Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex.

The development of protein subunit vaccines to combat some of the world's deadliest pathogens such as a malaria parasite, Plasmodium falciparum, is stalled, due in part to the inability to induce and sustain high-titer antibody responses. Here, we show the induction of persistent, high-titer antibody responses to recombinant Pfs25H, a human malarial transmission-blocking protein vaccine candidate, after chemical conjugation to the outer-membrane protein complex (OMPC) of Neisseria meningitidis serogroup B and adsorption to aluminum hydroxyphosphate. In mice, the Pfs25H-OMPC conjugate vaccine was >1,000 times more potent in generating anti-Pfs25H ELISA reactivity than a similar 0.5-microg dose of Pfs25H alone in Montanide ISA720, a water-in-oil adjuvant. The immune enhancement requires covalent conjugation between Pfs25H and the OMPC, given that physically mixed Pfs25H and OMPC on aluminum hydroxyphosphate failed to induce greater activity than the nonconjugated Pfs25H on aluminum hydroxyphosphate. The conjugate vaccine Pfs25H-OMPC also was highly immunogenic in rabbits and rhesus monkeys. In rhesus monkeys, the antibody responses were sustained over 18 months, at which time another vaccination with nonconjugated Pfs25H induced strong anamnestic responses. The vaccine-induced anti-Pfs25-specific antibodies in all animal species blocked the transmission of parasites to mosquitoes. Protein antigen conjugation to OMPC or other protein carrier may have general application to a spectrum of protein subunit vaccines to increase immunogenicity without the need for potentially reactogenic adjuvants.

Pharmaceutical and immunological evaluation of human papillomavirus viruslike particle as an antigen carrier.

We report the preparation and the immunogenicity of a conjugate vaccine obtained by chemically conjugating a variant of the extracellular peptide fragment of influenza type A M2 protein to the human papillomavirus (HPV) viruslike particle (VLP). Conjugates comprised of approximately 4,000 copies of the antigenic peptide per VLP are obtained as the result of the reaction between a C-terminal cysteine residue on the peptide and the maleimide-activated HPV VLP. The resulting conjugates have an average particle size slightly larger than the carrier and present enhanced overall stability against chemical and thermal-induced denaturation. The M2-HPV VLP conjugates lost the binding affinity for anti-HPV conformational antibodies but retained reactivity to a M2-specific monoclonal antibody. The conjugate vaccine formulated with aluminum adjuvant and delivered in two doses of 30-ng peptide was found to be highly immunogenic and conferred good protection against lethal challenge of influenza virus in mice. These results suggest that HPV VLP can be used as a carrier for synthetic or small antigens for the development of subunit vaccines.

Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys.

A universal influenza virus vaccine that does not require frequent updates and/or annual immunizations will offer significant advantages over current seasonal flu vaccines. The highly conserved influenza virus A M2 membrane protein has been previously suggested as a potential antigen target for such a vaccine. Here, we report systematic evaluation of M2 peptide conjugate vaccines (synthetic peptides of M2 extracellular domain conjugated to keyhole limpet hemocyanin (KLH) or Neisseria meningitidis outer membrane protein complex (OMPC)) in mice, ferrets, and rhesus monkeys. The conjugate vaccines were highly immunogenic in all species tested and were able to confer both protection against lethal challenge of either H1N1 or H3N1 virus in mice and reduce viral shedding in the lower respiratory tracts of mice and ferrets. The protection against lethal challenge in mice could also be achieved by passive transfer of monkey sera containing high M2 antibody titers. In addition, we showed that M2 antisera were cross reactive with M2 peptides derived from a wide range of human influenza A strains, but they failed to react with M2 peptides of the pathogenic H5N1 virus (A/Hong Kong/97). The data presented here will permit better understanding of the potential of an M2-based vaccine approach.