Antiviral Research 118 (2015) 1–9

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Reconstitution and characterization of antibody repertoires of HIV-1-infected ‘‘elite neutralizers’’ Zehua Sun a, Jingjing Li a, Xintao Hu b,1, Yiming Shao b, Mei-Yun Zhang a,c,⇑ a

Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong, China Division of Research on Virology and Immunology, National Center for AIDS/STD Control and Prevention, China CDC, Beijing 102206, China c Liver Disease Institute, Shenzhen Third People’s Hospital, Shenzhen 518112, China b

a r t i c l e

i n f o

Article history: Received 9 August 2014 Revised 23 January 2015 Accepted 16 February 2015 Available online 12 March 2015 Keywords: HIV Antibody ELISA Neutralization Cell–cell fusion ADCC

a b s t r a c t Around 3–5% HIV-1-infected individuals develop high titers of broadly neutralizing HIV-1 antibodies (bnAbs) during chronic infection. However, monoclonal antibodies (mAbs) isolated from such ‘‘elite neutralizers’’ do not, in most cases, depict the serum IgGs in neutralizing the virus. We hypothesize that HIV-1-specific antibodies in infected subjects may work in a population manner in containing the virus in vivo, and in vitro reconstituted antibody repertoires of ‘‘elite neutralizers’’ may mimic the sera in binding and neutralizing the virus. This study aims to investigate the antibody repertoires of three such ‘‘elite neutralizers’’ by reconstituting the immune antibody repertories in vitro followed by a comparative study of the recombinant library IgGs with the corresponding serum IgGs. We found that the recombinant library IgGs were much weaker than the serum IgGs in binding to envelope glycoproteins (Envs) and in neutralizing the virus and inhibiting Env-mediated cell–cell fusion. However, the sorted libraries composing of HIV-1-specific neutralizing antibodies (nAbs) in the three recombinant libraries exhibited comparable binding and inhibitory activities, as well as antibody-dependent cell-mediated cytotoxicity (ADCC), to the serum IgGs. The sorted library IgGs further showed neutralization profiles which are similar to those of the serum IgGs, but they were overall less potent than the serum IgGs. The sorted library IgGs and the serum IgGs bound weakly to the resurfaced Env gp120, RSC3, and did not bind to the CD4 binding site (CD4bs) knock-out mutant, DRSC3. Profiling with VRC01 binding site knock-out site mutants of gp120BaL indicates that, if there are any CD4bs bnAbs in these sera, they are more likely b12-like, but not VRC01-class bnAbs. Our results suggest that HIV-1-specific Ab-expressing B cells, especially potent nAb-expressing B cells may not be rich in the B cell repertoires of ‘‘elite neutralizers’’, but they may be highly active in producing nAbs in vivo. In vitro reconstituted HIV-1 nAb repertoires of ‘‘elite neutralizers’’ may be used in passive immunization to prevent HIV-1 infection. HIV-1 vaccine immunogens may be designed to target multiple neutralizing determinants to stimulate multiple B cell populations. HIV-1-specific antibodies induced by such immunogens may work in combination or synergistically in containing the virus. Ó 2015 Published by Elsevier B.V.

1. Introduction Impaired production of HIV-1 bnAbs during natural exposure to virus or vaccine protocols is a hallmark of HIV-1 infection in

Abbreviations: PBMCs, peripheral blood mononuclear cells; bnAbs, broadly neutralizing antibodies; bnmAbs, broadly neutralizing HIV-1 human monoclonal antibodies; ADCC, antibody-dependent cell-mediated cytotoxicity; FBS, fetal bovine serum. ⇑ Corresponding author at: Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pok Fu Lam, Hong Kong, China. Tel.: +852 28183685; fax: +852 28177805. E-mail address: [email protected] (M.-Y. Zhang). 1 Current address: Human Retrovirus Pathogenesis Section, National Cancer Institute, Frederick, MD, USA. http://dx.doi.org/10.1016/j.antiviral.2015.02.006 0166-3542/Ó 2015 Published by Elsevier B.V.

humans. About 3–5% HIV-1-infected individuals do develop high titers of HIV-1 bnAbs during chronic infection (Li et al., 2007; Scheid et al., 2009). Such sera potently neutralized HIV-1 primary isolates from various clades and have been used to treat HIV-1 infection. But treatment with patient IgGs purified from patient sera may have a high risk of coinfection with other blood-borne pathogens. We and also others have been isolating broadly neutralizing HIV-1 human monoclonal antibodies (bnmAbs) from HIV-1-infected ‘‘elite neutralizers’’ whose sera exhibit broadly neutralizing activity. Such bnmAbs may be used for passive immunization and their epitopes may be used for vaccine immunogen design. Many bnmAbs have been identified so far, most of which were isolated from the memory B cell pools of ‘‘elite neutralizers’’ in the recent three years by single cell sorting followed by single cell

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PCR and cloning or single cell sorting in combination with highthroughput screening for broadly neutralizing antibody clones. Known HIV-1 bnmAbs can be categorized into four groups based on their epitopes (West et al., 2014): (1) anti-CD4 binding site (CD4bs), including b12 (Burton et al., 1994), m14 (Zhang et al., 2004), HJ16 (Corti et al., 2010), VRC01-3 (Wu et al., 2010), VRC01PG04 and VRC-CH30-35 (Wu et al., 2011), 3BNC60, 12A12 and 8ANC131 (Scheid et al., 2009), and NIH45-46 (Scheid et al., 2011); (2) anti-MPER (membrane proximity external region) of gp41, including 2F5, 4E10 (Purtscher et al., 1994; Stiegler et al., 2001), Z13e1 (Nelson et al., 2007), m66.6 (Zhu et al., 2011), and 10e8 (Huang et al., 2012); (3) anti-glycan/variable loops (V2 or V3) on Env, including anti-glycan/V2 bnmAbs PG9/16 (Walker et al., 2009) and PGT141-5 (Walker et al., 2011), and anti-glycan/V3 bnmAbs 2G12 (Scanlan et al., 2002), PGT121-3, 125-8, 130-1 and PGT135-7 (Walker et al., 2011); and (4) anti-gp120/gp41 interface, i.e. m43 (Zhang et al., 2012), 8ANC195 (Scharf et al., 2014) and PGT151-158 (Blattner et al., 2014; Falkowska et al., 2014). Some of the known bnmAbs exhibit high potency and broad spectrum of neutralization, and a single or a few bnmAbs isolated from the same ‘‘elite neutralizers’’ can largely recapitulate the neutralization profile of the corresponding patient serum, such as, VRC01-class bnmAbs and PGT series. These bnmAbs showed sequence and structural convergence compared with the antibodies identified by deep sequencing of the antibody repertories of the same ‘‘elite neutralizers’’ (Scheid et al., 2011; Walker et al., 2011; Wu et al., 2011). However, in most cases, a single or a few neutralizing HIV1 human mAbs isolated from ‘‘elite neutralizers’’ do not fully recapitulate the neutralization profiles of the corresponding broadly neutralizing sera (Scheid et al., 2009; Zhang et al., 2004, 2012). Those isolated HIV-1 mAbs are broadly cross-reactive, but have modest neutralization potency and limited breadth of neutralization spectrum. This indicates that high potency of the broadly neutralizing sera of ‘‘elite neutralizers’’ may not be attributed to a single bnmAb, or to antibodies targeting a single structural determinant. The finding that non-neutralizing HIV-1-specific antibodies exhibited inhibition of viral replication in vivo (Florese et al., 2009) suggests the importance of antibody effector function, especially antibody-dependent cell-mediated cytotoxicity (ADCC), in containing the virus in vivo. ADCC is a main route of protective immunity to infectious diseases. It was reported that HIV-1 bnmAb b12 confers protection in rhesus macaque through ADCC (Hessell et al., 2007). Based on these observations, we hypothesize that HIV-specific antibodies may work in a population manner in containing the virus in vivo. In vitro reconstituted HIV-specific antibody repertoires of ‘‘elite neutralizers’’ may mimic the polyclonal sera in binding and neutralizing the virus, and may be used in passive immunization for prevention and treatment of HIV-1 infection. In this study, we reconstituted in vitro three antibody repertoires using the total RNA prepared from the B cells or memory B cells of three ‘‘elite neutralizers’’ and characterized the recombinant library IgGs and nAb-sorted library IgGs for various biofunctions in comparison with the corresponding serum IgGs. 2. Materials and methods 2.1. Cells, plasmids, proteins and viruses 293T cell line was purchased from ATCC. TZM-bl, TF228 and Sup-T1 cell lines and HIV-1 isolates were obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP) (Division of AIDS, National Institute of Allergy and Infectious Diseases). TZM-bl was maintained in DMEM containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 lg/ ml streptomycin.TF228 was maintained in RPMI1640 containing the same supplements. 293F cell line was purchased from

Invitrogen and cultivated in 293 Free-style medium. Clade B0 chronically infected and treat-naïve patient samples, 1-1, 1-3 and 1-4, were obtained from National Center for AIDS/STD Control and Prevention, China CDC (Beijing, China). Heparinized whole blood samples were used to isolate human PBMCs by Ficoll density gradient separation and IgGs by Protein A affinity purification. All these experiments were approved by ethical committees of the respective institutes, and conducted according to local guidelines and regulations. BaL gp120 site mutants, D368R, N279A and G459E, were generated in our laboratory by using site-directed mutagenesis kit (Stratagene). RSC3 and DRSC3 expressing plasmids were gifts from Peter Kwong and John Mascola at VRC, NIAID, NIH. Recombinant wild type (WT) Envs and Env mutants, as well as HIV1 mAbs, including CD4 binding site (CD4bs) mAbs IgG1s b12 and VRC01 and CD4 induced (CD4i) mAb IgG1 X5, were produced in our laboratory using 293F transient transfection system (Invitrogen) and ProteinA affinity purification. Fab X5 and Fab 1-3 was expressed in Escherichia coli strain HB2151 and purified by Protein G affinity purification. 2.2. Construction of IgG library Patient peripheral B cells or memory B cells were isolated from the PBMCs as follow: Patient PBMCs were washed with PBS containing 2% FBS, and stained with APC conjugated to anti-CD19 for sorting total B cells, or co-stained with APC conjugated to anti-CD19 and PE conjugated to anti-CD27 for sorting memory B cells by using FACSAria III sorter. Unstained PBMCs and single fluorescence stained PBMCs were used as controls in cell sorting. Total RNA and mRNA were prepared from the B cells of patient 1-1, the PBMCs of patient 1-3 and the memory B-cells of patient 1-4 using total RNA and mRNA isolation kits (Qiagen). First-strand cDNAs were synthesized using oligo dT (Invitrogen) according to the instructions from the manufacturer. A panel of primer pairs were designed according to V-base (human antibody sequence database, http://vbase.mrc-cpe.cam.ac.uk/) and strategies used by Scheid et al. (2009) (Table S1). Human antibody heavy chain variable regions (VHs) and light chains (LCs) were PCR amplified from the cDNAs using the designed primers and PCR cycles: 94 °C for 5 min, followed by 10 cycles of 95 °C for 15 s, 45 °C for 30 s and 72 °C for 45 s, and 20 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 45 s, and final extension at 72 °C for 10 min. VHs and LCs were then digested withSacI/XbaI and HindIII/EcoRI, respectively, and ligated to a mammalian expression vector PDR12 containing human IgG1 constant regions (CH1-3). Ligation products were desalted and eletroporated into TG1 electroporation competent cells, resulting in recombinant full-length IgG libraries, designated Lib 1-1, 1-3 and 1-4. 2.3. Binding assay One lg/ml of recombinant Env gp120 or gp140 or Env mutants in pH8.3 coating buffer was coated on Maxi-sorp ELISA plates by incubation at 4 °C overnight. The plates were blocked with 2.5% skim milk in PBS (MPBS) by incubation at 37 °C for 1 h.Threefold serially diluted IgGs in MPBS were added to the plates and incubated at 37 °C for 1 h. Bound IgGs were detected using HRP conjugated to anti-human Fc as secondary antibody and TMB as substrate. The optical density at 450 nm (OD450nm) was measured after color development at RT for 20 min. 2.4. TZM-bl neutralization assay A standardized TZM-bl cell line-based Env-pseudotyped neutralization assay was used as previously described (Yuan et al., 2011a,b). Briefly, Env-pseudotyped viruses were prepared by cotransfection of 50–80% confluent HEK 293T cells with

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pNL4-3.luc.E-R- and HIV-1 Env plasmid using polyethylenimine. 8 h post transfection, medium was changed to growth medium containing 10% FBS. Cells were allowed to grow for additional 40 h. The supernatant was harvested, centrifuged at 16,000 rpm for 5 min at 4 °C, and filtered through a 0.45 lm pore filter (Millipore). Neutralization assays were carried out in triplicate by pre-incubation of 50 ll of threefold serially diluted IgGs with 50 ll of pseudovirus suspension at 37 °C for 60 min. Virus– antibody mixtures were then added to TZM-bl cells in 96-well culture plates (Costar, Corning) and incubated at 37 °C with 5% CO2 for 48 h. Cells were then washed with PBS and lysed with 80 ll lysis buffer for 30 min. 30 ll of luciferin (Promega) were added to each well and luminescence readings were measured by PE Victor3 luminometer. Antibody concentrations that lead to 50% and 20% neutralization (IC50s and IC20s, lg/ml) were determined by using GraphPad prism software. 2.5. Cell–cell fusion assay A cell–cell fusion assay was carried out using TF228 cells stably expressing HIV-1 IIIB gp160 as target cells and the CD4 positive T cell line Sup-T1 as effector cells. Cells were cultured in fresh RPMI1640 containing 10% FBS overnight. 5  105 TF228 cells were pre-incubated with IgGs at different concentrations at RT for 30 min. 5000–10,000 effector and target cells at a ratio of 1:1 were then mixed and incubated 37 °C for 20 h in CO2 incubator. Syncytia were counted post co-incubation for 4 h, 20 h and 48 h. One tailed unpaired t-test was used for statistical analysis by using Prism 5.0 software. 2.6. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay A flow cytometry-based ADCC assay was carried out using TF228 cells as target cells and healthy human PBMCs as effector cells at an E/T ratio of 50 (Srivastava et al., 2013; Zaritskaya et al., 2010). Two fluorescent dyes were used to discriminate live and dead cells. PKH67, a membrane labeling dye, was used to specifically identify the target cells. PKH-67 binds to the cell membrane, and the dye remains on the cell membrane, even after cell death, avoiding cross-contamination with effector cells. 7-Amino-actinomycin-D (7-AAD) is excluded by viable cells, but can penetrate the cell membrane of dead or dying cells, and intercalate into double stranded DNA. TF228 cells were stained with PKH-67 for 10 min at RT and 3 ml FBS added to stop the reaction. Pooled PBMCs were freshly prepared from five healthy donors and suspended in RPMI 1640 containing 100 U/ml penicillin and 100 lg/ml streptomycin. 1.0  104 labeled target cells were dispensed in 50 ll of RPMI 1640 medium in round-bottomed 96-well plate. 50 ll of diluted antibodies in triplicate were added to the wells and the plate incubated at 37 °C for 15 min in CO2 incubator. 50 ll of unlabeled pooled PBMCs at a concentration of 1  107 cells/ml were added to each well and the plate incubated at 37 °C for 4 h in CO2 incubator. One ll of 7-AAD solution was added to the wells and the plate incubated at 4 °C in dark for 15 min. Cell mixtures were then analyzed by flow cytometry. A total of 5000 target cells were acquired. Percent cell death were determined by software analysis of four identifiable cell populations, live effector cells (no dye), dead effector cells (7-AAD only), live target cells (PKH-67 only) and dead target cells (PKH-67 and 7-AAD). Percent ADCC was calculated as [(% experimental lysis  % spontaneous lysis)/(% maximum lysis  % spontaneous lysis)]  100, in which ‘‘% spontaneous lysis’’ referred to percent lysis of target cells incubated with effector cells in the absence of IgGs, and ‘‘% maximum lysis’’ referred to percent lysis of target cells following heat shock by incubating the cells at 85 °C for 30 min.

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3. Results 3.1. Reconstitution of antibody repertoires of three ‘‘elite neutralizers’’ in vitro We have previously screened a panel of 103 serum samples from clade B0 chronically infected, treat-naïve former plasma donor cohort in central China by TZM-bl neutralization assay against a panel of 23 tier 2 and 3 Env-pseudotyped viruses from clade A, B, C, cross-clade CRF01_AE and CRF07_BC. We identified ‘‘elite neutralizers’’ based on both neutralization magnitude (geometric mean ID50 (GMT) > 100) and breadth (>95%). Two ‘‘elite neutralizers’’, dubbed patient 1-1 and 1-3, had more than 10 years of HIV-1 infection and were identified as rapid and slow progressor, respectively, based on the clinical parameters (Hu et al., 2012). The neutralization potency and breadth of patient sera 1-1 and 1-3 were 144/96% (top 4) and 101/96% (top 5), respectively. We constructed two antibody Fab libraries using the mRNAs of the PBMCs from patient 1-1 and 1-3 and panned the two phagedisplayed Fab libraries against recombinant Env trimers, but did not isolate monoclonal antibodies (mAbs) that recapitulated the patient sera in neutralizing the virus. We decided to investigate the antibody repertories of these two ‘‘elite neutralizers’’ in this study. The third ‘‘elite neutralizer’’, dubbed patient 1-4, was screened from an injection drug user population in western China with at least 8 years of HIV-1 infection. The neutralization magnitude and breath of patient serum 1-4 was 127/100% for a large panel of 30 tier 2 and 3 Env-pseudotyped viruses from clade A, B, C, cross-clade CRF01_AE and CRF07_BC (unpublished data). Patient 1-4 was diagnosed with AIDS based on the clinical parameters. Three recombinant full-length IgG libraries, designated Lib 1-1, 1-3 and 1-4, were constructed using the total RNA prepared from 1  105 B cells of patient 1-1, 5  106 PBMCs of patient 1-3 and 1  105 memory B cells of patient 1-4, respectively. First strand of cDNA was synthesized and the VHs and LCs were PCR amplified and subsequently cloned to pDR12. The resultant Lib 1-1, 1-3 and 1-4 contain 6  106, 1  107 and 4  106 individual clones, respectively. Twenty clones were randomly picked up from each IgG library and DNA sequenced. No repeated sequences were found, suggesting good quality (diversity) of the libraries. Maxi-prep plasmids from the three IgG libraries were used to transiently transfect 293F cells to express soluble IgGs. Recombinant library IgGs were purified from the culture supernatant and used in various assays in comparison with purified serum IgGs from the same ‘‘elite neutralizers’’. 3.2. Recombinant library IgGs are not comparable to the serum IgGs in binding to recombinant Envs and in neutralizing the virus and inhibiting Env-mediated cell–cell fusion Recombinant library IgGs were tested by ELISA for binding to recombinant Envs derived from clade B isolates, including Env gp140 trimer derived from YU2 and SF162, oligomeric gp140s from JRFL and 89.6, and monomeric gp120 from BaL and JRFL. Library IgGs showed overall much weaker binding to the recombinant Envs than the serum IgGs (Fig. 1). We then tested the library IgGs and the serum IgGs in a standardized TZM-bl neutralization assay against a panel of nine HIV-1 clade B or cross-clade BC isolates from tier 1, 2 and 3 viruses (Table 1). The recombinant library IgGs did not neutralize these isolates at the highest concentration tested (150 lg/ml), while the serum IgGs neutralized some of the isolates (Table 1). Furthermore, compared to the serum IgGs, the recombinant library IgGs exhibited significantly low activity in inhibiting Env-mediated cell–cell fusion (Fig. 2).

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Fig. 1. Binding of the recombinant library IgGs and the serum IgGs to recombinant Envs by ELISA. Various recombinant Envs were coated on Maxi-sorp ELISA plates. Threefold serially diluted IgGs were added to the plates and bound IgGs were detected using HRP conjugated to anti-human Fc as secondary antibody and TMB as substrate. The OD450nm was measured after color development at RT for 20 min.

Table 1 Neutralization activity of the recombinant library IgGs, the sorted library IgGs and the serum IgGs by a standardized TZM-bl neutralization assay. Threefold serially diluted IgGs with a start concentration of 150 lg/ml were tested in the TZM-bl assay. Each dilution was tested in triplicate. Antibody concentrations that lead to 50% and 20% neutralization (IC50s and IC20s, lg/ml) were determined by using GraphPad prism software. IgG1 b12 was included as control. HIV-1 isolates

Tier 1A, Clade B

Tier 1B, Clade B

Tier 2, Cross_clade BC

SF162

JRFL

JRCSF

CH110

CH117

CH181

CH70

CH115

CH120

IgG sample Ser 1-1 Ser 1-3 Ser 1-4 Lib 1-1 Lib 1-3 Lib 1-4 Lib 1-1S Lib 1-3S Lib 1-4S IgG1 b12

IC50 (lg/ml) 12.68 7.17 10.37 >150 >150 >150 66.13 >150 83.34 150 >150 >150 >150 >150 >150 >150 0.07

>150 >150 148.68 >150 >150 >150 >150 >150 >150 0.85

>150 >150 72.9 >150 >150 >150 >150 >150 >150 >50

ND ND ND >150 >150 >150 >150 >150 118.3 >50

>150 >150 86.29 >150 >150 >150 >150 >150 >150 >50

>150 >150 116.9 >150 >150 >150 >150 >150 >150 >50

>150 >150 >150 >150 >150 >150 >150 >150 >150 >50

>150 >150 >150 >150 >150 >150 >150 >150 >150 >50

IgG sample Ser 1-1 Ser 1-3 Ser 1-4 Lib 1-1 Lib 1-3 Lib 1-4 Lib 1-1S Lib 1-3S Lib 1-4S IgG1 b12

IC20 (lg/ml) 3.61 2.09 150 >150 6.43 57.1 19.31 150 >150 >150 >150 150 90 150 >150 >150 >150 >150 107.09 150 >150 0.16 >150 >150 >150 >150 >150 79.38 >50

ND ND ND >150 >150 >150 >150 >150 79.1 >50

>150 >150 27.77 >150 >150 >150 >150 >150 >150 1.3

>150 >150 49.67 >150 >150 >150 >150 >150 119.57 >50

>150 >150 1.85 >150 >150 >150 >150 >150 83.96 9.97

>150 >150 11.93 >150 >150 >150 >150 >150 >150 49.66

3.3. NAb-sorted library IgGs are comparable to the serum IgGs in binding to recombinant Envs and inhibiting Env-mediated cell–cell fusion, but are overall less potent than the serum IgGs in neutralizing the virus To investigate whether HIV-1-specific nAb repertoires in the recombinant library IgGs are comparable to the serum IgGs in

Tier 3, Cross_clade BC

binding to Envs and neutralizing the virus, we subcloned the VHs and LCs to a modified pDR12, designated pTM, containing human IgM transmembrane domain and cytoplasmic tail downstream the human IgG1 CH1-3, which allows cell surface display of IgGs. Three recombinant pTM-IgG library plasmids were used to transiently transfect TZM-bl cells. The cells were subsequently infected with HIV-1 pseudovirus and then sorted for low or no luciferase

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Fig. 2. Inhibition of syncytium formation by the recombinant library IgGs and the serum IgGs in cell–cell fusion assay. TF228 cells stably expressing HIV-1 IIIB gp160 were used as target cells and the CD4 positive T cell line Sup-T1 as effector cells. 5000–10,000 effector and target cells at a ratio of 1:1 were mixed and incubated 37 °C for 20 h in CO2 incubator. Syncytia were counted post co-incubation for 4 h, 20 h and 48 h. One tailed unpaired t-test was used for statistical analysis by using Prism 5.0 software.

expressing cells with less or no virus entry compared to the controls transfected with non-HIV antibody gene containing pTM plasmid DNA (manuscript in preparation). The plasmids were extracted from the sorted TZM-bl cells and the inserts transferred back to pDR12, resulting in three sorted full-length IgG sublibraries, designated Lib 1-1S, 1-3S and 1-4S. Recombinant IgGs from the sorted libraries were tested for binding and neutralization activities in comparison with the serum IgGs. We found that the sorted library IgGs were comparable to the serum IgGs in binding to recombinant Envs (Fig. 3), and inhibiting Env-mediated cell–cell fusion (Fig. 4). All three sorted library IgG samples showed improved neutralization activity compared to the unsorted recombinant library IgGs. However, they were still weaker than the serum IgGs in neutralizing this panel of HIV-1 isolates (Table 1). 3.4. NAb-sorted library IgGs are comparable to the serum IgGs in ADCC We further tested the sorted library IgGs and the serum IgGs for ADCC using TF228 cell line stably expressing IIIB gp160 as target cells and human PBMCs from healthy donors as effector cells. All IgG samples were tested at four different concentrations and no significant difference was observed in paired analyses between the sorted library IgGs and the corresponding serum IgGs (Fig. 5). All IgG samples had low ADCC compared to mAb 2F5. Nevertheless, serum 1-3 and 1-4 IgGs and their sorted library IgGs showed relatively high ADCC compared to serum 1-1 IgG and its sorted library IgGs. Polyclonal Fab 1-3 from previously constructed recombinant Fab 1-3 library and serum IgGs from healthy individuals were included in the ADCC assay, and they did not exhibit ADCC as expected.

3.5. The sorted library IgGs and the serum IgGs may contain b12-like CD4bs Abs, but not VRC01-class bnAbs We further characterized the sorted library IgGs and the serum IgGs for binding to neutralizing determinants in the CD4bs. We tested all IgG samples for binding to resurfaced Env gp120, RSC3, and its CD4bs knock-out mutant, DRSC3. All three sorted library IgGs and the serum IgGs bound weakly to RSC3, and did not bind to DRSC3 (Fig. 6). We further tested these IgG samples for binding to wild type gp120BaL and its VRC01 binding site knock-out site mutants, including D368R (CD4bs), N279A (loop D) and G459E (loop V5) mutants. Known bnmAbs b12 and VRC01 were included as controls in the binding assay. Unlike VRC01, all IgG samples bound well to loop D site mutant, N279A, which is similar to b12. Serum 1-4 IgGs and sorted library 1-1 IgGs bound equally well to wild type gp120BaL and the three site mutants (Fig. 7). These results suggest that CD4bs bnAbs, if any, may not be dominant in these samples. If CD4bs bnAbs do exist in these samples, they are more likely b12-like, but not VRC01-class bnAbs. 4. Discussion In this study, we investigated the antibody repertoires of three ‘‘elite neutralizers’’ whose sera exhibited broadly neutralizing activity by constructing recombinant full-length IgG libraries followed by a comparative study of the in vitro reconstituted antibody repertoires with the corresponding sera for binding and neutralization activities. We found that the recombinant library IgGs were not comparable to the serum IgGs in binding to Envs and in neutralizing the virus and inhibiting Env-mediated

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Fig. 3. Binding of the sorted recombinant library IgGs and the serum IgGs to recombinant Envs by ELISA. The same binding assay was used as described in Fig. 1.

Fig. 4. Inhibition of syncytium formation by the sorted library IgGs and the serum IgGs relative to the control in the absence of IgGs. The same syncytium assay was used as described in Fig. 2. All IgG samples were tested at 50 lg/ml.

cell–cell fusion, while nAb-sorted library IgGs exhibited comparable binding and inhibitory activities, as well as ADCC, to the serum IgGs, but the sorted library IgGs were still overall less potent than the serum IgGs in neutralizing the virus. Further analysis indicates that CD4bs bnAbs, if any, may not be dominant in these ‘‘elite neutralizers’’, and they are more likely b12-like, but not VRC01-class bnAbs. Our results indicate that B cells that express HIV-1-specific Abs, especially nAbs, may not be rich in the B cell repertoires of ‘‘elite neutralizers’’, but nAbexpressing B cells may be highly active at translational level to produce Abs, which leads to the richness of nAbs in the serum IgGs, but not in the recombinant library IgGs. The antibody (clone) profiles in the recombinant antibody libraries most likely represent the B cell profiles in the patient PBMCs, but they do not

represent the antibody (protein) profiles in the patient sera. NAbsorted library IgGs are expected to contain HIV-1-specific nAbs that inhibit virus entry. Indeed, the sorted library IgGs are comparable to the serum IgGs in binding to recombinant Envs and in inhibiting Env-mediated cell–cell fusion, but the sorted library IgGs still do not fully recapitulate the neutralization profiles of the serum IgGs. This may be partially attributed to the CD4i Abs in the sorted library IgGs. CD4i Abs is often rich in patient sera. Although CD4i Abs cannot neutralize free virus in TZM-bl neutralization assay, they can potently block virus entry when displayed on the surface of permissive cells. Therefore, the sorted library IgGs may contain certain amount of CD4i Abs. Removing CD4i Abs from the sorted libraries may facilitate a better mimicry of the serum IgGs by the sorted library IgGs.

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Fig. 5. Percent ADCC of the sorted library IgGs and the serum IgGs. A flow cytometry-based ADCC assay was carried out using TF228 cells as target cells and healthy human PBMCs as effector cells at an E/T ratio of 50. TF228 cells stained with PKH-67 were dispensed in round-bottomed 96-well plate. Serially diluted antibodies in triplicate were added to the wells and the plate incubated at 37 °C for 15 min. Freshly prepared PBMCs were then added to the wells and the plate incubated at 37 °C for 4 h. Cell mixtures were then stained with 7-AAD solution by incubation at 4 °C in dark for 15 min and subsequently analyzed by flow cytometry. Percent cell death was determined and percent ADCC calculated as described in Section 2.

Fig. 6. Binding of the sorted library IgGs and the serum IgGs to recombinant RSC3 and DRSC3. Recombinant RSC3 or DRSC3 were coated on microwell plates. Threefold serially diluted IgGs were added to the plates and bound IgGs were detected using HRP conjugated to anti-human Fc as secondary antibody and TMB as substrate. The OD450nm was measured after color development at RT for 20 min.

We compared the sorted library IgGs with the serum IgGs for ADCC activity. ADCC for an antibody varies depending on the binding affinity of the Fc for the Fc receptor expressed on the effector cell and the epitope the antibody recognizes. In this study, all recombinant library IgGs have the same IgG1 heavy chain constant region, therefore, any difference in ADCC may be attributed to the different epitopes antibodies bind to. Serum 1-1 IgGs exhibited

lower ADCC than serum 1-2 and 1-4 IgGs, which may partially explain why patient 1-1 is a rapid progressor. But when we analyzed the composition of nAbs in these IgG samples, we did not observe significant difference in the amount of possible CD4bs nAbs, suggesting that the relatively high ADCC activity in the serum 1-3 and 1-4 IgGs or the sorted 1-3 and 1-4 IgGs may come from non-CD4bs nAbs and/or non-neutralizing HIV-1-specific Abs.

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Fig. 7. Binding of the sorted library IgGs and the serum IgGs to recombinant gp120BaL and VRC01 binding site knock-out site mutants. Recombinant gp120BaL and site mutants D368R (CDbs), N279A (loop D) and G459E (V5) were coated on microwell plates. Threefold serially diluted serum IgGs and the sorted library IgGs, as well as IgG1s b12 and VRC01, were added to the plates. Bound IgGs were detected using HRP conjugated to anti-human Fc as secondary antibody and TMB as substrate. The OD450nm was measured after color development at RT for 20 min.

We did not find significant difference in ADCC between the sorted library IgGs and the corresponding serum IgGs at the same antibody concentration, suggesting that the sorted library IgGs may nicely mimic the serum IgGs in eliminating HIV-1-infected cells and containing the virus in vivo, most likely through a route of ADCC. These results may have implications for vaccine development. If HIV-1 nAb-expressing B cells are not rich in the B cell repertoires, vaccine immunogens may be designed to target multiple neutralizing determinants to stimulate multiple B cell populations. Polyclonal HIV-1-specific antibodies induced by such immunogens may work in combination or synergistically in containing the virus in vivo through Fab-mediated direct neutralization of free virus and Fc-mediated effector function for elimination of infected cells. Acknowledgements We wish to thank Peter Kwong, Tongqing Zhou, Zhi-Yong Yang and John Mascola for providing recombinant plasmids encoding RSC3, DRSC3, gp140SF162 and gp140yu2 trimer, Dennis Burton for b12-expressing plasmids, Dimiter Dimitrov, Xiaodong Xiao and Jianqing Xu for helpful discussions. This work was supported by Hong Kong Health and Medical Research Fund (HMRF) (# 11101052) and General Research Fund (GRF) (# 785112), and China 12th 5-year Mega project for HIV/AIDS (# 2012ZX10001006) to M-Y. Z.

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