Two antibodies show broad, synergistic neutralization against SARS-CoV-2 variants by inducing conformational change within the RBD.

Continual evolution of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) virus has allowed for its gradual evasion of neutralizing antibodies (nAbs) produced in response to natural infection or vaccination. The rapid nature of these changes has incited a need for the development of superior broad nAbs (bnAbs) and/or the rational design of an antibody cocktail that can protect against the mutated virus strain. Here, we report two angiotensin-converting enzyme 2 competing nAbs-8H12 and 3E2-with synergistic neutralization but evaded by some Omicron subvariants. Cryo-electron microscopy reveals the two nAbs synergistic neutralizing virus through a rigorous pairing permitted by rearrangement of the 472-489 loop in the receptor-binding domain to avoid steric clashing. Bispecific antibodies based on these two nAbs tremendously extend the neutralizing breadth and restore neutralization against recent variants including currently dominant XBB.1.5. Together, these findings expand our understanding of the potential strategies for the neutralization of SARS-CoV-2 variants toward the design of broad-acting antibody therapeutics and vaccines.

Characterization and immunogenicity of SARS-CoV-2 spike proteins with varied glycosylation.

The ongoing coronavirus disease-19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drastically changed our way of life and continues to have an unmitigated socioeconomic impact across the globe. Research into potential vaccine design and production is focused on the spike (S) protein of the virus, which is critical for virus entry into host cells. Yet, whether the degree of glycosylation in the S protein is associated with vaccine efficacy remains unclear. Here, we first optimized the expression of the S protein in mammalian cells. While we found no significant discrepancy in purity, homogeneity, or receptor binding ability among S proteins derived from 293F cells (referred to as 293F S-2P), 293S GnTI- cells (defective in N-acetylglucosaminyl transferase I enzyme; 293S S-2P), or TN-5B1-4 insect cells (Bac S-2P), there was significant variation in the glycosylation patterns and thermal stability of the proteins. Compared with the partially glycosylated 293S S-2P or Bac S-2P, the fully glycosylated 293F S-2P exhibited higher binding reactivity to convalescent sera. In addition, 293F S-2P induced higher IgG and neutralizing antibody titres than 293S or Bac S-2P in mice. Furthermore, a prime-boost-boost regimen, using a combined immunization of S-2P proteins with various degrees of glycosylation, elicited a more robust neutralizing antibody response than a single S-2P alone. Collectively, this study provides insight into ways to design a more effective SARS-CoV-2 immunogen.

Endodomain truncation of the HIV-1 envelope protein improves the packaging efficiency of pseudoviruses.

HIV-1 remains one of the most devastating infectious pathogens without available vaccines. A valid neutralization assay using multiple representative virus strains is prerequisite for antibody response analysis in HIV-1 vaccine development, where HIV pseudoviruses (PsVs) commonly serve as surrogate agents for the authentic HIV, offering a safer manipulation in Biosafety Level 2+. However, PsV production is of low efficiency and is unstable in this field. Here, we optimize PsV production conditions via the use of alternative host cells, packaging ratios and gene truncation. We show that a 153-aa truncation of the endodomain substantially enhances the packaging efficiency of HIV PsVs, providing 4 to 25 times higher infection titers than the full-length Env. Further, we obtained a robust HIV-1 PsV panel covering 12 representative global strains for neutralization assay testing. This work sheds light on how to optimize HIV PsV packaging, and provides functional insight into the cytoplasmic domain of HIV-1.