OAS-1a, TLR2, and TRIM30 as well as a negative role for C1q in regulation of CXCL9
and E-selectin. These data suggest that CP and AP of complement actually modulate rather than
synergistically amplify these responses. Furthermore, we found that many of these responses are
suppressed by the presence of functional mCR1/2 inclusing ADAR, ICAM, MyD88, OAS-1a,
SOCS-1, TLR2, TLR3, TRAF2bp, and VCAM.
We and others have shown that many cytokines and chemokines are induced as a result
of intravenous Ad administration.[67,103,120,121] We show here that at 6 hpi, Ad inductions of
G-CSF, MCP-1, and RANTES are dependent on the presence of functional C3. We were
however unable to find a distinct role for CP or AP in these responses. While the variability of
the mice and sensitivity of the assay played roles in our inability to delineate specific roles for
CP or AP in mediating these responses, the results may suggest redundancy in the CP and AP

44

pathways when sensing Ads, a hypothesis that can only be confirmed in future, more expansive
studies. However, we were able to show that mCR1/2 is responsible for down-regulating G-CSF,
MCP-1, and RANTES.
Given that cytokine and chemokine secretion modulates T and B cell proliferation and
activation and, as we have shown, many Ad-induced chemokines and cytokines are complement
dependent, we sought to elucidate the role of complement in initiating humoral immune
responses. Here we show that induction of Ad specific neutralizing antibody is dependent on
functional C3. While we could not determine the impact of CP (C1q) or AP (FB) on induction of
Ad specific neutralizing antibody due to assay insensitivity, there are indications that both CP
and AP are required for maximal antibody induction. For example, further analysis revealed that
induction of total anti-Ad IgG and IgG3 was dependent upon functional C3 and FB.
Additionally, analysis of immunoglobulin sub-isotypes revealed that CP (C1q-KO)-deficient
mice were unable to stimulate titers of IgG1 or IgG2c equivalent to wildtype mice, indicating a
role for CP in stimulating the maximal anti-Ad antibody response. We found striking differences
in Th1/Th2 antibody ratios in C3-KO animals. These data suggest that complement plays a
significant role in modulating the balance between the Th1 and Th2 antibody responses to Ad
vectors. This dramatic skewing of the IgG2a/IgG1 ratio could be responsible for the observed
decrease in anti-Ad neutralizing antibody titers in these animals, as it has been shown that a
balanced IgG2a/IgG1 ratio is associated with improved protection.[122] However, it is possible
that C3-KO mice produce more IgG2c compensatory to the lack of C3, as IgG2c (analogous to
IgG2a in function) is known to assist in the binding of C3 to a pathogen. While functionally C3
was required for an anti-Ad IgG response, complement does not interfere with anti-transgene
humoral inductions. In fact, transgene specific antibody titers were actually higher in C3-KO

45

mice. On the other hand, similar Ad treatments of mCR1/2-KO mice induced lower titers of
transgene specific antibody. Decreased antigen expression by Ads in these latter animals is likely
responsible for mCR1/2-KO animal’s low titers of anti-transgene antibody as we observed
equivalent transduction but lower levels of transgene expression at 24 hpi and 28 dpi (data not
shown). It is possible that the altered cytokoine and chemokine responses indirectly resulted in
decreased CMV-driven expression of the transgene from the Ad vector, a phenomenon
previously noted by our group in other studies.[100]
Unexpectedly, mCR1/2-KO mice had Ad specific neutralizing antibody titers equivalent
to mock injection despite the presence of stronger Ad-induced innate responses. Lack of anti-Ad
neutralizing antibody titers in both mCR1/2-KO and C3-KO mice positively correlated with lack
of B cell activation as measured by analyzing percentages of splenocytes that are CD19+ CD69+
(data not shown). Since Ad treated C3-KO exhibited decreased levels of proinflammatory
cytokines while mCR1/2-KO demonstrated increased levels in many of the same cytokines when
compared to wildtype mice, cytokine levels per se cannot be the reason for the lack of anti-Ad
neutralizing antibody. Instead, we propose a mechanism where C3-opsonization of Adenovirus
allows for interaction with mCR1/2 on B cells activating the B cell to produce anti-Ad
neutralizing antibody. Therefore, blockade of C3 opsonized Ad from interaction with CR1/2
(like in C3-KO mice) could be preventing activation of B cells that would otherwise produce
anti-Ad capsid antibody while still allowing induction of transgene specific antibody responses
by T cell dependent B cell activation.
Despite the necessity of complement to have been activated before DAF can “decay” C3
convertases, we were able to provide evidence that recombinant DAF fusion to the Ad-capsid
can prevent complement activation. As evidenced by intravenous injection of “DAF-displaying”

46

Ads inhibition of complement mediated responses inclusive of observed reductions in proinflammatory gene expression and decreased cytokine release when compared to intravenous
injection of control Ads.
These data cumulatively describe rAd interactions with multiple components of the
complement system and their involvement in generating humoral responses. We have confirmed
the roles of C3, C4, FB and mCR1/2 in regulating multiple Ad-induced innate immune
responses. Furthermore, we describe a novel Ad successfully engineered to actively reduce Adtrigged innate immune responses by modulating interactions of the Ad capsid with the
complement system. It is important to understand these multiple interactions, as this knowledge
will foster safer and more efficacious use of Ad-based vectors in the same vein as our “DAFdisplaying” Ad vector.

47

Chapter 3:
Efficacy when utilizing Adenovirus serotype 4 and 5 vaccines expressing Circumsporozoite
protein in naïve and Ad5 immune mice

48

3.1 Introduction:
Despite use of prophylactic medications and vector control, malaria continues to be one
of the world’s most deadly health concerns claiming the lives of almost 1 million people
annually. The protozoan parasite, P. falciparum, accounts for about 90% of these deaths.[1]
Numerous P. falciparum targeted vaccine studies are currently underway in efforts to eliminate
this dangerous killer. The P. falciparum derived CSP is the most studied and commonly used
antigen for the purpose of developing a vaccine against malaria.[30,123,124,125,126] CSP is
abundant on the surface of the sporozoite, and is also present in the plasma membrane and
cytosol of plasmodium infected hepatocytes.
Of the several malaria vaccine vectors that target CSP, the most successful to date is a
vaccine formulation that consists of a novel fusion protein between the Hepatitis B surface
protein (HBsAg) and CSP, and additional adjuvants. This formulation, referred to as
RTS,S/AS01B, is currently in a phase 3 clinical trial.[127] This vaccine has been able to confer
protection to 56% of vaccinated individuals.[36,37,38,39,44,46,124] Although promising, the
results also suggest that more potent immune responses may be required to achieve higher levels
of protection. For this reason other vectors and immunogenic strategies incorporating CSP are
being pursued in efforts to develop a highly efficacious, malaria specific vaccine.
Recombinant adenovirus serotype 5 (rAd5) based vaccines are important in this regard as
they have been confirmed to elicit potent adaptive responses against expressed
transgenes.[71,128,129] Multiple studies have utilized rAd5s genetically engineered to express
CSP in human and mouse models of malaria.[75,90,126] However, pre-existing Ad5 immunity is
common in regions where malaria is endemic, and the presence of neutralizing antibodies against
Ad5 has been shown to hinder Ad5 based vaccine efficacy.[83,84,85] We and others have

49

hypothesized that the use of alternative serotype based rAds may induce improved immunogenic
responses to antigens irrespective of pre-existing Ad5 immunity, for example in HIV vaccine
development.[130,131] Use of alternative serotype based Ad vectors can serve other important
purposes aside from stimulating immune responses in Ad5 immune patients. Heterologous prime
boost regimens where the prime and boost vaccinations are derived from two different Ad
serotypes based vaccines can provide greater inductions of immunity than homologous prime
boosting with a single Ad serotype based vaccine.[47,92,97]
In this context, Ad4 based vectors may be promising for use in malaria specific
applications. The efficacy and safety of Ad4 vaccine platforms has been established. For
instance, as the principal serotype causing Acute Respiratory Disease (ARD) in military recruits,
an orally administered, live Ad4 virus was utilized for decades in vaccinations of recruits against
ARD.[132,133,134,135] More recently, Ad4 based vaccines have been successfully utilized in
HIV vaccine strategies in dog and chimpanzee models.[130,131] Here we analyze the ability of
an Ad4 based malaria specific vaccine expressing CSP to stimulate potent immune responses
when used in homologous or heterologus prime boost regimens with an Ad5 vaccine also
expressing CSP, both in the context of Ad5 naïve and Ad5 immune animals.

50

3.2 Results:
rAds of serotype 4 and serotype 5 were engineered to express a codon optimized form of
CSP using methods previously described.[100,136] Four vaccination regimens were utilized; 1.
Ad5-CSP/Ad5-CSP, 2. Ad5-CSP/Ad4-CSP, 3. Ad4-CSP/Ad4-CSP, and 4. Ad4-CSP/Ad5-CSP,
where the Ad serotype used in the priming vaccination is immediately followed by the serotype
of the boosting vaccination in each vaccine regimen or group. Initially, (day 0) Ad naïve
BALB/cJ mice were injected IM with either Ad4-CSP or Ad5-CSP (1x10

10

vp/mouse) (n=10).

14 days later 5 mice from each treatment group received a homologous boost (same Ad-CSP
serotype vaccine) of 1x10

10

vp/mouse, the other 5 mice from the same group received a

heterologous boosting vaccination of 1x10

10

vp/mouse with the alternative Ad-CSP serotype

vaccine. 28 days after the priming vaccinations, splenocytes were harvested from the animals
and stimulated with the CSP derived peptide (NYDNAGTNL) and the number of IFNγ secreting
splenocytes were quantified by ELISpot. While every vaccine treatment resulted in a significant
increase in the numbers of CSP responsive INFγ secreting splenocytes when compared to nonvaccinated animals, the Ad4-CSP/Ad5-CSP heterologous prime boosting vaccine treatment
group induced significantly higher numbers of IFNγ secreting splenocytes than any other
treatment group (Figure 9A). These results were further supported by intracellular staining with
antibodies against CD3, CD8, and IFNγ, as the percentage of CSP responsive CD3+ CD8+
IFNγ+ cells present in splenocytes derived from mice vaccinated with the Ad4-CSP/Ad5-CSP
regimen were significantly higher when compared to splenocytes from animals treated with the
other vaccination strategies (Figure 9B). Intracellular staining was also performed to enumerate

51

Figure 9: Ad4-CSP/Ad5-CSP heterologous prime boost results in improved quality of T cell
10
10
response. A prime injection of 1x10 vp/mouse Ad4-CSP followed by a boost of 1x10
vp/mouse of Ad5-CSP resulted in significantly more IFNγ secretion by splenocytes measured by
ELISpot (A) and CD3+ CD8+ T cells measured by flow cytometry (B). Ad4-CSP/Ad5-CSP was
the only treatment to stimulate a significantly higher percentage of TNFα production as
compared to unvaccinated animals (C). Cells were stained with CD3-APC-Cy7, CD8-Alexa
flour700, TNFα-PE-Cy7, IFNγ-FITC, and Granzyme B-APC. Bars represent ± standard error.
Statistical analysis was completed using One Way ANOVA with Student-Newman-Keuls posthoc test, *, **, *** denotes significance over naïve, P<0.05, P<0.01, P<0.001.

52

the frequency of TNFα and Granzyme B producing CD8+ T cells present in the spleens of the
variously vaccinated animals. Again, the Ad4-CSP/Ad5-CSP experimental vaccination regimen
appeared to confer the most robust immune responses against CSP, as it was the only treatment
to induce significantly higher percentages of CSP responsive CD3+ CD8+ TNFα+ cells as
compared to non-vaccinated animals (Figure 9C). Interestingly, none of the vaccination
strategies induced significantly higher percentages of CSP responsive CD3+ CD8+ Granzyme
B+ cells as compared to non-vaccinated animals; however, animals from the Ad5-CSP/Ad4-CSP
vaccination group had significantly lower percentages of CD3+, CD8+, Granzyme B+ T cells as
compared to all other treatment groups (Figure 9D).
As detected by use of the NYDNAGTNL tetramer, each of the vaccination regimens
induced significantly higher percentages of CSP specific CD3+ CD8+ T cells in the spleen as
compared with non-vaccinated control animals (p<0.001) (Figure 10A). Of the four groups, the
Ad4-CSP/Ad5-CSP heterologous prime boosting regimen induced the lowest percentage of
CD3+ CD8+ tet+ T cells, a decrease that was statistically significant as compared to both the
Ad5-CSP/Ad5CSP and the Ad5-CSP/Ad4-CSP treatment groups (p<0.01; p<0.05 respectively).
When peripheral blood mononuclear cells (PBMCs) from the vaccinated mice were similarly
analyzed, again all groups of vaccinated mice had significantly increased numbers of CD3+
CD8+ tet+ T cells present as compared to non-vaccinated mice. However, the Ad4-CSP/Ad4CSP treated animals elicited the lowest percentages of CD3+ CD8+ tet+ T cells of the four
groups, this decrease reaching statistical significance when this group was compared to both the
Ad5-CSP/Ad5-CSP and Ad5-CSP/Ad4-CSP treatment groups (p<0.05 for each group) (Figure
10B).

53

Figure 10: Ad5-CSP/Ad5-CSP vaccination resulted in higher percentage of tetramer positive
CD8+ T cells than Ad4-CSP/Ad5-CSP in the spleen. Splenocytes and PBMCs were collected
two weeks after final vaccination. All vaccination regimens resulted in significantly higher
percentage of CD3+ CD8+ NYDNAGTNL tetramer positive T cells in the spleen (A) and
circulating blood (B) as measured by flow cytometry, cells were stained with CD8-Alexa
flour700, CD3-APC-Cy7, and CSP (NYD)-Tetramer-PE. Ad5-CSP/Ad5-CSP stimulated a
higher percentage of CD3+ CD8+ tet+ than Ad4-CSP/Ad5-CSP treated animals in the spleen (A)
and higher percentage of CD3+ CD8+ tet+ than Ad4-CSP/Ad4-CSP in the circulating blood (B).
Bars represent ± standard error. Statistical analysis was completed using One Way ANOVA with
Student-Newman-Keuls post-hoc test, *, **, *** denotes significance over naïve, P<0.05,
P<0.01, P<0.001.

54

Ad vectors are known to elicit strong Tem cell responses thought to be due to more
persistent antigen production. This is important in the context of a malaria vaccine as Tem cell
responses have been shown to be beneficial in protecting against liver stage malaria.[25] We
compared the magnitude of CSP-specific central memory and effector memory CD8+ T cell
responses that each of the various prime boost regimens induced in splenocytes and PBMCs
harvested 14 days after the boosting vaccinations. All prime boost regimens demonstrated much
higher percentages of CSP specific Tcm and Tem cells than was observed in non-vaccinated
animals as indicated by the percent of CD127+ CD62L+ and CD127+ CD62L- tet+ T cells
present in the splenocytes (Figure 11B-C). We also analyzed the percentage of CSP specific Tcm
and Tem cells circulating in the blood and found the Ad5-CSP/Ad4-CSP vaccination group was
the only group that had a significantly higher percentage of CSP specific Tcm cells in circulating
blood when compared to non-vaccinated animals, while all vaccinated animals had higher
percentages of CSP specific Tem cells present in this compartment as compared to nonvaccinated animals (Figure 11D-E). When we analyzed memory phenotypes by gating on
tetramer positive cells first and then gating for CD127 and CD62L we found that the tetramer
positive cells of all groups had similar memory phenotypes as defined by comparison of the
percentages of tet+ cells that were Tem and those that were Tcm cell (data not shown).
Splenocytes from all treatments were analyzed for the presence of anti-Ad4 and/or antiAd5 antigen specific IFNγ secreting T cells by ELISpot. There was no significant cross
stimulation between the two serotypes detected by this assay, as animals that received Ad4CSP/Ad4-CSP treatment had significantly less Ad5 specific IFNγ secreting cells than all other
vaccination regimens, and were not significantly different than naïve animals. Likewise, animals
that were vaccinated with Ad5-CSP/Ad5-CSP had significantly less Ad4 specific IFNγ secreting

55

Figure 11: Memory responses triggered by vaccination with homologous and heterologous
prime boost regimens utilizing Ad4-CSP and Ad5-CSP in Ad naïve mice. Splenocytes (B-C) and
PBMCs (D-E) were collected two weeks after final vaccination. Cells were stained for CD62LV450, CD127-PerCP Cy5.5, and CSP (NYD) tet-PE. CSP specific central memory T cells were
determined as CD62L+ CD127+ cells that are tet+ and effector memory cells are CD62Llo
CD127+ cells that are tet+. Provided above is an example of gating (A). Bars represent ±
standard error. Statistical analysis was completed using One Way ANOVA with StudentNewman-Keuls post-hoc test, **, *** denotes significance over naïve, P<0.01, P<0.001.

56

Figure 12: Ad4-CSP/Ad4-CSP and Ad5-CSP/Ad5-CSP vaccinated animals have no significant
cross stimulation of splenocytes. Splenocytes were collected 14 days post final vaccination and
were stimulated with either heat inactivated Ad4-Null or heat inactivated Ad5-Null. Animals
treated with Ad5-CSP/Ad5-CSP were not significantly different from naïve animals when
stimulated with heat inactivated Ad4-CSP as measured by IFNγ secretion by ELISpot. Likewise,
animals treated with Ad4-CSP/Ad4-CSP were not significantly different from naïve animals
when stimulated with heat inactivated Ad5-CSP as measured by IFNγ secretion by ELISpot.
Bars represent ± standard error. Statistical analysis was completed using One Way ANOVA with
Student-Newman-Keuls post-hoc test, *, **, *** denotes significance over naïve, P<0.05,
P<0.01, P<0.001.
57

cells than animals that received Ad4-CSP injections and were also not significantly different than
naïve animals (Figure 12).
We measured how prime boost vaccinations combining Ad4-CSP and Ad5-CSP might
affect CSP specific antibody production as compared to homologous prime boosts using the
same vectors. Plasma was collected from BALB/cJ mice injected with the four prime boost
regimens and 28 days post initial injection was tested by ELISA for total anti-CSP IgG antibody
levels. Mice from the Ad4-CSP/Ad4-CSP vaccination group demonstrated significantly lower
plasma levels of IgG anti-CSP relative to unvaccinated animals at the 1:100 dilutions (p<0.05)
(Figure 13). All other vaccination regimens induced significantly higher levels of anti-CSP IgG
as compared to both the non-vaccinated animals and animals receiving the Ad4-CSP/Ad4-CSP
regimen (p<0.001) (Figure 13). Similar trends were observed when sub-isotyping analysis was
performed for anti-CSP IgG1, IgG2a, IgG2b, and IgG3 levels (Figure 14). We also analyzed the
IgG2a/IgG1 ratio as an indirect assessment of Th1 vs. Th2 immune responses in animals treated
with the vaccine regimens; however the Th1/Th2 ratio was not significantly different with use of
any of the vaccination regimens (Figure 15).
To assess the efficacy of Ad4 based vaccination regimens to induce functional, CSP
specific cytolytic T cell responses, we measured CSP specific cytotoxic T lymphocyte killing in
vivo. BALB/cJ mice were vaccinated with the homologous and heterologous prime boost
regimes as described above. 28 days after the initial vaccination, splenocytes from naïve mice
were collected and incubated with either a high concentration of CFSE (10µM) and
NYDNAGTNL peptide or a low concentration of CFSE (1µM) and a non-specific peptide.
Stained and peptide pulsed splenocytes were then mixed at equal quantities and injected
intravenously into vaccinated or non-vaccinated animals. After 18 hours, CSP specific cell

58

Figure 13: All vaccinations stimulated significantly higher anti-CSP total IgG than unvaccinated
and AD4-CSP/Ad4-CSP vaccination in Ad naïve animals. Plasma was collected 14 days post the
final vaccination. Plasma was diluted 1:100, 1:200, and 1:400 and measured for total IgG against
CSP by ELISA. Bars represent ± standard error. Statistical analysis was completed using One
Way ANOVA with Student-Newman-Keuls post-hoc test, *, **, *** denotes significance over
naïve, P<0.05, P<0.01, P<0.001. ### denotes significance over Ad4-CSP/Ad4CSP treatment,
P<0.001.

59

Figure 14: Sub-isotype analysis of IgG antibody from plasma of mice vaccinated with
heterologous and homologous prime boost regimens utilizing Ad4-CSP and Ad5-CSP. Plasma
was collected 14 days post final vaccination. The amount of CSP specific subisotype IgG1 (A),
IgG2a (B), IgG2b (C), and IgG3 (D) were analyzed by ELISA. Bars represent ± standard error.
Statistical analysis was completed using One Way ANOVA with Student-Newman-Keuls posthoc test, *, **, *** denotes significance over naïve, P<0.05, P<0.01, P<0.001.

60

Figure 15: Th1 to Th2 ratio (IgG2a/IgG1) of plasma from vaccinated Ad naïve animals. Plasma
was collected 14 days post final vaccination. The amount of CSP specific IgG subisotypes was
measured by ELISA. Th1 to Th2 ratio was determined by dividing O.D. values from IgG2a and
IgG1. Bars represent ± standard error. Statistical analysis was completed using One Way
ANOVA with Student-Newman-Keuls post-hoc test, * denotes significance over naïve, P<0.05.

61

killing was measured in the spleens of the vaccinated animals by flow cytometry. Only animals
that received the Ad5-CSP/Ad5-CSP and Ad4-CSP/Ad5-CSP vaccination regimens achieved
significantly elevated levels of CSP specific cell killing as compared to non-vaccinated animals
(p<0.01) (Figure 16).
Given the high seroprevalence of wildtype Ad5 in adults living in malaria endemic
regions, we also analyzed the ability of these homologous and heterologous prime boost vaccine
regimens to elicit potent CSP specific adaptive responses in animals that were made Ad5
immune prior to receipt of the various vaccine regimens. BALB/cJ mice received two injections
14 days apart of 1x10

10

vp/mouse of an Ad5 vector that does not express a transgene (Ad5-

Null). It has been previously demonstrated that two immunizations with 1x10

10

vps of rAd5-

Null vector induced Ad5 neutralizing antibodies titers that were >1:200, a level that closely
parallels levels of pre-existing Ad5 immunity noted in human populations.[60] 14 days after the
last injection of Ad5-Null, Ad5-immune animals received 1x10

10

vp/mouse prime injection of

either Ad4-CSP or Ad5-CSP followed by either a heterologous or homologous boost 14 days
after the initial priming vaccination. 28 days after the prime vaccination plasma, PBMCs, and
splenocytes were collected. Splenocytes were stimulated as before with NYDNAGTNL and were
analyzed for CSP specific IFNγ secreting cells by ELISpot. Ad5-CSP/Ad4-CSP, Ad4-CSP/Ad4CSP, and Ad4-CSP/Ad5-CSP vaccinated Ad5 immune animals all had significantly higher
numbers of NYDNAGTNL responsive IFNγ secreting cells present when compared to the Ad5CSP/Ad5-CSP cohort or the non-vaccinated animals (Figure 17). However, as compared to Ad5
naive animals, overall induction of NYDNAGTNL responsive, IFNγ secreting splenocytes was
notably diminished in Ad5 immune animals despite use of Ad4-CSP in some of the regimens

62

Figure 16: Ad5-CSP/Ad5-CSP and Ad4-CSP/Ad5-CSP both stimulated more percent specific
killing than unvaccinated animals. 14 days post vaccination splenocytes from naïve animals were
pulsed with either NYDNAGTNL and high concentration of CFSE or non-specific peptide and
low concentration of CFSE. Stained splenocytes were combined in equal amounts and roughly 8
million cells were injected into vaccinated animals IV. After 20 hours splenocytes from
vaccinated mice were collected and analyzed by flow cytometry to assess the amount of
NYDNAGTNL specific killing. % Specific killing = 1((%CFSEhigh/%CFSElow)immunized/(%CFSEhigh/CFSElow)non-immunized. Bars represent ±
standard error. Statistical analysis was completed using One Way ANOVA with StudentNewman-Keuls post-hoc test, **denotes significance over naïve, P<0.01.

63

Figure 17: IFNγ secretion of cells from Ad5 immune mice vaccinated with heterologous and
homologous prime boost regimens utilizing Ad4-CSP and Ad5-CSP. All vaccinations were
capable of stimulating significantly more IFNγ secreting cells than unvaccinated and Ad5CSP/Ad5-CSP vaccinated Ad5 immune animals. Splenocytes were collected 14 days after final
vaccination. Splenocytes were then stimulated with CSP dominant antigen NYDNAGTNL and
IFNγ secretion was measure by ELISpot. Bars represent ± standard error. Statistical analysis was
completed using One Way ANOVA with Student-Newman-Keuls post-hoc test, *, ** denotes
significance over naïve, P<0.05, P<0.01.

64

(Table 4). The reductions prevented detection of significant differences between the treatments
when ICS of the splenocytes for IFNγ, TNFα, and Granzyme B was undertaken (Figure 18A-C).
We analyzed PBMCs and splenocytes for CD3+ CD8+ T cells that were CSP peptide
tetramer binding by flow cytometry and found that all vaccinated Ad5 immune animals,
including Ad5-CSP/Ad5-CSP vaccinated animals, had a significantly higher percentage of CSP
specific CD3+ CD8+ tet+ T cells present in both the spleen and peripheral blood (Figure 19AB). All treatments including Ad5-CSP/Ad5-CSP also had significantly higher percentages of
tetramer positive Tcm cells when compared to the non-vaccinated animals in both the spleen and
in the peripheral blood (Figure 20B, D). Only mice from the Ad5-CSP/Ad-5CSP treatment group
had higher frequencies of CSP specific Tem cells in their spleens as compared to non vaccinated
mice (Figure 20C). Ad5-CSP/Ad5-CSP, Ad5-CSP/Ad4-CSP, and Ad4-CSP/Ad4-CSP
vaccination groups all stimulated significantly more Tem cells in the peripheral blood than nonvaccinated and Ad4-CSP/Ad-5CSP vaccinated, Ad5 immune-animals (Figure 20E). When we
analyzed T cells for memory phenotypes we found that homologous prime boost vaccinations
biased the T cell responses toward Tcm rather than Tem cell phenotype memory in Ad5 immune
mice (Figure 21). We also evaluated the in vivo cytolytic activity of CD8+ T cells in Ad5 preimmune mice. No significant increase in percent specific killing was observed in any treatment
groups when compared to unvaccinated Ad5 immune animals (data not shown).We analyzed
undiluted plasma collected from unvaccinated animals and Ad5 immune animals from each
vaccination regimen for anti-CSP total IgG by ELISA. From the undiluted plasma we found that
Ad5-CSP/Ad4-CSP, Ad4-CSP/Ad4-CSP, and Ad4-CSP/A5-CSP vaccinated animals all had
significantly more CSP specific total IgG than non-vaccinated animals (p<0.001) and the Ad5
immune animals homologously vaccinated with Ad5-CSP (p<0.001) (Figure 22).

65

Table 4: Decreased Mean Spot Forming Cells in Ad5 Immune animals. All vaccinations,
inclusive of homologous Ad4-CSP prime boost, elicited fewer IFNγ secreting splenocytes in
Ad5 immune animals as measured by ELISpot. The table displays the mean numbers of spot
forming cells per 500,000 splenocytes from spleens of Ad5 naïve and Ad5 immune mice treated
with each prime boost regimen.

66

Figure 18: CD8+ T cell activation in Ad5 immune animals vaccinated with heterologous or
homologous prime boost regimens utilizing Ad4-CSP and Ad5-CSP. Splenocytes were collected
from vaccinated animals 14 days post the final vaccination. Cells were stained with CD8-Alexa
flour700, CD3-APC-Cy7, TNFα-PE-Cy7, IFNγ-FITC, and Granzyme B-APC and analyzed by
flow cytometry for INFγ secreting CD3+ CD8+ T cells (A), TNFα secreting CD3+ CD8+ T cells
(B), and granzyme B+ CD3+ CD8+ T cells (C). Bars represent ± standard error. Statistical
analysis was completed using One Way ANOVA with Student-Newman-Keuls post-hoc test, *,
**, *** denotes significance over naïve, P<0.05, P<0.01, P<0.001.

67

Figure 19: All vaccinations in Ad5 immune animals resulted in significantly higher percentages
of CD8+ CSP tetramer positive cells than unvaccinated Ad5 immune animals. Splenocytes and
PBMCs were collected two weeks after final vaccination. All vaccination regimens resulted in
significantly higher percentage of CD3+ CD8+ NYDNAGTNL tetramer positive T cells in the
spleen (A) and circulating blood (B) as measured by flow cytometry, cells were stained with
CD8-Alexa flour700, CD3-APC-Cy7, and CSP (NYD)-Tetramer-PE. Bars represent ± standard
error. Statistical analysis was completed using One Way ANOVA with Student-Newman-Keuls
post-hoc test, * denotes significance over naïve, P<0.05.

68

Figure 20: Memory responses triggered by vaccination with homologous and heterologous
prime boost regimens utilizing Ad4-CSP and Ad5-CSP in Ad5 immune animals. Splenocytes (BC) and PBMCs (D-E) were collected two weeks after final vaccination. Cells were stained for
CD62L-V450, CD127-PerCP Cy5.5, and CSP (NYD) tet-PE. CSP specific central memory T
cells were determined as CD62L+ CD127+ cells that are tet+ and effector memory cells are
CD62Llo CD127+ cells that are tet+. Provided above is an example of gating (A). Bars
represent ± standard error. Statistical analysis was completed using One Way ANOVA with
Student-Newman-Keuls post-hoc test, *, **, *** denotes significance over naïve, P<0.05,
P<0.01, P<0.001.

69

70

Figure 21 cont’d: Memory Phenotype. Homologous prime boost regimens favor a Tcm cell
phenotype in the peripheral blood of Ad5 immmune mice. Memory phenotype was defined as
percent of CSP (NYD) tetramer positive cells that are Tem cells (CD62Llo CD127+) and
percentage that are Tcm cells (CD62L+ CD127+) as opposed to percent of Tem and Tcm cells
that are Tet+. PBMCs were collected on day 14 post final injection and stained according to the
above defined memory phenpotype. Example of gating appears above the graphs (A). Percentage
of Tcm Cells was significantly higher PBMCs from homologously boosted animals in Ad5
immune mice (B). There was no significant difference in the percentage of Tem cells present
between any of the groups (C). Bars represent ± standard error. Statistical analysis was
completed using One Way ANOVA with Student-Newman-Keuls post-hoc Test.

71

Figure 22: All vaccinations stimulated significantly higher anti-CSP total IgG than unvaccinated
and Ad5-CSP/Ad5-CSP vaccination in Ad5 immune animals. Plasma was collected 14 days post
the final vaccination. Plasma was measured undiluted for total IgG against CSP by ELISA. Serial
dilutions were not possible as the undiluted plasma data point required the majority of the plasma
collected from an animal. Bars represent ± standard error. Statistical analysis was completed
using One Way ANOVA with Student-Newman-Keuls post-hoc test, *** denotes significance
over naïve, P<0.001.

72

3.3 Discussion:
Ad4 has many qualities that make it a desirable choice as a vaccine platform, inclusing an
ability to induce robust early innate responses and a high rate of infectivity.[100] Ad4 also has a
long history of use as a vaccine vector, dating back to 1971 when Ad4 was used as an enteric live
Ad4 vaccine by the military to vaccinate recruits against acute respiratory
disease.[132,133,134,135] For these reasons Ad4 has already been utilized as a potential HIV
vaccine vector in several large animal HIV models.[130,131] In this study, we chose to
investigate how Ad4 based vaccines targeting malaria might be incorporated into malaria vaccine
regimens, either in isolation, or in combination with a first generation Ad5 vaccine platform.
The combination of a priming vaccination of Ad4-CSP boosted by Ad5-CSP in Ad5
naïve animals results in induction of higher levels of activated CD8+ T cells than any other
vaccination regimen used in this study. The activated T cells induced by an Ad4-CSP priming
vaccination boosted by Ad5-CSP were also capable of potent CSP specific killing to levels that
are equivalent to use of Ad5-CSP homologous vaccinations, despite the fact that animals
homologously vaccinated with Ad5-CSP had higher levels of CSP specific CD8+ T cells
detectable by staining with antibodies for CD3, CD8, and tetramer specific for CSP. These data
suggest that combined use of Ad4-CSP priming followed by an Ad5-CSP boosting vaccination
induced more efficient cytotoxic T cell killers than those induced by homologous prime boost of
Ad5-CSP. If the aim is to provide a large quantity of CSP reactive T cells, a homologous prime
boost vaccination of Ad5-CSP should be utilized. However, if one wishes to elicit IFNγ and
TNFα secreting T cells specific for CSP a priming vaccination of Ad4-CSP followed by boosting
vaccination of Ad5-CSP should be selected.

73

While Ad4-CSP provided benefit when utilized as a priming vaccination prior to boosting
with Ad5-CSP, Ad4-CSP was not as capable as Ad5-CSP at stimulating potent CSP specific
immune responses when utilized in a homologous prime boost regimen. Additionally, boosting a
prime of Ad5-CSP with Ad4-CSP induced very poor CSP specific immune responses in general.
Diminished induction by Ad4 based vaccines of transgene-specific IgG has been previously
observed by us, and the effect was suggested to be a result of the Ad4 capsid inducing high
levels of IFN-β, interfering with the CMV promoter used to drive expression of the transgene.
Interference with the CMV promoter may ultimately reduce the length of time the CSP antigen is
expressed from Ad4 vaccine platforms, and may explain the decrease in efficacy when Ad4-CSP
is utilized in isolation or as a boosting vaccination in our current studies.[100]
To obtain protection from liver stage malaria, the presence of Tem cells are thought to be
an essential element and a significant correlate to predicting vaccine efficacy.[25] Among our
vaccination regimens, the induction of CSP specific Tem cell and Tcm cells were grossly similar
when Ad4 or Ad5 based CSP vaccine treatments were conducted in Ad naïve animals. However,
vaccination regimens did not perform equally when we tested in vivo cytotoxicity, as only the
animals receiving an Ad4-CSP priming vaccination boosted by Ad5-CSP, or animals receiving
the homologous Ad5-CSP prime-boost vaccination regimens resulted in detection of
significantly improved levels of CSP specific cell killing, as compared to non-vaccinated
animals. Since 14 days post vaccination is within the time frame when peak of CD8+ effector T
cell responses may be present, and CD8+ T cell contraction usually does not take place until
after three weeks post vaccination, it is likely that the observed CSP specific killing is a result of
the lingering presence of CD8+ effector T cells, rather than induction of Tem cells.

74

Another reason we undertook these studies was to determine if the use of serologically
distinct Ad4 based malaria targeted vaccines might allow for improved induction of CSP
immune responses, relative to repeated use of Ad5 in Ad5 immune animals. Indeed, Ad4-CSP
was capable of stimulating the induction of significantly more CSP antigen specific IFNγ
secreting splenocytes, as well as higher levels of anti-CSP antibodies when incorporated into
prime boost regimens in Ad5 immune mice (as compared to use of the Ad5-CSP vaccine), albeit
to levels well below what was observed when the Ad4-CSP vaccine was utilized in Ad5 naïve
animals. Furthermore, a more stringent functional analysis suggested that use of Ad4 in Ad5
immune animals also did not result in improved induction of CSP specific cytotoxic activity as
compared to non-vaccinated animals. Although Ads are segregated into subgroups based on antisera neutralization there is evidence that T cell responses can react across subgroups.[137] These
cross reactive T cells could be responsible for the decrease of immunogenicity observed in Ad5
immune animals homologously vaccinated with Ad4-CSP. Therefore, a priori assumptions that
different subgroups of adenovirus are not cross reactive must be reconsidered in light of data
demonstrating diminished immunogenicity when using some alternative serotypes in Ad5
immune animals. Likely, mild cross reactivity not measured by conventional means (such as the
neutralizing antibody and ELISpot based assays used in our study) is still capable of diminishing
immunogenicity of two very distinct serotypes on the bases of perhaps only a few cross reactive
epitopes.[138]
Prior immunity to Ad5 did not appear to affect the ability of any of the Ad4-CSP or Ad5CSP vaccination regimens to induce CSP specific CD8+ T cells. The percentages of CSP
specific tetramer positive CD8+ T cells observed in Ad5 immune animals were similar to
percentages observed in Ad naive animals. All vaccinations appeared to increase the percentage

75

of CD8+ T cells specific to the CSP epitope NYDNAGTNL in Ad5 immune animals in spite of
the observed ablation in cytokine production by these same cells. Similarly, all vaccinations
except an Ad4-CSP prime boosted by Ad5-CSP resulted in high percentages of CSP specific
Tem cells in the circulating blood. Heterologous prime boost vaccinations even appear to trend
toward a Tem cell phenotype while homologous vaccinations biased toward a Tcm cell
phenotype. However, none of these responses correlated with evidence of improved in vivo CSP
specific cytotoxic T cell killing when either of these vectors was deployed into Ad5 immune
animals. Ad5 cross reactivity with Ad4 appears to result in the ablation of IFNγ and TNFα
secreting CSP specific cytotoxic T cells induction by Ad4-CSP based vaccines, despite allowing
for the induction of high percentages of CSP specific T cells.
Our data shows that there exists a complex interaction between immune responses
triggered by a rAd4 (subgroup E) and those triggered by rAd5 (subgroup C), each expressing the
same malaria antigen, in this instance, CSP. While combined use of Ad4-CSP priming
vaccinations with Ad5-CSP boosting vaccinations results in the induction of greater numbers of
CSP responsive cytokine secreting, cytotoxic T cells in Ad5 naïve animals, there appears to be
interference between the two seemingly distinct Ad subgroups, resulting in diminished
inductions of transgene specific immune responses in Ad5 immune animals despite the use of the
Ad4 platform. Future studies need to be performed to further elucidate the mechanism behind
Ad4’s decreased ability to stimulate immune responses in an Ad5 immune background. Based on
our results it is important that future use of other alternative serotypes be scrutinized under
similarly stringent assay conditions to ascertain their true effectiveness in overcoming preexisting Ad5 immunity.

76

Acknowledgments: We wish to thank Michigan State University Laboratory Animal support
facilities for their assistance in the humane care and maintenance of the animals utilized in this
work, the NIH Core Tetramer Facility at Emory University for manufacturing the
NYDNAGTNL tetramer, and the Michigan State University flow cytometry facility for their
assistance with the multiple experiments.

77

Chapter 4:
Vaccine platforms combining Circumsporozoite protein and potent immune modulators,
rEA or EAT-2, paradoxically result in opposing immune responses

This chapter is the edited version of a research article that was published in Public Library of
Science, Volume 6, Issue 8 (e24147), August 2011
Authors: Nathaniel J. Schuldt*, Yasser A. Aldhamen*, Daniel M. Appledorn, Sergey S.
Seregin, Youssef Kousa, Sarah Godbehere, Andrea Amalfitano
* indicates both authors contributed equally to this manuscript
Nathaniel J. Schuldt: Engineered Ad5-CSP virus, performed all injections, ELISA, ELISpot
assays, in vivo CTL assay, and wrote the manuscript.

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4.1 Introduction:
Some of the most successful malaria vaccine studies to date have attempted to induce
adaptive immune responses to the P. falciparum CSP. Induction of potent cellular immune
responses to CSP by a prophylactic malaria vaccine could potentially eradicate both sporozoites
and infected hepatocytes, potentially stopping the infection before clinical symptoms occur. The
leading malaria subunit vaccine, RTS,S/AS01B, stimulates both anti-CSP antibody as well as a
CSP specific cytotoxic T cell response, although initial formulations without the adjuvant
AS01B stimulated less cytotoxic T cells and were less effective. These results underscore the
importance of potent CSP specific CD8+ T cell responses. Multiple studies have also
demonstrated the importance of CD8+ T cell responses in combating murine malaria
infections.[22,26,59,139] It has even been shown that passive transfer of CD8+ T cells specific
for murine malaria CSP antigen resulted in 100% survival upon sporozoite challenge.[26]
Furthermore, Ads expressing murine malaria derived CSP have been shown to be capable of
providing cytotoxic T cell mediated inhibition of parasite liver stage development up to 93%.[59]
rAds are particularly good at stimulating CD8+ T cell responses against a transgene. Ad
induction of potent innate responses through TLR signaling can be exploited to drive robust
adaptive responses against a transgene.[71,72] Many studies, outlined previously in this
dissertation, have demonstrated Ads abilities to induce potent CMI responses. Although Ad
induced responses have not yet provided significant protection from malaria in humans.[81] The
high seroprevalence of Ad5 throughout Africa has been implicated as a reason for poor Ad5
based malaria vaccine protection, which has led many to pursue alternative Ad serotypes. This
reaction may be imprudent given recently published data showing low seroprevalence of Ad5 in
children of Africa, which make up the primary group at risk for death from malaria.[48]

79

Alternative serotypes could still be utilized to create potent boosting vaccinations and should
continue to be studied for this purpose. However, as our studies and those of others have
confirmed, the utilization of alternative serotype rAds or chimp derived rAds as a vaccine
platform may be dangerous due to the increased innate toxicity of non-Ad5 rAds and crossreactivity between sub-groups.[101] Therefore, improving the capability of rAd5 vaccines to
induce more potent antigen specific adaptive immune responses is a high priority in the drive to
find an efficacious malaria vaccine. In this study we sought to improve CSP specific CMI
responses induced by CSP-expressing rAd5s by co-expression of innate immune response
modulating proteins by the vaccine platform.
The innate immune system plays an integral role in augmenting and/or shaping the
induction of antigen specific adaptive immune responses.[63] A group of cellular receptors that
recognize a variety of pathogen derived antigens, known as the TLRs, play a crucial role in
identifying PAMPs, and then augmenting adaptive responses to those PAMPs. We have
previously confirmed that rAds ability to induce innate and adaptive responses are dependent
upon several TLR’s, and that many of these responses are primarily dependent upon MyD88
functionality.[69,140] Given data demonstrating TLR adaptor molecules can enhance vaccine
induced adaptive responses to viral and tumor antigens, we hypothesized that further stimulation
of TLR pathways by inclusion of TLR agonists could potentially improve adaptive responses to a
rAd expressed antigen.[141] To this end we demonstrated that when rAd5 vaccines engineered to
express a novel TLR agonist derived from Eimeria tenella, rEA, are co-administered with rAd5
vaccines expressing a target antigen there was significant improvement in the ability of the
vaccine to induce antigen specific cellular immune responses.[142]

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rEA was discovered while searching bovine small intestine for anti-cancer agents. They
uncovered a non-bovine protein capable of stimulating the secretion of large amounts of IL-12
from murine dendritic cells in vitro, as well as increased systemic levels of IL-12 and other Th1
cytokines in vivo after intra-peritoneal injection of the protein in mice.[143] The protein was
later found to be homologous to a highly conserved Eimeria surface protein with unknown
function, although it has been suggested the protein’s purpose may be to modulate the immune
system.[143] Uses of rEA in murine in vivo models of disease have been shown to increase
protective immunity and human clinical trials have demonstrated rEA induced innate immune
responses.[144,145] Given our previous research demonstrating improved adaptive responses to
an Ad expressed antigen following co-injection of an Ad expressing rEA, we hypothesized that
use of this platform could likewise induce adaptive responses to CSP.[142]
Similarly, we recently discovered that augmentation of the adaptive immune response can
also be achieved by the expression of an adaptor protein found in dendritic cells called Ewing’s
sarcoma-related transcript-2 (EAT-2) that signals through signaling lymphocyte activation
molecule (SLAM) receptors. When Ad-EAT-2 is co-injected with a rAd5 expressing HIV
antigen (Gag) improved antigen specific T cell responses are observed including, increased
antigen specific proliferation, IFNγ secretion, and improved cytotoxic activity.[136,146] SLAM
family of receptors are expressed in hemopoietic cells and signal through homotypic self-sensing
by their extracellular domains to activate immune cells.[147,148] EAT-2 is a member of the
SLAM associated adaptor family of protein and is only found in dendritic cells, macrophages,
and natural killer cells.[147,148] EAT-2 signaling has yet to be fully elucidated; however, it is
known that EAT-2 possesses a src homology 2 domain (SH2) that interacts with the
immunoreceptor tyrosine-based switch motif (ITSM) of the SLAM receptor as well as CD2-like

81

receptor activating cytotoxic cells (CRACC) to ultimately activate the cell.[149] It has been
shown that expression of EAT-2 from a rAd5 results in more mature and activated dendritic cells
that lead to an enhanced T cell response.[146] The observed enhanced T cell responses are likely
due to improved synapses between dendritic cells and T cells mediated by the presence of more
co-stimulatory cell surface receptors present on the dendritic cell surface. Further studies will
need to be performed to assess this platforms ability to confer protection.
In this study, we determined what the impact of modulation of innate immune responses
during CSP presentation would have upon induction of subsequent CSP specific immune
responses in vivo. Unexpectedly, use of a TLR agonist uncovered a potent immunosuppressive
activity inherent to the combined use of rEA and CSP, an activity that mitigated induction of any
CSP specific adaptive immune responses. Fortunately, expression of the SLAM receptors
adaptor protein EAT-2 overcame and enlightened possible mechanisms underlying the
paradoxical CSP immunosuppressive activity we uncovered when stimulating TLR pathways.

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4.2 Results:
CSP expressed from rAd5 based vaccines can induce CSP specific B and T cell responses
A rAd5 based vaccine expressing a codon optimized form of the CSP (Ad5-CSP) was
constructed (Figure 23), and a dose study was initially performed to assess at what dose optimal
CS specific B and T cell responses could be detected. BALB/cJ mice were intra-muscularly (IM)
7

9

injected with varying doses of Ad5-CSP ranging from 1x10 to 1x10 vps per animal. At 14 dpi
splenocytes derived from the vaccinated animals were harvested, and exposed to an
immunodominant CSP derived peptide (NYDNAGTNL). Significantly increased numbers of
7

IFNγ secreting splenocytes were noted in Ad5-CSP vaccinated mice treated with 5.0x10 to
9

8

1.0x10 vps, with peak numbers achieved at a dose of 1.0x10 vps/mouse. Higher Ad5-CSP
doses resulted in a trend of decreasing, though not significantly less, numbers of spot forming
cells (SFCs) (Figure 24A). This phenomenon has also been observed by other groups, however
an explanation for this phenomenon has yet to be forwarded.[77,150] These finding were further
supported in individual splenocytes derived from the vaccinated animals, where CD8+ T cell
IFNγ, TNFα, and IL-2 levels were measured by intracellular staining (ICS) using flow
8

cytometry. IFNγ and TNFα production peaked at the 5.0x10 vps/mouse with similar decreasing
9

trend occurring at 1.0x10 vps/mouse. IL-2 producing cells were much lower in percentage, with
7

the greatest numbers being observed as the 5.0x10 vps/mouse dose (Figure 24B). We will
further discuss the importance of these findings in the Discussion section.
To determine if Ad5-CSP is also capable of stimulating B cell responses specific to the
CSP, plasma was collected from the vaccinated mice and assayed by a IgG CSP specific ELISA

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Figure 23: Ad5-CSP construction. Recombinant Ad5-CSP was constructed by creating a codon
optimized CSP sequence flanked by NheI sites in a pGA4 plasmid. The sequence was excised
with the Nhe1 and cloned into a pShuttle containing a CMV expression cassette. The resulting
plasmid was linearized with PmeI and recombined with pAdeasyI Ad5 vector in BJ 5183 cells.
pAd5-CSP was then purified and linearized with PacI enzyme and transfected into HEK 293
cells from which Ad5-CSP was purified using cesium gradients.

84

85

Figure 24 cont’d: Ad5-CSP Stimulates CSP specific T and B cell responses. CSP specific
immune responses increase in an Ad5-CSP dose dependent manner. BALB/cJ mice (n=3) were
7
9
injected IM with Ad5-CSP ranging from 1x10 to 1x10 vps/mouse, increasing by half logs. 14
days post injection splenocytes and plasma were collected. (A) ELISpot assays were performed
to quantify IFNγ secreting cells from splenocytes stimulated with CSP peptide, NYDNAGTNL,
ex vivo. (B) IFNγ, TNFα, and IL-2 expression by splenocyte derived CD3+ CD8+ T cells was
analyzed by flow cytometry following ex vivo stimulation with NYDNAGTNL. (C) Total IgG
against CSP was assessed by ELISA. The bars represent mean ± SD. Statistical analysis was
completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, *,**,***
denotes significance over naïve, p<0.05, p<0.01, p<0.001.

86

at 14 dpi. Significant increases in CS specific IgG were detected in all mice treated with Ad57

CSP with a peak response occurring at 5.0x10 vps/mouse, demonstrating Ad5-CSP is capable
of stimulating a B cell response against CSP even at the lowest dose used in the study (Figure
24C).
The use of a TLR agonist unexpectedly reduces CSP specific cellular immune responses
Previous experiments confirm that expressing the TLR agonist rEA from an Ad vector
stimulates innate immune responses during Ad mediated vaccination, responses that positively
correlated with improved induction of antigen specific adaptive immune responses against
several antigens, such as the HIV antigen, Gag.[142] In this study, we sought to utilize rEA to
improve induction of CSP specific immune responses. We first confirmed that expression of rEA
along with CSP facilitated induction of pro-inflammatory innate immune responses, responses
we had noted in our previous studies of rEA.[142] Plasma cytokine levels at 6 hours post
injection (hpi) in mice co-injected intravenously (IV) with either 3.75x10
3.75x10

10

10

vps of Ad5-CSP and

vps Ad-GFP/rEA were compared to responses measured after identical co-injections

utilizing an Ad-GFP expressing vector (that does not express rEA) as a control (Figure 25) . We
observed significantly higher levels of IL-6, IL-12(p40), G-CSF, MCP-1, MIP-1β, RANTES,
KC, and TNFα in mice treated with Ad5-CSP+Ad-GFP/rEA as compared to control virus treated
animals, as well as, mock infected animals (Figure 25).
To assess the impact that these early increases in cytokine and chemokine responses had
7

on cell mediated immune (CMI) responses to CSP we IM co-injected 5x10 vps/mouse of Ad57

CSP and 5x10 vps/mouse of Ad-GFP/rEA and compared the induction of CS specific adaptive
7

immune responses to those noted in our control animals receiving 5x10 vps/mouse of Ad5-CSP
87

Figure 25: TLR agonist, rEA, induced innate cytokines 6 hours post injection. Co-injection of
Ad-GFP/rEA and Ad5-CSP stimulated robust expression of innate cytokines and chemokines as
10
compared to the control vaccine. BALB/cJ mice were injected IV with either 3.75x10
10

vps/mouse of Ad5-CSP+Ad-GFP or 3.75x10 vps/mouse Ad-GFPrEA+Ad5-CSP. Plasma was
collected at 6 hours post injection. Plasma cytokine/chemokine levels were measured with a
mouse multiplexed bead array based quantitative system. The bars represent mean ± SD.
Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls posthoc test, *,** denotes significance between treatments, p<0.05, p<0.01.

88

7

and 5x10 vps/mouse of Ad-GFP IM, or mock infected mice. Splenocytes derived from mock
vaccinated animals did not show the presence of CSP specific CMI responses while Ad5CSP+Ad-GFP confirmed induction of CSP specific CMI responses using ELISpot analysis
(p<0.05) (Figure 26A). However, despite the rEA enhanced activation of the innate immune
responses noted in Figure 25, ELISpot analysis of splenocytes derived from Ad5-CSP+AdGFP/rEA vaccinated animals confirmed a profound lack of induction of average CSP specific
CMI responses, responses that were essentially identical to CS responses measured in naïve mice
(p>0.05) (Figure 26A). Previously we have not observed an ablation of CMI responses when
CSP was co-administered with Ads expressing other antigens at these low doses, further
suggesting that this effect may be specific to simultaneous TLR stimulation (Figure 27). Despite
there being no significant differences between CSP responses in Ad5-CSP+Ad-GFP/rEA treated
animals and naïve animals, we did note that in one Ad5-CSP+Ad-GFP/rEA animal there was
some evidence of an elevated CSP specific response, independently verifying that this group did
in fact receive viable Ad5-CSP vector (Figure 26A).
Augmentation of the innate immune responses via SLAM adaptor EAT-2, improves CSP
specific T-Cell responses
Based upon the loss of CSP responsiveness after utilizing TLR mediated augmentation
along with CSP antigen vaccination we hypothesized that the CSP may have an ability to
mitigate induction of beneficial innate immune responses in the context of excessive, TLR
pathway mediated activation as the ablated immune responses were only observed after Ad6

GFP/rEA doses exceeded 5x10 vp/mouse (Figure 28). To attempt to test this hypothesis, we
made use of a recently described, alternative method for augmenting induction of antigen

89

Figure 26: Immuno-modulating proteins conversely affect IFNγ secreting splenocytes. Covaccination with Ad5-CSP and Ad-EAT2 dramatically increases IFNγ secreting splenocytes in
response to stimulation with CSP epitope, NYDNAGTNL. BALB/cJ mice were injected IM with
7
7
7
either 5x10 vps/mouse of Ad5-CSP and 5x10 vps/mouse Ad-GFP or 5x10 vps/mouse of Ad57

CSP and 5x10 vps/mouse of either (A) Ad-GFP/rEA (n=5) or (B) Ad-EAT2 (n=6). Splenocytes
were collected 14 days post co-injection. ELISpot were performed on the splenocytes of these
mice stimulated with NYDNAGTNL peptide to assess the amount of IFNγ secreting cells. The
bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a
Student-Newman-Keuls post-hoc test,* Denotes significance over naïve animals, p<0.05.
Representative figures of two independent experiments.

90

Figure 27: CSP expression does not interfere with antigen specific immune responses against
other transgenes at low doses. Co-vaccination with Ad-gag+Ad5-CSP did not result in decreased
5
gag specific immune responses.BALB/cJ mice were injected with 5x10 vp/mouse of Ad-gag
7

5

7

and 5x10 vp/mouse of Ad5-CSP or 5x10 vp/mouse of Ad-gag and 5x10 vp/mouse of AdGFP. Splenocytes were collected 14 dpi and assayed by ELISpot for CSP peptide
(NYDNAGTNL) specific IFNγ secretion (A) or gag peptide (AMQMLKETI) specific IFNγ
secretion (B). The bars represent mean ± SD. Statistical analysis for Supplemental Figure 3A
included other peptides tested from the peptide library that are not displayed in the graph. Two
Way ANOVA with Student-Newman-Keuls post-hoc test (A) or One Way ANOVA with a
Student-Newman-Keuls post-hoc test (B) were utilized for statistical analysis. **,*** denotes
significance between treatments, p<0.01, p<0.001.

91

7

Figure 28: Ad-GFP/rEA combined with 5x10 vp/mouse of Ad5-CSP begins to display a
6

diminished CSP specific CMI response after a dose of 5x10 vp/mouse. Only after the dose of
6

Ad-GFP/rEA exceeds 5x10 vp/mouse do we observe a diminished CS specific CMI response
7

when combined with 5x10 vp/mouse of Ad5-CSP. BALB/cJ mice were injected with doses
6

8

7

ranging from 5x10 to 5x10 vp/mouse of Ad-GFP/rEA combined with 5x10 vp/mouse of Ad5CSP. Splenocytes were collected 14 dpi and were analyzed by flow cytometry for
NYDNAGTNL tetramer+ CD3+ and CD8+ cells (A) or ELISpot for CSP specific IFNγ secretion
(B). Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls
post-hoc test, *** denotes significance between treatments, p<0.01, p<0.001.

92

specific adaptive immune responses, utilizing Ad mediated co-expression of a SLAM receptor
signaling pathway adaptor, EAT-2, along with a targeted antigen.[146] To accomplish this we
7

7

co-injected 5x10 vps/mouse of Ad5-CSP and 5x10 vps/mouse of Ad-EAT2, and compared the
induction of CS specific adaptive immune responses to those noted in the control mice receiving
7

7

5x10 vps/mouse of Ad5-CSP and 5x10 vps/mouse of Ad-GFP IM, as well as mock vaccinated
mice. Again, splenocytes were collected at 14 dpi and stimulated with the CS derived peptide
NYDNAGTNL ex vivo. In dramatic contrast to our previous results utilizing the Ad-GFP/rEA
and Ad5-CSP vaccination strategy, splenocytes from mice co-treated with Ad5-CSP and AdEAT2 had significantly more IFNγ secreting cells than splenocytes from both mock injected
mice as well as mice co-treated with the control vaccine (p<0.05) (Figure 26B). Given these
results, we sought to further characterize the EAT-2 dependent improvement in CS specific
immune responses by flow cytometry. Peripheral blood mononuclear cells (PBMC) derived from
the vaccinated animals were stained with CD3 and CD8 fluorescent antibodies, as well as a
NYDNAGTNL peptide loaded tetramer. Ad5-CSP+Ad-EAT2 treated mice had significantly
higher percentages of CSP specific tetramer positive CD8+cells present in their PBMCs than the
percentage noted in the Ad5-CSP+Ad-GFP control group (p<0.001) (Figure 29A). CD3+ CD8+
splenocytes were additionally analyzed for IFNγ and perforin by ICS using flow cytometry. The
percent of CD3+ CD8+ cells that secreted IFNγ was significantly higher in Ad5-CSP+Ad-EAT2
treated mice as compared to Ad5-CSP+Ad-GFP treated control (p<0.05) (Figure 29B). The
percent of CSP peptide specific CD3+ CD8+ perforin+ cells also tended to be higher in animals
given the Ad-EAT2+Ad5-CSP vaccination cocktails however this did not reach statistical
significance (Figure 29C). To confirm that the differences in the responses observed are not a

93

Figure 29: Co-expression of CSP and EAT-2 stimulates more potent CSP specific CMI
responses. Co-vaccination with Ad5-CSP and Ad-EAT2 resulted in increased NYDNAGTNL
tetramer positive CD8+ T cells as well as improved IFNγ secretion from CD8+ T cells. BALB/cJ
7
7
mice (n=6) were co-injected IM with 5x10 vps/mouse of Ad5-CSP and 5x10 vps/mouse of Ad7

7

EAT2 or 5x10 vps/mouse of Ad5-CSP and 5x10 vps/mouse of Ad-GFP. (A) Peripheral Blood
Mononuclear Cells (PBMCs) were stained with CD8-Alexa Flour700, CD3-APC-Cy7, and CSP
(NYD)-Tetramer. (B-C) Intracellular staining was performed on splenocytes after stimulation
with NYDNAGTNL peptide. Cells were stained with CD8-Alexa Flour700, CD3-APC-Cy7,
ViViD, IFNg-APC, and Perforin-PE antibodies. The bars represent mean ± SD. Statistical
analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, *,
**, *** denotes significance over naïve animals, p<0. 05, p<0. 01, p<0.001.

94

result of GFP antigens competing with CSP antigens, but are in fact a direct result of the
expression of EAT-2 we injected mice with either Ad5-CSP+Ad-GFP or Ad5-CSP + an empty
Ad vector (Ad-Null). We observed no differences between the treatments, indicating GFP does
not interfere with induction of CSP specific CMI responses (Figure 30).
Increased breadth of CMI responses to a pathogen derived protein has been shown to be
beneficial relative to eventual protection against actual pathogen challenge.[151,152,153] To
detect CMI responses against other peptides present within the CSP (and therefore to gauge the
breadth of response against the whole CSP) we generated a CSP specific peptide library. This
library consists of 15 mer peptides that overlap each other by 5 amino acids and spans the nonrepeating regions of the full length CSP. At 14 dpi, pooled splenocytes derived from the control
or experimental groups of vaccinated animals were stimulated ex vivo with one 15mer peptide
per well. Mice co-vaccinated with Ad5-CSP and Ad-GFP/rEA had an overall lower breadth of
response as is evident by the number of wells with more than 15 spots (Figure 31A). In contrast
to the response seen in rEA treated animals, animals co-vaccinated with Ad5-CSP and Ad-EAT2
demonstrated a dramatic increase in breadth of response to CS derived peptides when similarly
analyzed (Figure 31B).
Co-injection of Ad5-CSP and Ad-EAT2 improves the cytolytic activity of CSP specific T
cells in vivo
To better assess the functional consequence of the improved CS specific CMI responses
noted by expression of EAT-2, we stimulated splenocytes from naïve mice, mice vaccinated with
the control vaccine, and mice vaccinated with Ad5-CSP+Ad-EAT2 with NYDNAGTNL ex vivo,
then analyzed them by flow cytometry for CD3+, CD8+ T cells that were also positive for a
degranulation marker, CD107a. Both control treated and Ad5-CSP+Ad-EAT2 treated mice

95

Figure 30: Expression of GFP does not interfere with CSP specific CMI responses. Co-injection
of Ad-GFP does not interfere with Ad5-CSP initiated CSP specific CMI responses. BALB/cJ
7
mice were co-injected with 5x10 vp/mouse of Ad-GFP and 5x107 vp/mouse of Ad5-CSP or
7

5x107 vp/mouse of Ad-Null and 5x10 vp/mouse of Ad5-CSP. Splenocytes were collected 14
dpi and cells were measured for NYDNAGTNL tet+, CD3+, CD8+ T-cells. Both treatments had
a higher percentage of CSP specific tet+, CD3+, CD8+ T-cells than Naïve with no difference
observed between Ad5-CSP+Ad-Null and Ad5-CSP+Ad-GFP. Statistical analysis was
completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, *** denotes
significance between treatments, p<0.01, p<0.001.

96

97

Figure 31 cont’d: Co-expression of CSP and EAT-2 increases the breadth of response against
CSP. Increased breadth of response against CSP epitopes was observed in mice co-vaccinated
with Ad5-CSP and Ad-EAT2 as compared to the control vaccine. Splenocytes from groups of
five BALB/cJ mice were collected and pooled together from 14 days post injection with either
innate modulating treatments or control. ELISpots were performed to measure IFNγ secreting
cells when stimulated with a CSP peptide library made up of 52 15mers that overlap by 5 a.a. on
either side. Wells that contained more than 15 spots were counted and compared between
7
treatment groups (inset). (A) Mice were co-injected IM with 5x10 vps/mouse of Ad5-CSP and
7

7

7

5x10 vps/mouse of Ad-GFP/rEA or 5x10 vps/mouse of Ad5-CSP and 5x10 vps/mouse of Ad7

7

GFP. (B) Mice were co-injected IM with 5x10 vps/mouse of Ad5-CSP and 5x10 vps/mouse of
7

7

Ad-EAT2 or 5x10 vps/mouse of Ad5-CSP and 5x10 vps/mouse of Ad-GFP. As a negative
conrol, naïve splenocytes were also tested against paired peptides from the peptide pool, with an
average background of spots per paired peptides being only 2.2 spots.

98

demonstrated significantly higher number of CD8+, CD107a+ T cells than those quantified in
naïve mice, indicating increased ability of CD8+ T cells to express granules when stimulated
with a CSP epitope (Figure 32). However, the assay was not sensitive enough to measure a
difference between the control vaccinated mice and Ad5-CSP+Ad-EAT2 vaccinated mice. We
then conducted a more sensitive in vivo CTL assay.[154] Mice were co-vaccinated with either
8

8

8

1x10 vp/mouse of Ad5-CSP and 1x10 vps/mouse of Ad-GFP or 1x10 vp/mouse of Ad5-CSP
8

and 1x10 vps/mouse of Ad-EAT2. 14 days later vaccinated mice were treated with CFSE
labeled splenocytes that had been incubated with either the NYDNAGTNL peptide, or a nonspecific control peptide, and the elimination of NYDNAGTNL pulsed cells (CFSEhigh cells)
was measured by flow cytometry. Based on the calculated percent specific killing, animals
vaccinated with Ad5-CSP+Ad-EAT2 were more effective at killing cells exposed to the
NYDNAGTNL peptide than animals vaccinated with the control Ad5-CSP vaccine (Figure 33).
Induction of CSP specific antibody responses by Ad5-CSP vaccines augmented by rEA or
EAT-2 expressing rAds
CSP antibody specific ELISAs were also performed on plasma derived from Ad5CSP+Ad-GFP/rEA and Ad5-CSP+Ad-GFP treated animals. CSP specific total IgG antibody
levels in control vaccine treated animals were significantly elevated (p<0.05) as compared to
naïve animals. However, there was again no significant difference observed in Ad5-CSP+AdGFP/rEA treated animals when compared to naïve animals (p<0.05) (Figure 34A). Conversely,
plasma collected from Ad5-CSP+Ad-EAT2 treated animals had significantly higher levels of
CSP specific IgG as compared to levels detected in naïve mice (p<0.05) (Figure 34B). However,
the mice receiving the control vaccine treatment had higher total CSP specific IgG levels than
naïve and Ad5-CSP+Ad-EAT2 treated animals (p<0.05) (Figure 34B). Further isotyping of IgG
99

Figure 32: Improved degranulation of CD8+ T cells in mice co-vaccinated with Ad5-CSP and
Ad-EAT2. Degranulation marker, CD107a, expression in CD8+ T cell from mice co-vaccinated
with Ad5-CSP+Ad-EAT2 or Ad5-CSP+Ad-GFP. Splenocytes were collected from BALB/cJ
7
7
mice 14 days post co-injection of either 5x10 vps of Ad5-CSP and 5x10 vps of Ad-GFP or
7

7

6

5x10 vps of Ad5-CSP and 5x10 vps of Ad-EAT2. 2×10 splenocytes from naive or mice covaccinated with either treatment were stimulated with 2ug NYD-peptide at 37°C for 3 days.
Cells were then washed with FACS buffer and stained with CD8-Alexa700, CD107-FITC
antibodies and viability dye (ViViD) and ran on LSR-II. % of live CD107+ CD3+ T cells is
shown. The bars represent mean ± SD. Statistical analysis was completed using One Way
ANOVA with a Student-Newman-Keuls post-hoc test,* Indicates significance over naïve p<0.05

100

Figure 33: Co-expression of CSP and EAT-2 increases cytolytic activity of CSP specific T cells.
Co-vaccination of mice with Ad5-CSP and Ad-EAT2 increased specific killing cells pulsed with
8
CSP peptides. BALB/cJ mice (n=4) were co-injected IM with either 1x10 vps/mouse Ad5-CSP
8
8
8
and 1x10 vps/mouse Ad-GFP or 1x10 vps/mouse Ad5-CSP and 1x10 vps/mouse Ad-EAT2
on Day 0. Day 14 splenocytes were collected from naïve mice and pulsed with either
NYDNAGTNL peptide or an irrelevant peptide. NYDNAGTNL pulsed splenocytes were stained
with a high concentration of CFSE while splenocytes pulsed with irrelevant peptide were stained
with a low concentration of CFSE. Stained splenocytes were then combined in equivalent doses.
8 million cells were then injected IV into naïve, Ad5-CSP+Ad-GFP co-vaccinated, or Ad5CSP+Ad-EAT2 co-vaccinated mice. After 18 hrs splenocytes from these mice were collected
and analyzed by flow cytometry to assess the amount of NYDNAGTNL specific killing. %
Specific killing = 1-((%CFSEHigh/%CFSELow)immunized/(%CFSEHigh/CFSELow)nonimmunized). * denotes significant difference between treatments p<0.05.

101

was performed, the ratios of Th1 to Th2 antibody (IgG2a/IgG1) in mice treated with AdEAT2+Ad5-CSP were similar to the ratio of Th1 to Th2 antibody in control treated mice in all
dilution except 1:400, indicating that expression of EAT-2 did not induce a Th1 or Th2 bias in
these mice at 14 dpi as measured by this assay (Figure 35). In addition, when measured by ICS,
there was no significant difference in the number of likely CD4+ IFNγ expressing T-cells, as the
number of CD8- CD3+ T cells in Ad5-CSP+Ad-EAT2 treated animals and were similar to the
numbers of these cells noted in Ad5-CSP+Ad-GFP treated animals (Figure 36).

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Figure 34: Induction of CSP specific antibody responses by Ad5-CSP vaccines augmented by
rEA or EAT-2. Total IgG antibody against CSP is ablated in Ad5-CSP+Ad-GFP/rEA covaccinated mice while Ad5-CSP+Ad-EAT2 co-vaccinated mice demonstrated significantly more
7
CSP specific IgG than naïve animals. BALB/cJ mice (n=5) were co-injected IM with 5x10
7

7

vps/mouse of Ad5-CSP and 5x10 vps/mouse of Ad-GFP or 5x10 vps/mouse of Ad5-CSP and
7

5x10 vps/mouse of either (A) Ad-GFP/rEA or (B) Ad-EAT2. Plasma was collected at day 14.
Total IgG against CSP in the plasma was measured by ELISA. The bars represent mean ± SD.
Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls posthoc test, * Denotes significance over naïve p<0.05. † Denotes significant difference between
treatments p<0.05.
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Figure 35: Sub-isotype analysis of IgG antibody from plasma of mice co-vaccinated with Ad57
CSP and Ad-EAT2. BALB/cJ mice (n=6) were co-injected i.m. with 5x10 vps of Ad5-CSP and
7

7

7

5x10 vps of Ad-GFP or 5x10 vps of Ad5-CSP and 5x10 vps of Ad-EAT2. Plasma was
collected at day 14. The amount of CSP specific IgG subisotypes, IgG1 (A) and IgG2a (B) was
measured by ELISA. The ratio of IgG2a/IgG1 was calculated to indicate the Th1 to Th2 response
ratio (C). The bars represent mean ± SD. Statistical analysis for sub-isotyping (A,B) was
completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test and standard ttest was performed for Th1 to Th2 ratios (C),* indicates significance over naïve p<0.05. †
Indicates significance between treatments p<0.05.

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Figure 36: CD3+ CD8- IFNγ+ cells respond similarly to both vaccine regimens. Co-vaccination
with Ad5-CSP and Ad-EAT2 resulted in similar IFNγ secretion from CD3+ CD8- T cells.
7
7
BALB/cJ mice (n=6) were co-injected IM with 5x10 vps/mouse of Ad5-CSP and 5x10
7

7

vps/mouse of Ad-EAT2 or 5x10 vps/mouse of Ad5-CSP and 5x10 vps/mouse of Ad-GFP.
Splenocytes were stimulation with NYDNAGTNL peptide. Cells were stained with CD8-Alexa
Flour700, CD3-APC-Cy7, ViViD, and IFNg-APC. The bars represent mean ± SD. Statistical
analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test.

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4.3 Discussion:
Our earlier works and those of others suggest that activation of the innate immune system
can play an important role in beneficially augmenting subsequent antigen specific adaptive
immune responses.[68,69,72,142,155] For example, we previously augmented CMI responses
against HIV-Gag by co-injecting a rAd5 vector expressing HIV-Gag with a rAd5 vector
expressing a TLR agonist, rEA.[142] Similarly, co-injecting a rAd5 vector expressing HIV-Gag
with a rAd5 vector expressing the SLAM receptors adaptor protein EAT-2 also augmented
induction of innate immune responses, and improved the induction of HIV-Gag specific T cell
responses.[146] As a new approach to increasing the potency of malaria specific vaccines, we
now describe the use of adenoviral based vaccines engineered to express malaria derived
proteins, simultaneously administered with rAds expressing proteins known to modulate the
innate immune system. Most importantly, we have confirmed that rAd mediated expression of a
SLAM pathway derived adaptor (EAT-2) can significantly augment the induction of malaria
antigen (CSP) specific CMI responses. This was verified based upon ELISpost analysis of
splenocytes (both as to their responsiveness to immunodominant peptides, as well the breadth of
these responses to the full length CSP), ICS staining of cells for IFNγ, and most importantly by a
CSP specific in vivo CTL functional assay. EAT-2 expressing vaccines should be considered for
use in future malaria vaccine trials attempting to boost malaria antigen specific CMI responses.
Furthermore, EAT-2 co-expression allowed for the induction of CS specific antibody responses
as well.
In contrast, co-vaccination of mice with a rAd vaccine expressing a TLR agonist
simultaneously with a rAd expressing CSP, actually had the opposite effect, and completely
mitigated induction of CSP specific adaptive humoral and cellular immune responses, as

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compared to responses typically induced by the rAd vaccines expressing CSP alone. There could
be numerous reasons for these unexpected, paradoxical and potentially disturbing results. A
simple reason could be that the increase in pro-inflammatory cytokines caused by rAd mediated
expression of the TLR agonist, rEA, could be influencing expression of CSP from the rAd5
vector. However, this effect would have likely been observed in our previous studies utilizing the
same vector combinations, as well as the same TLR or SLAM receptors derived adaptors, but a
different target antigen (HIV-Gag). Those studies also confirmed induction of similar innate
immune responses to those noted in this study.[142,146] It is more logical that the CSP somehow
negatively interacts with immune pathways excessively activated by TLR agonists such as rEA,
resulting in a complete ablation of CSP specific CMI responses. This immunosuppressive
activity of CSP appears to only be unveiled after excessive stimulation of TLR pathways, as our
use of EAT-2 demonstrated not only avoidance of CS immunosuppressive activity, but also
allowed for enhanced induction of CS specific adaptive immune responses.
The CSP has been specifically confirmed to be capable of outcompeting the transcription
factor NF-κB for binding to the nuclear transport protein, importan α, resulting in the
downregulation of at least forty NF-κB controlled genes.[156] CSP was also shown to inhibit
NF-κB entry into the nucleus by 75%.[156] As NF-κB is known to control numerous genes
involved in pro-inflammatory immune responses, one hypothesis may be that the CSP can
downregulate excessive (TLR-driven) NF-κB transcriptome responses, and result in a
dramatically diminished acute inflammatory response, thereby blunting subsequent CSP antigen
specific adaptive immune responses.[157] This may make biological sense, as infection of
hepatocytes by malaria sporozoites has been shown to induce the activation of NF-κB in a
MyD88 specific manner.[158] Expression of CSP by the parasite may have evolved to counteract

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this inflammatory response and prevent excessive induction of malaria specific adaptive immune
responses in the infected host. In support of our hypothesis that CSP mediated NF-κB
interference ablates adaptive responses, recent studies on an immunosuppressive drug
(dehydroxymethylepoxyquinomicin) that specifically interferes with the NF-κB-importan α
interaction was shown at lower doses to only modestly affect IL-6 and TNFα levels, while
dramatically affecting Th1 expansion, results paralleling those noted in our experiments.[159]
These notions may also explain our findings, as well results previously reported by
others.[77,150] Those studies and ours verify that at very high doses, rAd vaccines expressing
CSP also show a trend toward diminished induction of CSP specific CMI responses (Figure
24).[77,150] Multiple studies have shown that Ad vectors can also induce NF-κB.[102,103]
Quite possibly, the CSP immunosuppressive effects are not uncovered until an “NF-κB
activation threshold” has been broached, in this instance by use of excessively high doses of rAd
vaccines expressing CSP, or by using more modest doses of the Ad vaccine coupled with potent
TLR activation. Further studies will need to be performed to elucidate whether this or other
mechanisms may be responsible for our results. Regardless, our data demonstrate the need to
consider the impact the inclusion of CSP derived peptides, or the entire protein along with other
immunostimulatory compounds may have upon present and future malaria specific vaccines.
Taken together with recent data demonstrating that protection from malaria challenge can be
independent of CSP suggests that the use of CSP in certain malaria vaccine formulations will
have to be carefully considered.[30,31]
In contrast to co-expression of the TLR agonist, co-expression of EAT-2 and CSP
eventuated in the enhanced induction of CMI responses to the CSP, relative to the use of the
Ad5-CSP vaccine alone. We have also previously observed a potent CMI response against HIV

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derived Gag in mice treated with Ad-EAT2+Ad-Gag.[146] Like TLRs, activation of the SLAM
receptor pathway in DCs and macrophages can also enhance the production of pro-inflammatory
cytokines.[160]
The biochemical mechanism and intracellular signaling pathway behind EAT-2’s ability
to function as a T cell (and possibly a B cell) stimulator in the face of CSP over expression is not
fully elucidated, but is a question that has been unveiled by our studies. SLAM associated
proteins like EAT-2 are known to play a role in several novel immunomodulatory pathways,
including the SLAM, CD22, and FcγRIIB.[161,162,163] These pathways may not be subject to
the immune suppressive actions of CSP possibly by virtue of its specific mode of action relative
to NF-κB and/or TLR activation pathways described earlier.
It has been established that greater numbers of CD8+ T cells are required to police
infected hepatocytes and achieve long term protective immunity against malaria, emphasizing
the importance of inducing a large population of CD8+ T cells capable of killing.[24] There is
some evidence that improved protection is also related to increased breadth of the CMI response
in addition to the potency of the CMI response.[151,152,153] Here, as an accessory to the
increased CMI response, we have demonstrated Ad-EAT2s ability to stimulate increased T cell
responses against multiple CSP epitopes. We not only observed an increase in the percentage of
CSP specific CD8+ T cells, but also improved in vivo CTL killing of CS pulsed splenocytes from
mice treated with Ad5-CSP+Ad-EAT2. The use of EAT-2 to augment CSP specific functional
CD8+ T cells may be of greatest importance in killing Plasmodium infected hepatocytes, as these
types of responses are not only positively correlated with protective capability, but also may
outweigh the need for induction of malaria antigen specific antibody responses.[22,24,26,164]

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Improvements over sole use of Ad5-CSP to induce CSP antigen specific B cell responses
were not achieved in mice treated with either Ad5-CSP vaccine cocktail. However, covaccination with the Ad5-CSP and Ad-EAT2 vectors at least prevented the loss of induction of
CS specific antibody responses noted after use of the Ad-GFP/rEA and Ad5-CSP vaccine
combination. These results did not appear to be due to a skewing from Th2 to Th1 type antibody
response, as measured by IgG1/IgG2a ratios, there were also no observed differences in IFNγ
secreting CD8- CD3+ T cells between treatment groups. Further research will need to be
performed to elucidate the reasons behind the observed antibody responses.
The importance of stimulating a strong cytotoxic T cell response against P.falciparum
infected hepatocytes is vital in creating a subunit based vaccine that is protective against malaria.
With this study we have successfully stimulated a CMI response to CSP that can overcome CSP
related adaptive immune response ablation and is even more potent than the previous generation
of rAd5s expressing CSP. Incorporation of this new vaccine platform into ongoing or future
malaria vaccine trials could potentially achieve the levels of prophylaxis needed to protect
vulnerable populations against natural malaria infections. Future studies will need to be
performed to assess this platforms ability to protect larger animals challenged with malaria.

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Acknowledgements: We wish to thank Michigan State University Laboratory Animal support
facilities for their assistance in the humane care and maintenance of the animals utilized in this
work, the NIH Core Tetramer Facility at Emory University for manufacturing the
NYDNAGTNL tetramer, and the Michigan State University flow cytometry facility for their
assistance with the multiple experiments.

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Chapter 5:
Capability of advanced generation, Adenovirus based malaria vaccines to prevent malaria
infection.

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5.1 Introduction
Previous attempts at putative malaria vaccines have all come up short in either their
ability to generate long lasting protection to malaria infections, or in their inability to be
practically manufactured and distributed. Use of irradiated sporozoite based vaccine
formulations has proven that protection is achievable, reaching levels of protection greater than
90%.[19] However, irradiated sporozoites cannot survive long outside of the mosquito salivary
gland, and they do not endure freeze thaw cycles well.[23] Delivery of sporozoites via bites from
irradiated infected mosquitoes can prolong the lifespan of the sporozoite, but it is not a practical
vaccination method as many hundreds of bites are required to achieve protection.[18,19,21] On
the other hand, subunit vaccines (like the leading subunit malaria vaccine candidate (RTS,S)
consisting of a pseudo virion made up of HBsAg-CSP) can be easily manufactured and
administered, although no subunit vaccine has reached the levels of protection that irradiated
sporozoites confer. In fact, early formulations of RTS,S were poorly immunogenic and
stimulated a predominantly humoral response against CSP.[45] It was only after potent adjuvants
aimed at improving CD8+ T cell responses were incorporated that the RTS,S platform was
capable achieving 56% protection from naturally occurring malaria.[44]
While many disease vaccine platforms require potent antibody responses to achieve
protection, protection from malaria appears to be mediated by a potent CD8+ T cell response
against the liver stage. In fact, irradiated sporozoite mediated protection from malaria challenge
has repeatedly been shown to be primarily due to CD8+ T cell responses against the liver
stage.[22,24,26] Initially, Ad based vaccine vectors seemed like an excellent candidate to
stimulate the potent CD8+ T cell responses against liver stage antigens that are required to
achieve protection from malaria. Mice vaccinated with a single injection of Ad expressing P.

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yoelii CSP even exhibited 93% inhibition of liver stage development following sporozoite
challenge with P. yoelii.[59] However, despite Ad vectors natural ability to induce potent CD8+
T cell responses against a trangene, no Ad-based malaria vaccine has achieved improved
protection over the leading malaria vaccine candidate, RTS,S.
Our previous works have demonstrated that Ad mediated induction of CD8+ T cells can
be further augmented with the incorporation of a potent immunomodulator.[136,146] The
expression of SLAM receptor adaptor protein, EAT-2, from an Ad co-injected with an Ad
expressing CSP resulted in augmented induction of innate immune responses, which ultimately
led to improved cytotoxic T cell responses against CSP.[136] Importantly, we have undertaken
functional analysis to prove that this platform creates a cytotoxic T cell response that is more
effective at killing cells displaying epitopes of CSP in vivo than the control vaccine.[136] Here,
we assess this platforms ability to confer protection from live parasite malaria challenge in a
mouse model. In doing so, we also assess the validity of our in vivo cytotoxic T lymphocyte
assay to predict efficacy of a putative malaria vaccine.

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5.2 Results:
Construction and validation of Ad5-PbCSP
We constructed an Ad5 vector expressing Plasmodium berghei CSP from a CMV
promoter (Ad5-PbCSP) in the same manner as previously described for Ad5 expressing P.
falciparum (Ad5-PfCSP).[136] We then confirmed that this construct responds similarly in
BALB/cJ mice as our previous experiments with Ad5-PfCSP. BALB/cJ mice were co-vaccinated
7

7

with 5x10 vp/mouse of Ad5-EAT2 and 5x10 vp/mouse of Ad5-PbCSP ( Ad5-EAT2+Ad57

7

PbCSP ) or 5x10 vp/mouse of Ad5-Null and 5x10 vp/mouse Ad5-PbCSP (Ad5-Null+Ad5PbCSP) (n=5). 14 dpi splenocytes were harvested and stimulated with a P. berghei CSP
dominant epitope (SYIPSAEKI) overnight. ELISpots were then performed measuring IFNγ
secreting splenocytes. Ad5-EAT2+Ad5-PbCSP co-vaccinated animals had significantly more
IFNγ secreting splenocytes than both unvaccinated and Ad5-Null+Ad5-PbCSP treated mice
(p<0.001 and p<0.01 respectively) (Figure 37A). Likewise, when we analyzed CD8+ T cells
specific for P. berghei CSP by staining with a SYIPSAEKI tetramer and antibodies for CD3 and
CD8, we found Ad5-EAT2+Ad5-PbCSP co-vaccinated animals had significantly higher
percentages of tetramer positive CD8+ T cells than both unvaccinated and Ad5-Null+Ad5PbCSP treated mice (p<0.05) ( Figure 37B).
P. berghei Challenge:
We next sought to determine our vaccine’s ability to confer protection from malaria
challenge in mice bitten with P. berghei ANKA strain infected mosquitoes. We obtained P.
berghei ANKA strain engineered to express GFP from New York University Insectory.
Although we were able to detect increased numbers of P. berghei CSP specific IFNγ secreting

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Figure 37: Co-vaccination with Ad5-PbCSP and Ad5-EAT2 drastically increases PbCSP
7
specific CMI responses. BALB/cJ mice were injected IM with either 5x10 vps/mouse of Ad57

7

7

PbCSP and 5x10 vps/mouse of Ad5-Null or 5x10 vps/mouse of Ad5-PbCSP and 5x10
vps/mouse Ad5-EAT2 (n=5). Splenocytes were collected 14 days post co-injection. (A) ELISpot
was performed on the splenocytes of these mice stimulated with SYIPSAEKI peptide to assess
the amount of IFNγ secreting cells. (B) Splenocytes were also stained with a SYIPSAEKI
tetramer and antibodies for CD3 and CD8 to determine CD8+ T cells specific for PbCSP. The
bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a
Student-Newman-Keuls post-hoc test,*,**,*** Denotes significance over naïve animals, p<0.05,
p<0.01, and p<0.001.
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splenocytes and increased percentages of CD8+ tetramer positive cells in animals co-vaccinated
with Ad5-EAT2+Ad5-PbCSP as compared to unvaccinated and Ad5-Null+Ad5-PbCSP covaccinated animals, we were unable to detect differences in protective efficacy when BALB/cJ
8

8

mice were co-vaccinated with 1x10 vp/mouse of Ad5-EAT2 and 1x10 vp/mouse of Ad58

8

PbCSP (Ad5-EAT2+Ad5-PbCSP) or 1x10 vp/mouse of Ad5-Null and 1x10 vp/mouse Ad5PbCSP (Ad5-Null+Ad5-PbCSP ) (n=10). We selected a slightly higher dose of vaccine to better
optimize CSP specific CMI responses based on our previously published Ad5-PfCSP dose curve
(Figure 24). 14 dpi mice were anesthetized and placed over a net covered cup containing 12-15
infected mosquitoes. Mosquitoes were allowed to bite for 9 minutes repositioning the mice every
3 minutes. 7 and 14 days post challenge mice were bled via tail snip. Two drops of blood were
collected from each mouse; one was used to make a thin blood smear and the other drop was
collected in 500 µL of Alsever’s solution. Blood smears were photographed under confocal
microscopy filtered for GFP expressing cells and were subsequently stained with Giemsa stain
and analyzed under microscopy for percent infected red blood cells. Blood collected in Alsever’s
solution was analyzed by flow cytometry for FITC fluorescing cells.
Our data supports previously published data demonstrating that Giemsa stain assessed
percent parisitemia tightly correlates with flow cytomery measured percent parasitemia
following challenge with GFP fluorescing P.berghei (Figure 38).[165] We observed no
differences in percent parasitemia between non-vaccinated, Ad5-Null+Ad5-PbCSP vaccinated,
or Ad5-EAT2+Ad5-PbCSP vaccinated animals by any assay at either time point (Figure 39).
BALB/cJ mice are particularly susceptible to P. berghei infection.[166] It appears that allowing
12-15 mosquitoes per mouse to bite for 9 minutes results in an unrealistically high dose of
parasite.
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Figure 38: Giemsa stain strongly correlates with % FITC+ red blood cells. 7 and 14 days post
challenge with P. berghei ANKA expressing GFP blood was collected via tail snip. Thin blood
smears were stained with Giemsa stain. Infected red blood cells were counted on each frame and
divided by average red blood cells per frame to determine % parasitemia (y-axis). Red blood
cells were analyzed by flow cytometry for % FITC+ cells (x-axis). Giemsa stain was founf to
strongly correlate with % FITC+ red blood cells (r=).

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Figure 39: Parasitemia was similar between treatments at 7 and 14 days post challenge. Blood
was collected via tail snip at 7 and 14 days post challenge with P. berghei ANKA expressing
GFP and analyzed by flow cytometry for FITC+ red blood cells. Statistical analysis was
completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test.

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This high dose of parasite challenged overwhelmed the ability of all vaccinations tested to
prevent parasite growth.

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5.3 Discussion:
We were able to detect increased numbers of P. berghei CSP specific IFNγ secreting
splenocytes and increased percentages of CD8+ tetramer positive cells in animals co-vaccinated
with Ad5-EAT2+Ad5-PbCSP as compared to non-vaccinated and Ad5-Null+Ad5-PbCSP covaccinated animals. However, we were unable to detect differences in protective efficacy
between any of the vaccinated or unvaccinated groups after parasite challenge. Likely the
unrealistically high dose of parasites used in these challenge experiments overwhelmed the
protective capability of both vaccination strategies. BALB/cJ mice are particularly susceptible to
P. berghei infection and it has been demonstrated that a single bite can result in over 80%
infection of naïve BALB/cJ mice.[166] It is also possible that the vaccine may not be able to
protect against live infections despite the induction of potent CSP specific cytotoxic T cell
responses. The decreased humoral responses associated with the Ad5-EAT2+Ad5-CSP platform
could be limiting the vaccines efficacy. While multiple studies demonstrate the need for potent
CD8+ T cell responses, strong humoral responses may still be required for an effective CSP
based malaria vaccine. Based on these results it is evident that functional, real world assays
should be undertaken to determine putative malaria vaccine efficacy. Future experiments will be
conducted repeating the experiments outlined above using fewer P. berghei infected mosquitoes
per mouse in an effort to more realistically simulate real world infections. Alternatively,
sporozoites can be harvested and injected IV in order to more closely regulate the amount of
sporozoites administered per mouse. We could also switch mouse strains to a less susceptible
strain of mice like Swiss Webster outbred mice to further our ability to discern if use of immune
modulation via Ad mediated EAT-2 expression can prevent malaria parasite growth in
vaccinated animals. Should the use of Ad5-EAT2+Ad5-CSP prove not to be effective as a single

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injection it might still be utilized to improve CSP CD8+ T cells responses in heterologous prime
boost regimens with more antibody biased malaria vaccines like RTS,S.
Use of GFP expressing P. berghei parasites proved to be a consistent and easy method for
measuring percent parasitemia of challenged mice by analyzing FITC+ red blood cells by flow
cytometry. Measuring percent FITC+ red blood cells by flow cytometry eliminates some human
error and objectivity in measuring percent parasitemia. This method could be an important tool
in future parasite challenge experiments.

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Acknowledgements: We wish to thank Michigan State University Laboratory Animal support
facilities for their assistance in the humane care and maintenance of the animals utilized in this
work, the NIH Core Tetramer Facility at Emory University for manufacturing the
NYDNAGTNL tetramer, the Michigan State University flow cytometry facility, Michigan State
University Histocore, and the Michigan State University Microscopy lab for their assistance with
the multiple experiments.

123

Chapter 6:
Summary and future directions

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6.1 Ad interactions with the innate immune system:
Adenovirus is commonly used as a vector for two very disparate purposes. As a gene
delivery vector, Ad interactions with the innate immune system is a detriment to its efficacy,
while Ad mediated induction of innate immune responses can be exploited for stimulation of
more potent adaptive immune responses to a targeted antigen in vaccine platforms. My research
has helped to elucidate multiple complex interactions between Adenovirus and the innate
immune system. These realizations can be used to potentially improve both gene delivery and
vaccine vector functions of rAds. For example, we have uncovered potential targets that can be
used to suppress or augment immune responses.
We have experimentally proven that Ad induced liver transcriptome dysregulation is
complement dependent, but we were unable to delineate specific roles of either the classical or
alternative complement pathways in transcriptome responses aside from a role for C4 in TLR2
transcription.[71] We did however find that the dysregulation of some genes are actually
negatively regulated by CP and AP components (C1q and FB respectively). We also confirmed
that many of the observed Ad-induced transcriptome changes are negatively regulated by
mCR1/2. Similarly, inductions of some cytokines and chemokines by systemic delivery of
adenovirus were also found to be complement dependent and are negatively regulated by
mCR1/2.[167]
Primarily via the use of complement protein knockout mice, when we analyzed induction
of anti-Ad neutralizing antibodies we found functional C3 is required for production of anti-Ad
neutralizing antibody. Further, inductions of total anti-Ad IgG and IgG3 were both found to be
dependent on FB and C3.[71] We also uncovered a substantial role for complement in
modulating the balance between Th1 Th2 antibody responses. However, while complement is

125

required for maximal anti-Ad humoral responses, we did not detect a role for complement in
anti-transgene humoral responses.[71] When we looked at the role of mCR1/2 in stimulation of
anti-Ad humoral responses we found that despite mCR1/2’s negative role in controlling multiple
genes involved in inflammation and cytokines, mCR1/2 is actually required for induction of
maximal anti-Ad antibody responses, inclusing anti-Ad neutralizing antibody responses. Unlike
what we observed in C3-KO, anti-transgene humoral responses are decreased in mCR1/2-KO
mice.[167]
We hypothesize that lack of functional mCR1/2 allows for unchecked secretion of
cytokines, which interferes with trangene expression and might explain the decreased antitransgene humoral responses. As for mCR1/2’s involvement in anti-Ad humoral responses we
determined mCR1/2 could be playing an important role in activating B cells opsonized with C3
(data not shown).[167]
Given our findings we sought to modulate Ad-induced innate immune responses by
incorporating a negative regulator of complement (DAF) similar to mCR1/2 into the Ad-caspid.
We fused a reoriented DAF to adenovirus capsid protein IX in order to display DAF in a more
natural conformation. Use of this novel Ad vector successfully inhibited multiple Ad-induced
complement mediated innate immune responses validating our previously discussed data. This
novel Ad vector could prove to be a valuable tool in gene therapy applications.[168]
6.2 Studies of alternative Ad serotypes:
Our explication of Ad interactions with the innate immune system and its affect on
adaptive responses can also be exploited for vaccine platforms. Our research further explores
Ads for use as a vaccine vector using malaria as a model platform. First, we sought to overcome
a commonly cited problem with utilizing Ad5 as a malaria vaccine vector, namely the high

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seroprevalence of pre-existing Ad5 immunity of humans residing in malaria endemic regions.
We attempted to utilize an alternative serotype to Ad5 that has been shown to be more
immunogenic and has a history of use as a vaccine vector, Ad4. We engineered Ad4 and Ad5
vectors to express the most commonly used malaria antigen for vaccine applications (CSP) and
examined how their distinctive stimulation of the innate immune system could be used in
homologous and heterologous prime boost regimens in both Ad5 naïve and Ad5 immune
backgrounds.
We found that the combined use of an Ad4-CSP prime followed by a boost with Ad5CSP resulted in more efficient inductions of CSP specific cytotoxic T cells in Ad5 naïve animals,
requiring fewer CSP specific CD8+ T cells to achieve the same level of CSP specific killing in
vivo as homologous prime boost of Ad5-CSP. This knowledge could be used to design more
effective heterologous prime boost vaccination regimens. However, when we looked at the
efficacy of the Ad5 based malaria vaccine platform in Ad5 immune animals we observed
dramatically ablated responses despite these viruses residing in different subgroups, based upon
classical anti-sera neutralization categorization strategies.[100] This data suggests that separation
of Ads based on anti-sera neutralization properties alone may not be sufficient to determine their
levels of cross-reactivity when utilized in these types of applications. This critical finding
unearths a serious concern with using alternative serotypes as replacements for Ad5 in Ad5
immune patients. Future studies should similarly and stringently analyze use of alternative
serotype Ads to properly evaluate their potential for efficacy in Ad5-immune patients. Future
studies comparing T cell antigens of Ads from different subgroups might result in the discovery
of more immunologically distinct Ads that can be utilized to improve heterologous prime boost
vaccine responses, as well as Ad-induced antigen specific immune responses in Ad5 immune

127

patients. Knowledge of specific antigens that are cross-reactive between different Ad subgroups
could also allow for the engineering of Ad vectors that have genetically eliminated or altered the
cross-reactive antigens. Likewise, further elucidation of Ad4 induced innate immune responses
could uncover the Ad4 antigens responsible for the stimulation of the early innate cytokines that
interfere with CMV promoter expression, the elimination of which could potentially improve
Ad4-based vaccine vector efficacy.
6.3 Immunomodulation:
We then sought to improve upon Ad5 based malaria vaccine inductions of adaptive
responses against CSP by use of innate immune modulators. Since Ad5 has been shown to
activate the innate immune system through TLRs and has the ability to stimulates potent CD8+ T
cell responses against a transgene, we hypothesized that further stimulation of TLRs would result
in increased CD8+ T cell responses to CSP. To test this we engineered an Ad expressing a potent
TLR agonist called rEA (Ad-GFP/rEA) and co-injected it with Ad5-CSP. Perplexingly, we found
that despite large increases in secretion of multiple innate cytokines, adaptive responses to CSP
were paradoxically ablated. It is possible that the increase in pro-inflammatory cytokines
interfered with CSP expression. However, if this were the case we would have observed similar
ablation in previous studies where we expressed an HIV derived protein instead of CSP as
similar cytokine inductions were observed.[142] It is more logical that CSP negatively interacts
with the specific immune pathway that is excessively activated by the TLR agonist (rEA).
CSP has been shown to interfere with NF-κB nuclear translocation by outcompeting NFκB for important α, although how this interference can affect adaptive responses has not been
shown.[169] We hypothesize that CSP mediated downregulation of the TLR-driven excessive
NF-κB transcriptome response dramatically diminishes acute inflammatory responses therefore

128

resulting in ablated CSP specific adaptive immune responses. In support of our hypothesis,
recent studies on an immunosuppressive drug (dehydroxymethylepoxyquinomicin) that
specifically interferes with the NF-κB-importan α interaction was shown at lower doses to only
modestly affect IL-6 and TNFα levels, while dramatically affecting Th1 expansion, results
paralleling those noted in our experiments.[159] Here we uncover an important
immunosuppressive function of CSP through inadvertent stimulation of the very pathway CSP
suppresses. Future experiments could be undertaken to further elucidate CSP
immunosuppression mechanisms by measuring CSP interference with NF-κB nuclear
translocation and the resulting impact on a wider array of cytokines than was measured in our
experiments. Deletion or mutation of CSP’s nuclear localization site could also be undertaken in
future CSP-based malaria vaccines to eliminate CSP immunosuppression. Additionally, TLR
agonists, like rEA, might still have great benefit in non-CSP malaria vaccine platforms and
should continue to be studied.
We then substituted a NF-κB independent immunomodulator, called EAT-2, in an
attempt to bypass CSP mediated immunosuppression of the NF-κB pathway. We found
simultaneous injection of Ads expressing EAT-2 and Ad5-CSP vectors successfully stimulated
dramatic increases in CSP specific activation, percentages of CSP specific CD8+ T cells, and
CSP specific cytotoxic activity in vivo despite CSP interference with NF-κB nuclear
translocation. Like TLRs, activation of the SLAM receptor pathway in DCs and macrophages
can also enhance the production of pro-inflammatory cytokines.[160] The biochemical
mechanism and intracellular signaling pathway behind EAT-2 mediated activation of adaptive
responses is not fully elucidated. SLAM associated proteins like EAT-2 are known to play a role
in several novel immunomodulatory pathways, including the SLAM, CD22, and

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FcγRIIB.[161,162,163] These pathways may be distinct from NF-κB and/or TLR activation
pathways; therefore they may not be subject to the immune suppressive actions of CSP. Future
studies could be performed elucidating EAT-2 signaling mechanisms and their role in dendritic
cell and macrophage maturation.
6.4 Challenge study:
Based on our results we attempted to assess ability of the Ad5-EAT2 with Ad5-CSP
platform to provide protection from parasite infection in a mouse model of malaria. We
constructed and validated that the Ad5-PbCSP virus performs just as Ad5-PfCSP did when coinjected with Ad5-EAT2 as measured by ELISpot and tetramer staining. After co-vaccination
with Ad5-EAT2+Ad5-PbCSP or a control vaccine, mice were challenged via bites from
mosquitoes infected with P. berghei ANKA expressing GFP (12-15 mosquitoes per mouse).
Percent parasitemia was measured at 7 and 14 days post parasite challenge. Through this
research we have proved that the use of flow cytometry to measure parasitemia after challenge
with GFP expressing sporozoites is a valuable tool that eliminates some human error and
objectivity. However, the dose of parasite used proved to be high, as we were unable to detect
any differences between the two vaccination treatments or unvaccinated mice since both
vaccinations were overwhelmed. It is also possible that the vaccines may not be able to protect
against live infections despite the induction of potent T cell responses. This finding underlines
the importance of including functional, real world assays in such studies. Future studies using
lower doses of the parasite may allow for the vaccines to prevent infection, and/or confirm
improved efficacy of Ad malaria vaccines expressing EAT-2. We might also consider the use of
less susceptible animals, like Swiss Webster outbred mice, as an alternative to the highly
susceptible BALB/cJ mice in future experiments. The potential of this vaccine to stimulate

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potent CSP specific cytotoxic T cell responses when utilized in heterologous prime boost
regimens with potent anti-CSP antibody stimulating vaccines, like RTS,S, should also be
examined as combined use could result in improved responses over either vaccine when used
alone. Should use of Ad5-EAT2+Ad5-CSP prove to be protective in these studies, we could
move on to non-human primate models and ultimately human safety and efficacy clinical trials
as has been done by GSK with their RTS,S malaria vaccine.

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Chapter 7:
Material and methods

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Materials and Methods:
Vector construction:
Ad-LacZ: The recombinant adenoviral vectors, rAd5-LacZand rAd5-GFP, are vectors carrying
either CMV-LacZ or CMV-GFP transgene expression cassettes that replace the Ad E1 region of
the [E3-]Ad genome, and were grown to high titers on human 293 cells as previously
described.[170] Purification consisted of harvesting infected cell lysates, DNase and RNase
treatment, and cesium chloride density gradient bandings as per the method of Ng and
Graham.[171] The purified vector preparation was extensively dialyzed against 10 mM Tris (pH
8.0), and was stored in 1% sucrose/PBS at _80 1C. The vector preparation was determined to be
free of replication competent Ad by PCR using E1-specific primers and titered by SDS
disruption and by OD260 spectrophotometry essentially as previously described.10,12 The titer
was further evaluated by in vitro transduction of 293 cells and by the enumeration of bacterial bgalactosidase staining cells as previously described, and the viral particle/bacterial bgalactosidase transducing unit titer was approximately 8:1 (data not shown).[172,173]

Ad5-GFP-IX-dDAF_REO: The N-terminal cDNA coding for the N-terminal domain (entire
320 amino acids: 35-354, DAF-CCPR1-4) of the human DAF gene was subcloned in-frame into
the C-terminus of pIX. CCPR1-4 of DAF was PCR derived using following amplification
primers tailed with NheI sites: (DAF-F 5’-gctagcgactgtggccttcccccagatgtacc-3’, DAF-R 5’gctagcacctgaagtggttccacttcctttatttgg-3’). The NheI tailed PCR product, amplified from a human
DAF cDNA clone (ATCC# MGC-5192), was subcloned in-frame into the C-terminus of viral
protein IX into pShuttle-IX/NheI, the latter constructed in our laboratory by introducing NheI
recognition site at the C-terminus of capsid protein IX (just upstream of normal pIX stop codon)

133

as previously described.[174] The plasmid so obtained we refer to as pShuttle-IX-DAF, was
linearised with PmeI restriction enzyme and homologously recombined with the rest of the Ad5
vector genome present in the plasmid pAdEasyI as previously described, yielding pAd-IXDAF.[53]
We have also displayed DAF in a more native context (DAF_REO): N-terminuspIX-Cterminus-fusion/C-terminus-DAF-N-terminus. This required synthetic production (Geneart,
Regensburg, Germany) of the DNA molecule encoding for the DAF amino acid sequence
corresponding to 3’-5’ DAF (i.e. aa sequence was reversed and displayed in the C-terminus of
pIX) as described above. A GFP expression cassette was inserted into the MCS of the pShuttleIX-DAF (or pShuttle-IX-DAF_REO) as previously described.[171] All viruses were found to be
RCA free both by RCA PCR (E1 region amplification) and direct sequencing, methods as
previously described.[175] All Ads have also been tested for the presence of bacterial endotoxin
as previously described and were found to contain <0.15 EU per ml.

Ad5-CSP: The Open Reading Frame (ORF) of the P. falciparum CSP gene, composed of a
codon optimized consensus of several P. falciparum CSP sequences (Figure 40), was
incorporated into plasmid pGA4 (GENEART, Burlingame, CA) and excised from pGA4 using
endonuclease NheI (NEB, Ipswich, MA). The excised portion was subcloned into the pAd
Shuttle vector containing a CMV expression cassette. The resulting pAd5-CSP shuttle plasmid
was linearized with PmeI restriction enzyme and homologously recombined with the pAdEasyI
Ad5 vector genome as previously described yielding pAd5-CSP.[176] Virus was amplified in
HEK293 cells. Ad5-CSP virus was purified using a CsCl2 gradient as previously described.[171]
Direct sequencing and restriction enzyme mapping were carried out to confirm the fidelity of the

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Figure 40: CSP sequence. The CSP sequence utilized for constructing the Ad5-CSP vaccine was
designed based on several known CSP sequences. The NYDNAGTNL peptide’s location is
underlined in the sequence. Bold font within the sequence indicates the repeat region of CSP.
The location of the Thrombospondin-like Type 1 repeat region (TSR domain) is indicated by
gray font.

135

CSP sequence. The same CSP consensus sequence was incorporated into an adenovirus serotype
4 vector. Ad4 vector construction was performed as previously described for other transgenes
inserted into an Ad4 recombination based production system.[100] Construction of, Ad5-GFP,
Ad5-GFP/rEA, and Ad5-EAT2 was performed as previously described.[71,142,146]

Animal procedures: All animal procedures were approved by the Michigan State University
Institutional Animal Care and Use Committee (IACUC). Adult BALB/cJ mice, adult C57BL/6
mice, and B6.129S4-C3tmlCrr (C3-KO) mice were purchased from Jackson Laboratory (Bar
Harbor, ME, USA). FB-deficient mice, on the C57BL/6 background, were a generous gift of Dr
Alex Szalai (University of Alabama). C4-KO and C1q-KO mice were a generous gift from Dr
Garnett Kelsoe (Duke University Medical Center). mCR1/2-KO mice in C57BL/6 background
were a kind gift from Dr. Tedder, (Duke University Medical Center).[177,178] Intravenous
injections were performed via retro-orbital sinus in a volume of 200μL of PBS. Intramuscular
(IM) injections were performed by injection into the tibialis anterior of the right hindlimb. Total
injected volume of IM injections was 20 l. When required, mice were anesthetized with a nose
cone containing isoflurane. Splenocytes and plasma were collected. All procedures with rAds
were performed under BSL-2, and all vector treated animals were maintained in ABSL-2
conditions. Care for mice was provided in accordance with PHS and AAALAC standards.

ELISA: ELISA-based antibody assays were completed as previously described.[71] Highbinding flat bottom 96-well plates were coated with 0.2μg of purified CSP per well in a volume
of 100μL and incubated overnight at 4C. Plates were washed with PBS-Tween (0.05%) then
blocked with blocking buffer (3% bovine serum albumin) for 1 hour at room temperature.

136

Plasma was diluted (1:50, 1:100, 1:200, 1:400) in blocking buffer.and added to the wells and
incubated for 1 hour at room temperature. Wells were washed with PBS-Tween (0.05%) and
HRP antibody (Bio-Rad) was added at 1:4000 dilution in PBS-Tween. Tetramethylbenzidine
(TMB) (Sigma-Aldich) was added to each well and the reaction was stopped with 1N phosphoric
acid. Plates are read at 450nm in a microplate spectrophotometer. Subisotyping tittering was
completed with a hybridoma subisotyping kit (Calbiochem, La Jolla, CA) with plasma dilutions
of 1:50, 1:100. 1:200. 1:400. Statistical analyses were performed using Student t-test.

Cytokine and chemokine analysis: Utilization of the 23-plex Bio-Rad cytokine assay system
(Hercules, CA, USA) was complicated by lot-to-lot variations during the course of our studies.
We therefore designed a 7-plex multiplex-based assay system to more accurately determine
cytokine/chemokine plasma concentrations as per the manufacturer’s instructions (BioRad) by Luminex 100 technology (Luminex, Austin, TX, USA) essentially as previously
described.[103] The presence of the following cytokine and chemokines were simultaneously
queried in each plasma sample: IL-6, IL-12 (p40), G-CSF, KC, MIP-1b and RANTES. Statistics
were completed using Student’s t-test.

qRT-PCR analyses: To determine relative levels of a specific RNA transcript, tissues were
snap-frozen in liquid nitrogen and RNA was harvested from E100 mg of frozen tissue using
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s protocol. Following
RNA isolation, reverse transcription was performed on 180 ng of total RNA using SuperScript II
(Invitrogen) reverse transcriptase and random hexamers (Applied Biosystems, Foster

137

City, CA, USA) as per the manufacturer’s protocol excluding RNaseOUT. Reverse transcription
(RT) reactions were diluted to a total volume of 60 ml, and 2 ml was used as the template in the
subsequent PCR reactions. Primers were designed using Primer Bank web based-software
(http://pga.mgh.harvard.edu/primerbank/). Primers used for amplification have been previously
described.[67,101] Quantitative polymerase chain reaction (qPCR) was carried out on an ABI
7900HT Fast Real-Time PCR System using SYBR Green PCR Mastermix (Applied Biosystems)
in a 15 ml reaction. All PCRs were subjected to the following procedure: 95 1C for 10 min
followed by 40 cycles of 95 1C for 15 s followed by 60 1C for 1 min. The comparative Ct
method was used to determine relative gene expression using glyceraldehydes 3-phosphate
dehydrogenase (GAPDH) to standardize expression levels across all samples. Relative
expression increases were calculated based on levels of a respective transcript quantified in
mock-injected animals of the same genotype. Statistical analyses were completed using Student’s
t-test comparing C57BL/6-treated animals with each respective genotype individually.

Neutralizing Antibody: Neutralizing antibody assays were performed as previously
3

described.[71] 2x10 HEK293 cells were plated in 125 µL of complete medie (Dulbecco’s
modified Eagle’s medium (DMEM)), supplemented with 10% fetal bovine serum and
penicillin/streptomycin/fungizone (PSF) and incubated overnight at 37°C, 5% CO2. Plasma was
heat inactivated for 60 minutes at 56°C then brought to room temperature before dilutions were
6

made in 100 µL and added to the appropriate wells. 1.3x10 viral particals were then added to
plasma dilutITER 96 AQueous One solution (Promega, Madison, WI, USA) was added to each
well and incubated for 2 hours at 37°C, 5% CO2. 150 µL of media was removed into a clean
microtiter plate and read at 492 nm in a spectrophotometer.
138

Isolation of lymphocytes: Splenocytes from individual mice were prepared by physical
disruption of the spleen. The spleen was passed through a sterile 40μm nylon mesh cell strainer
(Fisher Scientific, Pittsburgh, PA). Red blood cells were lysed using ACK lysis buffer
(Invitrogen, Carlsbad, CA) remaining cells were resuspended in RPMI 1640 supplemented with
10% FBS and penicillin/streptomycin/fungizone.[142]

ELISPOT analysis: ELISpots were performed in accordance to manufacturer’s protocol using
the Ready-set Go IFNγ mouse ELISpot kit produced by eBiosciences (San Diego, CA).
Splenocytes were stimulated ex vivo with 4μg/mL of the >98% pure CSP immunodominant
peptide NYDNAGTNL (amino acids 43-51 of the CSP sequence) (GenScript Piscataway,
NJ).[179] A library of 15mers overlapping by 5 amino acids spanning the entire CSP nonrepeating region was constructed and also used to stimulate splenocytes ex vivo (Biosynthesis
Inc., Lewisville, TX). Spots were counted and photographed by an automated ELISPOT reader
system (Cellular Technology, Cleveland, OH). Ready-set Go IFNγ and IL-2 mouse ELISPOT
kits purchased from eBioscience (San Diego, CA).

Cell staining and flow cytometry: Splenocytes were stained with various combinations of the
following antibodies: PE-CD69, (3 µg/ml), FITC-CD8a, APC-CD3, APC-Cy7-CD3, Alexa
Floure700-CD8a, PerCpCy5.5-CD19, PE-Cy7-NK1.1, PE-Cy7-TNFα, APC-IFNγ (4 µg/ml),
PerCpCy5.5-CD127, PE-Cy7-CSP (NYD) tetramer, V450-CD62L, Granzyme B- (4 µg/ml) (All
obtained from BD Biosciences, San Diego, CA) and PerCpCy5.5-IL-2 (4 µg/ml) (BioLegend,
San Diego, CA). Cells were incubated on ice with the appropriate antibodies for 30 minutes,

139

washed, and sorted using an LSR II instrument and analyzed using FlowJo software. For
intracellular cytokines staining, cells were surface stained, fixed with 2% formaldehyde
(Polysciences, Warrington, PA), permeabilized with 0.2% Saponin (Sigma-Aldrich, St. Louis,
MO), and stained for intracellular cytokines. Large cells and debris were excluded in the
forward- and side-scatter plot, to minimize background levels of staining caused by nonspecific
binding of antibodies; we initially stained the cells with CD16/32 FcR III/II antibody. In addition
we included the violet fluorescent reactive dye (ViViD, Invitorgen) as a viability marker to
exclude dead cells from the analysis.[180] Blood was isolated by retro-orbital bleeds and PBMCs
were isolated using Lympholyte-Mammal (Cedarlane, Burlington NC).Tetramer staining of
PBMCs was completed using a PE conjugated MHC-I (H2d) tetramer folded with the
NYDNAGTNL peptide generated at the NIH Tetramer Core Facility.

In vivo CTL assay: BALB/cJ were co-vaccinated with equivalent doses of Ad5-CSP and either
Ad-GFP or Ad-EAT2 (totaling 2×108 vps). At 14 days, syngeneic splenocytes were isolated and
pulsed with either an irrelevant peptide or peptide specific to the P. falciparum circumsporozoite
antigen (NYDNAGTNL) for 1 hour at 37ºC. Irrelevant peptide pulsed cells were stained with
1μM CFSE (CFSELow) while CSP-peptides pulsed cells were stained with 10μM CFSE
(CFSEHigh). Naïve and immunized mice were injected with equivalent amount of both
CFSELow and CFSEHigh stained cells via the retro-orbital sinus. After 18-24 hours splenocytes
were harvested and sorted on an LSRII flow cytometer. FlowJo software was used to determine
percentages of CFSE stained cells. % Specific killing = 1-((% CFSEHigh / % CFSELow)
immunized / (% CFSEHigh / % CFSELow) non-immunized).

140

Statistical analysis: Statistically significant differences in ELISpot assays were determined
using either Two Way ANOVA with a Bonferroni post-hoc test or a One Way ANOVA with a
Student-Newman-Keuls post-hoc test (p value < 0.05). For ELISA analysis, a t-test was used to
assess significance between treatments. For multiparameter flow cytometry, a One Way
ANOVA with a Student-Newman-Keuls post-hoc test was used. For in vivo CTL assay, a One
Way ANOVA with a Student-Newman-Keuls post-hoc test was used. All graphs in this paper are
presented as Mean ± SD with the exception of graphs of ELISA data which use Mean ± SE.
GraphPad Prism software was utilized for statistical analysis.

P. berghei challenge: P. berghei ANKA GFP expressing parasite infected mosquitoes were
purchased from New York University Insectory (New York, NY, USA) Mosquitoes were starved
for 2 hours prior to be allowed to feed on BALB/cJ mice. BALB/cJ mice were anesthetized with
80 mg ketamine per kg weight and placed over a netted cup containing 12-15 mosquitoes in a
dimly lit room. Mice were repositioned every 3 minutes for a total of 9 minutes.

Microscopy: Mice were anesthetized via isoflurane inhalation and 1mm was cut from the very
end of the tail. One drop of blood was used to make a thin blood smear on a glass slide and was
allowed to dry completely. Pictures of dried thin blood smears were then taken under 600X
confocal microscopy filtered for green fluorescent protein. Three pictures were taken per slide.
Sections were selected based on similar red blood cell confluence. Average red blood cells per
frame were counted. Then the total green fluorescent cells per frame were counted. Percent
parasitemia was calculated as number of green cells divided by average red blood cells per

141

frame. One Way ANOVA with a Student-Newman-Keuls post-hoc test was used to calculate
statistical significance.

Geimsa Stain: Mice were anesthetized via isoflurane inhalation and 1mm was cut from the very
end of the tail. One drop of blood was used to make a thin blood smear on a glass slide and was
allowed to dry completely. Slides were fixed by submerging them in 100% methanol for 1
minute. Slides were allowed to dry and were then submerged in 10% Giemsa stain diluted in
deionized water for 50 minutes (GIBCO, Grand Island, New York, USA). Slides were than
rinsed with deionized water and allowed to dry. Pictures of slides were taken at 400X. Average
red blood cells per frame were counted. Then the total stained cells per frame were counted.
Percent parasitemia was calculated as number of stained cells divided by average red blood cells
per frame. One Way ANOVA with a Student-Newman-Keuls post-hoc test was used to calculate
statistical significance.

Flow cytometry measurement of parasitemia: Mice were anesthetized via isoflurane
inhalation and 1mm was cut from the very end of the tail. One drop of blood was collected in
500 µL of Alsever’s solution. Diluted blood was run on flow cytometry gating first by size then
for FITC+ cells. One Way ANOVA with a Student-Newman-Keuls post-hoc test was used to
calculate statistical significance.

142

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