Malaria Transmission Blocking Vaccine Discovery

抗原发现的重点是使用从自然暴露于疟疾的个体收集的人类血清样本，这些样本似乎能够产生有效的免疫力，防止配子体血症或阻止寄生虫向蚊子传播，作为确定候选疫苗抗原的工具。具体来说，将具有目标活性的血清样本或抗体与缺乏该活性的血清/抗体进行比较，因为它们能够选择或识别恶性疟原虫的单个重组蛋白结构。然后将通过差异筛选鉴定的重组蛋白制备为免疫原，并测试其诱导有效的抗配子细胞或传递阻断抗体的能力。

Antigen discovery has focused on using human serum samples collected from individuals that are naturally exposed to malaria and appear to develop effective immunity that prevents gametocytemia or blocks parasite transmission to mosquitoes, as tools to identify candidate vaccine antigens. Specifically, serum samples or antibodies that have activity of interest are compared to sera/antibodies that lack this activity, for their ability to select or recognize individual recombinant proteins constructs of P. falciparum. Recombinant proteins identified through differential screening are then prepared as immunogens and tested for their ability to induce effective anti-gametocyte or transmission-blocking antibodies. From our publication this year, we report the following advances in FY2020: 1. Tentokam BCN, Amaratunga C, Alani NAH, MacDonald NJ, Narum DL, Salinas ND, Kwan JL, Suon S, Sreng S, Pereira DB, Tolia NH, Fujiwara RT, Bueno LL, Duffy PE, Coelho CH. Naturally Acquired Antibody Response to Malaria Transmission Blocking Vaccine Candidate Pvs230 Domain 1. Frontiers in Immunology. 2019 Oct 4;10:2295. doi: 10.3389/fimmu.2019.02295. We collected sera or plasma samples from P. vivax-infected subjects from Brazil (n = 70) and Cambodia (n = 79) to assess antibody responses to domain 1 of the gametocyte/gamete stage protein Pvs230 (Pvs230D1M). We found that: -- 27.1% (19/70) and 26.6% (21/79) of subjects from Brazil and Cambodia, respectively, presented with detectable antibody responses to Pvs230D1M antigen. --The most frequent subclasses elicited in response to Pvs230D1M were IgG1 and IgG3. Although age did not correlate significantly with Pvs230D1M antibody levels overall, we observed significant differences between age strata. --Hemoglobin concentration inversely correlated with Pvs230D1M antibody levels in Brazil, but not in Cambodia. Additionally, we analyzed the antibody response against Pfs230D1M, the P. falciparum ortholog of Pvs230D1M. --We detected antibodies to Pfs230D1M in 7.2 and 16.5% of Brazilian and Cambodian P. vivax-infected subjects. Depletion of Pvs230D1M IgG did not impair the response to Pfs230D1M, suggesting pre-exposure to P. falciparum, or co-infection. --We also analyzed IgG responses to sporozoite protein PvCSP (11.4 and 41.8% in Brazil and Cambodia, respectively) and to merozoite protein PvDBP-RII (67.1 and 48.1% in Brazil and Cambodia, respectively), whose titers also inversely correlated with hemoglobin concentration only in Brazil. These data establish patterns of seroreactivity to sexual stage Pvs230D1M and show similar antibody responses among P. vivax-infected subjects from regions of differing transmission intensity in Brazil and Cambodia. Our unpublished progress during this reporting period includes the following advances: In FY2020 A fusion protein of Pfs230D1 and Pfs48/45D3 was expressed in mammalian cells. Production of Pfs230D1-Pfs48/45D3 was scaled, purified and milligram quantities per liter of cells was produced. The fusion protein had reactivity to Pfs230D1 human monoclonal antibody LMIV-230-01 and the Pfs48/45 mouse monoclonal antibody 3E12. Both antibodies have TBV activity measured in SMFA suggesting the fusion protein is retaining these critical epitopes for activity. Rabbits were immunized with the fusion or Pfs230D1 alone. High titers against the fusion were attained and antibodies against Pfs48/45D3 are present after depletion of the Pfs230D1 antibodies. The Pfs230D1-3 fragment was generated in milligram quantities and mice were immunized. High titers against the fragment were obtained and the serum had strong SMFA activity that was comparable or greater than Pfs230D1 alone. Human serum from Mali was screened for enhanced reactivity to Pfs230D1-3 over Pfs230D1 in order to find suitable candidates that have antibodies against Pfs230D2-3 in order to develop human monoclonal antibodies against these other domains as well as Pfs230D1. In the initial screen, one subject was identified that had higher Pfs230D1-3 titers over Pfs230D1 titers, suggesting that subject has antibodies against Pfs230D2-3. Other samples are being screened. Additional larger fragments of Pfs230 were expressed in mammalian cell expression systems. Small scale transient transfection demonstrated that Pfs230D1-5 and Pfs230D1-6 were expressed as a secreted protein as demonstrated by Western blot. These larger recombinant fragments of Pfs230 will similarly be used to survey naturally acquired human antibody responses and subsequently to generate human monoclonal antibodies.