Envelope residue 375 substitutions in simian–humanimmunodeficiency viruses enhance CD4 binding andreplication in rhesus macaquesHui Lia, Shuyi Wanga, Rui Konga, Wenge Dinga, Fang-Hua Leea, Zahra Parkera, Eunlim Kima, Gerald H. Learna,Paul Hahna, Ben Policicchiob, Egidio Brocca-Cofanob, Claire Deleagec, Xingpei Haoc, Gwo-Yu Chuangd, Jason Gormand,Matthew Gardnere, Mark G. Lewisf, Theodora Hatziioannoug, Sampa Santrah, Cristian Apetreib, Ivona Pandreab,S. Munir Alami, Hua-Xin Liaoi, Xiaoying Sheni, Georgia D. Tomarasi, Michael Farzane, Elena Chertovac,Brandon F. Keelec, Jacob D. Estesc, Jeffrey D. Lifsonc, Robert W. Domsj, David C. Montefiorii, Barton F. Haynesi,Joseph G. Sodroskik, Peter D. Kwongc, Beatrice H. Hahna,1, and George M. Shawa,1

aDepartment of Medicine, University of Pennsylvania, Philadelphia, PA 19104; bCenter for Vaccine Research, University of Pittsburgh, Pittsburgh, PA 15261;cAIDS and Cancer Virus Program, Leidos Biomedical Research Inc., Frederick National Laboratory, Frederick, MD 21702; dVaccine Research Center, NationalInstitute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; eDepartment of Infectious Disease, Scripps ResearchInstitute, Jupiter, FL 33458; fBioqual, Inc., Rockville, MD 20852; gAaron Diamond AIDS Research Center, Rockefeller University, New York, NY 10016; hCenterof Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA 02215; iDepartment of Medicine, Duke University, Durham, NC 27710;jDepartment of Pathology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104; and kDepartment of Cancer Immunology and Virology, Dana-FarberCancer Institute, Harvard Medical School, Boston, MA 02215

Contributed by Beatrice H. Hahn, April 28, 2016 (sent for review March 30, 2016; reviewed by Shiu-Lok Hu, Paul Luciw, and Leo Stamatatos)

Most simian–human immunodeficiency viruses (SHIVs) bearing enve-lope (Env) glycoproteins from primary HIV-1 strains fail to infectrhesus macaques (RMs). We hypothesized that inefficient Env bind-ing to rhesus CD4 (rhCD4) limits virus entry and replication and couldbe enhanced by substituting naturally occurring simian immunode-ficiency virus Env residues at position 375, which resides at a criticallocation in the CD4-binding pocket and is under strong positive evo-lutionary pressure across the broad spectrum of primate lentiviruses.SHIVs containing primary or transmitted/founder HIV-1 subtype A, B,C, or D Envs with genotypic variants at residue 375 were constructedand analyzed in vitro and in vivo. Bulky hydrophobic or basic aminoacids substituted for serine-375 enhanced Env affinity for rhCD4,virus entry into cells bearing rhCD4, and virus replication in primaryrhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated withsubtype A, B, C, or D SHIVs all became productively infected withdifferent Env375 variants—S, M, Y, H, W, or F—that were differen-tially selected in different Env backbones. Notably, SHIVs replicatedpersistently at titers comparable to HIV-1 in humans and elicitedautologous neutralizing antibody responses typical of HIV-1. Sevenanimals succumbed to AIDS. These findings identify Env–rhCD4 bind-ing as a critical determinant for productive SHIV infection in RMs andvalidate a novel and generalizable strategy for constructing SHIVswith Env glycoproteins of interest, including those that in humanselicit broadly neutralizing antibodies or bind particular Ig germ-lineB-cell receptors.

HIV/AIDS | SHIV | CD4 | HIV-1 envelope

Simian–human immunodeficiency viruses (SHIVs), which expressthe envelope of HIV-1 within the context of simian immuno-

deficiency virus of macaque (SIVmac) structural and regulatoryelements, have broad potential applications for HIV/AIDS vaccine,treatment (cure), and pathogenesis research (1, 2). SHIVs havebeen used to assess the protective effects of neutralizing or anti-body-dependent cell-mediated cytotoxicity conferring antibodies(3–7), the elicitation of broadly neutralizing antibodies (bNAbs)(8–11), the efficacy of broadly neutralizing monoclonal antibodies(mAbs) in preclinical trials with suppressive and curative intent (12,13), and virus–host interactions underlying virus transmission, im-munodeficiency, and neurocognitive impairment (1, 14–22). De-spite these varied and important applications of SHIVs to HIV/AIDS research, it is widely recognized that most SHIVs developedthus far have significant shortcomings as a model system for HIV-1

infection of humans (reviewed in ref. 1). These limitations areoften related to properties of the envelope (Env), which in mostSHIVs does not faithfully reflect primary HIV-1 strains or trans-mitted/founder (T/F) viruses (23, 24). HIV-1 Env, and its coun-terparts in naturally infected chimpanzees (SIVcpz) and gorillas(SIVgor), have evolved over millennia to infect great ape and,more recently, human CD4 T cells, but not CD4 T cells of lowerprimates, including Indian rhesus macaques (RMs; Macacamulatta) (25–27). Thus, most HIV-1 Envs do not bindMacaca spp.CD4 efficiently. Overbaugh and coworkers (26) provided a partialmolecular explanation for this inefficiency by identifying a distinguishing

Significance

Simian–human immunodeficiency viruses (SHIVs) are an invaluabletool for assessing HIV-1 vaccines, developing therapeutic “cure”strategies, and understanding viral immunopathogenesis. How-ever, only limited success has been achieved in creating SHIVs thatincorporate HIV-1 envelopes (Envs) that retain the antigenic fea-tures of clinically relevant viruses. Here we focus on a critical resi-due of the CD4-binding region, Env375, which is under strongpositive selection across the broad range of primate lentiviruses.We find that genotypic variation of residue 375 allows for thecreation of pathogenic SHIVs that retain the antigenicity, tier 2neutralization sensitivity, and persistence properties characteristicof primary HIV-1 strains. Taken together, our findings suggest anew paradigm for SHIV design and modeling with important ap-plications to HIV-1 vaccine, cure, and pathogenesis research.

Author contributions: H.L., R.W.D., B.F.H., J.G.S., P.D.K., B.H.H., and G.M.S. designed re-search; H.L., S.W., R.K., W.D., F.-H.L., Z.P., E.K., G.H.L., P.H., B.P., E.B.-C., C.D., X.H., G.-Y.C., J.G.,M.G.L., T.H., S.S., C.A., I.P., S.M.A., H.-X.L., X.S., G.D.T., E.C., B.F.K., J.D.E., J.D.L., and D.C.M.performed research; M.G. and, M.F. contributed new reagents/analytic tools; H.L., R.W.D.,D.C.M., J.G.S., P.D.K., B.H.H., and G.M.S. analyzed data; and H.L., B.F.H., J.G.S., P.D.K., B.H.H.,and G.M.S. wrote the paper.

Reviewers: S.-L.H., University of Washington; P.L., University of California, Davis; and L.S.,Fred Hutchinson Cancer Research Center.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database (accession nos. KU955514 and KU958484–KU958489).1To whom correspondence may be addressed. Email: bhahn@upenn.edu or shawg@upenn.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1606636113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1606636113 PNAS | Published online May 31, 2016 | E3413–E3422

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polymorphism at position 39 in rhesus and pigtailed macaqueCD4 compared with human CD4. By necessity, then, SHIVsdeveloped thus far contain HIV-1 Envs derived from one of foursources: (i) laboratory-adapted CXCR4-tropic HIV-1 strains(28); (ii) viruses passaged in vitro so as to acquire Env adapta-tions necessary for efficient infection of rhCD4 T cells, but at thecost of reduced conformational masking and protection fromantibody neutralization (29–31); (iii) in vivo passaged andadapted virus swarms or clones (14, 15, 17–21, 32–34); or (iv)random screens of primary HIV-1 strains for rare viruses thatencode Envs that fortuitously bind rhCD4 and allow for rhesuscell entry and replication (35–38).The construction and development schemes of SHIVs have

also been complicated. Many SHIVs derive from the originalSHIV–HXBc2 construction of Sodroski and coworkers whereHIV-1 HXBc2 tat, rev, and env sequences were subcloned intoSIVmac239 (28, 39, 40). SHIV–HXBc2 was further modified bysubstitution of the env gene from the dual CCR5/CXCR4 tropicHIV-1 89.6 strain, followed by additional passages in RMs,resulting in a highly pathogenic CXCR4 tropic phenotype (14,15, 20). Henceforth, a molecular clone (SHIV–KB9) of thisstrain served as a preferred backbone for SHIV constructions,including four recent reports (35–38). Other SHIV constructionshave been similarly complicated (9, 18, 21, 29, 30, 32, 33).We took a different approach to SHIV design based on the

hypothesis that a critical determinant of successful SHIV replicationin RMs is efficient binding of Env to rhCD4. We further surmisedthat optimization of the SIVmac backbone and simplification of theHIV-1 insert strategy would enhance the chances of success. Ourstrategy was thus to: (i) molecularly clone a new SIVmac251-derivedT/F virus, pSIVmac766 (41, 42), that lacked any of four mutationsfound in SIVmac239 known to be suboptimal for replication (39,43); (ii) substitute into this vector complete tat–rev–vpu–env(gp160) cassettes of T/F or primary HIV-1 strains; and (iii) modifyEnv residue 375, which is part of the CD4-binding pocket andfacilitates CD4 engagement (44), to encode genotypic variants(Met, M; Tyr, Y; His, H; Trp, W; and Phe, F) found naturally atthis position across the broad evolutionary spectrum of primatelentiviruses (25) (www.hiv.lanl.gov/) (Fig. S1A). Here we engineerand test six new SHIVs containing Env glycoproteins from primaryor T/F strains of HIV-1 subtypes A, B, C, and D, each containingsix different Env375 variants. We then describe a vector platformand refinements to the design strategy that can streamline thedevelopment of next-generation “designer SHIVs.”

ResultsSelection of Env Residue 375. HIV-1 Env binding to the CD4 re-ceptor involves multiple structural rearrangements as Env transitionsfrom a prefusion closed state (45), through a single CD4-boundintermediate (46), to a more fully engaged Env trimer capable ofrecognizing the coreceptor (47). Although CD4 39I is the criticalalteration in rhCD4 impeding HIV-1 Env binding (Fig. S2A)(26), the corresponding residue in huCD4 (39N) does not actuallyinteract directly with HIV-1 Env, either in the prefusion closedstate or in the CD4-bound state (48) (Fig. S2 B–E). Instead, it liesproximal to the critical phenylalanine 43 (Phe-43) residue, whichmakes contact with Env residues in the Phe-43 binding pocket (48).In rhCD4, modeling suggested that 39I could subtly alter the angleof the Phe-43 binding loop (Fig. S2G).Because structural rearrangements in HIV-1 Env associated

with CD4 binding can be affected by large alterations in side-chainvolume, we analyzed SIV and HIV-1 group M Env residuesproximal to CD4 in the ligand-bound conformation for differencesin amino acid volume (Fig. S1B). We observed large differences attwo sites: residue 375, where a Ser in HIV-1 corresponds to alarger, generally hydrophobic amino acid in SIVs (Fig. S1B); andresidue 458. Of these, Env375 is closer to residue 43 of CD4 inboth prefusion closed and CD4-bound conformations (Fig. S2 B

and C). Moreover, in the HIV-1 YU2 backbone, a 375W sub-stitution for 375S allows Env to more readily sample CD4-boundconformations and enhances its affinity for human CD4 (25, 44, 49,50). These findings, along with phylogenetic evidence of positiveevolutionary selection at Env375 (Fig. S1A), suggested to us thatsubstitution of Env375S by residues containing larger hydrophobicor basic, space-filling side chains (e.g., M, Y, H, W, or F) mightenhance rhCD4 binding and SHIV replication in RMs.

SHIV Design. We cloned a replication-competent, pathogenic T/Fprovirus designated SIVmac766 (Fig. 1A) from a RM infected byatraumatic rectal inoculation with an early passage isolate ofSIVmac251 (41, 42). Complete tat–rev–vpu–env(gp160) cassettesfrom a prototypic primary subtype B strain YU2 (51) and from agenetically divergent T/F subtype D HIV-1 strain 191859 (52)were exchanged for the corresponding region of SIVmac766(Fig. 1 B and C). Site-directed mutagenesis was used to modify Envresidue 375 from Ser to Met, Tyr, His, Trp, or Phe in each env. Insubsequent SHIV designs, we incorporated a carboxy terminalgp41 tail exchange of 58 amino acids of SIVmac766 gp41 for thehomologous 33 amino acids of HIV-1 (SHIV.D.191859.dCT in Fig.1D and Fig. S3), because this substitution occurred spontaneouslyin infected RMs and enhanced SHIV replication in vitro and invivo (see below).We used a similar design strategy to exchange all or portions

of tat–rev–vpu–env(gp160) from other T/F HIV-1 strains into theSIVmac766 backbone, but a number of these constructs failed togenerate infectious virus. We attributed this inconsistency todifferences in the reading-frame organization of different HIV-1inserts relative to the SIVmac766 vpr/tat overlap, resulting inuntoward effects on RNA splicing and protein expression or to

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Fig. 1. SHIV construction scheme. (A–C) A complete tat–rev–vpu–env(gp160)segment of HIV-1 B.YU2 or D.191859 was substituted for the correspondingregion in SIVmac766. (D and E) SHIV.D.191859.dCT, which lost cloning sitelinkers (yellow bars in C) and the carboxy terminal 33 amino acids of HIV-1 gp41but with the carboxy terminus of SIVmac766 gp41 joined in-frame (Figs. S3 andS5A), served as a platform for further SHIV constructions containing vpu–env–gp140 segments of HIV-1 strains CH505, CH848, and BG505. Met-M, Tyr-Y,His-H, Trp-W, and Phe-F were exchanged for the wild-type Ser-S residue atposition 375 in Env. SIVmac766 and SHIV sequences are publicly available(GenBank accession nos. KU955514 and KU958484-9).

E3414 | www.pnas.org/cgi/doi/10.1073/pnas.1606636113 Li et al.

strain-specific differences in HIV-1 Tat or gp41, which must interactwith cognate motifs of SIVmac766 (i.e., the RNA TAR elementand Gag matrix protein, respectively). To avoid these potentialcomplicating factors and to develop a more consistent and reliablestrategy for generating SHIVs with a high likelihood of replicativesuccess, we refined our approach to exchange just the HIV-1 vpu–env(gp140) or env(gp140) cassettes into the highly infectious andreplicative prototypic clone SHIV.D.191859.dCT (Fig. 1 D and Eand Fig. S3). Importantly, this construction scheme allowed for theexchange of complete gp120 and gp41 extracellular and membranespanning domains, which contain the epitopes for all neutralizingantibodies (NAbs). This strategy was used to generate SHIVscontaining vpu–env(gp140) sequences from T/F HIV-1 subtype Cstrains CH505 and CH848 (53) and T/F subtype A strain BG505(54). These three viruses were of particular interest because theyrepresented additional HIV-1 subtypes, had elicited bNAbs in theirrespective human hosts (53, 54), and included the widely studiedpreclinical vaccine strains CH505 and BG505 (53, 55, 56). For allSHIVs (Fig. 1 B–E), we modified residue Env375S to encode Met,Tyr, His, Trp, or Phe to determine which residue was preferred forrhCD4 binding, infection, and replication in the respective Envbackgrounds. In addition, for SHIV.A.BG505, we modified residueEnv332T, which corresponds to the wild-type T/F virus (57)(GenBank accession no. DQ208458), to Env332N that is presentin the BG505 SOSIP.664 (58–60). All 36 SHIVs were sequence-confirmed.

Envelope Binding to Human and Rhesus CD4. A total of 36 SHIVDNAs (B.YU2, D.191859, C.CH505, C.CH848, A.BG505.332T,and A.BG505.332N, each containing six genotypic variants at Env375)were transfected into 293T cells for virus generation and biologicalanalysis. In every instance, virions were efficiently produced, generallyin the range of ∼1010 virions per mL, as determined by quantificationof vRNA by real-time RT-PCR or p27 antigen by ELISA(Dataset S1). All 36 SHIVs were highly infectious in TZM-blcells, which bear huCD4 and huCCR5 on their surface (61), withinfectivity to particle (I:P) ratios of 0.001–0.00002. When grownin primary rhCD4 T cells, these same SHIVs with favorableEnv375 substitutions reached I:P ratios as high as 0.02 whentitered in TZM-bl cells and 0.0006 in primary, activated rhCD4 Tcells (Dataset S1).We tested the effects of Env375 variation on the binding of Env

to human and rhesus CD4 by three different methods. The firstassay examined the ability of soluble rhCD4-Ig or huCD4-Ig to bindthe functional Env trimer on infectious SHIVs and inhibit virusentry into TZM-bl cells (Fig. 2A). Overall, B.YU2 Env375S wassubstantially more sensitive to huCD4-Ig and rhCD4-Ig than werewild-type Env375S alleles from the other SHIVs, which is consistentwith previous studies showing that YU2 Env-huCD4 affinity ishigh, thereby allowing it to use low levels of CD4 on humanT cells, macrophages, and microglia for entry (25, 44, 50, 62). Arank order of SHIV sensitivity to huCD4-Ig and rhCD4-Ig wasB.YU2 > D.191859 > C.CH505 > C.CH848 > A.BG505.332N andA.BG505.332T. For any particular Env375 substitution in any ofthe Env backbones, huCD4-Ig neutralized SHIVs at ∼100-foldlower concentrations than did rhCD4-Ig, indicating a substantiallyhigher Env affinity for huCD4 than for rhCD4. Envs containing thewild-type 375S residue were least sensitive to rhCD4-Ig or huCD4-Igentry inhibition. Substitutions at Env375 led to increased sensitivity torhCD4-Ig and huCD4-Ig inhibition by rank orderW,Y>H,F>M> S.As a group, Envs bearing the 375 Y, W, H, or F residueswere ∼10- to 100-fold more sensitive to rhCD4-Ig and huCD4-Igthan were Env375S or Env375M. Analyses using soluble huCD4and rhCD4 gave similar results. These findings indicate thatsubstitution of nonpolar aliphatic, bulky hydrophobic, or basicaromatic amino acids at Env375 substantially increases the affinityof the functional HIV-1 Env trimer for rhesus and human CD4.

We next analyzed whether Env375 mutations affected the bind-ing and entry of SHIVs in cells expressing rhesus or human CD4 ontheir surface (Fig. 2B). A canine cell line, ZB5, stably transduced toexpress rhCD4 and rhCCR5 at levels comparable to huCD4 andhuCCR5 on TZM-bl cells, was used to assess SHIV binding andentry. In ZB5 cells, SHIV B.YU2 was the only wild-type 375Svariant capable of achieving significant levels of infectivity. Thisresult correlated with the enhanced affinity of the SHIV B.YU2Env375S allele for soluble rhCD4-Ig (Fig. 2A). The 375M sub-stitution in all six SHIV backbones had either no effect or increasedvirus infectivity modestly (twofold to fourfold) (Fig. 2B). This resultagain correlated with the unchanged or modestly enhanced sensi-tivity of the Env375M allele to soluble rhCD4-Ig (Fig. 2A). Incontrast, the 375Y/H/W/F substitutions increased virus infectivity inZB5 cells markedly (10- to 1,000-fold) in all SHIV Env backbonesexcept B.YU2, where the effect was more modest. Overall, a gen-eral trend was observed between enhanced entry of SHIV Env375variants into ZB5 cells and enhanced sensitivity to entry inhibitionby soluble rhesus CD4-Ig, although this correlation was imperfect.These findings suggested that a threshold for SHIV Env–rhCD4binding must be reached in order for virus infection to occur, butthat additional postbinding events contribute to efficient virus entryand replication (see below).As a third measure of Env–CD4 binding, we tested by surface

plasmon resonance (SPR) the affinity of rhCD4-Ig and huCD4-Igfor monomeric HIV-1 Env gp120 containing each of the sixEnv375 variants and expressed in 293F cells. Fig. 2C shows rep-resentative SPR plots for binding of three D.191859 Env375

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genotypic variants to rhesus and human CD4-Ig, and Dataset S2summarizes results for all six Env375 variants of D.191859, B.YU2,and C.CH505. For D.191859 gp120, the association rate ka forrhCD4-Ig was not affected by residue 375 substitutions and rangedfrom 0.75 × 104 to 1.0 × 104 M−1s−1, whereas the dissociation ratekd ranged from 10.6 × 10−4 s−1 for 375S to 1.63 × 10−4 s−1 for 375W(Dataset S2). From these values, the binding constants (Kd) couldbe determined as follows: S375, 138 nM;M375, 105 nM; Y375, 78 nM;H375, 43 nM; F375, 25 nM; and W375, 21 nM (Fig. 2C andDataset S2). For huCD4-Ig,Kd values were as follows: S375, 34 nM;M375, 16 nM; Y375, 12 nM; H375, 7 nM; F375, 4 nM; andW375, 4 nM, again reflecting an overall higher affinity of thesubtype D.191859 Env gp120 for huCD4 compared with rhCD4.Similar trends were observed for YU2 and CH505 Env gp120(Dataset S2). In all cases, HIV-1 Envs had higher affinity forhuCD4 than for rhCD4, and substitution of 375S by 375M, 375H,375Y, 375F, or 375W led to higher binding affinity to human andrhesus CD4.

Neutralization Sensitivity of SHIVs with Env375 Mutations.We performeda comprehensive analysis of the sensitivity of all 36 SHIVs to a largepanel of neutralizing mAbs, polyclonal anti-HIV antibodies in patientsera, and the entry fusion inhibitor T1249. The objective was to de-termine whether amino acid substitutions at residue 375 altered Envantigenicity, neutralization sensitivity, or fusion efficiency. As con-trols, we analyzed the corresponding HIV-1 Envs containing the wild-type 375S residue expressed from full-length T/F HIV-1 proviralgenomes or as Env pseudotypes. In all six SHIV Env backbones,there were little or no appreciable effects of residue 375 substitutionson antigenicity or neutralization sensitivity for any mAb or immuneplasma tested (Fig. 3 and Fig. S4). This result included as many as sixdifferent CD4bs mAbs (b12, VRC01, 3BNC117, NIH 45-46 G54W,CH103, and CH31); seven V1V2 mAbs (PG9, PG16, PGDM1400,PGT145, CH01, VRC26.08, and VRC26.25); four V3 high mannosepatch mAbs (PGT121, PGT126, PGT128, and 2G12); a gp120/gp41interface mAb (8ANC195); membrane proximal external region(MPER) mAbs (4E10, 10E8, and 2F5); V3 epitope directed mAbs(3869, 3074, and 447–52D); and CD4-induced (CD4i) mAbs (17b,21c, and 19e). There were also no effects of Env375 substitutions onsensitivity to T1249, which indirectly assesses fusion efficiency (63).Importantly, we observed that the V3 and CD4i mAbs (64) did notneutralize any of the wild type SHIVs or their Env375 variants asidefrom modest activity against B.YU2 (Fig. 3 and Fig. S4). Thesefindings, together with the consistent neutralization patterns of theother mAbs shown in Fig. 3 and Fig. S4, including V1V2 mAbs thatrecognize quaternary epitopes, are strong indications that the 36SHIVs retain native-like Env trimer conformations and intact con-formational masking (65).For most SHIV – antibody combinations, we observed IC50 values

very similar to the corresponding wild type HIV-1 Envs. Theexceptions were enhanced SHIV.B.YU2 and SHIV.D.191859sensitivity to mAbs 4E10, 2F5, 10E8, and 8ANC195. These differ-ences may reflect the fact that the carboxy terminus of gp41 inSHIVs B.YU2 and D.191859 (Fig. 1 B and C and Fig. S3) is ofHIV-1 origin and must interact with the Gag matrix of SIVmac766,whereas for the HIV-1 controls the carboxy terminus of gp41and the Gag matrix are both of HIV-1 derivation. This gp41–Gag matrix mismatch was not a confounding factor for SHIVsA.BG505.332N.dCT, A.BG505.332T.dCT, C.CH505.dCT orC.CH848.dCT because they contain chimeric Envs where thecarboxy terminus of gp41 is of SIVmac766 derivation (Fig. 1Eand Fig. S3). We show below that SHIVs containing an SIVmacgp41 terminus have higher Env:Gag ratios, higher I:P ratios, andhigher replication rates in rhCD4 T cells compared with SHIVsthat contain an HIV-1 gp41 terminus. Thus, the gp41–Gag matrixmismatch for SHIVs B.YU2 and D.191859 (but not for the otherSHIVs) may alter slightly the per virion number and geometry ofthe Env trimer complex as it is tethered to the virus membrane or

retard virus fusion sufficiently to allow for greater neutralizationactivity to be exerted by MPER and gp120/gp41 interface mAbs.

Replication of SHIVs in Human and Rhesus CD4 T Cells. Env375 vari-ants of SHIVs B.YU2, D.191859, C.CH505.dCT, C.CH848.dCT,A.BG505.332N.dCT, and A.BG505.332T.dCT were tested for repli-cation in primary, activated human and rhesus CD4 T cells (Fig. 4).All 36 SHIVs replicated in human cells efficiently with the wild-typeEnv375S generally equaling or exceeding other Env375 variants. Thisresult is not surprising, because Env375S is the residue found mostcommonly in HIV-1 group M strains (Fig. S1). In contrast, theEnv375 variants exhibited very different patterns of replication inrhCD4 T cells. For SHIVs D.191859, C.CH505.dCT, C.CH848.dCT,A.BG505.332N.dCT, and A.BG505.332T.dCT, replication of thewild type Env375S variant was very low or undetectable (Fig. 4).For SHIV.B.YU2, the 375S variant replicated comparably withthe other Env375 variants. The Env375M variant of SHIVsB.YU2 and D.191859 (but not of C.CH505.dCT, C.CH848.dCT,A.BG505.332N.dCT, and A.BG505.332T.dCT) also replicatedwell in rhCD4 T cells. In all six SHIV Env backbones, the 375Y/H/Wvariants replicated well in rhCD4 T cells, generally to levelscomparable to those observed in huCD4 T cells, whereas Env375Freplication was variable. Thus, there was a good overall corre-lation among the different SHIV Env375 variants for enhancedbinding to rhCD4 (Fig. 2 A and C and Dataset S2), enhancedentry into cells bearing rhCD4 on their surface (Fig. 2B), andenhanced replication in primary activated rhCD4 T cells (Fig. 4).

Replication of SHIVs in RMs. Given the encouraging in vitro replica-tion data, we inoculated a total of 24 RMs with SHIVs by intravenousor intrarectal routes (Fig. 5). Because we were initially uncertain ofthe likelihood of productive infection, we depleted a subset of RMs

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E3416 | www.pnas.org/cgi/doi/10.1073/pnas.1606636113 Li et al.

of CD8+ cells by infusing mAb MT807R1 on the day of virusinoculation. Fig. 5A shows results for two CD8-depleted RMsinoculated intravenously with equal mixtures of the six Env375variants of SHIV.D.191859 and two other RMs inoculated in-travenously with equal mixtures of the six Env375 variants ofSHIV.B.YU2. Persistent viremia was observed for 62–86 wk inall four animals, with substantially higher viral load setpoints forSHIV.D.191859 than for SHIV.B.YU2. Both SHIV.D.191859-infected animals developed AIDS at weeks 62 (RM138) and 86(RM131) and were euthanized, whereas SHIV.B.YU2 animalsremained healthy beyond 86 wk.To determine which Env375 variants best supported virus repli-

cation in the SHIV.D.191859 and SHIV.B.YU2 backbones, weperformed deep sequencing by Illumina MiSeq (Fig. 5F) and agenome-wide analysis by single genome sequencing (SGS) (Fig.S5A) (66). MiSeq analysis at a depth of >2,500 vRNA sequencesshowed that both SHIV.D.191859-inoculated animals were initiallyinfected by SHIVs bearing Env375M, -Y, -H, and -F, but not byEnv375S or -W (Fig. 5F). Env375M replicated preferentially in bothanimals at weeks 3, 10, and 20 after infection. In SHIV.B.YU2-inoculated animals, all six Env375 variants led initially to pro-ductive infection, although Env375S, and to a lesser extentEnv375M, replicated preferentially at all time points (Fig. 5F).SGS confirmed and extended these findings, demonstrating thatsequence variation at weeks 2 and 4 after infection was essentiallyrandom across the entire genome, aside from preferential replica-tion of Env375M, -Y, -H, and -F variants (Fig. S5A). The SGS datathus make the important point that during the initial 4-wk period ofinfection when virus replication is maximal, there is no apparentvirus adaptation or selection besides that involving the Env375substitution. This finding means that SHIV D.191859.Env375M,-Y, -H, -F variants are well fit from the outset and do not requireany additional mutations across their entire 9-Kb genomes toachieve efficient replication in RMs. By week 10, there wasevidence of strong selection for Env375M with additional

nonsynonymous mutations in Tat and Nef (RM131) and Vpx,Vpr, and Nef (RM138) corresponding to known or predictedcytotoxic T-cell epitopes (Fig. S5A).A striking finding in the week 10 and 20 sequences of both

RM131 and RM138 was a highly selected deletion of 104–125 nt ofthe carboxy terminus of HIV-1 gp41, including an 11-nt multiplecloning site used to introduce the HIV-1 tat–rev–vpu–env cassette(Fig. S5A). This deletion led to an in-frame juxtaposition of thecorresponding gp41 carboxy terminus of SIVmac766 encoded inthe nef overlap (Fig. S3). A 14- to 19-nt deletion of the multiplecloning site at the 5′ junction of the HIV-1 tat–rev–vpu–env cassettealso occurred. Similar, but smaller and less prevalent, deletionsoccurred in plasma viral sequences from RM72 and RM74. Wehypothesized that the substitution of the SIVmac766 gp41 carboxyterminus for the corresponding region of HIV-1 Env contributed to

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Fig. 5. SHIV replication and selection in RMs. (A) CD8-depleted RMs wereinoculated intravenously with a mixture of six Env375 variants (50 ng ofp27Ag of each variant) of SHIV D.191859 (RM131 and RM138) or SHIV B.YU2(RM72 and RM74). (B) Non-CD8-depleted RMs were inoculated intravenouslyor intrarectally with a mixture of SHIV D.191859 variants with or without aS375M substitution and with or without an SIV-for-HIV gp41 carboxy ter-minal tail exchange (50 ng of p27Ag of each variant intravenously; 500 ngof p27Ag of each variant intrarectally). (C and D) CD8-depleted RMs wereinoculated intravenously with a mixture of six Env375 variants of SHIVC.CH505.dCT or SHIV C.CH848.dCT, respectively (50 ng of p27Ag of eachvariant, blue; 500 ng of p27Ag of each variant, red). (E) Non-CD8-depletedRMs inoculated intravenously with a mixture of six Env375 variants of SHIVA.BG505.dCT with or without a T332N substitution (50 ng of p27Ag of eachvariant; see J for inocula). (F–J) Sequence analysis of SHIV inocula and SHIVevolution in infected RMs. Sequence depth was >2,500 sequences for eachMiSeq analysis (F and H–J) and ≥24 for SGS. The 293T-derived virus stock wasused for all inoculations. Animal deaths are indicated by a red cross.

Li et al. PNAS | Published online May 31, 2016 | E3417

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the persistent high-level replication observed in the twoSHIV.D.191859-infected animals by enhancing Env gp41–Gagmatrix interactions and leading to higher virion Env content,higher I:P ratios, and enhanced virus replication. We found sup-port for this hypothesis by showing that virion Env:Gag ratioswere elevated by 1.5- to 2.5-fold (Fig. S5B), I:P ratios were in-creased by 37- to 100-fold (Dataset S1), and replication in hu-man and rhesus CD4 T cells was increased by at least 10-fold(Fig. S5C) in SHIV.D.191859.dCT (Fig. 1D), compared withSHIV.D.191859 (Fig. 1C). Similar in vivo adaptations have beenseen previously in other SHIVs that contained redundant HIV-1and SIVmac239 gp41 carboxy-terminal coding sequences (20).Next, we tested directly the replication fitness advantage afforded

to SHIV.D.191859 by the SIV/HIV gp41 tail exchange and theSer-to-Met substitution at Env375 and, in the same experiment,the effects of intravenous vs. intrarectal virus inoculation andCD8+ T-cell depletion (Fig. 5B). Thus, we inoculated six non-CD8-depleted RMs by intravenous (n = 2 RMs) or intrarectal(n = 4 RMs) routes with SHIV.D.191859 containing an equalmixture of Env375M.dCT, Env375S.dCT, Env375M (withoutdCT), and Env375S (without dCT). All RMs became infectedafter a single inoculation. Early viral replication kinetics weresimilar between animals infected by intravenous vs. intrarectalroutes. Peak virus titers occurred at day 17 in both groups ofanimals and were indistinguishable in magnitude (range: 1.8 ×106 to 1.3 × 107 vRNA copies per mL; mean ± SEM: 6.0 × 106 ±1.9 × 106). Setpoint viremia titers were ∼100-fold higher in an-imals that were subjected to CD8+ cell depletion (Fig. 5A)compared with nondepleted animals (mean: 4.9 × 105 vs. 4.3 × 103;P < 0.0001) (Fig. 5B). SGS was used to determine which Env allelesreplicated preferentially 3 wk after infection by intravenous andintrarectal routes in the non-CD8-depleted animals. All six animalsshowed an essentially identical pattern of selection: Of a total of 210env sequences analyzed, 210 contained the SIV-for-HIV gp41 car-boxy terminal tail substitution, and 209 of 210 sequences containedEnv375M (Fig. 5G). Otherwise, sequence variation was again ran-dom across the complete Env gp160 and 3′ half genome. Takingthese data together, we found that in eight animals infected withEnv variants of SHIV.D.191859, there was strong selection favoringreplication of SHIV.D.191859.375M.dCT and no evidence of earlyvirus selection otherwise. For this reason, as well as the persistentreplication kinetics for >1 y, SHIV.D.191859.375M.dCT (Fig. 1D)was selected as a new molecular platform for further SHIV con-

structions, which included Envs from C.CH505, C.CH848, andA.BG505 (Fig. 1E).Four CD8 T-cell–depleted RMs were inoculated intravenously

with a SHIV.C.CH505.dCT.Env375 pool (Fig. 5C) and four otherswith a SHIV.C.CH848.dCT.Env375 pool (Fig. 5D). All eight animalsbecame productively infected and exhibited indistinguishableearly viral replication kinetics. One animal (RM6073) infectedby SHIV.C.CH505.dCT died 3 wk after infection from an un-related allergic event; a second SHIV.C.CH505-infected animal(RM6070) was euthanized at 36 wk after infection because of arapidly progressive AIDS-defining high-grade lymphoma, and athird SHIV.C.CH505-infected animal (RM6069) was euthanizedat 40 wk after infection with wasting, weight loss, rhesus cyto-megalovirus (RhCMV) colitis, and Pneumocystis carinii pneumo-nia (PCP), all AIDS-defining conditions. Two animals (RM6161and RM6024) infected by SHIV.C.CH848 developed AIDS withwasting, weight loss, RhCMV colitis, anemia, and thrombocyto-penia 10 and 14 wk after infection and were euthanized. The otheranimals remained healthy through the latest follow-up time points(52 and 36 wk, respectively) and had lower viral load setpointsthan did animals that progressed to AIDS. To determine whichEnv375 variants supported preferential virus replication in vivo,we performed MiSeq analyses on plasma vRNA 2–10 wk afterinfection. In SHIV.C.CH505-infected animals, Env375Y, H, W,and F comprised nearly 100% of the circulating virus at 2 wk, butby 10 wk, Env375H predominated at 87–99%, with minor frac-tions of 375Y and 375F (Fig. 5H). In SHIV.C.CH848-infectedanimals 4 wk after infection, Env 375H predominated at 54–80%with the remainder comprising nearly entirely 375W (Fig. 5I).Finally, we inoculated six non-CD8-depleted animals intravenously

with a SHIV.A.BG505.332T.dCT Env375 pool (n = 2 RMs), aSHIV.A.BG505.332N.dCT Env375 pool (n = 2 RMs), or an equalmixture of all 12 SHIV variants (n = 2 RMs). All six animalsbecame productively infected and exhibited similar acute viralreplication kinetics (Fig. 5E). Peak viral loads ranged from 9.6 ×105 to 7.2 × 107 vRNA copies/mL, and at 8 wk after infection, viralloads ranged from 6.62 × 102 to 1.1 × 106 vRNA copies/mL AnimalRM6447, with the highest setpoint virus load, developed wasting,weight loss, pancytopenia, RhCMV reactivation, colitis, and PCP,necessitating euthanasia at week 9, whereas the other five animalsremained clinically healthy through the most recent sampling timepoint at week 12. In all six animals, Env375Y replicated mostefficiently (83–95%) followed by Env375W (3–15%) (Fig. 5J).Interestingly, in the two RMs inoculated with SHIVs containingboth Env332T and Env332N, the two variants replicated nearlyequally in the initial 4 wk, indicating that there is no fitnessadvantage associated with the gain or loss of this antigenicallyimportant site within the BG505.Env375Y background.

Immunopathogenesis of SHIV Infections. In total, we studied SHIVinfections in 24 RMs: 12 with CD8 depletion and 12 without. SixCD8-depleted animals developed AIDS, compared with onenon–CD8-depleted animal (P < 0.05), and all were euthanizedand necropsied (Figs. S6 and S7 and Datasets S3 and S4). Threeanimals died of AIDS within 3 mo of infection (RM6424, RM6161,and RM6447) and were considered “rapid progressors” (67). Oneof these animals, RM6447, is notable because it was not subjectedto CD8 T-cell depletion before virus inoculation. All three rapidprogressors had evidence of multiorgan RhCMV disease (DatasetS4), with RM6447 also demonstrating PCP (Fig. S6D). Two ani-mals (RM6069 and RM6070) died at 9 mo after infection and twoothers at 15 and 21 mo (RM138 and RM131), which is more typicalof SIV infection (34, 67). All four of these latter animals had AIDS-defining conditions, including wasting, weight loss, multiorganRhCMV disease, PCP or atypical mycobacterial infection (Figs. S6and S7 and Dataset S4). We obtained virus isolates from terminalperipheral blood mononuclear cell samples from all seven animals

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E3418 | www.pnas.org/cgi/doi/10.1073/pnas.1606636113 Li et al.

that died with AIDS to characterize virus coreceptor use. In eachcase, R5 tropism was retained (Fig. S8).Peripheral blood CD4 and CD8 T-cell counts, along with effector

memory, transitional, central memory, and naïve subsets, wereassessed in all 24 RMs (Dataset S3). Animals that developed AIDSexhibited greater declines in absolute CD4 T-cell counts, CD4/CD8ratios, and CD4 Tem populations compared with animals that didnot develop AIDS. At their terminal blood draw, animals that de-veloped AIDS had marked depressions in all CD4 T-cell subsets.Analysis of gut-associated lymphoid tissue (GALT) revealed earlyand sustained loss of lamina propria CD4 T cells and marked GItract pathology, including GI tract damage evidenced by enteritis/colitis, neutrophil infiltration, crypt abscesses, crypt and villusblunting, and, in one case, infiltration with foamy macrophages, alesion characteristic for atypical mycobacteria infection (Figs. S6and S7 and Dataset S4). Analysis of lymph nodes revealed pathologyassociated with either asymptomatic SIV disease, such as follicularhyperplasia with confluent germinal centers, or lesions characteristicof classic progressive HIV/SIV disease, including CD4 T-cell loss,lymph node fibrosis, and follicular involution and depletion as-sociated with amyloid deposits in the germinal centers. Necropsyfindings in the seven animals that died with AIDS also includedinterstitial pneumonia, lymphoid infiltrates around trachea andbronchioles, glomerulonephritis and glomerulosclerosis, lymphoidinfiltrates in the kidney cortical and medullary areas, well-definedgranulomas in the liver consistent with atypical mycobacterial in-fection, and inflammatory infiltrates around portal and centralveins (Figs. S6 and S7 and Dataset S4).Western blot immunoreactivity to core and Env proteins was

assessed at 4 and 10 wk after infection, corresponding to a timeframe that in humans is associated with anti–HIV-1 antibodyseroconversion (68). Western blot reactivity to diagnostic Env andGag proteins was strongly positive in all but three animals (RM195,RM6424, and RM6161) (Dataset S3). Two of these animals hadentirely negative Western immunoblots, which was associated withhigh peak and setpoint plasma virus titers and accelerated deathdue to AIDS at weeks 10 and 14. Similar findings have beenreported in a subset of SIV-infected RMs (67) and in rare humanHIV-1 infections (69). Anti-SIVp27 binding antibody titers wereassessed at weeks 10 and 32 after infection. Results were stronglyreactive for all animals, except RM6424 and RM6161 (Dataset S3).NAb responses were assessed in 13 RMs with at least 4 mo follow-up postinfection against a standardized global panel of tier 1 (n =3) and tier 2 (n = 17) virus strains corresponding to HIV-1 sub-types A, B, C, D, and circulating recombinant-form CRF01_AE(Dataset S5) (70, 71). Among SHIV D.191859-infected (n = 8),B.YU2-infected (n = 2), and C.CH505-infected (n = 3) animals,12 of 13 had reciprocal autologous NAb titers (IC50), rangingfrom 25 to 7,911 (Datasets S3 and S5). There was a statisticallysignificant positive correlation between viral load setpoint andmaximum autologous NAb titer (Spearman rs = 0.7, P < 0.05).Among 10 SHIV D.191859- or B.YU2-infected animals that weresampled for >1 y, 10 had reciprocal plasma IC50 NAb titers tothree heterologous tier 1 viruses ranging from 34 to >43,740; 10had titers against at least one heterologous tier 2 virus, three ani-mals neutralized two heterologous tier 2 viruses, and one or moreanimals neutralized four, five, or seven heterologous tier 2 viruses.Reciprocal IC50 NAb titers against heterologous tier 2 viruses weregenerally low, ranging from 20 to 557.

DiscussionOverbaugh and coworkers recently reviewed the history of SHIVdevelopment, the limitations of current SHIVs, and the still largelyunrealized potential of SHIVs to advance vaccine, cure, andpathogenesis research (1). They, along with other authors (35–38),concluded that a paramount objective continues to be the devel-opment of pathogenic SHIVs that encode env sequences fromcirculating T/F HIV-1 variants and establish persistent infection

without passage or adaptation in macaques. The present reportgoes a long way toward reaching this goal.We prospectively identified six primary HIV-1 Envs for SHIV

development. In each case, we were successful in constructingSHIVs that replicated well in RMs with plasma viral load kinetics(Fig. 5) remarkably similar to acute and early chronic HIV-1infection in humans (72, 73). In other recent studies, we createda seventh SHIV, this one from a T/F HIV-1 subtype C virus Ce1176(74), that is part of a widely used 12-member global neutraliza-tion panel developed by Montefiori, Seaman, and coworkers (71).SHIV.C.Ce1176 replicated efficiently in primary human and rhesusCD4 T cells in vitro and productively infected five of five non-CD8-depleted RMs after intravenous challenge. Thus, using the strategyoutlined in Fig. 1, we have created seven consecutive SHIVs fromprimary or T/F subtype A, B, C, and D HIV-1 viruses that infectand replicate efficiently and persistently in RMs, in some casesleading to AIDS pathology and death. This finding contrasts withfour recent reports (35–38) that used a conventional KB9 SHIVdesign strategy (20), where the vast majority of 91 SHIVs con-structed from primary HIV-1 Envs failed to replicate efficiently orpersistently in vivo. Not only is the success rate of the strategydescribed in the present report far higher, but it has the advantageof allowing for strategic, prospective targeting of desirable HIV-1Envs for SHIV construction and analysis, which was highlightedin the present study by SHIVs containing T/F Envs corre-sponding to HIV-1 CH505 (53, 55, 75), CH848, and BG505 (54,57–60), all of which evolved in their human hosts to elicit bNAbs.The SHIVs described herein also exhibit particularly favorablebiological and antigenic properties that distinguish them frommost previously described SHIVs (3, 9, 18, 21, 29–33, 35–38, 76).Specifically, they exhibit sensitivity to a wide spectrum of broadlyneutralizing mAbs that target the five principal sites of bNAbrecognition (77, 78), while retaining effective conformationalmasking typical of primary or T/F virus strains (23, 65).If substitution of Env375 with variant residues is the sine qua non

of our SHIV design strategy, what, then, is the mechanistic basis forenhanced rhCD4 binding and cell entry? The native, unligandedHIV-1 Env trimer is conformationally flexible or “metastable” (25,44, 49, 50, 79–81), and movement of the Env trimer from its unli-ganded state to the CD4-bound conformation must be carefullyregulated, because premature triggering of the unliganded glyco-protein complex to downstream conformations results in functionalinactivation (82, 83). Structural and molecular biological studies haverevealed a “layered” inner domain gp120 architecture that facilitatesmovement among alternative Env conformations required for virusentry (25, 50, 80, 84). Env gp120 refolds itself upon binding CD4,with the inner, outer, and bridging sheet domains brought intoproximity at the interface with CD4. This process creates a ∼150-Å3

cavity between HIV-1 gp120 and CD4 bounded by Phe-43 of CD4,which makes numerous contacts with conserved gp120 residues, al-though Env375 is not one of them (48, 85). Env375 does, however,abut the Phe-43 cavity, as do other highly conserved residues, manyof them containing aromatic side chains. These Phe-43 cavity-liningresidues contribute to an aromatic array that helps stabilize the CD4-bound conformation. Substitution of Env375W for Env375S in HIV-1YU2 gp120 fills the Phe-43 cavity and allows the glycoprotein tosample conformations closer to the CD4-bound state spontaneously(25, 44, 50, 84). This process leads to enhanced affinity for CD4,largely due to a decrease in the off-rate of the bound Env–CD4complex (50). We hypothesized that substitution of the naturallyoccurring HIV-1 Env375S by certain larger space-occupying sidechains would enhance Env gp120 binding to rhesus CD4 and facil-itate virus entry in rhesus cells in a similar manner. Molecularmodeling of HIV-1 gp120 in complex with human and rhesus CD4(Fig. S2), together with three independent lines of empirical evi-dence—neutralization of infectious virus by soluble rhCD4 (Fig. 2A),entry of virus into rhCD4-bearing target cells (Fig. 2B), and directanalysis of Env binding to rhCD4 by SPR (Fig. 2C)—support this

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hypothesis. Thus, we conclude that the mechanism by which thedifferent SHIV Env375 amino acid substitutions enhance rhCD4binding is likely to be similar to that described for enhanced bindingof the YU2 Env375W variant to human CD4 (25, 44, 50, 84).Factors contributing to successful replication of SHIV variants

in rhesus cells in vitro and in vivo, however, likely extend beyondthe initial binding event of Env to rhCD4. One of these factors isthe “intrinsic reactivity” or “responsiveness” of Env to CD4 engage-ment, which reflects the product of CD4 binding and downstreamconsequences of CD4 binding that allow the Env glycoprotein tonegotiate transitions to lower-energy states, including those responsiblefor cell entry (25, 44, 50, 80, 82, 84, 86, 87). Env responsiveness to CD4influences the ability of HIV-1 to infect cells with different levels ofCD4 (e.g., macrophages) or variants of CD4 (e.g., CD4 fromother species) and the sensitivity of HIV-1 to sCD4. The respon-siveness of HIV-1 to CD4 can be altered by generic changes in Env,which also affect the sensitivity of the virus to antibody neutrali-zation (31), or by more selective changes, as in the case of Env375variants. We hypothesized that SHIVs must attain a certain level ofEnv responsiveness to rhCD4 to infect RM CD4 T cells andreplicate efficiently in vivo, but without leading to abortivedownstream conformations that prevent infection. A corollary isthat, because natural HIV-1 Env variants exhibit different levelsof CD4 responsiveness, different degrees of compensation con-ferred by changes in residue 375 will be required for optimaladaptation to rhCD4. To test these notions, we used the sensi-tivity of SHIVs containing wild-type Env375S to human andrhesus sCD4-Ig as an indicator of the baseline CD4 responsivenessand determined whether the size of residue 375 side chains con-tributing to efficient virus replication in rhCD4 T cells in vitro andin vivo correlated inversely. The answer was affirmative (Fig. 6).The results shown support the hypothesis that to replicate effi-ciently in monkeys, SHIVs with lower baseline CD4 responsiveness,such as C.CH505, C.CH848, and A.BG505, require residue Env375substitutions that result in proportionally greater CD4 responsiveness.Conversely, SHIVs with higher levels of baseline CD4 responsiveness(i.e., that exhibit greater sensitivity to sCD4), such as B.YU2 andD.191859, require residue Env375 substitutions that result in smallerchanges in CD4 responsiveness. These observations suggestthat a major force driving the selective replication of SHIVEnv375 variants in RMs—375S for B.YU2, 375M for D.191859,375H for C.CH505 and C.CH848, and 375Y for BG505.332Tand BG505.332N—is the requirement to achieve both adequaterhCD4 binding and enhanced Env responsiveness to CD4 withoutaltering other essential properties of the native Env trimer, includingconformational masking and orderly downstream conformationalchanges leading to virus–cell membrane fusion.Although CD8 depletion in half of the study animals limited

our ability to fully evaluate the natural history and immunopa-thogenesis of SHIV infections, the findings from analysis ofplasma viral load kinetics and peripheral blood T-cell subsets inall animals at multiple time points, biopsies of lymph nodes andGALT at early and late time points, and immunopathologicalexaminations at necropsy, leave no doubt as to the pathogenicityof the Env375-modified T/F SHIVs. Plasma viral load kinetics inacute and early chronic phases of infection (Fig. 5) were nearlyindistinguishable from human infection by HIV-1 (72, 73). AllCD4 T-cell subsets, not just CD4 naïve cells, underwent gradualdecline; GALT was selectively depleted of CD4 T cells early ininfection and did not recover; and R5 tropism of SHIVs did notchange to X4 tropism, even in animals that died of acceleratedAIDS within the first 3 mo of infection (Fig. S8). AIDS-definingevents included wasting, CD4 T-cell lymphopenia, pancytopenia,PCP, atypical mycobacterial infections, RhCMV reactivation anddisease, and high-grade lymphoma (Dataset S4). Of note, thedevelopment of NAbs in SHIV-infected RMs resembled that inhumans infected by HIV-1 with respect to kinetics, titers, breadth,and potency. In SHIV infections, autologous tier 2 NAbs developed

in most RMs to reciprocal IC50 titers between 25 and 8,000 in thefirst 3–6 mo of infection (Datasets S3 and S5), whereas heterologoustier 2 NAbs developed sporadically and generally to only low titersafter 1 y of infection (Dataset S5). Further studies with additionalSHIVs, larger numbers of infected animals, and longer follow-upperiods will be needed to assess the similarities and differencesin NAb responses in RMs and humans to viruses bearing primaryHIV-1 Envs.Designer SHIVs bearing selected amino acid substitutions at

residue 375 can be an enabling tool for HIV vaccine and cure re-search. As challenge stocks for active and passive preclinical vac-cines studies, we now have in hand well-pedigreed SHIVs thatexpress primary or T/F subtype A, B, C, and D Envs. These SHIVshave been expanded in primary activated rhCD4 T cells in tissueculture to high titers (∼109 virions per milliliter) and exhibit highinfectivity-to-particle ratios when measured on TZM-bl cells(∼0.01) and primary rhCD4 T cells (∼0.0005) (Dataset S1). Theyalso transmit efficiently in vivo across mucosal surfaces, includingrectal, penile (glans and foreskin), or vaginal without hormonalmanipulation. This finding makes possible RM modeling of vaccineprevention against all of the principal routes of HIV-1 acquisitionusing SHIVs with biologically relevant Envs. Importantly, the SHIVC.CH505, C.CH848, D.191859, and A.BG505 challenge stockscontain no discernible in vitro adaptations that alter their nativeantigenicity or neutralization sensitivity, a problem that has plaguedother SHIV challenge viruses (12, 29, 31–33, 35, 38). With respectto immunogen design, by strategically targeting for SHIV con-struction primary or T/F HIV-1 Envs with unique properties such asthe ability to bind unmutated common ancestor germ-line Ig re-ceptors of human bNAb lineages [e.g., CD4bs bNAb lineages byHIV-1 CH505 (53, 55, 75) or V1/V2 bNAb lineages by HIV-1CRF_AG_250, ZM233, WITO4160, and CAP256 (88, 89)] or toelicit bNAbs in humans [e.g., HIV-1 CH505, CH848, and BG505(53–55, 75)], critical questions regarding the inherent immunoge-nicity of particular Env glycoproteins, contributions of rhesus hostgenetics to the elicitation of NAbs and bNAbs, and the role ofstochastic vs. deterministic events in antibody elicitation can beparsed. In this way, analysis of virus–antibody coevolution can po-tentially guide structure-based (56, 90, 91) or viral lineage-based(92, 93) immunogen design. Finally, further model development todocument authentic HIV-1–like SHIV persistence and pathogene-sis and susceptibility to suppression by combination antiretroviraltherapy should set the stage for these viruses to facilitate the pre-clinical evaluation of HIV-1 Env targeted therapeutic interventionsin the context of HIV-1 cure research.

Materials and MethodsNonhuman Primates. Indian RMs were housed and studied at the University ofPittsburgh or Bioqual, according to the Association for Assessment and Ac-creditation of Laboratory Animal Care standards. Experiments were ap-proved by the University of Pittsburgh and Bioqual Institutional Animal Careand Use Committees (SI Materials and Methods).

SHIV Constructions. The SIVmac766 clone (GenBank accession no. KU955514)was modified by replacing the SIV genome between the stop codon of vprand start codon nef with a linker containing the unique restriction enzymesites AgeI and AclI. A complete HIV-1 tat/rev/vpu/env(gp160) cassette wasinserted (SI Materials and Methods).

SHIV Infection of Primary Rhesus and Human CD4 T Cells. SHIV stocks weregenerated in 293T cells. Virus imput was normalized to ∼1,000 virions (0.1 pgof p27Ag) per cell (SI Materials and Methods).

SHIV Entry Assays. SHIV entry was assessed in primary activated human andrhesus CD4 T cells and in cell lines expressing huCD4 and huCCR5 [TZM-bl cells(61, 94)] or rhCD4 and rhCCR5 (ZB5 cells) (SI Materials and Methods).

SHIV Neutralization Assays. Neutralization was assessed in TZM-bl cells by astandardized method (61, 70, 71, 94).

E3420 | www.pnas.org/cgi/doi/10.1073/pnas.1606636113 Li et al.

SPR. Three HIV-1 Envs with residue 375 substitutions were analyzed, as de-scribed (SI Materials and Methods).

Virion-Associated Env:Gag Ratio Measurements. Virus was purified by filtrationand density gradient centrifugation. Quantitative measurements of viral p27(CA) and gp120 protein were performed by dual-color fluorescent protein gelanalysis (41, 95) (SI Materials and Methods).

Viral Sequencing. SGS and MiSeq sequences were generated as describedpreviously (23, 66) (SI Materials and Methods).

Immunohistochemistry. Immunohistological stains were performed as describedpreviously (96).

Coreceptor Use. Coreceptor use was determined in TZM-bl cells by selectiveblockade using Maraviroc (CCR5), AMD3100 (CXCR4), or a mixture of in-hibitors (66) (SI Materials and Methods).

Phylogenetic Analyses. Lentiviral pol genes were aligned by using clustalw(v2.1) (97) and analyzed by using PhyML v3.0 (98) (SI Materials and Methods).

Viral and Immunodiagnostics. Quantitative and qualitative analyses of virus,antibodies, and cells were performed as described (SI Materials and Methods).

Molecular Modeling. Structural analysis was performed by using PyMOL (99) andMolprobity (45, 100–102). Mutants for rhCD4 or residue 375 were generated insilico by using the mutagenesis tool in PyMOL (SI Materials and Methods).

Statistical Analyses. Unpaired t and Fisher exact tests and Spearman rank cor-relations were calculated by using Prism 6 software (SI Materials and Methods).

ACKNOWLEDGMENTS.WethankC. Xu, T. He, T. Dunsmore, G.H. Richter, K. Raehtz,J. Roser, R. Fast, K. Oswald, R. Shoemaker, J. Bess, and A. Meyer for technicalassistance. This project was funded in part by Bill & Melinda Gates Founda-tion Grants OPP37874 and OPP1145046; and NIH Grants AI100645, AI088564,AI094604, AI087383, AI078788, AI045008, and HHSN261200800001E.

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