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. 2012;8(8):e1002890.
doi: 10.1371/journal.ppat.1002890. Epub 2012 Aug 23.

ADCC develops over time during persistent infection with live-attenuated SIV and is associated with complete protection against SIV(mac)251 challenge

Michael D Alpert  1 , Jackson D Harvey, W Anderson Lauer, R Keith Reeves, Michael Piatak Jr, Angela Carville, Keith G Mansfield, Jeffrey D Lifson, Wenjun Li, Ronald C Desrosiers, R Paul Johnson, David T Evans
Affiliations

ADCC develops over time during persistent infection with live-attenuated SIV and is associated with complete protection against SIV(mac)251 challenge

Michael D Alpert et al. PLoS Pathog. 2012.

Abstract

Live-attenuated strains of simian immunodeficiency virus (SIV) routinely confer apparent sterilizing immunity against pathogenic SIV challenge in rhesus macaques. Understanding the mechanisms of protection by live-attenuated SIV may provide important insights into the immune responses needed for protection against HIV-1. Here we investigated the development of antibodies that are functional against neutralization-resistant SIV challenge strains, and tested the hypothesis that these antibodies are associated with protection. In the absence of detectable neutralizing antibodies, Env-specific antibody-dependent cell-mediated cytotoxicity (ADCC) emerged by three weeks after inoculation with SIVΔnef, increased progressively over time, and was proportional to SIVΔnef replication. Persistent infection with SIVΔnef elicited significantly higher ADCC titers than immunization with a non-persistent SIV strain that is limited to a single cycle of infection. ADCC titers were higher against viruses matched to the vaccine strain in Env, but were measurable against viruses expressing heterologous Env proteins. In two separate experiments, which took advantage of either the strain-specificity or the time-dependent maturation of immunity to overcome complete protection against SIV(mac)251 challenge, measures of ADCC activity were higher among the SIVΔnef-inoculated macaques that remained uninfected than among those that became infected. These observations show that features of the antibody response elicited by SIVΔnef are consistent with hallmarks of protection by live-attenuated SIV, and reveal an association between Env-specific antibodies that direct ADCC and apparent sterilizing protection by SIVΔnef.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Development of neutralizing antibody and ADCC titers in macaques inoculated with SIVmac239Δnef.
Plasma collected from 10 animals at 0, 3, 5, 7, 13 or 15, and 21 weeks after inoculation with SIVmac239Δnef was evaluated for its capacity to neutralize SIVmac239 (A) and to direct ADCC against SIVmac239-infected cells (B). The loss of relative light units (RLU) indicates the loss of virus-infected cells during an 8-hour incubation in the presence of plasma and an NK cell line. Target cells infected by SHIVSF162P3 served as a negative control for all ADCC assays (gray). Dashed lines indicate 50% activity. Neutralizing antibody titers were compared with 50% ADCC titers (C), and with AUC values for ADCC (D). An odds ratio (OR) for the probability of detecting neutralization per log10 increase in 50% ADCC titer, or per 1 AUC unit increase in ADCC activity, was estimated by logistic regression.
Figure 2
Figure 2. ADCC titers elicited by SIVmac239Δnef versus single-cycle SIV.
Plasma samples collected on weeks 2 and 12 after a series of inoculations with single-cycle SIV were titered for ADCC against SIVmac239-infected cells (A). Target cells infected with SHIVSF162P3 served as a negative control (gray). Dashed lines indicate 50% activity. Geometric mean vaccine strain viral loads reflecting virus particles produced in vivo after inoculation with SIVmac239Δnef or single-cycle SIV are shown (B). Animals in Group A were inoculated 3 times with single-cycle SIV that was trans-complemented with the vesicular stomatitis virus glycoprotein (VSV G), whereas the animals in Group B were inoculated 6 times with single-cycle SIV that was not trans-complemented . The 50% ADCC titers (C) and the AUC values for ADCC (D) elicited by SIVmac239Δnef were significantly higher than those elicited by single-cycle SIV (2-tailed Mann-Whitney U tests, P = 0.0062 to P<0.0001). Binding titers measured by ELISA against SIVmac239 gp120 were correlated with 50% ADCC titers (E), and with AUC values for ADCC (F). Binding titers against SIVmac239 gp140 were also correlated with 50% ADCC titers (G), and with AUC values for ADCC (H).
Figure 3
Figure 3. ADCC against viruses matched or mismatched to the vaccine strain in Env.
Sera drawn 0, 6, or 22 weeks after inoculation with SIVmac239Δnef (A) or with the recombinant vaccine strain SIVmac239Δnef/E543-3env (B) were tested for ADCC against target cells infected with SIVmac239 (black), SIVmac239/E543-3env (green), or SHIVSF162P3 (gray). Dashed lines indicate 50% activity.
Figure 4
Figure 4. Relationship between the extent of vaccine strain replication and ADCC activity.
The extent of SIVmac239Δnef or SIVmac239Δnef/E543-3env replication was estimated from the area under the curve (AUC) of log10-transformed vaccine strain viral loads over weeks 0 through 21 or 22, and compared to ADCC activity at week 21 or 22. Vaccine strain viral load AUC values were correlated with 50% ADCC titers (A) against Env-matched (RS = 0.68, P<0.0001) and Env-mismatched (RS = 0.55, P = 0.006) SIV strains, and also with AUC values for ADCC activity (B) against Env-matched (RS = 0.64, P<0.0001) and Env-mismatched (RS = 0.42, P = 0.0421) SIV strains. Linear regression lines are drawn.
Figure 5
Figure 5. Neutralization and ADCC on the day of intravenous challenge with SIVmac251NE.
Macaques were challenged with an intravenous dose of SIVmac251NE on week 46 after inoculation with SIVmac239Δnef or SIVmac239Δnef/E543-3env. Sera collected the day of challenge were evaluated for neutralization of SIVmac251NE (A) and ADCC against SIVmac251NE-infected cells (B). Solid black symbols indicate animals that became infected by SIVmac251NE. Dashed lines indicate 50% activity. Target cells infected with SHIVSF162P3 served as a negative control for ADCC assays (gray). Differences in 50% ADCC titers were not significant (C). However, AUC values for ADCC were higher among the immunized animals that remained uninfected versus the immunized animals that became infected (2-tailed Mann-Whitney U test, P = 0.0091) (D). None of these macaques had the MHC class I alleles Mamu-A*01, -B*08 or -B*17 associated with reduced viral replication –.
Figure 6
Figure 6. Neutralization and ADCC on the day of high-dose vaginal challenge with SIVmac251UCD.
At 5, 20, or 40 weeks after inoculation with SIVmac239Δnef, groups of 6 macaques each were challenged by high-dose vaginal inoculation with SIVmac251UCD. Serum collected the day of challenge was evaluated for neutralization of SIVmac251UCD (A–C) and ADCC against SIVmac251UCD-infected cells (D–F). Symbols appear in color for immunized macaques that remained uninfected by SIVmac251UCD, and in black for the immunized animals that became infected. Target cells infected with SHIVSF162P3 served as a negative control for ADCC assays (gray). Dashed lines indicate 50% activity. The groups challenged on weeks 5, 20, and 40 were combined for statistical analysis (G–J). The SIVmac239Δnef-immunized macaques remaining uninfected by SIVmac251UCD had higher 50% ADCC titers than those that became infected (2-tailed Mann-Whitney U test, P = 0.0487) (G). A similar but non-significant trend was observed in AUC values for ADCC (2-tailed Mann-Whitney U test, P = 0.0761) (H). Non-significant trends were in the direction of lower peak SIVmac251UCD viral loads for animals with higher 50% ADCC titers (RS = −0.4615, P = 0.1124) (I) and higher AUC measurements of ADCC activity (RS = −0.4560, P = 0.1173) (J). Linear regression lines are shown. The macaque with the lowest ADCC titers among those remaining uninfected was the only animal possessing the protective combination of MHC class I alleles Mamu-A*01 and -B*17 .
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