Human and hamster sera correlate well in identifying antigenic drift among SARS-CoV-2 variants, including JN.1.

Antigenic assessments of SARS-CoV-2 variants inform decisions to update COVID-19 vaccines. Primary infection sera are often used for assessments, but such sera are rare due to population immunity from SARS-CoV-2 infections and COVID-19 vaccinations. Here, we show that neutralization titers and breadth of matched human and hamster pre-Omicron variant primary infection sera correlate well and generate similar antigenic maps. The hamster antigenic map shows modest antigenic drift among XBB sub-lineage variants, with JN.1 and BA.4/BA.5 variants within the XBB cluster, but with fivefold to sixfold antigenic differences between these variants and XBB.1.5. Compared to sera following only ancestral or bivalent COVID-19 vaccinations, or with post-vaccination infections, XBB.1.5 booster sera had the broadest neutralization against XBB sub-lineage variants, although a fivefold titer difference was still observed between JN.1 and XBB.1.5 variants. These findings suggest that antibody coverage of antigenically divergent JN.1 could be improved with a matched vaccine antigen.IMPORTANCEUpdates to COVID-19 vaccine antigens depend on assessing how much vaccine antigens differ antigenically from newer SARS-CoV-2 variants. Human sera from single variant infections are ideal for discriminating antigenic differences among variants, but such primary infection sera are now rare due to high population immunity. It remains unclear whether sera from experimentally infected animals could substitute for human sera for antigenic assessments. This report shows that neutralization titers of variant-matched human and hamster primary infection sera correlate well and recognize variants similarly, indicating that hamster sera can be a proxy for human sera for antigenic assessments. We further show that human sera following an XBB.1.5 booster vaccine broadly neutralized XBB sub-lineage variants but titers were fivefold lower against the more recent JN.1 variant. These findings support updating the current COVID-19 vaccine variant composition and developing a framework for assessing antigenic differences in future variants using hamster primary infection sera.

Effects of N-glycan modifications on spike expression, virus infectivity, and neutralization sensitivity in ancestral compared to Omicron SARS-CoV-2 variants.

The SARS-CoV-2 spike glycoprotein has 22 potential N-linked glycosylation sites per monomer that are highly conserved among diverse variants, but how individual glycans affect virus entry and neutralization of Omicron variants has not been extensively characterized. Here we compared the effects of specific glycan deletions or modifications in the Omicron BA.1 and D614G spikes on spike expression, processing, and incorporation into pseudoviruses, as well as on virus infectivity and neutralization by therapeutic antibodies. We found that loss of potential glycans at spike residues N717 and N801 each conferred a loss of pseudovirus infectivity for Omicron but not for D614G or Delta variants. This decrease in infectivity correlated with decreased spike processing and incorporation into Omicron pseudoviruses. Oligomannose-enriched Omicron pseudoviruses generated in GnTI- cells or in the presence of kifunensine were non-infectious, whereas D614G or Delta pseudoviruses generated under similar conditions remained infectious. Similarly, growth of live (authentic) SARS-CoV-2 in the presence of kifunensine resulted in a greater reduction of titers for the BA.1.1 variant than Delta or D614G variants relative to their respective, untreated controls. Finally, we found that loss of some N-glycans, including N343 and N234, increased the maximum percent neutralization by the class 3 S309 monoclonal antibody against D614G but not BA.1 variants, while these glycan deletions altered the neutralization potency of the class 1 COV2-2196 and Etesevimab monoclonal antibodies without affecting maximum percent neutralization. The maximum neutralization by some antibodies also varied with the glycan composition, with oligomannose-enriched pseudoviruses conferring the highest percent neutralization. These results highlight differences in the interactions between glycans and residues among SARS-CoV-2 variants that can affect spike expression, virus infectivity, and susceptibility of variants to antibody neutralization.

D-dimer and CoV-2 spike-immune complexes contribute to the production of PGE2 and proinflammatory cytokines in monocytes.

An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8. The DD-induced PGE2 and inflammatory cytokines were enhanced significantly by co-treatment with immune complexes (IC) of SARS CoV-2 recombinant S protein or of pseudovirus containing SARS CoV-2 S protein (PVCoV-2) coated with spike-specific chimeric monoclonal antibody (MAb) containing mouse variable and human Fc regions. The production of PGE2 and cytokines in monocytes activated with DD and ICs was sensitive to the inhibitors of ß2 integrin and FcγRIIa, and to the inhibitors of calcium signaling, Mitogen-Activated Protein Kinase (MAPK) pathway, and tyrosine-protein kinase. Importantly, strong increase in PGE2 and in IL-6/IL-8/IL-1ß cytokines was observed in monocytes activated with DD in the presence of IC of PVCoV-2 coated with plasma from hospitalized COVID-19 patients but not from healthy donors. The IC of PVCoV-2 with convalescent plasma induced much lower levels of PGE2 and cytokines compared with plasma from hospitalized COVID-19 patients. PGE2 and IL-6/IL-8 cytokines produced in monocytes activated with plasma-containing IC, correlated well with the levels of spike binding antibodies and not with neutralizing antibody titers. Our study suggests that a combination of high levels of DD and high titers of spike-binding antibodies that can form IC with SARS CoV-2 viral particles might accelerate the inflammatory status of lung infiltrating monocytes leading to increased lung pathology in patients with severe form of COVID-19.

Bivalent Coronavirus Disease 2019 Vaccine Antibody Responses to Omicron Variants Suggest That Responses to Divergent Variants Would Be Improved With Matched Vaccine Antigens.

We compared neutralizing antibody responses to BA.4/5, BQ.1.1, XBB, and XBB.1.5 Omicron severe acute respiratory syndrome coronavirus 2 variants after a bivalent or ancestral coronavirus disease 2019 (COVID-19) messenger RNA booster vaccine or postvaccination infection. We found that the bivalent booster elicited moderately high antibody titers against BA.4/5 that were approximately 2-fold higher against all Omicron variants than titers elicited by the monovalent booster. The bivalent booster elicited low but similar titers against both XBB and XBB.1.5 variants. These findings inform risk assessments for future COVID-19 vaccine recommendations and suggest that updated COVID-19 vaccines containing matched vaccine antigens to circulating divergent variants may be needed.

Characterization of Entry Pathways, Species-Specific Angiotensin-Converting Enzyme 2 Residues Determining Entry, and Antibody Neutralization Evasion of Omicron BA.1, BA.1.1, BA.2, and BA.3 Variants.

The SARS-CoV-2 Omicron variants were first detected in November 2021, and several Omicron lineages (BA.1, BA.2, BA.3, BA.4, and BA.5) have since rapidly emerged. Studies characterizing the mechanisms of Omicron variant infection and sensitivity to neutralizing antibodies induced upon vaccination are ongoing by several groups. In the present study, we used pseudoviruses to show that the transmembrane serine protease 2 (TMPRSS2) enhances infection of BA.1, BA.1.1, BA.2, and BA.3 Omicron variants to a lesser extent than ancestral D614G. We further show that Omicron variants have higher sensitivity to inhibition by soluble angiotensin-converting enzyme 2 (ACE2) and the endosomal inhibitor chloroquine compared to D614G. The Omicron variants also more efficiently used ACE2 receptors from 9 out of 10 animal species tested, and unlike the D614G variant, used mouse ACE2 due to the Q493R and Q498R spike substitutions. Finally, neutralization of the Omicron variants by antibodies induced by three doses of Pfizer/BNT162b2 mRNA vaccine was 7- to 8-fold less potent than the D614G. These results provide insights into the transmissibility and immune evasion capacity of the emerging Omicron variants to curb their ongoing spread. IMPORTANCE The ongoing emergence of SARS-CoV-2 Omicron variants with an extensive number of spike mutations poses a significant public health and zoonotic concern due to enhanced transmission fitness and escape from neutralizing antibodies. We studied three Omicron lineage variants (BA.1, BA.2, and BA.3) and found that transmembrane serine protease 2 has less influence on Omicron entry into cells than on D614G, and Omicron exhibits greater sensitivity to endosomal entry inhibition compared to D614G. In addition, Omicron displays more efficient usage of diverse animal species ACE2 receptors than D614G. Furthermore, due to Q493R/Q498R substitutions in spike, Omicron, but not D614G, can use the mouse ACE2 receptor. Finally, three doses of Pfizer/BNT162b2 mRNA vaccination elicit high neutralization titers against Omicron variants, although the neutralization titers are still 7- to 8-fold lower those that against D614G. These results may give insights into the transmissibility and immune evasion capacity of the emerging Omicron variants to curb their ongoing spread.

Key Substitutions in the Spike Protein of SARS-CoV-2 Variants Can Predict Resistance to Monoclonal Antibodies, but Other Substitutions Can Modify the Effects.

Mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can compromise the effectiveness of therapeutic antibodies. Most clinical-stage therapeutic antibodies target the spike receptor binding domain (RBD), but variants often have multiple mutations in several spike regions. To help predict antibody potency against emerging variants, we evaluated 25 clinical-stage therapeutic antibodies for neutralization activity against 60 pseudoviruses bearing spikes with single or multiple substitutions in several spike domains, including the full set of substitutions in B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.429 (epsilon), B.1.526 (iota), A.23.1, and R.1 variants. We found that 14 of 15 single antibodies were vulnerable to at least one RBD substitution, but most combination and polyclonal therapeutic antibodies remained potent. Key substitutions in variants with multiple spike substitutions predicted resistance, but the degree of resistance could be modified in unpredictable ways by other spike substitutions that may reside outside the RBD. These findings highlight the importance of assessing antibody potency in the context of all substitutions in a variant and show that epistatic interactions in spike can modify virus susceptibility to therapeutic antibodies. IMPORTANCE Therapeutic antibodies are effective in preventing severe disease from SARS-CoV-2 infection (COVID-19), but their effectiveness may be reduced by virus variants with mutations affecting the spike protein. To help predict resistance to therapeutic antibodies in emerging variants, we profiled resistance patterns of 25 antibody products in late stages of clinical development against a large panel of variants that include single and multiple substitutions found in the spike protein. We found that the presence of a key substitution in variants with multiple spike substitutions can predict resistance against a variant but that other substitutions can affect the degree of resistance in unpredictable ways. These findings highlight complex interactions among substitutions in the spike protein affecting virus neutralization and, potentially, virus entry into cells.

Durability of Antibody Response and Frequency of SARS-CoV-2 Infection 6 Months after COVID-19 Vaccination in Healthcare Workers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies decay but persist 6 months postvaccination; lower levels of neutralizing titers persist against Delta than wild-type virus. Of 227 vaccinated healthcare workers tested, only 2 experienced outpatient symptomatic breakthrough infections, despite 59/227 exhibiting serologic evidence of SARS-CoV-2 infection, defined as presence of nucleocapsid protein antibodies.

Comparison of A(H3N2) Neutralizing Antibody Responses Elicited by 2018-2019 Season Quadrivalent Influenza Vaccines Derived from Eggs, Cells, and Recombinant Hemagglutinin.

BACKGROUND: Low vaccine effectiveness against A(H3N2) influenza in seasons with little antigenic drift has been attributed to substitutions in hemagglutinin (HA) acquired during vaccine virus propagation in eggs. Clinical trials comparing recombinant HA vaccine (rHA) and cell-derived inactivated influenza vaccine (IIV) to egg-derived IIVs provide opportunities to assess how egg-adaptive substitutions influence HA immunogenicity. METHODS: Neutralization titers in pre- and postimmunization sera from 133 adults immunized with 1 of 3 types of influenza vaccines in a randomized, open-label trial during the 2018-2019 influenza season were measured against egg- and cell-derived A/Singapore/INFIMH-16-0019/2016-like and circulating A(H3N2) influenza viruses using HA pseudoviruses. RESULTS: All vaccines elicited neutralizing antibodies to all H3 vaccine antigens, but the rHA vaccine elicited the highest titers and seroconversion rates against all strains tested. Egg- and cell-derived IIVs elicited responses similar to each other. Preimmunization titers against H3 HA pseudoviruses containing egg-adaptive substitutions T160K and L194P were high, but lower against H3 HA pseudoviruses without those substitutions. All vaccines boosted neutralization titers against HA pseudoviruses with egg-adaptive substitutions, but poorly neutralized wild-type 2019-2020 A/Kansas/14/2017 (H3N2) HA pseudoviruses. CONCLUSION: Egg- and cell-derived 2018-2019 season influenza vaccines elicited similar neutralization titers and response rates, indicating that the cell-derived vaccine did not improve immunogenicity against the A(H3N2) viruses. The higher responses after rHA vaccination may be due to its higher HA content. All vaccines boosted titers to HA with egg-adaptive substitutions, suggesting boosting from past antigens or better exposure of HA epitopes. Studies comparing immunogenicity and effectiveness of different influenza vaccines across many seasons are needed.

Neutralizing and Neuraminidase Antibodies Correlate With Protection Against Influenza During a Late Season A/H3N2 Outbreak Among Unvaccinated Military Recruits.

BACKGROUND: Antibodies that inhibit hemagglutination have long been considered a correlate of protection against influenza, but these antibodies are only a subset of potentially protective antibodies. Neutralizing and neuraminidase antibodies may also contribute to protection, but data on their associations with protection are limited. METHODS: We measured preoutbreak hemagglutinin pseudovirus neutralization (PVN) and neuraminidase inhibition (NAI) antibody titers in unvaccinated military recruits who experienced an H3N2 influenza outbreak during training. We conducted a case-control study to investigate the association between titers and protection against influenza illness or H3N2-associated pneumonia using logistic regression. RESULTS: With every 2-fold increase in PVN titer, the odds of medically attended polymerase chain reaction-confirmed H3N2 infection (H3N2+) decreased by 41% (odds ratio [OR], 0.59; 95% confidence interval [CI], .45 to .77; P < .001). Among those who were H3N2+, the odds for pneumonia decreased by 52% (OR, 0.48; CI, .25 to .91; P = .0249). With every 2-fold increase in NAI titer, the odds of medically attended H3N2 infection decreased by 32% (OR, 0.68; 95% CI, .53 to .87; P = .0028), but there was no association between NAI titers and H3N2-associated pneumonia. There was also no synergistic effect of PVN and NAI antibodies. CONCLUSIONS: PVN and NAI titers were independently associated with reduced risk of influenza illness. NAI titers associated with protection had greater breadth of reactivity to drifted strains than PVN titers. These findings show that PVN and NAI titers are valuable biomarkers for assessing the odds of influenza infection.

Mutations That Increase the Stability of the Postfusion gp41 Conformation of the HIV-1 Envelope Glycoprotein Are Selected by both an X4 and R5 HIV-1 Virus To Escape Fusion Inhibitors Corresponding to Heptad Repeat 1 of gp41, but the gp120 Adaptive Mutations Differ between the Two Viruses.

Binding of the gp120 surface subunit of the envelope glycoprotein (Env) of HIV-1 to CD4 and chemokine receptors on target cells triggers refolding of the gp41 transmembrane subunit into a six-helix bundle (6HB) that promotes fusion between virus and host cell membranes. To elucidate details of Env entry and potential differences between viruses that use CXCR4 (X4) or CCR5 (R5) coreceptors, we generated viruses that are resistant to peptide fusion inhibitors corresponding to the first heptad repeat region (HR1) of gp41 that target fusion-intermediate conformations of Env. Previously we reported that an R5 virus selected two resistance pathways, each defined by an early gp41 resistance mutation in either HR1 or the second heptad repeat (HR2), to escape inhibition by an HR1 peptide, but preferentially selected the HR1 pathway to escape inhibition by a trimer-stabilized HR1 peptide. Here, we report that an X4 virus selected the same HR1 and HR2 resistance pathways as the R5 virus to escape inhibition by the HR1 peptide. However, in contrast to the R5 virus, the X4 virus selected a unique mutation in HR2 to escape inhibition by the trimer-stabilized peptide. Significantly, both of these X4 and R5 viruses acquired gp41 resistance mutations that improved the thermostability of the six-helix bundle, but they selected different gp120 adaptive mutations. These findings show that these X4 and R5 viruses use a similar resistance mechanism to escape from HR1 peptide inhibition but different gp120-gp41 interactions to regulate Env conformational changes.IMPORTANCE HIV-1 fuses with cells when the gp41 subunit of Env refolds into a 6HB after binding to cellular receptors. Peptides corresponding to HR1 or HR2 interrupt gp41 refolding and inhibit HIV infection. Previously, we found that a CCR5 coreceptor-tropic HIV-1 acquired a key HR1 or HR2 resistance mutation to escape HR1 peptide inhibitors but only the key HR1 mutation to escape a trimer-stabilized HR1 peptide inhibitor. Here, we report that a CXCR4 coreceptor-tropic HIV-1 selected the same key HR1 or HR2 mutations to escape inhibition by the HR1 peptide but different combinations of HR1 and HR2 mutations to escape the trimer-stabilized HR1 peptide. All gp41 mutations enhance 6HB stability to outcompete inhibitors, but gp120 adaptive mutations differed between these R5 and X4 viruses, providing new insights into gp120-gp41 functional interactions affecting Env refolding during HIV entry.