IFNγ insufficiency during mouse intra-vaginal Chlamydia trachomatis infection exacerbates alternative activation in macrophages with compromised CD40 functions.

Chlamydia trachomatis (C.tr), an obligate intracellular pathogen, causes asymptomatic genital infections in women and is a leading cause of preventable blindness. We have developed in vivo mouse models of acute and chronic C. trachomatis genital infection to explore the significance of macrophage-directed response in mediating immune activation/suppression. Our findings reveal that during chronic and repeated C. trachomatis infections, Th1 response is abated while Treg response is enhanced. Additionally, an increase in exhaustion (PD1, CTLA4) and anergic (Klrg3, Tim3) T cell markers is observed during chronic infection. We have also observed that M2 macrophages with low CD40 expression promote Th2 and Treg differentiation leading to sustained C. trachomatis genital infection. Macrophages infected with C. trachomatis or treated with supernatant of infected epithelial cells drive them to an M2 phenotype. C. trachomatis infection prevents the increase in CD40 expression as observed in western blots and flow cytometric analysis. Insufficient IFNγ, as observed during chronic infection, leads to incomplete clearance of bacteria and poor immune activation. C. trachomatis decapacitates IFNγ responsiveness in macrophages via hampering IFNγRI and IFNγRII expression which can be correlated with poor expression of MHC-II, CD40, iNOS and NO release even following IFNγ supplementation. M2 macrophages during C. trachomatis infection express low CD40 rendering immunosuppressive, Th2 and Treg differentiation which could not be reverted even by IFNγ supplementation. The alternative macrophages also harbour high bacterial load and are poor responders to IFNγ, thus promoting immunosuppression. In summary, C. trachomatis modulates the innate immune cells, attenuating the anti-chlamydial functions of T cells in a manner that involves decreased CD40 expression on macrophages.

Poly(I:C) and R848 ligands show better adjuvanticity to induce B and T cell responses against the antigen(s).

Poly(I:C) and R848, synthetic ligands that activate Toll-like receptor 3 (TLR3) and TLR7/8 respectively, have been well-established for their ability to stimulate the immune system and induce antigen-specific immune responses. These ligands are capable of inducing the production of cytokines and chemokines, and hence support the activation and differentiation of B and T cells. We saw the long-lasting and perdurable immune responses by these adjuvants essentially required for an efficacious subunit vaccine. In this study, we investigated the potential of poly(I:C) and R848 to elicit B and T cell responses to the OVA antigen. We assessed the stimulatory effects of these ligands on the immune system, their impact on B and T cell activation, and their ability to enhanced generation of B and T cells. Collectively, our findings contribute to the understanding how poly(I:C) and R848 can be utilized as an adjuvant system to enhance immune responses to protein-based subunit vaccines. In the end, this work provides insights for the development of novel vaccination strategies and improving the vaccine efficacy. Present work shall help formulate newer strategies for subunit vaccines to address the infectious diseases.

Role of natural killer and B cell interaction in inducing pathogen specific immune responses.

The innate lymphoid cell (ILC) system comprising of the circulating and tissue-resident cells is known to clear infectious pathogens, establish immune homeostasis as well as confer antitumor immunity. Human natural killer cells (hNKs) and other ILCs carry out mopping of the infectious pathogens and perform cytolytic activity regulated by the non-adaptive immune system. The NK cells generate immunological memory and rapid recall response tightly regulated by the adaptive immunity. The interaction of NK and B cell, and its role to induce the pathogen specific immunity is not fully understood. Hence, present article sheds light on the interaction between NK and B cells and resulting immune responses in the infectious diseases. The immune responses elicited by the NK-B cell interaction is of particular importance for developing therapeutic vaccines against the infectious pathogens. Further, experimental evidences suggest the immune-response driven by NK cell population elicits the host-specific antibodies and memory B cells. Also, recently developed humanized immune system (HIS) mice and their importance in to understanding the NK-B cell interaction and resulting pathogen specific immunity has been discussed.

TCR-signals downstream adversely correlate with the survival signals of memory CD8+ T cells under homeostasis.

The significance of self-peptide-MHC-I/TCR (SMT) interaction in the survival of CD8+ T cells during naïve- and developmental-stages is well documented. However, the same for the memory stage is contentious. Previous studies have attempted to address the issue using MHC-I or TCR deficient systems, but inconsistent findings with memory CD8+ T cells of different TCR specificities have complicated the interpretation. Differential presence and/or processing of TCR-signals downstream in memory CD8+ T cells of different TCR specificities could be thought of as a reason. In this study, we examined the TCR-signals downstream in memory CD8+ T cells and compared them to the presence of survival-related signals (Annexin-V, Bcl-2, and Ki-67). We categorically tracked foreign antigen-experienced memory CD8+ T (TM) cells generated after Plasmodium pre-erythrocytic-stage malaria infection in C57BL/6 mice. Interestingly, we found that memory CD8+ T cells had more TCR-signals downstream than naive cells. We reasoned and attributed the increased expression of cell adhesion molecules to the enhanced TCR-signaling. TCR-signals downstream correlate more closely with survival signals in naive CD8+ T cells than with death signals in TM cells. Further investigation using antigen-specific CD8+ T cells and diverse infection systems would aid in conceptualizing the findings.

Infectious sporozoite challenge modulates radiation attenuated sporozoite vaccine-induced memory CD8+ T cells for better survival characteristics.

Radiation attenuated sporozoite (RAS), a whole-parasite vaccine approach, provides sterile protection against malaria. However, RAS immunization does not confer protection for long, and that has been correlated with the waning parasite-induced memory CD8+ T-cell responses. Interestingly, an intermittent infectious (wild type) sporozoite challenge to the RAS-vaccinated mice lengthened the protection period from 6 to 18 months. Herein, we have studied the changes induced by the infectious sporozoites in RAS-induced memory CD8+ T cells for conferring lengthened protection. We observed that the infectious sporozoite challenge boosted the frequency of foreign antigen-experienced memory CD8+ T cells. In those CD8+ T cells, it has reduced the Annexin-V reactivity, raised Bcl-2 expression, and also more cells undergo homeostatic proliferation (Ki-67+ ). It has also scaled down the frequency of Nur77 and CX3CR1 high expressing cells in those memory CD8+ T-cell populations which we further correlated with better survival signals.

Development of Stable Chimeric IL-15 for Trans-Presentation by the Antigen Presenting Cells.

IL-15 is one of the important biologics considered for vaccine adjuvant and treatment of cancer. However, a short half-life and poor bioavailability limit its therapeutic potential. Herein, we have structured IL-15 into a chimeric protein to improve its half-life enabling greater bioavailability for longer periods. We have covalently linked IL-15 with IgG2 base to make the IL-15 a stable chimeric protein, which also increased its serum half-life by 40 fold. The dimeric structure of this kind of IgG based biologics has greater stability, resistance to proteolytic cleavage, and less frequent dosing schedule with minimum dosage for achieving the desired response compared to that of their monomeric forms. The structured chimeric IL-15 naturally forms a dimer, and retains its affinity for binding to its receptor, IL-15Rß. Moreover, with the focused action of the structured chimeric IL-15, antigen-presenting cells (APC) would transpresent chimeric IL-15 along with antigen to the T cell, that will help the generation of quantitatively and qualitatively better antigen-specific memory T cells. In vitro and in vivo studies demonstrate the biological activity of chimeric IL-15 with respect to its ability to induce IL-15 signaling and modulating CD8+ T cell response in favor of memory generation. Thus, a longer half-life, dimeric nature, and anticipated focused transpresentation by APCs to the T cells will make chimeric IL-15 a super-agonist for memory CD8+ T cell responses.

Transdermal Immunization of Elastic Liposome-Laden Recombinant Chimeric Fusion Protein of P. falciparum (PfMSP-Fu24) Mounts Protective Immune Response.

Transdermal immunization exhibits poor immunogenic responses due to poor permeability of antigens through the skin. Elastic liposomes, the ultradeformable nanoscale lipid vesicles, overcome the permeability issues and prove a versatile nanocarrier for transcutaneous delivery of protein, peptide, and nucleic acid antigens. Elastic liposome-mediated subcutaneous delivery of chimeric fusion protein (PfMSP-Fu24) of Plasmodium falciparum exhibited improved immunogenic responses. Elastic liposomes-mediated immunization of PfMSP-Fu24 conferred immunity to the asexual blood-stage infection. Present study is an attempt to compare the protective immune response mounted by the PfMSP-Fu24 upon administered through transdermal and intramuscular routes. Humoral and cell-mediated immune (CMI) response elicited by topical and intramuscularly administered PfMSP-Fu24-laden elastic liposomes (EL-PfMSP-Fu24) were compared and normalized with the vehicle control. Sizeable immune responses were seen with the transcutaneously immunized EL-PfMSP-Fu24 and compared with those elicited with intramuscularly administered antigen. Our results show significant IgG isotype subclass (IgG1and IgG3) response of specific antibody levels as well as cell-mediated immunity (CMI) activating factor (IFN-γ), a crucial player in conferring resistance to blood-stage malaria in mice receiving EL-PfMSP-Fu24 through transdermal route as compared to the intramuscularly administered formulation. Heightened immune response obtained by the vaccination of EL-PfMSP-Fu24 was complemented by the quantification of the transcript (mRNA) levels cell-mediated (IFN-γ, IL-4), and regulatory immune response (IL-10) in the lymph nodes and spleen. Collectively, elastic liposomes prove their immune-adjuvant property as they evoke sizeable and perdurable immune response against PfMSP-Fu24 and justify its potential for the improved vaccine delivery to inducing both humoral and CM immune response.

RNA-loaded dendritic cells: more than a tour de force in cancer therapeutics.

A wide array of therapeutic strategies has been implemented against cancers, yet their clinical benefit is limited. The lack of clinical efficacy of the conventional treatment options might be due to the inept immune competency of the patients. Dendritic cells (DCs) have a vital role in initiating and directing immune responses and have been frequently used as delivery vehicles in clinical research. The recent clinical data suggest the potential use of DCs pulsed with nucleic acid, especially with RNA holds a great potential as an immunotherapeutic measure with compare to other cancer therapeutics. This review mainly deals with the DCs and their role in transfection with RNA in cancer immunotherapy.

Parasite load stemming from immunization route determines the duration of liver-stage immunity.

Immunization with radiation-attenuated Plasmodium sporozoites (RAS) induces sterile and long-lasting protective immunity. Although intravenous (IV) route of RAS immunization is reported to induce superior immunity compared to intradermal (ID) injection, its role in the maintenance of sterile immunity is yet to be understood. We investigated whether the route of homologous sporozoite challenge of Plasmodium berghei (Pb) RAS-immunized mice would influence the longevity of protection. C57BL/6 mice immunized with Pb-RAS by IV were 100% protected upon primary IV/ID sporozoite challenge. In contrast, ID immunization resulted in 80% protection, regardless of primary challenge route. Interestingly, the route of primary challenge was found to bring difference in the maintenance of sterile protection. While IV Pb RAS-immunized mice remained protected at all challenges regardless of the route of primary challenge, ID Pb-RAS-immunized mice receiving ID primary challenge became parasitaemic upon secondary IV challenge. Significantly, primary IV challenge of Pb RAS ID-immunized mice resulted in 80% and 50% survival at secondary and tertiary challenges, respectively. According to phenotypically diverse liver CD8+ T cells, the percentages and the numbers of both CD8+ T effector memory and resident memory cells were significantly higher in IV than in ID Pb RAS-immunized mice. IFN-γ-producing CD8+ T cells specific to Pb TRAP130 and MIP-4-Kb-17 were also found significantly higher in IV mice than in ID mice. The enhanced T-cell generation and the longevity of protection appear to be dependent on the parasite load during challenge when infection is tolerated under suboptimal CD8+ T-cell response.

Influence of Length and Amino Acid Composition on Dimer Formation of Immunoglobulin based Chimera.

BACKGROUND: The dimeric immunoglobulin (Ig) chimeras used for drug targeting and delivery are preferred biologics over their monomeric forms. Designing these Ig chimeras involves critical selection of a suitable Ig base that ensures dimer formation. In the present study, we systematically analyzed several factors that influence the formation of dimeric chimera. We designed and predicted 608 cytokine-Ig chimeras where we tested the contributions of (1) different domains of Ig constant heavy chain, (2) length of partner proteins, (3) amino acid (AA) composition and (4) position of cysteine in the formation of homodimer. METHOD: The sequences of various Ig and cytokines were procured from Uniprot database, fused and submitted to COTH (CO-THreader) server for the prediction of dimer formation. Contributions of different domains of Ig constant heavy chain, length of chimeric proteins, AA composition and position of cysteine to the homodimer formation of 608 cytokine-Ig chimeras were tested. Various in silico approaches were adopted for validating the in silico findings. Experimentally we also validated our approach by expressing the chimeric design of shorter cytokine with Ig domain in CHO cells and analyzing the protein by SDS-PAGE. RESULTS: Our results advocate that while the CH1 region and the Hinge region of Ig heavy chain are critical, the length of partner proteins also crucially influences homodimer formation of the Ig-based chimera. We also report that the CH1 domain of Ig is not required for dimer formation of Ig based chimera in the presence of larger partner proteins. For shorter partner proteins fused to CH2-CH3, careful selection of partner sequence is critical, particularly the hydrophobic AA composition, cysteine content & their positions, disulphide bond formation property, and the linker sequences. We validated our in silico observation by various bioinformatics tools and checked the ability of chimeras to bind with the receptors of native protein by docking studies. As a proof of concept, we have expressed the chimeric proteins in CHO cells and found that our design favors the synthesis of dimeric proteins. CONCLUSION: Our structural prediction study suggests that extra amino acids in the range of 15-20 added to the CH2 domain of Ig is a critical requirement to make homodimer. This information from our study will have implication in designing efficacious homodimeric chimera.

Leptospira in the different ecological niches of the tribal union territory of India.

INTRODUCTION: Leptospirosis is a widespread zoonotic disease, which has a medical and veterinary importance, and also a commonly re-emerging infectious disease. The main causes of transmission are known; however, the respective prominence of each of the components and the respective environmental risk factors are obscure. METHODOLOGY: Present study was conducted in the different locations (urban/rural/tribal) of the union territory of Dadra and Nagar Haveli (UT of Dadra and Nagar Haveli). Periodical sample collection approach was used to collect the samples from May 2016 to April 2017, to determine the persistence of leptospiral contamination of the environmental sources. RESULTS: The PCR detection and isolation of Leptospira revealed that pathogenic leptospires were present in water, soil and animal urine. The highest positivity was reported from the water of household drainage in urban areas and in the water from rice field of rural areas. CONCLUSIONS: The data obtained from the present study may help and guiding in developing preventive measures for leptospirosis in the UT of Dadra and Nagar Haveli.

Frequent inoculations with radiation attenuated sporozoite is essential for inducing sterile protection that correlates with a threshold level of Plasmodia liver-stage specific CD8+ T cells.

Whole sporozoite vaccine (WSV) is shown to induce sterile protection that targets Plasmodium liver-stage infection. There are many underlying issues associated with induction of effective sterile protracted protection. In this study, we have addressed how the alterations in successive vaccine regimen could possibly affect the induction of sterile protection. We have demonstrated that the pattern of vaccination with RAS (radiation attenuated sporozoites) induces varying degrees of protection among B6 mice. Animals receiving four successive doses generated 100% sterile protection. However, three successive doses, though with the same parasite inoculum as four doses, could induce sterile protection in ∼50% mice. Interestingly, mice immunized with the same 3 doses, but with longer gap, could not survive the challenge. We demonstrate that degree of protection correlates with the frequencies of IFN-γ+ and multifunctional (IFN-γ+ CD107a+) CD8+ TEM cells present in liver. The failure to achieve protective threshold frequency of these cells in liver might make the host more vulnerable to parasite infection during infectious sporozoite challenge.

Human IDO-competent, long-lived immunoregulatory dendritic cells induced by intracellular pathogen, and their fate in humanized mice.

Targeting of myeloid-dendritic cell receptor DC-SIGN by numerous chronic infectious agents, including Porphyromonas gingivalis, is shown to drive-differentiation of monocytes into dysfunctional mDCs. These mDCs exhibit alterations of their fine-tuned homeostatic function and contribute to dysregulated immune-responses. Here, we utilize P. gingivalis mutant strains to show that pathogen-differentiated mDCs from primary human-monocytes display anti-apoptotic profile, exhibited by elevated phosphorylated-Foxo1, phosphorylated-Akt1, and decreased Bim-expression. This results in an overall inhibition of DC-apoptosis. Direct stimulation of complex component CD40 on DCs leads to activation of Akt1, suggesting CD40 involvement in anti-apoptotic effects observed. Further, these DCs drove dampened CD8+ T-cell and Th1/Th17 effector-responses while inducing CD25+Foxp3+CD127- Tregs. In vitro Treg induction was mediated by DC expression of indoleamine 2,3-dioxygenase, and was confirmed in IDO-KO mouse model. Pathogen-infected &CMFDA-labeled MoDCs long-lasting survival was confirmed in a huMoDC reconstituted humanized mice. In conclusion, our data implicate PDDCs as an important target for resolution of chronic infection.

Interleukin 15: A key cytokine for immunotherapy.

Interleukin (IL)-15, a member of the immunoregulatory cytokines family, is a pluripotent molecule with therapeutic potential. It is predominantly expressed by the myeloid cells, as well as other cell types. IL-15 serves multiple functions including dictating T cell response, regulating tissue repair and B cell homing, modulating inflammation, and activating NK cells. Among cytokines, IL-15 is unique because of its wide expression, tightly regulated secretion, trans-presentation, and therapeutic potential. IL-15 has been investigated for its therapeutic potential for the induction and maintenance of T cell responses. In addition, IL-15 can be targeted by antibody- or mutant IL-15 therapy to reduce inflammation. Its multifaceted biological applications are crucial in immunotherapy. In this article, we review the functions, expression, and regulation of IL-15 for designing an improved IL-15-based therapy targeting the IL-15 signaling pathway.

Transdermal immunization of P. falciparum surface antigen (MSP-119) via elastic liposomes confers robust immunogenicity.

As transdermal immunization results in poor immunogenicity, which is attributed to poor permeability of antigens through the skin, we believed ultradeformable lipid vesicles (elastic liposome) might address the challenges encountered during transdermal immunization. The elastic liposome, versatile carrier, proves better vehicle for transcutaneous delivery of protein, peptide and nucleic acid antigens. Our recently published article (1) is suggestive of improved immunogenicity of carboxyl-terminal 19 kDa fragment of merozoite surface protein-1 (PfMSP-119) of Plasmodium falciparum when administered subcutaneously via elastic liposomes ( Fig. 1 ).

Elastic liposome-mediated transdermal immunization enhanced the immunogenicity of P. falciparum surface antigen, MSP-119.

Transdermal immunization results in poor immunogenicity, which can be attributed to poor permeability of antigens through the skin. Therefore, elastic liposome, ultradeformable lipid vesicles, may overcome the challenges faced during transdermal immunization. This versatile carrier proves better vehicle for transcutaneous delivery of protein, peptide and nucleic acid antigens. The present results are suggestive of improved immunogenicity of carboxyl-terminal 19 kDa fragment of merozoite surface protein-1 (PfMSP-119) of Plasmodium falciparum when administered subcutaneously through elastic liposomes. The prepared elastic liposomes were characterized with respect to vesicles shape and surface morphology, size and size distribution, entrapment efficiency, elasticity, stability and in vitro release. Humoral and cell-mediated immune (CMI) response elicited by topically applied PfMSP-119-loaded elastic liposomes, intramuscularly administered alum-adsorbed PfMSP-119 solution, and topically applied PfMSP-119-loaded conventional liposomes were compared and normalized with vehicle control. Results suggest greater transcutaneous immunization via elastic liposomes, and induced robust and perdurable IgG-specific antibody and cytophilic isotype responses. We report to have achieved sizeable CMI activating factor (IFNγ), a crucial player in conferring resistance to asexual blood stage malaria, responses with elastic liposomes when compared with other formulations. The fluorescence microscopy and histopathology results are suggestive of prominent skin permeation and biodistribution, and demonstrate efficient delivery of malaria antigen via elastic liposomes to immunocompetent Langerhans cells (LC) and lymphatics. In conclusion, elastic liposomal formulation provided greater entrapment efficiency, enhanced penetration and heightened and long-lasting immune response. Moreover, effective immunoadjuvant property of this carrier justifies its potential for improved vaccine delivery, and opens new avenues to explore further on the development of malaria vaccine.

Resolution of infection promotes a state of dormancy and long survival of CD4 memory T cells.

Memory T cells survive throughout the lifetime of an individual and are protective upon recall. It is not clear how memory T cells can live so long. Here, we demonstrate that at the resolution of a viral infection, low levels of antigen are captured by B cells and presented to specific CD4(+) memory T cells to render a state of unresponsiveness. We demonstrate in two systems that this process occurs naturally during the fall of antigen and is associated with a global gene expression program initiated with the clearance of antigen. Our study suggests that in the absence of antigen, a state of dormancy associated with low-energy utilization and proliferation can help memory CD4(+) T cells to survive nearly throughout the lifetime of mice. The dormant CD4(+) memory T cells become activated by stimulatory signals generated by a subsequent infection. We propose that quiescence might be a mechanism necessary to regulate long-term survival of CD4 memory T cells and to prevent cross-reactivity to self, hence autoimmunity.

A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes.

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4(+) T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: human leukocyte antigen-DR1 (HLA-DR1), HLA-DM and cathepsins. Our reductionist system successfully identified the physiologically selected immunodominant epitopes of two model antigens: hemagglutinin-1 (HA1) from influenza virus (A/Texas/1/77) and type II collagen (CII). When applied for identification of new epitopes from a recombinant liver-stage antigen of malaria falciparum (LSA-NRC) or HA1 from H5N1 influenza virus ('avian flu'), the system selected single epitopes from each protein that were confirmed to be immunodominant by their capacity to activate CD4(+) T cells from H5N1-immunized HLA-DR1-transgenic mice and LSA-NRC-vaccinated HLA-DR1-positive human volunteers. Thus, we provide a new tool for the identification of physiologically relevant helper T cell epitopes from antigens.

Suboptimal engagement of the T-cell receptor by a variety of peptide-MHC ligands triggers T-cell anergy.

T cells recognize antigen via the T-cell receptor (TCR) and produce a spectrum of responses that range from activation to anergy or cell death. The variety of outcomes may be dictated by the strength of the signals transmitted upon cognate recognition of the TCR. The physiological outcome of TCR engagement is determined by several factors, including the avidity of the ligand for TCR, the duration of engagement, and the presence and nature of accessory molecules present on antigen-presenting cells (APCs). In this review, we discuss a model of anergy induced by presentation of low densities of peptide-major histocompatibility complex (MHC) ligand in CD4(+) T cells and compare it to anergy induced by altered peptide ligands in an effort to identify a unifying mechanism. We suggest that altered peptide ligand (APL) and low densities of agonist ligands induce anergy by engaging less than optimal numbers of TCRs. The physiological impacts of anergy in memory CD4(+) T cells are discussed.

Staphylococcal enterotoxin A induces small clusters of HLA-DR1 on B cells.

The superantigen SEA causes non-specific hyperactivation of T and B cells at low concentrations. Studies of mutants or soluble proteins suggest SEA is bivalent for its ligand, MHC class II. However, the interaction between these molecules on intact cells is unknown. On primary mouse B cells expressing the MHC class II allele HLA-DR1, measurements of Förster Resonance Energy Transfer between HLA-DR1 molecules on SEA-treated cells indicated specific clustering, not observed in untreated or monovalent superantigen treated cells. Tomographic visualization and electron microscopy of immunogold-labeled SEA-treated B cells revealed small clusters of surface HLA-DR1 (< or = 4 gold labels). These results present direct visual evidence of SEA-mediated clustering of MHC class II molecules on treated antigen presenting cells, and provide a new structural approach to addressing problems of this nature.