Charalambos D. Partidos

Prediction and identification of a T cell epitope in the fusion protein of measles virus immunodominant in mice and humans

Amino acid residues 288 to 302 of the fusion protein of measles virus were predicted by a variety of methods to represent a putative T cell epitope. This sequence was synthesized and the peptide was injected into mice of six inbred strains to test this possibility. Lymphocytes from peptide-immunized mice from all six H-2 disparate strains were able to mount a proliferative response following in vitro culture with the peptide. In addition, lymphocytes from three strains also proliferated in the presence of live measles virus. The peptide also behaved as a B cell epitope in that immunization with free peptide in adjuvant resulted in anti-peptide antibody production in all mouse strains. However, these antibodies did not react with the virus in either a solid-phase immunoassay or a virus neutralization assay. Peripheral blood lymphocytes from 10 laboratory personnel with a prior history of exposure to measles virus were tested in a proliferation assay with the peptide and with the virus. Lymphocytes from all 10 individuals proliferated in response to culture with the virus and those from eight responded to the peptide. These results give further support to the concept of permissive interaction of antigenic peptides with a wide range of class II major histocompatibility complex molecules both in mice and man and indicate the possibility of designing peptides that could be used as components of a synthetic vaccine for use in man.

Immune responses in mice following immunization with chimeric synthetic peptides representing B and T cell epitopes of measles virus proteins.

The immunogenicity of chimeric peptides produced by collinear synthesis to contain both T and B cell epitopes from the fusion protein and the haemagglutinin of measles virus was studied in mice. The T cell epitope used was from the fusion protein (residues 288 to 302), which has been shown to be promiscuous in its binding to mouse major histocompatibility complex molecules. This epitope was coupled by (i) a glycine-glycine spacer to a B cell epitope from the fusion protein (residues 404 to 414) and (ii) either its amino or carboxy terminus to a neutralizing antibody epitope from the haemagglutinin (residues 188 to 199). The results obtained show that such chimeric peptides can indeed function as complete immunogens in a range of mouse strains of different H-2 haplotype, and can induce the production of antibodies which bind to the fusion protein and to measles virus. Furthermore, it was shown that the orientation of the T cell epitope with respect to the B cell epitope had a significant effect upon the immunogenicity and antigenic specificity of the chimera. This work gives further support to the concept of rationally designed synthetic peptide vaccines.

The effect of orientation of epitopes on the immunogenicity of chimeric synthetic peptides representing measles virus protein sequences

We have studied the influence of the orientation of T- and B-cell epitopes on the immunogenicity of chimeric synthetic peptides in terms of the ability of the T-cell epitope to provide help for the production of antibody to the B-cell epitope. A T-cell epitope from the fusion protein of measles virus (288-302), previously shown to act as a T-helper epitope in a panel of six inbred mouse strains, was co-linearly synthesized at either the amino- or carboxyl- terminus of a B-cell epitope from the haemagglutinin of the virus (188-199) with or without the inclusion of a glycine-glycine spacer. The four chimeric peptides were used to immunize a panel of five mouse strains and induced good anti-chimera antibody responses. In general, the chimeras in which the T-cell epitope was amino-terminal to the B-cell epitope induced antibodies which bound well to the B-cell epitope whereas the carboxyl-terminal orientation of the T-cell epitope with respect to the B-cell epitope failed to induce such antibody. These latter chimeras induced the production of antibodies which preferentially bound to the T-cell epitope. The inclusion of the gly-gly spacer in the chimeras did not enhance their immunogenicity nor did it increase antibody titres to the B-cell epitope. The affinity of the anti-peptide antibodies was markedly influenced by the orientation of the epitopes in the chimeras. The antibody elicited by the peptide in which the T-cell epitope was amino terminal to the B-cell epitope had significantly higher affinity for the B-cell epitope than that induced by immunization with the peptide in the reverse orientation.

Biodegradable microparticles as a delivery system for measles virus cytotoxic T cell epitopes

Cytotoxic T-cell (CTL) responses are likely to be important for the clearance of a measles virus (MV) infection. To induce CTL responses. replicating vectors have generally been used but the use of such vectors in humans mav be problematic, and immunization with synthetic peptides may be more appropriate. We have investigated the potential of poly(lactide-co-glycolide)(PLG) microparticles as a delivery system for a CTL epitope representing residues 51-59 from MV nucleoprotein. After a single intraperitoneal injection in saline of the encapsulated epitope, CTL responses to the homologous peptide and MV were detected over a period of 4 months. Responses reached a maximum 30 days after priming and were maintained at high levels for 120 days. These responses were higher than those observed when the CTL epitope was administered in saline or as an emulsion in Incomplete Freunds Adjuvant. The pronounced immunostimulatory effect of microparticles, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic peptide immunogens.

INDUCTION OF MEASLES VIRUS-SPECIFIC CYTOTOXIC T-CELL RESPONSES AFTER INTRANASAL IMMUNIZATION WITH SYNTHETIC PEPTIDES

We have investigated the structural requirements for the induction of cytotoxic T‐cell responses (CTL) in vivo after intranasal immunization with an immunodominant CTL epitope from the nucleoprotein of measles virus (MV). For the induction of CTL responses, covalent linkage of the CTL epitope to a helper T‐cell epitope was required and the orientation of the epitopes influenced the immunogenicity of the CTL epitope. The presence of two copies as compared with one copy of a T‐helper epitope, rendered the CTL epitope more immunogenic and resulted in the in vivo induction of MV‐specific CTLs without the need for an adjuvant. The role of CTL responses to this epitope in protection after intranasal administration was evaluated in a mouse model against challenge with a neuroadapted strain of MV. Although a decreased mortality in the peptide immunized compared with that in unimmunized mice was observed, the protection achieved was not significant. These findings highlight the importance of the rational design of synthetic immunogens for the induction of CTL responses and the potential of the intranasal route for immunization.

Specificity of antibodies reactive with hepatitis B surface antigen following immunization with synthetic peptides

The amino acid sequence 139-147 from hepatitis B surface antigen (HBsAg) has previously been shown to represent a B-cell epitope with potential as a component of a synthetic peptide vaccine against hepatitis B. In this paper, two regions of HBsAg which act as T-cell epitopes in inbred mice have been identified (residues 23-34 and residues 160-171). The ability of synthetic peptides representing these epitopes to provide help for the production of antibody against the 139-147 epitope has been assessed following their co-linear synthesis with the B-cell epitope and following co-immunization of the peptides in an uncoupled form. Both these strategies result in the induction of anti-peptide antibodies which specifically react with recombinant HBsAg. The results presented give further support to the concept that synthetic peptides representing appropriately chosen B- and T-cell epitopes from HBsAg could form the basis of a synthetic vaccine against hepatitis B.

Induction of systemic immune responses to measles virus synthetic peptides administered intranasally

A systemic antibody response was induced when a chimeric peptide containing two copies of a promiscuous T-cell epitope and one copy of a B-cell epitope (TTB) from the fusion protein of measles virus (MV) was administered to mice intranasally without adjuvant. A higher antibody titre was produced when the peptide was administered intranasally with cholera toxin B subunit (CTB) as an adjuvant and these antibodies crossreacted with the MV. Furthermore, splenocytes from intranasally immunized mice proliferated in vitro in the presence of the TTB peptide. The immune response following intranasal immunization with the peptide was influenced by the MHC haplotype of the strain of mice used. Thus CBA and BALB/c mice were high responders whereas C57BL/6 mice were low responders. Although peptide administered intranasally with CTB to CBA mice induced an immune response, no significant protection was observed against intra-cranial challenge with canine distemper virus which is antigenically related to MV.

Identification of helper T cell antigenic sites in mice from the haemagglutinin glycoprotein of measles virus

The aim of this study was to define the helper T cell epitopes on the haemagglutinin (H) of measles virus (MV) in BALB/c (H-2d) and TO (H-2s) mice. A panel of 55 synthetic peptides (15-mers, overlapping by five amino acids) representing 92.2% of the H protein were synthesized and tested for immunogenicity and ability to stimulate MV-primed lymphocytes in vitro. The results obtained show that mouse lymphocytes respond to defined regions of the H protein which differ according to mouse strain. Virus-primed lymphocytes from BALB/c mice responded in vitro to peptides 7, 38, 39 and 44 whereas lymphocytes from virus-primed TO mice responded only to peptide 39. When mice of both strains were immunized with the peptides, a number of peptides induced proliferative responses, showing that the T cell repertoire for epitopes on the H protein is broader than that following immunization with virus. In BALB/c mice, lymphocytes primed to peptides 37, 39, 40, 42 and 43 responded in vitro to MV and in TO mice, lymphocytes primed to peptides 14, 32, 39, 40 and 49 responded to the virus. Thus in both strains of mice peptide 39 behaved as a dominant T cell epitope following immunization with virus or peptides. When the results obtained experimentally were compared with sequences predicted to be T cell epitopes by a number of algorithms, the concordance was limited.

Mucosal immunization with a measles virus CTL epitope encapsulated in biodegradable PLG microparticles

The immunogenicity of a cytotoxic T cell epitope (CTL) representing residues 52-60 from measles virus (MV) nucleoprotein, encapsulated in poly(lactide-co-glycolide) (PLG) microparticles was evaluated after mucosal immunization. After intranasal administration of the encapsulated CTL epitope linked at the carboxyl terminus of two copies of a T-helper epitope (TT-NP6), peptide-specific and MV-specific CTL responses were detected in splenocytes. However, these responses were lower than the responses observed when the TT-NP6 peptide was administered intranasally in saline or using CTB as an adjuvant. Intranasal coadministration of the encapsulated TT-NP6 peptide with CTB did not result in any significant potentiation of the CTL responses. The effectiveness of biodegradable PLG microparticles for mucosal delivery of CTL epitopes, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic immunogens. However, further work is needed to define the requirements for effective absorption by the nasal epithelium.

Fine specificity of the antibody response to a synthetic peptide from the fusion protein and protection against measles virus-induced encephalitis in a mouse model.

A synthetic peptide representing residues 397-420 from the measles virus (MV) fusion (F) protein was tested for its structure, immunogenicity and protective capacity against intracerebral challenge with a neuroadapted strain of MV. Analysis of the peptide by mass spectrometry showed that it was linear, despite the presence of two cysteine residues in the sequence. Circular dichroism spectroscopy highlighted a weak preference for the peptide to adopt an alpha-helical conformation. The peptide was shown to be immunogenic in BALB/c and C57BL/6 mice after intraperitoneal immunization in Freunds adjuvant, and anti-peptide antibodies from both strains of mice reacted with the MV as a solid phase antigen on an ELISA plate. When the fine specificity of the anti-peptide antibody response was examined using overlapping 8-mer peptides, serum antibodies from BALB/c mice recognized the region between residues 407-417 whereas antibodies from C57BL/6 mice recognized the region 408-420 of the 397-420 peptide sequence. Although anti-397-420 antibodies had no demonstrable neutralizing activity, protection against challenge with a neuroadapted strain of MV was demonstrated following active immunization with peptide in C57BL/6 mice or after passive transfer of anti-peptide antibodies in BALB/c mice. These findings highlight the importance of the 397-420 region in the induction of protective antibodies in the MV encephalitis mouse model, and suggest that this epitope might be a good candidate for inclusion in a future MV synthetic peptide vaccine.