Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites

Abstract

Introduction: Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease.

Methods: To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining.

Results: A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens.

Discussion: Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

Keywords: P. duboscqi; antigen; immune response; saliva; vaccine.

Figure 1

Timeline of Sand Fly Feeding (SFF) and blood collection. Healthy volunteers (n=14) were exposed to bites of uninfected P. duboscqi sand fly on a bi-weekly basis for the first two months and once every 2 months for the following 10 months, with an optional recall exposure 6 months after completion. Blood collections were performed to assess development of the specific humoral and cellular immune responses.

Figure 2

Demonstration of the sealed feeding apparatus. The feeding device containing ten (10) uninfected female sand flies was individually attached to the arm of each volunteer for 20 minutes.

Figure 3

Number of completed sand fly exposures. This shows the number of P. duboscqi exposures that each participant completed. Recall means challenge after a 6 month hiatus post exposure. Each color represents one participant.

Figure 4

Immediate and delayed skin reactions post feeding. (A) Represents the number and type of skin reactions immediately after feeding for each exposure time. (B) Represents the number and type of skin reactions after one week post P. duboscqi challenge for each exposure time.

Figure 5

Serial immediate skin reactions and systemic immune responses after P. duboscqi feeding on participant #21 (removed from further sand fly challenge after 4^th exposure). (A.1) Participant arm at exposure 1; (A.2) Participant arm at exposure 2; (A.3) Participant arm immediately after removing feeder at exposure 4; (A.4) Participant arm 5 minutes after removing feeder at exposure 4; (B) Plasma levels of IgG antibodies to P. duboscqi saliva of participant at different time points. (C) IFN-gamma (pg/ml) production to P. duboscqi SGH and recombinant proteins at various timepoints.

Figure 6

Delayed large local reaction from participant #16 after two exposures (different arms) of P. duboscqi bites. (A) Participant arm at exposure 1; (B) Participant arm at exposure 2; (C) Participant arm 2 days after exposure 2; (D) Participant arm 7 days after exposure 2.

Figure 7

Pruritus reported by participant. Number and percentage (%) of participants with pruritus at each exposure time. Each color represents one participant (n=14).

Figure 8

P. duboscqi challenge, 48 hour assessment including skin biopsy. Cellular infiltrate characteristics and bite site appearance from two participants that received the previous P. duboscqi exposure 2 months prior (now at exposures 8 and 9), hematoxylin/eosin staining (H&E) and immunohistochemical (IHC) staining.

Figure 9

P. duboscqi challenge, 48 hour assessment including skin biopsy. Skin biopsy characteristics and bite site appearance from two participants that had received the previous P. duboscqi exposure 6 months prior, hematoxylin/eosin staining (H&E) and immunohistochemical (IHC) staining.

Figure 10

Immunoreactivity to Pd salivary protein. (A) Plasma levels of IgG antibodies to P. duboscqi saliva in experimentally exposed human volunteers (n=14) at different time points. Pre-exposure is before sand fly bite exposure. The * represents the statistical significance between the time points when compared to pre-exposure. (B) Western blot of sand fly salivary proteins recognized by IgG (diluted 1:5000) from plasma (diluted 1:100) of volunteers exposed to the bite of P. duboscqi. One donor from the NIH blood bank was used as a negative control and one donor from a Mali endemic area was used as a positive control. Arrows represent some immunodominant proteins (~ 62 and 32kDa).

Figure 11

Characterization of the human cellular immune response to sand fly saliva or salivary recombinant proteins in volunteers experimentally exposed to P. duboscqi bites. (A) IFN-γ production (pg/ml) by Peripheral Blood Mononuclear cells (PBMCs) in response to a stimulation with SGH and recombinant salivary P. duboscqi proteins (PduM02, PduM10, PduM34, PduM35, PduM49 and PduM73) in exposed volunteers (n=13) versus blood bank donors (n=6). (B) IL-10 production (pg/ml) by Peripheral Blood Mononuclear cells (PBMCs) in response to stimulation with SGH and recombinant salivary P. duboscqi proteins (PduM02, PduM10, PduM34, PduM35, PduM49 and PduM73) in exposed volunteers (n=13) versus blood bank donors (n=2). Solid lines indicate the mean of each group. The * represents the statistical significance between SGH compared to the indicated recombinant protein. The # represents the statistical significance between stimuli versus controls.