Parzych Elizabeth M, Miura Kazutoyo, Ramanathan Aarti, Long Carole A, Burns James M
Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
Malaria Immunology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.
Infect Immun. 2017 Dec 19;86(1). doi: 10.1128/IAI.00486-17. Print 2018 Jan.
Challenges with the production and suboptimal immunogenicity of malaria vaccine candidates have slowed the development of a multiantigen vaccine. Attempting to resolve these issues, we focused on the use of highly immunogenic merozoite surface protein 8 (MSP8) as a vaccine carrier protein. Previously, we showed that a genetic fusion of the C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP1) to MSP8 (MSP8) facilitated antigen production and folding and the induction of neutralizing antibodies to conformational B cell epitopes of MSP1 Here, using the MSP1/8 construct, we further optimized the recombinant MSP8 (rMSP8) carrier by the introduction of two cysteine-to-serine substitutions (CΔS) to improve the yield of the monomeric product. We then sought to test the broad applicability of this approach using the transmission-blocking vaccine candidate s25. The production of rs25-based vaccines has presented challenges. Antibodies directed against the four highly constrained epidermal growth factor (EGF)-like domains of s25 block sexual-stage development in mosquitoes. The sequence encoding mature s25 was codon harmonized for expression in We produced a rs25-MSP8 fusion protein [rs25/8(CΔS)] as well as unfused, mature rs25. rs25 was purified with a modest yield but required the incorporation of refolding protocols to obtain a proper conformation. In comparison, chimeric rs25/8(CΔS) was expressed and easily purified, with the s25 domain bearing the proper conformation without renaturation. Both antigens were immunogenic in rabbits, inducing IgG that bound native s25 and exhibited potent transmission-reducing activity. These data further demonstrate the utility of MSP8 as a parasite-specific carrier protein to enhance the production of complex malaria vaccine targets.
疟疾候选疫苗在生产方面面临挑战且免疫原性欠佳，这延缓了多抗原疫苗的研发进程。为解决这些问题，我们聚焦于使用免疫原性强的裂殖子表面蛋白8（MSP8）作为疫苗载体蛋白。此前，我们发现裂殖子表面蛋白1（MSP1）的C端19-kDa片段与MSP8进行基因融合（MSP1/8）有助于抗原的产生、折叠以及诱导针对MSP1构象性B细胞表位的中和抗体。在此，利用MSP1/8构建体，我们通过引入两个半胱氨酸到丝氨酸的替换（CΔS）进一步优化重组MSP8（rMSP8）载体，以提高单体产物的产量。然后，我们试图使用传播阻断候选疫苗s25来测试这种方法的广泛适用性。基于rs25的疫苗生产面临挑战。针对s25四个高度保守的表皮生长因子（EGF）样结构域的抗体可阻断蚊子体内性发育阶段。编码成熟s25的序列进行了密码子优化以在[具体表达系统未给出]中表达。我们制备了rs25-MSP8融合蛋白[rs25/8(CΔS)]以及未融合的成熟rs25。rs25纯化产量适中，但需要采用重折叠方案以获得正确构象。相比之下，嵌合的rs25/8(CΔS)易于表达和纯化，s25结构域无需复性即具有正确构象。两种抗原在兔体内均具有免疫原性，诱导产生的IgG能结合天然s25并表现出强大的传播抑制活性。这些数据进一步证明了MSP8作为寄生虫特异性载体蛋白在提高复杂疟疾疫苗靶点产量方面的效用。
