Chakraborty Debajyoti, Singh Randhir, Rajmani Raju S, Kumar Sahil, Ringe Rajesh P, Varadarajan Raghavan
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Mynvax Private Limited, 3rd Floor, Brigade MLR Centre, No.50, Vani Vilas Road, Basavanagudi, Bengaluru 560004, India.
Vaccines (Basel). 2025 Mar 14;13(3):315. doi: 10.3390/vaccines13030315.
As with many viral fusion proteins, the native conformation of SARS-CoV-2 Spike is metastable. Most COVID-19 vaccines utilize a stabilized Spike (Spike-2P) containing two proline substitutions, and subsequently, a further stabilized variant with four additional proline substitutions, Spike-6P, has been developed. In an alternative approach, we introduced two aspartic acid residues (2D) in the HR1 region of Spike at positions that are exposed and buried in the pre- and postfusion states, respectively, to destabilize the postfusion conformation. The recombinant protein constructs were expressed in a mammalian cell culture and characterized for their yield and antigenicity, and the formulations were then used to immunize hamsters. After two immunizations, the hamsters were challenged with live B.1.351 SARS-CoV-2 virus for an evaluation of the protective efficacy. The introduction of the two aspartic acid mutations resulted in an approximately six-fold increase in expression, comparable to that in Spike-2P. When the 2D mutations were combined with the above four proline mutations (Spike-4P-2D), this led to a further three- to four-fold enhancement of protein expression, similar to that seen in Spike-6P. When formulated with the oil-in-water emulsion adjuvant Sepivac SWE, the 2P, 2D, 6P, and 4P-2D Spike variants all protected female hamsters against heterologous challenge with the B.1.351 SARS-CoV-2 virus and elicited high titers of neutralizing antibodies. We suggest that destabilization of the postfusion conformation through the introduction of charged amino acids at sites that are exposed in the pre- and buried in the postfusion conformation offers a general strategy to enhance the yield and stability of the native, prefusion conformation of viral surface proteins.
与许多病毒融合蛋白一样，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白的天然构象是亚稳态的。大多数2019冠状病毒病（COVID-19）疫苗使用含有两个脯氨酸替代的稳定化刺突蛋白（Spike-2P），随后，又开发出了一种具有另外四个脯氨酸替代的进一步稳定化变体Spike-6P。在另一种方法中，我们在刺突蛋白的HR1区域引入了两个天冬氨酸残基（2D），其位置分别在融合前和融合后状态下暴露和掩埋，以破坏融合后构象的稳定性。重组蛋白构建体在哺乳动物细胞培养物中表达，并对其产量和抗原性进行表征，然后将制剂用于免疫仓鼠。两次免疫后，用活的B.1.351 SARS-CoV-2病毒攻击仓鼠，以评估保护效果。引入两个天冬氨酸突变导致表达增加约六倍，与Spike-2P中的表达相当。当2D突变与上述四个脯氨酸突变（Spike-4P-2D）结合时，这导致蛋白质表达进一步提高三到四倍，类似于Spike-6P中的情况。当与水包油乳液佐剂Sepivac SWE配制时，2P、2D、6P和4P-2D刺突蛋白变体均能保护雌性仓鼠免受B.1.351 SARS-CoV-2病毒的异源攻击，并诱导产生高滴度的中和抗体。我们认为，通过在融合前暴露而在融合后掩埋的位点引入带电荷氨基酸来破坏融合后构象的稳定性，为提高病毒表面蛋白天然融合前构象的产量和稳定性提供了一种通用策略。
