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Objective: The recent success of a Plasmodium falciparum malaria vaccine consisting of circumsporozoite (CS) protein (CSP) T and B cell epitopes has rekindled interest in the development of a pre-erythrocytic vaccine. Our goal was to design an efficient delivery system for known neutralizing epitopes. Methods: Well-characterized CSP-specific neutralizing B cell epitopes and a ‘universal’ T cell epitope were combined with a particulate carrier platform, the hepatitis B core antigen (HBcAg), to produce a novel pre-erythrocytic vaccine candidate. Results: The vaccine candidate V12.PF3.1 is a potent immunogen in mice, eliciting unprecedented levels (greater than 106 titers) of sporozoite-binding antibodies after only two doses. The antisporozoite antibodies are long-lasting and represent all IgG isotypes, and antibody production is not genetically restricted. CSP-specific CD4+ T cells are also primed by V12.PF3.1 immunization in a majority of murine strains. Furthermore, the hybrid HBcAg-CS particles can be produced inexpensively in bacterial expression systems. Conclusion: These characteristics suggest that V12.PF3.1 represents an efficient and economical P. falciparum vaccine candidate for use separately or in combination with other formulations.

The hepatitis B virus (HBV) core gene codes for two partially colinear antigens: a secreted antigen (HBeAg) and the particulate core antigen (HBcAg), which assembles to form subviral particles and in virions contains the viral genome and polymerase. In this review we summarize data on the immune recognition of HBc/eA and recent progress in the use of HBcAg as a carrier moiety for heterologous epitopes. During HBV infection, HBcAg and HBeAg are important targets of antiviral immunity. HBcAg and HBeAg are serologically distinct but share all characterized T-cell epitopes. The particulate HBcAg can elicit T-cell-independent as well as T-cell-dependent antibody responses, HBeAg is a strictly T-cell-dependent antigen. Neonatal tolerance to maternally derived circulating HBeAg may facilitate chronic HBV infection after vertical transmission of HBV. In a murine transgenic model, HBc/eAg-specific Th1 cells were more readily anergized, whereas Th2 cells more easily escaped tolerization. In human HBV infection, acute adult HBV infection with subsequent virus elimination was characterized by Th1-like alpha-HBV serum IgG subtype distribution, whereas a Th2-like distribution of IgG subtypes was observed during chronic infection. During chronic infection, core gene mutants which abolish HBeAg synthesis were frequently observed. To exploit the unusual immunogenicity of particulate HBcAg as a vaccine carrier moiety, insertion sites for foreign epitopes were defined in recombinant expression systems. While fusion of epitopes to the N-terminus required a linker sequence for surface accessibility, both fusion to the N-terminus and to the C-terminus was compatible with particle assembly and preserved the native antigenicity and immunogenicity of HBcAg. Epitope insertion at an immunodominant internal site of HBcAg reduced the HBcAg immunogenicity and antigenicity and most drastically enhanced the immunogenicity of the inserted foreign epitopes. This internal site of HBcAg was used to express circumsporozoite antigen (CS) repeat epitopes of two rodent malaria parasites and of Plasmodium falciparum. Purified hybrid HBcAg-CS proteins were particulate and displayed CS antigenicity as well as reduced native HBc antigenicity. Immunization of several mouse strains with HBcAg-CS hybrid particles resulted in high-titered serum anti-CS antibodies representing all murine IgG isotypes and protected BALB/c mice against plasmodial challenge. Immunization of mice with HBcAg or HBcAg-CS particles formulated on alum, complete Freund's or incomplete Freund's adjuvant resulted in equivalent anti-CS and anti-HBc serum antibody titers. Preexisting immunity to HBcAg did not significantly alter the immunogenicity of hybrid HBcAg particles suggesting that carrier-specific immune suppression does not limit the use of hybrid HBcAg with internal insertions. Immunization with HBcAg-CS particles universally primed HBcAg-specific T cells and in addition CS-specific T cells were if the insert contained a CS-specific T-cell site for the corresponding murine MHC class II haplotype. The internal amino acid position in HBcAg is therefore permissive for the inclusion of heterologous T-helper as well as B-cell epitopes.