Michael J. Francis

Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein

Synthetic vaccines for viral diseases can use defined regions of viral proteins as immunogens1,2: the peptide sequence of amino acids 141–160 of the VP1 protein of foot and mouth disease virus (FMDV) elicits virus-neutralizing antibodies to protect guinea pigs, cattle and pigs either when coupled to a carrier protein or when administered in liposomes or in incomplete Freunds adjuvant3–5. The immune response to these peptides is much lower than that to complete virus particles1 and the same sequence fused to the N terminus of β-galactosidase did not produce a more potent immunogen than synthetic peptide alone6. We report here an expression system for immunogenic epitopes linked to a carrier protein, hepatitis B core antigen, to form part of a virus-like complex which can present these epitopes to the immune system at high density. The immunogenicity of these structures approaches that of FMDV particles.

Foreign epitopes in immunodominant regions of hepatitis B core particles are highly immunogenic and conformationally restricted

The presentation of heterologous amino acid sequences on the surface of hepatitis B core antigen (HBcAg) particles has been studied using a defined linear neutralization site from human rhinovirus (HRV). Previous work has shown that fusion particles, in which the HRV peptide sequence is linked to the amino terminus of the HBcAg protein, induce excellent immune responses in experimental animals. Using predictive models of HBcAg particulate structure and the approximate location of the major immunogenic regions we have designed and constructed bacterial expression vectors which direct synthesis of chimeric particles in which heterologous sequences are presented within an immunodominant area on the particle. Immunological responses to the heterologous peptide sequence are improved by at least tenfold when compared with amino terminal fusions of the same peptide sequence to HBcAg. Moreover, the restriction placed on the heterologous peptide by its linkage at both ends within the HBcAg protein results in a more constrained structure. In the case of the rhinovirus peptide sequence this results in an antigenic conformation more closely resembling that on the native virus particle. Such a system lends itself well as a general approach to the induction of high titre antibodies against defined epitopes.

Presentation and immunogenicity of viral epitopes on the surface of hybrid hepatitis B virus core particles produced in bacteria

We recently reported the enhanced immunogenicity of a peptide epitope when it was presented as a fusion protein with hepatitis B core antigen. In those experiments the fusion protein was expressed in vaccinia virus. We have now refined the system so that large amounts of highly immunogenic particles can be produced using a simple bacterial expression system. We describe the expression of three different viral epitopes as chimeric particles that induce good antibody responses to each epitope after one dose of low amounts of antigen. Finally we demonstrate that the immunogenicity is a reflection of both T helper cell sites within the core protein and also the particulate nature of the immunogens.

Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy

Rheumatoid arthritis (RA) is a chronic, inflammatory synovitis whose pathogenesis may involve autoimmune mechanisms. Anergy is a state of T-cell nonresponsiveness characterized by downregulated IL-2 production. Paradoxically, RA T cells are hyporesponsive and proliferate poorly to antigens and mitogens, thus sharing some characteristics with anergic T cells. We analyzed the molecular basis of anergy in cloned human CD4+ T cells using differential display RT-PCR and subsequently examined the levels of differentially expressed transcripts in RA and, as control, reactive arthritis (ReA) synovium. Several transcriptional events were common to anergic T cells and RA synovium. These included downregulation of CALMODULIN:, which is critical to T-cell activation, and of cellular apoptosis susceptibility protein, which may mediate resistance to apoptosis in RA. Transcription of CALMODULIN: in RA synovium was less than 1% of that in ReA and was lower in RA synovial fluid mononuclear cells than in paired PBMCs. Following anti-TNF-alpha therapy in vivo, RA PBMC CALMODULIN: transcripts increased five- to tenfold. Pharmacological calmodulin blockade in vitro impaired antigen-specific proliferation. These data provide a link between reduced CALMODULIN: transcription and impaired T-cell responsiveness in RA. The identification of transcriptional changes common to anergic and RA synovial T cells should help interpret some of the characteristic RA cellular defects.

A Synthetic Peptide Which Elicits Neutralizing Antibody against Human Rhinovirus Type 2

Synthetic peptides corresponding to six predicted immunogenic sites on human rhinovirus type 2 (HRV2) have been tested for their reactivity with an anti-virion antibody and for their ability to elicit neutralizing antibody. Four of the peptides reacted with HRV2 antiserum in an indirect ELISA. Rabbit antisera produced to three of these four peptides, one each from VP1, VP2 and VP3, reacted with the virus in an indirect ELISA and with the corresponding proteins by Western blotting. Furthermore, antiserum to one of the peptides, designed to cover the neutralization epitope NIm-II on VP2, not only reacted well in a sandwich ELISA and in an immunoprecipitation test but also neutralized virus infectivity.

Removal of the cleavage site of recombinant feline immunodeficiency virus envelope protein facilitates incorporation of the surface glycoprotein in immune-stimulating complexes

Recombinant vaccinia viruses were constructed that expressed the complete env gene of feline immunodeficiency virus with or without the nucleotide sequence encoding the cleavage site between the surface (SU) protein and the transmembrane (TM) protein. The removal of this cleavage site resulted in the expression of a 150K protein that was processed into a 130K protein and was not cleaved into the SU and the TM proteins. Removal of the cleavage site also facilitated incorporation of the SU protein in immune-stimulating complexes (iscoms). Antibody responses to both an SU and a TM peptide representing two immunodominant B cell epitopes were measured. These were higher in cats immunized with iscoms prepared from the cleavage site-deleted envelope protein than in cats immunized with iscoms prepared from the native envelope protein or immunized with the envelope protein and the adjuvant Quil A.

Feline immunodeficiency virus subunit vaccines that induce virus neutralising antibodies but no protection against challenge infection

Three experimental vaccines against feline immunodeficiency virus (FIV), all based on viral antigens presented via immune stimulating complexes (iscoms), were tested for their capacity to induce protection in cats from FIV infection. The respective vaccines consisted of FIV propagated in Crandell feline kidney (CrFK) cells (FIV-iscoms); FIV-iscoms spiked with recombinant vaccinia virus expressed FIV envelope glycoprotein incorporated into iscoms (FIV-iscoms + vGR657x15-iscoms) and vGR657x15-iscoms spiked with recombinant FIV Gag protein incorporated into iscoms (vGR657x15-iscoms + FIV-Gag-iscoms). Simian immunodeficiency virus envelope glycoprotein incorporated into iscoms, iscoms prepared with uninfected CrFK cells, and PBS served as controls. All cats vaccinated with vGR657x15-iscoms combined with FIV-iscoms or FIV-Gag-iscoms developed Env-specific plasma antibody responses. These antibodies neutralised FIV infection in CrFK cells, but failed to neutralise FIV infection in primary feline thymocytes. FIV-iscoms induced poor Env-specific responses and only one out of six cats developed antibodies that neutralised FIV in the CrFK cell based assay. Four weeks after challenge all cats proved to be infected, showing that none of the vaccine preparations provided protection. In contrast, 2 weeks after infection, virus infected peripheral blood mononuclear cells were only observed in cats vaccinated with FIV-iscoms + vGR657x15-iscoms or CrFK-iscoms and to a lesser extent in cats vaccinated with FIV-iscoms and vGR657x15-iscoms + FIV-Gag-iscoms, but not in cats vaccinated with SIV-iscoms or PBS. The differences found in cell associated virus loads amongst the respective groups are discussed in the light of antibody mediated enhancement of infectivity and protective effects provided by Gag-specific T cell responses.

Qualitative and quantitative differences in the immune response to foot-and-mouth disease virus antigens and synthetic peptides.

In cross-immunization studies using foot-and-mouth disease virus (FMDV) antigens and a synthetic peptide, from a region within virus coat protein VP1, it has been shown that intact virus will prime the immune system for intact virus, virus subunits and synthetic peptide but not for disrupted virus. In contrast, peptide will prime for a response to peptide and virus subunits but not to intact virus or disrupted virus. Furthermore, studies on antibody populations in anti-virus and anti-peptide antisera demonstrated clear differences in the nature of the antibody response to the two antigens. This result is reflected in protection studies carried out on animals immunized with virus particles or peptides where there is a clearer correlation between in vitro neutralization and protection in vivo following peptide immunization. Thus, it has been shown that there are major qualitative and quantitative differences in the immune response to the FMDV particle and synthetic peptide.

Proliferative Responses of T cells Primed Against Human Rhinovirus to other Rhinovirus Serotypes

Lymphocytes from mice immunized with human rhinovirus (HRV) serotypes 1A or 15 proliferated in vitro in response to HRV and the activated cells were shown to be helper T (Th) cells. Lymphocytes from mice primed with HRV-1A responded to seven of eight heterologous virus serotypes, the responses to other minor cell receptor group viruses being greater than to those belonging to the major cell receptor group. A similar bias was seen with cells from mice primed with HRV-15 in that they responded preferentially to other major receptor group viruses. This pattern of cross-serotype recognition was shown to be similar in three inbred mouse strains and was not dependent upon the major histocompatibility complex haplotype. These results have revealed that there are determinants within the viral proteins of a number of serotypes of HRV that are recognized by Th cells primed against a single HRV serotype. Thus, at the level of Th cell recognition of HRV, a cross-serotype reactivity is seen which is not reflected in the B cell antibody response to virus, which is generally highly serotype-specific.