We have used several established and new approaches to map the serum NAb specificities in 19 donors, mostly infected with non-clade B viruses, who exhibit remarkably broad and potent serum neutralizing activity. This study extends our previous serum mapping studies, which only focused on a small number of patients with relatively limited serum neutralization breadth and potency 
. Here, we found that the broad serum neutralizing activity in about one third of the donors in our cohort could not be efficiently adsorbed with recombinant monomeric gp120 or recombinantly trimerized gp140, suggesting the possible presence of bNAbs that target quaternary epitopes. Interestingly, in a subset of donors, the broad serum neutralizing activity was more efficiently adsorbed with recombinantly trimerized gp140 than monomeric gp120, indicating the presence of NAbs directed against gp120 epitopes that are somewhat better exposed on the YU2 gp140-foldon trimer or that the NAbs are directed against gp41. A caveat associated with these conclusions is that a single Env protein was used for the serum adsorption studies; it is possible that some of the broadly neutralizing specificities in these sera would react differently with Env proteins derived from other isolates.
MPER-directed neutralizing activity was detected in 30% of the donors, but these NAbs did not substantially contribute to the overall breadth and potency observed in the sera. Indeed, the broad and potent serum neutralizing activity for most donors in our cohort could either be adsorbed with monomeric gp120 and/or was abrogated by specific mutations located in gp120, further suggesting that gp41-directed NAb specificities do not mediate the overall serum neutralization breadth and potency in these donors. This result is in agreement with previous serum mapping studies that suggest NAbs directed against the MPER rarely mediate broad and potent serum neutralization 
Previous serum mapping studies have shown that, in some individuals, NAbs directed against the CD4bs or CRbs mediate broad serum neutralization 
. Furthermore, four broadly neutralizing CD4bs-directed mAbs have been isolated from HIV-1 infected donors 
, [43,Wu et al., submitted]
. To evaluate whether CD4bs or CRbs-directed NAbs were mediating the serum neutralization breadth and potency in these donors, we performed serum adsorptions in the presence or absence of saturating concentrations of the non-neutralizing CD4bs-directed mAb b6. We found that, in 5 out of the 17 donors, a significant proportion of the overall broad and/or potent serum neutralizing activity was mediated by receptor binding site-directed NAbs, and in one donor, nearly all of the broad serum neutralizing activity could be attributed to the b6-competed fraction. In two donors, similar results were obtained when a CD4bs-altered mutant (D368R) was used for adsorptions, indicating that the NAbs contributing to the serum neutralization breadth in these donors are directed against the CD4bs.
Although serum adsorption studies provide valuable insight into the epitopes targeted by NAbs that react with recombinant Env or linear peptides, defining the epitopes recognized by trimer-specific antibodies requires the use of functional assays. By testing the sera for neutralizing activity against a large panel of JR-CSF pseudovirus mutants incorporating single amino acid substitutions, we were able to define the epitopes recognized by the bNAb specificities in the context of the native trimer. Interestingly, we found that single amino acid mutations in Env frequently generated viruses that were far less sensitive to serum neutralization than the wild-type JR-CSF virus. Incorporation of these single substitutions into a cross-clade pseudovirus panel similarly generated viruses that were far more resistant to neutralization by certain sera than the corresponding wild-type viruses. These findings suggest that the broad and potent serum neutralizing activity in these donors is mediated by a limited number of antibody specificities. Notably, for the donor from whom PG9 and PG16 were isolated, the neutralization properties of the serum mirrored that of PG9 and PG16. These results complement the previous observation that PG9 and PG16 could recapitulate this donor's broad and potent serum neutralization against most isolates 
. Thus, it appears highly likely that PG9, PG16, and related antibodies mediate this donor's serum neutralization breadth and potency. In the context of the single amino acid variant viruses, it should be noted that although it is most likely that the substitutions directly define residues involved in bNAb binding, it is also possible that the effects are due to transmitted effects from substitutions distant from the neutralizing epitope.
Interestingly, for 9 out of the 19 donors, the potent cross-clade serum neutralizing activity was mediated by NAbs dependent on specific glycans for epitope recognition. In four of these donors, the glycan at position 160 at the base of the V2 loop was critical for serum neutralization breadth and potency, suggesting the presence of NAbs that target epitopes overlapping that of PG9 and PG16. To evaluate the contribution of PG9 and PG16-like antibodies to broad and potent serum neutralizing activity, we used the following criteria: 1) the broad and potent serum neutralizing activity could not be efficiently adsorbed with recombinant Env proteins, 2) the N-linked glycan at position 160 was essential for serum neutralization breadth and potency, and 3) the broad and potent serum neutralizing activity was diminished against pseudoviruses produced in the presence of the glycan processing inhibitor kifunensine. Based on these criteria, PG9 and PG16-like NAbs were identified in approximately 21% of the donors we studied, demonstrating that this specificity is relatively common in donors that develop broad and potent serum neutralization.
The I165A substitution located in the V2 loop of gp120 abrogated broad serum neutralizing activity in 4 out of the 19 donors studied. Given that the trimer-specific NAbs 2909, 2.2G, and 2.3E require this amino acid for potent neutralizing activity, and that the broad serum neutralizing activity in these 4 donors could not be adsorbed with monomeric gp120, it appears highly likely that these bNAb specificities also target epitopes preferentially expressed on trimeric HIV-1 Env. Indeed, this epitope may be linked to the epitope recognized by PG9 and PG16.
We found that the N-linked glycan at position 332 was critical for broad and potent serum neutralizing activity in approximately 25% of donors. Considering that this N-linked glycan is also important for formation of the 2G12 epitope, we investigated whether 2G12-like antibodies were mediating the serum neutralization breadth and potency in any of these donors. Notably, the presence of 2G12-like antibodies in HIV-1 positive sera has also been discussed recently by others 
. In one donor, a large fraction of the broad serum neutralizing activity cross-reacted with a yeast glycoprotein that expresses homogenous Man8
carbohydrates and binds 2G12 with high affinity, suggesting the presence of bNAbs that bind directly to the glycan shield. This donor's serum neutralizing activity was also dependent on the presence of the N-linked glycan at position 295, a second N-linked glycan required for 2G12 recognition 
. Interestingly, the sera from this individual did not inhibit 2G12 binding to gp120, suggesting that this bNAb specificity may bind to a glycan epitope distinct from that of 2G12. Considering that protein-carbohydrate interactions are typically weak, perhaps this antibody specificity gains the necessary avidity by cross-linking two protomers within a trimer 
. Hence, low affinity binding to monomeric gp120 may also explain the lack of competition with 2G12. Another observation worth noting is that only a single individual in our study was found to have bNAb specificities targeting the glycan shield, indicating that these types of antibodies rarely mediate serum neutralization breadth and potency. However, it has recently been shown that 2G12 is unusually efficient in protection relative to its neutralizing ability 
, suggesting that the glycan shield may have advantages as a vaccine target. Thus, it will be of interest to isolate the glycan-specific NAbs from this donor and test their protective efficacy to determine whether this is a general property of NAbs directed against the glycan shield. For the remaining four donors that exhibited N332A sensitivity, adsorption with TM-Pst1 coupled beads had no effect on the neutralizing activity of the sera; it therefore appears likely that the NAbs mediating serum neutralization breadth and potency in these donors bind to protein epitopes that are conformationally dependent on the glycan at position 332.
The observation that only a limited number of antibody specificities mediate serum neutralization breadth and potency in these donors contrasts with the results of a previous study by Scheid et. al in which no single broadly neutralizing monoclonal antibodies were isolated using single B cell sorting 
. The authors of that study concluded that the serum neutralization breadth in their donors was due to the combined activity of a large number of antibody specificities that individually display limited breadth and potency. Indeed, this may be the case for certain donors, particularly those with relatively weak serum neutralization breadth and potency. However, in donors with extraordinarily potent serum neutralization, it appears that this activity is usually only associated with one or a few different specificities. Of note, serum neutralization breadth found to be mediated by a single specificity may in fact require multiple different NAbs circulating in the plasma that recognize overlapping targets within the same region. For example, a number of different CD4bs-directed NAbs may mediate broad serum neutralization in donors where all of the serum neutralizing activity can be mapped to this region.
In summary, the data presented here show that the unusually potent cross-clade serum neutralizing activity observed in a selection of donors is mediated by a small number of antibody specificities that target conserved regions of Env to a significant extent (Figure S7
and ). Antibodies dependent on specific glycans for Env recognition were found to be responsible for this activity in approximately half of the donors we studied; 21% of these donors targeted epitopes overlapping those of PG9 and PG16 and 25% targeted epitopes that were dependent on the presence of an N-linked glycan at position 332. CD4bs and/or CRbs-directed NAbs contributed to serum neutralization breadth in 25% of donors, NAbs sensitive to the I165A substitution were identified in 21% of donors, and MPER-directed NAbs were present in a subset of donors but did not make a substantial contribution to the overall serum neutralization breadth and potency. Future studies, aimed at isolating and characterizing broadly neutralizing monoclonal antibodies from these donors, will be important for the molecular definition of the broadly neutralizing epitopes recognized by the donors.
Summary of the predominant NAb specificities that mediate serum neutralization breath and potency across all donors.