Identifying the immune responses that mediate protection by live-attenuated SIV and understanding their induction may inspire strategies for engineering a safe and effective vaccine against HIV-1. We hypothesized that antibody functions other than neutralization contribute to the protective immunity provided by live-attenuated SIV against wild-type pathogenic SIV challenge. Here we demonstrate that properties of the antibody response reflected in ADCC titers mirror hallmarks of protection by live-attenuated SIV. The protective immunity conferred by live-attenuated SIV increases over time 
, is usually incomplete against heterologous challenge 
, and is greater for vaccine strains that replicate at higher levels 
. In accordance with these observations, our data indicate that ADCC titers increase progressively over time, are lower against viruses expressing heterologous Env proteins, and are proportional to the extent of vaccine strain replication. Furthermore, in two different challenge experiments, measures of ADCC activity were associated with protection against infection by SIVmac
In one experiment, macaques inoculated with SIVmac239Δnef or SIVmac239Δnef/E543-3env that remained uninfected after intravenous challenge with SIVmac251NE had higher AUC values for ADCC than those that became infected. In another experiment, animals that remained uninfected after high-dose vaginal challenge with SIVmac251UCD at different time points after inoculation with SIVmac239Δnef had higher 50% ADCC titers than those that became infected. Differences in AUC values for ADCC were significant in one experiment, whereas differences in 50% ADCC titers were significant in the other, perhaps reflecting the limited power to detect differences using small numbers of infected versus uninfected animals. Additional differences between the two studies, including the greater resistance of SIVmac251UCD than SIVmac251NE to antibodies, the greater length of time allowed for the maturation of antibody responses prior to challenge with SIVmac251NE (46 weeks) than SIVmac251UCD (5, 20, and 40 weeks), and the effect of mismatches in Env between SIVmac239Δnef/E543-3env and SIVmac251NE, may also have contributed to the detection of differences in AUC values for ADCC against SIVmac251NE. Although differences between the two SIVmac251 challenge experiments may have favored one method of data analysis over the other, measures of ADCC activity were associated with complete protection in both experiments.
While the relationship between ADCC activity and the outcome of challenge suggests that these antibodies contribute to protection, correlation does not establish causation. In addition to ADCC, Fc receptor crosslinking stimulates the secretion of molecules that promote lymphocyte homing and activation, and that may inhibit virus replication 
. The antibodies that direct ADCC may also mediate effector functions through complement fixation 
. Furthermore, ADCC assays may measure antibodies that block virus infection at concentrations present in vivo
, but are undetectable using conventional neutralization assays. Therefore, although ADCC may be an important effector mechanism 
, ADCC could also be a surrogate for other effector mechanisms that contribute to protection. Mechanisms of immunity not mediated by antibodies may also covary with ADCC activity. For instance, T cell, antibody, and innate immune responses may all be affected by the extent of antigenic stimulation. It is conceivable that the observed relationships are due to differences that exist among animals inoculated with SIVmac
, or among animals challenged five, twenty, and forty weeks after inoculation with SIVmac
. Thus, while our findings implicate antibodies in protection by live-attenuated SIV, they do not preclude a role for other immune responses.
More than one type of immune response elicited by live-attenuated SIV may be necessary for complete protection against SIVmac
251 challenge. Passive transfer experiments in different live-attenuated SIV vaccine models have yielded mixed results regarding the ability of antibodies alone to protect against SIV infection, demonstrating complete protection in one study 
, and no protection in another 
. In contrast to the absence of detectable neutralizing antibodies in the completely protected animals in this study, relatively high concentrations of neutralizing monoclonal antibodies were necessary to protect macaques against SHIV infection in passive transfer experiments 
. T cell responses present in macaques inoculated with SIVΔnef
, but absent in macaques that received antibodies passively, may help to explain these differences in neutralizing antibody titers required for complete protection.
Our observations are in agreement with other reports that have associated antibody responses with protection. The Robert-Guroff laboratory, and others, have associated lower viral loads after infection with higher ADCC activity measured using target cells that were coated with monomeric gp120 
, recombinant gp140 
, or infected with T cell line-adapted SIV 
. However, in these studies, ADCC was not associated with protection from infection, or measured using target cells infected with neutralization-resistant viruses. Nevertheless, antibodies that bound recombinant forms of gp120 by ELISA and that neutralized neutralization-sensitive SIV strains were associated with a reduced rate of infection 
. Consistent with these observations on vaccine protection, a recent study on mother-to-child transmission of HIV-1 found that the breast milk of mothers whose newborns remained uninfected contained antibodies with higher ADCC activity against gp120-coated target cells 
. In the context of vaccination with different live-attenuated strains of SIV, antibody avidity was also associated with resistance to infection and lower post-challenge viral loads after vaginal challenge with SIVmac
. Likewise, neutralization of SIVmac
at a 1
4 dilution of serum was associated with protection in a combined group of animals that remained uninfected or strongly controlled SIVmac
viral loads 
. Taken together, these studies support a role for antibodies in protective immunity.
Interest in antibody functions other than neutralization has recently increased as a result of the RV144 trial, in which a modest reduction in the rate of HIV-1 infection was reported among recipients of a recombinant canarypox vector prime and gp120 protein boost vaccine 
. Virus-specific CD8+
T cell responses were not measurably different between vaccinated and unvaccinated trial participants. Whereas antibodies capable of neutralizing primary HIV-1 isolates were also undetectable among vaccinated individuals, gp120-binding titers were consistently detectable by ELISA. Functions of antibodies other than neutralization have therefore been postulated to potentially be responsible for protection in the RV144 trial 
. Among six primary variables in the immune correlates analysis of the RV144 trial, IgG titers to the V2 region of gp120 were associated with protection, whereas Env-specific IgA antibodies were associated with a higher risk of infection 
. There was also a non-significant trend towards a lower risk of HIV-1 infection among vaccine recipients with higher ADCC activity using the assay described here. This relationship reached borderline statistical significance after excluding subjects with Env-specific IgA in plasma 
. These observations further support a role for antibodies in vaccine protection against immunodeficiency virus infection.
Persistent expression of Env may be essential to elicit protective antibody responses. The progressive increases in ADCC activity over time, and the considerably higher ADCC activity elicited by SIVmac
versus single-cycle SIV, imply that the persistent antigenic stimulation provided by ongoing SIVmac
replication is important for the development of high ADCC titers. Differences in the maturation of antibody responses may also contribute to the better protection provided by SIVΔnef
in comparison to single-cycle SIV 
. Furthermore, a longer period of persistent infection with SIVΔnef
was required for ADCC titers against SIV strains expressing heterologous Env proteins to reach the levels observed at an earlier time point against the Env-matched strain. Persistent Env expression may therefore be required to elicit antibodies with high and broadly reactive ADCC activity against circulating HIV-1 strains with diverse neutralization-resistant Env proteins.
A vaccine against HIV-1 must contend with a degree of sequence variation that typically renders neutralizing sera ineffective against heterologous HIV-1 strains isolated from other people 
. The Env proteins of SIVmac
239 and SIVsm
E543-3 differ in amino acid sequence by 18%, which approximates the median difference between the Env proteins of individual HIV-1 isolates within a clade 
. Therefore, the ADCC titers observed against cells infected with neutralization-resistant Env-mismatched target viruses suggest that antibodies may have broader efficacy against heterologous HIV-1 isolates than is generally revealed by neutralization assays.
In summary, we show that properties of the antibody response elicited by SIVΔnef mirror hallmarks of protection by live-attenuated SIV, and that ADCC activity is associated with apparent sterilizing protection against SIVmac251. These observations support a role for antibodies in protection by live-attenuated SIV, despite the paradoxical absence of detectable neutralizing antibody titers against the challenge virus in most fully protected animals. The temporal analyses of ADCC activity against both Env-matched and Env-mismatched viruses, and the significantly higher ADCC titers observed in SIVΔnef-infected animals than in animals repeatedly immunized with single-cycle SIV, suggest that persistent Env expression may be necessary to drive the maturation of high-titer, broadly reactive antibody responses. Therefore, strategies designed to persistently stimulate Env-specific antibodies may significantly improve the efficacy of vaccines against HIV-1.