In-silico development of multi-epitope subunit vaccine against lymphatic filariasis.

The World Health Organization in 2022 reported that more than 863 million people in 50 countries are at risk of developing lymphatic filariasis (LF), a disease caused by parasitic infection. Immune responses to parasites suggest that the development of a prophylactic vaccine against LF is possible. Using a reverse vaccinology approach, the current study identified Trehalose-6-phosphatase (TPP) as a potential vaccine candidate among 15 reported vaccine antigens for B. malayi. High-ranking B and T-cell epitopes in the Trehalose-6-phosphatase (TPP) were shortlisted using online servers for subsequent analysis. We selected these peptides to construct a vaccine model using I-TASSER and GalaxyRefine server. The vaccine construct showed favorable physicochemical properties, high antigenicity, no allergenicity, no toxicity, and high stability. Structural validation using the Ramachandran plot showed that 98% of the residues were in favorable or mostly allowed regions. Molecular docking and simulation showed a strong binding affinity and stability of the subunit vaccine with toll-like receptor 4 (TLR4). Furthermore, the subunit vaccine showed a strong IgG/IgM response, with the disappearance of the antigen. We propose that our vaccine construct should be further evaluated using cellular and animal models to develop a vaccine that is safe and effective against LF.Communicated by Ramaswamy H. Sarma.

Current Status of ß-Thalassemic Burden in India.

Thalassemia is a major public health concern in India. The thalassemic burden in India is high, with an estimated 100,000 patients diagnosed with ß-thalassemia syndrome. However, the exact number is unknown because of the absence of National Registries for patients. India alone contributes to approximately 25% of the global ß-thalassemia burden. A possible option to control this burden is to endorse education and awareness programs, compulsory prenatal screening, and develop suitable facilities for genetic counseling, and availability of cost-effective diagnostic tests in India, especially in rural areas. In addition to the various clinical complications associated with thalassemia, lifelong intervention creates mental and physical trauma in patients and their relatives. Government and nongovernment organizations have initiated screening programs to prevent thalassemia. However, prenatal screening is not mandatory, and the reachability of screening programs in rural areas is yet to begin. This review article will discuss the progress in thalassemia research in India, including its prevalence, spectrum of ß-thalassemia mutations, preventive and therapeutic measures, and awareness programs. More importantly, we will discuss the need and roadmap to strengthen prevention programs in India.

Genomic Index of Sensitivity to Chemotherapy for Triple Negative Breast Cancer.

OBJECTIVE: Patients with triple-negative breast cancer (TNBC) frequently develop resistance to chemotherapy. Studies have shown that microRNAs (miRNAs) are often aberrantly expressed in TNBC and are associated with drug resistance. However, a prognostic strategy that correlates miRNAs with chemotherapy resistance remains largely unknown. METHODS: To identify breast cancer chemoresistance-associated miRNAs, the miRNA microarray dataset GSE71142 was downloaded from the Gene Expression Omnibus database. Differentially expressed miRNAs (DE-miRNAs) in chemoresistant groups were identified using the LIMMA package in R. Potential target genes were predicted using the miRTarBase 9. Functional and pathway enrichment analyses was done using WebGestalt. A protein-protein interaction network was visualized using Cytoscape software. The top six hub genes regulated by DE-miRNAs were identified using the random forest model. The chemotherapy resistance index (CRI) in TNBC was defined as sum of the median expression levels of the top six hub genes. The association of CRI with distant relapse risk was evaluated using point-biserial correlation coefficient in the validation cohorts of patients with TNBC. The correlation between CRI and cumulative hazard rate was estimated using the Cox model, and the predicted rate of distant relapse was obtained from the Breslow-type estimator of the survival function. All statistical computations were performed using Origin2019b. RESULTS: A total of 12 DE-miRNAs were screened, including six upregulated and six downregulated miRNAs in chemoresistant breast cancer tissues compared with chemosensitive tissues. Based on fold changes, miR-214-3p, miR-4758-3p, miR-200c-3p, miR-4254, miR-140-3p, and miR-24-3p were the top six most upregulated miRNAs, whereas miR-142-5p, miR-146-5p, miR-1268b, miR-1275, miR-4447, and miR-4472 were the top six most downregulated miRNAs. The top three hub genes for upregulated miRNAs were RAC1, MYC, and CCND1 and for downregulated miRNAs were IL-6, SOCS1, and PDGFRA. CRI was significantly associated with the risk of distant relapse. CONCLUSION: CRI predicted survival benefits with reduced hazard rate.

Potential status of A1 and A2 variants of bovine beta-casein gene in milk samples of Indian cattle breeds.

This study aimed to determine the polymorphism in 7th exon of beta-casein gene (CSN2) gene in seven domestic (Kosali, Tharparkar, Gangatiri, Sahiwal, Gir, Khariar, Motu) and two exotic cattle breeds (Jersey and Holstein-Friesian). Genomic DNA was extracted from 1000 milk samples, and the C > A polymorphism in CSN2 was determined using the tetra-primer amplification refractory mutation system-polymerase chain reaction method. In all Indigenous cattle breeds, the mean frequency of A1A2 and A2A2 genotypes was 0.19 and 0.80, respectively. The A1A1 genotype was absent in all seven domestic cattle breeds. The frequency of the A2A2 genotype was highest in the Gir breed (0.93). However, the Sahiwal, Tharparkar, and Motu breeds also had a higher frequency of A2A2 genotype compared to other breeds. In contrast, Gangatiri breed of India showed lowest frequency of A2A2 genotype. The mean A1 and A2 allele frequency was 0.09 and 0.91, respectively. In exotic breeds, the mean frequencies of the A1A1, A1A2, and A2A2 genotypes were 0.42, 0.55, and 0.03, respectively. Similarly, the mean A1 and A2 allele frequency was 0.69 and 0.31, respectively. This study suggests the high potential of Gir, Sahiwal, Tharparkar, and Motu cattle for A2 milk production since they carry a favorable A2 genotype.

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.

Establishment of a three­dimensional triculture model on the novel AXTEX­4D™ platform.

Cancer can be fatal if it is not treated in a timely manner; therefore, there is a high demand for more specific oncology drugs. Unfortunately, drugs showing positive responses on a two­dimensional (2D) culture platform do not often show the same effect in clinical trials. Therefore, three­dimensional (3D) culture platforms are garnering attention since they more closely mimic the tumor microenvironment (TME). The TME stimulates metastasis and drug resistance, and serves an essential role in tumor formation. An accurate understanding of tumor­stroma interactions is undoubtedly required to improve the response of patients to therapeutic strategies, and cancer therapeutic strategies that do not account for the stroma are considered inadequate. It should be noted that 3D monoculture systems do not completely mimic the TME since other cells in the 3D culture are missing, such as fibroblast or endothelial cells, which are essential components of the stroma; therefore, it is essential to develop advanced 3D culture systems. The present study aimed to develop a versatile triculture model that mimics the native TME; therefore, it could aid in high­throughput screening of chemotherapeutic drugs against cancer by evaluating their effects on tumor progression and cell cytotoxicity. The present study demonstrated the use of the AXTEX­4D™ platform in developing triculture tissueoids composed of MCF­7, human umbilical vein endothelial cells and MRC5 cells, and compared it with a 3D monoculture model (MCF­7) and a 2D culture model. The triculture model was validated for proliferation, ECM markers and T­cell infiltration by confocal microscopy. Alamar Blue assay demonstrated that triculture tissueoids exhibited higher drug resistance than the other two models, thus demonstrating their use in the screening of oncology drugs.

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.

PRAK-03202: A triple antigen virus-like particle vaccine candidate against SARS CoV-2.

The rapid development of safe and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is a necessary response to coronavirus outbreak. Here, we developed PRAK-03202, the world's first triple antigen virus-like particle vaccine candidate, by cloning and transforming SARS-CoV-2 gene segments into a highly characterized S. cerevisiae-based D-Crypt™ platform, which induced SARS CoV-2 specific neutralizing antibodies in BALB/c mice. Immunization using three different doses of PRAK-03202 induced an antigen-specific (spike, envelope, and membrane proteins) humoral response and neutralizing potential. Peripheral blood mononuclear cells from convalescent patients showed lymphocyte proliferation and elevated interferon levels suggestive of epitope conservation and induction of T helper 1-biased cellular immune response when exposed to PRAK-03202. These data support further clinical development and testing of PRAK-03202 for use in humans.

Corrigendum to "PRAK-03202: A triple antigen virus-like particle vaccine candidate against SARS CoV-2"[Heliyon 7 (10) (October 2021) e08124].

[This corrects the article DOI: 10.1016/j.heliyon.2021.e08124.].

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.

Single nucleotide polymorphisms in TNF-alpha, TNFR2 gene and TNF-alpha production in Asian Indians.

Polymorphisms in the TNF-alpha and TNF receptor type 2 (TNFR2) genes in Asian Indians are not well understood. We investigated single nucleotide polymorphisms in the TNF-alpha 5'-flanking promoter/enhancer region and in exons 6, 9 and 10 of TNFR2 gene by PCR-restriction length polymorphism (PCR-RFLP) and PCR-sequence specific primer (SSP) techniques, respectively. The results showed single bi-allelic polymorphism in TNF-alpha (-308G > A) and in TNFR2 exons 6 (676 T > G) and 9 (1176 G > A). Additionally, three bi-allelic polymorphisms (1663 G > A, 1668G > T and 1690C > T) were observed in the exon 10 of TNFR2 gene. The distribution of polymorphic alleles distinctly differed in Aryan and Dravidian Indian communities. The TNF-alpha and TNFR2 genotype and allele frequencies of Asian Indians stand closer to other Asian populations but distant from Caucasians. Furthermore, TNF-alpha -308 GA genotype was associated with significantly higher production of TNF-alpha as compared to GG genotype. These polymorphisms may be related to variation in TNF-alpha and TNFR2 expression during immune response to various stimuli in Asian Indians.

Association of TNF-alpha and TNFR1 promoters and 3' UTR region of TNFR2 gene polymorphisms with genetic susceptibility to tobacco-related oral carcinoma in Asian Indians.

Tobacco-related oral squamous cell carcinoma is a common malignancy in Asian people. It accounts for almost 40% of cancers among Indian men and 3% in the Western world. Smokeless tobacco has been shown to induce tumor necrosis factor-alpha (TNF-alpha), which, along with its receptors, is over-expressed in people with oral carcinoma. Single nucleotide polymorphisms (SNPs) in TNF-alpha and TNF receptor genes may affect their expression and may be a potential determinant of susceptibility to tobacco-related oral carcinomas. We assessed SNPs in TNF-alpha(-308, -238) and TNF receptor 1 (TNFR1; -609) promoters by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and at four sites of TNF receptor 2 gene (TNFR2; exon 9 site 1176; exon 10 sites 1663, 1668 and 1690) by PCR-sequence-specific primers (PCR-SSP) techniques, respectively, in 94 patients and 130 healthy controls. TNF-alpha-308 G allele was significantly lower (Pc=0.004; OR=3.85), whereas A allele was significantly higher (Pc=0.004; OR=0.25) in patients compared with controls. No significant change was observed at -238 promoter site between the two groups. In the case of TNF receptors, both TNFR1 -609 TT (Pc=0.006; OR=15.3) and TNFR2 1690 CT (Pc=0.018; OR=5.6) genotypes were significantly lower in patients compared with controls. It seems that TNF-alpha-308 G/A may be related to susceptibility, whereas -609 TT TNFR1 and 1690 C/T TNFR2 SNPs may be protective to tobacco-related oral squamous cell carcinoma. These SNPs may be useful as a marker for high-risk groups among Asian Indians.