Protection of CD4-IgG2-treated rhesus macaques in a high-dose SIVmac239 challenge experiment.

<p>To maintain serum concentrations, CD4-IgG2 (or control human polyclonal IgG) was administered subcutaneously over a two-week period by an ALZET osmotic pump. Animals were challenged intrarectally with a single high dose inoculum (3–5×10³ TCID₅₀) of SIVmac239 3-days after initiation of CD4-IgG2 administration. (<b>A</b>) Viral loads for animals treated with 200 mg of control polyclonal human IgG as a function of time following SIVmac239 challenge. All control animals became infected. (<b>B</b>) Viral loads for animals administered 20 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Three out of 6 animals were fully protected and one infected animal showed delayed primary viremia. Due to a technical problem with the ALZET osmotic pump, one of the protected animals (98045) did not receive the full dose of 20 mg but this animal did not become infected. (<b>C</b>) Viral loads for animals administered 200 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Five out of 7 animals were protected and showed no sign of infection at any time point. The minimum detection level was 125 SIV RNA copies/ml with a 95% confidence level. Open symbol indicates protected animal, closed symbol indicates infected animal.</p

Plasma concentration of CD4-IgG2 in treated animals.

<p>Animals administered 200 mg of CD4-IgG2 showed plasma concentrations in the range of 500 to 1400 ng/ml at the time of challenge. No apparent correlation between plasma concentration and protection was observed. The CD4-IgG2 concentration at the time of challenge in animals administered 20 mg was 100 ng/ml for 1 animal and below the limit of detection (8 ng/ml) for 2 animals. Serum samples from the remaining 3 animals administered 20 mg were unavailable for this analysis.</p

Anti-human CD4 response in animals treated with CD4-IgG2.

<p>Animal sera were tested in a human CD4-specific ELISA to detect macaque antibody responses against CD4-IgG2. Serum samples were tested up to 23 days post-viral challenge and no responses were detected before day 15, indicating that the animal protection outcome was independent of a response against human CD4. Serum samples from 3 animals administered 20 mg CD4-IgG2 were unavailable for this analysis.</p

Administration of GML (or placebo) for all study animals was once per day, except on each challenge day when treatment was given 1 hour prior to each of two separate inoculations of SIVmac251, resulting in two doses.

<p>There were 3 cohorts consisting of both treatment and control animals indicated by degree of shading (cohort 1 = lightest; cohort 2 = medium; cohort 3 = darker). Cohorts only differed in the scheduled time of pre-treatment and first and second challenge days. All cohorts received the same dose continuously for the time indicated. In addition, viral loads for uninfected animals were followed for at least one year after challenge 2.</p><p>Administration of GML (or placebo) for all study animals was once per day, except on each challenge day when treatment was given 1 hour prior to each of two separate inoculations of SIVmac251, resulting in two doses.</p

SIVmac251-specific CD8 T cell responses in selected animals of the study.

<p>^aND: Not Done</p><p>^bEC: Elite Controller</p><p>^cOI: Occult Infection.</p><p>SIVmac251-specific CD8 T cell responses in selected animals of the study.</p

The SIVmac251-Specific CD8 T Cells of Animal R04133 (OI) at 6 Months Following the Second Challenge Possess Cytotoxic Capability.

<p>Freshly isolated PBMC were stimulated with either overlapping 15-mer oligopeptides of SIVmac251 Gag aa1-291 (<i>a-c</i>) or Nef aa1-264 (<i>d-f</i>) and stained for IFN-gamma and CD107a. Plots <i>g-I</i>, show simultaneously measured background data. Bivariate plots display events gated for CD8 antigens within the lymphocyte gate.</p

CD4 T Cell Responses Against SIVmac251 Gag After the Second Viral Challenge.

<p>Freshly isolated PBMC were stimulated with overlapping 15-mer oligopeptides of SIVmac251 Gag aa1-291 and stained for the presence of intracellular cytokines IFN-gamma and IL-2. Bivariate plots display events gated for CD4 antigen expression within the lymphocyte gate. Comparison from simultaneously acquired data from animals classified from their VL as OI (R04133: VLs of 1.37 and 7.08 x 10² copy eq/ml at respectively weeks 3 and 4 post second challenge; VLs thereafter below LOD), elite controller animal (R02037: peak viral load at week 2 post-infection is 8.46 x 10⁶ vRNA copy eq/ml, followed by set-point viral load below detection level from eight week post-infection), and an uninfected animal (R04073). Frequency of IFN-gamma and/or IL-2 positive CD4⁺ lymphocytes at <i>a-c</i>, day 0; <i>d-f</i>, day 28; and <i>g-I</i>, 3 months after the first viral challenge. Frequency of IFN-gamma and/or IL-2 positive CD4⁺ lymphocytes at <i>j-l</i>, day 0; <i>m-o</i>, day 28; and <i>p-r</i>, 3 months after the second viral challenge.</p

GML Pharmacokinetics (PK) and Indirect Mechanism of Action.

<p><i>A</i>, <i>B</i> PK of daily administration of GML on suppressing MIP-3 alpha and IL-8 levels in CVF. Means and SD indicated for three animals. <i>C</i>, GML levels in CVF in treated animals and controls immediately after application and 1, 4 and 8 hours later. Arrow indicates the level prior to challenge at the one-hour time point in animal R01026. LOD = Limits of Detection = 7ug/ml, indicated by a dotted line. The Y-axis scale is discontinuous to show levels of GML immediately after administration.</p

SIV-specific CD8⁺ T cells from LTNP/EC mediate greater lysis of SIV-infected CD4⁺ T-cell targets compared with progressors.

<p>GrB target cell activity (<b>A</b>) and infected CD4 elimination (ICE) (<b>B</b>) are shown for LTNP/EC (n = 10, GrB target cell activity; n = 11, ICE) and progressors (n = 11). Horizontal bars represent the median values. <b>C.</b> Correlation between ICE and GrB target cell activity (n = 22) was determined by the Spearman rank method. Red, blue and cyan dots represent LTNP/EC, progressors and one SIV-uninfected animal, respectively.</p

SIV-specific CD8⁺ T cell cytotoxicity measured by granzyme B delivery or Infected CD4 Elimination (ICE).

<p><b>A.</b> The top panels show granzyme B (GrB) target cell activity representative of a “high responder”. The bottom panels show GrB target cell activity representative of a “low responder”. Values indicate percentages of targets with increased fluorescence due to GrB substrate cleavage. Background GrB target cell activity measured in response to uninfected targets (left column) was subtracted from responses measured against infected targets (right column) to determine net GrB target cell activity (red values). <b>B.</b> ICE values calculated based on p27 expression (sum of the upper quadrants) as described in the Methods, are shown in red for the same “high responder” (78.8%, top row) and “low responder” (22.3%, bottom row) as shown in A. Quadrant values indicate percentages of gated targets. In all experiments, CD4⁺ T cell lines were used as targets. CD8⁺ T cells that had been stimulated with SIV-infected targets for 6 days were used as effectors. GrB target cell activity and ICE were calculated after 1 hour of incubation of effectors and plated at an E∶T ratio of 25∶1.</p