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Multidrug resistant Shigella is one of the leading causes of mortality in children and infants. Availability of vaccine could prevent the Shigella infection and reduce the mortality. Conventional approaches of vaccine development against shigellosis have not resulted in desirable vaccine. As shigellosis may be caused by multiple strains and serotypes, there is a need to develop a multivalent vaccine, capable of providing protection against multiple Shigella strains. To develop broad spectrum vaccine, we had previously derived a pool of conserved epitopes against Shigella by using multiple immunoinformatic tools. In this study, the identified conserved epitopes derived from the Outer Membrane Proteins A and C of Shigella were chemically synthesized, and the EpiMix made up of 5 epitopes coupled to a carrier protein, ovalbumin was developed and validated for its immunogenicity. The intramuscular immunization with EpiMix in Balb/c mice led to increase in EpiMix specific serum IgG, and significant increase in fecal IgA as well as in IL-4, IL-2and IFN-γ levels. Further, the EpiMix immunized mice showed protection when challenged against S. flexneri ATCC 12022 using the intraperitoneal route. Moreover, the analysis of cytokine profile and IFN-γ/IL4 ratio in post Shigella challenge immunized mice suggested the high levels of IFN-γ levels and possible dominance of Th1 response, playing pivotal role in the elimination of Shigella. Collectively, the results demonstrate the immunogenic potential and protective efficacy of the EpiMix in the murine shigellosis model. However, the detailed study and further optimisation of epitopes would substantiate the prospective use of EpiMix as a prophylactic candidate for vaccination.

During antiretroviral therapy (ART), most of the human immunodeficiency virus (HIV) reservoirs persist in the B cell follicles (BCFs) of lymphoid tissue. Thus, for HIV cure strategies, it is critical to generate cytolytic CD8+ T cells that home to BCF, reduce the reservoir burden, and maintain strong antiviral responses in the absence of ART. Here, using a chronic simian immunodeficiency virus (SIV)/rhesus macaque model, we showed that therapeutic vaccination under ART using a CD40L plus TLR7 agonist–adjuvanted DNA/modified vaccinia Ankara vaccine regimen induced robust and highly functional, SIV-specific CD4+ and CD8+ T cell responses. In addition, the vaccination induced SIV-specific CD8+ T cells in the lymph nodes (LNs) that could home to BCF. Administration of PD-1 blockade before initiation of ART and during vaccination markedly increased the frequency of granzyme B+ perforin+ CD8+ T cells in the blood and LN, enhanced their localization in germinal centers of BCF, and reduced the viral reservoir. After ART interruption, the vaccine + anti–PD-1 antibody–treated animals, compared with the vaccine alone and ART alone control animals, displayed preservation of the granzyme B+ CD8+ T cells in the T cell zone and BCF of LN, maintained high SIV antigen-recognition breadth, showed control of reemerging viremia, and improved survival. Our findings revealed that PD-1 blockade enhanced the therapeutic benefits of SIV vaccination by improving and sustaining the function and localization of vaccine-induced CD8+ T cells to BCF and decreasing viral reservoirs in lymphoid tissue. This work has potential implications for the development of curative HIV strategies.

Highlights • LacVax® OmpA elicited humoral and cellular immune responses in BALB/c mice. • Oral immunization with LacVax® OmpA elicited sIgA, a hallmark of mucosal immunity. • LacVax® OmpA immunized mice were protected against Shigella induced dysenteriae. • First report outlining protective potential of L. lactis based vaccine for Shigella. • IgG, sIgA, high levels of INF-γ and IL-10 correlated with protection from Shigella., Shigellosis is an acute invasive disease of the lower intestine, which afflicts millions of people worldwide with an estimated one million fatalities per annum. Despite of extensive research during the last two decades, a vaccine against multi-drug resistant Shigella is not yet available in the market. To provide a safe, effective and broad-spectrum vaccine against Shigella, we explored food grade bacteria Lactococcus lactis (L. lactis) for the delivery of conserved antigenic protein; Outer membrane protein A (OmpA) to the mucosal sites for effective elicitation of systemic and mucosal immunity. We have previously confirmed the immunogenic potential of recombinant L. lactis expressing OmpA (LacVax® OmpA) in BALB/c mice. In the present study, we have characterized the humoral and cellular immune profile of LacVax® OmpA and assessed its protective efficacy using a newly developed human like murine shigellosis model. The significant increase in OmpA specific serum IgG, fecal sIgA and a Th1 dominant immune response (indicated by high INF-γ/IL-4 ratio) in LacVax® OmpA immunized mice revealed successful activation of humoral and cellular immunity. The LacVax® OmpA immunized animals were also protected from human-like shigellosis when challenged with S. flexneri 2a ATCC 12022. The antigen specific serum IgG, fecal sIgA, INF-γ and IL-10 levels were found to be the significant correlates of protection. Collectively these results suggest that the LacVax® OmpA is a promising prophylactic candidate against shigellosis. However, the protective efficacy of LacVax® OmpA in the higher animals would further strengthen its future application in humans.

Immunoinformatic plays a pivotal role in vaccine design and development. While traditional methods are exclusively depended on immunological experiments, they are less effective, relatively expensive, and time-consuming. However, recent advances in the field of immunoinformatics have provided innovative tools for the rational design of vaccine candidates. This approach allows the selection of immunodominant regions from the sequence of whole genome of a pathogen. The identified immunodominant region could be used to develop potential vaccine candidates that can trigger protective immune responses in the host. At present, epitope-based vaccine is an attractive concept which has been successfully trailed to develop vaccines against a number of pathogens. In this chapter, we outline the methodology and workflow of how to deploy immunoinformatics tools in order to identify immunodominant epitopes using Shigella as a model organism. The immunodominant epitopes, derived from S. flexneri 2a using this workflow, were validated using in vivo model, indicating the robustness of the outlined workflow.

Immunoinformatic plays a pivotal role in vaccine design and development. While traditional methods are exclusively depended on immunological experiments, they are less effective, relatively expensive, and time-consuming. However, recent advances in the field of immunoinformatics have provided innovative tools for the rational design of vaccine candidates. This approach allows the selection of immunodominant regions from the sequence of whole genome of a pathogen. The identified immunodominant region could be used to develop potential vaccine candidates that can trigger protective immune responses in the host. At present, epitope-based vaccine is an attractive concept which has been successfully trailed to develop vaccines against a number of pathogens. In this chapter, we outline the methodology and workflow of how to deploy immunoinformatics tools in order to identify immunodominant epitopes using Shigella as a model organism. The immunodominant epitopes, derived from S. flexneri 2a using this workflow, were validated using in vivo model, indicating the robustness of the outlined workflow.

The non-invasive food grade Lactococcus lactis (L. lactis) represents a safe and attractive alternative to invasive pathogens for the delivery of plasmid DNA at mucosal sites. We have earlier shown the DNA delivery potential of r-L. lactis harboring DNA vaccine reporter plasmid; pPERDBY in vitro. In the present work, we examined in vivo delivery potential of food grade non-invasive r-L. lactis::pPERDBY (LacVax® DNA-I) in BALB/c mice. Moreover, using EGFP as a model antigen, we also characterized and compared the immune response elicited by LacVax® DNA-I with other conventional vaccination approaches using protein and naked DNA immunization. The presence of antigen-specific serum IgG and fecal secretory IgA (sIgA) antibodies demonstrated in vivo DNA delivery and immune elicitation potential of the developed LacVax® DNA-I. As compared with intramuscular injection, oral delivery of pPERDBY via L. lactis resulted in a significantly rapid increase in IgG and higher sIgA titers, indicating the immunogenic and immunostimulatory properties of the LacVax® DNA-I. The needle-free immunization with LacVax® DNA-I led to increased production of IL-4, an indicator of Th2 screwed response. To the best of our knowledge, this report for the first time outlines comparison of orally administered LacVax® DNA-I with other conventional vaccination approaches.

Food grade Lactococcus lactis has been widely used as an antigen and DNA delivery vehicle. We have previously reported the use of non-invasive L. lactis to deliver the newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, construction of dual recombinant L. lactis expressing internalin A of Listeria monocytogenes and harboring pPERDBY (LL InlA + pPERDBY) to enhance the efficiency of delivery of DNA by L. lactis is outlined. After confirmation and validation of LL InlA + pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around threefold increases in the number of enhanced green fluorescent protein-expressing Caco-2 cells. These findings reinforce the prospective application of invasive strain of L. lactis for delivery of DNA/RNA and antigens.

Shigellosis, a major cause of mortality and morbidity, requires development of effective intervention strategy for which animal model mimicking human pathology is essential. Among various animal models for shigellosis, mice being more convenient have been used wherein intraperitoneal and intranasal routes are preferred. With the aim to comprehend the comparative pathophysiological indicators, we have examined relatively high and low dose of Shigella flexneri administered through intraperitoneal and intranasal routes in mice. Characterization of these two models along with the resulting pathophysiology of shigellosis adds to our understanding and offers suitable models appropriate to the objectives of the study.

Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article supports the hypothesis that in the absence of invasive proteins or relevant chemical treatment, L. lactis was found efficient in delivering DNA to mammalian cells.

The non-invasive food grade Lactococcus lactis (L. lactis) represents a safe and attractive alternative to invasive pathogens for the delivery of plasmid DNA at mucosal sites. We have earlier shown the DNA delivery potential of r-L. lactis harboring DNA vaccine reporter plasmid; pPERDBY in vitro. In the present work, we examined in vivo delivery potential of food grade non-invasive r-L. lactis::pPERDBY (LacVax

The extensive demand of large scale therapeutic protein production in Chinese Hamster Ovarian cell lines increased the need of developing efficient and cost effective transfection reagent. Polyethylenimine has recently emerged as an attractive alternative to currently employed conventional transfection methods. In the present report, we attempted to develop inexpensive and easy to prepare polyethylenimine-based transfection reagent and compared its transfection efficiency with calcium phosphate, electroporation and lipoplex mediated transfection methods. Our results indicated highest transfection efficiency with in-house prepared polyplexes as marked by maximum percentage of enhanced cyan fluorescence protein positive cells. These findings contribute towards the development of economical and effective solution for high protein expression in Chinese hamster ovarian system.

Food grade Lactococcus lactis has been widely used as an antigen and DNA delivery vehicle. We have previously reported the use of non-invasive L. lactis to deliver the newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, construction of dual recombinant L. lactis expressing internalin A of Listeria monocytogenes and harboring pPERDBY (LL InlA + pPERDBY) to enhance the efficiency of delivery of DNA by L. lactis is outlined. After confirmation and validation of LL InlA + pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around threefold increases in the number of enhanced green fluorescent protein-expressing Caco-2 cells. These findings reinforce the prospective application of invasive strain of L. lactis for delivery of DNA/RNA and antigens.

Aims To evaluate the comparative immunogenic potential of food grade Lactococcus lactis (L. lactis) expressing Outer membrane protein A (OmpA) of Shigella dysenteriae type-1 (SD-1) when administered either orally or intranasally. Methods and Results OmpA of SD-1 was cloned and expressed first in Escherichia coli (E. coli) and then in L. lactis. Presence of recombinant gene was confirmed by restriction enzyme digestion and Immunoblot analysis. Using immobilized metal affinity chromatography (IMAC), OmpA was purified from recombinant E. coli BL21 (DE3) and subcutaneously administered in BALB/c mice. Detection of OmpA specific IgG antibodies by enzyme linked immunosorbent assay (ELISA) confirmed the immunogenicity of OmpA. In order to establish r-L. lactis as a mucosal delivery vehicle, it was administered orally and nasally in BALB/c mice. Serum IgG and fecal IgA were assessed through ELISA to compare the relative potential of immunization routes and immunogenic potential of r-L. lactis. Immunization via the oral route proved superior to intranasal exposure. Conclusion Recombinant L. lactis expressing OmpA of SD-1 was found to be immunogenic. Oral administration of r-L. lactis elicited higher systemic and mucosal immune response when compared to the nasal route. Significance and Impact of the Study Using food grade recombinant L. lactis has implications in the development of a prophylactic against multi-drug resistant Shigella, which can be used as a prospective vaccine candidate. Evaluating mucosal routes of immunization demonstrated that the oral route of administration elicited better immune response against OmpA of Shigella. This article is protected by copyright. All rights reserved.

ObjectivesTo investigate whether dietary calcium intake will modify the relationship between serum 25-hydroxyvitamin D3 (25OHD) with intact serum parathyroid hormone (PTH) concentrations in apparently healthy Indian adolescents.Study designCross-sectional study.Setting and participantsApparently healthy adolescents aged 10–14 years (n=181), from Gujarat, western India. Study conducted from January 2012 to March 2014.MethodsSerum 25OHD concentrations and intact serum PTH concentrations (both using chemiluminescent microparticle immunoassay) were measured. Diet was recorded through 24 h diet recall and calcium intake was computed (C-diet V.2.1). To assess relationship between 25OHD and PTH, data were dichotomised according to median calcium intakes (520 mg/day) and relationship between serum 25OHD and PTH in the two subgroups was plotted.ResultsSubjects with calcium intakes above median (>520 mg/day) had lower intact serum PTH values for given serum 25OHD concentration while those with calcium intakes below median (ConclusionsDietary calcium intake modifies the relationship between serum 25OHD concentrations and intact serum PTH concentrations. Thus, dietary calcium intake should be taken into account when assessing an individual's vitamin D status.

Potential use of Lactococcus lactis (L. lactis) as a heterologous protein expression host as well as for delivery of multiple therapeutic proteins has been investigated extensively using Nisin Inducible Controlled Expression (NICE) system. Optimum inducible expression of heterologous protein by NICE system in L. lactis depends on multiple factors. To study the unexplored role of factors affecting heterologous protein expression in L. lactis using NICE, the present study outlines the optimization of various key parameters such as inducer concentration, host's proteases and precipitating agent using Outer membrane protein A (OmpA). For efficient expression and secretion of OmpA, pSEC:OmpA vector was successfully constructed. To circumvent the troubles encountered during detection of expressed OmpA, the precipitating agent was switched from TCA to methanol. Nevertheless, detection was achieved accompanied by degraded protein products. Speculating the accountability of observed degradation at higher inducer concentration, different nisin concentrations were evaluated. Lower nisin concentrations were found desirable for optimum expression of OmpA. Consistently observed degradation was eliminated by incorporation of protease inhibitor cocktail which inhibits intracellular proteases and expression in VEL1153 (NZ9000 ΔhtrA) strain which inhibits extracellular protease leading to optimum expression of OmpA. Versatility and complexity of NICE system in L. lactis requires fine-tuning of target protein specific parameters for optimum expression.

Context: Fluorosis is controlled by the duration of fluoride exposure and calcium and Vitamin D nutrition status. Aim: To examine (a) prevalence of dental and skeletal fluorosis in adolescents from upper, middle, and lower socioeconomic strata (SES) and (b) association of fluorosis with calcium intake and Vitamin D status. Settings and Design: A cross-sectional study conducted in 10–13.9 years apparently healthy adolescents (n = 90), from different SES of Patan (Gujarat, India). Materials and Methods: Dental fluorosis was graded as mild, moderate, and severe. Radiographs of the right hand and wrist were examined and graded. Serum 25 hydroxyvitamin D3 (25OHD) and parathyroid hormone concentrations were measured. Diet was recorded (24 h recall) and calcium intake was computed (C-diet V-2.1, 2013, Xenios Technologies Pvt. Ltd). Statistical Analysis: Generalized linear model was used to analyze relationships between fluorosis, SES, serum 25OHD concentration, and calcium intake. Results: Fluorosis was predominant in lower SES (17% had both dental and radiological features whereas 73% had dental fluorosis); no skeletal deformities were observed. Mean 25OHD concentrations and dietary calcium were 26.3 ± 4.9, 23.4 ± 4.7, and 18.6 ± 4 ng/ml and 441.2 ± 227.6, 484.3 ± 160.9, and 749.2 ± 245.4 mg/day, respectively, for lower, middle, and upper SES (P < 0.05). Fluorosis and SES showed a significant association (exponential β = 2.5, P = 0.01) as compared to upper SES, middle SES adolescents were at 1.3 times while lower SES adolescents were at 2.5 times higher risk. Serum 25OHD concentrations (P = 0.937) and dietary calcium intake (P = 0.825) did not show a significant association with fluorosis. Conclusion: Fluorosis was more common in lower SES adolescents, probably due to the lack of access to bottled water. Relatively adequate calcium intake and serum 25OHD concentrations may have increased the efficiency of dietary calcium absorption, thus preventing severe fluorosis.