There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS– coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective ...antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)–interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.
Cross-species transmission of viruses from wildlife animal reservoirs poses a marked threat to human and animal health
. Bats have been recognized as one of the most important reservoirs for emerging ...viruses and the transmission of a coronavirus that originated in bats to humans via intermediate hosts was responsible for the high-impact emerging zoonosis, severe acute respiratory syndrome (SARS)
. Here we provide virological, epidemiological, evolutionary and experimental evidence that a novel HKU2-related bat coronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), is the aetiological agent that was responsible for a large-scale outbreak of fatal disease in pigs in China that has caused the death of 24,693 piglets across four farms. Notably, the outbreak began in Guangdong province in the vicinity of the origin of the SARS pandemic. Furthermore, we identified SADS-related CoVs with 96-98% sequence identity in 9.8% (58 out of 591) of anal swabs collected from bats in Guangdong province during 2013-2016, predominantly in horseshoe bats (Rhinolophus spp.) that are known reservoirs of SARS-related CoVs. We found that there were striking similarities between the SADS and SARS outbreaks in geographical, temporal, ecological and aetiological settings. This study highlights the importance of identifying coronavirus diversity and distribution in bats to mitigate future outbreaks that could threaten livestock, public health and economic growth.
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.
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.
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.
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.
Department of Biotechnology India, Council of Scientific and Industrial Research India, and Fondation Botnar.
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.
•A SARS-CoV-2 protein subunit based vaccine design with immune-focusing approach•A protein scaffold is used to display the native-like conformation of the selected epitope.•Characterization of the vaccine candidate showed that it is well folded.•The vaccine candidate induces robust antibody response in immunized mice.•It also induced significant T cell response but mild neutralizing antibody response
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.
In the current study, we evaluated the efficacy of Ayush-64 (A64), a polyherbal formulation containing Alstonia scholaris (L.) R. Br. (A. scholaris), Caesalpinia crista L. (C. crista), Picrorhiza ...kurroa Royle ex Benth (P. kurroa), and Swertia chirata (Roxb.) H. Karst. (S. chirata) against COVID-19 in a Syrian hamster infection model. Preventative use of A64 resulted in the late-phase recovery of body weight loss in severe acquired respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected hamsters, suppression of pro-inflammatory cytokines, and blunted pulmonary pathology. In addition, we also investigated the efficacy of individual ingredients of A64, viz., A. scholaris, C. crista, P. kurroa, and S. chirata, in the hamster model. The hamster challenge data showed robust anti-viral and immunomodulatory potential in A. scholaris, followed by P. kurroa. However, C. crista and S. chirata of A64 showed prominent immunomodulatory potential without limiting the lung viral load. In order to better understand the immunomodulatory potential of these herbal extracts, we used an in vitro assay of helper T cell differentiation and found that A. scholaris mediated a more profound suppression of Th1, Th2, and Th17 cell differentiation as compared to A64 and other ingredients. Taken together, our animal study data identifies the ameliorative potential of A64 in mitigating coronavirus disease-19 (COVID-19) pulmonary pathology. A. scholaris, a constituent extract of A64, showed relatively higher anti-viral and immunomodulatory potential against COVID-19. The present study warrants further investigations to identify the active pharmaceutical ingredients of A. scholaris for further studies.
Highly mutated SARS-CoV-2 is known aetiological factor for COVID-19. Here, we have demonstrated that the receptor binding domain (RBD) of the spike protein can interact with human dipeptidyl ...peptidase 4 (DPP4) to facilitate virus entry, in addition to the usual route of ACE2-RBD binding. Significant number of residues of RBD makes hydrogen bonds and hydrophobic interactions with α/β-hydrolase domain of DPP4. With this observation, we created a strategy to combat COVID-19 by circumventing the catalytic activity of DPP4 using its inhibitors. Sitagliptin, linagliptin or in combination disavowed RBD to establish a heterodimer complex with both DPP4 and ACE2 which is requisite strategy for virus entry into the cells. Both gliptins not only impede DPP4 activity, but also prevent ACE2-RBD interaction, crucial for virus growth. Sitagliptin, and linagliptin alone or in combination have avidity to impede the growth of pan-SARS-CoV-2 variants including original SARS-CoV-2, alpha, beta, delta, and kappa in a dose dependent manner. However, these drugs were unable to alter enzymatic activity of PLpro and Mpro. We conclude that viruses hijack DPP4 for cell invasion via RBD binding. Impeding RBD interaction with both DPP4 and ACE2 selectively by sitagliptin and linagliptin is an potential strategy for efficiently preventing viral replication.
Pleotropic efficacy of sitagliptin, and linagliptin to impede pan-variants of SARS-CoV-2 infections. Strategy I: Establishing RBD of pan-variants of SARS-CoV-2 can interact with DPP4 in addition to ACE2 for the viral entry. Strategy II: Sitagliptin, and linagliptin, alone or in combination can suppress the growth of SARS-CoV-2 variants including most common VOCs like original SARS-CoV-2, alpha, beta, delta and kappa clade by impeding the DPP4 and RBD interaction. Strategy III: Both sitagliptin and linagliptin exhibit pleotropic effects as a result of gliptins' capacity to target ACE2-RBD interaction. Display omitted
The COVID-19 pandemic caused by SARS-CoV-2, lead to mild to severe respiratory illness and resulted in 6.9 million deaths worldwide. Although vaccines are effective in preventing COVID-19, they may ...not be sufficient to protect immunocompromised individuals from this respiratory illness. Moreover, novel emerging variants of SARS-CoV-2 pose a risk of new COVID-19 waves. Therefore, identification of effective antivirals is critical in controlling SARS and other coronaviruses, such as MERS-CoV. We show that Fangchinoline (Fcn), a bisbenzylisoquinoline alkaloid, inhibits replication of SARS-CoV, SARS-CoV-2, and MERS-CoV in a range of in vitro assays, by blocking entry. Therapeutic use of Fcn inhibited viral loads in the lungs, and suppressed associated airway inflammation in hACE2. Tg mice and Syrian hamster infected with SARS-CoV-2. Combination of Fcn with remdesivir (RDV) or an anti-leprosy drug, Clofazimine, exhibited synergistic antiviral activity. Compared to Fcn, its synthetic derivative, MK-04-003, more effectively inhibited SARS-CoV-2 and its variants B.1.617.2 and BA.5 in mice. Taken together these data demonstrate that Fcn is a pan beta coronavirus inhibitor, which possibly can be used to combat novel emerging coronavirus diseases.
Coronavirus‐induced disease‐19 (COVID‐19), caused by SARS‐CoV‐2, is still a major global health challenge. Human endogenous retroviruses (HERVs) represent retroviral elements that were integrated ...into the ancestral human genome. HERVs are important in embryonic development as well as in the manifestation of diseases, including cancer, inflammation, and viral infections. Here, we analyze the expression of several HERVs in SARS‐CoV‐2‐infected cells and observe increased activity of HERV‐E, HERV‐V, HERV‐FRD, HERV‐MER34, HERV‐W, and HERV‐K‐HML2. In contrast, the HERV‐R envelope is downregulated in cell‐based models and PBMCs of COVID‐19 patients. Overexpression of HERV‐R inhibits SARS‐CoV‐2 replication, suggesting its antiviral activity. Further analyses demonstrate the role of the extracellular signal‐regulated kinase (ERK) in regulating HERV‐R antiviral activity. Lastly, our data indicate that the crosstalk between ERK and p38 MAPK controls the synthesis of the HERV‐R envelope protein, which in turn modulates SARS‐CoV‐2 replication. These findings suggest the role of the HERV‐R envelope as a prosurvival host factor against SARS‐CoV‐2 and illustrate a possible advantage of integration and evolutionary maintenance of retroviral elements in the human genome.
Synopsis
HERV‐R‐Env (envelope) levels are reduced upon SARS‐CoV‐2 infection in cell‐based models and COVID‐19 patient samples. HERV‐R‐Env inhibits SARS‐CoV‐2 replication, suggesting its antiviral function, which is mediated by the ERK pathway.
SARS‐CoV‐2 infection reduces HERV‐R‐Env levels.
HERV‐R‐Env inhibits SARS‐CoV‐2 replication.
The HERV‐R‐Env antiviral function against SARS‐CoV‐2 is mediated by activation of the ERK pathway.
SARS‐CoV‐2 antagonizes the antiviral function of HERV‐R‐Env by upregulating the p38 MAPK activity and concomitantly downregulating the ERK activity.
HERV‐R‐Env (envelope) levels are reduced upon SARS‐CoV‐2 infection in cell‐based models and COVID‐19 patient samples. HERV‐R‐Env inhibits SARS‐CoV‐2 replication, suggesting its antiviral function, which is mediated by the ERK pathway.
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.
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