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A cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis.

Nieuwenhuizen NE, Meter JM, Horsnell WG, Hoving JC, Fick L, Sharp MF, Darby MG, Parihar SP, Brombacher F, Lopata AL - PLoS Negl Trop Dis (2013)

Bottom Line: Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting.Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens.The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology, Cape Town Component, and Institute of Infectious Diseases and Molecular Medicine, Medical Research Council, Division of Immunology, Faculty of Health Science, University of Cape Town, Cape Town, South Africa. nnieuwen@gmail.com

ABSTRACT

Background: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection.

Methodology/principal findings: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens.

Conclusion: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.

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Related in: MedlinePlus

Passive immunization with Anti-Hb reduces worm burdens in Nippostrongylus brasiliensis infected mice.Mice (n = 5–8) were injected with 200 µg of 4E8g (Anti-Hb) at day −1 and infected with 500 N. brasiliensis L3 at Day 0. A) Worm burdens in the lung at different timepoints post infection. Bars represent the mean and error bars show standard error of the mean (SEM). B) Worm burdens in the intestine at different timepoints post infection. Bars represent the mean and error bars show SEM. C–D) Mesenteric lymph node cells from individual mice at day 7 (n = 5–8) were restimulated with C) anti-CD3 or D) N. brasiliensis somatic extract for 72 hours and cytokines were measured in the supernatant by ELISA. Bars represent the mean and error bars show SEM.E) Mouse mast cell proteases in serum at day 7. Statistical analysis was performed in GraphPad Prism using the unpaired Student's t test (*, p≤0.05; **, p≤0.01; ***, p≤0.001).
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pntd-0002395-g008: Passive immunization with Anti-Hb reduces worm burdens in Nippostrongylus brasiliensis infected mice.Mice (n = 5–8) were injected with 200 µg of 4E8g (Anti-Hb) at day −1 and infected with 500 N. brasiliensis L3 at Day 0. A) Worm burdens in the lung at different timepoints post infection. Bars represent the mean and error bars show standard error of the mean (SEM). B) Worm burdens in the intestine at different timepoints post infection. Bars represent the mean and error bars show SEM. C–D) Mesenteric lymph node cells from individual mice at day 7 (n = 5–8) were restimulated with C) anti-CD3 or D) N. brasiliensis somatic extract for 72 hours and cytokines were measured in the supernatant by ELISA. Bars represent the mean and error bars show SEM.E) Mouse mast cell proteases in serum at day 7. Statistical analysis was performed in GraphPad Prism using the unpaired Student's t test (*, p≤0.05; **, p≤0.01; ***, p≤0.001).

Mentions: Nematode and vertebrate haemoglobins are very dissimilar [23] (Figure 7A), with nematode haemoglobins showing only 10–15% homology to vertebrate haemoglobins [13] 4E8g was shown not to bind mouse haemoglobin by ELISA and western blotting (Figure 7B). To determine whether nematode haemoglobin could be targeted for immunization strategies, mice were injected with 4E8g one day prior to N. brasiliensis infection. Parasite burdens were counted in the lungs and intestine at different timepoints post infection. N. brasiliensis L3 migrate to the lungs after injection and moult into L4 larvae between 19–32 hours [25] The L4 remain in the lung till approximately 50 hours post infection before penetrating the alveoli, being coughed up and swallowed into the intestine. In the intestine they moult into adult worms at about 90–108 hours post infection. The number of parasites in the lungs was similar in anti-Hb and isotype control treated groups at 24 h and 48 h post infection (Figure 8A). Parasite numbers were low in the lungs in both groups at 72 h due to migration out of the lungs, although in anti-Hb treated mice the parasite numbers were significantly lower. In the intestine, equivalent worm burdens were found in both groups of mice at 72 h post infection. However at both day 5 and day 7 post infection, worm burdens were significantly reduced in anti-Hb treated mice. Protection was associated with increased levels of MMCP-1, a marker of mast cell degranulation, in the serum (Figure 8B) and increased production of Th2 cytokines by restimulated mesenteric lymph node cells (Figure 8 C–F). Together these data suggest that anti-Hb primarily confers protection against the adult worm, either directly or by priming the immune system to increase expulsion or destruction of the parasites.


A cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis.

Nieuwenhuizen NE, Meter JM, Horsnell WG, Hoving JC, Fick L, Sharp MF, Darby MG, Parihar SP, Brombacher F, Lopata AL - PLoS Negl Trop Dis (2013)

Passive immunization with Anti-Hb reduces worm burdens in Nippostrongylus brasiliensis infected mice.Mice (n = 5–8) were injected with 200 µg of 4E8g (Anti-Hb) at day −1 and infected with 500 N. brasiliensis L3 at Day 0. A) Worm burdens in the lung at different timepoints post infection. Bars represent the mean and error bars show standard error of the mean (SEM). B) Worm burdens in the intestine at different timepoints post infection. Bars represent the mean and error bars show SEM. C–D) Mesenteric lymph node cells from individual mice at day 7 (n = 5–8) were restimulated with C) anti-CD3 or D) N. brasiliensis somatic extract for 72 hours and cytokines were measured in the supernatant by ELISA. Bars represent the mean and error bars show SEM.E) Mouse mast cell proteases in serum at day 7. Statistical analysis was performed in GraphPad Prism using the unpaired Student's t test (*, p≤0.05; **, p≤0.01; ***, p≤0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757078&req=5

pntd-0002395-g008: Passive immunization with Anti-Hb reduces worm burdens in Nippostrongylus brasiliensis infected mice.Mice (n = 5–8) were injected with 200 µg of 4E8g (Anti-Hb) at day −1 and infected with 500 N. brasiliensis L3 at Day 0. A) Worm burdens in the lung at different timepoints post infection. Bars represent the mean and error bars show standard error of the mean (SEM). B) Worm burdens in the intestine at different timepoints post infection. Bars represent the mean and error bars show SEM. C–D) Mesenteric lymph node cells from individual mice at day 7 (n = 5–8) were restimulated with C) anti-CD3 or D) N. brasiliensis somatic extract for 72 hours and cytokines were measured in the supernatant by ELISA. Bars represent the mean and error bars show SEM.E) Mouse mast cell proteases in serum at day 7. Statistical analysis was performed in GraphPad Prism using the unpaired Student's t test (*, p≤0.05; **, p≤0.01; ***, p≤0.001).
Mentions: Nematode and vertebrate haemoglobins are very dissimilar [23] (Figure 7A), with nematode haemoglobins showing only 10–15% homology to vertebrate haemoglobins [13] 4E8g was shown not to bind mouse haemoglobin by ELISA and western blotting (Figure 7B). To determine whether nematode haemoglobin could be targeted for immunization strategies, mice were injected with 4E8g one day prior to N. brasiliensis infection. Parasite burdens were counted in the lungs and intestine at different timepoints post infection. N. brasiliensis L3 migrate to the lungs after injection and moult into L4 larvae between 19–32 hours [25] The L4 remain in the lung till approximately 50 hours post infection before penetrating the alveoli, being coughed up and swallowed into the intestine. In the intestine they moult into adult worms at about 90–108 hours post infection. The number of parasites in the lungs was similar in anti-Hb and isotype control treated groups at 24 h and 48 h post infection (Figure 8A). Parasite numbers were low in the lungs in both groups at 72 h due to migration out of the lungs, although in anti-Hb treated mice the parasite numbers were significantly lower. In the intestine, equivalent worm burdens were found in both groups of mice at 72 h post infection. However at both day 5 and day 7 post infection, worm burdens were significantly reduced in anti-Hb treated mice. Protection was associated with increased levels of MMCP-1, a marker of mast cell degranulation, in the serum (Figure 8B) and increased production of Th2 cytokines by restimulated mesenteric lymph node cells (Figure 8 C–F). Together these data suggest that anti-Hb primarily confers protection against the adult worm, either directly or by priming the immune system to increase expulsion or destruction of the parasites.

Bottom Line: Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting.Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens.The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology, Cape Town Component, and Institute of Infectious Diseases and Molecular Medicine, Medical Research Council, Division of Immunology, Faculty of Health Science, University of Cape Town, Cape Town, South Africa. nnieuwen@gmail.com

ABSTRACT

Background: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection.

Methodology/principal findings: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens.

Conclusion: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.

Show MeSH
Related in: MedlinePlus