Comparison of the immune responses induced by chimeric alphavirus-vectored and formalin-inactivated alum-precipitated measles vaccines in mice

Abstract

A variety of vaccine platforms are under study for development of new vaccines for measles. Problems with past measles vaccines are incompletely understood and underscore the need to understand the types of immune responses induced by different types of vaccines. Detailed immune response evaluation is most easily performed in mice. Although mice are not susceptible to infection with wild type or vaccine strains of measles virus, they can be used for comparative evaluation of the immune responses to measles vaccines of other types. In this study we compared the immune responses in mice to a new protective alphavirus replicon particle vaccine expressing the measles virus hemagglutinin (VEE/SIN-H) with a non-protective formalin-inactivated, alum-precipitated measles vaccine (FI-MV). MV-specific IgG levels were similar, but VEE/SIN-H antibody was high avidity IgG2a with neutralizing activity while FI-MV antibody was low-avidity IgG1 without neutralizing activity. FI-MV antibody was primarily against the nucleoprotein with no priming to H. Germinal centers appeared, peaked and resolved later for FI-MV. Lymph node MV antibody-secreting cells were more numerous after FI-MV than VEE/SIN-H, but were similar in the bone marrow. VEE/SIN-H-induced T cells produced IFN-gamma and IL-4 both spontaneously ex vivo and after stimulation, while FI-MV-induced T cells produced IL-4 only after stimulation. In summary, VEE/SIN-H induced a balanced T cell response and high avidity neutralizing IgG2a while FI-MV induced a type 2 T cell response, abundant plasmablasts, late germinal centers and low avidity non-neutralizing IgG1 against the nucleoprotein.

Figure 1. Measles virus-specific antibody response to immunization.

Sera collected from individual mice immunized with VEE/SIN-H or FI-MV were assessed for quantity and quality of MV-specific antibody. MV-specific total IgG (A), IgG1 (B) and IgG2a (C) were measured by EIA. Avidity of MV-specific antibody was evaluated by a modified EIA and data are presented as an avidity index (D). Fifty percent plaque reduction neutralization titers (PRNT) for the Chicago-1 strain of MV on Vero cells are expressed as geometric means (E). Data points represent the mean +/- S.D. of three individual mice. (* P<0.05; Student's t test)

Figure 2. Germinal center formation after immunization.

Popliteal draining lymph nodes were harvested at day 7 after injection with VEE/SIN-H, FI-MV, SRBCs or PBS. Cryosections (10µm) were stained with PNA-FITC for GC B cells (green) and with PE-conjugated antibody to IgD for follicular B cells (red). Representative images are 200× magnification (A). GCs detected by histology were enumerated and presented as the mean +/− S.D. of at least 5 sections from one mouse (n.d. = none detected) (B).

Figure 3. Kinetics of the germinal center response after immunization.

Cells from popliteal draining lymph nodes harvested at various times after immunization with VEE/SIN-H, FI-MV, SRBCs (positive control) or PBS were analyzed for GC B cells by flow cytometry. Cells were stained with antibody to CD19 and PNA at day 7 (A) and periodically over an 80-day time course (B). All flow cytometry data points represent values from cells pooled from 2 mice.

Figure 4. Development of antibody-secreting cells in draining lymph nodes and bone marrow after immunization.

At various times after immunization, cells from draining popliteal LNs and bone marrow were collected and analyzed by IgG ELISpot. Total IgG-secreting cells were measured in the draining LNs (A) and the bone marrow (C), in addition to MV-specific ASCs (B and D). ELISpot plate images of bone marrow aspirates assayed for MV-specific ASCs at day 80 after immunization (E). The spot area for MV-specific IgG ASCs from the bone marrow at different times after immunization (F). Wells were loaded with 5.0×10⁵ unfractionated bone marrow cells. Assays were performed in triplicate (error bars represent S.D. of assay replicates) with cells pooled from 3 mice.

Figure 5. Protein-specific antibody responses to vaccine antigens.

Serum IgG specific for MV H (A), F (B) and N (C) proteins over a 125-day time course measured by EIA. H-specific ASCs from the bone marrow measured by ELISpot at day 125, represented as an index of total spot number multiplied by spot area (D). Serum antibody data points represent the mean +/− S.D. of 3 individual mice. IgG ELISpot data are generated from cells pooled from 3 mice.

Figure 6. Measles virus-specific T cell responses after immunization.

IFN-γ (A) and IL-4–secreting cells (B) from the dLN were measured at day 7 after immunization with VEE/SIN-H or FI-MV. Media-only stimulation represents ex vivo spontaneous secretion. This value has been subtracted from the MVL stimulation values. At days 7, 14 and 21 after immunization, cells from dLNs (C, D) and the spleen (E, F) were evaluated for IFNγ and IL-4 secretion after ex vivo stimulation with MVL antigen in ELISpot assays. Spot-forming cells are per 5×10⁵ total cells. Assays were performed in triplicate (error bars represent S.D. of assay replicates) with cells pooled from 3 mice.

Figure 7. Protein-specific T cell responses after immunization.

Cytokine secretion as a readout for recognition of class-II restricted CD4⁺ T cell MV epitopes for H (H1, H2), F (F1, F2) and N (N1) proteins, as well as complete peptide pools for H and F proteins, was measured for IFN-γ (A, C, E) and IL-4 (B, D, F) in cells from the dLN by ELISpot at 1, 2 and 3 weeks after immunization. Negative controls were an irrelevant influenza HA peptide (I1 - I-E^d) and media-only and ConA-stimulated cells (not shown) served as a positive control (n.d. = not determined). Negative controls (media-only) were subtracted to discount cells that spontaneously produced cytokines and to emphasize antigen-specific reactivity. Spot-forming cells are per 5×10⁵ total cells. Assays were performed in triplicate (error bars represent S.D. of assay replicates) with cells pooled from 3 mice.

Figure 8. Hemagglutinin-specific antibody-secreting cell recall responses.

Mice immunized with FI-MV, VEE/SIN-H or mock-immunized with PBS were given VEE/SIN-H at day 81 after primary immunization, bled at days 4 and 8 post-secondary immunization and sacrificed at day 12. EIAs were performed to measure serum antibody with reactivity against MVL (* P<0.05; Student's t test) (A) and H (B) induced in the recall response up to 12 days after administering VEE/SIN-H. ASCs in the draining LNs were assayed for total IgG and MV-specific antibody secretion by ELISpot (C, D) 12 days after secondary immunization. Serum antibody data points represent the mean +/− S.D. of 3 individual mice. IgG ELISpots were performed in triplicate (error bars represent S.D. of assay replicates) with cells pooled from 3 mice.