Limits...
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.

Show MeSH

Related in: MedlinePlus

Amino acid alignment of Anisakis haemoglobin with haemoglobins of Ascaris, Pseudoterranova and Nippostrongylus brasilensis, and phylogenetic analysis.A) The cDNA sequence of Anisakis haemoglobin was obtained by degenerate PCR and RACE-PCR (accession number: JX860676) and the amino acid sequence was deduced. Alignment of sequences was carried out using MUSCLE (http://www.ebi.ac.uk/Tools/muscle/index.html). The hydrophobic leader portion (sequence not obtained in Anisakis) and the C-terminal tail (important for assembly of octamers in Ascaris) are boxed in black, while hydrophobic haem-binding regions are boxed in red. The haemoglobin protein consists of two homologous domains, illustrated by dashed and dotted lines. The B10 tyrosine, E7 distal glutamine and F8 proximal histidine, important in binding of oxygen, are conserved between the three nematodes and are marked with boxes. B) A range of haemoglobin sequences across different species was obtained by BLAST search. The evolutionary history was inferred using the Minimum Evolution method [21]. The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 0. The Neighbor-joining algorithm was used to generate the initial tree. The analysis involved 26 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 64 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3757078&req=5

pntd-0002395-g003: Amino acid alignment of Anisakis haemoglobin with haemoglobins of Ascaris, Pseudoterranova and Nippostrongylus brasilensis, and phylogenetic analysis.A) The cDNA sequence of Anisakis haemoglobin was obtained by degenerate PCR and RACE-PCR (accession number: JX860676) and the amino acid sequence was deduced. Alignment of sequences was carried out using MUSCLE (http://www.ebi.ac.uk/Tools/muscle/index.html). The hydrophobic leader portion (sequence not obtained in Anisakis) and the C-terminal tail (important for assembly of octamers in Ascaris) are boxed in black, while hydrophobic haem-binding regions are boxed in red. The haemoglobin protein consists of two homologous domains, illustrated by dashed and dotted lines. The B10 tyrosine, E7 distal glutamine and F8 proximal histidine, important in binding of oxygen, are conserved between the three nematodes and are marked with boxes. B) A range of haemoglobin sequences across different species was obtained by BLAST search. The evolutionary history was inferred using the Minimum Evolution method [21]. The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 0. The Neighbor-joining algorithm was used to generate the initial tree. The analysis involved 26 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 64 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.

Mentions: Degenerate PCR resulted in a partial nucleotide sequence of 428 bp, which was used to design gene specific primers for 5′ and 3′ rapid amplification of cDNA ends. Despite repeated RACE reactions, the sequence for the initial 22 amino acids in the 5′ region was not obtained. However, complete sequencing at the 3′ end demonstrated that Anisakis haemoglobin is similar to Pseudoterranova haemoglobin and lacks the four –HKEE repeats of Ascaris haemoglobin that promote the assembly of tetramers [12] (Figure 3A). Like both Ascaris and Pseudoterranova haemoglobins, the Anisakis haemoglobin consists of two similar domains, each with a haem binding site, and has the B10 tyrosine, E7 glutamine and F8 histidine that are important for high oxygen avidity [12]. 4E8g also recognized the more distantly related N. brasiliensis haemoglobin (Figure 1B). Unlike ascarid haemoglobins, which contain two similar domains, N.brasiliensis haemoglobin contains one domain which occurs in two isoforms, a body globin isoform and a cuticular globin isoform [23]. This is thought to be due to gene duplication event similar to the one that resulted in the two domain gene of ascarid haemoglobins [23]. This one-domain protein tends to dimerize to a 37 kDa protein, as seen in Figure 1B, and shows high similarity to the second domain of Anisakis haemoglobin, explaining why the monoclonal antibody is cross-reactive (Figure 3A). A phylogenetic tree assembled using published haemoglobin sequences from various organisms shows that Anisakis haemoglobin branches off with Pseudoterranova haemoglobin, but is also closely related to Ascaris haemoglobin (Figure 3B).


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)

Amino acid alignment of Anisakis haemoglobin with haemoglobins of Ascaris, Pseudoterranova and Nippostrongylus brasilensis, and phylogenetic analysis.A) The cDNA sequence of Anisakis haemoglobin was obtained by degenerate PCR and RACE-PCR (accession number: JX860676) and the amino acid sequence was deduced. Alignment of sequences was carried out using MUSCLE (http://www.ebi.ac.uk/Tools/muscle/index.html). The hydrophobic leader portion (sequence not obtained in Anisakis) and the C-terminal tail (important for assembly of octamers in Ascaris) are boxed in black, while hydrophobic haem-binding regions are boxed in red. The haemoglobin protein consists of two homologous domains, illustrated by dashed and dotted lines. The B10 tyrosine, E7 distal glutamine and F8 proximal histidine, important in binding of oxygen, are conserved between the three nematodes and are marked with boxes. B) A range of haemoglobin sequences across different species was obtained by BLAST search. The evolutionary history was inferred using the Minimum Evolution method [21]. The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 0. The Neighbor-joining algorithm was used to generate the initial tree. The analysis involved 26 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 64 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002395-g003: Amino acid alignment of Anisakis haemoglobin with haemoglobins of Ascaris, Pseudoterranova and Nippostrongylus brasilensis, and phylogenetic analysis.A) The cDNA sequence of Anisakis haemoglobin was obtained by degenerate PCR and RACE-PCR (accession number: JX860676) and the amino acid sequence was deduced. Alignment of sequences was carried out using MUSCLE (http://www.ebi.ac.uk/Tools/muscle/index.html). The hydrophobic leader portion (sequence not obtained in Anisakis) and the C-terminal tail (important for assembly of octamers in Ascaris) are boxed in black, while hydrophobic haem-binding regions are boxed in red. The haemoglobin protein consists of two homologous domains, illustrated by dashed and dotted lines. The B10 tyrosine, E7 distal glutamine and F8 proximal histidine, important in binding of oxygen, are conserved between the three nematodes and are marked with boxes. B) A range of haemoglobin sequences across different species was obtained by BLAST search. The evolutionary history was inferred using the Minimum Evolution method [21]. The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 0. The Neighbor-joining algorithm was used to generate the initial tree. The analysis involved 26 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 64 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.
Mentions: Degenerate PCR resulted in a partial nucleotide sequence of 428 bp, which was used to design gene specific primers for 5′ and 3′ rapid amplification of cDNA ends. Despite repeated RACE reactions, the sequence for the initial 22 amino acids in the 5′ region was not obtained. However, complete sequencing at the 3′ end demonstrated that Anisakis haemoglobin is similar to Pseudoterranova haemoglobin and lacks the four –HKEE repeats of Ascaris haemoglobin that promote the assembly of tetramers [12] (Figure 3A). Like both Ascaris and Pseudoterranova haemoglobins, the Anisakis haemoglobin consists of two similar domains, each with a haem binding site, and has the B10 tyrosine, E7 glutamine and F8 histidine that are important for high oxygen avidity [12]. 4E8g also recognized the more distantly related N. brasiliensis haemoglobin (Figure 1B). Unlike ascarid haemoglobins, which contain two similar domains, N.brasiliensis haemoglobin contains one domain which occurs in two isoforms, a body globin isoform and a cuticular globin isoform [23]. This is thought to be due to gene duplication event similar to the one that resulted in the two domain gene of ascarid haemoglobins [23]. This one-domain protein tends to dimerize to a 37 kDa protein, as seen in Figure 1B, and shows high similarity to the second domain of Anisakis haemoglobin, explaining why the monoclonal antibody is cross-reactive (Figure 3A). A phylogenetic tree assembled using published haemoglobin sequences from various organisms shows that Anisakis haemoglobin branches off with Pseudoterranova haemoglobin, but is also closely related to Ascaris haemoglobin (Figure 3B).

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