Longitudinal waning of mRNA vaccine-induced neutralizing antibodies against SARS-CoV-2 detected by an LFIA rapid test

While mRNA vaccines against SARS-CoV-2 were highly efficacious against severe illness and hospitalization, they seem to be less effective in preventing infection months after vaccination, especially with the Delta variant. Breakthrough infections might be due to higher infectivity of the variants, relaxed protective measures by the general public in “COVID-19 fatigue”, and/or waning immunity post-vaccination. Determining the neutralizing antibody levels in a longitudinal manner may address this issue, but technical complexity of classic assays precludes easy detection and quick answers. We developed a lateral flow immunoassay NeutraXpress™ (commercial name of the test kit by Antagen Diagnostics, Inc.), and tested fingertip blood samples of subjects receiving either Moderna or Pfizer vaccines at various time points. With this device, we confirmed the reported clinical findings that mRNA vaccine-induced neutralizing antibodies quickly wane after 3–6 months. Thus, using rapid tests to monitor neutralizing antibody status could help identify individuals at risk, prevent breakthrough infections and guide social behavior to curtail the spread of COVID-19. Statement of Significance. Mounting evidence suggests that mRNA vaccine-induced neutralizing antibody titres against SARS-CoV-2 wane in 3–6 months. Quick identification of fully vaccinated persons with high risk of breakthrough infections is key to control the COVID-19 pandemic. The described LFIA device having a control/sample dual-lane design serves this purpose with successful field-test data.

Assessing SARS-CoV-2 Neutralizing Antibodies after BNT162b2 Vaccination and Their Correlation with SARS-CoV-2 IgG Anti-S1, Anti-RBD and Anti-S2 Serological Titers

The humoral response through neutralizing antibodies (NAbs) is a key component of the immune response to COVID-19. However, the plaque reduction neutralization test (PRNT), the gold standard for determining NAbs, is technically demanding, time-consuming and requires BSL-3 conditions. Correlating the NAbs and total antibodies levels, assessed by generalized and automated serological tests, is crucial. Through a commercial surrogate virus neutralization test (sVNT), we aimed to evaluate the production of SARS-CoV-2 NAbs in a set of vaccinated healthcare workers and to correlate these NAbs with the SARS-CoV-2 IgG anti-S1, anti-RBD and anti-S2 serological titers. We found that 6 months after vaccination, only 74% maintain NAbs for the Wuhan strain/UK variant (V1) and 47% maintain NAbs for the South African and Brazil variants (V2). Through Spearman’s correlation, we found the following correlations between the percentage of inhibition of NAbs and the SARS-CoV-2 IgG II Quant (Abbott Laboratories, Chicago, IL, USA) and BioPlex 2200 SARS-CoV-2 IgG Panel (Bio-Rad, Hercules, CA, USA) immunoassays: rho = 0.87 (V1) and rho = 0.73 (V2) for anti-S1 assessed by Abbott assay; rho = 0.77 (V1) and rho = 0.72 (V2) for anti-S1, rho = 0.88 (V1) and rho = 0.82 (V2) for anti-RBD, and rho = 0.68 (V1) and rho = 0.60 (V2) for anti-S2 assessed by BioPlex assay (p < 0.001 for all). In conclusion, we found a strong correlation between this fast, user-friendly, mobile and bio-safe sVNT and the serological immunoassays.

Duration of immunity against COVID-19 after vaccination in Indian subcontinent

Coronavirus disease (COVID-19) continues to be a major health concern leading to substantial mortality and morbidity across the world. Vaccination is effective in reducing the severity and associated mortality. Data pertaining to the duration of immunity, antibody waning and the optimal timing of booster dose administration is limited. In this cross-sectional study, we assessed the antibody levels in healthcare workers who were fully vaccinated after obtaining Institutional ethics committee approval and informed consent. Whole blood was collected and enumeration of S1/S2 neutralizing antibody levels was carried out using LIAISON SARS-COV-2 S1/S2 IgG assay. A total of 1636 individuals who were vaccinated with Covaxin or Covishield were included. Of these, 52% were males with a median age of 29 years. Diabetes and Hypertension was noted in 2.32% (38/1636) and 2.87% (47/1636) of the individuals. Spike neutralizing antibodies were below the detectable range (<15 AU/ml) in 6.0% (98/1636) of the individuals. Decline in neutralizing antibody was seen in 30% of the individuals above 40 years of age with comorbidities (diabetes and hypertension) after 6 months. These individuals may be prioritized for a booster dose at 6 months.

Inflammatory signals from fatty bone marrow supports the early stages of DNMT3a driven clonal hematopoiesis

Age related cancer is not only due to the random accumulation of mutations, but also how phenotypes are selected by the aging environment. While fatty bone marrow (FBM), is one of the hallmarks of bone marrow ageing, it is unknown whether FBM can modify the evolution of the early stages of leukemia and clonal hematopoiesis (CH). To address this question, we established FBM mice models and transplanted both human and mice preleukemic hematopoietic stem cells (PreL-HSCs) carrying DNMT3A mutations. We demonstrate that castration which models age related andropenia result in FBM. A significant increase in self-renewal was found when DNMT3AMut-preL-HSPCs were exposed to FBM. To better understand the mechanisms of the FBM-preL-HSPCs interaction, we performed single cell RNA-sequencing on HSPCs three days after FBM exposure. A 20-50 fold increase in DNMT3AMut-preL-HSCs was observed under FBM conditions in comparison to other conditions. PreL-HSPCs exposed to FBM exhibited an activated inflammatory signaling (IL-6 and INFγ). Cytokine analysis of BM fluid demonstrated increased IL-6 levels under FBM conditions. Anti-IL-6 neutralizing antibodies significantly reduced the selective advantage of DNMT3AMut-preL-HSPCs exposed to FBM. Overall, age related paracrine FBM inflammatory signals promote DNMT3A-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 receptor.

Spike-Dependent Opsonization Indicates Both Dose-Dependent Inhibition of Phagocytosis and That Non-Neutralizing Antibodies Can Confer Protection to SARS-CoV-2

Spike-specific antibodies are central to effective COVID19 immunity. Research efforts have focused on antibodies that neutralize the ACE2-Spike interaction but not on non-neutralizing antibodies. Antibody-dependent phagocytosis is an immune mechanism enhanced by opsonization, where typically, more bound antibodies trigger a stronger phagocyte response. Here, we show that Spike-specific antibodies, dependent on concentration, can either enhance or reduce Spike-bead phagocytosis by monocytes independently of the antibody neutralization potential. Surprisingly, we find that both convalescent patient plasma and patient-derived monoclonal antibodies lead to maximum opsonization already at low levels of bound antibodies and is reduced as antibody binding to Spike protein increases. Moreover, we show that this Spike-dependent modulation of opsonization correlate with the outcome in an experimental SARS-CoV-2 infection model. These results suggest that the levels of anti-Spike antibodies could influence monocyte-mediated immune functions and propose that non-neutralizing antibodies could confer protection to SARS-CoV-2 infection by mediating phagocytosis.

Extremely potent monoclonal antibodies neutralize Omicron and other SARS-CoV-2 variants

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity1. This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies1,2. Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe3. Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.

The SARS-CoV-2 Omicron (B.1.1.529) variant exhibits altered pathogenicity, transmissibility, and fitness in the golden Syrian hamster model

The newly emerging SARS-CoV-2 Omicron (B.1.1.529) variant first identified in South Africa in November 2021 is characterized by an unusual number of amino acid mutations in its spike that renders existing vaccines and therapeutic monoclonal antibodies dramatically less effective. The in vivo pathogenicity, transmissibility, and fitness of this new Variant of Concerns are unknown. We investigated these virological attributes of the Omicron variant in comparison with those of the currently dominant Delta (B.1.617.2) variant in the golden Syrian hamster COVID-19 model. Omicron-infected hamsters developed significantly less body weight losses, clinical scores, respiratory tract viral burdens, cytokine/chemokine dysregulation, and tissue damages than Delta-infected hamsters. The Omicron and Delta variant were both highly transmissible (100% vs 100%) via contact transmission. Importantly, the Omicron variant consistently demonstrated about 10-20% higher transmissibility than the already-highly transmissible Delta variant in repeated non-contact transmission studies (overall: 30/36 vs 24/36, 83.3% vs 66.7%). The Delta variant displayed higher fitness advantage than the Omicron variant without selection pressure in both in vitro and in vivo competition models. However, this scenario drastically changed once immune selection pressure with neutralizing antibodies active against the Delta variant but poorly active against the Omicron variant were introduced, with the Omicron variant significantly outcompeting the Delta variant. Taken together, our findings demonstrated that while the Omicron variant is less pathogenic than the Delta variant, it is highly transmissible and can outcompete the Delta variant under immune selection pressure. Next-generation vaccines and antivirals effective against this new VOC are urgently needed.

An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer

The protective human antibody response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus focuses on the spike (S) protein which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope (supersite) on the N terminal domain (NTD). Here, using the single B cell technology LIBRA-seq, we isolated a large panel of NTD-reactive and SARS-CoV-2 neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies to the NTD supersite commonly are encoded by the IGHV1-24 gene, forming a genetic cluster that represents a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited cell-to-cell spread of virus in culture, and conferred protection in human ACE2 transgenic mice against SARS-CoV-2 challenge. This study provides insight about antibody targeting of the S protein trimer interface region, suggesting this region may be a site of virus vulnerability.

Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity

Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN-𝛾 and IL-10-expressing memory SARS-CoV-2 spike-specific CD4+ T cells than previously naive individuals. While additional vaccination could increase humoral memory, it did not recapitulate the distinct CD4+ T cell cytokine profile in previously naive individuals. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.