Single-cell Masked Autoencoder: An Accurate and Interpretable Automated Immunophenotyper (preprint)

High-throughput single-cell cytometry data are crucial for understanding immune system\'s involvement in diseases and responses to treatment. Traditional methods for annotating cytometry data, specifically manual gating and clustering, face challenges in scalability, robustness, and accuracy. In this study, we propose a single-cell masked autoencoder (scMAE), which offers an automated solution for immunophenotyping tasks including cell type annotation. The scMAE model is designed to uphold user-defined cell type definitions, thereby facilitating easier interpretation and cross-study comparisons. The scMAE model operates on a pre-train and fine-tune approach. In the pre-training phase, scMAE employs Masked Single-cell Modelling (MScM) to learn relationships between protein markers in immune cells solely based on protein expression, without relying on prior information such as cell identity and cell type-specific marker proteins. Subsequently, the pre-trained scMAE is fine-tuned on multiple specialized tasks via task-specific supervised learning. The pre-trained scMAE addresses the shortcomings of manual gating and clustering methods by providing accurate and interpretable predictions. Through validation across multiple cohorts, we demonstrate that scMAE effectively identifies co-occurrence patterns of bound labeled antibodies, delivers accurate and interpretable cellular immunophenotyping, and improves the prediction of subject metadata status. Specifically, we evaluated scMAE for cell type annotation and imputation at the cellular-level and SARS-CoV-2 infection prediction, secondary immune response prediction against COVID-19, and prediction the infection stage in the COVID-19 progression at the subject-level. The introduction of scMAE marks a significant step forward in immunology research, particularly in large-scale and high-throughput human immune profiling. It offers new possibilities for predicting and interpretating cellular-level and subject-level phenotypes in both health and disease.

Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants (preprint)

The spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to accumulate substitutions, leading to breakthrough infections of vaccinated individuals and prompting the development of updated booster vaccines. Here, we determined the specificity and functionality of antibody and B cell responses following exposure to BA.5 and XBB variants in individuals who received ancestral SARS-CoV-2 mRNA vaccines. BA.5 exposures elicited antibody responses that primarily targeted epitopes conserved between the BA.5 and ancestral spike, with poor reactivity to the XBB.1.5 variant. XBB exposures also elicited antibody responses that targeted epitopes conserved between the XBB.1.5 and ancestral spike. However, unlike BA.5, a single XBB exposure elicited low levels of XBB.1.5-specific antibodies and B cells in some individuals. Pre-existing cross-reactive B cells and antibodies were correlated with stronger overall responses to XBB but weaker XBB-specific responses, suggesting that baseline immunity influences the activation of variant-specific SARS-CoV-2 responses. HighlightsO_LIVariant breakthrough infections boost ancestral cross-reactive antibodies and B cells C_LIO_LIFirst and second BA.5 exposures fail to elicit variant-specific antibodies and B cells C_LIO_LIXBB infections and monovalent vaccinations elicit XBB.1.5-specific responses in some individuals C_LIO_LIXBB.1.5-specific responses correlate with low levels of pre-existing humoral immunity C_LI

Prior vaccination enhances immune responses during SARS-CoV-2 breakthrough infection with early activation of memory T cells followed by production of potent neutralizing antibodies (preprint)

SARS-CoV-2 infection of vaccinated individuals is increasingly common but rarely results in severe disease, likely due to the enhanced potency and accelerated kinetics of memory immune responses. However, there have been few opportunities to rigorously study early recall responses during human viral infection. To better understand human immune memory and identify potential mediators of lasting vaccine efficacy, we used high-dimensional flow cytometry and SARS-CoV-2 antigen probes to examine immune responses in longitudinal samples from vaccinated individuals infected during the Omicron wave. These studies revealed heightened Spike-specific responses during infection of vaccinated compared to unvaccinated individuals. Spike-specific CD4 T cells and plasmablasts expanded and CD8 T cells were robustly activated during the first week. In contrast, memory B cell activation, neutralizing antibody production, and primary responses to non-Spike antigens occurred during the second week. Collectively, these data demonstrate the functionality of vaccine-primed immune memory and highlight memory T cells as rapid responders during SARS-CoV-2 infection.

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine (preprint)

Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for ~9-10 months after the primary 2-dose mRNA vaccine series, as well as for ~3 months after a 3rd mRNA vaccine dose. Notably, antibody decay slowed significantly between 6- and 9-months post-primary vaccination, essentially stabilizing at the time of the 3rd dose. Antibody quality also continued to improve for at least 9 months after primary 2-dose vaccination. Spike- and RBD-specific memory B cells were stable through 9 months post-vaccination with no evidence of decline over time, and ~40-50% of RBD-specific memory B cells were capable of simultaneously recognizing the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells induced by the first 2 doses of mRNA vaccine were boosted significantly by a 3rd dose and the magnitude of this boosting was similar to memory B cells specific for other variants. Pre-3rd dose memory B cell frequencies correlated with the increase in neutralizing antibody titers after the 3rd dose. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit reactivation of immunological memory and constrain further antibody boosting by mRNA vaccines. These data provide a deeper understanding of how the quantity and quality of antibody and memory B cell responses change over time and number of antigen exposures. These data also provide insight into potential immune dynamics following recall responses to additional vaccine doses or post-vaccination infections.

SARS-CoV-2 infections elicit higher levels of original antigenic sin antibodies compared to SARS-CoV-2 mRNA vaccinations (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines elicit higher levels of antibodies compared to natural SARS-CoV-2 infections in most individuals; however, the specificities of antibodies elicited by vaccination versus infection remain incompletely understood. Here, we characterized the magnitude and specificity of SARS-CoV-2 spike-reactive antibodies from 10 acutely infected health care workers and 23 participants who received mRNA-based SARS-CoV-2 vaccines. We found that infection and primary mRNA vaccination elicited S1 and S2-reactive antibodies, while secondary vaccination boosted mostly S1 antibodies. Using magnetic bead-based absorption assays, we found that SARS-CoV-2 infections elicited a large proportion of original antigenic sin-like antibodies that bound efficiently to common seasonal human coronaviruses but poorly to SARS-CoV-2. In converse, vaccination only modestly boosted antibodies reactive to common seasonal human coronaviruses and these antibodies bound efficiently to SARS-CoV-2. Our data indicate that SARS-CoV-2 mRNA vaccinations elicit fundamentally different antibody responses compared to SARS-CoV-2 infections. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/21264363v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@3e4acdorg.highwire.dtl.DTLVardef@1a0e83eorg.highwire.dtl.DTLVardef@ef4468org.highwire.dtl.DTLVardef@7441c4_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LISARS-CoV-2 mRNA vaccines elicit higher levels of antibodies compared to SARS-CoV-2 infections C_LIO_LIThe first dose of an mRNA vaccine generates both S1 and S2 responses while the second dose boosts primarily S1-specific antibodies C_LIO_LISARS-CoV-2 infections, but not mRNA vaccinations, elicit high levels of antibodies that bind strongly to seasonal coronaviruses but weakly to SARS-CoV-2 C_LI

Germinal center responses to SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals (preprint)

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. In this study, through a fine-needle-aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant (KTX) recipients. We found that, unlike healthy subjects, KTX recipients presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cells, SARS-CoV-2 receptor-binding-domain-specific memory B cells and neutralizing antibodies. KTX recipients also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals, and suggest a GC-origin for certain humoral and memory B cell responses following mRNA vaccination.

mRNA Vaccination Induces Durable Immune Memory to SARS-CoV-2 with Continued Evolution to Variants of Concern (preprint)

SARS-CoV-2 mRNA vaccines have shown remarkable efficacy, especially in preventing severe illness and hospitalization. However, the emergence of several variants of concern and reports of declining antibody levels have raised uncertainty about the durability of immune memory following vaccination. In this study, we longitudinally profiled both antibody and cellular immune responses in SARS-CoV-2 naive and recovered individuals from pre-vaccine baseline to 6 months post-mRNA vaccination. Antibody and neutralizing titers decayed from peak levels but remained detectable in all subjects at 6 months post-vaccination. Functional memory B cell responses, including those specific for the receptor binding domain (RBD) of the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants, were also efficiently generated by mRNA vaccination and continued to increase in frequency between 3 and 6 months post-vaccination. Notably, most memory B cells induced by mRNA vaccines were capable of cross-binding variants of concern, and B cell receptor sequencing revealed significantly more hypermutation in these RBD variant-binding clones compared to clones that exclusively bound wild-type RBD. Moreover, the percent of variant cross-binding memory B cells was higher in vaccinees than individuals who recovered from mild COVID-19. mRNA vaccination also generated antigen-specific CD8+ T cells and durable memory CD4+ T cells in most individuals, with early CD4+ T cell responses correlating with humoral immunity at later timepoints. These findings demonstrate robust, multi-component humoral and cellular immune memory to SARS-CoV-2 and current variants of concern for at least 6 months after mRNA vaccination. Finally, we observed that boosting of pre-existing immunity with mRNA vaccination in SARS-CoV-2 recovered individuals primarily increased antibody responses in the short-term without significantly altering antibody decay rates or long-term B and T cell memory. Together, this study provides insights into the generation and evolution of vaccine-induced immunity to SARS-CoV-2, including variants of concern, and has implications for future booster strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/457229v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@16c64b1org.highwire.dtl.DTLVardef@146ca3aorg.highwire.dtl.DTLVardef@86b7edorg.highwire.dtl.DTLVardef@956879_HPS_FORMAT_FIGEXP M_FIG C_FIG

Altered cellular and humoral immune responses following SARS-CoV-2 mRNA vaccination in patients with multiple sclerosis on anti-CD20 therapy. (preprint)

SARS-CoV-2 mRNA vaccination in healthy individuals generates effective immune protection against COVID-19. Little is known, however, about the SARS-CoV-2 mRNA vaccine-induced responses in immunosuppressed patients. We investigated induction of antigen-specific antibody, B cell and T cell responses in patients with multiple sclerosis on anti-CD20 (MS-aCD20) monotherapy following SARS-CoV-2 mRNA vaccination. Treatment with aCD20 significantly reduced Spike and RBD specific antibody and memory B cell responses in most patients, an effect that was ameliorated with longer duration from last aCD20 treatment and extent of B cell reconstitution. In contrast, all MS-aCD20 patients generated antigen-specific CD4 and CD8 T-cell responses following vaccination. However, treatment with aCD20 skewed these responses compromising circulating Tfh responses and augmenting CD8 T cell induction, while largely preserving Th1 priming. These data also revealed underlying features of coordinated immune responses following mRNA vaccination. Specifically, the MS-aCD20 patients who failed to generate anti-RBD IgG had the most severe defect in cTfh cell responses and more robust CD8 T cell responses compared to those who generated anti-RBD IgG, whose T cell responses were more similar to healthy controls. These data define the nature of SARS-CoV-2 vaccine-induced immune landscape in aCD20-treated patients, and provide insights into coordinated mRNA vaccine-induced immune responses in humans. Our findings have implications for clinical decision-making, patient education and public health policy for patients treated with aCD20 and other immunosuppressed patients.

Rapid induction of antigen-specific CD4+ T cells guides coordinated humoral and cellular immune responses to SARS-CoV-2 mRNA vaccination (preprint)

The SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses in healthy individuals following mRNA vaccination. Vaccination induced rapid near-maximal antigen-specific CD4+ T cell responses in all subjects after the first vaccine dose. CD8+ T cell responses developed gradually after the first and second dose and were variable. Vaccine-induced T cells had central memory characteristics and included both Tfh and Th1 subsets, similar to natural infection. Th1 and Tfh responses following the first dose predicted post-boost CD8+ T cell and neutralizing antibody levels, respectively. Integrated analysis of 26 antigen-specific T cell and humoral responses revealed coordinated features of the immune response to vaccination. Lastly, whereas booster vaccination improved CD4+ and CD8+ T cell responses in SARS-CoV-2 naive subjects, the second vaccine dose had little effect on T cell responses in SARS-CoV-2 recovered individuals. Thus, longitudinal analysis revealed robust T cell responses to mRNA vaccination and highlighted early induction of antigen-specific CD4+ T cells.

Longitudinal Analysis Reveals Distinct Antibody and Memory B Cell Responses in SARS-CoV2 Naive and Recovered Individuals Following mRNA Vaccination (preprint)

Novel mRNA vaccines for SARS-CoV2 have been authorized for emergency use and are currently being administered to millions of individuals worldwide. Despite their efficacy in clinical trials, there is limited data on vaccine-induced immune responses in individuals with a prior SARS-CoV2 infection compared to SARS-CoV2 naive subjects. Moreover, how mRNA vaccines impact the development of antibodies as well as memory B cells in COVID-19 experienced versus COVID-19 naive subjects remains poorly understood. In this study, we evaluated antibody responses and antigen-specific memory B cell responses over time in 33 SARS-CoV2 naive and 11 SARS-CoV2 recovered subjects. mRNA vaccination induced significant antibody and memory B cell responses against full-length SARS-CoV2 spike protein and the spike receptor binding domain (RBD). SARS-CoV2 naive individuals benefitted from both doses of mRNA vaccine with additional increases in antibodies and memory B cells following booster immunization. In contrast, SARS-CoV2 recovered individuals had a significant immune response after the first dose with no increase in circulating antibodies or antigen-specific memory B cells after the second dose. Moreover, the magnitude of the memory B cell response induced by vaccination was lower in older individuals, revealing an age-dependence to mRNA vaccine-induced B cell memory. Side effects also tended to associate with post-boost antibody levels, but not with post-boost memory B cells, suggesting that side effect severity may be a surrogate of short-term antibody responses. The frequency of pre-vaccine antigen-specific memory B cells in SARS-CoV2 recovered individuals strongly correlated with post-vaccine antibody levels, supporting a key role for memory B cells in humoral recall responses to SARS-CoV2. This observation may have relevance for future booster vaccines and for responses to viral variants that partially escape pre-existing antibodies and require new humoral responses to be generated from memory B cells. Finally, post-boost antibody levels were not correlated with post-boost memory responses in SARS-CoV2 naive individuals, indicating that short-term antibody levels and memory B cells are complementary immunological endpoints that should be examined in tandem when evaluating vaccine response. Together, our data provide evidence of both serological response and immunological memory following mRNA vaccination that is distinct based on prior SARS-CoV2 exposure. These findings may inform vaccine distribution in a resource-limited setting.