Systemic neutralizing antibodies induced by long interval mucosally primed systemically boosted immunization correlate with protection from mucosal SHIV challenge

Abstract

Immune correlates of vaccine protection from HIV-1 infection would provide important milestones to guide HIV-1 vaccine development. In a proof of concept study using mucosal priming and systemic boosting, the titer of neutralizing antibodies in sera was found to correlate with protection of mucosally exposed rhesus macaques from SHIV infection. Mucosal priming consisted of two sequential immunizations at 12-week intervals with replicating host range mutants of adenovirus type 5 (Ad5hr) expressing the HIV-1(89.6p) env gene. Following boosting with either heterologous recombinant protein or alphavirus replicons at 12-week intervals animals were intrarectally exposed to infectious doses of the CCR5 tropic SHIV(SF162p4). Heterologous mucosal prime systemic boost immunization elicited neutralizing antibodies (Nabs), antibody-dependent cytotoxicity (ADCC), and specific patterns of antibody binding to envelope peptides. Vaccine induced protection did not correlate with the type of boost nor T-cell responses, but rather with the Nab titer prior to exposure.

FIG. 1

Development of humoral binding responses pre and post SHIV_SF162p4 challenge. A: Titers of binding antibodies for each animal against peptide pools covering the gp120 of 89.6p or SHIV_SF162p4 or against SIV Gag peptides were detected by ELISA. Binding titers were measured 2 weeks after each immunization and at various time points post-challenge. Black arrows indicate immunizations and the white arrow indicates IR SHIV_SF162p4 challenge. B: Pepscan analysis of sera from the individual animals. Reactivity with all overlapping 15-mer peptides of HIV-1_SF162 gp120 were tested at a serum dilution of 1:100. C: Quality (Antibody Avidity Index: HN₄SCN concentration in M) of antibody responses induced by various immunization regimen. Sera before 1^st immunization and after the 4^th immunization (= 2 weeks pre-challenge) are shown.

FIG. 1

Development of humoral binding responses pre and post SHIV_SF162p4 challenge. A: Titers of binding antibodies for each animal against peptide pools covering the gp120 of 89.6p or SHIV_SF162p4 or against SIV Gag peptides were detected by ELISA. Binding titers were measured 2 weeks after each immunization and at various time points post-challenge. Black arrows indicate immunizations and the white arrow indicates IR SHIV_SF162p4 challenge. B: Pepscan analysis of sera from the individual animals. Reactivity with all overlapping 15-mer peptides of HIV-1_SF162 gp120 were tested at a serum dilution of 1:100. C: Quality (Antibody Avidity Index: HN₄SCN concentration in M) of antibody responses induced by various immunization regimen. Sera before 1^st immunization and after the 4^th immunization (= 2 weeks pre-challenge) are shown.

FIG. 2

Development of functional humoral responses pre and post SHIV_SF162p4 challenge. A: Neutralizing antibody responses against pseudovirus SHIV_SF162p4 cl5.1B were evaluated throughout the immunization schedule. The serum dilutions giving 50% inhibition of SHIV_SF162p4 cl5.1B infection of TZM-bl cells (Wei et al., 2003) are shown. Black arrows indicate immunizations and the white arrow indicates IR SHIV_SF162p4 challenge. B: ADCC using targets coated with 89.6P gp140 protein and SF₁₆₂ gp120 protein. Expressed are titers giving specific lysis of target cells. The cut off for positive activity and determination of endpoint titer was 15.62% and was calculated from the mean of the means of all dilutions at week 0 for each sample plus 3 S.D. C: Avidity of anti-o-gp140ΔV2 antibodies. Preserum and serum from week 42 were analysed for avidity.

FIG 3

T-cell ELISpot responses elicited over time. Shown are IFN-γ (upper panels), IL-2 (middle panels), and IL-4 (lower panels) ELISpots from individual animals immunized with Ad5hr vectors followed by recombinant glycoprotein (group 1) as a booster, VEE/SIN replicons (group 2) or MF59 only or empty VEE/SIN replicons (group 3). Background responses (mean numbers of SFC plus twice the standard deviations of triplicate assays with medium alone) were substracted. Responses after stimulation with either pp of 89.6p gp120 or SF162 gp120) are presented as the number of SFC per 10⁶ PBMC. Arrows indicate immunizations, arrow head indicate the challenge. Bold lines indicate protected animals. The Y-ax scale for IFN-γ differs from IL-2 and IL-4 Y-axes.

FIG. 4

Plasma viremia following IR challenge with SHIV_SF162p4 of all animals from the recombinant glycoprotein boost group (upper panel), the VEE/SIN boost group (middle panel) and the control group (lower panel).

FIG. 5

Reduction in virus load under the curve up to week 16 post challenge and correlation with neutralizing antibodies. A: Area under the curve (of FIG. 4) up to week 16 post challenge. The horizontal lines indicate the median level of area under the curve of each group. Differences in area under the curve (log-transformed values) were analyzed and compared using the Kruskal-Wallis test. (H=8.312; p=0.0157). B: Correlation between the neutralizing antibody titers at the day of challenge and the reduction in area under the curves (of FIG. 4).