Recombinant modified vaccinia virus Ankara provides durable protection against disease caused by an immunodeficiency virus as well as long-term immunity to an orthopoxvirus in a non-human primate

Abstract

Recombinant and non-recombinant modified vaccinia virus Ankara (MVA) strains are currently in clinical trials as human immunodeficiency virus-1 (HIV) and attenuated smallpox vaccines, respectively. Here we tested the ability of a recombinant MVA delivered by alternative needle-free routes (intramuscular, intradermal, or into the palatine tonsil) to protect against immunodeficiency and orthopoxvirus diseases in a non-human primate model. Rhesus macaques were immunized twice 1 month apart with MVA expressing 5 genes from a pathogenic simian human immunodeficiency virus (SHIV)/89.6P and challenged intrarectally 9 months later with the pathogenic SHIV/89.6P and intravenously 2.7 years later with monkeypox virus. Irrespective of the route of vaccine delivery, binding and neutralizing antibodies and CD8 responses to SHIV and orthopoxvirus proteins were induced and the monkeys were successively protected against the diseases caused by the challenge viruses in unimmunized controls as determined by viral loads and clinical signs. These non-human primate studies support the clinical testing of recombinant MVA as an HIV vaccine and further demonstrate that MVA can provide long-term poxvirus immunity, essential for use as an alternative smallpox vaccine.

Fig. 1

Structure of MVA/KB9-5, expression of SHIV genes, and study protocol. (A) Env and tat from SHIV/KB9 (gray boxes) and gagpol and nef from SIVmac239 (white boxes) were inserted into deletions II and III (black boxes) of MVA to form MVA/KB9-5. Chimeric Tatnef protein was expressed under control of the modified P7.5 promoter; Env and Gagpol were expressed under control of the modified H5 promoter. (B) SHIV antigens were immunoprecipitated from cells infected with MVA/KB9-5 with: monoclonal antibody T8 (α-env), SIV polyclonal serum E544 (α-SIV), and HIV-1BH10 Tat antiserum (α-tat). Precursor and cleavage products for Env and Gag are shown. The Tatnef protein was precipitated by both Tat and Nef (E544) antibodies. (C) Study timeline.

Fig. 2

Temporal immune responses induced by vaccination with MVA/KB9-5 and challenge with SHIV/89.6P. (A) Serum antibody endpoint titers were measured by ELISA with KB9 gp140 captured via antibody to the C-terminus of gp120. Average titers for each immunization group are shown with standard errors. (B) SHIV/89.6P neutralizing antibody titers were determined in an MT-2 cell-killing assay. Titers are the reciprocal of the dilution giving 50% neutralization; averages for each group are shown with standard errors. (C) The percent of CD8 T-cells that were positive for the gag CM9 peptide was measured by tetramer binding in the MamuA*01⁺ animals; averages for each group are shown. (D) Frequencies of Gag, Env, Tat and Nef specific cells was measured by IFN-γ ELISpots. Fresh PBMC were stimulated for 36 h with pools of approximately 10 peptides. Averages for each group are shown as follows: PT, white bars; ID, light gray bars; IM, dark gray bars; naïve (unimmunized) black bars. Arrowheads indicate times of MVA immunization; stars indicate day of challenge with SHIV/89.6P.

Fig. 3

CD4 T cell counts after challenge with SHIV/89.6P. CD4 cell counts of individual animals are shown in the four lower panels with animal deaths indicated by a cross (all naïve, one P.T.-immunized animal). The geometric mean for all groups is shown in the top panel.

Fig. 4

SHIV viral load. The number of copies of SHIV RNA per ml of plasma was determined by quantitative PCR (Cline et al., 2005). Data from individual animals are shown in the four lower panels; geometric means for all groups are shown in the top panel.

Fig. 5

Temporal VACV antibody responses. (A, B, C) Serum antibody titers were determined by ELISA against purified VACV MV particles, purified VACV L1 protein, and purified VACV B5 protein. Average titers with standard errors are shown for animals immunized by each route: PT, diamond; ID, square, IM, triangle. Times of MVA immunization are shown with arrowheads; star indicates day of challenge with SHIV/89.6P. (D) Neutralizing antibody titers were determined using a flow cytometric assay that measures reduction in infectivity of a VACV expressing EGFP (Earl et al., 2003). Symbols as above. (E) Percent neutralization of VACV EV was determined in the presence of excess L1 Mab 7D11. Serum samples from individual animals were assayed and the averages for animals in each group are indicated as: PT, light gray; IM, medium gray; ID, black. Vaccinia virus immune globulin (VIG) was used as a positive control. (F) A comet reduction assay was performed using VACV strain IHD-J. Serum samples from all animals were assayed and one representative example from each immunization group is shown.

Fig. 6

INF-γ producing MVA-specific CD8 cells. Quantitation of VACV-specific IFN- γ producing cells was performed on cryopreserved PBMC after infection with MVA overnight. Each dot represents one animal with positive values (>2-fold background) located above the shaded area. The average for each time point is denoted by a line.

Fig. 7

MPXV challenge. (A) Following challenge with MPXV, the number of MPXV genomes per ml of blood was determined by quantitative TacMan PCR. Average values for MVA-immunized and naïve groups are shown. (B) The number of lesions induced by MPXV challenge was enumerated every 3 days for 2 weeks after challenge. The number of lesions found on each animal is shown.