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An important aspect of malaria vaccine development is the identification of an appropriate adjuvant which is both capable of stimulating a protective immune response and safe for use by humans. Here, we investigated the feasibility of using novel immunostimulatory molecules as adjuvants combined with a crude antigen preparation and coadsorbed to aluminum hydroxide (alum) as a vaccine against blood-stage Plasmodium chabaudi AS malaria. Prior to challenge infection, immunization of genetically susceptible A/J mice with the combination of malaria antigen plus recombinant interleukin-12 (IL-12) in alum induced a Th1 immune response with production of high levels of gamma interferon (IFN-gamma) and diminished IL-4 levels by spleen cells stimulated in vitro with parasite antigen compared to mice immunized with antigen alone, antigen in alum, or antigen plus IL-12. Mice immunized with malaria antigen plus recombinant IL-12 in alum had high levels of total malaria-specific antibody and immunoglobulin G2a. Compared to unimmunized mice, immunization with antigen plus IL-12 in alum induced the highest level of protective immunity against challenge infection with P. chabaudi AS, which was evident as a significantly decreased peak parasitemia level and 100% survival. Protective immunity was dependent on CD4(+) T cells, IFN-gamma, and B cells and was long-lasting. Replacement of IL-12 as an adjuvant by synthetic oligodeoxynucleotides (ODN) containing CpG motifs induced a similar level of vaccine-induced protection against challenge infection with P. chabaudi AS. These results illustrate that it is possible to enhance the potency of a crude malaria antigen preparation delivered in alum by inclusion of immunostimulatory molecules, such as IL-12 or CpG-ODN.

Interleukin-10 (IL-10) is thought to play an important role in the regulation of microbial immunity. While T-cell-derived IL-10 has been shown to suppress cell-mediated immunity, there has been debate as to whether antigen presenting cell (APC)-derived cytokine can perform the same function in vivo. To assess the influence of APC-produced IL-10 on host resistance to mycobacterial infection, transgenic mice expressing human IL-10 under the control of the major histocompatibility complex class II promoter (hu10Tg) were infected with Mycobacterium avium, and bacterial burdens and immune responses were compared with those observed in wild-type (wt) animals. Hu10Tg mice harbored substantially higher numbers of M. avium and succumbed 16 to 18 weeks postinfection. The granulomas in infected hu10Tg mice showed marked increases in both acid-fast bacilli and host macrophages. In addition, these animals displayed a dramatic increase in hepatic fibrosis. The increased susceptibility of the hu10Tg mice to M. avium infection is independent of T-cell-produced endogenous murine IL-10, since bacterial burdens in mice derived by crossing hu10Tg mice with murine IL-10-deficient mice were not significantly different from those in hu10Tg mice. Importantly, gamma interferon (IFN-gamma) responses were not decreased in the infected transgenic animals from those in wt animals, suggesting the normal development of Th1 effector cells. In contrast, mycobacterium-induced macrophage apoptosis as well as production of TNF, nitric oxide, and IL-12p40 were strongly inhibited in hu10Tg mice. Together, these data indicate that APC-derived IL-10 can exert a major inhibitory effect on control of mycobacterial infection by a mechanism involving the suppression of macrophage effector function and apoptosis.

Vaccination of mice with yeast-secreted Plasmodium yoelii-derived 19-kilodalton merozoite surface protein 1 (yMSP1(19)) has been shown to afford protection from challenge with a lethal strain of P. yoelii. Sterile immunity can be achieved when MSP1(19) is emulsified in Freund adjuvant but not when it is adsorbed to aluminum hydroxide gel (alum). Because complete Freund adjuvant is not an acceptable adjuvant for use in humans, alternative adjuvants must be identified for formulating MSP1(19) as a vaccine for use in humans. To determine whether oligodeoxynucleotides with CpG motifs (ODN), reported to be a powerful new class of adjuvants, could enhance the immunogenicity of yMSP1(19), C57BL/6 mice were vaccinated either with yMSP1(19) formulated with Freund adjuvant, with alum, or with ODN plus alum and challenged intravenously with P. yoelii 17XL asexual blood-stage parasites. Adsorption of immunogen and adjuvant to alum was optimized by adjusting buffer (phosphate versus acetate) and pH. We found that the adjuvant combination of ODN plus alum with yMSP1(19), injected intraperitoneally (i.p.), increased immunoglobulin G (IgG) yMSP1(19)-specific antibody production 12-fold over Freund adjuvant given i.p., 3-fold over Freund adjuvant given subcutaneously (s.c.), 300-fold over alum given i.p., and 48-fold over alum given s.c. The predominant antibody isotype in the group receiving alum-ODN-yMSP1(19) was IgG1. Increased antibody levels correlated to protection from a challenge with P. yoelii 17XL. Supernatant cytokine levels of gamma interferon in yMSP1(19)-stimulated splenocytes were dramatically elevated in the alum-ODN-yMSP1(19) group. Interleukin-10 (IL-10) levels were also elevated; however, no IL-5 was detected. The cytokine profile, as well as the predominant IgG1 antibody isotype, suggests the protective immune response was a mixed Th1/Th2 response.

We have previously shown that specific-pathogen-free interleukin-10 (IL-10)-deficient (IL-10 KO) mice reconstituted with Helicobacter hepaticus develop severe colitis associated with a Th1-type cytokine response. In the present study, we formally demonstrate that IL-12 is crucial for disease induction, because mice deficient for both IL-10 and IL-12 p40 show no intestinal pathology following H. hepaticus infection. By using monoclonal antibodies (MAbs) to IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), we have further analyzed the role of these cytokines in the maintenance of the Th1 response and inflammation in IL-10 KO mice with established H. hepaticus-induced colitis. Treatment of infected colitic IL-10 KO mice with anti-IL-12 p40 resulted in markedly reduced intestinal inflammation, colonic IFN-gamma, TNF-alpha, and inducible nitric oxide synthase (iNOS) mRNA levels, and H. hepaticus-specific IFN-gamma secretion by mesenteric lymph node (MLN) cells compared to the findings in control MAb-treated mice. Moreover, the diminished pathology was associated with decreased numbers of colonic CD3(+) T cells and significantly reduced frequencies of Helicobacter-reactive CD4(+) Th1 cells in MLN. In contrast, anti-IFN-gamma and/or anti-TNF-alpha had no effect on intestinal inflammation in IL-10 KO mice with established colitis. Using IL-10/IFN-gamma double-deficient mice, we further show that IFN-gamma is not required for the development of colitis following H. hepaticus infection. MLN cells from infected IL-10/IFN-gamma KO animals secreted elevated amounts of IL-12 and TNF-alpha following bacterial antigen stimulation, indicating alternative pathways of disease induction. Taken together, our results demonstrate a crucial role for IL-12 in both inducing and sustaining intestinal inflammation through recruitment and maintenance of a pool of pathogenic Th1 cells.

We present here a novel approach to identify T-cell antigens from any infectious agent by use of a library of purified recombinant proteins. Essential features of this strategy include (i) a highly efficient cDNA cloning system which negatively selects against nonrecombinant transformants by making use of the bacterial EcoK restriction system, (ii) affinity staining of cDNA clones expressing recombinant proteins, and (iii) a procedure of simultaneous purification of recombinant proteins from large numbers of isolated clones (representing the protein library) in a single step from pools consisting of up to 24 individual clones. The feasibility of the screening system was confirmed by constructing a protein library of the human parasite Schistosoma mansoni. The recombinant antigens of this library were used to stimulate CD4(+) T cells derived from the axillary lymph nodes of mice vaccinated with irradiated cercariae. In initial screening experiments, we detected parasite-specific proliferation and gamma interferon (IFN-gamma) secretion in response to several pools of cDNA clones. Further analysis of one particular pool revealed that only one of its constituents stimulated considerable IFN-gamma secretion by CD4(+) T cells and that the expressed antigen is identical to a small fragment of myosin heavy chain.

Mice rendered deficient in interleukin-10 (IL-10) by gene targeting (IL-10(-/-) mice) develop chronic enterocolitis resembling human inflammatory bowel disease (IBD) when maintained in conventional animal facilities. However, they display a minimal and delayed intestinal inflammatory response when reared under specific-pathogen-free (SPF) conditions, suggesting the involvement of a microbial component in pathogenesis. We show here that experimental infection with a single bacterial agent, Helicobacter hepaticus, induces chronic colitis in SPF-reared IL-10(-/-) mice and that the disease is accompanied by a type 1 cytokine response (gamma interferon [IFN-gamma], tumor necrosis factor alpha, and nitric oxide) detected by restimulation of spleen and mesenteric lymph node cells with a soluble H. hepaticus antigen (Ag) preparation. In contrast, wild-type (WT) animals infected with the same bacteria did not develop disease and produced IL-10 as the dominant cytokine in response to Helicobacter Ag. Strong H. hepaticus-reactive antibody responses as measured by Ag-specific total immunoglobulin G (IgG), IgG1, IgG2a, IgG2b, IgG3, and IgA were observed in both WT and IL-10(-/-) mice. In vivo neutralization of IFN-gamma or IL-12 resulted in a significant reduction of intestinal inflammation in H. hepaticus-infected IL-10(-/-) mice, suggesting an important role for these cytokines in the development of colitis in the model. Taken together, these microbial reconstitution experiments formally establish that a defined bacterial agent can serve as the immunological target in the development of large bowel inflammation in IL-10(-/-) mice and argue that in nonimmunocompromised hosts IL-10 stimulated in response to intestinal flora is important in preventing IBD.

The granulomatous response to schistosome eggs is a CD4 T-cell-dependent, Th2-cytokine-dominated immunopathologic response. As infection proceeds to chronicity, both granuloma formation and egg-induced cytokine production become downregulated, and previous experiments have implicated CD8 T cells in this process. One mechanism by which CD8 T cells could suppress immunopathology is through the production of the counterregulatory cytokine gamma interferon (IFN-gamma), but no in vivo evidence exists to directly support this hypothesis. In this study, we analyzed hepatic granuloma formation and egg-induced cytokine production in Schistosoma mansoni-infected gene knockout mice deficient in either CD8 lymphocytes or IFN-gamma. Surprisingly, we found that neither immunologic component plays an essential function in the control of granuloma and cytokine responses during either the acute or chronic stage of infection. Thus, other mechanisms may be more important in the regulation of immunopathology in schistosomiasis.