Deficiency of Leishmania phosphoglycans influences the magnitude but does not affect the quality of secondary (memory) anti-Leishmania immunity

Despite inducing very low IFN-γ response and highly attenuated in vivo, infection of mice with phosphoglycan (PG) deficient Leishmania major (lpg2-) induces protection against virulent L. major challenge. Here, we show that mice infected with lpg2- L. major generate Leishmania-specific memory T cells. However, in vitro and in vivo proliferation, IL-10 and IFN-γ production by lpg2- induced memory cells were impaired in comparison to those induced by wild type (WT) parasites. Interestingly, TNF recall response was comparable to WT infected mice. Despite the impaired proliferation and IFN-γ response, lpg2- infected mice were protected against virulent L. major challenge and their T cells mediated efficient infection-induced immunity. In vivo depletion and neutralization studies with mAbs demonstrated that lpg2- L. major-induced resistance was strongly dependent on IFN-γ, but independent of TNF and CD8(+) T cells. Collectively, these data show that the effectiveness of secondary anti-Leishmania immunity depends on the quality (and not the magnitude) of IFN-γ response. These observations provide further support for consideration of lpg2- L. major as a live-attenuated candidate for leishmanization in humans since it protects strongly against virulent challenge, without inducing pathology in infected animals

Vector Transmission of Leishmania Abrogates Vaccine-Induced Protective Immunity

Numerous experimental vaccines have been developed to protect against the cutaneous and visceral forms of leishmaniasis caused by infection with the obligate intracellular protozoan Leishmania, but a human vaccine still does not exist. Remarkably, the efficacy of anti-Leishmania vaccines has never been fully evaluated under experimental conditions following natural vector transmission by infected sand fly bite. The only immunization strategy known to protect humans against natural exposure is “leishmanization,” in which viable L. major parasites are intentionally inoculated into a selected site in the skin. We employed mice with healed L. major infections to mimic leishmanization, and found tissue-seeking, cytokine-producing CD4+ T cells specific for Leishmania at the site of challenge by infected sand fly bite within 24 hours, and these mice were highly resistant to sand fly transmitted infection. In contrast, mice vaccinated with a killed vaccine comprised of autoclaved L. major antigen (ALM)+CpG oligodeoxynucleotides that protected against needle inoculation of parasites, showed delayed expression of protective immunity and failed to protect against infected sand fly challenge. Two-photon intra-vital microscopy and flow cytometric analysis revealed that sand fly, but not needle challenge, resulted in the maintenance of a localized neutrophilic response at the inoculation site, and removal of neutrophils following vector transmission led to increased parasite-specific immune responses and promoted the efficacy of the killed vaccine. These observations identify the critical immunological factors influencing vaccine efficacy following natural transmission of Leishmania

Study of Leishmania pathogenesis in mice : experimental considerations

Although leishmaniases are endemic in 98 countries, they are still considered neglected tropical diseases. Leishmaniases are characterized by the emergence of new virulent and asymptomatic strains of Leishmania spp. and, as a consequence, by a very diverse clinical spectrum. To fight more efficiently these parasites, the mechanisms of host defense and of parasite virulence need to be thoroughly investigated. To this aim, animal models are widely used. However, the results obtained with these models are influenced by several experimental parameters, such as the mouse genetic background, parasite genotype, inoculation route/infection site, parasite dose and phlebotome saliva. In this review, we propose an update on their influence in the two main clinical forms of the disease: cutaneous and visceral leishmaniases

KSAC, a Defined Leishmania Antigen, plus Adjuvant Protects against the Virulence of L. major Transmitted by Its Natural Vector Phlebotomus duboscqi

Leishmaniasis is a neglected disease caused by the Leishmania parasite and transmitted by the bite of an infective sand fly. Despite the importance of this disease there is no vaccine available for humans. Studies have shown that vector-transmitted infections are more virulent, promoting parasite establishment and abrogating protection observed against needle-injected parasites in vaccinated mice. KSAC and L110f, derived from Leishmania-based polyproteins, protected mice against the needle-injected parasites. Here, we tested the two molecules for their capacity to protect mice against cutaneous leishmaniasis transmitted by an infective sand fly. Our results show that KSAC, but not L110f, confers protection against Leishmania transmitted by sand fly bites where protection was correlated to a strong immune response to Leishmania antigens by memory T cells before and after sand fly transmission of the parasite. This is the first report of a Leishmania-based vaccine that confers protection against a virulent sand fly challenge. Our results support the importance of screening Leishmania vaccine candidates using infective sand flies before moving forward with the costly steps of vaccine development

Cerebral Changes Occurring in Arginase and Dimethylarginine Dimethylaminohydrolase (DDAH) in a Rat Model of Sleeping Sickness

Involvement of nitric oxide (NO) in the pathophysiology of human African trypanosomiasis (HAT) was analyzed in a HAT animal model (rat infected with Trypanosoma brucei brucei). With this model, it was previously reported that trypanosomes were capable of limiting trypanocidal properties carried by NO by decreasing its blood concentration. It was also observed that brain NO concentration, contrary to blood, increases throughout the infection process. The present approach analyses the brain impairments occurring in the regulations exerted by arginase and N(G), N(G)-dimethylarginine dimethylaminohydrolase (DDAH) on NO Synthases (NOS). In this respect: (i) cerebral enzymatic activities, mRNA and protein expression of arginase and DDAH were determined; (ii) immunohistochemical distribution and morphometric parameters of cells expressing DDAH-1 and DDAH-2 isoforms were examined within the diencephalon; (iii) amino acid profiles relating to NOS/arginase/DDAH pathways were established.Arginase and DDAH activities together with mRNA (RT-PCR) and protein (western-blot) expressions were determined in diencephalic brain structures of healthy or infected rats at various days post-infection (D5, D10, D16, D22). While arginase activity remained constant, that of DDAH increased at D10 (+65%) and D16 (+51%) in agreement with western-blot and amino acids data (liquid chromatography tandem-mass spectrometry). Only DDAH-2 isoform appeared to be up-regulated at the transcriptional level throughout the infection process. Immunohistochemical staining further revealed that DDAH-1 and DDAH-2 are contained within interneurons and neurons, respectively.In the brain of infected animals, the lack of change observed in arginase activity indicates that polyamine production is not enhanced. Increases in DDAH-2 isoform may contribute to the overproduction of NO. These changes are at variance with those reported in the periphery. As a whole, the above processes may ensure additive protection against trypanosome entry into the brain, i.e., maintenance of NO trypanocidal pressure and limitation of polyamine production, necessary for trypanosome growth

CD8+ T Cells as a Source of IFN-γ Production in Human Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) is usually a self-healing skin lesion caused by different species of Leishmania parasite. Resistance and susceptibility of mice to Leishmania major infection is associated with two types of CD4+ T lymphocytes development: Th1 type response with production of cytokine IFN-γ is associated with resistance, whereas Th2 type response with production of cytokines IL-4 and IL-5 is associated with susceptibility. A clear Th1/Th2 dichotomy similar to murine model is not defined in human leishmaniasis and we need as much information as possible to define marker(s) of protection. We purified CD4+/CD8+ T cells, stimulated them with Leishmania antigens and analysed gene and protein expression of Th1/Th2 cytokines in volunteers with a history of self-healing CL who are presumed to be protected against further Leishmania infection. We have seen significant upregulation of IFN-γ gene expression and high IFN-γ production in the Leishmania stimulated CD4+ T cells and CD8+ T cells. We concluded that both antigen-specific IFN-γ producing CD4+ Th1 cells and IFN-γ producing CD8+ T cells contribute to the long term protection in individuals with a history of CL. This proves the importance of CD8+ T cells as a source of IFN-γ in Th1-like immune responses

Potentiating Effects of MPL on DSPC Bearing Cationic Liposomes Promote Recombinant GP63 Vaccine Efficacy: High Immunogenicity and Protection

Visceral leishmaniasis (VL), a vector-transmitted disease caused by Leishmania donovani, is potentially fatal if left untreated. Vaccination against VL has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine is pressing for the control of the disease. Earlier, we observed protective efficacy using leishmanial antigen (Ag) in the presence of either cationic liposomes or monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) against experimental VL through the intraperitoneal (i.p.) route of administration in the mouse model. However, this route of immunization is not adequate for human use. For this work, we developed vaccine formulations combining cationic liposomes with MPL-TDM using recombinant GP63 (rGP63) as protein Ag through the clinically relevant subcutaneous (s.c.) route. Two s.c. injections with rGP63 in association with cationic liposomes and MPL-TDM showed enhanced immune responses that further resulted in high protective levels against VL in the mouse model. This validates the combined use of MPL-TDM as an immunopotentiator and liposomes as a suitable vaccine delivery system

Effector Memory Th1 CD4 T Cells Are Maintained in a Mouse Model of Chronic Malaria

Protection against malaria often decays in the absence of infection, suggesting that protective immunological memory depends on stimulation. Here we have used CD4+ T cells from a transgenic mouse carrying a T cell receptor specific for a malaria protein, Merozoite Surface Protein-1, to investigate memory in a Plasmodium chabaudi infection. CD4+ memory T cells (CD44hiIL-7Rα+) developed during the chronic infection, and were readily distinguishable from effector (CD62LloIL-7Rα−) cells in acute infection. On the basis of cell surface phenotype, we classified memory CD4+ T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62LloCD27+) dominated the chronic infection. We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells. In adoptive transfer, CD44hi memory cells from chronically infected mice were more effective at delaying and reducing parasitemia and pathology than memory cells from drug-treated mice without chronic infection, and contained a greater proportion of effector cells producing IFN-γ and TNFα, which may have contributed to the enhanced protection. These findings may explain the observation that in humans with chronic malaria, activated effector memory cells are best maintained in conditions of repeated exposure

A Comprehensive Genetic Analysis of Candidate Genes Regulating Response to Trypanosoma congolense Infection in Mice

About one-third of cattle in sub-Saharan Africa are at risk of contracting “Nagana”—a disease caused by Trypanosoma parasites similar to those that cause human “Sleeping Sickness.” Laboratory mice can also be infected by trypanosomes, and different mouse breeds show varying levels of susceptibility to infection, similar to what is seen between different breeds of cattle. Survival time after infection is controlled by the underlying genetics of the mouse breed, and previous studies have localised three genomic regions that regulate this trait. These three “Quantitative Trait Loci” (QTL), which have been called Tir1, Tir2 and Tir3 (for Trypanosoma Infection Response 1–3) are well defined, but nevertheless still contain over one thousand genes, any number of which may be influencing survival. This study has aimed to identify the specific differences associated with genes that are controlling mouse survival after T. congolense infection. We have applied a series of analyses to existing datasets, and combined them with novel sequencing, and other genetic data to create short lists of genes that share polymorphisms across susceptible mouse breeds, including two promising “candidate genes”: Pram1 at Tir1 and Cd244 at Tir3. These genes can now be tested to confirm their effect on response to trypanosome infection

Induction of Protective CD4+ T Cell-Mediated Immunity by a Leishmania Peptide Delivered in Recombinant Influenza Viruses

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4+ Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK158–173 CD4+ peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK158–173-specific CD4+ T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK158–173 triggers LACK158–173-specific Th1-biased CD4+ T cell responses within an appropriate cytokine milieu (IFN-γ, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2–4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-γ-producing CD4+ T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK158–173 led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-γ producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity