A chimeric alphavirus replicon particle vaccine expressing the hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques from measles

Abstract

Measles remains a major cause of child mortality, in part due to an inability to vaccinate young infants with the current live attenuated virus vaccine (LAV). To explore new approaches to infant vaccination, chimeric Venezuelan equine encephalitis/Sindbis virus (VEE/SIN) replicon particles were used to express the hemagglutinin (H) and fusion (F) proteins of measles virus (MV). Juvenile rhesus macaques vaccinated intradermally with a single dose of VEE/SIN expressing H or H and F proteins (VEE/SIN-H or VEE/SIN-H+F, respectively) developed high titers of MV-specific neutralizing antibody and gamma-interferon (IFN-gamma)-producing T cells. Infant macaques vaccinated with two doses of VEE/SIN-H+F also developed neutralizing antibody and IFN-gamma-producing T cells. Control animals were vaccinated with LAV or with a formalin-inactivated measles vaccine (FIMV). Neutralizing antibody remained above the protective level for more than 1 year after vaccination with VEE/SIN-H, VEE/SIN-H+F, or LAV. When challenged with wild-type MV 12 to 17 months after vaccination, all vaccinated juvenile and infant monkeys vaccinated with VEE/SIN-H, VEE/SIN-H+F, and LAV were protected from rash and viremia, while FIMV-vaccinated monkeys were not. Antibody was boosted by challenge in all groups. T-cell responses to challenge were biphasic, with peaks at 7 to 25 days and at 90 to 110 days in all groups, except for the LAV group. Recrudescent T-cell activity coincided with the presence of MV RNA in peripheral blood mononuclear cells. We conclude that VEE/SIN expressing H or H and F induces durable immune responses that protect from measles and offers a promising new approach for measles vaccination. The viral and immunological factors associated with long-term control of MV replication require further investigation.

FIG. 1.

Antibody responses of juvenile macaques to VEE/SIN, FIMV, and LAV measles vaccines. Groups of three monkeys were vaccinated with a single i.d injection of VEE/SIN-H (10⁸ particles) alone or of VEE/SIN-H (10⁸ particles) plus VEE/SIN-F (3 × 10⁶ particles). Control monkeys received three 0.5-ml i.m. injections of FIMV (arrows; n = 2) or a single i.m. injection of 5,000 PFU of LAV (n = 2). (A) MV-specific antibody was measured by PRN assay on Vero cells. Values are plotted as geometric means ± standard error of the means (SEM). The predicted protective level of PRN antibody (120 mIU/ml) is shown with a dashed line. (B) MV-specific IgG was measured by EIA with plasma diluted 1:400 and plates coated with a lysate of MV-infected cells. (C) H-specific IgG was measured by EIA with plasma diluted 1:100 and plates coated with a lysate of L cells expressing H. (D) Avidity of IgG for the H protein was assessed by disruption of antibody binding with 0 to 3 M NH₄SCN, and an avidity index was calculated. (E) N-specific IgG was measured by EIA with plasma diluted 1:1,000 and plates coated with baculovirus-expressed N protein. (F) Avidity of IgG for the N protein was assessed as described for panel D. Values are plotted as means ± SEM.

FIG. 2.

T-cell responses of juvenile macaques to VEE/SIN, FIMV, and LAV measles vaccines. Monkeys were immunized as described in the legend of Fig. 1. MV H or F peptide-induced production of IFN-γ and IL-4 was measured by ELISPOT assay. The peaks of the IFN-γ-producing cell response (A) and the IL-4-producing cell response (B) to pooled H and F peptides and the time course of the IFN-γ response to H peptides (C) and F peptides (D) are shown. Data are shown as numbers of SFCs/10⁶ PBMCs. Values are plotted as means ± SEM. ***, P = 0.0008.

FIG. 3.

Disease after challenge. All juvenile macaques were challenged intratracheally with wild-type MV Bilthoven (10⁴ TCID₅₀) 17 months after immunization and monitored for development of a rash. Only FIMV-immunized control monkeys (24R and 26R) developed measles. A rash was evident on the face (A and B) and the extremities and lower abdomen (C and D). Sections of rash biopsy specimens from these animals were stained with hematoxylin and eosin (E) and with antibody to the MV N protein (F).

FIG. 4.

Viremia and antibody responses after challenge. Viremia was measured by cocultivation of serially diluted PBMCs with B95-8 cells (A). MV-specific IgM was measured by EIA with plasma diluted 1:200 (B). Neutralizing antibody to MV was measured by PRN assay (C). MV-specific IgG was measured by EIA with plasma diluted 1:400 (D). The means ± SEM are shown.

FIG. 5.

T-cell responses after challenge. IFN-γ (A and B) and IL-4 (C and D) responses to stimulation by pools of MV H peptides (A, C) and F peptides (B, D) were assayed by ELISPOT assay after challenge. Values are mean numbers of SFCs/10⁶ PBMCs ± SEM.

FIG. 6.

MV RNA in PBMCs after challenge. RNA was extracted from 2 × 10⁶ PBMCs and analyzed by quantitative RT-PCR for MV N and GAPDH gene sequences. Copy numbers were calculated and normalized to GAPDH.

FIG. 7.

Immune responses of infant macaques to VEE/SIN-H+F measles vaccine. Infant macaques 1 to 2 months old (n = 3) were vaccinated i.d. with two doses of VEE/SIN-H (10⁸ particles) plus VEE/SIN-F (3 × 10⁶ particles) at 0 and 8 weeks. (A) Neutralizing antibody was measured by PRN assay. The predicted protective level of PRN antibody (120 mIU/ml) is indicated with a dashed line. Values are plotted as geometric means ± SEM. (B) MV-specific IgG (plasma diluted 1:400) and (C) H-specific IgG (plasma diluted 1:100) were measured by EIA. The mean OD values ± SEM are shown. (D) Avidity of IgG for the H protein was assessed by disruption of antibody binding with 0 to 3 M NH₄SCN, and an avidity index was calculated. IFN-γ responses to stimulation with MV H peptide pools (E) or F peptide pools (F) were assayed by ELISPOT assays. The means ± SEM for each group are shown.

FIG. 8.

Immune responses of VEE/SIN-H+F-vaccinated infant macaques after challenge. Vaccinated monkeys were challenged intratracheally at 1 year with 10⁴ TCID₅₀ of the Bilthoven wild-type strain of MV. (A) Neutralizing antibody was measured by PRN assay. (B) MV-specific IgG was measured by EIA. The mean OD values ± SEM for plasma diluted 1:400 are shown. IFN-γ responses to stimulation by peptide pools of MV H protein (C) or F protein (D) were assayed by ELISPOT assay. Values are mean numbers of SFCs/10⁶ PBMCs ± SEM.