Prevention of infection by a granulocyte-macrophage colony-stimulating factor co-expressing DNA/modified vaccinia Ankara simian immunodeficiency virus vaccine

Abstract

A simian immunodeficiency virus (SIV) vaccine coexpressing granulocyte-macrophage colony stimulating factor (GM-CSF) prevented infection in 71% of macaques that received 12 rectal challenges. The SIVsmE660 challenge had the tropism of incident human immunodeficiency virus (HIV) infections and a similar genetic distance from the SIV239 vaccine as intraclade HIV isolates. The heterologous prime-boost vaccine regimen used recombinant DNA for priming and recombinant modified vaccinia Ankara for boosting. Co-expression of GM-CSF in the DNA prime enhanced the avidity of elicited immunoglobulin G for SIV envelope glycoproteins, the titers of neutralizing antibody for easy-to-neutralize SIV isolates, and antibody-dependent cellular cytotoxicity. Impressively, the co-expressed GM-CSF increased vaccine-induced prevention of infection from 25% in the non-GM-CSF co-expressing vaccine group to 71% in the GM-CSF co-expressing vaccine group. The prevention of infection showed a strong correlation with the avidity of the elicited Env-specific antibody for the Env of the SIVsmE660 challenge virus (r = 0.9; P < .0001).

Figure 1.

Schematics of SIV239 DNA and recombinant modified vaccinia Ankara (MVA) vaccines. Transcriptional control elements are shaded. For the DNA vaccines, transcription is initiated by the cytomegalovirus immediate early promoter (CMVIE) including intron A and terminated by the bovine growth hormone polyadenylation sequence (BGHpA). For the MVA vaccine, transcription is under the control of the p7.5 (env) and mH5 (gag-pol) promoters. gag, Pr, RT, tat, rev, and env are sequences encoding the group-specific antigens, protease, reverse transcriptase, transcriptional activator, regulatory protein, and envelope glycoprotein, respectively, of SIV239. Xs indicate inactivating point mutations in reverse transcriptase and packaging sequences in gag. D, SIV239 DNA vaccine; Dg, GM-CSF co-expressing SIV239 DNA vaccine; M, SIV239 MVA vaccine.

Figure 2.

Humoral immune responses elicited by the granulocyte-macrophage colony stimulating factor (GM-CSF)–adjuvanted and nonadjuvanted DNA/ modified vaccinia Ankara (MVA) vaccines. DNA priming immunizations were administered at weeks 0 and 8, and MVA booster immunizations were administered at weeks 16 and 24. A, Env-specific immunoglobulin (Ig) G responses measured in serum at before immunization and at weeks 2, 10, 18, 21, 26, and 37 in the trial. Micrograms of IgG are estimated relative to a standard curve of rhesus IgG. Values are medians ± interquartile ranges. B, Tukey plots presenting Env-specific IgA responses in rectal secretions before immunization, 2 weeks after the indicated immunizations, and before challenge. IgA is presented as Env-specific IgA divided by total IgA. C, Avidity indices for elicited IgG for the SIV239 Env of the immunogen and the SIVE660 Env of the challenge measured at 2 weeks after the second MVA immunization. Avidity indices increased with time in the trial and further increased after infection. D, Neutralization titers for pseudotypes with 2 Envs molecularly cloned from the genetically diverse SIVE660 stock. Titers for SIVE660.11 were determined 2 weeks after the second MVA boost and, for SIVE660.17, at 13 weeks after the second MVA boost. Differences in overall titers for the 2 tested isolates reflect differences in the susceptibility of different isolates from the E660 quasi-species to neutralization and differences in the timing of assays. Titers are the reciprocal for the dilution of serum achieving an inhibitory dose of 50% (ID₅₀) in the TZM-bl assay. E, Antibody-dependent cellular cytotoxicity titers for SIVmac239 gp120 coated CEM.NKR_CCR5 cells at 2 weeks after the second MVA boost. In panels C–E, box plots present median and 25th and 75th percentiles for responses. Target Envs and the significance for differences between the DDMM and DgDgMM regimens are indicated above the box plots. Statistical comparisons were made using a 2-sided Wilcoxon rank-sum test. Data for the unadjuvanted DNA (DDMM) regimen are presented in turquoise, and those for the Gm-CSF-adjuvanted DNA (DgDgMM) regimen are presented in fuchsia.

Figure 3.

Cellular immune responses elicited by the granulocyte-macrophage colony stimulating factor (GM-CSF)–adjuvanted and nonadjuvanted DNA/ modified vaccinia Ankara (MVA) vaccines. DNA priming immunizations were administered at weeks 0 and 8, and MVA booster immunizations were administered at weeks 16 and 24. Shown are Vaccine-elicited CD4⁺ T cell responses (A) and CD8⁺ T-cell responses (B) before immunization and at weeks 2, 10, 17, 21, 25, and 37 in the trial. Responses are shown as interferon (IFN)–γ secreting cells measured by intracellular cytokine staining (ICS) after Gag and Env peptide stimulation of peripheral blood mononuclear cells (PBMCs). Gray boxes represent the background for detection. Also shown are the breadth of vaccine-elicited IFN-γ secreting CD4⁺ T-cell responses (C) and CD8⁺ T-cell responses (D) measured by ICS of PBMCs stimulated with 13 Gag and 11 Env peptide pools at 1 week after the first and second MVA immunizations. E and F, Polyfunctionality for cytokine production by elicited CD4⁺ and CD8+ T-cell responses at 1 week after the second MVA immunization. Boolean analyses were used to determine the frequencies of IFN-γ–, interleukin-2–, and tumor necrosis factor–α–producing cells responding to Gag and Env. Only those responses that were ≥0.07% of total cytokine-positive cells were considered for analysis. The box plots present the median and interquartile ranges for the percentage of responding cells (as a proportion of total cytokine positive cells) producing 1, 2, or 3 cytokines. Patterns of cytokine production for individual subsets of single or double producers were overall similar (data not shown). Data for the unadjuvanted DNA (DDMM) regimen are presented in turquoise, and those for the Gm-CSF-adjuvanted DNA (DgDgMM) regimen are presented in fuchsia.

Figure 4.

Co-expressed granulocyte-macrophage colony stimulating factor enhances protection against infection from 25% to 71%. A, Kaplan-Meier curve for number of challenges to infection. P = .003 is the significance for the difference in number of challenges to infection between the GM-CSF-adjuvanted DNA (DgDgMM) and unvaccinated group (by log-rank Mandel-Cox test). B, Temporal post challenge viremia in animals that became infected. Infection dates are adjusted, with week 1 being the first week an infection was detected. Data are presented as means ± 1 standard deviation to show the differences in overall levels of viremia in the groups. The gray box represents the background for detection of viral RNA in the quantitative real-time polymerase chain reaction test. Data for the unadjuvanted DNA (DDMM) regimen are presented in turquoise, and those for the DgDgMM regimen are presented in fuchsia.

Figure 5.

Absence of anamnestic antibody responses in repeatedly challenged animals that did not become infected. A, Absence of a detectable anamnestic Env-specific immunoglobulin (Ig) A response in uninfected rhesus macaques at various weeks after the last challenge. B, Strong anamestic IgA responses for Env in vaccinated animals that became infected. C, Absence of a detectable anamnestic IgG response for Env in uninfected rhesus macaques at various weeks after the last challenge. D, Strong anamnestic IgG responses for Env in vaccinated animals that became infected. Data are presented as medians ± interquartile ranges. The gray boxes represent backgrounds for detection. Data for the unadjuvanted DNA (DDMM) regimen are presented in turquoise, and those for the Gm-CSF-adjuvanted DNA (DgDgMM) regimen are presented in fuchsia.

Figure 6.

Avidity of the vaccine-elicited immunoglobulin (Ig) G for the Env of the challenge virus correlates with protection. A, Significant correlation between avidity of the elicited IgG for the SIVE660 Env of the challenge virus and the number of challenges to infection. Data are presented as the mean ± 1 standard deviation for 3 independent assays. Animals that did not become infected by the 12 challenges are plotted at 14 challenges. Correlations were done using the 2-sided Spearman rank order statistical analysis. B, The TRIM5α genotype of vaccinated rhesus macaques does not restrict the number of challenges to infection r, restrictive TRIM5α genotype (homozygous or heterozygous for TRIM5αTFP or CYPA); s, susceptible genotype (homozygous for TRIM5αQ); m, moderately susceptible (heterozygous for a restrictive and permissive allele). Animals that were not infected by the 12 challenges are plotted at challenge 14.