David E. Lanar

A Nonadjuvanted Polypeptide Nanoparticle Vaccine Confers Long-Lasting Protection against Rodent Malaria

We have designed and produced a prototypic malaria vaccine based on a highly versatile self-assembling polypeptide nanoparticle (SAPN) platform that can repetitively display antigenic epitopes. We used this platform to display a tandem repeat of the B cell immunodominant repeat epitope (DPPPPNPN)2D of the malaria parasite Plasmodium berghei circumsporozoite protein. Administered in saline, without the need for a heterologous adjuvant, the SAPN construct P4c-Mal conferred a long-lived, protective immune response to mice with a broad range of genetically distinct immune backgrounds including the H-2b, H-2d, and H-2k alleles. Immunized mice produced a CD4+ T cell-dependent, high-titer, long-lasting, high-avidity Ab response against the B cell epitope. Mice were protected against an initial challenge of parasites up to 6 mo after the last immunization or for up to 15 mo against a second challenge after an initial challenge of parasites had successfully been cleared. Furthermore, we demonstrate that the SAPN platform not only functions to deliver an ordered repetitive array of B cell peptide epitopes but operates as a classical immunological carrier to provide cognate help to the P4c-Mal-specific B cells.

Process development and analysis of liver-stage antigen 1, a preerythrocyte-stage protein-based vaccine for Plasmodium falciparum

ABSTRACT Plasmodium falciparum liver-stage antigen 1 (LSA-1) is expressed solely in infected hepatocytes and is thought to have a role in liver schizogony and merozoite release. Specific humoral, cellular, and cytokine immune responses to LSA-1 are well documented, with epitopes identified that correlate with antibody production, proliferative T-cell responses, or cytokine induction. With the goal of developing a vaccine against this preerythrocyte-stage protein, we undertook the good manufacturing practices (GMP) manufacture of a recombinant LSA-1 construct, LSA-NRC, incorporating the N- and C-terminal regions of the protein and two of the centrally placed 17-amino-acid repeats. To improve the protein yield, a method of codon harmonization was employed to reengineer the gene sequence for expression in Escherichia coli. A 300-liter GMP fermentation produced 8 kg of bacterial cell paste, and a three-step column chromatographic method yielded 8 mg of purified antigen per g of paste. The final bulk protein was >98% pure, demonstrated long-term stability, and contained <0.005 endotoxin units per 50 μg of protein. To accomplish the initial stages of evaluation of this protein as a human-use vaccine against malaria, we immunized rabbits and mice with LSA-NRC in Montanide ISA 720. New Zealand White rabbits and A/J (H-2K) mice produced high-titer antibodies that recognized liver-stage parasites in infected cultured human hepatocytes. Gamma interferon-producing cells, which have been associated with LSA-1-mediated protection, were detected in splenocytes harvested from immunized mice. Finally, sera taken from people living in a region where malaria is holoendemic recognized LSA-NRC by Western blotting.

Murine Immune Responses to Liver-Stage Antigen 1 Protein FMP011, a Malaria Vaccine Candidate, Delivered with Adjuvant AS01B or AS02A

ABSTRACT Liver-stage antigen 1 (LSA1) is expressed by Plasmodium falciparum only during the intrahepatic cell stage of the parasites development. Immunoepidemiological studies in regions where malaria is endemic suggested an association between the level of LSA1-specific humoral and cell-mediated immune responses and susceptibility to clinical malaria. A recombinant LSA1 protein, FMP011, has been manufactured as a preerythrocytic vaccine to induce an immune response that would have the effect of controlling parasitemia and disease in humans. To evaluate the immunogenicity of FMP011, we analyzed the immune response of three inbred strains of mice to antigen immunization using two different adjuvant formulations, AS01B and AS02A. We report here the ability of BALB/c and A/J mice, but not C57BL/6J mice, to mount FMP011-specific humoral (antibody titer) and cellular (gamma interferon [IFN-γ] production) responses following immunization with FMP011 formulated in AS01B or AS02A. Immunization of BALB/c and A/J mice with FMP011/AS01B induced more antigen-specific IFN-γ-producing splenocytes than immunization with FMP011/AS02A. A slightly higher titer of antibody was induced using AS02A than AS01B in both strains. C57BL/6J mice did not respond with any detectable FMP011-specific IFN-γ splenocytes or antibody when immunized with FMP011 in AS01B or AS02A. Intracellular staining of cells isolated from FMP011/AS01B-immunized BALB/c mice indicated that CD4+ cells, but not CD8+ cells, were the main IFN-γ-producing splenocyte. However, inclusion of blocking anti-CD4+ antibody during the in vitro restimulation ELISpot analysis failed to completely abolish IFN-γ production, indicating that while CD4+ T cells were the major source of IFN-γ, other cell types also were involved.