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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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Related in: MedlinePlus

Depletion of neutrophils following sand fly bite enhances the efficacy of ALM+CpG vaccination.AMC, ALM+CpG vaccinated, or healed mice were exposed to the bites of 4 L.m.-infected sand flies and subsequently treated with GL113 control IgG (open symbols) or NIMP-R14 neutrophil-depleting Ab (closed symbols) at 3.5, 9 and 14 days following sand fly bite. (A and B) Representative dot plot of CD11b gated, Ly6-G and F4/80 expressing ear cells (A) and the total number of CD11b+Ly-6G+F4/80− neutrophils and CD11b+Ly6G−F4/80+ macrophage/monocytes per ear (n = 4) (B), among ALM+CpG vaccinated mice 2.5 days following Ab treatment and 6 days following exposure to infected sand flies. (*) p = 0.03 versus GL113. (C) Frequency of TNF-α+ and/or IFN-γ+ cells among CD3+CD4+-gated T cells after DC or DC+Ag re-stimulation of ear derived cells 15 days following exposure to infected sand fly bites. Analysis of parasite numbers per ear (D) and lesion diameter (E) among the indicated groups 28 days following exposure to infected sand fly bites. Each data point represents an individual ear in three pooled experiments in which animals from AMC and ALM+CpG groups were treated with GL113 or NIMP-R14 mAb. In 4D: (‡‡‡‡) p<0.0001 versus AMC(NIMP-R14); (**) p = 0.002, (*) p = 0.044 versus ALM+CpG(GL113); (#) p = 0.009, (###) p = 0.0004 versus AMC(GL113). In 4E (‡‡‡‡) p<0.0001 versus AMC(NIMP-R14); (*) p = 0.011 versus ALM+CpG(GL113).
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ppat-1000484-g004: Depletion of neutrophils following sand fly bite enhances the efficacy of ALM+CpG vaccination.AMC, ALM+CpG vaccinated, or healed mice were exposed to the bites of 4 L.m.-infected sand flies and subsequently treated with GL113 control IgG (open symbols) or NIMP-R14 neutrophil-depleting Ab (closed symbols) at 3.5, 9 and 14 days following sand fly bite. (A and B) Representative dot plot of CD11b gated, Ly6-G and F4/80 expressing ear cells (A) and the total number of CD11b+Ly-6G+F4/80− neutrophils and CD11b+Ly6G−F4/80+ macrophage/monocytes per ear (n = 4) (B), among ALM+CpG vaccinated mice 2.5 days following Ab treatment and 6 days following exposure to infected sand flies. (*) p = 0.03 versus GL113. (C) Frequency of TNF-α+ and/or IFN-γ+ cells among CD3+CD4+-gated T cells after DC or DC+Ag re-stimulation of ear derived cells 15 days following exposure to infected sand fly bites. Analysis of parasite numbers per ear (D) and lesion diameter (E) among the indicated groups 28 days following exposure to infected sand fly bites. Each data point represents an individual ear in three pooled experiments in which animals from AMC and ALM+CpG groups were treated with GL113 or NIMP-R14 mAb. In 4D: (‡‡‡‡) p<0.0001 versus AMC(NIMP-R14); (**) p = 0.002, (*) p = 0.044 versus ALM+CpG(GL113); (#) p = 0.009, (###) p = 0.0004 versus AMC(GL113). In 4E (‡‡‡‡) p<0.0001 versus AMC(NIMP-R14); (*) p = 0.011 versus ALM+CpG(GL113).

Mentions: We explored the possibility that neutrophil depletion might rescue the ability of the killed vaccine to confer protection against sand fly transmitted infection. As neutrophils are important for the early establishment of sand fly transmitted infections, their depletion at the time of challenge would, as previously shown [34], promote early resistance and compromise infection even in the naïve mice. Thus, the mice were left untreated for the first 3.5 days following sand fly transmission, then treated on days 3.5, 9, and 14, with a neutrophil depleting Ab [38],[39] or control IgG to mimic the loss of neutrophils observed following needle inoculation, but not sand fly transmission. Analysis of CD11b+Ly-6G+F4/80− neutrophils and CD11b+Ly-6G−F4/80+ macrophages/monocytes at the site of infection 6 days post-transmission revealed that the neutrophil depletion was both specific and efficient (Figure 4, A and B). At 2 weeks post-transmission, the neutrophil depletion promoted stronger Ag-specific IFN-γ and TNF-α responses in the ALM+CpG vaccinated mice (Figure 4C). More importantly, the neutrophil depletion enhanced the efficacy of the killed vaccine. Analysis of extensive data pooled from three independent experiments revealed that on day 28 post-transmission, the neutrophil depleted, ALM+CpG-vaccinated mice showed a highly significant reduction in parasite load compared with neutrophil depleted, naïve mice (p<0.0001), as well as control treated, ALM+CpG vaccinated mice (p = 0.002), and indistinguishable from that in healed animals (Figure 4D). The enhanced parasite clearance in neutrophil depleted, ALM+CpG vaccinated mice was associated with a significant reduction in lesion size compared with neutrophil depleted, naïve mice (p<0.0001) and control treated, ALM+CpG vaccinated mice (p = 0.01) (Figure 4E). Importantly, the neutrophil-depleted, naïve controls did not exhibit lower parasite loads compared with their control treated counterparts, suggesting the effect of neutrophil depletion after the initial establishment of infection, and during the extended period of neutrophil recruitment following transmission by bite, was specific to the vaccine setting.


Vector transmission of leishmania abrogates vaccine-induced protective immunity.

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

Depletion of neutrophils following sand fly bite enhances the efficacy of ALM+CpG vaccination.AMC, ALM+CpG vaccinated, or healed mice were exposed to the bites of 4 L.m.-infected sand flies and subsequently treated with GL113 control IgG (open symbols) or NIMP-R14 neutrophil-depleting Ab (closed symbols) at 3.5, 9 and 14 days following sand fly bite. (A and B) Representative dot plot of CD11b gated, Ly6-G and F4/80 expressing ear cells (A) and the total number of CD11b+Ly-6G+F4/80− neutrophils and CD11b+Ly6G−F4/80+ macrophage/monocytes per ear (n = 4) (B), among ALM+CpG vaccinated mice 2.5 days following Ab treatment and 6 days following exposure to infected sand flies. (*) p = 0.03 versus GL113. (C) Frequency of TNF-α+ and/or IFN-γ+ cells among CD3+CD4+-gated T cells after DC or DC+Ag re-stimulation of ear derived cells 15 days following exposure to infected sand fly bites. Analysis of parasite numbers per ear (D) and lesion diameter (E) among the indicated groups 28 days following exposure to infected sand fly bites. Each data point represents an individual ear in three pooled experiments in which animals from AMC and ALM+CpG groups were treated with GL113 or NIMP-R14 mAb. In 4D: (‡‡‡‡) p<0.0001 versus AMC(NIMP-R14); (**) p = 0.002, (*) p = 0.044 versus ALM+CpG(GL113); (#) p = 0.009, (###) p = 0.0004 versus AMC(GL113). In 4E (‡‡‡‡) p<0.0001 versus AMC(NIMP-R14); (*) p = 0.011 versus ALM+CpG(GL113).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691580&req=5

ppat-1000484-g004: Depletion of neutrophils following sand fly bite enhances the efficacy of ALM+CpG vaccination.AMC, ALM+CpG vaccinated, or healed mice were exposed to the bites of 4 L.m.-infected sand flies and subsequently treated with GL113 control IgG (open symbols) or NIMP-R14 neutrophil-depleting Ab (closed symbols) at 3.5, 9 and 14 days following sand fly bite. (A and B) Representative dot plot of CD11b gated, Ly6-G and F4/80 expressing ear cells (A) and the total number of CD11b+Ly-6G+F4/80− neutrophils and CD11b+Ly6G−F4/80+ macrophage/monocytes per ear (n = 4) (B), among ALM+CpG vaccinated mice 2.5 days following Ab treatment and 6 days following exposure to infected sand flies. (*) p = 0.03 versus GL113. (C) Frequency of TNF-α+ and/or IFN-γ+ cells among CD3+CD4+-gated T cells after DC or DC+Ag re-stimulation of ear derived cells 15 days following exposure to infected sand fly bites. Analysis of parasite numbers per ear (D) and lesion diameter (E) among the indicated groups 28 days following exposure to infected sand fly bites. Each data point represents an individual ear in three pooled experiments in which animals from AMC and ALM+CpG groups were treated with GL113 or NIMP-R14 mAb. In 4D: (‡‡‡‡) p<0.0001 versus AMC(NIMP-R14); (**) p = 0.002, (*) p = 0.044 versus ALM+CpG(GL113); (#) p = 0.009, (###) p = 0.0004 versus AMC(GL113). In 4E (‡‡‡‡) p<0.0001 versus AMC(NIMP-R14); (*) p = 0.011 versus ALM+CpG(GL113).
Mentions: We explored the possibility that neutrophil depletion might rescue the ability of the killed vaccine to confer protection against sand fly transmitted infection. As neutrophils are important for the early establishment of sand fly transmitted infections, their depletion at the time of challenge would, as previously shown [34], promote early resistance and compromise infection even in the naïve mice. Thus, the mice were left untreated for the first 3.5 days following sand fly transmission, then treated on days 3.5, 9, and 14, with a neutrophil depleting Ab [38],[39] or control IgG to mimic the loss of neutrophils observed following needle inoculation, but not sand fly transmission. Analysis of CD11b+Ly-6G+F4/80− neutrophils and CD11b+Ly-6G−F4/80+ macrophages/monocytes at the site of infection 6 days post-transmission revealed that the neutrophil depletion was both specific and efficient (Figure 4, A and B). At 2 weeks post-transmission, the neutrophil depletion promoted stronger Ag-specific IFN-γ and TNF-α responses in the ALM+CpG vaccinated mice (Figure 4C). More importantly, the neutrophil depletion enhanced the efficacy of the killed vaccine. Analysis of extensive data pooled from three independent experiments revealed that on day 28 post-transmission, the neutrophil depleted, ALM+CpG-vaccinated mice showed a highly significant reduction in parasite load compared with neutrophil depleted, naïve mice (p<0.0001), as well as control treated, ALM+CpG vaccinated mice (p = 0.002), and indistinguishable from that in healed animals (Figure 4D). The enhanced parasite clearance in neutrophil depleted, ALM+CpG vaccinated mice was associated with a significant reduction in lesion size compared with neutrophil depleted, naïve mice (p<0.0001) and control treated, ALM+CpG vaccinated mice (p = 0.01) (Figure 4E). Importantly, the neutrophil-depleted, naïve controls did not exhibit lower parasite loads compared with their control treated counterparts, suggesting the effect of neutrophil depletion after the initial establishment of infection, and during the extended period of neutrophil recruitment following transmission by bite, was specific to the vaccine setting.

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