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.

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.

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.

Waning natural and vaccine-induced immunity leading to reinfection with SARS-CoV-2 Omicron variant.

We have investigated six COVID-19 recovered cases with two doses of Covishield vaccination followed by reinfection. The primary SARS-CoV-2 infection found to occur with B.1 and reinfection with Omicron BA.1 and BA.2 variants. The genomic characterization and duration between two infections confirms these cases as SARS-CoV-2 reinfection. The immune response determined at different time intervals demonstrated boost post two dose vaccination, decline in pre-reinfection sera post 7 months and rise post reinfection. In conclusion, it was observed that these cases got SARS-CoV-2 reinfection with declined hybrid immunity acquired from primary infection and two dose covishield vaccination. This findings suggests the need to protect the community through booster dose of vaccination and prevent further infections following personal hygiene and non-pharmaceutical interventions.

Serosurvey for Nipah virus in bat population of southern part of India.

Nipah virus (NiV) is one of the priority pathogens with pandemic potential. Though the spread is far slower than SARS-CoV-2, case fatality is the biggest concern. Fruit bats belonging to genus Pteropus are identified to be the main reservoir of the virus causing sporadic cases and outbreaks in Malaysia, Bangladesh and India. The sudden emergence of Nipah in Kerala, India during 2018-2019 has been astonishing with respect to its introduction in the unaffected areas. With this, active Nipah virus surveillance was conducted among bat populations in Southern part of India viz., Karnataka, Kerala, Tamil Nadu, Telangana, Puducherry and Odisha during January-November 2019. Throat swabs/rectal swabs (n = 573) collected from Pteropus medius and Rousettus leschenaultii bat species and sera of Pteropus medius bats (n = 255) were screened to detect the presence of Nipah viral RNA and anti-Nipah IgG antibodies respectively. Of 255 P. medius bats sera samples, 51 bats (20%) captured from Karnataka, Kerala, Tamil Nadu and Puducherry demonstrated presence of anti-Nipah IgG antibodies. However, the presence of virus couldn't be detected in any of the bat specimens. The recent emergence of Nipah virus in Kerala in September 2021 warrants further surveillance of Nipah virus among bat populations from the affected and remaining states of India.

Isolation and Genomic Characterization of SARS-CoV-2 Omicron Variant Obtained from Human Clinical Specimens.

Due to the failure of virus isolation of the Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, an initial in vivo and subsequent in vitro approach was utilized for the isolation of the virus. A total of 74 oropharyngeal/nasopharyngeal specimens were collected from SARS-CoV-2 positive international travellers and a contact case at Delhi and Mumbai, India. All the specimens were sequenced using next-generation sequencing and simultaneously inoculated onto Vero CCL-81 cells for virus isolation. Subsequently, two omicron positive specimens were inoculated into Syrian hamsters for two passages. The initial passage of the positive hamster specimens was inoculated onto Vero CCL-81 cells. The clinical specimens, hamster specimens, and Vero CCL-81 passages were sequenced to assess the mutational changes in different host species. The replication of the Omicron variant in hamsters was confirmed with the presence of a high viral load in nasal turbinate and lung specimens of both passages. The successful isolation of the virus from hamster specimens with Vero CCL-81 was observed with cytopathic effect in infected cells and high viral load in the cell suspension. The genome analysis revealed the presence of L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene of hamster passage sequences and an absence of V17I mutation in E gene in hamster passage sequences, unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequences, suggesting intact pathogenicity of the virus isolate. Our data demonstrated successful isolation of the Omicron variant with the in vivo method first followed by in vitro method. The virus isolate could be used in the future to explore different aspects of the Omicron variant.

Nipah Virus Outbreak in Kerala State, India Amidst of COVID-19 Pandemic.

We report here a Nipah virus (NiV) outbreak in Kozhikode district of Kerala state, India, which had caused fatal encephalitis in a 12-year-old boy and the outbreak response, which led to the successful containment of the disease and the related investigations. Quantitative real-time reverse transcription (RT)-PCR, ELISA-based antibody detection, and whole genome sequencing (WGS) were performed to confirm the NiV infection. Contacts of the index case were traced and isolated based on risk categorization. Bats from the areas near the epicenter of the outbreak were sampled for throat swabs, rectal swabs, and blood samples for NiV screening by real-time RT-PCR and anti-NiV bat immunoglobulin G (IgG) ELISA. A plaque reduction neutralization test was performed for the detection of neutralizing antibodies. Nipah viral RNA could be detected from blood, bronchial wash, endotracheal (ET) secretion, and cerebrospinal fluid (CSF) and anti-NiV immunoglobulin M (IgM) antibodies from the serum sample of the index case. Rapid establishment of an onsite NiV diagnostic facility and contact tracing helped in quick containment of the outbreak. NiV sequences retrieved from the clinical specimen of the index case formed a sub-cluster with the earlier reported Nipah I genotype sequences from India with more than 95% similarity. Anti-NiV IgG positivity could be detected in 21% of Pteropus medius (P. medius) and 37.73% of Rousettus leschenaultia (R. leschenaultia). Neutralizing antibodies against NiV could be detected in P. medius. Stringent surveillance and awareness campaigns need to be implemented in the area to reduce human-bat interactions and minimize spillover events, which can lead to sporadic outbreaks of NiV.

Detection and isolation of SARS-CoV-2 Eta variant from the international travelers and local residents of India.

International travel has been the major source for the rapid spread of new SARS-CoV-2 variants across the globe. During SARS-CoV-2 genomic surveillance, a total of 212 SARS-CoV-2 positive clinical specimens were sequenced using next-generation sequencing. A complete SARS-CoV-2 genome could be retrieved from 90 clinical specimens. Of them, 14 sequences belonged to the Eta variant from clinical specimens of international travelers (n = 12) and local residents (n = 2) of India, and 76 belonged to other SARS-CoV-2 variants. Of all the Eta-positive specimens, the virus isolates were obtained from the clinical specimens of six international travelers. Many variants of interest have been found to cause substantial community transmission or cluster infections. The detection of this variant with lethal E484K mutation across the globe and India necessitates persistent genomic surveillance of the SARS-CoV-2 variants, which would aid in taking preventive action.

Isolation and characterization of SARS-CoV-2 Beta variant from UAE travelers.

BACKGROUND: The emergence of SARS-CoV-2 variants in places where the virus is uncontained poses a global threat from the perspective of public health and vaccine efficacy. Travel has been important factor for the easy spread of SARS-CoV-2 variants worldwide. India has also observed the importation of SARS-CoV-2 variants through international travelers. METHODS: In this study, we have collected the oropharyngeal and nasopharyngeal swab specimens from 58 individuals with travel history from United Arab Emirates (UAE), East, West and South Africa, Qatar, Ukraine and Saudi Arabia arrived in India during February-March 2021. The clinical specimens were initially screened for SARS-CoV-2 using Real time RT-PCR. All the specimens were inoculated on to Vero CCL-81 cells for virus isolation. The viral isolates were further sequenced using Next-Generation Sequencing. RESULTS: All 58 cases were tested positive for SARS-CoV-2 using Real time RT-PCR. Four specimens showed progressive infectivity with fusion of the infected cells with neighboring cells leading to large mass of cells. Replication competent virus was confirmed from culture supernatant of the passage 2 using Real time RT-PCR. Two plaque purified SARS-CoV-2 isolates demonstrated high viral RNA load of 3.8-7.5 × 1011 and 1.1-1.6 × 1011 at passage 4 and 5 respectively. Nucleotide variations along with amino acid changes were also observed among these two isolates at passage 2-5. All four cases were male with no symptoms and co-morbidity. The sequence analysis has shown two different clusters, first cluster with nucleotide deletions in the ORF1ab and the spike, while second cluster with deletions in spike region. The viral isolates demonstrated 99.88-99.96% nucleotide identity with the representative sequences of Beta variant (B.1.351). CONCLUSION: These findings suggest easier transmission of SARS-CoV-2 variants with human mobility through international travel. The isolated Beta variant would be useful to determine the protective efficacy of the currently available and upcoming COVID-19 vaccines in India.

A case with SARS-CoV-2 reinfection from India.

A healthcare worker presented with fever, cough, headache and tested positive by SARS-CoV-2 real time reverse transcriptase polymerase chain reaction (qRT-PCR). He got admitted to hospital and recovered after 14 days. After 2 months, as a screening protocol considering the high risk setup he got tested and again found to be positive for SARS-CoV-2 by qRT-PCR. Our patient had detectable levels of Anti-SARS-CoV-2 IgG antibodies during the reinfection but found negative for Neutralizing antibodies (NAb). Our findings suggest that the person after the initial infection might not develop the desired protective immunity to prevent the reinfection as demonstrated by absence of NAb.

Community transmission of SARS-CoV-2 with B.1.1.7 lineage in Mumbai, India.

The B.1.1.7 (Alpha) variant has been detected in Mumbai, India during February 2021. Subsequently, we retrieved 43 sequences from specimens of 51 COVID-19 cases from Mumbai. The sequence analysis revealed that the cases were mainly affected with Alpha variant which suggests its role in community transmission of SARS-CoV-2 in Mumbai, India.