Loop-directed antibodies PG9 and PG16 have similar breadth but PG16 is more potent
PG9 and PG16, the first of the ‘second-generation’ monoclonal antibodies described, are somatic variants that target distinct but partially overlapping quaternary epitopes on gp120 that include the V1, V2, and V3 loops [30,36,37]. As expected, we observed a high degree of concordance between these two bNAbs, with only 2.2% (5/227) of isolates resistant to one but sensitive to the other. When mothers and their infected infants were analyzed separately, we found PG9 neutralized all viruses from 72% (33/46) of patients (i.e. 72% coverage), whereas PG16 had 67% (31/46) coverage of our cohort (Table 2). When results were pooled by transmission-pair (mother and infant considered together) we observed 100% concordance with 43% (10/23) of pairs harboring resistance to both antibodies and the remaining 57% (13/23) of pairs harboring virus fully sensitive to both (Figs 2a,b and 3a, b). PG16 was approximately 4.4× more potent than PG9 by IC50 ratio (data not shown).
Potent CD4 binding-site antibodies VRC01, NIH45–46, and 3BNC117 display similar breadth and potency
VRC01, VRC03, and NIH45–46 (4546) were isolated from the same patient using different sets of PCR primers, and target the CD4 binding site (CD4bs) on gp120 [29,31]. 8ANC195 and 3BNC117, also targeting the CD4bs, were isolated from two additional patients . VRC03 and 8ANC195, both of which have been described as having limited breadth and potency relative to the other CD4bs bNAbs [29,31], also had minimal activity in our cohort and were excluded from further analysis (data not shown).
In the per-patient analysis, VRC01 had complete coverage (neutralizing all envelopes from a given patient) of 65% (30/46) of patients, 4546 had 50% (23/46) coverage, and 3BNC117 had 57% (26/46) coverage (Table 2). VRC01, 4546, and 3BNC117 also had similar breadth in the more conservative per-pair analysis, with overall coverage of 48% (VRC01), 35% (4546), and 39% (3BNC117) (Figs 2c–e and 3a,b). There was also significant overlap, with most transmission-pairs that harbored virus resistant to one CD4bs bNAb also harboring variants resistant to the other two, though not in every case. On a per-clone basis, all three bNAbs were similarly potent, with median IC50 ratios of 0.85–1.00 (data not shown).
Engineered bNAb NIH45–46G54W has significantly improved breadth and potency relative to other CD4bs antibodies
NIH45–46G54W (4546W) is the first example of structure-based engineering of a neutralizing antibody. It is a variant of NIH45–46 designed to improve breadth and potency by inserting a tryptophan into the ‘Phe43 pocket’, a critical site involved in gp120 binding to CD4. The design and binding properties of 4546W have been described . The glycine-to-tryptophan substitution increased coverage of this antibody relative to the parental variant, from 50% (23/46) to 78% (36/46) in the per-patient analysis (Table 2), and doubled it from 35% (8/23) to 70% (16/23) in the more conservative per-pair analysis (Figs 2f and 3a, b). In our cohort, 4546W was approximately 2.7-fold more potent (by IC50 ratio) than 4546, and approximately 3.7 and 6.1× more potent than 3BNC117 and VRC01, respectively. Importantly, no isolates previously sensitive to 4546 were found to be resistant to 4546W.
A combination of NIH45–46G54W and PG16 achieves the highest coverage overall and against early-transmitted isolates
We next sought to determine which combination of a loop-directed and CD4bs-directed bNAb would give the best overall coverage with the highest neutralization potency. PG9 and PG16 had equivalent coverage, but PG16 neutralized with substantially greater potency. Of the CD4bs bNAbs tested, 4546W had clear advantages in both breadth and potency in our conservative per-pair analysis. The combination of PG16 and 4546W had effective per-pair coverage of 87%, the highest of any two-antibody combination available. PG16 and VRC01 was the next most effective combination, with 78% overall coverage in the per-pair analysis (Fig. 3d).
Since we had data on a large number of early transmitted isolates, we examined the baseline coverage of these antibodies both alone and in combination to simulate a prophylactic intervention. Against all 68 early transmitted isolates (from 23 transmission pairs) we found antibody coverage was within a similar range (65–78%) for all bNAbs tested (Fig. 3c). The combination of PG16 with either VRC01 or 4546W offered the best coverage, with all variants from 96% of infants (22/23) susceptible to at least one bNAb. Both 4546 and 3BNC117 achieved 87% coverage in conjunction with PG16 (Fig. 3e). Considering the median IC50 ratios of just the early-transmitted isolates, 4546W was approximately 2.1× more potent than parental 4546 and approximately 5× more potent than VRC01.
Minor variants resistant to NIH45–46G54W are uncommon and associated with resistance to other CD4bs bNAbs
The ability to detect pre-existing minor variants that harbor resistance is a critical factor in the success or failure of treatment with existing antiretroviral drugs including entry inhibitors such as maraviroc [38–40]. Thus, whereas broad sampling at shallow depth gives a good estimate of the prevalence of dominant resistance, it cannot determine the frequency of minor variants that could, under selection, rapidly outgrow, and compromise the effectiveness of an inhibitor. Whereas we do not consider a median sampling depth of 10 clones per pair to be exhaustive, this cohort does give us the opportunity to probe for minority variants within quasispecies resistant to these new antibodies in a way that has not been previously described.
When we set a ‘low frequency’ cut-off of 20% or less, we identified only three pairs harboring minor variants resistant to 4546, 4546W, four with minor variants resistant to 3BNC117, and six pairs harboring a minority population resistant to VRC01. Importantly, the presence of a minor population resistant to 4546W was associated with low and/or high frequency resistance to at least two of the other CD4bs antibodies. In contrast, there were several cases where minor variants resistant to 4546, 3BNC117, or VRC01 were detected in the absence of resistance to multiple other CD4bs antibodies (Fig. 2c–f).
High intra-pair and intra-patient variation and its effect on the relationship between sampling depth and coverage
We observed a surprising degree of intra-pair variation in susceptibility with all tested antibodies, with a median fold difference of 14.7–38.1× between the most resistant and most sensitive clone within a pair (data not shown). In one example (Subj. 13) sampling of 10 unique clones identified a 265-fold difference in IC50 for PG9, without detecting outright resistance (range 0.0024–0.6361 μg/ml) (Fig. 2a).
Given the high degree of intra-patient variation, we next estimated the probability of detecting at least one resistant variant for each transmission pair over a range of sampling depths and constructed a plot showing estimated coverage with a sampling depth ranging from 1 to 10 clones per pair (Fig. 3f). Coverage estimates produced by sampling only a single clone per pair ranged from 62–87%, with PG9, PG16, and 4546W scoring above 80% and VRC01 scoring 79%. These coverage estimates are at least 20% higher than our actual results at a median of 10 clones per pair, which are still likely to be overestimates.
We found that loop-targeted antibodies PG9 and PG16 neutralized envelopes from our cohort of MTCT pairs with 57% coverage in our conservative per-patient analysis. Resistance to one PG antibody was highly correlated with resistance to the other, which is expected given that they are somatic variants targeting distinct but overlapping epitopes. PG16 neutralized viruses from this cohort more potently than PG9 by almost half a log10, suggesting it would be the better choice for initial clinical studies, at least for prevention of MTCT (pMTCT) in a population predominantly infected with subtype C.
Qualitatively, the battery of CD4-binding site antibodies fell into three categories: VRC03 and 8ANC195 performed poorly; VRC01, 3BNC117, and 4546 performed moderately well and neutralized with similar potencies; and 4546W had the broadest coverage and highest neutralization potency of any CD4bs antibody in our study. Additionally, minor variants resistant to 4546W were uncommon, and patients harboring 4546W-resistant envelopes typically harbored variants resistant to the other CD4bs antibodies as well. A caveat is that our median sampling depth of 10 clones per transmission pair, whereas more extensive than other studies, is not definitive, and likely underestimates the true prevalence of pre-existing resistance to these reagents. Our data argue in favor of using 4546W (or a further modified variant) for future clinical studies, since resistance to 4546W appears to be rare and associated with resistance to the CD4bs antibody class in general. Studies of additional CD4 binding site antibodies would be required to confirm this characteristic of 4546W. This observation is compatible with a more conserved mechanism of neutralization for 4546W (via the ‘Phe43 pocket’) and a higher genetic barrier to resistance, but our study did not directly address these issues.
Our data indicate that the combination of 4546W and PG16 is likely to be the most effective pair of antibodies for pMTCT, and perhaps preventive studies in general in subtype C-infected populations. This combination has the greatest breadth in both the conservative per-pair analysis and the restricted subset of early transmitted isolates (neutralizing all tested variants from 22/23 infected infants). Moreover, these antibodies had the highest neutralization potency (measured by IC50) of all the antibodies we tested from their respective classes. Several other CD4bs antibodies (VRC01, 3BNC117, and 4546) also had relatively broad coverage of the early transmitted isolates, but at 2–5× reduced potency compared to 4546W.
Women in our study were HIV-positive at the time of enrollment and seroconversion dates were not available. Their low CD4 cell counts suggest late-stage chronic infection, which is generally associated with greater genetic diversity, likely contributing to the frequent detection of pre-existing bNAb-resistant variants. Envelopes from the 23 epidemiologically linked infants exhibited the restricted genetic diversity typical of recent infection, and were better covered by the tested bNAbs. The generalizability of our findings to other forms of transmission is unclear and warrants further investigation.
The study represents, to our knowledge, the deepest sampling of HIV-infected persons in the context of second-generation bNAbs. Our data are qualitatively similar to a recent study  that sampled approximately 200 patients from multiple clades at a median depth of 1 clone per patient and found the combination of a loop-targeted (PG series) and CD4bs antibody (VRC01, VRC-PG04) had very good coverage of a diverse panel of global strains, though that study did not include the 4546W variant for comparison. It is important to emphasize here the heterogeneity of our data and the effect of sampling depth on coverage estimates that we observed. Antibody susceptibility within some patient-pairs differed by several orders of magnitude, which indicates substantial phenotypic variation exists even within patients described by our analyses as fully sensitive. At a sampling depth of 1 clone per pair, our results would be very similar to that study , with VRC01, PG9 and PG16 all achieving coverage in the approximately 75–85% range at an IC50 less than 10 μg/ml, whereas our actual median sampling depth of 10 clones identified resistant variants in an additional 25–30% of patients. Given that even minor resistant variants in the 2% prevalence range have been implicated in virologic failure with other therapeutic agents, including the entry inhibitor maraviroc [39,40], our data encourage tempering of expectations. HIV's propensity for rapid escape, due to the exceptional plasticity of its envelope, suggests modified reagents that raise the genetic barrier to resistance may have a disproportionate advantage against diverse quasispecies. Our current inability to screen for bNAb resistance using high-throughput genetic methods requires isolates to be phenotypically characterized. Thus, it will be important to pair studies like this one with broader surveys such as the one referenced above .
Considering the difficulty the field has experienced in immunogen design for the induction of broadly neutralizing antibodies, and the consequent interest in using a gene therapy-based approach to bypass the immune system, we see no reason to constrain future proof-of-principle immunotherapy or immunoprophylaxis studies to ‘naturally’ occurring antibodies only. In this context, our data indicate that engineered antibody 4546W is the most broad and potent CD4 binding site antibody currently available in the setting of HIV-1 subtype C pMTCT, and it, or an even more potent variant, should be considered for inclusion in any future clinical trial.
We thank the participants and staff of the Zambia Exclusive Breastfeeding Study, without whose generosity none of this work would have been possible. We also thank the NIH AIDS Research and Reference Reagent Program and Drs M. Sharp and D. Finzi for their support of this project.
Author contributions: Designed experiments: K.J.N., G.M.A. Performed experiments: K.J.N., C.C. Analyzed data: K.J.N., C.C., E.R.S., L.H., L.K. Contributed reagents/materials/tools: L.H., M.S., C.K., D.M.T., J.I.M., L.K. Wrote manuscript: K.J.N., G.M.A., L.K.
Funding: This work was supported by the National Institute of Child Health and Development (R01 HD 39611, R01 HD 40777, R01 HD 57617) and the International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) U01 AI 68632, and the University of Washington Center for AIDS Research Computational biology Core (P30 AI 27757). G.M.A. is an Elizabeth Glaser Pediatric AIDS Foundation Scientist.
Overall support for IMPAACT was provided by the National Institute of Allergy and Infectious Diseases (NIAID) (U01 AI 68632), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the National Institute of Mental Health (NIMH) (AI 68632).
Additional funding provided by the University of Washington Center for AIDS Research (CFAR), an NIH-funded program (P30 AI 27757) which is supported by the following NIH Institutes and Centers (NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, NIA).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2013 Lippincott Williams & Wilkins, Inc.
AIDS; antibodies; HIV; neutralization; paediatrics; prevention of mother-to-child transmission; vaccine