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，用于鼻内免疫接种SARS-CoV-2。疫苗载体表达融合前稳定的SARS-CoV-2 S蛋白，并被设计为在人类中高度减毒，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的浅表层复制，诱导对SARS-CoV-2 S蛋白的强烈的局部粘膜和全身免疫应答，包括病毒中和血清抗体，以及多功能SARS-CoV-2 S特异性CD 8+和CD 4 + T细胞的全身和粘膜应答。 儿童SARS-CoV-2感染，虽然通常是轻微的，但与大量的发病率有关，并有助于传播动力学。我们基于表达融合前稳定（S-2 P）SARS-CoV-2 S刺突蛋白（SARS-CoV-2的主要保护性和中和抗原）的可复制嵌合牛/人副流感病毒3型（B/HPIV 3），开发了用于婴儿和儿童的抗2019冠状病毒病（COVID-19）鼻内活载体候选疫苗。融合前稳定化增加了体外B/HPIV 3的S表达。在仓鼠中，单次鼻内给药B/HPIV 3/S-2 P诱导高水平的血清SARS-CoV-2中和抗体以及血清SARS-CoV-2 S蛋白的伊加和IgG。血清抗体对SARS-CoV-2的A、B.1.1.7和B.1.351谱系表现出广泛的中和活性。 免疫后4周，仓鼠鼻内用4.5 log 10 50%组织培养感染剂量（TCID 50）的SARS-CoV-2进行攻击。在B/HPIV 3空载体免疫的仓鼠中，SARS-CoV-2在肺中复制至平均滴度6.6 log 10 TCID 50/g，在鼻组织中复制至平均滴度7 log 10 TCID 50/g，并诱导中度体重减轻。在B/HPIV 3/S-2 P免疫的仓鼠中，在鼻组织和肺中未检测到感染性SARS-CoV-2攻击病毒;用B/HPIV 3/S-2 P免疫保护SARS-CoV-2攻击后的体重减轻。 基于这些结果，表达融合前稳定形式的S蛋白的B/HPIV 3是保护婴儿和幼儿抵抗HPIV 3和SARS-CoV-2的有希望的疫苗候选物（Liu X，Luongo C，Matsuoka Y，Park HS，桑托斯C，Yang L，摩尔IN，Afroz S，约翰逊RF，Lafont BAP，Martens C，Best SM，Munster VJ，冬青J，Yewdell JW，Le Nouen C，Munir S，布赫霍尔茨UJ.一个单一的鼻内剂量的减毒副流感病毒载体SARS-CoV-2疫苗是保护仓鼠。美国国家科学院院刊。2021;118（50））。基于B/HPIV 3的先导候选疫苗的临床试验材料是根据cGMP生产的，用于在1期研究中评价安全性和免疫原性。 所有年龄组都需要能够限制SARS-CoV-2在呼吸道复制的单剂量疫苗，以帮助控制COVID-19。除了上述B/HPIV 3/S-2 P候选疫苗外，我们正在开发基于载体平台的候选疫苗，这些载体平台在人体中不存在预先存在的抗载体免疫力，包括禽副粘病毒3型（APMV 3）。在临床前研究中，我们发现表达融合前稳定的S-6P版本的S蛋白的APMV 3（APMV 3/S-6P）在含胚鸡蛋中复制至高滴度，并且是遗传稳定的。在仓鼠中，单次鼻内给药APMV 3/S-6P诱导了对S蛋白及其受体结合域的强烈血清IgG和伊加应答，以及对疫苗匹配的SARS-CoV-2分离株WA 1/2020（谱系A）的强烈血清中和抗体应答。来自APMV 3/S-6P免疫的仓鼠的血清也有效地中和了所关注的α和β变体。用WA 1/2020攻击的免疫仓鼠没有表现出在空载体免疫对照中观察到的体重减轻和肺部炎症;与对照中的大量复制相比，免疫动物的上呼吸道和下呼吸道中的SARS-CoV-2复制较低或不可检测。因此，单次鼻内剂量的APMV 3/S-6 P具有高度免疫原性和针对SARS-CoV-2攻击的保护性，表明APMV 3/S-6 P适合于临床开发（Park HS，Matsuoka Y，Luongo C，Yang L，桑托斯C，Liu X，Ahlers LRH，摩尔IN，Afroz S，约翰逊RF，Lafont BAP，Dorward DW，Fischer ER，Martens C，Samal SK，Munir S，布赫霍尔茨UJ，Le Nouen C.表达SARS-CoV-2刺突蛋白的禽副粘病毒3型鼻内免疫保护仓鼠对抗SARS-CoV-2。NPJ疫苗。2022;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).