Envelope residue 375 substitutions in simian–human PNAS PLUS immunodeficiency viruses enhance CD4 binding and replication in rhesus macaques
Hui 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, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; eDepartment of Infectious Disease, Scripps Research Institute, Jupiter, FL 33458; fBioqual, Inc., Rockville, MD 20852; gAaron Diamond AIDS Research Center, Rockefeller University, New York, NY 10016; hCenter of 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-Farber Cancer 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- infection of humans (reviewed in ref. 1). These limitations are lope (Env) glycoproteins from primary HIV-1 strains fail to infect often related to properties of the envelope (Env), which in most rhesus macaques (RMs). We hypothesized that inefficient Env bind- SHIVs does not faithfully reflect primary HIV-1 strains or trans- ing to rhesus CD4 (rhCD4) limits virus entry and replication and could mitted/founder (T/F) viruses (23, 24). HIV-1 Env, and its coun- be enhanced by substituting naturally occurring simian immunode- terparts in naturally infected chimpanzees (SIVcpz) and gorillas ficiency virus Env residues at position 375, which resides at a critical (SIVgor), have evolved over millennia to infect great ape and, location in the CD4-binding pocket and is under strong positive evo- lutionary pressure across the broad spectrum of primate lentiviruses. more recently, human CD4 T cells, but not CD4 T cells of lower SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, primates, including Indian rhesus macaques (RMs; Macaca – C, or D Envs with genotypic variants at residue 375 were constructed mulatta)(25 27). Thus, most HIV-1 Envs do not bind Macaca spp. and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino CD4 efficiently. Overbaugh and coworkers (26) provided a partial acids substituted for serine-375 enhanced Env affinity for rhCD4, molecular explanation for this inefficiency by identifying a distinguishing virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody- Significance mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with Simian–human immunodeficiency viruses (SHIVs) are an invaluable — — different Env375 variants S, M, Y, H, W, or F that were differen- tool for assessing HIV-1 vaccines, developing therapeutic “cure” tially selected in different Env backbones. Notably, SHIVs replicated strategies, and understanding viral immunopathogenesis. How- persistently at titers comparable to HIV-1 in humans and elicited ever, only limited success has been achieved in creating SHIVs that autologous neutralizing antibody responses typical of HIV-1. Seven incorporate HIV-1 envelopes (Envs) that retain the antigenic fea- – animals succumbed to AIDS. These findings identify Env rhCD4 bind- tures of clinically relevant viruses. Here we focus on a critical resi- ing as a critical determinant for productive SHIV infection in RMs and due of the CD4-binding region, Env375, which is under strong validate a novel and generalizable strategy for constructing SHIVs positive selection across the broad range of primate lentiviruses. with Env glycoproteins of interest, including those that in humans We find that genotypic variation of residue 375 allows for the elicit broadly neutralizing antibodies or bind particular Ig germ-line creation of pathogenic SHIVs that retain the antigenicity, tier 2 B-cell receptors. neutralization sensitivity, and persistence properties characteristic of primary HIV-1 strains. Taken together, our findings suggest a HIV/AIDS | SHIV | CD4 | HIV-1 envelope new paradigm for SHIV design and modeling with important ap- plications to HIV-1 vaccine, cure, and pathogenesis research. MEDICAL SCIENCES imian–human immunodeficiency viruses (SHIVs), which express Sthe envelope of HIV-1 within the context of simian immuno- 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- deficiency virus of macaque (SIVmac) structural and regulatory 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. elements, have broad potential applications for HIV/AIDS vaccine, performed research; M.G. and, M.F. contributed new reagents/analytic tools; H.L., R.W.D., treatment (cure), and pathogenesis research (1, 2). SHIVs have 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., been used to assess the protective effects of neutralizing or anti- and G.M.S. wrote the paper. body-dependent cell-mediated cytotoxicity conferring antibodies Reviewers: S.-L.H., University of Washington; P.L., University of California, Davis; and L.S., (3–7), the elicitation of broadly neutralizing antibodies (bNAbs) Fred Hutchinson Cancer Research Center. (8–11), the efficacy of broadly neutralizing monoclonal antibodies The authors declare no conflict of interest. (mAbs) in preclinical trials with suppressive and curative intent (12, Freely available online through the PNAS open access option. 13), and virus–host interactions underlying virus transmission, im- Data deposition: The sequences reported in this paper have been deposited in the Gen- Bank database (accession nos. KU955514 and KU958484–KU958489). munodeficiency, and neurocognitive impairment (1, 14–22). De- 1To whom correspondence may be addressed. Email: [email protected] or shawg@upenn. spite these varied and important applications of SHIVs to HIV/ edu. AIDS research, it is widely recognized that most SHIVs developed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. thus far have significant shortcomings as a model system for HIV-1 1073/pnas.1606636113/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1606636113 PNAS | Published online May 31, 2016 | E3413–E3422 Downloaded by guest on September 26, 2021 polymorphism at position 39 in rhesus and pigtailed macaque A CD4 compared with human CD4. By necessity, then, SHIVs SIVmac766 developed thus far contain HIV-1 Envs derived from one of four 1 RU5 Vpx Vpr gp120 gp41 U3 R sources: (i) laboratory-adapted CXCR4-tropic HIV-1 strains 2 Tat PR RT RNase IN Nef 3 (28); (ii) viruses passaged in vitro so as to acquire Env adapta- Read frame Rev MA CA NCp6 Vif tions necessary for efficient infection of rhCD4 T cells, but at the B cost of reduced conformational masking and protection from SHIV.B.YU2 375 S/M/Y/H/W/F – 1 antibody neutralization (29 31); (iii) in vivo passaged and RU5 Vpx Vpr gp120 gp41 U3 R – – 2 Tat adapted virus swarms or clones (14, 15, 17 21, 32 34); or (iv) PR RT RNase IN Nef
vpu random screens of primary HIV-1 strains for rare viruses that Read frame 3 Rev MA CA NCp6 Vif Vpu encode Envs that fortuitously bind rhCD4 and allow for rhesus C cell entry and replication (35–38). SHIV.D.191859 375 S/M/Y/H/W/F 1 The construction and development schemes of SHIVs have RU5 Vpx Vpr gp120 gp41 U3 R 2 Tat also been complicated. Many SHIVs derive from the original PR RT RNase IN Nef
SHIV–HXBc2 construction of Sodroski and coworkers where Read frame 3 Rev MA CA NCp6 Vif Vpu HIV-1 HXBc2 tat, rev, and env sequences were subcloned into D SHIV.D.191859.dCT 375 S/M/Y/H/W/F SIVmac239 (28, 39, 40). SHIV–HXBc2 was further modified by Vpu 1 Rev substitution of the env gene from the dual CCR5/CXCR4 tropic RU5 Vpx Vpr U3 R 2 HIV-1 89.6 strain, followed by additional passages in RMs, PR RT RNase IN SP gp120 gp41
Read frame 3 Tat resulting in a highly pathogenic CXCR4 tropic phenotype (14, MA CA NCp6 Vif Nef 15, 20). Henceforth, a molecular clone (SHIV–KB9) of this E SHIV.C.CH505.dCT/SHIV.C.CH848.dCT/SHIV.A.BG505.dCT 375 S/M/Y/H/W/F strain served as a preferred backbone for SHIV constructions, Vpu 1 Rev including four recent reports (35–38). Other SHIV constructions RU5 Vpx Vpr U3 R 2 env have been similarly complicated (9, 18, 21, 29, 30, 32, 33). PR RT RNase IN SP gp120 gp41
Read frame 3 Tat We took a different approach to SHIV design based on the MA CA NCp6 Vif Nef hypothesis that a critical determinant of successful SHIV replication in RMs is efficient binding of Env to rhCD4. We further surmised Fig. 1. SHIV construction scheme. (A–C)Acompletetat–rev–vpu–env(gp160) that optimization of the SIVmac backbone and simplification of the segment of HIV-1 B.YU2 or D.191859 was substituted for the corresponding HIV-1 insert strategy would enhance the chances of success. Our region in SIVmac766. (D and E) SHIV.D.191859.dCT, which lost cloning site linkers (yellow bars in C) and the carboxy terminal 33 amino acids of HIV-1 gp41 strategy was thus to: (i) molecularly clone a new SIVmac251-derived but with the carboxy terminus of SIVmac766 gp41 joined in-frame (Figs. S3 and T/F virus, pSIVmac766 (41, 42), that lacked any of four mutations S5A), served as a platform for further SHIV constructions containing vpu– found in SIVmac239 known to be suboptimal for replication (39, env–gp140 segments of HIV-1 strains CH505, CH848, and BG505. Met-M, Tyr-Y, 43); (ii) substitute into this vector complete tat–rev–vpu–env His-H, Trp-W, and Phe-F were exchanged for the wild-type Ser-S residue at (gp160) cassettes of T/F or primary HIV-1 strains; and (iii) modify position 375 in Env. SIVmac766 and SHIV sequences are publicly available Env residue 375, which is part of the CD4-binding pocket and (GenBank accession nos. KU955514 and KU958484-9). facilitates CD4 engagement (44), to encode genotypic variants (Met, M; Tyr, Y; His, H; Trp, W; and Phe, F) found naturally at this position across the broad evolutionary spectrum of primate and C). Moreover, in the HIV-1 YU2 backbone, a 375W sub- lentiviruses (25) (www.hiv.lanl.gov/)(Fig. S1A). Here we engineer stitution for 375S allows Env to more readily sample CD4-bound and test six new SHIVs containing Env glycoproteins from primary conformations and enhances its affinity for human CD4 (25, 44, 49, or T/F strains of HIV-1 subtypes A, B, C, and D, each containing 50). These findings, along with phylogenetic evidence of positive six different Env375 variants. We then describe a vector platform evolutionary selection at Env375 (Fig. S1A), suggested to us that and refinements to the design strategy that can streamline the substitution of Env375S by residues containing larger hydrophobic development of next-generation “designer SHIVs.” or basic, space-filling side chains (e.g., M, Y, H, W, or F) might enhance rhCD4 binding and SHIV replication in RMs. Results Selection of Env Residue 375. HIV-1 Env binding to the CD4 re- SHIV Design. We cloned a replication-competent, pathogenic T/F ceptor involves multiple structural rearrangements as Env transitions provirus designated SIVmac766 (Fig. 1A) from a RM infected by from a prefusion closed state (45), through a single CD4-bound atraumatic rectal inoculation with an early passage isolate of – – – intermediate (46), to a more fully engaged Env trimer capable of SIVmac251 (41, 42). Complete tat rev vpu env(gp160) cassettes recognizing the coreceptor (47). Although CD4 39I is the critical from a prototypic primary subtype B strain YU2 (51) and from a alteration in rhCD4 impeding HIV-1 Env binding (Fig. S2A) genetically divergent T/F subtype D HIV-1 strain 191859 (52) (26), the corresponding residue in huCD4 (39N) does not actually were exchanged for the corresponding region of SIVmac766 interact directly with HIV-1 Env, either in the prefusion closed (Fig. 1 B and C). Site-directed mutagenesis was used to modify Env state or in the CD4-bound state (48) (Fig. S2 B–E). Instead, it lies residue 375 from Ser to Met, Tyr, His, Trp, or Phe in each env.In proximal to the critical phenylalanine 43 (Phe-43) residue, which subsequent SHIV designs, we incorporated a carboxy terminal makes contact with Env residues in the Phe-43 binding pocket (48). gp41 tail exchange of 58 amino acids of SIVmac766 gp41 for the In rhCD4, modeling suggested that 39I could subtly alter the angle homologous 33 amino acids of HIV-1 (SHIV.D.191859.dCT in Fig. of the Phe-43 binding loop (Fig. S2G). 1D and Fig. S3), because this substitution occurred spontaneously Because structural rearrangements in HIV-1 Env associated in infected RMs and enhanced SHIV replication in vitro and in with CD4 binding can be affected by large alterations in side-chain vivo (see below). volume, we analyzed SIV and HIV-1 group M Env residues We used a similar design strategy to exchange all or portions proximal to CD4 in the ligand-bound conformation for differences of tat–rev–vpu–env(gp160) from other T/F HIV-1 strains into the in amino acid volume (Fig. S1B). We observed large differences at SIVmac766 backbone, but a number of these constructs failed to two sites: residue 375, where a Ser in HIV-1 corresponds to a generate infectious virus. We attributed this inconsistency to larger, generally hydrophobic amino acid in SIVs (Fig. S1B); and differences in the reading-frame organization of different HIV-1 residue 458. Of these, Env375 is closer to residue 43 of CD4 in inserts relative to the SIVmac766 vpr/tat overlap, resulting in both prefusion closed and CD4-bound conformations (Fig. S2 B untoward effects on RNA splicing and protein expression or to
E3414 | www.pnas.org/cgi/doi/10.1073/pnas.1606636113 Li et al. Downloaded by guest on September 26, 2021 strain-specific differences in HIV-1 Tat or gp41, which must interact PNAS PLUS A SHIV.B.YU2 SHIV.D.191859 SHIV.C.CH505 SHIV.C.CH848 SHIV.A.BG505.332N huCD4-Ig huCD4-Ig huCD4-Ig huCD4-Ig huCD4-Ig with cognate motifs of SIVmac766 (i.e., the RNA TAR element 100 and Gag matrix protein, respectively). To avoid these potential 75 50 complicating factors and to develop a more consistent and reliable 25 strategy for generating SHIVs with a high likelihood of replicative Infectivity (%) 1 1 1 1 1 10 10 10 10 10 0.1 100 0.1 100 0.1 100 0.1 100 0.1 100 – 0.01 1000 0.01 1000 0.01 1000 0.01 1000 0.01 1000 success, we refined our approach to exchange just the HIV-1 vpu SHIV.B.YU2 SHIV.D.191859 SHIV.C.CH505 SHIV.C.CH848 SHIV.A.BG505.332N rhCD4-Ig rhCD4-Ig rhCD4-Ig rhCD4-Ig rhCD4-Ig env(gp140) or env(gp140) cassettes into the highly infectious and 100 75 replicative prototypic clone SHIV.D.191859.dCT (Fig. 1 D and E 50 and Fig. S3). Importantly, this construction scheme allowed for the 25 Infectivity (%)
1 1 1 1 1 10 10 10 exchange of complete gp120 and gp41 extracellular and membrane 0.1 100 0.1 10 0.1 100 0.1 100 0.1 10 0.01 0.01 100 0.01 1000 0.01 0.01 100 μg/ml 1000 μg/ml 1000 μg/ml μg/ml 1000 μg/ml 1000 spanning domains, which contain the epitopes for all neutralizing HIV-1 375S SHIV 375S SHIV 375M SHIV 375Y SHIV 375H SHIV 375W SHIV 375F
SHIV.A.BG505.332N antibodies (NAbs). This strategy was used to generate SHIVs B4000 SHIV.B.YU2 4000 SHIV.D.191859 SHIV.C.CH505 SHIV.C.CH848 4000 TZM-bl TZM-bl 20000 TZM-bl 20000 TZM-bl TZM-bl containing vpu–env(gp140) sequences from T/F HIV-1 subtype C 3000 3000 15000 15000 3000 2000 2000 10000 10000 2000
strains CH505 and CH848 (53) and T/F subtype A strain BG505 RLU (54). These three viruses were of particular interest because they 1000 1000 5000 5000 1000 represented additional HIV-1 subtypes, had elicited bNAbs in their S M Y H WF S M Y H W F S M Y H W F S M Y H WF S M Y H W F SHIV.B.YU2 SHIV.D.191859 SHIV.C.CH505 3000 3000 8000 4000 SHIV.C.CH848 3000 SHIV.A.BG505.332N respective human hosts (53, 54), and included the widely studied ZB5 ZB5 ZB5 ZB5 ZB5 6000 3000 2000 2000 2000 preclinical vaccine strains CH505 and BG505 (53, 55, 56). For all 4000 2000
RLU 1000 1000 1000 SHIVs (Fig. 1 B–E), we modified residue Env375S to encode Met, 2000 1000 Tyr, His, Trp, or Phe to determine which residue was preferred for C S M Y H W F S M Y H W F S M Y HWF S M YHWF S M YHW F rhCD4 binding, infection, and replication in the respective Env 250 huCD4-Ig 250 huCD4-Ig 250 huCD4-Ig 15μg/ml 15μg/ml gp120 375W 200 gp120 375S 15μg/ml 200 gp120 375M 200 backgrounds. In addition, for SHIV.A.BG505, we modified residue Kd=34 nM Kd=16 nM 10μg/ml Kd=4 nM 10μg/ml Env332T, which corresponds to the wild-type T/F virus (57) 150 10μg/ml 150 150 100 5μg/ml 100 5μg/ml 100 5μg/ml
(GenBank accession no. DQ208458), to Env332N that is present 50 2μg/ml 50 2μg/ml 50 2μg/ml – Response (RU) in the BG505 SOSIP.664 (58 60). All 36 SHIVs were sequence- 0 200 400 600 0 200 400 600 0 200 400 600 120 confirmed. 120 rhCD4-Ig rhCD4-Ig 120 rhCD4-Ig 25μg/ml 100 gp120 375S 100 gp120 375M 100 gp120 375W Kd=21 nM 15μg/ml 80 Kd=138 nM 80 Kd=105 nM 25μg/ml 80 25μg/ml Envelope Binding to Human and Rhesus CD4. A total of 36 SHIV 60 60 15μg/ml 60 10μg/ml 40 15μg/ml 40 10μg/ml 40 5μg/ml 10μg/ml DNAs (B.YU2, D.191859, C.CH505, C.CH848, A.BG505.332T, 5μg/ml 20 5μg/ml 20 20 2μg/ml and A.BG505.332N, each containing six genotypic variants at Env375) Response (RU) 2μg/ml 2μg/ml 0 200 400 600 0 200 400 600 0 200 400 600 were transfected into 293T cells for virus generation and biological Time Time Time analysis. In every instance, virions were efficiently produced, generally Fig. 2. SHIV Env binding to human and rhesus CD4. (A) Inhibition of SHIV 10 in the range of ∼10 virions per mL, as determined by quantification entry into TZM-bl cells by human and rhesus CD4-Ig. Controls included wild-type of vRNA by real-time RT-PCR or p27 antigen by ELISA HIV-1 Env375S expressed from infectious molecular proviral clones or as Env- (Dataset S1). All 36 SHIVs were highly infectious in TZM-bl pseudotyped particles. (B) SHIV entry into TZM-bl or ZB5 cells. RLU, relative light cells, which bear huCD4 and huCCR5 on their surface (61), with units. Variance is expressed as SD. (C) SPR analysis of HIV-1 D.191859 gp120 infectivity to particle (I:P) ratios of 0.001–0.00002. When grown Env375 variants to human and rhesus CD4-Ig. Plots are of representative ex- in primary rhCD4 T cells, these same SHIVs with favorable periments replicated three times. Env375 substitutions reached I:P ratios as high as 0.02 when titered in TZM-bl cells and 0.0006 in primary, activated rhCD4 T We next analyzed whether Env375 mutations affected the bind- cells (Dataset S1). ing and entry of SHIVs in cells expressing rhesus or human CD4 on We tested the effects of Env375 variation on the binding of Env their surface (Fig. 2B). A canine cell line, ZB5, stably transduced to to human and rhesus CD4 by three different methods. The first express rhCD4 and rhCCR5 at levels comparable to huCD4 and assay examined the ability of soluble rhCD4-Ig or huCD4-Ig to bind huCCR5 on TZM-bl cells, was used to assess SHIV binding and the functional Env trimer on infectious SHIVs and inhibit virus entry. In ZB5 cells, SHIV B.YU2 was the only wild-type 375S entry into TZM-bl cells (Fig. 2A). Overall, B.YU2 Env375S was variant capable of achieving significant levels of infectivity. This substantially more sensitive to huCD4-Ig and rhCD4-Ig than were result correlated with the enhanced affinity of the SHIV B.YU2 wild-type Env375S alleles from the other SHIVs, which is consistent Env375S allele for soluble rhCD4-Ig (Fig. 2A). The 375M sub- with previous studies showing that YU2 Env-huCD4 affinity is stitution in all six SHIV backbones had either no effect or increased high, thereby allowing it to use low levels of CD4 on human virus infectivity modestly (twofold to fourfold) (Fig. 2B). This result
T cells, macrophages, and microglia for entry (25, 44, 50, 62). A again correlated with the unchanged or modestly enhanced sensi- MEDICAL SCIENCES rank order of SHIV sensitivity to huCD4-Ig and rhCD4-Ig was tivity of the Env375M allele to soluble rhCD4-Ig (Fig. 2A). In B.YU2 > D.191859 > C.CH505 > C.CH848 > A.BG505.332N and contrast, the 375Y/H/W/F substitutions increased virus infectivity in A.BG505.332T. For any particular Env375 substitution in any of ZB5 cells markedly (10- to 1,000-fold) in all SHIV Env backbones the Env backbones, huCD4-Ig neutralized SHIVs at ∼100-fold except B.YU2, where the effect was more modest. Overall, a gen- lower concentrations than did rhCD4-Ig, indicating a substantially eral trend was observed between enhanced entry of SHIV Env375 higher Env affinity for huCD4 than for rhCD4. Envs containing the variants into ZB5 cells and enhanced sensitivity to entry inhibition wild-type 375S residue were least sensitive to rhCD4-Ig or huCD4-Ig by soluble rhesus CD4-Ig, although this correlation was imperfect. entry inhibition. Substitutions at Env375 led to increased sensitivity to These findings suggested that a threshold for SHIV Env–rhCD4 rhCD4-Ig and huCD4-Ig inhibition by rank order W,Y > H,F > M > S. binding must be reached in order for virus infection to occur, but As a group, Envs bearing the 375 Y, W, H, or F residues that additional postbinding events contribute to efficient virus entry were ∼10- to 100-fold more sensitive to rhCD4-Ig and huCD4-Ig and replication (see below). than were Env375S or Env375M. Analyses using soluble huCD4 As a third measure of Env–CD4 binding, we tested by surface and rhCD4 gave similar results. These findings indicate that plasmon resonance (SPR) the affinity of rhCD4-Ig and huCD4-Ig substitution of nonpolar aliphatic, bulky hydrophobic, or basic for monomeric HIV-1 Env gp120 containing each of the six aromatic amino acids at Env375 substantially increases the affinity Env375 variants and expressed in 293F cells. Fig. 2C shows rep- of the functional HIV-1 Env trimer for rhesus and human CD4. resentative SPR plots for binding of three D.191859 Env375
Li et al. PNAS | Published online May 31, 2016 | E3415 Downloaded by guest on September 26, 2021 genotypic variants to rhesus and human CD4-Ig, and Dataset S2 b12 VRC01 3BNC117 NIH45-46 CH103 summarizes results for all six Env375 variants of D.191859, B.YU2, 100 G54W 75
and C.CH505. For D.191859 gp120, the association rate ka for 50 rhCD4-Ig was not affected by residue 375 substitutions and ranged − − 25 × 4 × 4 1 1 Infectivity (%) from 0.75 10 to 1.0 10 M s , whereas the dissociation rate 0 −4 −1 −4 −1 kd ranged from 10.6 × 10 s for 375S to 1.63 × 10 s for 375W PG9 PG16 PGDM1400 PGT145 8ANC195 100 (Dataset S2). From these values, the binding constants (Kd) could be determined as follows: S375, 138 nM; M375, 105 nM; Y375, 78 nM; 75 H375, 43 nM; F375, 25 nM; and W375, 21 nM (Fig. 2C and 50 25 Infectivity (%) Dataset S2). For huCD4-Ig, Kd values were as follows: S375, 34 nM; 0 M375, 16 nM; Y375, 12 nM; H375, 7 nM; F375, 4 nM; and PGT121 PGT126 PGT128 2G12 4E10 W375, 4 nM, again reflecting an overall higher affinity of the 100 subtype D.191859 Env gp120 for huCD4 compared with rhCD4. 75 Similar trends were observed for YU2 and CH505 Env gp120 50 25
(Dataset S2). In all cases, HIV-1 Envs had higher affinity for Infectivity (%) huCD4 than for rhCD4, and substitution of 375S by 375M, 375H, 0 10E8 2F5 3869 3074 447-52D 375Y, 375F, or 375W led to higher binding affinity to human and 100
rhesus CD4. 75 50 Neutralization Sensitivity of SHIVs with Env375 Mutations. We performed 25 Infectivity (%) 0 a comprehensive analysis of the sensitivity of all 36 SHIVs to a large 1 1 0.1 10 0.1 10 panel of neutralizing mAbs, polyclonal anti-HIV antibodies in patient 17b 21c 19e 0.01 0.01 sera, and the entry fusion inhibitor T1249. The objective was to de- 100 CH1754 HIVIG-C T1249 75 100 75 termine whether amino acid substitutions at residue 375 altered Env 50 50 antigenicity, neutralization sensitivity, or fusion efficiency. As con- 25 25 Infectivity (%) trols, we analyzed the corresponding HIV-1 Envs containing the wild- 0 0 1 1 1 1 1 10 10 10 10 0.1 10 0.1 0.1 0.1 0.1 0.010.1 100 0.01 100 0.01 0.01 0.01 0.0010.01 1000 type 375S residue expressed from full-length T/F HIV-1 proviral 0.0001 genomes or as Env pseudotypes. In all six SHIV Env backbones, HIV-1 375S SHIV 375S SHIV 375M SHIV 375Y SHIV 375H SHIV 375W SHIV 375F there were little or no appreciable effects of residue 375 substitutions Fig. 3. Neutralization of SHIV.BG505.332N.dCT Env375 variants in TZM-bl on antigenicity or neutralization sensitivity for any mAb or immune cells by mAbs, plasma from an HIV-1 patient with bNAbs (CH1754), HIV-1 plasma tested (Fig. 3 and Fig. S4). This result included as many as six immune globulin (HIVIG-C), or the fusion inhibitor T1249. Concentrations on different CD4bs mAbs (b12, VRC01, 3BNC117, NIH 45-46 G54W, the x axis are expressed in micrograms per milliliter except for CH1754, CH103, and CH31); seven V1V2 mAbs (PG9, PG16, PGDM1400, which is expressed as a plasma dilution. Plots are of representative experi- PGT145, CH01, VRC26.08, and VRC26.25); four V3 high mannose ments replicated three times. patch mAbs (PGT121, PGT126, PGT128, and 2G12); a gp120/gp41 interface mAb (8ANC195); membrane proximal external region (MPER) mAbs (4E10, 10E8, and 2F5); V3 epitope directed mAbs retard virus fusion sufficiently to allow for greater neutralization (3869, 3074, and 447–52D); and CD4-induced (CD4i) mAbs (17b, activity to be exerted by MPER and gp120/gp41 interface mAbs. 21c, and 19e). There were also no effects of Env375 substitutions on sensitivity to T1249, which indirectly assesses fusion efficiency (63). Replication of SHIVs in Human and Rhesus CD4 T Cells. Env375 vari- Importantly, we observed that the V3 and CD4i mAbs (64) did not ants of SHIVs B.YU2, D.191859, C.CH505.dCT, C.CH848.dCT, neutralize any of the wild type SHIVs or their Env375 variants aside A.BG505.332N.dCT, and A.BG505.332T.dCT were tested for repli- from modest activity against B.YU2 (Fig. 3 and Fig. S4). These cation in primary, activated human and rhesus CD4 T cells (Fig. 4). findings, together with the consistent neutralization patterns of the All 36 SHIVs replicated in human cells efficiently with the wild-type other mAbs shown in Fig. 3 and Fig. S4, including V1V2 mAbs that Env375S generally equaling or exceeding other Env375 variants. This recognize quaternary epitopes, are strong indications that the 36 result is not surprising, because Env375S is the residue found most SHIVs retain native-like Env trimer conformations and intact con- commonly in HIV-1 group M strains (Fig. S1). In contrast, the formational masking (65). Env375 variants exhibited very different patterns of replication in For most SHIV – antibody combinations, we observed IC50 values rhCD4 T cells. For SHIVs D.191859, C.CH505.dCT, C.CH848.dCT, very similar to the corresponding wild type HIV-1 Envs. The A.BG505.332N.dCT, and A.BG505.332T.dCT, replication of the exceptions were enhanced SHIV.B.YU2 and SHIV.D.191859 wild type Env375S variant was very low or undetectable (Fig. 4). sensitivity to mAbs 4E10, 2F5, 10E8, and 8ANC195. These differ- For SHIV.B.YU2, the 375S variant replicated comparably with ences may reflect the fact that the carboxy terminus of gp41 in the other Env375 variants. The Env375M variant of SHIVs SHIVs B.YU2 and D.191859 (Fig. 1 B and C and Fig. S3)isof B.YU2 and D.191859 (but not of C.CH505.dCT, C.CH848.dCT, HIV-1 origin and must interact with the Gag matrix of SIVmac766, A.BG505.332N.dCT, and A.BG505.332T.dCT) also replicated whereas for the HIV-1 controls the carboxy terminus of gp41 well in rhCD4 T cells. In all six SHIV Env backbones, the 375Y/H/W and the Gag matrix are both of HIV-1 derivation. This gp41– variants replicated well in rhCD4 T cells, generally to levels Gag matrix mismatch was not a confounding factor for SHIVs comparable to those observed in huCD4 T cells, whereas Env375F A.BG505.332N.dCT, A.BG505.332T.dCT, C.CH505.dCT or replication was variable. Thus, there was a good overall corre- C.CH848.dCT because they contain chimeric Envs where the lation among the different SHIV Env375 variants for enhanced carboxy terminus of gp41 is of SIVmac766 derivation (Fig. 1E binding to rhCD4 (Fig. 2 A and C and Dataset S2), enhanced and Fig. S3). We show below that SHIVs containing an SIVmac entry into cells bearing rhCD4 on their surface (Fig. 2B), and gp41 terminus have higher Env:Gag ratios, higher I:P ratios, and enhanced replication in primary activated rhCD4 T cells (Fig. 4). higher replication rates in rhCD4 T cells compared with SHIVs that contain an HIV-1 gp41 terminus. Thus, the gp41–Gag matrix Replication of SHIVs in RMs. Given the encouraging in vitro replica- mismatch for SHIVs B.YU2 and D.191859 (but not for the other tion data, we inoculated a total of 24 RMs with SHIVs by intravenous SHIVs) may alter slightly the per virion number and geometry of or intrarectal routes (Fig. 5). Because we were initially uncertain of the Env trimer complex as it is tethered to the virus membrane or the likelihood of productive infection, we depleted a subset of RMs
E3416 | www.pnas.org/cgi/doi/10.1073/pnas.1606636113 Li et al. Downloaded by guest on September 26, 2021 SHIV.D.191859 SHIV.B.YU2 nonsynonymous mutations in Tat and Nef (RM131) and Vpx, PNAS PLUS 150 150 Human Rhesus Human Rhesus CD4 CD4 CD4 CD4 Vpr, and Nef (RM138) corresponding to known or predicted 125 125 cytotoxic T-cell epitopes (Fig. S5A). 100 100 A striking finding in the week 10 and 20 sequences of both 75 75 RM131 and RM138 was a highly selected deletion of 104–125 nt of 50 50 the carboxy terminus of HIV-1 gp41, including an 11-nt multiple p27 ng /m l 25 25 cloning site used to introduce the HIV-1 tat–rev–vpu–env cassette 0 0 (Fig. S5A). This deletion led to an in-frame juxtaposition of the SHIV.C.CH505.dCT SHIV.C.CH848.dCT corresponding gp41 carboxy terminus of SIVmac766 encoded in 200 350 Human Rhesus Human Rhesus the nef overlap (Fig. S3). A 14- to 19-nt deletion of the multiple CD4 CD4 CD4 CD4 300 125 ′ – – – 150 cloning site at the 5 junction of the HIV-1 tat rev vpu env cassette 250 100 also occurred. Similar, but smaller and less prevalent, deletions 200 100 75 occurred in plasma viral sequences from RM72 and RM74. We 150 50 hypothesized that the substitution of the SIVmac766 gp41 carboxy 100
p27 ng50 /m l 50 25 terminus for the corresponding region of HIV-1 Env contributed to
0 0
SHIV.A.BG505.332T.dCT SHIV.A.BG505.332N.dCT 250 300 Human Rhesus Human Rhesus CD4 CD4 CD4 CD4 SHIV.D.191859 250 A SHIV.B.YU2 200 109 F 8 RM131 RM72 10 RM138 RM74 200 7 150 10 RM131 RM138 RM72 RM74 106 150 5 10 wk2 S 100 104 M 100 3 Y p27 ng /m l 10 wk3 H 50 102 W 50 vRNA copies/ml 1 F 10 wk10 0 0 10 20 30 40 50 60 70 80 90 100 36912 36912 36912 36912 Weeks post infection wk20 Days post infection Days post infection B 8 375S.dCT 375S.dCT 375S.dCT 10 RM130 (IV) RM194 (IR) G S M Y H W F 7 RM133 (IV) RM195 (IR) 10 RM196 (IR) 106 RM197 (IR) Fig. 4. Replication of SHIV Env375 variants in primary human and rhesus CD4 T 105 104 RM130 RM133 RM194 3 cells. Imput virus was normalized by p27Ag (SI Materials and Methods). All vari- 10 0 4000 0 4000 0 4000 ants replicated efficiently in human cells, but only some variants replicated well in 102 vRNA copies/ml 101 rhesus cells. Plots are of representative experiments replicated three times. RM195 10 20 30 40 50 60 RM196 RM197 Weeks post infection C 0 4000 0 4000 0 4000 + 109 Base number Base number Base number of CD8 cells by infusing mAb MT807R1 on the day of virus 108 6069 107 6070 H SHIV.C.CH505 6 6072 inoculation. Fig. 5A shows results for two CD8-depleted RMs 10 6073 105 inoculated intravenously with equal mixtures of the six Env375 104 300 ng 3000 ng 103 variants of SHIV.D.191859 and two other RMs inoculated in- 2 10 6069 6070 6072 6073 vRNA copies/ml 101 travenously with equal mixtures of the six Env375 variants of wk2 10 20 30 40 50 60 SHIV.B.YU2. Persistent viremia was observed for 62–86 wk in Weeks post infection D wk10 all four animals, with substantially higher viral load setpoints for 109 108 6161 SHIV.D.191859 than for SHIV.B.YU2. Both SHIV.D.191859- 107 6163 SHIV.C.CH848 6 6167 I 10 6424 infected animals developed AIDS at weeks 62 (RM138) and 86 105 104 300 ng 3000 ng (RM131) and were euthanized, whereas SHIV.B.YU2 animals 103 2 10 6161 6163 6167 6424
remained healthy beyond 86 wk. vRNA copies/ml 101 wk4 To determine which Env375 variants best supported virus repli- 10 20 30 40 50 60 cation in the SHIV.D.191859 and SHIV.B.YU2 backbones, we E Weeks post infection 109 SHIV.A.BG505 8 6434 J performed deep sequencing by Illumina MiSeq (Fig. 5F)anda 10 Inoculum A Inoculum B Inoculum C 7 6442 10 6446 332 375 N 332 375 332 375 genome-wide analysis by single genome sequencing (SGS) (Fig. 106 6447 T > 105 6454 other S5A) (66). MiSeq analysis at a depth of 2,500 vRNA sequences 104 43335 103 showed that both SHIV.D.191859-inoculated animals were initially 102 6434 6442 6446 6447 6454 43335 infected by SHIVs bearing Env375M, -Y, -H, and -F, but not by vRNA copies/ml 101 wk4 5 10 15 Env375S or -W (Fig. 5F). Env375M replicated preferentially in both Weeks post infection MEDICAL SCIENCES animals at weeks 3, 10, and 20 after infection. In SHIV.B.YU2- inoculated animals, all six Env375 variants led initially to pro- Fig. 5. SHIV replication and selection in RMs. (A) CD8-depleted RMs were ductive infection, although Env375S, and to a lesser extent inoculated intravenously with a mixture of six Env375 variants (50 ng of p27Ag of each variant) of SHIV D.191859 (RM131 and RM138) or SHIV B.YU2 Env375M, replicated preferentially at all time points (Fig. 5F). (RM72 and RM74). (B) Non-CD8-depleted RMs were inoculated intravenously SGS confirmed and extended these findings, demonstrating that or intrarectally with a mixture of SHIV D.191859 variants with or without a sequence variation at weeks 2 and 4 after infection was essentially S375M substitution and with or without an SIV-for-HIV gp41 carboxy ter- random across the entire genome, aside from preferential replica- minal tail exchange (50 ng of p27Ag of each variant intravenously; 500 ng tion of Env375M, -Y, -H, and -F variants (Fig. S5A). The SGS data of p27Ag of each variant intrarectally). (C and D) CD8-depleted RMs were thus make the important point that during the initial 4-wk period of inoculated intravenously with a mixture of six Env375 variants of SHIV infection when virus replication is maximal, there is no apparent C.CH505.dCT or SHIV C.CH848.dCT, respectively (50 ng of p27Ag of each virus adaptation or selection besides that involving the Env375 variant, blue; 500 ng of p27Ag of each variant, red). (E) Non-CD8-depleted substitution. This finding means that SHIV D.191859.Env375M, RMs inoculated intravenously with a mixture of six Env375 variants of SHIV A.BG505.dCT with or without a T332N substitution (50 ng of p27Ag of each -Y, -H, -F variants are well fit from the outset and do not require variant; see J for inocula). (F–J) Sequence analysis of SHIV inocula and SHIV any additional mutations across their entire 9-Kb genomes to evolution in infected RMs. Sequence depth was >2,500 sequences for each achieve efficient replication in RMs. By week 10, there was MiSeq analysis (F and H–J) and ≥24 for SGS. The 293T-derived virus stock was evidence of strong selection for Env375M with additional used for all inoculations. Animal deaths are indicated by a red cross.
Li et al. PNAS | Published online May 31, 2016 | E3417 Downloaded by guest on September 26, 2021 structions, which included Envs from C.CH505, C.CH848, and 40 A.BG505 A.BG505 (Fig. 1E). 35 Four CD8 T-cell–depleted RMs were inoculated intravenously , μg/ml]
50 with a SHIV.C.CH505.dCT.Env375 pool (Fig. 5C) and four others 30 with a SHIV.C.CH848.dCT.Env375 pool (Fig. 5D). All eight animals became productively infected and exhibited indistinguishable 25 C.CH848 early viral replication kinetics. One animal (RM6073) infected 20 by SHIV.C.CH505.dCT died 3 wk after infection from an un- related allergic event; a second SHIV.C.CH505-infected animal 15 (RM6070) was euthanized at 36 wk after infection because of a D.191859 10 rapidly progressive AIDS-defining high-grade lymphoma, and a third SHIV.C.CH505-infected animal (RM6069) was euthanized 5 at 40 wk after infection with wasting, weight loss, rhesus cyto- megalovirus (RhCMV) colitis, and Pneumocystis carinii pneumo- 0 C.CH505 nia (PCP), all AIDS-defining conditions. Two animals (RM6161
Sensitivity of Env 375S to huCD4-Ig [IC B.YU2 and RM6024) infected by SHIV.C.CH848 developed AIDS with S
E3418 | www.pnas.org/cgi/doi/10.1073/pnas.1606636113 Li et al. Downloaded by guest on September 26, 2021 that died with AIDS to characterize virus coreceptor use. In each without passage or adaptation in macaques. The present report PNAS PLUS case, R5 tropism was retained (Fig. S8). goes a long way toward reaching this goal. Peripheral blood CD4 and CD8 T-cell counts, along with effector We prospectively identified six primary HIV-1 Envs for SHIV memory, transitional, central memory, and naïve subsets, were development. In each case, we were successful in constructing assessed in all 24 RMs (Dataset S3). Animals that developed AIDS SHIVs that replicated well in RMs with plasma viral load kinetics exhibited greater declines in absolute CD4 T-cell counts, CD4/CD8 (Fig. 5) remarkably similar to acute and early chronic HIV-1 ratios, and CD4 Tem populations compared with animals that did infection in humans (72, 73). In other recent studies, we created not develop AIDS. At their terminal blood draw, animals that de- a seventh SHIV, this one from a T/F HIV-1 subtype C virus Ce1176 veloped AIDS had marked depressions in all CD4 T-cell subsets. (74), that is part of a widely used 12-member global neutraliza- Analysis of gut-associated lymphoid tissue (GALT) revealed early tion panel developed by Montefiori, Seaman, and coworkers (71). and sustained loss of lamina propria CD4 T cells and marked GI SHIV.C.Ce1176 replicated efficiently in primary human and rhesus tract pathology, including GI tract damage evidenced by enteritis/ CD4 T cells in vitro and productively infected five of five non-CD8- colitis, neutrophil infiltration, crypt abscesses, crypt and villus depleted RMs after intravenous challenge. Thus, using the strategy blunting, and, in one case, infiltration with foamy macrophages, a outlined in Fig. 1, we have created seven consecutive SHIVs from lesion characteristic for atypical mycobacteria infection (Figs. S6 primary or T/F subtype A, B, C, and D HIV-1 viruses that infect and S7 and Dataset S4). Analysis of lymph nodes revealed pathology and replicate efficiently and persistently in RMs, in some cases associated with either asymptomatic SIV disease, such as follicular leading to AIDS pathology and death. This finding contrasts with hyperplasia with confluent germinal centers, or lesions characteristic four recent reports (35–38) that used a conventional KB9 SHIV of classic progressive HIV/SIV disease, including CD4 T-cell loss, design strategy (20), where the vast majority of 91 SHIVs con- lymph node fibrosis, and follicular involution and depletion as- structed from primary HIV-1 Envs failed to replicate efficiently or sociated with amyloid deposits in the germinal centers. Necropsy persistently in vivo. Not only is the success rate of the strategy findings in the seven animals that died with AIDS also included described in the present report far higher, but it has the advantage interstitial pneumonia, lymphoid infiltrates around trachea and of allowing for strategic, prospective targeting of desirable HIV-1 bronchioles, glomerulonephritis and glomerulosclerosis, lymphoid Envs for SHIV construction and analysis, which was highlighted infiltrates in the kidney cortical and medullary areas, well-defined in the present study by SHIVs containing T/F Envs corre- granulomas in the liver consistent with atypical mycobacterial in- sponding to HIV-1 CH505 (53, 55, 75), CH848, and BG505 (54, fection, and inflammatory infiltrates around portal and central 57–60), all of which evolved in their human hosts to elicit bNAbs. veins (Figs. S6 and S7 and Dataset S4). The SHIVs described herein also exhibit particularly favorable Western blot immunoreactivity to core and Env proteins was biological and antigenic properties that distinguish them from assessed at 4 and 10 wk after infection, corresponding to a time most previously described SHIVs (3, 9, 18, 21, 29–33, 35–38, 76). frame that in humans is associated with anti–HIV-1 antibody Specifically, they exhibit sensitivity to a wide spectrum of broadly seroconversion (68). Western blot reactivity to diagnostic Env and neutralizing mAbs that target the five principal sites of bNAb Gag proteins was strongly positive in all but three animals (RM195, recognition (77, 78), while retaining effective conformational RM6424, and RM6161) (Dataset S3). Two of these animals had masking typical of primary or T/F virus strains (23, 65). entirely negative Western immunoblots, which was associated with If substitution of Env375 with variant residues is the sine qua non high peak and setpoint plasma virus titers and accelerated death of our SHIV design strategy, what, then, is the mechanistic basis for due to AIDS at weeks 10 and 14. Similar findings have been enhanced rhCD4 binding and cell entry? The native, unliganded reported in a subset of SIV-infected RMs (67) and in rare human HIV-1 Env trimer is conformationally flexible or “metastable” (25, HIV-1 infections (69). Anti-SIVp27 binding antibody titers were 44, 49, 50, 79–81), and movement of the Env trimer from its unli- assessed at weeks 10 and 32 after infection. Results were strongly ganded state to the CD4-bound conformation must be carefully reactive for all animals, except RM6424 and RM6161 (Dataset S3). regulated, because premature triggering of the unliganded glyco- NAb responses were assessed in 13 RMs with at least 4 mo follow- protein complex to downstream conformations results in functional up postinfection against a standardized global panel of tier 1 (n = inactivation (82, 83). Structural and molecular biological studies have 3) and tier 2 (n = 17) virus strains corresponding to HIV-1 sub- revealed a “layered” inner domain gp120 architecture that facilitates types A, B, C, D, and circulating recombinant-form CRF01_AE movement among alternative Env conformations required for virus (Dataset S5) (70, 71). Among SHIV D.191859-infected (n = 8), entry (25, 50, 80, 84). Env gp120 refolds itself upon binding CD4, B.YU2-infected (n = 2), and C.CH505-infected (n = 3) animals, with the inner, outer, and bridging sheet domains brought into 3 12 of 13 had reciprocal autologous NAb titers (IC50), ranging proximity at the interface with CD4. This process creates a ∼150-Å from 25 to 7,911 (Datasets S3 and S5). There was a statistically cavity between HIV-1 gp120 and CD4 bounded by Phe-43 of CD4, significant positive correlation between viral load setpoint and which makes numerous contacts with conserved gp120 residues, al- maximum autologous NAb titer (Spearman rs = 0.7, P < 0.05). though Env375 is not one of them (48, 85). Env375 does, however,
Among 10 SHIV D.191859- or B.YU2-infected animals that were abut the Phe-43 cavity, as do other highly conserved residues, many MEDICAL SCIENCES sampled for >1 y, 10 had reciprocal plasma IC50 NAb titers to of them containing aromatic side chains. These Phe-43 cavity-lining three heterologous tier 1 viruses ranging from 34 to >43,740; 10 residues contribute to an aromatic array that helps stabilize the CD4- had titers against at least one heterologous tier 2 virus, three ani- bound conformation. Substitution of Env375W for Env375S in HIV-1 mals neutralized two heterologous tier 2 viruses, and one or more YU2 gp120 fills the Phe-43 cavity and allows the glycoprotein to animals neutralized four, five, or seven heterologous tier 2 viruses. sample conformations closer to the CD4-bound state spontaneously Reciprocal IC50 NAb titers against heterologous tier 2 viruses were (25, 44, 50, 84). This process leads to enhanced affinity for CD4, generally low, ranging from 20 to 557. largely due to a decrease in the off-rate of the bound Env–CD4 complex (50). We hypothesized that substitution of the naturally Discussion occurring HIV-1 Env375S by certain larger space-occupying side Overbaugh and coworkers recently reviewed the history of SHIV chains would enhance Env gp120 binding to rhesus CD4 and facil- development, the limitations of current SHIVs, and the still largely itate virus entry in rhesus cells in a similar manner. Molecular unrealized potential of SHIVs to advance vaccine, cure, and modeling of HIV-1 gp120 in complex with human and rhesus CD4 pathogenesis research (1). They, along with other authors (35–38), (Fig. S2), together with three independent lines of empirical evi- concluded that a paramount objective continues to be the devel- dence—neutralization of infectious virus by soluble rhCD4 (Fig. 2A), opment of pathogenic SHIVs that encode env sequences from entry of virus into rhCD4-bearing target cells (Fig. 2B), and direct circulating T/F HIV-1 variants and establish persistent infection analysis of Env binding to rhCD4 by SPR (Fig. 2C)—support this
Li et al. PNAS | Published online May 31, 2016 | E3419 Downloaded by guest on September 26, 2021 hypothesis. Thus, we conclude that the mechanism by which the in most RMs to reciprocal IC50 titers between 25 and 8,000 in the different SHIV Env375 amino acid substitutions enhance rhCD4 first 3–6 mo of infection (Datasets S3 and S5), whereas heterologous binding is likely to be similar to that described for enhanced binding tier 2 NAbs developed sporadically and generally to only low titers of the YU2 Env375W variant to human CD4 (25, 44, 50, 84). after 1 y of infection (Dataset S5). Further studies with additional Factors contributing to successful replication of SHIV variants SHIVs, larger numbers of infected animals, and longer follow-up in rhesus cells in vitro and in vivo, however, likely extend beyond periods will be needed to assess the similarities and differences the initial binding event of Env to rhCD4. One of these factors is in NAb responses in RMs and humans to viruses bearing primary the “intrinsic reactivity” or “responsiveness” of Env to CD4 engage- HIV-1 Envs. ment, which reflects the product of CD4 binding and downstream Designer SHIVs bearing selected amino acid substitutions at consequences of CD4 binding that allow the Env glycoprotein to residue 375 can be an enabling tool for HIV vaccine and cure re- negotiate transitions to lower-energy states, including those responsible search. As challenge stocks for active and passive preclinical vac- for cell entry (25, 44, 50, 80, 82, 84, 86, 87). Env responsiveness to CD4 cines studies, we now have in hand well-pedigreed SHIVs that influences the ability of HIV-1 to infect cells with different levels of express primary or T/F subtype A, B, C, and D Envs. These SHIVs CD4 (e.g., macrophages) or variants of CD4 (e.g., CD4 from have been expanded in primary activated rhCD4 T cells in tissue other species) and the sensitivity of HIV-1 to sCD4. The respon- culture to high titers (∼109 virions per milliliter) and exhibit high siveness of HIV-1 to CD4 can be altered by generic changes in Env, infectivity-to-particle ratios when measured on TZM-bl cells which also affect the sensitivity of the virus to antibody neutrali- (∼0.01) and primary rhCD4 T cells (∼0.0005) (Dataset S1). They zation (31), or by more selective changes, as in the case of Env375 also transmit efficiently in vivo across mucosal surfaces, including variants. We hypothesized that SHIVs must attain a certain level of rectal, penile (glans and foreskin), or vaginal without hormonal Env responsiveness to rhCD4 to infect RM CD4 T cells and manipulation. This finding makes possible RM modeling of vaccine replicate efficiently in vivo, but without leading to abortive prevention against all of the principal routes of HIV-1 acquisition downstream conformations that prevent infection. A corollary is using SHIVs with biologically relevant Envs. Importantly, the SHIV that, because natural HIV-1 Env variants exhibit different levels C.CH505, C.CH848, D.191859, and A.BG505 challenge stocks of CD4 responsiveness, different degrees of compensation con- contain no discernible in vitro adaptations that alter their native ferred by changes in residue 375 will be required for optimal antigenicity or neutralization sensitivity, a problem that has plagued adaptation to rhCD4. To test these notions, we used the sensi- other SHIV challenge viruses (12, 29, 31–33, 35, 38). With respect tivity of SHIVs containing wild-type Env375S to human and to immunogen design, by strategically targeting for SHIV con- rhesus sCD4-Ig as an indicator of the baseline CD4 responsiveness struction primary or T/F HIV-1 Envs with unique properties such as and determined whether the size of residue 375 side chains con- the ability to bind unmutated common ancestor germ-line Ig re- tributing to efficient virus replication in rhCD4 T cells in vitro and ceptors of human bNAb lineages [e.g., CD4bs bNAb lineages by in vivo correlated inversely. The answer was affirmative (Fig. 6). HIV-1 CH505 (53, 55, 75) or V1/V2 bNAb lineages by HIV-1 The results shown support the hypothesis that to replicate effi- CRF_AG_250, ZM233, WITO4160, and CAP256 (88, 89)] or to ciently in monkeys, SHIVs with lower baseline CD4 responsiveness, elicit bNAbs in humans [e.g., HIV-1 CH505, CH848, and BG505 such as C.CH505, C.CH848, and A.BG505, require residue Env375 (53–55, 75)], critical questions regarding the inherent immunoge- substitutions that result in proportionally greater CD4 responsiveness. nicity of particular Env glycoproteins, contributions of rhesus host Conversely, SHIVs with higher levels of baseline CD4 responsiveness genetics to the elicitation of NAbs and bNAbs, and the role of (i.e., that exhibit greater sensitivity to sCD4), such as B.YU2 and stochastic vs. deterministic events in antibody elicitation can be D.191859, require residue Env375 substitutions that result in smaller parsed. In this way, analysis of virus–antibody coevolution can po- changes in CD4 responsiveness. These observations suggest tentially guide structure-based (56, 90, 91) or viral lineage-based that a major force driving the selective replication of SHIV — (92, 93) immunogen design. Finally, further model development to Env375 variants in RMs 375S for B.YU2, 375M for D.191859, – 375H for C.CH505 and C.CH848, and 375Y for BG505.332T document authentic HIV-1 like SHIV persistence and pathogene- and BG505.332N—is the requirement to achieve both adequate sis and susceptibility to suppression by combination antiretroviral rhCD4 binding and enhanced Env responsiveness to CD4 without therapy should set the stage for these viruses to facilitate the pre- altering other essential properties of the native Env trimer, including clinical evaluation of HIV-1 Env targeted therapeutic interventions conformational masking and orderly downstream conformational in the context of HIV-1 cure research. – changes leading to virus cell membrane fusion. Materials and Methods Although CD8 depletion in half of the study animals limited our ability to fully evaluate the natural history and immunopa- Nonhuman Primates. Indian RMs were housed and studied at the University of Pittsburgh or Bioqual, according to the Association for Assessment and Ac- thogenesis of SHIV infections, the findings from analysis of creditation of Laboratory Animal Care standards. Experiments were ap- plasma viral load kinetics and peripheral blood T-cell subsets in proved by the University of Pittsburgh and Bioqual Institutional Animal Care all animals at multiple time points, biopsies of lymph nodes and and Use Committees (SI Materials and Methods). GALT at early and late time points, and immunopathological examinations at necropsy, leave no doubt as to the pathogenicity SHIV Constructions. The SIVmac766 clone (GenBank accession no. KU955514) of the Env375-modified T/F SHIVs. Plasma viral load kinetics in was modified by replacing the SIV genome between the stop codon of vpr acute and early chronic phases of infection (Fig. 5) were nearly and start codon nef with a linker containing the unique restriction enzyme indistinguishable from human infection by HIV-1 (72, 73). All sites AgeI and AclI. A complete HIV-1 tat/rev/vpu/env(gp160) cassette was CD4 T-cell subsets, not just CD4 naïve cells, underwent gradual inserted (SI Materials and Methods). decline; GALT was selectively depleted of CD4 T cells early in SHIV Infection of Primary Rhesus and Human CD4 T Cells. SHIV stocks were infection and did not recover; and R5 tropism of SHIVs did not ∼ change to X4 tropism, even in animals that died of accelerated generated in 293T cells. Virus imput was normalized to 1,000 virions (0.1 pg of p27Ag) per cell (SI Materials and Methods). AIDS within the first 3 mo of infection (Fig. S8). AIDS-defining events included wasting, CD4 T-cell lymphopenia, pancytopenia, SHIV Entry Assays. SHIV entry was assessed in primary activated human and PCP, atypical mycobacterial infections, RhCMV reactivation and rhesus CD4 T cells and in cell lines expressing huCD4 and huCCR5 [TZM-bl cells disease, and high-grade lymphoma (Dataset S4). Of note, the (61, 94)] or rhCD4 and rhCCR5 (ZB5 cells) (SI Materials and Methods). development of NAbs in SHIV-infected RMs resembled that in humans infected by HIV-1 with respect to kinetics, titers, breadth, SHIV Neutralization Assays. Neutralization was assessed in TZM-bl cells by a and potency. In SHIV infections, autologous tier 2 NAbs developed standardized method (61, 70, 71, 94).
E3420 | www.pnas.org/cgi/doi/10.1073/pnas.1606636113 Li et al. Downloaded by guest on September 26, 2021 SPR. Three HIV-1 Envs with residue 375 substitutions were analyzed, as de- Phylogenetic Analyses. Lentiviral pol genes were aligned by using clustalw PNAS PLUS scribed (SI Materials and Methods). (v2.1) (97) and analyzed by using PhyML v3.0 (98) (SI Materials and Methods).
Virion-Associated Env:Gag Ratio Measurements. Virus was purified by filtration Viral and Immunodiagnostics. Quantitative and qualitative analyses of virus, and density gradient centrifugation. Quantitative measurements of viral p27 antibodies, and cells were performed as described (SI Materials and Methods). (CA) and gp120 protein were performed by dual-color fluorescent protein gel analysis (41, 95) (SI Materials and Methods). Molecular Modeling. Structural analysis was performed by using PyMOL (99) and Molprobity (45, 100–102). Mutants for rhCD4 or residue 375 were generated in Viral Sequencing. SGS and MiSeq sequences were generated as described silico by using the mutagenesis tool in PyMOL (SI Materials and Methods). previously (23, 66) (SI Materials and Methods). Statistical Analyses. Unpaired t and Fisher exact tests and Spearman rank cor- Immunohistochemistry. Immunohistological stains were performed as described relations were calculated by using Prism 6 software (SI Materials and Methods). previously (96). ACKNOWLEDGMENTS. We thank C. Xu, T. He, T. Dunsmore, G. H. Richter, K. Raehtz, J. Roser, R. Fast, K. Oswald, R. Shoemaker, J. Bess, and A. Meyer for technical Coreceptor Use. Coreceptor use was determined in TZM-bl cells by selective assistance. This project was funded in part by Bill & Melinda Gates Founda- blockade using Maraviroc (CCR5), AMD3100 (CXCR4), or a mixture of in- tion Grants OPP37874 and OPP1145046; and NIH Grants AI100645, AI088564, hibitors (66) (SI Materials and Methods). AI094604, AI087383, AI078788, AI045008, and HHSN261200800001E.
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