Despite two decades of vaccine research efforts, the discovery of an immunogen able to induce broadly neutralizing antibodies (bNAbs) that mediate potent cross-clade neutralizing activity, has not been reached yet [1–4]. However, recent findings have inspired optimism and might have paved the way to reach this goal. First, it has become clear that some HIV-1-infected individuals develop bNAbs [5,6]. This means that the relevant epitope(s) exist toward which a specific response can be mounted, at least in some individuals. Second, the results of passive immunization experiments in animal models have demonstrated that human monoclonal bNAbs (b12, 2G12, 2F5, 4E10) can fully protect from HIV-1 infection [7–12]. Third, the association of several technological advances recently allowed the identification of highly potent human monoclonal (HuMo) bNAbs (particularly the PG, PGT and VRC series) that are 10-fold to 100-fold more potent than the HuMo bNAbs cited above [13–16]. Interestingly enough, several studies suggested that broad and potent serum neutralizing activity in most of the patients with bNAbs arises through a limited number of specificities that correspond to the targets of the human monoclonal bNAbs [17–20].
All the concepts and studies described above concerned HIV-1 of group M. HIV-1 has been classified into four groups, M, N, O and P [21–25]. HIV-1 group M has spread worldwide and is responsible for the AIDS pandemic, whereas variants of the other groups remained restricted geographically to their region of emergence from wild reservoirs, mainly Cameroon, and have a much lower epidemiological burden [23,24,26–33]. Although highly divergent, a few studies using a limited number of group O isolates have suggested that neutralizing epitopes might be conserved between group M and group O [34–36]. Such intergroup conserved neutralizing epitopes would be ideal components for an AIDS vaccine that would induce bNAbs. The aim of our study was to revisit the intergroup neutralization based on a large panel of viruses representative of the divergent groups M, O, N and P, and the identification of new highly potent HuMo bNAbs.
Twelve HIV-1 strains related to groups O, N and P were used (see table, Supplemental Digital Content 1, http://links.lww.com/QAD/A308, which describes their characteristics). In addition, we included three group M (subtype B) viruses. The MN strain was used as a prototype of T-cell line adapted variant highly sensitive to neutralization (tier 1). The two other M viruses (FRO, BIG) were primary isolates with moderate or low sensitivity to neutralization (tier 2–3) . The virus stocks for the neutralization assays were prepared as described .
Twelve sera were obtained from patients infected by HIV-1 group O variants , one serum from a group P-infected patient (RBF168)  and 10 sera from group M infected blood donors, including two sera (H112 and H285) previously identified as having bNAbs (unpublished data) (Table 1). In addition a broadly neutralizing serum issued from a clade B-infected elite neutralizer (LTNP 08003) was used .
Neutralization assays were performed as described  using 100 TCID50 of each virus and three-fold serial dilutions of each heat-inactivated serum (starting at 1/20) or of each HuMo bNAbs (starting at 50 μg/ml for b12, 2G12, 2F5 and 4E10, and at 10 μg/ml for PG9, PG16, VRC01, VRC03 and HJ16). The results were expressed as the mean values of the assays performed in duplicate.
Sequence analysis and structural modeling
V1/V2 sequence alignments were performed using Clustal W from the BioEdit package (www.mbio.ncsu.edu/bioedit/bioedit.html). On the basis of the sequence homology of YBF30 with CAP45, the recently defined structure of the V1/V2 domain of HIV-1 gp120 in complex with PG9  was used to model the structure of the YBF30 V1/V2-PG9 complex (see legend of figure, Supplemental Digital Content 2, http://links.lww.com/QAD/A308, that describes the structural modeling).
Cross-group neutralization by human sera
Although the human sera displayed a wide heterogeneity of neutralizing activity, cross-group neutralization was clearly observed (Table 1). On the basis of the IC50 values, six, four, one and four of the 12 group O sera cross-neutralized, respectively, the tier two group M strain FRO, the tier 1 group M strain MN, the group N strain YBF30 and the group P strain RBF168. Sera from the group M elite neutralizer (LTNP 08003) and H285 presented a neutralizing activity against all the viruses tested. Three other M sera (H112, H122, H154) cross-neutralized from four to six group O primary isolates, the group N strain and, except H154, the group P strain. Interestingly enough, the three group M sera that neutralized the three M strains showed the broadest cross-neutralization activity (H112, H285 and LTNP 08003). The serum from the group P patient (RBF168) did not show any neutralizing activity, including autologous neutralization.
Cross-group neutralization by human monoclonal broadly neutralizing antibodies
The HuMo bNAbs did not show any cross-group neutralization, except PG9 and PG16. Two groups O primary isolates (BCF02 and RBF189; Table 1 and Fig. 1a) were neutralized by both PG9 and PG16 (IC50: 0.23–7.62 μg/ml), and one group O primary isolate (BCF03) was neutralized by PG9 only (IC50: 9.39 μg/ml). However, their neutralization sensitivity was low since the IC90 was detectable only for PG9 and for only one primary isolate (BCF02). In contrast, the group N primary isolate (YBF30) was highly sensitive to neutralization by PG9 (IC50: 0.28 μg/ml; IC90: 4.28 μg/ml) and PG16 (IC50: <0.12 μg/ml; IC90: 4.99 μg/ml) (Table 1). The intergroup M/O recombinant strain RBF208 was sensitive to neutralization by 2F5 and VRC01. It can be easily explained by the fact that the env gene of this recombinant strain is related to group M.
Conservation of residues involved in the PG9 epitope
To try to understand the molecular basis of viral sensitivity or resistance to PG9/PG16, we performed gp120 sequence alignment analyses. We focused on residues 154-184 of V1V2 (HXB2-relative residue numbering), covering the entire PG9 epitope [15,40–42] (Fig. 1b). We searched for the conservation of residues interacting with PG9 in CAP45 and ZM109 strains, including two glycosylated residues Asn156 or Asn173 (N-Gly156 or N-Gly173) and Asn160 (N-Gly160), and a stretch of cationic residues of strand C making specific electrostatic interactions with negatively charged residues on the complementary determining region 3 (CDR H3) of the PG9 heavy chain . We completed this analysis by looking for the conservation of residues whose mutagenesis was shown to confer PG9 resistance in the context of JR-CSF  or ConC .
All sequences from group O and P primary isolates lack the N-Gly156 and the Tyr173 residue predicted to stabilize the positioning of N-Gly156. In addition, sequences of group O primary isolates possess an acidic residue at position 170 (Glu170) that could repel the negatively charged sulphated tyrosines of PG9. These observations could explain the resistance or low sensitivity of the group P and of most group O primary isolates to both PG9 and PG16. In contrast, residues essential for PG9 binding were conserved in the sequence of the PG9-sensitive group N primary isolate (YBF30) and for most of the available sequences of other group N primary isolates of the Los Alamos HIV sequence database (www.hiv.lanl.gov/content/index), except for N1_FR_2011 primary isolates whose sequence lacks N-Gly160 (Fig. 1b). This was confirmed by modeling the three-dimensional structure of the V1/V2 domain of YBF30 complexed with PG9 (see Figure, Supplemental Digital Content 2, http://links.lww.com/QAD/A308). N-Gly156 and 160 form major interactions with PG9. In addition, interactions between strand C of YBF30 and the CDR H3 of PG9 are made by side-chains of cationic lysines of YBF30 (Lys168, Lys169 and Lys170) forming electrostatic interactions with negatively charged residues of PG9 including sulphated tyrosines Tyr100G, Tyr100H and Asp100I.
The PG9 resistance of the intergroup M/O recombinant strain RBF208 could be explained by the lack of the Tyr173 residue predicted to stabilize the positioning of N-Gly156. In contrast the group M strains BIG and FRO were PG9 resistant albeit the key residues were identical or similar to those of the susceptible strain JR-CSF. It is interesting to note that BIG remains sensitive to PG16, suggesting that residues outside of the structure identified-epitope could differentially affect the PG9 or PG16 sensitivity.
The identification of antigenic targets of bNAbs that would be conserved among highly divergent HIV variants could be useful in the conception of an HIV vaccine. A few studies have suggested that neutralizing epitopes might be conserved between group M and group O HIV-1 variants [34–36]. We wanted to revisit this question using a larger panel of isolates, including groups N and P viruses, as well as a large panel of human sera and several HuMo bNAbs [13–16]. We have confirmed that sera from HIV-1-infected individuals may share cross-neutralizing activities against group M, N, O and P primary HIV-1 isolates. Interestingly enough, the sera from group M-infected patients that were the most efficient at neutralizing group M viruses were also the most efficient to cross-neutralize the primary isolates from the three outgroups O, N and P, suggesting that trans-groups conserved epitopes were targeted.
We then asked whether the use of monoclonal antibodies would help to identify conserved antigenic targets of cross-group neutralization. Ferrantelli et al.  previously showed that b12, 2F5 and 4E10 neutralized a few group O isolates. We did not confirm this finding, as none of them neutralized the group O isolates or the prototypes of groups N and P. The discrepancy might be attributable to either the nature of the strains or of the neutralization assays. The recently described highly efficient HuMo bNAbs that target the CD4 binding site, VRC01, VRC03 and HJ16, did not neutralize any primary isolate of the three outgroups N, O and P.
In contrast, PG9 and PG16 demonstrated their capability to cross-neutralize various HIV-1 groups, even at a very low concentration. Two groups O primary isolates and the group N prototype strain YBF30 were neutralized by both HuMo bNAbs, YBF30 being particularly sensitive. It has been shown that PG9 and PG16 are quaternary–structure–preferring V1/V2-directed antibodies . On the basis of the knowledge acquired through the structure of the V1/V2 domain of HIV-1 gp120 in complex with PG9 , the antigenic conservation of PG9 binding site between group M and particularly group N was explained by the conservation of key residues including two glycosylated residues, N156/N173 and N160, and a stretch of cationic residues of strand C of the four-strand β-sheet domain. In addition, we confirmed the interaction between PG9 and these key residues of the YBF30 V1/V2 by molecular modeling. The quaternary-structure-preferring V1/V2-directed antibodies are among the most common bNAbs in sera from infected donors [20,42]. We demonstrated in the present study that the targets of such antibodies are conserved between HIV-1 groups, at least group M and group N; group O at a lesser extent. Taken all together, these data suggest that the PG9/PG16 epitopes would be essential components for an HIV vaccine immunogen able to induce bNAbs.
We thank Pascal Poignard and IAVI for providing us with PG9 and PG16, Syria Laperche for providing us with the samples from blood donors, and Sylvie Brunet for her technical assistance. The Following reagents were obtained through the NIH AIDS Research and Reference Program, Division of AIDS, NIAID, NIH: VRC01 (catalog number 12033) and VRC03 (catalog number 12032) from Dr John Mascola, and HJ16 (catalog number 12138) from Dr Antonio Lanzavecchia. b12, 2G12, 4E10 and 2F5 were purchased from Polymun Scientific (Vienna, Austria).
M.B. and F.B. conceived and designed the study; M.B., E.-Y.G., and T.M. performed the experiments; J.C.P., E.A., and F.S. selected and characterized the samples related to groups N, O, and P; M.B., E.-Y.G., T.M., and F.B. analyzed the data; M.B. and F.B. wrote the manuscript. All authors received the manuscript and approved it for publication.
This work was supported by the Agence Nationale de Recherche sur le SIDA et les hépatites (ANRS, Paris, France). E.-Y.G. was supported by a postdoctoral fellowship from the ANRS.
Conflicts of interest
There are no conflicts of interest.
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