SARS-CoV-2 Omicron boosting induces de novo B cell response in humans (preprint)

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. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells (MBCs). 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 naive 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.

SARS-CoV-2 booster vaccination rescues attenuated IgG1 memory B cell response in primary antibody deficiency patients (preprint)

SARS-CoV-2 vaccines have proven effective in eliciting an immune response capable of providing protective immunity in healthy individuals. However, whether SARS-CoV-2 vaccination induces a long-lived immune response in immunocompromised individuals is poorly understood. Primary antibody deficiency (PAD) syndromes are among the most common immunodeficiency disorders in adults and are characterized by an impaired ability to mount robust antibody responses following infection or vaccination. Here, we present data from a prospective study in which we analyzed the B and T cell response in PAD patients following SARS-COV-2 vaccination. Unexpectedly, individuals with PAD syndromes mounted a SARS-CoV-2 specific B and CD4+ T cell response that was comparable in magnitude to healthy individuals. Many individuals with PAD syndromes displayed reduced IgG1+ and CD11c+ memory B cell responses following the primary vaccination series. However, the IgG1 class-switching defect was largely rescued following mRNA booster vaccination. Boosting also elicited an increase in the SARS-CoV-2-specific B and T cell response and the development of Omicron-specific memory B cells in COVID-19-naive PAD patients. Together, these data indicate that SARS-CoV-2 vaccines elicit memory B and T cells in PAD patients that may contribute to long-term protective immunity.

mRNA vaccine boosting enhances antibody responses against SARS-CoV-2 Omicron variant in patients with antibody deficiency syndromes (preprint)

Patients with primary antibody deficiency syndromes (PAD) have poor humoral immune responses requiring immunoglobulin replacement therapy. We followed PAD patients after SARS-CoV-2 vaccination by evaluating their immunoglobulin replacement products and serum for anti-spike binding, Fc{gamma}R binding, and neutralizing activities. Immunoglobulin replacement products had low anti-spike and receptor binding domain (RBD) titers and neutralizing activity. In COVID-19-naive PAD patients, anti-spike and RBD titers increased after mRNA vaccination but decreased to pre-immunization levels by 90 days. Patients vaccinated after SARS-CoV-2 infection developed higher responses comparable to healthy donors. Most vaccinated PAD patients had serum neutralizing antibody titers above an estimated correlate of protection against ancestral SARS-CoV-2 and Delta virus but not against Omicron virus, although this was improved by boosting. Thus, currently used immunoglobulin replacement products likely have limited protective activity, and immunization and boosting of PAD patients with mRNA vaccines should confer at least short-term immunity against SARS-CoV-2 variants, including Omicron.

Germinal centre-driven maturation of B cell response to SARS-CoV-2 vaccination (preprint)

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). Induction of the latter is a hallmark of durable immunity after vaccination. SARS-CoV-2 mRNA vaccination induces a robust GC response in humans, 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.

Glucocorticoids and B Cell Depleting Agents Substantially Impair Immunogenicity of mRNA Vaccines to SARS-CoV-2 (preprint)

BackgroundIndividuals with chronic inflammatory diseases (CID) are frequently treated with immunosuppressive medications that can increase their risk of severe COVID-19. While novel mRNA-based SARS-CoV-2 vaccination platforms provide robust protection in immunocompetent individuals, the immunogenicity in CID patients on immunosuppression is not well established. Therefore, determining the effectiveness of SARS-CoV-2 vaccines in the setting of immunosuppression is essential to risk-stratify CID patients with impaired protection and provide clinical guidance regarding medication management. MethodsWe conducted a prospective assessment of mRNA-based vaccine immunogenicity in 133 adults with CIDs and 53 immunocompetent controls. Blood from participants over 18 years of age was collected before initial immunization and 1-2 weeks after the second immunization. Serum anti-SARS-CoV-2 spike (S) IgG+ binding, neutralizing antibody titers, and circulating S-specific plasmablasts were quantified to assess the magnitude and quality of the humoral response following vaccination. ResultsCompared to immunocompetent controls, a three-fold reduction in anti-S IgG titers (P=0.009) and SARS-CoV-2 neutralization (p<0.0001) were observed in CID patients. B cell depletion and glucocorticoids exerted the strongest effect with a 36- and 10-fold reduction in humoral responses, respectively (p<0.0001). Janus kinase inhibitors and antimetabolites, including methotrexate, also blunted antibody titers in multivariate regression analysis (P<0.0001, P=0.0023, respectively). Other targeted therapies, such as TNF inhibitors, IL-12/23 inhibitors, and integrin inhibitors, had only modest impacts on antibody formation and neutralization. ConclusionsCID patients treated with immunosuppressive therapies exhibit impaired SARS-CoV-2 vaccine-induced immunity, with glucocorticoids and B cell depletion therapy more severely impeding optimal responses.

The plasmablast response to SARS-CoV-2 mRNA vaccination is dominated by non-neutralizing antibodies that target both the NTD and the RBD (preprint)

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast derived mAbs from subjects 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, that 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 spike proteins. 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 E484K.

Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization (preprint)

Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of most COVID-19 vaccines and being developed as therapeutics, escape mutations could compromise such countermeasures. To define the immune-mediated mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor binding domain (RBD) to generate 48 escape mutants. These variants were mapped onto the RBD structure and evaluated for cross-resistance by convalescent human plasma. Although each mAb had unique resistance profiles, many shared residues within an epitope, as several variants were resistant to multiple mAbs. Remarkably, we identified mutants that escaped neutralization by convalescent human sera, suggesting that some humans induce a narrow repertoire of neutralizing antibodies. By comparing the antibody-mediated mutational landscape in S protein with sequence variation in circulating SARS-CoV-2 strains, we identified single amino acid substitutions that could attenuate neutralizing immune responses in some humans.

in vitro: Natural Compounds (Thymol, Carvacrol, Hesperidine, And Thymoquinone) Against SARS-CoV2 Strain Isolated From Egyptian Patients (preprint)

The current pandemic of the coronavirus disease-2019 (COVID-19) has badly affected our life during the year 2020. SARS-CoV-2 is the primary causative agent of the newly emerged pandemic. Natural flavonoids, Terpenoid and Thymoquinone are tested against different viral and host-cell protein targets. These natural compounds have a good history in treating Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV). Molecular docking combined with cytotoxicity and plaque reduction assay is used to test the natural compounds against different viral (Spike, RdRp, and Mpro) and host-cell (TMPRSS II, keap 1, and ACE2) targets. The results demonstrate the binding possibility of the natural compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) to the viral main protease (Mpro). Some of these natural compounds were approved to start clinical trail from Egypt Center for Research and Regenerative Medicine ECRRM IRB (Certificate No.IRB00012517)

Targeted Immunosuppression Distinguishes COVID-19 from Influenza in Moderate and Severe Disease (preprint)

Coronavirus disease 2019 (COVID-19) is characterized by a high incidence of acute respiratory failure. The underlying immunopathology of that failure and how it compares to other causes of severe respiratory distress, such as influenza virus infection, are not fully understood. Here we addressed this by developing a prospective observational cohort of COVID-19 and influenza subjects with varying degrees of disease severity and assessing the quality and magnitude of their immune responses at the cellular and protein level. Additionally, we performed single-cell RNA transcriptional profiling of peripheral blood mononuclear cells from select subjects. The cohort consists of 79 COVID-19 subjects, 26 influenza subjects, and 15 control subjects, including 35 COVID-19 and 7 influenza subjects with acute respiratory failure. While COVID-19 subjects exhibited largely equivalent or greater activated lymphocyte counts compared to influenza subjects, they had fewer monocytes and lower surface HLA-class II expression on monocytes compared to influenza subjects and controls. At least two distinct immune profiles were observed by cytokine levels in severe COVID-19 patients: 3 of 71 patients were characterized by extreme inflammation, with greater than or equal to ~50% of the 35 cytokines measured greater than 2 standard deviations from the mean level of other severe patients (both influenza and COVID-19); the other immune profile, which characterized 68 of 71 subjects, had a mixed inflammatory signature, where 28 of 35 cytokines in COVID-19 patients had lower mean cytokine levels, though not all were statistically significant. Only 2 cytokines were higher in COVID-19 subjects compared to influenza subjects (IL-6 and IL-8). Influenza and COVID-19 patients could be distinguished statistically based on cytokine module expression, particularly after controlling for the significant effects of age on cytokine expression, but again with lower levels of most cytokines in COVID-19 subjects. Further, high circulating levels of IL-1RA and IL-6 were associated with increased odds of intubation in the combined influenza and COVID-19 cohort [OR = 3.93 and 4.30, respectively] as well as among only COVID-19 patients. Single cell transcriptional profiling of COVID-19 and influenza subjects with respiratory failure identified profound suppression in type I and type II interferon signaling in COVID-19 patients across multiple clusters. In contrast, COVID-19 cell clusters were enriched for alterations in metabolic, stress, and apoptotic pathways. These alterations were consistent with an increased glucocorticoid response in COVID-19 patients compared to influenza. When considered across the spectrum of innate and adaptive immune profiles, the immune pathologies underlying severe influenza and COVID-19 are substantially distinct. The majority of COVID-19 patients with acute respiratory failure do not have a cytokine storm phenotype but instead exhibit profound type I and type II IFN immunosuppression when compared to patients with acute influenza. Upregulation of a small number of inflammatory mediators, including IL-6, predicts acute respiratory failure in both COVID-19 and influenza patients.