Protection of rhesus monkeys against infection with minimally pathogenic simian-human immunodeficiency virus: correlations with neutralizing antibodies and cytotoxic T cells

Abstract

We studied the capacity of active immunization of rhesus monkeys with HIV-1 envelope protein (Env) to induce primary virus cross-reactive neutralizing antibodies to prevent infection following intravenous challenge with simian-human immunodeficiency virus (SHIV). Monkeys were immunized with the human immunodeficiency type 1 (HIV-1) strain R2 Env. Initially, the Env was expressed in vivo by an alphavirus replicon particle system, and then it was administered as soluble oligomeric gp140. Concurrently, groups of monkeys received expression vectors that encoded either simian immunodeficiency virus (SIV) gag/pol genes or no SIV genes in vivo to test the additional protective benefit of concurrent induction of virus-specific cell-mediated immune (CMI) responses. Groups of control monkeys received either the gag/pol regimen or sham immunizations. The antibodies induced by the Env immunization regimen neutralized diverse primary HIV-1 strains. Similarly, potent CMI responses were induced by the gag/pol regimen, as measured by gamma interferon enzyme-linked immunospot assays. Differences in the responses among groups of monkeys strongly suggested that there was interference between the Env and gag/pol immunization regimens. Complete protection of some of the monkeys against infection after intravenous challenge with the partially pathogenic SHIV(DH12R (Clone 7)) was associated independently with both neutralizing antibody and CMI responses. Protection was associated with SHIV(DH12 (Clone 7)) serum neutralizing antibody titers of > or =1:80 or with cellular immune responses corresponding to >2,000 spot forming cells per 10(6) peripheral blood mononuclear cells. Immunization was also associated with a reduction in the magnitude and duration of virus load. Induction of cross-reactive, primary HIV-1-neutralizing antibodies is feasible and, when potent, may result in complete protection against infection with a heterologous challenge virus strain.

FIG. 1.

Schematic diagram of immunization and challenge regimens administered to Chinese-origin Macaca mulatta. Six adult rhesus monkeys were included in each group. VEE-R2env indicates VEE-RP expressing HIV-1env_R2 gene. VEE indicates control replicons not expressing foreign genes. VEE-R2env and VEE replicon particle preparations were administered at doses of 10^6.5 FFU/ml, 0.5 ml subcutaneously in the inguinal region and 0.5 ml intravenously at each time point. Sequential doses were administered on alternate sides. DNA gag/pol indicates the codon-optimized SIV_mac239 gag/pol gene in DNA plasmid expression vector. Doses of 5 mg in 1 ml each were given intramuscularly in the leg. R2 gp140 indicates gp140_R2 purified from supernatants of cell cultures infected with vaccinia virus expressing the glycoprotein. It was administered in 300-μg doses in QS-21 or 400-μg doses in RiBi adjuvant, as indicated, intramuscularly in the leg. MVA gag/pol indicates MVA expressing SIV_{mac251 (Clone J5)} gag/pol. MVA is the control MVA. Doses of MVA were 5 × 10⁸ PFU given intradermally in the lateral thigh. VEE-gag/pol and VEE-gag indicate VEE replicons expressing SIV_{mac251 (Clone J5)} gag/pol or gag gene-coding sequences, respectively. Doses of 10⁶ or 10⁷ FFU, respectively, were given, half intravenously and half intradermally in the inguinal area.

FIG. 2.

SIV Gag peptide-specific cellular immune responses of monkeys to immunization with DNA and MVA vectors expressing the SIV-gag/pol gene. Responses were measured in IFN-γ ELISPOT assays, and results are shown as numbers of SFC per 10⁶ PBMC. Responses were considered positive if there were greater than 55 SFC/10⁶ PBMC and results were at least four times the background level. Only those responses that were rated as positive are included in the graphs. PBMC were stimulated in aliquots with nine different pools, each consisting of a mixture of 15-mer peptides. Collectively, the pools included sequences homologous to the full Gag amino acid sequence. Heights of bars indicate cumulative number of SFC for all positive responses, with background subtracted, at the time points indicated.

FIG. 3.

Env_R2 gp140 binding antibody responses of rhesus macaques immunized with VEE-RP expressing gp160Δ CT_R2 and then boosted with soluble gp140_R2 in QS21 adjuvant. Oligomeric gp140_R2 was used as solid phase reagent in an ELISA. Open symbols indicate responses of individual monkeys, and closed symbols indicate geometric mean titers for each group.

FIG. 4.

Neutralizing antibody responses induced by immunization of rhesus monkeys with R2 envelope immunogens. (A and B) Assays were performed by using pseudotyped luciferase reporter viruses. In panel A, sera from sequential time points during the study period were assayed for neutralization of HIV-1_SF162, HIV-1_R2, and SHIV_{DH12R (Clone 7)}. Results shown are geometric means for sera from animals in group I (♦), group III (▪), and group IV (▴). In panel B, pools of sera from five or six monkeys in each of groups II (open bars), group III (filled bars), and group IV (striped bars) were tested in pseudotyped virus neutralization assays against the HIV-1 strains shown. Neutralization titers are the endpoint dilutions that resulted in >50% inhibition of virus infection. (C) HIV-1 neutralization measured by using replication-competent viruses and selected sera from monkeys in groups I, III, and IV. Neutralization titers are the endpoint dilutions of sera that mediated >80% inhibition of virus infection. Sera used for panels B and C were from week 51 of the study.

FIG. 5.

Titration of SHIV_{DH12R (Clone 7)} pool in Chinese-origin rhesus monkeys. Individual monkeys were inoculated with 1,000 (▪, ▴), 100 (▾, ♦), or 10 (•, □) TCID₅₀ of SHIV_{DH12R (Clone 7)}. Additional monkeys received inocula in each of the following amounts (two monkeys for each dose): 1, 0.1, and 0.01 TCID₅₀; results for these monkeys are not shown, since none developed infection detected by our testing. (A) Viral RNA copies per milliliter of plasma at times after inoculation, as determined by reverse transcription-PCR. (B) CD4/CD8 ratios at indicated times after infection.

FIG. 6.

Responses of immunized and control monkeys to intravenous challenge with SHIV_{DH12R (Clone 7)}. (Left column graphs) Viral RNA copies per milliliter of plasma, determined as described in the legend to Fig. 4. (Middle column) Numbers of CD4⁺ cells per milliliter of peripheral blood. (Right column) Changes in SHIV_{DH12R (Clone 7)} neutralization titers from before challenge to 12 weeks after SHIV challenge.

FIG. 7.

Analyses of differences between virus loads of immunized and control monkeys. (A) Areas under curves for each monkey were calculated, and mean areas were determined. (B) Mean number of days of positivity in assay for plasma viral RNA per group is shown. Results in each panel were compared by a Student's t test with Excel.

FIG. 8.

Correlation between SHIV_{DH12R(Clone 7)}-neutralizing antibody titer and protection from infection after intravenous challenge with the same SHIV. Titers were determined on testing of sera taken at week 51 of the study.