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Bacille-Calmette-Guerin (BCG) has variable efficacy as an adult tuberculosis (TB) vaccine but can reduce the incidence and severity of TB infection in humans. We have engineered modified vaccinia Ankara (MVA) strain vaccine constructs to express the secreted mycobacterial proteins Ag85A and ESAT-6 (MVA-AE) and evaluated their immunogenicity and protective efficacy as mucosal booster vaccines for BCG given subcutaneously in early life. Intranasal delivery of MVA-AE to young adult mice induced CD4(+) and CD8(+) T cell responses to both Ag85A and ESAT-6 in lung mucosae. These responses were markedly enhanced in mice that had been primed neonatally with BCG prior to intranasal MVA-AE immunization (BCG/MVA-AE), as evidenced by numbers of pulmonary Ag85A-, ESAT-6-, and PPD-specific CD4(+) and CD8(+) T cells and by their capacity to secrete multiple antimicrobial factors, including IFNγ, IL-2 and IL-17. Moreover, MVA-AE boosting generated multifunctional lung CD4(+) T cells responding to ESAT-6, which were not, as expected, detected in control mice given BCG, and elevated Ag85A-specific circulating antibody responses. After aerosol challenge with M. tuberculosis H37Rv (Mtb), the BCG/MVA-AE group had significantly reduced mycobacterial burden in the lungs, compared with either BCG primed mice boosted with control MVA or mice given only BCG. These data indicate that intranasal delivery of MVA-AE can boost BCG-induced Th1 and Th17-based immunity locally in the lungs and improve the protective efficacy of neonatally-administered BCG against M. tuberculosis infection.

Pneumocystis pneumonia is a life-threatening opportunistic fungal infection observed in individuals with severe immunodeficiencies, such as AIDS. Molecules with the ability to bind β-glucan and signal at Fcγ receptors enhance defense against Pneumocystis f. sp. murina, though it is unclear whether antibodies reactive with fungal cell wall carbohydrates are induced during Pneumocystis infection. We observed that systemic and lung mucosal immunoglobulins cross-reactive with β-glucan and chitosan/chitin are generated after Pneumocystis infection, with increased quantities within the lung mucosal fluid after challenge. While IgG responses against Pneumocystis protein antigens are markedly CD4+ T cell dependent, CD4+ T cell depletion did not impact quantities of IgG cross-reactive with β-glucan or chitosan/chitin in the serum or mucosa after challenge. Notably, lung mucosal quantities of IgA cross-reactive with β-glucan or chitosan/chitin are decreased in the setting of CD4+ T cell deficiency, occurring in the setting of concurrent reduced quantities of active transforming growth factor β, while mucosal IgM is significantly increased in the setting of CD4+ T cell deficiency. Interleukin-21 receptor deficiency does not lead to reduction in mucosal IgA reactive with fungal carbohydrate antigens after Pneumocystis challenge. These studies demonstrate differential CD4+ T cell-dependent regulation of mucosal antibody responses against β-glucan and chitosan/chitin after Pneumocystis challenge, suggesting that different B cell subsets may be responsible for the generation of these antibody responses, and suggest a potential immune response against fungi that may be operative in the setting of CD4+ T cell-related immunodeficiency.

Tuberculosis remains a major public health hazard worldwide, with neonates and young infants potentially more susceptible to infection than adults. BCG, the only vaccine currently available, provides some protection against tuberculous meningitis in children but variable efficacy in adults, and is not safe to use in immune compromised individuals. A safe and effective vaccine that could be given early in life, and that could also potentiate subsequent booster immunization, would represent a significant advance. To test this proposition, we have generated gene-based vaccine vectors expressing Ag85B from Mycobacterium tuberculosis (Mtb) and designed experiments to test their immunogenicity and protective efficacy particularly when given in heterologous prime-boost combination, with the initial DNA vaccine component given soon after birth. Intradermal delivery of DNA vaccines elicited Th1-based immune responses against Ag85B in neonatal mice but did not protect them from subsequent aerosol challenge with virulent Mtb H37Rv. Recombinant adenovirus vectors encoding Ag85B, given via the intranasal route at six weeks of age, generated moderate immune responses and were poorly protective. However, neonatal DNA priming following by mucosal boosting with recombinant adenovirus generated strong immune responses, as evidenced by strong Ag85B-specific CD4+ and CD8+ T cell responses, both in the lung-associated lymph nodes and the spleen, by the quality of these responding cells (assessed by their capacity to secrete multiple antimicrobial factors), and by improved protection, as indicated by reduced bacterial burden in the lungs following pulmonary TB challenge. These results suggest that neonatal immunization with gene-based vaccines may create a favorable immunological environment that potentiates the pulmonary mucosal boosting effects of a subsequent heterologous vector vaccine encoding the same antigen. Our data indicate that immunization early in life with mycobacterial antigens in an appropriate vaccine setting can prime for protective immunity against Mtb.

Pneumocystis pneumonia is a life-threatening opportunistic fungal infection observed in individuals with severe immunodeficiencies, such as AIDS. Molecules with the ability to bind β-glucan and signal at Fcγ receptors enhance defense against Pneumocystis f. sp. murina, though it is unclear whether antibodies reactive with fungal cell wall carbohydrates are induced during Pneumocystis infection. We observed that systemic and lung mucosal immunoglobulins cross-reactive with β-glucan and chitosan/chitin are generated after Pneumocystis infection, with increased quantities within the lung mucosal fluid after challenge. While IgG responses against Pneumocystis protein antigens are markedly CD4(+) T cell dependent, CD4(+) T cell depletion did not impact quantities of IgG cross-reactive with β-glucan or chitosan/chitin in the serum or mucosa after challenge. Notably, lung mucosal quantities of IgA cross-reactive with β-glucan or chitosan/chitin are decreased in the setting of CD4(+) T cell deficiency, occurring in the setting of concurrent reduced quantities of active transforming growth factor β, while mucosal IgM is significantly increased in the setting of CD4(+) T cell deficiency. Interleukin-21 receptor deficiency does not lead to reduction in mucosal IgA reactive with fungal carbohydrate antigens after Pneumocystis challenge. These studies demonstrate differential CD4(+) T cell-dependent regulation of mucosal antibody responses against β-glucan and chitosan/chitin after Pneumocystis challenge, suggesting that different B cell subsets may be responsible for the generation of these antibody responses, and suggest a potential immune response against fungi that may be operative in the setting of CD4(+) T cell-related immunodeficiency.

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

Impairment of host immunity, particularly CD4+ T cell deficiency, presents significant complications for vaccine immunogenicity and efficacy. CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor superfamily (TNFSF), is an important co-stimulatory molecule and, through interactions with its cognate receptor CD40, plays a pivotal role in the generation of host immune responses. Exploitation of CD40L and its receptor CD40 could provide a means to enhance and potentially restore protective immune responses in CD4+ T cell deficiency. To investigate the potential adjuvanticity of CD40L, we constructed recombinant plasmid DNA and adenoviral (Ad) vaccine vectors expressing murine CD40L and the mycobacterial protein antigen 85B (Ag85B). Co-immunization of mice with CD40L and Ag85B by intranasal or intramuscular prime-boosting led to route-dependent enhancement of the magnitude of vaccine-induced circulating and lung mucosal CD4+ and CD8+ T cell responses in both normal (CD4-replete) and CD4+ T cell deficient animals, including polyfunctional T cell responses. The presence of CD40L alone was insufficient to enhance or restore CD4+ T cell responses in CD4-ablated animals; however, in partially-depleted animals, co-immunization with Ag85B and CD40L was capable of eliciting enhanced T cell responses, similar to those observed in normal animals, when compared to those given vaccine antigen alone. In summary, these findings show that CD40L has the capacity to enhance the magnitude of vaccine-induced polyfunctional T cell responses in CD4+ T cell deficient mice, and warrants further study as an adjuvant for immunization against opportunistic pathogens in individuals with CD4+ T cell deficiency.

Mycobacterium tuberculosis H37Rv (Mtb) excludes phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) while preventing lysosomal fusion in macrophages (MPhis). The antigen 85A deficient (Delta fbpA) mutant of Mtb was vaccinogenic in mice and the mechanisms of attenuation were compared with MPhis infected with H37Rv and BCG. Delta fbpA contained reduced amounts of trehalose 6, 6, dimycolate and induced minimal levels of SOCS-1 in MPhis. Blockade of oxidants enhanced the growth of Delta fbpA in MPhis that correlated with increased colocalization with phox and iNOS. Green fluorescent protein-expressing strains within MPhis or purified phagosomes were analysed for endosomal traffick with immunofluorescence and Western blot. Delta fbpA phagosomes were enriched for rab5, rab11, LAMP-1 and Hck suggesting enhanced fusion with early, recycling and late endosomes in MPhis compared with BCG or H37Rv. Delta fbpA phagosomes were thus more mature than H37Rv or BCG although, they failed to acquire rab7 and CD63 preventing lysosomal fusion. Finally, Delta fbpA infected MPhis and dendritic cells (DCs) showed an enhanced MHC-II and CD1d expression and primed immune T cells to release more IFN-gamma compared with those infected with BCG and H37Rv. Delta fbpA was thus more immunogenic in MPhis and DCs because of an enhanced susceptibility to oxidants and increased maturation.

Flagellin has been tested as a protein-based vaccine adjuvant, with the majority of studies focused on antibody responses. Here, we evaluated the adjuvant activity of flagellin for both cellular and humoral immune responses in BALB/c mice in the setting of gene-based immunization, and have made several novel observations. DNA vaccines and adenovirus (Ad) vectors were engineered to encode mycobacterial protein Ag85B, with or without flagellin of Salmonella typhimurium (FliC). DNA-encoded flagellin given IM enhanced splenic CD4+ and CD8+ T cell responses to co-expressed vaccine antigen, including memory responses. Boosting either IM or intranasally with Ad vectors expressing Ag85B without flagellin led to durable enhancement of Ag85B-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with significantly improved protection against challenge with pathogenic aerosolized M. tuberculosis. However, inclusion of flagellin in both DNA prime and Ad booster vaccines induced localized pulmonary inflammation and transient weight loss, with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in levels of mucosal CD4+ and CD8+ T cell responses following IM DNA/IN Ad mucosal prime-boosting, although antibody responses were not diminished. These findings indicate that flagellin has differential and route-dependent adjuvant activity when included as a component of systemic or mucosally-delivered gene-based prime-boost immunization. Clear adjuvant activity for both T and B cell responses was observed when flagellin was included in the DNA priming vaccine, but side effects occurred when given in an Ad boosting vector, particularly via the pulmonary route.

Complement C5-deficient (C5−/−) macrophages derived from B.10 congenic mice were found to be defective in killing intracellular Mycobacterium tuberculosis (MTB). They were bacteriostatic after activation with IFN-γ alone but bactericidal in the combined presence of IFN-γ and C5-derived C5a anaphylatoxin that was deficient among these macrophages. Reduced killing correlated with a decreased production of reactive oxygen species (ROS) in the C5−/− macrophages measured using fluorescent probes. Furthermore, a lack of colocalization of p47phox protein of the NADPH oxidase (phox) complex with GFP-expressing MTB (gfpMTB) indicated a defective assembly of the phox complex on phagosomes. Reconstitution with C5a, a known ROS activator, enhanced the assembly of phox complex on the phagosomes as well as the production of ROS that inhibited the growth of MTB. Protein kinase C (PKC) isoforms are involved in the phosphorylation and translocation of p47phox onto bacterial phagosomes. Western blot analysis demonstrated a defective phosphorylation of PKC (α, β, δ) and PKC-ζ in the cytosol of C5−/− macrophages compared with C5 intact (C5+/+) macrophages. Furthermore, in situ fluorescent labeling of phagosomes indicated that PKC-β and PKC-ζ were the isoforms that are not phosphorylated in C5−/− macrophages. Because Fc receptor-mediated phox assembly was normal in both C5−/− and C5+/+ macrophages, the defect in phox assembly around MTB phagosomes was specific to C5 deficiency. Reduced bactericidal function of C5−/− macrophages thus appears to be due to a defective assembly and production of ROS that prevents effective killing of intracellular MTB.

Antigen three-dimensional structure potentially limits antigen processing and presentation to helper T-cell epitopes. The association of helper T-cell epitopes with the mobile loop in Hsp10s from mycobacteria and bacteriophage T4 suggests that the mobile loop facilitates proteolytic processing and presentation of adjacent sequences. Sites of initial proteolytic cleavage were mapped in divergent Hsp10s after treatment with a variety of proteases including cathepsin S. Each protease preferentially cleaved the Hsp10s in the mobile loop. Flexibility in the 22-residue mobile loop most probably allows it to conform to protease active sites. Three variants of the bacteriophage T4 Hsp10 were constructed with deletions in the mobile loop to test the hypothesis that shorter loops would be less sensitive to proteolysis. The two largest deletions effectively inhibited proteolysis by several proteases. Circular dichroism spectra and chemical cross-linking of the deletion variants indicate that the secondary and quaternary structures of the variants are native-like, and all three variants were more thermostable than the wild-type Hsp10. Local structural flexibility appears to be a general requirement for proteolytic sensitivity, and thus, it could be an important factor in antigen processing and helper T-cell epitope immunogenicity.

Antigen three-dimensional structure potentially limits the access of endoproteolytic processing enzymes to cleavage sites and of class II major histocompatibility antigen-presenting proteins to helper T-cell epitopes. Helper T-cell epitopes in bacteriophage T4 Hsp10 have been mapped by restimulation of splenocytes from CBA/J and C57BL/6J mice immunized in conjunction with mutant (R192G) heat-labile enterotoxin from Escherichia coli. Promiscuously immunogenic sequences were associated with unstable loops in the three-dimensional structure of T4 Hsp10. The immunodominant sequence lies on the N-terminal flank of the 22-residue mobile loop, which is sensitive to proteolysis in divergent Hsp10s. Several mobile loop deletions that inhibited proteolysis in vitro caused global changes in the helper T-cell epitope map. A mobile loop deletion that strongly stabilized the protein dramatically reduced the immunogenicity of the flanking immunodominant helper T-cell epitope, although the protein retained good overall immunogenicity. Antisera against the mobile loop deletion variants exhibited increased cross-reactivity, most especially the antisera against the strongly stabilized variant. The results support the hypothesis that unstable loops promote the presentation of flanking epitopes and suggest that loop deletion could be a general strategy to increase the breadth and strength of an immune response.

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulent M. tuberculosis H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β), in response to PPD, despite the demonstration of higher serum levels of TNF-α and TGF-β after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-β 4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance to M. tuberculosis , in part, by altering the cytokine profile to favor macrophage deactivation.