Malaria Transmission Blocking Vaccine Discovery

抗原发现的重点是使用从自然接触疟疾的人身上收集的人类血清样本，作为识别候选疫苗抗原的工具。这些人似乎具有有效的免疫力，可以防止配子细胞症或阻止寄生虫传播给蚊子。具体地说，将具有感兴趣活性的血清样本或抗体与缺乏这种活性的血清/抗体进行比较，以了解它们选择或识别恶性疟原虫个别重组蛋白结构的能力。然后，通过差异筛选鉴定的重组蛋白被制备成免疫原，并测试它们诱导有效的抗配子体或传播阻断抗体的能力。 从我们今年的出版物来看，我们在2020财年报告了以下进展： 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.对疟疾传播阻断疫苗候选Pvs230的自然获得性抗体反应1.免疫学前沿。2019年10月4日；10：2295。DOI：10.3389/fimmu.2019.02295. 我们收集了来自巴西(n=70)和柬埔寨(n=79)间日疟原虫感染者的血清或血浆样本，以评估对配子/配子阶段蛋白Pvs230(Pvs230D1M)结构域1的抗体反应。我们发现： 巴西和柬埔寨分别有27.1%(19/70)和26.6%(21/79)的受试者对Pvs230D1M抗原有可检测到的抗体反应。 --对Pvs230D1M最常见的亚类是IgG1和IgG3。虽然年龄与Pvs230D1M抗体水平总体上没有显著相关性，但我们观察到不同年龄层之间的显著差异。 --在巴西，血红蛋白浓度与Pvs230D1M抗体水平呈负相关，但在柬埔寨则不然。此外，我们还分析了恶性疟原虫Pvs230D1M的同源基因Pfs230D1M的抗体反应。 --我们在7.2%和16.5%的巴西和柬埔寨间日疟原虫感染者中检测到Pfs230D1M抗体。Pvs230D1M免疫球蛋白耗尽不影响对Pfs230D1M的反应，提示预先暴露于恶性疟原虫或合并感染。 --我们还分析了对子孢子蛋白PvCSP(在巴西和柬埔寨分别为11.4%和41.8%)和裂殖子蛋白PvDBP-RII(在巴西和柬埔寨分别为67.1%和48.1%)的免疫球蛋白反应，只有在巴西，这两种蛋白的滴度与血红蛋白浓度呈负相关。 这些数据建立了对性阶段Pvs230D1M的血清反应性模式，并显示了来自巴西和柬埔寨不同传播强度地区的间日疟原虫感染者之间类似的抗体反应。 在本报告所述期间，我们未公布的进展包括以下进展： 在2020财年，Pfs230D1和Pfs48/45d3融合蛋白在哺乳动物细胞中得到了表达。对Pfs230d1-Pfs48/45d3的生产进行了规模化、提纯，并生产了每升细胞的毫克量。该融合蛋白与Pfs230D1人源单抗LMIV-230-01和Pfs48/45鼠单抗3E12具有较强的结合活性。两种抗体都在SMFA中检测到了TBV活性，这表明融合蛋白保留了这些关键的活性表位。用融合蛋白Pfs230D_1和Pfs230D_1单独免疫兔。在去除Pfs230D1抗体后，获得了高滴度的融合抗体，并产生了抗Pfs48/45d3的抗体。 获得毫克量的Pfs230D1-3片段，免疫小鼠。获得了高滴度的抗该片段，且该血清具有与Pfs230D1相当或更高的SMFA活性。对来自马里的人血清进行筛选，以提高对Pfs230D1-3的反应性，以便找到具有抗Pfs230D2-3抗体的合适候选者，以便开发出针对这些其他结构域以及Pfs230D1的人源单抗。在最初的筛查中，一名受试者的Pfs230D1-3滴度高于Pfs230D1效价，表明该受试者有针对Pfs230D2-3的抗体。其他样本正在进行筛选。在哺乳动物细胞表达系统中还表达了Pfs230的其他较大片段。小规模瞬时转染法证明Pfs230d1-5和Pfs230d1-6以分泌型蛋白的形式表达。这些较大的Pfs230重组片段将同样用于检测自然获得的人类抗体反应，并随后产生人类单抗。

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.