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Using a large animal model, we evaluated whether delivery of influenza vaccine via its mucosal site of infection could improve vaccine effectiveness. Unexpectedly, pulmonary immunization with extremely low antigen doses (0.04 μg influenza) induced serum antibody levels equivalent to those resulting from a current human vaccine equivalent (15 μg unadjuvanted influenza, subcutaneously) and vastly superior lung mucosal antibodies. Induction of this potent response following lung vaccination was dependent on addition of ISCOMATRIX adjuvant and deep lung delivery. Functional antibody activity, marked by hemagglutination inhibition, was only present in the lungs of animals that received adjuvanted vaccine via the lungs, suggesting this approach could potentially translate to improved protection. The 375-fold reduction in antigen dose and improved mucosal antibody responses, compared to the current vaccine, suggests that mucosal delivery via the pulmonary route may be particularly relevant in the event of an influenza pandemic, when vaccine supplies are unlikely to meet demand.Mucosal Immunology (2008) 1, 489–496; doi:10.1038/mi.2008.59; published online 24 September 2008

Using a large animal model, we evaluated whether delivery of influenza vaccine via its mucosal site of infection could improve vaccine effectiveness. Unexpectedly, pulmonary immunization with extremely low antigen doses (0.04 μg influenza) induced serum antibody levels equivalent to those resulting from a current human vaccine equivalent (15 μg unadjuvanted influenza, subcutaneously) and vastly superior lung mucosal antibodies. Induction of this potent response following lung vaccination was dependent on addition of ISCOMATRIX adjuvant and deep lung delivery. Functional antibody activity, marked by hemagglutination inhibition, was only present in the lungs of animals that received adjuvanted vaccine via the lungs, suggesting this approach could potentially translate to improved protection. The 375-fold reduction in antigen dose and improved mucosal antibody responses, compared to the current vaccine, suggests that mucosal delivery via the pulmonary route may be particularly relevant in the event of an influenza pandemic, when vaccine supplies are unlikely to meet demand.

A large-scale DNA vaccination trial was performed with sheep to investigate whether an antigen targeted by CTLA-4 enhanced and accelerated the humoral immune response. Vaccination with genetically detoxified phospholipase D (DeltaPLD) has been shown to be effective, at least partially, against Corynebacterium pseudotuberculosis, the causal agent of caseous lymphadenitis in sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used to direct the fusion antigens to sites of immune induction. Here we demonstrated that targeting DeltaPLD as a CTLA-4 fusion protein significantly enhanced the speed, magnitude, and longevity of the antibody response compared to that obtained with DNA encoding DeltaPLD. While all groups of sheep vaccinated with DNA encoding DeltaPLD were afforded better protection against an experimental challenge with C. pseudotuberculosis than those immunized with an irrelevant plasmid or those left unimmunized, the best protection was provided by the targeted DNA vaccine. We propose that targeting antigens to antigen-presenting cells offers a generic strategy for enhancing the efficacy of DNA vaccines.

This study was performed to test the hypothesis that anti-endometrial, lymphocyte-mediated cytotoxicity and natural killer (NK) activity are reduced in baboons with endometriosis when compared to animals with a normal pelvis. Lymphocyte-mediated cytotoxicity was determined in 28 baboons (15 with endometriosis, 13 with normal pelvis) and NK cell activity was evaluated in 42 baboons (31 with endometriosis, 11 with normal pelvis). Anti-endometrial lymphocyte-mediated cytotoxicity was determined by a 20 h assay with effector-target ratios of 50:1 and 25:1. The NK activity (K562 cell line as target) was simultaneously measured in all animals during a 4 h assay with effector:target ratios of 200:1, 100:1, 50:1, 25:1, 12:1, 6:1 and 3:1. Statistical analysis was performed using analysis of variance, paired rank, Mann-Whitney, Kruskal-Wallis and Fisher exact tests where appropriate. Lymphocyte-mediated cytotoxicity was significantly lower (P < 0.025) in baboons with endometriosis (mean 5.9 +/- 8.7%, median 0%, range 0-26%) than in animals with a normal pelvis (mean 22.9 +/- 23.0%, median 7%, range 0-78%). This difference could be explained by the absence of cytotoxicity in baboons with moderate to severe endometriosis, probably due to high spontaneous release of 51Cr from labelled target cells. When stricter criteria were used and only animals with a labelling index (maximal/spontaneous release) of > or = 1.7 were analysed (n = 11), the anti-endometrial cytotoxicity was comparable between baboons with and without endometriosis. NK cell activity was also comparable in primates with and without endometriosis. In conclusion, no difference in lymphocyte-mediated cytotoxicity and NK cell activity was observed between baboons with and without endometriosis.

Leishmania major promastigote surface antigen-2 complex (PSA-2) comprises a family of three similar but distinct polypeptides. The three PSA-2 polypeptides were purified from cultured promastigotes by a combination of detergent phase separation and monoclonal antibody affinity chromatography. Intraperitoneal vaccination of C3H/He mice with PSA-2 with Corynebacterium parvum as an adjuvant resulted in complete protection from lesion development after challenge infection with virulent L. major. Significant protection was also obtained in the genetically susceptible BALB/cH-2k and BALB/c mice. One of the PSA-2 genes was cloned and expressed in both Escherichia coli and Leishmania mexicana promastigotes. Vaccination with the recombinant PSA-2 purified from E. coli did not confer protection, in contrast to the L. mexicana-derived recombinant PSA-2, which provided excellent protection. CD4+ T cells isolated from the spleens of vaccinated mice produced large amounts of gamma interferon but no detectable interleukin 4 upon stimulation with PSA-2 in vitro. Limiting dilution analysis showed a marked increase in the precursor frequency of PSA-2-specific gamma interferon-secreting CD4+ T cells. No substantial change in precursor frequency was observed for interleukin 4-secreting T cells in the vaccinated mice. A CD4+ PSA-2 specific T-cell line generated from splenocytes of a vaccinated mouse produces a cytokine pattern consistent with a TH1 phenotype. Intravenous injection of this line into naive mice reduced significantly the parasite burden upon challenge infection. Taken together, the data suggest that vaccination with PSA-2 induces a TH1 type of immune response which protects mice from L. major infection. Moreover, a single recombinant PSA-2 polypeptide derived from a genomic clone can also vaccinate, provided that the structural form of the antigen is near native.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Leishmania major promastigote surface antigen-2 complex (PSA-2) comprises a family of three similar but distinct polypeptides. The three PSA-2 polypeptides were purified from cultured promastigotes by a combination of detergent phase separation and monoclonal antibody affinity chromatography. Intraperitoneal vaccination of C3H/He mice with PSA-2 with Corynebacterium parvum as an adjuvant resulted in complete protection from lesion development after challenge infection with virulent L. major. Significant protection was also obtained in the genetically susceptible BALB/cH-2k and BALB/c mice. One of the PSA-2 genes was cloned and expressed in both Escherichia coli and Leishmania mexicana promastigotes. Vaccination with the recombinant PSA-2 purified from E. coli did not confer protection, in contrast to the L. mexicana-derived recombinant PSA-2, which provided excellent protection. CD4+ T cells isolated from the spleens of vaccinated mice produced large amounts of gamma interferon but no detectable interleukin 4 upon stimulation with PSA-2 in vitro. Limiting dilution analysis showed a marked increase in the precursor frequency of PSA-2-specific gamma interferon-secreting CD4+ T cells. No substantial change in precursor frequency was observed for interleukin 4-secreting T cells in the vaccinated mice. A CD4+ PSA-2 specific T-cell line generated from splenocytes of a vaccinated mouse produces a cytokine pattern consistent with a TH1 phenotype. Intravenous injection of this line into naive mice reduced significantly the parasite burden upon challenge infection. Taken together, the data suggest that vaccination with PSA-2 induces a TH1 type of immune response which protects mice from L. major infection. Moreover, a single recombinant PSA-2 polypeptide derived from a genomic clone can also vaccinate, provided that the structural form of the antigen is near native.

This study was performed to test the hypothesis that anti-endometrial, lymphocyte-mediated cytotoxicity and natural killer (NK) activity are reduced in baboons with endometriosis when compared to animals with a normal pelvis. Lymphocyte-mediated cytotoxicity was determined in 28 baboons (15 with endometriosis, 13 with normal pelvis) and NK cell activity was evaluated in 42 baboons (31 with endometriosis, 11 with normal pelvis). Anti-endometrial lymphocyte-mediated cytotoxicity was determined by a 20 h assay with effector-target ratios of 50: 1 and 25: 1. The NK activity (K562 cell line as target) was simultaneously measured in all animals during a 4 h assay with effector:target ratios of 200: 1, 100: 1, 50: 1, 25: 1, 12: 1, 6: 1 and 3: 1. Statistical analysis was performed using analysis of variance, paired rank, Mann—Whitney, Kruskal—Wallis and Fisher exact tests where appropriate. Lymphocyte-mediated cytotoxicity was significantly lower (P < 0.025) in baboons with endometriosis (mean 5.9 ± 8.7 %, median 0%, range 0–26%) than in animals with a normal pelvis (mean 22.9 ± 23.0 %, median 7%, range 0–78 %). This difference could be explained by the absence of cytotoxicity in baboons with moderate to severe endometriosis, probably due to high spontaneous release of 51Cr from labelled target cells. When stricter criteria were used and only animals with a labelling index (maximal/spontaneous release) of ≥1.7 were analysed (n = 11), the anti-endometrial cytotoxicity was comparable between baboons with and without endometriosis. NK cell activity was also comparable in primates with and without endometriosis. In conclusion, no difference in lymphocyte-mediated cytotoxicity and NK cell activity was observed between baboons with and without endometriosis.