CD4(+) T-lymphocyte and immunoglobulin G2 responses in calves immunized with Anaplasma marginale outer membranes and protected against homologous challenge

Abstract

Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4(+) T cells, and CD4(+) T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.

FIG. 1

Nested PCR analysis of the msp-5 gene in A. marginale membrane-immunized calves and nonimmunized control calves after challenge with A. marginale. Nested PCR was performed with primers specific for msp-5 by using distilled water as a negative control (lane 1) and DNA prepared from the following animals: a bovine persistently infected with the Florida strain (positive control; lane 2), immunized calves 96BO5 (lane 3), 96BO6 (lane 4), and 96BO9 (lane 5), and control calves 96B19 (lane 6), 96B20 (lane 7), and 96B21 (lane 8). Nested PCR products (345 bp) were visualized in a 2% agarose gel following electrophoresis. Molecular weight markers (M) consisting of a 100-bp DNA ladder and prominently displaying the 600-bp fragment were included in the gel.

FIG. 2

Dose-dependent proliferation of PBMC from A. marginale-immunized calves to A. marginale homogenate and outer membrane antigens. PBMC were obtained 10 days following the third immunization with membranes prepared from the Florida strain of A. marginale and were assayed for proliferation against medium or 1 to 25 μg of URBC (open circles)/ml, 0.2 to 25 μg of Florida strain homogenate (solid circles)/ml, 0.2 to 25 μg of outer membranes purified from Florida strain organisms (open triangles)/ml. PBMC were cultured for 6 days in triplicate with antigen, radiolabeled, and harvested. Results are presented as mean counts per minute from replicate cultures ± 1 SEM.

FIG. 3

Proliferative responses of PBMC from A. marginale-immunized calves to homogenates prepared from homologous Florida (FL) and heterologous Virginia (VA), Washington O (WA O), Washington C (WA C), and Idaho (ID) strains of A. marginale and from A. ovis. PBMC were obtained approximately 5 months following the last immunization and were assayed for proliferation against URBC membrane antigen or homogenates of the indicated A. marginale strains or A. ovis. PBMC were cultured for 6 days in triplicate with antigen, radiolabeled, and harvested, and the results for stimulation with the optimal concentration of protein (0.4 μg/ml for calf 96BO6 and 10.0 μg/ml for calves 96BO5 and 96BO9) or medium are presented as the mean counts per minute of replicate cultures ± 1 SEM.

FIG. 4

Proliferative responses of PBMC from A. marginale-immunized calves to purified native MSP-1, MSP-2, and MSP-3. PBMC were obtained at approximately 2 (calf 96BO5) or 5 (calves 96BO6 and 96BO9) months following the last immunization and were assayed for proliferation against the different MSPs isolated from the Florida strain of A. marginale. PBMC were cultured for 6 days in triplicate with antigen, radiolabeled, and harvested. Results for stimulation with 4.4 μg of MSP antigen/ml, 2.0 μg of URBC antigen/ml, or medium are presented as the mean counts per minute of replicate cultures ± 1 SEM.

FIG. 5

Dose-dependent, antigen-specific proliferative responses of short-term T-cell lines from A. marginale-immunized calves. T-cell lines were established at 4 weeks after the primary antigen immunization (2 weeks after the second immunization) and cultured for either 2 (calf 96BO6) or 4 (calves 96BO5 and 96BO9) weeks prior to assay. T cells were cultured for 4 days in duplicate with medium or 0.04 to 5.0 μg of A. marginale Florida homogenate (solid circles) or B. bovis membrane antigen (open circles)/ml, radiolabeled, and harvested. Results are presented as the mean counts per minute of duplicate cultures ± 1 SEM.