Diverse HIV-1 Isolates Antigen on HIV-1 Particles and Neutralize Antibodies That React with a Cellular GB Virus Type C Envelope

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GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates This information is current as of October 2, 2021. Emma L. Mohr, Jinhua Xiang, James H. McLinden, Thomas M. Kaufman, Qing Chang, David C. Montefiori, Donna Klinzman and Jack T. Stapleton J Immunol 2010; 185:4496-4505; Prepublished online 8

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates

Emma L. Mohr,*,†,1 Jinhua Xiang,*,1 James H. McLinden,* Thomas M. Kaufman,* Qing Chang,* David C. Monteﬁori,‡ Donna Klinzman,* and Jack T. Stapleton*,†

Broadly neutralizing Abs to HIV-1 are well described; however, identiﬁcation of Ags that elicit these Abs has proven difﬁcult. Per- sistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus Downloaded from particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1– pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune- precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that http://www.jimmunol.org/ cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs. The Journal of Immunology, 2010, 185: 4496–4505.

uman immunodeficiency virus-1 vaccine development treatment (Fuzeon) (5, 6). 2F5 and 4E10 Abs are polyspecific and has relied primarily on the use of HIV-1 proteins as cross-react with cellular Ags including several lipids (7–12). Al- H immunogens in an attempt to elicit either neutralizing Abs though Ags that interact with these Abs have been identified, active or cellular immune responses to prevent or modify HIV-related immunization with gp41, MPER, or T-20 does not elicit broadly

disease (reviewed in Refs. 1 and 2). Because of the high replica- neutralizing HIV Abs (13, 14). Clearly, new approaches to HIV-1 by guest on October 2, 2021 tive rate of HIV-1 and the error-prone RNA-dependent DNA vaccines are needed (1, 2). polymerase, neutralization and T cell escape mutants are generated GB virus type C (GBV-C)is a common human infection that is not on a daily if not hourly basis in infected individuals. Broadly clearly associated with any disease. The virus replicates in B and neutralizing human HIV mAbs (hmAbs) have been isolated from T lymphocytes including CD4+ and CD8+ T cell subsets (15; HIV-infected individuals (3), including hmAbs directed against reviewed in Ref. 16). Because of shared modes of transmission, the gp120 that interfere with CD4 binding (e.g., 2G12) or that react prevalence of GBV-C in HIV-infected people is high (17–42%) with the membrane proximal ectodomain region (MPER) of gp41 (17). Several studies and a meta-analysis of studies including 1294 (e.g., 2F5 and 4E10) (3, 4). These Abs also react with a 36-residue HIV-infected individuals found that persistent GBV-C infection is peptide that overlaps with the MPER called T-20 (5). T-20 inhibits associated with prolonged survival (18–22). GBV-C infection is also HIV replication by preventing virus envelope fusion with the cell associated with decreased maternal-to-child HIV-1 transmission (23, membrane, and T-20 is an effective and licensed antiretroviral 24). Abs to GBV-C are usually not detected during viremia; however,

*Division of Infectious Diseases, Department of Internal Medicine, Iowa City Veterans Address correspondence and reprint requests to Dr. Jack Stapleton, Department of Affairs Medical Center; †Interdisciplinary Program in Molecular and Cellular Biology, Internal Medicine, SW54, General Hospital, 200 Hawkins Drive, Iowa City, IA University of Iowa, Iowa City, IA 52242; and ‡Department of Surgery, Duke University, 52242. E-mail address: [email protected] Durham, NC, 27708 Abbreviations used in this paper: AZT, azidothymidine; BHK, baby hamster kidney; 1E.L.M. and J.X. contributed equally to this work. CHO, Chinese hamster ovary; GBV-C, GB virus type C; GBVpp, GBV-C enveloped retrovirus pseudoparticle; HIV , HIV-enveloped retrovirus pseudoparticle; HIV , Received for publication June 14, 2010. Accepted for publication August 3, 2010. pp ppHIV HIV-enveloped retrovirus pseudoparticle; hmAb, human mAb; HOS, human osteo- This work was supported in part by grants from the Department of Veterans Affairs, carcoma; I, IgG; IC, isotype control; IP, immune precipitation; IPV, the amount of Veterans Health Administration, Ofﬁce of Research and Development (Merit Review input virus used in an immune-precipitated experiment; M, the lane containing the to J.X. and J.T.S.), the National Institutes of Health (Grant R01 AI-58740 to J.T.S.), m.w. marker; MPER, membrane proximal ectodomain region; MV, mumps virus; NA, a Grand Challenges grant from the Bill and Melinda Gates Foundation (to J.X.), and not applicable (not obtained from NARRRP); NARRRP, National Institutes of Health the Biological Sciences Funding Program from the University of Iowa (to J.X.). E.L.M. AIDS Research and Reference Reagent Program; NS5A, GBV-C nonstructural protein received support from the University of Iowa Medical Scientist Training Program and 5A; P, positive; Pep, rabbit anti–GBV-C E2 peptide IgG; Post, postimmune rabbit IgG; from the Virology T-32 Training Grant. R5, CCR5 tropic; RLU, relative light unit; S, serum; Sup, supernatant; UIVC, isolate obtained from University of Iowa Virology Clinic; VS, virostat; VSV, vesicular sto- Portions of this work were presented at the International Meeting of the Institute matitis virus; VSV , VSV-enveloped retrovirus pseudoparticle; X4, CXCR4 Human Virology, Baltimore, MD (October 31–November 4, 2004); the International ppHIV tropic; YFV, yellow fever virus. AIDS Society Meeting (July 22–25, 2007) in Sydney, New South Wales, Australia; the Conference on Retroviruses and Opportunistic Infections, Montreal, Quebec, Canada (February 8–11, 2009); and the Keystone HIV Vaccines Conference, Banff, Alberta, Canada (March 21–26, 2010). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001980 The Journal of Immunology 4497

Table I. Characteristics of HIV isolates used in neutralization assays

Isolate Isolate Name Tropism Derivationa Clade Catalog No.b Cell Types Studied 1 HIV-1 92UGO31 R5 Clinical A 1741 PBMC 2 AZT intermediatec R5-X4 Clinical B 1073 PBMC 3 HIV-1 92UGO29 X4 Clinical A 1650 MT-2; PBMC 4 HIV-1JR-CSF R5 Laboratory B 394 PBMC 5 HIV-1IIIB/H9 X4 Laboratory B 398 MT-2; PBMC 6 HIV-1xxBru-JF X4 Laboratory B 2969 PBMC 7 HIV-1ELI X4 Laboratory D 2521 MT-2; PBMC 8 NFN-HAS (pNL4-3) X4 Laboratory B NA MT-2; PBMC 9 NFN-SX-HSA (pNL4-3) R5 Laboratory B NA PBMC 10 UIVC R5 Clinical B NA MT-2; PBMC 11 UIVC R5/X4 Clinical B NA MT-2; PBMC aDerivation refers to whether HIV isolate is a laboratory-adapted isolate or a clinical isolate. bNARRRP catalog number. Isolate name from NARRRP data sheet. cIsolate was initially mixed tropic (R5-X4), but following ampliﬁcation in MT-2, cells reverted to CXCR4 tropic. NA, not applicable (not obtained from NARRRP); R5, CCR5 tropic; UIVC, isolate obtained from University of Iowa Virology Clinic; X4, CXCR4 tropic.

following clearance of GBV-C, Abs specific for the envelope gly- complex. M6 binds to six amino acids within E2 if there are four Downloaded from coprotein (E2) are identified. Consequently, GBV-C E2 Ab serves as or eight amino acids added to the C or N termini, respectively, sug- a marker of prior infection (reviewed in Ref. 16). Although persistent gesting that there is a size and sequence requirement for interaction GBV-C viremia is associated with the best survival in epidemio- (26). A GBV-C E2 peptide encompassing this epitope has been logical studies (25), one study found that subjects without viremia proposed to be involved in GBV-C–cell membrane fusion, based on who have GBV-C E2 Abs survived longer than those without E2 Abs findings that it forms an amphipathic helix in the presence of lipids

(20). Human GBV-C E2 Abs and all but one of characterized GBV-C and model membranes (28, 29). In addition, another E2 peptide that http://www.jimmunol.org/ E2 murine mAbs are conformation dependent (26). One mAb (M6) overlaps the putative fusion peptide prevents oligomerization of the recognizes a linear epitope on E2 (27); however, the interaction is HIV-1 gp41 fusion peptide and membrane fusion in an in vitro model (30). Finally, incubation of PBMCs or CD4+ T cell lines with the GBV-C envelope glycoprotein E2 competitively inhibits HIV-1 entry invitro (31, 32), raising the possibility that there is structural mimicry between GBV-C E2 and HIV-1 particles, or between E2 and a cell surface molecule carried on the HIV particle that is involved in HIV-1 entry. If this is true, Abs directed against GBV-C E2 may interfere

with HIVattachment or fusion and potentially modify HIV-1 disease by guest on October 2, 2021 progression.

FIGURE 2. GBV-C E2 protein elicits HIV-1–neutralizing Abs in mice. FIGURE 1. Ab to the GBV-C envelope glycoprotein E2 neutralizes Mouse sera (diluted 1/5000) from two of three mice immunized with HIV-1 in vitro. Human serum (S) diluted 1/100 obtained from GBV-C E2 rGBV-C E2 protein reacted with CHO cell culture supernatant containing Ab-positive (P) subjects (n = 3; SP1, SP2, and SP3), IgG (I) puriﬁed from rE2 but not supernatant (Sup) from CHO cells not expressing E2. Pooled these sera (I-P1, I-P2, and I-P3; 10 mg/ml), murine mAbs [M1(VS) and preimmune murine sera diluted 1/5000 did not react with E2 (A). There M6], and human HIV-1 mAbs (2G12, 2F5, and 4E10) (5 mg/ml for all was insufﬁcient serum from the third mouse to test by immunoblot. The mAbs) neutralized CCR5- and CXCR4-tropic HIV-1 isolates in primary concentrations of HIV p24 Ag produced in MT-2 cells 5 d following in- human PBMCs compared with no Ab controls, whereas E2 Ab-negative cubation with preimmune sera (1/1000) or postimmune (GBV-C E2 Ab human sera (SN1 and SN2), IgG (I-N1 and I-N2), or murine isotype positive) sera are shown in B. Data represent the average HIV p24 con- control (IC) Ab did not (A). HIV p24 Ag concentration was measured 5 d centration released by cells following neutralization with the three mouse postinfection. The dose relationship between the human IgG preparations serum samples performed at the dilutions shown. The dashed line shows is shown (B). the amount of HIV-1 p24 Ag produced in the no Ab control cells. 4498 GBV-C E2 Abs NEUTRALIZE HIV-1

We examined naturally occurring human and experimentally Cells and viruses induced murine and rabbit GBV-C E2 Abs and found that they cross- PBMCs were isolated from blood obtained from healthy donors. All human reacted with and neutralized diverse HIV-1 isolates. They also subjects provided written informed consent, and the project was approved by precipitated pseudotyped HIV-1 gag particles independent of the the University of Iowa Institutional Review Board. CD4+ cell lines (MT-2 viral envelope used. In contrast, the Abs did not neutralize or pre- and human osteosarcoma [HOS] cells expressing human CD4, CCR5, and cipitate mumps virus or yellow fever virus (YFV), and they did not CXCR4) were maintained as described previously (34–36). The HOS cells were obtained from NARRRP and also contained GFP under the control of neutralize vesicular stomatitis virus (VSV) particles, suggesting that HIV-1 long terminal repeat (catalog numbers 3685 and 3944). Baby hamster the Ag is of cellular origin and that it is present on HIV-1 gag kidney (BHK) 21 cells were maintained in DMEM containing 10% FCS and particles. Thus, immunization with GBV-C E2 protein elicited antibiotics. Table I summarizes the infectious HIV-1 isolates used in these broadly neutralizing HIV Abs, a long sought-after goal in HIV-1 studies. Brieﬂy, using the nomenclature described in the data sheets for vaccine development.

Materials and Methods Ags and Abs GBV-C E2 protein truncated to remove the C-terminal transmembrane domain (nt 1167–2162 based on the infectious clone isolate; GenBank number AF121950) (33) was expressed in Chinese hamster ovary (CHO) cells as described previously (26). The CHO cells were adapted for serum- free growth, and supernatants containing GBV-C E2 protein were concen- Downloaded from trated on an Amicon CP10 concentrator (Millipore, Billerica, MA). Three BALB/C mice were immunized with 25 mg rGBV-C E2 protein mixed with IFA, followed by two immunizations of E2 in IFA at 2-wk intervals. Rabbits were immunized with 50 mg E2 in IFA (Invitrogen, Carlsbad, CA) and boosted 6 and 12 wk later by the Iowa State University Hybridoma Facility (Ames, IA). Sera were collected before immunization (preimmune) and at various intervals after immunization for analysis and IgG puriﬁcation as described previously (26). A synthetic peptide representing GBV-C E2 http://www.jimmunol.org/ sequences (GGAGLTGGFYEPLVRRC) was provided by Dr. O. Sharma (National Institutes of Health [NIH] AIDS Research and Reference Reagent Program [NARRRP], Rockville, MD). Rabbits were immunized with KLH- conjugated E2 peptide (100 mg) in IFA and boosted 2, 7, and 9 wk later. HIV-, HCV- and HBsAg-negative sera were obtained from healthy blood donors. Anti-E2 Ab status was assessed by ELISA (mPlate anti-HGenv test; provided by Dr. G. Hess, Roche Diagnostics, Mannheim, Germany). Murine anti–GBV-C E2 mAb M1(virostat [VS]) was purchased (Virostat, Portland, ME), whereas M6 and M11 (Roche Laboratories, Penzberg, Germany) were provided by Dr. A. Engel (Roche Diagnostics). Isotype control Abs were purchased from Sigma-Aldrich (St. Louis, MO). M1(VS) and M11 recognize by guest on October 2, 2021 conformation-dependent epitopes, whereas M6 recognizes a linear epitope on GBV-C E2 (26). E2 immunoblot were performed as described previously (26). HIV-neutralizing hmAbs 2F5, 2G12, and 4E10 were obtained from NARRRP (catalog numbers 1475, 1476, and 10091, respectively).

FIGURE 4. Rabbit anti–GBV-C E2 Abs neutralize infectious HIV-1 particles and HIV-1–pseudotyped HIV-1 particles but not HIV-1–pseudotyped VSV particles. Rabbit anti–GBV-C E2 IgG from the four rabbits (R1, R2, R3, and R4) neutralized HIV replication in MT-2 cells compared with no Ab, rabbit preimmune IgG, and control Abs (anti-hepatitis B surface Ag and anti–GBV-C NS5A protein). pp , 0.001 for R1–R4 versus all controls (A). Postimmune rabbit IgG (Post) was maintained in the FIGURE 3. GBV-C E2 protein and antigenic peptide elicits E2 Abs in rab- media or was removed from media following washing of cells postin- bits. Rabbit serum (n = 2) obtained following immunization with GBV-C E2 fection. HIV-1 replication was monitored at day 5 postinfection (B). pp , peptide (rPep-post) or GBV-C E2 protein (n = 4; R1, R2, R3, and R4) reacted 0.01; †p , 0.05, respectively, when to preimmune IgG. NA, no Ab control. with GBV-C E2 protein by ELISA (A), whereas the preimmune peptide (rPep- Postimmune rabbit IgG (rabbit 2, R2) neutralized HIVenv-pseudotyped pre) and preimmune E2 sera (E2-pre) did not. All rabbit Abs reacted with E2 defective HIV-1 particles [HIVppHIV E2(R2)] but not HIVenv pseudotyped protein by immunoblot analysis (B). M6 is a control murine mAb that recognizes defective VSV particles (VSVppHIV), whereas preimmune rabbit IgG did a linear epitope on GBV-C E2. M, the lane containing the m.w. marker; Pep, not neutralize either (A). †p , 0.005 compared with no Ab control; ‡p , rabbit anti–GBV-C E2 peptide IgG. 0.001 compared with no Ab control and preimmune (C). The Journal of Immunology 4499

Table II. Anti–GBV-C E2 Abs neutralize diverse HIV isolates

HIV Isolate Number (see Table I for characteristics)

Ab Description Ab Identiﬁcation 1234567891011 Human anti-E2 polyclonal IgG I-P1 8.1a 3.4 2.9 1.6 2.2 2.3 4.1 3.5 2.8 3.7 5.1 I-P2 2.6 8.3 I-P3 5.8 1.7 ,10 ,10 Rabbit anti-E2 protein polyclonal IgG R1b,c 4.3 5.0 R2 1.5 2.1 1.1 3.1 1.6 ,10 ,10 ,10 ,10 1.5 1.2 R3 9.3 10 R4 5.6 4.9 ,10 ,10 ,10 ,10 ,10 2.1 1.7 Rabbit anti-E2 peptide polyclonal IgG rPep-1c >50 >50 >50 >50 >50 >50 >50 rPep-2 >50 >50 >50 >50 Mouse anti-E2 monoclonal IgG M1 (VS) 1.4 2.5 ,5 ,5 ,5 ,5 ,5 ,5 ,5 M5 ,5 ,5 M6 1.8 1.1 Human anti-HIV monoclonal IgG 2F5 1.7 4E10 1.3 2G12 >5 0.2

IC50 results .5 mg/ml for mAbs or .30 mg/ml for polyclonal Abs are in bold. aNeutralization assays were performed in PBMCs for R5 isolates and MT-2 cells for X4 isolates. b Downloaded from Concentration (micrograms per milliliter) required to reduce HIV infectivity by 50% compared with control Ab (IC50). cRabbits were immunized with either rGBV-C E2 protein (anti-E2) or the 17-aa peptide Ag recognized by the M6 murine mAb (anti-rPep) as described in Materials and Methods. Blank, not tested. The highest or lowest concentration tested is equal to or more than or less than, respectively.

HIV-1 isolates obtained from NARRRP, we studied the following viruses: at 4˚C. Alternatively, 293T cells were transfected with pHXB2 env-expressing R5 isolates—HIV-1 92UGO31 (clade A; catalog number 1741), HIVJR-CSF plasmid and tranduced with defective VSV particles with a GFP reporter gene (clade B; catalog number 394), and NFN-SX-heat stabile Ag (HSA) (clade provided by Dr. W. Maury (University of Iowa). Pseudotyped VSV particles http://www.jimmunol.org/ B); and X4 isolates—HIV-1 92UGO29 (clade A; catalog number 1650), were collected from supernatants and filtered with a 0.45-mm filter prior to azidothymidine (AZT) intermediate (clade B; catalog number 1073), HIV- use. 1IIIB/H9 (clade B; catalog number 398), HIVxxBru-JF (clade B; catalog number 2969), HIVELI (clade D; catalog number 2521), and NFN-HSA (pNL4.3 backbone; clade B). Infectious cDNA clones of two HIV-1 iso- Viral infections and neutralization assays lates were provided by Dr. B. Jamieson (University of California, Los The HIV-1 inocula (2 ng/ml p24 Ag per infection) or defective HIVor VSV Angeles, Los Angeles, CA): NFN-HSA (derived from pNL4.3 and ex- particles (produced in 293T) were incubated with a range of concentrations of pressing murine HSA [also known as CD24]) and NFN-SX-HSA (pNL4.3 GBV-CE2 Ab-positive sera, IgG preparations or mAbs for 1 h at 37˚C prior to env replaced with an R5-tropic env gene (37, 38). Two clinical isolates were adding to CD4+ T cell or HOS cell cultures. GBV-C E2 Ab-negative sera, E2 obtained and characterized from the University of Iowa HIV/AIDS Clinic Ab-negative IgG preparations, and isotype control mAbs served as the (Iowa City, IA). HIV stock viruses were prepared in MT-2 cells (X4 iso- negative control Abs. Inocula were removed, CD4+ cells (PBMCs or MT-2 by guest on October 2, 2021 lates), PBMCs (X4 and R5 isolates), or HOS cells as indicated in Results. as described in Results) were washed after 4 h, and culture supernatants were YFV (vaccine strain, 17D; Sanofi-Pasteur, Swiftwater, PA) was propagated collected postinfection for measurement of HIV-1 replication by measuring in BHK21 cells or MT-2 cells. The mumps virus was propagated in Vero HIV-1 p24 Ag (ELISA) or reporter gene expression 72 h posttransduction Cells or MT-2 cells. as described previously (19, 35). Pseudoparticle transduction was deter- Pseudotype virus production mined by measuring luciferase by the Bright-Glo Luciferase Assay System (Promega, Madison, WI) or GFP expression by flow cytometry. The con- 293T cells were cotransfected with pNL4-3.Luc.R-E2 (luciferase reporter centration of each Ab preparation (micrograms per milliliter) required to inserted into pNL4-3 nef gene) and a plasmid that expresses the envelope reduce HIV-1 p24 Ag release into culture supernatants by 50% was calcu- glycoproteins of VSV (VSV-G), HIV (pHXB2-env), GBV-C E1-E2 (nt lated (IC50). YFVand mumps virus infectious titers (tissue culture infective 555–2479), or no viral envelope protein using CaCl2 as described pre- dose 50%) were determined by terminal dilution in BHK21 cells or viously (39). Particles were collected 72 h posttransfection, filtered with Vero cells, respectively (40, 41), and YFV and mumps viral RNA concen- a 0.45-mm filter, and concentrated by centrifugation at 12,000 rpm for 20 h trations were measured by real-time PCR as described previously (42, 43).

Table III. Interassay variation in GBV-C E2 Ab neutralization

ID50 in TZM-bl Cellsa Virus Isolates

MN SS1196.01 QH0692.42 PAVO 0.04 6535 0.03 REJO 0.67 THRO 0.18 Du123 0.06 Du156 0.12 Du172 0.17 Du422.01 SIVmac Ab (B) (B) (B) (B) (B) (B) (B) (C) (C) (C) (C) 239/CS) M1 (VS) 3.9 4.2 4.3 4.4 4 >5 4 2.3 2.5 2.7 2.7 2.6 M5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 >5 M6 >>5>5>5>5>5>5>5>5>5 4.8 >5 rPep-1 >50 >50 >50 >50 >50 45 >50 41.7 30.3 43.5 41.7 >50 rPep-2 >50 >50 >50 >50 >50 >50 .50 27.8 21.3 38.5 27.8 >50 I-N2 >50 >50 45.5 >50 >50 >50 >50 34.5 25.6 38.5 29.4 >50 I-N1 >50 >50 >50 >50 >50 >50 >50 43.5 35.7 >50 >50 >50 I-P1 >50 >50 >50 >50 >50 >50 >50 27.8 18.9 37 40 >50 I-P2 ,1.9 >50 8.3 >50 38.5 6.8 >50 >50 >50 >50 >50 >50 I-P3 21.7 >50 >50 >50 >50 37 >50 26.3 23.3 40 29.4 34.5

IC50 results .5 mg/ml for mAbs or .30 mg/ml for polyclonal Abs are in bold. aValues represent concentration (micrograms per milliliter) at which RLUs were reduced 50% compared with virus control wells (no test sample). All HIV-1 isolates, except MN, are molecularly cloned pseudoviruses produced by transfection in 293T cells. M1(VS), M5, and M6 are murine anti–GBV-C E2 mAbs. rPep-1 and rPep-2 are rabbit anti-E2 peptide Abs (see Materials and Methods). I-N1 and I-N2 are two human IgG preparations from blood donors without E2 Ab, and I-P1, I-P2, and I-P3 are human IgG preparations from three blood donors with E2 Ab. 4500 GBV-C E2 Abs NEUTRALIZE HIV-1

All neutralization experiments were performed in triplicate and in- abilization using the BD Cytofix/Cytoperm kit as recommended by the dependently repeated at least once with consistent results. manufacturer (BD Biosciences). Cells were washed, and PE-labeled anti- For some experiments, the standardized neutralization of HIV envelope- mouse secondary Ab (Southern Biotechnology Associates, Birmingham, pseudotyped HIV particles was assessed after a single round of infection in AL) was applied for 30 min at 4˚C. Ab binding was assessed by flow TZM-bl cells as described previously (36). A total of 200 tissue culture cytometry as described previously (44, 45). infective dose 50% of virus was incubated with serial dilutions of test samples for 1 h at 37˚C in 96-well flat-bottom culture plates. Freshly Statistics trypsinized cells were added to each well. No Ab control and cell only controls served as the background controls. Cells were lysed, and lumi- Statistics were performed using SigmaStat software V3.11 (Jandel Scientific, nescence was measured 48 h after transduction using a Victor 2 lumin- Chicago, IL). t tests were used for direct comparisons for individual data ometer. The serum concentration or IgG concentration that resulted in a 50% points, and results on experiments monitoring multiple days were analyzed reduction in relative light units (RLUs) compared with virus control wells by ANOVA. after subtraction of background RLUs was calculated (36). HIV-1 and SIV envelopes used to pseudotype HIV-1 retroviral particles Results included SS1196.01, QH0692.42, PAVO.04, 6535.03, REJO.67, THRO.18, GBV-C E2 Abs neutralize HIV-1 infection Du123.06, Du156.12, Du172.17, Du422.01, and control HIV, and SIV isolates included HIVMN (propagated in H9 cells) and SIVmac239/CS Heat-inactivated sera from healthy blood donors with and without generated in PBMCs. In addition, HIV isolate SF162.LS was propagated GBV-C E2 Ab were tested for HIV-1–neutralizing activity. E2 in PBMCs and used in one TZM-bl experiment. Ab-positive, but not E2 Ab-negative, sera (diluted 1/100) neutral- Immune precipitation ized a CCR5-tropic HIV isolate (R5, isolate 1) (Table I) and a CXCR4-tropic HIV isolate (X4, isolate 2) (Table I) in a PBMC- HIV-1, YFV, mumps virus, or defective HIV-1 pseudoparticles were mixed based neutralization assay. The percent inhibition compared with no with Abs at various concentrations and incubated while mixing overnight at Downloaded from 4˚C. All three viruses (HIV-1, YFV, and mumps) were prepared in PBMCs Ab control is shown in Fig. 1A. Sera were maintained in culture and pseudoparticles were prepared in 293T cells. Heat-killed, formalin-fixed media (1/100). None of these healthy blood donor sera reacted with Staphylococcus aureus cells with a coat of protein A (Pansorbin; Calbio- denatured HIV-1 proteins in Western blot analyses (data not shown). chem, San Diego, CA) were added and incubated for 2 h at 4˚C. Cells were To determine whether the effect was due to soluble factors in pelleted, washed three times in TBS or PBS containing 0.02% Tween 20, and resuspended in PBS, and viral particle precipitation was measured (p24 Ag serum or whether it was due to serum Abs, IgGs purified from the ELISA or real-time PCR for YFV and mumps RNA as above). For the E2 serum samples were tested for HIV-1 neutralization activity in competition assay, GBV-CE2 or E2-negative cell supernatants were incubated PBMCs (concentration 10 mg/ml) (Fig. 1A). The three human GBV-C http://www.jimmunol.org/ with E2 Ab for 1 h at 37˚C prior to incubating the Ab with virus particles. E2 Ab-positive IgG preparations neutralized HIV-1 in MT-2 cells in a dose-dependent manner (Fig. 1B). Three murine GBV-C E2 mAbs ELISA M1(VS), M5, and M6 also inhibited HIV-1 isolates (concentration GBV-C E2 Ab reactivities with GBV-C E2 were assessed using a GBV-C 5 mg/ml) in the PBMC-based assay (Fig. 1A). Three broadly neu- mAb E2 capture assay. Plates were coated with E2-specific mAb (M5) in tralizing human HIV-1 mAbs (2G12, 2F5, and 4E10; 5 mg/ml) (Fig. carbonate buffer (pH 9.6) overnight at 4˚C, washed with PBS containing 0.02% Tween 20 (PBST), and blocked with PBS containing 1% BSA 1A) served as positive control Abs. (PBSA) at 4˚C overnight or 1 h at 37˚C. E2 Ag was applied at 1 mg/ml in PBSA and allowed to adsorb for 1 h at 37˚C. Plates were washed with PBST, GBV-C E2 immunogenicity by guest on October 2, 2021 and serial dilutions of Abs in PBSAwere applied and incubated at 37˚C for 1 Mice and rabbits were immunized with rGBV-C E2 protein to de- h. Following washing, bound rabbit Abs were detected using alkaline phosphatase-labeled goat anti-rabbit Fc Ab (Sigma-Aldrich). Plates were termine whether it elicited HIV-1–neutralizing Abs. Murine serum washed, p-nitrophenylphosphate substrate was added, and the absorbance obtained 10 d after the third E2 immunization demonstrated anti-E2 (405 nm) was measured on a Bio-Rad model 680 microplate reader. Pre- reactivity in a mAb capture ELISA, whereas the preimmune sera vious studies found that human anti–GBV-C E2 Abs bound to E2 that was did not (data not shown). Sufficient sera were available to test two captured by M5 mAb (26, 27). of the three mice by immunoblot assay, and both reacted with rE2 Flow cytometry (Fig. 2A). All three murine sera neutralized the infectivity of HIV-1 MT-2 cells and PBMCs were incubated with the anti–GBV-C M6 mAb or (isolate 2) (Table I) in MT-2 cells compared with preimmune sera, IgG2a isotype control Ab (BD Biosciences, Franklin Lakes, NJ) for 1 h at as measured by HIV-1 p24 Ag release into culture supernatants 4˚C. Ab was added to cells that were untreated or following cell perme- 5 d post-HIV infection (Fig. 2B).

Table IV. Comparison of HIV neutralization in TZM-bl cells and PBMCs

ID50 in TZM-bl Cells (mg/ml)a ID50 in PBMCs (mg/ml)b

SF162.LS HIV-1 R5 HIV-1 X4 HIV-1 R5 HIV-1 X4 Sample Clade B Isolate 1 Isolate 2 Isolate 1 Isolate 2 I-N2 >45 >45 >45 >45 >45 I-P1 >45 >45 >45 >45 40 I-P2 >45 >45 >45 915 I-P3 >45 >45 >45 14 19 M1 (VS) 2.6 4.0 3.2 1 2 M5 >50 >50 >50 38 M6 >50 >50 >50 17 >50 TriMAbc 0.04 >25 0.58 2 1

IC50 results .5 mg/ml for mAbs or .30 mg/ml for polyclonal Abs are in bold. HIV isolates 1 (R5) and 2 (X4) refer to isolates described in Table I. aValues represent the Ab concentration (micrograms per milliliter) at which RLUs were reduced 50% compared with virus control wells (no test sample). bValues represent the Ab concentration (micrograms per milliliter) at which p24 Gag Ag synthesis was reduced by 50% relative to virus control wells (no test sample). cTriMAb is a positive control of three HIV-1 human neutralizing mAbs. The Journal of Immunology 4501

GBV-C E2 protein and the GBV-C 17-aa E2 peptide were im- surface Ag did not inhibit HIV-1 infection compared with the no Ab munogenic in rabbits as well, as sera obtained from all rabbits re- control (Fig. 4A). All polyclonal Abs were used at a concentration acted with E2 in a mAb capture ELISA (Fig. 3A) and by immunoblot of 10 mg/ml, and the Ab was maintained in the media following analysis (Fig. 3B), whereas preimmune sera did not. Postimmune HIV-1 inoculation of cells. anti–GBV-C E2 IgG (n = 4 rabbits) significantly inhibited HIV-1 To ascertain whether the rabbit IgG inhibited the HIV-1 p24 Ag (isolate 2) (Table I) replication in MT-2 cells 3 and 6 d postinfection ELISA,HIV-1wasincubatedwithnoAborpre-orpostimmunerabbit (Fig. 4A). In contrast, preimmune rabbit IgG, IgG purified from IgG (10 mg/ml each), and the concentration was assessed. There was rabbit sera following immunization with a different GBV-C protein no difference between the no Ab control (11,107 pg/ml) and the pre- (NS5A) (46), or polyclonal goat IgG containing Ab to hepatitis B (11,219 pg/ml) and postimmune (11,106 pg/ml) (ANOVA; p = 0.99). In addition, incubation of MT-2 cells and PBMCs with rabbit preimmune or anti-E2 IgG did not alter cell number or viability during 6 d in culture (data not shown). HIV-1 inhibition was diminished when Abs were not maintained in the cell culture media (Fig. 4B). Specifically, HIV replication was inhibited by .99% in MT-2cells if E2 Ab was maintained in the media (p , 0.01); however, HIV-1 replication inhibition was diminished when Ab was removed after inoculation. Initial infection appears to be incompletely inhibited by the Abs, and HIV-1 cell-to-cell spread is suppressed in the presence of GBV-C E2 Abs. Single cycle replication assays allow assessment of viral en- Downloaded from velope specificity and provide information regarding which step(s) of HIV replication is inhibited. HIV-1 envelope protein (gp160) was used to pseudotype defective HIV-1 (gag) particles or defective VSV particles generated in 293T cells, and rabbit anti–GBV-C E2 IgG was assessed for neutralizing activity in a HOS cell-based assay. Anti–GBV-C E2 IgG preparation neutralized HIV-1–pseu- http://www.jimmunol.org/ dotyped retrovirus particles in a dose-dependent fashion; however, this Ab did not neutralize HIV-1–pseudotyped VSV particles (Fig. 4C). Preimmune rabbit IgG did not neutralize either particle type. These data suggest that the anti–GBV-C E2 Abs neutralize HIV-1 particles independently from the HIV-1 envelope proteins. HIV-1 neutralization by GBV-C E2 Abs is assay dependent The breadth and potency of HIV-1–neutralizing activity present in by guest on October 2, 2021 GBV-C E2 Ab preparations was examined in virus replication assays using a diverse panel of HIV-1 isolates including clinical and laboratory derived R5 and 34 isolates and representing clades A, B, and D (Table I). The Ab concentration (micrograms per milliliter) required to inhibit HIV replication by 50% (IC50) for human polyclonal anti-E2 Ab, rabbit anti-E2 Ab, rabbit anti-E2 peptide Ab, and murine mAbs M1(VS), M5, and M6 is shown in Table II. All anti-E2 Abs possessed neutralizing activity against all isolates tested except the rabbit anti-E2 peptide Ab (,50% reduction at 50 mg/ml Ab concentration). Three HIV-1–neutralizing hmAbs served as control Abs (2F5, 4E10, and 2G12). Because endotoxin con- tamination of Ab may lead to non–specific-neutralizing activity against HIV-1 (47), all of the rabbit IgG preparations were tested for endotoxin (assay; Cape Cod Associates, Woods Hole, MA) and were found to be endotoxin free (,0.25 EU/ml). FIGURE 5. Rabbit anti–GBV-C E2 Abs precipitate HIV particles. HIV- Some HIV-neutralizing Abs perform differently in neutralization 1 particles were immune precipitated (IP) by the postimmune rabbit IgG assays that use different cell types (48, 49). To see whether this were (R1, R2, R3, and R4) but not preimmune (pre) rabbit anti-E2 IgG (A; true for the GBV-C E2 Abs, a standardized TZM-bl assay was used pp , 0.01 compared with preimmune IgG). CHO cell culture super- to examine neutralization of a panel of HIV-enveloped pseudo- natants containing E2 protein inhibited IP in a dose-dependent manner, particles (36, 50, 51). Differences in neutralization in the TMZ- whereas CHO cell culture supernatants without E2 did not (A). The bl cells and the PBMC-, MT-2–, and HOS-based assays were ob- protein concentration represents the total protein in culture supernatants served (Table III). The murine mAb M5 did not neutralize any of and not E2-specific protein. Defective HIV gag particles displaying HIV, the isolates tested in TZM-bl cells, and M6 mAb neutralized only VSV, or GBV-C envelope glycoproteins, or HIV-1 particles with no virus one HIV envelopes (Du422.01) at concentrations of ,5 mg/ml envelope glycoproteins were precipitated by postimmune rabbit anti-E2 (Table III). In contrast, the murine M1(VS) mAb was broadly IgG and not by preimmune rabbit IgG (B; pp , 0.01 compared with preimmune IgG; †p , 0.05). YFV and mumps virus (MV) were not neutralizing and neutralized all viruses HIV-1 envelopes studied, neutralized following incubation with pre- or postimmune (post) rabbit E2 except REJO.67 in the TZM-bl cell assay (Table III). The mAb M1 IgG (left panel; C), and they were not IP by postimmune rabbit anti-E2 (VS) was also active against an SIV macaque envelope (SIVmac IgG (right panel; C). NA, no Ab control; IPV, the amount of input virus 239/CS), further suggesting that the interaction is independent of used in IP experiments. HIV-1 envelope glycoproteins. Although the rabbit anti-peptide 4502 GBV-C E2 Abs NEUTRALIZE HIV-1

IgG and E2-positive human IgG neutralized some HIV and SIV parations precipitated infectious HIV-1 particles prepared in isolates at high concentrations (generally .20 mg/ml) (Table III), HOS cell lines (Fig. 5A), suggesting that the interaction is con- the specificity of this is unclear, because the E2-negative IgG also formation dependent. Immune precipitation was inhibited by CHO inhibited some isolates at high concentration (Table III). One of the cell culture supernatant fluid containing rE2 protein in a dose- HIV-negative blood donor human anti-E2 IgG preparations (I-P2) dependent manner but not CHO supernatant without E2 (Fig. 5A). neutralized three HIV isolates at ,10 mg/ml, indicating a more Rabbit anti–GBV-C E2 IgG precipitated retrovirus particles narrow neutralization effect on HIV-1 envelope-containing par- displaying HIVenv, GBV-Cenv, or VSV envelope glycoprotein ticles in the TZM-bl cell neutralization assay (Table III). Because significantly more than preimmune IgG or no Ab control (Fig. 5B). these are polyclonal human Abs, the relative concentration of Ab In addition, defective HIV particles with no viral envelope protein that reacts with E2 is considerably lower than the HIV-neutralizing were precipitated by the rabbit anti–GBV-C E2 Abs (Fig. 5B), in- IC50 concentration. dicating that precipitation did not involve the viral envelope pro- To determine whether the differences were related to the neu- teins. GBV-C–enveloped HIV pseudoparticles (GBV-Cpp) were tralization system used or to the viruses studied, human anti-E2 IgG precipitated to a significantly greater extent than the other defective and the three murine anti–GBV-C E2 mAbs were retested at Duke particles by anti-E2 Ab, suggesting that, in addition to the gag University (Durham, NC) in PBMC-based assays using HIV-1 particle Ag recognized by these Abs, interactions with the GBV-C isolates 1 and 2 (Table I). Two of the three human GBV-C E2 E2 protein contributed to the immune precipitation (Fig. 5B). The Ab-positive IgG preparations neutralized the R5 and 34 isolate at murine mAb M6 and the human anti-gp120 (2G12) mAb pre- ,20 mg/ml (I-P2 and I-P3), and the three murine E2 mAbs neu- cipitated HIV-1–pseudotyped gag particles (HIVpp) more than the tralized HIV-1 in PBMC assays (Table IV). The TriMAb control IgG2a and human polyclonal IgG control Abs (data not shown). (2G12, 4E10, and 2F5) did not neutralize the HIV-1 R5 isolate However, the percentage of input of defective HIVpp and GBV-Cpp Downloaded from (Table I, isolate 1) in TZM-bl cells but did in PBMCs. Of the anti– precipitated by all of these mAbs was very low (,2% of input GBV-C E2 mAbs, only M6 did not neutralize well in PBMCs, virus), suggesting that their reactivity with gag particles is sig- with a very high IC50 value for the isolate 1 (17 mg/ml) and no nificantly weaker than the rabbit polyclonal anti–GBV-C E2 Ab. activity against the 34 isolate number 2. The rabbit GBV-C E2 Abs did not precipitate or neutralize YFV or mumps virus prepared in MT-2 cells (Fig. 5C). Like HIV-1, the GBV-C E2 Abs precipitate HIV-1 particles mumps virus has a class I envelope glycoprotein. YFV prepared in http://www.jimmunol.org/ As noted above, naturally occurring human GBV-C E2 Abs are Vero cells, and mumps prepared in BHK cells were also not neu- conformation dependent (26), although the rabbit anti-E2 Abs tralized or precipitated by the E2 Abs (data not shown). Taken to- recognized denatured E2. The human GBV-C E2 Abs and the rabbit gether, the data suggest that the anti-E2 Ab interactions are not de- anti-E2 Abs did not react with denatured HIV-1 proteins (Refs. 52– pendent on HIV-1 or other viral envelope glycoproteins and that the 54 and data not shown). In contrast, the rabbit anti-E2 IgG pre- antigenic site is not exposed on flavivirus or orthomyxovirus particles. by guest on October 2, 2021

FIGURE 6. Anti–GBV-C E2 Abs do not inhibit HIV- 1 by interacting with cell plasma membrane but interfere with HIV-1 cellular attachment. HIV replication was not altered when MT-2 cells were preincubated with no Ab (NA), pre- (Pre), or post-E2 (Post) rabbit IgG (10 mg/ml) (A). Incubation of HIV-1 particles with rabbit postimmune (anti–GBV-C E2 positive) IgG inhibited HIV-1 particle attachment to MT-2 cells compared with no Ab or preimmune IgG controls in a dose-dependent manner (B; see Materials and Methods)(pp , 0.01 compared with preimmune IgG; †p , 0.05). Surface staining of intact MT-2 cells and PBMCs by the murine anti-E2 M6 mAb is shown in C and E, respectively. M6 reactivity increased with both MT-2 cells and PBMCs following ﬁxation and permeabilization (D and F, respectively). IC, IgG2a isotype control Ab. The Journal of Immunology 4503

Anti–GBV-C E2 Abs interfere with HIV-1 particle binding but not YFV or mumps viruses. Immune precipitation was indepen- To determine whether GBV-C E2 Abs neutralized HIV-1 by dent of the viral envelope, because retrovirus particles with HIV-1, binding to cell plasma membrane Ags and blocking either HIV-1 VSV, GBV-C, or no envelope protein were precipitated. attachment or entry, MT-2 cells were incubated with no Ab, E2- Anti–GBV-C E2 Abs interacted with cellular Ag(s) following negative rabbit IgG (preimmune; 10 mg/ml), or anti–GBV-C E2 cell permeabilization, suggesting that anti-E2 Abs cross-react with rabbit IgG for 2 h at 4˚C (postimmune; 10 mg/ml). Cells were an Ag(s) that is not exposed on the cell surface. Together with the washed, and HIV-1 was added prior to warming the cells to 37˚C. neutralization and immune precipitation data, these data indicate HIV replication was monitored. Preincubation of cells with rabbit that the anti-E2 Abs neutralize and precipitate HIV-1 particles via GBV-C E2 Ab did not inhibit HIV-1 replication compared with interactions with a cellular Ag that is present or enriched on HIV-1 controls (Fig. 6A). Thus, if the Ag interacting with GBV-C E2 Abs gag particles. The Ab interactions appear to be similar to the well- is of cellular origin, it is either not exposed on the surface of MT- characterized and broadly neutralizing HIV-1 Abs 2F5 and 4E10, 2 cells, or it is expressed in a different confirmation. which react with both HIV-1 gp41 peptides and permeabilized cells To determine the step of HIV replication that is inhibited by (10). These Abs also react with a variety of phosphatidylinositols and GBV-C E2 Abs, HIV-1 was mixed with rabbit E2 Abs for 1 h prior lipids (10). Studies are under way to further characterize the cellular to addition to PBMCs for 2 h at 4˚C. Cells were washed to remove Ag recognized by the anti–GBV-C E2 Abs (J.H. McLinden, J. Xiang, nonspecifically bound HIV-1 particles, and the amount of HIV-1 E.L. Mohr, T.M. Kaufman, Q. Chang, and J.T. Stapleton, manuscript bound to the cell was measured by p24 Ag ELISA. Rabbit anti– in preparation). GBV-C E2 Ab blocked HIV-1 binding in a dose-dependent HIV-1 neutralization required incubation of anti-E2 Abs with the viral particles before interaction with the cell surface, because the manner, whereas preimmune IgG did not (Fig. 6B). The murine Downloaded from mAb 6 did not interact with intact MT-2 cells or PBMCs as Abs did not bind to cells, and they did not block entry following measured by flow cytometry (Fig. 6C,6E). However, following viral attachment in single cycle infections. Preincubation of the E2 fixation and permeabilization, M6 reacted with MT-2 cells and Abs with HIV-1 particles reduced virus-cell binding; thus, HIV-1 PBMCs (Fig. 6D,6F). Similarly, M6 did not react with intact replication is inhibited at least in part by decreasing HIV-1 attach- CHO cells but did following fixation and permeabilization (data ment (Fig. 6B). Of note, the GBV-C E2 peptide recognized by the not shown). These data indicate that the E2 Abs react with cellular M6 mAb did not elicit HIV-1–neutralizing Abs nor neutralize HIV-1 http://www.jimmunol.org/ Ags that are not highly exposed on the cell surface. infectivity, whereas the rE2 protein did, illustrating that the peptide To determine whether the anti–GBV-C E2 Abs inhibited HIV is insufficient to induce neutralizing HIV-1 Abs. Previous studies entry in addition to inhibiting HIVattachment, HIV-1 was incubated found that immunization with the MPER peptide sequence or T-20 with cells for 2 h at 4˚C to allow attachment. Following attach- is not immunogenic unless the peptide is presented as part of a struc- ment, preimmune rabbit IgG, rabbit anti-E2, or rabbit anti-E2 Fab tural particle (e.g., viral particles or liposomes) (9, 11, 55, 56). fragments were added to the cells at 4˚C and cells were warmed to 37˚C. One hour later, the medium was replaced with medium with or without the rabbit IgG or Fab fragments. In cells maintained in anti-E2 IgG or Fab fragments prepared from the polyclonal anti- by guest on October 2, 2021 E2 IgG preparation, HIV-1 replication was inhibited in a dose- dependent manner (Fig. 7A). HIV inhibition was not related to steric interactions between the Fc portion of the anti-E2 IgG. Similar to what was observed when anti-E2 IgG was removed from the media (Fig. 4B), inhibition was lost when anti-E2 Fab fragments were removed from the media. These data cannot distinguish between inhibition of entry during initial infection or subsequent inhibition of HIV-1 cell-to-cell spread. To assess this in a single cycle of infection, HIV-1 envelope pseudotyped gag particles generated in 293T cells were added to HOS cells expressing human CD4, CXCR4, and CCR5. Pre- or postimmune (anti-E2) rabbit IgG was added to the cells following viral attachment for 2 h at 4˚C, and the cells were warmed to 37˚C to allow viral entry. Luciferase activity was measured 72 h later, and HIV-1 transduction was not inhibited (Fig. 7B). Thus, once HIV particles bound to CD4, access to the cross-reacting Ag was blocked, and the Abs were not able to block subsequent entry steps. Discussion Naturally occurring GBV-C E2 Abs from HIV-negative blood donors and experimentally induced GBV-C E2 Abs neutralized FIGURE 7. GBV-C Abs do not block HIV-1 entry following HIV-1 HIV-1 infection in vitro. The Abs inhibited HIV-1 isolates obtained attachment to cells. Following attachment of HIV particles to MT-2 cells from patients from geographically diverse regions and representing for 2 h at 4˚C, rabbit anti–GBV-C E2 Ab or Fab fragments were added to three separate HIV-1 clades. Neutralization was independent of cells for 1 h. Following washing, Abs were included in the media entry coreceptor usage (CCR5 or CXCR4), and similar to other (maintain) or not (remove). Cells were warmed to 37˚C, and HIV-1 p24 Ag release was measured 3 d later (A). HIV envelope pseudotyped defective characterized Abs, neutralization differed when neutralization assays HIV-1 particles were similarly analyzed in HOS cells. Following attach- used different cellular substrates. VSV particles displaying HIV-1 ment at 4˚C, pre- or postimmune rabbit GBV-C E2 IgG was added at the envelope glycoproteins, YFVs, and mumps viruses were not neu- concentrations indicated (B). Cells were warmed to 37˚C, and transduction tralized by the anti–GBV-C E2 Abs. Anti–GBV-C E2 Abs also (luciferase activity) was measured 72 h later and compared to the virus precipitated infectious HIV-1 particles and defective HIV-1 particles only control (B). 4504 GBV-C E2 Abs NEUTRALIZE HIV-1

GBV-C E2 protein inhibits HIV-1 entry in vitro through an un- intermediate isolate from Dr. Douglas Richman, HIVJR-CSF from Dr. Irvin known mechanism (31, 32). Thus, either viremia with GBV-C or the Chen, HTLV-IIIB/H9 from Dr. Robert Gallo, HIVxxBru-JF from Dr. John presence of E2 Ab may influence HIV-1 replication. Viremia Mellors and Chaofu Shi, and HIVELI from Dr. Jean-Marie Bechet and Dr. appears to have the greatest impact on survival in HIV-infected Luc Montagnier. Catalog numbers are shown in Materials and Methods.We people (20), perhaps because of a dose effect. GBV-C concentra- also thank Dr. Opendra Sharma (NARRRP) for providing the GBV-C synthetic peptides and Drs. Georg Hess and Alfred Engel (Roche Diagnostics and tion in infected humans is typically .50 million genome equivalents/ Laboratories) for providing GBV-C E2 ELISA kits and M6 mAb. We also ml plasma (57), and GBV-C is produced by B lymphocytes and thank Shawn Roach (University of Iowa) for assistance with the figures. both CD4+ and CD8+ T lymphocytes (15). Because each virus particle is predicted to have multiple copies of E2 protein on the surface, lymphocytes are constantly exposed to low concentrations of Disclosures E2 protein. In contrast, the concentration of GBV-C E2 Abs in The authors have no financial conflicts of interest. humans varies over time and may drop to levels below the limit of detection during longitudinal follow up (20, 53). It is likely that the E2 Ab concentration is an important variable in determining the References 1. Fauci, A. S., M. I. Johnston, C. W. Dieffenbach, D. R. Burton, S. M. Hammer, magnitude of any potential clinical effect of GBV-C E2 Abs. J. A. Hoxie, M. Martin, J. Overbaugh, D. I. Watkins, A. Mahmoud, and No significant amino acid sequence homology between GBV-C W. C. Greene. 2008. HIV vaccine research: the way forward. Science 321: 530–532. E2 and either HIV-1 or cellular proteins was identified in a protein- 2. Johnston, M. I., and A. S. Fauci. 2008. An HIV vaccine—challenges and prospects. N. Engl. J. Med. 359: 888–890. protein basic local alignment search tool search (http://blast.ncbi. 3. Buchacher, A., R. Predl, K. Strutzenberger, W. Steinfellner, A. Trkola, nlm.nih.gov/Blast.cgi). Although several studies have identified M. Purtscher, G. Gruber, C. Tauer, F. Steindl, A. Jungbauer, et al. 1994. Gen- ∼ eration of human monoclonal antibodies against HIV-1 proteins; electrofusion Downloaded from HIV-1–neutralizing Abs in 20% of humans with HIV-1 infection and Epstein-Barr virus transformation for peripheral blood lymphocyte immor- and a lower percentage in HIV-uninfected people (58–64), there talization. AIDS Res. Hum. Retroviruses 10: 359–369. are few data suggesting a beneficial clinical course in those with 4. Dennison, S. M., S. M. Stewart, K. C. Stempel, H.-X. Liao, B. F. Haynes, and S. M. Alam. 2009. Stable docking of neutralizing human immunodeficiency HIV-neutralizing Abs compared with those without. In contrast, virus type 1 gp41 membrane-proximal external region monoclonal antibodies a modest association between GBV-C E2 Ab and prolonged sur- 2F5 and 4E10 is dependent on the membrane immersion depth of their epitope vival was observed by Tillmann et al. (20). There are clearly neu- regions. J. Virol. 83: 10211–10223. 5. Wild, C., T. Greenwell, and T. Matthews. 1993. A synthetic peptide from HIV-1 http://www.jimmunol.org/ tralizing Abs present in HIV-1–infected people that are not due to gp41 is a potent inhibitor of virus-mediated cell-cell fusion. AIDS Res. Hum. GBV-C E2 Abs; thus, measuring survival on the basis of the pres- Retroviruses 9: 1051–1053. 6. Ferrantelli, F., R. Hofmann-Lehmann, R. A. Rasmussen, T. Wang, W. Xu, ence of HIV-1–neutralizing Abs independent of the presence of P.-L. Li, D. C. Montefiori, L. A. Cavacini, H. Katinger, G. Stiegler, et al. GBV-C E2 Ab will confound the analysis. It will be interesting to 2003. Post-exposure prophylaxis with human monoclonal antibodies pre- determine what proportion of HIV-1–neutralizing Abs in HIV- vented SHIV89.6P infection or disease in neonatal macaques. AIDS 17: 301–309. infected individuals are directed against the GBV-C E2 protein, 7. Sańchez-Martıńez, S., M. Lorizate, K. Hermann, R. Kunert, G. Basan˜ez, and and studies to address this question are under way. J. L. Nieva. 2006. Specific phospholipid recognition by human immunodeficiency One concern related to the use of non–HIV-1 Ags to invoke HIV- virus type-1 neutralizing anti-gp41 2F5 antibody. FEBS Lett. 580: 2395–2399. 8. Haynes, B. F., and S. M. Alam. 2008. HIV-1 hides an Achilles’ heel in virion 1–neutralizing Abs is that these Ags may induce autoantibodies.

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