Peptides used as carriers in immunogenic constructs suitable for development of synthetic vaccines

The invention relates to conjugates of poorly immunogenic antigens, e.g., peptides, proteins and polysaccharides, with a synthetic peptide carrier constituting a T cell epitope derived from the sequence of E. coli hsp65 (GroEL), or an analog thereof, said peptide or analog being capable of increasing substantially the immunogenicity of the poorly immunogenic antigen. A suitable peptide according to the invention is Pep278e, which corresponds to positions 437-453 of the E. coli hsp65 molecule.

This is a continuation of Application No. PCT/US95/06575 filed May 24, 
1995, which claims priority benefits of Israel Application No. 109790 
filed May 25, 1994. 
FIELD OF THE INVENTION 
A synthetic peptide, is described having an amino acid sequence 
corresponding to that of a T cell epitope of the heat shock protein 65 of 
E. coli (hereinafter GroEL) and its analogs able to be recognized in 
association with a range of mouse major histocompatibility complex (MHC) 
molecules. Said peptide or its analogs can be used as synthetic carriers 
in the preparation of immunogenic conjugates consisting of said peptides 
and a natural or synthetic hapten derived from a pathogenic agent of 
interest. 
BACKGROUND OF THE INVENTION 
Immunization against infection caused by pathogenic microorganism 
(bacteria, viruses and parasites) is generally achieved by inoculating an 
individual with the natural antigen (attenuated or killed microorganism) 
or parts of said infectious agent (for example detoxified microbial 
products) in order to stimulate a protective immune response able to 
neutralize the pathogenic microbe or its deleterious effects. 
Limited availability of the natural antigenic substance, risks involved in 
handling pathogenic material as well as storage problems stimulated the 
interest in the development of subunit vaccines. Isolated protective 
epitopes nevertheless are often characterized by their poor 
immunogenicity. The carbohydrate capsules of bacteria are an example of 
such coats: They are not easily recognized by T cells and therefore the 
immune response to these antigens is deprived of T cell help, T cell 
memory, IgG class switch, and affinity maturation. Such an immune response 
is inefficient and resistance to infection with bacteria encoated with 
carbohydrate capsules is not easily obtained by vaccination, with 
bacterial carbohydrates. Peptide epitopes too may be poorly immunogenic, 
the absence of a T cell epitope and the genetically restricted immune 
response being the reason. 
It is now well established that most antigens require T cell help to induce 
B cells to produce antibodies. Conjugating a "helper" or T cell 
determinant to a B cell-specific antigen was shown to induce humoral 
immune responses to the coupled B cell epitope. The discovery by Avery & 
Goebel (1929) that coupling of polysaccharides to protein carriers 
increases immunogenicity has recently been used for the preparation of 
vaccines for human use. Both in humans and in rodents these conjugates 
behave like T cell dependent antigens by exhibition of immunological 
memory. There are similarities between conjugate polysaccharide vaccines 
and protein carrier-hapten systems. Thus the capsular polysaccharide (CPS) 
conjugates are able to induce protective levels of CPS antibodies in 
infants, while CPS alone is not. It is possible that the superior 
immunogenicity of conjugates compared to that of pure polysaccharides is 
due to the help by carrier-specific T cells, as has been demonstrated in 
the carrier-hapten system in rodents. 
In most cases, T cell independent (T-ind) antigens have been coupled to 
large immunogenic carrier proteins such as tetanus toxoid, cholera toxin 
or diphtheria toxoid. Nevertheless, besides dosage limitations and the 
risk of sensitization to the carrier itself, as reported for tetanus 
toxoid, the immunological response to high molecular weight carrier 
molecules harboring stimulatory as well as suppressive T cell epitopes is 
not very predictable. It has been shown that the antibody response to a 
hapten coupled to a carrier protein can also be inhibited when the 
recipient has been previously immunized with the unmodified protein. This 
phenomenon has been termed carrier-induced epitope suppression and was 
recently demonstrated to occur with a number of hapten-protein conjugates 
(Herzenberg & Tokuhisa, 1982). Since the development of more potent 
conjugate vaccines against a large number of extremely infectious 
organisms is still important, efforts are being made to search for more 
appropriate carrier molecules providing the needed T cell epitopes. 
Universally immunogenic T cell epitopes, defined by specific peptides with 
sharply outlined immunological characteristics, might represent a new 
generation of such alternative molecules. T cell epitopes of various sorts 
have been used for this purpose before. However, to trigger a strong 
memory response when the host meets the infectious agent after 
vaccination, the T cell carrier epitope should be present along with the 
specific B cell epitope. This fact would seem to require that a different 
T cell carrier be used for each infectious agent. Highly abundant proteins 
well recognized by the immune system might be an appropriate source for 
peptides serving this purpose. 
Studies using a wide variety of proteins, both those closely related to 
self and those phylogenetically distantly related, have shown that the 
majority of T cells are focused onto a few immunodominant epitopes with a 
minority responding to other, subdominant determinants. This hierarchy of 
determinant utilization by T cells could result from a combination of 
factors including differential affinities for the available MHC molecules, 
the diversity of the T cell repertoire, internal competition for 
MHC-binding sites and fine differences in processing (Babitt et al, 1985; 
Kappler et al, 1987; Brett et al, 1988) 
Evidence is accumulating that proteins belonging to the family of heat 
shock proteins (hsp's) are major antigens of many pathogens (Young et al, 
1988). Hsp's were first described and later named due to their production 
by cells exposed to sudden elevations in temperature. The hsp's include 
proteins of various molecular weights,. including 20 kD, 60 kD, 65-68 kD, 
70 kD, 90 kD, 110 kD, and others. It is now apparent that hsp's are 
induced in all cells by many different environmental insults, including 
oxidative injury, nutrient depletion and infection with intracellular 
pathogens; the hsp response enables the cell to survive under otherwise 
unfavorable conditions. Although cellular stress increases the synthesis 
of hsp's, many hsp's are also constitutively expressed and play an 
essential role in normal cell function. The hsp response is ubiquitous 
throughout the pro- and eukaryotic kingdoms and hsp's belong to some of 
the most conserved molecules. 
Hsp65, as a representative member of the proteins belonging to the hsp 
family, can be considered to be a dominant antigen because infection or 
immunization with many different bacteria induces antibodies and T cells 
specific for the hsp65 molecule (Young et al, 1988). In mice immunized 
with Mycobacterium tuberculosis, 20% of all T cells which respond to the 
bacterium, are specific for hsp65. Interestingly, T cells with reactivity 
to hsp65 have also been identified in normal healthy individuals lacking 
any clinical signs of disease (Munk et al, 1988). 
Lussow et al. (1990) showed that priming of mice with live Mycobacterium 
tuberculosis var.bovis (BCG) and immunization with the repetitive malaria 
synthetic peptide (NANP).sub.40, conjugated to purified protein derivative 
(PPD), led to the induction of high and long-lasting titers of 
anti-peptide IgG antibodies. Later on, Lussow et al. (1991) reported that 
the mycobacterial hsp65 as well as the hsp65 of E. coli (GroEL) acted as 
carrier molecules in mice, previously primed with BCG, for the induction 
of high and long-lasting titers of IgG against the repetitive malaria 
synthetic peptide (NANP).sub.40. Anti-peptide antibodies were induced when 
the malaria peptide, conjugated to the mycobacterial or E. coli hsp, was 
given in the absence of any adjuvants. 
Barrios et al. (1992) have shown that mice immunized with peptides or 
oligosaccharides conjugated to the 70 kD hsp produced high titers of IgG 
antibodies in the absence of any previous priming with BCG. The 
anti-peptide antibody response persisted for at least 1 year. This 
adjuvant-free carrier effect of the 70 kD hsp was T cell dependent, since 
no anti-peptide nor anti70 kD IgG antibodies were induced in athymic nu/nu 
mice. Previous immunization of mice with the 65 kD or 70 kD hsp did not 
have any negative effect on the induction of anti-peptide IgG antibodies 
after immunization with hsp-peptide conjugates in the absence of 
adjuvants. Furthermore, preimmunization with the 65 kD hsp could 
substitute for BCG in providing an effective priming for the induction of 
anti-(NANP).sub.40 antibodies. The carrier effect of mycobacterial hsp65 
and hsp70 for conjugated peptides was demonstrated also in non-human 
primates (Perraut et al, 1993). 
It can be assumed that some T cell epitopes within the sequence of the 
bacterial hsp65 protein show immunodominance and are able to induce 
immunological memory, whereas others do not express privileged 
immunological recognition or are involved in the induction of autoimmune 
diseases. Distinguishing between functionally different T cell epitopes, 
binding to several different MHC molecules, may lead to the identification 
of universally immunogenic peptides, which can qualify as safe, defined, 
and potent alternatives for carrier molecules of T-ind antigens. 
Israel Patent Application No. 102687 of the same applicants describes 
specific T cell epitopes of human hsp65, and analogs thereof, conjugated 
to poorly immunogenic molecules. 
None of the above mentioned references describes specific T cell epitopes 
derived from the sequence of hsp65 of E. coli (GroEL) conjugated to poorly 
immunogenic molecules. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a method for enhancing 
the immunogenicity of poorly immunogenic antigen molecules, thus 
converting them to suitable antigens for immunization. 
For this purpose, the present invention provides conjugates of a poorly 
immunogenic antigen and a synthetic peptide carrier constituting a T cell 
epitope derived from the sequence of E. coli hsp65 (GroEL) or an analog 
thereof, said peptide or analog being capable of increasing substantially 
the immunogenicity of the poorly immunogenic antigen. 
Any peptide, or analog thereof, derived from GroEL constituting a T cell 
epitope and able to increase substantially the immunogenicity cm the 
poorly immunogenic antigen, can be used in the invention. 
A preferred peptide according to the invention, herein designated 278e, 
corresponds to positions 437-453 of the GroEL molecule, and has the 
sequence: 
##STR1## 
The poorly immunogenic antigen molecule may be a peptide, a polypeptide or 
a protein, e.g., a peptide derived from HIV virus or from malaria antigen, 
or a bacterial polysaccharide, e.g., capsular polysaccharides from 
Haemophilus influenzas type b, Streptococcus pneumoniae, Neisseria 
meningitidis, group B Streptococci, E. coli type Kl, Salmonella, such as 
Salmonella typhi, etc. 
The carrier peptide is covalently linked to the poorly immunogenic antigen 
molecule, either directly or through a spacer. 
The invention further relates to vaccines comprising a conjugate of the 
invention or a mixture of the poorly immunogenic antigen and the suitable 
peptide carrier. 
In another embodiment, the invention relates to a method of immunization of 
a mammalian host which comprises administering to said host an effective 
amount of a conjugate of the invention, or co-administering effective 
amounts of a poorly immunogenic antigen molecule and of a synthetic 
peptide carrier constituting a T cell epitope derived from the sequence of 
GroEL, or an analog thereof, said peptide or analog being able to enhance 
substantially the immunogenicity of the poorly immunogenic antigen.

DETAILED DESCRIPTION OF THE INVENTION 
Preferred conjugates according to the invention are formed by covalently 
linking peptide 278e with a bacterial polysaccharide, e.g., the capsular 
polysaccharide (CPS) Vi of Salmonella typhi, hereinafter referred to as Vi 
or Vi-fragments, a linear homopolymer of poly-.alpha.-(1-4)GalNAc variably 
O-acetylated at the C.sub.3 -position, as shown in scheme 1. The native Vi 
molecule has a molecular weight of about 3.times.10.sup.3 kD (Vi). 
Vi-fragments (about 45 kD) are prepared by ultrasonic cleavage, which does 
not alter the structure of its monomeric units and which produces a 
relatively homogeneous polysaccharide (Stone & Szu, 1988). Vi/Vi-fragments 
alone, like other CPSs, do not elicit a booster response in mammals, 
either in animals or in humans, when reinjected, but its immunogenicity is 
increased when presented as a conjugate according to the invention coupled 
to a suitable peptide derived from GroEL or an analog thereof, or in a 
mixture with such a peptide or analog. Reinjection of the Vi-peptide 
conjugate induces an increase in the level of anti-Vi antibodies (booster 
effect), which are mainly represented by the IgG isotype. 
Peptide 278e of the present invention is clearly distinct from peptides 
278h and 278m of above-mentioned Patent Application No. 102687. 
278e N E D Q N V G I K V A L R A M E A (SEQ ID NO:1) 
278h N E D Q K I G I E I I K R T L K I (SEQ ID NO:2) 
278m N E D Q K I G I E I I K R A L K I (SEQ ID NO:3) 
Peptide 278e is a highly charged and hydrophobic molecule. Thus, 5 out of 
17 constituent amino acids are ionized (3 negatively and 2 positively) at 
physiological pH. Five amino acid residues are hydrophobic. In addition, 3 
residues are amidated and capable of establishing substantial hydrogen 
bonding. The peptide is further characterized as possessing a polar 
negatively-charged N-terminal domain, a polar charged C-terminal domain 
and a highly hydrophobic core. 278e can be modified while retaining 
activity. In order to preserve activity, however, its overall structural 
features should be maintained. Thus, positions 2, 3 and 16 can be either 
occupied by either E or D, and positions 9 and 13 by either K or R. 
Conservation of the charge at positions 9 and 13 (positive to negative and 
vice-versa) will lead to active peptides. A hydrogen bond forming amino 
acid, preferably N and Q, should occupy positions 1 and 4. 
Hydrophobicity at positions 6, 8, 10, 12 and 15 should be maintained by 
incorporating hydrophobic amino acids, natural, e.g., I, L, V, M or F, or 
unnatural, e.g., norleucine (Nle) or norvaline (Nva). 
The term "analogs" in the present invention relates to peptides obtained by 
replacement, deletion or addition of amino acid residues to the sequence 
of the T cell epitope, as long as they have the capability of enhancing 
substantially the immunogenicity of poorly immunogenic antigen molecules. 
Analogs, in the case of peptide 278e, are peptides such that at least 70%, 
preferably 90-100%, of the electric properties and of the hydrophobicity 
of the peptide molecule are conserved. These peptides can be obtained 
according to the instructions in the paragraph herein before. 
The peptides according to the invention may have all the optically active 
amino acid residues in L or D form, or some of the amino acid residues are 
in L and others are in D form. 
By "substantially increasing the immunogenicity of a poorly immunogenic 
antigen molecule" it is meant to comprise both the induction of an 
increase in the level of antibodies against said antigen as well as the 
presentation of said antibodies as mainly of the IgG isotype. 
The peptide carrier may be linked to the antigen molecule directly or 
through a spacer. 
A direct link between the peptide and Vi or Vi-fragments is shown in Scheme 
1 herein, where the conjugate 
##STR2## 
is obtained by Procedure 1 as described hereafter. 
The spacer may have the formula --O--R--CO or --NH--R--CO, thus forming an 
ester or amide, respectively, with the carboxyl group of Vi or 
Vi-fragments and a peptide bond with the terminal amino group of the 
peptide; or --NH--R--CH.sub.2 --, wherein R is a saturated or unsaturated 
hydrocarbon chain optionally substituted and/or interrupted by one or more 
aromatic radicals or by heteroatoms such as O, S or N. Preferably, R is an 
aliphatic hydrocarbon chain containing 3-16 carbon atoms, such as the 
residue of .epsilon.-aminocaproic acid. 
##STR3## 
The conjugate of the formula: 
##STR4## 
in which Ac is acetyl, AC is the residue of .epsilon.-aminocaproic acid, 
Pep is the residue of the peptide carrier 278e or an analog thereof and 
the saccharide residue represents a repeating unit of the Vi capsular 
polysaccharide (Vi or vi-fragments) of Salmonella typhi, may be prepared 
by Procedure 2 depicted in Scheme 1 and described in detail hereafter. 
The conjugates wherein the spacer is --NH--R--CH.sub.2 -- are obtained by 
reduction of --NH--R--CO-- groups or by alkylation cm the peptidic amino 
terminus with --NH--R--CH.sub.2 --X, when X is an appropriate leaving 
group such as an halide. 
The invention further relates to vaccines comprising a conjugate of the 
invention. These vaccines may be administered by any suitable route, e.g., 
orally or via the subcutaneous route in suitable vehicles for human and 
veterinary purposes. 
The invention will now be illustrated by the following non-limiting 
examples: 
EXAMPLES 
In the examples, the following materials and methods will be used. 
Materials & Methods 
a. Materials: All solvents and chemicals were of analytical grade and 
obtained from Aldrich, U.S.A., unless otherwise mentioned. 
b. Peptide synthesis: Peptide 278e was prepared with an automated 
synthesizer (Applied Biosystem model 430A, Germany) using the company's 
protocols for t-butyloxycarbonyl (BOC) strategy (Kent et al, 1984). 
The following control peptides were synthesized: Peptide 278h corresponding 
to positions 458-474 of the human hsp65 molecule, 278m corresponding to 
positions 458-474 of the murine hsp65, and 278cox corresponding to 
positions 437-453 of the Coxiella burnetti hsp65 protein, said control 
peptides having the sequences depicted below: 
278h N E D Q K I G I E I I K R T L K I (SEQ ID NO:2) 
278m N E D Q K I G I E I I K R A L K I (SEQ ID NO:3) 
278cox N E D Q R V G V E I A R R A M A Y (SEQ ID NO:4) 
A further control peptide, AcR259-271, corresponds to positions 259-271 of 
the murine acetylcholine receptor .alpha.-chain and has the sequence: 
V I V E L I P S T S S A V 
This peptide is recognized by T cells in the context of MHC class II 
molecules of the H-2d haplotype. 
c. Reversed-phase HPLC: The purity of the peptide products was estimated by 
using the analytical HPLC column RP18 (Merck, Darmstadt, Germany) 
employing the SP8750 liquid chromatography system equipped with a SP8733 
variable wavelength detector in water-acetonitrile gradients containing 
0.1% trifluoroacetic acid (TFA). The effluents were monitored by UV 
absorbance at 220 nm. Acetonitrile of HPLC grade was purchased from Merck 
(Darmstadt, Germany). Peptides were further analyzed by amino acid 
analysis. 
d. Vi: The Vi purified from Citrobacter freundii WR7011 (kindly donated by 
J. B. Robbins and S. C. Szu, National Institute of Health, Bethesda, Md., 
U.S.A.) contained &lt;1% (each) protein, nucleic acid, and 
lipopolysaccharide. The molecular size of the Vi was estimated to be 
3.times.10.sup.3 kD. The Vi-fragments of about 45 kD were prepared by 
ultrasonic irradiation and were kindly provided by Dominique Schulz 
(Pasteur-Merieux, France). 
e. Coupling of Vi and Vi-fragments with peptide: 
Procedure 1 (see scheme 1) Conjugation of Vi/Vi-fragments and peptide 
without a spacer. One part of Vi/Vi-fragment and one part of peptide were 
dissolved in a minimal volume of double distilled water (ddw) and 
incubated for 12 hours at room temperature (RT) at pH 6 in the presence of 
two parts water-soluble carbodiimide (CDI; 
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride). After 
dialysis of the reaction mixture, the peptide density in the conjugate was 
determined by amino acid analysis and the sugar content of the construct 
estimated by Fourier transformed infrared spectroscopy (FTIR). 
Procedure 2 (see scheme 1) Coupling of Vi/Vi-fragments and peptide 
following extension of peptide chain by a spacer in solution. In order to 
activate the carboxyl-function of the tBoc-.epsilon.-aminocaproic acid 
(t-Boc-AC) by N-hydroxysuccinimide, 1 mmol t-Boc-AC was mixed with 1.15 
mmol N-hydroxysuccinimide in a minimal volume of dioxane (Merck, Germany); 
1.15 mmol N,N'-dicyclohexylcarbodiimide (DCC) dissolved in dioxane was 
added, and after 3 hours the reaction mixture was filtered and washed with 
dioxane. 0.1 mmol of the desired peptide was dissolved in a small amount 
of ddw and mixed with 0.2 mmol KHCO.sub.3, (Merck). The solution, in 
dioxane, of the N-hydroxysuccinimide ester of t-BocAC and the prepared 
peptide solution were mixed and reacted for 1 hour with vigorous mixing. 
The reaction mixture was then diluted with ddw (10 ml), cooled and 
acidified with 1N KHSO.sub.4, solution. The product was extracted by ethyl 
acetate. The organic solution was washed with ddw, dried over Na.sub.2 
SO.sub.4, and evaporated to dryness. After drying the product for 2 hours 
over P.sub.2 O.sub.5, dissolving it with 4-5 ml TFA and reacting for 10 
minutes, the liquid was evaporated in vacuum at 30.degree. C. The compound 
was washed twice with CH.sub.2 Cl.sub.2 and the fluid evaporated before 
drying 2-3 hours over P.sub.2 O.sub.5. Subsequently, the peptide-AC 
product was dissolved in ddw and the pH adjusted to 8. Five mg 
N-hydroxysuccinimide ester (prepared as described in Procedure 2 of Patent 
Application No. 102687) of Vi/Vi-fragments were added. After several hours 
of incubation, the resulting Vi-AC-Peptide conjugate was dialysed against 
ddw. The peptide density in the conjugate was estimated by amino acid 
analysis. 
f. Immunization: Female mice belonging to different strains, 2-3 months 
old, were immunized subcutaneously (sc), two times 4 weeks apart with 
Vi/Vi-fragment alone or the Vi/Vi-fragment-conjugate. The injected amount 
of antigen varied from experiment to experiment and is indicated in the 
figures. The used adjuvant was in all cases IFA. Mice from each 
experimental group were bled 12 days after each injection. 
g. Serology: Vi/Vi-fragment antibody levels elicited in mice with native or 
conjugated Vi, were determined by an enzyme-linked immunosorbent assay 
(ELISA). Since negatively-charged polysaccharides do not attach well to 
the polystyrene commonly used in the solid-phase ELISA, positively charged 
methylated bovine serum albumin (BSA) was used to coat Vi/Vi-fragments on 
the solid surface with very little non-specific binding. In detail, 0.5 mg 
Vi were dissolved in 1 ml PBS and stirred for 1 hour at room temperature. 
Ten mg methylated BSA (Sigma) were suspended in 1 ml H.sub.2 O and the 
obtained solution filtered on a 0.8 .mu.m filter. To prepare the coating 
solution, 1 ml of dissolved polysaccharide was stirred for 20 minutes at 
room temperature with 50 .mu.l of the methylated BSA solution and 
subsequently diluted 1:20 in PBS. Nunclon delta Si microwell plates were 
coated for 3 hours at 37.degree. C. with 100 .mu.l coating solution per 
well (2.5 .mu.g Vi/well). The plates wee washed five times with PBS 
containing 0.33% Brij35 (Sigma) and blocked with a solution of PBS and 1% 
dried skimmed milk for 2 hours at 37.degree. C. After washing, 100 .mu.l 
aliquots of diluted unknown sera and of diluted standard serum (dilution 
buffer containing 1% skimmed milk and 0.33% Brij35 in PBS) were added and 
the plates were incubated for 1 hour at 37.degree. C. Reference and test 
sera were applied to the plates in duplicate. The non-bound antibodies 
were removed by washing and a 1:5000 dilution of goat anti-mouse IgG 
Fab.sub.2 -alkaline phosphatase conjugate (Sigma), in the case of the test 
sera, and rabbit anti-horse IgG Fab.sub.2 enzyme conjugate, in the case of 
the standard serum, was added to the plates (100 .mu.l/well). After an 
incubation of 2 hours at 37.degree. C., the plates were washed and the 
bound antibody visualized by the addition of 100 .mu.l substrate solution 
containing 0.6 mg/ml of p-nitrophenylphosphate (Sigma) in 
diethanolamine-H.sub.2 O pH 9.8. The enzyme reaction was stopped 20 
minutes later by the addition of 10 .mu.l 5N NaOH per well. Optical 
densities were read at 405 nm. The anti-Vi standard serum Burro 260, 
containing 550 mg Vi antibody/ml, was prepared by multiple intravenous 
injections of formalin-fixed Salmonella typhi Ty2 (kindly donated by J. B. 
Robbins and S. C. Szu, NIH, Maryland, U.S.A.). The results obtained are 
expressed as optical density read at 405 nm. 
h. Lymph node Proliferation after peptide-immunization: 
Groups of 3 mice of the designated mouse strain were immunized sc into the 
footpads with 20 .mu.g peptide emulsified in 0.2 ml IFA/PBS (0.1 ml/foot). 
Draining lymph nodes were taken 10 days later. Lymph node cells (LNC) of 
immunized mice 5.times.10.sup.6 /well) were cultured in the presence of 
different antigens. Cultures were set up in 200 .mu.l Eagles medium 
supplemented with 2 mM glutamine, nonessential amino acids, 1 mM sodium 
pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin, 5.times.10.sup.5 M 
.beta.-mercaptoethanol (Fluka AG, Buchs, Switzerland) containing 1% of 
syngeneic normal mouse serum, in round bottom microtiter plates (Falcon). 
After four-five days incubation, .sup.3 H-thymidine (0.5 mCi of 5 Ci/mmol, 
Nuclear Research Center, Negev, Israel) was added. Sixteen hours later, 
cells were harvested and radioactivity was counted. Results are expressed 
as counts per minute (cpm) or as stimulation indices (SI). The SI was 
defined as the ratio of the mean cpm of test cultures (with antigen) and 
the mean cpm of control cultures (without antigen). 
EXAMPLES 
Example 1 
Preparation of Vi-peptide conjugates 
Conjugates of Vi/Vi-fragments with peptide 278e and the control peptides 
were prepared as described above. 
The composition of the Vi-peptide conjugates is summarized in Table 1. The 
results presented in Table 1 indicate that the molar ratio of peptide per 
sugar monomer was variable. Peptide doses of 0.8-2.2 .mu.g injected per 
mouse as sugar-peptide conjugate were shown to be most effective. 
Example 2 
Lymph node cell proliferation to peptide 278e in different mouse strains 
with varying major histocompatibility complex MHC) background. 
2.1. Lymph node proliferation after immunization with free carrier peptide. 
In order to test if peptide 278e can be recognized by the immune system in 
the context of different alleles of the murine MHC, 2-3 month old female 
mice (three animals per group) were injected sc with 20 .mu.g of free 
peptide 278e emulsified in IFA as described in Material & Methods herein 
and specific proliferation of lymph node cells to peptide 278e and control 
peptides. 
As shown in FIG. 1, LNCs of BALB/c (H-2d) mice inoculated with peptide 278e 
showed clear specific proliferative responses to the latter whereas no 
proliferation occurred to control peptide 278m and 278cox. Thus, LNCs 
primed with peptide 278e do not cross-react with the homologous 
self-peptide 278m derived from the sequence of murine hsp65. 
FIG. 2a-c demonstrates that peptide 278e was also recognized in the three 
different congenic B10 mouse strains. LNCs of B10.RIII mice (H-2.sup.r) 
(FIG. 2a), B10.BR mice (H-2.sup.k) (FIG. 2b) and B10.S mice (H-2.sup.s) 
(FIG. 2c) showed significant higher proliferative responses to peptide 
278e in the designated peptide concentrations than to the control peptide 
AcR259-271. 
2.2. Lymph node cell proliferation to peptide 278e after immunization with 
peptide 278e conjugated to Vi-fragments. To analyze if coupling of peptide 
278e to the polysaccharide Vi-fragments changes its antigenic structure, 
the LNC response to the peptide alone was tested after immunization with 
the sugar-peptide conjugate. FIG. 3 and FIG. 4 distinctly show that LNCs 
elicited by Vi-fragments-278e in BALB/c mice can recognize the 
unconjugated peptide when immunized with 2 .mu.g Vi-fragments/mouse (FIG. 
3) or 20 .mu.g Vi-fragments/mouse (FIG. 4) as sugar-peptide conjugate (for 
the belonging injected peptide amount see Table 1). 
TABLE 1 
______________________________________ 
Peptide amount injected per 
Vi-fragment-peptide conjugate 
2 .mu.g Vi-fragment .mu.g! 
______________________________________ 
Vi-fragments-278e 
0.8 
Vi-fragments-278m 
1.8 
Vi-fragments-278h 
2.2 
______________________________________ 
Example 3 
Antigenicity of Vi-fragments conjugated to peptide 278e. To examine if 
peptide 278e conjugated to Vi-fragments can enhance the immune response to 
this T-ind antigen, the immune response to the sugar was studied after 
inoculation of five BALB/c mice with the sugar-peptide conjugate. FIG. 5 
clearly demonstrates that peptide 278e covalently linked to Vi-fragments 
can enhance the sugar-specific IgG antibody production substantially. 
Immunizing mice with a second dose of the conjugate gave rise to a strong 
booster effect indicating the involvement of T cells in the sugar-specific 
immune response. Inoculating BALB/c mice with the unconjugated 
polysaccharide only induced negligible levels of specific antibodies. The 
immune response induced by Vi-fragments-278e is compared to that elicited 
by the sugar conjugated to peptide 278h and 278m. 
The above experiments offer evidence that peptide 278e can be recognized in 
association with a wide range of alleles of murine MHC molecules and can 
be used as carrier epitope for inducing enhanced immune responses to 
poorly immunogenic molecules. This evidence may be summarized a follows: 
(i) Primed LNCs of mouse strains with varying genetic MHC-background were 
able to recognize peptide 278e by exhibiting specific proliferative 
responses. 
(ii) Conjugating peptide 278e to Vi-fragments did not change its antigenic 
structure since LNCs primed with 278e coupled to the polysaccharide were 
still able to recognize the unbound peptide in an in vitro lymph node 
proliferation assay. 
(iii) The immunogenicity of the Vi-fragments was increased when presented 
to the immune system as a conjugate coupled to peptide 278e. 
The fact that LNCs that were primed with peptide 278e were not 
cross-reacting with the mouse homologue peptide 278m, indicates that 
peptide 278e used as carrier epitope probably will not induce immune 
responses directed to self components. 
Since the immune response to peptide 278e seems not to be genetically 
restricted in mice, this synthetic peptide and analogs thereof might be 
used as universal carriers for the preparation of immunogenic conjugates 
to provide protective immunity against different pathogenic agents and can 
be suitable for the development of synthetic vaccines. 
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168:357-373 (1988) 
--Herzenberg, L. A. & Tokuhisa T., J. Exp. Med., 155: 1730-1740 (1982) 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..17 
(D) OTHER INFORMATION: /label=Pep278e 
/note= "Corresponds to positions 437-453 of the E. coli 
hsp65 molecule." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AsnGluAspGlnAsnValGlyIleLysValAlaLeuArgAlaMetGlu 
151015 
Ala 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..17 
(D) OTHER INFORMATION: /label=Pep278h 
/note= "Corresponds to positions 458-474 of the human 
hsp65 molecule." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AsnGluAspGlnLysIleGlyIleGluIleIleLysArgThrLeuLys 
151015 
Ile 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..17 
(D) OTHER INFORMATION: /label=Pep278m 
/note= "Corresponds to positions 458-474 of the murine 
hsp65 protein." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AsnGluAspGlnLysIleGlyIleGluIleIleLysArgAlaLeuLys 
151015 
Ile 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..17 
(D) OTHER INFORMATION: /label=Pep278cox 
/note= "Corresponds to positions 437-453 of Coxiella 
burnetti hsp65 protein." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GluGlyAspGluAlaThrGlyAlaAsnIleValLysValAlaLeuGlu 
151015 
Ala 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..13 
(D) OTHER INFORMATION: /label=AcR259-271 
/note= "Corresponds to positions 259-271 of the murine 
acetyl-choline receptor `-chain." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ValIleValGluLeuIleProSerThrSerSerAlaVal 
1510 
__________________________________________________________________________