BA.1, BA.2 and BA.2.75 variants show comparable replication kinetics, reduced impact on epithelial barrier and elicit cross-neutralizing antibodies.

The Omicron variant of SARS-CoV-2 is capable of infecting unvaccinated, vaccinated and previously-infected individuals due to its ability to evade neutralization by antibodies. With multiple sub-lineages of Omicron emerging in the last 12 months, there is inadequate information on the quantitative antibody response generated upon natural infection with Omicron variant and whether these antibodies offer cross-protection against other sub-lineages of Omicron variant. In this study, we characterized the growth kinetics of Kappa, Delta and Omicron variants of SARS-CoV-2 in Calu-3 cells. Relatively higher amounts infectious virus titers, cytopathic effect and disruption of epithelial barrier functions was observed with Delta variant whereas infection with Omicron sub-lineages led to a more robust induction of interferon pathway, lower level of virus replication and mild effect on epithelial barrier. The replication kinetics of BA.1, BA.2 and BA.2.75 sub-lineages of the Omicron variant were comparable in cell culture and natural infection in a subset of individuals led to a significant increase in binding and neutralizing antibodies to the Delta variant and all the three sub-lineages of Omicron but the level of neutralizing antibodies were lowest against the BA.2.75 variant. Finally, we show that Cu2+, Zn2+ and Fe2+ salts inhibited in vitro RdRp activity but only Cu2+ and Fe2+ inhibited both the Delta and Omicron variants in cell culture. Thus, our results suggest that high levels of interferons induced upon infection with Omicron variant may counter virus replication and spread. Waning neutralizing antibody titers rendered subjects susceptible to infection by Omicron variants and natural Omicron infection elicits neutralizing antibodies that can cross-react with other sub-lineages of Omicron and other variants of concern.

A broadly neutralizing monoclonal antibody overcomes the mutational landscape of emerging SARS-CoV-2 variants of concern.

The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic monoclonal antibodies (mAbs). Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concern (VOCs) with high efficiency, including the highly transmissible Omicron. The crystal structure of the P4A2 Fab:RBD complex revealed that the residues of the RBD that interact with P4A2 are a part of the ACE2-receptor-binding motif and are not mutated in any of the VOCs. The pan coronavirus pseudotyped neutralization assay confirmed that the P4A2 mAb is specific for SARS-CoV-2 and its VOCs. Passive administration of P4A2 to K18-hACE2 transgenic mice conferred protection, both prophylactically and therapeutically, against challenge with VOCs. Overall, our data shows that, the P4A2 mAb has immense therapeutic potential to neutralize the current circulating VOCs. Due to the overlap between the P4A2 epitope and ACE2 binding site on spike-RBD, P4A2 may also be highly effective against a number of future variants.

Test concordance and diagnostic accuracy of three serological assays for detection of anti-SARS-CoV-2 antibody: result from a population-based sero-epidemiological study in Delhi.

BACKGROUND: Several methodological tests are available to detect SARS-CoV-2 antibody. Tests are mostly used in the aid of diagnosis or for serological assessment. No tests are fully confirmatory and have variable level of diagnostic ability. We aimed at assessing agreement with three serological tests: quantitative anti receptor binding domain ELISA (Q-RBD), qualitative ELISA (WANTAI SARS-CoV-2 Ab) and qualitative chemiluminescence assay (CLIA). METHODS: This study was a part of a large population based sero-epidemiological cohort study. Participants aged 1 year or older were included from 25 randomly selected clusters each in Delhi urban (urban resettlement colony of South Delhi district) and Delhi rural (villages in Faridabad district, Haryana). Three type of tests were applied to all the baseline blood samples. Result of the three tests were evaluated by estimating the total agreement and kappa value. RESULTS: Total 3491 blood samples collected from March to September, 2021, out of which 1700 (48.7%) from urban and 1791 (51.3%) from rural. Overall 44.1% of participants were male. The proportion of sero-positivity were 78.1%, 75.2% and 31.8% by Wantai, QRBD and CLIA tests respectively. The total agreement between Wantai and QRBD was 94.5%, 53.1% between Wantai and CLIA, and 56.8% between QRBD and CLIA. The kappa value between these three tests were 0.84 (95% CI 0.80-0.87), 0.22 (95% CI 0.19-0.24) and 0.26 (95% CI 0.23-0.28). CONCLUSIONS: There was strong concordance between Wantai and QRBD test. Agreement between CLIA with other two tests was low. Wantai and QRBD tests measuring the antibody to same S protein can be used with high agreement based on the relevant scenario.

Antibody Response to SARS-CoV-2 among COVID-19 Confirmed Cases and Correlates with Neutralizing Assay in a Subgroup of Patients in Delhi National Capital Region, India.

Background: The plaque reduction neutralization test (PRNT) is the gold standard to detect the neutralizing capacity of serum antibodies. Neutralizing antibodies confer protection against further infection. The present study measured the antibody level against SARS-CoV-2 among laboratory-confirmed COVID-19 cases and evaluated whether the presence of anti-SARS-CoV-2 antibodies indicates virus neutralizing capacity. Methods: One hundred COVID-19 confirmed cases were recruited. Their sociodemographic details and history of COVID-19 vaccination, contact with positive COVID-19 cases, and symptoms were ascertained using a self-developed semi-structured interview schedule. Serum samples of the participants were collected within three months from the date of the positive report of COVID-19. The presence of anti-SARS-CoV-2 antibodies (IgA, IgG and IgM antibodies), receptor binding domain antibodies (anti-RBD), and neutralizing antibodies were measured. Findings: Almost all the participants had anti-SARS-CoV-2 antibodies (IgA, IgG and IgM) (99%) and anti-RBD IgG antibodies (97%). However, only 69% had neutralizing antibodies against SARS-CoV-2. Anti-RBD antibody levels were significantly higher among participants having neutralizing antibodies compared with those who did not. Interpretation: The present study highlights that the presence of antibodies against SARS-CoV-2, or the presence of anti-RBD antibodies does not necessarily imply the presence of neutralizing antibodies.

Serological prevalence of SARS-CoV-2 antibody among children and young age group (between 2 and 17 years) in India: An interim result from a large multicentric population-based seroepidemiological study.

Background: Estimating seroepidemiolgical prevalence of SARS-CoV-2 antibody is an essential public health strategy. There is insufficient evidence of prevalence among those belonging to young age population in India. Objective: To compare the SARS-CoV-2 seropositivity rate between children and adults in selected sites from India. Materials and Methods: This was a multicentric population-based seroepidemiological study conducted in selected urban and rural areas of five sites selected from four states (Delhi, Odisha, Uttar Pradesh, Tripura) of India. Participants aged ≥1 year were included from different clusters of each area. Total serum antibody against SARS-CoV-2 virus was assessed qualitatively by using a standard enzyme-linked immunosorbent assay (ELISA) kit. Results: Data collection period was from 15 March 2021 to 10 June 2021. Total available data was of 4509 participants, of whom 700 were <18 years of age and 3809 were ≥18 years of age. The site-wise number of available data among those aged 2-17 years was 92, 189, 165, 146 and 108 for the sites of Delhi urban, Delhi rural, Bhubaneswar rural, Gorakhpur rural and Agartala rural area, respectively. The seroprevalence was 55.7% in the <18 years age group and 63.5% in the ≥18 years age group. The prevalence among female children was 58% and among male children was 53%. Conclusion: SARS-CoV-2 seropositivity rate among children was high and comparable to that of the adult population. Hence, it is unlikely that any future third wave by prevailing SARS-CoV-2 variant would disproportionately infect children 2 years or older.

Pre-existing antibody levels negatively correlate with antibody titers after a single dose of BBV152 vaccination.

Many adults in India have received at least one dose of COVID-19 vaccine with or without a prior history SARS-CoV-2 infection. However, there is limited information on the effect of prior immunity on antibody response upon vaccination in India. As immunization of individuals continues, we aimed to assess whether pre-existing antibodies are further boosted by a single dose of BBV152, an inactivated SARS-CoV-2 vaccine, and, if these antibodies can neutralize SARS-CoV-2 Delta and Omicron variants. Here we show that natural infection during the second wave in 2021 led to generation of neutralizing antibodies against other lineages of SARS-CoV-2 including the Omicron variant, albeit at a significantly lower level for the latter. A single dose of BBV152 boosted antibody titers against the Delta and the Omicron variants but the antibody levels remained low against the Omicron variant. Boosting of antibodies showed negative correlation with baseline neutralizing antibody titers.

Sub-optimal neutralisation of omicron (B.1.1.529) variant by antibodies induced by vaccine alone or SARS-CoV-2 Infection plus vaccine (hybrid immunity) post 6-months.

BACKGROUND: Rapid spread of the omicron SARS-CoV-2 variant despite extensive vaccination suggests immune escape. The neutralising ability of different vaccines alone or with natural SARS-CoV-2 infection against omicron is not well-known. METHODS: In this cross-sectional study, we tested the ability of vaccine and natural infection induced antibodies to neutralise omicron variant in a live virus neutralisation assay in four groups of individuals: (i) ChAdOx1 nCoV-19 vaccination, (ii) ChAdOx1 nCoV-19 vaccination plus prior SARS-CoV-2 infection, (iii) vaccination with inactivated virus vaccine (BBV152), and (iv) BBV152 vaccination plus prior SARS-CoV-2 infection. Primary outcome was fold-change in virus neutralisation titre against omicron compared with ancestral virus. FINDINGS: We included 80 subjects. The geometric mean titre (GMT) of the 50% focus reduction neutralisation test (FRNT50) was 380·4 (95% CI: 221·1, 654·7) against the ancestral virus with BBV152 vaccination and 379·3 (95% CI: 185·6, 775·2) with ChAdOx1 nCov-19 vaccination alone. GMT for vaccination plus infection groups were 806·1 (95% CI: 478·5, 1357·8) and 1526·2 (95% CI: 853·2, 2730·0), respectively. Against omicron variant, only 5 out of 20 in both BBV152 and ChAdOx1 nCoV-19 vaccine only groups, 6 out of 20 in BBV152 plus prior SARS-CoV-2 infection group, and 9 out of 20 in ChAdOx1 nCoV-19 plus prior SARS-CoV-2 infection group exhibited neutralisation titres above the lower limit of quantification (1:20) suggesting better neutralisation with prior infection. A reduction of 26·6 and 25·7 fold in FRNT50 titres against Omicron compared to ancestral SARS-CoV-2 strain was observed for individuals without prior SARS-CoV-2 infection vaccinated with BBV152 and ChAdOx1 nCoV-19, respectively. The corresponding reduction was 57·1 and 58·1 fold, respectively, for vaccinated individuals with prior infection. The 50% neutralisation titre against omicron demonstrated moderate correlation with serum anti-RBD IgG levels [Spearman r: 0·58 (0·41, 0·71)]. INTERPRETATION: Significant reduction in the neutralising ability of both vaccine-induced and vaccine plus infection-induced antibodies was observed for omicron variant which might explain immune escape. FUNDING: Department of Biotechnology, India; Bill & Melinda Gates Foundation, USA.

Humoral cross-reactivity towards SARS-CoV-2 in young children with acute respiratory infection with low-pathogenicity coronaviruses.

Background: SARS-CoV-2 infection in children frequently leads to only asymptomatic and mild infections. It has been suggested that frequent infections due to low-pathogenicity coronaviruses in children, impart immunity against SARS-CoV-2 in this age group. Methods: From a prospective birth cohort study prior to the pandemic, we identified children with proven low-pathogenicity coronavirus infections. Convalescent sera from these children were tested for antibodies against respective seasonal coronaviruses (OC43, NL63, and 229E) and SARS-CoV-2 by immunofluorescence and virus microneutralization assay respectively. Results: Forty-two children with proven seasonal coronavirus infection were included. Convalescent sera from these samples demonstrated antibodies against the respective seasonal coronaviruses. Of these, 40 serum samples showed no significant neutralization of SARS-CoV-2, while 2 samples showed inconclusive results. Conclusion: These findings suggest that the antibodies generated in low-pathogenicity coronavirus infections offer no protection from SARS-CoV-2 infection in young children.

Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences.

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.

Resolution of viral load in mild COVID-19 patients is associated with both innate and adaptive immune responses.

Over 90% of the COVID-19 patients manifest mild/moderate symptoms or are asymptomatic. Although comorbidities and dysregulation of immune response have been implicated in severe COVID-19, the host factors that associate with asymptomatic or mild infections have not been characterized. We have collected serial samples from 23 hospitalized COVID-19 patients with mild symptoms and measured the kinetics of SARS-CoV-2 viral load in respiratory samples and markers of inflammation in serum samples. We monitored seroconversion during the acute phase of illness and quantitated the amount of total IgG against the receptor-binding domain of SARS-CoV-2 and estimated the virus neutralization potential of these antibodies. Viral load decreased by day 8 in all the patients but the detection of viral RNA in saliva samples did not correlate well with viral RNA detection in nasopharyngeal/oropharyngeal swab samples. 25% of the virus-positive patients had no detectable neutralizing antibodies in the serum and in other cases, the efficiency of antibodies to neutralize SARS-CoV-2 B1.1.7 strain was lower as compared to the circulating virus isolate. Decrease in viral load coincided with increase in neutralizing antibodies and interferon levels in serum. Most patients showed no increase in inflammatory cytokines such as IL-1ß or IL-6, however, elevated levels of IL-7 and other inflammatory mediators such as TNF-α and IL-8 was observed. These data suggest that most mild infections are associated with absence of inflammation coupled with an active innate immune response, T-cell activation and neutralizing antibodies.

A novel G-quadruplex aptamer-based spike trimeric antigen test for the detection of SARS-CoV-2.

The recent SARS-CoV-2 outbreak has been declared a global health emergency. It will take years to vaccinate the whole population to protect them from this deadly virus, hence the management of SARS-CoV-2 largely depends on the widespread availability of an accurate diagnostic test. Toward addressing the unmet need of a reliable diagnostic test in the current work by utilizing the power of Systematic Evolution of Ligands by EXponential enrichment, a 44-mer G-quadruplex-forming DNA aptamer against spike trimer antigen of SARS-CoV-2 was identified. The lead aptamer candidate (S14) was characterized thoroughly for its binding, selectivity, affinity, structure, and batch-to-batch variability by utilizing various biochemical, biophysical, and in silico techniques. S14 has demonstrated a low nanomolar KD, confirming its tight binding to a spike antigen of SARS-CoV-2. S14 can detect as low as 2 nM of antigen. The clinical evaluation of S14 aptamer on nasopharyngeal swab specimens (n = 232) has displayed a highly discriminatory response between SARS-CoV-2 infected individuals from the non-infected one with a sensitivity and specificity of ∼91% and 98%, respectively. Importantly, S14 aptamer-based test has evinced a comparable performance with that of RT-PCR-based assay. Altogether, this study established the utility of aptamer technology for the detection of SARS-CoV-2.

Epidemiology of Coronavirus Infection in Children and Their Impact on Lung Health: Finding From a Birth Cohort Study.

In this birth cohort, coronavirus acute respiratory infection was detected in 6.5% of the episodes; the commonest strain was OC43, followed by NL63, HKU1, and 229E. Children with coronavirus acute respiratory infection during infancy had significantly decreased forced expiratory volume in 0.5 seconds, forced expiratory flow between 25% and 75% of forced vital capacity, and peak expiratory flow at 3 years of age.

Visual Detection of SARS-CoV-2 RNA by Conventional PCR-Induced Generation of DNAzyme Sensor.

The gold standard for the diagnosis of SARS-CoV-2, the causative agent of COVID-19, is real-time polymerase chain reaction (PCR), which is labor-intensive, expensive, and not widely available in resource-poor settings. Therefore, it is imperative to develop novel, accurate, affordable, and easily accessible assays/sensors to diagnose and isolate COVID-19 cases. To address this unmet need, we utilized the catalytic potential of peroxidase-like DNAzyme and developed a simple visual detection assay for SARS-CoV-2 RNA using a conventional thermal cycler by the PCR-induced generation of DNAzyme sensor. The performance of RT-PCR DNAzyme-based sensor was comparable to that of real-time PCR. The pilot scale validation of RT-PCR DNAzyme-based sensor has shown ~100% sensitivity and specificity in clinical specimens (nasopharyngeal swab, n = 34), with a good correlation (Spearman r = 0.799) with the Ct-value of fluorescence probe-based real-time PCR. These findings clearly indicate the potential of this inexpensive, sensitive, and specific molecular diagnostic test to extend our testing capabilities for the detection of SARS-CoV-2 to curtail COVID-19 transmission.

Respiratory Syncytial Virus-Induced Oxidative Stress Leads to an Increase in Labile Zinc Pools in Lung Epithelial Cells.

Zinc supplementation in cell culture has been shown to inhibit various viruses, like herpes simplex virus, rotavirus, severe acute respiratory syndrome (SARS) coronavirus, rhinovirus, and respiratory syncytial virus (RSV). However, whether zinc plays a direct antiviral role in viral infections and whether viruses have adopted strategies to modulate zinc homeostasis have not been investigated. Results from clinical trials of zinc supplementation in infections indicate that zinc supplementation may be beneficial in a pathogen- or disease-specific manner, further underscoring the importance of understanding the interaction between zinc homeostasis and virus infections at the molecular level. We investigated the effect of RSV infection on zinc homeostasis and show that RSV infection in lung epithelial cells leads to modulation of zinc homeostasis. The intracellular labile zinc pool increases upon RSV infection in a multiplicity of infection (MOI)-dependent fashion. Small interfering RNA (siRNA)-mediated knockdown of the ubiquitous zinc uptake transporter ZIP1 suggests that labile zinc levels are increased due to the increased uptake by RSV-infected cells as an antiviral response. Adding zinc to culture medium after RSV infection led to significant inhibition of RSV titers, whereas depletion of zinc by a zinc chelator, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) led to an increase in RSV titers. The inhibitory effect of zinc was specific, as other divalent cations had no effect on RSV titers. Both RSV infection and zinc chelation by TPEN led to reactive oxygen species (ROS) induction, whereas addition of zinc blocked ROS induction. These results suggest a molecular link between RSV infection, zinc homeostasis, and oxidative-stress pathways and provide new insights for developing strategies to counter RSV infection.IMPORTANCE Zinc deficiency rates in developing countries range from 20 to 30%, and zinc supplementation trials have been shown to correct clinical manifestations attributed to zinc deficiency, but the outcomes in the case of respiratory infections have been inconsistent. We aimed at understanding the role of zinc homeostasis in respiratory syncytial virus (RSV) infection. Infection of lung epithelial cell lines or primary small-airway epithelial cells led to an increase in labile zinc pools, which was due to increased uptake of zinc. Zinc supplementation inhibited RSV replication, whereas zinc chelation had an opposing effect, leading to increases in RSV titers. Increases in labile zinc in RSV-infected cells coincided with induction of reactive oxygen species (ROS). Both zinc depletion and addition of exogenous ROS led to enhanced RSV infection, whereas addition of the antioxidant inhibited RSV, suggesting that zinc is part of an interplay between RSV-induced oxidative stress and the host response to maintain redox balance.