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.

抗原发现的重点是使用从自然暴露于疟疾的个体中收集的人血清样品，并且似乎会产生有效的免疫力，从而阻止配子细胞血症或阻止寄生虫传播到蚊子作为识别候选疫苗抗原的工具。具体而言，将具有感兴趣活性的血清样品或抗体与缺乏这种活性的血清/抗体进行了比较，因为它们可以选择或识别恶性疟原虫的单个重组蛋白构建体。然后，通过鉴定筛查鉴定出的重组蛋白作为免疫原子制备，并测试了它们诱导有效的抗甲状腺细胞或传播阻断抗体的能力。 从今年的出版物中，我们报告了2020财年的以下进展： 1。TentokamBCN，Amaratunga C，Alani Nah，Macdonald NJ，Narum DL，Salinas ND，Kwan JL，Suon S，Sreng S，Sreng S，Pereira DB，Pereira DB，Peria NH，Tolia NH，Fujiiwara RT，Bueno LL，Bueno LL，Duffy PE，Coelho，Coelho Ch。自然获得的抗体反应对疟疾传播阻断疫苗候选PVS230域1。免疫学领域。 2019年10月4日； 10：2295。 doi：10.3389/fimmu.2019.02295。 我们从巴西（n = 70）和柬埔寨（n = 79）中收集了来自疟原虫感染的受试者的血清或血浆样品，以评估对配子/配子级蛋白质PVS230（PVS230D1M）的结构域1的抗体反应。我们发现： - 分别来自巴西和柬埔寨的受试者27.1％（19/70）和26.6％（21/79），对PVS230D1M抗原具有可检测的抗体反应。 - 响应PVS230D1M引起的最常见的子类是IgG1和IgG3。尽管年龄与PVS230D1M抗体的总体水平没有显着相关，但我们观察到年龄层之间的显着差异。 - 血红蛋白浓度与巴西的PVS230D1M抗体水平成反比，但在柬埔寨却与不相关。此外，我们分析了针对PFS230D1M的抗体反应，即PVS230D1M的恶性疟原虫直系同源物。 - 我们在7.2中检测到对PFS230D1M的抗体，而巴西和柬埔寨P. Vivax感染的受试者的抗体。 PVS230D1M IgG的耗竭不会损害对PFS230D1M的反应，这表明预先暴露于恶性疟原虫或共感染。 - 我们还分析了IgG对Sporozoite蛋白PVCSP的反应（分别为巴西和柬埔寨的11.4和41.8％），以及对Merozoite蛋白PVDBP-RII（67.1和48.1％）（分别在巴西和柬埔寨），其滴滴员的滴虫（67.1和48.1％），他们的滴滴失所也与HymoBIN相关。仅集中在巴西。 这些数据建立了对性阶段PVS230D1M的血清反应性模式，并在巴西和柬埔寨不同传播强度区域的体内感染受试者中显示出相似的抗体反应。 在此报告期内，我们未发表的进展包括以下进展： 在2020财年，PFS230D1和PFS48/45D3的融合蛋白在哺乳动物细胞中表达。将PFS230D1-PFS48/45D3的生产缩放，纯化和每升细胞的毫克数量。融合蛋白对PFS230D1人单克隆抗体LMIV-230-01和PFS48/45小鼠单克隆抗体3E12具有反应性。两种抗体都具有在SMFA中测得的TBV活性，这表明融合蛋白保留了这些关键表位以进行活性。单独使用融合或PFS230D1对兔子进行免疫。在PFS230D1抗体耗尽后，存在针对融合的高滴度，并存在针对PFS48/45D3的抗体。 PFS230D1-3的片段以毫克数量产生，并免疫小鼠。获得了针对片段的高滴度，并且血清具有强大的SMFA活性，仅比单独的PFS230D1大。从PFS230D1上筛选了来自Mali的人血清，以提高对PFS230D1-3的反应性，以便找到适合针对PFS230D2-3抗体的候选者，以开发针对这些其他领域的人类单克隆抗体以及PFS230D1。在最初的屏幕中，确定了一个受试者在PFS230D1滴度上具有较高的PFS230D1-3滴度，这表明受试者具有针对PFS230D2-3的抗体。其他样本正在筛选。 PFS230的其他较大片段在哺乳动物细胞表达系统中表达。小规模的瞬时转染表明PFS230D1-5和PFS230D1-6被表达为分泌的蛋白质，如Western Blot所证明。 PFS230的这些较大的重组片段将类似地用于调查自然获得的人类抗体反应，然后随后生成人类单克隆抗体。