Memory B cell responses to Omicron subvariants after SARS-CoV-2 mRNA breakthrough infection in humans.

Individuals who receive a third mRNA vaccine dose show enhanced protection against severe COVID-19, but little is known about the impact of breakthrough infections on memory responses. Here, we examine the memory antibodies that develop after a third or fourth antigenic exposure by Delta or Omicron BA.1 infection, respectively. A third exposure to antigen by Delta breakthrough increases the number of memory B cells that produce antibodies with comparable potency and breadth to a third mRNA vaccine dose. A fourth antigenic exposure with Omicron BA.1 infection increased variant-specific plasma antibody and memory B cell responses. However, the fourth exposure did not increase the overall frequency of memory B cells or their general potency or breadth compared to a third mRNA vaccine dose. In conclusion, a third antigenic exposure by Delta infection elicits strain-specific memory responses and increases in the overall potency and breadth of the memory B cells. In contrast, the effects of a fourth antigenic exposure with Omicron BA.1 are limited to increased strain-specific memory with little effect on the potency or breadth of memory B cell antibodies. The results suggest that the effect of strain-specific boosting on memory B cell compartment may be limited.

Humoral immunity to SARS-CoV-2 elicited by combination COVID-19 vaccination regimens.

The SARS-CoV-2 pandemic prompted a global vaccination effort and the development of numerous COVID-19 vaccines at an unprecedented scale and pace. As a result, current COVID-19 vaccination regimens comprise diverse vaccine modalities, immunogen combinations, and dosing intervals. Here, we compare vaccine-specific antibody and memory B cell responses following two-dose mRNA, single-dose Ad26.COV.2S, and two-dose ChAdOx1, or combination ChAdOx1/mRNA vaccination. Plasma-neutralizing activity, as well as the magnitude, clonal composition, and antibody maturation of the RBD-specific memory B cell compartments, showed substantial differences between the vaccination regimens. While individual monoclonal antibodies derived from memory B cells exhibited similar binding affinities and neutralizing potency against Wuhan-Hu-1 SARS-CoV-2, there were significant differences in epitope specificity and neutralizing breadth against viral variants of concern. Although the ChAdOx1 vaccine was inferior to mRNA and Ad26.COV.2S in several respects, biochemical and structural analyses revealed enrichment in a subgroup of memory B cell neutralizing antibodies with distinct RBD-binding properties resulting in remarkable potency and breadth.

Severe Acute Respiratory Syndrome Coronavirus 2 Neutralization After Messenger RNA Vaccination and Variant Breakthrough Infection.

The emergence of severe acute respiratory syndrome coronavirus 2 variants that have greater transmissibility and resistance to neutralizing antibodies has increased the incidence of breakthrough infections. We show that breakthrough infection increases neutralizing antibody titers to varying degrees depending on the nature of the breakthrough variant and the number of vaccine doses previously administered. Omicron breakthrough infection resulted in neutralizing antibody titers that were the highest across all groups, particularly against Omicron.

Antibody evolution to SARS-CoV-2 after single-dose Ad26.COV2.S vaccine in humans.

The single-dose Ad.26.COV.2 (Janssen) vaccine elicits lower levels of neutralizing antibodies and shows more limited efficacy in protection against infection than either of the two available mRNA vaccines. In addition, Ad.26.COV.2 has been less effective in protection against severe disease during the Omicron surge. Here, we examined the memory B cell response to single-dose Ad.26.COV.2 vaccination. Compared with mRNA vaccines, Ad.26.COV.2 recipients had significantly lower numbers of RBD-specific memory B cells 1.5 or 6 mo after vaccination. Despite the lower numbers, the overall quality of the memory B cell responses appears to be similar, such that memory antibodies elicited by both vaccine types show comparable neutralizing potency against SARS-CoV-2 Wuhan-Hu-1, Delta, and Omicron BA.1 variants. The data help explain why boosting Ad.26.COV.2 vaccine recipients with mRNA vaccines is effective and why the Ad26.COV2.S vaccine can maintain some protective efficacy against severe disease during the Omicron surge.

Plasma and memory antibody responses to Gamma SARS-CoV-2 provide limited cross-protection to other variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a global problem in part because of the emergence of variants of concern that evade neutralization by antibodies elicited by prior infection or vaccination. Here we report on human neutralizing antibody and memory responses to the Gamma variant in a cohort of hospitalized individuals. Plasma from infected individuals potently neutralized viruses pseudotyped with Gamma SARS-CoV-2 spike protein, but neutralizing activity against Wuhan-Hu-1-1, Beta, Delta, or Omicron was significantly lower. Monoclonal antibodies from memory B cells also neutralized Gamma and Beta pseudoviruses more effectively than Wuhan-Hu-1. 69% and 34% of Gamma-neutralizing antibodies failed to neutralize Delta or Wuhan-Hu-1. Although Class 1 and 2 antibodies dominate the response to Wuhan-Hu-1 or Beta, 54% of antibodies elicited by Gamma infection recognized Class 3 epitopes. The results have implications for variant-specific vaccines and infections, suggesting that exposure to variants generally provides more limited protection to other variants.

Antibody and Memory B-Cell Immunity in a Heterogeneously SARS-CoV-2-Infected and -Vaccinated Population.

Global population immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is accumulating through heterogeneous combinations of infection and vaccination. Vaccine distribution in low- and middle-income countries has been variable and reliant on diverse vaccine platforms. We studied B-cell immunity in Mexico, a middle-income country where five different vaccines have been deployed to populations with high SARS-CoV-2 incidences. Levels of antibodies that bound a stabilized prefusion spike trimer, neutralizing antibody titers, and memory B-cell expansion correlated with each other across vaccine platforms. Nevertheless, the vaccines elicited variable levels of B-cell immunity, and the majority of recipients had undetectable neutralizing activity against the recently emergent omicron variant. SARS-CoV-2 infection, experienced before or after vaccination, potentiated B-cell immune responses and enabled the generation of neutralizing activity against omicron and SARS-CoV for all vaccines in nearly all individuals. These findings suggest that broad population immunity to SARS-CoV-2 will eventually be achieved but by heterogeneous paths. IMPORTANCE The majority of studies on SARS-CoV-2 vaccine-elicited immunity and immune evasion have focused on single vaccines corresponding to those distributed in high-income countries. However, in low- and middle-income countries, vaccine deployment has been far less uniform. It is therefore important to determine the levels of immunity elicited by vaccines that have been deployed globally. Such data should help inform policy. Thus, this paper is very much a "real-world" study that focuses on a middle-income country, Mexico, in which five different vaccines based on mRNA, adenovirus, and inactivated-virus platforms have been extensively deployed, while (as documented in our study) SARS-CoV-2 variants with increasing degrees of immune evasiveness have propagated in the Mexican population, culminating in the recent emergence of B.1.1.529 (omicron).

SARS-CoV-2 neutralization after mRNA vaccination and variant breakthrough infection

The emergence of SARS-CoV-2 variants that have greater transmissibility and resistance to neutralizing antibodies has increased the incidence of breakthrough infections. We show that breakthrough infection increases neutralizing antibody titers to varying degrees depending on the nature of the breakthrough variant and the number of vaccine doses previously administered. Omicron breakthrough infection resulted in neutralizing antibody titers that were the highest across, particularly against omicron.

Increased memory B cell potency and breadth after a SARS-CoV-2 mRNA boost.

The Omicron variant of SARS-CoV-2 infected many vaccinated and convalescent individuals1-3. Despite the reduced protection from infection, individuals who received three doses of an mRNA vaccine were highly protected from more serious consequences of infection4. Here we examine the memory B cell repertoire in a longitudinal cohort of individuals receiving three mRNA vaccine doses5,6. We find that the third dose is accompanied by an increase in, and evolution of, receptor-binding domain (RBD)-specific memory B cells. The increase is due to expansion of memory B cell clones that were present after the second dose as well as the emergence of new clones. The antibodies encoded by these cells showed significantly increased potency and breadth when compared with antibodies obtained after the second dose. Notably, the increase in potency was especially evident among newly developing clones of memory cells, which differed from persisting clones in targeting more conserved regions of the RBD. Overall, more than 50% of the analysed neutralizing antibodies in the memory compartment after the third mRNA vaccine dose neutralized the Omicron variant. Thus, individuals receiving three doses of an mRNA vaccine have a diverse memory B cell repertoire that can respond rapidly and produce antibodies capable of clearing even diversified variants such as Omicron. These data help to explain why a third dose of a vaccine that was not specifically designed to protect against variants is effective against variant-induced serious disease.

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.

Anti-SARS-CoV-2 receptor-binding domain antibody evolution after mRNA vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces B cell responses that continue to evolve for at least a year. During that time, memory B cells express increasingly broad and potent antibodies that are resistant to mutations found in variants of concern1. As a result, vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals with currently available mRNA vaccines produces high levels of plasma neutralizing activity against all variants tested1,2. Here we examine memory B cell evolution five months after vaccination with either Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) mRNA vaccine in a cohort of SARS-CoV-2-naive individuals. Between prime and boost, memory B cells produce antibodies that evolve increased neutralizing activity, but there is no further increase in potency or breadth thereafter. Instead, memory B cells that emerge five months after vaccination of naive individuals express antibodies that are similar to those that dominate the initial response. While individual memory antibodies selected over time by natural infection have greater potency and breadth than antibodies elicited by vaccination, the overall neutralizing potency of plasma is greater following vaccination. These results suggest that boosting vaccinated individuals with currently available mRNA vaccines will increase plasma neutralizing activity but may not produce antibodies with equivalent breadth to those obtained by vaccinating convalescent individuals.

High genetic barrier to SARS-CoV-2 polyclonal neutralizing antibody escape.

The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in individuals who are SARS-CoV-2 convalescent and vaccinated are key determinants of neutralization breadth and the genetic barrier to viral escape1-4. Using HIV-1 pseudotypes and plasma selection experiments with vesicular stomatitis virus/SARS-CoV-2 chimaeras5, here we show that multiple neutralizing epitopes, within and outside the receptor-binding domain, are variably targeted by human polyclonal antibodies. Antibody targets coincide with spike sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic 'polymutant' spike protein pseudotypes that resisted polyclonal antibody neutralization to a similar degree as circulating variants of concern. By aggregating variant of concern-associated and antibody-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in the SARS-CoV-2 spike protein are sufficient to generate pseudotypes with near-complete resistance to the polyclonal neutralizing antibodies generated by individuals who are convalescent or recipients who received an mRNA vaccine. However, plasma from individuals who had been infected and subsequently received mRNA vaccination neutralized pseudotypes bearing this highly resistant SARS-CoV-2 polymutant spike, or diverse sarbecovirus spike proteins. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against potential future sarbecovirus pandemics.

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.

Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses.

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.