Vaccine In Murine Model Research Articles

Intranasal Peptide-Based FpvA-KLH Conjugate Vaccine Protects Mice From Pseudomonas aeruginosa Acute Murine Pneumonia.

Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic respiratory infections associated with morbidity and mortality, especially in patients with cystic fibrosis. Vaccination against P. aeruginosa before colonization may be a solution against these infections and improve the quality of life of at-risk patients. To develop a vaccine against P. aeruginosa, we formulated a novel peptide-based P. aeruginosa subunit vaccine based on the extracellular regions of one of its major siderophore receptors, FpvA. We evaluated the effectiveness and immunogenicity of the FpvA peptides conjugated to keyhole limpet hemocyanin (KLH) with the adjuvant curdlan in a murine vaccination and challenge model. Immunization with the FpvA-KLH vaccine decreased the bacterial burden and lung edema after P. aeruginosa challenge. Vaccination with FpvA-KLH lead to antigen-specific IgG and IgM antibodies in sera, and IgA antibodies in lung supernatant. FpvA-KLH immunized mice had an increase in recruitment of CD11b+ dendritic cells as well as resident memory CD4+ T cells in the lungs compared to non-vaccinated challenged mice. Splenocytes isolated from vaccinated animals showed that the FpvA-KLH vaccine with the adjuvant curdlan induces antigen-specific IL-17 production and leads to a Th17 type of immune response. These results indicate that the intranasal FpvA-KLH conjugate vaccine can elicit both mucosal and systemic immune responses. These observations suggest that the intranasal peptide-based FpvA-KLH conjugate vaccine with curdlan is a potential vaccine candidate against P. aeruginosa pneumonia.

Effect of pleurotus sajor-caju polysaccharide encapsulated in poly D, L lactide-co-glycolide nanoparticles for HPV vaccine in murine model

Objective(s): In the current work, poly D, L lactide-co-glycolide (PLGA) particles were applied for a viral vaccine for the delivery of antigens in cytosolic pathway by increasing the antigen presentation to T-lymphocytes. HPV-E7 protein with PLGA particles has been reported as a potent adjuvant for HPV vaccine by encapsulating protein into the PLGA particles. Polysaccharide from Pleurotus sajor-caju was also applied as a potent immunomodulator. Materials and Methods: HPV-E7 protein and Pleurotus sajor-caju polysaccharides (PSC) were encapsulated into PLGA nanoparticles. This combination comprised a strategy to induce helper and cytotoxic T-lymphocytes (CTL) expansion. Mice antibodies and T-lymphocyte expansion were investigated in comparison between encapsulated E7 protein into PLGA nanoparticles (E7PLGA) and E7 protein with PSC encapsulated into PLGA nanoparticles (PSC-E7PLGA). Results: The results showed that E7PLGA and PSC-E7PLGA could induce antibody response to HPV. The PSC-E7PLGA could increase the level of viral antigen-specific IgG antibodies. The cellular immune responses are also significantly enhanced by expansion of helper T-lymphocytes and CTL. PSC-E7PLGA was shown to be significantly higher in immunomodulating activity than E7PLGA. Conclusion: Thus, the encapsulation of polysaccharide and HPV-E7 antigen into PLGA nanoparticles is a strategy for development of HPV vaccine.

Flagellin is a Th1 polarizing factor for human CD4+ T cells and induces protection in a murine neonatal vaccination model of rotavirus infection

Neonates have an increased susceptibility to infections, particularly those caused by intracellular pathogens, leading to high morbidity and mortality rates. This is partly because of a poor response of neonatal CD4+ T cells, leading to deficient antibody production and a low production of IFN-γ, resulting in deficient elimination of intracellular pathogens. The poor memory response of human neonates has underpinned the need for improving vaccine formulations. Molecular adjuvants that improve the response of neonatal lymphocytes, such as the ligands of toll-like receptors (TLRs), are attractive candidates. Among them, flagellin, the TLR5 ligand, is effective at very low doses; prior immunity to flagellin does not impair its adjuvant activity. Human CD4+ and CD8+ T cells express TLR5. We found that flagellin induces the expression of IFN-γ, IL-1β and IL-12 in mononuclear cells from human neonate and adult donors. When human naïve CD4+ T cells were activated in the presence of flagellin, there was high level of expression of IFN-γ in both neonates and adults. Furthermore, flagellin induced IFN-γ production in Th1 cells obtained from adult donors; in the Th2 population, it inhibited IL-4 cytokine production. Flagellin also promoted expression of the IFN-γ receptor in naive CD4+ T cells from neonates and adults. To test the adjuvant capacity of flagellin in vivo, we used a murine neonate vaccination model for infection with rotavirus, a pathogen responsible for severe diarrhea in young infants. Using the conserved VP6 antigen, we observed an 80% protection against rotavirus infection in the presence of flagellin, but only in those mice previously primed in the neonatal period. Our data suggest that flagellin could be an attractive adjuvant for achieving a Th1 response.

Human Milk Oligosaccharide 2'-Fucosyllactose Improves Innate and Adaptive Immunity in an Influenza-Specific Murine Vaccination Model.

Human milk is uniquely suited to provide optimal nutrition and immune protection to infants. Human milk oligosaccharides are structural complex and diverse consisting of short chain and long chain oligosaccharides typically present in a 9:1 ratio. 2'-Fucosyllactose (2'FL) is one of the most prominent short chain oligosaccharides and is associated with anti-infective capacity of human milk. To determine the effect of 2'FL on vaccination responsiveness (both innate and adaptive) in a murine influenza vaccination model and elucidate mechanisms involved. A dose range of 0.25-5% (w/w) dietary 2'FL was provided to 6-week-old female C57Bl/6JOlaHsd mice 2 weeks prior primary and booster vaccination until the end of the experiment. Intradermal (i.d.) challenge was performed to measure the vaccine-specific delayed-type hypersensitivity (DTH). Antigen-specific antibody levels in serum as well as immune cell populations within several organs were evaluated using ELISA and flow cytometry, respectively. In an ex vivo restimulation assay, spleen cells were cocultured with influenza-loaded bone marrow-derived dendritic cells (BMDCs) to study the effects of 2'FL on vaccine-specific CD4+ and CD8+ T-cell proliferation and cytokine secretions. Furthermore, the direct immune regulatory effects of 2'FL were confirmed using in vitro BMDCs T-cell cocultures. Dietary 2'FL significantly (p < 0.05) enhanced vaccine specific DTH responses accompanied by increased serum levels of vaccine-specific immunoglobulin (Ig) G1 and IgG2a in a dose-dependent manner. Consistently, increased activation marker (CD27) expression on splenic B-cells was detected in mice receiving 2'FL as compared to control mice. Moreover, proliferation of vaccine-specific CD4+ and CD8+ T-cells, as well as interferon-γ production after ex vivo restimulation were significantly increased in spleen cells of mice receiving 2'FL as compared to control mice, which were in line with changes detected within dendritic cell populations. Finally, we confirmed a direct effect of 2'FL on the maturation status and antigen presenting capacity of BMDCs. Dietary intervention with 2'FL improves both humoral and cellular immune responses to vaccination in mice, which might be attributed in part to the direct effects of 2'FL on immune cell differentiation.

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination.

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.

Lambda phage-based vaccine induces antitumor immunity in hepatocellular carcinoma

Background and aimsHepatocellular carcinoma (HCC) is a difficult to treat tumor with a poor prognosis. Aspartate β-hydroxylase (ASPH) is a highly conserved enzyme overexpressed on the cell surface of both murine and human HCC cells.MethodsWe evaluated therapeutic effects of nanoparticle lambda (λ) phage vaccine constructs against ASPH expressing murine liver tumors. Mice were immunized before and after subcutaneous implantation of a syngeneic BNL HCC cell line. Antitumor actively was assessed by generation of antigen specific cellular immune responses and the identification of tumor infiltrating lymphocytes.ResultsProphylactic and therapeutic immunization significantly delayed HCC growth and progression. ASPH-antigen specific CD4+ and CD8+ lymphocytes were identified in the spleen of tumor bearing mice and cytotoxicity was directed against ASPH expressing BNL HCC cells. Furthermore, vaccination generated antigen specific Th1 and Th2 cytokine secretion by immune cells. There was widespread necrosis with infiltration of CD3+ and CD8+ T cells in HCC tumors of λ phage vaccinated mice compared to controls. Moreover, further confirmation of anti-tumor effects on ASPH expressing tumor cell growth were obtained in another murine syngeneic vaccine model with pulmonary metastases.ConclusionsThese observations suggest that ASPH may serve as a highly antigenic target for immunotherapy.

Immunogenicity and protective efficacy of inactivated equine influenza (H3N8) virus vaccine in murine model

Equine influenza viruses (EIVs) are responsible for acute contagious respiratory infection in equines and the disease remains a major threat for equine population throughout the world despite vaccination strategies in place. The present study was aimed to assess the suitability of BALB/c mice as a potential small animal model for preliminary screening of EI vaccine candidates. For this, we evaluated the immunogenicity and protective efficacy of an inactivated EIV (H3N8) vaccine in BALB/c mouse model after challenge with homologous H3N8 virus (Clade 2 virus, Florida sublineage) through serology, clinical signs, gross and histopathology lesions with grading, immunohistochemistry and virus quantification. Serological responses in immunized mice were evaluated by haemagglutination inhibition assay (HAI) and antibodies were subtyped by ELISA. The vaccine induced optimum protective antibody titre on 49 dpi along with balanced Th1/Th2 responses. Immunized mice were well protected against EIV challenge as evident by significant rise in serum antibody titre which concurred with mild clinical signs, early recovery, lower gross and histopathological lesions score, less severe intensity of viral antigen distribution, restricted virus replication in respiratory tract and less virus detection in nasal washes for short duration. The duration of the viral load was also lower and only for brief period as compared to unvaccinated challenged mice. In conclusion, induction of H3N8 specific antibody response and protection against H3N8 challenge proves that egg grown inactivated H3N8 whole virus vaccine would provide an effective intercession against H3N8 virus. In addition, BALB/c mouse can serve as an attractive tool for adjudging protective efficacy of vaccine candidates prior to final testing in equines.

Molecular and histopathological evaluation of the efficacy of Saponifiedand Freund’s incomplete adjuvanted paratuberculosiskilled vaccine in murine model

Johne’s disease (JD) is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Vaccination is regarded as the only cost effective method for controlling JD. Experimental trial for 11 months was carried out by dividing mice into four different groups. Group I- saponified MAP killed vaccine; Group II- Freund’s incomplete adjuvant (FIA) MAP killed vaccine; Group III-saponin adjuvant control; Group IV- FIA control. Mice of all the groups were challenged with 108 colony forming unit live MAP organisms on 40th day after vaccination. A total of 188 samples (52 faecal, 76 sera and 60 tissue samples) were collected from different groups of mice at different interval for monitoring MAP load by Ziehl Neelsen’s staining and nested PCR along with tissue histopathology. Both the vaccinated groups (Group I and II) showed encouraging results in comparison to adjuvant control groups. There was reduction in faecal shedding (p less than 0.01), tissue load of MAP and less granuloma formation in both the vaccinated groups, however mice vaccinated with saponified MAP antigen gave better result in comparison to FIA vaccinated mice. Results of this study suggested that, saponified MAP killed vaccine in particular might reduce the overall burden of JD and show promising result in future.

PEG modified liposomes containing CRX-601 adjuvant in combination with methylglycol chitosan enhance the murine sublingual immune response to influenza vaccination

The mucosa is the primary point of entry for pathogens making it an important vaccination site to produce a protective mucosal immune response. While the sublingual (SL) mucosa presents several barriers to vaccine penetration, its unique anatomy and physiology makes it one of the best options for mucosal vaccination. Efficient and directed delivery of adjuvants and antigens to appropriate immune mediators in the SL tissue will aid in development of effective SL vaccines against infectious diseases. Herein we demonstrate a robust immune response against influenza antigens co-delivered sublingually with engineered liposomes carrying the synthetic Toll-like receptor-4 agonist, CRX-601. Liposome modification with PEG copolymers (Pluronics), phospholipid-PEG conjugates and chitosan were evaluated for their ability to generate an immune response in a SL murine influenza vaccine model. Phospholipid-PEG conjugates were more effective than Pluronic copolymers in generating stable, surface neutral liposomes. SL vaccination with surface modified liposomes carrying CRX-601 adjuvant generated significant improvements in flu-specific responses compared with unmodified liposomes. Furthermore, the coating of modified liposomes with methylglycol chitosan produced the most effective flu-specific immune response. These results demonstrate efficient SL vaccine delivery utilizing a combination of a muco-adhesive and surface neutral liposomes to achieve a robust mucosal and systemic immune response.

Abstract 2514: Activity of a novel Foxp3-tumor cell vaccine in a murine model of renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and represents 80% of the cases. Treatment options for patients with advanced disease are limited. Targeted molecular therapies have shown survival benefit but most patients develop therapy resistance overtime. Preclinical and clinical studies indicate that immunotherapies are effective and may play a role in the treatment of advanced RCC. However, the response rate is ∼ 20%, with a limited number of patients achieving durable remission. A major barrier to vaccine therapy is the presence of immunosuppressive cell populations including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). In the current study, we designed a tumor cell and Tregs dual-targeting vaccine by inducing Foxp3 expression in tumor cells, and tested this vaccine along with an unmodified vaccine in an orthotopic murine model of RCC, RENCA. We observed that both RENCA cell vaccine and RENCA Foxp3 cell vaccine prevented tumor growth in 6 out 10 animals. Interestingly, in an intervention setting, the RENCA Foxp3 tumor cell vaccine was superior to the RENCA tumor cell vaccine with a significant anti-tumor activity (45% inhibition of tumor weights) (p&amp;lt;0.01). Moreover, RENCA Foxp3 tumor cell vaccine reduced the number of tumor infiltrating Tregs (42% reduction, p&amp;lt;0.05) and Treg Foxp3 expression both in peripheral blood and tumor infiltrates. In summary, our results suggest that a strategy targeting Tregs along with vaccine therapy or a dual targeting vaccine strategy may be beneficial in established RCC. We also observed that suppressive myeloid cell (SMCs) populations dominate the established tumor microenvironment and we are currently testing the combination of the dual-targeting vaccine with a SMC-targeting agent. Citation Format: Li Shen, Ashley Orillion, Remi Adelaiye, Eric Ciamporcero, Swathi Ramakrishnan, Roberto Pili. Activity of a novel Foxp3-tumor cell vaccine in a murine model of renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2514. doi:10.1158/1538-7445.AM2015-2514

Modulation of influenza vaccine immune responses using an epidermal growth factor receptor kinase inhibitor.

Systemic use of epidermal growth factor receptor inhibitors (EGFRIs) has been shown to alter MHC expression and that of several chemokines, and to enhance immune cell recruitment into human skin. We hypothesized that EGFRIs may have value as cutaneous immune response modifiers, and determined the effects of topical application of an irreversible EGFRI on a well-established murine model of influenza vaccination. We found that a single topical application of an EGFRI led to increased levels of antibodies that inhibit influenza mediated hemagglutination and viral cytopathic effects. The topically applied EGFRI significantly enhanced the generation of vaccine-specific IL-4 and IFN-γ producing cells within skin-draining lymph nodes as early as one week following vaccination. The EGFRI/vaccine group showed a twelve-fold reduction in detectable pulmonary viral load four days after infection as compared to the vaccine alone control group. The reduction in the lung viral titers correlated with the survival rate, which demonstrated 100% protection in the EGFRI/vaccine immunized group but only 65% protection in the mice immunized with vaccine alone. These findings are significant because they demonstrate that inhibition of defined signaling pathways within the skin using small molecule kinase inhibitors provides a novel approach to enhance immune responses to vaccines.

Chloroquine neither eliminates liver stage parasites nor delays their development in a murine Chemoprophylaxis Vaccination model.

Chemoprophylaxis Vaccination (CVac) confers long lasting sterile protection against homologous parasite strains in humans, and involves inoculation of infectious sporozoites (SPZ) under drug cover. CVac using the drug chloroquine (CQ) induces pre-erythrocytic immunity in humans that includes antibody to SPZ and T-cell responses to liver stage (LS) parasites. The mechanism by which CVac with CQ induces strong protective immunity is not understood as untreated infections do not confer protection. CQ kills blood stage parasites, but its effect on LS parasites is poorly studied. Here we hypothesized that CQ may prolong or perturb LS development of Plasmodium, as a potential explanation for enhanced pre-erythrocytic immune responses. Balb/c mice with or without CQ prophylaxis were infected with sporozoite forms of a luciferase-expressing rodent parasite, Plasmodium yoelii-Luc (Py-Luc). Mice that received primaquine, a drug that kills LS parasites, served as a positive control of drug effect. Parasite burden in liver was measured both by bioluminescence and by qRT-PCR quantification of parasite transcript. Time to appearance of parasites in the blood was monitored by microscopic analysis of Giemsa-stained thick and thin blood smears. The parasite load in livers of CQ-treated and untreated mice did not significantly differ at any of the time points studied. Parasites appeared in the blood smears of both CQ-treated and untreated mice 3 days after infection. Taken together, our findings confirm that CQ neither eliminates LS parasites nor delays their development. Further investigations into the mechanism of CQ-induced protection after CVac are required, and may give insights relevant to drug and vaccine development.

Plasmids encoding protein aggregation domains act as molecular adjuvants for DNA vaccines.

DNA vaccines provide high tolerability and safety but commonly suffer from suboptimal immunogenicity. We previously reported that a plasmid vector (pATRex), encoding the DNA sequence for the von Willebrand I/A domain of the tumor endothelial marker-8 (TEM8) when given in combination with plasmid-encoded tumor antigens acted as a powerful molecular adjuvant enhancing immunity against breast and melanoma tumors. In the present study we addressed two unsolved issues; would the adjuvant action of pATRex extend to a DNA vaccine against infectious disease and, if so, what is the mechanistic basis for pATRex adjuvant action? Here we show in a murine malaria vaccine model that co-administration of pATRex potentiates antibody production elicited by an intramuscular injection of plasmid encoding Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5). pATRex enhanced the B-cell response and induced increased IgG1 production consistent with TH2 polarization of the DNA vaccine response. To explore the mechanism of adjuvant action, cells were transfected in vitro with pATRex and this resulted in formation of insoluble intracellular aggregates and apoptotic cell death. Using a structural modeling approach we identified a short peptide sequence (α3-β4) within ATRex responsible for protein aggregation and confirmed that transfection of cells with plasmid encoding this self-assembling peptide similarly triggered intracellular aggregates, caspase activation and cell death. Plasmids encoding aggregation-promoting domains induce formation of insoluble intracellular aggregates that trigger caspase activation and apoptotic cell death leading to activation of the innate immune system thereby acting as genetic adjuvants.

Evaluation of efficacy of saponin and freund's incomplete adjuvanted paratuberculosis vaccine in murine model

Aim: subspp. (MAP) adjuvanted with saponin, Group II- for FIC, Group III- Saponin control and Gr IV- FIC control). Faecal shedding, changes in body weight, presence of local reactions and clinical symptoms, were evaluated. Serological assays for humoral and cell mediated immune response were determined by agar gel immunodiffusion (AGID) and nitric oxide test (NO) respectively. A total of 76 serum samples, 52 faecal samples and 20 tissue samples were collected at different intervals during the study period. ZN staining; AGID test and nitric oxide test were employed for analysis of samples. : A total of 38 samples (37 faecal and 1 tissue sample) and 18 serum samples were recorded as positive by ZN staining and AGID test respectively from all four groups of mice. Significant decrease in faecal shedding of organisms was observed in Group I mice vaccinated with saponin adjuvanted vaccine in comparison to Group II. Gain in body weight of Group I mice was recorded as higher than Group II. NO test revealed increase in the concentration of NO level in both vaccinated group but more significant in Group I. The tests results obtained revealed that killed MAP vaccine adjuvanted with saponin have higher protective efficacy in terms of reduced faecal shedding, gain in body weight, less side effects, absence of attributable specific symptoms of Johne's disease, etc. in comparison to killed MAP vaccine adjuvanted with FIC. The present study also reveals that AGID and NO estimation tests are specific tests which can be employed for diagnosis and comparison of efficacy of paratuberculosis vaccine. MAP ( subspecies

Inhibition of STAT6 during vaccination with formalin-inactivated RSV prevents induction of Th2-cell-biased airway disease.

The pattern of immune response to a vaccine antigen can influence both efficacy and adverse events. Th2-cell-deviated responses have been implicated in both human and murine susceptibility to enhanced disease following formalin-inactivated (FI) vaccines for measles and RSV. In this study, we used the Th2-cell-deviated murine model of FI-RSV vaccination to test the ability of a dominant negative, cell-penetrating peptide inhibitor of STAT6 (STAT6 inhibitory peptide (IP)) to modulate the vaccine-induced predisposition to exaggerated inflammation during later RSV infection. Intranasal delivery of STAT6-IP in BALB/c mice at the time of distal intramuscular FI-RSV vaccination (Early Intervention) markedly decreased vaccine-enhanced, Th2-cell-dependent pathology upon subsequent RSV challenge. Administration of the STAT6-IP at the time of RSV challenge (Late Intervention) had no effect. Following RSV challenge, the STAT6-IP-treated mice in the Early Intervention group had lower airway eosinophils, increased lung IFN-γ levels, as well as increased IFN-γ-secreting CD4(+) and CD8(+) cells in the lungs. Our findings demonstrate the feasibility of targeting intracellular signaling pathways as a new way to modulate vaccine-induced responses.

Mucosal adjuvant activity of IL-2 presenting spores of bacillus subtilis in a murine model of Helicobacter pylori vaccination.

The endospores of Bacillus subtilis are now widely used as a platform for presentation of heterologous proteins and due to their safety record and high resistance to harsh environmental conditions can be considered as potential vehicles for oral vaccination. In this research we show that recombinant B. subtilis spores presenting a fragment of the Helicobacter acinonychis UreB protein and expressing the ureB gene under vegetative promoter elicit a strong cellular immune response in orally immunized mice when co-administered with spores presenting IL-2. We show for the first time the successful application of two types of recombinant spores, one carrying an antigen and the other an adjuvant, in a single oral immunization.

In Vivo and In Vitro Characterization of the Immune Stimulating Activity of the Neisserial Porin PorB

Vaccines play a vital role in modern medicine. The development of novel vaccines for emerging and resistant pathogens has been aided in recent years by the use of novel adjuvants in subunit vaccines. A deeper understanding of the molecular pathways behind adjuvanticity is required to better select immunostimulatory molecules for use in individual vaccines. To this end, we have undertaken a study of the essential signaling pathways involved in the innate and adaptive immune responses to the Neisseria meningitidis outer membrane protein Porin B (PorB). We have previously demonstrated that PorB is an agonist of Toll-Like Receptor 2 (TLR2) and acts as an adjuvant in vaccines for protein, carbohydrate and lipopolysaccharide antigens using murine models. Here we demonstrate NFκB translocation following stimulation with PorB only occurs in the presence of TLR2. IL-6 and TNF-α secretion was shown to be MAPK dependent. Surface expression of activation markers on macrophages, including CD40, CD69, and CD86, was increased following PorB stimulation in vitro. Interestingly, some upregulation of CD54 and CD69 was still observed in macrophages obtained from TLR2 KO mice, indicating a possible non-TLR2 mediated activation pathway induced by PorB. In a murine vaccination model, using ovalbumin as the antigen and PorB as the adjuvant, a decreased antigen-specific IgG production was observed in TLR2 KO mice; adjuvant-dependent increased IgG production was entirely ablated in MyD88 KO mice. These observations demonstrate the importance of the above pathways to the adjuvant activity of PorB. The potential TLR2 independent effect is currently being explored.

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model

In this study, we investigated the potential molecular and immunological differences of a recombinant fusion protein (Hybrid-1), comprising of the immunodominant antigens Ag85B and ESAT-6 from Mycobacterium tuberculosis, derived from two different expression systems, namely Mycobacterium smegmatis and Escherichia coli. The fusion protein was successfully expressed and purified from both bacterial hosts and analyzed for any host-dependent post-translational modifications that might affect the immunogenicity of the protein. We investigated the immunogenicity of Hybrid-1 expressed in the two host species in a murine vaccination model, together with a reference standard Hybrid-1 (expressed in E. coli) from the Statens Serum Institut. No evidence of any post-translation modification was found in the M. smegmatis-derived Hybrid-1 fusion protein, nor were there any significant differences in the T-cell responses obtained to the three antigens analyzed. In conclusion, the Hybrid-1 fusion protein was successfully expressed in a homologous expression system using M. smegmatis and this system is worth considering as a primary source for vaccination trials, as it provided protein of excellent yield, stability and free from lipopolysaccharide.

Protective immunity using MPL-A and autoclaved Leishmania donovani as adjuvants along with a cocktail vaccine in murine model of visceral leishmaniasis

The current study is an extension of our previous study where we tested the protective efficacy of gp63 and Hsp70 against murine visceral leishmaniasis. The cocktail vaccine was combined with MPL-A and ALD adjuvants and the protection afforded by the three vaccines was compared. Inbred BALB/c mice were immunized twice at an interval of two weeks with the vaccine formulations. Two weeks after the booster, they were challenged with 10(7) promastigotes of Leishmania donovani and sacrificed on 30, 60 and 90 days post infection/challenge. The protective efficacy of vaccines was analyzed by assessment of the hepatic and splenic parasite burden and generation of cellular and humoral immune responses. The immunized animals revealed a significant reduction in parasite burden as compared to the infected controls. These animals also showed heightened DTH response, increased generation of IgG2a, IFN-γ and IL-2 by spleen cells. This was also accompanied by a decrease in the levels of IgG1 and IL-10. Mice immunized with gp63+Hsp70+MPL-A exhibited significantly greater protection in comparison to those immunized with gp63+Hsp70+ALD.

Regulation of TB Vaccine-Induced Airway Luminal T Cells by Respiratory Exposure to Endotoxin

Tuberculosis (TB) vaccine-induced airway luminal T cells (ALT) have recently been shown to be critical to host defense against pulmonary TB. However, the mechanisms that maintain memory ALT remain poorly understood. In particular, whether respiratory mucosal exposure to environmental agents such as endotoxin may regulate the size of vaccine-induced ALT population is still unclear. Using a murine model of respiratory genetic TB vaccination and respiratory LPS exposure, we have addressed this issue in the current study. We have found that single or repeated LPS exposure increases the number of antigen-specific ALT which are capable of robust secondary responses to pulmonary mycobacterial challenge. To investigate the potential mechanisms by which LPS exposure modulates the ALT population, we have examined the role of ALT proliferation and peripheral T cell recruitment. We have found that LPS exposure-increased ALT is not dependent on increased ALT proliferation as respiratory LPS exposure does not significantly increase the rate of proliferation of ALT. But rather, we find it to be dependent upon the recruitment of peripheral T cells into the airway lumen as blockade of peripheral T cell supplies markedly reduces the initially increased ALT. Thus, our data suggest that environmental exposure to airborne agents such as endotoxin has a profound modulatory effect on TB vaccine-elicited T cells within the respiratory tract. Our study provides a new, M.tb antigen-independent mechanism by which the respiratory mucosal anti-TB memory T cells may be maintained.