In 1998 and 1999, two Phase III human clinical trials were conducted by VaxGen (VaxGen Inc, South San Francisco, CA) to test the efficacy of the AIDSVAX™ HIV vaccine. The AIDSVAX vaccine consisted of a bivalent immunogen derived from the recombinant envelope glycoprotein gp120 of HIV-1 subtypes B and E (strains MN and A244) in VAX003, and recombinant gp120 molecules from subtype B (strains MN and GNE8) in VAX004 
. The choice of the HIV-1 strains was made based on phylogenetic analysis to cover the majority of HIV strains present in the regions where the clinical trials were conducted 
. In the VAX003 and VAX004 randomized double-blind, placebo-controlled Phase III clinical trials, a total of 7963 volunteers were enrolled, and 611 of them were infected with HIV-1 during the study 
. The human subjects in the VAX003 and VAX004 trials thus represent existing, well-characterized placebo-controlled vaccination research cohorts.
AIDSVAX vaccination failed to broadly protect the overall study population (6.7% infection rate in the vaccinees, compared to 7.0% in the placebo group, p>0.1), although certain subgroups (non-Hispanic ethnic minorities, i.e. Asians and Africans) may have experienced protection 
. Protection, if it occurred, may have correlated with higher titers of antibodies in those groups to the matched viral strains, MN, GNE8, or A244, but the gp120 amino acid sequences of the infecting viruses were substantially different from those in the immunogens 
, making the detection of a protective effect by traditional means extremely difficult.
The third variable loop (V3 loop) of the HIV-1 surface envelope glycoprotein (gp120) is an immunogenic region of the viral envelope 
. The V3 loop is known to contain epitopes that induce both broadly and narrowly cross-reactive neutralizing antibodies 
. After the VAX004 clinical trial, only one linear, one-dimensional (1D), sequence-defined V3 loop region was evaluated as a putative antibody-targeted viral epitope – the GPGRAF motif, presented in the V3 loop of the gp120's from MN and GNE8 strains. The presence of this sequence motif in infecting viruses did not affect the degree of correlation between antibody levels or HIV-1 incidence 
. No crystal structure exists of a monoclonal antibody specific for the entire GPGRAF sequence, and a linear peptide like GPGRAF present in an immunogen may give rise to many nAbs with diverse binding modes overlapping within the GPGRAF peptide. Furthermore, the activities of nAbs that engage epitopes not completely included in the GPGRAF fragment but encompassing amino acid atoms in nearby areas of the V3 loop crown were missed. Thus, the published study using the GPGRAF fragment 
was not truly epitope-specific and could have detected only a fraction of the currently known V3 epitopes defined by 3D structures of V3-Ab crystallographic complexes.
HIV vaccine-induced immune responses that are undetectable by laboratory tests may be detected via “sieve” effects, wherein HIV acquisition is partially blocked (only certain viruses matched to the immune response are blocked). Prior attempts at HIV vaccine trial sieve analysis have only focused on linear T-cell epitopes, as defining conformational epitopes in linear DNA or amino acid sequences presents a significant technical challenge to traditional sieve analysis 
. We attempted to meet this challenge with a sieve analysis of the true conformational epitope-specific human nAb response to immunization with the AIDSVAX vaccines using a novel computational structural biology method for the detection of three-dimensionally (3D) -defined conformational epitopes in gp120. The three-dimensional conformational epitopes are projected into one-dimensional sequence space via sensitive and specific “signature motifs” defined using a panel of seven anti-V3 monoclonal antibodies' crystal structures 
. Briefly, in this method, a conformational 3D-epitope is defined by a number of sequentially-disparate but 3D space-clustered amino acids positions (a sequence motif representing the signature of the 3D conformational epitope: “signature motif”). The amino acids are restricted to those for which their side chains are engaged in buried atomic contacts within the 3D molecular surface of a monoclonal nAb in the crystallographic complex of the nAb with its cognate bound viral peptide 
. The resolving of the 3D conformational epitope shape bound by the specific monoclonal nAb into a simple sequence motif affords rapid and accurate classification of any library of gp120 viral sequences into those that contain the specific conformational 3D epitope and those that do not.
In the present study, we report application of this methodology in a novel form of sieve analysis 
, analyzing the library of sequences of the viruses infecting vaccine and placebo recipients in the VAX003 and VAX004 clinical trials (published by Global Solutions for Infectious Diseases (GSID)). Specifically, we classified the viruses infecting placebo and vaccinated patients into those that contain certain conformational epitopes and those that do not using the epitope motifs targeted by seven anti-V3-loop neutralizing monoclonal antibodies 
. The exploratory metric used was that one or more of the epitopes would show a lower rate of occurrence in viruses infecting vaccinated subjects as compared to viruses infecting placebo subjects, potentially indicating that the vaccine imposed immune selection pressure that reduced the occurrence of infection of the vaccinated subjects with viruses bearing those epitopes.