In vivo delivery of pPERDBY to BALB/c mice by LacVax® DNA-I and comparison of elicited immune response with conventional immunization methods.

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.

Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY.

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.

Factors Affecting Inducible Expression of Outer Membrane Protein A (OmpA) of Shigella dysenteriae Type-1 in Lactococcus lactis Using Nisin Inducible Controlled Expression (NICE).

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.

The ß2-adrenergic receptor (ADRB2) entrains circadian gene oscillation and diurnal responses to virus infection in CD8 + T cells.

Adaptive immune cells are regulated by circadian rhythms (CR) under both steady state conditions and during responses to infection. Cytolytic CD8 + T cells display variable responses to infection depending upon the time of day of exposure. However, the neuronal signals that entrain these cyclic behaviors remain unknown. Immune cells express a variety of neurotransmitter receptors including nicotinic, glucocorticoid, and adrenergic receptors. Here, we demonstrate that the ß2-adrenergic receptor (ADRB2) regulates the periodic oscillation of select core clock genes, such as Per2 and Bmal1 , and selective loss of the Adrb2 gene dramatically perturbs the normal diurnal oscillation of clock gene expression in CD8 + T cells. Consequently, their circadian-regulated anti-viral response is dysregulated, and the diurnal development of CD8 + T cells into variegated populations of cytolytic T cell (CTL) effectors is dramatically altered in the absence of ADRB2 signaling. Thus, the Adrb2 directly entrains core clock gene oscillation and regulates CR-dependent T cell responses to virus infection as a function of time-of-day of pathogen exposure. One Sentence Summary: The ß2-adrenergic receptor regulates circadian gene oscillation and downstream daily timing of cytolytic T cell responses to virus infection.

Impact of norepinephrine on immunity and oxidative metabolism in sepsis.

Sepsis is a major health problem in the United States (US), constituting a leading contributor to mortality among critically ill patients. Despite advances in treatment the underlying pathophysiology of sepsis remains elusive. Reactive oxygen species (ROS) have a significant role in antimicrobial host defense and inflammation and its dysregulation leads to maladaptive responses because of excessive inflammation. There is growing evidence for crosstalk between the central nervous system and the immune system in response to infection. The hypothalamic-pituitary and adrenal axis and the sympathetic nervous system are the two major pathways that mediate this interaction. Epinephrine (Epi) and norepinephrine (NE), respectively are the effectors of these interactions. Upon stimulation, NE is released from sympathetic nerve terminals locally within lymphoid organs and activate adrenoreceptors expressed on immune cells. Similarly, epinephrine secreted from the adrenal gland which is released systemically also exerts influence on immune cells. However, understanding the specific impact of neuroimmunity is still in its infancy. In this review, we focus on the sympathetic nervous system, specifically the role the neurotransmitter norepinephrine has on immune cells. Norepinephrine has been shown to modulate immune cell responses leading to increased anti-inflammatory and blunting of pro-inflammatory effects. Furthermore, there is evidence to suggest that norepinephrine is involved in regulating oxidative metabolism in immune cells. This review attempts to summarize the known effects of norepinephrine on immune cell response and oxidative metabolism in response to infection.

EpiMix Based Novel Vaccine Candidate for Shigella: Evidence of Prophylactic Immunity in Balb/c Mice.

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.

Adrenergic regulation of immune cell function and inflammation.

The sympathetic nervous system integrates the functions of multiple organ systems by regulating their autonomic physiological activities. The immune system is regulated both locally and systemically by the neurotransmitters epinephrine and norepinephrine secreted by the adrenal gland and local sympathetic neurons. Immune cells respond by activation of adrenergic receptors, primarily the ß2-adrenergic receptor, which signal through heterotrimeric G-proteins. Depending upon the cell type, adrenergic signaling regulates a variety of functions in immune cells ranging from cellular migration to cytokine secretion. Furthermore, due to the diurnal oscillation of systemic norepinephrine levels, various immune functions follow a circadian rhythmic pattern. This review will highlight recent advances in our understanding of how the sympathetic nervous system regulates both innate and adaptive immune functions and how this regulation is linked to circadian rhythms.

Oral immunization with LacVax® OmpA induces protective immune response against Shigella flexneri 2a ATCC 12022 in a murine model.

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.

Shigellosis murine model established by intraperitoneal and intranasal route of administration: a comparative comprehension overview.

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.