moms ONTHE macaw. 0F '

RAMES VACCINE DERWED FROM RABBIT BRAIN

than for m. Dam. of Ph. D.
MlCHiGAN STATE UNIVERSITY.
Rasher? .EL Gauthier
1956

THESXS

This is to certify that the

thesis entitled
Studies on the Purification of

Rabies Vaccine Derived from Rabbit Brain

presented by

Robert, J. Gauthier

has been accepted towards fulfillment
of the requirements for

M degree in Microbiology

;W

(Major professor

 

/’>/

Date May 23) 1-956

0-169

STUDIES ON THE PURIFICATION OF RABIES VACCINE

DERIVED FROM RABBIT BRAIN

by

Robert J. Gauthier
AN ABSTRACT

submitted to the School for Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Micrdbiology and Public Health

1956

v f (7

 

It has long been known that antirabic vaccines occasionally produce
allergic encephalitis or other severe central nervous system reactions
in vaccinated individuals. Although the cause of these reactions is not
clear, most workers believe it to be an allergic reaction to the brain
tissue contained in the vaccine. It is generally believed that post-
vaccinal reactions could'be greatly reduced if the vaccines were freed
of most of the non-specific brain tissue. The vaccines must, however,
be highly antigenic.

This investigation was undertaken to develOp an acceptable method
for the purification of rabies vaccine derived from rabbit brain. Our
procedures employed zinc precipitation of the antigen followed by selective
dissociation of the zinc complex.

These procedures, which incorporated pH changes, failed to purify
the rabies vaccine without appreciably decreasing its antigenicity.
Although attempts to purify the vaccine were unsuccessful, significant
data were Obtained concerning the nature of the rabies antigen.

Under the conditions employed, it was found that the rabies antigen ’
was largely insoluble or poorly dispersed in aqueous and saline media.
This was thought to be due either to the intimate association of the
antigen with the brain substances or to its chemical composition. In
any case, this insolubility was the apparent reason for the inability
of the zinc to precipitate the bulk of the antigen from the vaccine.

Sonic oscillation, under the conditions employed, greatly increased

the antigenicity of the rabies vaccine. However, no comparable increase

   

 

 

 

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was found in its antigen solUbility. The liberation of some insoluble
antigen, which was zinc precipitable, followed sonic disintegration of
the tissue cells.

Repeated freezing and thawing of the rabies vaccine resulted in
the complete destruction of the antigenic component. Similarly, the
extraction of the vaccine with ether or a mixture of ether and ethanol
at low temperatures resulted in a great loss of antigenicity. There
appears to be some similarity between the rabies antigen and lipid-
protein combinations.

It was concluded that before any acceptable method of rabies
vaccine purification could be developed, it will be necessary first
to more adequately determine the chemical prOperties of the inactivated
rabies virus. The intimate association of the antigen with brain sub-
stances or protective chemical complexes has defied most physical and

chemical methods of separation without denaturation of the antigen.

 

STUDIES ON THE PURIFICATION OF RABIES VACCINE

DERIVED FROM RABBIT BRAIN

by

RObert J. Gauthier

A THESIS

submitted to the School for Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of MicrobiolOgy and Public Health

1956

I any; 7

3/4133

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation to the
Michigan Department of Health for making this study possible. He is
also greatly indebted to Dr. H. J. Stafseth of the Michigan State
University under whose supervision this investigation was undertaken,
and to Dr. R. Y. Gottshall and Dr. H. D. Anderson of the Michigan
Department of Health for advice and guidance given. The author
extends his sincere thanks to Dr. K. B. MCCall, Dr. R. J. Driesens,
William Gebhard, Zelma Ozolins and Thelma Scott for their valuable

assistance.

II.

III-

VII.

TABLE OF CONTENTS

INTRODUCTION........................................
HISTORICAL REVIEW...................................
MATERIALS AND METHODS...............................
A. Rabies Virus....................................
B. Preparation of'Rabies Vaccine...................
C. Methods of Purification.........................
D. Antigenicity Testing............................
E. Chemical Analysis...............................
RESULTS.............................................
DISCUSSION..........................................
SUMMARY.............................................
LITERATURE CITED....................................

APPEMHOOOOOOOO0.000.000.0000.00000COCOOOOOOOOOO.o.

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Table

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III

VII

LIST OF TABLES

pH Modifications of Purification Procedure of
Chapman and Surgenor.......................oo..o

Results of Purification Procedure of Martin
and ChapmmIOOOO0.000000000000000000000.0.0....000

Studies on Rabies Antigen Solubility. . . . . . . . . . . . . . .

Effect of Repeated Freezing and Thawing on
Rabies VaCCineOOOoooooooooo00.000000000000000...

Effect of Sonic Vibration on Rabies Vaccine . . . . . . . .

Results of Purification Procedure of Martin
and Chapman on Sonic Vibrated Rabies Vaccine... .

Effects of Ether and Ether-Ethanol Extraction on
Rabies Vaccine..................................

Page

22

23
25

26
27

28

3O

I. INTRODUCTION

One of the main prdblems of rabies research is to produce a vaccine
which will be highly antigenic and free from the agents which cause
postvaccinal reactions.

It has long been known that antirabic vaccines occasionally produce
paralytic accidents. The cause of these reactions is not clear; however,
most workers believe it to be an allergic reaction to the brain tissue
contained in the vaccine. Results of the studies of Stuart 33 al. (1928),
Rivers gt 2;. (1933), Lewis (1933) and Kabat gt El‘ (l9h7) show that brain
tissue functions as an organ-Specific instead of a species-specific
antigen. Paralysis caused by rabies vaccination must, therefore, be
considered as a specific sensitization to brain material. It is generally
believed that the maximum reduction in postvaccinal reactions could be
obtained if the non-Specific brain tissue could be removed from the vaccine.
The vaccine must, however, be highly antigenic.

The zinc precipitation method for the fractionation of plasma des-
cribed by Cohn (1953) was modified by Martin (1951+), Chapman (1951;), and
Smolens (1955) for the purification and concentration of viruses.

COX.SE.§l' (l9h7) reported rabies virus purification by means of
alcohol precipitation. Harris (l9h8) employed ether extraction and Bell
.22.§l' (l9h9) etherabenzene extraction for the purification of rabies
virus.

The purpose of this study is to investigate possible methods of
rabies vaccine purification employing some of the virus purification

procedures.

II. HISTORICAL REVIEW

Rabies has been known in EurOpe and Asia since ancient times. Rabies
was described in dogs and domestic animals by Aristotle. He Observed that
other dogs bitten by rabid dogs likewise became made. Galen (200 A.D.)
gave one of the earliest medical descriptions of the disease as follows:
"Hydrophdbia is a disease that follows the bite of a mad dog and is
accompanied by an aversion to drink liquids, convulsions and hiccoughs.
Sometimes maniacal attacks supervene" (Castiglioni, l9hl).

According to Johnson (19h8) rabies was known in western EurOpe as
early as 1271, at which time it was prevalent among wolves in France.

The disease appeared in Italy in 1708 in epizootic prOportions in dogs
and by 1728 spread to most of the major cities of Hungary, Germany and
France. Mullett (19h5) stated that rabies was known in England in 1613,
but not in epizootic preportions until 173%. .

In the United States rabies was known as early as 1753 in Virginia,
1762 in North Carolina and by 1785 spread through New England (Johnson,
l9h8).

Rabies was shown to be infectious by Zinke (180%) by the inoculation
of saliva of rabid dogs into normal dOgs. The work of Zinke, cited by
Webster (19u2) was not available for study. Orfila (1817) specifically
incriminated the saliva as the source of rabies infection (Mettler, 19h7).
Galtier (1879) introduced the use of domesticated rabbits for the study

of rabies.

 

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Pasteur (1885) successfully develOped a rabies prephylaxis consist-
ing of the subcutaneous injection of a fixed attenuated virus preparation
obtained from an emulsion of the spinal cords of rabbits dead of rabies.
This vaccine was ad0pted as a routine procedure in medical centers
throughout the world.

Many modifications of the Pasteur method have been developed since
that time. Among these have been the dilution method of Hagyes (1897)
and the glycerol containing vaccine of Calmette (1891).

Fermi (1908) introduced the use of phenol in the treatment of tissue
suspensions of fixed virus for the production of a vaccine. Semple (1911)
produced a phenol killed rabies vaccine that was completely non-infectious
and yet effective as an immunizing agent.

It was soon determined, however, that rabies vaccine could occasion-
ally produce an allergic encephalitis or other severe central nervous
system reactions in vaccinated individuals.

The incidence of paralytic accidents as a result of antirabic vaccina-
tion was shown by McKendrick (19h0) to be one in 8,887 for phenol-killed
virus vaccines, one in 3,398 for the attenuated virus vaccines and one in
3,19h for diluted virus vaccines.

In the tenth analytical report on antirabies treatment the occurrence
of postvaccinal paralysis was shown by Greenwood (l9h5) to be one in 8,517
for phenol-killed virus vaccines, one in 3,375 for the attenuated virus
vaccines and one in 3,A35 for the diluted virus vaccines.

Redewill and Underwood (l9h7) report the incidence of severe post-
vaccinal complication following rabies vaccination at one per 1,19h persons

treated in Los Angeles County during a five year period (19h0-19h5 inclusive).

In Los.Ange1es County and City, Pait and Pearson (19h9) reported nine
cases of postvaccinal encephalitis among 5,500 treated persons, an inci-
dence of one in 600. The authors indicated that in this area the possi-
bility of acquiring postvaccinal reactions as a result of antirabies
treatment is approximately twice as great as that of acquiring rabies
from known dog bites.

Cook £3 21. (1955) reported that postvaccinal reactions occurred at
the rate of one in 527 treated persons in a group of 8,h30 treated with
phenolized rabies vaccine in Texas from 19h9 through 1953. Appelbaum (1953)
reported the incidence of encephalomyelitis as a result of rabies vaccina-
tion at one accident to 2,025 persons treated in New York City Department
of Health Clinics. Here the risk of rabies from a'bite was shown by the
author to be greater than that of encephalitis from vaccination.

It is generally believed, however, that many minor cases of paralytic
accidents fail to be reported and that the true incidence is higher than
any published data would indicate (Remlinger, 1927).

Published statistical data on the occurrence of postvaccinal reactions
following rabies vaccination in Michigan was not available.

Although rabies vaccines containing nerve tissue have been used exten-
sively, they cannot be considered entirely safe for use in either man or
animal since myelitis or encephalitis occasionally occurs (Burkhart st 21.
1950). The possibility of such reactions from the injection of nerve tissue
must be balanced against any advantage attained by the use of such vaccines.

The vaccine which has been most widely employed within recent years
has been the Semple type vaccine, which is a killed vaccine prepared from
'brain tissue, the inactivating agent being phenol. This is, of course, one

of the crudest types of antigenic material.

The cause of paralytic accidents resulting from antirabic treatment
have, in the past, been ascribed to infection with the fixed virus con-
tained in the vaccine by Fielder (1916), Busson (1926) and Van Stockum
(1935). Bassoe and Grinker (1930), however, believed the reactions were
due to a separate virus entity. Marsden and Hurst (1932) also stated
that the postvaccinal reaction is entitled to recognition as an indepen-
dent clinical and pathological entity. It was Observed, however, that
these reactions continued to occur even when completely killed vaccines
were employed.

Some of the earlier workers, on the other hand, were of the Opinion
that the reactions were of an allergic nature. Stuart and Krikorian (1928)
believed that the reactions resulted from the introduction of nerve sub-
stance during the process of immunization. Stimson (1910) suggested that
it was an allergic reaction to foreign nerve protein, while Cornwall (1918)
stated that the reactions were anaphylactic in nature, the antigen being
either normal brain matter or the products of metabolism of the rabies
organism.

It has more recently'been indicated that the encephalitic reactions
are allergic in nature. The neurologic lesions are believed to arise
from an allergic reaction of the patient to the nervous tissue in the
vaccine.

In determining the incidence of allergy among those receiving rabies
prophylaxis, Horack (1939) was able to demonstrate allergy in 87.5 per cent
of the paralytic cases and 33 per cent in cases showing no neurological

symptoms.

Symptoms and path010gical changes in the central nervous system,
similar to those produced in postvaccinal reactions, were shown by Freund
‘gt §l° (19h7) to result from serial injections of normal brain tissue with
adjuvants in guinea pigs. Similar results were Obtained by Jervis and
KOprowski (19118) and KOprowski and Jervis (191t8) .

Some workers have been able to produce paralysis in animals by means
of repeated injections of either homologous or heterologous brain material.
Schwentker and Rivers (193A) demonstrated paralysis in rabbits by means of
repeated injections of normal rabbit brain tissue. Kabat gt g1. (19h7)
and Morgan (19h7) were able to show encephalomyelitis in monkeys as a
result of injections with normal monkey brain. Rivers, Sprunt and Berry
(1933) and Rivers and Schwentker (1935) reported encephalomyelitis in
monkeys as a result of injections of normal rabbit brain and extracts of
normal rabbit brain.

Olitsky and Tal (1952) and Tel and Olitsky (1952), however, have shown
that the proteolipides, isolated from'brain by Folch and Lees (1951a, 1951b)
are capable of bringing about acute disseminated encephalomyelitis in mice
indistinguishable from the condition induced by the inoculation of whole
brain tissue. Goldstein 239 3;. (1953) demonstrated the same reaction with
guinea pigs.

There are indications that the postvaccinal reactions are due to anti-
body to the injected brain material reacting with the tissues of the nervous
system of the susceptible animal, thereby producing the encephalitis Or
other nervous system reaction.

Schwentker and Rivers (1931+) determined that brain tissue under proper
conditions functions as a complete antigen and is capable of producing

complement-fixating antibodies in rabbits which are organ-Specific

rather than species-specific. Lewis (1933) demonstrated the presence of
antiebrain antibodies in experimental animals injected with'brain sub-
stance. These antibodies to brain tissue were also shown by Kirk and
Ecker (l9h9) to be produced in humans receiving antirabies vaccine.
Kbprowski and LaBell (1950) demonstrated the development of complement-
fixating antibodies to brain tissue in the serums of 50 per cent of 3h
persons receiving Semple vaccine.

These Observations strengthen the hypothesis that postvaccinal
reactions are in some manner associated with the deve10pment of specific
antibodies for brain.

The cause of the postvaccinal reactions is not clear. However, it
is believed that either the removal of the encephalogenic factor from
the vaccine or the use of a vaccine which does not contain brain tissue
would prObably eliminate the danger of such reactions (Jervis, 195A).

Rabies virus Obtained from suspensions of infected tissue may be
purified to some degree by the common methods of selective precipitation
of serum.proteins. Cox gt g1. (19h7) reported purification and concen-
tration of rabies virus by means of alcohol precipitation. Tagaya gt El'
(1953), however, does not recommend alcohol precipitation because repeated
treatment results in a great loss of virus activity, but favors repeated
acid precipitation. Behrens 33 El‘ (1939) reported purification of rabies
virus by precipitating the tissue protein at its isoelectric point.

In addition, Warren gt a1. (19h9) succeeded in precipitating rabies
virus with protamine sulphate and.Mu11er (1950) and Sawai gt 2;. (1959)

purified rabies virus with the application of ion exchange resins.

 

 

Apparently none of the foregoing methods Of rabies virus purifi-
cation has been adaptable to quantity production of purified rabies
vaccines.

Bell, Wright and Habel (1919), however, described a method for the
removal of the encephalogenic factor from brain tissue suspensions by
the fractionation of'benzene-ether treated infected brain tissue with
calcium acetate. Paterson gt El. (1953) indicated that protamine sul-
fate appeared to sediment most, if not all, of the encephalogenic
activity of‘both rabbit and mouse brain material. Harris (19h8)
employed ether at low temperatures to extract the fats and lipids from
infected brain material. D'Silva 33 El‘ (1951) and Hottle and Peers
(195h), however, advocate the use of centrifugation of brain tissue
suspensions in distilled water to remove a large part of the encephalo-
genic factor from rabies vaccine.

Cohn and his workers (1953) successfully purified and fractionated
the various important proteins of human plasma employing the interaction
of the proteins with heavy metals. Chapman and Surgenor (1951+) utilized
this method for the precipitation, concentration and purification of
viruses. The method proved successful for the extraction of several
kinds of viruses from several kinds of tissues and tissue fluids. 'With
the zinc precipitation method, they were able successfully to precipitate
and extract the Rous sarcoma virus from infected allantoic and amniotic
fluids and from aqueous and saline extracts of tumors, embryonic liver,
chorioallantoic and amniotic membranes. This method was modified by
Martin and Chapman (195h) to purify and concentrate the PR8 influenza

virus from infected allantoic fluids. In addition, modifications of

 

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this method have been applied to the concentration and purification of
Polio viruses from kidney tissue by Smolens 33 21. (1955).

It is generally assumed that viruses are protein in nature. There-
fore, it should be possible to separate them from tissues by the same
method which enabled the fractionation of plasma. Some viruses can be
concentrated from.infected tissues as metal complexes and following the
dissociation of the metal-virus complex, the virus is Obtained without
measurable loss of yield or potency.

The use of brain tissue in rabies vaccine production is both
practical and economical. The method of purification of rabies vaccine
should, therefore, be amenable to the use of brain tissue as the start-
ing material even though it contains excessive amounts of extraneous
material and a high concentration of nonviral materials which prObably
have properties similar to those of the virus.

It is not within the sc0pe of this study to purify the living rabies
virus pg; gg,‘but rather to reduce the concentration of non-specific

materials contained in the vaccine without appreciably decreasing its

antigenicity.

III. MATERIALS AND METHODS

A. Rabies Virus

 

The fixed virus strain employed in this study was received from the
Department of Health in Detroit, Michigan in 1932. The Detroit Department
of Health Obtained the virus from.Dr. F. G. Novy of the Pasteur Institute
at the University of Michigan in Ann.Arbor, Michigan, who in turn received
the virus from.Dr. Louis Pasteur. The virus employed represents 196
passages at the Michigan Department of Health.

The virus was prepared as an emulsion containing 20 per cent rabbit
brain tissue in a diluent of two per cent horse serum in distilled water.
The infected rabbit brain was ground in a chilled Waring'Blendor for five
minutes and dispensed in 3-ml amounts in ampoules. The ampoules were
flame-sealed and the contents were shell frozen in a dry ice-alcohol'bath.
The ampoules were stored at -500C. until needed.

When needed an ampoule was removed from storage, the contents thawed
and 2.5 ml of the emulsion was diluted with 2.5 ml of two per cent horse
serum in distilled water to give a ten per cent tissue concentration. This
suspension was centrifuged at 3500 r.p.m. for 15 minutes and the supernate

was drawn off and diluted to l x 10’3 for rabbit passage.
B. Preparation of Rabies Vaccine

Normal adult rabbits were injected intracerebrally through the Opening
about one-half inch posterior to the outer canthus of the eye with 0.2 ml

of a 1 x 10"3 dilution of the fixed virus by means of a 20 gauge needle

10

with a short bevel. After the rabbits showed symptoms of rabies and had
been prostrate for 2h hours, which was usually on the fourth and fifth
day after inoculation, they were sacrificed by injecting air into the
ear vein.

The body of the rabbit was then sheathed in a cloth saturated in
five per cent phenol solution and the hair on the head was also wet down
with five per cent phenol solution.

An incision was made down the mid-line of the head and the skin folded
back. Care was exercised not to cut the ear canals, inasmuch as large
numbers of bacteria are present in this area. The skull was opened by
means of bone forceps, the brain removed and placed in a sterile tared
jar. Each brain was tested for sterility by removing a small piece of
brain tissue from each of two different portions of the brain and placing
them into tubes of National Institutes of Health sterility test medium.
After sterility tubes had been seeded, the brain was weighed and placed
in storage at -50°C.

The infected brains, kept in glass jars, were thawed in a bath of
five per cent phenol solution and then transferred to the sterile hopper
of a colloid mill, the rotor of the colloid mill having been previously
sterilized with ten per cent phenol and thoroughly rinsed with sterile
distilled water.

A portable circulating refrigeration bath was connected to the mill
and cold (-5°C.) ethylene glycol circulated through the hopper jacket.

Sufficient saline was also placed in the mill hopper to give a 50
per cent tissue emulsion. The brains were emulsified by grinding for two
minutes. The suspension was then sampled to determine its virus content.
After sampling, a calculated volume of phenolized saline was added to the

50 per cent emulsion in a hopper and the grinding continued for another two

'1'!

minutes. This #0 per cent emulsion, containing 0.8 per cent phenol, was
drained from the mill hOpper into gallon bottles. Merthiolate was added
to a contentration of 1:10,000 and the #0 per cent brain emulsion was
allowed to attenuate at room temperature (20-250C.) for 21 days. The
emulsion was agitated gently several times daily. A

After 21 days the vaccine was diluted to 6.66 per cent tissue with
0.85 per cent saline and screened through 100 and l50-mesh monel metal
screens. Additional merthiolate was added to give a final concentration
of l:10,000.

The vaccine was sampled and six mice were injected intracerebrally
with 0.03 ml to determine if complete attenuation had occurred. The mice
were Observed for symptoms of fixed virus rabies for a period of 1M days.
When the virus was shown to be non-viable by mouse test, the vaccine was
sampled for antigenicity and safety test in rabbits. Two rabbits were
injected intracerebrally with 0.25 ml each in the same manner as used for
injection of rabbits for production purpose. The rabbits were Observed

1h days for symptoms of fixed virus paralysis.
C. Methods of Purification

The purification methods employed in these studies were based upon
the use of a 6.66 per cent rabbit brain tissue vaccine as the starting
material. This vaccine is representative of that produced by the Michigan
Department of Health.

1. Zinc precipitation methods.
a. Method of Chapman and Surgenor. Lot I was purified according to
the tentative method of Chapman and Surgenor (1953) for the

extraction and purification of viruses.

12

A schematic diagram of the procedure is as follows:

6.66 per cent tissue vaccine

Zinc reagent (0.025 M Zn)

 

 

 

 

 

g pH 7.1+ 0°C.
l‘ . \
Zinc precipitated Zinc Solubles
Complex
wgsh (0.005 M Zn + 0.02 M glycine)
r- 0 0' == Solubles
ppt
Zinc acetate inowater (0.001 M.Zn)
F. PH 5'5 ' 5'6 O C‘ :— Extract I
PPt
Zinc acetate in saline (0.00% Zn in
r 0.15 M NaCl) pH 5.5 - 5.6 0 c. ’ Extract II
91313
_ 0.1 M Sodium citrate 0°C. __ Purified virus
r_ 'I' extract
Residue

All glassware, reagents and vaccine suspensions were kept at
00 to hOC. throughout the precipitation and extraction procedures.

In addition, the entire procedure was carried out in a refrigerated
bath and centrifuge. The temperature never exceeded 5°C.

The vaccine suspension was gently stirred and the zinc reagent
added drOpwise in the ratio of 0.25 ml zinc reagent per 5 ml of vaccine
suspension. (After the complete addition of the zinc reagent and
adjustment of the pH to 7.h with l N NaOH, the mixture was allowed

to equilibriate for an hour at 0°C. with occasional stirring. The

13

mixture was centrifuged at 3000 r.p.m. for 30 minutes and the
supernate removed (zinc Solubles).

The precipitate was resuspended in the wash reagent at 0°C.
and centrifuged at 1500 r.p.m. for 30 minutes and the supernate
removed (Solubles).

The precipitate was resuspended in the zinc acetate in water
solution. The pH was carefully adjusted to 5.5 - 5.6 by bubbling
C02 through the mixture while the suspension remained at 0°C.*

The mixture was centrifuged at 1500 r.p.m. and the supernate
removed (Extract I). The precipitate was resuspended in the zinc
acetate in saline solution, while the suspension was held at 0°C.
The pH was again carefully adjusted to 5.5 - 5.6 by bubbling 002
through the mixture. The mixture was centrifuged at 1500 r.p.m.
for 30 minutes and the supernate removed (Extract II). The
precipitate was dissolved in a 0.1 M sodium citrate solution to
extract the antigen from any remaining insoluble materials. The
mixture was centrifuged at 3000 r.p.m. and the supernate recovered.

The supernate represented the purified vaccine extract and
was designated IeA.

Modifications of zinc method. Lots II, III and IV represent certain
modifications of the procedure for precipitating and extracting the

rabies antigen (virus) as advocated by Chapman and Surgenor.

 

* A small 002 generator was made by adding water to dry ice and
washing the released gas in water.

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The precipitation procedure for Lot II was altered. The pH
of the starting material was not adjusted to 7.h with.Na0H. The
pH of the starting material itself was 6.8.

In processing Lot III, the following modifications were made
in the extraction procedure:

Lot IIIeA. The zinc acetate in water extraction was carried out
at pH 5.8.

The zinc acetate in saline extraction was carried out
at pH 6.1.

Lot IIIéB. The zinc acetate in saline extraction was carried out
at pH 5.8.

Lot III-C. The zinc acetate in water extraction was carried out
at pH 5.8.

Instead of zinc acetate in saline, the precipitate was
extracted with buffered saline at pH 5.9.

The precipitation procedure was also altered for Lot IV. The
pH of the starting material was adjusted to 7.8 and the diluent for
the material was distilled water.

Method of Martin and Chapman. A 100 ml sample of 6.66 per cent
vaccine was treated in accordance with the procedure of Martin and
Chapman (l95h) for the purification of influenza virus.

The sample was zinc precipitated in the same manner as described
for the method of Chapman and Surgenor. The precipitate, however,
was repeatedly extracted with.l M glycine at pH 7.3. The extraction
procedure was carried out first with 100 ml of glycine and then with
three successive MO-ml volumes of glycine. The material was centri-
fuged each time at 3000 r.p.m. fOr one hour at 3°C. The extracts
were designated as Lots VeA, VéB, V-C and V-D, respectively. The

residue of the final extraction was made up to the original volume

15

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of the starting material with saline. This residue vaccine was
labeled Lot VeE.

Vaccine antigen solubility study. A sample of the starting 6.66
per cent tissue vaccine made in saline was centrifuged at 3°C. for
30 minutes at 3500 r.p.m. The supernatant fluid was drawn off and
designated Lot A-l.

A sample of the starting vaccine made in distilled water was
also centrifuged at 3°C. for 30 minutes at 3500 r.p.m. and the
supernatant fluid was drawn off and designated as Lot B-l.

After centrifugation the residues of the above vaccine samples
were made up to their original volumes with their respective
diluents. These products were designated as Lots.A and.B,

respectively.

2. Physical methods of cellular disintegration.

a.

Sonic oscillation. Sonic vibration was applied to 50 ml samples of

 

the vaccines. In order to determine the optimal time exposure to
sonic vibration the samples were sonerated at various time intervals.
The material was sonerated in a 10-k.c. Raytheon oscillator. The
sonic oscillator cup containing the vaccine was continuously cooled
'by passing cold (~10°C.) ethylene glycol through the oscillator cup
jacket.

The time of exposure of the vaccine to sonic vibration was 2, h,
6, 8, 10 and 1h minutes. These samples were designated as Lots VI—2,
VI-u, v1-6, VI-8, VI-lO and VI-lh, respectively.
Freezes-thaw technique. A 100-m1 sample of the vaccine was disinte-
grated by alternately freezing and thawing. The brain tissue

suspension was frozen rapidly by means of an alcohol-dry ice bath,

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followed by rapid thawing in a 37°C. water bath. This procedure
was repeated 20 times. This material was centrifuged in a refriger-
ated centrifuge at approximately 3000 r.p.m. for 30 minutes. The
supernatant fluid was drawn off and the residue was made up to its
original volume with physiological saline. The products were then
designated as Lots VII-A and VII-B, respectively.

Slow alternate freezing and thawing of a 100-ml sample of the
vaccine was carried out 20 times. The suspension was frozen in a
mechanical freezer at -50°c. and thawed at room temperatures. The
material was centrifuged at 3000 r.p.m. for 30 minutes. The super-
natant fluid was drawn off and the residue was made up to the
original volume with saline. The products were designated as

Lots VII-C and VII-D, respectively.

3. Combined physical and chemical procedures.

a.

Sonic oscillation - zinc precipitation. Lot VIII represents a
1004m1 sample of starting vaccine which was treated in accordance
with the method of Martin and Chapman (19510 after sonic vibrating
for two minutes at approximately 10-k.c.

The sample of sonic-vibrated vaccine was zinc precipitated'by
adding zinc diglycinate reagent drOpwise while gently stirring at
0°C. The reagent was added in the ratio of 0.25 ml per 5 ml of
vaccine suspension. .After the complete addition of the zinc reagent
the mixture was adjusted to pH 7.h with l N NaOH and allowed to come
to equilibrium for one hour. The mixture was centrifuged at 3000
r.p.m. for 30 minutes and the supernate removed.

The precipitate was repeatedly extracted with 1 M.g1ycine at

pH 7.3. The extraction procedure was first carried out with a 100-ml

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b.

C.

quantity of glycine and then with three successive hO-ml amounts.
The mixture was centrifuged each time at 3000 r.p.m. for one hour
at 3°C. The extracts were designated as Lots VIIIeA, VIIIéB,
VIII-C and‘VIIIéD, respectively. The residue of the final
extraction was made up to its original volume with saline.

This material was designated as Lot VIIIéE.

Effect of sonic treatment on antigen solubility. .A sample of
the starting vaccine in saline, which was sonic vibrated for
six minutes, was centrifuged for 30 minutes at 3500 r.p.m. The
supernatant fluid was drawn off and designated Lot C-l.

The residue of the above sample was made up to its original

volume with saline. This product was designated Lot C.

Ether extracted vaccine. .An extraction procedure modified from
that of Harris (19MB) was followed, using ten volumes of ethyl
ether as the extraction solvent. This was cooled to a tempera-
ture of about -50°C. and then the 6.66 per cent tissue vaccine
at Just above its freezing point was added drOpwise with gentle
stirring. Thereafter the mixture was brought to ~10°C. over a
period of several hours in a refrigerated‘bath. The ether was
drawn off and the residue made up to volume with saline. This

vaccine was then designated as Lot IXeA.

18

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d. Ether-ethanol extracted vaccine. Ten volumes of an extraction
solvent consisting of three parts ethyl ether and one part ethanol
was employed in this extraction. The conditions employed were the

same as those above. The residue vaccine was designated as Lot IXAB.
_D. Antigenicity Testing

The potency (protective value) of the vaccine was determined in
accordance with the Minimuijequirements for Rabies Vaccine of the National
Institutes of Health (1953) .

This test was based on the use of white Swiss mice approximately four
weeks old, uniform in weight (ll-15 gms) and of one sex.

Three or more dilutions of the vaccine under test were prepared using
fivefold increments. The diluent employed was either saline or distilled
water.

{At least ten mice were injected intraperitoneally with 0.5 ml of each
dilution of the vaccine. Two doses were given to each mouse one week apart.
Enough control mice were set aside at the time of the injection of the first
dose of vaccine, so that an adequate titration of the challenge virus could
be made. .At least ten mice were used for each dilution of challenge virus.
The challenge virus was supplied by the National Institutes of Health.

At the time of challenge of the test mice, the control mice were divided
into groups of at least ten mice and 0.03 ml of tenfold dilutions of the
challenge virus injected intracerebrally. The dilutions were usually 10'6,
10'7, 10"8 and 10-9. These control groups were inoculated with the challenge
virus only after all the test mice had been inoculated.

All of the test mice were injected intracerebrally with 0.03 ml of a

10"6 dilution of the challenge virus 1% days after the first dose of vaccine.

19

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This dilution of challenge virus usually gives challenge doses of 5 to
50 LDSO'

All mice were observed for 1h days from the time of the challenge
injection. Only those deaths occurring after the fifth day were considered
as rabies deaths. The mice which became paralyzed, but survived the lh-day
Observation period, were considered to have died with rabies.

Fifty per cent end-points were determined for the test vaccines and
the controls by the method of Reed and Muench (1938). The end-points were
calculated as an ED50 of vaccine in milligrams of original brain tissue
which will protect 50 per cent of the mice. The LD50 of challenge virus
received by the immunized mice is calculated by dividing the dilution of
virus used as the test dose by the 50 per cent end-point dilution of virus

in control mice as calculated by the method of Reed and Muench.

E. Chemical Analysis

 

The relative degree of rabies vaccine purification attained was based
upon the reduction of protein nitrogen, total solids, and total ether-
soluble lipids.

Protein nitrogen was determined by precipitation with 30 per cent
trichloracetic acid followed by micro-Kjeldahl analysis of the nitrogen
by a modification of the method of Ma and Zuazaga (19h2).

Ether-soluble lipid was determined by continuous extraction in a
micro-Soxhlet apparatus, following the procedure described in Methods of
Analysis, Association of Official Agricultural Chemists.

Total solids were determined by drying samples to constant weight

at 1050 to 110°C.

20

 

 

IV. RESULTS

The application of the zinc precipitation and extraction procedure,
advocated'by Chapman and Surgenor for the purification of viruses, proved
unsuccessful for the purification of rabies vaccine derived from.rabbit
brain. Only a small amount of the antigen of the original vaccine could
be recovered in the purified form.

Failure to purify the vaccine without appreciably decreasing its
antigenicity suggested that certain modifications of the method of
precipitating and extracting the antigen should be made. These modi-
fications were directed primarily at changing the hydrogen-ion concentrations.

The results of these alterations, as well as those of the original
method, are shown in summary form.in Table I. It will be noted that
regardless of the pH of the zinc precipitating procedure or the treat-
ment of the zinc-protein complex, only a small amount of antigen of the
original vaccine could be Obtained.

The zinc precipitation and extraction method of Martin and Chapman
gave only slightly'better results with respect to antigen yield. The
results of the application of this method are shown in Table II. Inasmuch
as the majority of the antigen of the starting vaccine can be accounted for
in the various extractions, no measurable amount of antigen denaturation
took.place. This would indicate, then, that either the zinc failed to
precipitate the majority of the antigen or that the glycine failed to

efficiently extract the antigen from the zinc complex. In the repeated

21

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1) VII?!

TABLE II

RESULTS OF PURIFICATION PROCEDURE OF MARTIN AND CHAPMAN

 

 

SUMMARY*
Antigenicity W
Lot ED§O Antigen.Recovery
Control 1.70 mg --
Extract V—A 10.0 17
Extract V-B 10.0 17
Extract V-C 8.92 19
Extract V-D >10 . O 4 17
Residue V-E 5.28 32.2

 

 

 

* Original data presented in Appendix ii

extractions of the zinc complex with glycine, substances which bind zinc
least avidly are removed first, and those which bind zinc most strongly
are removed last.

The results indicate, however, that most of the antigenic substance,
if it were zinc precipitated, is not removed in any single extraction. If
the zinc did not precipitate the major portion of the antigen, the glycine
solutions merely extracted that portion of antigen which is soluble in the
medium. The relatively constant amount of antigen Obtained per extraction
seems to indicate this, and that if the residue was again extracted, the
residue would be reduced by a similar amount of antigen.

The zinc precipitating method was employed by others primarily for

the precipitation and extraction of viruses from allantoic and amniotic

23

   

 

 

 

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fluids and from aqueous and saline extracts of tissue. In view of this,
it seems that the virus must be largely soluble or highly dispersed in
the medium.in order to be precipitated by zinc and subsequently extracted.

Brain tissue suspensions, which constitute the starting rabies vaccine,
differ from allantoic fluid or aqueous and saline extracts of tissue in
respect to solubility. It seems logical to assume that the solubility of
the antigen plays an important role in the ability of the zinc to
precipitate it.

Results of the study to determine the amount of antigen that can be
expected to be present in the aqueous and saline extracts of rabies vaccine
are shown in Table III. The extracts contained little antigen as compared
to the entire vaccine. Approximately nine per cent of the total antigen
was recovered in the aqueous extract. The saline extract of the vaccine,
however, contained even less antigen. Approximately four per cent yield
of antigen was Obtained from the original vaccine in the saline extract.

These observations serve to point out the fact that since only a
small amount of antigen is present in either the saline or aqueous extracts,
it can be assumed that the majority of the antigenic component of rabies
vaccine is associated with the small particles of brain tissue. subse-
quently, this may indicate the reason for the apparent inability of the
zinc to precipitate the bulk of the antigen from the suspension. The
zinc is not effective in disrupting the cells or particles, but merely
precipitates the material in solution.

The extraction of soluble antigen from tissue cells or particles,
then, can only follow the further destruction of the cellular membranes,

since these must be largely impermeable to the antigen.

2h

TABLE III

STUDIES ON RABIES ANTTGEN SOLUBILITY

 

 

 

 

SUMMARY*
1.1:!" _._ r M
Antigenicity Percent Total
Lot Epic Antigen Recovery
Control A 0.50 mg --
A-l
(Saline Extract) 12.60 1+
B-l
(Aqueous Extract) 3.19 9
Control C 0.828 --
C-l
(Sonic treated Extract) _26.90 3

 

 

 

*Original data presented in Appendix iii

In view of the above Observations, several methods of mechanical
disruption of the cells or tissue particles were attempted on the starting
brain tissue vaccine. The methods included slow and rapid freeze-thaw
techniques and sonic oscillation.

The simplest and'best method for the destruction of cellular mem-
branes is considered to be disintegration by repeated freezing and thawing.

The effects of repeated freezing and thawing on the starting vaccine
are shown in Table IV. Complete denaturation of the antigen, as measured
by the mouse potency test, resulted from repeated slow freezing and
thawing. .Almost complete inactivation of the antigen was Observed with

the repeated rapid freeze and thaw technique.

25

TABLE IV

EFFECT OF REPEATED FREEZING AND THAWING ON RABIES VACCINE

 

 

SUMMARY*
=r_ gr Antigenicity
Lot ED5O Antigen Recovery
Control 0.668 --
VIeA Not measurable O
VIéB Not measurable O
VI-C Not measurable 0
VI-D ' Not measurable O

 

 

 

* Original data presented in Appendix iv

The effects of sonic oscillation, on the other hand, present quite
a different picture. The results, shown in Table V, indicate the effect.
of sonic vibration on the antigenicity of the starting vaccine in relation
to the time of exposure. It appears evident that sonic vibration for
periods exceeding six minutes decreases the antigenicity of the vaccine
appreciably. This is undoubtedly a denaturing effect which could be
caused by mechanical agitation or thermal effects. HOwever, sonic
vibration for periods less than six minutes increased the antigenicity
of the vaccine considerably. It appears reasonable to assume that the
breaking up of the small particles or cells of tissue releases more
antigen (virus), which results in a higher potency than would otherwise

be attained with the vaccine.

26

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TEBLE V

EFFECT OF SONIC VIBRATION ON RABIES VACCINE

 

SUMMARY*
WW
Lot Exposure Antigenicity ED 0 Control Vaccine/

in Minutes ED5O ED50 Test Vaccine

Control -- 1.07 mg ~-

VII-2 2 0.56u 1.90
VII-h u 0.66h 1.61
VII-6 6 0.60M 1.77
VII-8 8 1.17 0.92
Control -- 1.18 --

VII-10 10 1. 311 0.88
VII-1h 1h 1.39 0.85

 

 

 

 

*Original data presented in.Appendix v

This Observation suggested the possibility of Obtaining a higher
antigen yield from the starting vaccine, if the vaccine was first sonerated
for the Optimum time exposure before being precipitated by zinc. Inasmuch
as the glycine extraction procedure appeared to be somewhat better than
that of the Chapman and Surgenor method, the former method of extraction
was employed.

As indicated in Table VI, the use of sonic oscillation in conjunction
with zinc precipitation and repeated extractions with glycine increased
the yield of purified vaccine to some extent. The yield represented an

approximate twofold increase in the antigen recovered in the first glycine

27

 

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extract. This amount of antigen, however, does not approach the minimum

required to insure an adequate degree of protection.

Further, subsequent

extractions were not proportionately increased in antigen, indicating

that increased solubility of the antigen had not necessarily been attained.

The antigenicity of the residue decreased in direct prOportion to the

increase found in the first glycine extract.

This may indicate that the

further disintegration of the cells permits more antigen to be liberated

in the form of an insoluble component which could not be precipitated

from the non-sonerated vaccine due to its association with tissue cells.

This does not mean that the antigen is rendered more soluble in the dis-

persing medium,'but that it is made more available to the zinc.

TABLE VI

RESULTS OF PURIFICATION PROCEDURE OF MARTIN AND CHAPMAN

ON SONIC VIBRATED RABIES VACCINE

 

 

SUMMARY*
==I
Antigenicity Percent Total
Lot ED50 .Antigen Recovery

Control 1.70 mg -—

Extract VIII-A 11.92 311.6
Extract VIII-B >10.0 <17.0
Extract VIII-C 7.88 21.6
Extract VIII-D A>lO.O ‘<17.0
Residue VIII-E 10.0 17.0

 

 

 

*Original data presented in Appendix vi

28

 

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On the basis of this Observation, a further study on the solubility
of the antigen was made. This study was conducted on a sonic vibrated
sample of vaccine. The data of this study, also presented in Table III,
indicate that the amount of soluble antigen present in the saline extract
of the sonic vibrated sample was no greater than that present in the
saline extract of the regular starting vaccine. Regardless of further
disintegration of the cells by sonic oscillation, the solubility of the
antigen remained relatively unchanged.

The results of attempts to free the starting rabies vaccine from
the majority of the non-specific proteins and lipids of brain matter by
employing ether and an ether-ethanol mixture as extractants are given in
Table VII. No reduction in protein nitrogen was Observed with either
extractant. The ether extracted approximately #8 per cent of the lipids,
and the ether-ethanol mixture extracted approximately 7h per cent. A
comparable reduction in total solids was observed. Both techniques,
however, greatly decreased the antigenicity of the vaccine.

These results indicate only that a mixture of ether and ethanol is
more effective in removing the lipids from the vaccine than is ether
alone. This may be due, in part, to the destructive action of ethanol
on the cellular structures and/or the splitting of lipOprotein complexes
by the ethanol.

Various chemical determinations (total nitrogen, total solids and
total lipids) were performed on most of the purified vaccines studied.
Since these vaccines were of insignificant antigenicity, the inclusion

of these findings was not warranted.

29

 

 

 

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V. DISCUSSION

Chapman and Surgenor (195H) were successful in purifying viruses
from allantoic and amniotic fluids and from aqueous and saline extracts'
of tissues. They employed a modification of the zinc precipitation method
of Cohn (1953) fOr the purification of plasma proteins. A modification of
this method was also used by Martin and Chapman (1951;) for the purification
of influenza virus from allantoic fluid.

These methods of virus purification, as well as modifications of the
zinc precipitation methods, were applied without success in this study to
the purification of rabies vaccine derived from rabbit brain.

In dispersing media, such as allantoic and amniotic fluids or extracts
of tissue, the zinc precipitable substances apparently can be readily
precipitated as zinc complexes. The precipitable substances can be re-
garded, therefore, as being largely soluble or highly dispersed in the
medium. In any case, the substances are free to react with the zinc.

The small degree of solubility exhibited by the rabies antigen in
aqueous and saline extracts of the vaccine indicates that the major portion
of the antigen is associated with the minute tissue particles or the tissue
cells. It was also Observed that the antigenicity of the vaccine was
enhanced when tissues were disintegrated by sonic oscillation. The
saline extract of sonic treated vaccine, however, demonstrated no compar-
able increase in antigenicity, indicating the apparent insolubility of
the antigen.

It was further Observed that when sonic treated vaccine was zinc
precipitated and extracted with glycine, more antigen was recovered than

when this treatment was applied to the regular starting vaccine. This

31

indicated the possibility of having an insoluble antigen which is
liberated, to some extent, from the brain cells or substances by sonic
oscillation and subsequently preCipitated by the zinc.

The action of the phenol, employed as the rabies virus inactivating
agent, must also be considered in regard to the insolubility of the rabies
antigen. The rabies virus may be complexed or denatured by the phenOl or
otherwise rendered more insoluble by its action.

Most denatured proteins are insoluble. However, some proteins can
be rendered insoluble without any loss of biological activity. Johnson
(19h8) stated that live rabies virus is no better in invoking an immune
response than an inactivated or killed virus. Inactivation of the virus,
then, does not necessarily result in loss of antigenicity, although the
inactivating agent could render it more insoluble. It may be pointed
out, however, that some workers were able to maintain the Flury strain
of living rabies virus in chick embryos and were able to induce biological
and immunological changes that were significant contributions to the
preparation of a commercial vaccine of high antigenicity.

It has been Observed that a large portion of the live rabies virus
is found in the sediment of macerated brain tissue as shown by regrinding
the sediment and titrating the supernate. The virus particle may combine
with and remain fixed to host tissue components as do certain other virus
particles (Curnen and Horsfall, 19h6). Similarly, the antigenicity of
phenol-inactivated rabies vaccine is largely found in the sediment.

Attempts to purify the rabies vaccine by zinc precipitation of the
antigen followed by selective dissociation of the zinc complex was

apparently accomplished only on that portion of the antigenic component

32

which can be considered to be free to react with the zinc. The zinc is
prObably not effective in disrupting the tissue particles or the chemical
complexes which may have been formed with the phenol.

Whereas many proteins remain in the native state when their solutions
are frozen and thawed repeatedly, the solubility and biological properties
of lipOproteins are affected by such treatment. It is well known that
the lipOproteins of plasma are denatured upon repeated freezing and
thawing (McFarlane 19112, Haurowitz 1950) . Johnson (19u8) stated that
repeated freezing and thawing of live rabies virus suspensions results
in the loss of infectivity. It was also Observed in this study that
the antigenic component of rabies vaccine or the inactivated virus was
completely destroyed by repeated freezing and thawing.

A considerable portion of the lipids of rabies vaccine cannot be
extracted by treatment with ether. This may be due to the presence of
lipOprotein complexes. The lipOproteins are, however, split by the
action of ethanol. Inasmuch as the temperatures of the ether and ethanol
extractions employed in this study never exceeded ~100C, it was felt that
thermal denaturation of the rabies antigen was not indicated. However,
when ether was employed as the extraction agent for the removal of lipids
from rabies vaccine, almost complete loss of antigen resulted. A similar
loss of antigen resulted from the use of an ether-ethanol mixture, although
the amount of lipid extracted was considerably greater. This only indi-
cates that the ether-ethanol mixture is a better lipid extracting agent
than ether alone. However, there is the possibility that the use of

ether at -50°C. may have partially denatured the rabies antigen as was

33

shown with the repeated freeze and thaw technique. MOFarlane (19h2)
employed a method of freezing and thawing with ether to split lipOproteins.

Some purified viruses have been found to contain significant amounts
of lipid. Equine encephalomyelitis virus was found to contain large
amounts of lipid in the form of phospholipid, cholesterol and neutral
fat (Beard, l9h5). Influenza virus was found to contain lipid, in
addition to nucleOprotein (Knight, 19A7). Although data on the chemical
nature of the rabies virus are not available, it is possible that this
virus also contains lipids and that the action of lipid solvents dena-
tures the virus. It has been shown that the rabies virus is only
moderately resistant to ether, but very resistant to phenol (Johnson, l9h8).

In addition to the denaturation of the rabies antigen and virus by
lipid solvents, it has been shOwn that the rabies antigen, as well as the
active virus, is denatured by repeated freezing and thawing. This pheno-
menon is characteristic of lipOproteins. It is also possible that lipids,
in some manner, may protect the rabies antigen or virus in some chemical
combination.

It is the author's Opinion that before any acceptable method of
rabies vaccine purification can be accomplished, it will be necessary
first to characterize chemically the inactivated rabies virus. The
intimate association of the antigen with brain substances or protective
chemical complexes has defied most physical and chemical methods of

separation without denaturation of the antigen.

3h

VI. SUMMARY

The zinc precipitation and~extraction procedures advocated by Chapman
and Surgenor (195A) and Martin and Chapman (195h) fOr the purification of
certain viruses were unsuccessful when applied to the purification of
rabies vaccine derived from rabbit brain. Modifications of the method
likewise proved unsatisfactory. .Although attempts to_purify the rabies
vaccine without appreciably decreasing its antigenicity failed, significant
information was Obtained concerning the antigen.

The rabies antigen is largely insoluble or poorly dispersed in aqueous
and saline solutions. This is due either to the intimate association of the
antigen with the brain substances or to its chemical composition. This
insolubility is thought to be the primary cause for the failure of the
zinc to precipitate the major portion of the antigen from the vaccine.

Sonic oscillation for short intervals greatly increases the antigen-
icity of the rabies vaccine. However, no comparable increase is found in
its antigen solubility. The liberation of some insoluble antigen, which
was zinc precipitable, follows sonic disintegration of the tissue cells.

Repeated freezing and thawing of rabies vaccine results in the complete
destruction of the antigenic component. Similarly, the extraction of the
vaccine with ether or a mixture of ether and ethanol at low temperatures
results in a great loss of antigenicity. The similarity of the rabies
antigen to lipid-protein combinations is discussed.

It is concluded that the chemical characteristics of the inactivated
rabies virus must first be more adequately determined before an acceptable

method of rabies vaccine purification can be develOped.

35

 

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VII. LITERATURE CITED

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cations following antirabies vaccination. J..Am. Med. Assoc.,'151,
188-1910

ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS 19h5 Official and Tenta-
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BASSOE, P. AND GRINKER, R. 1930 Human rabies and rabies vaccine
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DELL, J., WRIGHT, J., AND HABEL, K. 1919 Rabies vaccine freed of the
factor causing allergic encephalitis. Proc. Soc. Exptl. Biol. and
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BURKHART, R., JERVIS, 0., AND KOPROWSKI, H. 1950 Postvaccinal paralysis
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£2, 31.

BUSSON, B. 1926 Blatternschutz-und Tollwutinfektion. Wien. Klin.
Wchnschr., 39, 1183-1185.

CALMETTE, A. 1891 Notes sur la rage en Indo-Chine. Ann. Inst. Pasteur,
2’ 633-6’4'1 o

CASTTGLIONI, A. 19A1 A history of medicine (trans. by E. B. Krumbhaar),
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CHAPMAN, S. S. AND SURGENOR, D. M. 1953 Personal communication.

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Rous sarcoma virus using a new plasma protein fractionation method.

Bacteriol. Proc., 195h, 85.

COHN, E. J., SURGENOR, D. M., SCHMID, K5, BATCHELOR, W. H., ISLIKER, H. C.,
- AND ALAMERI, E. H. 1953 The interaction of plasma proteins with heavy
metals and with alkaline earths, with Specific anions and specific
steroids, with specific polysaccharides and with the formed elements
of the blood. Discussions of the Faraday Soc., 13, 176-189.

36

 

 

COOK, E. B. M., STEARNS, c., FEILD, J., AND IRONS, J. v. 1955 Report on
the use of phenolized rabies vaccine in Texas from.19h9 through 1953.
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”CORNWALL, J. W. 1918 Anaphylactic reactions in the course of antirabic
treatment. Indian J. Med. Research, é, 237-2h7.

-COX, HO‘RO’ VAN DER SCHEER, J0) AISTON, SO,A.ND BOWL, E0 1914‘? The
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CURNEN, E. c. AND HORSFALL, F. L., JR. 1916 Studies on pneumonia virus
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D'SILVA, C. B., BROOKS, A. G., THOMAS, A. K., AND AHUJA, M. L. 1951
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FERMI, C. 1908 Uber die Immunisierung gegen Wutkrankheit. Ztschr. f.
IIng 1.10 InfektionSkI‘o, 8, 233-2760

FIELDER, F. S. 1916 Paralysis during Pasteur antirabic treatment. J.
Am. Med. Assoc., 66, 1769-177h.

FOLCH-PI, J., ASCOLI, I., LEES, M., MEATH, J. A., AND LeBARON, F. N. 1951
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FOLCH-PI, J. AND LEES, M. 1951 Proteolipides, a new type of tissue lipo-
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FREUND, J., STERN, E. R., AND PISANI, T. M. 19%? Isoallergic encephalo—
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and mycdbacteria in water-in-oil emulsion. J. Immunol., 51, 179.

GALTIER, V. .1879. Etudes sur la rage. Compt. Rend. Acad. Sci., 82! hhh-hh6.

GOLDSTEIN, N. P., KOLB, L. C., MASON, H. L., SOYRE, G. P. AND KARLSON, A. G.
1953 Relation of hom010gous brain proteolipide to allergic encephalo-

myelitis in guinea pigs. Neurol., 3, 609-6lh.

GREENWOOD, M. 19h5 Tenth report on data of anti-rabies treatments supplied
by Pasteur Institutes. Bull. Health Org. League of Nations, 12, 301-36h.

37

 

 

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HARRIS, D. L. 19% Purified rabies vaccine. Science, 128, 158.

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HOGYES , IL. 1897 Lyssa in spezielle 'Path010gie und Therapie (Nothnagel ,
11.), Alfred Holder, Wien, 5, pt. 5, Abt. 2, 1-211-0.

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to antirabic treatment. Am. J. Med. Sci., 1 , 672-682.

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encephalomyelitis in the dog associated with antirabies vaccination.

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JERVIS, G. A. AND KOPROWSKI, H. 19118 EXperimental allergic encephalo-
myelitis. J. Neurol. Path. Neurol., 1, 309.

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LEWIS, J. H. 1933 Immunologic specificity of brain tissue. J. Immunol.,
25, 193-211.

38

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/

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39'

 

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RIVERS, T. M., SPRUNT, D. H. AND BERRY, G. P. 1933 Observations on
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SCHWENTKER, F. F. AND RIVERS, T. M. 1931+ The antibody response of
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1+0

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Appendix i

Results of Challenging Vaccinated Mice:

 

 

 

Lot Vaccine Eizize S/T Reconstructed totals Percent ED
Dilution __mg. 8 D Mgptality 5°
1:6.66 10.0 17/18 A3 1
1:33.3 2.0 12/18 2A 7 23

Conirol 1:166.5 o.h 9/18 12 16 57 0.556 mg

1:832.5 0.08 3/18 3 31
1:6.66 10.0 12/18 21 6 22
1:33.3 2.0 7/18 9 17 65 3.50 mg

I-A 1:166.5 o.h 2/16 2 33
1:832.5 0.08 0/18 0 51

 

 

 

 

 

 

 

 

Results of Challenging Control Mice:

 

 

Dilution Reconstructed Totals Percent
of Virugggi S/T "—"—'_§__—‘T"'"ET‘“‘_"' Mortality
10"6 1/12 1 19
10'7 7/12 8 8 5o
10‘8 9/12 17 3
10'9 12/12 29 0

 

 

 

 

 

Challenge dose of virus = 10.0 LDSO

A2

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lot Vaccine Eizifle S/T ’Reconstructed Totals Percent ED50
Dilution mg. S D Mortality
1:33.3 2.0 17/18 26 1 3
Coiirol 1:166.5 0.h 6/18 9 13 59 0.518 mg
l:832.5 0.08 3/18 3 28
1:6.66 10.0 6/20 16 1h A7
1:33.3 2.0 3/20 10 31 76 8.h6 mg
II—A 1:166.5 0.h 6/19 7 AA
1:832.5 0.08 1/20 1 63
Results of Challenging Control Mice:
BEER; s/T “st—+935“;— M32323,
10"6 0/12 0 19
10'7 5/12 5 7 57
10'8 12/12 17 o o

 

 

 

 

 

Challenge dose of virus = 13.3 LD50

43

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lot Vaccine Eizifie S/T Reconstructed Totals Percent ED
Dilution 495. s D Mortality 5°
1:6.66 10.0 17/18 35 1
III 1:33.3 2.0 1A/17 18 A 18
Control
1:166.5 0.A A/17 A 17 81 0.880 mg
1:832.5 0.08 0/18 0 35
1:6.66 10.0 7/10 11 3 21
III-A 1:33.3 2.0 A/lo A 9 69 3.78 mg
1:166.5 0.A 0/10 0 l9
1:6.66 10.0 2/8 8 6 A3
III-B 1:33.3 2.0 A/10 6 12 67 6.2A mg
1:166.5 0.A 2/10 2 20
1:6.66 10.0 7/10 9 3 30
III-C , 1:33.3 2.0 1/9 2 11 85 5.56 mg
1:166.5 0.A 1/10 1 20
Results of Challenging Control Mice:
iii-1313.32 s/T Re°°§5trmd ”3‘3“” M53232,
10"6 0/12 0 2A
10"7 2/12 2 12 86
10‘8 10/12 12 2 17

 

 

 

 

 

Challenge dose of virus =

33.2 LD

50

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Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

Brain
Lot Vaccine tissue S/T Reconstructed Totals Percent ED50
Dilution r_mg. S D Mortality
1:A 25.0 11/12 23 1
1:20 5.0 5/12 12 8 A0
Iv
1:100 1.0 5/12 7 15 68
1:500 0.2 2/12 2 25 2.81 mg
1:A 25.0 6/12 6 6 50 25.0 mg
1:20 5.0 0/12 0 18
IV-A
1:100 1.0 0/12 0 3o
1:500 0.2 0/12 0 A2
Results of Challenging Control Mice:
Dilution Reconstructed Totals Percent
of Virug s/T s D Mortality
10"6 0/12 0 23
10"7 3/12 3 11 79
10'8 , 10/12 13 2 13

 

 

 

 

 

Challenge dose of virus = 27.h LD50

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Appendix ii

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Brain
Lot Vaccine tissue S/T Reconstructed Totals Percent ED
Dilution mg. 3 D Mortality 5°
1:6.66 . 10.0 12/12 18 0
v
Control 1:33.3 2.0 5/10 6 5 A5
1:166.5 0.A 1/12 1 16 9A 1.70 mg
1:6.66 10.0 6/12 6 6 50 10.0
v-A 1: 33 .3 2.0 0/12 0 18
1: 166.5 0.A 0/10 0 28
1:6.66 10.0 3/11 8 8 50 10.0
v-3 1: 33. 3 2.0 3/11 5 16
1:166.5 0.A 2/12 2 26
1:6.66 10.0 6/12 7 6 A6
v-0 1: 33.3 2.0 1/12 1 17 9A 8.92
1:166.5 0.A 0/11 0 28
1:6.66 10.0 A/ll 5 7 58 >10.0
V-D B333 2 .0 1/12 1 l8
1:166.5 0.A 0/11 0 29
1:6.66 10.0 6/11 10 5 33
V-E 1: 33. 3 2.0 3/11 A 13 76 5.28
1: 166. 5 0.A 1/12 1 2A
A6

 

Results of Challenging Control Mice:

 

 

 

Dilution Reconstructed Totals Percent
of Virus S/T s D MortalitL
10"6 0 /12 0 22
10’7 2/12 2 10 83
10"8 11/11 13 0 o

 

 

 

 

 

Challenge dose of virus = 25.0 LD

50

1+7

 

Appendix iii

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lot Vaccine giggle S /T Reconstructed Totals Percent EDSO
Dilution mg . S D Mortality
1:6.66 10.0 13/13 30 0
Confirol 1:33.3 2.0 11/12 17 1 6
1:166.5 0.A 6/13 6 8 57 0.50 mg
Undiluted 66.6 10/13 17 3 15
A-l 1:6.66 10.0 6/12 7 9 56 12.60 mg
1:33.3 2.0 1/13 1 21
Results of Challenging Control Mice:
*LDilution Reconstructed Totals Percent
of Virus s/T s D Mortality
10-6 0/12 0 21
10‘7 A/12 A 9 69
10"8 11/12 15 1 6
10"9 12/12 27 0

 

 

 

 

 

Challenge dose of virus = 20.0 LD50

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lot Vaccine Eizifie S/T Reconstructed Totals Percent ED50
Dilution __mg. S , D Mgrtality
1:100 1.0 16/23 31 7 18
Conirol 1:500 0.2 7/23 15 23 60 0.292 mg
1:2500 0.0A 8/22 8 37
1:20 5.0 11/22 18 11 38
1:100 1.0 A/23 7 30 81 3.19 mg
3.1 1:500 0.2 2/23 3 51
1:2500 0.0A 1/23 1 73
Results of Challenging Control Mice:
Dilution Reconstructed Totals Percent
of Virus S/T s D ” Mortality
10'6 1/12 1 17 9A
10"7 8/12 9 6 A0
10‘8 10/12 19 2
10"9 12/12 31 0

 

 

 

 

 

Challenge dose of virus = 6.51 LD50

A9

 

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lot Vaccine gigige S/T Reconstructed Totals Percent ED
Dilution . """7§“""“"IT"“' Mortality 5°
1:6.66 10.0 10/12 2A 2
C 1:33.3 2.0 10/12 1A A 22
Control
1:166.5 0.A A/ll A 11 73 0.828 mg
1:832.5 0.08 0/12 0 23
Undiluted 66.6 9/12 12 3 20
1:6.66 10.0 2/13 3 1A 82 26.9 mg
C-l 1:33.3 2.0 1/13 1 26
1:166.5 0.A 0/13 0 39
Results of Challenging Control Mice:
Dilution Reconstructed Totals Percent
of Virus S/T ""'?§""""‘73"“‘ Mggpglity
10"6 0/12 0 21
10'7 A/12 A 9 69
10'8 11/12 15 1 6
10‘9 12/12 27 0
Challenge dose of virus = 20.0 LD50
50
I.

Appendix iv

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Brain
Lot Vaccine tissue S/T Reconstrgfited Totals Percent ED 0
Dilution mg. S D Mprtality 5
1:6.66 10.0 15/15 35 0
VI
Control 1:33.3 2.0 15/16 20 1 5
1:166.5 0.A 5/16 5 12 71 0.668 mg
VI-A 1:6.66 10.0 0/16
Undiluted 66.6 6/15
VI-B 1:6.66 10.0 1/1A
1:33.3 2.0 0/16
VI-C Undiluted 66.6 0/12
VI-D Undiluted 66.6 0/12
1:6.66 10.0 0/11
Results of Challenging Control Mice:
Dilution Reconstructed Totals Percent
of Virus S/T Mortality
10'6 0/12 0 21
10'7 5/12 5 9 6A
10'8 10/12 15 2 l2

 

 

 

 

Challenge dose of virus = 18.6 LD50

51

Appendix v

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Brain
Lot Vaccine tissue S/T Reconstructed Totals Percent ED 0
Dilution mg. S D Mprtality 5
1:6.66 10.0 11/11 20 0
VII

Control 1:33.3 2.0 6/10 9 A 31
1:166.5 0.A 3/11 3 12 80 1.07 mg
1:6.66 10.0 10/10 2A 0
1:33.3 2.0 8/10 1A 2 l3

VII-2
1:166.5 0.A 3/10 6 9 60 0.56A mg
1:832.5 0.08 3/10 3 l6
1:6.66 10.0 12/12 25 0
1:33.3 2.0 9/11 13 2 13

VII-A
1:166.5 0.A A/lo A 8 67 0.66A mg
1:832.5 0.08 0/11 0 19
1:6.66 10.0 8/10 2A 2
1:33.3 2.0 9/12 16 5 2A

VII-6
1:166.5 0.A 6/11 7 lo 59 0.60A mg
1:832.5 0.08 1/12 1 21
1:6.66 10.0 10/10 19 0
1:33.3 2.0 6/11 9 5 36

VII-8
1:166.5 0.A 3/9 3 11 78 1.17 mg
1:832.5 0.08 0/12 0 23

52

 

Results of Challenging Control Mice:

 

 

Dilution Reconstructed Totals Percent
of Virus s/T ""'E?""""'IT“"' Mgrtality
10'6 . 0/11 0 19
10'7 A/12 A 8 67
10'8 11/11 15 0 0

 

 

 

 

 

Challenge dose of virus = 18.0 LD5o

Results of Challenging Vaccinated Mice:

 

 

 

 

Brain
Lot Vaccine tissue S/T Reconstructed Totals Percent EDSO
Dilution mg. S D Mortality
1:6.66 10.0 16/16 3A 0
VII
Control 1:33.3 2.0 15/16 18 1 6
1:166.5 0.A 3/16 3 1A 82 1.18 mg
1:6.66 10.0 12/16 26 A
1:33.3 2.0 11/16 1A 9 39
VII-10
1:166.5 0.A 3/16 A 21 8A 1.3A mg
1:832.5 0.08 1/16 1 36
1:6.66 10.0 13/16 26 3
VII-1A 1:33.3 2.0 11/16 13 8 38
1:166.5 0.A 2/16 2 22 92 1.39 mg

 

 

 

 

 

 

 

 

Results of Challenging Control Mice:

 

 

Dilution Reconstructed Totals Percent

of Virus s/T L""ET"'7 D Mortality
10"6 0/12 0 22 '
10-7 5/12 5 10 67
10‘8 9/12 1A 3 18

 

 

 

 

 

Challenge dose of virus = 2.2 LD5O

53

 

Appendix vi

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Brain ,
Lot Vaccine tissue S/T Reconstructed Totals Percent ED 0
Dilution mg. S D Mortality 5
1:6.66 10.0 12/12 18 0
VIII
Control 1:33.3 2.0 5/10 6 5 A5
1:166.5 0.A 1/12 1 16 9A 1.70 mg
1:6.66 10.0 8/9 8 1 11
VIII-A 1:33.3 2.0 0/11 0 12 100 A.92 mg
1: 166.5 0.A 0/11 0 23
1:6.66 10.0 3/12 8 9 53 10.0 mg
VIIIiB 1:33.3 2.0 3/11 5 l7
1:166.5 0.A 2/11 2 26
1:6.66 10.0 3/11 9 8 A7
VIII-C 1:33.3 2.0 5/10 6 13 68 7.88 mg
1:166.5 0.A 1/12 1 2A
1:6.66 10.0 3/12 A 8 67 10.0 mg
VIII-D 1:33.3 2.0 1/12 1 19
1:166.5 0.A 0/12 0 31
1:6.66 10.0 A/12 8 8 50 10.0 mg
VIII-E 1:33.3 2.0 2/12 A 18
1:166.5 0.A 2/12 2 28
5A

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Results of Challenging Control Mice:

 

 

Dilution Reconstructed Totals Percent
of Virus S/T s | D jortality
10'6 0/12 0 22
10"7 2 /12 2 10 83
10‘8 11/11 13 0 0

 

 

 

 

Challenge dose of virus = 25.0 LD50

55

Appendix vii

Results of Challenging Vaccinated Mice:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Brain
Lot Vaccine tissue S/T Reconstructed Totals Percent ED
Dilution mg. S D Mortality 5°
1:6.66 10.0 13/16 29 3
1x
Control 1:33.3 2.0 ll/l6 16 8 33
1:166.5 0.A 5/16 5 19 79 1.10 mg
undiluted 33.3 7/16 15 9 37
1:6.66 5.0 5/16 8 20 71 13.50 mg
Ix-A
1:33.3 1.0 3/16 3 33
1:166.5 0.2 0/16 0 A9
undiluted 33.3 7/16 13 9 Al
1:6.66 5.0 5/16 6 20 77 16.75 mg
IXéB
1:33.3 1.0 0/16 1 20
1:166.5 0.2 1/16 1 21
Results of Challenging Control Mice:
-‘7Di1ution Reconstructed Totals Percent
' of Virus SAT S D Mortality
10"6 0/12 0 25
10"7 1/12 1 13 93
10"8 10/12 11 2 15

 

Challenge dose of virus = 35.6 LDSO

56

 

i

 

 

 

 

 

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Appendix viii

Zinc Reaggnt
0.25 M.g1ycine
0.1 M zinc oxide
0.A- M zinc acetate
To 125 m1 hot distilled water 18.77 gm glycine was dissolved.
The mixture was heated but not‘boiled. The mixture was stirred
constantly and 8.1% gm zinc oxide dusted into the hot glycine.
The zinc oxide was added slowly and in small portions so that it
goes completely into solution before another aliquot was added.
In this way the complete conversion to zinc diglycinate was obtained
and the resultant solution was clear.
To 300 ml distilled water 87.8 gm zinc acetate (Zn(02H302)2.2H20)
was dissolved. This solution was added to the zinc diglycinate
prepared above. The final volume was adjusted to 1 liter with distilled

water. The solution was tightly steppered and refrigerated.

57

ROOM usr. om

 

      

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61

    

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