Paramyxoviruses as Vaccine Vectors Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

我们正在开发副粘病毒载体疫苗，用于对SARS-CoV-2的鼻内免疫。疫苗载体表达SARS-CoV-2 S蛋白的预融合稳定型版本，设计用于在人类中高度减毒，同时在SARS-CoV-2的主要感染部位保持高水平的免疫原性。病毒载体在呼吸道上皮浅层复制，诱导对SARS-CoV-2 S蛋白产生强烈的局部和系统免疫反应，包括病毒中和血清抗体，以及多功能SARS-CoV-2 S特异性CD8+和CD4+T细胞的系统和粘膜反应。 儿童SARS-CoV-2感染虽然一般较轻，但与相当大的发病率有关，并有助于传播动态。我们基于可复制的牛/人3型副流感病毒嵌合病毒(B/HPIV3)表达融合稳定(S-2P)的SARS-CoV-2 S刺突蛋白(SARS-CoV-2)的主要保护和中和抗原，开发了针对SARS-CoV-2的候选鼻腔活载体疫苗(新冠肺炎)。灌流稳定可增加B/HPIV3在体外对S的表达。在金黄地鼠中，一次鼻腔注射B/HPIV3/S-2P可诱导产生高水平的SARS-CoV-2中和抗体，并能诱导血清中抗SARS-CoV-2 S蛋白的IgA和Ig G水平升高。血清抗体对A、B.1.1.7和B.1.351三个谱系的SARS-CoV-2具有广泛的中和活性。免疫后4周，以4.5log10 50%组织培养感染量(TCID50)攻击金黄地鼠。在B/HPIV3空载体免疫的仓鼠中，SARS-CoV-2在肺部复制的平均滴度为6.6log10 TCID50/g，在鼻腔组织复制的平均滴度为7log10 TCID50/g，并导致中度体重下降。在B/HPIV3/S-2P免疫的仓鼠中，鼻组织和肺组织中未检测到传染性SARS-CoV-2攻击病毒，B/HPIV3/S-2P免疫对SARS-CoV-2攻击后的体重减轻有保护作用。根据这些结果，表达融合稳定版本的S蛋白的B/HPIV3是一种有希望的候选疫苗，可用于保护婴幼儿免受HPIV3和SARS-CoV-2的感染(刘X、Luongo C、Matsuoka Y、Park HS、Santos C、杨L、Moore In、AFroz S、Johnson RF、乐峰BAP、Marten C、Best SM、Munster VJ、Holly J、Yewdell JW、Le Nouen C、Munir S、Buchholz UJ)。一剂鼻腔注射减毒副流感病毒载体的SARS-CoV-2疫苗对仓鼠有保护作用。美利坚合众国国家科学院院刊。2021年；118(50))。以B/HPIV3为基础的候选疫苗的临床试验材料是在cGMP下制作的，用于在第一阶段研究中评估安全性和免疫原性。 所有年龄段都需要能够限制SARS-CoV-2在呼吸道复制的单剂疫苗，以帮助控制新冠肺炎的努力。除了上述B/HPIV3/S-2P候选疫苗外，我们还在开发基于载体平台的候选载体疫苗，这些平台在人类中没有预先存在的抗媒介免疫，包括禽副粘病毒3型(APMV3)。在一项临床前研究中，我们发现，表达融合稳定的S蛋白S-6P版本的APMV3(APMV3/S-6P)在鸡胚中复制到高滴度，并且遗传稳定。在仓鼠中，一次鼻腔注射APMV3/S-6P可诱导出对S蛋白及其受体结合域的强免疫球蛋白和免疫球蛋白A反应，以及对疫苗匹配的SARS冠状病毒WA1/2020分离株(A系)的强血清中和抗体反应。来自APMV3/S-6P免疫仓鼠的血清也有效地中和了令人担忧的Alpha和Beta变体。用WA1/2020免疫的仓鼠没有表现出空载体免疫对照中观察到的体重减轻和肺部炎症；与对照动物的大量复制相比，免疫动物的上呼吸道和下呼吸道中的SARS-CoV-2复制较低或检测不到。因此，单次鼻腔注射APMV3/S-6P对SARS-CoV-2攻击具有高度的免疫原性和保护性，提示APMV3/S-6P适合临床应用(Park HS，Matsuoka Y，Luongo C，杨L，Santos C，Liu X，Ahler LRH，Moore IN，AFroz S，Johnson RF，Lafont BAP，Dorward DW，Fischer，Marten C，Samal SK，Munir S，Buchholz UJ，Le Nouen C)。NPJ疫苗。2022年；7(1)：72)。

We are developing paramyxovirus vectored vaccines for intranasal immunization against SARS-CoV-2. The vaccine vectors express prefusion-stabilized versions of the SARS-CoV-2 S protein and are designed to be highly attenuated in humans while maintaining a high level of immunogenicity at the primary sites of infection of SARS-CoV-2. The viral vectors replicate in the superficial layers of the respiratory epithelium, inducing strong local mucosal and systemic immune responses to the SARS-CoV-2 S protein, including virus-neutralizing serum antibodies, and a systemic and mucosal response of multifunctional SARS-CoV-2 S-specific CD8+ and CD4+ T cells. Pediatric SARS-CoV-2 infections, though generally mild, are associated with substantial morbidity and contribute to transmission dynamics. We developed live intranasal vector vaccine candidates for infants and children against coronavirus disease-2019 (COVID-19) based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) expressing the prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced high levels of serum SARS-CoV-2-neutralizing antibodies, and serum IgA and IgG to SARS-CoV-2 S protein. Serum antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 4.5 log10 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 6.6 log10 TCID50/g in lungs and 7 log10 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-2P-immunized hamsters, infectious SARS-CoV-2 challenge virus was undetectable in nasal tissues and lungs; immunization with B/HPIV3/S-2P protected against weight loss after SARS-CoV-2 challenge. Based on these results, B/HPIV3 expressing a prefusion-stabilized version of the S protein is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2 (Liu X, Luongo C, Matsuoka Y, Park HS, Santos C, Yang L, Moore IN, Afroz S, Johnson RF, Lafont BAP, Martens C, Best SM, Munster VJ, Holly J, Yewdell JW, Le Nouen C, Munir S, Buchholz UJ. A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters. Proceedings of the National Academy of Sciences of the United States of America. 2021;118(50)). Clinical trial material of the lead B/HPIV3-based vaccine candidate was manufactured under cGMP for evaluation of safety and immunogenicity in a Phase 1 study. Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts towards control of COVID-19. In addition to the B/HPIV3/S-2P candidate described above, we are developing vector vaccine candidates based on vector platforms without pre-existing anti-vector immunity in humans, including avian paramyxovirus type 3 (APMV3). In a preclinical study, we found that APMV3 expressing the prefusion-stabilized S-6P version of the S protein (APMV3/S-6P) replicated to high titers in embryonated chicken eggs and was genetically stable. In hamsters, a single intranasal dose of APMV3/S-6P induced strong serum IgG and IgA responses to the S protein and its receptor-binding domain, and strong serum neutralizing antibody responses to the vaccine-matched SARS-CoV-2 isolate WA1/2020 (lineage A). Sera from APMV3/S-6P-immunized hamsters also efficiently neutralized Alpha and Beta variants of concern. Immunized hamsters challenged with WA1/2020 did not exhibit the weight loss and lung inflammation observed in empty vector-immunized controls; SARS-CoV-2 replication in the upper and lower respiratory tract of immunized animals was low or undetectable compared to the substantial replication in controls. Thus, a single intranasal dose of APMV3/S-6P was highly immunogenic and protective against SARS-CoV-2 challenge, suggesting that APMV3/S-6P is suitable for clinical development (Park HS, Matsuoka Y, Luongo C, Yang L, Santos C, Liu X, Ahlers LRH, Moore IN, Afroz S, Johnson RF, Lafont BAP, Dorward DW, Fischer ER, Martens C, Samal SK, Munir S, Buchholz UJ, Le Nouen C. Intranasal immunization with avian paramyxovirus type 3 expressing SARS-CoV-2 spike protein protects hamsters against SARS-CoV-2. NPJ Vaccines. 2022;7(1):72).