A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis : First-in-human trial of ChAd63-KH

BACKGROUND: Visceral leishmaniasis (VL or kala azar) is the most serious form of human leishmaniasis, responsible for over 20,000 deaths annually, and post kala azar dermal leishmaniasis (PKDL) is a stigmatizing skin condition that often occurs in patients after successful treatment for VL. Lack of effective or appropriately targeted cell mediated immunity, including CD8+ T cell responses, underlies the progression of VL and progression to PKDL, and can limit the therapeutic efficacy of anti-leishmanial drugs. Hence, in addition to the need for prophylactic vaccines against leishmaniasis, the development of therapeutic vaccines for use alone or in combined immuno-chemotherapy has been identified as an unmet clinical need. Here, we report the first clinical trial of a third-generation leishmaniasis vaccine, developed intentionally to induce Leishmania-specific CD8+ T cells. METHODS: We conducted a first-in-human dose escalation Phase I trial in 20 healthy volunteers to assess the safety, tolerability and immunogenicity of a prime-only adenoviral vaccine for human VL and PKDL. ChAd63-KH is a replication defective simian adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB. Uniquely, the latter was engineered to reflect repeat domain polymorphisms and arrangements identified from clinical isolates. We monitored innate immune responses by whole blood RNA-Seq and antigen specific CD8+ T cell responses by IFNγ ELISPOT and intracellular flow cytometry. FINDINGS: ChAd63-KH was safe at intramuscular doses of 1x1010 and 7.5x1010 vp. Whole blood transcriptomic profiling indicated that ChAd63-KH induced innate immune responses characterized by an interferon signature and the presence of activated dendritic cells. Broad and quantitatively robust CD8+ T cell responses were induced by vaccination in 100% (20/20) of vaccinated subjects. CONCLUSION: The results of this study support the further development of ChAd63-KH as a novel third generation vaccine for VL and PKDL. TRIAL REGISTRATION: This clinical trial (LEISH1) was registered at EudraCT (2012-005596-14) and ISRCTN (07766359)

Overview of cellular responses during an asymptomatic <i>L. donovani</i> infection.

<p>Infected macrophages can produce TNF and IL-1β in response to <i>L. donovani</i> infection as part of the innate immune response. However, DC IL-12 production in response to <i>L. donovani</i> infection is required to drive the differentiation of antigen-specific CD4⁺ T cells into IFNγ- and TNF-producing Th1 cells. These cells can activate infected macrophages and monocytes to produce ROI and RNI that kill intracellular parasites. There are also reports in humans that Th17 and Th22 cells develop in asymptomatic, infected individuals, possibly driven by IL-23 and IL-6. However, the antiparasitic mechanism mediated by these CD4⁺ T cell subsets following <i>L. donovani</i> infection remains unknown. Although parasite-specific antibodies are readily detected in asymptomatic individuals, their role, if any, in control of infection and protection against reinfection is unknown. Abbreviations: MO, monocyte; Mφ, macrophage.</p

Overview of cellular responses during a chronic <i>L. donovani</i> infection.

<p>During an established <i>L. donovani</i> infection, a subset of regulatory DCs in the spleen can produce IL-10 that promotes the expansion of IL-10-producing regulatory T cells (Tr1), as well as inhibiting antimicrobial mechanisms in macrophages and other phagocytic cells (including suppression of ROI and RNI generation). IL-27 produced by regulatory DCs and macrophages, along with T cell–derived IL-21, can drive the differentiation of Th1 cells into Tr1 cells, as well as inhibit Th17 development. IL-10 produced by Tr1 cells can suppress antigen presentation, contributing to T cell dysfunction, as well as down-regulate CD4⁺ T cell IFNγ production. There has been a report that IL-10 can also be produced by Treg cells in the BM of VL patients. Although uptake of infected neutrophils undergoing apoptosis by macrophages contributes to the establishment of <i>L. major</i> infection in mice, no such mechanism has yet been described during <i>L. donovani</i> infection. Abbreviations: N, neutrophil.</p

Mice lacking programmed cell death-1 show a role for CD8+ T cells in long-term immunity against blood-stage malaria

Even after years of experiencing malaria, caused by infection with Plasmodium species, individuals still have incomplete immunity and develop low-density parasitemia on re-infection. Previous studies using the P. chabaudi (Pch) mouse model to understand the reason for chronic malaria, found that mice with a deletion of programmed cell death-1 (PD-1KO) generate sterile immunity unlike wild type (WT) mice. Here we investigated if the mechanism underlying this defect during acute immunity also impacts on long-term immunity. We infected WT and PD-1KO mice with Pch-malaria and measured protection as well as immune responses against re-infections, 15 or 20 weeks after the original infection had cleared. WT mice showed approximately 1% parasitemia compared to sterile immunity in PD-1KO mice on re-infection. An examination of the mechanisms of immunity behind this long-term protection in PD-1KO mice showed a key role for parasite-specific CD8 + T cells even when CD4 + T cells and B cells responded to re-infection. These studies indicate that long-term CD8 + T cell-meditated protection requires consideration for future malaria vaccine design, as part of a multi-cell type response

Type I Interferons Suppress Anti-parasiticImmunity and Can Be Targeted to Improve Treatment of Visceral Leishmaniasis

Type I interferons (IFNs) play critical roles in anti-viral and anti-tumor immunity. However, they also suppress protective immune responses in some infectious diseases.This work was made possible through NIH Tropical Medicine Research Centers (TMRC) grant U19 AI074321 and Queensland State Government funding. The research was supported by grants and fellowships from the National Health and Medical Research Council of Australia (NHMRC; 1037304, 1058685, 1132975, and 1154265) as well as Australian post-graduate awards through Griffith University Institute of Glycomics and School of Natural Sciences (to P.B. and S.S.N., respectively), junior research fellowships from the Indian Council of Medical Research (to S.S.S. and S.B.C.), and an INSPIRE Faculty fellowship (LBSM-109/IF-14 to R.K.) provided by the Indian government Department of Science and Technology (DST)

Coinfection with blood-stage plasmodium promotes systemic type I interferon production during pneumovirus infection but impairs inflammation and viral control in the lung

Acute lower respiratory tract infections (ALRTI) are the leading cause of global childhood mortality, with human respiratory syncytial virus (hRSV) being a major cause of viral ALRTI in young children worldwide. In sub-Saharan Africa, many young children experience severe illnesses due to hRSV or Plasmodium infection. Although the incidence of malaria in this region has decreased in recent years, there remains a significant opportunity for coinfection. Recent data show that febrile young children infected with Plasmodium are often concurrently infected with respiratory viral pathogens but are less likely to suffer from pneumonia than are non-Plasmodium-infected children. Here, we hypothesized that blood-stage Plasmodium infection modulates pulmonary inflammatory responses to a viral pathogen but does not aid its control in the lung. To test this, we established a novel coinfection model in which mice were simultaneously infected with pneumovirus of mice (PVM) (to model hRSV) and blood-stage Plasmodium chabaudi chabaudi AS (PcAS) parasites. We found that PcAS infection was unaffected by coinfection with PVM. In contrast, PVM-associated weight loss, pulmonary cytokine responses, and immune cell recruitment to the airways were substantially reduced by coinfection with PcAS. Importantly, PcAS coinfection facilitated greater viral dissemination throughout the lung. Although Plasmodium coinfection induced low levels of systemic interleukin-10 (IL-10), this regulatory cytokine played no role in the modulation of lung inflammation or viral dissemination. Instead, we found that Plasmodium coinfection drove an early systemic beta interferon (IFN-beta) response. Therefore, we propose that blood-stage Plasmodium coinfection may exacerbate viral dissemination and impair inflammation in the lung by dysregulating type I IFN-dependent responses to respiratory viruses

Galectin-1 Impairs the Generation of Anti-Parasitic Th1 Cell Responses in the Liver during Experimental Visceral Leishmaniasis

Many infectious diseases are characterized by the development of immunoregulatory pathways that contribute to pathogen persistence and associated disease symptoms. In diseases caused by intracellular parasites, such as visceral leishmaniasis (VL), various immune modulators have the capacity to negatively impact protective CD4(+) T cell functions. Galectin-1 is widely expressed on immune cells and has previously been shown to suppress inflammatory responses and promote the development of CD4(+) T cells with immunoregulatory characteristics. Here, we investigated the role of galectin-1 in experimental VL caused by infection of C57BL/6 mice with Leishmania donovani. Mice lacking galectin-1 expression exhibited enhanced tissue-specific control of parasite growth in the liver, associated with an augmented Th1 cell response. However, unlike reports in other experimental models, we found little role for galectin-1 in the generation of IL-10-producing Th1 (Tr1) cells, and instead report that galectin-1 suppressed hepatic Th1 cell development. Furthermore, we found relatively early effects of galectin-1 deficiency on parasite growth, suggesting involvement of innate immune cells. However, experiments investigating the impact of galectin-1 deficiency on dendritic cells indicated that they were not responsible for the phenotypes observed in galectin-1-deficient mice. Instead, studies examining galectin-1 expression by CD4(+) T cells supported a T cell intrinsic role for galectin-1 in the suppression of hepatic Th1 cell development during experimental VL. Together, our findings provide new information on the roles of galectin-1 during parasitic infection and indicate an important role for this molecule in tissue-specific Th1 cell development, but not CD4(+) T cell IL-10 production

Galectin-1 Impairs the Generation of Anti-Parasitic Th1 Cell Responses in the Liver during Experimental Visceral Leishmaniasis

Many infectious diseases are characterized by the development of immunoregulatory pathways that contribute to pathogen persistence and associated disease symptoms. In diseases caused by intracellular parasites, such as visceral leishmaniasis (VL), various immune modulators have the capacity to negatively impact protective CD4+ T cell functions. Galectin-1 is widely expressed on immune cells and has previously been shown to suppress inflammatory responses and promote the development of CD4+ T cells with immunoregulatory characteristics. Here, we investigated the role of galectin-1 in experimental VL caused by infection of C57BL/6 mice with Leishmania donovani. Mice lacking galectin-1 expression exhibited enhanced tissue-specific control of parasite growth in the liver, associated with an augmented Th1 cell response. However, unlike reports in other experimental models, we found little role for galectin-1 in the generation of IL-10-producing Th1 (Tr1) cells, and instead report that galectin-1 suppressed hepatic Th1 cell development. Furthermore, we found relatively early effects of galectin-1 deficiency on parasite growth, suggesting involvement of innate immune cells. However, experiments investigating the impact of galectin-1 deficiency on dendritic cells indicated that they were not responsible for the phenotypes observed in galectin-1-deficient mice. Instead, studies examining galectin-1 expression by CD4+ T cells supported a T cell intrinsic role for galectin-1 in the suppression of hepatic Th1 cell development during experimental VL. Together, our findings provide new information on the roles of galectin-1 during parasitic infection and indicate an important role for this molecule in tissue-specific Th1 cell development, but not CD4+ T cell IL-10 production

Distinct roles for CD4+ Foxp3+ regulatory T cells and IL-10–mediated immunoregulatory mechanisms during experimental visceral Leishmaniasis caused by Leishmania donovani

The outcome of intracellular parasitic infection can be determined by the immunoregulatory activities of natural regulatory CD4 Foxp3 T (Treg) cells and the anti-inflammatory cytokine IL-10. These mechanisms protect tissue but can also suppress antiparasitic CD4 T cell responses. The specific contribution of these regulatory pathways during human parasitic diseases remains unclear. In this study, we investigated the roles of Treg cells and IL-10 during experimental visceral leishmaniasis caused by infection of C57BL/6 mice. We report only a limited contribution of Treg cells in suppressing antiparasitic immunity, but important roles in delaying the development of splenic pathology and restricting leukocyte expansion. We next employed a range of cell-specific, IL-10- and IL-10R-deficient mice and found these Treg cell functions were independent of IL-10. Instead, conventional CD4 T cells and dendritic cells were the most important cellular sources of IL-10, and the absence of IL-10 in either cell population resulted in greater control of parasite growth but also caused accelerated breakdown in splenic microarchitecture. We also found that T cells, dendritic cells, and other myeloid cells were the main IL-10-responding cells because in the absence of IL-10R expression by these cell populations, there was greater expansion of parasite-specific CD4 T cell responses associated with improved control of parasite growth. Again, however, there was also an accelerated breakdown in splenic microarchitecture in these animals. Together, these findings identify distinct, cell-specific, immunoregulatory networks established during experimental visceral leishmaniasis that could be manipulated for clinical advantage

Programmed death-1 ligand 2-mediated regulation of the PD-L1 to PD-1 axis is essential for establishing CD4⁺ T cell immunity

Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to "deactivate" T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4⁺ T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling