Vaccine requirements for sustained cellular immunity to an intracellular parasitic infection.

The humoral immunity induced by many viral and bacterial vaccines mediates protection that is maintained over a long period of time. In contrast, for other intracellular infections (such as with Leishmania major or Mycobacterium tuberculosis) for which cell-mediated immunity is required for protection, the mechanisms for developing durable responses after vaccination have not been well defined. Here we demonstrate that vaccination with plasmid DNA encoding a specific leishmanial antigen is more effective than leishmanial protein plus recombinant IL-12 in eliciting long-term immunity capable of controlling L. major infection. We also show that leishmanial protein plus IL-12 DNA produces an immunity that lasts much longer than does immunity elicited by leishmanial protein plus IL-12 protein, indicating that the persistence of IL-12 may be the essential determinant in maintaining durable cell-mediated immune responses for an intracellular parasitic infection.

CD40 ligand is not essential for induction of type 1 cytokine responses or protective immunity after primary or secondary infection with histoplasma capsulatum.

The induction of type 1 immune responses (interleukin [IL]-12, interferon [IFN]-gamma) has been shown to be important in mediating protection against many intracellular infections including Histoplasma capsulatum. Costimulatory molecules such as CD40 ligand (CD40L) have been shown to be a central regulator of type 1 responses in vivo. To study the role of CD40L in mediating protection against infection with H. capsulatum, CD40L-deficient (CD40L-/-) and CD40L+/+ mice were infected with H. capsulatum and assessed for various parameters. After a lethal challenge of H. capsulatum, CD40L-/- mice were not substantially different from CD40L+/+ mice in terms of mortality, fungal burden, or production of IFN-gamma, IL-12, nitric oxide, or tumor necrosis factor alpha. Moreover, CD40L-/- mice treated with anti-IFN-gamma or anti-IL-12 at the time of infection had accelerated mortality, providing further evidence that IL-12 and IFN-gamma are produced in vivo in the absence of CD40L. In addition, CD40L-/- mice infected with a sublethal dose of H. capsulatum survived infection, whereas all mice infected with the same dose and treated with anti-IFN-gamma had accelerated mortality, demonstrating that IFN-gamma but not CD40L was essential for primary immunity to H. capsulatum infection. Interestingly, depletion of either CD4+ or CD8+ T cells resulted in accelerated mortality in CD40L-/- mice, suggesting a critical role for these cells in response to infection. Finally, CD40L-/- mice initially infected with a sublethal dose of H. capsulatum were protected from secondary infection with a lethal dose of H. capsulatum, demonstrating that CD40L is not required for the maintenance of memory immunity.

Activation events during thymic selection.

During their differentiation in the mouse thymus, CD4+8- cells undergo several of the sequential changes observed upon normal activation of mature, peripheral CD4+ lymphocytes. Expression of CD69, an early activation marker, is first observed on a minority of cells at the T cell receptor (TCR)lo/med double-positive stage, is maximal (50-90%) on heat-stable antigen (HSA)hi TCRhi double-positive, HSAhi TCRmed CD4+8lo, and HSAhi TCRhi CD4+8- cells, and is downmodulated at the mature HSAlo CD4+8- stage. In contrast, CD44, a late activation marker, is selectively expressed at the HSAlo stage. The set of lymphokines that CD4+8- thymocytes can produce upon stimulation also characteristically expands from mainly interleukin 2 (IL-2) at the HSAhi stage, to IL-2 and very large amounts of IL-4, IL-5, IL-10, and interferon gamma (IFN-gamma) at the HSAlo stage. 1 in 30 HSAlo CD4+8- adult thymocytes secrete IL-4 upon stimulation through their TCR. This frequency is 25% of the frequency of IL-2 producers, about 100-fold above that of peripheral (mainly resting) CD4+ T cells. With time after their generation in organ culture, CD4+8- thymocytes lose their capacity to secrete IL-4, IL-5, and IFN-gamma, but not IL-2. Similarly, the frequency of IL-4, but not of IL-2, producers progressively decreases after emigration to the periphery as judged by direct comparison between thymic and splenic CD4+ cells in newborns, or by following the fate of intrathymically labeled CD4+8- cells in adults after their migration to the spleen. This sequence suggests that thymic selection results from an activation process rather than a simple rescue from death at the double-positive stage, and shows that the functional changes induced after intrathymic activation, although transient, are still evident after export to the periphery.

The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice.

To study the factors that determine whether CD4+ T cells produce interleukin 4 (IL-4) or interferon gamma (IFN-gamma) upon stimulation we used a system allowing naive T cells to be primed in vitro by specific antigen. Dense CD4+ T cells were purified from mice that expressed transgenes encoding a T cell receptor specific for pigeon cytochrome C peptide 88-104 in association with I-Ek. These T cells produced very limited amounts of IL-4 and IFN-gamma upon immediate challenge with 88-104 and antigen-presenting cells (APC). However, after an initial "priming" culture in which they were incubated for 4 d in the presence of 88-104, APC, and 1,000 U/ml IL-4, the T cells acquired the capacity to produce substantial amounts of IL-4 upon rechallenge but made very little IFN-gamma. Cells primed in the absence of IL-4 produced IFN-gamma upon rechallenge but virtually no IL-4. The inhibitory effect of IL-4 on IFN-gamma production did not appear to be mediated by the induction of IL-10 production since IL-10 addition to initial cultures did not suppress priming for IFN-gamma production, nor did anti-IL-10 block the inhibitory effect of IL-4. IFN-gamma itself did not increase priming for IFN-gamma production, nor did anti-IFN-gamma reduce such priming. IFN-gamma did, however, diminish priming for IL-4 production when limiting amounts of IL-4 (100 U/ml) were used in the initial culture. The dominant effect of IL-4 in determining the lymphokine-producing phenotype of primed cells was observed with dendritic cells (DC), activated B cells, and I-Ek-transfected fibroblasts as APC. However, the different APC did vary in their potency, with DC being superior to activated B cells, which were superior to transfected fibroblasts.

CD28-mediated costimulation of interleukin 2 (IL-2) production plays a critical role in T cell priming for IL-4 and interferon gamma production.

Naive T cells require interleukin 4 (IL-4) to develop into IL-4-producing T cells and IL-4 blocks development of such cells into interferon gamma (IFN-gamma) producers. Prior studies in accessory cell-independent priming systems using antireceptor antibodies as agonists have demonstrated that IL-2 is also necessary for the development of IL-4-producing cells under these culture conditions. Here we have examined the role of IL-2 and the CD28 costimulation pathway in priming for IL-4 and IFN-gamma production using a more physiologic model. This involved antigen presentation by accessory cells to naive CD4+ T cells from transgenic mice whose cells express a T cell receptor (TCR) specific for a cytochrome c peptide in association with I-Ek. With splenic antigen-presenting cells (APCs), inhibition of CD28 costimulation by the fusion protein CTLA4-immunoglobulin (Ig) blocked effective priming. Similarly, transfected fibroblasts expressing both MHC class II and the CD28 ligand B7 could prime for IL-4 production and such priming also was blocked by CTLA4-Ig. However, APCs deficient in CD28 ligands also could prime TCR transgenic T cells to become IL-4 producers if an exogenous source of IL-2, as well as IL-4, was provided, and the inhibition of priming seen with splenic or transfected fibroblast APCs in the presence of CTLA4-Ig could be reversed by addition of IL-2. Likewise, priming for IFN-gamma production could be blocked by CTLA4-Ig and reversed by IL-2. Thus, we conclude that IL-2 plays a critical role in priming naive CD4+ T cells to become IL-4 or IFN-gamma producers. Engagement of the CD28 pathway, although normally important in such priming, is unnecessary in the presence of exogenous IL-2.

Cytokine interactions in human immunodeficiency virus-infected individuals: roles of interleukin (IL)-2, IL-12, and IL-15.

Cytokines have been shown to be powerful regulators of the immune response. In this study, we analyze the effect that the newly recognized cytokine interleukin (IL)-15 has on proliferation and cytokine induction using peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells from patients infected with human immunodeficiency virus (HIV) who are at various stages in their disease. We observed that IL-15 enhances the proliferative response in a dose-dependent manner from PBMCs of HIV-infected individuals when stimulated by polyclonal mitogen, tetanus toxoid, or HIV-specific antigen. The effects of exogenous IL-15 are substantially diminished by adding a neutralizing antibody to the beta chain of the IL-2 receptor. Moreover, the ability of IL-15 to increase proliferation is enhanced by the presence of endogenous IL-2 produced in the cultures. The effect that exogenous IL-15 had on IL-2, IL-4, and interferon (IFN)-gamma induction from PBMC's or CD4+ T cells in response to mitogen or tetanus toxoid was also examined. This was compared to the effect that exogenous IL-2 and IL-12 had under the same conditions. Addition of IL-2 or IL-15 to short-term in vitro cultures of either PBMCs or CD4+ T cells had little effect on IL-2, IL-4, or IFN-gamma production. By contrast, IL-12 caused substantial enhancement of both IL-2 and IFN-gamma production from these cultures. The role that endogenous cytokines have on IFN-gamma induction was also studied. Addition of a neutralizing antibody to the alpha chain of the IL-2 receptor or IL-12 to antigen stimulated cultures caused a striking decrease in IFN-gamma production. Neutralization of endogenous IL-15 also resulted in diminished IFN-gamma production from cultures stimulated with mitogen. IL-4 and IFN-gamma protein production by PBMCs and CD4+ T cells stimulated with mitogen was assessed to see if we could detect a specific bias of cytokine production. Small amounts of IL-4 were detected from CD4+ T cells but not PBMCs from most individuals tested. IFN-gamma and IL-2, however, were also produced from these same cultures. These results further elucidate the mechanism of cytokine regulation in HIV-infected individuals, and they provide evidence that IL-15 may be a useful immune modulator.

Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major.

To determine whether DNA immunization could elicit protective immunity to Leishmania major in susceptible BALB/c mice, cDNA for the cloned Leishmania antigen LACK was inserted into a euykaryotic expression vector downstream to the cytomegalovirus promoter. Susceptible BALB/c mice were then vaccinated subcutaneously with LACK DNA and challenged with L. major promastigotes. We compared the protective efficacy of LACK DNA vaccination with that of recombinant LACK protein in the presence or absence of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic protection was achieved by LACK DNA vaccination. The control of disease progression and parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon-gamma (IFN-gamma) production. Moreover, both the enhancement of IFN-gamma production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8(+) T cells at the time of vaccination or infection also abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8(+) T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.

Feasibility of direct venous inoculation of the radiation-attenuated Plasmodium falciparum whole sporozoite vaccine in children and infants in Siaya, western Kenya.

PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites, is administered by direct venous inoculation (DVI) for maximal efficacy against malaria. A critical issue for advancing vaccines that are administered intravenously is the ability to efficiently administer them across multiple age groups. As part of a pediatric safety, immunogenicity, and efficacy trial in western Kenya, we evaluated the feasibility and tolerability of DVI, including ease of venous access, injection time, and crying during the procedure across age groups. Part 1 was an age de-escalation, dose escalation trial in children aged 13 months-5 years and infants aged 5-12 months; part 2 was a vaccine efficacy trial including only infants, using the most skilled injectors from part 1. Injectors could use a vein viewer, if needed. A total of 1222 injections (target 0.5 mL) were initiated by DVI in 511 participants (36 were 5-9-year-olds, 65 were 13-59-month-olds, and 410 infants). The complete volume was injected in 1185/1222 (97.0%) vaccinations, 1083/1185 (91.4%) achieved with the first DVI. 474/511 (92.8%) participants received only complete injections, 27/511 (5.3%) received at least one partial injection (<0.5 mL), and in 10/511 (2.0%) venous access was not obtained. The rate of complete injections by single DVI for infants improved from 77.1% in part 1 to 92.8% in part 2. No crying occurred in 51/59 (86.4%) vaccinations in 5-9-year-olds, 25/86 (29.1%) vaccinations in 13-59-month-olds and 172/1067 (16.1%) vaccinations in infants. Mean administration time ranged from 2.6 to 4.6 minutes and was longer for younger age groups. These data show that vaccination by DVI was feasible and well tolerated in infants and children in this rural hospital in western Kenya, when performed by skilled injectors. We also report that shipping and storage in liquid nitrogen vapor phase was simple and efficient. (Clinicaltrials.gov NCT02687373).

IL-12 in conjunction with dendritic cells enhances antiviral CD8+ CTL responses in vitro.

CD8+ cytolytic T lymphocytes (CTLs) are important mediators for resistance to infections and malignant diseases. IL-12 enhances proliferative and cytolytic responses by killer cells, but its function in the generation of human antiviral CD8+ T cell responses has not been defined. We therefore evaluated the role of IL-12 in the generation of CTLs to influenza-infected dendritic cells. IL-12 was not detectable in supernatants of infected-dendritic cells, or during CTL generation. Furthermore, anti-IL-12 antibody did not block CTL generation. However, exogenous IL-12 (30-300 pg/ml) enhanced CD8+ T cell proliferative and cytolytic responses. The effect was greatest in individuals with weak reactivity to influenza virus or at antigen-presenting cell (APC):T cell ratios of 1:100 or less. IL-12 augmented interferon-gamma production during CTL generation. The CTL enhancing effects of the cytokine, however, could not be blocked by neutralizing anti-interferon-gamma antibody. Together with IL-12, antigen-pulsed dendritic cells may be a useful approach for boosting CTL responses against infectious agents and malignancies.

T helper cell differentiation in immune response.

Significant progress has been made in understanding the maturation of antigen-specific CD4+ T helper cell responses. Recent progress centers on the network of cytokines, accessory molecules, and cell types that shapes the differentiation of distinct T helper cell subsets. Use of transgenic and knockout mice and well characterized in vivo models have helped clarify the interdependence or independence of many of these complex factors.

DNA vaccines: a key for inducing long-term cellular immunity.

Over the past few years, major advances in several areas of immunology have provided a foundation for the rational design of vaccines against diseases requiring cellular immunity. Among these advances are the cellular mechanisms by which DNA vaccines can sustain long-term humoral and cellular immunity.

Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity.

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

High-dose IL-2 and IL-15 enhance the in vitro priming of naive CD4+ T cells for IFN-gamma but have differential effects on priming for IL-4.

Cytokines play an important role for the in vitro differentiation of naive CD4+ T cells into IL-4- or IFN-gamma-producing cells. The presence of both IL-4 and IL-2 is required to prime cells for IL-4 in vitro. Using purified CD4+/LECAM-1high T cells from TCR transgenic mice as naive responder cells, the role of IL-15 was studied to see if it functioned similarly to IL-2 with regard to IL-4 priming. Purified CD4+ T cells cultured in the presence of IL-4 and anti-IL-2 failed to prime cells for IL-4 production. Addition of IL-15 to these same cultures could not restore priming for IL-4, suggesting that IL-2 and IL-15 may have different functional properties during the in vitro differentiation of IL-4-producing cells. The role of IL-15 in priming for IFN-gamma was also examined. The addition of high doses of IL-15 to priming cultures resulted in a striking increase in the amount of IFN-gamma produced following restimulation. Similarly, addition of a relatively high dose of IL-2 also produced a significant enhancement of IFN-gamma production; however, as previously reported, the presence of IL-12 in priming cultures induced the greatest increase in IFN-gamma production, leaving it as the predominant controller of Th1 differentiation in physiologic situations. Finally, IL-15 was shown to increase proliferation of activated CD4+ T cell blasts but not of naive CD4+ T cells. Moreover, cultures containing both IL-12 and IL-15 showed greater proliferation than either cytokine alone, suggesting an additive effect between these cytokines.

Acquisition of lymphokine-producing phenotype by CD4+ T cells.

Naive CD4+ T cells when stimulated produce IL-2 as their major lymphokine. Upon priming, these cells develop into cells that produce either IFN gamma, TNF beta, and IL-2 or IL-4 and its congeners. The former cells are designated TH1-like, and the latter TH2-like. Here we review the regulation of the differentiation of naive CD4 cells into IFN gamma- or IL-4-producers. The dominant factors that determine such differentiation are lymphokines and other cytokines. IL-2 itself appears to be required for naive cells to develop into TH1- or TH2-like cells but is not deterministic of their differentiation fate. If IL-4 is also present during the priming period, the resultant CD4+ T cells produce IL-4 upon restimulation; the development of IFN gamma-producing cells is strikingly inhibited by IL-4. In the absence of IL-4, priming for IFN gamma-production occurs, but this is markedly enhanced by IL-12. The role of IFN gamma in enhancing priming for IFN gamma-production is not fully resolved. In some in vitro systems, it appears to act together with IL-12 to enhance such production. Anti-IFN gamma diminishes priming for IFN gamma production in vivo. Lymphokines also exert a "cross-regulatory" or inhibitory effect. As noted above, IL-4 strikingly diminishes priming for IFN gamma production, although this inhibitory effect is blunted in the presence of IL-12. IFN gamma similarly diminishes priming for IL-4 production; this effect is principally observed when low concentrations of IL-4 are used in the priming culture. Although other factors may play a role in the determination of lymphokine-producing phenotype, such as antigen dose, type of antigen-presenting cell, and expression of accessory molecules and hormones, these effects appear to be secondary to the dominant role of the lymphokines and cytokines.

Are differentiated human T helper cells reversible?

Cytokines and cytokine antagonists have important roles in controlling the type of immune response generated. These mediators have the most profound effects if used at the initiation of an immune response. For example, IL-4 and IL-12 are important determinants in the generation of Th1 and Th2 cells, respectively, in both murine and human systems. One critical question that relates to human pathogenesis is whether Th1- and Th2-type cells can be altered after disease is initiated. Data will be presented showing how cytokines can change the types of cytokines produced from established Th1 and Th2 cells. Moreover, intracellular staining of cytokines was done to more accurately define whether cytokine production was altered on a single cell basis. These studies may have implications for immune manipulation for established allergic or autoimmune diseases.

Vaccines against intracellular infections requiring cellular immunity.

Vaccines against a variety of infectious diseases represent one of the great triumphs of medicine. The immune correlates of protection induced by most current vaccines seem to be mediated by long-lived humoral immune responses. By contrast, there are no currently available vaccines that are uniformly effective for diseases such as HIV, malaria and tuberculosis, in which the cellular immune response might be crucial in mediating protection. Here we examine the mechanisms by which long-lived cellular immune responses are generated and maintained in vivo. We then discuss current approaches for vaccination against diseases in which cellular immune responses are important for protection.

The NZB X SWR model of lupus nephritis. I. Cross-reactive idiotypes of monoclonal anti-DNA antibodies in relation to antigenic specificity, charge, and allotype. Identification of interconnected idiotype families inherited from the normal SWR and the autoimmune NZB parents.

The incidence of lupus nephritis is low in autoimmune NZB mice, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. In a previous study we showed that anti-DNA auto-antibodies produced by the SNF1 mice were qualitatively different from those made by the NZB parents with respect to their isotype, charge, and antigenic specificity patterns. Here we studied idiotypic cross-reactions among the 65 monoclonal anti-DNA antibodies that were derived from four NZB and seven SNF1 mice. A library of 15 anti-idiotypic antibodies were prepared by immunizing rabbits with 15 monoclonal anti-DNA antibodies selected from the panel of 65. We identified 10 cross-reactive idiotype (CRI) families among this large collection of autoantibodies. Five of these CRI families were restricted to cationic anti-DNA antibodies that were exclusively of SNF1 origin, and the strongly cross-reacting members were predominantly IgG2b auto-antibodies with the allotype of the normal SWR parent. The cationic anti-DNA CRI families could be grouped into an interrelated cluster called the Id564 cluster. The other five anti-DNA CRI families were not restricted to any particular parental allotype or charge, although two of these CRI were shared exclusively by SNF1-derived autoantibodies and four of these CRI families could also be grouped into an idiotypically interrelated cluster called the Id512 cluster. In the case of seven out of the 10 CRI families, the idiotypic determinants detected were close to the antigen-binding site of the anti-DNA antibodies. The results indicate that the idiotypic repertoire of anti-DNA autoantibodies produced by the SNF1 mice is different from the NZB parents, and potentially pathogenic (cationic) antibodies produced by the SNF1 mice that are encoded by genes from the normal SWR parent can be identified as distinct CRI families. In the accompanying paper we demonstrate the role of these anti-DNA CRI families in the development of lupus nephritis.

Differential roles for IL-12 in the maintenance of immune responses in infectious versus autoimmune disease.

IL-12 has been shown to be important in the generation of a functional Th1 response in animal models of both infectious and autoimmune disease. Furthermore, the role of IL-12 in the maintenance of the immune responses in these diseases is now emerging. Herein, we discuss the idea that memory responses for certain infections may be IL-12 independent, whereas memory responses for specific autoimmune diseases still require IL-12 to maintain a pathogenic response.