Leveraging vaccination-induced protective antibodies to define conserved epitopes on influenza N2 neuraminidase.

There is growing appreciation for neuraminidase (NA) as an influenza vaccine target; however, its antigenicity remains poorly characterized. In this study, we isolated three broadly reactive N2 antibodies from the plasmablasts of a single vaccinee, including one that cross-reacts with NAs from seasonal H3N2 strains spanning five decades. Although these three antibodies have diverse germline usages, they recognize similar epitopes that are distant from the NA active site and instead involve the highly conserved underside of NA head domain. We also showed that all three antibodies confer prophylactic and therapeutic protection in vivo, due to both Fc effector functions and NA inhibition through steric hindrance. Additionally, the contribution of Fc effector functions to protection in vivo inversely correlates with viral growth inhibition activity in vitro. Overall, our findings advance the understanding of NA antibody response and provide important insights into the development of a broadly protective influenza vaccine.

Spatiotemporal development of the human T follicular helper cell response to Influenza vaccination.

We profiled blood and draining lymph node (LN) samples from human volunteers after influenza vaccination over two years to define evolution in the T follicular helper cell (TFH) response. We show LN TFH cells expanded in a clonal-manner during the first two weeks after vaccination and persisted within the LN for up to six months. LN and circulating TFH (cTFH) clonotypes overlapped but had distinct kinetics. LN TFH cell phenotypes were heterogeneous and mutable, first differentiating into pre-TFH during the month after vaccination before maturing into GC and IL-10+ TFH cells. TFH expansion, upregulation of glucose metabolism, and redifferentiation into GC TFH cells occurred with faster kinetics after re-vaccination in the second year. We identified several influenza-specific TFH clonal lineages, including multiple responses targeting internal influenza proteins, and show each TFH state is attainable within a lineage. This study demonstrates that human TFH cells form a durable and dynamic multi-tissue network.

Human anti-N1 monoclonal antibodies elicited by pandemic H1N1 virus infection broadly inhibit HxN1 viruses in vitro and in vivo.

Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our current understanding of NA antigenicity is incomplete. Here, we describe human monoclonal antibodies (mAbs) from a patient with a pandemic H1N1 virus infection in 2009. Two mAbs exhibited broad reactivity and inhibited NA enzyme activity of seasonal H1N1 viruses circulating before and after 2009, as well as viruses with avian or swine N1s. The mAbs provided robust protection from lethal challenge with human H1N1 and avian H5N1 viruses in mice, and both target an epitope on the lateral face of NA. In summary, we identified two broadly protective NA antibodies that share a novel epitope, inhibited NA activity, and provide protection against virus challenge in mice. Our work reaffirms that NA should be included as a target in future broadly protective or universal influenza virus vaccines.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Human NK cells confer protection against HIV-1 infection in humanized mice.

The role of NK cells against HIV-1 infections remains to be elucidated in vivo. While humanized mouse models potentially could be used to directly evaluate human NK cell responses during HIV-1 infection, improved functional development of human NK cells in these hosts is needed. Here, we report the humanized MISTRG-6-15 mouse model, in which NK cells were quick to expand and exhibit degranulation, cytotoxicity, and proinflammatory cytokine production in nonlymphoid organs upon HIV-1 infection but had reduced functionality in lymphoid organs. Although HIV-1 infection induced functional impairment of NK cells, antiretroviral therapy reinvigorated NK cells in response to HIV-1 rebound after analytic treatment interruption. Moreover, a broadly neutralizing antibody, PGT121, enhanced NK cell function in vivo, consistent with antibody-dependent cellular cytotoxicity. Monoclonal antibody depletion of NK cells resulted in higher viral loads in multiple nonlymphoid organs. Overall, our results in humanized MISTRG-6-15 mice demonstrated that NK cells provided direct anti-HIV-1 responses in vivo but were limited in their responses in lymphoid organs.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses of these vaccines and the development of new variant-derived ones 1-4 . SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells (MBCs) 5-9 . It remains unclear, however, whether the additional doses induce germinal centre (GC) reactions where reengaged B cells can further mature and whether variant-derived vaccines can elicit responses to novel epitopes specific to such variants. Here, we show that boosting with the original SARS- CoV-2 spike vaccine (mRNA-1273) or a B.1.351/B.1.617.2 (Beta/Delta) bivalent vaccine (mRNA-1273.213) induces robust spike-specific GC B cell responses in humans. The GC response persisted for at least eight weeks, leading to significantly more mutated antigen-specific MBC and bone marrow plasma cell compartments. Interrogation of MBC-derived spike-binding monoclonal antibodies (mAbs) isolated from individuals boosted with either mRNA-1273, mRNA-1273.213, or a monovalent Omicron BA.1-based vaccine (mRNA-1273.529) revealed a striking imprinting effect by the primary vaccination series, with all mAbs (n=769) recognizing the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted approach, we isolated mAbs that recognized the spike protein of the SARS-CoV-2 Omicron (BA.1) but not the original SARS-CoV-2 spike from the mRNA-1273.529 boosted individuals. The latter mAbs were less mutated and recognized novel epitopes within the spike protein, suggesting a naïve B cell origin. Thus, SARS-CoV-2 boosting in humans induce robust GC B cell responses, and immunization with an antigenically distant spike can overcome the antigenic imprinting by the primary vaccination series.

mRNA-1273 and Ad26.COV2.S vaccines protect against the B.1.621 variant of SARS-CoV-2.

BACKGROUND: Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, viral variants with greater transmissibility or immune-evasion properties have arisen, which could jeopardize recently deployed vaccine- and antibody-based countermeasures. METHODS: Here, we evaluated in mice and hamsters the efficacy of a pre-clinical version of the Moderna mRNA vaccine (mRNA-1273) and the Johnson & Johnson recombinant adenoviral-vectored vaccine (Ad26.COV2.S) against the B.1.621 (Mu) variant of SARS-CoV-2, which contains spike mutations T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N. FINDINGS: Immunization of 129S2 and K18-human ACE2 transgenic mice with the mRNA-1273 vaccine protected against weight loss, lung infection, and lung pathology after challenge with the B.1.621 or WA1/2020 N501Y/D614G SARS-CoV-2 strain. Similarly, immunization of 129S2 mice and Syrian hamsters with a high dose of Ad26.COV2.S reduced lung infection after B.1.621 virus challenge. CONCLUSIONS: Thus, immunity induced by the mRNA-1273 or Ad26.COV2.S vaccine can protect against the B.1.621 variant of SARS-CoV-2 in multiple animal models. FUNDING: This study was supported by the NIH (R01 AI157155 and U01 AI151810), NIAID Centers of Excellence for Influenza Research and Response [CEIRR] contracts 75N93021C00014 and 75N93021C00016, and the Collaborative Influenza Vaccine Innovation Centers [CIVIC] contract 75N93019C00051. It was also supported, in part, by the National Institutes of Allergy and Infectious Diseases Center for Research on Influenza Pathogenesis (HHSN272201400008C) and the Japan Program for Infectious Diseases Research and Infrastructure (JP21wm0125002) from the Japan Agency for Medical Research and Development (AMED).

Germinal centre-driven maturation of B cell response to mRNA vaccination.

Germinal centres (GC) are lymphoid structures in which B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells1-5 (BMPCs). SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans6-8. The fate of responding GC B cells as well as the functional consequences of such persistence remain unknown. Here, we detected SARS-CoV-2 spike protein-specific MBCs in 42 individuals who had received two doses of the SARS-CoV-2 mRNA vaccine BNT162b2 six month earlier. Spike-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs, lymph node GC B cells and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1,540 spike-specific B cell clones. On average, early blood spike-specific plasmablasts exhibited the lowest SHM frequencies. By contrast, SHM frequencies of spike-specific GC B cells increased by 3.5-fold within six months after vaccination. Spike-specific MBCs and BMPCs accumulated high levels of SHM, which corresponded with enhanced anti-spike antibody avidity in blood and enhanced affinity as well as neutralization capacity of BMPC-derived monoclonal antibodies. We report how the notable persistence of the GC reaction induced by SARS-CoV-2 mRNA vaccination in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus.

SARS-CoV-2 mRNA vaccination elicits a robust and persistent T follicular helper cell response in humans.

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

Germinal centre-driven maturation of B cell response to SARS-CoV-2 vaccination.

Germinal centres (GC) are lymphoid structures where vaccine-responding B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells (BMPCs) 1-4 . Induction of the latter is a hallmark of durable immunity after vaccination 5 . SARS-CoV-2 mRNA vaccination induces a robust GC response in humans 6-8 , but the maturation dynamics of GC B cells and propagation of their progeny throughout the B cell diaspora have not been elucidated. Here we show that anti-SARS-CoV-2 spike (S)-binding GC B cells were detectable in draining lymph nodes for at least six months in 10 out of 15 individuals who had received two doses of BNT162b2, a SARS-CoV-2 mRNA vaccine. Six months after vaccination, circulating S-binding MBCs were detected in all participants (n=42) and S-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of single-cell RNA sequencing of responding blood and lymph node B cells from eight participants and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1540 S-specific B cell clones. SHM accumulated along the B cell differentiation trajectory, with early blood plasmablasts showing the lowest frequencies, followed by MBCs and lymph node plasma cells whose SHM largely overlapped with GC B cells. By three months after vaccination, the frequency of SHM within GC B cells had doubled. Strikingly, S + BMPCs detected six months after vaccination accumulated the highest level of SHM, corresponding with significantly enhanced anti-S polyclonal antibody avidity in blood at that time point. This study documents the induction of affinity-matured BMPCs after two doses of SARS-CoV-2 mRNA vaccination in humans, providing a foundation for the sustained high efficacy observed with these vaccines.

Structural mechanism of SARS-CoV-2 neutralization by two murine antibodies targeting the RBD.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.

Neutralizing Monoclonal Antibodies That Target the Spike Receptor Binding Domain Confer Fc Receptor-Independent Protection against SARS-CoV-2 Infection in Syrian Hamsters.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is the main target for neutralizing antibodies. These antibodies can be elicited through immunization or passively transferred as therapeutics in the form of convalescent-phase sera or monoclonal antibodies (MAbs). Potently neutralizing antibodies are expected to confer protection; however, it is unclear whether weakly neutralizing antibodies contribute to protection. Also, their mechanism of action in vivo is incompletely understood. Here, we demonstrate that 2B04, an antibody with an ultrapotent neutralizing activity (50% inhibitory concentration [IC50] of 0.04 µg/ml), protects hamsters against SARS-CoV-2 in a prophylactic and therapeutic infection model. Protection is associated with reduced weight loss and viral loads in nasal turbinates and lungs after challenge. MAb 2B04 also blocked aerosol transmission of the virus to naive contacts. We next examined three additional MAbs (2C02, 2C03, and 2E06), recognizing distinct epitopes within the receptor binding domain of spike protein that possess either minimal (2C02 and 2E06, IC50 > 20 µg/ml) or weak (2C03, IC50 of 5 µg/ml) virus neutralization capacity in vitro. Only 2C03 protected Syrian hamsters from weight loss and reduced lung viral load after SARS-CoV-2 infection. Finally, we demonstrated that Fc-Fc receptor interactions were not required for protection when 2B04 and 2C03 were administered prophylactically. These findings inform the mechanism of protection and support the rational development of antibody-mediated protection against SARS-CoV-2 infections. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2, has resulted in the loss of millions of lives. Safe and effective vaccines are considered the ultimate remedy for the global social and economic disruption caused by the pandemic. However, a thorough understanding of the immune correlates of protection against this virus is lacking. Here, we characterized four different monoclonal antibodies and evaluated their ability to prevent or treat SARS-CoV-2 infection in Syrian hamsters. These antibodies varied in their ability to neutralize the virus in vitro. Prophylactic administration of potent and weakly neutralizing antibodies protected against SARS-CoV-2 infection, and this effect was Fc receptor independent. The potent neutralizing antibody also had therapeutic efficacy and eliminated onward aerosol transmission. In contrast, minimally neutralizing antibodies provided no protection against infection with SARS-CoV-2 in Syrian hamsters. Combined, these studies highlight the significance of weakly neutralizing antibodies in the protection against SARS-CoV-2 infection and associated disease.

A vaccine-induced public antibody protects against SARS-CoV-2 and emerging variants.

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.

SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD, RBD, and S2.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.

SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses.

SARS-CoV-2 mRNA-based vaccines are about 95% effective in preventing COVID-191-5. The dynamics of antibody-secreting plasmablasts and germinal centre B cells induced by these vaccines in humans remain unclear. Here we examined antigen-specific B cell responses in peripheral blood (n = 41) and draining lymph nodes in 14 individuals who had received 2 doses of BNT162b2, an mRNA-based vaccine that encodes the full-length SARS-CoV-2 spike (S) gene1. Circulating IgG- and IgA-secreting plasmablasts that target the S protein peaked one week after the second immunization and then declined, becoming undetectable three weeks later. These plasmablast responses preceded maximal levels of serum anti-S binding and neutralizing antibodies to an early circulating SARS-CoV-2 strain as well as emerging variants, especially in individuals who had previously been infected with SARS-CoV-2 (who produced the most robust serological responses). By examining fine needle aspirates of draining axillary lymph nodes, we identified germinal centre B cells that bound S protein in all participants who were sampled after primary immunization. High frequencies of S-binding germinal centre B cells and plasmablasts were sustained in these draining lymph nodes for at least 12 weeks after the booster immunization. S-binding monoclonal antibodies derived from germinal centre B cells predominantly targeted the receptor-binding domain of the S protein, and fewer clones bound to the N-terminal domain or to epitopes shared with the S proteins of the human betacoronaviruses OC43 and HKU1. These latter cross-reactive B cell clones had higher levels of somatic hypermutation as compared to those that recognized only the SARS-CoV-2 S protein, which suggests a memory B cell origin. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a persistent germinal centre B cell response, which enables the generation of robust humoral immunity.

SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans.

Long-lived bone marrow plasma cells (BMPCs) are a persistent and essential source of protective antibodies1-7. Individuals who have recovered from COVID-19 have a substantially lower risk of reinfection with SARS-CoV-28-10. Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived BMPCs may not be generated and humoral immunity against SARS-CoV-2 may be short-lived11-13. Here we show that in convalescent individuals who had experienced mild SARS-CoV-2 infections (n = 77), levels of serum anti-SARS-CoV-2 spike protein (S) antibodies declined rapidly in the first 4 months after infection and then more gradually over the following 7 months, remaining detectable at least 11 months after infection. Anti-S antibody titres correlated with the frequency of S-specific plasma cells in bone marrow aspirates from 18 individuals who had recovered from COVID-19 at 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy individuals with no history of SARS-CoV-2 infection. We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. Consistently, circulating resting memory B cells directed against SARS-CoV-2 S were detected in the convalescent individuals. Overall, our results indicate that mild infection with SARS-CoV-2 induces robust antigen-specific, long-lived humoral immune memory in humans.

A public vaccine-induced human antibody protects against SARS-CoV-2 and emerging variants.

The emergence of antigenically distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility is a public health threat. Some of these variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies, which principally target the receptor binding domain (RBD) on the virus spike glycoprotein. Here, we describe 2C08, a SARS-CoV-2 mRNA vaccine-induced germinal center B cell-derived human monoclonal antibody that binds to the receptor binding motif within the RBD. 2C08 broadly neutralizes SARS-CoV-2 variants with remarkable potency and reduces lung inflammation, viral load, and morbidity in hamsters challenged with either an ancestral SARS-CoV-2 strain or a recent variant of concern. Clonal analysis identified 2C08-like public clonotypes among B cell clones responding to SARS-CoV-2 infection or vaccination in at least 20 out of 78 individuals. Thus, 2C08-like antibodies can be readily induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern. ONE SENTENCE SUMMARY: Protection against SARS-CoV-2 variants by a potently neutralizing vaccine-induced human monoclonal antibody.

Evaluation of HIV-1 latency reversal and antibody-dependent viral clearance by quantification of singly spliced HIV-1 vpu/env mRNA.

The latent reservoir of HIV-1 is a major barrier for viral eradication. Potent HIV-1 broadly neutralizing antibodies (bNabs) have been used to prevent and treat HIV-1 infections in animal models and clinical trials. Combination of bNabs and latency-reversing agents (LRAs) is considered a promising approach for HIV-1 eradication. PCR-based assays that can rapidly and specifically measure singly spliced HIV-1 vpu/env mRNA are needed to evaluate the induction of the viral envelope production at the transcription level and bNab-mediated reservoir clearance. Here we reported a PCR-based method to accurately quantify the production of intracellular HIV-1 vpu/env mRNA. With the vpu/env assay, we determined the LRA combinations that could effectively induce vpu/env mRNA production in CD4+ T cells from ART-treated individuals. None of the tested LRAs were effective alone. A comparison between the quantitative viral outgrowth assay (Q-VOA) and the vpu/env assay showed that vpu/env mRNA production was closely associated with the reactivation of replication-competent HIV-1, suggesting that vpu/env mRNA was mainly produced by intact viruses. Finally, antibody-mediated in vitro killing in HIV-1-infected humanized mice demonstrated that the vpu/env assay could be used to measure the reduction of infected cells in tissues and was more accurate than the commonly used gag-based PCR assay which measured unspliced viral genomic RNA. In conclusion, the vpu/env assay allows convenient and accurate assessment of HIV-1 latency reversal and bNab-mediated therapeutic strategies.ImportanceHIV-1 persists in individuals on antiretroviral therapy (ART) due to the long-lived cellular reservoirs that contain dormant viruses. Recent discoveries of HIV-1-specific broadly neutralizing antibodies (bNabs) targeting HIV-1 Env protein rekindled the interest in antibody-mediated elimination of latent HIV-1. Latency-reversing agents (LRAs) together with HIV-1 bNabs is a possible strategy to clear residual viral reservoirs, which makes the evaluation of HIV-1 Env expression upon LRA treatment critical. We developed a PCR-based assay to quantify the production of intracellular HIV-1 vpu/env mRNA. Using patient CD4+ T cells, we found that induction of HIV-1 vpu/env mRNA required a combination of different LRAs. Using in vitro, ex vivo and humanized mouse models, we showed that the vpu/env assay could be used to measure antibody efficacy in clearing HIV-1 infection. These results suggest that the vpu/env assay can accurately evaluate HIV-1 reactivation and bNab-based therapeutic interventions.

Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic. Rapidly spreading SARS-CoV-2 variants may jeopardize newly introduced antibody and vaccine countermeasures. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera and human sera from recipients of the BNT162b2 mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, chimeric strains with South African or Brazilian spike genes and isogenic recombinant viral variants. Many highly neutralizing mAbs engaging the receptor-binding domain or N-terminal domain and most convalescent sera and mRNA vaccine-induced immune sera showed reduced inhibitory activity against viruses containing an E484K spike mutation. As antibodies binding to spike receptor-binding domain and N-terminal domain demonstrate diminished neutralization potency in vitro against some emerging variants, updated mAb cocktails targeting highly conserved regions, enhancement of mAb potency or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

The plasmablast response to SARS-CoV-2 mRNA vaccination is dominated by non-neutralizing antibodies and targets both the NTD and the RBD.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.