A sand fly salivary protein vaccine shows efficacy against vector-transmitted cutaneous leishmaniasis in nonhuman primates
TL;DR: It is demonstrated that a vaccine against sand fly salivary protein can protect nonhuman primate from leishmania infection and PdSP15 sequence and structure show no homology to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.
Abstract: Currently, there are no commercially available human vaccines against leishmaniasis. In rodents, cellular immunity to salivary proteins of sand fly vectors is associated to protection against leishmaniasis, making them worthy targets for further exploration as vaccines. We demonstrate that nonhuman primates (NHP) exposed to Phlebotomus duboscqi uninfected sand fly bites or immunized with salivary protein PdSP15 are protected against cutaneous leishmaniasis initiated by infected bites. Uninfected sand fly-exposed and 7 of 10 PdSP15-immunized rhesus macaques displayed a significant reduction in disease and parasite burden compared to controls. Protection correlated to the early appearance of Leishmania-specific CD4(+)IFN-I³(+) lymphocytes, suggesting that immunity to saliva or PdSP15 augments the host immune response to the parasites while maintaining minimal pathology. Notably, the 30% unprotected PdSP15-immunized NHP developed neither immunity to PdSP15 nor an accelerated Leishmania-specific immunity. Sera and peripheral blood mononuclear cells from individuals naturally exposed to P. duboscqi bites recognized PdSP15, demonstrating its immunogenicity in humans. PdSP15 sequence and structure show no homology to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.
Summary (3 min read)
- Leishmaniasis is a neglected tropical disease that affects the poorest of communities and comes only second to malaria and fourth among tropical parasitic diseases in mortality and morbidity, respectively (1).
- Experimentally, it has been shown that exposure to saliva through bites of uninfected sand flies or immunization with an appropriate salivary protein protects rodents against cutaneous and visceral leishmaniases (2–5).
- 5Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil.
- Some twothirds of new CL cases occur in six countries including Afghanistan, Algeria, Brazil, Colombia, Iran, and the SyrianArabRepublic (9).
- Exposure to uninfected sand fly bites protects NHP against sand fly–transmitted CL Next, the authors tested whether immunity generated to sand fly salivary proteins in NHP was protective against vector-transmitted CL.
- Exact measurements and P values for all the samples tested are presented in tables S1 and S2.
- Similar to uninfected sand fly–exposed NHP, both CD8+ and CD4+ lymphocytes produced Leishmania-specific IFN-g, but only the frequency of the latter was significantly higher in PdSP15–IFN-g+ NHP compared to controls (Fig. 3J and fig. S4C), reinforcing the conclusion that the protective immune response is mostly driven by CD4+ lymphocytes.
- This study was designed to investigate the efficacy of P. duboscqi sand fly salivary proteins as vaccine candidates against CL.
- The authors exposed rhesus macaques to uninfected sand fly bites or immunized them with an immunogenic sand fly salivary protein (PdSP15).
- For challenge experiments, the authors calculated their sample sizes to achieve statistically significant results if protection is at least 80% effective (a 0.835% probability of preventing or reducing the CL outcome) when considering that 90% of the naïve NHP would develop lesions after challenge.
- Data from available frozen sera and cryopreserved PBMCs from inhabitants of an endemic area where P. duboscqi is prevalent were used to assess the immunogenicity of PdSP15 in humans.
- The number of animals per group and per experiment is indicated in all figure legends.
- Rhesus macaques (Macaca mulatta, Indian strain) were housed in the Walter Reed Army Institute of Research vivarium where manipulations were conducted under protocol IEO02-09 approved by the Walter Reed Army Institute of Research Institute Animal Care and Use Committee and by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee under protocol LMVR12.
- All NHP were screened for good physical health and absence of antibodies reactive to P. duboscqi sand fly salivary proteins before enrollment into the animal protocol.
- During experimental manipulations, they were housed individually in stainless steel cages (cubic ~2 m), kept in environmentally controlled rooms with 10 to 15 air changes per hour, temperature range 18° to 29°C, relative humidity 70%, and light/dark 12:12-hour cycle.
- NHP were fed a staple diet of LabDiet Primate Chow #5038, supplemented by Prima Treats #F05709 (Bio-Serve) fresh fruits and vegetables with water available ad libitum from an automatic system.
- Macaques were consistently negative for SIV (simian immunodeficiency virus) and STLV (simian T lymphotrophic virus) when serologically tested annually, and quarterly, intradermal TB tests were negative.
Sand flies and SGH
- P. duboscqi sand flies originally from Mali, West Africa, and reared in the insectary facilities of the Laboratory of Malaria and Vector Research, NIAID, NIH, were used for the described experiments.
- For transmission experiments, 3- to 4-day-old sand flies were allowed to feed on blood containing L. major promastigotes as previously described (4).
- Sand flies with mature infections (11 to 15 days after blood feeding) were used to transmit Leishmania parasites to NHP.
- Five- to 7-day-old sand flies were used for preparation of SGH.
- Briefly, pools of 20 salivary glands were dissected in phosphate-buffered saline.
- L. majorWR 2885 strain was used to infect sand flies and for preparation of specific Leishmania antigen.
- This strain of parasites was recently isolated from a soldier deployed to Iraq as previously described (4).
- Leishmania antigen was prepared by harvesting 1 × 109 parasites from culture flasks and repeated freeze-thaw cycles.
- Twenty-three DNA plasmids coding to P. duboscqi salivary proteins were cloned in the VR2001-TOPO vector (Vical Inc.), and endotoxinfree DNA was purified as previously described (14).
Reverse antigen screening
- NHP were inoculated intradermally with 30 mg of the 23 distinct DNA plasmids, once, for reverse antigen experiments.
- Onemonth after the last uninfected sand fly bite exposure, NHP were injected intradermally using an insulin syringe with each of the DNA candidates, empty plasmid control, SGH, and bites from one uninfected sand fly in the inner thighs and/or chest.
- Twenty-four and 48 hours after the inoculations, reactions were recorded by measurement of the induration diameter suing a Vernier caliper.
- The structure of PdSP15 was determined by molecular replacement using a monomeric PdSP15b structure (56) as a search model in the program PHASER (57).
- Lesion sizewasmeasuredweekly as thediameter of the skin lesionusing adigitalVernier caliper .
Leishmanin skin test
- Animals were inoculated with 100 mg in 100 ml of the Leishmania antigen, intradermally in the left inner thigh.
- Measurements were taken with a Vernier caliper .
- Antibody measurements by enzyme-linked immunosorbent assay Microtiter plates were coated with P. duboscqi sand fly SGH (1 pair/ ml) or with rPdSP15 (2 mg/ml) overnight at 4 °C.
- Alkaline phosphatase substrate was added for 30 min, and absorbance was read at 405 nm in a spectrophotometer (Molecular Devices).
Histological analysis of DTH site
- DNA or RNA was extracted using QIAamp DNA Micro Kit or RNeasy Mini Kit, respectively, following the manufacturer’s instructions.
- Numbers of parasites in the skin were determined by SYBR Green real-time PCR assay and primers JW11-JW12 as targets for Leishmania amplification (59).
- Results are expressed as relative expression, where value of 1 is the normal skin baseline.
- Lines present in the scatterplot graphs represent the mean, and bar graphs depict means ± SEM or SD as indicated.
- Statistical differences between two groups were tested by t test (two-tailedMann-Whitney test).
- Correlations were tested by Spearman test, and dotted lines illustrate the 95% CI.
- Fig. S3. CD8+ lymphocytes are not critical for protection fromCL inNHPexposed to uninfected sand fly bites.
- Fig. S4. Anti-PdSP15 antibodies and CD8+ lymphocytes are not critical for protection from CL in PdSP15-immunized NHP.
REFERENCES AND NOTES
- R.G., C.T., andP.A.C. participated in laboratory and animal studies.
- Leishmaniasis is transmitted by the bite of infected phlebotomine sand flies, which also transfer some of demonstrate that a vaccine against sand fly salivary protein can protect nonhuman primate from leishmaniaal.
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Cites background from "A sand fly salivary protein vaccine..."
...Development of a commercially available vaccine for Old World CL should also be made a priority, as one does not currently exist even though it would enhance efficacy of disease and vector control programs [71,72]....
"A sand fly salivary protein vaccine..." refers background in this paper
...The presence of polyfunctional T cells has been associated with effectiveness of the protective immune response in several vaccine candidates for diverse infectious diseases such as leishmaniasis (36), smallpox (37), hepatitis C virus (38), and tuberculosis (TB) (39, 40)....
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