Mutations in S2 subunit of SARS-CoV-2 Omicron spike strongly influence its conformation, fusogenicity and neutralization sensitivity (preprint)

SARS-CoV-2 has remarkable ability to respond to and evolve against the selection pressure by host immunity exemplified by emergence of Omicron lineage. Here, we characterized the functional significance of mutations in Omicron spike. By systematic transfer of mutations in WT spike we assessed neutralization sensitivity, fusogenicity, and TMPRSS2-dependence for entry. The data revealed that the mutations in both S1 and S2 complement to make Omicron highly resistant. Strikingly, the mutations in Omicron S2 modulated the neutralization sensitivity to NTD- and RBD-antibodies, but not to S2 specific neutralizing antibodies, suggesting that the mutations in S2 were primarily acquired to gain resistance to S1-antibodies. Although all six mutations in S2 appeared to act in concert, D796Y showed greatest impact on neutralization sensitivity and rendered WT virus >100-fold resistant to S309, COVA2-17, and 4A8. S2 mutations greatly reduced the antigenicity for NAbs due to reduced exposure of epitopes. In terms of the entry pathway, S1 or S2 mutations only partially altered the entry phenotype of WT and required both sets of mutations for complete switch to endosomal route and loss of syncytia formation. In particular, N856K and L981F in Omicron reduced fusion capacity and explain why subsequent Omicron variants lost them to regain fusogenicity.

Hybrid immunity from SARS-CoV-2 delta variant surge induced low to undetectable levels of neutralizing antibodies against Omicron variant (preprint)

SARS-CoV-2 variants are emerging at frequent intervals with an ability to transmit faster and evade the immune responses. Most of Indian adults have received one or two doses of vaccination and, have also been infected naturally during the first and second waves. However, whether or not this hybrid immunity is protective against the emerging variants has not been determined. We found that a single dose of ChAdOx1 nCoV-19 vaccine in individuals with prior history of COVID-19 infection induced high levels of neutralizing antibodies which associated with protection from infection. Natural infection during the delta variant surge generated neutralizing antibodies against other lineages including the omicron variant. However, most of the subjects had undetectable levels of antibodies to neutralize omicron variant in the follow-up samples collected after six months suggesting that a large majority of people in India are at risk of infection by omicron variant due to waning antibody titers.

Relative Importance of Various Inflammatory Markers and Their Critical Thresholds for COVID-19 Mortality (preprint)

BackgroundVarious inflammatory markers are commonly assessed in many patients to help in the management of COVID-19 patients. It is not clear, though, how much risk of mortality their different levels of elevations entail, and which marker signifies more risk than others and how much. This study was undertaken to describe their levels and to answer these questions regarding eight inflammatory markers, namely, CRP, D-dimer, ferritin, IL-6, LDH, CPK, troponin-I. MethodsThe data were retrieved from the electronic records of 19852 CoViD-19 patients admitted to a chain of hospitals in north India from March 2020 to July 2021. Levels for most markers were available for more than 10,000 patients. In view of widely different ranges of values of different markers, we divided their values into quintiles (Qs) and studied the pattern of mortality and for running the logistic regression. In addition, logarithm transformation was also tried. The statistical distribution of the values was compared by Mann-Whitney test. Relative importance was judged by the mortality rates, area under the ROC curves (AUROCs), and the odds ratios. ResultsAlthough the mortality increased with decreasing ALC and increasing level of all the other markers, more than 70% survived even with levels in the extreme quintile. The adjusted odds ratio was the highest (7.62) for the Q5 levels of IL-6, closely followed by D-dimer (OR = 6.04). The AUROC was the highest (0.817) for LDH and the least (0.612) for CPK. However, the optimal cut-off for any marker could correctly classify not more than 80% deaths and the multivariable logistic regression could correctly classify patients with mortality in less than 24% cases. ConclusionAlthough elevated levels of all the markers and low values of ALC were significant risk factor but no firm evidence was available for any of the eight markers to be a major indicator of the mortality in COVID-19 unless they reach to a critical threshold. Among those studied, D-Dimer (>192 ng/mL) followed by IL-6 (>4.5 pg/mL) had stronger association with mortality even with moderate and higher end of the normal levels and LDH (>433 U/L) and troponin-I (>0.002ng/mL) with only steeply increased levels. Ferritin had modest association, and CPK, CRP and ALC were a relatively poor risk of mortality.

Clinicogenomic analysis of breakthrough infections by SARS CoV2 variants after ChAdOx1 nCoV- 19 vaccination in healthcare workers (preprint)

Background: India saw a massive surge and emergence of SARS CoV2 variants. We elucidated clinical and humoral immune response and genomic analysis of vaccine breakthrough (VBT) infections after ChAdOx1 nCoV-19 vaccine in healthcare workers (HCWs). Methods: The study was conducted on 1858 HCWs receiving two doses of ChAdOx1 nCoV- 19 vaccine. Serial blood samples were collected to measure SARS CoV2 IgG and neutralizing antibodies. 46 RT-PCR positive samples from VBT infections were subjected to whole genome sequencing (WGS). Results: Infection was confirmed in 219 (11.79%) HCWs of which 21.46% (47/219) were non-vaccinated, significantly more (p <0.001) than 9.52% (156/1639) vaccinated group. VBT infections were significantly higher in doctors and nurses compared to other hospital staff (p <0.001). Unvaccinated individuals had 1.57 times higher risk of infection compared to partially vaccinated (p 0.02) and 2.49 times than fully vaccinated (<0.001). Partially vaccinated were at higher risk of infection than fully vaccinated (RR 1.58,p 0.01). There were 3 (1.36%) severe cases and 1 death in unvaccinated group compared to none in the vaccinated. Non-response after 14 days of second dose was seen in 6.5% (3/46) and low antibody levels (1-4.62 S/CO) in 8.69% (4/46). Delta variant (B.1.617.2) was dominant (69.5%) and reinfection was documented in 4 (0.06%) HCWs. Conclusions: Nearly one in ten vaccinated HCWs can get infected, more so with only single dose (13.65%) than two doses (8.62%). Fully vaccinated are better protected with higher humoral immune response. Genomic analysis revealed an alarming rise of Delta variant (B.1.617.2) in VBT infections.

Plasma gradient of soluble urokinase-type plasminogen activator receptor is linked to pathogenic plasma proteome and immune transcriptome and stratifies outcomes in severe COVID-19 (preprint)

Disease caused by SARS-CoV-2 coronavirus (COVID-19) has resulted in significant morbidity and mortality world-wide. A systemic hyper-inflammation characterizes the severe COVID-19 disease often associated with acute respiratory distress syndrome (ARDS). Blood biomarkers capable of risk stratification are of great importance in effective triage and critical care of severe COVID-19 patients. In the present study we report higher plasma abundance of soluble urokinase-type plasminogen activator receptor (sUPAR), expressed by an abnormally expanded circulating myeloid cell population, in severe COVID-19 patients with ARDS. Plasma sUPAR level was found to be linked to a characteristic proteomic signature of plasma, linked to coagulation disorders and complement activation. Receiver operator characteristics curve analysis identified a cut-off value of sUPAR at 1996.809 pg/ml that could predict survival in our cohort (Odds ratio: 2.9286, 95% confidence interval 1.0427-8.2257). Lower sUPAR level than this threshold concentration was associated with a differential expression of the immune transcriptome as well as favourable clinical outcomes, both in terms of survival benefit (Hazard ratio: 0.3615, 95% confidence interval 0.1433-0.912) and faster disease remission in our patient cohort. Thus we identified sUPAR as a key pathogenic circulating molecule linking systemic hyperinflammation to the hypercoagulable state and stratifying clinical outcomes in severe COVID-19 patients with ARDS.

SARS-CoV-2 B.1.617.2 Delta variant emergence and vaccine breakthrough (preprint)

The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and has spread throughout India, displacing the B.1.1.7 (Alpha) variant and other pre-existing lineages. Mathematical modelling indicates that the growth advantage is most likely explained by a combination of increased transmissibility and immune evasion. Indeed in vitro, the delta variant is less sensitive to neutralising antibodies in sera from recovered individuals, with higher replication efficiency as compared to the Alpha variant. In an analysis of vaccine breakthrough in over 100 healthcare workers across three centres in India, the Delta variant not only dominates vaccine-breakthrough infections with higher respiratory viral loads compared to non-delta infections (Ct value of 16.5 versus 19), but also generates greater transmission between HCW as compared to B.1.1.7 or B.1.617.1 (p=0.02). In vitro, the Delta variant shows 8 fold approximately reduced sensitivity to vaccine-elicited antibodies compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against the SARS-CoV-2 Delta variant were significantly lower in participants vaccinated with ChadOx-1 as compared to BNT162b2 (GMT 3372 versus 654, p<0001). These combined epidemiological and in vitro data indicate that the dominance of the Delta variant in India has been most likely driven by a combination of evasion of neutralising antibodies in previously infected individuals and increased virus infectivity. Whilst severe disease in fully vaccinated HCW was rare, breakthrough transmission clusters in hospitals associated with the Delta variant are concerning and indicate that infection control measures need continue in the post-vaccination era.

Genomic characterization and Epidemiology of an emerging SARS-CoV-2 variant in Delhi, India (preprint)

In April 2021, after successfully enduring three waves of the SARS-CoV2 pandemic in 2020, and having reached population seropositivity of about 50%, Delhi, the national capital of India was overwhelmed by the fourth wave. Here, we trace viral, host, and social factors contributing to the scale and exponent of the fourth wave, when compared to preceding waves, in an epidemiological context. Genomic surveillance data from Delhi and surrounding states shows an early phase of the upsurge driven by the entry of the more transmissible B.1.1.7 variant of concern (VOC) into the region in January, with at least one B.1.1.7 super spreader event in February 2021, relatable to known mass gatherings over this period. This was followed by seeding of the B.1.617 VOC, which too is highly transmissible, with rapid expansion of B.1.617.2 sub-lineage outpacing all other lineages. This unprecedented growth of cases occurred in the background of high seropositivity, but with low median neutralizing antibody levels, in a serially sampled cohort. Vaccination breakthrough cases over this period were noted, disproportionately related to VOC in sequenced cases, but usually mild. We find that this surge of SARS-CoV2 infections in Delhi is best explained by the introduction of a new highly transmissible VOC, B.1.617.2, with likely immune-evasion properties; insufficient neutralizing immunity, despite high seropositivity; and social behavior that promoted transmission.

Severe Acute Respiratory Syndrome Coronavirus-2 genome sequence variations relate to morbidity and mortality in Coronavirus Disease-19 (preprint)

Outcome of infection with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) may depend on the host, virus or the host-virus interaction related factors. Complete SARS-CoV-2 genome was sequenced using Illumina and Nanopore platforms from naso-/oro-pharyngeal ri-bonucleic acid (RNA) specimens from COVID-19 patients of varying severity and outcomes, including patients with mild upper respiratory symptoms (n=35), severe disease admitted to intensive care with respiratory and gastrointestinal symptoms (n=21), fatal COVID-19 outcome (n=17) and asymptomatic (n=42). Of a number of genome variants observed, p.16L>L (Nsp1), p.39C>C (Nsp3), p.57Q>H (ORF3a), p.71Y>Y (Membrane glycoprotein), p.194S>L (Nucleocapsid protein) were observed in similar frequencies in different patient subgroups. However, seventeen other variants were observed only in symptomatic patients with severe and fatal COVID-19. Out of the latter, one was in the 5UTR (g.241C>T), eight were synonymous (p.14V>V and p.92L>L in Nsp1 protein, p.226D>D, p.253V>V, and p.305N>N in Nsp3, p.34G>G and p.79C>C in Nsp10 protein, p.789Y>Y in Spike protein), and eight were non-synonymous (p.106P>S, p.157V>F and p.159A>V in Nsp2, p.1197S>R and p.1198T>K in Nsp3, p.97A>V in RdRp, p.614D>G in Spike protein, p.13P>L in nucleocapsid). These were completely absent in the asymptomatic group. SARS-CoV-2 genome variations have a significant impact on COVID-19 presentation, severity and outcome.

RAY: CRISPR diagnostic for rapid and accurate detection of SARS-CoV2 variants on a paper strip (preprint)

The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations- some of which have recently been linked with increased viral load and prognostic complexities. Interestingly, several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qPCR) method. This necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. In this study, we show that the high specificity of Francisella novicida Cas9 (FnCas9) to point mismatches can be successfully adapted for the simultaneous detection of SARS-CoV2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the mutation N501Y (earlier shown to be present in the British N501Y.V1, South African N501Y.V2, and Brazilian N501Y.V3 variants of SARS-CoV2) within an hour using paper strip chemistry. The results were corroborated using deep sequencing. Our design principle can be rapidly adapted for other mutations, highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19.

The lethal triad: SARS-CoV-2 Spike, ACE2 and TMPRSS2. Mutations in host and pathogen may affect the course of pandemic. (preprint)

Variants of SARS-CoV-2 have been identified rapidly after the beginning of pandemic. One of them, involving the spike protein and called D614G, represents a substantial percentage of currently isolated strains. While research on this variant was ongoing worldwide, on December 20th 2020 the European Centre for Disease Prevention and Control reported a Threat Assessment Brief describing the emergence of a new variant of SARS-CoV-2, named B.1.1.7, harboring multiple mutations mostly affecting the Spike protein. This viral variant has been recently associated with a rapid increase in COVID-19 cases in South East England, with alarming implications for future virus transmission rates. Specifically, of the nine amino acid replacements that characterize the Spike in the emerging variant, four are found in the region between the Fusion Peptide and the RBD domain (namely the already known D614G, together with A570D, P681H, T716I), and one, N501Y, is found in the Spike Receptor Binding Domain - Receptor Binding Motif (RBD-RBM). In this study, by using in silico biology, we provide evidence that these amino acid replacements have dramatic effects on the interactions between SARS-CoV-2 Spike and the host ACE2 receptor or TMPRSS2, the protease that induces the fusogenic activity of Spike. Mostly, we show that these effects are strongly dependent on ACE2 and TMPRSS2 polymorphism, suggesting that dynamics of pandemics are strongly influenced not only by virus variation but also by host genetic background.

Surveillance of genetic diversity and evolution in locally transmitted SARS-CoV-2 in Pakistan during the first wave of the COVID-19 pandemic (preprint)

Surveillance of genetic diversity in the SARS-CoV-2 is extremely important to detect the emergence of more infectious and deadly strains of the virus. In this study, we monitored mutational events in the SARS-CoV-2 genome through whole genome sequencing. The samples (n=48) were collected from the hot spot regions of the metropolitan city Karachi, Pakistan during the four months (May 2020 to August 2020) of first wave of the COVID-19 pandemic. The data analysis highlighted 122 mutations, including 120 single nucleotide variations (SNV), and 2 deletions. Among the 122 mutations, there were 71 singletons, and 51 recurrent mutations. A total of 16 mutations, including 5 nonsynonymous mutations, were detected in spike protein. Notably, the spike protein missense mutation D614G was observed in 31 genomes. The phylogenetic analysis revealed majority of the genomes (36) classified as B lineage, where 2 genomes were from B.6 lineage, 5 genomes from B.1 ancestral lineage and remaining from B.1 sub-lineages. It was noteworthy that three clusters of B.1 sub-lineages were observed, including B.1.36 lineage (10 genomes), B.1.160 lineage (11 genomes), and B.1.255 lineage (5 genomes), which represent independent events of SARS-CoV-2 transmission within the city. The sub-lineage B.1.36 had higher representation from the Asian countries and the UK, B.1.160 correspond to the European countries with highest representation from the UK, Denmark, and lesser representation from India, Saudi Arabia, France and Switzerland, and the third sub-lineage (B.1.255) correspond to the USA. Collectively, our study provides meaningful insight into the evolution of SARS-CoV-2 lineages in spatio-temporal local transmission during the first wave of the pandemic.

Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation (preprint)

There is an urgent need to limit and stop the worldwide coronavirus disease 2019 (COVID-19) pandemic via quick development of efficient and safe vaccination methods. Plasmid DNA vaccines are one of the most remarkable vaccines that can be developed in a short term. pVAX1-SARS-CoV2-co, which is a plasmid DNA vaccine, was designed to express severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. The produced antibodies lead to Immunoreactions against S protein, anti-receptor-binding-domain, and neutralizing action of pVAX1-SARS-CoV2-co, as confirmed in a previous study. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine, a pyro-drive jet injector (PJI) was employed. PJI is an injection device that can adjust the injection pressure depending on various target tissues. Intradermally-adjusted PJI demonstrated that pVAX1-SARS-CoV2-co vaccine injection caused a strong production of anti-S protein antibodies, triggered immunoreactions and neutralizing actions against SARS-CoV-2. Moreover, a high dose of pVAX1-SARS-CoV2-co intradermal injection via PJI did not cause any serious disorders in the rat model. Finally, virus infection challenge in mice, confirmed that intradermally immunized (via PJI) mice were potently protected from COVID-19 infection. Thus, pVAX1-SARS-CoV2-co intradermal injection via PJI is a safe and promising vaccination method to overcome the COVID-19 pandemic.

Impacts of 203/204: RG>KR mutation in the N protein of SARS-CoV-2 (preprint)

We present a structure-based model of phosphorylation-dependent binding and sequestration of SARS-CoV-2 nucleocapsid protein and the impact of two consecutive amino acid changes R203K and G204R. Additionally, we studied how mutant strains affect HLA-specific antigen presentation and correlated these findings with HLA allelic population frequencies. We discovered RG>KR mutated SARS-CoV-2 expands the ability for differential expression of the N protein epitope on Major Histocompatibility Complexes (MHC) of varying Human Leukocyte Antigen (HLA) origin. The N protein LKR region K203, R204 of wild type (SARS-CoVs) and (SARS-CoV-2) observed HLA-A*30:01 and HLA-A*30:21, but mutant SARS-CoV-2 observed HLA-A*31:01 and HLA-A*68:01. Expression of HLA-A genotypes associated with the mutant strain occurred more frequently in all populations studied. ImportanceThe novel coronavirus known as SARS-CoV-2 causes a disease renowned as 2019-nCoV (or COVID-19). HLA allele frequencies worldwide could positively correlate with the severity of coronavirus cases and a high number of deaths.

Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2 (preprint)

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus. IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

Immunogenicity and protective efficacy of a highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative (preprint)

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 °C and were stable to long term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼ three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

COVID-19 Risk Stratification and Mortality Prediction in Hospitalized Indian Patients (preprint)

The clinical course of coronavirus disease 2019 (COVID-19) infection is highly variable with the vast majority recovering uneventfully but a small fraction progressing to severe disease and death. Appropriate and timely supportive care can reduce mortality and it is critical to evolve better patient risk stratification based on simple clinical data, so as to perform effective triage during strains on the healthcare infrastructure. This study presents risk stratification and mortality prediction models based on usual clinical data from 544 COVID-19 patients from New Delhi, India using machine learning methods. A Random Forest classifier yielded the best performance on risk stratification (F1 score of 0.81). A logistic regression model yielded the best performance on mortality prediction (F1 score of 0.71). Significant biomarkers for predicting risk and mortality were identified. Examination of the data in comparison to a similar dataset with a Wuhan cohort of 375 patients was undertaken to understand the much lower mortality rates in India and the possible reasons thereof. The comparison indicated higher survival rate in the Delhi cohort even when patients had similar parameters as the Wuhan patients who died. Steroid administration was very frequent in Delhi patients, especially in surviving patients whose biomarkers indicated severe disease. This study helps in identifying the high-risk patient population and suggests treatment protocols that may be useful in countries with high mortality rates.

Clinical and immunological benefits of convalescent plasma therapy in severe COVID-19: insights from a single center open label randomised control trial (preprint)

Introduction: A single center open label phase II randomised control trial was done to assess the pathogen and host-intrinsic factors influencing clinical and immunological benefits of passive immunization using convalescent plasma therapy (CPT), in addition to standard of care (SOC) therapy in severe COVID-19 patients, as compared to patients only on SOC therapy. Methods: Convalescent plasma was collected from patients recovered from COVID-19 following a screening protocol which also included measuring plasma anti SARS-CoV2 spike IgG content. Retrospectively, neutralizing antibody content was measured and proteome was characterized by LC-MS/MS for all convalescent plasma units that were transfused to patients. Severe COVID-19 patients with evidence for acute respiratory distress syndrome (ARDS) with PaO2/FiO2 ratio 100-300 (moderate ARDS) were recruited and randomised into two parallel arms of SOC and CPT, N=40 in each arm. Peripheral blood samples were collected on the day of enrolment (T1) followed by day3/4 (T2) and day 7 (T3). RT-PCR and sequencing was done for SARS-CoV2 RNA isolated from nasopharyngeal swabs collected at T1. A panel of cytokines and neutralizing antibody content were measured in plasma at all three timepoints. Patients were followed up for 30 days post-admission to assess the primary outcomes of all cause mortality and immunological correlates for clinical benefits. Results: While across all age-groups no statistically significant clinical benefit was registered for patients in the CPT arm, significant immediate mitigation of hypoxia, reduction in hospital stay as well as survival benefit was recorded in severe COVID-19 patients with ARDS aged less than 67 years receiving convalescent plasma therapy. In addition to its neutralizing antibody content a prominent effect of convalescent plasma on attenuation of systemic cytokine levels possibly contributed to its benefits. Conclusion: Precise targeting of severe COVID-19 patients is necessary for reaping the clinical benefits of convalescent plasma therapy.

Clinical Profile of First 1000 COVID-19 Cases Admitted at Tertiary Care Hospitals and the Correlates of their Mortality: An Indian Experience (preprint)

ObjectiveTo describe the clinical profile and factors leading to increased mortality in coronavirus disease (COVID-19) patients admitted to a group of hospitals in India. DesignA records-based study of the first 1000 patients with a positive result on real-time reverse transcriptase-polymerase-chain-reaction assay for SARS-CoV-2 admitted to our facilities. Various factors such as demographics, presenting symptoms, co-morbidities, ICU admission, oxygen requirement and ventilator therapy were studied. ResultsOf the 1000 patients, 24 patients were excluded due to lack of sufficient data. Of the remaining 976 in the early phase of the epidemic, males were admitted twice as much as females (67.1% and 32.9%, respectively). Mortality in this initial phase was 10.6% and slightly higher for males and steeply higher for older patients. More than 8% reported no symptoms and the most common presenting symptoms were fever (78.3%), productive cough (37.2%), and dyspnea (30.64%). More than one-half (53.6%) had no co-morbidity. The major co-morbidities were hypertension (23.7%), diabetes without (15.4%), and with complications (9.6%). The co-morbidities were associated with higher ICU admissions, greater use of ventilators as well as higher mortality. A total of 29.9% were admitted to the ICU, with a mortality rate of 32.2%. Mortality was steeply higher in those requiring ventilator support (55.4%) versus those who never required ventilation (1.4%). The total duration of hospital stay was just a day longer in patients admitted to the ICU than those who remained in wards. ConclusionMale patients above the age of 60 and with co-morbidities faced the highest rates of mortality. They should be admitted to the hospital in early stage of the disease and given aggressive treatment to help reduce the morbidity and mortality associated with COVID-19.

Kinetics of viral load, immunological mediators and characterization 1 of a SARS-CoV-2 isolate in mild COVID-19 patients during acute phase of infection (preprint)

Over 95% of the COVID-19 cases are mild-to-asymptomatic who contribute to disease transmission whereas most of the severe manifestations of the disease are observed in elderly and in patients with comorbidities and dysregulation of immune response has been implicated in severe clinical outcomes. However, it is unclear whether asymptomatic or mild infections are due to low viral load or lack of inflammation. We have measured the kinetics of SARS-CoV-2 viral load in the respiratory samples and serum markers of inflammation in hospitalized COVID-19 patients with mild symptoms. We observed a bi-phasic pattern of virus load which was eventually cleared in most patients at the time of discharge. Viral load in saliva samples from a subset of patients showed good correlation with nasopharyngeal samples. Serum interferon levels were downregulated during early stages of infection but peaked at later stages correlating with elevated levels of T-cell cytokines and other inflammatory mediators such as IL-6 and TNF-alpha which showed a bi-phasic pattern. The clinical recovery of patients correlated with decrease in viral load and increase in interferons and other cytokines which indicates an effective innate and adaptive immune function in mild infections. We further characterized one of the SARS-CoV-2 isolate by plaque purification and show that infection of lung epithelial cells (Calu-3) with this isolate led to cytopathic effect disrupting epithelial barrier function and tight junctions. Finally we showed that zinc was capable of inhibiting SARS-CoV-2 infection in this model suggesting a beneficial effect of zinc supplementation in COVID-19 infection.