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.

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.

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.

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.

Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells.

T cell populations derived from naive mice produce very small amounts of interleukin 4 (IL-4) in response to stimulation on anti-CD3-coated dishes. IL-4 production by such cells is mainly found among large- and intermediate-sized T cells and is dependent upon IL-2. Injection of anti-IgD into mice, a stimulus that leads to striking increases in serum levels of IgG1 and IgE, causes a striking increase in the IL-4-producing capacity of T cells. This increase is first observed 4 d after injection of anti-IgD. IL-4 production by T cells from anti-IgD-injected donors is mainly found among large- and intermediate-sized T cells. Small, dense T cells are poor producers of IL-4. The capacity of T cells from anti-IgD-injected donors to produce IL-4 is enhanced by addition of IL-2 and is largely, but not completely, inhibited by neutralization of in situ produced IL-2. These results indicate that the control of IL-4 production in T cells from naive and anti-IgD-injected donors is similar. However, it is possible that a portion of the IL-4-producing activity of T cells from activated donors is IL-2 independent. Although small T cells from naive donors have a very limited capacity to produce IL-4 in response to stimulation with anti-CD3, even in the presence of added IL-2, they can give rise to IL-4-producing cells upon in vitro culture on plates coated with anti-CD3 if both IL-2 and IL-4 are added. This leads to the appearance of IL-4-producing cells within 2 d. When analyzed after 5 d of culture by harvesting and re-exposure to anti-CD3-coated culture wells and IL-2, these cells have increased their IL-4-producing capacity by approximately 100-fold. The development of IL-4-producing cells in response to anti-CD3, IL-2, and IL-4 is not inhibited by interferon gamma (IFN-gamma), nor does IFN-gamma diminish IL-4 production by these cells upon challenge with anti-CD3 plus IL-2.

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.

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.

Experimental procedure for measuring and comparing head-neck-trunk posture and movements caused by different progressive addition lens designs.

This study demonstrates that appropriate measurement procedures can detect differences in head movement in a near reading task when using three different progressive addition lenses (PALs). The movements were measured using an anatomical reference system with a biomechanical rationale. This reference system was capable of representing rotations for comparing head flexion relative to trunk, head flexion relative to neck, head rotation relative to trunk and trunk flexion. The subject sample comprised 31 volunteers and three PAL designs with different viewing zones were selected. Significant differences were found between the lenses for three of the seven movement parameters examined. The differences occurred for both vertical and horizontal head movements and could be attributed to aspects of the PAL design. The measurement of the complete kinematic trunk-neck-head chain improved the number of differences that were found over those in previous studies. STATEMENT OF RELEVANCE: The study proposes a methodology based on a biomechanical rationale able to differentiate head-neck-trunk posture and movements caused by different progressive addition lens designs with minimum invasiveness. This methodology could also be applied to analyse the ergonomics of other devices that restrict the user's field of view, such as helmets, personal protective equipment or helmet-mounted displays for pilots. This analysis will allow designers to optimise designs offering higher comfort and performance.

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.

Pleural effusion after plasmapheresis in Waldenström's macroglobulinemia.

Transudative pleural effusion developed after plasmapheresis in a patient with Waldenström's macroglobulinemia and hyperviscosity. When albumin was added to the apheresis, the effusion resolved. We therefore suspect that rapid removal of high-molecular-weight IgM led to an abrupt decrease in intravascular colloid oncotic pressure and transudation of fluid into the pleural space. Plasmapheresis can cause pleural effusions in patients who were previously hemodynamically compensated at high levels of colloid oncotic pressure.

Dendritic cells as immunogens for human CTL responses.

The cellular requirements for generating potent human CD8+ CTLs to influenza A virus in vitro have been defined. Furthermore, we have developed improved methods for generating large numbers of DCs from non-proliferating progenitors. These developments have enabled the design of new strategies to elicit CTLs in vivo. For example, together with IL-12, antigen-pulsed DCs may be a useful approach for boosting CTL responses against infectious agents and malignancies. Our results also reopen the potential use of inactivated virus preparations as immunogens for CTL responses.

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.