CD4(+) T lymphocytes from calves immunized with Anaplasma marginale major surface protein 1 (MSP1), a heteromeric complex of MSP1a and MSP1b, preferentially recognize the MSP1a carboxyl terminus that is conserved among strains

Abstract

Native major surface protein 1 (MSP1) of the ehrlichial pathogen Anaplasma 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-gamma), which promotes switching to high-affinity immunoglobulin G (IgG) and activation of phagocytic cells to produce nitric oxide. Thus, an effective vaccine for A. marginale and 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-gamma 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.

FIG. 1

A. marginale MSP1-specific proliferation by PBMC of MSP1-immunized calves 87 (A), 93 (B), and 96 (C). PBMC obtained more than 1 year after immunization were cultured for 6 days with 0.4, 2, and 10 μg of A. marginale (FL strain) homogenate (A.marg), MSP1, or uninfected erythrocytes (URBC) per ml; radiolabeled; and counted. Results are presented as the mean ± 1 SD (error bars) of triplicate cultures.

FIG. 2

PBMC from calves immunized with MSP1a from the FL strain respond to heterologous strains of A. marginale. PBMC were cultured for 6 days with 1, 5, or 25 μg of A. marginale homogenate per ml; radiolabeled; and counted. Results are presented as the SI, calculated as the mean cpm of replicate cultures of PBMC cultured with antigen (5 μg/ml)/the mean cpm of PBMC cultured with medium. These results are representative of two or three independent experiments performed with each calf. Strain abbreviations: WA′., Washington′ Okaganon; S ID, South Idaho.

FIG. 3

PBMC from MSP1-immunized calves 87 (A), 93 (B), and 96 (C) preferentially respond to the MSP1a subunit. PBMC were tested for proliferation against 0.2, 1, or 5 μg of MSP1a and MSP1b (F1 to F4) per ml. Data are presented for antibody-affinity-purified MSP1a and MSP1bF1 as the mean cpm ± 1 SD (error bars) of triplicate cultures. There was no response to any of the MSP1b proteins tested (data are not shown for Ni²⁺ affinity-purified F2 to F4). These data are representative of at least three experiments performed with each calf.

FIG. 4

Short-term cell lines from MSP1-immunized calves 87 (A), 93 (B), and 96 (C) preferentially respond to MSP1a. Cell lines were cultured for 2 weeks with A. marginale homogenate (A.marg) (A), or PBMC were depleted of γδ T cells and cultured for 1 week with A. marginale and 1 week with MSP1 (B and C). Cell lines were tested in a 3-day proliferation assay against antigen (0.2 to 25 μg/ml). Results are presented as the mean cpm ± 1 SD (error bars) of duplicate cultures.

FIG. 5

A. marginale MSP1a-specific cell lines (87 [A], 93 [B], and 96 [C]) are MHC restricted. T-cell lines depleted of γδ T cells were cultured for 2 weeks with A. marginale and tested in a 3-day proliferation assay with A. marginale at a concentration of 10 μg/ml (black bars) or MSP1a at a concentration of 5 μg/ml (white bars) in the presence of autologous APC, APC matched for one DRB3-DQ haplotype, or mismatched APC. The DRB3 haplotypes of donor APC are indicated in parentheses. Results are presented as the mean cpm of duplicate cultures, after subtracting the mean cpm of T cells cultured with medium and respective APC. Error bars, SD.

FIG. 6

Proliferation and IFN-γ production by MSP1-specific lymphocytes in response to the C-terminal region of MSP1a. (A) PBMC were cultured for 1 week with A. marginale and tested for proliferation in a 3-day assay with antigen (0.4, 2, or 10 μg/ml) in triplicate cultures. Results are presented as the mean cpm ± 1 SD (error bars) of triplicate cultures stimulated with the optimal concentration of antigen, which was 10 μg/ml for calves 87 and 96 and 2 μg/ml for calf 93 lymphocytes, after subtracting the background cpm for cells cultured without antigen, and are representative of at least three experiments. (B) Cell lines were cultured for 1 week with A. marginale and for 1 week without antigen and then were restimulated for 72 h with autologous APC and 5 μg of MSP1a C-region or control MBP antigen per ml, and supernatants were tested for IFN-γ production by ELISA. Additional controls consisted of supernatants from APC stimulated with antigen.