This activity was compared with the breadth of a set of 10 human mAbs selected because they are considered broadly neutralizing and represent the known major neutralization epitopes on tier 2 viruses (Fig. 2d). We included the CD4bs bnAbs VRC01 [19,20], CH01 [16,21] and b12 , the glycan-dependent V1V2 bnAbs PG9 [22–24] and PGT145 , the V3/C3 glycan-dependent bnAb PGT128 , the glycan-specific bnAb 2G12  and the MPER bnAbs 2F5, 4E10  and 10E8 . This includes the older set of bnAbs that have lower potency and breadth but are still considered broadly neutralizing (b12, 2G12, 2F5, 4E10), as well as some of the more potent recently isolated bnAbs (VRC01, PGT128, 10E8). The bnAbs were tested against a panel of 119 tier 2 viruses, 103 of which were also in the panel of 219 used to evaluate the sera (Fig. 1b). At the highest concentration tested (50 μg/ml), 4E10, 10E8, VRC01, PG9 and PGT145 demonstrated the greatest breadth (75.7–97.3% of viruses), corresponding to 2.6–19.7% of sera using 50% neutralization cut-off (Fig. 2d–f, Table 1). Other bnAbs (e.g. PGT128, 2F5, CH01, b12, 2G12) neutralized 20.2–59.1% of the virus panel and this level of breadth was seen among 33.3–83.8% of the sera. Indeed, 77% of the sera exhibited greater breadth than the prototypic CD4bs bnAb, b12. The sera also exhibited substantial breadth when compared with several bnAbs using more stringent 80% neutralization values, especially when compared with more moderate concentrations of 1–5 μg/ml of the bnAbs (Table 1).
Notably, our results with b12 and 2G12 suggest that these two bnAbs may not be as broadly neutralizing as previously thought. We found that b12 (50 μg/ml) neutralized only 29.4% of all 119 viruses tested, including 57.1 and 33% of subtype B and C viruses, respectively. A previous large study estimated these values to be 50% (all), 72% (subtype B) and 67% (subtype C) . We also found that 2G12 (50 μg/ml) neutralized 20.2% of the 119 viruses tested, including 57.1 and 7.7% of subtype B and C viruses, respectively, whereas previous estimates were 41, 72 and 0%. These differences may reflect greater genetic diversity among the viruses in our panel than in previous panels.
Although it was not possible to directly compare the inhibitory concentration of monoclonal bnAbs with the inhibitory dilution of sera, we note that many serum neutralization titres were moderate in magnitude, often near or below the threshold shown to prevent infection after experimental challenge with R5, tier 2 simian HIV (SHIV) in nonhuman primates [27,28]. Nonetheless, our results demonstrate the potential to elicit measurable cross-neutralizing activity in most people at levels that exceed the best responses seen in HIV-1 vaccine efficacy trials [29,30]. Although it is not known what titres will be needed to protect against natural HIV-1 transmission, relatively low serum neutralization titres in the range of 1 : 20 to 1 : 50, which were often exceeded here, are protective against other viruses  and have been shown to impede HIV-1 replication and drive neutralization escape in humans , suggesting that they may have adequate potency for vaccines that aim to prevent HIV-1 infection.
We have not determined whether the serum neutralization breadth seen here is monoclonal or polyclonal in nature. In most cases examined, broad serum neutralization is explained by the presence of one or very few antibody specificities, each of which exhibits substantial breadth against tier 2 viruses [16,19,22,23,25,33–35]. Strain-specific neutralizing antibodies to other epitopes are also present in these sera; however, polyvalent mixtures of such strain-specific antibodies exhibit little breadth . Thus, detection of even moderate breadth against tier 2 viruses usually indicates the presence of antibodies to one or more of a small number of conserved vulnerable regions on the virus.
We note that moderate cross-neutralizing activity generally is not observed prior to 3 years of HIV-1 infection . Because we do not know the length of time of infection in our study participants, some individuals might have been infected for less than 3 years, which could have resulted in an underestimation of neutralization breadth. Also, serum neutralization potency and breadth can fluctuate over time during chronic infection. In a study of 155 chronically infected individuals sampled at two time points and tested against a panel of 12 tier-2 viruses, 30% had geometric mean titres that varied more than two-fold, and 6% had geometric mean titres that varied between three and six-fold (unpublished data). This dynamic nature of the bnAb response suggests that cross-sectional studies based on single samples may underestimate the potential of infected individuals to make bnAbs at some point during infection.
Overall, these observations provide a more accurate picture of the spectrum of neutralizing antibody responses that are possible in HIV-1 infected individuals. Sera with greatest breadth of neutralization are certainly of particular interest for detailed study. Still, most people are capable of making antibodies with more moderate breadth of coverage that would be deemed of value if elicited in a vaccine context. Studies of the relatively common HIV-1 infected individuals who have moderate breadth of neutralization could be valuable for understanding how to elicit similar responses via vaccination. An example of particular interest is the recent description of a clonal lineage of CD4bs-specific bnAbs that exhibits less breadth of neutralization but is also less mutated than other CD4bs bnAbs, and that may serve as a template to identify suitable immunogens that will re-elicit this lineage and generate neutralizing antibodies with similar breadth .
We thank Francine McCutchan, Carolyn Williamson, Beatrice Hahn, Ronald Swanstrom, Feng Gao, Jerome Kim and Miguel Thompson for molecular Env clones. We also thank Lynn Morris, Guy de Bruyn, Ramesh Paranjape, Pachamuthu Balakrishnan, Yiming Shao, Kunxue Hong, Hao Wu, Ning Li, Linqi Zhang, Hong Shang, Aine McKnight, Ruengpung Sutthent, Esper Kallas, Center For HIV/AIDS Vaccine Immunology, Centre for the AIDS Programme of Research in South Africa, International AIDS Vaccine Initiative, HIV Vaccine Trials Network, HIV Prevention Trials Network, Southern African National Blood Services, US Military HIV Research Program, Zambia-Emory HIV Research Project and the Bill and Melinda Gates Foundation's Collaboration for AIDS Vaccine Discovery for serum specimens from HIV-1 infected individuals. We thank Dennis Burton for b12, PG9, PGT128 and PGT145, Mark Connors for 10E8, Herman Katinger for 2G12, 2F5 and 4E10, and Barton Haynes for CH01 and longitudinal neutralization data from 155 chronic HIV-1 infected individuals. Finally, we thank Kelli Greene and Hongmei Gao for coordinating the acquisition of reagents and the activities involved in performing assays and quality assurance of the final data.
D.C.M. designed and led the CAVD Neutralization Serotype Discovery Project (NSDP) that provided the data for analysis. B.T.K. and P.H. designed and conducted the data analyses. All three wrote the initial draft of the manuscript. M.S.S., R.T.B. and J.R.M. supervised the majority of NSDP neutralization assays and assisted with the interpretation of results and manuscript writing.
This work was funded by a grant from the Bill & Melinda Gates Foundation (Collaboration for AIDS Vaccine Discovery) and by the Intramural Research Program of the Vaccine Research Center, NIAID, NIH.
There are no conflicts of interest.
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