Comparative immune profiling in survivors of the 2023 Nipah outbreak in Kerala state, India.

Immune profiling of Nipah virus (NiV) infection survivors is essential for advancing our understanding of NiV pathogenesis, improving diagnostic and therapeutic strategies, and guiding public health efforts to prevent future outbreaks. There is currently limited data available on the immune response to NiV infection. We aimed to elucidate the specific immune mechanisms involved in protection against NiV infection by analyzing the immune profiles of survivors of the Nipah outbreak in Kerala, India 2023. Immune cell populations were quantified and compared between survivors (up to 4 months post onset day of illness) and healthy controls. Statistical analysis was performed to explore associations between immune profiles and clinical outcomes. Immune signatures common to all three cases were: a heretofore undescribed persistent lymphopenia including the CD4+ Treg compartment with the relative expansion of memory Tregs; trends indicative of global leukopenic modulation were observed in monocytes and granulocytes including an expansion of putatively immunosuppressive low-density granulocytes described recently in the context of severe COVID-19; altered mucosal homing with respect to integrin beta-7 (ITGB7) expressing subsets; increased mobilization of activated T-cells (CD4+ and CD8+) and plasmablasts in the early phase of infection. Comparative analysis based on clinical presentation and outcome yielded lower initial viremia, increased activated T-cell responses, expanded plasmablasts, and restoration of ITGB7 expressing CD8+ T-cells as possible protective signatures. This longitudinal study delineates putative protective signatures associated with milder NiV disease. It emphasizes the need for the development of immunotherapeutic interventions such as monoclonal antibodies to blunt early viremia and ameliorate pathogenesis.

Neutralizing antibody responses to SARS-CoV-2 Omicron variants: Post six months following two-dose & three-dose vaccination of ChAdOx1 nCoV-19 or BBV152.

BACKGROUND OBJECTIVES: The Omicron sub-lineages are known to have higher infectivity, immune escape and lower virulence. During December 2022 - January 2023 and March - April 2023, India witnessed increased SARS-CoV-2 infections, mostly due to newer Omicron sub-lineages. With this unprecedented rise in cases, we assessed the neutralization potential of individuals vaccinated with ChAdOx1 nCoV (Covishield) and BBV152 (Covaxin) against emerging Omicron sub-lineages. METHODS: Neutralizing antibody responses were measured in the sera collected from individuals six months post-two doses (n=88) of Covishield (n=44) or Covaxin (n=44) and post-three doses (n=102) of Covishield (n=46) or Covaxin (n=56) booster dose against prototype B.1 strain, lineages of Omicron; XBB.1, BQ.1, BA.5.2 and BF.7. RESULTS: The sera of individuals collected six months after the two-dose and the three-dose demonstrated neutralizing activity against all variants. The neutralizing antibody (NAbs) level was highest against the prototype B.1 strain, followed by BA5.2 (5-6 fold lower), BF.7 (11-12 fold lower), BQ.1 (12 fold lower) and XBB.1 (18-22 fold lower). INTERPRETATION CONCLUSIONS: Persistence of NAb responses was comparable in individuals with two- and three-dose groups post six months of vaccination. Among the Omicron sub-variants, XBB.1 showed marked neutralization escape, thus pointing towards an eventual immune escape, which may cause more infections. Further, the correlation of study data with complete clinical profile of the participants along with observations for cell-mediated immunity may provide a clear picture for the sustained protection due to three-dose vaccination as well as hybrid immunity against the newer variants.

Clinico-epidemiological presentations and management of Nipah virus infection during the outbreak in Kozhikode district, Kerala state, India 2023.

India experienced its sixth Nipah virus (NiV) outbreak in September 2023 in the Kozhikode district of Kerala state. The NiV is primarily transmitted by spillover events from infected bats followed by human-to-human transmission. The clinical specimens were screened using real-time RT-PCR, and positive specimens were further characterized using next-generation sequencing. We describe here an in-depth clinical presentation and management of NiV-confirmed cases and outbreak containment activities. The current outbreak reported a total of six cases with two deaths, with a case fatality ratio of 33.33%. The cases had a mixed presentation of acute respiratory distress syndrome and encephalitis syndrome. Fever was a persistent presentation in all the cases. The Nipah viral RNA was detected in clinical specimens until the post-onset day of illness (POD) 14, with viral load in the range of 1.7-3.3 × 104 viral RNA copies/mL. The genomic analysis showed that the sequences from the current outbreak clustered into the Indian clade similar to the 2018 and 2019 outbreaks. This study highlights the vigilance of the health system to detect and effectively manage the clustering of cases with clinical presentations similar to NiV, which led to early detection and containment activities.

Cellular Immune Responses Against γ-Inactivated Antigen in the Recovered Cases of Kyasanur Forest Disease.

Kyasanur Forest Disease Virus (KFDV) is a tick-borne flavivirus that causes life-threatening hemorrhagic fever in humans with case fatality rates of 3-5%. Relatively little is known about the mechanism of its pathogenesis or host immune responses to KFDV infection. Here, we investigated KFDV-specific cellular immune responses in the recovered cases of Kyasanur Forest Disease (KFD). Peripheral blood mononuclear cells of the recovered KFD cases and healthy controls were exposed to γ-inactivated KFDV antigen ex vivo. The proliferation index was determined using an enzyme-linked immunosorbent assay-based lymphoproliferative assay. The frequencies of CD4+ and CD8+ T cells expressing intracellular interferon (IFN)-γ in response to stimulation with γ-inactivated KFDV antigen were determined using flow cytometry. A significant increase in lymphoproliferation and a high frequency of CD4+ and CD8+ T cells secreting IFN-γ against γ-inactivated KFDV antigen were found in the recovered KFD group compared to the healthy control group. In conclusion, the study indicated the generation of cellular immune responses in individuals who recovered from KFD and can be used as indicators of cellular immunity in KFD vaccine studies.

Clinical, epidemiological, and molecular investigation of Kyasanur forest disease from Karnataka state, India during 2018-2019.

BACKGROUND: In this study, we carried out an investigation of Kyasanur Forest Disease (KFD) suspected human cases reported in Karnataka state, India from December 2018 to June 2019. METHODS: The clinical samples of KFD suspected cases (n = 1955) from 14 districts of Karnataka were tested for KFD using real-time RT-PCR and IgM ELISA. Further, the KFD-negative samples were tested for IgM antibodies against dengue and chikungunya viruses. Monkey samples (n = 276) and tick pools (n = 11582) were also screened using real-time RT-PCR. KFD-positive samples were further analysed using next-generation sequencing along with clinico-epidemiological analysis. RESULTS: Of all, 173 (8.8%) cases tested positive for KFD either by real-time RT-PCR (n = 124), IgM ELISA (n = 53) or both tests (n = 4) from seven districts. Among KFD-negative cases, IgM antibody positivity was observed for dengue (2.6%), chikungunya (5.8%), dengue and chikungunya coinfection (3.7%). KFD cases peaked in January 2019 with fever, conjunctivitis, and myalgia as the predominant symptoms and a mortality of 4.6%. Among confirmed cases, 41% received a single dose and 20% received two doses of the KFD vaccine. Of the seven districts with KFDV positivity, Shivamogga and Hassan districts reported KFD viral RNA positivity in humans, monkeys, and ticks. Sequencing analysis of 2019 cases demonstrated a difference of less than 1.5% amino acid compared to prototype KFDV. CONCLUSION: Although the KFD has been endemic in many districts of Karnataka state, our study confirms the presence of KFDV for the first time in two new districts, i.e. Hassan and Mysore. A comparative analysis of KFDV infection among the KFD-vaccinated and non-vaccinated populations demonstrated an insignificant difference.

Broadly Reactive SARS-CoV-2-Specific T-Cell Response and Participation of Memory B and T Cells in Patients with Omicron COVID-19 Infection.

January 2022 onward, India witnessed a sudden increase in Omicron COVID-19 infections, having a mild course that prompted us to identify the key host factors/immune molecules modulating disease course/outcomes. The current study evaluated the percentages of lymphocyte subsets by flowcytometry, SARS-CoV-2 specific T-cell immune response by ELISPOT, estimation of plasma cytokine/chemokine levels on a Bio-plex Multiplex Immunoassay System and anti-SARS-CoV-2 IgG levels by enzyme-linked immunosorbent assay in 19 mild Omicron infected patients, 45 mild SARS-CoV-2 (2020) patients and 36 uninfected controls from India. Natural killer cells, B and memory B cells were high in vaccinated and total Omicron-infected patients groups compared to the mild SARS-CoV-2 (2020) patient group, while CD8+ T cells were high in total Omicron-infected patients group compared to the uninfected control group (p < 0.05 each). Omicron-infected patients had T-cell response against SARS-CoV-2 whole virus, S1 proteins (wild type and delta variant) in 10 out of 17 (59%), 10 out of 17 (59%), and 8 out of 17 (47%), respectively. The current study of Omicron-infected patients elucidates broadly reactive antibody, T-cell response, and participation of memory B and T cells induced by vaccination/natural infection. The limited effect of Omicron's mutations on T-cell response is suggestive of protection from severity. Pro-inflammatory IL-6, IFN-γ, chemokines CCL-2, CCL-3, CCL-4, CCL-5, and IL-8 as potential biomarkers of Omicron infection may have future diagnostic importance. The cellular immune response data in Omicron-infected patients with parental Omicron lineage could serve as a starting point to define the readouts of protective immunity against circulating Omicron subvariants.

First detection of Varicella Zoster Virus clade 9 cases in India during mpox surveillance.

BACKGROUND: The multi-country mpox outbreak across the globe has led to the systematic surveillance of mpox cases in India. During the surveillance of mpox, we encountered cases of Varicella Zoster Virus (VZV) in suspected mpox cases amongst children & adults. This study focused on the genomic characterization of VZV in India. METHODS: A total of 331 mpox suspected cases were tested for VZV through real-time PCR, and the positive samples were subjected to next-generation sequencing to retrieve the whole genome of VZV using CLC genomics software. Phylogenetic analysis has been done in MEGA 11.0 software to identify circulating clades. RESULT: Of the 331 suspected cases, 28 cases with vesicular rashes were found to be positive for VZV. The maximum genome could be retrieved from the clinical specimens of 16 cases with coverage greater than 98% when mapped with reference strain Dumas (NC 001348). The phylogenetic analyses of these sequences determined the circulation of clades 1, 5, and 9 in India. Further, the sequence analysis demonstrated non-synonymous single nucleotide polymorphism (SNPs) among specific ORF of VZV including ORF 14, ORF 22, ORF 36, ORF 37 and ORF 51. Although clade 1 and 5 has been reported earlier, the circulation of clade 9 of VZV has been determined for the first time in India. CONCLUSION: Although the circulation of different clades of VZV was reported from India, the presence of clade 9 was detected for the first time during the mpox surveillance.

Needle-free injection system delivery of ZyCoV-D DNA vaccine demonstrated improved immunogenicity and protective efficacy in rhesus macaques against SARS-CoV-2.

The apprehension of needles related to injection site pain, risk of transmitting bloodborne pathogens, and effective mass immunization have led to the development of a needle-free injection system (NFIS). Here, we evaluated the efficacy of the NFIS and needle injection system (NIS) for the delivery and immunogenicity of DNA vaccine candidate ZyCoV-D in rhesus macaques against SARS-CoV-2 infection. Briefly, 20 rhesus macaques were divided into 5 groups (4 animals each), that is, I (1 mg dose by NIS), II (2 mg dose by NIS), III (1 mg dose by NFIS), IV (2 mg dose by NFIS) and V (phosphate-buffer saline [PBS]). The macaques were immunized with the vaccine candidates/PBS intradermally on Days 0, 28, and 56. Subsequently, the animals were challenged with live SARS-CoV-2 after 15 weeks of the first immunization. Blood, nasal swab, throat swab, and bronchoalveolar lavage fluid specimens were collected on 0, 1, 3, 5, and 7 days post infection from each animal to determine immune response and viral clearance. Among all the five groups, 2 mg dose by NFIS elicited significant titers of IgG and neutralizing antibody after immunization with enhancement in their titers postvirus challenge. Besides this, it also induced increased lymphocyte proliferation and cytokine response. The minimal viral load post-SARS-CoV-2 challenge and significant immune response in the immunized animals demonstrated the efficiency of NFIS in delivering 2 mg ZyCoV-D vaccine candidate.

Clinical presentation, viral kinetics, and management of human monkeypox cases from New Delhi, India 2022.

We describe the clinical and demographic characteristics, virological follow-up, and management of five confirmed monkeypox cases from New Delhi, India without any international travel history. The viral load kinetics and viral clearance were estimated in oropharyngeal swabs (OPS), nasopharyngeal swabs (NPS), EDTA blood, serum, urine, and various lesion specimens on every fourth day of follow-up ranging from 5 to 24 post onset day (POD) of illness. All five cases presented with mild to moderate-grade intermittent fever, myalgia, and lesions on the genitals, groins, lower limb, trunk, and upper limb. Four cases had non-tender firm lymphadenopathy. No secondary complications or sexually transmitted infections were recorded in these cases except for the presence of viral hepatitis B infection marker hepatitis B virus surface antigen (HBsAg) in one case. All the cases were mild and had a good recovery. A higher viral load was detected in lesion fluid (POD 9), followed by lesion roof (POD 9), urine (POD 5), lesion base (POD 5), and OPS/NPS (POD 5). The monkeypox virus (MPXV) DNA was detected in clinical samples from 5th to 24th POD. These monkeypox cases without international travel history suggest the underdiagnosed monkeypox infection in the community. This emphasizes the need for active surveillance of MPXV in the high-risk population such as men having sex with men and female sex workers.

Evaluation of immunogenicity post two doses of inactivated SARS-CoV-2 vaccine, Covaxin after six months.

We have evaluated the immunogenicity of two dose of Covaxin given at a one-month interval to two adult populations, i.e. COVID-19 naïve-vaccinated individuals (n = 118) and COVID-19 recovered individuals (n = 128) with the vaccination. The immune response in the study population were assessed at three follow-ups, namely at one month post first dose, one and six months after the second dose. The persistence of S1RBD IgG and neutralizing antibodies for six months post vaccination was observed at different time intervals. The enhanced immune response was observed in both the participant groups. The study emphasizes the need for a booster dose post six months of vaccination.

Omicron BA.2 lineage predominance in severe acute respiratory syndrome coronavirus 2 positive cases during the third wave in North India.

Background: Recent studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reveal that Omicron variant BA.1 and sub-lineages have revived the concern over resistance to antiviral drugs and vaccine-induced immunity. The present study aims to analyze the clinical profile and genome characterization of the SARS-CoV-2 variant in eastern Uttar Pradesh (UP), North India. Methods: Whole-genome sequencing (WGS) was conducted for 146 SARS-CoV-2 samples obtained from individuals who tested coronavirus disease 2019 (COVID-19) positive between the period of 1 January 2022 and 24 February 2022, from three districts of eastern UP. The details regarding clinical and hospitalized status were captured through telephonic interviews after obtaining verbal informed consent. A maximum-likelihood phylogenetic tree was created for evolutionary analysis using MEGA7. Results: The mean age of study participants was 33.9 ± 13.1 years, with 73.5% accounting for male patients. Of the 98 cases contacted by telephone, 30 (30.6%) had a travel history (domestic/international), 16 (16.3%) reported having been infected with COVID-19 in past, 79 (80.6%) had symptoms, and seven had at least one comorbidity. Most of the sequences belonged to the Omicron variant, with BA.1 (6.2%), BA.1.1 (2.7%), BA.1.1.1 (0.7%), BA.1.1.7 (5.5%), BA.1.17.2 (0.7%), BA.1.18 (0.7%), BA.2 (30.8%), BA.2.10 (50.7%), BA.2.12 (0.7%), and B.1.617.2 (1.3%) lineages. BA.1 and BA.1.1 strains possess signature spike mutations S:A67V, S:T95I, S:R346K, S:S371L, S:G446S, S:G496S, S:T547K, S:N856K, and S:L981F, and BA.2 contains S:V213G, S:T376A, and S:D405N. Notably, ins214EPE (S1- N-Terminal domain) mutation was found in a significant number of Omicron BA.1 and sub-lineages. The overall Omicron BA.2 lineage was observed in 79.5% of women and 83.2% of men. Conclusion: The current study showed a predominance of the Omicron BA.2 variant outcompeting the BA.1 over a period in eastern UP. Most of the cases had a breakthrough infection following the recommended two doses of vaccine with four in five cases being symptomatic. There is a need to further explore the immune evasion properties of the Omicron variant.

Differential Cell Line Susceptibility to the SARS-CoV-2 Omicron BA.1.1 Variant of Concern.

The unique mutations of the SARS-CoV-2 Omicron variant are associated with increased transmissibility, immune escape, increased binding affinity to ACE-2, and increased viral load. Omicron exhibited a shift in tropism infecting the upper respiratory tract compared to other variants of concern which have tropism for the lower respiratory tract. The tropism of omicron variants in cell lines of different hosts and tissue origins still remains unclear. Considering this, we assessed the susceptibility of different cell lines to the SARS-CoV-2 omicron BA.1.1 variant and permissiveness among different cell lines for omicron replication. Susceptibility and permissiveness of a total of eleven cell lines, including six animal cell lines and five human cell lines for omicron BA.1.1 infection, were evaluated by infecting individual cell lines with omicron BA.1.1 isolate at a 0.1 multiplicity of infection. Virus replication was assessed by observation of cytopathic effects followed by viral load determination by real-time PCR assay and virus infectivity determination by TCID50 assay. The characteristic cytopathic effect, increased viral load, and productive omicron replication was detected in Vero CCL-81, Vero E6, Vero/hSLAM, MA-104, and Calu-3 cells. Although LLC MK-2 cells showed an increased TCID50 titer at the second infection, the viral load did not show much difference in both infections. Caco-2 cells did not show evident CPE, but they supported omicron replication at a low level. A549, RD, MRC-5, and BHK-21 cells supported omicron BA.1.1 replication without the CPE. This is the first study on the comparison of susceptibility of different cell lines to Omicron variant BA.1.1, which might be useful for future studies on emerging SARS-CoV-2 variants.

A case control investigation of COVID-19 associated mucormycosis in India.

BACKGROUND: Increased occurrence of mucormycosis during the second wave of COVID-19 pandemic in early 2021 in India prompted us to undertake a multi-site case-control investigation. The objectives were to examine the monthly trend of COVID-19 Associated Mucormycosis (CAM) cases among in-patients and to identify factors associated with development of CAM. METHODS: Eleven study sites were involved across India; archived records since 1st January 2021 till 30th September 2021 were used for trend analysis. The cases and controls were enrolled during 15th June 2021 to 30th September 2021. Data were collected using a semi-structured questionnaire. Among 1211 enrolled participants, 336 were CAM cases and 875 were COVID-19 positive non-mucormycosis controls. RESULTS: CAM-case admissions reached their peak in May 2021 like a satellite epidemic after a month of in-patient admission peak recorded due to COVID-19. The odds of developing CAM increased with the history of working in a dusty environment (adjusted odds ratio; aOR 3.24, 95% CI 1.34, 7.82), diabetes mellitus (aOR: 31.83, 95% CI 13.96, 72.63), longer duration of hospital stay (aOR: 1.06, 95% CI 1.02, 1.11) and use of methylprednisolone (aOR: 2.71, 95% CI 1.37, 5.37) following adjustment for age, gender, occupation, education, type of houses used for living, requirement of ventilatory support and route of steroid administration. Higher proportion of CAM cases required supplemental oxygen compared to the controls; use of non-rebreather mask (NRBM) was associated as a protective factor against mucormycosis compared to face masks (aOR: 0.18, 95% CI 0.08, 0.41). Genomic sequencing of archived respiratory samples revealed similar occurrences of Delta and Delta derivates of SARS-CoV-2 infection in both cases and controls. CONCLUSIONS: Appropriate management of hyperglycemia, judicious use of steroids and use of NRBM during oxygen supplementation among COVID-19 patients have the potential to reduce the risk of occurrence of mucormycosis. Avoiding exposure to dusty environment would add to such prevention efforts.