CD4⁺T-Lymphocyte and Immunoglobulin G2 Responses in Calves Immunized withAnaplasma marginaleOuter Membranes and Protected against Homologous Challenge

Abstract

Protective immunity against the ehrlichial pathogenAnaplasma 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.

Long‐term in vivo depletion of functional CD4+ T lymphocytes from calves requires both thymectomy and anti‐CD4 monoclonal antibody treatment

“…Proliferation assays were performed as described previously. 22 PBMC were cultured in replicates of six wells. Concanavalin A (Sigma) was added to positive control wells at ®nal concentrations of 0 .…”

“…23 Immunoblots Sodium dodecyl sulphate±polyacrylamide gel electrophoresis and immunoblots were performed with mAbs speci®c for bovine IgG1 and IgG2 (Serotec Ltd, Oxford, UK) to determine the subclass of the speci®c IgG response to ovalbumin, as described previously. 22 Antibody complement-mediated depletion assay PBMC were isolated by density gradient centrifugation then resuspended at a concentration of 2r10 7 cells/ml in PBS and incubated for 30 min at 4u with anti-CD4 mAb (IL-A11A; IgG2a) diluted to a ®nal concentration of 15 mg of mAb per ml. Cells were washed twice with PBS, then re-suspended in PBS and incubated for 30 min at 37u with an equal volume of a 1 : 8 ®nal dilution of rabbit complement (Pel-Freez Clinical Systems, LLC., Brown Deer, WI).…”

Cross-Protection Between Geographically DistinctAnaplasma marginaleIsolates Appears to be Constrained by Limited Antibody Responses

“…While the MSP5 cELISA assay is very useful for measuring seroconversion as an indicator of infection, it has limited application in estimating protective immunity. Measuring the IgG response against all MSPs, either by ELISA as used here, or by Western blot as in Brown et al (1998), appears to give more information on the likely degree of cross-protection induced by infection or vaccination.…”

“…The combination of activated macrophages and high levels of IgG2 would lead to increased opsonization and phagocytosis of the rickettsia. To corroborate this, Brown et al (1998) found that protection after vaccination with outer membranes was associated with CD4+ proliferation and IFNc production and high IgG2 levels. Anaplasma marginale infection itself leads to the down-regulation of high pre-challenge, vaccinationinduced, CD4 T-cell responses.…”

Contagious bovine pleuropneumonia: A rationale for the development of a mucosal sub-unit vaccine

“…CD4+ T-cells should contribute to protection against CBPP mainly through their cytokine network including IFNg that will enhance phagocytosis and cell killing ability and promote the expression of MHC class II molecules, thus increasing the host cell capacity for antigen processing and epitope presentation to T-lymphocytes [14]. A protective role for the IFNginduced IgG2 antibody, only able to promote killing by neutrophils in cattle, is also expected since neutrophil recruitment has been observed in CBPP lung lesions [15][16][17]. Besides, CD4+ T-cells play also a key role in facilitating B-cell proliferation and class-switching to give rise to antigen-specific IgAs in mucosal effector sites [5].…”