Composition of nasopharyngeal microbiota in individuals with SARS-COV-2 infection across three COVID-19 waves in India (preprint)

Background: SARS-COV-2 is an enveloped RNA virus that is responsible for the global pandemic COVID-19. The virus is reported to cause dysbiosis of the Human Nasopharyngeal microbiota, consequently regulating the host immunity and infection pathophysiology. The compositional change in microbial diversity due to the virus has been reported by independent authors in smaller cohorts and different geographical regions, with a few correlating with fungal and bacterial co-infections. Here, we study for the first time, the nasopharyngeal microbial diversity in the COVID-19 patients, across the three waves in India and explore its correlation with the causative virus variant (and/or the severity of symptoms, if any). Methods: We profiled the nasopharyngeal microbiota of 589 Indian subjects, across the three waves (First; n=181, Second; n=217, Third; n=191), which were further categorized as COVID-19 positives and COVID-19 negatives. These respective groups were further divided into subgroups based on the symptoms as Asymptomatic and Symptomatic. The nasopharyngeal swabs were collected from subjects providing samples for diagnostics purposes at the Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, India. Using high throughput 16S rRNA gene amplicon-based sequencing, we sequenced and profiled the nasopharyngeal DNA microbiome prior to subjecting them to diversity, composition and network analyses. Results: Patients infected with SARS-COV-2 showed a reduced microbial alpha diversity compared to the COVID-19 negatives, in a wave-dependent manner, as implicated by measuring the alpha diversity indices. Furthermore, the compositional change in the community was found to be significantly associated with the viral load as well as the severity of the symptoms observed in the patients. Preliminary taxonomic analysis indicated that, overall, Firmicutes, Proteobacteria, and Actinobacteriota were amongst the dominating Phyla, while Staphylococcaceae and Corynebacteriaceae were the most abundant Families. Also, the microbiota signatures of the first and third wave were more similar to each other at the phylum level compared to the second wave. However, the abundance of microbes varied greatly between the major groups i.e COVID-19 positives and the negatives at the family level, in the respective waves. A similar observation was made where both the commensals and pathobionts differed in abundance between the patient subgroups. Interestingly, the change in microbial network architecture from first to second wave was driven by opportunistic pathogens such as Paenibacillus, Peptostreptococcus, and Solobacterium while Leptotrichia and Actinomyces were noted to be taxonomic groups driving the changes during the third wave when compared to the second wave. Conclusion: In the Indian cohort examined, SARS-COV-2 infection perturbs the nasopharyngeal microbiome, resulting in lower & varied diversity in the niche, irrespective of the virus variant (& thus, the COVID wave) and the disease severity. Whether these changes assist in COVID-19 disease onset & progression, would be interesting to explore in the future.

SARS-CoV-2 Variant Delta Potently Suppresses Innate Immune Response and Evades Interferon-Activated Antiviral Responses (preprint)

ABSTRACT Delta variant of SARS-CoV-2 has caused more severe infections than its previous variants. We studied the host innate immune response to Delta, Alpha and two earlier variants to map the evolution of the recent ones. Our biochemical and transcriptomic studies reveal that Alpha and Delta have progressively evolved over the ancestral variants by silencing innate immune response, thereby limiting cytokine and chemokine production. Though Alpha silenced RLR pathway just as Delta, it failed to persistently silence the innate immune response unlike Delta. Both Alpha and Delta have evolved to resist IFN treatment while they are still susceptible to RLR activation, further highlighting the importance of RLR-mediated, IFN-independent mechanisms in restricting SARS-CoV-2. Our studies reveal that SARS-CoV-2 Delta has integrated multiple mechanisms to silence host innate immune response and evade IFN response. Delta’s silent replication and sustained suppression of host innate immune response, possibly resulting in delayed or reduced intervention by the adaptive immune response, could potentially contribute to the severe symptoms and poor recovery index associated with it.

Genomic analysis of SARS-CoV-2 breakthrough infections from Varanasi, India (preprint)

Studies worldwide have shown that the available vaccines are highly effective against SARS-CoV-2. However, there are growing laboratory reports that the newer variants of concerns (VOCs e.g. Alpha, Beta, Delta etc) may evade vaccine induced defense. In addition to that, there are few ground reports on health workers having breakthrough infections. In order to understand VOC driven breakthrough infection we investigated 14 individuals who tested positive for SARS-CoV-2 after being administered a single or double dose of Covishield (ChAdOx1, Serum Institute of India) from the city of Varanasi, which is located in the Indian state of Uttar Pradesh. Genomic analysis revealed that 78.6% (11/14) of the patients were infected with the B.1.617.2 (Delta) variant. Notably, the frequency (37%) of this variant in the region was significantly lower (p<0.01), suggesting that the vaccinated people were asymmetrically infected with the Delta variant. Most of the patients tested displayed mild symptoms, indicating that even a single dose of the vaccine can help in reducing the severity of the disease. However, more comprehensive epidemiological studies are required to understand the effectiveness of vaccines against the newer VOCs.

SARS-CoV-2 seroprevalence in the city of Hyderabad, India in early 2021 (preprint)

BackgroundCOVID-19 emerged as a global pandemic in 2020, rapidly spreading to most parts of the world. The proportion of infected individuals in a population can be reliably estimated via sero-surveillance, making it a valuable tool for planning control measures. We conducted a serosurvey study to investigate SARS-CoV-2 seroprevalence in the urban population of Hyderabad at the end of the first wave of infections. MethodsThe cross-sectional survey conducted in January 2021 included males and females aged 10 years and above, selected by multi-stage random sampling. 9363 samples were collected from 30 wards distributed over 6 zones of Hyderabad and tested for antibodies against SARS-CoV-2 nucleocapsid antigen. ResultsOverall seropositivity was 54.2%, ranging from 50-60% in most wards. Highest exposure appeared to be among 30-39y and 50-59y olds, with women showing greater seropositivity. Seropositivity increased with family size, with only marginal differences among people with varying levels of education. Seroprevalence was significantly lower among smokers. Only 11% of the survey subjects reported any COVID-19 symptoms, while 17% had appeared for Covid testing. ConclusionOver half the citys population was infected within a year of onset of the pandemic. However, [~]46% people were still susceptible, contributing to subsequent waves of infection. Highlights National level serosurveys under-estimate localised prevalence in dense urban areas SARS-CoV-2 seroprevalence in Hyderabad city was 54.2% after the first wave A large proportion of the population remains at risk over a year into the pandemic

Clinical outcomes in vaccinated individuals hospitalized with Delta variant of SARS-CoV-2 (preprint)

Emerging variants of SARS-CoV-2 with increased transmissibility or immune escape have been causing large outbreaks of COVID-19 infections across the world. As most of the vaccines currently in use have been derived from viral strains circulating in the early part of the pandemic, it becomes imperative to constantly assess the efficacy of these vaccines against emerging variants. In this hospital-based cohort study, we analysed clinical profiles and outcomes of 1161 COVID-19 hospitalized patients (vaccinated with COVISHIELD (ChAdOx1) or COVAXIN (BBV-152), n = 495 and unvaccinated n = 666) in Hyderabad, India between April 24th and May 31st 2021. Viral genome sequencing revealed that >90% of patients in both groups were harbouring the Delta variant (Pango lineage B.1.617.2) of SARS-CoV-2. Vaccinated individuals showed higher neutralizing antibodies (545+-1256 AU/ml Vs 51.1+-296 AU/ml; p<0.001) and significantly decreased Ferritin (392.26+-448.4 ng/mL Vs 544.82+-641.41 ng/mL; p<0.001) and LDH (559.45+-324.05 U/L Vs 644.99+- 294.03 U/L; p<0.001), when compared to the unvaccinated group. Severity of the disease (3.2% Vs 7.2%; p=0.0039) and requirement of ventilatory support (2.8% Vs 5.9%; p=0.0154) were significantly low in the vaccinated group despite the fact that these individuals had significantly higher age and risk factors. The rate of mortality was about 50% lower (2/132=1.51%) in the completely vaccinated breakthrough infections although mortality in individuals who had received a single dose was similar to the unvaccinated group (9/269=3.35% vs 23/666= 3.45%). Our results demonstrate that both COVISHIELD and COVAXIN are effective in preventing disease severity and mortality against the Delta variant in completely vaccinated hospitalized patients.

Host transcriptional response to SARS-CoV-2 infection in COVID-19 patients (preprint)

Background: One of the most perplexing aspects of infection with the SARS-CoV-2 virus has been the variable response elicited in its human hosts. Investigating the transcriptional changes in individuals affected by COVID-19 can help understand and predict the degree of illness and guide clinical outcomes in diverse backgrounds. Methods: Analysis of host transcriptome variations via RNA sequencing from naso/oropharyngeal swabs of COVID-19 patients. Results: We report strong upregulation of the innate immune response, especially type I interferon pathway, upon SARS-CoV-2 infection. Upregulated genes were subjected to a comparative meta-analysis using global datasets to identify a common network of interferon stimulated and viral response genes that mediate the host response and resolution of infection. A large proportion of mis-regulated genes showed a reduction in expression level, suggesting an overall decrease in host mRNA production. Significantly downregulated genes included those encoding olfactory, taste and neuro-sensory receptors. Many pro-inflammatory markers and cytokines were also downregulated or remained unchanged in the COVID-19 patients. Finally, a large number of non-coding RNAs were identified as down-regulated, with a few of the lncRNAs associated with functional roles in directing the response to viral infection. Conclusions: SARS-CoV-2 infection results in the robust activation of the innate immunity. Reduction of gene expression is well correlated with the clinical manifestations and symptoms of COVID-19 such as the loss of smell and taste, and myocardial and neurological complications. This study provides a critical dataset of genes that will enhance our understanding of the nature and prognosis of COVID-19.

Intra-host variability in global SARS-CoV-2 genomes as signatures of RNA editing: implications in viral and host response outcomes (preprint)

Since its zoonotic transmission in the human host, the SARS-CoV-2 virus has infected millions and has diversified extensively. A hallmark feature of viral system survival is their continuous evolution and adaptation within the host. RNA editing via APOBEC and ADAR family of enzymes has been recently implicated as the major driver of intra-host variability of the SARS-CoV-2 genomes. Analysis of the intra-host single-nucleotide variations (iSNVs) in SARS-CoV-2 genomes at spatio-temporal scales can provide insights on the consequence of RNA editing on the establishment, spread and functional outcomes of the virus. In this study, using 1,347 transcriptomes of COVID-19 infected patients across various populations, we find variable prevalence of iSNVs with distinctly higher levels in Indian population. Our results also suggest that iSNVs can likely establish variants in a population. These iSNVs may also contribute to key structural and functional changes in the Spike protein that confer antibody resistance.

Intranasal administration of SARS-CoV-2 neutralizing human antibody prevents infection in mice (preprint)

Prevention of SARS-CoV-2 infection at the point of nasal entry is a novel strategy that has the potential to help contain the ongoing pandemic. Using our proprietary technologies, we have engineered a human antibody that recognizes SARS-CoV-2 S1 spike protein with an enhanced affinity for mucin to improve the antibodys retention in respiratory mucosa. The modified antibody, when administered into mouse nostrils, was shown to block infection in mice that were exposed to high titer SARS-CoV-2 pseudovirus 10 hours after the initial antibody treatment. Our data show that the protection against SARS-CoV-2 infection is effective in both nasal and lung areas 7 days after viral exposure. The modified antibody is stable in a nasal spray formulation and maintains its SARS-CoV-2 neutralizing activity. Nasal spray of the modified antibody can be developed as an affordable and effective prophylactic product to protect people from infection by exposure to SARS-CoV-2 virus in the air. One-sentence summaryA Fc-modified human antibody prevents SARS-CoV-2 viral infection via nasal administration

A distinct phylogenetic cluster of Indian SARS-CoV-2 isolates (preprint)

From an isolated epidemic, COVID-19 has now emerged as a global pandemic. The availability of genomes in the public domain following the epidemic provides a unique opportunity to understand the evolution and spread of the SARS-CoV-2 virus across the globe. The availability of whole genomes from multiple states in India prompted us to analyse the phylogenetic clusters of genomes in India. We performed whole-genome sequencing for 64 genomes making a total of 361 genomes from India, followed by phylogenetic clustering, substitution analysis, and dating of the different phylogenetic clusters of viral genomes. We describe a distinct phylogenetic cluster (Clade I / A3i) of SARS-CoV-2 genomes from India, which encompasses 41% of all genomes sequenced and deposited in the public domain from multiple states in India. Globally 3.5% of genomes, which till date could not be mapped to any distinct known cluster fall in this newly defined clade. The cluster is characterized by a core set of shared genetic variants - C6312A (T2016K), C13730T (A88V/A97V), C23929T, and C28311T (P13L). Further, the cluster is also characterized by a nucleotide substitution rate of 1.4 x 10-3 variants per site per year, lower than the prevalent A2a cluster, and predominantly driven by variants in the E and N genes and relative sparing of the S gene. Epidemiological assessments suggest that the common ancestor emerged in the month of February 2020 and possibly resulted in an outbreak followed by countrywide spread, as evidenced by the low divergence of the genomes from across the country. To the best of our knowledge, this is the first comprehensive study characterizing the distinct and predominant cluster of SARS-CoV-2 in India.