Structural and functional characteristics of the SARS-CoV-2 Omicron subvariant BA.2 spike protein.

The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. Here, we have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and an animal model with previously prevalent variants. BA.2 S can fuse membranes slightly more efficiently than Omicron BA.1, but still less efficiently than other previous variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces, leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility of the Omicron subvariants.

Autologous neutralizing antibodies increase with early antiretroviral therapy and shape HIV rebound after treatment interruption.

Early initiation of antiretroviral therapy (ART) alters viral rebound kinetics after analytic treatment interruption (ATI) and may play a role in promoting HIV-1 remission. Autologous neutralizing antibodies (aNAbs) represent a key adaptive immune response in people living with HIV-1. We aimed to investigate the role of aNAbs in shaping post-ATI HIV-1 rebound variants. We performed single-genome amplification of HIV-1 env from pre-ART and post-ATI plasma samples of 12 individuals who initiated ART early after infection. aNAb activity was quantified using pseudoviruses derived from the most common plasma variant, and the serum dilution that inhibited 50% of viral infections was determined. aNAb responses matured while participants were on suppressive ART, because on-ART plasma and purified immunoglobulin G (IgG) demonstrated improved neutralizing activity against pre-ART HIV-1 strains when compared with pre-ART plasma or purified IgG. Post-ATI aNAb responses exerted selective pressure on the rebounding viruses, because the post-ATI HIV-1 strains were more resistant to post-ATI plasma neutralization compared with the pre-ART virus. Several pre-ATI features distinguished post-treatment controllers from noncontrollers, including an infecting HIV-1 sequence that was more similar to consensus HIV-1 subtype B, more restricted proviral diversity, and a stronger aNAb response. Post-treatment control was also associated with the evolution of distinct N-glycosylation profiles in the HIV-1 envelope. In summary, aNAb responses appeared to mature after early initiation of ART and applied selective pressure on rebounding viruses. The combination of aNAb activity with select HIV-1 sequence and reservoir features identified individuals with a greater chance of post-treatment control.

Antibody-mediated SARS-CoV-2 entry in cultured cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells by first engaging its cellular receptor angiotensin converting enzyme 2 (ACE2) to induce conformational changes in the virus-encoded spike protein and fusion between the viral and target cell membranes. Here, we report that certain monoclonal neutralizing antibodies against distinct epitopic regions of the receptor-binding domain of the spike can replace ACE2 to serve as a receptor and efficiently support membrane fusion and viral infectivity in vitro. These receptor-like antibodies can function in the form of a complex of their soluble immunoglobulin G with Fc-gamma receptor I, a chimera of their antigen-binding fragment with the transmembrane domain of ACE2 or a membrane-bound B cell receptor, indicating that ACE2 and its specific interaction with the spike protein are dispensable for SARS-CoV-2 entry. These results suggest that antibody responses against SARS-CoV-2 may help expand the viral tropism to otherwise nonpermissive cell types with potential implications for viral transmission and pathogenesis.

Subtle Longitudinal Alterations in Env Sequence Potentiate Differences in Sensitivity to Broadly Neutralizing Antibodies following Acute HIV-1 Subtype C Infection.

Broadly neutralizing antibodies (bNAbs) for HIV-1 prevention or cure strategies must inhibit transmitted/founder and reservoir viruses. Establishing sensitivity of circulating viruses to bNAbs and genetic patterns affecting neutralization variability may guide rational bNAbs selection for clinical development. We analyzed 326 single env genomes from nine individuals followed longitudinally following acute HIV-1 infection, with samples collected at ~1 week after the first detection of plasma viremia; 300 to 1,709 days postinfection but prior to initiating antiretroviral therapy (ART) (median = 724 days); and ~1 year post ART initiation. Sequences were assessed for phylogenetic relatedness, potential N- and O-linked glycosylation, and variable loop lengths (V1 to V5). A total of 43 env amplicons (median = 3 per patient per time point) were cloned into an expression vector and the TZM-bl assay was used to assess the neutralization profiles of 15 bNAbs targeting the CD4 binding site, V1/V2 region, V3 supersite, MPER, gp120/gp41 interface, and fusion peptide. At 1 µg/mL, the neutralization breadths were as follows: VRC07-LS and N6.LS (100%), VRC01 (86%), PGT151 (81%), 10-1074 and PGT121 (80%), and less than 70% for 10E8, 3BNC117, CAP256.VRC26, 4E10, PGDM1400, and N123-VRC34.01. Features associated with low sensitivity to V1/V2 and V3 bNAbs were higher potential glycosylation sites and/or relatively longer V1 and V4 domains, including known "signature" mutations. The study shows significant variability in the breadth and potency of bNAbs against circulating HIV-1 subtype C envelopes. VRC07-LS, N6.LS, VRC01, PGT151, 10-1074, and PGT121 display broad activity against subtype C variants, and major determinants of sensitivity to most bNAbs were within the V1/V4 domains. IMPORTANCE Broadly neutralizing antibodies (bNAbs) have potential clinical utility in HIV-1 prevention and cure strategies. However, bNAbs target diverse epitopes on the HIV-1 envelope and the virus may evolve to evade immune responses. It is therefore important to identify antibodies with broad activity in high prevalence settings, as well as the genetic patterns that may lead to neutralization escape. We investigated 15 bNAbs with diverse biophysical properties that target six epitopes of the HIV-1 Env glycoprotein for their ability to inhibit viruses that initiated infection, viruses circulating in plasma at chronic infection before antiretroviral treatment (ART), or viruses that were archived in the reservoir during ART in subtype C infected individuals in South Africa, a high burden country. We identify the antibodies most likely to be effective for clinical use in this setting and describe mutational patterns associated with neutralization escape from these antibodies.

Vaccine breakthrough infection leads to distinct profiles of neutralizing antibody responses by SARS-CoV-2 variant.

Protective immunity against SARS-CoV-2 infection after COVID-19 vaccination may differ by variant. We enrolled vaccinated (n = 39) and unvaccinated (n = 11) individuals with acute, symptomatic SARS-CoV-2 Delta or Omicron infection and performed SARS-CoV-2 viral load quantification, whole-genome sequencing, and variant-specific antibody characterization at the time of acute illness and convalescence. Viral load at the time of infection was inversely correlated with antibody binding and neutralizing antibody responses. Across all variants tested, convalescent neutralization titers in unvaccinated individuals were markedly lower than in vaccinated individuals. Increases in antibody titers and neutralizing activity occurred at convalescence in a variant-specific manner. For example, among individuals infected with the Delta variant, neutralizing antibody responses were weakest against BA.2, whereas infection with Omicron BA.1 variant generated a broader response against all tested variants, including BA.2.

Structural and functional characteristics of SARS-CoV-2 Omicron subvariant BA.2 spike.

The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. We have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and animal model with previously prevalent variants. BA.2 S can fuse membranes more efficiently than Omicron BA.1, mainly due to lack of a BA.1-specific mutation that may retard the receptor engagement, but still less efficiently than other variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility for the Omicron subvariants.

Structural and functional impact by SARS-CoV-2 Omicron spike mutations.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), bearing an unusually high number of mutations, has become a dominant strain in many countries within several weeks. We report here structural, functional, and antigenic properties of its full-length spike (S) protein with a native sequence in comparison with those of previously prevalent variants. Omicron S requires a substantially higher level of host receptor ACE2 for efficient membrane fusion than other variants, possibly explaining its unexpected cellular tropism. Mutations not only remodel the antigenic structure of the N-terminal domain of the S protein but also alter the surface of the receptor-binding domain in a way not seen in other variants, consistent with its remarkable resistance to neutralizing antibodies. These results suggest that Omicron S has acquired an extraordinary ability to evade host immunity by excessive mutations, which also compromise its fusogenic capability.

Vaccine Breakthrough Infection with the SARS-CoV-2 Delta or Omicron (BA.1) Variant Leads to Distinct Profiles of Neutralizing Antibody Responses.

There is increasing evidence that the risk of SARS-CoV-2 infection among vaccinated individuals is variant-specific, suggesting that protective immunity against SARS-CoV-2 may differ by variant. We enrolled vaccinated (n = 39) and unvaccinated (n = 11) individuals with acute, symptomatic SARS-CoV-2 Delta or Omicron infection and performed SARS-CoV-2 viral load quantification, whole-genome sequencing, and variant-specific antibody characterization at the time of acute illness and convalescence. Viral load at the time of infection was inversely correlated with antibody binding and neutralizing antibody responses. Increases in antibody titers and neutralizing activity occurred at convalescence in a variant-specific manner. Across all variants tested, convalescent neutralization titers in unvaccinated individuals were markedly lower than in vaccinated individuals. For individuals infected with the Delta variant, neutralizing antibody responses were weakest against BA.2, whereas infection with Omicron BA.1 variant generated a broader response against all tested variants, including BA.2.

Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant.

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report the structure, function, and antigenicity of its full-length spike (S) trimer as well as those of the Gamma and Kappa variants, and compare their characteristics with the G614, Alpha, and Beta variants. Delta S can fuse membranes more efficiently at low levels of cellular receptor angiotensin converting enzyme 2 (ACE2), and its pseudotyped viruses infect target cells substantially faster than the other five variants, possibly accounting for its heightened transmissibility. Each variant shows different rearrangement of the antigenic surface of the amino-terminal domain of the S protein but only makes produces changes in the receptor binding domain (RBD), making the RBD a better target for therapeutic antibodies.

Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant.

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report here structure, function and antigenicity of its full-length spike (S) trimer in comparison with those of other variants, including Gamma, Kappa, and previously characterized Alpha and Beta. Delta S can fuse membranes more efficiently at low levels of cellular receptor ACE2 and its pseudotyped viruses infect target cells substantially faster than all other variants tested, possibly accounting for its heightened transmissibility. Mutations of each variant rearrange the antigenic surface of the N-terminal domain of the S protein in a unique way, but only cause local changes in the receptor-binding domain, consistent with greater resistance particular to neutralizing antibodies. These results advance our molecular understanding of distinct properties of these viruses and may guide intervention strategies.

Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity-suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants.

Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants.

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains in the COVID-19 pandemic. We report here cryo-electron microscopy structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Amino acid substitutions in the B.1.1.7 protein increase both the accessibility of its receptor binding domain and the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement may account for the increased transmissibility. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, making it resistant to some potent neutralizing antibodies. These findings provide structural details on how SARS-CoV-2 has evolved to enhance viral fitness and immune evasion.

Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants.

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains that continue to fuel the COVID-19 pandemic despite intensive vaccination efforts throughout the world. We report here cryo-EM structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Mutations in the B.1.1.7 protein increase the accessibility of its receptor binding domain and also the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement can account for the increased transmissibility and risk of mortality as the variant may begin to infect efficiently infect additional cell types expressing low levels of ACE2. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, rendering complete resistance to some potent neutralizing antibodies. These findings provide structural details on how the wide spread of SARS-CoV-2 enables rapid evolution to enhance viral fitness and immune evasion. They may guide intervention strategies to control the pandemic.

Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with an unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded monoclonal antibodies (mAbs) from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found 7 major mAb competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of mAb-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. mAbs that competed for binding the original S isolate bound differentially to S variants, suggesting the protective importance of otherwise-redundant recognition. The results furnish a global atlas of the S-specific memory B cell repertoire and illustrate properties conferring robustness against emerging SARS-CoV-2 variants.

A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent.

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin-angiotensin system that modulates vascular functions. We report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of ~60 pM for the spike protein of SARS­CoV­2 (compared with 77 nM for monomeric ACE2 and 12-22 nM for dimeric ACE2 constructs), and its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the renin-angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARS­CoV­2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

Quick COVID-19 Healers Sustain Anti-SARS-CoV-2 Antibody Production.

Antibodies are key immune effectors that confer protection against pathogenic threats. The nature and longevity of the antibody response to SARS-CoV-2 infection are not well defined. We charted longitudinal antibody responses to SARS-CoV-2 in 92 subjects after symptomatic COVID-19. Antibody responses to SARS-CoV-2 are unimodally distributed over a broad range, with symptom severity correlating directly with virus-specific antibody magnitude. Seventy-six subjects followed longitudinally to ∼100 days demonstrated marked heterogeneity in antibody duration dynamics. Virus-specific IgG decayed substantially in most individuals, whereas a distinct subset had stable or increasing antibody levels in the same time frame despite similar initial antibody magnitudes. These individuals with increasing responses recovered rapidly from symptomatic COVID-19 disease, harbored increased somatic mutations in virus-specific memory B cell antibody genes, and had persistent higher frequencies of previously activated CD4+ T cells. These findings illuminate an efficient immune phenotype that connects symptom clearance speed to differential antibody durability dynamics.

A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent in vitro.

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that forms a dimer and serves as the cellular receptor for SARS-CoV-2. It is also a key negative regulator of the renin-angiotensin system (RAS), conserved in mammals, which modulates vascular functions. We report here the properties of a trimeric ACE2 variant, created by a structure-based approach, with binding affinity of ~60 pM for the spike (S) protein of SARS-CoV-2, while preserving the wildtype peptidase activity as well as the ability to block activation of angiotensin II receptor type 1 in the RAS. Moreover, the engineered ACE2 potently inhibits infection of SARS-CoV-2 in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

A minor population of macrophage-tropic HIV-1 variants is identified in recrudescing viremia following analytic treatment interruption.

HIV-1 persists in cellular reservoirs that can reignite viremia if antiretroviral therapy (ART) is interrupted. Therefore, insight into the nature of those reservoirs may be revealed from the composition of recrudescing viremia following treatment cessation. A minor population of macrophage-tropic (M-tropic) viruses was identified in a library of recombinant viruses constructed with individual envelope genes that were obtained from plasma of six individuals undergoing analytic treatment interruption (ATI). M-tropic viruses could also be enriched from post-ATI plasma using macrophage-specific (CD14) but not CD4+ T cell-specific (CD3) antibodies, suggesting that M-tropic viruses had a macrophage origin. Molecular clock analysis indicated that the establishment of M-tropic HIV-1 variants predated ATI. Collectively, these data suggest that macrophages are a viral reservoir in HIV-1-infected individuals on effective ART and that M-tropic variants can appear in rebounding viremia when treatment is interrupted. These findings have implications for the design of curative strategies for HIV-1.

Differential Outcomes following Optimization of Simian-Human Immunodeficiency Viruses from Clades AE, B, and C.

Simian-human immunodeficiency virus (SHIV) infection of rhesus monkeys is an important preclinical model for human immunodeficiency virus type 1 (HIV-1) vaccines, therapeutics, and cure strategies. SHIVs have been optimized by incorporating HIV-1 Env residue 375 mutations that mimic the bulky or hydrophobic residues typically found in simian immunodeficiency virus (SIV) Env to improve rhesus CD4 binding. We applied this strategy to three SHIV challenge stocks (SHIV-SF162p3, SHIV-AE16, and SHIV-325c) and observed three distinct outcomes. We constructed six Env375 variants (M, H, W, Y, F, and S) for each SHIV, and we performed a pool competition study in rhesus monkeys to define the optimal variant for each SHIV prior to generating large-scale challenge stocks. We identified SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH as the optimal variants. SHIV-SF162p3S could not be improved, as it already contained the optimal Env375 residue. SHIV-AE16W exhibited a similar replicative capacity to the parental SHIV-AE16 stock. In contrast, SHIV-325cH demonstrated a 2.6-log higher peak and 1.6-log higher setpoint viral loads than the parental SHIV-325c stock. These data demonstrate the diversity of potential outcomes following Env375 modification in SHIVs. Moreover, the clade C SHIV-325cH challenge stock may prove useful for evaluating prophylactic or therapeutic interventions against clade C HIV-1.IMPORTANCE We sought to enhance the infectivity of three SHIV stocks by optimization of a key residue in human immunodeficiency virus type 1 (HIV-1) Env (Env375). We developed the following three new simian-human immunodeficiency virus (SHIV) stocks: SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH. SHIV-SF162p3S could not be optimized, SHIV-AE16W proved comparable to the parental virus, and SHIV-325cH demonstrated markedly enhanced replicative capacity compared with the parental virus.

HIV-1 fusion inhibitors targeting the membrane-proximal external region of Env spikes.

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.