The Babesia bovis Merozoite Surface Antigen 1 Hypervariable Region Induces Surface-Reactive Antibodies That Block Merozoite Invasion

ABSTRACT A hypervariable region (HVR) previously identified in the carboxy-terminal one-third of the Babesia bovis variable merozoite surface antigen family was more extensively analyzed in merozoite surface antigen 1 (MSA-1) from 16 strains and isolates. The MSA-1 HVR is proline rich and contains three semiconserved motifs nearly identical to those described for the related family member MSA-2. Two MSA-1-specific monoclonal antibodies previously shown to be reactive with the merozoite surface bound to a recombinant construct encoding the HVR, indicating that the HVR is surface exposed and accessible to antibody binding. Importantly, these surface-reactive, HVR-specific monoclonal antibodies were capable of inhibiting merozoite infectivity of the host erythrocyte in vivo. The results indicate that the MSA-1 HVR is involved in erythrocyte invasion and suggest that selection of MSA-1 variants may be driven by invasion-blocking antibodies.

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Merozoite Surface Antigen 2 Proteins of Babesia bovis Vaccine Breakthrough Isolates Contain a Unique Hypervariable Region Composed of Degenerate Repeats

Infection and Immunity ◽

10.1128/iai.73.11.7180-7189.2005 ◽

2005 ◽

Vol 73 (11) ◽

pp. 7180-7189 ◽

Cited By ~ 48

Author(s):

Shawn J. Berens ◽

Kelly A. Brayton ◽

John B. Molloy ◽

Russell E. Bock ◽

Ala E. Lew ◽

...

Keyword(s):

Surface Antigen ◽

Protective Immunity ◽

Sequence Variation ◽

Immune Escape ◽

Hypervariable Region ◽

Babesia Bovis ◽

Erythrocyte Invasion ◽

Merozoite Surface Antigen ◽

Nucleotide Repeats ◽

Degenerate Nucleotide

ABSTRACT The merozoite surface antigen 2 (MSA-2) proteins of Babesia bovis are members of the variable merozoite surface antigen (VMSA) family that have been implicated in erythrocyte invasion and are important targets for antibody-mediated blocking of invasion. Extensive sequence variation in another VMSA member, MSA-1, has been shown in all vaccine breakthrough isolates. To test the hypothesis that the msa-2 genes of vaccine breakthrough isolates would also encode a diverse set of proteins, the complete msa-2 locus was characterized from 12 Australian B. bovis strains and isolates, including two vaccine strains and eight vaccine breakthrough isolates, and compared to the loci in previously and newly characterized American strains. In contrast to American strains, the msa-2 loci of all Australian strains and isolates examined contain, in addition to msa-2c, only a solitary gene (designated msa-2a/b) closely related to American strain msa-2a and msa-2b. Nevertheless, the proteins encoded by these genes are quite diverse both between and within geographic regions and harbor evidence of genetic exchange among other VMSA family members, including msa-1. Moreover, all but one of the Australian breakthrough isolate MSA-2a/b proteins is markedly different from the vaccine strain from which immune escape occurred, consistent with their role in strain-specific protective immunity. The densest distribution of polymorphisms occurs in a hypervariable region (HVR) within the carboxy third of the molecule that is highly proline rich. Variation in length and content of the HVR is primarily attributable to differences in the order and number of degenerate nucleotide repeats encoding three motifs of unknown function.

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Sequence Variation and Immunologic Cross-Reactivity among Babesia bovis Merozoite Surface Antigen 1 Proteins from Vaccine Strains and Vaccine Breakthrough Isolates

Infection and Immunity ◽

10.1128/iai.73.9.5388-5394.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5388-5394 ◽

Cited By ~ 36

Author(s):

Tanya LeRoith ◽

Kelly A. Brayton ◽

John B. Molloy ◽

Russell E. Bock ◽

Stephen A. Hines ◽

...

Keyword(s):

Vaccine Strain ◽

Surface Antigen ◽

Sequence Variation ◽

Antigenic Variation ◽

Cross Reactivity ◽

Babesia Bovis ◽

Extracellular Region ◽

Merozoite Surface Antigen ◽

The Difference ◽

Blocking Antibodies

ABSTRACT The Babesia bovis merozoite surface antigen 1 (MSA-1) is an immunodominant membrane glycoprotein that is the target of invasion-blocking antibodies. While antigenic variation has been demonstrated in MSA-1 among strains from distinct geographical areas, the extent of sequence variation within a region where it is endemic and the effect of variation on immunologic cross-reactivity have not been assessed. In this study, sequencing of MSA-1 from two Australian B. bovis vaccine strains and 14 breakthrough isolates from vaccinated animals demonstrated low sequence identity in the extracellular region of the molecule, ranging from 19.8 to 46.7% between the T vaccine strain and eight T vaccine breakthrough isolates, and from 18.7 to 99% between the K vaccine strain and six K vaccine breakthrough isolates. Although MSA-1 amino acid sequence varied substantially among strains, overall predicted regions of hydrophilicity and hydrophobicity in the extracellular domain were conserved in all strains examined, suggesting a conserved functional role for MSA-1 despite sequence polymorphism. Importantly, the antigenic variation created by sequence differences resulted in a lack of immunologic cross-reactivity among outbreak strains using sera from animals infected with the B. bovis vaccine strains. Additionally, sera from cattle hyperinfected with the Mexico strain of B. bovis and shown to be clinically immune did not cross-react with MSA-1 from any other isolate tested. The results indicate that isolates of B. bovis capable of evading vaccine-induced immunity contain an msa-1 gene that is significantly different from the msa-1 of the vaccine strain, and that the difference can result in a complete lack of cross-reactivity between MSA-1 from vaccine and breakthrough strains in immunized animals.

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Characterization of Allelic Variation in theBabesia bovis Merozoite Surface Antigen 1 (MSA-1) Locus and Identification of a Cross-Reactive Inhibition-Sensitive MSA-1 Epitope

Infection and Immunity ◽

10.1128/iai.68.12.6865-6870.2000 ◽

2000 ◽

Vol 68 (12) ◽

pp. 6865-6870 ◽

Cited By ~ 66

Author(s):

Carlos E. Suarez ◽

Monica Florin-Christensen ◽

Stephen A. Hines ◽

Guy H. Palmer ◽

Wendy C. Brown ◽

...

Keyword(s):

Surface Antigen ◽

Allelic Variation ◽

Sequence Divergence ◽

Amino Acid Sequences ◽

Babesia Bovis ◽

Erythrocyte Invasion ◽

Merozoite Surface Antigen ◽

Inhibitory Antibodies ◽

Limited Sequence

ABSTRACT The Babesia bovis merozoite surface antigen 1 (MSA-1), a member of the variable merozoite surface antigen (VMSA) family, is an immunodominant glycoprotein which elicits antibodies that inhibit erythrocyte invasion. While antigenic polymorphism is a general feature of vmsa genes, the molecular basis and extent ofmsa-1 sequence polymorphism have not been well characterized. In this study we defined the msa-1 locus in the biologically cloned Mexico Mo7 strain of B. bovis and identified the sequence differences between MSA-1 antigenically dissimilar strains. We then determined whether sequences conserved between distinct msa-1 alleles would induce cross-reactive CD4+ T lymphocytes or inhibitory antibodies. Themsa-1 locus in Mo7 contains a single msa-1 gene flanked by transcribed genes with no sequence homology to members of the VMSA gene family. Argentina B. bovis strains R1A and S2P have msa-1 genes with amino acid sequences that are 98.8% identical to each other, and antibodies against S2P MSA-1 cross-react with native R1A MSA-1. In contrast, identity between the Argentina and Mexico Mo7 msa-1 alleles is only 52%, with no continuous stretch of identity longer than 16 amino acids. Despite limited sequence conservation, antibodies against R1A MSA-1 were able to inhibit invasion of erythrocytes by Mo7 merozoites. The results indicate that inhibition-sensitive epitopes are conserved despite significant sequence divergence between Mexico and Argentina strain alleles and support a conserved functional role for polymorphic MSA-1 in erythrocyte invasion.

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Babesia bovis Merozoite Surface Antigen 2 Proteins Are Expressed on the Merozoite and Sporozoite Surface, and Specific Antibodies Inhibit Attachment and Invasion of Erythrocytes

Infection and Immunity ◽

10.1128/iai.70.11.6448-6455.2002 ◽

2002 ◽

Vol 70 (11) ◽

pp. 6448-6455 ◽

Cited By ~ 55

Author(s):

Juan Mosqueda ◽

Terry F. McElwain ◽

Guy H. Palmer

Keyword(s):

Surface Antigen ◽

Significant Inhibition ◽

Babesia Bovis ◽

Merozoite Invasion ◽

Specific Antibodies ◽

Surface Molecules ◽

Erythrocyte Invasion ◽

Merozoite Surface Antigen ◽

Initial Binding ◽

Bovine Erythrocytes

ABSTRACT The Babesia bovis merozoite surface antigen 2 (MSA-2) locus encodes four proteins, MSA-2a1, -2a2, -2b, and -2c. With the use of specific antibodies, each MSA-2 protein was shown to be expressed on the surface of live extracellular merozoites and coexpression on single merozoites was confirmed. Individual antisera against MSA-2a, MSA-2b, and MSA-2c significantly inhibited merozoite invasion of bovine erythrocytes. As tick-derived sporozoites also directly invade erythrocytes, expression of each MSA-2 protein on the sporozoite surface was examined and verified. Finally, statistically significant inhibition of sporozoite binding to the erythrocytes was demonstrated by using antisera specific for MSA-2a, MSA-2b, and MSA-2c. These results indicate an important role for MSA-2 proteins in the initial binding and invasion of host erythrocytes and support the hypothesis that sporozoites and merozoites use common surface molecules in erythrocyte invasion.

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Babesia bovis Merozoite Surface Antigen 1 and Rhoptry-Associated Protein 1 Are Expressed in Sporozoites, and Specific Antibodies Inhibit Sporozoite Attachment to Erythrocytes

Infection and Immunity ◽

10.1128/iai.70.3.1599-1603.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1599-1603 ◽

Cited By ~ 65

Author(s):

Juan Mosqueda ◽

Terry F. McElwain ◽

David Stiller ◽

Guy H. Palmer

Keyword(s):

Surface Antigen ◽

Babesia Bovis ◽

Specific Antibodies ◽

Erythrocyte Invasion ◽

Merozoite Surface Antigen

ABSTRACT We examined Babesia bovis sporozoites for the expression of two molecules, merozoite surface antigen 1 (MSA-1) and rhoptry-associated protein 1 (RAP-1), that are postulated to be involved in the invasion of host erythrocytes. Both MSA-1 and RAP-1 were transcribed and expressed in infectious sporozoites. Importantly, monospecific MSA-1 and RAP-1 antisera each inhibited sporozoite invasion of erythrocytes in vitro. This is the first identification of antigens expressed in Babesia sp. sporozoites and establishes that, at least in part, sporozoites and merozoites share common targets of antibody mediated inhibition of erythrocyte invasion.

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Vaccination-induced variation in the 140 kD merozoite surface antigen of Plasmodium knowlesi malaria.

Journal of Experimental Medicine ◽

10.1084/jem.165.2.359 ◽

1987 ◽

Vol 165 (2) ◽

pp. 359-367 ◽

Cited By ~ 31

Author(s):

F W Klotz ◽

D E Hudson ◽

H G Coon ◽

L H Miller

Keyword(s):

Monoclonal Antibodies ◽

Malaria Infection ◽

Rhesus Monkeys ◽

Plasmodium Knowlesi ◽

Rabbit Antiserum ◽

Antigenic Determinants ◽

Partial Protection ◽

Erythrocyte Invasion ◽

Merozoite Surface Antigen

Immunity to 143/140 kD schizont antigens of a monkey malaria, Plasmodium knowlesi, provides partial protection to lethal malaria infection in rhesus monkeys challenged with uncloned parasites. To determine the capacity of a cloned parasite to generate variants of the 143/140 kD antigens, immunized monkeys were challenged with a clone of P. knowlesi. Parasites recovered 8 d after inoculation with a cloned parasite retained the 143/140 kD antigens. Parasites recovered 30 d after challenge had undergone changes in the 143/140 kD antigens. Antibodies that block erythrocyte invasion in vitro of the inoculum parasites did not inhibit invasion of erythrocytes by two isolates recovered from the immunized monkeys. An isolate from one monkey recovered on day 30 contained clones expressing new 76/72 kD antigens reactive with rabbit antiserum against the 143/140 kD proteins, and other clones expressing no antigens crossreactive with antisera against the 143/140 kD proteins. An isolate from another monkey obtained 59 d after challenge expressed new antigens of 160/155, 115/113, and 87/85 kD. Using monoclonal antibodies, we found that epitopes were lost from the variant proteins, but we were unable to determine whether new epitopes had appeared. We conclude that clones of P. knowlesi can rapidly vary antigenic determinants on the 143/140 kD proteins in animals immunized with these antigens.

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A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

Antibodies ◽

10.3390/antib7040037 ◽

2018 ◽

Vol 7 (4) ◽

pp. 37 ◽

Cited By ~ 3

Author(s):

Jennifer Linden ◽

Kiel Telesford ◽

Samantha Shetty ◽

Paige Winokour ◽

Sylvia Haigh ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Monoclonal Antibodies ◽

Western Blot ◽

Clostridium Perfringens ◽

Specificity And Sensitivity ◽

Epsilon Toxin ◽

Blocking Antibodies

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

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