Multi-low-dose mucosal simian immunodeficiency virus SIVmac239 challenge of cynomolgus macaques immunized with "hyperattenuated" SIV constructs

Abstract

Hyperattenuated simian immunodeficiency virus SIVmac239-derived constructs Delta5-CMV and Delta6-CCI are an effort to render SIV incapable of, in practical terms, both reversion and recombination while maintaining the immune features of SIV as a retrovirus. Primary inoculation of cynomolgus macaques with 10(8) 50% tissue culture infective doses (TCID(50)) of Delta5-CMV or Delta6-CCI induced low-level humoral and cellular responses detectable in the absence of measureable in vivo replication. The first of three DNA boosts resulted in elevated gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) responses to Gag, Pol, and Env in the Delta5-CMV vaccine group compared to the Delta6-CCI vaccine group (P = 0.001). Weekly intrarectal challenge with a low dose of SIVmac239 followed by a dose escalation was conducted until all animals became infected. The mean peak viral load of the Delta5-CMV-vaccinated animals (3.7 x 10(5) copies/ml) was approximately 1 log unit lower than that of the control animals. More dramatically, the viral load set point of these animals was decreased by 3 log units compared to that of the controls (<50 versus 1.64 x 10(4) copies/ml; P < 0.0001). Seventy-five percent (6/8) of vaccine recipients controlled virus below 1,000 copies/ml for at least 6 months, with a subset controlling virus and maintaining substantial CD4 T-cell counts for close to 2 years of follow-up. The correlates of protection from SIV disease progression may lie in the rapidity and protective value of immune responses that occur early in primary SIV infection. Prior immunization with hyperattenuated SIVmac239, even if sterilizing immunity is not achieved, may allow a more advantageous host response.

FIG. 1.

Detection of SIV-specific antibody responses by Western blotting. SIV-specific antibody responses against vaccine constructs and following SIVmac239 infection were elicited. Humoral immune responses to the vaccines and those that developed following SIV infection were monitored by Western blotting with a commercial kit. Plasma samples were obtained postvaccination (A) and at 5 weeks postinfection (B). The qualitative assay allowed for detection of antibodies directed against the structural genes (Gag, Pol, and Env) expressed by both Δ5-CMV and Δ6-CCI. The relative positions of SIV-derived antigens are indicated by the arrows to the left of the gels. Positive (+ve) and negative (-ve) reactivity control lanes are indicated in addition to a positive serum control band present within each lane.

FIG. 2.

Magnitude of SIV-specific IFN-γ ELISPOT responses following immunization. SIV-specific cytotoxic T-cell responses elicited during vaccination of the Δ5-CMV vaccinated group (A) and the Δ6-CCI vaccinated group (B). ELISPOT responses to SIV antigens were measured for 39 peptide pools (twenty 15-mers/pool) at the times indicated during the vaccination phase (Table 1). Positive responses to individual pools that met our criteria were used to determine the number of SIV-specific IFN-γ-secreting T cells responding to each of the nine SIV genes. Gag peptide pools were employed at all time points examined, whereas peptide pools representing the other SIV genes were introduced only following DNA vaccinations (i.e., 77, 83, and 93 weeks postvaccination). Responses generated against peptides pools for Tat, Nef, Ref, Vif, Vpr, and Vpx were pooled and tallied as “Other”.

FIG. 3.

Longitudinal plasma viral RNA levels in vaccinated and control groups following intrarectal SIVmac239 challenge. Plasma viral RNA levels were determined by quantitative RT-PCR and transformed to log₁₀ copies/ml for analysis. (A) Viral loads are shown for each animal after challenge. The animal numbers are shown in abbreviated form (for instance, 61 for C97061M) (see text or Tables 2, 3, and 4 for animal numbers). (B) Peak viral loads were assessed within the first 2 weeks postinfection. Δ5-CMV-vaccinated animals demonstrated a reduction in peak viral loads of approximately 1 log unit compared to those of the Δ6-CCI-vaccinated or control animals. Peak values are represented as means ± standard errors of the means (SEMs) (error bars). NS, not significant. (C) Set point viral loads were significantly decreased in the Δ5-CMV-vaccinated group compared to the control group (∼3-log-unit decrease; P < 0.001). Data points represent the mean values for each group of animals postinfection ± SEMs. The smoothed curve is the estimated viral load following transformation with a smoothed local regression (LOESS) curve fit. Viral load set points as illustrated by the dotted lines were determined as the mean of data accrued between weeks 15 and 55 postinfection.

FIG. 4.

Summary of lymphocyte populations following SIVmac239 infection. T cells were enumerated as either CD3⁺ CD4⁺ or CD3⁺ CD8⁺ T-cell populations at various times postinfection. (A) Data represent the absolute CD4⁺ T-cell counts/μl of PBMCs for each individual animal. Δ5-CMV-vaccinated animals maintained normal CD4 counts throughout the study (>560/μl), with the exception of animal C98008M, whose CD4 count had dropped below 250/μl by week 69 postinfection. (B) CD8⁺ T-cell enumeration demonstrates a brief expansion within this cell population followed by return to near-normal levels throughout the study. Baseline cell counts for each population are represented as dashed lines and were determined as the mean counts for each group taken prior to initiation of challenge.

FIG. 5.

Acute peripheral CD4⁺ T-cell depletion following SIVmac239 infection. The loss of CD4 T lymphocytes was evaluated in PBMCs and expressed as the percentage remaining at each time point relative to baseline values. Data points represent the mean values for each group with error bars showing the SEMs. Linear regression analysis was employed to assess the rate of CD4 depletion within each group. At all time points assessed, the Δ5-CMV-vaccinated group maintained a greater fraction of peripheral T cells than either the Δ6-CCI-vaccinated or control group (P < 0.05).