Determinants of de novo B cell responses to drifted epitopes in post-vaccination SARS-CoV-2 infections.

Vaccine-induced immunity may impact subsequent de novo responses to drifted epitopes in SARS-CoV-2 variants, but this has been difficult to quantify due to the challenges in recruiting unvaccinated control groups whose first exposure to SARS-CoV-2 is a primary infection. Through local, statewide, and national SARS-CoV-2 testing programs, we were able to recruit cohorts of individuals who had recovered from either primary or post-vaccination infections by either the Delta or Omicron BA.1 variants. Regardless of variant, we observed greater Spike-specific and neutralizing antibody responses in post-vaccination infections than in those who were infected without prior vaccination. Through analysis of variant-specific memory B cells as markers of de novo responses, we observed that Delta and Omicron BA.1 infections led to a marked shift in immunodominance in which some drifted epitopes elicited minimal responses, even in primary infections. Prior immunity through vaccination had a small negative impact on these de novo responses, but this did not correlate with cross-reactive memory B cells, arguing against competitive inhibition of naïve B cells. We conclude that dampened de novo B cell responses against drifted epitopes are mostly a function of altered immunodominance hierarchies that are apparent even in primary infections, with a more modest contribution from pre-existing immunity, perhaps due to accelerated antigen clearance.

Sensitive SARS-CoV-2 salivary antibody assays for clinical saline gargle samples using smartphone-based competitive particle immunoassay platforms.

Antibody assay for SARS-CoV-2 has become increasingly important to track latent and asymptomatic infections, check the individual's immune status, and confirm vaccine efficacy and durability. However, current SARS-CoV-2 antibody assays require invasive blood collection, requiring a remote laboratory and a trained phlebotomist. Direct detection of SARS-CoV-2 antibodies from clinical saline gargle samples has been considered challenging due to the smaller number of antibodies in such specimens and the high limit of detection of currently available rapid tests. This work demonstrates simple and non-invasive methods for detecting SARS-CoV-2 salivary antibodies. Competitive particle immunoassays were developed on a paper microfluidic chip using the receptor-binding domain (RBD) antigens on spike proteins. Using a smartphone, they were monitored by counting the captured fluorescent particles or evaluating the capillary flow velocities. The limit of detection (LOD), cross-binding between alpha- and omicron-strains, and the effect of angiotensin-converting enzyme 2 (ACE2) presence were investigated. LODs were 1-5 ng/mL in both 10% and 1% saliva. Clinical saline gargle samples were assayed using both methods, showing a statistical difference between virus-negative and virus-positive samples, although the assays targeted antibodies. Only a small number of virus-positive samples were antibody-negative. The high assay sensitivity detected a small number of antibodies developed even during the early phase of infections. Overall, this work demonstrates the ability to detect SARS-CoV-2 salivary IgG antibodies on simple, cost-effective, portable platforms towards mitigating SARS-CoV-2 and potentially other respiratory viruses.

Sensitive, smartphone-based SARS-CoV-2 detection from clinical saline gargle samples.

Saliva specimens have drawn interest for diagnosing respiratory viral infections due to their ease of collection and decreased risk to healthcare providers. However, rapid and sensitive immunoassays have not yet been satisfactorily demonstrated for such specimens due to their viscosity and low viral loads. Using paper microfluidic chips and a smartphone-based fluorescence microscope, we developed a highly sensitive, low-cost immunofluorescence particulometric SARS-CoV-2 assay from clinical saline gargle samples. We demonstrated the limit of detection of 10 ag/µL. With easy-to-collect saline gargle samples, our clinical sensitivity, specificity, and accuracy were 100%, 86%, and 93%, respectively, for n = 27 human subjects with n = 13 RT-qPCR positives.

Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip.

Respiratory viruses, especially coronaviruses, have resulted in worldwide pandemics in the past couple of decades. Saliva-based paper microfluidic assays represent an opportunity for noninvasive and rapid screening, yet both the sample matrix and test method come with unique challenges. In this work, we demonstrated the rapid and sensitive detection of SARS-CoV-2 from saliva samples, which could be simpler and more comfortable for patients than existing methods. Furthermore, we systematically investigated the components of saliva samples that affected assay performance. Using only a smartphone, an antibody-conjugated particle suspension, and a paper microfluidic chip, we made the assay user-friendly with minimal processing. Unlike the previously established flow rate assays that depended solely on the flow rate or distance, this unique assay analyzes the flow profile to determine infection status. Particle-target immunoagglutination changed the surface tension and subsequently the capillary flow velocity profile. A smartphone camera automatically measured the flow profile using a Python script, which was not affected by ambient light variations. The limit of detection (LOD) was 1 fg/µL SARS-CoV-2 from 1% saliva samples and 10 fg/µL from simulated saline gargle samples (15% saliva and 0.9% saline). This method was highly specific as demonstrated using influenza A/H1N1. The sample-to-answer assay time was <15 min, including <1-min capillary flow time. The overall accuracy was 89% with relatively clean clinical saline gargle samples. Despite some limitations with turbid clinical samples, this method presents a potential solution for rapid mass testing techniques during any infectious disease outbreak as soon as the antibodies become available.

Immune responses to two and three doses of the BNT162b2 mRNA vaccine in adults with solid tumors.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have shown high efficacy, but immunocompromised participants were excluded from controlled clinical trials. In this study, we compared immune responses to the BNT162b2 mRNA Coronavirus Disease 2019 vaccine in patients with solid tumors (n = 53) who were on active cytotoxic anti-cancer therapy to a control cohort of participants without cancer (n = 50). Neutralizing antibodies were detected in 67% of patients with cancer after the first immunization, followed by a threefold increase in median titers after the second dose. Similar patterns were observed for spike protein-specific serum antibodies and T cells, but the magnitude of each of these responses was diminished relative to the control cohort. In most patients with cancer, we detected spike receptor-binding domain and other S1-specific memory B cell subsets as potential predictors of anamnestic responses to additional immunizations. We therefore initiated a phase 1 trial for 20 cancer cohort participants of a third vaccine dose of BNT162b2 ( NCT04936997 ); primary outcomes were immune responses, with a secondary outcome of safety. At 1 week after a third immunization, 16 participants demonstrated a median threefold increase in neutralizing antibody responses, but no improvement was observed in T cell responses. Adverse events were mild. These results suggest that a third dose of BNT162b2 is safe, improves humoral immunity against SARS-CoV-2 and could be immunologically beneficial for patients with cancer on active chemotherapy.

Immune Responses to COVID-19 mRNA Vaccines in Patients with Solid Tumors on Active, Immunosuppressive Cancer Therapy.

Vaccines against SARS-CoV-2 have shown high efficacy, but immunocompromised participants were excluded from controlled clinical trials. We compared immune responses to the Pfizer/BioNTech mRNA vaccine in solid tumor patients (n=53) on active cytotoxic anti-cancer therapy to a control cohort (n=50) as an observational study. Using live SARS-CoV-2 assays, neutralizing antibodies were detected in 67% and 80% of cancer patients after the first and second immunizations, respectively, with a 3-fold increase in median titers after the booster. Similar trends were observed in serum antibodies against the receptor-binding domain (RBD) and S2 regions of Spike protein, and in IFNγ+ Spike-specific T cells. Yet the magnitude of each of these responses was diminished relative to the control cohort. We therefore quantified RBD- and Spike S1-specific memory B cell subsets as predictors of anamnestic responses to additional immunizations. After the second vaccination, Spike-specific plasma cell-biased memory B cells were observed in most cancer patients at levels similar to those of the control cohort after the first immunization. We initiated an interventional phase 1 trial of a third booster shot (NCT04936997); primary outcomes were immune responses with a secondary outcome of safety. After a third immunization, the 20 participants demonstrated an increase in antibody responses, with a median 3-fold increase in virus-neutralizing titers. Yet no improvement was observed in T cell responses at 1 week after the booster immunization. There were mild adverse events, primarily injection site myalgia, with no serious adverse events after a month of follow-up. These results suggest that a third vaccination improves humoral immunity against COVID-19 in cancer patients on active chemotherapy with no severe adverse events.

An Early Pandemic Analysis of SARS-CoV-2 Population Structure and Dynamics in Arizona.

In December of 2019, a novel coronavirus, SARS-CoV-2, emerged in the city of Wuhan, China, causing severe morbidity and mortality. Since then, the virus has swept across the globe, causing millions of confirmed infections and hundreds of thousands of deaths. To better understand the nature of the pandemic and the introduction and spread of the virus in Arizona, we sequenced viral genomes from clinical samples tested at the TGen North Clinical Laboratory, the Arizona Department of Health Services, and those collected as part of community surveillance projects at Arizona State University and the University of Arizona. Phylogenetic analysis of 84 genomes from across Arizona revealed a minimum of 11 distinct introductions inferred to have occurred during February and March. We show that >80% of our sequences descend from strains that were initially circulating widely in Europe but have since dominated the outbreak in the United States. In addition, we show that the first reported case of community transmission in Arizona descended from the Washington state outbreak that was discovered in late February. Notably, none of the observed transmission clusters are epidemiologically linked to the original travel-related case in the state, suggesting successful early isolation and quarantine. Finally, we use molecular clock analyses to demonstrate a lack of identifiable, widespread cryptic transmission in Arizona prior to the middle of February 2020.IMPORTANCE As the COVID-19 pandemic swept across the United States, there was great differential impact on local and regional communities. One of the earliest and hardest hit regions was in New York, while at the same time Arizona (for example) had low incidence. That situation has changed dramatically, with Arizona now having the highest rate of disease increase in the country. Understanding the roots of the pandemic during the initial months is essential as the pandemic continues and reaches new heights. Genomic analysis and phylogenetic modeling of SARS-COV-2 in Arizona can help to reconstruct population composition and predict the earliest undetected introductions. This foundational work represents the basis for future analysis and understanding as the pandemic continues.