Successful efforts to control infectious diseases have often required the use of effective vaccines. The current global strategy for control of malaria, including elimination and eradication will also benefit from the development of an effective vaccine that interrupts malaria transmission. To this end, a vaccine that disrupts malaria transmission within the mosquito host has been investigated for several decades targeting a 25 kDa ookinete specific surface protein, identified as Pfs25. Phase 1 human trial results using a recombinant Pfs25H/Montanide ISA51 formulation demonstrated that human Pfs25 specific antibodies block parasite infectivity to mosquitoes; however, the extent of blocking was likely insufficient for an effective transmission blocking vaccine. To overcome the poor immunogenicity, processes to produce and characterize recombinant Pfs25H conjugated to a detoxified form of Pseudomonas aeruginosa exoprotein A (EPA) have been developed and used to manufacture a cGMP pilot lot for use in human clinical trials. The Pfs25-EPA conjugate appears as a nanoparticle with an average molar mass in solution of approximately 600 kDa by static light scattering with an average diameter 20 nm (range 10 to 40 nm) by dynamic light scattering. The molar ratio of Pfs25H to EPA is about 3 to 1 by amino acid analysis, respectively. Outbred mice immunized with the Pfs25-EPA conjugated nanoparticle formulated on Alhydrogel® had a 75 to 110 fold increase in Pfs25H specific antibodies when compared to an unconjugated Pfs25H/Alhydrogel® formulation. A phase 1 human trial using the Pfs25-EPA/Alhydrogel® formulation is ongoing in the United States.

Both snail and parasite genes determine the susceptibility of the snail Biomphalaria glabrata to infection with the trematode Schistosoma mansoni. To identify molecular markers associated with resistance to the parasite in the snail host, we performed genetic crosses between parasite-resistant and -susceptible isogenic snails. Because resistance to infection in adult snails is controlled by a single locus, DNA samples from individual F 2 and F 1 backcross progeny, segregating for either the resistant or susceptible phenotypes, were pooled (bulked segregant). Genotypes for both parents were determined with 205 arbitrary decamer primers by random amplified polymorphic DNA-PCR. Of the 205 primers, 144 were informative, and the relative allele frequencies between the pools for these primers were determined. Two primers, OPM-04 and OPZ-11, produced fragments in the resistant parent of one cross that were inherited in a dominant fashion in the resistant F 2 and backcross-bulked segregant progeny. Subsequent typing of DNA samples of individual progeny snails showed that the 1.2-kb marker amplified by primer OPM-04 and the 1.0-kb marker produced by primer OPZ-11 segregated in the same dominant fashion with the resistant phenotype. Sequence analysis of the 1.2-kb marker showed that it corresponds to a repetitive sequence in the snail genome with no homology to existing DNA sequences in the public databases. Analysis of the 1.0-kb marker showed that it also corresponds to a repetitive sequence in the B. glabrata genome that contains an imperfect ORF, with homology to retrovirus-related group-specific antigens (gag) polyprotein.

Chemical conjugation of polysaccharide to carrier proteins has been a successful strategy to generate potent vaccines against bacterial pathogens. We developed a similar approach for poorly immunogenic malaria protein antigens. Our lead candidates in clinical trials are the malaria transmission blocking vaccine antigens, Pfs25 and Pfs230D1, individually conjugated to the carrier protein Exoprotein A (EPA) through thioether chemistry. These conjugates form nanoparticles that show enhanced immunogenicity compared to unconjugated antigens. In this study, we examined the broad applicability of this technology as a vaccine development platform, by comparing the immunogenicity of conjugates prepared by four different chemistries using different malaria antigens (PfCSP, Pfs25 and Pfs230D1), and carriers such as EPA, TT and CRM197. Several conjugates were synthesized using thioether, amide, ADH and glutaraldehyde chemistries, characterized for average molecular weight and molecular weight distribution, and evaluated in mice for humoral immunogenicity. Conjugates made with the different chemistries, or with different carriers, showed no significant difference in immunogenicity towards the conjugated antigens. Since particle size can influence immunogenicity, we tested conjugates with different average size in the range of 16–73 nm diameter, and observed greater immunogenicity of smaller particles, with significant differences between 16 and 73 nm particles. These results demonstrate the multiple options with respect to carriers and chemistries that are available for protein-protein conjugate vaccine development.

Malaria transmission blocking vaccines (TBV) target the mosquito stage of parasite development by passive immunization of mosquitoes feeding on a vaccinated human. Through uptake of vaccine-induced antibodies in a blood meal, mosquito infection is halted and hence transmission to another human host is blocked. Pfs230 is a gametocyte and gamete surface antigen currently under clinical evaluation as a TBV candidate. We have previously shown that chemical conjugation of poorly immunogenic TBV antigens to Exoprotein A (EPA) can enhance their immunogenicity. Here, we assessed Outer Membrane Protein Complex (OMPC), a membrane vesicle derived from Neisseria meningitidis , as a carrier for Pfs230. We prepared Pfs230-OMPC conjugates with varying levels of antigen load and examined immunogenicity in mice. Chemical conjugation of Pfs230 to OMPC enhanced immunogenicity and functional activity of the Pfs230 antigen, and OMPC conjugates achieved 2-fold to 20-fold higher antibody titers than Pfs230-EPA/AdjuPhos ® at different doses. OMPC conjugates were highly immunogenic even at low doses, indicating a dose-sparing effect. EPA conjugates induced an IgG subclass profile biased towards a Th2 response, whereas OMPC conjugates induced a strong Th1-biased immune response with high levels of IgG2, which can benefit Pfs230 antibody functional activity, which depends on complement activation. OMPC is a promising carrier for Pfs230 vaccines.

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Infectious pancreatic necrosis (IPN) virus, the etiologic agent of infectious pancreatic necrosis in salmonid fish, causes significant losses to the aquaculture industry. The gene for the viral capsid protein (VP2) was cloned into a yeast expression vector and expressed in Saccharomyces cerevisae. Expression of the capsid gene in yeast resulted in formation of approximately 20nm subviral particles composed solely of VP2 protein. Anti-IPNV antibodies were detected in rainbow trout vaccinated either by injection of purified VP2-subviral particles (rVP2-SVP) or by feeding recombinant yeast expressing rVP2-SVP. Challenge of rVP2-SVP immunized trout with a heterologous IPNV strain and subsequent viral load determination demonstrated that both injection and orally vaccinated fish had lower IPNV loads than naive or sham-vaccinated fish. This study demonstrates the ability of rVP2-SVPs to induce a specific immune response and the ability of immunized fish to reduce the viral load after an experimentally induced IPNV infection.

A practical method is described for synthesizing conjugated protein nanoparticles using thioether (thiol-maleimide) cross-linking chemistry. This method fills the need for a reliable and reproducible synthesis of protein conjugate vaccines for preclinical studies, which can be adapted to produce comparable material for clinical studies. The described method appears to be generally applicable to the production of nanoparticles from a variety of soluble proteins having different structural features. Examples presented include single-component particles of the malarial antigens AMA1, CSP and Pfs25, and two component particles comprised of those antigens covalently cross-linked with the immunogenic carrier protein EPA (a detoxified form of exotoxin A from Pseudomonas aeruginosa). The average molar masses (Mw) of particles in the different preparations ranged from 487 kDa to 3,420 kDa, with hydrodynamic radii (Rh) ranging from 12.1 nm to 38.3 nm. The antigenic properties and secondary structures of the proteins within the particles appear to be largely intact, with no significant changes seen in their far UV circular dichroism spectra, or in their ability to bind conformation-dependent monoclonal antibodies. Mice vaccinated with mixed particles of Pfs25 or CSP and EPA generated significantly greater antigen-specific antibody levels compared with mice vaccinated with the respective unmodified monomeric antigens, validating the potential of antigen-EPA nanoparticles as vaccines.

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