AVIRULENT SALMONELLA EXPRESSING HYBRID HEPATITIS-B VIRUS CORE/PRE-S GENES FOR ORAL VACCINATION

This report reviews and extends data on the use of hepatitis B virus (HBV) core (HBcAg) particles as a carrier moiety for B-cell epitopes of the HBV envelope proteins1-4. Virus-neutralizing epitopes of the HBV pre-S region were inserted at the N-terminus, the N-terminus through a precore linker sequence, the C-terminus and an internal position of HBcAg by genetic engineering in Escherichia coli. The hybrid HBc/pre-S proteins were purified and their antigenicity and immunogenicity analysed. All purified HBc/pre-S particles were particulate. Pre-S epitopes inserted at the N-terminus through a precore polylinker, the truncated C-terminus and at the internal position between HBcAg amino acids 75 and 81 were accessible on the particle surface. N-terminal fusions required the presence of the linker sequence to become surface accessible and immunogenic. Fusions to the N- and C-termini of HBcAg did not interfere with HBcAg antigenicity and immunogenicity. In contrast, insertion al the internal site abrogated recognition of HBcAg by five out of six monoclonal antibodies and diminished recognition by human polyclonal anti-HBc antibodies as well as HBcAg immunogenicity. A pre-S(2) sequence fused to the C-terminus of HBcAg was surface accessible and weakly immunogenic. Pre-S(1) sequences fused to the N-terminus through a precore linker were surface accessible and highly immunogenic. The same sequence fused to the core methionine was not surface accessible or immunogenic. Insertion of the same pre-S(1) sequence at an internal position of HBcAg resulted in the most efficient anti-pre-S(1) antibody response. Hybrid HBc/pre-S particles were also expressed in avirulent aroA or DELTAcya DELTAcrp Salmonella typhimurium and Salmonella dublin. Oral immunization of mice with Salmonella expressing C-terminally fused hybrid HBc/pre-S(2) particles resulted in high-titred serum anti-HBc antibodies and low-titred anti-pre-S(2) antibodies. Oral immunization with DELTAcya DELTAcrp S. typhimurium expressing internally fused HBc/pre-S hybrid elicited high titred serum anti-pre-S(1) antibodies.