Malaria Transmission Blocking Vaccine Discovery

疟疾传播阻断疫苗的发现

• 批准号: 10272119
• 负责人: Patrick Duffy
• 金额: $ 130.2万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272119
• 关键词: Age; Antibodies; Antibody Response; Antigens; Area; Blocking Antibodies; Brazil; Cambodia; Cambodian; Cells; Chimeric Proteins; Culicidae; Data; Epitopes; Exposure to; Expression Library; Germ Cells; Goals; Hemoglobin; Human; IgG1; IgG3; Immunity; Immunize; Immunoglobulin G; Immunology; Individual; Investigation; Malaria; Mali; Mammalian Cell; Measures; Methods; Monoclonal Antibodies; Mus; Orthologous Gene; Oryctolagus cuniculus; Parasites; Pattern; Plasma; Plasmodium falciparum; Plasmodium vivax; Production; Proteins; Publications; Recombinant Proteins; Recombinants; Reporting; Salinas Valley; Sampling; Serum; Sporozoites; Surveys; System; Testing; Transfection; Vaccine Antigen; Vaccines; Western Blotting; Work; burden of illness; co-infection; frontier; human monoclonal antibodies; interest; malaria transmission; malaria transmission-blocking vaccine; milligram; novel; prevent; response; screening; tool; transmission process; transmission-blocking vaccine; vaccine candidate; vaccine discovery

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.

抗原发现集中于使用从自然暴露于疟疾的个体收集的人血清样品，并且似乎产生有效的免疫力，防止配子体血症或阻断寄生虫向蚊子的传播，作为鉴定候选疫苗抗原的工具。具体地，将具有感兴趣的活性的血清样品或抗体与缺乏该活性的血清/抗体进行比较，以确定它们选择或识别恶性疟原虫的单个重组蛋白构建体的能力。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体抗体或传播阻断抗体的能力。 自本年度刊发以来，我们报告二零二零财政年度的以下进展： 1. Tentokam BCN，Amaratunga C，Alani NAH，MacDonald NJ，Narum DL，Salinas ND，Kwan JL，Suon S，Sreng S，佩雷拉DB，Tolia NH，Fujiwara RT，Mrslo LL，Duffy PE，Coelho CH. Naturally Acquired Antibody Response to Malaria Transmission Blocking Vaccine Candidate Pvs 230 Domain 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）的间日疟原虫感染受试者的血清或血浆样品，以评估对配子母细胞/配子阶段蛋白Pvs 230（Pvs 230 D1 M）的结构域1的抗体应答。我们发现： 来自巴西和柬埔寨的受试者中分别有27.1%（19/70）和26.6%（21/79）对Pvs 230 D1 M抗原产生可检测的抗体应答。 --对Pvs 230 D1 M的应答中最常见的亚类是IgG 1和IgG 3。虽然年龄与Pvs 230 D1 M抗体水平总体上没有显著相关性，但我们观察到年龄层之间存在显著差异。 --在巴西，血红蛋白浓度与Pvs 230 D1 M抗体水平呈负相关，但在柬埔寨则不然。此外，我们分析了针对Pfs 230 D1 M（Pfs 230 D1 M的恶性疟原虫直系同源物）的抗体应答。 我们在7.2%和16.5%的巴西和柬埔寨间日疟原虫感染受试者中检测到Pfs 230 D1 M抗体。Pvs 230 D1 M IgG的耗尽并不损害对Pfs 230 D1 M的应答，这表明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（巴西和柬埔寨分别为11.4%和41.8%）和裂殖子蛋白PvDBP-RII（巴西和柬埔寨分别为67.1%和48.1%）的IgG应答，其滴度也仅与巴西的血红蛋白浓度呈负相关。 这些数据建立了性阶段Pvs 230 D1 M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受试者中相似的抗体应答。 我们在本报告所述期间未公布的进展包括以下进展： 在FY 2020中，在哺乳动物细胞中表达Pfs 230 D1和Pfs 48/45 D3的融合蛋白。Pfs 230 D1-Pfs 48/45 D3的生产按比例放大、纯化并生产毫克量/升细胞。该融合蛋白与Pfs 230 D1人源单克隆抗体LMIV-230-01和Pfs 48/45鼠源单克隆抗体3E 12具有反应性。两种抗体都具有在SMFA中测量的TBV活性，表明融合蛋白保留了这些活性的关键表位。用融合物或单独的Pfs 230 D1免疫家兔。获得了针对融合体的高滴度，并且在Pfs 230 D1抗体耗尽后存在针对Pfs 48/45 D3的抗体。 以毫克量产生Pfs 230 D1 -3片段并免疫小鼠。获得了针对该片段的高滴度，并且血清具有与单独的Pfs 230 D1相当或更高的强SMFA活性。筛选来自Mali的人血清对Pfs 230 D1 -3的反应性超过Pfs 230 D1，以找到具有抗Pfs 230 D2 -3的抗体的合适候选物，从而开发抗这些其它结构域以及Pfs 230 D1的人单克隆抗体。在初始筛选中，鉴定出一名受试者的Pfs 230 D1 -3滴度高于Pfs 230 D1滴度，表明该受试者具有抗Pfs 230 D2 -3的抗体。其他样本正在筛选中。在哺乳动物细胞表达系统中表达Pfs 230的另外的较大片段。小规模瞬时转染表明Pfs 230 D1 -5和Pfs 230 D1 -6以分泌蛋白形式表达。Pfs 230的这些较大的重组片段将类似地用于调查天然获得的人抗体应答，并随后产生人单克隆抗体。