broadly neutralizing antibodies targeting the hiv-1 ... · to 5.2 log siv rna copies/ml), and 75%...

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

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1Ragon Institute of Massachusetts General Hospital, Massachusetts Institute ofTechnology, and Harvard University, Cambridge, MA 02139, USA. 2Center for Virologyand Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA.3AIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick NationalLaboratory for Cancer Research, Frederick, MD 21702, USA. 4Los Alamos NationalLaboratory, Los Alamos, NM 87545, USA. 5Vaccine Research Center, National Instituteof Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20814,USA. 6The Scripps Research Institute, La Jolla, CA 92037, USA. 7National Institute forCommunicable Diseases of the National Health Laboratory Service and the Universityof the Witwatersrand, 2131 Johannesburg, South Africa. 8Centre for the AIDS Programmeof Research in South Africa, 4013 Durban, South Africa. 9Columbia University, New York,NY 10032, USA. *These authors contributed equally to this work.†Corresponding author. Email: [email protected]

Julg et al., Sci. Transl. Med. 9, eaal1321 (2017) 6 September 2017

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Broadly neutralizing antibodies targeting theHIV-1 envelope V2 apex confer protection against aclade C SHIV challengeBoris Julg,1,2* Lawrence J. Tartaglia,2* Brandon F. Keele,3 Kshitij Wagh,4 Amarendra Pegu,5

Devin Sok,6 Peter Abbink,2 Stephen D. Schmidt,5 Keyun Wang,5 Xuejun Chen,5 M. Gordon Joyce,5

Ivelin S. Georgiev,5 Misook Choe,5 Peter D. Kwong,5 Nicole A. Doria-Rose,5 Khoa Le,6

Mark K. Louder,5 Robert T. Bailer,5 Penny L. Moore,7,8 Bette Korber,4 Michael S. Seaman,2

Salim S. Abdool Karim,8,9 Lynn Morris,7,8 Richard A. Koup,5 John R. Mascola,5

Dennis R. Burton,1,6 Dan H. Barouch1,2†

Neutralizing antibodies to the V2 apex antigenic region of the HIV-1 envelope (Env) trimer are among the mostprevalent cross-reactive antibodies elicited by natural infection. Two recently described V2-specific antibodies,PGDM1400 and CAP256-VRC26.25, have demonstrated exquisite potency and neutralization breadth againstHIV-1. However, little data exist on the protective efficacy of V2-specific neutralizing antibodies. We created anovel SHIV-325c viral stock that included a clade C HIV-1 envelope and was susceptible to neutralization by bothof these antibodies. Rhesus macaques received a single infusion of either antibody at three different concentrations(2, 0.4, and 0.08 mg/kg) before challenge with SHIV-325c. PGDM1400 was fully protective at the 0.4 mg/kg dose,whereas CAP256-VRC26.25-LS was fully protective even at the 0.08 mg/kg dose, which correlated with its greaterin vitro neutralization potency against the challenge virus. Serum antibody concentrations required for protectionwere <0.75 mg/ml for CAP256-VRC26.25-LS. These data demonstrate unprecedented potency and protective effi-cacy of V2-specific neutralizing antibodies in nonhuman primates and validate V2 as a potential target for theprevention of HIV-1 infection in passive immunization strategies in humans.

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INTRODUCTIONDespite the efforts of the HIV-1 vaccine field, no HIV-1 envelope (Env)immunogen to date has been able to elicit antibodies with broadlyneutralizing activity (1). In contrast, many HIV-1–infected individualsproduce neutralizing antibodies with some degree of breadth duringthe course of infection (2–4). Over the past few years, several antibodiestargeting distinct epitopes of the HIV-1 envelope (Env) trimer and withpotent and broad activity against diverse clinical isolates have been iden-tified (5–8). In particular, neutralizing antibodies directed toward theCD4 binding site and the V3 region have shown promise in preclinicalstudies, in which single intravenous doses of antibodies protected rhesusmacaques against challenges with simian human immunodeficiency virus(SHIV) (9–12). In the absence of a vaccine that can elicit such broadlyneutralizing antibody (bNAb) responses, passive immunization withbNAbs is being explored for HIV-1 prevention strategies.

Although antibodies against several regions of the Env trimer havebeen described (6), neutralizing antibodies to the V2 apex antigenic

region of the HIV-1 Env trimer are among the most prevalent cross-reactive antibodies elicited during infection (13–15). The V1V2 region,which harbors multiple glycans and is highly sequence-diverse, is lo-cated at the Env apex and plays a vital role in the Env function by sta-bilizing the trimeric spike on the virion surface. It also shields V3 andthe co-receptor binding sites in the prefusion state and exposes themupon CD4 binding (16). Although these antibodies are common inHIV-1–infected individuals, we know very little about their ability toconfer protection against infection. In the recent RV144 HIV-1 vaccinestudy, binding antibodies against the V1V2 region were associated withreduced risk of infection (17).

To date, V2-directed bNAbs have been isolated from several donors,including the International AIDS Vaccine Initiative (IAVI) Protocol Gdonor 24 (PG9 and PG16) (18), the Center for HIV/AIDS VaccineImmunology (CHAVI) donor 0219 (CH01–CH04) (19), the Centrefor the AIDS Programme of Research in South Africa (CAPRISA)256 donor (CAP256-VRC26.01-33) (20, 21), and the IAVI ProtocolG donor 84 (PGT141–145 and PGDM1400–1412) (5, 22). These antibodiesbind to the intact trimer with a stoichiometry of one per trimer (20)and interact with glycans at N160 and, to a lesser extent, N156 (18).They also have a very long heavy-chain complementarity-determiningregion 3 (CDRH3), which enables them to effectively penetrate theglycan shield (21). For the present study, we selected two V2-specificmonoclonal antibodies (mAbs), CAP256-VRC26.25 and PGDM1400, fortheir exquisite potency and neutralization breadth. CAP256-VRC26.25neutralized 57% of global viral isolates and 70% of clade C isolateswith a median inhibitory concentration (IC50) of 0.001 mg/ml againstsensitive viruses (21, 23). Among the PGT145 antibody family, thesomatic variant PGDM1400 had a particularly broad and exceptionallypotent neutralization activity with 83% global coverage at a median IC50

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Virus Env clade

PGDM1400 CAP256-VRC26.25 PG9

IC50 IC80 IC50 IC80 IC50 IC80

SF162P3.Rh B >50 >50 >50 >50 >50 >50

BaLP4.Rh B >50 >50 >50 >50 0.552 >50

327c.Rh C >50 >50 >50 >50 >50 >50

327c.Hu C >50 >50 >50 >50 >50 >50

325c.Hu C 0.037 0.104 0.003 0.006 0.976 2.33

AD8EO.Rh B >50 >50 >50 >50 >50 >50

1157ipd3N4.Rh C 0.197 12.4 >50 >50 0.527 >50


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of 0.003 mg/ml (22). These V2-specific antibodies have superior in vitropotency compared to the V3 glycan–dependent antibodies PGT121 andPGT128 (5), which are among the most potent bNAbs described todate. However, the protective efficacy of V2-specific bNAbs againstpathogenic tier 2 SHIV challenges remains unexplored.

Here, we evaluated the protective efficacy of these V2-specificbNAbs against SHIV challenge in nonhuman primates. We created anovel SHIV-325c stock that included a clade C Env and against whichPGDM1400 and CAP256-VRC26.25 showed potent neutralizationactivity. Animals received a single infusion of PGDM1400 orCAP256-VRC26.25-LS (engineered with the Fc-LS mutation to increasein vivo half-life) at three different concentrations and were challengedwith SHIV-325c.

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RESULTSGeneration and characterization of SHIV-325cPGDM1400 and CAP256-VRC26.25 have been shown to neutralizeHIV-1 broadly and with high potency (21, 22), but they did notneutralize several commonly used SHIVs (Table 1). We thereforegenerated a novel challenge stock SHIV-325c by cloning a clade CHIV-1 env sequence from an early HIV-1–infected individual fromSouth Africa (24) into the SHIV KB9-AC backbone. Large-scale challengestocks were generated by inoculation of the 293T transfection–derivedsupernatants into primary human peripheral blood mononuclear cells(PBMCs), resulting in virus infectivity titers of 3.49 × 105 TCID50

(median tissue culture infectious dose)/ml and viral loads ranging from1.3 × 109 to 1.5 × 109 RNA copies/ml. We sequenced the env of theSHIV-325c challenge stock using single-genome amplification (SGA) andcompared it to the original HIV-1 env patient sequence (fig. S1). Thediversity of the viral stock was low (mean diversity, 0.06%; excludingAPOBEC-mutated sequences). Among 25 SGA-derived sequencesobtained from the SHIV-325c stock, 12 amplicons were 100% identicalto the original HIV-1 env consensus sequences, and 11 ampliconsshowed one to three sporadic nucleotide differences (mean divergence,0.03%; maximum divergence, 0.13%). Only one amplicon demonstratedseveral APOBEC-mutated sequences. These data show that the envsequences in the SHIV-325c stock were very similar to the originalHIV-1 env sequence.


We next evaluated the infectivity of the SHIV stock in vivo. Eightadult rhesus macaques were inoculated via the intrarectal route with1 ml of the SHIV-325c challenge stock. All animals developed productiveinfection (Fig. 1A) and exhibited robust peak viral loads ranging from 4.7to 6.7 log RNA copies/ml after inoculation. These viral loads werecomparable to those after SHIV-SF162P3 or SHIV-AD8 infection(25, 26). Viral loads declined and reached chronic set point levelsby about week 10. Set point viral loads varied among animals (2.2to 5.2 log SIV RNA copies/ml), and 75% (six of eight) of infectedanimals exhibited chronic viremia at week 14, with mean set pointviral loads of 3.9 log RNA copies/ml. During acute infection, meanCD4+ T cell numbers declined by 27% in SHIV-325c–inoculatedanimals (preinfection count, 1406 cells/mm3; postinfection nadir,1027 cells/mm3; P = 0.016, paired t test) (Fig. 1B) but did not subse-quently exhibit further decline.

When tested against a panel of 14 bNAbs, SHIV-325c exhibitedsensitivity to the V2 antibodies PG9, CAP256-VRC26.25, andPGDM1400 but was resistant to several other bNAbs, including theCD4 binding site antibodies VRC01 and 3BNC117, the V3 glycan

Table 1. Neutralization profiles of PGDM1400, CAP256-VRC26.25, and PG9 against SHIV challenge stocks. IC50 and IC80 values are in micrograms permilliliter. Values between 0.001 and 0.01 mg/ml are highlighted in red; between 0.01 and 0.1 mg/ml, orange; between 0.1 and 1 mg/ml, yellow; between 1 and 10 mg/ml,light green; and between 10 and 50 mg/ml, dark green. Virus suffix indicates PBMC cell type used for production: .Rh, rhesus PBMCs; .Hu, human PBMCs.

Fig. 1. Plasma viral loads and CD4+ T cell counts in rhesus macaques chal-lenged with SHIV-325c. (A) Plasma viral RNA (log RNA copies/ml) are shown foranimals that were challenged intrarectally with SHIV-325c. The red line representsthe mean log RNA copies/ml. (B) CD4+ T cell numbers in the same animals beforeand after SHIV-325c infection (P = 0.016, paired t test).

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antibody PGT128, and several membrane-proximal external region(MPER) and CD4i antibodies (Table 2). SHIV-325c sensitivity acrossthe tested bNAbs was comparable to the frequently used tier 2 SHIV-SF162P3, which demonstrated resistance against 7 of the 14 tested bNAbs.In contrast, SHIV-325c was more resistant than the tier 1 SHIV-BaL,which was neutralized by 11 of the 14 bNAbs, indicating that SHIV-325c had a tier 2 phenotype. SHIV-325c neutralization sensitivity toPGDM1400 and CAP256-VRC26.25 (IC80 of 0.104 and 0.006 mg/ml,respectively) was comparable to the median neutralization sensitivityof a multiclade panel of 208 HIV pseudoviruses (IC80 of 0.04 and0.03 mg/ml, respectively) (Fig. 2A and table S1). PGDM1400 and CAP256-VRC26.25 also neutralized SHIV-325c at a similar potency to a panelof clade C pseudoviruses (IC80 of 0.02 and 0.01 mg/ml, respectively)(Fig. 2B). No neutralization curve plateaus below 100% neutralizationwere observed with these three V2 antibodies against SHIV-325c(Fig. 3), unlike those previously described for PG9 against SHIV-BaL (11).

These data indicate that SHIV-325c had a tier 2 neutralizationphenotype and a representative neutralization profile for PGDM1400and CAP256-VRC26.25 as compared with a large panel of clade Cviruses, suggesting that it may be a useful challenge model for evaluatingthe protective activity of these antibodies.

In vivo protection against SHIV-325c challengeTo assess the protective efficacy of PGDM1400 and CAP256-VRC26.25-LSagainst SHIV-325c challenge, we performed an antibody titration chal-lenge study in rhesus macaques. CAP256-VRC26.25-LS is a variant ofCAP256-VRC26.25 that has been engineered to include the LS mutationin its Fc region that increases its in vivo half-life (27) from about 4.4 to8.8 days in rhesus macaques (fig. S2).

Thirty-five rhesus macaques were randomized to the followinggroups: two groups of five animals each received 2 or 0.4 mg/kg of


PGDM1400, and one group of four animals received 0.08 mg/kg ofPGDM1400. Three additional groups of four animals each received 2,0.4, or 0.08 mg/kg of CAP256-VRC26.25-LS. A control group of nineanimals received saline. Antibodies were administered intravenously24 hours before the animals were challenged via the intrarectal routewith a single high dose of SHIV-325c (500 TCID50). All animals in thecontrol group became infected with detectable viremia between days 7and 28, and the median peak viremia was 5.8 log viral copies/ml (Fig. 4A),confirming the robust infectivity of the novel SHIV-325c challengestock. In the high-dose group (2 mg/kg), one of five animals thatreceived PGDM1400 became infected and showed plasma viremiaon day 28 (Fig. 4B), whereas all of the CAP256-VRC26.25-LS pre-treated animals at the same dose were protected (Fig. 4C). At 0.4 mg/kg,no infections occurred in either group (Fig. 4, D and E). In the low-dosegroup (0.08 mg/kg), three of four animals that received PGDM1400were infected (Fig. 4F), whereas none of the animals that receivedCAP256-VRC26.25-LS were infected (Fig. 4G). These data demonstratethat the in vitro neutralization potency of these V2-specific antibodiestranslates into protective efficacy in vivo at doses of 0.08 to 0.4 mg/kg.

Pharmacokinetics of PGDM1400 and CAP256-VRC26.25-LSin vivoSerum samples were obtained throughout the study, and PGDM1400and CAP256-VRC26.25-LS concentrations were determined byenzyme-linked immunosorbent assay (ELISA). The results show thatthe animals that received PGDM1400 at 2, 0.4, and 0.08 mg/kg hadaverage serum antibody concentrations of 6.9, 2.5, and 0.22 mg/ml atthe time of challenge (Fig. 5). In contrast, serum concentrations ofCAP256-VRC26.25-LS were 19.8, 3.3, and 0.75 mg/ml for the animals thatreceived 2, 0.4, and 0.08 mg/kg of CAP256-VRC26.25-LS, respectively.Serum concentrations of CAP256-VRC26.25-LS were thus slightly higherthan PGDM1400 (P = 0.02 to 0.03 for all dose groups) at the time of

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Table 2. Neutralization properties of three different SHIV viruses in TZM-bl assays against a panel of bNAbs targeting distinct epitopes. IC50 and IC80values are in micrograms per milliliter. Values between 0.001 and 0.01 mg/ml are highlighted in red; between 0.01 and 0.1 mg/ml, orange; between 0.1 and 1 mg/ml,yellow; between 1 and 10 mg/ml, light green; and between 10 and 50 mg/ml, dark green.

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challenge, likely reflecting differences in distribution or elimination ofthese two antibodies. The half-life of PGDM1400 based on the level ofserum antibody decay (two-phase exponential decay) was calculated to be7.7 days for the 2-mg/kg dose group, 6.7 for the 0.4-mg/kg dose group,and 6.3 for the 0.08-mg/kg group. The half-life of CAP256-VRC26.25-LSin this study was calculated to be 10.3 days for the 2 mg/kg-dose group,9.9 days for the 0.4-mg/kg dose group, and 7.7 days for the 0.08-mg/kgdose group, consistent with the initial experiment (fig. S2). The higherserum concentrations and more potent neutralization activity ofCAP256-VRC26.25-LS compared to PGDM1400 against SHIV-325ccorrelated with its higher protective efficacy.

Sequence analysis of breakthrough virusesAlthough only 4 of 14 PGDM1400 pretreated animals developedplasma viremia, we were interested to examine potential escape/


resistance pathways in SHIV-325c after PGDM1400 administration.We therefore generated single-genome env sequences from plasmaviruses at the time of peak viremia after breakthrough infection.Overall, there was very little sequence variation (mean divergence,0.08 to 0.19%) between the challenge strain and the plasma viruses.We then focused on the variable loop 1 and 2 region, and no specific pat-tern of sequence variation was noticeable when comparing PGDM1400and saline control animals (Fig. 6). Position 167 in V2, a known resist-ance mutation for CAP256 lineage antibodies (18, 28, 29), showedD/N and rare D/E changes in several of the bNAb-pretreated animalsbut also in the saline control animals, and the D/N variant was alsofound in the challenge stock. Mutations at this position (D/A) also con-fer resistance to PGDM1400 (table S2), suggesting that the rare D/E var-iation seen in the PGDM1400-pretreated animals could be an escapepathway. Similarly, one PGDM1400-pretreated animal developed a

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Fig. 2. Neutralization profiles of PGDM1400 and CAP256-VRC26.25 againstmulticlade pseudoviruses. (A) Each bar represents the IC80 (mg/ml) of PGDM1400(top graph) or CAP256-VRC26.25 (bottom graph) against a single virus. Viruses areranked according to increasing IC80 values. Bars reaching the dotted line representIC80 values >50 mg/ml. The red bars highlight the IC80 values against SHIV-325c.(B) IC80 values for PGDM1400 and CAP256-VRC26.25 against SHIV-325c (red dot)and against the clade C pseudoviruses included in (A). The horizontal bars representthe mean of IC80 values of neutralizable pseudoviruses.

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D/E mutation at position 165, which has been associated with escapefrom CAP256 lineage antibodies (30). Although we do not see reducedneutralization susceptibility for PGDM1400 with an I/A mutationat this position (table S2), the effect of I/E needs to be evaluated. Inanimal M97, which received 2 mg/kg of PGDM1400 but neverthelessdeveloped high plasma viremia at day 28, no variant stood out in theV1V2 region, but a fixed mutation (F210L) was observed in all 27 ampli-cons generated from this animal (Fig. 6, mutation F210L is marked with •).This variant was absent in the challenge stock and was not detected inany other animal. Whether this mutation contributed to viral escapefrom PGDM1400 remains to be determined.

Complementary coverage of HIV-1 clade C viruses byV2- and V3-specific antibodiesPGDM1400 and CAP256-VRC26.25 both have potent neutralizationactivity against HIV-1 clade C viruses and exhibited 61 to 65% coverage


of a large panel of 200 clade C env pseudoviruses (median IC80 of 1.17and 0.19 mg/ml for PGDM1400 and CAP256-VRC26.25, respec-tively) (Fig. 7A). A previous study reported that the combination ofPGDM1400 with the V3 glycan–dependent antibody PGT121 provided98% coverage of multiclade viruses with a median IC50 of 0.007 mg/ml(22). We therefore determined whether the coverage of clade C viruseswould improve if PGDM1400 or CAP256-VRC26.25 was combinedwith PGT121. PGT121, which covers 65% of the clade C virus panelat a median IC80 of 1.85 mg/ml (23), targeted many of the “gaps” in viralcoverage of PGDM1400 and CAP256-VRC26.25 (Fig. 7A). PGT121plus CAP256-VRC26.25 as well as PGT121 plus PGDM1400 showedrobust coverage of >90% of clade C viruses and additive potency ascompared to single bNAbs (IC80 for combinations 0.017 and 0.07 mg/ml,respectively) (Fig. 7B).

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DISCUSSIONThe V2 region of the HIV-1 Env trimer is a common target of bNAbs,and some bNAbs directed against this epitope have demonstratedexquisite neutralization potency and breadth. Our findings demon-strate the capacity of the V2-specific antibodies PGDM1400 andCAP256-VRC26.25-LS to protect rhesus macaques at extremely lowtiters against high-dose challenge with SHIV-325c. Protection wasachieved at low serum antibody concentrations of <0.75 mg/ml forCAP256-VRC26.25-LS, consistent with the extraordinary neutraliza-tion potency of these bNAbs.

Protection at such low antibody concentrations has not previouslybeen reported (31). Previous studies have tested the protective capacityof several bNAbs, including the CD4 binding site mAbs VRC01 and3BNC117 as well as the V3 glycan–dependant bNAbs PGT121 and10–1074 against various SHIV strains (for example, SHIV-AD8EO,SHIV-SF162P3, and SHIV-BaL) (9–11). Average serum concentrationsat the time of challenge necessary for protection were variable but

Fig. 3. Neutralization of SHIV-325c by CAP256-VRC26.25, PGDM1400, andPG9. Neutralization was measured using replication-competent challenge stockSHIV-325c infection of TZM-bl cells.

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Fig. 4. Protective efficacy of PGDM1400and CAP256-VRC26.25-LS against SHIV-325c in rhesus macaques. Plasma viralRNA (vRNA) (log RNA copies/ml) are shownfor animals that received saline control (A),PGDM1400 (2 mg/kg) (B ) , CAP256-VRC26.25-LS (2 mg/kg) (C), PGDM1400 (0.4 mg/kg) (D), CAP256-VRC26.25-LS (0.4 mg/kg) (E), PGDM1400 (0.08 mg/kg) (F), or CAP256-VRC26.25-LS (0.08 mg/kg) (G).
The assay sensitivity limit was >50 RNA copies/ml.
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higher than that reported here (31). Thelowest serum antibody concentrationsthat conferred complete protection werewith PGT121 at 15 and 22 mg/ml againstSHIV-SF162P3 and SHIV-AD8EO chal-lenges, respectively, and partial or no pro-tection was seen at serum level of 1.8 mg/ml(9, 10). VRC01 only protected 4 of 10 ani-mals against the neutralization-sensitivetier 1 virus SHIV-BaL at serum concen-tration of 1.3 mg/ml, whereas 10E8 onlyprotected 3 of 6 animals against this virusat serum concentration of 1.8 mg/ml (11).The low doses of PGDM1400 and CAP256-VRC26.25-LS required for protection sug-gest that they could be feasibly developedfor subcutaneous administration (32).

No previous studies have assessed theprotective efficacy of V2-specific bNAbsagainst tier 2 SHIV challenges, and thus,the present studies help validate V2 asa protective target for bNAbs. PG9 isthe only other V2-specific bNAb that

Fig. 5. Serum concentrations of CAP256-VRC26.25-LS and PGDM1400 in antibody-treated animals. Serumantibody concentrations of CAP256-VRC26.25-LS (A) and PGDM1400 (B) were determined by ELISA. The dottedvertical line marks the day of SHIV challenge.


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Fig. 6. Analysis of SHIV-325c V1V2 envelope sequences in breakthrough infections. Single-genome envelope amplification was performed with plasma viral RNAon day 28. For one saline control animal, H515, viral amplification failed and therefore is not shown here. The env sequence at the top presents the SHIV-325c molecularclone inoculum. The column on the right reports the frequency of distinct amplicons (based on unique sequence variations in V1V2) per total env amplicons obtainedfrom a given animal.

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has been evaluated for protective efficacy to date in nonhuman pri-mates. Partial protection was achieved at 5 mg/kg, and no protectionwas observed at 0.3 mg/kg (serum antibody levels at day of challengewere 3.7 and 0.28 mg/ml, respectively) against the neutralization-sensitive tier 1 virus SHIV-BaL, despite a mean IC50 of 0.06 mg/mlagainst SHIV-BaL (11). It remains to be determined whether the in-ability of PG9 to achieve 100% neutralization of the challenge stockby TZM-bl assays in vitro (33) may have affected its protective efficacyin vivo.

SHIV-325c was neutralized by PGDM1400 and CAP256-VRC26.25 at IC80 values that were comparable to the medianIC80 values against large global virus panels. In addition to theirexquisite potency, both PGDM1400 and CAP256-VRC26.25 have60 to 65% (IC80) of 200 tested clade C viruses. The pattern of resistantviruses was relatively similar for these two V2-specific bNAbs,suggesting that these antibodies could be combined with otherbNAbs that target different epitopes to improve coverage.PGT121 has demonstrated potent antiviral activity in both therapeuticand protection studies in rhesus macaques (9, 34) and shows strikingcomplementary coverage with both PGDM1400 and CAP256-VRC26.25-LS here. This complementarity increases viral coverage ofthese antibody combinations to >90% of clade C viruses (23), suggestingthat PGT121 may be a useful antibody to coadminister with eitherCAP256-VRC26.25-LS or PGDM1400 to prevent cladeCHIV-1 infectionin South Africa.

This study has several limitations: We did not measure anti-PGDM1400 or anti–CAP256-VRC26.25-LS antibodies in this studyto determine whether bNAb levels were affected. Although xenogeneicresponses against human antibodies are commonly induced inmacaques (27), no anti-antibody responses have been observedto date in clinical studies with VRC01 and 3BNC117 (35, 36).Both bNAbs protected at low serum antibody concentrations againstour clade C SHIV, and although PGDM1400 and CAP256-VRC26.25are active against 57 to 83% of global viral isolates (20–22), we did notassess in vivo protection against non–clade C SHIVs. Furthermore, theaddition of PGT121 to either PGDM1400 or CAP256-VRC26.25increased viral coverage of clade C virus strains in vitro, but a small per-centage of viruses remained resistant to both antibodies. A fully protectivebNAb cocktail might therefore require three bNAbs to fully cover globalviral diversity.

V1V2 and V3 are in close spatial proximity (37, 38), and thus, ithas been suggested that coadministration of antibodies targeting theseregions (for example, PGT121 together with either PGDM1400 orCAP256-VRC26.25-LS) might affect each antibody’s binding kinetics.Previous data have suggested a certain level of competition betweenPGT121 and the V2-specific antibody PG9 (5), but this was notobserved for PGDM1400, which showed very little to no competitionwith PGT121 (22). Similarly, binding of CAP256-VRC26.08, which isfrom the same lineage as CAP256-VRC26.25, was only minimallyreduced by PGT121 in a standard binding competition assay (20).These data suggest that, overall, no significant reduction in antibodybinding should be expected when these V2- and V3-specific bNAbsare coadministered.

In summary, our data demonstrate that potent protective efficacycan be achieved with bNAbs that target the HIV-1 env trimer apex atvery low antibody doses. PGDM1400 and CAP256-VRC26.25-LS,particularly when combined with a V3 glycan–dependent antibodysuch as PGT121, should therefore be explored in clinical trials forHIV-1 prevention.

Fig. 7. Potency and breadth profiles of single and combination bNAbs against200 clade C HIV-1 Env pseudoviruses. (A) Heat maps of IC80 values for singlebNAbs and bNAb combinations. Rows represent Env pseudoviruses, and columnsrepresent single and combination bNAbs. Darker hues of red indicate more potentneutralization, and gray cells indicate IC80 above threshold. (B) Potency-breadthcurves for single bNAbs and bNAb combinations are shown. IC80 scores for combina-tions and single bNAbs were compared using Wilcoxon rank-sum test. Antibody a,PGDM1400; antibody b, CAP256-VRC26.25; antibody c, PGT121.

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MATERIALS AND METHODSAnimals and study designThe overall objective of this study was to investigate the protectiveefficacy of two HIV-1 envelope V2 apex–specific antibodies against aclade C SHIV challenge in rhesus macaques. Two additional substudieswere performed (i) to characterize infectivity and pathogenicity of theSHIV-325c challenge stock in rhesus macaques and (ii) to determinethe pharmacokinetics and half-life of CAP256-VRC26.25 with the LSmutation in rhesus macaques. No randomization or blinding wasperformed for any study. Saline controls were used for the main protectionstudy to measure the protective efficacy of a single infusion of PGDM1400or CAP256-VRC26.25-LS at different doses. For this study design, aminimum of four animals per group was used similar to previous studies(10, 11, 39). The number of animals analyzed is stated in the figurelegends. Primary data for the substudies can be found in table S3.SHIV-325c infection studyEight Indian-origin, outbred, young adult, male and female, experimentallynaïve rhesus macaques (Macaca mulatta) that did not express the class Ialleles Mamu-A*01, Mamu-B*08, and Mamu-B*17 associated withspontaneous virological control were housed at Bioqual Inc. Animalswere atraumatically challenged via the intrarectal route with 500TCID50 of SHIV-325c. All animals were monitored for viral loadsand CD4+ T cell counts. The animal studies were approved by theappropriate Institutional Animal Care and Use Committee (IACUC).The study was carried out in strict accordance with the recommendationsin the Guide for the Care and Use of Laboratory Animals of the NationalInstitutes of Health (NIH).CAP256-VRC26.25 pharmacokinetic studyAll animal experiments were reviewed and approved by the AnimalCare and Use Committee of the Vaccine Research Center, NationalInstitute of Allergy and Infectious Diseases (NIAID), NIH, and allanimals were housed and cared for in accordance with local, state, federal,and institute policies in an American Association for Accreditation ofLaboratory Animal Care–accredited facility at the NIH. Eight Indian-origin rhesus macaques were administered low-endotoxin antibodypreparations [<1 EU (endotoxin unit)/mg] intravenously at 10 mg/kgof body weight. Whole-blood samples were collected before injectionand at multiple time points until week 4 after administration.SHIV-325c protection studyThirty-five Indian-origin, outbred, young adult, male and female,experimentally naïve rhesus macaques that did not express the classI alleles Mamu-A*01, Mamu-B*08, and Mamu-B*17 associatedwith spontaneous virological control were housed at Bioqual Inc.and Alphagenesis Inc. Animals were randomly allocated to the differentantibody dose and saline control groups. The antibody or saline wasadministered intravenously 24 hours before the animals wereatraumatically challenged via the intrarectal route with 500 TCID50 ofSHIV-325c. Serum samples for antibody detection and viral loaddetermination were obtained at week −1 and days 0, 1, 3, 7, 14, 28,42, and 56. The animal studies were approved by the appropriateIACUC. The study was carried out in strict accordance with therecommendations in the Guide for the Care and Use of LaboratoryAnimals of the NIH.

Construction of SHIV-325c molecular cloneSHIV-325c was generated from an env sequence derived from an earlyHIV-1–infected individual, CA325 (24, 40). The env sequence was synthe-sized by GeneArt (Invitrogen) and inserted into the KB9-AC plasmid asdescribed (40, 41). The plasmid was sequenced and transfected into 293T


cells using LipoD293 (SignaGen Laboratories). Cell culture supernatantswere collected after 48 hours and clarified through a 0.45-mm filter.

Generation of large-scale SHIV stocksFor the generation of the SHIV-325c virus stock, PBMCs were isolatedfrom 120 ml of deidentified human buffy coats that were purchased. Cellculture supernatants harvested from transiently transfected 293T cellswere used to inoculate to ConA-stimulated PBMCs in the presenceof human interleukin-2 (20 U/ml; AIDS Research and Reference ReagentProgram). The medium was replaced and collected every 3 days. Virus wasquantified by SIV p27 ELISA (Zeptometrix), and the TCID50 wasdetermined in TZM-bl cells (40). Virus was aliquoted and stored at −80°C.

Antibody productionPGDM1400 was generated as previously described (5) and purified byProtein A affinity matrix (GE Healthcare). CAP256-VRC26.25-LS waspurified after transient transfection of Expi293 cells (Invitrogen)with expression vectors encoding for its heavy and light chains.Phosphate-buffered saline (PBS) was used as control in this study.The CAP256-VRC26.25-LS antibody included the Fc region mutation(LS) (27) that increased circulating half-life.

Synthesis and purification of the CNE58-strandC-CAP256.SUSOSIP trimer for ELISAHIV-1 gp140 SOSIP-type molecule based on the clade C strainCNE58 (42–44), including the “SOS” mutations (A>501C andT605C), the isoleucine-to-proline mutation at residue 559 (I559P),the mutation of the cleavage site to 6R (REKR to RRRRRR), and thetruncation of the C terminus to residue 664 (all HIV-1 Env numberingaccording to the HX nomenclature), was used in this study. The CNE58SOSIP construct also used a chimera strategy by using the gp41 and Nand C termini of BG>505 and a part of the V1V2 C strand (residues 166to 173) from the CAP256.SU strain (20, 28) and a C-terminal six–aminoacid glycine-serine linker, HRV-3c cleavage site, His8 purification tag(GGSGGSGLEVLFQGPGHHHHHHHH). The HIV-1 SOSIP moleculewas expressed as previously described (45) by cotransfecting with furin inhuman embryonic kidney–293F cells using HIV-1 SOSIP DNA and furinplasmid DNA. Transfection supernatants were harvested after 6 days, andthe CNE58-strandC-CAP256.SU HIV-1 trimer supernatant was passedover a nickel–nitrilotriacetic acid affinity column. The eluate was con-centrated, and the peak corresponding to trimeric HIV-1 Env was iden-tified using size exclusion chromatography, pooled, and concentrated.

Enzyme-linked immunosorbent assayPGDM1400 and CAP256-VRC26.25-LS antibody concentration in serumwas determined by ELISA as described (46) using the BG505 SOSIP trimeror the novel CNE58-strandC-CAP256.SU SOSIP trimer, respectively.Briefly, microtiter plates were coated with SOSIP trimer (1 mg/ml) andincubated overnight at 4°C. The plates were washed with PBS/0.05%Tween 20 and blocked with PBS/1% casein (Pierce). After blocking,serial dilution of serum samples was added to the plate and incubatedfor 2 hours at 37°C. Binding was detected with a horseradish peroxidase–conjugated goat anti-human immunoglobulin G (IgG) secondary antibody(Fisher Scientific) and visualized with SureBlue tetramethylbenzidinemicrowell peroxidase (KPL Research Products).

Neutralization assaysNeutralization of diverse, replication-competent SHIV challengestocks (including SHIV-325c) or Env pseudoviruses was evaluated

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in vitro by using TZM-bl target cells and a luciferase reporter assay asdescribed (11, 47, 48). Briefly, HIV-1 Env pseudoviruses were generatedby transfection in 293T cells, and SHIV challenge stocks were producedby transfection of 293T cells with infectious molecular clone plasmids,followed by propagation in human or rhesus PBMCs, as describedabove and as indicated in Table 1. The rhesus PBMC–derived stockof SHIV-AD8 was provided by M. Martin (12), and SHIV-1157ipd3N4was obtained through the AIDS Reagent Program, Division of AIDS,NIAID, NIH from R. Ruprecht. SHIV stocks or HIV-1 pseudoviruseswere incubated with the antibody for 30 min at 37°C before TZM-blcells were added. The protease inhibitor indinavir was added to assayswith SHIV stocks to a final concentration of 1 mM to limit infection oftarget cells to a single round of viral replication. Luciferase expressionwas quantified 48 hours after infection upon cell lysis and the additionof luciferin substrate (Promega).

SGA and analysisSGA followed by direct sequencing of the Env gene was used to eliminateTaq-induced errors and in vitro recombination as described (49). Briefly,viral RNA was isolated from plasma using a QIAamp viral RNA kit(Qiagen). Reverse transcription of RNA to single-stranded complementaryDNA (cDNA) was performed using SuperScript III Reverse Transcriptaseaccording to the manufacturer’s recommendations (Invitrogen). In brief, acDNA reaction of 1× RT buffer, 0.5 mM of each deoxynucleotidetriphosphate (dNTP), 5 mM dithiothreitol, RNase OUT [RNase (recombinantribonuclease) inhibitor] (2 U/ml), SuperScript III Reverse Transcriptase(10 U/ml), and 0.25mM antisense primer EnvB3out 5′-TTGCTACTTGT-GATTGCTCCATGT-3′ was performed. RNA, primers, and dNTPs wereheated at 65°C for 5 min and then chilled on ice for 1 min; then, the entirereaction was incubated at >50°C for 60 min, followed by 55°C for anadditional 60 min. Finally, the reaction was heat-inactivated at 70°Cfor 15 min and then treated with 1 ml of RNase H each at 37°C for 20min. Then, cDNA templates were serially diluted until only a fraction(~25%) of amplicons are polymerase chain reaction (PCR)–positiveunder the following PCR conditions: PCR amplification was carriedout using the Platinum Taq (Invitrogen) with 1× buffer, 2 mMMgCl2,0.2 mM each dNTP, 0.2 mM each primer, and 0.025 U/ml PlatinumTaq polymerase. The primers for the first-round PCR were EnvB5out5′-TAGAGCCCTGGAAGCATCCAGGAAG-3′ and EnvB3out 5′-TTGCTACTTGTGATTGCTCCATGT-3′. The primers for the second-round PCR were EnvB5in 5′-CACCTTAGGCATCTCCTATGGCAG-GAAGAAG-3′ and EnvB3in 5′-GTCTCGAGATACTGCTCCCACCC-3′. The cycler parameters were 94°C for 2 min, followed by 35 cycles of94°C for 15 s, 55°C for 30 s, and 68°C for 4 min, followed by a finalextension of 68°C for 10 min. The product of the first-round PCR (1 ml)was subsequently used as a template in the second-round PCR under thesame conditions but with a total of 45 cycles. All PCR-positive ampliconswere directly sequenced using BigDye Terminator chemistry (AppliedBiosystems). Any sequence with evidence of double peaks was excludedfrom further analysis.

Statistical analysesAnalyses of independent data were performed by two-tailedMann-WhitneyU tests,Wilcoxon rank-sum test, or paired t test. P values less than 0.05were considered significant. Combination IC80 titers were calculated usingthe Bliss-Hill model, which has been shown to provide accurate predictionsof combination neutralization properties using those of individual bNAbs(23). Statistical analyses were performed using GraphPad Prism or the Statsmodule in Scipy (www.scipy.org2015).


SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/406/eaal1321/DC1Fig. S1. Highlighter amino acid sequence alignment of env derived from the SHIV-325c stockand the parental HIV-1 env.Fig. S2. Serum concentration of CAP256-VRC26.25-LS and CAP256-VRC26.25 in naïve,uninfected rhesus macaques.Table S1. IC80 (mg/ml) values of PGDM1400 and CAP256-VRC26.25 against SHIV-325c and amulticlade panel of 208 HIV-1 pseudoviruses.Table S2. Neutralization of JR-CSF alanine variants by PGDM1400 or PG9.Table S3. Primary data for Fig. 1 and fig. S2.

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Acknowledgments: We thank M. Lewis and W. Rinaldi for clinical conduct of the animalstudies and J. Baalwa, D. Ellenberger, F. Gao, B. Hahn, K. Hong, J. Kim, F. McCutchan,D. Montefiori, L. Morris, J. Overbaugh, E. Sanders-Buell, G. Shaw, R. Swanstrom, M. Thomson,S. Tovanabutra, C. Williamson, and L. Zhang for the HIV-1 envelope plasmids. Funding: Thiswork was supported by the NIH (AI106408, AI096040, AI100663, AI124377, AI126603, andHHSN261200800001E), amfAR (109219), the Ragon Institute of Massachusetts General


Hospital, Massachusetts Institute of Technology, and Harvard University, and theintramural research program of the Vaccine Research Center, NIAID, NIH. The contentof this publication does not necessarily reflect the views or policies of the U.S. Departmentof Health and Human Services, nor does the mention of trade names, commercialproducts, or organizations imply endorsement by the U.S. government. Authorcontributions: Project planning was performed by D.H.B., D.R.B., J.R.M., R.A.K., L.M., andS.S.A.K; the SHIV-325c was developed by L.J.T. and P.A.; antibodies were generated byA.P., P.L.M., D.S., and X.C.; viral neutralization assays were performed and analyzed byM.K.L., R.T.B., A.P., D.S., S.D.S., N.A.D-R., and K.L.; env sequences were generated andanalyzed by B.F.K.; plasma viral loads were measured by P.A.; pharmacokinetic ELISA wasperformed by A.P., D.S., and K. Wang; synthesis and purification of CNE58-strandC-CAP256.SUSOSIP trimer for ELISA were performed by X.C., M.G.J., I.S.G., M.C., and P.D.K.; PGDM1400alanine scanning mutants were produced by D.S.; bNAb coverage/breadth analysis wasperformed by K. Wagh, M.S.S., and B.K.; and the manuscript was written by B.J., D.R.B., L.M.,J.R.M., and D.H.B. Competing interests: The authors declare that they have no competinginterests. Data and materials availability: The data presented in this paper are tabulated inthe main paper and in the Supplementary Materials. Materials are available with anappropriate material transfer agreement.

Submitted 25 October 2016Resubmitted 15 March 2017Accepted 14 August 2017Published 6 September 201710.1126/scitranslmed.aal1321

Citation: B. Julg, L. J. Tartaglia, B. F. Keele, K. Wagh, A. Pegu, D. Sok, P. Abbink, S. D. Schmidt, K. Wang, X. Chen, M. G. Joyce, I. S. Georgiev, M. Choe, P. D. Kwong, N. A. Doria-Rose, K. Le, M. K. Louder, R. T. Bailer, P. L. Moore, B. Korber, M. S. Seaman, S. S. Abdool Karim, L. Morris, R. A. Koup, J. R. Mascola, D. R. Burton, D. H. Barouch, Broadly neutralizing antibodies targeting the HIV-1 envelope V2 apex confer protection against a clade C SHIV challenge. Sci. Transl. Med. 9, eaal1321 (2017).

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against a clade C SHIV challengeBroadly neutralizing antibodies targeting the HIV-1 envelope V2 apex confer protection

Abdool Karim, Lynn Morris, Richard A. Koup, John R. Mascola, Dennis R. Burton and Dan H. BarouchDoria-Rose, Khoa Le, Mark K. Louder, Robert T. Bailer, Penny L. Moore, Bette Korber, Michael S. Seaman, Salim S.D. Schmidt, Keyun Wang, Xuejun Chen, M. Gordon Joyce, Ivelin S. Georgiev, Misook Choe, Peter D. Kwong, Nicole A. Boris Julg, Lawrence J. Tartaglia, Brandon F. Keele, Kshitij Wagh, Amarendra Pegu, Devin Sok, Peter Abbink, Stephen

DOI: 10.1126/scitranslmed.aal1321, eaal1321.9Sci Transl Med

antibody, suggesting that passive immunization with these types of antibodies might be protective in people.with a novel clade C SHIV strain and observed protection at very low concentrations of circulating neutralizingantibodies given prophylactically could prevent infection from taking place. They tested this in nonhuman primates

reasoned that potent anti-V2 HIVet al.neutralizing antibodies, such as the V2 loop of the envelope protein. Julg HIV is thought to be an elusive virus, but there are multiple epitopes on HIV that can be targeted by

Potently protective antibodies

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broadly neutralizing antibodies targeting the hiv-1 ... · to 5.2 log siv rna copies/ml), and 75%...

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