Vaccine-Induced Measles Virus-Specific T Cells Do Not Prevent Infection or Disease but Facilitate Subsequent Clearance of Viral RNA

MeV-specific antibody responses after nebulized (Neb) and intramuscular (IM) immunization with LAV. Total MeV-specific IgG (A) and MeV H-, F-, and N-specific IgG (D to F) were determined by enzyme immunoassays using plates coated with lysates from MeV-infected Vero cells (A), MeV H-expressing L cells (D), MeV F-expressing L cells (E), or baculovirus-expressed MeV N (F). Values are plotted as means + SEM of optical density (OD). (B) Neutralizing antibody determined by plaque reduction neutralization test. The predicted protective level of antibody (120 mIU/ml) is indicated with a dashed line. Values are plotted as geometric means + SEM. (C) Avidity of MeV-specific IgG assessed by disruption of antibody binding with 0.5 to 3.0 M ammonium thiocyanate (NH₄SCN). Values are plotted as means ± SEM of the avidity index, calculated as the concentration of NH₄SCN at which 50% of the bound antibody was eluted. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (Student’s t test).

Viremia, blood leukocyte counts, antibody responses, and levels of viral RNA after wild-type MeV challenge. (A) Infectious virus in the blood quantified by cocultivation of PBMCs with B95-8 or Vero/hSLAM cells. Infectious MeV was not detected in i.m.-immunized (IM) animals but was cleared by 14 days in nebulizer-immunized (Neb) animals and by 17 days in naive macaques. TCID₅₀, 50% tissue culture infectious dose. Differences between the naive and nebulizer-immunized groups were not significant. (B and C) Absolute numbers of circulating lymphocytes and monocytes. (D) MeV-specific IgM determined by enzyme immunoassay using plates coated with MeV-infected Vero cell lysate. IgM was detected in the nebulizer-immunized and naive animals but not in i.m.-immunized animals. (E) Neutralizing antibody determined by plaque reduction neutralization test (PRNT). Values are plotted as geometric means + SEM. Titers were higher in nebulizer-immunized than naive and i.m.-immunized animals on day 14 (nebulizer immunized versus naive, P < 0.05; nebulizer immunized versus i.m. immunized, P < 0.01). Titers were higher in i.m.-immunized than in nebulizer-immunized and naive animals on days 0 and 7 (P < 0.001, two-way ANOVA with Bonferroni posttest). (F) MeV RNA quantified by qRT-PCR on total RNA extracted from 2 × 10⁶ PBMCs. RNA was amplified with MeV N-specific primers and measured with an N-specific probe. Results were normalized to GAPDH. No MeV RNA was detectable in i.m.-immunized macaques. MeV RNA was cleared by 24 days in nebulizer-immunized macaques and by 70 days in naive macaques. Values are shown as means + SEM.

MeV-specific T-cell responses after challenge with wild-type MeV. Shown are T-cell responses 7 (A), 14 (B), and 35 (C) days after challenge as assessed by IFN-γ ELISpot. PBMCs were stimulated with overlapping peptides from the hemagglutinin (H), fusion (F), or nucleocapsid (N) proteins. Numbers of MeV protein-specific spot-forming cells (SFCs) were calculated by averaging duplicate wells and subtracting nonspecific responses. Nebulizer-immunized macaques had a more robust T-cell response than i.m.-immunized (IM) or naive macaques 14 and 35 days after infection (one-way ANOVA with Bonferroni’s multiple comparison tests). ***, P < 0.001; **, P < 0.01; *, P < 0.05. (D) Correlation of the dynamics of the N-specific T-cell response and MeV RNA load. Shown is the group average of the number of N-specific SFCs (blue, orange, and green symbols) plotted against MeV N RNA load in PBMCs (gray symbols). Nebulizer-immunized animals showed a biphasic pattern of IFN-γ-producing T cells with a first peak at 14 days and a second peak at 35 days after challenge. The MeV RNA load and magnitude of T-cell responses showed an inverse relationship.

Functionality of MeV-specific T cells after challenge of nebulizer-immunized and naive macaques with wild-type MeV analyzed by intracellular cytokine staining and multicolor flow cytometry. A total of 10⁶ fresh PBMCs were stimulated with medium or pooled H (A and B) or N (C and D) peptides (1 µg/ml) in the presence of anti-CD107a antibody, brefeldin A, and Golgistop for 12 h. Cells expressing IFN-γ, TNF, IL-2, or CD107 were gated from CD3⁺ CD4⁺ cells and CD3⁺ CD8⁺ cells. Subsets of cells expressing each functional marker were analyzed by Boolean gating. Seven CD4⁺ subsets and 15 CD8⁺ subsets were identified. Subsets that express one (gray), two (yellow), three (orange), or four (red) different functional markers were grouped. The frequency of each subset within CD4⁺ or CD8⁺ T cells was calculated by subtracting nonspecific responses and averaging results from animals in the naive (A and C; orange) or nebulizer-immunized (B and D; blue) groups and shown in the bar chart. The functional composition of CD4⁺ and CD8⁺ T-cell responses is shown in pie charts. More polyfunctional H- and N-specific CD4⁺ and N-specific CD8⁺ T cells were present in nebulizer-immunized than naive macaques.