Pathogenesis studies on COVID-19 and development of a VLP-forming mRNA vaccine against SARS-CoV2

在我们的基于共配制的Env+Gag mRNA的HIV-1疫苗平台成功以产生非感染性VLP之后（Zhang等人，27：2234，2021），与Moderna，Inc.我们已经为SARS-CoV-2设计了一个类似的疫苗平台。迄今取得的主要成果包括： 1. 我们合成了不同的尾部修饰的SARS-CoV 2包膜刺突（S），以促进同源刺突-Gag相互作用和与慢病毒核心蛋白的VLP形成。为了促进含有慢病毒（HIV-1或SIV）核心蛋白的VLP的组装，我们设计了通过用HIV-1或SIV gp 41 CT替换天然尾来修饰其胞质尾（CT）的S蛋白。 基于其促进更有效的表面膜表达的能力，选择了SIV尾（Sst）的截短形式。 Moderna公司已经按照我们的设计产生了所有特定的mRNA。 2. 在体外，我们测试了用修饰的刺突蛋白生产VLP的效率。将刺突蛋白mRNA与编码HIV-1或SIV Gag或Gag-Pol的mRNA以所有可能的组合共转染到哺乳动物细胞中。收获细胞外VLP并通过蔗糖垫上的超离心浓缩。已经使用广泛的物理和免疫学测定，包括通过mAb捕获病毒体，然后进行ELISA用于慢病毒核心抗原定量、Western印迹等，对产生的VLP进行了广泛的定量和定性表征。 3. 在体内，我们测试了我们的杂交SARS-CoV-2-SIV VLP形成疫苗在BALB/c小鼠中的免疫原性。在第0、4和16周，用SSt+gag mRNA或单独的Set接种动物。与单独的SSt mRNA相比，VLP形成疫苗（Sst+Gag）在所有时间点诱导更高滴度的刺突结合抗体和自体中和抗体。此外，用SSt+gag mRNA免疫的小鼠产生了有效对抗所关注的不同变体的中和抗体。 这些数据表明，Gag/VLP mRNA平台可以成功应用于针对不同试剂的疫苗，以预防全球相关的传染病。 我们对发病机制的研究导致了与COVID-19患者疾病严重程度相关的循环抗PF 4抗体的鉴定。引发COVID-19最严重并发症（通常是致命的）的致病机制在很大程度上仍然未知。严重的COVID-19通常与血栓形成和血小板减少症有关，这让人想起两种危及生命的综合征的临床表现：肝素诱导的血小板减少症（HIT），发生在心脏或骨科手术后接受高剂量肝素治疗的患者中，以及疫苗诱导的血栓形成伴血小板减少症（VITT），这是腺病毒载体COVID-19疫苗的罕见并发症。HIT和VITT综合征的致病机制都涉及靶向α-趋化因子血小板因子4（PF 4或CXCL 4）中的部分隐蔽表位的自身抗体的激发，其在与肝素或其他聚阴离子分子结合时完全显示。使用临床验证的免疫测定法，我们评估了100名患有中度或重度疾病（WHO评分，4至10）的COVID-19住院患者，25名急诊科（艾德）的急性COVID-19病例和65名康复期个体中PF 4抗体的存在。在100名（95.0%）住院COVID-19患者中检测到抗PF 4抗体，无论既往是否接受肝素治疗，平均光密度值为0.871 ± 0.405（范围0.177-2.706）。相比之下，因与COVID-19无关的严重急性呼吸道疾病住院的患者的水平明显较低。在高比例的COVID-19患者中，检测的所有三种同种型（IgG、IgM和伊加）同时升高。在男性中检测到的抗体水平高于女性，在非洲裔美国人和西班牙裔美国人中检测到的抗体水平高于白色高加索人。抗PF 4抗体水平与最大疾病严重程度评分和住院期间循环血小板计数显著降低相关。在从COVID-19恢复的个体中，抗体水平恢复到接近正常值。来自COVID-19患者的血清诱导的血小板活化水平高于来自健康献血者的血清，但结果与抗PF 4抗体水平无关。 这些结果表明，绝大多数严重COVID-19患者会产生抗PF 4抗体，这可能在COVID-19的临床并发症中发挥作用，并对最严重病例的治疗管理产生影响。

Following the success of our HIV-1 vaccine platform based on co-formulated Env+Gag mRNA in order to produce non-infectious VLPs (Zhang et al., Nat. Med. 27:2234, 2021), in collaboration with Moderna, Inc., we have designed a similar vaccine platform for SARS-CoV-2. The major results obtained thus far include the following: 1. We synthesized different tail-modified SARS-CoV2 envelope spikes (S) in order to facilitate cognate Spike-Gag interaction and VLP formation with lentivirus core proteins. To promote the assembly of VLPs containing lentivirus (HIV-1 or SIV) core proteins, we have designed S proteins modified in their cytoplasmic tails (CT) by replacement of the natural tail with either the HIV-1 or SIV gp41 CT. A truncated form of the SIV tail (Sst) has been selected based on its ability to promote a more efficient surface membrane expression. Moderna Inc. has produced all the specific mRNAs according to our design. 2. In vitro, we tested the efficiency of production of VLPs with the modified spike proteins. The spike protein mRNAs were co-transfected into mammalian cells in all possible combinations with mRNAs encoding HIV-1 or SIV Gag or Gag-Pol. The extracellular VLPs were harvested and concentrated by ultracentrifugation on sucrose cushions. The produced VLPs have been extensively characterized both quantitatively and qualitatively using a wide array of physical and immunological assays, including virion capture by mAbs followed by ELISA for lentivirus core antigen quantification, Western blot and others. 3. In vivo, we tested the immunogenicity of our hybrid SARS-CoV-2-SIV VLP-forming vaccine in BALB/c mice. The animals were vaccinated with SSt+gag mRNA or Set alone at 0, 4 and 16 weeks. The VLP-forming vaccine (Sst+Gag) induced higher titers of Spike-binding and autologous neutralizing antibodies at all time points compared to SSt mRNA alone. Furthermore, mice immunized with SSt+gag mRNA developed neutralizing antibodies effective against different variants of concern. These data demonstrate that the Gag/VLP mRNA platform can be successfully applied to vaccines against different agents for the prevention of infectious diseases of global relevance. Our research on pathogenesis led to the identification of circulating anti-PF4 antibodies associated with disease severity in patients with COVID-19. The pathogenic mechanisms that trigger the most severe complications of COVID-19, which are often fatal, are still largely unknown. Severe COVID-19 is frequently associated with thrombosis and thrombocytopenia, which are reminiscent of the clinical presentation of two life-threatening syndromes: heparin-induced thrombocytopenia (HIT), which develops in patients treated with high doses of heparin after cardiac or orthopedic surgery, and vaccine-induced thrombosis with thrombocytopenia (VITT), which is a rare complication of adenovirus-vectored COVID-19 vaccines. The pathogenic mechanism of both the HIT and VITT syndromes involves the elicitation of autoantibodies that target partially cryptic epitopes in the a-chemokine platelet factor 4 (PF4 or CXCL4), which are fully revealed upon binding to heparin or other polyanionic molecules. Using a clinically validated immunoassay, we evaluated the presence of antibodies to PF4 in 100 hospitalized patients with COVID-19 with moderate or severe disease (WHO score, 4 to 10), 25 acute COVID-19 cases visiting the Emergency Department (ED) and 65 convalescent individuals. Anti-PF4 antibodies were detected in 95 of 100 (95.0%) hospitalized COVID-19 patients irrespective of prior heparin treatment, with a mean optical density value of 0.871 0.405 (range, 0.177-2.706). In contrast, patients hospitalized for severe acute respiratory disease unrelated to COVID-19 had markedly lower levels. In a high proportion of COVID-19 patients, all three isotypes tested (IgG, IgM and IgA) were simultaneously elevated. Higher antibody levels were detected in males than in females, and in African Americans and Hispanics than in White Caucasians. Anti-PF4 antibody levels were correlated with the maximum disease severity score and with significant reductions in circulating platelet counts during hospitalization. In individuals convalescent from COVID-19, the antibody levels returned to near-normal values. Sera from COVID-19 patients induced higher levels of platelet activation than sera from healthy blood donors, but the results were not correlated with the levels of anti-PF4 antibodies. These results demonstrate that the vast majority of patients with severe COVID-19 develop anti-PF4 antibodies, which may play a role in the clinical complications of COVID-19, and have implications for the therapeutic management of the most severe cases.