Abstract Brucella is a typical facultative intracellular bacterium that can cause zoonotic infections. For Brucella, it is difficult to eliminate with current medical treatment. Therefore, a multi-epitope vaccine (MEV) should be designed to prevent Brucella infection. For this purpose, we applied the reverse vaccinology approach from Omp10, Omp25, Omp31 and BtpB. Finally, we obtained 13 cytotoxic T lymphocyte (CTL) epitopes, 17 helper T lymphocyte (HTL) epitopes, 9 linear B cell epitopes, and 2 conformational B cell epitopes for further study. To keep the protein folded normally, we linked AAY, GPGPG, and KK to CTL epitopes, HTL epitopes, and B cell epitopes, respectively. The N-terminal of the vaccine peptide is supplemented with appropriate adjuvants to enhance immunogenicity. To evaluate its immunogenicity, stability, safety, and feasibility, a final MEV containing 806 amino acids was constructed by linking linkers and adjuvants. In addition, molecular docking and molecular dynamics simulations were performed to verify the affinity and stability of the MEV-TLR4. Then, codon adaptation and in silico cloning studies were carried out to identify the possible codons for expressing the MEV. In animal experiments, the results demonstrated that the MEV had high immunogenicity. Collectively, this study provided a theoretical basis for the development of a Brucella vaccine. 

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Design of a multi-epitope vaccine candidate against Brucella melitensis

COVID-19 caused, as of September, 1rst, 2022, 599,825,400 confirmed cases, including 6,469,458 deaths. Currently used vaccines reduced severity and mortality but not virus transmission or reinfection by different strains. They are based on the Spike protein of the Wuhan reference virus, which although highly antigenic suffered many mutations in SARS-CoV-2 variants, escaping vaccine-generated immune responses. Multiepitope vaccines based on 100% conserved epitopes of multiple proteins of all SARS-CoV-2 variants, rather than a single highly mutating antigen, could offer more long-lasting protection. In this study, a multiepitope multivariant vaccine was designed using immunoinformatics and in silico approaches. It is composed of highly promiscuous and strong HLA binding CD4+ and CD8+ T cell epitopes of the S, M, N, E, ORF1ab, ORF 6 and ORF8 proteins. Based on the analysis of one genome per WHO clade, the epitopes were 100% conserved among the Wuhan-Hu1, Alpha, Beta, Gamma, Delta, Omicron, Mµ, Zeta, Lambda and R1 variants. An extended epitope-conservancy analysis performed using GISAID metadata of 3,630,666 SARS-CoV-2 genomes of these variants and the additional genomes of the Epsilon, Lota, Theta, Eta, Kappa and GH490 R clades, confirmed the high conservancy of the epitopes. All but one of the CD4 peptides showed a level of conservation greater than 97% among all genomes. All but one of the CD8 epitopes showed a level of conservation greater than 96% among all genomes, with the vast majority greater than 99%. A multiepitope and multivariant recombinant vaccine was designed and it was stable, mildly hydrophobic and non-toxic. The vaccine has good molecular docking with TLR4 and promoted, without adjuvant, strong B and Th1 memory immune responses and secretion of high levels of IL-2, IFN-γ, lower levels of IL-12, TGF-β and IL-10, and no IL-6. Experimental in vivo studies should validate the vaccine's further use as preventive tool with cross-protective properties. 

/pdf/a-novel-vaccine-based-on-sars-cov-2-cd4-and-cd8-t-cell-2a18ae6q.pdf

A novel vaccine based on SARS-CoV-2 CD4+ and CD8+ T cell conserved epitopes from variants Alpha to Omicron

The present study focused on phytofabrication of selenium nanoparticles (SeNPs) from Carica papaya extract and exploration of their multi-biofunctional features. Total phenolics and flavonoids of C. papaya fruit extract were determined as 23.30 ± 1.88 mg gallic acid equivalents and 19.21 ± 0.44 mg quercetin equivalents per gram, respectively, which suggested that C. papaya fruit extract could be a competitive reducing and stabilizing agent during phytofabrication of nanoparticles. UV–Vis and FTIR spectroscopy showed the formation of SeNPs from sodium selenite, which could be related to the reducing and stabilizing activities of C. papaya fruit extract. The SeNPs were found to be stable with a Zeta potential of −32 mV. The average hydrodynamic size of SeNPs was found as 159 nm by dynamic light scattering. The SeNPs showed a broader XRD pattern with no sharp Bragg’s peaks and found to be amorphous. SEM showed that SeNPs were spherical in shape and EDX pattern showed that SeNPs were made up of Se (71.81%), C (11.41%), and O (14.88%). The HR-TEM picture showed that SeNPs were spherical in morphology and have a size range of 101–137 nm. The SeNPs exhibited potent antioxidant activity and their EC50 values (effective concentration required to inhibit 50% of radicals) were 45.65 ± 2.01 and 43.06 ± 3.80 μg/ml in DPPH and ABTS assays, respectively. The antimicrobial action of SeNPs was found as a broad spectrum and suppressed microbial pathogens in ascending order: fungi > Gram-positive bacteria > Gram-negative bacteria. The SeNPs have been demonstrated to reduce the growth and ochratoxin A (OTA) of mycotoxigenic Aspergillus ochraceus and Penicillium verrucosum at 40 μg/ml in broth culture, which is noteworthy. The SeNPs reduced cancer cell proliferation (RAW 264.7, Caco-2, MCF-7, and IMR-32) more preferentially than normal cells (Vero), found to be highly biocompatible. Lower doses of SeNPs (up to 50 μg/ml) were shown to be less toxic and did not cause death in Danio rerio (zebrafish) embryos, implying that lower doses of SeNPs could be beneficial for biological purposes. The present study concluded that phytofabricated SeNPs have multiple biofunctional properties, including antioxidant, antimicrobial, antimycotoxin, and anticancer activities, as well as high biocompatibility.

/pdf/multi-biofunctional-properties-of-phytofabricated-selenium-3jou7izn.pdf

Multi-Biofunctional Properties of Phytofabricated Selenium Nanoparticles From Carica papaya Fruit Extract: Antioxidant, Antimicrobial, Antimycotoxin, Anticancer, and Biocompatibility

A subtractive proteomics and immunoinformatics approach towards designing a potential multi-epitope vaccine against pathogenic Listeriamonocytogenes.

Staphylococcus aureus is a serious pathogen unleashing its virulence through several classes of exotoxins such as hemolysins and enterotoxins. In this study, we designed a novel multi-antigen subunit vaccine which can induce innate, humoral and cellular immune responses. Alpha hemolysin, enterotoxins A and B were selected as protective antigens for combining into a triple antigen chimeric protein (HAB). Immunoinformatics analysis predicted HAB protein as a suitable vaccine candidate for inducing both humoral and cellular immune responses. Tertiary structure of the HAB protein was predicted and validated through computational approaches. Docking studies were performed between the HAB protein and mice TLR2 receptor. Furthermore, we constructed and generated recombinant HAB (r-HAB) protein in E. coli and studied its toxicity, immunogenicity and protective efficacy in a mouse model. Triple antigen chimeric protein (r-HAB) was found to be highly immunogenic in mouse as the anti-r-HAB hyperimmune serum was strongly reactive to all three native exotoxins on Western blot. In vitro toxin neutralization assay using anti-r-HAB antibodies demonstrated > 75% neutralization of toxins on RAW 264.7 cell line. Active immunization with r-HAB toxoid gave ~ 83% protection against 2 × lethal dosage of secreted exotoxins. The protection was mediated by induction of strong antibody responses that neutralized the toxins. Passive immunization with anti-r-HAB antibodies gave ~ 50% protection from lethal challenge. In conclusion, in vitro and in vivo testing of r-HAB found the molecule to be nontoxic, highly immunogenic and induced excellent protection towards native toxins in actively immunized and partial protection to passively immunized mice groups. • HAB protein was computationally designed to induce humoral and cellular responses.
 • r-HAB protein was found to be nontoxic, immunogenic and protective in mouse model.
 • r-HAB conferred protection against lethal challenge in active and passive immunization.

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Immunoinformatics analysis and evaluation of recombinant chimeric triple antigen toxoid (r-HAB) against Staphylococcus aureus toxaemia in mouse model.

https://link.springer.com/content/pdf/10.1186/s43141-021-00160-z.pdf

An immunoinformatics approach for the design of a multi-epitope vaccine targeting super antigen TSST-1 of Staphylococcus aureus.

Lungs and the other regions of respiratory tract are not sterile. Respiratory system consists of separate and balanced microbial communities called respiratory microbiome. Respiratory microbiome is the set of diverse microbial communities residing on the mucosal surface of respiratory tract. The presence of microbiota in respiratory tract is identified by 16S rRNA amplicon sequencing and the diversity of microbes in the respiratory tract was clearly understood. Microbiome equilibrium is maintained in healthy state of an individual. The composition of microbiome gets altered during disease. During health conditions the shape of microbiome is maintained by three detrimental factors: microbial immigration, emigration, and relative reproduction of microbes. Sometimes balance among these three factors is disturbed and the constancy of microbiota is affected. This condition is called as respiratory dysbiosis. There are certain factors such as smoking, antibiotics, chronic respiratory disorders (CRDs), and lung transplantation that contribute to imbalance and dynamic change of microbiome in respiratory tract. In this chapter, we discussed briefly the changes in microbial communities in response to these factors.

Dynamics of Respiratory Microbiome Profiles Contributes to Imbalance and Lung Dysbiosis in the Respiratory Tract

Most of the commercial ruminant feeds comprise Corn meal, Groundnut cake, Maize etc., which contain anti-nutritional factors called Phytates. They have high binding affinity to cationic minerals. This results in decrease in activity of the cattle by making minerals unavailable for absorption in the intestine. Many of the feed industries add Phytase enzyme that degrades the Phytic acid. Addition of Phytase enzyme is very complex method and increases the cost of the feed. If Phytase is not added, Phytic acid enters the ecosystem through the dung and due to microbial degradation, the Phytic acid gets converted into Phosphates. Phosphate abundance in water bodies leads to Eutrophication, which is a serious ecological issue. And also, in many parts of the country, post-harvest Paddy straw is burnt unused. This intentional stubble burning causes air pollution. We combined paddy straw and Azolla along with jaggery to prepare animal feed supplement. The feed pellets were evaluated for their nutritional composition and efficacy. The efficacy of the developed feed was evaluated by feeding it to milch cows and compared with regular feed for a period of 15 days. The Azolla-Hay feed improved the milk yield and also quality of the milk in comparison to the control group. This study revealed that Azolla in combination with paddy straw powder can be low-cost feed alternative especially during the lean period where the availability of feed is scarce.

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Harish Babu Kolla

Papers

An immunoinformatics approach for the design of a multi-epitope vaccine targeting super antigen TSST-1 of Staphylococcus aureus.

Immunoinformatics analysis and evaluation of recombinant chimeric triple antigen toxoid (r-HAB) against Staphylococcus aureus toxaemia in mouse model.

Dynamics of Respiratory Microbiome Profiles Contributes to Imbalance and Lung Dysbiosis in the Respiratory Tract

Nutri rich cattle feed (NuCa feed) supplement with improved fiber content free of anti-nutritional factor Phytic acid

Optimization and evaluation of a multiplex PCR assay for detection of Staphylococcus aureus and its major virulence genes for assessing food safety