Evaluation of OMPC as a delivery platform for Transmission Blocking Vaccine antigens We evaluated OMPC as a delivery platform for TBV antigens, synthesizing several conjugates of Pfs25 and Pfs230 by two different synthetic methods and with varying levels of antigen load, to investigate the effect of these parameters on the immunogenicity of the conjugates. Conjugates were evaluated in mouse immunogenicity studies and found to have high levels of antibody in response to the conjugated antigens. Level of antigen load did not have a significant effect on immunogenicity of the conjugates. Comparison of the Pfs25M-OMPC conjugate with corresponding EPA conjugate showed significantly higher antibody response at lower (0.1 g) doses and comparable antibody response at higher (0.5g) dose. Functional analysis of the immune sera by Standard Membrane Feed assay demonstrated high inhibition of oocyst formation in the infected mosquitos. Development of a Bivalent Conjugate Vaccine Candidate against Malaria Transmission and Typhoid Fever Malaria and typhoid fever are co-endemic in large parts of the world, particularly in tropical areas. We therefore explored the possibility of generating a bivalent vaccine against Plasmodium falciparum malaria and typhoid fever, which are co-endemic in many parts of the world, by conjugating Vi polysaccharide, an approved antigen in typhoid vaccine, to Pfs25, a malaria transmission blocking vaccine antigen in clinical trials. Vi-Pfs25 conjugates induced strong immune responses against both Vi and Pfs25 in mice, whereas the unconjugated antigens are poorly immunogenic. Functional assays of immune sera revealed potent transmission blocking activity mediated by anti-Pfs25 antibody and serum bactericidal activity due to anti-Vi antibody. Conjugation modified the IgG isotype distribution of antisera, inducing a Th2 polarized immune response against both antigens. This conjugate may be further developed as a bivalent vaccine to concurrently target malaria and typhoid fever. Protein-protein conjugate nanoparticles for malaria antigen delivery and enhanced mmunogenicity 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. 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 demonstrating the multiple options with respect to carriers and chemistries that are available for protein-protein conjugate vaccine development. 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. Additionally, in collaboration with FinaBio, and Scarab Biotechnologies, we are currently evaluating other carriers, Tetanus toxin heavy chain (TThc), and EcoCRM (a version of CRM197 expressed in E. Coli) as potential carriers. Current activities involve synthesizing conjugates of TThc and EcoCRM with TBV antigens and evaluating them in mouse immunogenicity studies to test their utility as carriers of TBV antigens and to examine how they compare with EPA. Evaluation of mRNA technology for malaria antigens: In FY2018, we established a collaboration with CureVac, Germany to test the immunogenicity of LMIVs malaria antigens in CureVacs RNActive technology platform. Antigen delivery using mRNA technology has generated considerable excitement in the vaccine field as a technology that can rapidly generate vaccine candidates for clinical testing. This technology is now being tested in a number of clinical trials by CureVac and Moderna Therapeutics; both have their proprietary technologies for designing and manufacturing potent mRNAs for vaccine. We are working with CureVac to construct mRNA for our TBV and PMV antigens. CureVac will generate the mRNA constructs and LMIV will evaluate their immunogenicity in mouse and NHP models. Needle-free vaccine delivery: In FY2018, we established a collaboration with Takeda Pharmaceuticals, Japan to evaluate their proprietary Microneedle Patch delivery technology for delivery of our conjugate immunogens for transmission blocking vaccine. Takedas dissolving microneedle is a technology for vaccine delivery that has a number of attractive features useful for malaria vaccines. Administration of Microneedle patches do not require a skilled medical professional or can be self-administered. It avoids needle use by eliminating accidental needle injuries and pain associated with needle delivery. It also does not require cold-chain transport and storage, thereby reducing the cost of mass immunization campaigns. We are currently working with Takeda to perform the first animal study involving the microneedle delivery. Takeda will generate microneedle patches with our antigens and we will evaluate them in mouse immunogenicity studies at LMIV. Conjugates of Alternate TBV antigens - Pfs48/45: In FY2018, we are continuing our collaboration with Radboud University to evaluate if conjugation to protein carriers can enhance the immunogenicity and functional activity of their TBV antigen, Pfs48/45. During FY 2018, we synthesized EPA conjugates of Pfs48/45 6-Cys domain and their mouse immunogenicity study is currently ongoing. Conjugates of Alternate TBV antigens - Pfs47: In FY2018 we initiated an intramural collaboration with Carolina Barillas group (NIAID) to test an alternate TBV antigen, Pfs47. This collaboration will evaluate if conjugation to a protein carrier can enhance the immunogenicity and functional activity of Pfs47. Pfs47 will be produced by Barillas group and we will conjugate it to EPA and evaluate its immunogenicity in animal models. If found efficacious, this could be tested in combination with our Pfs230 conjugate to determine if combination of two antigens will enhance TBV activity.

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An, So Jung; Scaria, Puthupparampil V; Chen, Beth et al. (2018) Development of a bivalent conjugate vaccine candidate against malaria transmission and typhoid fever. Vaccine 36:2978-2984
Radtke, Andrea J; Anderson, Charles F; Riteau, Nicolas et al. (2017) Adjuvant and carrier protein-dependent T-cell priming promotes a robust antibody response against the Plasmodium falciparum Pfs25 vaccine candidate. Sci Rep 7:40312
Scaria, Puthupparampil V; Chen, Beth; Rowe, Christopher G et al. (2017) Protein-protein conjugate nanoparticles for malaria antigen delivery and enhanced immunogenicity. PLoS One 12:e0190312
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