Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1 337 1 1 5
VACCINATION AGAINST RAP~.~-RELATED VIRUSES
This invention was made under grants from the National
Institutes of Health. The United States Government has certain
rights in the invention.
TECHNICAL ~LD OF THE INVENTION
This invention relates to rabies virus and rabies-related
viruses. More particularly it relates to methods of immllni~ine host
anim~l.c to protect against infections with rabies and rabies-related
viruses.
BACKGROUND OF THE INVENTION
Rabies virus continues to be endemic in most areas of the
world. It causes an acute central nervous system ~lice~ce which is
normally fatal to hllm~n.c and domestic and wild ~nim~lc.
The virus structure is bullet-sh~ped~ consisting of a nucleo-
capsid core surrounded by a membrane envelope. The nucleocapsid
is comprised of a single, non-segmented strand of RNA together with
RNA transcriptase (L), phosphoprotein (NS), and nucleoprotein (N).
The N protein, which is the major portion of the nucleocapsid, is
noncovalently bound to the RNA to form the helical ribonucleo-
protein (RNP) complex. Two viral proteins are associated with the
13371 15
viral envelope, the major surface antigen (G) which is glyco~sylated
and the matrix protein (M) which is thought to be located on the
inner leaflet of the lipid bilayer, associating with both the
C-terminal dom~in of the membrane-in~serted G protein and the RNP
structure.
Virus-neutralizing antibodies raised in ~nim~l.c again~st rabies
virus, only recognize the G protein. The level of antibody
production has been thought to correlate with the degree of
protection afforded against live virus infection. See Crick, Post
Graduate Medical Journal, Vol. 49, p. 551 (1973) and Sikes, et al.,
Journal of American Veterinary Medical Association, Vol. 150, p.
1491 (1971). However, there are indications that antibody alone is
not sufficient to protect from viral infection. For instance, passive
immunization with anti-rabies antibodies without a vaccine as
po~st e~L,o~lre therapy,`does not decrease the probability of infec-
tion. Nicholco~, et al., Journal of Infect. Diseases, Vol. 140, p. 176
(1979). In addition, there exist effective vaccine~s which do not
induce high level antibody production. One such vaccine is produced
by Norden Labs.
It i~s p~ccihle that resistance to infection by rabies virus may
require both virus-neutralizing antibodies and effector T-cell
responses. Both the G protein and the nucleoprotein (N) have been
shown to stimulate proliferation of rabies antigen-specific T cell
lines. Most such T cell lines respond strongly to N protein and less
strongly to G protein. A minority of rabies reactive T cell lines
respond to G protein, but not at all to N protein. Celis et al.,
Journal of Immunology, Vol. 136, pp. 692-697, (1986). It has not been
shown previously that N protein provides any protection against
rabies infection in immunocompetent ~nim~ls or hllm~n.c
3 1 3371 1 5
Current vaccines against rabies consist of an inactivated
rabies virus. These vaccines do not provide any cross-protection
against the rabies-related virus Mokola. Preparation of current
vaccines involves growth of the virus on permissive animal cells or
embryos. This method of production requires the h~n-llinE of the
virus by workers which entails an undesirable risk of infection.
Further, some inactivated virus vaccines can cause adverse side
effects, such as demyelinating allergic encephalitis and systemic
reactions, in a proportion of the vaccine recipients.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method
for inducing protective immunity to rabies and rabies-related
viruses.
It is another object of the present invention to provide a
rabies-related virus vaccine which does not require growth of
viruses.
It is yet another object of the present invention to provide a
rabies-related virus vaccine which can be chemically synth~-ci7ed.
It is still another object of the present invention to provide a
rabies-related virus vaccine which can be produced in a micro-
organism.
It is an object of the present invention to provide a
rabies-related virus vaccine which stimulates proliferation of both T
and B cells.
It is another object of the present invention to provide a
method of inducing protective immunity to rabies-related virus
which does not cause demyelinating allergic encephalitis.
- 1 337 1 1 5
In accordance with these objects, the present
invention provides an improved method for inducing
protective immunity to rabies-related viruses comprising
administering a priming injection of an antigen and one
or more booster injections, the improvement comprising
employing a polypeptide having amino acid sequence
homology to rabies-related virus nucleoprotein as said
priming injection, said polypeptide being substantially
free of rabies-related virus glycoprotein, said
polypeptide having the ability to induce proliferation of
rabies-related virus-specific human T cells.
In another embodiment of the present invention, a
biologically pure sample of a polypeptide is provided
capable of inducing a proliferative response in
rabies-related virus-specific human T cells, said
polypeptide having sequence homology with the
nucleoprotein of rabies-related virus and having the
ability to induce protective immunity to rabies-related
viruses .
In yet another embodiment of the present invention a
method is provided for inducing protective immunity to a
rabies-related virus comprising administering an
injection of a preparation comprising a polypeptide
having sequence homology with the nucleoprotein of a
rabies-related virus, said polypeptide being capable of
inducing a proliferative response in rabies-related
virus-specific human T cells, said polypeptide being
substantially free of rabies-related virus glycoprotein.
According to an aspect of the invention, a method of
inducing protective immunity to rabies-related virus
comprises:
administering to a human or domestic animal as a
priming injection a preparation which comprises the
nucleoprotein of a rabies-related virus, the preparation
1337115
4a
is substantially free of rabies-related virus
glycoprotein; and
administering to the human or domestic animal one or
more booster injections which comprises an inactivated
rabies virus.
According to another aspect of the invention, a
method of inducing protective immunity to rabies-related
virus consisting essentially of the step of:
administering to a human or domestic animal one
injection of a preparation which comprises the
nucleoprotein of a rabies-related virus, the preparation
is substantially free of rabies-related virus
glycoprotein.
According to a further aspect of the invention, a
method of inducing protective immunity to rabies-related
virus consisting essentially of:
administering to a human or domestic animal a
priming injection which comprises the nucleoprotein of a
rabies-related virus, the nucleoprotein is substantially
free of rabies-related virus glycoprotein; and
administering to the human or domestic animal one
booster injection which comprises the nucleoprotein of a
rabies-related virus, the nucleoprotein is substantially
free of rabies-related virus glycoprotein.
The vaccines of this invention can be produced
without growing the rabies virus and they do not cause
the adverse side effects associated with inactivated
virus vaccines. In addition, the vaccine provides
protection against heterologous rabies-related virus
strains.
- 13371 15
DETAILED DESCRIPTION OF THE INVENTION
The inventors have found that the nucleoprotein of rabies
virus, a protein which is internal to the membranous envelope of the
virus, can serve as an effective inducer of both humoral and ce~ r
immllne respon~ses. Further, a particular region of the nucleoprotein
(N protein), amino acid residues 313-337, has been found to contain
an epitope identified by certain anti-rabies antibodies. In addition,
this isolated region of the protein (as well as two other regions,
cu-,esL,onding to amino acid rP-si-luP-s 369-383 and 394-408) is able to
stimulate proliferation of rabies antigen-specific human T celLs in
the presence of irradiated autologous mono~llclear cells. Other
suitable peptides may be found which are able to stimulate
proliferation of rabies antigen-specific human T cells and which can
provide protective immunity to rabies infection. Stimulation of
proliferation of T celLs can be tested according to the method of
Celis et al, J. Immunol. 56:426-433 (1985), and in Example 6 below.
Protective immunity can be tested according to standard methods
known in the art and described in Example 7 below.
As normally practiced in the art, rabies prophylaxis is
performed by a series of injections, the first one being termed a
priming injection and subsequent ones termed boosters. Methods for
minictering such injections, including al,prop-iate formulations,
dosages, anatomical localizations and timing are generally known in
the medical and veterinary sciences. Generally, the dosage desired
is such that will induce a protective response without causing side
effects or immlJne tolerance. Standard inactivated rabies virus
vaccine, for example, may be used in the practice of the present
invention as a booster inoculation. The inactivation of virus i~s
generally performed by treatment with a chemical, such as
-6- ~ 337~ ~ 5
beta-propiolactone. Such a vaccine is av~ hle, for example, from
Institut Merieux, Lyon, France.
For the purposes of the present invention the term ~rabies-
related virus~ is meant to encompass both rabies as well as
rabies-related viruses such as the Mokola and European bat virus
(e.g. the Duvenhage 6 strain). This definition is used because of the
finding that the vaccines of the present invention provide
cross-protection to heterologous virus strains. This is a great
benefit provided by the present invention over prior vaccines.
Subunits of the rabies-related virus, such as N protein or RNP
complex, may be prepared and purified according to known methods.
See, for example, Virology 124:330 (1983) and J. Virol. ~:241 (19~1).
Even purer preparations of N protein can be obtained by preparative
gel electrophoresis, as described in J. Virol, 44:596 (1982). Other
purification steps may be used, as are known in the art.
The useful polypeptides of the present invention can be most
easily prepared by chemical synthesis, according to the solid phase
method described by Merrifield, Adv. Enzymol., 32:221-296 (1969).
Alternatively, proteolysis of N protein can be accomplished using
enzymes or chemicals, e.g., trypsin, 3-bromo-3-methyl-2 [(2-nitro-
phenyl) thiol] -3 H-indole skatole, cyanogen bromide, or protease
V8. Fragments af ter cleavage can be separated by gel electro-
phoresis, for example. Although many fragments can be found
which are reactive with monoclonal antibodies raised against rabies
N protein, only some f ragments are also able to stimulate
rabies-specific T cell proliferation. The inventors have found one
polypeptide to be particularly useful among a set of proteolytic
cleavage products of N protein. This fragment, termed N-V12b, was
originally produced by protease V-8 treatment of N protein, and
later chemically syn~h~-si7ed. Fragments N-V10c and D30 were also
- -- 1 337 1 1 5
found to stimulate T cell proliferation, although to a lesser degree.
Fragment N-V12b corresponds to the amino acids 313-337 of N
protein; fragment N-V10c co~ onds to amino acids 369-383; and
fragment D30 corr~ol ds to amino acids 394-405. The sequences
are shown below in Table 4. The sequence of the N protein has been
previously determined by Tordo et al., Nucleic Acids Research, Vol.
14, pp. 2671-2683 (1986).
When the synthetic peptides are coupled to a protein, e.g.
keyhole limpet hemocyanin, either via thiol groups (Biochemistry
18:690-697 (1979)) or via amino groups with glutar~ ldehyde, they
are found to stimulate anti-N protein antibodies in rabbits.
However, the antibody titer was lower in anti-N-V12b serum than in
anti-N-VlOc serum.
Although the sequences of the polypeptides are defined herein
with particularity (Table 4) it will be apparent to one skilled in the
art that some of the amino acids can be conservatively substituted
without losing the beneficial characteristics. It is also apparent to
one skilled in the art that changes in length can be made without
altering the antigenicity. The minimllm nllmher of amino acids
necessary to comprise the epitopes has not been determined.
The polypeptides of the present invention can also be
prepared through genetic enEineering. That is to say that parts or
all of the N gene can be cloned into suitable expression vectors, as
are known in the art. Organisms transformed with the cloned gene
or portion thereof can be grown to produce the polypeptide
products. Recovery of the polypeptide products is within the
ordinary skill of the art. Comhin~tions or concatemers of antigenic
polypeptide fragments can also be used. These can be made by
synthesis, çlo~ine, or post-synthetic covalent bo~line.
1 337 1 1 5
--8--
Liposomes, which can be employed in the practice of the
present invention, are known in the art. They are used as a
macromolecular carrier for the polypeptides to ensure antigenicity.
Other proteins or synthetic nanoparticles may also be used as
carriers. In the case of RNP a~lminictration no carrier is required.
Liposomes may be formed, e.g., according to the method of
Thiholeau, ~Genetic variation among influenza viruses," Acad. Press,
N.Y. (1981) p. 587. Generally, a mixture of lipids in particular
ratios, such as ph~-sphatidyl rholine~ cholesterol, and lysophos-
phatidyl choline, are mixed under conditions to form closed lipid
vesicles. Many such conditions and methods are known in the art.
In the practice of the present invention the polypeptides are
coupled to a saturated fatty acid having from about 15 to about 21
carbon atoms, before mixing with the lipid composition to form
liposomes. Suitable fatty acids include, palmitic, stearic and oleic
acids. Conjugates may be formed by the method of Hopp, Molecular
Immunology, 21:13-16 (1984), wherein peptide fragment spacers of
gly-gly-lys-(NH2)2 are added to the N-terminus of the polypeptide,
and the alpha and e~ilon amino groups of lysine are used to couple
with the fatty acids.
The biologically pure s~mples of the polypeptides of the
present invention are substantially f ree of glycoprotein of rabies
virus. They are sufficiently pure that after injection into mice,
rabies glycoprotein~pecific antibody or T cells are not induced. In
addition, rabies glycoprotein specific T cells would not be stimulated
in vitro to proliferate in the presence of the preparation.
The following examples are illustrative only and are not
intended to limit the scope of the invention. The invention is
defined by the claims appended below.
9 133ill5
EXAMPLE 1
This example describes the selection of a multiple variant
virus of rabies virus.
Seven anti-glycoprotein monoclonal antibodies c~p~hle of
neutralizing parent CVS-11 rabies virus were used to sequentially
select antigenic variants. Serial dilutions of virus were mixed with
monoclonal antibody diluted 1:100, overlayed with nutrient agar, and
after 4 to 5 days of incubation at 35C in a 5% carbon lioxide
atmosphere, plaques were selected. Viruses able to replicate in the
presence of the monoclonal antibodies used for their selection were
recovered. The seventh generation variant CVS-V7 was not
neutralized by any of 40 diiferent rabies virus-specific neutralizing
monoclonal antibody. The nucleoprotein antigen of the CVS-V7 virus
rem~ined immllnologically indistinguishable from that of the CVS-11
parent virus.
EXAMPLE 2
This ex~mple shows the protection afforded by vaccination
with the CVS-V7 virus, as well as the virus neutralizing antibodies it
induced against parent strain CVS-11.
Vaccines prepared f rom the CVS-V7 variant viruses, were
used to immunize mice. Groups of nine 4-week old female ICR mice
were immllni7pd with 0.2 ml of 5 serial dilutions (2000-3 ng) of the
CVS-V7 inactivated virus vaccine on days 0 and 7. The inactivated
virus vaccine was prepared with beta-propiolactone and adjusted to
a protein concentration of 100 ug/ml. The viruses had been grown
on BHK 21 cell monolayers and purified as described in Journal of
Virology, Vol. 21, pp. 626-635 (1977). On day 14, vaccinated and
unvaccinated control mice were bled and infected intracerebrally
1 337 1 1 5
-10-
with 0.03 ml (50 MIC LD50) of CVS-11. (CVS-11 parent virus was
used in the ch~llenge because the CVS-V~ variant virus was not
pathogenic for adult mice.) The ~nim~l.c were observed for 3 weeks
and mortalities were recorded daily. The effective dose was
calculated for each vaccine as described in Atansiu, P., ~Quanti-
tative assay and potency test of antirabies serum," In: Laboratory
Techniques In Rabies, 2nd Ed., Geneva: World Health Organization,
1966, pp. 16~-~2. The neutralizing activity of the mouse immune
sera for CVS-ll was determined as described in Lumio et al., Lance,
Vol. i, p. 3~8 (1986).
The geometric mean tighter (GMT) was calculated for each
vaccination group. Multiple group comparisons for differences in
GMT were tested by one way analysis of variance and two-group
GMT were compared statistically by a one-tailed test.
As can be seen in Table 1, virus neutralizing activity as well
as protection from death were provided by the CVS-V7 vaccine. As
the CVS-V~ virus, from which the vaccine was prepared, was not
neutralized by any of 40 rabies glycoprotein-specific monoclonal
antiho~liP-s and yet was able to confer protection against ch~llPnge
with the parental CVS-11 strain, it is concluded that the glyco-
protein is not the sole factor in determining the relative efficacy of
rabies prophylaxis.
EXAMPLE 3
This example demonstrates the protection afforded and the
virus neutralizing antibody induced by means of rabies virus subunit
vaccines which are formulated with liposomes.
The glycoprotein and ribonucleoprotein (RNP) were purified
from the CVS-V~ variant virus as described in Diet7chold et al.,
CVS-V7-G- CVS-V7-N- CVS-V7-(N+G)-
Vaccine CVS-V7-Virus Liposome Liposome Liposome
Vaccine VNA against Mortality VNA agalnst MortalityVNA a~ainstIViortallty VNA agalnstiviortallty
Concen- GMT (range) Rate GiviT (range) Rate GMT (range) GMT (range)
tration
[ng]
10000 (27o-l62o) 0/7 (<10-810) 1/s 7/7 588 1/6
2000 (60 540) 2/7 (290 540) 2/7 0 7/7 (260 810) 1/7
400 (60-540 3/7 (<10 180) 4/~ 7/7 323 1/7
(<10-540) 3/7 (211o 60) 3/6 0 7/7 (<10-540) 1/7
1~ (<10-540) 3/7 (<10 30) 5/6 o 7/7 (<10-50) 5/7
Effective 33.3 ng 588 ng 45 ng
(ED50)
i
Table 1 - Protective activities of an inactivated CV~ S-V7 virus vaccine and CVS-V7 subunit vaccines
against an i.c. chalienge infection with CV~l 1 virus.
-12- 1 337 1 1 5
Isolation and Purification of a Polymeric Form of the Glycoprotein
of Rabies Virus, J. Gen. Virol. 1978, 40 131-135 and Schneider et al.,
Rabies Group-Specific Ribonucleoprotein Antigen and a Test System
for Grouping and Typing of Rhabdoviruses 1973, J. Viral. 11, 748-755.
The proteins were inserted into liposomes as described in Thibodeau,
Genetic Variation Among Influenze Viruses, Acad. Press, N.Y. 1981,
p. 587. The amount of rabies protein liposomes used per injection is
expressed as weight of the total complex. Mice were injected
intracerebrally on days O and 7 and ch~llenged at day 14 as described
in Example 2. The resulting mortality rates and amount of virus
neutralizing antibody produced are shown in Table 1.
The RNP incorporated into liposomes induced no detectable
VNA against CVS-ll nor any detectable protection from death. The
CVS-V7-derived G protein incorporated into liposomes induced low
titers of VNA and poor protection from death. However, when the
RNP was combined with G protein, the treatment was significantly
better than either protein alone, and produced results roughly
comparable to those obtained with the whole virus vaccine. This
too, shows the importance of N protein in immunity to rabies.
EXAMPLE 4
This ex~mple demor~ctrates that rabies virus RNP can
augment the function of B cells producing neutralizing antibody.
Groups of mice were primed with either 5 ug of N protein
(from the ERA strain of rabies) plus complete Freund's adjuvant
(CFA) or CFA alone. Ten days after priming both groups of An;m~l~
received serial dilutions of inactivated rabies virus vaccine or
isolated rabies glycoprotein. Mice that were primed with RNP plus
CFA and boosted with rabies virus vaccine developed significantly
higher VNA titers than those mice primed with CFA alone and then
1 337 1 1 5
-13-
boosted with a rabies virus vaccine. The results are shown in Table
2. In addition, the effective dose of rabies virus vaccine was
determined and found to be about 10-fold lower in the mice which
had been primed with RNP.
The mice which received booster vaccinations consisting of
just rabies virus glycoprotein developed only low levels of VNA and
were poorly protected against a lethal ch~lle~Ee infection with
rabies virus, which can be seen in the right half of Table 2. This
suggests that there must be common antigens between the priming
and boosting immllni7ations in order to induce an effective immune
response.
The mortality rates shown in Table 2 were determined by
ch~llengin~ all mice with an intracerebral innoculation with 50 MIC
LD50 Of rabies virus and observing the survival up to 3 weeks
post-infection.
EXAMPLE 5
This ~x~mplPs demonctrates that protective immunity can be
induced using rabies virus RNP to an intramuscular (I.M.) ch~llpn~e
infection with homologous or heterologous rabies virus strains.
Purified RNP of the ERA strain of rabies or of the
rabies-related strain Mokola was used to immunize Balb/c mice
against an (I.M.) ch~llenEe with the rabies strain CVS-24. The RNP
was isolated and purified according to the method of Schn~ider, et
al., J. Virol., Vol. 11, pp. ~48-~55 (19~3). Groups of 10 to 20 mice
were immunized intraperitoneally (I.P.) with ERA-or Mokola-RNP
plus CFA, or with CFA alone, or subcutaneollcly (S.C.) with
ERA-RNP alone. Four weeks after immunization, the mice were
ch~ nged (I.M.) with eight mouse I.M. LD50 f CVS-24 rabies virus
Booster immunization with Booster immunization with
Vaccine ERA-virus ERA~
tration
Priming with Priming with Priming with Priming with
(ng) ERA-N + CfA CFA ERA N + CFA CFA
VNA a~aalnst Mortallty Vna a~alr6t Mortdlty VNA a~al~t Mortallty VNA agalr~t Mortallty
CVS-l r Rate CVS-ll I?ate CVS~ ate CVS-l 1 Rate
GMT (tan~e) GM~ (rar~e) GMT (rar~e) GlvlT (rrar~e)
5000 (1~C~4~0) 3/7 (<10-180) 5/5 (6C~80) / 28 6/7
489 39 6/7 25 6/7 ~ 7/7
(6C~1620) 4/7 (10-180) (10-60) (c10~60)
138 10 13 1.3
200 (20-1620) 3/7 (<lO~S0) 6 n (10-20) 6/7 (<1C~20) 7/7
34 26 4 1.3
(<1C~270) 5/6 (20~60) 6/7(<1C~20) 7/7 (<10-20) 7/7
8 (<10-540) 6/7 (<1C~30) 7/7(<1C~20) 7/7 6/6
Effective ~,~
oDs5eO) ~25r~3 >5000ng ~5000 ng ,5000 n~
~able 2 - Effect of RNP - priming on VNA tite~ and mortality rates
1 337 1 1 5
-15-
(see Table 3) or two mouse I.M. LD50 f rabies-related virus
Duvenhage 6 (see Table 4).
As can be seen in Table 3, 80% of the mice that received
ERA-RNP plus CFA intraperitoneally or ERA-RNP alone subcatane-
ously survived the ch~llenee. In addition, 90% of the mice that were
immunized intraperitoneally with Mokola-RNP plus CFA survived
the ch~llen~e infection. In contrast only 10% of the mice that
received only complete Freund Adjuvant (CFA) succumbed to rabies.
It is known that neutralizing antibody produced against the
Mokola virus does not neutralize rabies virus; therefore, this
experiment clearly demonstrates that the protection conferred by
RNP is not due to neutralizing antibody which may have been
induced by very small, undetectable amounts of glycoprotein. In
addition, the results shown in Table 4 demonstrate that RNP from
both the ERA strain and from the Mokola virus induce protective
immunity against the rabies-related European bat virus strain
Duvenhage 6. Taken together these results demonstrate that RNP
purified from rabies virus and rabies-related viruses can induce
protective immunity against heterologous viruses.
EXAMPLE 6
The synthetic peptides shown below in Table 5 were
synth~-ci7ed according to the method of Merrified, cited above. In a
typical coupling reaction, the tboc group of the amino terminus was
removed with 50% trifluoroacetic acid (TFA). After neutralization
with N, N-diisopropylethylamine (DIEA), a 4- to 6-molar excess of
preformed tboc-amino acid-pentafluorophenylester (Kisfaludy et al.,
Liebigs Ann. Chem. pp. 1421-1429 (19~3)) and a 1-molar equivalent
of DIEA in dichloromethane (1:1) were added. After b~hbling with
N2 gas for 1.5-2 h, the resin was analyzed for the presence of free
1 337 1 1 5
- 1 6 -
amino groups as described by Kaiser et al. (Anal. Biochem.,
34:595-598 (1970)). Couplings were repeated until less than 1% free
NH2 groups were found.
Peptides were cleaved and deblocked with HF/thio~nicol (10:1)
at 0C for 30 min, and the peptide was extracted with 0.1 M
NH4HCO3 and lyophili7ed~ The crude peptide was then dissolved in
0.1 M NH4HCO3 and purified on a BioGelT~ P4 column calibrated
with 0.1 M NH4HCO3. The eluted peptide was then applied to a
Vydac~ 2P C18 reverse-phase column and eluted with methanol-
water (8:2). The elution of the peptide was monitored with a UV
detector at 214 nm. To verify the amino acid sequence, aliquots of
the peptide were subjected to amino acid analysis and amino acid
sequencing.
The peptides were tested for the ability to stimulate T-cell
proliferation in the presence of antigen-presenting populations of
cells of the same HLA-DR type. At concentrations of 0.4 ug/ml to
25 ug/ml of protein, fragments N-V12b and N-VlOc, and N protein
isolated from ERA strain virus, stimulated proliferation of the
T-cells. Hepatitis B antigens, used as controls, caused no
stimulation. At low concentrations, N-V12b was more stimulatory
than N protein, whereas at higher concentrations the reverse was
true.
The rabies specific T-cell lines were isolated and tested as
described in Celis et al., J. Immunol. 56:426-433 (1985).
EXAMPLE 7
This e~mrle demonstrates the protective c~p~hilities of the
synthetic peptides N-V10 and N-V12b against the rabies strain
CVS-24.
1 33 7 1 1 5 TABLE 3
Immunization of Balb/C mice with Rabies-RNP and
Mokola-RNP again~t an l.M. challenge with CVS-24
Antigen Route of Mortality (%)
Immunization
+ CFA 2/10 (20)
10 ug ERA-RNP S.C. 2/10 (20)
10 ug MOK-RNP l.P. 1/10 (10)
+ CFA
CFA l.P. 18/20 (90)
1 3371 1 5 -i~- T~BLE 4
Immunization of Balb/C mice with ERA-RNP and
Mokola-RNP against an l.M. challence with DUV 6 virus
Antigen Immunization Mortality (%)
10 ug ERA-RNP + CFA l.P. 0/10 (0)
10 ug Mok-RNP + CFA l.P. 1/10 (10)
CFA l.P. 6/10 (60)
13311 15
- 1 9
TABLE 5
N-VlOc NH2-tyr-glu-ala-ala-glu-leu-thr-lys-thr-asp-val-
ala-leu-ala-asp.
N-V12b NH2-his-phe-val-gly-cys-tyr-met-gly-glu-val-arg-
ser-leu-asn-ala-thr-val-ile-ala-ala-cys-ala-pro-
his-glu.
D-30 NH2-tyr-phe-ser-gly-glu-thr-arg-ser-pro-glu-ala-
val-tyr-thr-arg.
-
1 3371 1 5
-20-
Groups of 5 to 10 mice were immunized intraperitoneally with
N-V12b-liposomes plus CFA, N-VlOc-liposomes plus CFA, or lipo-
somes plus CFA. The mice were then ch~llenged with various
amounts of CVS-24 virus.
The lirllsomes were formed by the method of Thibodeau,
Genetic Variations Among Influenza Viruses, Acad. Press, N.Y., 58~
(1981). The peptides were incorporated into liposomes as described
above in ~x~mple 3. To facilitate the incorporation of the peptides
into liposomes, palmitic acid was linked to the amino terminal end
of the peptide as described by Hopp, Mol. Immunol., Vol. 21, pp.
13-16, 1984. In experiment No. 1, two times the mouse I.M. LD50
was used as ch~llen~e. As shown in Table 6, 88% of the mice that
received the N-V12b-liposome vaccine survived while none of the
mice immunized by N-VlOc-liposome survived three weeks after the
ch~llenge.
In experiment No. 2, four times the mo~Lse I.M. LD50 wa,s used
as a ch~llenge. Sixty-two percent of the mice immunized with
peptide N-V12b-lip~some vaccine survived while only 12% of the
mice which received the control vaccine of liposomes plus CFA
survived the ch~llenge.
In experiment No. 3, 8 times the mouse I.M. LD50 was used as
a ch~ nge. Sixty percent of the mice immunized with the
N-V12b-lip~some vaccine survived the ch~llenEe, while only 10 and
20 percent respectively of the mice immunized with N-V10
liposomes plus CFA and liposome control vaccine plus CFA survived.
Thus, peptide N-V12b provides good protection from rabies
virus ~h~llen~e when incor~orated into liposomes and administered
with CFA. Peptide N-VlOc however does not provide good
-2l- l 3371 l 5 TABLE 6
Protective activitie~ of N-Y1 2b peptide liposomes in mice
- to i.m. challen~e with CVS-24
Experiment #1, i.m. challenge with 2 MIM LD 50
Concentratlon (u~) Vacclne R te
15 N-V10-Liposomes +-CFA 5J5
15 N-V1 2b-Liposomes + CFA 1 /8
Experiment #2, i.m. challenge with 4 MIM LD
so
Vacclne Vacclne Mortallty
Concentratlon (u~) ~ a e
N-V12b-Liposomes + CFA 3/8
--- Liposome ~ CFA 7/8
Experiment #3, I.m. challenge with 8 MIM LD
Concentratlon (u~) Vacclne R te
15 N-V12b-Liposomes + CFA 4/10
15 N-V10 Liposomes + CFA 9/10
--- Liposomes +CFA 8/10
-Z2- l 337 1 1 5
protection against rabies virus ch~llenge~ even though it is able to
stimulate rabies specific T cell proliferation.
EXAMPLE 8
Peptides consisting of 15 amino acids were synth~-si~ed which
together correspond to the entire amino acid sequence of the ERA-N
protein. These were synth~si~ed a,s described above in Example 6.
Each peptide was screened for T cell proliferative activity in vitro
and for protective activity in vivo as described above for peptides
N-V12b and N-V10c.
The results are displayed in Table ~. Peptide nllmher 30D
demon~strated both significant T cell stimulatory activity as well a~s
partial but significant protection against rabies viru~s ch~llenge.
Peptide D30 protected 60% of the immunized mouse population from
a ch~llenge of 8 times the mouse I.M. LD50 of CVS-24 viru~s.
TI~BLE 7
13371 15
-23-
T-Cell Stimulatory and Protective Activities of
synthetic N-peptides
Peptide No.T-cell Stimulatory Activity Mor~lit~ b~ Averace Su~.val Tima Days
Liposo- e ctr. - / ~ b ' .-
/- ~ . .
+ / "b ' .-
- /' ~- O
- / ~b .9
+ t` / b1 ) .1
O~lu
- 7/l
- / b
+ /' 'o
- /' ~1 ~.1
_/ ~, . 7
+ / "b . 3
- / b .5
- / ,~ 0.2
/- , 9.
+ -/- b 9
+ / % 1 .
++ , /'0~ 1
~ 2~ +++ 4/9 ( %)
V1 2b- - 711~ . ~%)
V1 b- I - 1 / 1 ( %)
V12"- 1 - 1 (
V1 2~- /
V1 C +++ 9/1 (9 ~) .'
