Measles virus-specific CD4 T-cell activity does not correlate with protection against lung infection or viral clearance

Abstract

Acute measles in children can be prevented by immunization with the live attenuated measles vaccine virus. Although immunization is able to induce CD4 and CD8 T cells as well as neutralizing antibodies, only the latter have been correlated with protective immunity. CD8 T cells, however, have been documented to be important in viral clearance in the respiratory tract, whereas CD4 T cells have been shown to be protective in a mouse encephalitis model. In order to investigate the CD4 T-cell response in infection of the respiratory tract, we have defined a T-cell epitope in the hemagglutinin (H) protein for immunization and developed a monoclonal antibody for depletion of CD4 T cells in the cotton rat model. Although the kinetics of CD4 T-cell development correlated with clearance of virus, the depletion of CD4 T cells during the primary infection did not influence viral titers in lung tissue. Immunization with the H epitope induced a CD4 T-cell response but did not protect against infection. Immunization in the presence of maternal antibodies resulted in the development of a CD4 T-cell response which (in the absence of neutralizing antibodies) did not protect against infection. In summary, CD4 T cells do not seem to protect against infection after immunization and do not participate in clearance of virus infection from lung tissue during measles virus infection. We speculate that the major role of CD4 T cells is to control and clear virus infection from other affected organs like the brain.

FIG. 1.

Kinetics of MV-specific CD4 T-cell development in cotton rats during infection. The proliferation of MV-specific CD4 T cells was measured by stimulation of cotton rat spleen cells at different time points after infection with UV-inactivated measles virus. An SI above 2 was considered to be positive. The kinetics of viral titers (dotted line) is depicted schematically and is based on previously published data (13).

FIG. 2.

Immunohistochemical staining of the B-cell and T-cell areas of cotton rat lymph nodes. Lymph nodes were stained with antibodies against CD79a, CD3, and CD4. The anti-CD79a antibody clearly stains the B-cell areas (germinal centers; brown, left); the anti-CD3 antiserum stains the T-cell areas (brown, middle) in the white pulp. CR-CD4 does not stain the B-cell area but does stain the T cell-area (black, right). The magnifications for the lymph node are ×100 (top row) and ×400 (bottom row).

FIG. 3.

Staining cells of lymphoid cells with CR-CD4. Lymph node cells, thymic cells, and spleen cells (bottom left) were stained with the CR-CD4 antibody, and the staining was analyzed by flow cytometry. The thin black line indicates values for cell populations stained with an isotype control and secondary antibody, and the thick gray line indicates values for cells stained with CR-CD4 and secondary antibody. After depletion of animals with CR-CD4, spleen cells were stained with CR-CD4 (bottom right, gray line) and compared to spleen cells from an animal not depleted of CR-CD4 (bottom right, black line).

FIG. 4.

Inhibition of MV-specific T-cell proliferation by antibodies against MHC-II and CD4. Spleen cells from MV-immune animals were stimulated with UV-inactivated MV (proliferation in counts per minute) and cultured in the presence or absence of various concentrations of antibody. Antibody 13/4 cross-reacts with cotton rat MHC-II (A), CR-CD4 recognizes cotton rat CD4 (B), and 13/42 recognizes human but not cotton rat CD46 (C). nd, not determined.

FIG. 5.

Determination of CD4 T-cell epitopes on MV hemagglutinin by peptide scanning. Cotton rats were immunized intradermally with plasmid pSC-N which expresses the nucleocapsid protein, with pCG-F1 which expresses the fusion protein, or pCG-H5 which expresses the MV hemagglutinin, as described previously (29). Spleen cells were stimulated with peptides 15 amino acid long that overlapped by 5 amino acids. The threshold for positive proliferation was set as three times the lowest counts (broken line). The one-peptide or two-peptide combinations which stimulated T-cell proliferation are signified by an asterisk. For the hemagglutinin (H) only one peptide corresponding to amino acids 553 to 567 on the hemagglutinin protein stimulated T-cell proliferation; on the fusion (F) protein amino acids 348 to 372 were recognized, and on the nucleocapsid (N) protein amino acids 261 to 285 and 348 to 372 were recognized.

FIG. 6.

Immunization in the presence of maternal antibodies. One day after the transfer of an equivalent of 1 ml of antibody with a neutralization titer of 320, 160, 80, or 0 of human MV-specific antibodies, groups of four cotton rats were immunized i.p. with 10⁵ PFU of MV (Edm strain). Seven weeks later, serum samples were taken, and animals were challenged i.n. with MV (HU2 strain). Five days later, T-cell proliferation was measured from spleen cells (C), and virus titers were determined from lung tissues (D). Serum samples were analyzed by ELISA (A) and neutralization assay (B). Immunization in the presence of NTs of 320 and 160 did not result in neutralizing antibody titers above the threshold level of 10. The asterisk indicates significant differences compared to animals immunized in the absence of MV-specific antibody (0) for ELISA titers (at least P < 0.002) and NTs (at least P < 0.001); for virus titers, the asterisk indicates significant differences compared to nonimmunized animals (P < 0.002).