Potential of a novel flagellin epitope as a broad-spectrum vaccine candidate against enteric fever.

Relentless emergence of antibiotic resistant Salmonella strains, coupled with the drawbacks associated with currently available vaccines against enteric fever, warrants an urgent need to look for new vaccine candidates. Out of the multiple virulence factors harbored by Salmonella, flagella are regarded as one of the most important targets of innate as well as adaptive immune response. Individual Salmonella serotypes alternate between expression of two different antigenic forms encoded by fliC and fljB genes, respectively thereby employing this as a strategy to escape the host immune response. In the present study, using various immunoinformatic approaches, a flagellin epitope, present in both antigenic forms of typhoidal Salmonellae has been targeted. Following B-cell epitope and B-cell derived T-cell epitope prediction and interaction studies with major histocompatibility complexes using molecular docking, a peptide epitope was selected. Further, it was screened for its presence in majority of typhoidal serovars along with other useful attributes, in silico. Thereafter, safety studies were performed with the synthesized peptide. Subsequently, immunization studies were carried out using S. Typhi as well as S. Paratyphi A induced murine peritonitis model. Active immunization with peptide-BSA conjugate resulted in 75% and 80% mice survival following lethal challenge with S. Typhi and S. Paratyphi A respectively, along with a significant IgG antibody titer, thereby highlighting its immunogenic potential. Reduced bacterial burden in vital organs along with improved histoarchitecture and cytokine levels further substantiated the protective efficacy of the proposed candidate. Passive immunization studies with the candidate verified the protective efficacy of the generated antibodies against lethal challenge of bacteria in mice. Given the endemic nature of enteric fever and the antigenic variability observed in Salmonella serotypes, present study highlights the importance of using a vaccine candidate, which, along with generating a strong immune response, also exhibits a broad coverage against both, S. Typhi as well as S. Paratyphi A strains.

Diet, Gut Microbiota and COVID-19.

Worldwide, millions of individuals have been affected by the prevailing SARS-CoV-2. Therefore, a robust immune system remains indispensable, as an immunocompromised host status has proven to be fatal. In the absence of any specific antiviral drug/vaccine, COVID-19 related drug repurposing along with various other non-pharmacological measures coupled with lockdown have been employed to combat this infection. In this context, a plant based rich fiber diet, which happens to be consumed by a majority of the Indian population, appears to be advantageous, as it replenishes the host gut microbiota with beneficial microbes thereby leading to a symbiotic association conferring various health benefits to the host including enhanced immunity. Further, implementation of the lockdown which has proven to be a good non-pharmacological measure, seems to have resulted in consumption of home cooked healthy diet, thereby enriching the beneficial microflora in the gut, which might have resulted in better prognosis of COVID-19 patients in India in comparison to that observed in the western countries.

Peptides as adjuvants for ampicillin and oxacillin against methicillin-resistant Staphylococcus aureus (MRSA).

Fast emerging antibiotic resistance in pathogens requires special attention for strengthening the reservoir of antimicrobial compounds. In view of this, several peptides with known antimicrobial activities have been reported to enhance the efficacy of antibiotics against multidrug resistant (MDR) pathogens. In the present study, potential of peptides having distinct mechanism of action, if any, was evaluated to improve the efficacy of conventional antibiotics against methicillin-resistant S. aureus (MRSA). After primary screening of six peptides, two peptides namely T3 and T4 showing very high minimum inhibitory concentrations (MICs) were selected to assess their role in altering the MICs of antibiotics to which the pathogen was resistant. In the presence of the peptides, the MICs of the antibiotics were found to be reduced as per the fractional inhibitory concentration indices (FICI) and time kill assay. These observations prompted us to look for their mechanism of action. The effect of peptides on the morphology of pathogen by field emission scanning electron microscopy (FE-SEM) revealed no damage to the cells at the sub-inhibitory concentrations of the peptide which correlated well with the higher MIC of the peptide, indicating no direct impact on the pathogen. However, dielectric spectroscopy, confocal microscopy and flow cytometry confirmed the interaction and localization of peptides with the bacterial membrane. The peptides were also found to inhibit efflux of ethidium bromide which is the substrate for many proteins involved in efflux system. Therefore, it is speculated that the peptides after interacting with the membrane of the pathogen might have resulted in the inhibition of the efflux of antibiotics thereby reducing their effective concentrations. The study thus suggests that peptides with no antimicrobial activity of their own, can also enhance the efficacy of the antibiotics by interacting with the pathogen thereby, acting as adjuvants for the antibiotics.

Dual immunization with CdtB protein and flagellin epitope offers augmented protection against enteric fever in mice.

AIMS: Present study has explored the protective response of dual immunization using two different antigenic entities (i.e. flagellin epitope and cytolethal distending toxin subunit B (CdtB) protein) against lethal challenge of typhoidal serovars in a murine model. MAIN METHODS: In-vitro immunogenicity of flagellin epitope-BSA conjugate and CdtB protein was confirmed using Indirect ELISA of typhoid positive patients' sera. Further, both entities were administered intraperitoneally in mice individually or in combination, followed by lethal challenge of typhoidal Salmonellae. Various parameters were analysed such as bacterial burden, mice survival, histopathological analysis, cytokine analysis and immunophenotyping. Serum samples obtained from the immunized mice were used for passive immunization studies, wherein mice survival and mechanism of action of the generated antibodies was studied. KEY FINDINGS: Active immunization studies using the combination of both entities demonstrated improved mice survival after lethal challenge with typhoidal Salmonellae, reduced bacterial burden in organs, expression of immunophenotypic markers in splenocytes and restored tissue histoarchitecture. When used in combination, the effective doses of both the candidates reduced which may be attributed to multiprong approach used by the immune system to recognize Salmonella. Passive immunization studies further determined the protective efficacy of generated antibodies by different mechanisms such as complement mediated bactericidal action, swarming inhibition and increased phagocytic uptake. SIGNIFICANCE: Present study is the first phase of the proof-of-concept which may prove to be beneficial in developing an effective bi-functional vaccine candidate to render protection against both Vi-positive as well as Vi-negative Salmonella strains.

Reverse engineering approach: a step towards a new era of vaccinology with special reference to Salmonella.

INTRODUCTION: Salmonella is responsible for causing enteric fever, septicemia, and gastroenteritis in humans. Due to high disease burden and emergence of multi- and extensively drug-resistant Salmonella strains, it is becoming difficult to treat the infection with existing battery of antibiotics as we are not able to discover newer antibiotics at the same pace at which the pathogens are acquiring resistance. Though vaccines against Salmonella are available commercially, they have limited efficacy. Advancements in genome sequencing technologies and immunoinformatics approaches have solved the problem significantly by giving rise to a new era of vaccine designing, i.e. 'Reverse engineering.' Reverse engineering/vaccinology has expedited the vaccine identification process. Using this approach, multiple potential proteins/epitopes can be identified and constructed as a single entity to tackle enteric fever. AREAS COVERED: This review provides details of reverse engineering approach and discusses various protein and epitope-based vaccine candidates identified using this approach against typhoidal Salmonella. EXPERT OPINION: Reverse engineering approach holds great promise for developing strategies to tackle the pathogen(s) by overcoming the limitations posed by existing vaccines. Progressive advancements in the arena of reverse vaccinology, structural biology, and systems biology combined with an improved understanding of host-pathogen interactions are essential components to design new-generation vaccines.

Cocktail of carbohydrases from Aspergillus niger: an economical and eco-friendly option for biofilm clearance from biopolymer surfaces.

Biofilm formation on both biotic and abiotic surfaces accounts for a major factor in spread of antimicrobial resistance. Due to their ubiquitous nature, biofilms are of great concern for environment as well as human health. In the present study, an integrated process for the co-production of a cocktail of carbohydrases from a natural variant of Aspergillus niger was designed. The enzyme cocktail was found to have a noteworthy potential to eradicate/disperse the biofilms of selected pathogens. For application of enzymes as an antibiofilm agent, the enzyme productivities were enhanced by statistical modelling using response surface methodology (RSM). The antibiofilm potential of the enzyme cocktail was studied in terms of (i) in vitro cell dispersal assay (ii) release of reducing sugars from the biofilm polysaccharides (iii) the effect of enzyme treatment on biofilm cells and architecture by confocal laser scanning microscopy (CLSM). Potential of the enzyme cocktail to disrupt/disperse the biofilm of selected pathogens from biopolymer surfaces was also assessed by field emission scanning electron microscopy (FESEM) analysis. Further, their usage in conjunction with antibiotics was assessed and it was inferred from the results that the use of enzyme cocktail augmented the efficacy of the antibiotics. The study thus provides promising insights into the prospect of using multiple carbohydrases for management of heterogeneous biofilms formed in natural and clinical settings.

Aptamer functionalized MoS2-rGO nanocomposite based biosensor for the detection of Vi antigen.

We report a novel aptamer functionalized MoS2-rGO based electrochemical method for Vi polysaccharide antigen mediated detection of enteric fever. Herein, highly selective anti-Vi aptamers were screened from a pool of oligonucleotides using a microtitre based SELEX approach and characterized for its specificity and stability. The MoS2-rGO nanocomposite was synthesized using a liquid assisted exfoliation by taking optimum ratio of MoS2 and rGO. The nanocomposite presented synergistic effect owing to easy biomolecular functionalization and enhanced conductivity. The screened anti-Vi aptamers were embedded on the MoS2-rGO nanocomposite via thiol linkage to give a stable biointerface. The developed aptasensor was characterized and further evaluated for its performance with different concentrations of Vi antigen using ferrocene labeled boronic acid as an electroactive probe. The aptasensor responded linearly in the range between 0.1 ng mL-1 to 1000 ng mL-1with a detection limit of 100 pg mL-1, and did not show any cross-reactivity with other bacterial polysaccharides indicating high specificity. The applicability of the developed aptasensor was further validated in urine and sera specimens spiked with Vi antigen.