Enhancement of CD8+ T cell immunity in the lung by CpG oligodeoxynucleotides increases protective efficacy of a modified vaccinia Ankara vaccine against lethal poxvirus infection even in a CD4-deficient host

Immunostimulatory CpG oligodeoxynucleotides (ODN) have proven effective as adjuvants for protein-based vaccines, but their impact on immune responses induced by live viral vectors is not known. We found that addition of CpG ODN to modified vaccinia Ankara (MVA) markedly improved the induction of longer-lasting adaptive protective immunity in BALB/c mice against intranasal pathogenic vaccinia virus (Western Reserve; WR). Protection was mediated primarily by CD8(+) T cells in the lung, as determined by CD8-depletion studies, protection in B cell-deficient mice, and greater protection correlating with CD8(+) IFN-gamma-producing cells in the lung but not with those in the spleen. Intranasal immunization was more effective at inducing CD8(+) T cell immunity in the lung, and protection, than i.m. immunization. Addition of CpG ODN increased the CD8(+) response but not the Ab response. Depletion of CD4 T cells before vaccination with MVA significantly diminished protection against pathogenic WR virus. However, CpG ODN delivered with MVA was able to substitute for CD4 help and protected CD4-depleted mice against WR vaccinia challenge. This study demonstrates for the first time a protective adjuvant effect of CpG ODN for a live viral vector vaccine that may overcome CD4 deficiency in the induction of protective CD8(+) T cell-mediated immunity.     

Macrophages, CD4+ or CD8+ cells are each sufficient for protection against Chlamydia pneumoniae infection through their ability to secrete IFN-gamma

By using a T, B, or NK cell-deficient mouse strain (recombinase-activating gene (RAG)-1(-/-)/common cytokine receptor gamma-chain (gamma(C)R)), and T and B cell and IFN-gamma-deficient (RAG-1(-/-)/IFN-gamma(-/-)) mice, we have studied the generation of immunity against infection by Chlamydia pneumoniae. We found that IFN-gamma secreted by innate-cell populations protect against C. pneumoniae infection. However, NK cells were not needed for such IFN-gamma-dependent innate immune protection. Inoculation of wild type, but not IFN-gamma(-/-) bone marrow-derived macrophages protected RAG-1(-/-)/IFN-gamma(-/-) mice against C. pneumoniae infection. In line, pulmonary macrophages from RAG-1(-/-) C. pneumoniae-infected mice expressed IFN-gamma mRNA. Reconstitution of RAG-1(-/-)/gamma(c)R(-/-) or RAG-1(-/-)/IFN-gamma(-/-) mice with CD4(+) or CD8(+) cells by i.v. transfer of FACS sorted wild type spleen cells (SC) increased resistance to C. pneumoniae infection. On the contrary, no protection was observed upon transfer of IFN-gamma(-/-) CD4(+) or IFN-gamma(-/-) CD8(+) SC. T cell-dependent protection against C. pneumoniae was weaker when IFN-gammaR(-/-) CD4(+) or IFN-gammaR(-/-) CD8(+) SC were inoculated into RAG-1(-/-)/IFN-gamma(-/-) mice. Thus both nonlymphoid and T cell-derived IFN-gamma can play a central and complementary role in protection against C. pneumoniae. IFN-gamma secreted by nonlymphoid cells was not required for T cell-mediated protection against C. pneumoniae; however, IFN-gamma regulated T cell protective functions.     

Impaired protection against Mycobacterium bovis bacillus Calmette-Guerin infection in IL-15-deficient mice

To investigate the potential role of endogenous IL-15 in mycobacterial infection, we examined protective immunity in IL-15-deficient (IL-15(-/-)) mice after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or recombinant OVA-expressing BCG (rBCG-OVA). IL-15(-/-) mice exhibited an impaired protection in the lung on day 120 after BCG infection as assessed by bacterial growth. CD4(+) Th1 response capable of producing IFN-gamma was normally detected in spleen and lung of IL-15(-/-) mice on day 120 after infection. Although Ag-specific CD8 responses capable of producing IFN-gamma and exhibiting cytotoxic activity were detected in the lung on day 21 after infection with rBCG-OVA, the responses were severely impaired on days 70 and 120 in IL-15(-/-) mice. The degree of proliferation of Ag-specific CD8(+) T cells in IL-15(-/-) mice was similar to that in wild-type mice during the course of infection with rBCG-OVA, whereas sensitivity to apoptosis of Ag-specific CD8(+) T cells significantly increased in IL-15(-/-) mice. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung.     

DNA vaccines against chronic lung infections by Pseudomonas aeruginosa

Vaccines containing outer membrane protein F (OprF) of Pseudomonas aeruginosa are effective in reducing lesion severity in a mouse pulmonary chronic infection model. One OprF-based vaccine, called F/I, contains carboxy oprF sequences fused to oprI in an expression vector. When delivered three times biolistically by gene gun, the F/I vaccine induces protection that is antibody-mediated in outbred mice. To demonstrate the role of F/I-induced antibody-mediated immunity, B-cell-deficient [B(-)] and B-cell-intact [B(+)] mice were immunized with F/I, challenged with Pseudomonas, and examined for lesion severity. As expected, F/I-immunized B(+) mice had fewer and less severe lesions than vector-immunized B(+) mice. However, surprisingly, F/I- and vector-immunized B(-) mice were equally protected to levels similar to F/I-immunized B(+) mice. Examination of immune cell populations and cytokine levels indicated a relative increase in the quantity of CD3+ T-lymphocytes in vector- or F/I-immunized and challenged B(-) mice compared to B(+) mice. These data indicate the protective role played by cell-mediated immunity in B(-) mice, which supports our hypothesis that cell-mediated immunity can play an important role in protection against P. aeruginosa.     

Protective and memory immunity to Histoplasma capsulatum in the absence of IL-10

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10(-/-) mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-alpha or IFN-gamma, but not GM-CSF, abrogated protection in naive IL-10(-/-) mice; mAb to TNF-alpha, but not IFN-gamma or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10(-/-) mice was altered in those given mAb to IFN-gamma or TNF-alpha. More Gr-1(+) and Mac-3(+) cells were present in lungs of IL-10(-/-) mice given mAb to IFN-gamma, and treatment with mAb to TNF-alpha sharply reduced the number of CD8(+) cells in lungs of IL-10(-/-) mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3(+), CD44(high), CD62(low), and IFN-gamma(+) cells in IL-10(-/-) mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10(-/-) mice were required to mediate protection upon adoptive transfer into infected TCR alphabeta(-/-) mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-alpha and/or IFN-gamma. Memory cell generation was transiently increased in IL-10(-/-) mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.     

Recombinant attenuated Toxoplasma gondii expressing the Plasmodium yoelii circumsporozoite protein provides highly effective priming for CD8+ T cell-dependent protective immunity against malaria

The protozoan parasite Toxoplasma gondii elicits strong cell-mediated immunity against itself as well as nonspecific resistance against other pathogens and tumors. For this reason, we asked whether recombinant Toxoplasma could be utilized as an effective vaccine vehicle for inducing immunity against heterologous microbial infections. The circumsporozoite protein (PyCSP) of Plasmodium yoelii was engineered into a T. gondii temperature-sensitive strain (ts-4), a mutant that induces complete protection against virulent Toxoplasma challenge. When administered to mice in a single dose, a recombinant ts-4 (CSC3) that both secretes and expresses surface PyCSP induced strong anti-CSP Ab responses, with an isotype distribution pattern similar to that stimulated by the T. gondii carrier. When challenged with P. yoelii sporozoites during the first month after CSC3 vaccination, these animals displayed substantial levels of nonspecific resistance attributable entirely to the T. gondii carrier. Nevertheless, after the nonspecific protection had waned, high levels (up to 79%) of specific immunity against sporozoite challenge were achieved by boosting the animals with recombinant vaccinia virus expressing PyCSP. These CSC3-primed PyCSP-vaccinia-boosted mice displayed high frequencies of splenic PyCSP-specific IFN-gamma-producing cells, as well as CD8+ T cell-dependent cytolytic activity. In vivo depletion of CD8+ lymphocytes at the time of challenge completely ablated protective immunity in the T. gondii-primed/vaccinia-boosted animals, while neutralization of IFN-gamma or IL-12 caused a partial but significant reduction in resistance. Together these findings establish the efficacy of recombinant attenuated Toxoplasma as a vaccine vehicle for priming CD8+-dependent cell-mediated immunity.     

TCR vaccines against a murine T cell lymphoma: a primary role for antibodies of the IgG2c class in tumor protection

Tumor-associated proteins can act as effective immunotherapeutic targets. Immunization with tumor TCR protein conjugated to the immunogenic protein keyhole limpet hemocyanin (KLH) protects mice from tumor challenge with the murine T cell lymphoma C6VL. The immune mechanisms responsible for this tumor protection are of interest for designing more effective vaccine strategies. Previous studies using depletion experiments had suggested a CD8-mediated component of protection induced by TCR-KLH vaccines. In this study we used CD8alpha knockout, micro MT, and FcgammaR knockout mice to investigate the relative roles of CD8+ T cells and Ab in protective immunity induced by TCR-KLH immunization. We found that CD8+ T cells are not required for tumor protection, although they may contribute to protection. Vaccine-induced Abs are sufficient to mediate protection against this murine T cell lymphoma through an FcR-dependent mechanism. This was confirmed with Ab transfers, which protect challenged mice. Additionally, recombinase-activating gene 1(-/-) splenocytes can mediate Ab-dependent cellular cytotoxicity against this tumor in the presence of bound anti-TCR Abs. IFN-gamma knockout mice demonstrated a requirement for IFN-gamma, probably via generation of IgG2c Abs, in vaccine-induced tumor protection. IFN-gamma knockout mice were not protected by immunization and had a severe impairment in IgG2c Ab production in response to immunization. Although mock-depleted anti-TCR Abs could transfer tumor protection, IgG2c-deficient anti-TCR Abs were unable to transfer tumor protection to wild-type mice. These results suggest that TCR-KLH vaccine-induced tumor protection in the C6VL system is primarily attributable to the induction of IgG2c Abs and humoral immunity.     

Osteopontin is not required for the development of Th1 responses and viral immunity

Osteopontin (OPN) has been defined as a key cytokine promoting the release of IL-12 and hence inducing the development of protective cell-mediated immunity to viruses and intracellular pathogens. To further characterize the role of OPN in antiviral immunity, OPN-deficient (OPN-/-) mice were analyzed after infection with influenza virus and vaccinia virus. Surprisingly, we found that viral clearance, lung inflammation, and recruitment of effector T cells to the lung were unaffected in OPN-/- mice after influenza infection. Furthermore, effector status of T cells was normal as demonstrated by normal IFN-gamma production and CTL lytic activity. Moreover, activation and Th1 differentiation of naive TCR transgenic CD4+ T cells by dendritic cells and cognate Ag was normal in the absence of OPN in vitro. Contrary to a previous report, we found that OPN-/- mice mounted a normal immune response to Listeria monocytogenes. In conclusion, OPN is dispensable for antiviral immune responses against influenza virus and vaccinia virus.     

The complexity of protective immunity against liver-stage malaria

Sterile protective immunity against challenge with Plasmodium spp. sporozoites can be induced in multiple model systems and humans by immunization with radiation-attenuated Plasmodium spp. sporozoites. The infected hepatocyte has been established as the primary target of this protection, but the underlying mechanisms have not been completely defined. Abs, CD8+ T cells, CD4+ T cells, cytokines (including IFN-gamma and IL-12), and NO have all been implicated as critical effectors. Here, we have investigated the mechanisms of protective immunity induced by immunization with different vaccine delivery systems (irradiated sporozoites, plasmid DNA, synthetic peptide/adjuvant, and multiple Ag peptide) in genetically distinct inbred strains, genetically modified mice, and outbred mice. We establish that there is a marked diversity of T cell-dependent immune responses that mediate sterile protective immunity against liver-stage malaria. Furthermore, we demonstrate that distinct mechanisms of protection are induced in different strains of inbred mice by a single method of immunization, and in the same strain by different methods of immunization. These data underscore the complexity of the murine host response to a parasitic infection and suggest that an outbred human population may behave similarly. Data nevertheless suggest that a pre-erythrocytic-stage vaccine should be designed to induce CD8+ T cell- and IFN-gamma-mediated immune responses and that IFN-gamma responses may represent an in vitro correlate of pre-erythrocytic-stage protective immunity.     

Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses

In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss. CTL-mediated viral clearance was not dependent on IFN-gamma since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance. Depletion of IFN-gamma did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance. Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge. Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV. These studies emphasize that protective and immunoregulatory CD8(+) CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease.     

Protective and therapeutic efficacy study of divalent fusion protein rL7/L12-Omp25 against B. abortus 544 in presence of IFNγ

Brucella as intracellular pathogen requires a coordinate interaction between Th1 subset of gamma interferon-secreting CD4 T cells and CD8 T cells for optimal protective immunity. It was previously recognized that L7/L12 as T cell-reactive antigen from the pathogen. On other hand, Omp25 was found as another antigen to provide protection against the Brucella infection by eliciting both Th1 and Th2 type of immune responses in mice. Here, we analyzed the prophylactic and therapeutic efficacy of a divalent fusion protein (rL7/L12-Omp25) comprising these two promising immunogens of Brucella in the presence of murine IFN-gamma in mice against B. abortus 544 challenge. rIFN-gamma with rL7/L12-Omp25 resulted in superior immune response when compared to the animal vaccine strain B. abortus S19. The vaccine candidate caused dominance of IgG1 over IgG2a and upregulated cytokine secretion (IFN-gamma, TNF-α, and IL-10) among immunized mice. Moreover, the antigen in combination with murine IFN-gamma elicited stronger cell-mediated immune response among the immunized animals when compared to standard vaccine (S19). The registered log protection unit among challenged mice with B. abortus 544 pathogen was 2.16, p = 0.0001 when rL7/L12-Omp25 was administered alone and 2.4, p = 0.0001 when it was administered along with rIFN-gamma. However, the molecule upon administration with murine IFN-gamma imparted very minimal or no therapeutic effect against brucellosis. To conclude, our study demonstrates the potential of rL7/L12-Omp25 as an immunogen of prospective and efficient prophylaxis as it is capable of eliciting both cell-mediated and humoral immune responses against brucellosis.

     

IL-12 is required for induction but not maintenance of protective, memory responses to Blastomyces dermatitidis: implications for vaccine development in immune-deficient hosts

Cellular immunity mediated by T lymphocytes, in particular CD4(+) and CD8(+) type 1 (T1) cells, is the main defense against pathogenic fungi. IL-12 initiates T1 cell development and cell-mediated immunity, but it is unclear whether IL-12 contributes to the maintenance of an antifungal T1 response. In this study, we addressed the role of IL-12 for vaccine-induced memory T cell development against experimental pulmonary blastomycosis. CD4(+) T cells absolutely required IL-12 to control a live genetically engineered attenuated strain of Blastomyces dermatitidis given s.c. as a vaccine, whereas CD8(+) T cells were significantly less dependent on IL-12. Despite differential dependency of T cell subsets on IL-12 during vaccination, neither subset acquired memory immunity in the absence of IL-12. In contrast, adoptive transfer of immune CD4 T cells from wild-type mice into IL-12(-/-) mice showed that CD4(+) T1 memory cells sustained a T1 cytokine profile and remained protective over a period of 6 mo posttransfer. Similarly, memory CD8 cells elicited in IL-12(-/-) mice with killed yeast and transient rIL-12 treatment (during vaccination) remained durable and protective after animals were rested for 3 mo. In conclusion, these studies demonstrate that once CD4 and CD8 cells have acquired a protective T1 phenotype they no longer require the presence of IL-12 to maintain antifungal protective memory.     

Cutting Edge: IFN-gamma-producing CD4 T lymphocytes mediate spore-induced immunity to capsulated Bacillus anthracis

Virulent strains of Bacillus anthracis produce immunomodulating toxins and an antiphagocytic capsule. The toxin component-protective Ag is a key target of the antianthrax immune response that induces production of toxin-neutralizing Abs. Coimmunization with spores enhances the antitoxin vaccine, and inactivated spores alone confer measurable protection. We aimed to identify the mechanisms of protection induced in inactivated-spore immunized mice that function independently of the toxin/antitoxin vaccine system. This goal was addressed with humoral and CD4 T lymphocyte transfer, in vivo depletion of CD4 T lymphocytes and IFN-gamma, and Ab-deficient (muMT(-/-)) or IFN-gamma-insensitive (IFN-gammaR(-/-)) mice. We found that humoral immunity did not protect from nontoxinogenic capsulated bacteria, whereas a cellular immune response by IFN-gamma-producing CD4 T lymphocytes protected mice. These results are the first evidence of protective cellular immunity against capsulated B. anthracis and suggest that future antianthrax vaccines should strive to augment cellular adaptive immunity.     

Cutting edge: antilisterial activity of CD8+ T cells derived from TNF-deficient and TNF/perforin double-deficient mice

The mechanisms by which CD8+ T cells mediate immunity against bacterial pathogens remain largely unknown. Perforin-dependent cytolysis plays a role, but is not required for CD8+ T cell-mediated immunity against Listeria monocytogenes. TNF is essential for CD8+ T cell immunity to L. monocytogenes, but the cellular source of TNF is undefined. TNF-deficient and TNF/perforin double-deficient mice were used to generate CD8+ T cells specific for an L. monocytogenes-derived Ag. Wild-type and TNF-deficient CD8+ T cells mediated antilisterial immunity in wild-type but not TNF-deficient host mice, revealing that CD8+ T cell-derived TNF is not required for CD8+ T cell-mediated antilisterial immunity, but demonstrating a role for TNF derived from other cell types. TNF/perforin double-deficient CD8+ T cells mediated antilisterial immunity in the liver, but not in the spleen, of wild-type recipient mice, suggesting that perforin-independent immunity in the spleen requires CD8+ T cell-derived TNF.     

Effective DNA vaccination against listeriosis by prime/boost inoculation with the gene gun.

Protective immunity against Listeria monocytogenes strongly depends on CD8+ T lymphocytes, and both IFN-gamma secretion and target cell killing are considered relevant to protection. We analyzed whether we could induce a protective type 1 immune response by DNA vaccination with the gene gun using plasmids encoding for two immunodominant listerial Ags, listeriolysin and p60. To induce a Th1 response, we 1) coprecipitated a plasmid encoding for GM-CSF, 2) employed a prime/boost vaccination schedule with a 45-day interval, and 3) coinjected oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. DNA immunization of BALB/c mice with plasmids encoding for listeriolysin (pChly) and p60 (pCiap) efficiently induced MHC class I-restricted, Ag-specific CD8+ T cells that produced IFN-gamma. Coinjection of CpG-ODN significantly increased the frequency of specific IFN-gamma-secreting T cells. Although pChly induced specific CD8+ T cells expressing CTL activity, it failed to stimulate CD4+ T cells. Only pCiap induced significant CD4+ T cell and humoral responses, which were predominantly of Th2 type. Vaccination with either plasmid induced protective immunity against listerial challenge, and coinjection of CpG ODN improved vaccine efficacy in some situations. This study demonstrates the feasibility of gene gun administration of plasmid DNA for inducing immunity against an intracellular pathogen for which protection primarily depends on type 1 CD8+ T cells.     

IL-10 from regulatory T cells determines vaccine efficacy in murine Leishmania major infection

Leishmaniasis affects 12 million people, but there are no vaccines. Immunological correlates of vaccine efficacy are unclear. Polarized Th1 vs Th2 responses in Leishmania major-infected mice suggested that a shift in balance from IL-4 to IFN-gamma was the key to vaccine success. Recently, a role for IL-10 and regulatory T cells in parasite persistence was demonstrated, prompting re-evaluation of vaccine-induced immunity. We compared DNA/modified vaccinia virus Ankara heterologous prime-boost with Leishmania homolog of the receptor for activated C kinase (LACK) or tryparedoxin peroxidase (TRYP). Both induced low IL-4 and high IFN-gamma prechallenge. Strikingly, high prechallenge CD4 T cell-derived IL-10 predicted vaccine failure using LACK, whereas low IL-10 predicted protection with TRYP. The ratio of IFN-gamma:IL-10 was thus a clear prechallenge indicator of vaccine success. Challenge infection caused further polarization to high IL-10/low IFN-gamma with LACK and low IL-10/high IFN-gamma with TRYP. Ex vivo quantitative RT-PCR and in vitro depletion and suppression experiments demonstrated that Ag-driven CD4+ CD25+ T regulatory 1-like cells were the primary source of IL-10 in LACK-vaccinated mice. Anti-IL-10R treatment in vivo demonstrated that IL-10 was functional in determining vaccine failure, rendering LACK protective in the presence of high IFN-gamma/low IL-5 responses.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.     

Interleukin-23 restores immunity to Mycobacterium tuberculosis infection in IL-12p40-deficient mice and is not required for the development of IL-17-secreting T cell responses

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-gamma and their recruitment to the site of infection. The development of more efficient vaccines against tuberculosis requires detailed understanding of the induction and maintenance of T cell immunity. Cytokines important for the development of cell-mediated immunity include IL-12 and IL-23, which share the p40 subunit and the IL-12Rbeta1 signaling chain. To explore the differential effect of IL-12 and IL-23 during M. tuberculosis infection, we used plasmids expressing IL-23 (p2AIL-23) or IL-12 (p2AIL-12) alone in dendritic cells or macrophages from IL-12p40(-/-) mice. In the absence of the IL-12/IL-23 axis, immunization with a DNA vaccine expressing the M. tuberculosis Ag85B induced a limited Ag-specific T cell response and no control of M. tuberculosis infection. Co-delivery of p2AIL-23 or p2AIL-12 with DNA85B induced strong proliferative and IFN-gamma-secreting T cell responses equivalent to those observed in wild-type mice immunized with DNA85B. This response resulted in partial protection against aerosol M. tuberculosis; however, the protective effect was less than in wild-type mice owing to the requirement for IL-12 or IL-23 for the optimal expansion of IFN-gamma-secreting T cells. Interestingly, bacillus Calmette-Guérin immune T cells generated in the absence of IL-12 or IL-23 were deficient in IFN-gamma production, but exhibited a robust IL-17 secretion associated with a degree of protection against pulmonary infection. Therefore, exogenous IL-23 can complement IL-12 deficiency for the initial expansion of Ag-specific T cells and is not essential for the development of potentially protective IL-17-secreting T cells.     

Antigen-specific CD4+ T cells produce sufficient IFN-gamma to mediate robust protective immunity against genital Chlamydia muridarum infection

Chlamydia has been shown to evade host-specific IFN-gamma-mediated bacterial killing; however, IFN-gamma-deficient mice exhibit suboptimal late phase vaginal Chlamydia muridarum clearance, greater dissemination, and oviduct pathology. These findings introduce constraints in understanding results from murine chlamydial vaccination studies in context of potential implications to humans. In this study, we used mice deficient in either IFN-gamma or the IFN-gamma receptor for intranasal vaccination with a defined secreted chlamydial Ag, chlamydial protease-like activity factor (CPAF), plus CpG and examined the role of IFN-gamma derived from adoptively transferred Ag-specific CD4+ T cells in protective immunity against genital C. muridarum infection. We found that early Ag-specific IFN-gamma induction and CD4+ T cell infiltration correlates with the onset of genital chlamydial clearance. Adoptively transferred IFN-gamma competent CPAF-specific CD4+ T cells failed to enhance the resolution of genital chlamydial infection within recipient IFN-gamma receptor-deficient mice. Conversely, IFN-gamma production from adoptively transferred CPAF-specific CD4+ T cells was sufficient in IFN-gamma-deficient mice to induce early resolution of infection and reduction of subsequent pathology. These results provide the first direct evidence that enhanced anti-C. muridarum protective immunity induced by Ag-specific CD4+ T cells is dependent upon IFN-gamma signaling and that such cells produce sufficient IFN-gamma to mediate the protective effects. Additionally, MHC class II pathway was sufficient for induction of robust protective anti-C. muridarum immunity. Thus, targeting soluble candidate Ags via MHC class II to CD4+ T cells may be a viable vaccine strategy to induce optimal IFN-gamma production for effective protective immunity against human genital chlamydial infection.     

Lymphatic tracing and T cell responses following oral vaccination with live Mycobacterium bovis (BCG)

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis bacille Calmette-Guérin (BCG) expands a subset of interferon-gamma (IFN-gamma)-secreting T cells and mediates protection against aerosol mycobacterial challenge. We have traced the movement of the live vaccine through the regional lymphatics of mice and monitored the resultant immune response. Six hours after oral vaccination BCG was detected in low numbers systemically and in draining lymphatic tissue. However, after 48 h, BCG was predominantly associated with alimentary tract lymphatic tissues, such as the cervical and mesenteric lymph nodes and Peyer's patches. Lymphocytes that produced IFN-gamma in response to PPD-B or BCG-pulsed dendritic cells predominated in the spleen and were almost exclusively CD4(+), CD44(+) and CD62L(-), thus resembling an effector memory T cell population. Despite the fact that an oral route was used for immunization, splenic IFN-gamma-secreting T cells in vaccinated mice did not express the mucosal homing antigens alpha(4)beta(7) integrin or alphaIEL (CD103). However, a proportion of BCG-specific CD4(+) T cells expressed the CD29 integrin (beta(1)) chain, potentially involved in lung homing function. Thus, oral priming with M. bovis BCG appears to induce a subset of spleen-resident CD4(+) T cells with the potential to provide protective immunity in the lung.