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A Limited Number of Antibody Specificities Mediate Broad and Potent Serum Neutralization in Selected HIV-1 Infected Individuals

A Limited Number of Antibody Specificities Mediate Broad and Potent Serum Neutralization in Selected HIV-1 Infected Individuals
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  A Limited Number of Antibody Specificities MediateBroad and Potent Serum Neutralization in Selected HIV-1Infected Individuals Laura M. Walker 1 , Melissa D. Simek  2 , Frances Priddy 2 , Johannes S. Gach 1 , Denise Wagner 3 , Michael B.Zwick  1 , Sanjay K. Phogat 3 , Pascal Poignard 1 , Dennis R. Burton 1,4 * 1 Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California, United States of America, 2 International AIDS Vaccine Initiative, New York, New York, United States of America,  3 IAVI AIDS Vaccine Design and Development Laboratory, Brooklyn, New York,United States of America,  4 Ragon Institute of MGH, MIT, and Harvard, Boston, Massachusetts, United States of America Abstract A protective vaccine against HIV-1 will likely require the elicitation of a broadly neutralizing antibody (bNAb) response.Although the development of an immunogen that elicits such antibodies remains elusive, a proportion of HIV-1 infectedindividuals evolve broadly neutralizing serum responses over time, demonstrating that the human immune system canrecognize and generate NAbs to conserved epitopes on the virus. Understanding the specificities that mediate broadneutralization will provide insight into which epitopes should be targeted for immunogen design and aid in the isolation of broadly neutralizing monoclonal antibodies from these donors. Here, we have used a number of new and establishedtechnologies to map the bNAb specificities in the sera of 19 donors who exhibit among the most potent cross-clade serumneutralizing activities observed to date. The results suggest that broad and potent serum neutralization arises in mostdonors through a limited number of specificities (1–2 per donor). The major targets recognized are an epitope defined bythe bNAbs PG9 and PG16 that is associated with conserved regions of the V1, V2 and V3 loops, an epitope overlapping theCD4 binding site and possibly the coreceptor binding site, an epitope sensitive to a loss of the glycan at N332 and distinctfrom that recognized by the bNAb 2G12 and an epitope sensitive to an I165A substitution. In approximately half of thedonors, key N-linked glycans were critical for expression of the epitopes recognized by the bNAb specificities in the sera. Citation:  Walker LM, Simek MD, Priddy F, Gach JS, Wagner D, et al. (2010) A Limited Number of Antibody Specificities Mediate Broad and Potent SerumNeutralization in Selected HIV-1 Infected Individuals. PLoS Pathog 6(8): e1001028. doi:10.1371/journal.ppat.1001028 Editor:  Alexandra Trkola, University of Zurich, Switzerland Received  March 25, 2010;  Accepted  July 1, 2010;  Published  August 5, 2010 Copyright:    2010 Walker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  This work was supported by funds from the International AIDS Vaccine Initiative, NIAID AI33292, and the Ragon Institute of MGH, MIT, and Harvard. Thefunders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests:  The authors declare competing financial interests. LMW, SKP, PP, and DRB are inventors on a patent describing the human broadlyneutralizing antibodies PG9 and PG16 (U.S. provisional patent application numbers USSN 61/161,010; USSN 61/165,829; and USSN 61/224,739).* E-mail: Introduction The hallmark of most successful anti-viral vaccines is the abilityto induce neutralizing antibodies [1,2,3,4]. For HIV-1, NAbs havebeen shown to provide protection against viral challenge in non-human primate models [5,6,7,8,9,10,11,12,13], suggesting that a vaccine capable of inducing similar types of antibodies wouldprovide benefit upon exposure to the virus. However, due to theextraordinary genetic diversity of the HIV-1, a successful vaccinewill require the induction of antibodies that neutralize a widespectrum of global circulating viral isolates, i.e. broadly neutral-izing antibodies (bNAbs) [14]. Unfortunately, the development of an immunogen capable of eliciting bNAbs has not been met withsuccess to date. Importantly, although NAbs generated during natural HIV-1 infection usually target immunodominant variableregions of the virus, recent studies have shown that 10–30% of infected individuals develop moderate to broadly neutralizing sera[15,16,17,18]. These individuals are of considerable interest froma vaccine standpoint; understanding the antibody specificities thatmediate potent cross-clade serum neutralizing activity mayilluminate potential targets for HIV-1 immunogen design. Inaddition, knowledge of the epitopes targeted by the bNAbs canassist in the design of reagents, ‘‘baits’’, to facilitate the isolation of broadly neutralizing monoclonal antibodies (bnMAbs) from thesedonors. BnMAbs can be used in molecular studies to help direct vaccine design [19,20,21].Several studies have previously been performed to systemati-cally analyze the NAb specificities in HIV-1 positive seradisplaying varying degrees of neutralization breadth and potency[15,16,17,18,22,23,24]. In all of these studies, a series of complementary methods, such as selective removal of certainantibody specificities using antigen-coated beads, inhibition of neutralizing activity using linear peptides, and the use of chimeric viruses displaying specific epitopes, were used to define theepitopes targeted by NAbs in broadly neutralizing sera. Althoughthe breadth of serum neutralization could rarely be mappedexclusively to a single epitope, several sera appeared to containCD4bs and co-receptor binding site (CRbs)-specific antibodies thatcontributed to the overall breadth of serum neutralizing activity[16,17,22,25]. In a minority of cases, sera were found to containNAbs to the membrane-proximal external region (MPER)[16,17,23]. Arguably, one of the most significant results fromthese studies was that a substantial fraction of the serum NAbsappeared to target unidentified viral epitopes. Considering that PLoS Pathogens | 1 August 2010 | Volume 6 | Issue 8 | e1001028  most of the reagents used for characterization were based onmonomeric gp120 and linear peptides, one possibility here is thatthe serum neutralization breadth is mediated by NAbs thatrecognize quaternary epitopes preferentially expressed on trimericEnv. Two recently described broad and potent NAbs, PG9 andPG16, fall into this category [26]. An important question that has arisen from serum studies concernsthe number of NAb specificities that mediate broad serumneutralization.Afewscenariosarepossible;broadserumneutralizationcould be mediated by a very large number of neutralizing antibodieswith limited breadth [27], a few relatively broad and potentneutralizing antibody specificities, or a single, extraordinarily broadand potent, neutralizing antibody specificity. Although these scenariosarenotmutuallyexclusive,thelattertwoaremoreattractiveintermsof  vaccine design, as it appears far more practical to focus the immuneresponse on a small number of conserved epitopes with a vaccinerather than on a large number of more variable epitopes.In a previous study, we screened sera from approximately 1,800HIV-1infecteddonorsfromThailand,Australia,theUnitedKingdom,the United States, and several sub-Saharan African countries forneutralizing activityand identified donors who exhibit among the mostbroad and potent neutralizing serum activity observed to date [15].The top 1% of samples screened, designated ‘‘elite neutralizers’’,displayedparticularlypotentserumneutralizingactivityagainstacross-clade pseudovirus panel. These donors are valuable for understanding the development of broad responses and for the isolation of broad andpotent neutralizing monoclonal antibodies. Notably, PG9 and PG16were isolated from an individual who ranked in the top 5% of donorsscreened [26]. In this study, we have used a number of established andnew techniques to map the broadly neutralizing antibody specificitiesin the serum of individuals who were ranked in the top 5% of neutralizersidentifiedinourpreviousstudy,includingeliteneutralizers.Importantly,sincemanyofourapproachesrelyontheuseoffunctionalassays, we defined the epitopes recognized by the broadly neutralizing serum antibodies in the context of the native trimer. Our resultsdemonstrate that the broad neutralization in the sera of most of theindividual donors can be associated with single or a small number of specificities. Across the donor panel, broad neutralization appearsassociated with 4–5 principal specificities. Results Serum neutralizing activity and demographics of selected donors  A total of 19 volunteers from diverse HIV-1 epidemics werecharacterized. Of the 19 volunteers, 63% were female, 26% maleand 11% unknown. The median age of all volunteers was 38 witha median CD4 count of 414 and median log viral load of 4.07. All volunteers were infected for at least 3 years. Of the 19 donorsanalyzed, 14 ranked in the top 1% of neutralizers identified in ourprevious report (elite neutralizers), and the remaining 5 ranked inthe top 5% of neutralizers. Figure S1 shows the serumneutralization profiles of the selected donors. Serum neutralizationwas assessed using an indicator cross-clade pseudovirus panel thathas previously been shown to be predictive of neutralizationbreadth and potency over a larger number of isolates [15]. Recombinant gp120 and/or gp140-reactive antibodiesmediate broad serum neutralization in 70% of donors  As a first approach, we used a previously described serumadsorption method to determine whether the NAb specificities inthese sera would react with recombinant monomeric gp120 [17].Eighteen sera, which all neutralized HIV-1 YU2, were adsorbedwith recombinant YU2 gp120 coupled beads or blank controlbeads (donor  # 37 was excluded from the analysis because theplasma did not neutralize YU2). After confirming depletionefficiencies by ELISAs, which showed that all detectable gp120-binding antibodies had been removed (Figure S2), the adsorbedfractions were tested for neutralizing activity against a cross-cladepseudovirus panel (Figure 1A). For ten donors (  # 74,  # 36,  # 20, # 51,  # 26,  # 33,  # 57,  # 17,  # 14, and  # 23), most of the broadserum neutralizing activity was removed after gp120 adsorption,indicating that the serum neutralization breadth could beattributed to gp120-reactive NAbs. In contrast, for the remaining eight donors (  # 21,  # 30,  # 39,  # 15,  # 31,  # 24,  # 56,  # 29), alarge proportion of the broad neutralizing serum activity wasretained after removal of the YU2 gp120-specific Abs, suggesting the presence of NAbs that recognize epitopes that are notexpressed on recombinant YU2 gp120. Based on these results, wenext sought to determine whether the NAb specificities in theserum would react with a recombinant trimerized Env protein.The YU2 gp140-foldon trimer was chosen for these studiesbecause it has been well characterized structurally and antigen-ically [28,29,30,31,32,33], and it has been previously used for theisolation of NAbs from HIV-1 infected patients [27]. Using thesame method as for gp120, we adsorbed the sera with YU2 gp140-foldon coupled beads or blank control beads (Figure 1B). Asexpected, we found that, if the broad neutralizing activity of aparticular serum could be adsorbed with YU2 gp120, it could alsobe adsorbed with YU2 gp140. However, for a subset of donors(  # 39,  # 21, and  # 15), the broad neutralizing activity of the serawas absorbed more efficiently with YU2 gp140 than YU2 gp120.This result may suggest that certain gp120 epitopes are betterpresented on the YU2 gp140-foldon trimer. Alternatively, asignificant fraction of the serum neutralization breadth in thesedonors could be mediated by NAbs directed against gp41. Anti-MPER NAbs do not mediate a major fraction of serum neutralization breadth and potency in the donorsstudied We next investigated the contribution of gp41-directed NAbs tobroad serum neutralizing activity. Since the MPER region of gp41contains the epitopes recognized by three broadly neutralizing monoclonal antibodies, 2F5, 4E10, and Z13e1, and is the onlyknown neutralizing determinant on gp41, we focused ondetermining whether NAbs directed against this region weremediating broad serum neutralizing activity. As a first step, wetested the sera for neutralizing activity against a chimeric HIV-2 virus containing the complete MPER region of gp41 [24]. Based Author Summary The development of an immunogen that elicits antibodiesthat neutralize a wide range of global circulating HIV-1isolates is a major goal of HIV-1 vaccine research.Unfortunately, even the most promising antibody-basedvaccine candidates have only induced NAb responses thatneutralize a limited number of these strains. However,recent studies have demonstrated that broad and potentNAb responses develop in the sera of a subset of HIV-1infected individuals, and studying the nature of theseresponses may provide clues for the design of new vaccineimmunogens. Here, we show that the broad neutralizationin the sera of most of the individual donors that westudied can be associated with single or a small number of specificities. Across the donor panel, broad neutralizationappears associated with 4–5 principal specificities. HIV-1 Broadly Neutralizing SeraPLoS Pathogens | 2 August 2010 | Volume 6 | Issue 8 | e1001028  on this assay, six sera appeared to contain MPER-reactive NAbs(Figure 2A). One of these donors (  # 36) also neutralized theparental HIV-2 virus, indicating that this donor may be co-infected with HIV-2 or contain anti-HIV-1 NAbs that cross-reactwith HIV-2. Notably, since HIV-2/MPER chimeras are 1 to 2-logs more sensitive to NAbs 4E10 and Z13e1 than HIV-1 primary Figure 1. Serum adsorptions with recombinant Env proteins.  Sera were adsorbed with YU2 gp120 or gp140-coupled beads or blank controlbeads and then tested for neutralizing activity against a cross-clade pseudovirus panel using TZM-bl target cells. A) Serum neutralizing activitydepleted on YU2 gp120. B) Serum neutralizing activity depleted on YU2 gp140. Percent neutralizing activity depleted on YU2 gp120 or YU2 gp140was calculated using the equation=(1 2 (IC 50 blank beads /IC 50 antigen-coated beads ))*100. Boxes are color coded as follows: Gray, 0–45%, yellow, 45–65%;orange, 65–85%; red, 85–100%. C) Serum neutralizing activity against the selected cross-clade pseudovirus panel. Serum IC 50  titers are color coded asfollows: gray squares, 1:300 . IC50 $ 1:100, yellow squares, 1:500 . IC50 $ 1:300; orange squares, 1:1500 . IC50 $ 1:500, red squares, IC50 $ 1:1500.doi:10.1371/journal.ppat.1001028.g001 Figure 2. Contribution of MPER-directed NAbs to broad serum neutralization.  A) Sera were tested for neutralizing activity against achimeric HIV-2 virus containing the complete MPER region of gp41 [24]. Serum IC 50  titers are expressed as 1/serum dilution. Boxes are color coded asfollows: white squares, IC 50 # 1:80; green squares, 1:200 . IC 50 . 1:80, yellow squares, 1:500 . IC 50 . 1:200. The IC 50 s of anti-MPER NAbs 2F5, 4E10, Z13e1were also determined, expressed in  m g/ml. The anti-Dengue antibody Den3 is included as a negative control. B) Serum neutralizing activity afteradsorption with MPER peptide-coated beads. Sera were adsorbed with full-length MPER peptide-coated beads or blank control beads and thentested for neutralizing activity against a cross-clade pseudovirus panel, as described previously [54]. Percent serum neutralizing activity depleted onthe MPER peptide was calculated using the equation=(1 2 (IC 50 blank beads /IC 50 MPER beads ))*100. C) Neutralizing activity of serum antibodies elutedfrom the MPER-coated beads. Functional Abs were eluted from the MPER-coupled beads by exposing the beads to a series of increasingly acidicconditions as described [17]. ELISA assays were used to determine the concentration of total IgG in the eluted fraction. Boxes are color coded asfollows: white squares, IC 50 . 20  m g/ml; green squares, 20 $ IC 50 . 5  m g/ml, yellow squares, 5 . IC 50 . 1  m g/ml.doi:10.1371/journal.ppat.1001028.g002HIV-1 Broadly Neutralizing SeraPLoS Pathogens | 3 August 2010 | Volume 6 | Issue 8 | e1001028  isolates [22], this assay may overestimate the contribution of anti-MPER antibodies to serum neutralization breadth and potency.Indeed, for all six of these donors, a substantial fraction of thebroad serum neutralization could be adsorbed with monomericgp120, demonstrating that anti-MPER NAbs probably do notdominate the overall serum neutralization breadth and potency.Nonetheless, to further investigate the contribution of anti-MPERNAbs to the serum neutralization breadth in these six donors, weadsorbed the sera with MPER-coupled beads or blank controlbeads, as described previously [23]. As above, the adsorbed serawere then tested for neutralizing activity against a cross-cladepseudovirus panel (Figure 2B). In addition, the MPER-specificantibodies were eluted off the beads and tested for neutralizing activity against the clade B isolate JR-CSF (Figure 2C). Asexpected, for all six donors, the neutralizing activity of the serumafter adsorption with MPER peptide-coated beads or blank beadswere comparable, indicating that anti-MPER NAbs do notmediate a major fraction of the serum neutralization breadthand potency. However, for three donors, weak to moderateneutralizing activity against JR-CSF was observed in the fractioneluted from the MPER peptide-coupled beads, suggesting thepresence of MPER-directed NAbs at low concentrations and/orlow neutralizing potency in these sera. CD4bs and CRbs-directed antibodies contribute to broadserum neutralization in approximately 25% of donors Previous serum mapping studies have evaluated CD4bs andCRbs-directed neutralizing activity in sera by performing serumadsorptions with gp120 point mutants that fail to react withexisting mAbs directed against these epitopes. However, a caveatto this approach is that some CD4bs or CRbs NAbs may beinsensitive or only partially sensitive to these particular mutations.Notably, a recently reported broadly neutralizing CD4bs-directedNAb binds to the D368R gp120 variant, often considered aprototypic non-CD4bs Ab binding gp120, with higher affinitythan the wild-type (WT) gp120 molecule [34]. Therefore, as analternative to use of the D368R gp120 variant, we developed aserum adsorption method based on antibody competition. Using this method, serum adsorptions to Env are performed in thepresence of saturating concentrations of a non-neutralizing competitor mAb (the competitor Ab must be non-neutralizing so its presence will not affect the results of the neutralizationassay). In principle, Abs directed against epitopes overlapping that of the competitor Ab will fail to bind to the Env-coatedbeads. Since the non-neutralizing CD4bs-directed mAb b6 hasbeen shown to compete with both CD4bs and CRbs-directed Absfor binding to gp120 [35], b6 was used as a competitor in theseexperiments.To first validate this method, we adsorbed b12 (a CD4bs-directed bNAb) with YU2 gp140-coated beads in the presence orabsence of saturating concentrations of b6 or blank control beads.Indeed, all of the b12 neutralizing activity could be adsorbed withYU2 gp140-coated beads, but none of neutralizing activity couldbe adsorbed when the assay was performed in the presence of saturating concentrations of b6 (Figure S3).We next performed the assay using donor sera (Figure 3A). Forone donor (  # 23), adsorption of the broad serum neutralizing activity with YU2 gp140 was completely inhibited by b6, suggesting that CD4bs or CRbs NAbs dominate the serum neutralizationbreadth and potency in this individual. For 4 additional donors, b6inhibited 50–70% of broad serum neutralization indicating thatCD4bs or CRbs Abs contribute significantly to the overall serumneutralization breadth and potency but not exclusively. For theremaining 12 donors, none or only a small fraction of the broadserum neutralization was blocked by b6, suggesting that CD4bs andCRbs-directed NAbs are of minor importance to the broadneutralizing responses in these individuals. A caveat to note isthat anti-CD4bs or anti-CRbs NAbs that mediate the serumneutralization against some isolates (but not others) could bepresent in these 12 sera, but their epitopes may not be properlyexpressed in the context of YU2 gp140 used in the adsorptionexperiments. Figure 3. Analysis of CD4bs and CRbs-directed neutralizing activity.  A) Sera were tested for neutralizing activity after adsorption with YU2gp140-coupled beads in the presence or absence of saturating concentrations of mAb b6 or blank control beads. Percent gp140-directedneutralization to the CD4bs or CRbs was calculated using the equation=[(% neutralization absorbed by gp140 beads 2 % neutralization absorbed bygp140 beads in the presence of excess b6)/% neutralization absorbed by gp140 beads] *100. B) Sera were tested for neutralizing activity afteradsorption with YU2 gp120 D368R coated beads or blank control beads. Percent gp120-directed neutralization sensitive to the D368R mutation wascalculated using the equation=[(% neutralization absorbed by gp120 beads 2 % neutralization absorbed by gp120 D368R beads)/% neutralizationabsorbed by gp120 beads] *100. Boxes are color coded as follows: Gray, 0–45%, yellow, 45–65%; orange, 65–85%; red, 85–100%.doi:10.1371/journal.ppat.1001028.g003HIV-1 Broadly Neutralizing SeraPLoS Pathogens | 4 August 2010 | Volume 6 | Issue 8 | e1001028  Since the b6-blocking approach does not discriminate betweenCD4bs and CRbs-directed NAbs, we also performed serumadsorptions with a D368R gp120 variant that fails to bind CD4and most, although not all, CD4bs-directed mAbs [25]. In twocases, a positive correlation was observed between the b6-inhibition adsorptions and the D368R adsorptions, indicating that the NAbs contributing to serum neutralization breadth inthese donors are directed against the CD4bs (Figure 3B).However, for donors  # 20 and  # 57, the NAb specificitiesmediating serum neutralization breadth and potency competedwith mAb b6 for gp140 binding yet did not exhibit sensitivity tothe D368R mutation. It is possible that these NAbs are directedagainst the CRbs or novel epitopes that overlap the b6 epitope. Alternatively, these NAbs may be directed against the CD4bs butare insensitive to the D368R substitution. Dissecting the epitope fine specificities of serum NAbsusing a large panel of mutant pseudoviruses The inability to adsorb a significant fraction of the broad serumneutralization with recombinant Env proteins in approximatelyone third of the donors prompted us to develop mapping strategiesbased on functional assays. As a first approach, we tested all of thesera for neutralizing activity against approximately 100 JR-CSFpseudoviruses incorporating single amino acid substitutions(Figure 4 and Table S1). In principle, if the serum neutralizationagainst JR-CSF were mediated by a small number of NAbspecificities, this activity would be diminished against pseudo- viruses incorporating mutations that disrupt the epitopes targetedby these bNAbs. Indeed, for approximately 75% of the donors,potent serum neutralization against JR-CSF was abrogated bysingle amino acid substitutions. Notably, for the donor from whomPG9 and PG16 were isolated (  # 24), the N160K substitution in thecontext of JR-CSF Env resulted in complete viral escape fromserum neutralization. Considering that this glycan is essential forPG9 and PG16 neutralizing activity [26], but does not affect thebinding or neutralization profiles of any of the other Abs we tested(Table S2), this result suggests that this donor’s potent serumneutralization against JR-CSF is entirely mediated by PG9, PG16,and similar antibodies. The N160K mutation also diminishedserum neutralization against JR-CSF in four additional donors(  # 56, # 29, # 31, and # 21), suggesting the presence of NAbs thattarget epitopes overlapping that of PG9 and PG16 in these sera. Alanine mutations in other regions of the V1/V2 and V3 loopsalso abrogated serum neutralizing activity against JR-CSF in thesedonors, further suggesting involvement of these regions in forming the epitopes recognized by these NAbs. Additionally, the broadand potent serum neutralizing activity in the five sera above couldbe not be efficiently adsorbed with monomeric gp120 orrecombinantly trimerized gp140, indicating that these NAbs bindpoorly to recombinant Env proteins. However, it is worth noting that a small number of gp120s have been identified that reactweakly with PG9 [26], suggesting that it may be possible to adsorbthese donor sera on certain gp120s.In five different donors (  # 17,  # 51,  # 26,  # 14,  # 33), the N-linked glycan at position 332 at the base of the V3 loop of gp120was critical for potent serum neutralization against JR-CSF. Sincethis glycan is also critical for 2G12 recognition [36], but does notsignificantly affect the neutralization profiles of other neutralizing mAbs we tested (Table S2), this result raised the possibility that thedonor sera target epitopes overlapping that of 2G12. Interestingly,2G12 also requires the glycan at position 295 for neutralizing activity, and one of the donors (  # 33) also exhibited sensitivity tothis mutation. The glycan-dependent nature of the epitopestargeted by the NAbs in these sera is discussed below. Interes-tingly, a significant fraction of the broad serum neutralizing activity in all five of these sera could be adsorbed with monomericgp120 and trimeric gp140, indicating that the epitopes targeted bythese NAbs are expressed on recombinant forms of Env. Figure 4. Effects of single amino acid substitutions on serum neutralizing activity against JR-CSF.  Sera were tested for neutralizingactivity against 97 JR-CSF pseudoviruses incorporating single amino acid substitutions. Percent serum neutralizing activity sensitive to the indicatedsubstitution was calculated using the equation=(1 2 (IC 50 variant /IC 50 WT ))*100. Boxes are color coded as follows: yellow, 20–50%; orange, 50–80%; red,80–100%. The first and second rows designate the gp120 domain and the single amino acid substitution, respectively. JR-CSF pseudovirus variantsthat were similarly sensitive as wild type JR-CSF to all of the sera or variants that were globally sensitive to serum neutralization are not shown but arelisted in Table S1.doi:10.1371/journal.ppat.1001028.g004HIV-1 Broadly Neutralizing SeraPLoS Pathogens | 5 August 2010 | Volume 6 | Issue 8 | e1001028
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