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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

Expression of haemoglobin in Anisakis pegreffii.A–D) A. pegreffii L3 collected from Thyrsites atun were formalin fixed and stained with 4E8g (Anti-Hb). E–F) A. pegreffii L3 sections were stained with isotype control antibody (mouse IgG1). Detection was carried out using the anti-mouse IgG detection system with DAB. Haemoglobin is stained in brown. Counterstaining was performed with haemotoxylin. All photos taken at 200× magnification.
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pntd-0002395-g004: Expression of haemoglobin in Anisakis pegreffii.A–D) A. pegreffii L3 collected from Thyrsites atun were formalin fixed and stained with 4E8g (Anti-Hb). E–F) A. pegreffii L3 sections were stained with isotype control antibody (mouse IgG1). Detection was carried out using the anti-mouse IgG detection system with DAB. Haemoglobin is stained in brown. Counterstaining was performed with haemotoxylin. All photos taken at 200× magnification.

Mentions: The monoclonal antibody 4E8g allowed for immunohistochemical staining to identify the location of haemoglobin expression in Anisakis, Ascaris and N. brasiliensis (Figures 4–6). Staining of Anisakis sections confirmed immunoblotting results, indicating that Anisakis haemoglobin is an excretory-secretary protein (Figure 4). Haemoglobin is highly concentrated in the intestinal lumen of the Anisakis larvae (Figure 4A,B), but is also found in the surrounding muscle cells and just below the cuticle. The staining demonstrates that haemoglobin is an ES product that is expressed in the gut and passes via the excretory glands into the environment (Figure 4C,D). Sections stained with isotype control antibody did not show any staining in these areas (Figure 4 E–F). Ascaris haemoglobin also appears to be expressed from the gut through excretory cells (Figure 5) and is also found within muscle cells. Similarly, in N. brasiliensis worms, haemoglobin was associated with excretory glands near the intestine (Figure 6A, B), suggesting that this nematode species may also secrete haemoglobin. Staining of haemoglobin in cross-sections of N. brasiliensis worms that were present in infected mouse small intestine also demonstrated the presence of haemoglobin in peripheral areas such as the fluid-filled median zone of the cuticle (Figure 6B, C).


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)

Expression of haemoglobin in Anisakis pegreffii.A–D) A. pegreffii L3 collected from Thyrsites atun were formalin fixed and stained with 4E8g (Anti-Hb). E–F) A. pegreffii L3 sections were stained with isotype control antibody (mouse IgG1). Detection was carried out using the anti-mouse IgG detection system with DAB. Haemoglobin is stained in brown. Counterstaining was performed with haemotoxylin. All photos taken at 200× magnification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757078&req=5

pntd-0002395-g004: Expression of haemoglobin in Anisakis pegreffii.A–D) A. pegreffii L3 collected from Thyrsites atun were formalin fixed and stained with 4E8g (Anti-Hb). E–F) A. pegreffii L3 sections were stained with isotype control antibody (mouse IgG1). Detection was carried out using the anti-mouse IgG detection system with DAB. Haemoglobin is stained in brown. Counterstaining was performed with haemotoxylin. All photos taken at 200× magnification.
Mentions: The monoclonal antibody 4E8g allowed for immunohistochemical staining to identify the location of haemoglobin expression in Anisakis, Ascaris and N. brasiliensis (Figures 4–6). Staining of Anisakis sections confirmed immunoblotting results, indicating that Anisakis haemoglobin is an excretory-secretary protein (Figure 4). Haemoglobin is highly concentrated in the intestinal lumen of the Anisakis larvae (Figure 4A,B), but is also found in the surrounding muscle cells and just below the cuticle. The staining demonstrates that haemoglobin is an ES product that is expressed in the gut and passes via the excretory glands into the environment (Figure 4C,D). Sections stained with isotype control antibody did not show any staining in these areas (Figure 4 E–F). Ascaris haemoglobin also appears to be expressed from the gut through excretory cells (Figure 5) and is also found within muscle cells. Similarly, in N. brasiliensis worms, haemoglobin was associated with excretory glands near the intestine (Figure 6A, B), suggesting that this nematode species may also secrete haemoglobin. Staining of haemoglobin in cross-sections of N. brasiliensis worms that were present in infected mouse small intestine also demonstrated the presence of haemoglobin in peripheral areas such as the fluid-filled median zone of the cuticle (Figure 6B, C).

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