Boosting Vaccine Response in Autoimmune Rheumatic Disease Patients With Inadequate Seroconversion: An Analysis of the Immunogenicity of Vector-Based and Inactivated Vaccines.

BACKGROUND: An additional dose of COVID-19 vaccine is being offered to vaccinated people, especially those immunocompromised. The most widely available vaccines in India are the adenoviral vector-based AZD1222 (ChAdOx1 nCoV-19) and the heat-inactivated (BBV152). This study investigated the efficacy of both vaccines in patients with autoimmune rheumatic diseases (AIRD). OBJECTIVES:  To compare final anti-SARS-CoV-2 antibody titers, neutralization of pseudovirions by these antibodies, and T cell responses between patients of AIRD who had received the third dose of AZD1222 and BBV152 vaccines. METHODS: Patients with stable AIRD who had completed two doses of COVID-19 vaccination but had a suboptimal response (anti-receptor binding domain (RBD) antibody<212) were randomized (1:1) to receive either AZD1222 or BBV152 as a booster dose. Patients with previous hybrid immunity or those who developed COVID-19 during the trial were excluded. Antibody titers, neutralization of Wuhan and Omicron pseudovirions, and interferon release by T cells (enzyme-linked immunosorbent spot (ELISpot)) in response to the Spike antigen were measured four weeks after this booster dose. RESULTS: 146 were screened, 91 were randomized, and 67 were analyzed per protocol. The third dose improved antibody titers (p<0.001), neutralization of the Wuhan strain (p<0.001), and T cell interferon release (p<0.001) but not neutralization of the Omicron strain (p=0.24). Antibody titers were higher (p<0.005) after ADZ1222 boost (2,414 IU (interquartile range (IQR): 330-10,315)) than BBV1222 (347.7 IU (0.4-973)). Neutralization of the Wuhan stain was better (AZD1222: 76.6%(23.0-95.45) versus BBV152 (32.7% (0-78.9), p=0.03 by ANCOVA). Neutralization of Omicron (0 (0-28.4) vs 0 (0-4.8)) and T cell interferon release (57.0 IU (23.5-95) vs 50.5 IU (13.2-139)) were similar. CONCLUSION: The third dose improved all parameters of immunogenicity in AIRD patients with previous inadequate responses except Omicron neutralization. The vector-based vaccine exhibits notable efficacy, particularly in antibody titers and neutralizing the Wuhan strain. TRIAL REGISTRATION: CTRI/2021/12/038928.

Targeted Sanger Sequencing of a Cluster of COVID-19 Cases in the Surgical ICU of a Non-COVID Hospital: Lessons Learned.

Small clusters of infection due to SARS-CoV-2 in a non-COVID-19 healthcare facility can disrupt services. Here, we investigated a cluster of SARS-CoV-2 cases by targeted Sanger sequencing and clinical epidemiological methods in a non-COVID-19 super-specialty hospital. Epidemiological data were collected in a blinded manner using a proforma to find the risk factors associated with infection. Targeted Sanger sequencing of the spike protein receptor binding domain (RBD) coding region was performed on all the available real-time reverse transcription polymerase chain reaction (RT-PCR)-positive samples that included a patient, his mother, and 11 healthcare workers (HCWs) to determine any genomic variations in the samples from the cluster. All positive cases were due to the Delta variant. However, it detected a unique mutation, N501I, in the RBD region of the SARS-CoV-2 strains. The viral genome extracted from the mother's sample lacked the mutation, thus excluding her from the cluster and pointing out that the outbreak was nosocomial, leading to a focus on infection control measures. Though whole genome sequencing is more universally accepted, in this study, targeted sanger sequencing provided a rapid and cost-effective solution to correctly delineate between the actual cases that form the cluster and other community cases in a pandemic situation.

A high-throughput screening system for SARS-CoV-2 entry inhibition, syncytia formation and cell toxicity.

BACKGROUND: The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell is mediated through the binding of the SARS-CoV-2 Spike protein via the receptor binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2). Identifying compounds that inhibit Spike-ACE2 binding would be a promising and safe antiviral approach against COVID-19. METHODS: In this study, we used a BSL-2 compatible replication-competent vesicular stomatitis virus (VSV) expressing Spike protein of SARS-CoV-2 with eGFP reporter system (VSV-eGFP-SARS-CoV-2) in a recombinant permissive cell system for high-throughput screening of viral entry blockers. The SARS-CoV-2 permissive reporter system encompasses cells that stably express hACE2-tagged cerulean and H2B tagged with mCherry, as a marker of nuclear condensation, which also enables imaging of fused cells among infected EGFP positive cells and could provide real-time information on syncytia formation. RESULTS: A limited high-throughput screening identified six natural products that markedly inhibited VSV-eGFP-SARS-CoV-2 with minimum toxicity. Further studies of Spike-S1 binding using the permissive cells showed Scillaren A and 17-Aminodemethoxygeldanamycin could inhibit S1 binding to ACE2 among the six leads. A real-time imaging revealed delayed inhibition of syncytia by Scillaren A, Proscillaridin, Acetoxycycloheximide and complete inhibition by Didemnin B indicating that the assay is a reliable platform for any image-based drug screening. CONCLUSION: A BSL-2 compatible assay system that is equivalent to the infectious SARS-CoV-2 is a promising tool for high-throughput screening of large compound libraries for viral entry inhibitors against SARS-CoV-2 along with toxicity and effects on syncytia. Studies using clinical isolates of SARS-CoV-2 are warranted to confirm the antiviral potency of the leads and the utility of the screening system.

A rapid bead-based assay for screening of SARS-CoV-2 neutralizing antibodies.

Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost-effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. We describe a bead-based screening assay for S1-neutralization using recombinant fluorescent proteins of hACE2 and SARS-CoV2-S1, immobilized on solid beads employing nanobodies/metal-affinity tags. Nanobody-mediated capture of SARS-CoV-2-Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single-color fluorescent imaging of ACE2-GFPSpark binding to His-tagged S1-Receptor Binding Domain (RBD-His) immobilized beads. The second approach is dual-color imaging of soluble ACE2-GFPSpark to S1-Orange Fluorescent Protein (S1-OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening.