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One of the fundamental mechanisms developed by the host to contain the highly infectious and rapidly proliferating SARS coronavirus is elevation of body temperature, a natural fallout of which is Heat Shock Protein (HSP) over-expression. Here, for the first time, we demonstrate that the SARS-CoV-2 virus exploits the host Hsp70 chaperone for its entry and propagation and blocking it can combat the infection. SARS-CoV-2 infection as well as febrile temperature enhanced Hsp70 overexpression in host Vero E6 cells. In turn, Hsp70 overexpression elevated the host cell autophagic response that is a prerequisite for viral propagation. Suppressive and prophylactic treatment of Vero E6 cells with HSP70 inhibitor PES-Cl, a small molecule derivative of Pifithrin μ, abrogated viral infection more potently than the currently used drug Remdesivir by suppressing host HSP70 and autophagic response. In conclusion, our study not only provides a fundamental insight into the role of host Hsp70 in SARS-CoV-2 pathogenesis, it paves the way for the development of potent and irresistible anti-viral therapeutics.

The COVID-19 pandemic caused by SARS-CoV-2 has a significant burden on the economy and healthcare around the world. Vaccines are the most effective tools to fight infectious diseases by containing the spread of the disease. The current vaccines against SARS-CoV-2 are mostly based on the spike protein of SARS-CoV-2, which is large and has many immune-dominant non-neutralizing epitopes that may effectively skew the antibody response towards non-neutralizing antibodies. Here, we have explored the possibility of immune-focusing the receptor binding motif (RBM) of the spike protein of SARS-CoV-2 that induces mostly neutralizing antibodies in natural infection or in vacinees. The result shows that the scaffolded RBM can bind to Angiotensin Converting Enzyme 2 (ACE2) although with low affinity and induces a strong antibody response in mice. The immunized sera can bind both, the receptor binding domain (RBD) and the spike protein, which holds the RBM in its natural context. Sera from the immunized mice showed robust interferon γ response but poor neutralization of SARS-CoV-2 suggesting presence of a predominant T cell epitope on scaffolded RBM. Together, we provide a strategy for inducing strong antigenic T cell response which could be exploited further for future vaccine designing and development against SARS-CoV-2 infection.

Coronavirus induced disease-19 (COVID-19), caused by the SARS-CoV-2 remains a major global health challenge. Human endogenous retroviruses (HERVs) represent retroviral elements that got integrated into the ancestral human genome. HERVs are important in development and diseases, including cancer, inflammation and viral infections. Here, we analyzed the expression of several HERVs in SARS-CoV-2 infected cells and observed increased activity of HERV-E, HERV-V, HERV-FRD, HERV-MER34, HERV-W and HERV-KHML2. In contrast, HERV-R-envelope was downregulated in cell-based models and COVID-19 patient PBMCs. HERV-R overexpression inhibited SARS-CoV-2 replication, suggesting its antiviral action. Further studies demonstrated the role of extracellular signal-regulated kinase (ERK) in regulating HERV-R antiviral activity. Cross-talk between the ERK and p38 MAPK controls HERV-R envelope synthesis, which in turn modulates the replication of SARS-CoV-2. These findings establish the importance of HERV-R envelope as a host restriction factor against SARS-CoV-2 and illustrate the advantage of integration and evolutionary maintenance of retroviral-elements in the human genome.

Severe coronavirus disease (COVID-19) is accompanied by acute respiratory distress syndrome and pulmonary pathology, and is presented mostly with an inflammatory cytokine release, a dysregulated immune response, a skewed neutrophil/lymphocyte ratio, and a hypercoagulable state. Though vaccinations have proved effective in reducing the COVID-19-related mortality, the limitation of the use of vaccine against immunocompromised individuals, those with comorbidity, and emerging variants remains a concern. In the current study, we investigate for the first time the efficacy of the

The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic mAbs. Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concerns (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 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 and will be highly effective against future variants as well due to its unique mode of binding.

The underlying factors contributing to the evolution of SARS-CoV-2-specific T cell responses during COVID-19 infection remain unidentified. To address this, we characterized innate and adaptive immune responses with metabolomic profiling longitudinally at three different time points (0-3, 7-9, and 14-16 days post-COVID-19 positivity) from young mildly symptomatic active COVID-19 patients infected during the first wave in mid-2020. We observed that anti-RBD IgG and viral neutralization are significantly reduced against the Delta variant compared to the ancestral strain. In contrast, compared to the ancestral strain, T cell responses remain preserved against the delta and omicron variants. We determined innate immune responses during the early stage of active infection in response to TLR 3/7/8 mediated activation in PBMCs and serum metabolomic profiling. Correlation analysis indicated PBMCs-derived proinflammatory cytokines, IL-18, IL-1β, and IL-23, and the abundance of plasma metabolites involved in arginine biosynthesis were predictive of a robust SARS-CoV-2-specific Th1 response at a later stage (two weeks after PCR positivity). These observations may contribute to designing effective vaccines and adjuvants that promote innate immune responses and metabolites to induce long-lasting anti-SARS-CoV-2 specific T cells response.

Although efficacious vaccines have significantly reduced the morbidity and mortality due to COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of two highly potently neutralizing mAbs (THSC20.HVTR04 and THSC20.HVTR26) from an Indian convalescent donor, that neutralize SARS-CoV-2 VOCs at picomolar concentrations including the delta variant (B.1.617.2). These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein thereby preventing the virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness.

Immunization is expected to confer protection against infection and severe disease for vaccines while reducing risks to unimmunized populations by inhibiting transmission. Here, based on serial serological studies of an observational cohort of healthcare workers, we show that during a Severe Acute Respiratory Syndrome -Coronavirus 2 Delta-variant outbreak in Delhi, 25.3% (95% Confidence Interval 16.9-35.2) of previously uninfected, ChAdOx1-nCoV19 double vaccinated, healthcare workers were infected within less than two months, based on serology. Induction of anti-spike response was similar between groups with breakthrough infection (541 U/ml, Inter Quartile Range 374) and without (342 U/ml, Inter Quartile Range 497), as was the induction of neutralization activity to wildtype. This was not vaccine failure since vaccine effectiveness estimate based on infection rates in an unvaccinated cohort were about 70% and most infections were asymptomatic. We find that while ChAdOx1-nCoV19 vaccination remains effective in preventing severe infections, it is unlikely to be completely able to block transmission and provide herd immunity.

Summary Background SARS-CoV-2 variants of concern (VOCs) have threatened COVID-19 vaccine effectiveness. We aimed to assess the effectiveness of the ChAdOx1 nCoV-19 vaccine, predominantly against the delta (B.1.617.2) variant, in addition to the cellular immune response to vaccination. Methods We did a test-negative, case-control study at two medical research centres in Faridabad, India. All individuals who had a positive RT-PCR test for SARS-CoV-2 infection between April 1, 2021, and May 31, 2021, were included as cases and individuals who had a negative RT-PCR test were included as controls after matching with cases on calendar week of RT-PCR test. The primary outcome was effectiveness of complete vaccination with the ChAdOx1 nCoV-19 vaccine against laboratory-confirmed SARS-CoV-2 infection. The secondary outcomes were effectiveness of a single dose against SARS-CoV-2 infection and effectiveness of a single dose and complete vaccination against moderate-to-severe disease among infected individuals. Additionally, we tested in-vitro live-virus neutralisation and T-cell immune responses to the spike protein of the wild-type SARS-CoV-2 and VOCs among healthy (anti-nucleocapsid antibody negative) recipients of the ChAdOx1 nCoV-19 vaccine. Findings Of 2379 cases of confirmed SARS-CoV-2 infection, 85 (3·6%) were fully vaccinated compared with 168 (8·5%) of 1981 controls (adjusted OR [aOR] 0·37 [95% CI 0·28–0·48]), giving a vaccine effectiveness against SARS-CoV-2 infection of 63·1% (95% CI 51·5–72·1). 157 (6·4%) of 2451 of cases and 181 (9·1%) of 1994) controls had received a single dose of the ChAdOx1 nCoV-19 vaccine (aOR 0·54 [95% CI 0·42–0·68]), thus vaccine effectiveness of a single dose against SARS-CoV-2 infection was 46·2% (95% CI 31·6–57·7). One of 84 cases with moderate-to-severe COVID-19 was fully vaccinated compared with 84 of 2295 cases with mild COVID-19 (aOR 0·19 [95% CI 0·01–0·90]), giving a vaccine effectiveness of complete vaccination against moderate-to-severe disease of 81·5% (95% CI 9·9–99·0). The effectiveness of a single dose against moderate-to-severe disease was 79·2% (95% CI 46·1–94·0); four of 87 individuals with moderate-to-severe COVID-19 had received a single dose compared with 153 of 2364 participants with mild disease (aOR 0·20 [95% CI 0·06–0·54]). Among 49 healthy, fully vaccinated individuals, neutralising antibody responses were lower against the alpha (B.1.1.7; geometric mean titre 244·7 [95% CI 151·8–394·4]), beta (B.1.351; 97·6 [61·2–155·8]), kappa (B.1.617.1; 112·8 [72·7–175·0]), and delta (88·4 [61·2–127·8]) variants than against wild-type SARS-CoV-2 (599·4 [376·9–953·2]). However, the antigen-specific CD4 and CD8 T-cell responses were conserved against both the delta variant and wild-type SARS-CoV-2. Interpretation The ChAdOx1 nCoV-19 vaccine remained effective against moderate-to-severe COVID-19, even during a surge that was dominated by the highly transmissible delta variant of SARS-CoV-2. Spike-specific T-cell responses were maintained against the delta variant. Such cellular immune protection might compensate for waning humoral immunity. Funding Department of Biotechnology India, Council of Scientific and Industrial Research India, and Fondation Botnar.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection initiates with viral entry in the upper respiratory tract, leading to coronavirus disease 2019 (COVID-19). Severe COVID-19 is characterized by pulmonary pathologies associated with respiratory failure. Thus, therapeutics aimed at inhibiting the entry of the virus or its internalization in the upper respiratory tract are of interest. Herein, we report the prophylactic application of two intranasal formulations provided by the National Medicinal Plant Board (NMPB), Anu oil and til tailya, in the hamster model of SARS-CoV-2 infection. Prophylactic intra-nasal instillation of these oil formulations exhibited reduced viral load in lungs and resulted in reduced body weight loss and lung-pneumonitis. In line with reduced viral load, histopathological analysis revealed a reduction in lung pathology in the Anu oil group as compared to the control infected group. However, the til tailya group did not show a significant reduction in lung pathology. Furthermore, molecular analysis using mRNA expression profiling indicated reduced expression of pro-inflammatory cytokine genes, including Th1 and Th17 cytokines for both the intranasal formulations as a result of decreased viral load. Together, the prophylactic intranasal application of Anu oil seems to be useful in limiting both viral load and severity in SARS-CoV2 infection in the hamster model.

Cross-reactivity among the two diverse viruses is believed to originate from the concept of antibodies recognizing similar epitopes on the two viral surfaces. Cross-reactive antibody responses have been seen in previous variants of SARS and SARS-CoV-2, but little is known about the cross reactivity with other similar RNA viruses like HIV-1. In the present study, we examined the reactivity the SARS-CoV-2 directed antibodies, via spike, immunized mice sera and demonstrated whether they conferred any cross-reactive neutralization against HIV-1. Our findings show that SARS-CoV-2 spike immunized mice antibodies cross-react with the HIV-1 Env protein. Cross-neutralization among the two viruses is uncommon, suggesting the presence of a non-neutralizing antibody response to conserved epitopes amongst the two viruses. Our results indicate, that SARS-CoV-2 spike antibody cross reactivity is targeted towards the gp41 region of the HIV-1 Env (gp160) protein. Overall, our investigation not only answers a crucial question about the understanding of cross-reactive epitopes of antibodies generated in different viral infections, but also provides critical evidence for developing vaccine immunogens and novel treatment strategies with enhanced efficacy capable of recognising diverse pathogens with similar antigenic features.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection initiates with viral entry in upper respiratory tract leading to coronavirus disease 2019 (Covid-19). Severe Covid-19 is characterized by pulmonary pathologies associated with respiratory failure. Thus, therapeutics aimed at inhibiting entry of the virus or its internalization in the upper respiratory tract, are of interest. Herein, we report the prophylactic application of two intra-nasal formulations provided by the National Medicinal Plant Board (NMPB), Anu oil and Til tailya in SARS-CoV2 infection hamster model. Prophylactic nasal instillation of these oil formulations exhibited reduced viral load in lungs, and resulted in reduced body weight loss and pneumonitis. In line with reduced viral load, histopathlogical analysis revealed a reduction in lung pathology in Anu oil group as compared to the control infected group. However, Til tailya group did not show a significant reduction in lung pathology. Furthermore, molecular analysis using mRNA expression profiling indicated reduced expression of pro-inflammatory cytokines genes, including Th1 and Th17 cytokines for both the intra-nasal formulations as a result of decreased viral load. Together, the prophylactic intra-nasal application of Annu oil seems to be useful in limiting both the viral load and disease severity disease in SARS-CoV2 infection in hamster model.

Natural reservoir hosts can sustain infection of pathogens without succumbing to overt disease. Multiple bat species host a plethora of viruses, pathogenic to other mammals, without clinical symptoms. Here, we detail infection of bat primary cells, immune cells, and cell lines with Dengue virus. While antibodies and viral RNA were previously detected in wild bats, their ability to sustain infection is not conclusive. Old-world fruitbat cells can be infected, producing high titres of virus with limited cellular responses. In addition, there is minimal interferon (IFN) response in cells infected with MOIs leading to dengue production. The ability to support in vitro replication/production raises the possibility of bats as a transient host in the life cycle of dengue or similar flaviviruses. New antibody serology evidence from Asia/Pacific highlights the previous exposure and raises awareness that bats may be involved in flavivirus dynamics and infection of other hosts.

Bats are special in their ability to host emerging viruses. As the only flying mammal, bats endure high metabolic rates yet exhibit elongated lifespans. It is currently unclear whether these unique features are interlinked. The important inflammasome sensor, NLR family pyrin domain containing 3 (NLRP3), has been linked to both viral-induced and age-related inflammation. Here, we report significantly dampened activation of the NLRP3 inflammasome in bat primary immune cells compared to human or mouse counterparts. Lower induction of apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and secretion of interleukin-1β in response to both 'sterile' stimuli and infection with multiple zoonotic viruses including influenza A virus (-single-stranded (ss) RNA), Melaka virus (PRV3M, double-stranded RNA) and Middle East respiratory syndrome coronavirus (+ssRNA) was observed. Importantly, this reduction of inflammation had no impact on the overall viral loads. We identified dampened transcriptional priming, a novel splice variant and an altered leucine-rich repeat domain of bat NLRP3 as the cause. Our results elucidate an important mechanism through which bats dampen inflammation with implications for longevity and unique viral reservoir status.

Immunization is expected to confer protection against infection and severe disease for vaccinees, while reducing risks to unimmunized populations by inhibiting transmission. Here, based on serial serological studies, we show that during a severe SARS-CoV2 Delta-variant outbreak in Delhi, 25.3% (95% CI 16.9 - 35.2) of previously uninfected, ChAdOx1-nCoV19 double vaccinated, healthcare-workers (HCW) were infected within a period of less than two months, based on serology. Induction of anti-spike response was similar between groups with breakthrough infection (541 U/ml, IQR 374) or not (342 U/ml, IQR 497), as was induction of neutralization activity to wildtype. Most infections were unrecognized. The Delta-variant thus causes frequent unrecognized breakthrough infections in adequately immunized subjects, reducing any herd-effect of immunity, and requiring reinstatement of preventive measures such as masking.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is a positive-strand RNA virus. Viral genome is capped at the 5’-end, followed by an untranslated region (UTR). There is poly-A tail at 3’-end, preceded by an UTR. Self-interaction between the RNA regulatory elements present within 5’- and 3’-UTRs as well as their interaction with host/virus-encoded proteins mediate the function of 5’- and 3’-UTRs. Using RNA-protein interaction detection (RaPID) assay coupled to liquid chromatography with tandem mass-spectrometry, we identified host interaction partners of SARS-CoV-2 5’- and 3’-UTRs and generated an RNA-protein interaction network. By combining these data with the previously known protein-protein interaction data proposed to be involved in virus replication, we generated the RNA-protein-protein interaction (RPPI) network, likely to be essential for controlling SARS-CoV-2 replication. Notably, bioinformatics analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism. Lysosome-associated membrane protein-2a (Lamp2a) was one of the host proteins that interact with the 5’-UTR. Further studies showed that Lamp2 level is upregulated in SARS-CoV-2 infected cells and overexpression of Lamp2a and Lamp2b variants reduced viral RNA level in infected cells and vice versa. In summary, our study provides an useful resource of SARS-CoV-2 5’- and 3’-UTR binding proteins and reveal the antiviral function of host Lamp2 protein.ImportanceReplication of a positive-strand RNA virus involves an RNA-protein complex consisting of viral genomic RNA, host RNA(s), virus-encoded proteins and host proteins. Dissecting out individual components of the replication complex will help decode the mechanism of viral replication. 5’- and 3’-UTRs in positive-strand RNA viruses play essential regulatory roles in virus replication. Here, we identified the host proteins that associate with the UTRs of SARS-CoV-2, combined those data with the previously known protein-protein interaction data (expected to be involved in virus replication) and generated the RNA-protein-protein interaction (RPPI) network. Analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism, which are important for virus replication. Analysis of one of the interaction partners of the 5’-UTR (Lamp2a) demonstrated its antiviral role in SARS-CoV-2 infected cells. Collectively, our study provides a resource of SARS-CoV-2 UTR-binding proteins and identifies an antiviral role of host Lamp2a protein.

Background: The emergence of SARS-CoV-2 variants of concern (VoC) has threatened the effectiveness of vaccination due to decreased neutralisation ability of the vaccine generated antibodies. Our objective was to assess ChAdOx1 nCoV-19 vaccine effectiveness during the massive surge from 1st April to 31stMay 2021 predominantly due to the more infectious B·1·617·2 (Delta) in India. Methods: We conducted a test-negative case-control study to assess the effectiveness of ChAdOx1 nCoV-19 vaccine. Cases were RT-PCR positive for SARS-CoV-2 infection. In addition, we tested live virus neutralisation and cellular immune responses against VoC among healthy recipients of ChAdOx1 nCoV-19 vaccine. The outcomes were effectiveness of ChAdOx1 nCoV-19 against infection and severe coronavirus disease-19, virus neutralisation effectivity and T-cell responses against the VoC among healthy vaccine recipients. Findings: Of the 2766 cases of confirmed SARS-CoV-2 infection, 3·1% were fully vaccinated compared with 7·1% of the 2377 controls giving an adjusted OR of 0·37 (95%CI 0·28, 0·48); this translated to 63·1% (95%CI 51·5, 72·1) vaccine effectiveness against SARS-CoV-2 B·1·617·2 variant, seen in 90% of the infected population as confirmed by whole-genome virus sequencing. Full vaccination prevented moderate-severe COVID-19 in 81·5% (95%CI: 9·9, 99·0). The effectiveness of single-dose vaccine was 46·2% (95%CI: 31·6, 57·7) against infection but 79·2% (95%CI: 46·1, 94·0) in preventing moderate-severe Covid-19. Among healthy vaccinated persons, plasma live virus neutralisation was 2·5-6·8 fold lower against B·1·1·7, B·1·351, B·1·617·1 and B·1·617·2 being lowest against B·1·617·2. However, T-cell responses were preserved against the recombinant mutant receptor binding domain antigens suggesting cell-mediated immune protection. Interpretation: Despite significantly reduced virus neutralisation, the effectiveness of ChAdOx1 nCoV-19 vaccine was 63% against B·1·617·2 infection and 81·5% in preventing severe disease. Spike specific T cells responses against virus variants were maintained and might contribute to immune protection following vaccination. Funding Information: Department of Biotechnology, Government of India; Council for Scientific and Industrial Research, India; Fondation Botnar. Declaration of Interests: None to declare. Ethics Approval Statement: The studies were approved by the Institute Ethics Committees of the partnering institutions.

The newly emerged novel coronavirus, SARS-CoV-2, the causative agent of COVID-19 has proven to be a threat to the human race globally, thus, vaccine development against SARS-CoV-2 is an unmet need driving mass vaccination efforts. The receptor binding domain of the spike protein of this coronavirus has multiple neutralizing epitopes and is associated with viral entry. Here we have designed and characterized the SARS-CoV-2 spike protein fragment 330-526 as receptor binding domain 330-526 (RBD330-526) with two native glycosylation sites (N331 and N343); as a potential subunit vaccine candidate. We initially characterized RBD330-526 biochemically and investigated its thermal stability, humoral and T cell immune response of various RBD protein formulations (with or without adjuvant) to evaluate the inherent immunogenicity and immunomodulatory effect. Our result showed that the purified RBD immunogen is stable up to 72 h, without any apparent loss in affinity or specificity of interaction with the ACE2 receptor. Upon immunization in mice, RBD generates a high titer humoral response, elevated IFN-γ producing CD4+ cells, cytotoxic T cells, and robust neutralizing antibodies against live SARS-CoV-2 virus. Our results collectively support the potential of RBD330-526 as a promising vaccine candidate against SARS-CoV-2.

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100°C when lyophilized, upto 70°C in solution and stable for over four weeks at 37°C. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Nucleic acid real-time PCR test has become the main diagnostic tool for SARS‐CoV‐2 infection, and there are also drawbacks to these real-time PCR test kits. Additionally, it has reported high false-negative rates. A reliable and rapid test method is urgently required to briskly detect antibody response in a large number of infected patients and ensure prompt care for patients. Therefore, we report here a Luminex bead based multiplexed immunoassay to serologically diagnose the SARS-CoV2 infection and detect IgM and IgG response in COVID-19 patients. The technology can provide sensitive and specific detection using a low amount of serum samples in high-throughput screening platforms.

In the absence of serum panels from individuals with absolute defined infection status, sera from experimentally infected monkeys were generated to cover all four infection scenarios with Zika or dengue virus alone as well as Zika infection followed by dengue and dengue infection followed by Zika. Our data indicated that no currently available single assay platform is sufficiently specific to serologically determine Zika positivity in the presence of prior exposure to dengue and vice versa.

The spike protein of the SARS-CoV-2 virus is the foremost target for the designing of vaccines and therapeutic antibodies and also acts as a crucial antigen in the assessment of COVID-19 immune responses. The enveloped viruses; such as SARS-CoV-2, Human Immunodeficiency Virus-1 (HIV-1) and influenza, often hijack host-cell glycosylation pathways and influence pathobiology and immune selection. These glycan motifs can lead to either immune evasion or viral neutralization by the production of cross-reactive antibodies that can lead to antibody-dependent enhancement (ADE) of infection. Potential cross-protection from influenza vaccine has also been reported in COVID-19 infected individuals in several epidemiological studies recently; however, the scientific basis for these observations remains elusive. Herein, we show that the anti-SARS-CoV2 antibodies cross-reacts with the Hemagglutinin (HA) protein. This phenomenon is common to both the sera from convalescent SARS-CoV-2 donors and spike immunized mice, although these antibodies were unable to cross-neutralize, suggesting the presence of a non-neutralizing antibody response. Epitope mapping suggests that the cross-reactive antibodies are targeted towards glycan epitopes of the SARS-CoV-2 spike and HA. Overall, our findings address the cross-reactive responses, although non-neutralizing, elicited against RNA viruses and warrant further studies to investigate whether such non-neutralizing antibody responses can contribute to effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or ADE.

Dromedary camels are important reservoir hosts of various coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) that cause human infections. CoV genomes regularly undergo recombination during infection as observed in bat SARS-related CoVs. Here we report for the first time that only a small proportion of MERS-CoV receptor-binding domain positive (RBD) of spike protein positive camel sera in Kenya were also seropositive to MERS-CoV nucleocapsid (NP). In contrast, many of them contain antibodies against bat HKU8-related (HKU8r)-CoVs. Among 584 camel samples that were positive against MERS-CoV RBD, we found only 0.48 (8.22%) samples were also positive for NP. Furthermore, we found bat HKU8r-CoV NP antibody in 73 (12.5%) of the MERS-CoV RBD positive and NP negative samples, yet found only 3 (0.43%) of the HKU8r-CoV S1 antibody in the same samples. These findings may indicate co-infection with MERS-CoV and a HKU8r-CoV in camels. It may also raise the possibility of the circulation of a recombinant coronavirus virus with the spike of MERS-CoV and the NP of a HKU8r-CoV in Kenya. We failed to find molecular evidence of an HKU8r-CoV or a putative recombinant virus. Our findings should alert other investigators to look for molecular evidence of HKU8r-CoV or recombinants.

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

To determine whether Pteropine orthoreovirus (PRV) exposure has occurred in Singapore, we tested 856 individuals from an existing serum panel collected from 2005-2013. After an initial screen with luciferase immunoprecipitation system and secondary confirmation with virus neutralization test, we identified at least seven individuals with specific antibodies against PRV in both assays. Our findings confirm that PRV spillover into human populations is relatively common in this region of the world.

Bats are special in their ability to host emerging viruses. As the only flying mammal, bats endure high metabolic rates yet exhibit elongated lifespans. It is currently unclear whether these unique features are interlinked. The important inflammasome sensor, NLR family pyrin domain containing 3 (NLRP3), has been linked to both viral-induced and age-related inflammation. Here, we report significantly dampened activation of the NLRP3 inflammasome in bat primary immune cells compared to human or mouse counterparts. Lower induction of apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and secretion of interleukin-1β in response to both 'sterile' stimuli and infection with multiple zoonotic viruses including influenza A virus (-single-stranded (ss) RNA), Melaka virus (PRV3M, double-stranded RNA) and Middle East respiratory syndrome coronavirus (+ssRNA) was observed. Importantly, this reduction of inflammation had no impact on the overall viral loads. We identified dampened transcriptional priming, a novel splice variant and an altered leucine-rich repeat domain of bat NLRP3 as the cause. Our results elucidate an important mechanism through which bats dampen inflammation with implications for longevity and unique viral reservoir status.