This study represents the largest screening and evaluation of neutralization patterns to date in individuals infected with non-clade B HIV-1. The creation of an analytical selection algorithm and reduced virus screening panel to assess serum neutralization activity against multiple HIV-1 isolates representing several clade groups have enabled us to rapidly identify and prioritize HIV-1+
individuals who have a combination of both potency and breadth in neutralization. Our results confirm and extend observations that broad neutralizing serum activity is not uncommon in chronically HIV-1-infected individuals, with 34% of volunteers from the first 463 screened possessing broad neutralizing activity, defined as an IC50
titer of ≥100 to at least one pseudovirus from four clade groups (11
). One percent of all volunteers from approximately 1,800 screened exhibited elite activity, defined as the ability to neutralize on average more than one pseudovirus at an IC50
titer of ≥300 within a clade group and across at least four clade groups. For all elite neutralizers the average geometric mean IC50
titer was 500 (Table ). While it is not precisely known what level of Abs are required for protection against HIV-1 infection, recent work examining the efficacy of low b12 antibody titers against low-dose repeated pathogenic simian-human immunodeficiency virus challenge in macaques indicates that high concentrations of antibodies may not be needed to provide protective benefit (19
). The individuals identified as elite neutralizers in this study represent a new resource for the HIV vaccine community for the identification of novel monoclonal antibodies that are both broad and potent against HIV-1 and offers new hope for the creation of immunogens capable of eliciting bnMAbs at titers which could be effective in protection against HIV-1. By extension, these findings and the methods employed may also be useful in rational vaccine design for the identification of novel broad and potent neutralizing antibodies for other viral diseases, such as hepatitis C, for which hypervariability within the envelope is a major issue in vaccine design (13
). In addition, our results suggest that neutralizing activity across multiple geographic regions, with different spectra of circulating HIV-1, can be reliably assessed using a small panel of pseudoviruses. Such a panel can be easily incorporated into a high-throughput selection protocol for screening additional human sera and potentially for immunogen screening. Previous recommendations for the design and use of standard neutralization screening panels suggested the use of a multitier approach to screen potential vaccine immunogens (30
). The recommended multitier approach included the incorporation of vaccine strains and neutralization-sensitive viruses (tier 1), heterologous viruses matching the genetic subtype(s) of the vaccine (tier 2), and a multiclade panel comprised of six tier 2 viruses of each genetic subtype, excluding the genetic subtype(s) evaluated in tier 2 (tier 3). In addition, it was suggested that only viruses representing recently transmitted isolates or viruses collected within the first year of infection should be evaluated in this multitier approach to account for viral and antigenic drift (30
We found that the inclusion of NL4-3, a tier 1 virus representing a laboratory-adapted strain of HIV-1, provided no benefit in helping to predict the overall sera potency to other pseudoviruses representing primary HIV-1 isolates. While NL4-3 is a representative tier 1 neutralization-sensitive virus, by extension our findings suggest that tier 1 viruses may provide little to no information regarding neutralization activity to primary isolates and should only be used as an assay control. Our results also indicate that there is no difference in neutralization patterns between viruses isolated during chronic or acute infection or viruses that represent recently transmitted HIV-1 isolates. Individuals whose sera possessed both broad and potent activity, as determined by score 1, neutralized both chronic and acute viruses and more recently transmitted isolates at similar frequencies and potencies (Fig. ). These findings indicate that although significant HIV-1 envelope variation occurs within an individual over the course of the infection, broadly neutralizing antibodies that can recognize divergent epitopes both within an individual and across HIV-1 epidemics are generated. Therefore, the selection of pseudoviruses for incorporation in standardized neutralization screening panels probably need not take into account collection year and stage of HIV-1 infection but should focus more on the overall resistance profile of the pseudovirus as measured in serum and against the current identified monoclonal antibodies, b12, 4E10, 2F5, and 2G12.
In addition, our findings suggest that screening panels which primarily include heterologous viruses within a clade group and not across clade groups (tier 2) may overestimate the overall breadth and potency of a potential immunogen across multiple HIV-1 epidemics. We observed that volunteers' sera tended to preferentially neutralize at higher potency pseudoviruses that were closer in phylogenetic origin to the infecting strain and that having a response to viruses that represented the presumed infecting clade group only correlated with a response to other clade groups 66% of the time. It should be noted that this preferential neutralization does not suggest clade-specific serotypes, as many of the volunteers were also found to have a high prevalence of broadly neutralizing antibodies to pseudoviruses from other clades, although at low titers (Table ). Therefore, a panel that only includes heterologous viruses matching the genetic subtype(s) of either the vaccine candidate or the virus of the HIV-1-infected individual may only be useful in measuring change in breadth within a clade group and not across clade groups. This may be of benefit if one is trying to identify immunogens that possess neutralization characteristics that are more clade restricted, such as 2G12 and 2F5, but not immunogens that elicit antibodies that are more cross-clade, such as b12 and 4E10. Collectively, these findings suggest that the best method to rapidly identify bnMAbs that are both broad and potent against primary HIV-1 isolates from diverse regions would be by using a panel of heterologous isolates of a moderately resistant profile crossing the spectrum of clades. Although our current reduced panel meets this criterion, it should be noted that all of the pseudoviruses identified in the regression analysis were by chance somewhat b12 sensitive. We are currently examining all individuals identified as having either broad or elite neutralizing activity on the previously identified panel 3, including b12-resistant pseudoviruses, to ensure that sample selection was not skewed toward donors with a greater contribution of “b12-like” (CD4b) antibodies to neutralization.
One limitation of the current study is the lack of understanding of the specificities accounting for the broadly neutralizing activity seen within both our broad and elite neutralizers and also the host factors responsible for generating and maintaining such a response. Recent work done by Connors, Binley, and Stamatatos et al. found that although CD4-binding site antibodies are found in individuals with broadly neutralizing antibodies, cross-neutralization activity and epitope specificity in many of their volunteers could not be mapped to currently known neutralization epitopes. In addition, time since infection and the presence of low to moderate viremia have been suggested as strong clinical predictors for the development of broadly neutralizing antibodies(1
). Examination of each of these findings in order to identify possible new mechanisms and epitopes that are responsible for the production of broadly neutralizing activity primarily in elite neutralizers is currently under way with our study population.
In summary, this study represents the establishment of a high-throughput screening method for the identification of elite neutralizers and is the first study to date to identify elite neutralizers representative of diverse HIV-1 clade regions. Identification of the specificities responsible for the broad and potent neutralization activity seen in these elite neutralizers may provide new insight and guidance for rational HIV-1 vaccine design.