Validation of a heat-inducible Ixodes scapularis HSP70 promoter and developing a tick-specific 3xP3 promoter sequence in ISE6 cells.

Ixodes scapularis is an important vector of many pathogens, including the causative agent of Lyme disease, tick-borne encephalitis, and anaplasmosis. The study of gene function in I. scapularis and other ticks has been hampered by the lack of genetic tools, such as an inducible promoter to permit temporal control over transgenes encoding protein or double-stranded RNA expression. Studies of vector-pathogen relationships would also benefit from the capability to activate anti-pathogen genes at different times during pathogen infection and dissemination. We have characterized an intergenic sequence upstream of the heat shock protein 70 (HSP70) gene that can drive Renilla luciferase expression and mCherry fluorescence in the I. scapularis cell line ISE6. In another construct, we replaced the Drosophila melanogaster minimal HSP70 promoter in the synthetic 3xP3 promoter with a minimal portion of the I. scapularis HSP70 promoter and generated an I. scapularis specific 3xP3 (Is3xP3) promoter. Both promoter constructs, IsHSP70 and Is3xP3, allow for heat-inducible expression of mCherry fluorescence in ISE6 cells with an approximately 10-fold increase in the percentage of fluorescent positive cells upon exposure to a 2 h heat shock. These promoters described here will be valuable tools for gene function studies and temporal control of gene expression, including anti-pathogen genes.

Antiviral innate immune memory in alveolar macrophages following SARS-CoV-2 infection.

Pathogen encounter results in long-lasting epigenetic imprinting that shapes diseases caused by heterologous pathogens. The breadth of this innate immune memory is of particular interest in the context of respiratory pathogens with increased pandemic potential and wide-ranging impact on global health. Here, we investigated epigenetic imprinting across cell lineages in a disease relevant murine model of SARS-CoV-2 recovery. Past SARS-CoV-2 infection resulted in increased chromatin accessibility of type I interferon (IFN-I) related transcription factors in airway-resident macrophages. Mechanistically, establishment of this innate immune memory required viral pattern recognition and canonical IFN-I signaling and augmented secondary antiviral responses. Past SARS-CoV-2 infection ameliorated disease caused by the heterologous respiratory pathogen influenza A virus. Insights into innate immune memory and how it affects subsequent infections with heterologous pathogens to influence disease pathology could facilitate the development of broadly effective therapeutic strategies.

Heteromultimeric sarbecovirus receptor binding domain immunogens primarily generate variant-specific neutralizing antibodies.

Vaccination will likely be a key component of strategies to curtail or prevent future sarbecovirus pandemics and to reduce the prevalence of infection and disease by future SARS-CoV-2 variants. A "pan-sarbecovirus" vaccine, that provides maximum possible mitigation of human disease, should elicit neutralizing antibodies with maximum possible breadth. By positioning multiple different receptor binding domain (RBD) antigens in close proximity on a single immunogen, it is postulated that cross-reactive B cell receptors might be selectively engaged. Heteromultimeric vaccines could therefore elicit individual antibodies that neutralize a broad range of viral species. Here, we use model systems to investigate the ability of multimeric sarbecovirus RBD immunogens to expand cross-reactive B cells and elicit broadly reactive antibodies. Homomultimeric RBD immunogens generated higher serum neutralizing antibody titers than the equivalent monomeric immunogens, while heteromultimeric RBD immunogens generated neutralizing antibodies recognizing each RBD component. Moreover, RBD heterodimers elicited a greater fraction of cross-reactive germinal center B cells and cross-reactive RBD binding antibodies than did homodimers. However, when serum antibodies from RBD heterodimer-immunized mice were depleted using one RBD component, neutralization activity against the homologous viral pseudotype was removed, but neutralization activity against pseudotypes corresponding to the other RBD component was unaffected. Overall, simply combining divergent RBDs in a single immunogen generates largely separate sets of individual RBD-specific neutralizing serum antibodies that are mostly incapable of neutralizing viruses that diverge from the immunogen components.

Characterization of Live-Attenuated Powassan Virus Vaccine Candidates Identifies an Efficacious Prime-Boost Strategy for Mitigating Powassan Virus Disease in a Murine Model.

Powassan virus (POWV) is an emerging tick-borne virus and cause of lethal encephalitis in humans. The lack of treatment or prevention strategies for POWV disease underscores the need for an effective POWV vaccine. Here, we took two independent approaches to develop vaccine candidates. First, we recoded the POWV genome to increase the dinucleotide frequencies of CpG and UpA to potentially attenuate the virus by raising its susceptibility to host innate immune factors, such as the zinc-finger antiviral protein (ZAP). Secondly, we took advantage of the live-attenuated yellow fever virus vaccine 17D strain (YFV-17D) as a vector to express the structural genes pre-membrane (prM) and envelope (E) of POWV. The chimeric YFV-17D-POWV vaccine candidate was further attenuated for in vivo application by removing an N-linked glycosylation site within the nonstructural protein (NS)1 of YFV-17D. This live-attenuated chimeric vaccine candidate significantly protected mice from POWV disease, conferring a 70% survival rate after lethal challenge when administered in a homologous two-dose regimen. Importantly, when given in a heterologous prime-boost vaccination scheme, in which vaccination with the initial chimeric virus was followed by a protein boost with the envelope protein domain III (EDIII), 100% of the mice were protected without showing any signs of morbidity. Combinations of this live-attenuated chimeric YFV-17D-POWV vaccine candidate with an EDIII protein boost warrant further studies for the development of an effective vaccine strategy for the prevention of POWV disease.

Clonal replacement sustains long-lived germinal centers primed by respiratory viruses.

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures.

IL-6/STAT3 axis dictates the PNPLA3-mediated susceptibility to non-alcoholic fatty liver disease.

BACKGROUND & AIMS: A number of genetic polymorphisms have been associated with susceptibility to or protection against non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain unknown. Here, we focused on the rs738409 C>G single nucleotide polymorphism (SNP), which produces the I148M variant of patatin-like phospholipase domain-containing protein 3 (PNPLA3) and is strongly associated with NAFLD. METHODS: To enable mechanistic dissection, we developed a human pluripotent stem cell (hPSC)-derived multicellular liver culture by incorporating hPSC-derived hepatocytes, hepatic stellate cells, and macrophages. We first applied this liver culture to model NAFLD by utilising a lipotoxic milieu reflecting the circulating levels of disease risk factors in affected individuals. We then created an isogenic pair of liver cultures differing only at rs738049 and compared NAFLD phenotype development. RESULTS: Our hPSC-derived liver culture recapitulated many key characteristics of NAFLD development and progression including lipid accumulation and oxidative stress, inflammatory response, and stellate cell activation. Under the lipotoxic conditions, the I148M variant caused the enhanced development of NAFLD phenotypes. These differences were associated with elevated IL-6/signal transducer and activator of transcription 3 (STAT3) activity in liver cultures, consistent with transcriptomic data of liver biopsies from individuals carrying the rs738409 SNP. Dampening IL-6/STAT3 activity alleviated the I148M-mediated susceptibility to NAFLD, whereas boosting it in wild-type liver cultures enhanced NAFLD development. Finally, we attributed this elevated IL-6/STAT3 activity in liver cultures carrying the rs738409 SNP to increased NF-κB activity. CONCLUSIONS: Our study thus reveals a potential causal link between elevated IL-6/STAT3 activity and 148M-mediated susceptibility to NAFLD. IMPACT AND IMPLICATIONS: An increasing number of genetic variants manifest in non-alcoholic fatty liver disease (NAFLD) development and progression; however, the underlying mechanisms remain elusive. To study these variants in human-relevant systems, we developed an induced pluripotent stem cell-derived multicellular liver culture and focused on a common genetic variant (i.e. rs738409 in PNPLA3). Our findings not only provide mechanistic insight, but also a potential therapeutic strategy for NAFLD driven by this genetic variant in PNPLA3. Our liver culture is therefore a useful platform for exploring genetic variants in NAFLD development.

A minimally-edited mouse model for infection with multiple SARS-CoV-2 strains.

Efficient mouse models to study SARS-CoV-2 infection are critical for the development and assessment of vaccines and therapeutic approaches to mitigate the current pandemic and prevent reemergence of COVID-19. While the first generation of mouse models allowed SARS-CoV-2 infection and pathogenesis, they relied on ectopic expression and non-physiological levels of human angiotensin-converting enzyme 2 (hACE2). Here we generated a mouse model carrying the minimal set of modifications necessary for productive infection with multiple strains of SARS-CoV-2. Substitution of only three amino acids in the otherwise native mouse Ace2 locus (Ace2 TripleMutant or Ace2™), was sufficient to render mice susceptible to both SARS-CoV-2 strains USA-WA1/2020 and B.1.1.529 (Omicron). Infected Ace2™ mice exhibited weight loss and lung damage and inflammation, similar to COVID-19 patients. Previous exposure to USA-WA1/2020 or mRNA vaccination generated memory B cells that participated in plasmablast responses during breakthrough B.1.1.529 infection. Thus, the Ace2™ mouse replicates human disease after SARS-CoV-2 infection and provides a tool to study immune responses to sequential infections in mice.

Cell-impermeable staurosporine analog targets extracellular kinases to inhibit HSV and SARS-CoV-2.

Herpes simplex virus (HSV) receptor engagement activates phospholipid scramblase triggering Akt translocation to the outer leaflet of the plasma membrane where its subsequent phosphorylation promotes viral entry. We hypothesize that this previously unrecognized outside-inside signaling pathway is employed by other viruses and that cell-impermeable kinase inhibitors could provide novel antivirals. We synthesized a cell-impermeable analog of staurosporine, CIMSS, which inhibited outer membrane HSV-induced Akt phosphorylation and blocked viral entry without inducing apoptosis. CIMSS also blocked the phosphorylation of 3-phosphoinositide dependent protein kinase 1 and phospholipase C gamma, which were both detected at the outer leaflet following HSV exposure. Moreover, vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike protein (VSV-S), but not native VSV or VSV pseudotyped with Ebola virus glycoprotein, triggered this scramblase-Akt outer membrane signaling pathway. VSV-S and native SARS-CoV-2 infection were inhibited by CIMSS. Thus, CIMSS uncovered unique extracellular kinase processes linked to HSV and SARS-CoV-2 entry.

The risk of COVID-19 death is much greater and age dependent with type I IFN autoantibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection fatality rate (IFR) doubles with every 5 y of age from childhood onward. Circulating autoantibodies neutralizing IFN-α, IFN-ω, and/or IFN-ß are found in ∼20% of deceased patients across age groups, and in ∼1% of individuals aged <70 y and in >4% of those >70 y old in the general population. With a sample of 1,261 unvaccinated deceased patients and 34,159 individuals of the general population sampled before the pandemic, we estimated both IFR and relative risk of death (RRD) across age groups for individuals carrying autoantibodies neutralizing type I IFNs, relative to noncarriers. The RRD associated with any combination of autoantibodies was higher in subjects under 70 y old. For autoantibodies neutralizing IFN-α2 or IFN-ω, the RRDs were 17.0 (95% CI: 11.7 to 24.7) and 5.8 (4.5 to 7.4) for individuals <70 y and ≥70 y old, respectively, whereas, for autoantibodies neutralizing both molecules, the RRDs were 188.3 (44.8 to 774.4) and 7.2 (5.0 to 10.3), respectively. In contrast, IFRs increased with age, ranging from 0.17% (0.12 to 0.31) for individuals <40 y old to 26.7% (20.3 to 35.2) for those ≥80 y old for autoantibodies neutralizing IFN-α2 or IFN-ω, and from 0.84% (0.31 to 8.28) to 40.5% (27.82 to 61.20) for autoantibodies neutralizing both. Autoantibodies against type I IFNs increase IFRs, and are associated with high RRDs, especially when neutralizing both IFN-α2 and IFN-ω. Remarkably, IFRs increase with age, whereas RRDs decrease with age. Autoimmunity to type I IFNs is a strong and common predictor of COVID-19 death.

Analysis of memory B cells identifies conserved neutralizing epitopes on the N-terminal domain of variant SARS-Cov-2 spike proteins.

SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor-binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for antibodies. Here, we use NTD-specific probes to capture anti-NTD memory B cells in a longitudinal cohort of infected individuals, some of whom were vaccinated. We found 6 complementation groups of neutralizing antibodies. 58% targeted epitopes outside the NTD supersite, 58% neutralized either Gamma or Omicron, and 14% were broad neutralizers that also neutralized Omicron. Structural characterization revealed that broadly active antibodies targeted three epitopes outside the NTD supersite including a class that recognized both the NTD and SD2 domain. Rapid recruitment of memory B cells producing these antibodies into the plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants, including Omicron.

Conserved Neutralizing Epitopes on the N-Terminal Domain of Variant SARS-CoV-2 Spike Proteins.

SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for the antibody system. Neutralizing antibodies targeting the RBD bind to several different sites on this domain. In contrast, most neutralizing antibodies to NTD characterized to date bind to a single supersite, however these antibodies were obtained by methods that were not NTD specific. Here we use NTD specific probes to focus on anti-NTD memory B cells in a cohort of pre-omicron infected individuals some of which were also vaccinated. Of 275 NTD binding antibodies tested 103 neutralized at least one of three tested strains: Wuhan-Hu-1, Gamma, or PMS20, a synthetic variant which is extensively mutated in the NTD supersite. Among the 43 neutralizing antibodies that were further characterized, we found 6 complementation groups based on competition binding experiments. 58% targeted epitopes outside the NTD supersite, and 58% neutralized either Gamma or Omicron, but only 14% were broad neutralizers. Three of the broad neutralizers were characterized structurally. C1520 and C1791 recognize epitopes on opposite faces of the NTD with a distinct binding pose relative to previously described antibodies allowing for greater potency and cross-reactivity with 7 different variants including Beta, Delta, Gamma and Omicron. Antibody C1717 represents a previously uncharacterized class of NTD-directed antibodies that recognizes the viral membrane proximal side of the NTD and SD2 domain, leading to cross-neutralization of Beta, Gamma and Omicron. We conclude SARS-CoV-2 infection and/or Wuhan-Hu-1 mRNA vaccination produces a diverse collection of memory B cells that produce anti-NTD antibodies some of which can neutralize variants of concern. Rapid recruitment of these cells into the antibody secreting plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants including omicron.

The risk of COVID-19 death is much greater and age-dependent with type I IFN autoantibodies.

SARS-CoV-2 infection fatality rate (IFR) doubles with every five years of age from childhood onward. Circulating autoantibodies neutralizing IFN-α, IFN-ω, and/or IFN-ß are found in ~20% of deceased patients across age groups. In the general population, they are found in ~1% of individuals aged 20-70 years and in >4% of those >70 years old. With a sample of 1,261 deceased patients and 34,159 uninfected individuals, we estimated both IFR and relative risk of death (RRD) across age groups for individuals carrying autoantibodies neutralizing type I IFNs, relative to non-carriers. For autoantibodies neutralizing IFN-α2 or IFN-ω, the RRD was 17.0[95% CI:11.7-24.7] for individuals under 70 years old and 5.8[4.5-7.4] for individuals aged 70 and over, whereas, for autoantibodies neutralizing both molecules, the RRD was 188.3[44.8-774.4] and 7.2[5.0-10.3], respectively. IFRs increased with age, from 0.17%[0.12-0.31] for individuals <40 years old to 26.7%[20.3-35.2] for those ≥80 years old for autoantibodies neutralizing IFN-α2 or IFN-ω, and from 0.84%[0.31-8.28] to 40.5%[27.82-61.20] for the same two age groups, for autoantibodies neutralizing both molecules. Autoantibodies against type I IFNs increase IFRs, and are associated with high RRDs, particularly those neutralizing both IFN-α2 and -ω. Remarkably, IFR increases with age, whereas RRD decreases with age. Autoimmunity to type I IFNs appears to be second only to age among common predictors of COVID-19 death.

Flavivirus-host interactions: an expanding network of proviral and antiviral factors.

Flaviviruses are zoonotic pathogens transmitted by the bite of infected mosquitos and ticks and represent a constant burden to human health. Here we review recent literature aimed at uncovering how flaviviruses interact with the cells that they infect. A better understanding of these interactions may ultimately lead to novel therapeutic targets. We highlight several studies that employed low-biased methods to discover new protein-protein, protein-RNA, and genetic interactions, and spotlight recent work characterizing the host protein, TMEM41B, which has been shown to be critical for infection by diverse flaviviruses and coronaviruses.

Metabolites with SARS-CoV-2 Inhibitory Activity Identified from Human Microbiome Commensals.

The COVID-19 pandemic has highlighted the need to identify additional antiviral small molecules to complement existing therapies. Although increasing evidence suggests that metabolites produced by the human microbiome have diverse biological activities, their antiviral properties remain poorly explored. Using a cell-based SARS-CoV-2 infection assay, we screened culture broth extracts from a collection of phylogenetically diverse human-associated bacteria for the production of small molecules with antiviral activity. Bioassay-guided fractionation uncovered three bacterial metabolites capable of inhibiting SARS-CoV-2 infection. This included the nucleoside analogue N6-(Δ2-isopentenyl)adenosine, the 5-hydroxytryptamine receptor agonist tryptamine, and the pyrazine 2,5-bis(3-indolylmethyl)pyrazine. The most potent of these, N6-(Δ2-isopentenyl)adenosine, had a 50% inhibitory concentration (IC50) of 2 µM. These natural antiviral compounds exhibit structural and functional similarities to synthetic drugs that have been clinically examined for use against COVID-19. Our discovery of structurally diverse metabolites with anti-SARS-CoV-2 activity from screening a small fraction of the bacteria reported to be associated with the human microbiome suggests that continued exploration of phylogenetically diverse human-associated bacteria is likely to uncover additional small molecules that inhibit SARS-CoV-2 as well as other viral infections. IMPORTANCE The continued prevalence of COVID-19 and the emergence of new variants has once again put the spotlight on the need for the identification of SARS-CoV-2 antivirals. The human microbiome produces an array of small molecules with bioactivities (e.g., host receptor ligands), but its ability to produce antiviral small molecules is relatively underexplored. Here, using a cell-based screening platform, we describe the isolation of three microbiome-derived metabolites that are able to prevent SARS-CoV-2 infection in vitro. These molecules display structural similarities to synthetic drugs that have been explored for the treatment of COVID-19, and these results suggest that the microbiome may be a fruitful source of the discovery of small molecules with antiviral activities.

Fc-engineered antibody therapeutics with improved anti-SARS-CoV-2 efficacy.

Monoclonal antibodies with neutralizing activity against SARS-CoV-2 have demonstrated clinical benefits in cases of mild-to-moderate SARS-CoV-2 infection, substantially reducing the risk for hospitalization and severe disease1-4. Treatment generally requires the administration of high doses of these monoclonal antibodies and has limited efficacy in preventing disease complications or mortality among hospitalized patients with COVID-195. Here we report the development and evaluation of anti-SARS-CoV-2 monoclonal antibodies with optimized Fc domains that show superior potency for prevention or treatment of COVID-19. Using several animal disease models of COVID-196,7, we demonstrate that selective engagement of activating Fcγ receptors results in improved efficacy in both preventing and treating disease-induced weight loss and mortality, significantly reducing the dose required to confer full protection against SARS-CoV-2 challenge and for treatment of pre-infected animals. Our results highlight the importance of Fcγ receptor pathways in driving antibody-mediated antiviral immunity and exclude the possibility of pathogenic or disease-enhancing effects of Fcγ receptor engagement of anti-SARS-CoV-2 antibodies upon infection. These findings have important implications for the development of Fc-engineered monoclonal antibodies with optimal Fc-effector function and improved clinical efficacy against COVID-19 disease.

Autoantibodies neutralizing type I IFNs are present in ~4% of uninfected individuals over 70 years old and account for ~20% of COVID-19 deaths.

Circulating autoantibodies (auto-Abs) neutralizing high concentrations (10 ng/mL, in plasma diluted 1 to 10) of IFN-α and/or -ω are found in about 10% of patients with critical COVID-19 pneumonia, but not in subjects with asymptomatic infections. We detect auto-Abs neutralizing 100-fold lower, more physiological, concentrations of IFN-α and/or -ω (100 pg/mL, in 1/10 dilutions of plasma) in 13.6% of 3,595 patients with critical COVID-19, including 21% of 374 patients > 80 years, and 6.5% of 522 patients with severe COVID-19. These antibodies are also detected in 18% of the 1,124 deceased patients (aged 20 days-99 years; mean: 70 years). Moreover, another 1.3% of patients with critical COVID-19 and 0.9% of the deceased patients have auto-Abs neutralizing high concentrations of IFN-ß. We also show, in a sample of 34,159 uninfected subjects from the general population, that auto-Abs neutralizing high concentrations of IFN-α and/or -ω are present in 0.18% of individuals between 18 and 69 years, 1.1% between 70 and 79 years, and 3.4% >80 years. Moreover, the proportion of subjects carrying auto-Abs neutralizing lower concentrations is greater in a subsample of 10,778 uninfected individuals: 1% of individuals <70 years, 2.3% between 70 and 80 years, and 6.3% >80 years. By contrast, auto-Abs neutralizing IFN-ß do not become more frequent with age. Auto-Abs neutralizing type I IFNs predate SARS-CoV-2 infection and sharply increase in prevalence after the age of 70 years. They account for about 20% of both critical COVID-19 cases in the over-80s, and total fatal COVID-19 cases.

Fc-engineered antibody therapeutics with improved efficacy against COVID-19.

Monoclonal antibodies (mAbs) with neutralizing activity against SARS-CoV-2 have demonstrated clinical benefit in cases of mild to moderate SARS-CoV-2 infection, substantially reducing the risk for hospitalization and severe disease1-4. Treatment generally requires the administration of high doses of these mAbs with limited efficacy in preventing disease complications or mortality among hospitalized COVID-19 patients5. Here we report the development and evaluation of Fc-optimized anti-SARS-CoV-2 mAbs with superior potency to prevent or treat COVID-19 disease. In several animal models of COVID-19 disease6,7, we demonstrate that selective engagement of activating FcγRs results in improved efficacy in both preventing and treating disease-induced weight loss and mortality, significantly reducing the dose required to confer full protection upon SARS-CoV-2 challenge and treatment of pre-infected animals. Our results highlight the importance of FcγR pathways in driving antibody-mediated antiviral immunity, while excluding any pathogenic or disease-enhancing effects of FcγR engagement of anti-SARS-CoV-2 antibodies upon infection. These findings have important implications for the development of Fc-engineered mAbs with optimal Fc effector function and improved clinical efficacy against COVID-19 disease.

Naturally enhanced neutralizing breadth to SARS-CoV-2 after one year.

Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies 1,2 . Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines 3,4 . In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naïve individuals 2,5-8 . The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern 4,9 . In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

Naturally enhanced neutralizing breadth against SARS-CoV-2 one year after infection.

More than one year after its inception, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains difficult to control despite the availability of several working vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination, antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable between 6 and 12 months after infection. Vaccination increases all components of the humoral response and, as expected, results in serum neutralizing activities against variants of concern similar to or greater than the neutralizing activity against the original Wuhan Hu-1 strain achieved by vaccination of naive individuals2,5-8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in the variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand markedly after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.