Immune Evasion, Infectivity, and Fusogenicity of SARS-CoV-2 Omicron BA.2.86 and FLip Variants (preprint)

Evolution of SARS-CoV-2 requires the reassessment of current vaccine measures. Here, we characterized BA.2.86 and the XBB-lineage variant FLip by investigating their neutralization alongside D614G, BA.1, BA.2, BA.4/5, XBB.1.5, and EG.5.1 by sera from 3-dose vaccinated and bivalent vaccinated healthcare workers, XBB.1.5-wave infected first responders, and monoclonal antibody (mAb) S309. We assessed the biology of the variant Spikes by measuring viral infectivity and membrane fusogenicity. BA.2.86 is less immune evasive compared to FLip and other XBB variants, consistent with antigenic distances. Importantly, distinct from XBB variants, mAb S309 was unable to neutralize BA.2.86, likely due to a D339H mutation based on modeling. BA.2.86 had relatively high fusogenicity and infectivity in CaLu-3 cells but low fusion and infectivity in 293T-ACE2 cells compared to some XBB variants, suggesting a potentially differences conformational stability of BA.2.86 Spike. Overall, our study underscores the importance of SARS-CoV-2 variant surveillance and the need for updated COVID-19 vaccines.

Immune Evasion and Membrane Fusion of SARS-CoV-2 XBB Subvariants EG.5.1 and XBB.2.3 (preprint)

Immune evasion by SARS-CoV-2 paired with immune imprinting from monovalent mRNA vaccines has resulted in attenuated neutralizing antibody responses against Omicron subvariants. In this study, we characterized two new XBB variants rising in circulation: EG.5.1 and XBB.2.3, for their ability of neutralization and syncytia formation. We determined the neutralizing antibody in sera of individuals that received a bivalent mRNA vaccine booster, BA.4/5 wave infection, or XBB.1.5 wave infection. Bivalent vaccination-induced antibodies neutralized efficiently ancestral D614G, but to a much less extent, two new EG.5.1 and XBB.2.3 variants. In fact, the enhanced neutralization escape of EG.5.1 appeared to be driven by its key defining mutation XBB.1.5-F456L. Notably, infection by BA.4/5 or XBB.1.5 afforded little, if any, neutralization against EG.5.1, XBB.2.3 and previous XBB variants, especially in unvaccinated individuals, with average neutralizing antibody titers near the limit of detection. Additionally, we investigated the infectivity, fusion activity, and processing of variant spikes for EG.5.1 and XBB.2.3 in HEK293T-ACE2 and CaLu-3 cells, but found no significant differences compared to earlier XBB variants. Overall, our findings highlight the continued immune evasion of new Omicron subvariants and, more importantly, the need to reformulate mRNA vaccines to include XBB spikes for better protection.

Extraordinary Evasion of Neutralizing Antibody Response by Omicron XBB.1.5, CH.1.1 and CA.3.1 Variants (preprint)

Newly emerging Omicron subvariants continue to emerge around the world, presenting potential challenges to current vaccination strategies. This study investigates the extent of neutralizing antibody escape by new subvariants XBB.1.5, CH.1.1, and CA.3.1, as well as their impacts on spike protein biology. Our results demonstrated a nearly complete escape of these variants from neutralizing antibodies stimulated by three doses of mRNA vaccine, but neutralization was rescued by a bivalent booster. However, CH.1.1 and CA.3.1 variants were highly resistant to both monovalent and bivalent mRNA vaccinations. We also assessed neutralization by sera from individuals infected during the BA.4/5 wave of infection and observed similar trends of immune escape. In these cohorts, XBB.1.5 did not exhibit enhanced neutralization resistance over the recently dominant BQ.1.1 variant. Notably, the spike proteins of XBB.1.5, CH.1.1, and CA.3.1 all exhibited increased fusogenicity compared to BA.2, correlating with enhanced S processing. Overall, our results support the administration of new bivalent mRNA vaccines, especially in fighting against newly emerged Omicron subvariants, as well as the need for continued surveillance of Omicron subvariants.

Distinct Neutralizing Antibody Escape of SARS-CoV-2 Omicron Subvariants BQ.1, BQ.1.1, BA.4.6, BF.7 and BA.2.75.2 (preprint)

Continued evolution of SARS-CoV-2 has led to the emergence of several new Omicron subvariants including BQ.1, BQ.1.1, BA.4.6, BF.7 and BA.2.75.2. Here we examine the neutralization resistance of these subvariants as well as their ancestral BA.4/5, BA.2.75 and D614G variants against sera from 3-dose vaccinated health care workers, hospitalized BA.1-wave patients, and BA.5-wave patients. We found enhanced neutralization resistance in all new subvariants, especially the BQ.1 and BQ.1.1 subvariants driven by a key N460K mutation and to a lesser extent, R346T and K444T mutations, as well as the BA.2.75.2 subvariant driven largely by its key F486S mutation. The BQ.1 and BQ.1.1 subvariants also exhibited enhanced fusogenicity and S processing dictated by the key N460K mutation. Interestingly, the BA.2.75.2 subvariant saw an enhancement by the F486S mutation and a reduction by the D1199N mutation to its fusogenicity and S processing resulting in minimal overall change. Molecular modelling revealed the mechanisms of receptor-binding and non-receptor binding monoclonal antibody-mediated immune evasion by R346T, K444T, F486S and D1199N mutations. Altogether, these findings shed light on the concerning evolution of newly emerging SARS-CoV-2 Omicron subvariants.

Determinants and Mechanisms of the Low Fusogenicity and Endosomal Entry of Omicron Subvariants (preprint)

The rapid spread and strong immune evasion of the SARS-CoV-2 Omicron subvariants has raised serious concerns for the global COVID-19 pandemic. These new variants exhibit reduced fusogenicity and increased endosomal entry pathway utilization compared to the ancestral D614G variant, the underlying mechanisms of which remain elusive. Here we show that the C-terminal S1 mutations of the BA.1.1 subvariant, H655Y and T547K, critically govern the low fusogenicity of Omicron. Notably, H655Y also dictates the enhanced endosome entry pathway utilization. Mechanistically, T547K and H655Y likely stabilize the spike trimer conformation, as shown by increased molecular interactions in structural modeling as well as reduced S1 shedding. Importantly, the H655Y mutation also determines the low fusogenicity and high dependence on the endosomal entry pathway of other Omicron subvariants, including BA.2, BA.2.12.1, BA.4/5 and BA.2.75. These results uncover mechanisms governing Omicron subvariant entry and provide insights into altered Omicron tissue tropism and pathogenesis.

Evasion of Neutralizing Antibody Response by the SARS-CoV-2 BA.2.75 Variant (preprint)

The newly emerged BA.2.75 SARS-CoV-2 variant exhibits an alarming 9 additional mutations in its spike (S) protein compared to the ancestral BA.2 variant. Here we examine the neutralizing antibody escape of BA.2.75 in mRNA-vaccinated and BA.1-infected individuals, as well as the molecular basis underlying functional changes in the S protein. Notably, BA.2.75 exhibits enhanced neutralization resistance over BA.2, but less than the BA.4/5 variant. The G446S and N460K mutations of BA.2.75 are primarily responsible for its enhanced resistance to neutralizing antibodies. The R493Q mutation, a reversion to the prototype sequence, reduces BA.2.75 neutralization resistance. The mutational impact is consistent with their locations in common neutralizing antibody epitopes. Further, the BA.2.75 variant shows enhanced cell-cell fusion over BA.2, driven largely by the N460K mutation, which enhances S processing. Structural modeling revealed a new receptor contact introduced by N460K, supporting a mechanism of potentiated receptor utilization and syncytia formation.

Durability of the Neutralizing Antibody Response to mRNA Booster Vaccination Against SARS-CoV-2 BA.2.12.1 and BA.4/5 Variants (preprint)

The recent emergence of the SARS-CoV-2 BA.4/5 and BA.2.12.1 variants has led to rising COVID-19 case numbers and concerns over the continued efficacy of mRNA booster vaccination. Here we examine the durability of neutralizing antibody (nAb) responses against these SARS-CoV-2 Omicron subvariants in a cohort of health care workers 1-40 weeks after mRNA booster dose administration. Neutralizing antibody titers fell by ~1.5-fold 4-6 months and by ~2.5-fold 7-9 months after booster dose, with average nAb titers falling by 11-15% every 30 days, far more stable than two dose induced immunity. Notably, nAb titers from booster recipients against SARS-CoV-2 BA.1, BA.2.12.1, and BA.4/5 variants were ~4.7-, 7.6-, and 13.4-fold lower than against the ancestral D614G spike. However, the rate of waning of booster dose immunity was comparable across variants. Importantly, individuals reporting prior infection with SARS-CoV-2 exhibited significantly higher nAb titers compared to those without breakthrough infection. Collectively, these results highlight the broad and stable neutralizing antibody response induced by mRNA booster dose administration, implicating a significant role of virus evolution to evade nAb specificity, versus waning humoral immunity, in increasing rates of breakthrough infection.

Differential Evasion of Delta and Omicron Immunity and Enhanced Fusogenicity of SARS-CoV-2 Omicron BA.4/5 and BA.2.12.1 Subvariants (preprint)

The rising case numbers of the SARS-CoV-2 Omicron BA.4, BA.5, and BA.2.12.1 subvariants has generated serious concern about the course of the pandemic. Here we examine the neutralization resistance, infectivity, processing, and fusogenicity of spike from the BA.4/5 and BA.2.12.1 SARS-CoV-2 variants compared with other Omicron subvariants and Delta. Critically, we found that the new Omicron subvariants BA.4/5 and BA.2.12.1 were more resistant to neutralization by mRNA-vaccinated and boosted health care worker sera and Omicron-BA.1-wave patient sera than were the BA.1 and BA.2 variants. Interestingly, Delta-wave patient sera neutralized more efficiently against not only Delta but also BA.4/5 and BA.2.12.1 variants that also contain substitutions at position L452, similar to Delta. The BA.4/5 and BA.2.12.1 variants also exhibited higher fusogenicity, and increased spike processing, dependent on the L452 substitution. These results highlight the key role of the L452R and L452Q mutations in BA.4/5 and BA.2.12.1 subvariants.

mRNA Booster Vaccines Elicit Strong Protection Against SARS-CoV-2 Omicron Variant in Cancer Patients (preprint)

Following its emergence in late November of 2020, the SARS-CoV-2 Omicron (B.1.1.529) variant has caused major global public health concerns. We recently demonstrated that in healthy adults the Omicron variant exhibits strong resistance to immunity induced by two doses of the mRNA vaccines, but a booster mRNA vaccine dose can provide strong protection against Omicron. However, it is currently unknown how well these mRNA vaccine boosters protect immunocompromised groups, including cancer patients, from the Omicron variant. Here we show that (1) neutralizing antibody (nAb) titers against the Delta and Omicron variants in cancer patients after two-dose mRNA vaccines are 4.2-fold and 21.3-fold lower, respectively, compared to the ancestral D614G, and (2) nAb titers against the Delta and Omicron variants in boosted cancer patients are 3.6-fold and 5.1-fold lower, respectively, compared to D614G. Our findings highlight the effectiveness and need for booster vaccination strategies in immunocompromised groups including cancer patients to protect from the Omicron variant.

Neutralization and Stability of SARS-CoV-2 Omicron Variant (preprint)

The SARS-CoV-2 B.1.1.529/Omicron variant was first characterized in South Africa and was swiftly designated a variant of concern. Of great concern is its high number of mutations, including 30-40 mutations in the virus spike (S) protein compared to 7-10 for other variants. Some of these mutations have been shown to enhance escape from vaccine-induced immunity, while others remain uncharacterized. Additionally, reports of increasing frequencies of the Omicron variant may indicate a higher rate of transmission compared to other variants. However, the transmissibility of Omicron and its degree of resistance to vaccine-induced immunity remain unclear. Here we show that Omicron exhibits significant immune evasion compared to other variants, but antibody neutralization is largely restored by mRNA vaccine booster doses. Additionally, the Omicron spike exhibits reduced receptor binding, cell-cell fusion, S1 subunit shedding, but increased cell-to-cell transmission, and homology modeling indicates a more stable closed S structure. These findings suggest dual immune evasion strategies for Omicron, due to altered epitopes and reduced exposure of the S receptor binding domain, coupled with enhanced transmissibility due to enhanced S protein stability. These results highlight the importance of booster vaccine doses for maintaining protection against the Omicron variant, and provide mechanistic insight into the altered functionality of the Omicron spike protein.

Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers and convalescent plasma donors: a cohort study using a rapid and sensitive high-throughput neutralization assay (preprint)

Rapid and specific antibody testing is crucial for improved understanding, control, and treatment of COVID-19 pathogenesis. Herein, we describe and apply a rapid, sensitive, and accurate virus neutralization assay for SARS-CoV-2 antibodies. The new assay is based on an HIV-1 lentiviral vector that contains a secreted intron Gaussia luciferase or secreted Nano-luciferase reporter cassette, pseudotyped with the SARS-CoV-2 spike (S) glycoprotein, and is validated with a plaque reduction assay using an authentic, infectious SARS-CoV-2 strain. The new assay was used to evaluate SARS-CoV-2 antibodies in serum from individuals with a broad range of COVID-19 symptoms, including intensive care unit (ICU) patients, health care workers (HCWs), and convalescent plasma donors. The highest neutralizing antibody titers were observed among ICU patients, followed by general hospitalized patients, HCWs and convalescent plasma donors. Our study highlights a wide phenotypic variation in human antibody responses against SARS-CoV-2, and demonstrates the efficacy of a novel lentivirus pseudotype assay for high-throughput serological surveys of neutralizing antibody titers in large cohorts.