Reverse vaccinology & immunoinformatics approach to design a multiepitope vaccine (CV3Ag-antiMRSA) against methicillin resistant Staphylococcus aureus (MRSA) - a pathogen affecting both human and animal health.

Infections caused by drug resistant bacteria is a silent detrimental pandemic affecting the global health care profoundly. Methicillin resistant Staphylococcus aureus (MRSA) is a pathogen that causes serious infections in different settings (community, hospital & veterinary) whose treatment remains highly challenging due to its powerful characteristics (antibiotic resistance strategies, virulence factors). In this study, we used reverse vaccinology (RV) approach and designed an immunogenic multi epitope vaccine (CV3Ag-antiMRSA) targeting three potential antigen candidates viz., mecA encoding transpeptidase (PBP2a) protein responsible for conferring methicillin resistance and two virulence determinants - hlgA encoding gamma-hemolysin component A (a pore forming toxin) and isdB encoding iron regulated surface determinant B (heme transport component that allows S. aureus to scavenge iron from host hemoglobin and myoglobin). We employed an array of immunoinformatic tools/server to identify and use immunogenic epitopes (B cell and MHC class) to develop the chimeric subunit vaccine V4 (CV3Ag-antiMRSA) with immune modulating adjuvant and linkers. Based on different parameters, the vaccine construct V4 (CV3Ag-antiMRSA) was determined to be suitable vaccine (antigenic and non-allergen). Molecular docking and simulation of CV3Ag-antiMRSA with Toll Like Receptor (TLR2) predicted its immuno-stimulating potential. Finally, in silico cloning of CV3Ag-antiMRSA construct into pet28a and pet30 vector displayed its feasibility for the heterologous expression in the E. coli expression system. This vaccine candidate (CV3Ag-antiMRSA) designed based on the MRSA genomes obtained from both animal and human hosts can be experimentally validated and thereby contribute to vaccine development to impart protection to both animal and human health.Communicated by Ramaswamy H. Sarma.

Route of Vaccine Administration Influences the Impact of Fms-Like Tyrosine Kinase 3 Ligand (Flt3L) on Chlamydial-Specific Protective Immune Responses.

We tested the hypothesis that the impact of the Fms-like tyrosine kinase 3-ligand (Flt3L; FL) on recombinant Vibrio cholerae ghost (rVCG) vaccine-induced chlamydial immunity is influenced by route of vaccine delivery. Female C57BL/6J mice were immunized rectally (IR) or intramuscularly (IM) with rVCG co-expressing the Chlamydia trachomatis PmpD and PorB proteins (rVCG- PmpD/PorB) with and without FL or glycoprotein D of HSV-2 (rVCG-gD2) as antigen control. Vaccine evaluation was based on measurement of T cell proliferation, Th1/Th2 cytokine, and humoral responses at systemic and mucosal compartments, and protection against intravaginal challenge infection. Results revealed that high levels of CD4+ T cell-mediated and humoral immune responses, were elicited in mice as a function of both IR and IM immunization. Unexpectedly, co-administration of vaccine with FL enhanced specific Th1-type cytokine levels and T cell proliferative responses following IR but not IM immunization. While administration of vaccine with FL enhanced the specific mucosal and systemic IgA antibody responses following both immunization routes, IgG2c responses were not enhanced following IR delivery. The vaccine-induced immune effectors protected mice against live heterologous C. muridarum infection irrespective of route of vaccine administration, with the regimen incorporating FL having a protective advantage. Further evaluation showed that protection afforded by the FL adjuvanted vaccine was facilitated by CD4+ T cells, as indicated by reduction in the intensity and duration of genital chlamydial shedding by naïve mice following adoptive transfer of immune CD4+ T cells. Taken together, the results indicate that comparable protective immunity, which is enhanced by co-delivery with FL, is elicited in the female genital tract against Chlamydia infection after mucosal and systemic administration, highlighting the ability of FL to function as an effective immunostimulator at both mucosal and systemic sites. The differential modulation of humoral and cellular immune responses, and protective immunity afforded by the FL adjuvanted vaccine following IR administration indicates that the immunomodulatory impact of FL on chlamydial-specific immunity is influenced by the route of vaccine administration. Thus, targeting of VCG-based vaccines to antigen presenting cells by co-delivery with FL is a feasible immunization approach for inducing effective chlamydial immunity in the female genital tract.

A unique insight into the MiRNA profile during genital chlamydial infection.

BACKGROUND: Genital C. trachomatis infection may cause pelvic inflammatory disease (PID) that can lead to tubal factor infertility (TFI). Understanding the pathogenesis of chlamydial complications including the pathophysiological processes within the female host genital tract is important in preventing adverse pathology. MicroRNAs regulate several pathophysiological processes of infectious and non-infectious etiologies. In this study, we tested the hypothesis that the miRNA profile of single and repeat genital chlamydial infections will be different and that these differences will be time dependent. Thus, we analyzed and compared differentially expressed mice genital tract miRNAs after single and repeat chlamydia infections using a C. muridarum mouse model. Mice were sacrificed and their genital tract tissues were collected at 1, 2, 4, and 8 weeks after a single and repeat chlamydia infections. Histopathology, and miRNA sequencing were performed. RESULTS: Histopathology presentation showed that the oviduct and uterus of reinfected mice were more inflamed, distended and dilated compared to mice infected once. The miRNAs expression profile was different in the reproductive tissues after a reinfection, with a greater number of miRNAs expressed after reinfection. Also, the number of miRNAs expressed each week after chlamydia infection and reinfection varied, with weeks eight and one having the highest number of differentially expressed miRNAs for chlamydia infection and reinfection respectively. Ten miRNAs; mmu-miR-378b, mmu-miR-204-5p, mmu-miR-151-5p, mmu-miR-142-3p, mmu-miR-128-3p, mmu-miR-335-3p, mmu-miR-195a-3p, mmu-miR-142-5p, mmu-miR-106a-5p and mmu-miR-92a-3p were common in both primary chlamydia infection and reinfection. Pathway analysis showed that, amongst other functions, the differentially regulated miRNAs control pathways involved in cellular and tissue development, disease conditions and toxicity. CONCLUSIONS: This study provides insights into the changes in miRNA expression over time after chlamydia infection and reinfection, as well as the pathways they regulate to determine pathological outcomes. The miRNAs networks generated in our study shows that there are differences in the focus molecules involved in significant biological functions in chlamydia infection and reinfection, implying that chlamydial pathogenesis occurs differently for each type of infection and that this could be important when determining treatments regime and disease outcome. The study underscores the crucial role of host factors in chlamydia pathogenesis.

Rectal administration of a chlamydial subunit vaccine protects against genital infection and upper reproductive tract pathology in mice.

In this study, we tested the hypothesis that rectal immunization with a VCG-based chlamydial vaccine would cross-protect mice against heterologous genital Chlamydia trachomatis infection and Chlamydia-induced upper genital tract pathologies in mice. Female mice were immunized with a C. trachomatis serovar D-derived subunit vaccine or control or live serovar D elementary bodies (EBs) and the antigen-specific mucosal and systemic immune responses were characterized. Vaccine efficacy was determined by evaluating the intensity and duration of genital chlamydial shedding following intravaginal challenge with live serovar E chlamydiae. Protection against upper genital tract pathology was determined by assessing infertility and tubal inflammation. Rectal immunization elicited high levels of chlamydial-specific IFN-gamma-producing CD4 T cells and humoral immune responses in mucosal and systemic tissues. The elicited immune effectors cross-reacted with the serovar E chlamydial antigen and reduced the length and intensity of genital chlamydial shedding. Furthermore, immunization with the VCG-vaccine but not the rVCG-gD2 control reduced the incidence of tubal inflammation and protected mice against Chlamydia-induced infertility. These results highlight the potential of rectal immunization as a viable mucosal route for inducing protective immunity in the female genital tract.

Chlamydia abortus Pmp18.1 Induces IL-1ß Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways.

The polymorphic membrane protein D (Pmp18D) is a 160-kDa outer membrane protein that is conserved and plays an important role in Chlamydia abortus pathogenesis. We have identified an N-terminal fragment of Pmp18D (designated Pmp18.1) as a possible subunit vaccine antigen. In this study, we evaluated the vaccine potential of Pmp18.1 by investigating its ability to induce innate immune responses in dendritic cells and the signaling pathway(s) involved in rPmp18.1-induced IL-1ß secretion. We next investigated the immunomodulatory impact of VCG, in comparison with the more established Th1-promoting adjuvants, CpG and FL, on rPmp18.1-mediated innate immune activation. Finally, the effect of siRNA targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in DCs on IL-1ß cytokine secretion was also investigated. Bone marrow-derived dendritic cells (BMDCs) were stimulated with rPmp18.1 in the presence or absence of VCG or CpG or FL and the magnitude of cytokines produced was assessed using a multiplex cytokine ELISA assay. Expression of costimulatory molecules and Toll-like receptors (TLRs) was analyzed by flow cytometry. Quantitation of intracellular levels of myeloid differentiation factor 88 (MyD88), nuclear factor kappa beta (NF-κB p50/p65), and Caspase-1 was evaluated by Western immunoblotting analysis while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The results showed DC stimulation with rPmp18.1 provoked the secretion of proinflammatory cytokines and upregulated expression of TLRs and co-stimulatory molecules associated with DC maturation. These responses were significantly (p ≤ 0.001) enhanced by VCG but not CpG or FL. In addition, rPmp18.1 activated the expression of MyD88, NF-κB p50, and Caspase-1 as well as the nuclear expression of NF-κB p65 in treated DCs. Furthermore, targeting TLR4, MyD88, NF-κB p50, and Caspase-1 mRNA in BMDCs with siRNA significantly reduced their expression levels, resulting in decreased IL-1ß cytokine secretion, strongly suggesting their involvement in the rPmp18.1-induced IL-1ß cytokine secretion. Taken together, these results indicate that C. abortus Pmp18.1 induces IL-1ß secretion by TLR4 activation through the MyD88, NF-κB as well as the Caspase-1 signaling pathways and may be a potential C. abortus vaccine candidate. The vaccine potential of Pmp18.1 will subsequently be evaluated in an appropriate animal model, using VCG as an immunomodulator, following immunization and challenge.

Comparative evaluation of the protective efficacy of two formulations of a recombinant Chlamydia abortus subunit candidate vaccine in a mouse model.

Chlamydia abortus (C. abortus) is the causative agent of ovine enzootic abortion (OEA) and poses a zoonotic risk to pregnant women. Current live attenuated 1B vaccines are efficacious but cause disease in vaccinated animals and inactivated vaccines are only marginally protective. We tested the ability of a new C. abortus subunit vaccine candidate based on the conserved and immunogenic polymorphic membrane protein D (Pmp18D) formulated in CpG1826+FL (Fms-like tyrosine kinase 3 Ligand; Flt3L) or Vibrio cholerae ghosts (VCG) to induce innate and cross protective immunity against genital C. abortus infection. We found that delivery of rPmp18D with VCG was more effective than with CpG+FL in up-regulating the expression of molecules critically involved in T cell activation and differentiation, including MHC II, CD40, CD80, and CD86, activation of TLRs and NLRP3 inflammasome engagement, and secretion of IL-1ß and TNF-α but not IL-10 and IL-4. rVCG-Pmp18D-immunized mice elicited more robust antigen-specific IFN-γ, IgA and IgG2c antibody responses compared to CpG+FL-delivered rPmp18D. Based on the number of mice with positive vaginal cultures, length of vaginal shedding, and number of inclusion forming units recovered following challenge with the heterologous C. abortus strain B577, vaccine delivery with VCG induced superior protective immunity than delivery with a combination of CpG1826 and FL, a nasal DC-targeting adjuvant. These results demonstrate that the ability of VCG to enhance protective immunity against genital C. abortus infection is superior to that of CpG+FL adjuvants.

Vibrio cholerae ghosts (VCG) exert immunomodulatory effect on dendritic cells for enhanced antigen presentation and induction of protective immunity.

BACKGROUND: We previously showed that the Vibrio cholerae ghost platform (VCG; empty V. cholerae cell envelopes) is an effective delivery system for vaccine antigens promoting the induction of substantial immunity in the absence of external adjuvants. However, the mechanism by which these cell envelopes enhance immunity and stimulate a predominantly Th1 cellular and humoral immune response has not been elucidated. We hypothesized that the immunostimulatory ability of VCG involves dendritic cell (DC) activation. OBJECTIVE: The aims of this study were: a) to investigate the ability of DCs [using mouse bone marrow-derived DCs (BMDCs) as a model system] to take up and internalize VCGs; b) to evaluate the immunomodulatory effect of internalized VCGs on DC activation and maturation and their functional capacity to present chlamydial antigen to naïve and infection-sensitized CD4+ T cells and; c) to evaluate the ability of VCGs to enhance the protective immunity of a chlamydial antigen. RESULTS: VCGs were efficiently internalized by DCs without affecting their viability and modulated DC-mediated immune responses. VCG-pulsed DCs showed increased secretion of proinflammatory cytokines and expression of co-stimulatory molecules associated with DC maturation in response to stimulation with UV-irradiated chlamydial elementary bodies (UV-EBs). Furthermore, this interaction resulted in effective chlamydial antigen presentation to infection-sensitized but not naïve CD4+ T cells and enhancement of protective immunity. CONCLUSIONS: The present study demonstrated that VCGs activate DCs leading to the surface expression of co-stimulatory molecules associated with DC activation and maturation and enhancement of protective immunity induced by a chlamydial antigen. The results indicate that the immunoenhancing activity of VCG for increased T-cell activation against antigens is mediated, at least in part, through DC triggering. Thus, VCGs could be harnessed as immunomodulators to target antigens to DCs for enhancement of protective immunity against microbial infections.

An intracameral injection of antigen induces in situ chemokines and cytokines required for the generation of circulating immunoregulatory monocytes.

Anterior Chamber-Associated Immune Deviation (ACAID) induced by an intracameral injection of antigen generates antigen-specific regulatory splenic T cells that suppress specifically cell-mediated immunity specific for the injected antigen. Circulating F4/80(+) cells recovered from mice receiving an intracameral injection of antigen are thought to be ocular in origin and induce the development of thymic and splenic regulatory T cells. We have shown previously that after the intracameral injection of antigen there is a CCR2/CCL2-dependent infiltration of circulating F4/80(+) cells into the anterior chamber associated with the generation of circulating, ACAID-inducing F4/80(+) monocytes. Here we tested the hypothesis that the intracameral injection of antigen induces events in the anterior chamber that are associated with the induction of circulating immunoregulatory monocytes that induce the suppression of cell-mediated immunity. The intracameral injection of antigen resulted in aqueous humor (i) a time- dependent increase of CCL2 and CCL7, (ii) a transient increase in TNF-α, and (iii) an infiltration of CD11b(hi), Gr1(hi) and F4/80(+) as well as F4/80(-) and Gr1(hi) peripheral blood cells into the anterior chamber. Further characterization of these F4/80(+) cells revealed that they are Ly 6C(hi), LY6G(lo) or negative, 7/4 (LY6B)(hi), CD115(+), CD45(+), CD49B(+), and CD62 L(+). Antibody-mediated neutralization of TGF-ß in situ in the anterior chamber prevented the induction of circulating, ACAID-inducing monocytes and ACAID. These cells did not increase in the irides of ACAID-refractory CCR2-/- and CCL2-/- mice that received an intracameral injection of antigen. Our results extend our suggestion that ACAID is initiated as the result of a mild proinflammatory response to intracameral injection that results in the infiltration of a CCR2(+) subset of monocytes into the anterior chamber where there is a TGF-ß-dependent induction of an immunosuppressive phenotype in the infiltrated monocytes that recirculate to induce antigen-specific regulatory T cells.

Wolbachia symbiont infections induce strong cytoplasmic incompatibility in the tsetse fly Glossina morsitans.

Tsetse flies are vectors of the protozoan parasite African trypanosomes, which cause sleeping sickness disease in humans and nagana in livestock. Although there are no effective vaccines and efficacious drugs against this parasite, vector reduction methods have been successful in curbing the disease, especially for nagana. Potential vector control methods that do not involve use of chemicals is a genetic modification approach where flies engineered to be parasite resistant are allowed to replace their susceptible natural counterparts, and Sterile Insect technique (SIT) where males sterilized by chemical means are released to suppress female fecundity. The success of genetic modification approaches requires identification of strong drive systems to spread the desirable traits and the efficacy of SIT can be enhanced by identification of natural mating incompatibility. One such drive mechanism results from the cytoplasmic incompatibility (CI) phenomenon induced by the symbiont Wolbachia. CI can also be used to induce natural mating incompatibility between release males and natural populations. Although Wolbachia infections have been reported in tsetse, it has been a challenge to understand their functional biology as attempts to cure tsetse of Wolbachia infections by antibiotic treatment damages the obligate mutualistic symbiont (Wigglesworthia), without which the flies are sterile. Here, we developed aposymbiotic (symbiont-free) and fertile tsetse lines by dietary provisioning of tetracycline supplemented blood meals with yeast extract, which rescues Wigglesworthia-induced sterility. Our results reveal that Wolbachia infections confer strong CI during embryogenesis in Wolbachia-free (Gmm(Apo)) females when mated with Wolbachia-infected (Gmm(Wt)) males. These results are the first demonstration of the biological significance of Wolbachia infections in tsetse. Furthermore, when incorporated into a mathematical model, our results confirm that Wolbachia can be used successfully as a gene driver. This lays the foundation for new disease control methods including a population replacement approach with parasite resistant flies. Alternatively, the availability of males that are reproductively incompatible with natural populations can enhance the efficacy of the ongoing sterile insect technique (SIT) applications by eliminating the need for chemical irradiation.

The Induction of Circulating, ACAID-Inducing Monocytes Requires CCR2/CCL2-Dependent Migration of Circulating F4/80(+) Cells into the Anterior Chamber.

To determine the origin of peripheral blood mononulclear cells (PBMC) that activate regulatory T cells in anterior chamber-associated immune deviation (ACAID), fluorescein-labeled PBMC were intravenously injected into mice before the mice received an intracameral injection of antigen. Six-24 hr after intracameral injection, fluorescein-labeled PBMC increased in the iris. Twenty-four-48 hr labeled cells decreased in the iris and increased in the thymus and spleen. The entry of the labeled PBMC into the anterior chamber and subsequent production of PBMC that transfer ACAID required the expression of CCR2 by the PBMC and the production of the chemokine CCL2 by the recipient of the PBMC. The results suggest that the intracameral injection of antigen induces i) the infiltration of F4/80(+) PBMC into the AC, ii) where these PBMC are converted to a regulatory phenotype, and iii) recirculate to activate T cells that suppress cell-mediated immunity.

Anterior Chamber-Associated Immune Deviation (ACAID): An Acute Response to Ocular Insult Protects from Future Immune-Mediated Damage?

The "immune privilege" that inhibits immune defense mechanisms that could lead to damage to sensitive ocular tissue is based on the expression of immunosuppressive factors on ocular tissue and in ocular fluids. In addition to this environmental protection, the injection of antigen into the anterior chamber or infection in the anterior chamber induces a systemic suppression of potentially damaging cell-mediated and humoral responses to the antigen. Here we discuss evidence that suggests that Anterior Chamber-Associated Immune Deviation (ACAID)(a) is initiated by an ocular response to moderate inflammation that leads to a systemic immunoregulatory response. Injection into the anterior chamber induces a rise in TNF-α and MCP-1 in aqueous humor and an infiltration of circulating F4/80(+) monocytes that home to the iris. The induction of ACAID is dependent on this infiltration of circulating monocytes that eventually emigrate to the thymus and spleen where they induce regulatory T cells that inhibit the inductive or effector phases of a cell-mediated immune response. ACAID therefore protects the eye from the collateral damage of an immune response to infection by suppressing a future potentially damaging response to infection.

The obligate mutualist Wigglesworthia glossinidia influences reproduction, digestion, and immunity processes of its host, the tsetse fly.

Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis. Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia. The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis.