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Vector transmission of leishmania abrogates vaccine-induced protective immunity.

Peters NC, Kimblin N, Secundino N, Kamhawi S, Lawyer P, Sacks DL - PLoS Pathog. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
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.

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ALM+CpG vaccinated mice are not protected against infected sand fly challenge.Ears of AMC, ALM+CpG vaccinated (ALM), or healed mice were exposed to the bites of 4 L.m.-infected sand flies, or needle inoculated with L.m. metacyclic promastigotes and subsequently analyzed at the indicated time-points. (A and B) Parasite loads in individual ears 28 days following exposure to infected sand fly bite or needle inoculation with either 103 L.m. parasites from culture (A) or 5×103 L.m. parasites from infected sand flies (B). (****) p<0.0001 relative to AMC Needle inoculated; (‡‡‡) p = 0.0002, (‡‡‡‡) p<0.0001 relative to AMC sand fly inoculated; (§§§) p = 0.0005, (§§§§) p<0.0001 relative to ALM+CpG. (C and D) Intracellular staining for the frequency (top number) and total number per ear (bottom number) of IFN-γ+ (C) or TNF-α+ (D) CD4+TcRβ+ T cells after in-vitro re-stimulation of pooled ear-derived cells from the same groups of mice employed in 2A, with BMDC alone (DC) or DC+Ag. (E) Detection of IFN-γ by ELISA following in-vitro re-stimulation of ear derived cells with DC or DC+Ag. (‡) p = 0.010; (‡‡) 0.003<p<0.004; (‡‡‡‡) p<0.0001 relative to DC; (**) p = 0.004; (****) p<0.0001 relative to AMC DC+Ag. (F) Frequency of TNF-α+ and/or IFN-γ+ cells among CD3+CD4+-gated ear derived T cells from the indicated groups following re-stimulation with DC or DC+Ag.
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ppat-1000484-g002: ALM+CpG vaccinated mice are not protected against infected sand fly challenge.Ears of AMC, ALM+CpG vaccinated (ALM), or healed mice were exposed to the bites of 4 L.m.-infected sand flies, or needle inoculated with L.m. metacyclic promastigotes and subsequently analyzed at the indicated time-points. (A and B) Parasite loads in individual ears 28 days following exposure to infected sand fly bite or needle inoculation with either 103 L.m. parasites from culture (A) or 5×103 L.m. parasites from infected sand flies (B). (****) p<0.0001 relative to AMC Needle inoculated; (‡‡‡) p = 0.0002, (‡‡‡‡) p<0.0001 relative to AMC sand fly inoculated; (§§§) p = 0.0005, (§§§§) p<0.0001 relative to ALM+CpG. (C and D) Intracellular staining for the frequency (top number) and total number per ear (bottom number) of IFN-γ+ (C) or TNF-α+ (D) CD4+TcRβ+ T cells after in-vitro re-stimulation of pooled ear-derived cells from the same groups of mice employed in 2A, with BMDC alone (DC) or DC+Ag. (E) Detection of IFN-γ by ELISA following in-vitro re-stimulation of ear derived cells with DC or DC+Ag. (‡) p = 0.010; (‡‡) 0.003<p<0.004; (‡‡‡‡) p<0.0001 relative to DC; (**) p = 0.004; (****) p<0.0001 relative to AMC DC+Ag. (F) Frequency of TNF-α+ and/or IFN-γ+ cells among CD3+CD4+-gated ear derived T cells from the indicated groups following re-stimulation with DC or DC+Ag.

Mentions: Vaccination with autoclaved L. major (ALM), or a recombinant leishmania protein, plus CpG oligodeoxynucleotides (ODN) has been shown to effectively protect against needle challenge with L. major in mice [11],[13]. We therefore employed ALM+CpG to test the efficacy of a non-living vaccine against natural transmission. Mice vaccinated with ALM+CpG three times s.c. in the footpad at two week intervals, along with age-matched naïve controls and mice with healed primary lesions, were exposed to the bites of 4 infected sand flies twelve weeks following the last vaccine injection. Four weeks following infected sand fly exposure or needle inoculation, coincident with the time of peak parasitic load in naïve mice, parasite burden in the ear dermis was assessed. Mice with healed primary lesions again dramatically controlled parasite growth following exposure to the bites of infected sand flies (Figure 2A). In contrast, ALM+CpG vaccination conferred no protection against transmission by sand fly bite, despite conferring strong protection against needle inoculation. Ear lesion measurements obtained 4 weeks after infection also revealed a compromised benefit of the ALM+CpG vaccine against sand fly challenge (Figure S1). Note that despite the comparable parasitic loads in naïve mice following sand fly or needle challenge, the pathology associated with transmission by bite was far more severe.


Vector transmission of leishmania abrogates vaccine-induced protective immunity.

Peters NC, Kimblin N, Secundino N, Kamhawi S, Lawyer P, Sacks DL - PLoS Pathog. (2009)

ALM+CpG vaccinated mice are not protected against infected sand fly challenge.Ears of AMC, ALM+CpG vaccinated (ALM), or healed mice were exposed to the bites of 4 L.m.-infected sand flies, or needle inoculated with L.m. metacyclic promastigotes and subsequently analyzed at the indicated time-points. (A and B) Parasite loads in individual ears 28 days following exposure to infected sand fly bite or needle inoculation with either 103 L.m. parasites from culture (A) or 5×103 L.m. parasites from infected sand flies (B). (****) p<0.0001 relative to AMC Needle inoculated; (‡‡‡) p = 0.0002, (‡‡‡‡) p<0.0001 relative to AMC sand fly inoculated; (§§§) p = 0.0005, (§§§§) p<0.0001 relative to ALM+CpG. (C and D) Intracellular staining for the frequency (top number) and total number per ear (bottom number) of IFN-γ+ (C) or TNF-α+ (D) CD4+TcRβ+ T cells after in-vitro re-stimulation of pooled ear-derived cells from the same groups of mice employed in 2A, with BMDC alone (DC) or DC+Ag. (E) Detection of IFN-γ by ELISA following in-vitro re-stimulation of ear derived cells with DC or DC+Ag. (‡) p = 0.010; (‡‡) 0.003<p<0.004; (‡‡‡‡) p<0.0001 relative to DC; (**) p = 0.004; (****) p<0.0001 relative to AMC DC+Ag. (F) Frequency of TNF-α+ and/or IFN-γ+ cells among CD3+CD4+-gated ear derived T cells from the indicated groups following re-stimulation with DC or DC+Ag.
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Related In: Results  -  Collection

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ppat-1000484-g002: ALM+CpG vaccinated mice are not protected against infected sand fly challenge.Ears of AMC, ALM+CpG vaccinated (ALM), or healed mice were exposed to the bites of 4 L.m.-infected sand flies, or needle inoculated with L.m. metacyclic promastigotes and subsequently analyzed at the indicated time-points. (A and B) Parasite loads in individual ears 28 days following exposure to infected sand fly bite or needle inoculation with either 103 L.m. parasites from culture (A) or 5×103 L.m. parasites from infected sand flies (B). (****) p<0.0001 relative to AMC Needle inoculated; (‡‡‡) p = 0.0002, (‡‡‡‡) p<0.0001 relative to AMC sand fly inoculated; (§§§) p = 0.0005, (§§§§) p<0.0001 relative to ALM+CpG. (C and D) Intracellular staining for the frequency (top number) and total number per ear (bottom number) of IFN-γ+ (C) or TNF-α+ (D) CD4+TcRβ+ T cells after in-vitro re-stimulation of pooled ear-derived cells from the same groups of mice employed in 2A, with BMDC alone (DC) or DC+Ag. (E) Detection of IFN-γ by ELISA following in-vitro re-stimulation of ear derived cells with DC or DC+Ag. (‡) p = 0.010; (‡‡) 0.003<p<0.004; (‡‡‡‡) p<0.0001 relative to DC; (**) p = 0.004; (****) p<0.0001 relative to AMC DC+Ag. (F) Frequency of TNF-α+ and/or IFN-γ+ cells among CD3+CD4+-gated ear derived T cells from the indicated groups following re-stimulation with DC or DC+Ag.
Mentions: Vaccination with autoclaved L. major (ALM), or a recombinant leishmania protein, plus CpG oligodeoxynucleotides (ODN) has been shown to effectively protect against needle challenge with L. major in mice [11],[13]. We therefore employed ALM+CpG to test the efficacy of a non-living vaccine against natural transmission. Mice vaccinated with ALM+CpG three times s.c. in the footpad at two week intervals, along with age-matched naïve controls and mice with healed primary lesions, were exposed to the bites of 4 infected sand flies twelve weeks following the last vaccine injection. Four weeks following infected sand fly exposure or needle inoculation, coincident with the time of peak parasitic load in naïve mice, parasite burden in the ear dermis was assessed. Mice with healed primary lesions again dramatically controlled parasite growth following exposure to the bites of infected sand flies (Figure 2A). In contrast, ALM+CpG vaccination conferred no protection against transmission by sand fly bite, despite conferring strong protection against needle inoculation. Ear lesion measurements obtained 4 weeks after infection also revealed a compromised benefit of the ALM+CpG vaccine against sand fly challenge (Figure S1). Note that despite the comparable parasitic loads in naïve mice following sand fly or needle challenge, the pathology associated with transmission by bite was far more severe.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus