The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.     

Flow cytometric analysis of peptide binding to major histocampatibility complex class I for hepatitis C virus core T-cell epitopes

BACKGROUND/METHODS: To characterize the repertoire of T-cell epitopes on the hepatitis C virus (HCV) core protein, we studied major histocompatibility complex (MHC) class I binding of 75 decapeptides on 20 human B-cell lines and murine spleen cells using a flow cytometric assay. The results were compared with MHC class I stabilization on T2 cells, the SYFPEITHI algorithm, and known T-cell epitopes from the literature. RESULTS: Binding of peptides proved to be specific for MHC class I molecules. We observed peak fluorescence signals at positions amino acids (aa) 35-44, aa 87-96, aa 131-140, and aa 167-176 in virtually all HLA-A2-positive cell lines. These sites corresponded to T-cell epitopes predicted by SYFPEITHI and the positions of known T-cell epitopes, whereas T2 stabilization was at variance for two peptides. The assay was applied to HLA-A2-negative cells and murine spleen cells without further modification, and identified additional peptides, corresponding to known T-cell epitopes. CONCLUSIONS: Peptide binding to different MHC class I alleles can be mapped rapidly by a flow cytometric assay and enables a first orientation on the sites of possible T-cell epitopes. Application of this assay to HCV core suggests a rather limited repertoire of epitopes in the Caucasoid population.     

Interspecies major histocompatibility complex-restricted Th cell epitope on foot-and-mouth disease virus capsid protein VP4

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.     

Manipulation of epitope function by modification of peptide structure: a minireview.

We have explored various approaches to modify the immunrecognition of linear peptides representing sequential or continuous topographic B-cell or T-cell epitopes. For these studies, epitopes from herpes simplex virus (HSV) glycoprotein D (gD) and from mucin 1 and mucin 2 glycoproteins or T-cell epitopes from 16 kDa and 38 kDa proteins of Mycobacterium tuberculosis were selected. To increase antigenicity and immunogenicity we have prepared cyclic and chimaeric peptide variants as well as epitope peptides with altered flanking regions and epitope-carrier conjugates containing multiple epitope copies.     

Outer membrane protein PhoE as a carrier for the exposure of foreign antigenic determinants at the bacterial cell surface

PhoE protein is an abundant outer membrane protein of theEscherichia coli K-12 outer membrane. This protein can be used as an exposure system to produce foreign antigenic determinants and for their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature could be made in different cell surface-exposed regions of PhoE, without interfering with the assembly process of the mutant proteins into the outer membrane. Two antigenic determinants of the structural VP1 protein of foot-and-mouth disease virus were inserted in different combinations in four cell surface-exposed regions of PhoE. The epitopes were exposed at the bacterial cell surface and they keep their antigenic and immunogenic properties in this PhoE-associated conformation. Immunization of guinea pigs with one hybrid protein, containing a combination of the two epitopes inserted in the fourth exposed region, resulted in complete protection against challenge with the virus. A T-cell epitope of the 65 kDa heat shock protein ofMycobacterium tuberculosis was inserted in the fourth exposed region of PhoE and in vitro proliferation of two T-cell specific clones was demonstrated. Thus, the PhoE exposure system has been shown to be suitable for presentation of both B-cell and T-cell determinants to the immune system. Furthermore, good expression of the hybrid protein in attenuatedSalmonella strains, which can be used as live oral vaccines, was shown.Paper awarded the Kluyver Prize 1990 by the Netherlands Society of Microbiology to Dr. M.C. Agterberg     

Expression of foreign epitopes in P-fimbriae of Escherichia coli.

Summary Hypervariable regions (HRs) of the major subunit of F11 fimbriae were exploited for insertion of foreign epitopes. Two insertion vectors were created that contain a unique cloning site in HR1 or HR4 respectively. Several oligonucleotides, coding for antigenic determinants derived from different pathogens, were cloned in both insertion vectors. Hybrid fimbrial subunits were generally shown to be assembled in fimbriae when the length of the inserted peptide did not exceed 14 amino acids. The inserted peptides appeared to be exposed in the fimbrial filament. One hybrid fimbrial protein induced detectable levels of antibodies against the inserted epitope if injected into mice.     

Function in the Immune System of Antigenic Peptides from S-Antigen (Arrestin)

A significant amount of information concerning immunologic domains of an antigenic molecule can be obtained by studying its peptides. We describe a method for identifying and characterizing immunologically relevant T-cell and B-cell epitopes in S-antigen, a well-characterized, highly pathogenic retinal autoantigen for the induction of experimental autoimmune uveitis. The method involves the generation of peptide fragments by enzymatic treatment of native S-antigen and by the simultaneous synthesis of large numbers of peptides in small quantities for screening and testing. Peptides demonstrating T- or B-cell activity are then synthesized in large quantity for additional studies. Although useful information was obtained by the use of enzymatically generated peptides, synthetic peptides provided the greatest flexibility and specificity, allowing the precise localization of amino acid sequences of S-antigen required for a particular immunological function such as antibody binding, T-cell proliferative responses, pathogenicity, and the induction of tolerance. These studies have wide applicability to the study of other antigenic molecules and have led to a better understanding of the immune mechanisms involved in the pathogenesis of experimental autoimmune uveitis. This, in turn, provides a basis for the processes that may be occurring in certain forms of human uveitis.     

Activation of a heterogeneous hepatitis B (HB) core and e antigen-specific CD4+ T-cell population during seroconversion to anti-HBe and anti-HBs in hepatitis B virus infection.

Overcoming hepatitis B virus infection essentially depends on the appropriate immune response of the infected host. Among the hepatitis B virus antigens, the core (HBcAg) and e (HBeAg) proteins appear highly immunogenic and induce important lymphocyte effector functions. In order to investigate the importance of HBcAg/HBeAg-specific T lymphocytes in patients with acute and chronic hepatitis B and to identify immunodominant epitopes within the HBcAg/HBeAg, CD4+ T-cell responses to hepatitis B virus-encoded HBcAg and HBcAg/HBeAg-derived peptides were studied in 49 patients with acute and 39 patients with chronic hepatitis B. The results show a frequent antigen-specific CD4+ T-cell activation during acute hepatitis B infection, a rare HBcAg/HBeAg-specific CD4+ T-cell response among HBeAg+ chronic carriers, and no response in patients with anti-HBe+ chronic hepatitis. An increasing CD4+ T-cell response to HBcAg/HBeAg coincides with loss of HBeAg and hepatitis B virus surface antigen (HBsAg). Functional analysis of peptide-specific CD4+ T-cell clones revealed a heterogeneous population with respect to lymphokine production. Epitope mapping within the HBcAg/HBeAg peptide defined amino acids (aa) 1 to 25 and aa 61 to 85, irrespective of the HLA haplotype, as the predominant CD4+ T-cell recognition sites. Other important sequences could be identified in the amino-terminal part of the protein, aa 21 to 45, aa 41 to 65, and aa 81 to 105. The immunodominant epitopes are expressed in both proteins, HBcAg and HBeAg. Our findings lead to the conclusion that activation of CD4+ T lymphocytes by HBcAg/HBeAg is a prerequisite for viral elimination, and further studies have to focus on the question of how to enhance or induce this type of T-cell response in chronic carriers. The immunodominant viral sequences identified may have relevance to synthetic vaccine design and to the use of peptide T-cell sites as immunotherapeutic agents in chronic infection.     

The major subunit ClpG of Escherichia coli CS31A fibrillae as an expression vector for different combinations of two TGEV coronavirus epitopes

Previously, two B-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (TGEV), namely the C epitope (TGEV-C) amino acids (aa) 363–371 and the A epitope (TGEV-A) aa 522–531 of the spike S protein (TGEV-S), have been separately expressed on the CS31A fibrillae at the surface of Escherichia coli following insertion into a same region of ClpG. However, the resulting chimeras induced a marginal TGEV-neutralizing antibody (Ab) response in mice. Here, with the view to improving this response, we introduced TGEV-C alone or in different tandem association with TGEV-A (A::C or C::A) in twelve putatively exposed regions of ClpG. Among the 28 resulting engineered proteins only 15, carrying up to 51 extra aa, had not essentially disturbed the correct CS31A fibrillae formation process. Six partially permissive sites accepting only TGEV-C and three highly permissive sites tolerating A::C or C::A tandem peptide, were identified throughout ClpG. Intact bacteria or extracted CS31A hybrid fibrillae expressing TGEV epitopes at any of the permissive sites, were recognized by Ab directed against the foreign parent protein, providing a direct argument for exposure of the corresponding ClpG region at the cell surface and for antigenicity of the epitopes in the polymeric CS31A fibrillae context. The potential of CS31A fibrillae as carriers of the TGEV peptides indicates that there may be three positions (N terminus, aa 202–204 and 202–218) in ClpG which may turn out to be important fusion sites and therefore be relevant for the eventual design of TGEV vaccines. Unexpectedly, TGEV-A, whatever its position in ClpG, mediated the partial proteolytic degradation of the hybrid proteins, suggesting that it functions as a substrate for a cellular protease, and thereby that its suitability as a vaccine antigen candidate is doubtful.     

A structure-based approach for prediction of MHC-binding peptides

Identification of immunodominant peptides is the first step in the rational design of peptide vaccines aimed at T-cell immunity. The advances in sequencing techniques and the accumulation of many protein sequences without the purified protein challenge the development of computer algorithms to identify dominant T-cell epitopes based on sequence data alone. Here, we focus on antigenic peptides recognized by cytotoxic T cells. The selection of T-cell epitopes along a protein sequence is influenced by the specificity of each of the processing stages that precede antigen presentation. The most selective of these processing stages is the binding of the peptides to the major histocompatibility complex molecules, and therefore many of the predictive algorithms focus on this stage. Most of these algorithms are based on known binding peptides whose sequences have been used for the characterization of binding motifs or profiles. Here, we describe a structure-based algorithm that does not rely on previous binding data. It is based on observations from crystal structures that many of the bound peptides adopt similar conformations and placements within the MHC groove. The algorithm uses a structural template of the peptide in the MHC groove upon which peptide candidates are threaded and their fit to the MHC groove is evaluated by statistical pairwise potentials. It can rank all possible peptides along a protein sequence or within a suspected group of peptides, directing the experimental efforts towards the most promising peptides. This approach is especially useful when no previous peptide binding data are available.     

A novel strategy of epitope design in Neisseria gonorrhoeae

In spite of genome sequences of both human and N. gonorrhoeae in hand, vaccine for gonorrhea is yet not available. Due to availability of several host and pathogen genomes and numerous tools for in silico prediction of effective B-cell and T-cell epitopes; recent trend of vaccine designing has been shifted to peptide or epitope based vaccines that are more specific, safe, and easy to produce. In order to design and develop such a peptide vaccine against the pathogen, we adopted a novel computational approache based on sequence, structure, QSAR, and simulation methods along with fold level analysis to predict potential antigenic B-cell epitope derived T-cell epitopes from four vaccine targets of N. gonorrhoeae previously identified by us [Barh and Kumar (2009) In Silico Biology 9, 1-7]. Four epitopes, one from each protein, have been designed in such a way that each epitope is highly likely to bind maximum number of HLA molecules (comprising of both the MHC-I and II) and interacts with most frequent HLA alleles (A*0201, A*0204, B*2705, DRB1*0101, and DRB1*0401) in human population. Therefore our selected epitopes are highly potential to induce both the B-cell and T-cell mediated immune responses. Of course, these selected epitopes require further experimental validation.     

Selection of cryptic B-cell epitopes using informational analysis of protein sequences

Sub-unit vaccines are synthetic or recombinant peptides representing T- or B-cell epitopes of major protein antigens from a particular pathogen. Epitope selection requires the synthesis of peptides that overlap the protein sequences and screening for the most effective ones. In this study a new method of immunogenic peptide selection based on the analysis of information structure of protein sequences is suggested. The analysis of known B-cell epitope location in the information structure of Aspergillus fumigatus proteins Asp f 2 and Asp f 3 has shown that epitopes are scattered along the sequences of proteins for the exception of sites with Increased Degree Information Coordination (IDIC). Based on these results peptides from different allergens such as Asp f 2, Der p 1, and Fel d 1 were selected and produced in a recombinant form in the context of yeast virus-like particles (VLPs). Immunization of mice with VLPs containing peptides form allergens has induced the production of IgG able to recognize full-length antigens. This result suggests that the analysis of information structure of proteins can be used for the selection of peptides possessing cryptic B-cell epitope activity.     

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.     

Type 1 fimbriae of Escherichia coli as carriers of heterologous antigenic sequences

A strategy has been designed for the construction of recombinant bacterial strains which eventually may become useful as live vaccines and which may also be relevant for the preparation of conventional vaccines. The approach used is the fusion of small antigenic peptide sequences into specific segments of a protein whose location on the bacterial surface ensures that the recombinant organism is able to present the inserted antigen to the host (animal or human) infected by the bacterium. The chosen surface protein is a naturally occurring polymer of Escherichia coli, viz., type 1 fimbriae. The results obtained show that fusion of such foreign sequences into selected points of the structural protein of the fimbriae results in the production of functionally normal type 1 fimbriae. Furthermore, hybrid fimbriae carrying such small epitope sequences can be recognized by antibodies directed against the foreign parent protein. This observation is an important prerequisite for the eventual design of useful vaccines. The analysis of the fimbrial protein and its potential as a carrier of foreign peptides from hepatitis B surface antigen, foot-and-mouth disease virus and poliovirus indicated that there may be several positions in the protein which may turn out to be relevant for this purpose and be important fusion sites.     

Protective immunity to rabbit oral and cutaneous papillomaviruses by immunization with short peptides of L2, the minor capsid protein

The papillomavirus minor capsid protein, L2, has been shown to exhibit immunogenicity, whereby a variety of B-cell epitopes, predominantly in the amino terminus of L2, have been deduced. However, immunity to L2 in vivo has not been examined extensively. Notably, a common neutralization epitope for human papillomavirus (HPV) types 6 and 16 was mapped to amino acids (aa) 108 to 120. The objectives of this study were to derive antisera from rabbits using the corresponding sequences from rabbit viruses and to assess the ability of these peptides to protect against infection. Synthetic peptides consisting of two overlapping sequences each in the region of aa 94 to 122 of the rabbit oral (ROPV) and cottontail rabbit (CRPV) papillomaviruses were used to immunize rabbits. Rabbits were then infected with both ROPV and CRPV and monitored for the development of oral and cutaneous papillomas, respectively. Serum derived from rabbits immunized with either of the two peptides was shown to (i) react to purified L2 from the cognate virus, (ii) specifically recognize L2 within virus-infected cells, and (iii) neutralize virus in vitro. Following viral challenge, cutaneous papilloma growth was completely absent in rabbits immunized with either CRPV peptide. Likewise, ROPV peptide-immunized rabbits were protected from oral papillomatosis. Challenge of CRPV peptide-immune rabbits with the viral genome resulted in efficient papilloma growth, suggesting a neutralizing antibody-mediated mechanism of protection. These results afford in vivo evidence for the immunogenicity provided by a distinct region of L2 and further support previous evidence for the ability of this region to elicit antiviral immunity.     

Epitope mapping and evaluation of specificity of T-helper sites in four major antigenic peptides of chicken riboflavin carrier protein in outbred rats

This paper reviews our studies on synthetic peptides spanning the major antigenic determinants of the chicken riboflavin carrier protein (RCP; 219 AA). These determinants are composed of residues 4-24 (YGC), 64-83 (CED), 130-147 (GEN), and 200-219 (HAC) and function as minivaccines in terms of eliciting anti-peptide antibodies which recognize the native protein and are particularly promising contraceptive vaccine candidates. We have used 15-residue synthetic peptides to define short sequences involved in interaction with antibody and with T-cells. We have mapped the boundaries of T-cell epitopes of these peptides in outbred rats by immunizing the animals with each peptide and assaying the popliteal lymph node cell proliferation against a series of overlapping synthetic 15-mers covering the entire length of the individual peptides. The peptides YGC, GEN, and HAC harboured a single T-cell epitope each whereas the peptide CED exhibited bimodal response possessing two epitopes, one at N-terminus and the other at the C-terminus. These studies provide insight into the way in which an immunogen is viewed by the immune system. In addition, preferential T-cell helper function for B cells recognizing unique determinants on the same molecule was demonstrated. This information helps in exploiting synthetic peptides in the construction of designer immunogens which have potential as candidate vaccines.     

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.     

Proteomic scan for tyrosinase peptide antigenic pattern in vitiligo and melanoma: role of sequence similarity and HLA-DR1 affinity

Immune responses contribute to the pathogenesis of vitiligo and target melanoma sometimes associated with vitiligo-like depigmentation in some melanoma patients. We analyzed the sera from patients with vitiligo and cutaneous melanoma for reactivity toward tyrosinase peptide sequences 1) endowed with low level of similarity to human proteome, and 2) potentially able to bind HLA-DR1 Ags. We report that the tyrosinase autoantigen was immunorecognized with the same molecular pattern by sera from vitiligo and melanoma patients. Five autoantigen peptides composed the immunodominant anti-tyrosinase response: aa95-104FMGFNCGNCK; aa175-182 LFVWMHYY; aa176-190FVWMHYYVSMDALLG; aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC. All of the five antigenic peptides were characterized by being (or containing) a sequence with low similarity level to the self proteome. Sera from healthy subjects were responsive to aa95-104FMGFNCGNCK, aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC, but did not react with the aa175-182LFVWMHYY and aa176-190FVWMHYYVSMDALLG peptide sequences containing the copper-binding His180 and the oculocutaneous albinism I-A variant position F176. Our results indicate a clear-cut link between peptide immunogenicity and low similarity level of the corresponding amino acid sequence, and are an example of a comparative analysis that might allow to comprehensively distinguish the epitopic peptide sequences within a disease from those associated to natural autoantibodies. In particular, these data, for the first time, delineate the linear B epitope pattern on tyrosinase autoantigen and provide definitive evidence of humoral immune responses against tyrosinase.     

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.     

Synthetic immunogens constructed from T-cell and B-cell stimulating peptides (T:B chimeras): preferential stimulation of unique T- and B-cell specificities is influenced by immunogen configuration.

In our effort to develop synthetic immunogens as vaccines, we have focused on the combination of a known T-cell stimulating peptide with putative B-cell stimulating peptide epitopes derived from the sequences of respiratory syncytial (RS) virus proteins. The T-cell stimulating peptide consists of residues 45 through 60 of the 1A protein of RS virus, and it also contains an overlapping antibody binding (B-cell) site. Herein, we have combined the 1A T-cell stimulating peptide with a putative B-cell peptide epitope derived from the viral G glycoprotein using linear synthesis or using chemical crosslinking. The chimeric immunogens were compared to each other and to free peptides for their T- and B-cell stimulating properties. Both chimeras had potent T-cell stimulating and antibody-inducing activity. However, T-cells primed to free peptide differentially recognized the two chimeras and immunization with the chimeras primed T-cells with different specificity. Most strikingly, the two chimeras had opposite antibody-inducing properties: The chimera constructed by linear synthesis overwhelmingly elicited antibody directed against the G peptide, whereas the chimera constructed by chemical crosslinking overwhelmingly elicited antibody directed against the 1A peptide. Competition blocking studies revealed that the chimeras adopted different configurations in solution. The resulting antibody response, and hence the B-cell clone elicited, was consistent with the antibody accessibility of the individual peptide epitope.