A broadly protective CHO cell expressed recombinant spike protein subunit based vaccine (IMT-CVAX) against SARS-CoV-2 (preprint)

Protective immunity induced by COVID-19 vaccines is mediated mainly by spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report the development of a recombinant prefusion stabilized SARS-CoV-2 spike protein-subunit-based COVID-19 vaccine produced in the mammalian cell line. The gene encoding ectodomain (ECD) of the spike protein was engineered and cloned into Freedom pCHO 1.0, a mammalian expression vector, and subsequently expressed in the Chinese Hamster Ovary suspension cell line (CHO-S).The recombinant S protein ectodomain (hereafter referred to as IMT-CVAX) was purified using a combination of tangential flow filtration and liquid chromatography. Biochemical and biophysical characterization of IMT-CVAX was done to ensure its vital quality attributes. Intramuscular immunization of mice with two doses of adjuvanted IMT-CVAX elicited a strong anti-Spike IgG response.In pseudovirus-based assays, IMT-CVAX immune mice sera exhibited a broad-spectrum neutralization of several SARS-CoV-2 variants of concern (VoCs). Golden Syrian Hamster immunized with IMT-CVAX provided excellent protection against SARS-CoV-2 infection, and, hamster immune sera neutralized the live SARS-CoV-2 virus.The adjuvanted IMT-CVAX induced robust Tfh-cells response and germinal center (GC) reaction in human ACE2 receptor-expressing transgenic mice. The findings of this study may pave the way for developing next-generation protein subunit-based vaccines to combat the existing SARS-CoV-2 and its emerging VoCs. The IMT-CVAX is produced using a scalable process and can be used for large-scale vaccine production in an industrial setup.

Enhanced protective efficacy of a novel, thermostable, RBD-S2 fusion immunogen against SARS-CoV-2 and its variants (preprint)

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly protective antibody and T-cell responses. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, characterized, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Designed immunogens were expressed as soluble proteins with significantly higher purified yield than the Spike ectodomain. Squalene-in-water emulsion (SWE) formulated fusions were highly immunogenic. S2 immunization failed to elicit a neutralizing immune response but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant, while RBD-S2 fusions elicited broad neutralization including against the Clade 1a WIV-1 variant, and were highly efficacious. A lyophilized RBD-S2 fusion retained antigenicity and immunogenicity even after incubation at 37 degrees C for a month. In hamsters, SWE-formulated RBD-S2 showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations.

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.

UVC-based air disinfection system for rapid inactivation of SARS-CoV-2 present in the air (preprint)

The novel coronavirus disease 2019 (COVID-19) infections have rapidly spread throughout the world, and the virus has acquired an ability to spread via aerosols even at long distances. Hand washing, face-masking, and social distancing are the primary preventive measures against infections. With mounting scientific evidence, World Health Organisation (WHO) declared COVID-19 an air-borne disease. This ensued the need to disinfect air to reduce the transmission. Ultraviolet C (UVC) comprising the light radiation of 200-280 nm range is a commonly used method for inactivation of pathogens. The heating, ventilation, and air conditioning (HVAC) systems are not beneficial in closed spaces due to poor or no ability to damage circulating viruses. Therefore, standard infection-prevention practices coupled with a strategy to reduce infectious viral load in air substantially might be helpful in reducing virus transmissibility. In this study, we implemented UV light-based strategies to combat COVID-19 and future pandemics. We tested various disinfection protocols by using UVC-based air purification systems and currently installed such a system in workspaces, rushed out places, hospitals and healthcare facilities for surface, air, and water disinfection. In this study, we designed a prototype device to test the dose of UVC required to inactivate SARS-CoV-2 in aerosols and demonstrate that the radiation rapidly destroys the virus in aerosols. The UVC treatment renders the virus non-infectious due to chemical modification of nucleic acid. We also demonstrate that UVC treatment alters the Spike protein conformation that may further affect the infectivity of the virus. We show by using a mathematical model based on the experimental data that UVC-based air disinfection strategy can substantially reduce the risk of virus transmission. The systematic treatment by UVC of air in closed spaces via ventilation systems could be helpful in reducing the active viral load in the air.

Diphenylurea derivatives broadly inhibit SARS-CoV-2 and influenza A virus infections by perturbing endocytic trafficking (preprint)

Rapid evolution of SARS-CoV-2 and influenza A virus (IAV) poses enormous challenge in the development of broad-spectrum antivirals, effective against the existing and emerging viral strains. Virus entry through endocytosis represents an attractive target for drug development, as inhibition of this early infection step should block downstream infection processes and potentially inhibit viruses sharing the same entry route. Through high-content screening, we have identified diphenylurea derivatives (DPUDs) as a new class of endocytosis inhibitors, which broadly restricted entry and replication of several SARS-CoV-2 and IAV strains in tissue culture cells, without affecting cell viability. We found, DPUDs transported chloride ions into the cell, and interfered with the endocytic machinery by perturbing intracellular chloride homeostasis. Finally, we tested DPUDs in mice challenged with SARS-CoV-2 and IAV. Treatment with DPUDs led to remarkable body weight recovery, improved survival, and significant reduction in lung viral load, indicating their potential as broad-acting antivirals.

Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV2 (preprint)

BackgroundIndia has been amongst the most affected nations during the SARS-CoV2 pandemic, with sparse data on country-wide spread of asymptomatic infections and antibody persistence. This longitudinal cohort study was aimed to evaluate SARS-CoV2 sero-positivity rate as a marker of infection and evaluate temporal persistence of antibodies with neutralization capability and to infer possible risk factors for infection. MethodsCouncil of Scientific and Industrial Research, India (CSIR) with its more than 40 laboratories and centers in urban and semi-urban settings spread across the country piloted the pan country surveillance. 10427 adult individuals working in CSIR laboratories and their family members based on voluntary participation were assessed for antibody presence and stability was analyzed over 6 months utilizing qualitative Elecsys SARS CoV2 specific antibody kit and GENScript cPass SARS-CoV2 Neutralization Antibody Detection Kit. Along with demographic information, possible risk factors were evaluated through self to be filled online forms with data acquired on blood group type, occupation type, addiction and habits including smoking and alcohol, diet preferences, medical history and transport type utilized. Symptom history and information on possible contact and compliance with COVID 19 universal precautions was also obtained. Findings1058 individuals (10{middle dot}14%) had antibodies against SARS-CoV2. A follow-up on 346 sero-positive individuals after three months revealed stable to higher antibody levels against SARS-CoV2 but declining plasma activity for neutralizing SARS-CoV2 receptor binding domain and ACE2 interaction. A repeat sampling of 35 individuals, at six months, revealed declining antibody levels while the neutralizing activity remained stable compared to three months. Majority of sero-positive individuals (75%) did not recall even one of nine symptoms since March 2020. Fever was the most common symptom with one-fourth reporting loss of taste or smell. Significantly associated risks for sero-positivity (Odds Ratio, 95% CI, p value) were observed with usage of public transport (1{middle dot}79, 1{middle dot}43 - 2{middle dot}24, 2{middle dot}81561E-06), occupational responsibilities such as security, housekeeping personnel etc. (2{middle dot}23, 1{middle dot}92 - 2{middle dot}59, 6{middle dot}43969E-26), non-smokers (1{middle dot}52, 1{middle dot}16 - 1{middle dot}99, 0{middle dot}02) and non-vegetarianism (1{middle dot}67, 1{middle dot}41 - 1{middle dot}99, 3{middle dot}03821E-08). An iterative regression analysis was confirmatory and led to only modest changes to estimates. Predilections for sero-positivity was noted with specific ABO blood groups -O was associated with a lower risk. InterpretationIn a first-of-its-kind study from India, we report the sero-positivity in a country-wide cohort and identify variable susceptible associations for contacting infection. Serology and Neutralizing Antibody response provides much-sought-for general insights on the immune response to the virus among Indians and will be an important resource for designing vaccination strategies. FundingCouncil of Scientific and Industrial Research, India (CSIR)

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.