A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

A simple and rapid method to monitor the disassembly and reassembly of virus-like particles.

Protein fluorescence spectra (~300-440 nm) could be used as a simple and sensitive method to monitor the disassembly and reassembly of virus-like particles (VLPs). Insect cell expressed and purified HPV-16 L1 VLPs show significantly high fluorescence intensity, whereas the fluorescence is almost quenched after disassembly by adding the reducing agent. By removing the reducing agent, the fluorescence was restored to its original intensity, indicating the reassembly of VLPs. The data are consistent with enzyme-linked immunosorbent assay (ELISA) reactivity using conformation-specific mouse monoclonal antibody. The same method could be extended to VLPs of other viruses.

Development of recombinant single-chain variable fragment against hepatitis A virus and its use in quantification of hepatitis A antigen.

Phage display technology has been utilized for identification of specific binding molecules to an antigenic target thereby enabling the rapid generation and selection of high affinity, fully human antibodies directed towards disease target appropriate for antibody therapy. In the present study, single chain Fv antibody fragment (scFv) to hepatitis A virus (HAV) was selected from phage displayed antibody library constructed from peripheral blood lymphocytes (PBLs) of a vaccinated donor. The variable heavy (V(H)) and light chains (V(L)) were amplified using cDNA as template, assembled into scFv using splicing by overlap extension PCR (SOE PCR) and cloned into phagemid vector as a fusion for display of scFv on bacteriophage. The phage displaying antibody fragments were subjected to three rounds of panning with HAV antigen on solid phase. High affinity antibodies reactive to hepatitis A virus were identified by phage ELISA and cloned into a bacterial expression vector pET20b. The scFv was purified by immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The binding activity and specificity of the scFv was established by its non-reactivity towards other human viral antigens as determined by ELISA and immunoblot analysis. The scFv was further used in the development of an in-house IC-ELISA format in combination with a commercially available mouse monoclonal antibody for the quantification of hepatitis A virus antigen in human vaccine preparations. The adjusted r² values obtained by subjecting the values obtained by quantification of the NIBSC standards using the commercial and the in-house ELISA kits by regression analysis were 0.99 and 0.95. 39 vaccine samples were subjected to quantification using both the kits. Regressional statistical analysis through the origin of the samples indicated International Unit (IU) values of 0.0416x and 0.0419x, respectively for the commercial and in-house kit respectively.

Safety and immunogenicity of inactivated hepatitis-A vaccine developed by Human Biologicals Institute in two age groups of healthy subjects: A phase I open label study.

BACKGROUND: Hepatitis-A is an acute viral infection of the liver. Hepatitis-A virus has worldwide spread and is endemic in India. Though the disease is self-limiting in most cases, outbreaks are reported frequently from both developing and developed countries of the world. Severity and fatality occur more among infected symptomatic adults. The infection can be prevented with proper and timely immunization. This phase I, single arm, open label, multicenter trial was designed to assess the safety and immunogenicity of the inactivated hepatitis-A vaccine developed by Human Biologicals Institute when administered in a single dose in two age groups of healthy subjects. METHODS: This study was carried out in 55 subjects in two healthy age groups at two centers in India. Group A included subjects of 19-49 years and group B subjects of 12-18 years of age. Enrolled subjects received a single dose of inactivated hepatitis A vaccine. Blood samples were collected at baseline and 4-6 weeks after vaccination. Safety was assessed by collection and analysis of data on solicited and unsolicited adverse events and immunogenicity was assessed by estimating the seroconversion rate, seroprotection rate and the geometric mean titres of antibodies. RESULTS: Among the 55 subjects enrolled, 15 reported adverse events. No serious adverse event was reported. Pain at the injection site was the lone local adverse event. Systemic adverse events reported in Group A were: fatigue, headache, diarrhoea, fever, anorexia, nausea and upper respiratory tract infection, whereas there was no systemic event reported in Group B. There was 100% seroconversion and seroprotection and significant rise in antibody titre levels were observed in both the groups post vaccination. CONCLUSIONS: This study found HBI inactivated hepatitis-A vaccine to be safe and highly immunogenic when administered as a single dose in adolescent and adult subjects.

Assessment of packed bed bioreactor systems in the production of viral vaccines.

Vaccination is believed to be the most effective method for the prevention of infectious diseases. Thus it is imperative to develop cost effective and scalable process for the production of vaccines so as to make them affordable for mass use. In this study, performance of a novel disposable iCELLis fixed bed bioreactor system was investigated for the production of some viral vaccines like Rabies, Hepatitis-A and Chikungunya vaccines in comparison to conventional systems like the commercially available packed bed system and roller bottle system. Vero and MRC-5 cell substrates were evaluated for growth parameters in all the three systems maintaining similar seeding density, multiplicity of infection (MOI) and media components. It was observed that Vero cells showed similar growth in all the three bioreactors whereas MRC-5 cells showed better growth in iCELLis Nano system and roller bottle system. Subsequently, the virus infection and antigen production studies also revealed that for Hepatitis-A and Chikungunya iCELLis Nano bioreactor system was better to the commercial packed bed bioreactor and roller bottle systems. Although for rabies antigen production commercially available packed bed bioreactor system was found to be better. This study shows that different bioreactor platforms may be employed for viral vaccine production and iCELLis Nano is one of such new convenient and a stable platform for production of human viral vaccines.

Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein.

Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv) A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain) using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies.

Assessment of critical parameters in blood processing for the bovine interferon-gamma ELISPOT assay to detect Mycobacterium bovis infected cattle in India.

In vitro production of bovine interferon gamma (BoIFN-γ) cytokine from bovine peripheral blood mononuclear cells (PBMCs) can be detected using the most sensitive enzyme-linked immunosorbent spot (ELISPOT) assay. ELISPOT assays are dependent on the quantity and quality of PBMC preparations and hence contribute significantly to the performance of this assay. In order to standardise the methods for isolation of PBMCs, we compared two methods for the processing of bovine blood which included aliquots of blood that were stored in a horizontal position without dilution or agitation and aliquots of blood that were immediately diluted 1:1 with complete Rosewell Park Memorial Institute (RPMI) 1640 medium and stored in a horizontal position with gentle agitation. PBMCs were isolated at 2, 4, 6, 8 and 24 h and at 4°C and at 22°C ± 2°C. They were stimulated using tuberculosis-specific antigens, after which the ELISPOT assay was performed. Quantities of spot-forming cells (SFC) created by the release of BoIFN-γ in ELISPOT assays were significantly greater in the samples stored at 22°C ± 2°C than those held +4°C and the intensity of the signals dropped following processing after 6 h. A further drop in SFC was observed in those samples that had been stored undiluted and without agitation. These findings demonstrated that optimisation of PBMC isolation procedures can lead to increased sensitivity in the detection of BoIFN-γ using the ELISPOT assay, thus contributing to an enhanced diagnosis of bovine tuberculosis.

Immunocapture enzyme-linked immunosorbent assay for assessment of in vitro potency of recombinant hepatitis B vaccines.

Quantification of hepatitis B surface antigen (HBsAg) or relative in vitro potency in the final vaccines is a prerequisite for hepatitis B vaccine batch release. The commercial kit for automated analysis (AxSYM) is expensive, and an alternative is required for the estimation of HBsAg in hepatitis B vaccines. Mouse monoclonal antibodies (MAbs) specific for HBsAg were developed and characterized. One of the monoclonal antibodies (HBs06) was used in development of an immunocapture ELISA (IC-ELISA) as an unlabeled capture antibody and biotin-labeled detection antibody. The IC-ELISA was standardized and validated using experimental hepatitis B vaccine batches with various HBsAg concentrations per dose and commercial vaccines. The vaccine was treated with an alkaline solubilizer to desorb the HBsAg from Algel-adjuvanted vaccines before testing, and the sensitivity of the test was 5 ng/ml. A good correlation could be observed between the HBsAg estimates derived by both formats, except for the higher HBsAg concentration range, where the IC-ELISA format could estimate closer to the actual values than AxSYM. There was a significant correlation between the estimated relative potencies of the two methods. There was lack of correlation between the in vivo potency and the relative in vitro potency. However, the estimates of IC-ELISA were comparable to the in vivo values when compared with the estimates of AxSYM. The IC-ELISA can therefore be considered to be a reliable test for deriving in vitro relative potency and antigen concentration in vaccine batches for batch control and release.