VLP-ELISA for the Detection of IgG Antibodies against Spike, Envelope, and Membrane Antigens of SARS-CoV-2 in Indian Population.

BACKGROUND: Serological methods to conduct epidemiological survey are often directed only against the spike protein. To overcome this limitation, we have designed PRAK-03202, a virus-like particle (VLP), by inserting three antigens (Spike, envelope and membrane) of SARS-CoV-2 into a highly characterized S. cerevisiae-based D-Crypt™ platform. METHODS: Dot blot analysis was performed to confirm the presence of S, E, and M proteins in PRAK-03202. The number of particles in PRAK-03202 was measured using nanoparticle tracking analysis (NTA). The sensitivity of VLP-ELISA was evaluated in 100 COVID positive. PRAK-03202 was produced at a 5 L scale using fed-batch fermentation. RESULTS: Dot blot confirmed the presence of S, E, and M proteins in PRAK-03202. The number of particles in PRAK-03202 was 1.21 × 109 mL-1. In samples collected >14 days after symptom onset, the sensitivity, specificity, and accuracy of VLP-ELISA were 96%. We did not observe any significant differences in sensitivity, specificity, and accuracy when post-COVID-19 samples were used as negative controls compared to pre-COVID-samples. At a scale of 5 L, the total yield of PRAK-03202 was 100-120 mg/L. CONCLUSION: In conclusion, we have successfully developed an in-house VLP-ELISA to detect IgG antibodies against three antigens of SARS-CoV-2 as a simple and affordable alternative test.

Platforms, advances, and technical challenges in virus-like particles-based vaccines.

Viral infectious diseases threaten human health and global stability. Several vaccine platforms, such as DNA, mRNA, recombinant viral vectors, and virus-like particle-based vaccines have been developed to counter these viral infectious diseases. Virus-like particles (VLP) are considered real, present, licensed and successful vaccines against prevalent and emergent diseases due to their non-infectious nature, structural similarity with viruses, and high immunogenicity. However, only a few VLP-based vaccines have been commercialized, and the others are either in the clinical or preclinical phases. Notably, despite success in the preclinical phase, many vaccines are still struggling with small-scale fundamental research owing to technical difficulties. Successful production of VLP-based vaccines on a commercial scale requires a suitable platform and culture mode for large-scale production, optimization of transduction-related parameters, upstream and downstream processing, and monitoring of product quality at each step. In this review article, we focus on the advantages and disadvantages of various VLP-producing platforms, recent advances and technical challenges in VLP production, and the current status of VLP-based vaccine candidates at commercial, preclinical, and clinical levels.

Recapitulating Tumor Microenvironment Using AXTEX-4DTM for Accelerating Cancer Research and Drug Screening.

OBJECTIVE: The formation of three-dimensional spheroid tumor model using the scaffold-based platforms has been demonstrated over many years now. 3D tumor models are generated mainly in non-scalable culture systems, using synthetic and biological scaffolds. Many of these models fail to reflect the complex tumor microenvironment and do not allow long-term monitoring of tumor progression. This has resulted in inconsistent data in drug testing assays during preclinical and clinical studies. METHODS: To overcome these limitations, we have developed 3D tissueoids model by using novel AXTEX-4D platform. RESULTS: Cancer 3D tissueoids demonstrated the basic features of 3D cell culture with rapid attachment, proliferation, and longevity with contiguous cytoskeleton and hypoxic core. This study also demonstrated greater drug resistance in 3D-MCF-7 tissueoids in comparison to 2D monolayer cell culture. CONCLUSION: In conclusion, 3D-tissueoids are more responsive than 2D-cultured cells in simulating important tumor characteristics, anti-apoptotic features, and their resulting drug resistance.

AXTEX-4D: A Three-Dimensional Ex Vivo Platform for Preclinical Investigations of Immunotherapy Agents.

The latest advancements in oncology are majorly focused on immuno-oncology (I-O) therapies. However, only ∼7% of drugs are being approved from the preclinical discovery phase to phase 1. The most challenging issues in I-O are the development of active and efficient drugs in an economically feasible way and in a comparatively short time for testing and validation. This mandates an urgent need for the upgradation of preclinical screening models that closely mimic the in vivo tumor microenvironment (TME). The established and most common methods for investigating the tumoricidal activity of I-O drugs are either two-dimensional systems or primary tumor cells in standard tissue culture vessels. Unfortunately, they do not mimic the TME. Consequently, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell-mediated responses in reaction to the administration of I-O drugs. Despite many advantages of multicellular spheroids, challenges (e.g., incompatibility of quantitative assays with spheroid platforms) are still involved in the tedious procedures required for the spheroid culture that is holding back the biological community from adapting the well-recognized spheroid tissue models for studying drug delivery more widely. To this end, we have demonstrated the utility of the 3D ex vivo oncology model, developed on our novel AXTEX-4D™ platform to assess therapeutic efficacies of I-O drugs by investigating immune cell proliferation, migration, infiltration, cytokine profiling, and cytotoxicity of tumor tissueoids. The platform eliminates the need for additional biomolecules such as hydrogels and instead relies on the cancer cells themselves to create their own gradients and microenvironmental factors. In effect, the more comprehensive and ex vivo-like immune-oncology model developed on AXTEX-4D platform can be utilized for high-throughput screening of immunotherapeutic drugs.

Enhanced soluble production of biologically active recombinant human p38 mitogen-activated-protein kinase (MAPK) in Escherichia coli.

The conditions were optimized for maximum soluble yield of biologically active recombinant p38alpha mitogen activated protein kinase (MAPK) vis-à-vis insoluble fraction (inclusion body formation). This study reports a rapid, economical and single step purification process for the overproduction of GST tagged p38alpha MAPK. A yield of 18 mg of highly purified and soluble protein per liter of bacterial culture within 6 h timeframe was achieved. The purified protein was found to be biologically suitable for phosphorylation by upstream kinases and was catalytically active. We further demonstrated that our in-house p38alpha MAPK is more potent (>30%) than a commercially available enzyme.