Malaria Vaccines: TBV Antigens as Conjugates with Alternate Carriers

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

In FY19, LMIV scientists contributed to 2 publications on conjugate or particle vaccines, and we describe progress reported in those manuscripts here: 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 (Scaria PV, et al. npjVaccines). 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. Improvements in dimensional metrology and innovations in physical-chemical characterization of functionalized nanoparticles are critically important for the realization of enhanced performance and benefits of nanomaterials (Farkas N, Scaria PV, et al. Scientific Reports). Toward this goal, we propose a multi-technique measurement approach, in which correlated atomic force microscopy, dynamic light scattering, high performance liquid chromatography and mass spectroscopy measurements are used to assess molecular and structural properties of self-assembled polyplex nanoparticles with a core-shell structure. In this approach, measurement methods are first validated with a model system consisting of gold nanoparticles functionalized with synthetic polycationic branched polyethylenimine macromolecules. Shell thickness is measured by atomic force microscopy and dynamic light scattering, and the polyelectrolyte uptake determined by chromatographic separation and mass spectrometric analysis. Statistical correlation between size, structure and stability provide a basis for extending the methods to more complex self-assembly of nucleic acids and macromolecules via a condensation reaction. From these size and analytical chemical measurements, we obtain a comprehensive spatial description of these assemblies, obtain a detailed interpretation of the core-shell evolution, and identify regions of the parameter space where stable, discrete particle formation occurs. In unpublished work, we report below our progress on several ongoing projects: Further evaluation of OMPC as a delivery platform for Transmission Blocking Vaccine antigens: In FY2019, we continued the evaluation of OMPC as a delivery platform for TBV antigens. As noted above in our publication, mouse studies determined that OMPC conjugates provided superior immunogenic properties compared to control EPA conjugates of the same antigens. A qualitatively different, Th1-biased immune response was observed for OMPC conjugates as opposed to a Th2 response of EPA conjugates. Based on these findings, we have initiated evaluation of OMPC conjugates in nonhuman primates to determine their efficacy in this model and to evaluate the duration of immune response. Findings from the mouse immunogenicity studies were published during FY2019. Studies in nonhuman primates will evaluate the level of antibody response and different time points, up to a period of one year, assaying serum samples at different times points for their antibody titer and functional activity by Standard Membrane Feed assay. Evaluation of Alternate protein carriers for TBV antigens: We are currently evaluating alternate carriers and adjuvants. For this effort, we procured carriers from different commercial entities through collaborative agreements. In FY2019, we continued our collaboration with Fina Biosolutions to obtain EcoCRM (E. Coli produced CRM197) and TTHc (Tetanus Toxin heavy chain) for conjugation with Pfs230. We synthesized a series of conjugates of Pfs230 with EcoCRM, TTHC, CRM197 (from Pfenex) and TT (from SSI). These conjugates were tested in mouse immunogenicity studies and were found to generate strong immune responses. Based on these findings, additional collaborations were established with Scarab Genomics and Biofarma (Indonesia) to obtain clinically viable versions of CRM197 and TT respectively. These materials were obtained and conjugates were synthesized using these carriers; mouse studies to test these are ongoing, and preliminary results were presented at the ASTMH annual meeting in Oct 2018. Our work in this area has been accelerated through a partnership with MVI/PATH who is supporting preclinical conjugation/immunization studies of Pfs230D1 and Pfs230C1. Evaluation of mRNA technology for malaria antigens: In FY2019, we continued the collaboration with CureVac, Germany to test the immunogenicity of LMIVs malaria antigens in CureVacs RNActive technology platform. Antigen delivery using mRNA 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. In FY2019, CureVac generated a series of mRNA constructs for LMIVs TBV and pregnancy malaria antigens and tested their expression in mammalian cells. As part of this continuing collaboration, mouse immunogenicity studies will be performed at LMIV to test the immunogenicity and functional activity of these mRNA constructs. Needle-free vaccine delivery: In FY2019, we continued the collaboration established 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 microneedle delivery. In FY2019, we evaluated the compatibility of our conjugate antigens in polymer mix used for microneedle fabrication and found them compatible. We also established assays to quantify the antigen content in microneedle patches. This allows Takeda to fine tune microneedle fabrication. Based on these studies, Takeda generated a series of microneedle patches for animal studies. These patches are being evaluated in mouse immunogenicity studies at LMIV. Conjugates of Alternate TBV antigens - Pfs47: In FY2019 as part of the established intramural collaboration with Carolina Barillas group (NIAID) to test an alternate TBV antigen, Pfs47, we synthesized the EPA conjugate of Pfs47. This conjugate was evaluated in mouse immunogenicity studies. These studies indicated that chemical modification may alter the epitope important for functional activity. Based on these studies, other synthetic options are currently being pursued.