Specificity of Humoral and Cellular Immune Response against Recombinant Particles of Nucleocapsid Protein of Human Hepatitis B Virus in Rabbits

M. G. Isaguliants^1,2*, Yu. P. Kadoshnikov¹, T. I. Kalinina¹, V. D. Smirnov¹, and B. Wahren²

¹Ivanovsky Institute of Virology, ul. Gamalei 16, Moscow, 123098 Russia; fax: (095) 190-2867

²Department of Virology, Swedish Institute for Infectious Disease Control, S-10521, Stockholm, Sweden; fax: (46-8) 27-2231

^* To whom correspondence should be addressed.

Received September 5, 1997; Revision received November 20, 1997

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Nucleocapsid (core) protein of hepatitis B virus (HBcAg) induces potent cellular and humoral responses that have a clear protective potential. Rabbits were immunized by particles formed by recombinant molecules of HBcAg carrying N-terminally inserted heterologous sequences [1]. Specificity of humoral and cellular immune response against HBcAg and selection of HBcAg epitopes was surveyed. Immunological properties of the recombinant particles were similar to those of the original HBcAg. Recombinant particles were not toxic to the peripheral blood mononuclear cells (PBMC) of non-immune or HBcAg-immunized animals ex vivo. Proliferative response of PBMC (T-lymphocytes) to HBcAg in immunized animals increased in a concentration-dependent manner in the broad interval of HBcAg concentrations (10-10⁴ ng/ml). On the contrary, a narrow bell-shaped HBcAg dose-dependence curve was earlier observed for T-lymphocytes of donors immune to HBV after natural infection that was probably due to the cytotoxic effect of HBcAg on the expressing cells. Specificity of humoral and cellular immune response against HBcAg particles in the immunized animals and in natural infection with hepatitis B virus (HBV) was compared. Immunization with recombinant HBcAg particles induced potent anti-HBcAg antibody responses: high (up to 2·10⁷) titers of anti-HBcAg antibodies were reached. Appearance of anti-HBcAg antibodies was in every case preceded by an increasing T-cell response to the whole protein and HBcAg-derived peptides, thus mimicking immune responses during acute HBV infection in humans. A predominant universal (haplotype-independent) T-helper cell epitope (amino acid residues (aa) 61-85 of HBcAg (p61-85)) was recognized by T-cells of all animals. Transient antibody response against p61-85 was recorded during the early stages of immunization in spite of the fact that a major B-cell epitope localized in this region is supposed to be purely conformational. A sequence representing another cluster of immunodominant T-cell epitopes of mice and HBV infected humans, aa 121-140 (p121-140), was not immunogenic on the T-cell level. However, it appeared to be a potent B-cell immunogen, despite a common assumption that HBcAg and p121-140 are not cross-reactive at the B-cell level. A possibility that anti-p121-140 antibodies were induced by an exposed region of the native particulate HBcAg and not by the denatured protein molecules, was confirmed by recognition of the particulate HBcAg by antibodies specific to synthetic peptides representing aa 120-140 of HBcAg. The data point to the exposition of aa 121-140 on the surface of the particles.

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KEY WORDS: human hepatitis B virus, nucleocapsid (core) protein, recombinant particles, structure, immunogenicity, B-cell epitopes, T-cell epitopes

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