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.

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.

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.

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.

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.

Neutralizing Antibody Responses to Homologous and Heterologous H1 and H3 Influenza A Strains After Vaccination With Inactivated Trivalent Influenza Vaccine Vary With Age and Prior-year Vaccination.

BACKGROUND: Prior influenza immunity influences the homologous neutralizing antibody responses elicited by inactivated influenza vaccines (IIV), but neutralizing antibody responses to heterologous strains have not been extensively characterized. METHODS: We analyzed neutralizing antibody titers in individuals aged 1-88 who received the 2009-2010 season IIV before infection by or vaccination against the 2009 pandemic H1N1 virus. Neutralization titers to homologous and heterologous past, recent, and advanced H1 and H3 strains, as well as H2, H5, and H7 strains, were measured using influenza hemagglutinin pseudoviruses. We performed exploratory analyses based on age, prior-year IIV, and prevaccination titer, without controlling for Type I errors. RESULTS: IIV elicited neutralizing antibodies to past and advanced H1 and H3 strains, as well as to an H2 strain in individuals who were likely infected early in life. The neutralization of avian subtype viruses was rare, and there was no imprinting of neutralization responses to novel avian subtype viruses based on the influenza group. Compared to adults, children had higher seroresponse rates to homologous and heterologous strains, and their sera generated larger antigenic distances among strains. Seroresponse rates to homologous and heterologous strains were lower in subjects vaccinated with prior-year IIV, though postimmunization titers were generally high. CONCLUSIONS: IIV elicited neutralizing antibodies to heterologous H1 and H3 strains in all ages groups, but titers and seroresponse rates were usually higher in children. Prior-year vaccination with the same strains tended to blunt IIV neutralization responses to all strains in young and old age groups, yet postimmunization titers were high.

HIV-1 gp41 Residues Modulate CD4-Induced Conformational Changes in the Envelope Glycoprotein and Evolution of a Relaxed Conformation of gp120.

Entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by conformational changes in the envelope glycoprotein (Env) that are triggered by Env binding to cellular CD4 and chemokine receptors. These conformational changes involve the opening of the gp120 surface subunit, exposure of the fusion peptide in the gp41 transmembrane subunit, and refolding of the gp41 N- and C-terminal heptad repeat regions (HR1 and HR2) first into an extended prehairpin intermediate and then into a compact 6-helix bundle (6HB) that facilitates fusion between viral and host cell membranes. Previously, we reported that Envs resistant to HR1 peptide fusion inhibitors acquired key resistance mutations in either HR1 or HR2 that increased 6HB stability. Here, we identify residues in HR1 that contribute not only to fusion inhibitor resistance and 6HB stability but also to reduced reactivity to CD4-induced conformational changes that lead to 6HB formation. While all Envs show increased neutralization sensitivity to mimetic CD4 (mCD4), Envs with either the E560K or Q577R HR1 mutation reduced conformational reactivity to CD4 that resisted viral inactivation and triggering to the 6HB. Using a panel of monoclonal antibodies (mAbs), we further determined that Envs from both HR1 and HR2 resistance pathways exhibit a relaxed trimer conformation due to gp120 adaptive mutations in different regions of Env that segregate by resistance pathway. These findings highlight regions of cross talk between gp120 and gp41 and identify HR1 residues that play important roles in regulating CD4-induced conformational changes in Env.IMPORTANCE Binding of the HIV envelope glycoprotein (Env) to cellular CD4 and chemokine receptors triggers conformational changes in Env that mediate virus entry, but premature triggering of Env conformational changes leads to virus inactivation. Currently, we have a limited understanding of the network of residues that regulate Env conformational changes. Here, we identify residues in HR1 of gp41 that modulate conformational changes in response to gp120 binding to CD4 and show that the mutations in HR1 and HR2 that confer resistance to fusion inhibitors are associated with gp120 mutations in different regions of Env that confer a more open conformation. These findings contribute to our understanding of the regulation of Env conformational changes and efforts to design new entry inhibitors and stable Env vaccine immunogens.

Conformational Stability of the Hemagglutinin of H5N1 Influenza A Viruses Influences Susceptibility to Broadly Neutralizing Stem Antibodies.

Vaccines that elicit broadly neutralizing antibodies to the conserved stem of hemagglutinin (HA) are being developed as universal influenza vaccines that protect against influenza across multiple years. However, different influenza virus strains, even those in the same subtype with identical stem sequences, can vary in susceptibility to broadly neutralizing stem antibodies, and the reasons are not understood. Here we studied potential mechanisms underlying the differing sensitivities of a panel of H5N1 HA pseudoviruses to broadly neutralizing stem antibodies. We found that greater HA conformational stability, as measured by thermal inactivation and pH triggering of conformational changes, correlates with reduced neutralization sensitivity and antibody binding to HA under neutral- and low-pH conditions. Our data indicate that the conformational stability of HA is an important attribute of susceptibility to broadly neutralizing stem antibodies and is influenced by residues outside the stem antibody epitopes.IMPORTANCE The influenza virus surface glycoprotein hemagglutinin (HA) mediates virus attachment and membrane fusion between virus and host cells, allowing the viral core to enter the host cell cytoplasm for replication. Fusion occurs when HA undergoes low-pH-induced-conformational changes during endocytosis. Broadly neutralizing antibodies targeted to the conserved stem region of HA interfere with conformational changes required for fusion. Vaccines that elicit such antibodies are being developed as novel universal influenza vaccines for multiyear protection. We investigated why H5N1 HAs from different strains differ in their sensitivity to broadly neutralizing stem antibodies despite having conserved epitopes. We report that HA conformational stability due to residues outside the antibody binding site accounted for much of the variation in susceptibility to neutralization by stem antibodies. These findings highlight the importance of nonepitope residues in influencing neutralization sensitivity to stem antibodies and the complexities in developing universal vaccines targeting conserved epitopes in the HA stem.

Serum Samples From Middle-aged Adults Vaccinated Annually with Seasonal Influenza Vaccines Cross-neutralize Some Potential Pandemic Influenza Viruses.

We examined serum samples from adults ages 48-64 who received multiple seasonal influenza vaccines from 2004 to 2009 for cross-neutralizing antibodies to potential pandemic strains. Using pseudoviruses bearing various hemagglutinins (HA-pseudoviruses), we found serum neutralization titers (≥160) in 100% against A/Japan/305/1957 (H2N2), 53% against A/Hong Kong/1073/99 (H9N2), 56% against the H3N2 variant A/Indiana/08/11 (H3N2v), 11% against A/Hong Kong/G9/97 (H9N2), and 36% A/chicken/Hong Kong/SF4/01 (H6N1). None had titers >160 to A/Shanghai/2/13 (H7N9) or A/Netherlands/219/03 (H7N7). Thirty-six percent to 0% had neutralization titers to various H5N1 strains. Titers to H9, H6, and H5 HA-pseudoviruses correlated with each other, but not with H3N2v, suggesting group-specific cross-neutralization.

Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity.

UNLABELLED: Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop)-to-helix transition, which releases the fusion peptide at the HA2 N terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that the HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable than the HAs from other seasonal influenza virus strains. Here we show that residue 205 in HA1 and residue 399 in the B loop of HA2 (residue 72, HA2 numbering) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at a higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity. IMPORTANCE: Influenza virus hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, the influenza A virus HA is highly variable, necessitating frequent vaccine changes to match circulating strains. Sequence changes in HA affect not only antigenicity but also HA stability, which has important implications for vaccine production, as well as viral adaptation to hosts. HA from the pandemic 2009 H1N1 influenza A virus is less stable than other recent seasonal influenza virus HAs, but the molecular interactions that contribute to HA stability are not fully understood. Here we identify molecular interactions between specific residues in the surface and transmembrane subunits of HA that help regulate the HA conformational changes needed for HA stability and virus entry. These findings contribute to our understanding of the molecular mechanisms controlling HA function and antigen stability.

Resistance to N-peptide fusion inhibitors correlates with thermodynamic stability of the gp41 six-helix bundle but not HIV entry kinetics.

BACKGROUND: The HIV-1 envelope glycoprotein (Env) undergoes conformational changes that mediate fusion between virus and host cell membranes. These changes involve transient exposure of two heptad-repeat domains (HR1 and HR2) in the gp41 subunit and their subsequent self-assembly into a six-helix bundle (6HB) that drives fusion. Env residues and features that influence conformational changes and the rate of virus entry, however, are poorly understood. Peptides corresponding to HR1 and HR2 (N and C peptides, respectively) interrupt formation of the 6HB by binding to the heptad repeats of a fusion-intermediate conformation of Env, making the peptides valuable probes for studying Env conformational changes. RESULTS: Using a panel of Envs that are resistant to N-peptide fusion inhibitors, we investigated relationships between virus entry kinetics, 6HB stability, and resistance to peptide fusion inhibitors to elucidate how HR1 and HR2 mutations affect Env conformational changes and virus entry. We found that gp41 resistance mutations increased 6HB stability without increasing entry kinetics. Similarly, we show that increased 6HB thermodynamic stability does not correlate with increased entry kinetics. Thus, N-peptide fusion inhibitors do not necessarily select for Envs with faster entry kinetics, nor does faster entry kinetics predict decreased potency of peptide fusion inhibitors. CONCLUSIONS: These findings provide new insights into the relationship between 6HB stability and viral entry kinetics and mechanisms of resistance to inhibitors targeting fusion-intermediate conformations of Env. These studies further highlight how residues in HR1 and HR2 can influence virus entry by altering stability of the 6HB and possibly other conformations of Env that affect rate-limiting steps in HIV entry.

Antigenic cartography using hamster sera identifies SARS-CoV-2 JN.1 evasion seen in human XBB.1.5 booster sera.

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 five to six-fold 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 five-fold 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.

Trimeric, coiled-coil extension on peptide fusion inhibitor of HIV-1 influences selection of resistance pathways.

Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of viral fusion proteins can block infection of viruses in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that drives fusion between viral and host cell membranes. The 6HB of the HIV gp41 (endogenous bundle) consists of an HR1 coiled-coil trimer with grooves lined by antiparallel HR2 helices. HR1 peptides form coiled-coil oligomers that may bind to gp41 HR2 as trimers to form a heterologous 6HB (inhibitor bundle) or to gp41 HR1 as monomers or dimers to form a heterologous coiled coil. To gain insights into mechanisms of Env entry and inhibition by HR1 peptides, we compared resistance to a peptide corresponding to 36 residues in gp41 HR1 (N36) and the same peptide with a coiled-coil trimerization domain fused to its N terminus (IZN36) that stabilizes the trimer and increases inhibitor potency (Eckert, D. M., and Kim, P. S. (2001) Proc. Nat. Acad. Sci. U.S.A. 98, 11187-11192). Whereas N36 selected two genetic pathways with equal probability, each defined by an early mutation in either HR1 or HR2, IZN36 preferentially selected the HR1 pathway. Both pathways conferred cross-resistance to both peptides. Each HR mutation enhanced the thermostability of the endogenous 6HB, potentially allowing the virus to simultaneously escape inhibitors targeting either gp41 HR1 or HR2. These findings inform inhibitor design and identify regions of plasticity in the highly conserved gp41 that modulate virus entry and escape from HR1 peptide inhibitors.

Cross-neutralizing antibodies to pandemic 2009 H1N1 and recent seasonal H1N1 influenza A strains influenced by a mutation in hemagglutinin subunit 2.

Pandemic 2009 H1N1 influenza A virus (2009 H1N1) differs from H1N1 strains that circulated in the past 50 years, but resembles the A/New Jersey/1976 H1N1 strain used in the 1976 swine influenza vaccine. We investigated whether sera from persons immunized with the 1976 swine influenza or recent seasonal influenza vaccines, or both, neutralize 2009 H1N1. Using retroviral pseudovirions bearing hemagglutinins on their surface (HA-pseudotypes), we found that 77% of the sera collected in 1976 after immunization with the A/New Jersey/1976 H1N1 swine influenza vaccine neutralized 2009 H1N1. Forty five percent also neutralized A/New Caledonia/20/1999 H1N1, a strain used in seasonal influenza vaccines during the 2000/01-2006/07 seasons. Among adults aged 48-64 who received the swine influenza vaccine in 1976 and recent seasonal influenza vaccines during the 2004/05-2008/09 seasons, 83% had sera that neutralized 2009 H1N1. However, 68% of age-matched subjects who received the same seasonal influenza vaccines, but did not receive the 1976 swine influenza vaccine, also had sera that neutralized 2009 H1N1. Sera from both 1976 and contemporary cohorts frequently had cross-neutralizing antibodies to 2009 H1N1 and A/New Caledonia/20/1999 that mapped to hemagglutinin subunit 2 (HA2). A conservative mutation in HA2 corresponding to a residue in the A/Solomon Islands/3/2006 and A/Brisbane/59/2007 H1N1 strains that circulated in the 2006/07 and 2007/08 influenza seasons, respectively, abrogated this neutralization. These findings highlight a cross-neutralization determinant influenced by a point mutation in HA2 and suggest that HA2 may be evolving under direct or indirect immune pressure.

Selection with a peptide fusion inhibitor corresponding to the first heptad repeat of HIV-1 gp41 identifies two genetic pathways conferring cross-resistance to peptide fusion inhibitors corresponding to the first and second heptad repeats (HR1 and HR2) of gp41.

We generated four HIV-1 cultures that are resistant to a peptide fusion inhibitor corresponding to the first heptad repeat of gp41 in order to study mechanisms of resistance and gain insights into envelope glycoprotein-mediated membrane fusion. Two genetic pathways emerged that were defined by acquisition of a specific mutation in either the first or second heptad repeat region of gp41 (HR1 or the HR2, respectively). Each pathway was enriched in mutations that clustered in either HR2 and V3 or in HR1 and residues in or near CD4 contact sites. The gp41 mutations in both pathways not only accounted for resistance to the selecting HR1 peptide but also conferred cross-resistance to HR2 peptide fusion inhibitors and enhanced the stability of the six-helix bundle formed by the self-assembly of HR1 and HR2. The gp120 mutations alone enhanced fusion but did not appear to directly contribute to resistance. The implications of these findings for resistance mechanisms and regulation of envelope-mediated fusion are discussed.

Measuring Neutralizing Antibodies to SARS-CoV-2 Using Lentiviral Spike-Pseudoviruses.

Assays measuring neutralizing antibodies (nAbs) against SARS-CoV-2 are used to evaluate serological responses after SARS-CoV-2 infection and the potency of therapeutic antibodies and preventive vaccines. It is therefore imperative that neutralization assays be sensitive, specific, quantitative, and scalable for high throughput. Pseudoviruses are excellent surrogates for highly pathogenic viruses such as SARS-CoV-2 because they can be safely used to measure nAbs in a biosafety level-2 laboratory. In addition, pseudoviruses allow for easy introduction of mutations to study the effect of amino acid changes in the spike protein. In this chapter, we describe a recently optimized assay for measuring neutralizing antibodies to SARS-CoV-2 that uses a HIV-based lentiviral vector pseudotyped with the spike glycoprotein of SARS-CoV-2 to infect 293T cells stably expressing ACE2 and TMPRSS2.

SARS-CoV-2 BA.1 variant is neutralized by vaccine booster-elicited serum but evades most convalescent serum and therapeutic antibodies.

The rapid spread of the highly contagious Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) along with its high number of mutations in the spike gene has raised alarms about the effectiveness of current medical countermeasures. To address this concern, we measured the neutralization of the Omicron BA.1 variant pseudovirus by postvaccination serum samples after two and three immunizations with the Pfizer/BioNTech162b2 SARS-CoV-2 mRNA (Pfizer/BNT162b2) vaccine, convalescent serum samples from unvaccinated individuals infected by different variants, and clinical-stage therapeutic antibodies. We found that titers against the Omicron variant were low or undetectable after two immunizations and in many convalescent serum samples, regardless of the infecting variant. A booster vaccination increased titers more than 30-fold against Omicron to values comparable to those seen against the D614G variant after two immunizations. Neither age nor sex was associated with the differences in postvaccination antibody responses. We also evaluated 18 clinical-stage therapeutic antibody products and an antibody mimetic protein product obtained directly from the manufacturers. Five monoclonal antibodies, the antibody mimetic protein, three antibody cocktails, and two polyclonal antibody preparations retained measurable neutralization activity against Omicron with a varying degree of potency. Of these, only three retained potencies comparable to the D614G variant. Two therapeutic antibody cocktails in the tested panel that are authorized for emergency use in the United States did not neutralize Omicron. These findings underscore the potential benefit of mRNA vaccine boosters for protection against Omicron and the need for rapid development of antibody therapeutics that maintain potency against emerging variants.