Expression of Anaplasma marginale Major Surface Protein 2 Variants during Persistent Cyclic Rickettsemia

ABSTRACT Anaplasma marginale is an intraerythrocytic rickettsial pathogen of cattle in which infection persists for the life of the animal. Persistent A. marginale infection is characterized by repetitive rickettsemic cycles which we hypothesize reflect emergence of A. marginale antigenic variants. In this study, we determined whether variants of major surface protein 2 (MSP-2), a target of protective immunity encoded by a polymorphic multigene family, arise during persistent rickettsemia. By using a quantitative competitive PCR to identify rickettsemic cycles,msp-2 transcripts expressed in vivo were isolated from peak rickettsemia of sequential cycles. Cloning and sequencing ofmsp-2 cDNA revealed that genetic variants of MSP-2 emerge representing a minimum of four genetic variant types in each cycle during persistent infection. Two-color immunofluorescence using variant-specific antibody showed that emergence of MSP-2 variants resulted in expression of a minimum of three antigenic types of MSP-2 within one rickettsemic cycle. Therefore immune control of each cycle would require responses to an antigenically diverse A. marginale population. These findings demonstrate that polymorphic MSP-2 variants emerge during cyclic rickettsemia in persistent A. marginale infection and suggest that emergent variants play an important role in persistence.

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Interleukin-12 as an Adjuvant Promotes Immunoglobulin G and Type 1 Cytokine Recall Responses to Major Surface Protein 2 of the Ehrlichial Pathogen Anaplasma marginale

Infection and Immunity ◽

10.1128/iai.68.1.270-280.2000 ◽

2000 ◽

Vol 68 (1) ◽

pp. 270-280 ◽

Cited By ~ 30

Author(s):

Wenbin Tuo ◽

Guy H. Palmer ◽

Travis C. McGuire ◽

Daming Zhu ◽

Wendy C. Brown

Keyword(s):

Protective Immunity ◽

Mononuclear Cells ◽

Surface Protein ◽

Anaplasma Marginale ◽

Interleukin 12 ◽

Major Surface Protein ◽

Ifn Γ ◽

Major Surface ◽

Type 1 Cytokine

ABSTRACT Anaplasma marginale is a tick-transmitted pathogen of cattle closely related to the human ehrlichiae, Ehrlichia chaffeensis and the agent of human granulocytic ehrlichiosis (HGE). These pathogens have in common a structurally conserved outer membrane protein (OMP) designated the major surface protein 2 (MSP-2) in A. marginale and HGE and OMP-1 in E. chaffeensis. Protective immunity against ehrlichial pathogens is believed to require induction of gamma interferon (IFN-γ) and opsonizing immunoglobulin (Ig) subclasses directed against OMP epitopes that, in concert, activate macrophages for phagocytosis and killing. Because interleukin-12 (IL-12) acts as an adjuvant for protein immunization to induce IFN-γ and protective immunity against intracellular pathogens, we hypothesized that as an adjuvant with MSP-2, IL-12 would augment type 1 recall responses to A. marginale. IL-12 was coadsorbed with MSP-2 to alum and shown to significantly enhance IFN-γ production by lymph node cells (LNC) and LNC-derived CD4+ T-cell lines from immunized calves following recall stimulation with A. marginale. LNC proliferation and IL-2 production were also enhanced in IL-12-treated calves. Elevated recall proliferative responses by peripheral blood mononuclear cells were still evident 9 months after immunization. Serum IgG levels were consistently increased in IL-12 immunized calves, predominantly due to higher IgG1 responses. The results support the use of IL-12 coadsorbed with OMP of ehrlichial pathogens in alum to amplify both antibody and type-1 cytokine responses important for protective immunity.

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The immunization-induced antibody response to the Anaplasma marginale major surface protein 2 and its association with protective immunity

Vaccine ◽

10.1016/j.vaccine.2010.02.067 ◽

2010 ◽

Vol 28 (21) ◽

pp. 3741-3747 ◽

Cited By ~ 16

Author(s):

Susan M. Noh ◽

Yan Zhuang ◽

James E. Futse ◽

Wendy C. Brown ◽

Kelly A. Brayton ◽

...

Keyword(s):

Antibody Response ◽

Protective Immunity ◽

Surface Protein ◽

Anaplasma Marginale ◽

Major Surface Protein ◽

Major Surface

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CD4+ T Lymphocytes from Calves Immunized withAnaplasma marginale Major Surface Protein 1 (MSP1), a Heteromeric Complex of MSP1a and MSP1b, Preferentially Recognize the MSP1a Carboxyl Terminus That Is Conserved among Strains

Infection and Immunity ◽

10.1128/iai.69.11.6853-6862.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6853-6862 ◽

Cited By ~ 50

Author(s):

Wendy C. Brown ◽

Guy H. Palmer ◽

Harris A. Lewin ◽

Travis C. McGuire

Keyword(s):

T Lymphocytes ◽

T Lymphocyte ◽

Protective Immunity ◽

B Lymphocyte ◽

Surface Protein ◽

Specific Response ◽

Major Surface Protein ◽

Ifn Γ ◽

Major Surface ◽

Lymphocyte Responses

ABSTRACT Native major surface protein 1 (MSP1) of the ehrlichial pathogenAnaplasma marginale induces protective immunity in calves challenged with homologous and heterologous strains. MSP1 is a heteromeric complex of a single MSP1a protein covalently associated with MSP1b polypeptides, of which at least two (designated MSP1F1 and MSP1F3) in the Florida strain are expressed. Immunization with recombinant MSP1a and MSP1b alone or in combination fails to provide protection. The protective immunity in calves immunized with native MSP1 is associated with the development of opsonizing and neutralizing antibodies, but CD4+ T-lymphocyte responses have not been evaluated. CD4+ T lymphocytes participate in protective immunity to ehrlichial pathogens through production of gamma interferon (IFN-γ), which promotes switching to high-affinity immunoglobulin G (IgG) and activation of phagocytic cells to produce nitric oxide. Thus, an effective vaccine for A. marginaleand related organisms should contain both T- and B-lymphocyte epitopes that induce a strong memory response that can be recalled upon challenge with homologous and heterologous strains. This study was designed to determine the relative contributions of MSP1a and MSP1b proteins, which contain both variant and conserved amino acid sequences, in stimulating memory CD4+ T-lymphocyte responses in calves immunized with native MSP1. Peripheral blood mononuclear cells and CD4+ T-cell lines from MSP1-immunized calves proliferated vigorously in response to the immunizing strain (Florida) and heterologous strains of A. marginale. The conserved MSP1-specific response was preferentially directed to the carboxyl-terminal region of MSP1a, which stimulated high levels of IFN-γ production by CD4+ T cells. In contrast, there was either weak or no recognition of MSP1b proteins. Paradoxically, all calves developed high titers of IgG antibodies to both MSP1a and MSP1b polypeptides. These findings suggest that in calves immunized with MSP1 heteromeric complex, MSP1a-specific T lymphocytes may provide help to MSP1b-specific B lymphocytes. The data provide a basis for determining whether selected MSP1a CD4+ T-lymphocyte epitopes and selected MSP1a and MSP1b B-lymphocyte epitopes presented on the same molecule can stimulate a protective immune response.

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Anaplasma marginale Major Surface Protein 2 CD4+-T-Cell Epitopes Are Evenly Distributed in Conserved and Hypervariable Regions (HVR), Whereas Linear B-Cell Epitopes Are Predominantly Located in the HVR

Infection and Immunity ◽

10.1128/iai.72.12.7360-7366.2004 ◽

2004 ◽

Vol 72 (12) ◽

pp. 7360-7366 ◽

Cited By ~ 35

Author(s):

Jeffrey R. Abbott ◽

Guy H. Palmer ◽

Chris J. Howard ◽

Jayne C. Hope ◽

Wendy C. Brown

Keyword(s):

T Cell ◽

B Cell ◽

Surface Protein ◽

Anaplasma Marginale ◽

T Cell Epitopes ◽

B Cell Epitopes ◽

Major Surface Protein ◽

Hypervariable Regions ◽

Major Surface ◽

Linear B

ABSTRACT Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4+ T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.

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Genetic diversity of major surface protein 1a of Anaplasma marginale in dairy cattle

Infection Genetics and Evolution ◽

10.1016/j.meegid.2020.104608 ◽

2020 ◽

pp. 104608

Author(s):

Munir Aktas ◽

Sezayi Ozubek

Keyword(s):

Genetic Diversity ◽

Dairy Cattle ◽

Surface Protein ◽

Anaplasma Marginale ◽

Major Surface Protein ◽

Major Surface

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Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Memórias do Instituto Oswaldo Cruz ◽

10.1590/s0074-02762006000500005 ◽

2006 ◽

Vol 101 (5) ◽

pp. 511-516 ◽

Cited By ~ 7

Author(s):

Virgínia MG Silva ◽

Flábio R Araújo ◽

Claudio R Madruga ◽

Cleber O Soares ◽

Raul H Kessler ◽

...

Keyword(s):

Surface Protein ◽

Anaplasma Marginale ◽

Major Surface Protein ◽

Immunosorbent Assays ◽

Major Surface

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Phylogeography of New World isolates of Anaplasma marginale based on major surface protein sequences

Veterinary Microbiology ◽

10.1016/s0378-1135(02)00122-0 ◽

2002 ◽

Vol 88 (3) ◽

pp. 275-285 ◽

Cited By ~ 63

Author(s):

José de la Fuente ◽

Ronald A Van Den Bussche ◽

Jose C Garcia-Garcia ◽

Sergio D Rodrı́guez ◽

Miguel A Garcı́a ◽

...

Keyword(s):

New World ◽

Protein Sequences ◽

Surface Protein ◽

Anaplasma Marginale ◽

Major Surface Protein ◽

Major Surface

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Characterization of Anaplasma phagocytophilum Major Surface Protein 5 and the Extent of Its Cross-Reactivity with A. marginale

Clinical and Vaccine Immunology ◽

10.1128/cvi.00320-06 ◽

2007 ◽

Vol 14 (3) ◽

pp. 262-268 ◽

Cited By ~ 26

Author(s):

N. I. Strik ◽

A. R. Alleman ◽

A. F. Barbet ◽

H. L. Sorenson ◽

H. L. Wamsley ◽

...

Keyword(s):

United States ◽

Anaplasma Phagocytophilum ◽

Indirect Elisa ◽

Surface Protein ◽

Anaplasma Marginale ◽

The United States ◽

Cross Reactivity ◽

Serum Samples ◽

Major Surface Protein ◽

Major Surface

ABSTRACT Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

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