In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

International audiencePSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in severalLeishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice.We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in aL. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L.braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals weresubdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantuminfection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high orlow levels of IFN-c in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detectedin sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-c, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-a in more. No significant cytokine response wasobserved in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increasein CD4+ T cells producing IFN-c after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between thepercentage of IFN-c-producing CD4+ T cells and the released IFN-c. We showed that the LaPSA-38S protein was able toinduce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infectionindicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacityof Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infectio

Stochastic extinction and the selection of the transmission mode in microparasites.

International audienceStochastic fluctuations in the transmission process of microparasites generate a risk of parasite extinction that cannot be assessed by deterministic models, especially in host populations of small size. While this risk of extinction represents a strong selection pressure for microparasites, it is usually not clearly separated from the deterministic ones. We suggest here that this stochastic selection pressure can affect the selection of the transmission mode of microparasites. To avoid extinction, parasites should maximize their inter-population transmission to ensure frequent reintroductions. Since the types of contacts may differ if congeners belong to the same or distinct populations, strains that are mainly transmitted through inter-population contacts might be selected. To examine this assumption, we analyse the issue of the competition between two strains differing in their transmission mode using a stochastic metapopulation model in which hosts may display different behaviours inside and outside their populations. We show that stochastic selection pressures may drive parasite evolution towards a transmission mode that maximizes the persistence of the parasite. We study the conditions under which stochastic selection pressures may surpass the deterministic ones. Our results are illustrated by the cases of feline immunodeficiency virus in cats and of sexually transmitted diseases in mammals

Selection of endogenous reference genes for gene expression analysis in Leishmania major developmental stages

International audienceAt the era of post-genomics, gene expression analysis constitutes an important step for understanding the biological functions of genes. For this, reverse transcription and real-time polymerase chain reaction (RT-PCR) is one of the most accurate techniques available to date. Normalization with a proper internal control is critical for the generation of reliable results with biological significance. This is particularly true for pathogens, like Leishmania (L.) parasites, that alternate between different stages during their life cycle. In this study, we evaluate six different sequences for their potential as suitable internal control for the study of gene expression in three different developmental stages (procyclic and metacyclic promastigotes and amastigotes) of the parasite Leishmania major. Experiments were performed on RNA purified from three L. major isolates using the RT-PCR technique. Data analysis was performed using GeNorm and NormFinder programs. We could determine that a sequence encoding rRNA45 is the most stable in the three developmental stages of the parasite and can thus be used as a reference gene in gene expression studies in L. major

Immunomodulatory Effects of Four Leishmania infantum Potentially Excreted/Secreted Proteins on Human Dendritic Cells Differentiation and Maturation.

Leishmania parasites and some molecules they secrete are known to modulate innate immune responses through effects on dendritic cells (DCs) and macrophages. Here, we characterized four Leishmania infantum potentially excreted/secreted recombinant proteins (LipESP) identified in our laboratory: Elongation Factor 1 alpha (LiEF-1α), a proteasome regulatory ATPase (LiAAA-ATPase) and two novel proteins with unknown functions, which we termed LiP15 and LiP23, by investigating their effect on in vitro differentiation and maturation of human DCs and on cytokine production by DCs and monocytes. During DCs differentiation, LipESP led to a significant decrease in CD1a. LiP23 and LiEF-1α, induced a decrease of HLA-DR and an increase of CD86 surface expression, respectively. During maturation, an up-regulation of HLA-DR and CD80 was found in response to LiP15, LiP23 and LiAAA-ATPase, while an increase of CD40 expression was only observed in response to LiP15. All LipESP induced an over-expression of CD86 with significant differences between proteins. These proteins also induced significant IL-12p70 levels in immature DCs but not in monocytes. The LipESP-induced IL-12p70 production was significantly enhanced by a co-treatment with IFN-γ in both cell populations. TNF-α and IL-10 were induced in DCs and monocytes with higher levels observed for LiP15 and LiAAA-ATPase. However, LPS-induced cytokine production during DC maturation or in monocyte cultures was significantly down regulated by LipESP co-treatment. Our findings suggest that LipESP strongly interfere with DCs differentiation suggesting a possible involvement in mechanisms established by the parasite for its survival. These proteins also induce DCs maturation by up-regulating several costimulatory molecules and by inducing the production of proinflammatory cytokines, which is a prerequisite for T cell activation. However, the reduced ability of LipESP-stimulated DCs and monocytes to respond to lipopolysaccharide (LPS) that can be observed during human leishmaniasis, suggests that under certain circumstances LipESP may play a role in disease progression

Human cellular and humoral immune responses to Phlebotomus papatasi salivary gland antigens in endemic areas differing in prevalence of Leishmania major infection

International audienceBACKGROUND:Sand fly saliva compounds are able to elicit specific immune responses that have a significant role in Leishmania parasite establishment and disease outcome. Characterizing anti-saliva immune responses in individuals living in well defined leishmaniasis endemic areas would provide valuable insights regarding their effect on parasite transmission and establishment in humans.METHODOLOGY/PRINCIPAL FINDINGS:We explored the cellular and humoral immune responses to Phlebotomus (P.) papatasi salivary gland extracts (SGE) in individuals living in cutaneous leishmaniasis (CL) old or emerging foci (OF, EF). OF was characterized by a higher infection prevalence as assessed by higher proportions of leishmanin skin test (LST) positive individuals compared to EF. Subjects were further subdivided into healed, asymptomatic or naïve groups. We showed anti-SGE proliferation in less than 30% of the individuals, regardless of the immune status, in both foci. IFN-γ production was higher in OF and only observed in immune individuals from OF and naïve subjects from EF. Although IL-10 was not detected, addition of anti-human IL-10 antibodies revealed an increase in proliferation and IFN-γ production only in individuals from OF. The percentage of seropositive individuals was similar in immune and naïves groups but was significantly higher in OF. No correlation was observed between anti-saliva immune responses and LST response. High anti-SGE-IgG responses were associated with an increased risk of developing ZCL. No differences were observed for anti-SGE humoral or cellular responses among naïve individuals who converted or not their LST response or developed or not ZCL after the transmission season.CONCLUSIONS/SIGNIFICANCE:These data suggest that individuals living in an old focus characterized by a frequent exposure to sand fly bites and a high prevalence of infection, develop higher anti-saliva IgG responses and IFN-γ levels and a skew towards a Th2-type cellular response, probably in favor of parasite establishment, compared to those living in an emerging focus

Effect of LipESP on human DCs maturation.

<p>Immature DCs were stimulated with LipESP for 48h and analysis of CD40, HLA-DR, CD86 and CD80 expression was performed by cytometry. Non-stimulated or LPS-stimulated DCs were considered as control cultures. Results are expressed as mean ± standard deviation of percentages of positive cells (n = 8). *: The results are statistically significant (p<0.05), when compared to control cells.</p

Effect of LipESP on LPS-, IFN-γ-, or IFN-γ/LPS-induced cytokine production by human monocytes.

<p>Production of (a) IL-12p70, (b) IL-10 and (c) TNF-α were measured in monocytes supernatant by ELISA after 24 h of stimulation by LipESP in presence or absence of LPS (for IL-10 and TNF-α analysis) or after 12 h of priming with IFN-γ followed by 24 additional hours of stimulation by LipESP with or without LPS co-treatment (for IL-12 analysis). Results are expressed as mean ± standard deviation (n = 9). * Statistically significant difference between non stimulated and LipESP-, LPS-, and IFN-γ/LPS-stimulated monocytes (p<0·05); ** Statistically significant difference between IFN-γ stimulated and LipESP/IFN-γ-co-treated monocytes (p<0·05) for IL-12p70 and between LPS-stimulated and LipESP-LPS-co-treated monocytes (p<0·05) for IL-10 and TNF-α; *** Statistically significant difference between IFN-γ/LPS stimulated and LipESP/IFN-γ/LPS-co-treated monocytes (p<0·05).</p

Effect of LipESP on human DCs differentiation.

<p>After 6 days of differentiation in presence or absence of LipESP, cells were harvested and labeled with appropriate antibodies. Cells with media alone represent DCs differentiation in the absence of LipESP and are considered as control cultures. Results are expressed as mean ± standard deviation of percentages of positive cells (n = 7). *: The results are statistically significant (p<0.05), when compared to control cells.</p

Effect of LipESP on LPS-, IFN-γ-, or IFN-γ/LPS-induced cytokine production by human DCs.

<p>Production of (a) IL-12p70, (b) IL-10 and (c) TNF-α were measured in DCs supernatant by ELISA after 48 h of stimulation by LipESP in presence or absence of LPS or IFN-γ or IFN-γ/LPS. Results are expressed as mean ± standard deviation (n = 10). * Statistically significant difference between non stimulated and LipESP-, LPS-, IFN-γ and IFN-γ/LPS-stimulated DCs (p<0·05); ** statistically significant difference between LPS-stimulated and LipESP/LPS-co-treated DCs (p<0·05); *** statistically significant difference between IFN-γ stimulated and LipESP/IFN-γ-co-treated DCs (p<0·05).</p