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

Clinical and immunological parameters in individuals living in various emerging foci of <i>L</i>. <i>major</i> infection.

<p>Clinical and immunological parameters in individuals living in various emerging foci of <i>L</i>. <i>major</i> infection.</p

IgG response against <i>P</i>. <i>papatasi</i> salivary glands extracts in individuals living in old or emerging foci of <i>L</i>. <i>major</i> infection.

<p>Analysis of IgG response was performed in individuals from OF and EF (a) and LST+/Scar+ (healed), LST+/Scar- (asymptomatics) and LST-/Scar- (naïve) groups (b), in each focus. IgG antibodies were measured by ELISA. Results were expressed as relative OD (ROD) defined as the ratio of sample OD/mean OD of sera from negative controls. ROD superior or equal to 2 was considered positive. Negative sera were obtained from healthy controls living outside Tunisia in sand fly-free regions. Statistical significance was assigned to a value of p<0,05. Statistically significant differences between groups are showed for positive responders. Horizontal lines represent median ROD values and dotted lines represent cut-off level. The percentages of positive responders are presented for groups and foci.</p

IFN-γ levels induced by <i>P</i>. <i>papatasi</i> salivary glands extracts stimulation in the presence of an anti-IL-10 antibody, in individuals living in old or emerging foci of <i>L</i>. <i>major</i> infection.

<p>IFN-γ was quantified by ELISA, in the culture supernatants of PBMC stimulated with <i>P</i>. <i>papatasi</i> salivary gland extracts (1 gland/ml), with or without an anti-IL10 antibody (500ng/ml), during 72h. Analysis of IFN-γ production was performed in individuals from OF and EF (a) and LST+/Scar+ (healed), LST+/Scar- (asymptomatics) and LST-/Scar- (naïve) groups in OF (b) and EF (c). Statistical significance was assigned to a value of p<0.05. Statistically significant differences between stimulated and non-stimulated cultures and between groups are showed. Horizontal lines represent median IFN-γ values.</p

Clinical and immunological parameters in individuals living in old (OF) or emerging foci (EF) of <i>L</i>. <i>major</i> infection.

<p>Clinical and immunological parameters in individuals living in old (OF) or emerging foci (EF) of <i>L</i>. <i>major</i> infection.</p

Proliferative responses to <i>P</i>. <i>papatasi</i> salivary glands extracts in the presence of an anti-IL-10 antibody, in individuals living in old or emerging foci of <i>L</i>. <i>major</i> infection.

<p>PBMC were stimulated with <i>P</i>. <i>papatasi</i> salivary glands extracts (1gland/ml) with or without an anti-IL10 antibody (500ng/ml), for 5 days. Proliferation was analyzed in individuals from donors residing in an old (OF) or an emerging focus (EF) (a) and LST+/Scar+ (healed), LST+/Scar- (asymptomatics) and LST-/Scar- (naïve) groups (b), in each focus. Results were expressed as percentage of positive proliferative responders. The proliferative response was considered positive when SI was superior or equal to 2. Statistical significance was assigned to a value of p<0,05. Statistically significant differences between groups are showed.</p

Study design.

<p>Study design.</p

Proliferative responses to <i>P</i>. <i>papatasi</i> salivary glands extracts in individuals living in old or emerging foci of <i>L</i>. <i>major</i> infection.

<p>PBMC were stimulated for 5 days with <i>P</i>. <i>papatasi</i> salivary gland extracts (1gland/ml). Proliferation was analyzed in individuals from donors residing in an old (OF) or an emerging focus (EF) (a), LST+/Scar+ (healed), LST+/Scar- (asymptomatics) and LST-/Scar- (naïve) groups (b) and LST+, LST- groups (c), in each focus. Results were expressed as stimulation index (SI) obtained by dividing the mean counts of triplicates in antigen-stimulated cultures by the mean counts of triplicates in non-stimulated cultures. The proliferative response was considered positive when SI was superior or equal to 2. Statistical significance was assigned to a value of p<0,05. Horizontal lines represent median SI values and dotted lines represent cut-off level. The percentages of positive responders are presented for groups and foci.</p

Timeline showing the different steps of the prospective study.

<p>790 participants living in endemic areas of ZCL were followed up over 1 year throughout one season of <i>L</i>. <i>major</i> transmission. Parameters such as LST and the presence of typical scars were monitored at the beginning of the study and after the transmission season and the triggering of new cases. Peripheral blood samples were obtained from each donor at the beginning of the study.</p