Administration of recombinant rhesus interleukin-12 during acute simian immunodeficiency virus (SIV) infection leads to decreased viral loads associated with prolonged survival in SIVmac251-infected rhesus macaques

Abstract

The ability of recombinant rhesus interleukin-12 (rMamu-IL-12) administration during acute simian immunodeficiency virus SIVmac251 infection to influence the quality of the antiviral immune responses was assessed in rhesus macaques. Group I (n = 4) was the virus-only control group. Group II and III received a conditioning regimen of rMamu-IL-12 (10 and 20 microg/kg, respectively, subcutaneously [s.c.]) on days -2 and 0. Thereafter, group II received 2 microg of IL-12 per kg and group III received 10 microg/kg s.c. twice a week for 8 weeks. On day 0 all animals were infected with SIVmac251 intravenously. While all four group I animals and three of four group II animals died by 8 and 10 months post infection (p.i.), all four group III animals remained alive for >20 months p.i. The higher IL-12 dose led to lower plasma viral loads and markedly lower peripheral blood mononuclear cell and lymph node proviral DNA loads. During the acute viremia phase, the high-IL-12-dose monkeys showed an increase in CD3(-) CD8 alpha/alpha(+) and CD3(+) CD8 alpha/alpha(+) cells and, unlike the control and low-IL-12-dose animals, did not demonstrate an increase in CD4(+) CD45RA(+) CD62L(+) naive cells. The high-IL-12-dose animals also demonstrated that both CD8 alpha/alpha(+) and CD8 alpha/beta(+) cells produced antiviral factors early p.i., whereas only CD8 alpha/beta(+) cells retained this function late p.i. Long-term survival correlated with sustained high levels of SIV gag/pol and SIV env cytotoxic T lymphocytes and retention of high memory responses against nominal antigens. This is the first study to demonstrate the capacity of IL-12 to significantly protect macaques from SIV-induced disease, and it provides a useful model to more precisely identify correlates of virus-specific disease-protective responses.

FIG. 1.

Survival in months of the three groups of monkeys. Group I (n = 4) was SIVmac251-infected control monkeys, group II (n = 4) was SIV infected and given a loading dose of 10 μg of rMamu-IL-12 per kg s.c. on days −2 and 0 followed by 2 μg/kg s.c. twice a week for 8 weeks (low dose), and group III was SIV infected and given a loading dose of 20 μg of rMamu-IL-12 per kg s.c. on days −2 and 0 and 10 μg/kg s.c. twice a week for 8 weeks (high dose). Survival of group III monkeys was statistically significant (P < 0.001 by ANOVA at Tukey's 95% CI).

FIG. 2.

Sequential analysis of plasma viral loads in group I (A), group II (B), and group III (C) monkeys. Statistical analysis of the viral loads showed significantly lower (P < 0.004 by ANOVA at Tukey's 95% CI) plasma viral loads in group III monkeys than in group I monkeys.

FIG. 3.

Proviral DNA loads in PBMC samples and LNC from biopsies or at autopsy from the three groups of monkeys. The sensitivity of the assay was 1 copy per 10⁵ cells. (A) PBMC samples from group I at weeks 3 to 32, except monkey RYl-2, which died at week 20. (B) PBMC samples from group II monkeys obtained up to weeks 27, 12, 74, and 42. Monkey REn-4 is still alive. (C) PBMC samples from group III monkeys up to week 74 (still alive). (D) LNC from each of the three groups of monkeys obtained from biopsy samples at 3 weeks p.i. (sample 1); at 24 weeks p.i. or at autopsy (REk-4 at 12 weeks, RYl-2 at 20 weeks) (sample 2); and at 58 weeks p.i. or at autopsy for monkeys RDt-1, RJt-2, RRm-3, RCz-2, and R2318 (weeks 27 to 35) (sample 3). Statistical analysis of PBMC proviral DNA levels was performed by comparison of viral loads on individual days p.i. from each group of monkeys. Data for group III monkeys were statistically highly significantly different from those for group I monkeys for all data points (range, P < 0.00003 at week 27 to P < 0.04 at week 15). Similarly, the proviral DNA levels in LNC samples of the group III monkeys were also statistically significantly different from those for group I (P < 0.008, 0.01, and 0.0006 for samples 1, 2, and 3, respectively).

FIG. 4.

Plasma IFN-γ levels in samples from the three groups of monkeys collected at day −2 (prior to IL-12 administration) and at various times thereafter.

FIG. 5.

PBMC samples from groups I and III obtained prior to SIV infection and at 8, 14, and 18 weeks p.i. were assayed for levels of SIV Gag/Pol-specific (A) and SIV Env-specific (B) pCTLs as described in Materials and Methods. The pCTL values from group III versus group I at 8, 14, and 18 weeks are statistically significant (P < 0.0001) by the t test.

FIG. 6.

Cellular and humoral responses to Flu (A and B) and TT (C and D). PBMC and serum samples obtained from group I and group III prior to immunization with Flu, TT, or KLH and SIV infection (Pre Imm. or −4), after Flu, TT, and KLH immunization but prior to SIV infection (Post Imm. Pre SIV or −2 and 0) and at 12 and 20 weeks after SIV infection (Post SIV1 and Post SIV2, respectively) (A and C) or at the indicated months p.i. (B and D) were assayed for Flu-MP-specific net pCTL values (A) and their proliferative response to the P2 TT peptide (C) as described in Materials and Methods. The values of pCTLs for group III and the proliferative response to P2 TT peptide at week 12 p.i. compared to the values for group I are statistically significant, with a P value of 0.0001 (t test). Differences in humoral responses to Flu or TT between IL-12-conditioned (filled symbols) and control monkeys (open symbols) were not significant.