Engineering of IA and BiIA. Genes encoding single chains (scFv) of bnAbs including PG9, PG16, PGT128, VRC01, and Hu5A8 were prepared by automated codon optimization and DNA synthesis according to published sequences (7–9). Each scFv was constructed as a VL linked to a VH via a 15-mer (Gly 4 Ser) 3 linker (Figure 1). We fused scFvs of these bnAbs to the human immunoglobulin CH 2 -CH 3 (hIgG1-Fc, containing an E333V mutation according to the EU numbering) region. To create secretory antibodies, the secretory signal peptide of tissue plasminogen activator (tPA) was linked to the N-termini of the IAs using PCR-based techniques to enhance the protein expression and release. Two genes encoded BiIA-DG, each with 2 corresponding residue mutations to promote the dimerization of electrostatically matched Fc chains using the knobs-into-holes method (25, 37). BiIA-SG was engineered by fusion of scFv-PGT128 to the 5′ end of the scFv-Hu5A8-hIgG1-Fc backbone with a 20-mer (Gly 4 Ser) 4 linker in between (Figure 2). Because we used the original sequence of Hu5A8 but not the ibalizumab (28, 60), we kept the name Hu5A8 in this study.

IA or BiIA expression and purification. 293T cells (ATCC) were transfected with plasmids encoding various IA or BiIA genes and cultured for 72 hours at 37°C in a 5% CO 2 incubator after the transfection. IA-containing culture supernatants were harvested and centrifuged at 1,000 g for 10 minutes. IAs were purified immediately by affinity chromatography using Protein G–Agarose (Life Technologies) according to the manufacturer’s instructions. The purified IAs and BiIAs were concentrated by an Amicon ultracentrifuge filter device (molecular weight cutoff, 50 kDa; Millipore) to a volume of 0.2 ml in PBS (Life Technologies), and stored at –80°C. Only BiIA-DG was produced by cotransfection of 293T cells using equal amounts of 2 expression vectors.

Western blot analysis. The purity and molecular weights of IAs and BiIAs were analyzed in 10% SDS-PAGE separating gels with Coomassie Blue G-250 stain (ThermoScientific). The separated proteins were transferred onto PVDF membranes (Millipore) for antibody staining. The reducing and nonreducing conditions refer to the presence and absence of β-mercaptoethanol in the gel-loading buffer. The secondary antibody was an IRDye 800CW-conjugated goat anti–human IgG-Fc domain antibody (catalog 926-32232, Rockland). Protein bands were visualized using the Odyssey Image System (Li-COR).

Binding specificity of IA and BiIA by ELISA. Each well of high-binding 96-well plates (Costar) was coated with 50 ng gp120 or sCD4 overnight. After washing, the plates were blocked for 2 hours with the blocking buffer containing 2% BSA (catalog 10857, Affymetrix), 1 μM EDTA (catalog AM9260G, Life Technologies), and 0.05% Tween-PBS (catalog Sc-29113, Santa Cruz) and then incubated with serially diluted IA/BiIA for 60 minutes at 37°C. After washing, the HRP-conjugated goat anti–human detection antibody (catalog sc2907, Santa Cruz Biotechnology) was added for 1 hour as previously described (61), followed by washing and addition of 100 μl HRP chromogenic substrate 3,3′,5,5′-Tetramethylbenzidine (TMB) (catalog 860336, MilliporeSigma). The ODs were measured at 450 nm using the VICTOR3 1420 Multilabel Counter (PerkinElmer). The background values given by incubation of PBS alone in coated wells were subtracted. The positivity was determined when the OD value was 2-fold above the negative controls (e.g., normal serum). All experiments were performed in duplicate.

Surface plasmon resonance (SPR) analysis. The binding kinetics/affinity of IA and BiIA to gp120 and sCD4 were tested in PBS running buffer at 25°C by SPR analysis using a Biacore X100 machine (GE Healthcare) as previously described (25). HIV-1 JR-FL gp120 and sCD4 proteins (30 μg/ml) were covalently coupled to CM5 sensor chips (GE Healthcare) by amine chemistry at pH 5.0, resulting in an immobilization level of 2,000 response units (RUs) and 4,000 RUs, respectively. BiIA-SG, BiIA-DG, or parental IA was injected through flow cells at 2-fold dilutions starting from 125 nM at a rate of 30 μl/min with 3-minute association and 8-minute dissociation. The sensor surface was regenerated between each experiment with a 30-second injection of 10 mM glycine•HCl (pH 2.5) at a ﬂow rate of 30 μl/min. Binding curves were plotted after the subtraction of backgrounds. The 2:1 bivalent analyte model was used for the calculations according to the instructions.

HIV-1 neutralization assay. The Global Panel HIV-1 Env clones were obtained from the NIH AIDS Reagent Program (catalog 12670) (40). The in-house panel of 40 Env clones was generated by us or provided by our collaborator (H Shang, China Medical University, Shenyang, China). A standard inoculum of 200 TCID 50 of each pseudovirus was used to test the potency and breadth of various neutralizing antibodies as previously described (62–64). Pseudovirus neutralization was measured using a luciferase-based assay in GHOST(3)-X4R5 (catalog 3942, NIH AIDS Reagent Program) (35, 38). Each IA and BiIA was tested in duplicate with a 3-fold serial dilution. The 50% and 90% inhibitory concentrations (IC 50 and IC 90 ) of each IA and BiIA were calculated to reflect anti–HIV-1 potency. The synergistic effects of paired IAs were measured by a checkerboard synergy assay and calculated using the MacSynergy II software as previously described (36). Accordingly, the synergy volumes of –50 to 50, 50 to 100, and >100 in a synergy plot at the 95% CI were used to define additive, slight synergistic, and highly synergistic effects, respectively. For live HIV-1 neutralization, a standard viral inoculum of 200 TCID 50 was used for the assay in TZM-bl cells (catalog 8129, NIH AIDS Reagent Program).

Plasma BiIA-SG quantification by ELISA. For ELISA, 25 ng HIV-1 JR-FL gp120 in coating buffer (pH 9.6) was used to coat 96-well plates (Costar) at 4°C overnight. After washing, the plates were blocked for 1 hour with blocking buffer (5% skim milk in PBS) and then incubated with serially diluted BiIAs or BiIA-containing sera for 60 minutes at 37°C. After washing, goat anti–human secondary antibody conjugated with HRP (catalog sc2907, Santa Cruz Biotechnology) was added for 1 hour, followed by washing and adding 100 μl HRP chromogenic substrate 3,3′,5,5′-TMB (catalog T4444, MilliporeSigma). ODs were measured at 450 nm using the VICTOR3 1420 Multilabel Counter (PerkinElmer). All experiments were performed in duplicate.

Humanized mouse model. Animal procedures that might cause more than slight pain or distress were performed with appropriate sedation or anesthesia. Immunodeficient NSG mice were purchased from the Jackson Laboratory (catalog 005557). Humanized NSG-HuPBL mice were generated from 4- to 6-week-old NSG mice as we previously described (41).

Purity and half-life of BiIA-SG. The purity of BiIA-SG was measured by size exclusion–high-performance liquid chromatography (SEC-HPLC). BiIA-SG purified from CHO cells was injected i.p. in to 4 NSG-HuPBL mice at a dose of 20 mg/kg. ELISA was used to measure the serum concentration of BiIA-SG over a period of 7 days. The t 1/2 of BiIA-SG was computed as ln (2)/k, where k is a rate constant expressed reciprocally of the x axis time units by the 1-phase decay equation in the GraphPad software.

Intestinal flush and homogenates preparation. A single dose of 10 mg/kg BiIA-SG was i.p. injected into 4 NSG-HuPBL mice. Mucus, intestinal homogenates, and plasma specimens were collected 24 hours later as previously described (65). Sections (4 cm) from the small and large intestines were excised from NSG-HuPBL mice and flushed with 5 ml PBS using an 18-gauge needle (catalog NN-1825R, Terumo). Any remaining mucus was then manually extruded into the collection tube. The raw flush was vortexed for 15 seconds and centrifuged for 5 minutes at 16,000 g and the supernatant was separated. The flushed intestines were weighed and homogenized with 100 μl PBS. The homogenate was centrifuged for 5 minutes at 16,000 g and the supernatant was separated. The antibody concentration in small intestine and large intestine mucus and homogenate was detected by ELISA.

Fluorescence-activated cell sorting (FACS) analysis. Blood samples were collected from the facial veins of mice in Eppendorf tubes containing 50 μl anticoagulant (0.5 M EDTA) and were then centrifuged for 5 minutes at 1,150 g in a microcentrifuge. The plasma was stored for future analysis, and the cell pellets were resuspended in 2 ml of 1× RBC lysis buffer (BD Bioscience) and incubated on ice for at least 10 minutes to remove red blood cells. After the lysis, the cells were pelleted at 1,150 g in a microcentrifuge for 5 minutes at room temperature and stained for 60 minutes at 4°C with 100 μl of a cocktail containing 2 μl anti–human CD3-PB (catalog 300442/UCHT1), 2 μl anti–human CD4-PerCP-Cy5.5 (catalog 317428/OKT4), 2 μl anti–human CD8-PE (catalog 344706/SK1), and 2 μl anti–human CD45 PE/Cy7 antibodies (catalog 304016/H130) (all from Biolegend). The samples were washed with PBS supplemented with 2% fetal bovine serum and then were centrifuged at 800 g in a microcentrifuge for 5 minutes. The pelleted cells were resuspended in 300 μl wash buffer and analyzed on a FACSAria III flow cytometer (Becton-Dickinson). The samples were gated for human CD45 expression before analyzing the T cell subsets based on the CD3, CD4, and CD8 markers within this subset.

Immunofluorescence (IFA) staining of HIV-infected cells in tissues. Spleen and other tissues were immersed in 10% neutral buffered formalin (catalog Z2902, MilliporeSigma) for 24 hours. After the formalin fixation, the tissues were placed in 70% ethanol (Merck) and subsequently embedded with paraffin. Tissue sections (4-μm thick) were used for immunohistochemical staining for HIV-P24 detection using the Kal-1 murine monoclonal antibody (catalog M085701, Dako) as we previously described (41). Confocal images were obtained with a Carl Zeiss LSM 700 microscope using the ZEN 2012 software.

Viral RNA load measurement by quantitative RT-PCR (qRT-PCR). Viral RNA was extracted using the QIAamp viral RNA mini kit (Qiagen). Each RNA sample was reverse transcribed to 20 μl cDNA with the RT-PCR Prime Script Kit (Takara). The cDNA (2 μl) was used in a 20 μl qRT-PCR reaction with the TaqMan Universal PCR Master Mix (Life Technologies), a TaqMan probe (5′-FAM−CTCTCT CCTTCT AGCCTC−MGB-3′), and primers designed to target the P17 gene of HIV-1 (5′-TACTGA CGCTCT CGCACC-3′ and 5′-TCTCGA CGCAGG ACTCG-3′). The samples were run in triplicate on an Eppendorf Realplex4 Mastercycler (Eppendorf). The following cycling conditions were used: 1 cycle of 50°C for 2 minutes, 1 cycle of 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minutes. The virus titer was determined by comparison with a standard curve generated using RNA extracted from a serially diluted reference viral stock. The limit of detection was 500 copies per milliliter for HIV-1 JR-FL and HIV-1 BJZS7 .

HIV-1 proviral DNA measurement by digital PCR. Cell-associated HIV-1 proviral DNA was quantified by QuantStudio 3D digital PCR System (Life Technologies). Briefly, total cellular DNA was extracted using the QIAmp DNA Blood Mini Kit (Qiagen) and eluted in 40 μl volume. Purified DNA was quantified for HIV-1 proviral DNA by the QuantStudio 3D Digital PCR System using the primers, probe, and reaction conditions as mentioned above for post-RT HIV-1 RNA quantification. Human CCR5 DNA was quantified with primers (5′-ATGAT TCCTG GGAGA GACGC-3′ and 5′-AGCCA GGACG GTCAC CTT-3′) and the sequence-specific probe (5′-VIC-AACAC AGCCA CCACC CAAGT GATCA-TAMRA-3′). All PCR reactions contained 7.25 μl Quantstudio 3D digital PCR master mix v2 in 14.5 μl reaction volume. HIV-1 proviral load is reported as copies per cell justified by cell numbers from CCR5 copies.

In vivo efficacy of BiIA-SG in humanized mice. For PrEP experiments, 1 day before HIV-1 challenge, blood samples from NSG-HuPBL mice were subjected to flow cytometry to determine the baseline CD4/CD8 ratios. On the following day, 200 μg (10 mg/kg) BiIA-SG was injected i.p. into NSG-HuPBL mice (44). One hour later the mice were challenged through the i.p. route with 10 ng P24 of live HIV-1 JR-FL (466 TCID 50 ) or HIV-1 BJZS7 (640 TCID 50 ) diluted in 100 μl PBS (41). These mice were subjected to weekly blood sampling to monitor viral load, CD4/CD8 ratios, and P24+ cells, and finally were sacrificed for the detection of infected cells throughout the body. For cART treatment experiments, NSG-HuPBL mice were challenged through i.p. route with 10 ng P24 HIV-1 BJZS7 . Four days after HIV-1 challenge, mice were subjected to 4 different treatments: cART+BiIA-SG, cART, BiIA-SG, and placebo. The cART cocktail was prepared as previously described (22). Individual tablets of tenofovir disoproxil fumarate (TDF, Gilead), Lamivudine (3TC, Shandong Weifang Pharmaceutical Factory Co.), and Raltegravir (RAL, MSD Pharmaceuticals) were triturated into fine powder and suspended in 100 μl PBS. The cART cocktail (TDF 2.46 mg, 3TC 1.48 mg, RAL 1.23 mg) was administered by daily oral gavage to each mouse. BiIA-SG was i.p. injected (400 μg per mouse) every 4 days. All mice were subjected to weekly blood sampling to monitor viral load and were sacrificed 63 days after HIV-1 challenge.

AAV–BiIA-SG production and quantification. AAV–BiIA-SG production was conducted as previously described (15). An AAV Helper-Free System was purchased from Cell Biolabs. The pAAV-MCS plasmid containing the BiIA-SG transgene or the pAAV–IRES-hrGFP control was cotransfected into AAV-293T cells together with the helper vector pHELP (catalog 240071, Agilent Technologies) and pAAV-DJ (catalog VPK-430-DJ, Cell Biolabs) at a ratio of 1:1:1 using the polyethylenimine (PEI) transfection reagent (Polysciences Inc.) in Opti-MEM medium (Gibco). The AAV–BiIA-SG supernatant was collected at 48, 72, 96, and 120 hours after transfection, concentrated with 5× polyethylene glycol (PEG) 8000 (catalog 89510, MilliporeSigma) and finally purified by 1.37 g/ml cesium chloride centrifugation. The final AAV–BiIA-SG stock was dissolved in PBS, aliquoted, and stored at –80°C. Purified AAV–BiIA-SG was quantified by qPCR as previously described (15). Briefly, a frozen aliquot of AAV–BiIA-SG was treated with DNase I (MilliporeSigma) for 30 minutes at 37°C, and then quantified with SYBR Premix Ex Taq II (Takara) and primers specific for the CMV enhancer (AAV-Mono-CMV-F: CCATTG ACGTCA ATGGGT GGAGT and AAV-Mono-CMV-R: GCCAAG TAGGAA AGTCCC ATAAGG) or the BiIA-SG transgene (AAV-mono-F 383–406: GGACTC TGGTCA CTGTCA GCAGCG and AAV-mono-R 486–406: ACCCTT TCGCCC AGTGAG ACG). The samples were tested in triplicates on the ViiA 7 instrument (Applied Biosystems). The following PCR conditions were performed: 1 cycle of 50°C for 2 minutes, 1 cycle of 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The virus titer was determined by comparison with a standard curve generated using a purified DNA cut fragment after EcoRI/HindIII cut from the AAV-GFP vector. To evaluate the functional activity of the titered virus, AAV-HT1080 cells (catalog 240109, Agilent Technologies) were infected with AAV–BiIA-SG. The transgene expression was tested either by FACS on cell surface or by ELISA in cell supernatants after 6 days of infection.

Viral outgrowth assay (VOA). VOA assays were performed as previously described (66). Briefly, one million splenocytes from AAV-treated mice were seeded into each well of 24-well plates (Corning) in 500 μl RPMI 1640 culture medium (Life Technologies) containing 10% FBS (Life Technologies), and then stimulated by adding 1 μg/ml ionomycin (catalog I9657, MilliporeSigma) and 0.5 μg/ml phorbol 12-myristate 13-acetate (PMA, catalog P8139 Sigma-Aldrich). Stimulated splenocytes were cultured for 48 hours at 37°C, 5% CO 2 , and 100% humidity. Viral RNA copy numbers in culture supernatants were tested by qRT-PCR as mentioned above.

Cell adoptive transfer experiment. One million donor splenocytes in 100 μl PBS were delivered through i.p. injection into each healthy NSG-HuPBL mouse. Four weeks later, recipient mice were sacrificed for analysis.

Statistics. FACS data were analyzed with FlowJo 7.6 software. Graphs were generated with GraphPad Prism 5.01 software (GraphPad Software). Two-tailed Student’s t tests were used for group comparisons. P < 0.05 was considered statistically significant with necessary mean ± SEM or mean ± SD.

Study approval. All animal experimental procedures were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR 2786-12) at the Laboratory Animal Unit of The University of Hong Kong. The Laboratory Animal Unit is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International).