Fusogenicity and neutralization sensitivity of the SARS-CoV-2 Delta sublineage AY.4.2

SARS-CoV-2 lineages are continuously evolving. As of December 2021, the AY.4.2 Delta sub-lineage represented 20 % of sequenced strains in UK and has been detected in dozens of countries. It has since then been supplanted by the Omicron variant. AY.4.2 displays three additional mutations (T95I, Y145H and A222V) in the N-terminal domain (NTD) of the spike when compared to the original Delta variant (B.1.617.2) and remains poorly characterized. Here, we analyzed the fusogenicity of the AY.4.2 spike and the sensitivity of an authentic AY.4.2 isolate to neutralizing antibodies. The AY.4.2 spike exhibited similar fusogenicity and binding to ACE2 than Delta. The sensitivity of infectious AY.4.2 to a panel of monoclonal neutralizing antibodies was similar to Delta, except for the anti-RBD Imdevimab, which showed incomplete neutralization. Sensitivity of AY.4.2 to sera from individuals having received two or three doses of Pfizer or two doses of AstraZeneca vaccines was reduced by 1.7 to 2.1 fold, when compared to Delta. Our results suggest that mutations in the NTD remotely impair the efficacy of anti-RBD antibodies. The temporary spread of AY.4.2 was not associated with major changes in spike function but rather to a partially reduced neutralization sensitivity.

B.1.617.3 SARS CoV-2 spike E156G/Δ157-158 mutations contribute to reduced neutralization sensitivity and increased infectivity

SARS CoV-2 variants raise significant concerns due to their ability to cause vaccine breakthrough infections. Here, we sequence-characterized the spike gene, isolated from a breakthrough infection, that corresponded to B.1.617.3 lineage. Delineating the functional impact of spike mutations using reporter pseudoviruses (PV) revealed that N-terminal domain (NTD)-specific E156G/Δ157-158 contributed to increased infectivity and reduced sensitivity to ChAdOx1 nCoV-19 vaccine (CovishieldTM)-elicited neutralizing antibodies. A six-nucleotide deletion (467-472) in the spike coding region introduced this change in the NTD. We confirmed the presence of E156G/Δ157-158 in the RT-PCR-positive cases concurrently screened, in addition to other circulating spike (S1) mutations like T19R, T95I, L452R, E484Q, and D614G. Notably, E156G/Δ157-158 was present in more than 85% of the sequences reported from the USA, UK, and India in August 2021. The spike PV bearing combination of E156G/Δ157-158 and L452R further promoted infectivity and conferred immune evasion. Additionally, increased cell-to-cell fusion was observed when spike harbored E156G/Δ157-158, L452R, and E484Q, suggesting a combinatorial effect of these mutations. Notwithstanding, the plasma from a recovered individual robustly inhibited mutant spike PV, indicating the increased breadth of neutralization post-recovery. Our data highlights the importance of spike NTD-specific changes in determining infectivity and immune escape of variants.

Divergence of delta and beta variants and SARS-CoV-2 evolved in prolonged infection into distinct serological phenotypes

SARS-CoV-2 continues to evolve variants of concern (VOC) which escape antibody neutralization and have enhanced transmission. One variant may escape immunity elicited by another, and the delta VOC has been reported to escape beta elicited immunity. Systematic mapping of the serological distance of current and emerging variants will likely guide the design of vaccines which can target all variants. Here we isolated and serologically characterized SARS-CoV-2 which evolved from an ancestral strain in a person with advanced HIV disease and delayed SARS-CoV-2 clearance. This virus showed evolving escape from self antibody neutralization immunity and decreased Pfizer BNT162b2 vaccine neutralization sensitivity. We mapped neutralization of evolved virus and ancestral, beta and delta variant viruses by antibodies elicited by each VOC in SARS-CoV-2 convalescent individuals. Beta virus showed moderate (7-fold) and delta slight escape from neutralizing immunity elicited by ancestral virus infection. In contrast, delta virus had stronger escape from beta elicited immunity (12-fold), and beta virus even stronger escape from delta immunity (34-fold). Evolved virus had 9-fold escape from ancestral immunity, 27-fold escape from delta immunity, but was effectively neutralized by beta immunity. We conclude that beta and delta are serologically distant, further than each is from ancestral, and that virus evolved in prolonged infection during advanced HIV disease is serologically close to beta and far from delta. These results suggest that SARS-CoV-2 is diverging into distinct serological phenotypes and that vaccines tailored to one variant may become vulnerable to infections with another.

The continuous evolution of SARS-CoV-2 in South Africa: a new lineage with rapid accumulation of mutations of concern and global detection

SARS-CoV-2 variants of interest have been associated with increased transmissibility, neutralization resistance and disease severity. Ongoing SARS-CoV-2 genomic surveillance world-wide has improved our ability to rapidly identify such variants. Here we report the identification of a potential variant of interest assigned to the PANGO lineage C.1.2. This lineage was first identified in May 2021 and evolved from C.1, one of the lineages that dominated the first wave of SARS-CoV-2 infections in South Africa and was last detected in January 2021. C.1.2 has since been detected across the majority of the provinces in South Africa and in seven other countries spanning Africa, Europe, Asia and Oceania. The emergence of C.1.2 was associated with an increased substitution rate, as was previously observed with the emergence of the Alpha, Beta and Gamma variants of concern (VOCs). C.1.2 contains multiple substitutions (R190S, D215G, N484K, N501Y, H655Y and T859N) and deletions (Y144del, L242-A243del) within the spike protein, which have been observed in other VOCs and are associated with increased transmissibility and reduced neutralization sensitivity. Of greater concern is the accumulation of additional mutations (C136F, Y449H and N679K) which are also likely to impact neutralization sensitivity or furin cleavage and therefore replicative fitness. While the phenotypic characteristics and epidemiology of C.1.2 are being defined, it is important to highlight this lineage given its concerning constellations of mutations.

Different Neutralization Sensitivity of SARS-CoV-2 Cell-to-Cell and Cell-Free Modes of Infection to Convalescent Sera

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. These findings suggest that the cell-to-cell mode of SARS-CoV-2 transmission, for which the mechanisms are largely unknown, could be of great importance for treatment and prevention of COVID-19.

Virus Evolution and Neutralization Sensitivity in an HIV-1 Subtype B′ Infected Plasma Donor with Broadly Neutralizing Activity

We sought to analyze the evolutionary characteristics and neutralization sensitivity of viruses in a human immunodeficiency virus type 1 (HIV-1) subtype B′ infected plasma donor with broadly neutralizing activity, which may provide information for new broadly neutralizing antibodies (bNAbs) isolation and immunogen design. A total of 83 full-length envelope genes were obtained by single-genome amplification (SGA) from the patient’s plasma at three consecutive time points (2005, 2006, and 2008) spanning four years. In addition, 28 Env-pseudotyped viruses were constructed and their neutralization sensitivity to autologous plasma and several representative bNAbs were measured. Phylogenetic analysis showed that these env sequences formed two evolutionary clusters (Cluster I and II). Cluster I viruses vanished in 2006 and then appeared as recombinants two years later. In Cluster II viruses, the V1 length and N-glycosylation sites increased over the four years of the study period. Most viruses were sensitive to concurrent and subsequent autologous plasma, and to bNAbs, including 10E8, PGT121, VRC01, and 12A21, but all viruses were resistant to PGT135. Overall, 90% of Cluster I viruses were resistant to 2G12, while 94% of Cluster II viruses were sensitive to 2G12. We confirmed that HIV-1 continued to evolve even in the presence of bNAbs, and two virus clusters in this donor adopted different escape mechanisms under the same humoral immune pressure.

SARS CoV-2 escape variants exhibit differential infectivity and neutralization sensitivity to convalescent or post-vaccination sera

SUMMARYTowards eradicating COVID19, developing vaccines that induce high levels of neutralizing antibodies is a main goal. As counter measurements, viral escape mutants rapidly emerge and potentially compromise vaccine efficiency. Herein we monitored ability of convalescent or Pfizer-BTN162b2 post-vaccination sera to neutralize wide-type SARS-CoV2 or its UK-B.1.1.7 and SA-B.1.351 variants. Relative to convalescent sera, post-vaccination sera exhibited higher levels of neutralizing antibodies against wild-type or mutated viruses. However, while SARS-CoV2 wild-type and UK-N501Y were similarly neutralized by tested sera, the SA-N501Y/K417N/E484K variant moderately escaped neutralization. Significant contribution to infectivity and sensitivity to neutralization was attributed to each of the variants and their single or combined mutations, highlighting alternative mechanisms by which prevalent variants with either N501Y or E484K/K417N mutations spread. Our study validates the clinical significance of currently administered vaccines, but emphasizes that their efficacy may be compromised by circulated variants, urging the development of new ones with broader neutralization functions.