Rajesh Ravindran

Programming the magnitude and persistence of antibody responses with innate immunity

Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence indicates that they activate dendritic cells via Toll-like receptors (TLRs). For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed, activates dendritic cells via multiple TLRs to stimulate proinflammatory cytokines. Triggering specific combinations of TLRs in dendritic cells can induce synergistic production of cytokines, which results in enhanced T-cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that program such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus ligands that signal through TLR4 and TLR7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with nanoparticles containing antigens plus a single TLR ligand. Consistent with this there was enhanced persistence of germinal centres and of plasma-cell responses, which persisted in the lymph nodes for >1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma-cell response relative to that observed with single TLR ligands. Molecular profiling of activated B cells, isolated 7 days after immunization, indicated that there was early programming towards B-cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells, as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.

Activation of β-Catenin in Dendritic Cells Regulates Immunity Versus Tolerance in the Intestine

A Gut Feeling Special immune controls are necessary in the gut to prevent the immune system from reacting to the commensal microbiota and to food antigens. Dendritic cells (DCs) are important for maintaining gut tolerance because they help to keep T cells in an unresponsive state. However, in other environments, DCs activate T cells. What signals determine whether DCs induce T cell tolerance or activation? Manicassamy et al. (p. 849; see the Perspective by Mellman and Clausen) found that β-catenin–dependent signaling is required for maintaining DC-mediated gut tolerance in mice. Wnt ligands were expressed in the gut, and β-catenin signaling was activated in DCs in the small and large intestines but not in the spleen. When β-catenin was specifically deleted from DCs in mice, the frequency of regulatory T cells and anti-inflammatory cytokines was reduced, whereas the frequency of pro-inflammatory T helper 1 and T helper 17 cells and their associated cytokines was increased. Mice lacking β-catenin in dendritic cells also exhibited enhanced susceptibility in a mouse model of colitis. Gut-specific signaling pathways regulate immune cell–mediated tolerance in the intestine. Dendritic cells (DCs) play a vital role in initiating robust immunity against pathogens as well as maintaining immunological tolerance to self antigens. However, the intracellular signaling networks that program DCs to become tolerogenic remain unknown. We report here that the Wnt–β-catenin signaling in intestinal dendritic cells regulates the balance between inflammatory versus regulatory responses in the gut. β-catenin in intestinal dendritic cells was required for the expression of anti-inflammatory mediators such as retinoic acid–metabolizing enzymes, interleukin-10, and transforming growth factor–β, and the stimulation of regulatory T cell induction while suppressing inflammatory effector T cells. Furthermore, ablation of β-catenin expression in DCs enhanced inflammatory responses and disease in a mouse model of inflammatory bowel disease. Thus, β-catenin signaling programs DCs to a tolerogenic state, limiting the inflammatory response.

Toll-like receptor 2–dependent induction of vitamin A–metabolizing enzymes in dendritic cells promotes T regulatory responses and inhibits autoimmunity

Immune sensing of a microbe occurs via multiple receptors. How signals from different receptors are coordinated to yield a specific immune response is poorly understood. We show that two pathogen recognition receptors, Toll-like receptor 2 (TLR2) and dectin-1, recognizing the same microbial stimulus, stimulate distinct innate and adaptive responses. TLR2 signaling induced splenic dendritic cells (DCs) to express the retinoic acid metabolizing enzyme retinaldehyde dehydrogenase type 2 and interleukin-10 (IL-10) and to metabolize vitamin A and stimulate Foxp3+ T regulatory cells (Treg cells). Retinoic acid acted on DCs to induce suppressor of cytokine signaling-3 expression, which suppressed activation of p38 mitogen-activated protein kinase and proinflammatory cytokines. Consistent with this finding, TLR2 signaling induced Treg cells and suppressed IL-23 and T helper type 17 (TH17) and TH1-mediated autoimmune responses in vivo. In contrast, dectin-1 signaling mostly induced IL-23 and proinflammatory cytokines and augmented TH17 and TH1-mediated autoimmune responses in vivo. These data define a new mechanism for the systemic induction of retinoic acid and immune suppression against autoimmunity.

Vaccine Activation of the Nutrient Sensor GCN2 in Dendritic Cells Enhances Antigen Presentation

The Secret Life of a Vaccine Antigen-specific CD8÷ T cells play a central role in the adaptive immune response to viral infections and to cancer. Ravindran et al. (p. 313, published online 5 December) studied the successful yellow fever virus vaccine YF-17D to gain insight into its mechanism of action. The vaccine activated the nutrient deprivation sensor, GCN2 kinase, in dendritic cells. In transgenic mouse models, GCN2 activation promoted autophagy and antigen cross-presentation, enhancing the virus-specific CD8÷ T cell response. The findings suggest an important role for nutrient availability and autophagy in vaccine efficacy, which could aid more successful vaccine development. The success of the yellow fever vaccine is linked to the amino acid starvation pathway, which promotes adaptive immunity. The yellow fever vaccine YF-17D is one of the most successful vaccines ever developed in humans. Despite its efficacy and widespread use in more than 600 million people, the mechanisms by which it stimulates protective immunity remain poorly understood. Recent studies using systems biology approaches in humans have revealed that YF-17D–induced early expression of general control nonderepressible 2 kinase (GCN2) in the blood strongly correlates with the magnitude of the later CD8+ T cell response. We demonstrate a key role for virus-induced GCN2 activation in programming dendritic cells to initiate autophagy and enhanced antigen presentation to both CD4+ and CD8+ T cells. These results reveal an unappreciated link between virus-induced integrated stress response in dendritic cells and the adaptive immune response.

gp63 in stable cationic liposomes confers sustained vaccine immunity to susceptible BALB/c mice infected with Leishmania donovani.

ABSTRACT Visceral leishmaniasis is deadly if not treated, and development of a vaccine with long-term immunity remains a challenge. In this study, we showed that cationic distearoyl phosphatidylcholine (DSPC) liposomes, when used as vaccine adjuvant with the immunodominant 63-kDa glycoprotein (gp63) of Leishmania donovani promastigotes, induced significant protection against progressive visceral leishmaniasis in susceptible BALB/c mice. gp63 used without adjuvant elicited partial protection but in association with liposomes exhibited marked resistance in both the livers and spleens of the mice challenged 10 days after the last vaccination. The protective efficacy of liposomal gp63 vaccination was dose dependent, with 2.5 μg of protein showing optimal protection. The immunity conferred by this vaccine formulation was durable, as mice challenged 12 weeks after immunization were still protected, and the infection was controlled for at least 3 months postchallenge. Production of gamma interferon (IFN-γ) and interleukin-4 (IL-4) by splenic T cells, and of serum immunoglobulin G1 (IgG1) and IgG2a following immunization, suggested that a mixed Th1/Th2 response had been induced following immunization. However, control of disease progression and parasitic burden in mice vaccinated with gp63 in cationic DSPC liposomes was associated with enhancement of antigen-specific IFN-γ and downregulation of IL-4, demonstrating a Th1 bias. Long-term immunity elicited by this vaccine corresponded to, in addition to the presence of antigen-specific Th1, CD8+ T-cell responses. Our results demonstrated that stable cationic liposomes containing gp63 acted as a potent adjuvant for protein antigen to induce long-term protection against L. donovani that represents an alternative to DNA vaccination.

The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation

The integrated stress response (ISR) is a homeostatic mechanism by which eukaryotic cells sense and respond to stress-inducing signals, such as amino acid starvation. General controlled non-repressed (GCN2) kinase is a key orchestrator of the ISR, and modulates protein synthesis in response to amino acid starvation. Here we demonstrate in mice that GCN2 controls intestinal inflammation by suppressing inflammasome activation. Enhanced activation of ISR was observed in intestinal antigen presenting cells (APCs) and epithelial cells during amino acid starvation, or intestinal inflammation. Genetic deletion of Gcn2 (also known as Eif2ka4) in CD11c+ APCs or intestinal epithelial cells resulted in enhanced intestinal inflammation and T helper 17 cell (TH17) responses, owing to enhanced inflammasome activation and interleukin (IL)-1β production. This was caused by reduced autophagy in Gcn2−/− intestinal APCs and epithelial cells, leading to increased reactive oxygen species (ROS), a potent activator of inflammasomes. Thus, conditional ablation of Atg5 or Atg7 in intestinal APCs resulted in enhanced ROS and TH17 responses. Furthermore, in vivo blockade of ROS and IL-1β resulted in inhibition of TH17 responses and reduced inflammation in Gcn2−/− mice. Importantly, acute amino acid starvation suppressed intestinal inflammation via a mechanism dependent on GCN2. These results reveal a mechanism that couples amino acid sensing with control of intestinal inflammation via GCN2.

Immunity to viruses: learning from successful human vaccines

For more than a century, immunologists and vaccinologists have existed in parallel universes. Immunologists have for long reveled in using ‘model antigens’, such as chicken egg ovalbumin or nitrophenyl haptens, to study immune responses in model organisms such as mice. Such studies have yielded many seminal insights about the mechanisms of immune regulation, but their relevance to humans has been questioned. In another universe, vaccinologists have relied on human clinical trials to assess vaccine efficacy, but have done little to take advantage of such trials for studying the nature of immune responses to vaccination. The human model provides a nexus between these two universes, and recent studies have begun to use this model to study the molecular profile of innate and adaptive responses to vaccination. Such ‘systems vaccinology’ studies are beginning to provide mechanistic insights about innate and adaptive immunity in humans. Here, we present an overview of such studies, with particular examples from studies with the yellow fever and the seasonal influenza vaccines. Vaccination with the yellow fever vaccine causes a systemic acute viral infection and thus provides an attractive model to study innate and adaptive responses to a primary viral challenge. Vaccination with the live attenuated influenza vaccine causes a localized acute viral infection in mucosal tissues and induces a recall response, since most vaccinees have had prior exposure to influenza, and thus provides a unique opportunity to study innate and antigen‐specific memory responses in mucosal tissues and in the blood. Vaccination with the inactivated influenza vaccine offers a model to study immune responses to an inactivated immunogen. Studies with these and other vaccines are beginning to reunite the estranged fields of immunology and vaccinology, yielding unexpected insights about mechanisms of viral immunity. Vaccines that have been proven to be of immense benefit in saving lives offer us a new fringe benefit: lessons in viral immunology.

Progress in Vaccine Research and Possible Effector Mechanisms in Visceral Leishmaniasis

Visceral leishmaniasis represents a serious public health concern in endemic regions and is rapidly emerging as an opportunistic infection in HIV patients. The disease is difficult to diagnose and prevent, and available treatment is associated with toxicity and drug resistance. Even though significant headway has been made in the development of vaccines against cutaneous leishmaniasis, visceral leishmaniasis has received limited attention. The fact that a large proportion of the people living in endemic areas have self-resolving subclinical infection and individuals once recovered are immune to reinfection provides a rationale for designing immunoprophylactic strategies against visceral leishmaniasis. The primary aim of this paper is to review advances in vaccination strategies against visceral leishmaniasis, suggesting possible effector mechanism leading to resistance. It also covers the role of immunostimulators and gives an account of the adjuvants used against visceral leishmaniasis. Vaccine strategies in different established experimental models have also been dealt with which can provide potential leads for their application in humans. In light of the available observations made during the course of studies performed on experimental models of visceral leishmaniasis there is increasing evidence that a successful approach towards a vaccine involves the requirement of Th1 subset of CD4+ cells along with Th2, CD8+, and B cells. In this review we present the possible mechanism of interaction of these cells and their effector molecules in providing resistance against visceral leishmaniasis for the future design of effective vaccine against this disease.

Characterization of Immunoglobulin G and Its Subclass Response to Indian Kala-Azar Infection before and after Chemotherapy

ABSTRACT Serologic parameters of kala-azar were evaluated by Western blot analysis. Sera from kala-azar patients with confirmed diagnoses were screened for immunoglobulin G (IgG) and IgG subclass-specific reactivity against Leishmania donovani membrane antigen (LAg). Heterogenous LAg-specific IgG reactivity with numerous proteins with molecular masses ranging from 18 to 190 kDa was observed. Though the individual band patterns were varied, seven polypeptides of approximately 31, 34, 51, 63, 72, 91, and 120 kDa were immunoreactive with all the sera tested from kala-azar patients. The band patterns of the immunoblots of sera from patients after treatment and clinical cure with sodium antimony gluconate revealed a decrease in the frequency of the bands. Still, recognition of the 63- and 120-kDa bands was 100%, and the 55- and 91-kDa fractions were recognized in 93% of the sera from cured individuals. Among the IgG subclasses, IgG1 reacted with the greatest number of polypeptides. The 63-kDa protein was again detected by all of the IgG subclasses of all the sera tested. Other fractions recognized by the subclasses of more than 70% of the serum samples included those of 47, 51, 55, and 78 kDa. Following treatment, 63- and 51-kDa bands were the most reactive with the IgG subclasses. LAg-associated cross-reaction with other reference human antisera revealed a mild reactivity of the 63-kDa polypeptide with some of the serum samples from leprosy, malaria, typhoid, tuberculosis, and healthy controls. Western blot analysis of LAg entrapped in liposomes, strong vaccine candidates against experimental visceral leishmaniasis, revealed a more restricted band pattern. The 63-kDa fraction revealed by all pre- and posttreatment sera showed almost negligible levels of cross-reaction with sera from patients with other diseases or from healthy controls. These observations provide insight into induced immunity during kala-azar infection for future application.

Comparison of BCG, MPL and cationic liposome adjuvant systems in leishmanial antigen vaccine formulations against murine visceral leishmaniasis

BackgroundThe development of an effective vaccine against visceral leishmaniasis (VL) caused by Leishmania donovani is an essential aim for controlling the disease. Use of the right adjuvant is of fundamental importance in vaccine formulations for generation of effective cell-mediated immune response. Earlier we reported the protective efficacy of cationic liposome-associated L. donovani promastigote antigens (LAg) against experimental VL. The aim of the present study was to compare the effectiveness of two very promising adjuvants, Bacille Calmette-Guerin (BCG) and Monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) with cationic liposomes, in combination with LAg, to confer protection against murine VL.ResultsAll the three formulations afforded significant protection against L. donovani in both the visceral organs, liver and spleen. Although comparable level of protection was observed in BCG+LAg and MPL-TDM+LAg immunized mice, highest level of protection was exhibited by the liposomal LAg immunized group. Significant increase in anti-LAg IgG levels were detected in both MPL-TDM+LAg and liposomal LAg immunized animals with higher levels of IgG2a than IgG1. But BCG+LAg failed to induce any antibody response. As an index of cell-mediated immunity DTH responses were measured and significant response was observed in mice vaccinated with all the three different formulations. However, highest responses were observed with liposomal vaccine immunization. Comparative evaluation of IFN-γ and IL-4 responses in immunized mice revealed that MPL-TDM+LAg group produced the highest level of IFN-γ but lowest IL-4 level, while BCG+LAg demonstrated generation of suboptimum levels of both IFN-γ and IL-4 response. Elicitation of moderate levels of prechallenge IFN-γ along with optimum IL-4 corresponds with successful vaccination with liposomal LAg.ConclusionThis comparative study reveals greater effectiveness of the liposomal vaccine for protection against progressive VL in BALB/c. Again, evaluation of the immune responses by vaccination emphasizes the need of stimulation of potent cellular immunity based on both Th1 and Th2 cell responses to confer protection against VL.