Targeted Immuno-Antiretroviral to Promote Dual Protection against HIV: A Proof-of-Concept Study

The C–C motif chemokine receptor-5 (CCR5) expression on the T-cell surface is the prime barrier to HIV/AIDS eradication, as it promotes both active human immunodeficiency virus (HIV)-infection and latency; however, antiretrovirals (ARVs) suppress plasma viral loads to non-detectable levels. Keeping this in mind, we strategically designed a targeted ARVs-loaded nanoformulation that targets CCR5 expressing T-cells (e.g., CD4+ cells). Conceptually, CCR5-blocking and targeted ARV delivery would be a dual protection strategy to prevent HIV infection. For targeting CCR5+ T-cells, the nanoformulation was surface conjugated with anti-CCR5 monoclonal antibodies (CCR5 mAb) and loaded with dolutegravir+tenofovir alafenamide (D+T) ARVs to block HIV replication. The result demonstrated that the targeted-ARV nanoparticle’s multimeric CCR5 binding property improved its antigen-binding affinity, prolonged receptor binding, and ARV intracellular retention. Further, nanoformulation demonstrated high binding affinity to CCR5 expressing CD4+ cells, monocytes, and other CCR5+ T-cells. Finally, the short-term pre-exposure prophylaxis study demonstrated that prolonged CCR5 blockage and ARV presence further induced a “protective immune phenotype” with a boosted T-helper (Th), temporary memory (TM), and effector (E) sub-population. The proof-of-concept study that the targeted-ARV nanoformulation dual-action mechanism could provide a multifactorial solution toward achieving HIV “functional cure.”


Introduction
Antiretroviral therapy (ART) is the only treatment strategy for human immunodeficiency virus (HIV) infection. ART improves life expectancy by effectively controlling plasma viral load (pVL); however, it is unable to eradicate the virus. Patients have to commit to continual life-long ART. Additionally, ART stoppage can reactivate the latent virus; therefore, alternative ways are under investigation to search for potential candidates to 'functional-cure' HIV infection [1].
One of the essential targets of HIV research is the C-C motif chemokine receptor 5 (CCR5), a predominant co-receptor expressed on lymphocytes (such as CD4+ T-cells, latently infected cells, dendritic cells (DCs), and macrophages), responsible for HIV-1 cellular entrance [2]. Maraviroc, a CCR5 antagonist that blocks HIV entry by docking on the The first level of protection: Anti-CCR5 mAb+ ARVs loaded NP bound on the T-cell surface via CCR5 receptor (xfR5-D+T NP/CCR5 complex) blocks HIV interaction with CCR5 on the T-cell surface by two approaches, i.e., first, by blocking HIV from CCR5 binding and the second, providing steric hindrance to HIV virions due to nanoformulation crowding on T-cell surface (red block arrows) [30]. (B) The xfR5 D+T NP/CCR5 complex triggers the CCR5 trafficking pathway due to anti-CCR5 mAb binding with the CCR5+ T-cells [31], leading to the internalization of xfR5 D+T NP.
Within the endosome, the low pH induces the degradation of polymeric NPs, leading to the release of DTG (INSTI)and TAF (NRTI) into the cytoplasm. The second level of protection: the presence of an intercellular pool of TFV di-phosphates and DTG to prevent (C) naive cell HIV infection. (D) The latently HIV+ cells with free intracellular high-affinity DTG (INSTI), will bind with fresh integrase enzymes produced during the reactivation stage, resulting in a non-functional INSTI/integrase complex; therefore, fresh HIV virions thus produced will be with non-functional INSTIs, and, fresh virion will not be able to integrate viral DNA into the host genome. The ARV drug, i.e., dolutegravir (DTG, 98% purity), was purchased (BioChemPartner Co., Ltd., Shanghai, China), and tenofovir alafenamide (TAF, 100% purity) was a The first level of protection: Anti-CCR5 mAb+ ARVs loaded NP bound on the T-cell surface via CCR5 receptor (xfR5-D+T NP/CCR5 complex) blocks HIV interaction with CCR5 on the T-cell surface by two approaches, i.e., first, by blocking HIV from CCR5 binding and the second, providing steric hindrance to HIV virions due to nanoformulation crowding on T-cell surface (red block arrows) [30]. (B) The xfR5 D+T NP/CCR5 complex triggers the CCR5 trafficking pathway due to anti-CCR5 mAb binding with the CCR5+ T-cells [31], leading to the internalization of xfR5 D+T NP. Within the endosome, the low pH induces the degradation of polymeric NPs, leading to the release of DTG (INSTI)and TAF (NRTI) into the cytoplasm. The second level of protection: the presence of an intercellular pool of TFV di-phosphates and DTG to prevent (C) naive cell HIV infection. (D) The latently HIV+ cells with free intracellular high-affinity DTG (INSTI), will bind with fresh integrase enzymes produced during the reactivation stage, resulting in a non-functional INSTI/integrase complex; therefore, fresh HIV virions thus produced will be with non-functional INSTIs, and, fresh virion will not be able to integrate viral DNA into the host genome.  The ARV drug, i.e., dolutegravir (DTG, 98% purity), was purchased (BioChemPartner Co., Ltd., Shanghai, China), and tenofovir alafenamide (TAF, 100% purity) was a generous gift from Gilead Sciences Inc. (Foster City, CA, USA) under the MTA agreement. Internal standards, i.e., tenofovir-d6 (TFV-d6), TAF-d5, tenofovir-diphosphate-d6 (TFV-dp-d6), and dolutegravir-d4 (DTG-d4) were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada).

Primary Cells and Cell Lines
The TZM-bl cell line was obtained from the National Institutes of Health (NIH) acquired immunodeficiency syndrome (AIDS) reagent program. A JC53-bl (clone 13)/HeLa cell line phenotypically similar to HIV infecting cell type (stably overexpresses CD4 and CCR5 receptor) was used as an HIV-infection indicator cell type. TZM-bl cells were maintained in a complete DMEM medium (HiDMEM medium supplemented with 10% FBS and 1× AA) as standard protocol [14,15].

Production, Purification, and Characterization of xfR5 mAb
The modified high-affinity xfR5 mAb was produced and isolated from a hybridoma cell line, i.e., XF27/28/CCR5 43E2-AA (PTA-4054; ATCC repository) [32], by following the published method with modifications as described below [37]. Briefly, XF-CCR5 28/27 43E2AA hybridoma cells were seeded (at 10 6 /mL concentration) and maintained in antibody production inducing media, i.e., RPMI medium with 1 × AA, for several days until 50% cells were found to be compromised (cell death). The supernatant with soluble xfR5 mAb was harvested by pelleting out dead cells and debris. The soluble xfR5 mAb from the supernatant was isolated using HiTrap™ Protein-A HP prepacked column (GE Healthcare; Chicago, IL, USA) following standard manufacturer's protocol. The purity and concentration of the xfR5 mAbs were determined by the SDS-PAGE method and BCA assay using Pierce™ BCA Protein Assay Kit, following manufacturers' protocol. The xfR5 binding was evaluated based on the standard curve (linear regression analysis) from a known concentration of IgG4 isotype control mAb, as xfR5 is recombinant human CCR5 mAb with IgG4 isotype backbone [32].

ARV-Loaded NPs Formulation
The targeted nanoformulation was fabricated by following multiple steps. First, NHS functionalized DTG+TAF (D+T) loaded nanoformulation was obtained following the modi-Nanomaterials 2022, 12, 1942 5 of 23 fied oil-in-water emulsions phase inversion method [14,15]. Briefly, in the DCM organic phase, PLGA, PLGA-PEG-NHS, PEG-PLGA, and PF127, along with TAF and DTG, were dissolved at 1:1:2:2:4:4 ratios. TAF (at comparative ratio 2) was dissolved in PBS (aqueous phase). The aqueous phase was added dropwise to the organic phase under constant stirring conditions. The water-in-oil (w-o) emulsion obtained was sonicated as described below and then added dropwise to the three times higher volume of 1% PVA solution (aqueous phase) under high-speed stirring conditions. The above w-o-w emulsion was immediately probe-sonicated for 5 min on ice (setting: 90% Amplitude; pulse 0.9 cycle/bursts) with the help of a UP100H ultrasonic processor (Hielscher Inc. Mount Holly, NJ, USA). The organic phase from the o-w emulsion was completely evaporated overnight (O/N). The NHS functionalized D+T NPs were desiccated by lyophilization using Millrock LD85 lyophilizer (Kingston, NY, USA). The complete formulation method was carried out under the hood to maintain sterility during fabrication.

ARV NP Functionalization
The modified anti-CCR5 (xfR5) mAb was conjugated to the NHS functionalized D+T NPs by an amide bond [38]. Briefly, NHS ester-D+T NPs were dissociated in PBS (pH 7.4) as the NHS esters (4-5 h half-life of at pH 7.4). The xfR5 mAb in PBS has protonated amine groups. The protonated xfR5 mAb were added to NHS ester-D+T NPs at a 1:10 weight ratio under constant stirring at room temperature (RT), and the reaction proceeded for 2 h at RT. Immediately after the reaction, the unbound xfR5 mAb was washed off by dialysis using Float-A-Lyzer™ G2 Dialysis Devices (Thermo Fisher Scientific; Santa Clara, CA, USA) in cold PBS supplemented with 10mM hydroxylamine to quench any non-reacted NHS groups present on the xfR5-D+T NPs surface. Following three cold PBS buffer exchanges, xfR5-D+T NPs were collected and stored at 4 • C. The BCA assay evaluated the concentration of xfR5 mAb conjugated on the xfR5-D+T NPs surface. The PBS and D+T NPs were run in parallel during the BCA assay to obtain background values. The background values were subtracted from the mAb NPs absorbance value to obtain normalized mAb concentration of mAb on NPs.

CCR5 Targeted ARV NP Characterization
The physicochemical properties of the D+T NPs were evaluated based on dynamic light scattering (DLS), Fourier transform infrared (FT-IR) spectroscopy, and scanning electron microscopy. The size, surface charge, and polydispersity index (PDI) of the D+T NPs were determined by a ZetaPlus Zeta Potential Analyzer instrument (Brookhaven Instruments Corporation; Holtsville, NY, USA) following standardized methodology [14,15]. The DLS analysis was used to determine the D+T NPs size and polydispersity index (PDI), i.e., size homogeneity and distribution pattern. The zeta potential analysis identified the surface charge density on the D+T NPs. The D+T NP's surface NHS functionalization and xfR5 mAb binding via amide bond was evaluated on FT-IR spectroscopic analysis following a previously published method [39]. Briefly, the spectra of each sample in powder form were collected in the range 600-4000 cm −1 using 25 scans at a resolution of 4 cm −1 with% transmittance intensity mode and Happ-Genzel function apodization under IRPrestige-21 Fourier transform infrared spectrometer instrument (Shimadzu; Columbia, MD, USA). The data were analyzed using LabSolutions IR software (Shimadzu; Columbia MD, USA). By scanning electron microscopy, the morphology and shape of the D+T NPs were evaluated [40]. Briefly, D+T NPs were deposited on Whatman ® Nuclepore Track-Etch Membrane (~50 nm pore size) and air-dried for one day at RT under a chemical hood. The air-dried NPs membrane was sputter-coated with a thin layer (~3-5 nm thick) of chromium and imaged under a Hitachi S-4700 field-emission SEM (New York, NY, USA).
The % drug entrapment efficiency (%EE) of DTG and TAF in D+T NPs and xfR5-D+T NPs were evaluated by high-performance liquid chromatography (HPLC) instrument by following the published methodology [34,41,42]. Briefly, 1 mg of D+T NPs dissociated in 50 µL DMSO, and an appropriate amount of mobile phase (25 mM KH 2 PO 4 45%:ACN 55%) was added to obtain a 10% DMSO concentration in the final injection volume (20 µL). The same procedure was followed for the standard curve evaluation to prepare the D+T standard solutions (with each drug concentration from 0.5 to 0.0019 mg/mL). The chromatography separation was performed under an HPLC instrument (Shimadzu Scientific Instruments; Columbia, MD, USA) equipped with SIL-20AC auto-sampler, LC-20AB pumps, and SPD-20A UV/Visible detector, using Phenomenex ® C-18 (150 × 4.6 mm, particle size 5 µm) column (Torrance, CA, USA), under isocratic elution process. The mobile phase was maintained at 0.5 mL/min flow rate, temperature: 25 • C, and detection at 260 nm (retention time of 4 min for TAF and 6.3 min for DTG). The quantification of the drug was determined by evaluating the peak area under the curve (AUC) analysis at their respective retention time. The amount of TAF and DTG loaded in the D+T NPs was analyzed based on the standard curve construction (linear correlation, r 2 ≥ 0.99) respective from TAF and DTG standard concentration ranges from 0.5 mg/mL to 0.0019 mg/mL. The HPLC instrument illustrated inter-day and intra-day variability of <10%. The % encapsulation efficiency (%EE) of each drug in the D+T NPs batch was estimated by Equation (1). The data are presented as mean ± standard error of the mean (SEM) of three D+T NPs batches (n = 3).

%EE =
Amount o f drug entraped in NPs (mg) Amount o f drug used f or encapsulation (mg) × 100 (1)

Antibody Binding and Binding Affinity Evaluation
The binding affinity of isolated xfR5 mAb and xfR5-D+T NPs compared to wildtype CCR5 mAb (rabbit anti-human mAb; Bioss Inc.; Woburn, MA, USA) was evaluated by flow cytometry. The xfR5 mAb and wild-type Ab were conjugated with Cy3 dye by Cy3 NHS Ester Mono-Reactive CyDye (GE Healthcare; Chicago, IL, USA), following the manufacturer's protocol. The Cy3 dye to xfR5 mAb binding ratio was evaluated based on regression analysis of respective standards (i.e., 0.5 to 0.00625 mg/mL) data obtained from UV/vis spectroscopy and BCA assay. The batches with a 3:1 ratio of Cy3 dye to xfR5 mAb (Cy3-xfR5 mAb) and wild-type CCR5 mAb were considered for further studies. To study binding affinity by flow cytometry, Cy3-xfR5 mAb conjugated D+T NPs (Cy3-xfR5-D+T NPs) formulations were fabricated and characterized as described for xfR5-D+T NPs. Three independent batches were obtained and evaluated using the standardized formulation for further studies.
For the binding affinity of Cy3-xfR5 mAb and Cy3-xfR5-D+T NPs evaluation, TZM-bl cells (10 5 /well) and phytohemagglutinin (PHA, at 5 µg/mL) activated PBMCs (10 6 cells/well) were treated with Cy3-xfR5 mAb and Cy3-xfR5-D+T NPs at different concentrations (20, 10, 1, 0.1, 0.1 µg/mL of xfR5 concentration) O/N at 37 • C and 5% CO 2 atmosphere. In parallel, the wild-type Cy3-CCR5 mAb treated PBMCs at the same concentration and considered a control group. PHA is known to induce latent HIV-infected primary T-cells' reactivation [42]; therefore, to achieve latent HIV-infected primary T-cells phenotype (promote CD2+ T-cells), PHA-activated PBMCs were used. The treatment was washed off thrice with 1% BSA in PBS (PBA) solution by centrifugation (220× g for 10 min at 4 • C). As HIV-1 primarily infects CD4 T-cells, the binding affinity of xfR5 mAb and xfR5-D+T NPs compared to wild type anti-CCR5 mAbs to CD4+ T cells was evaluated by labeling the above-treated cells with anti-CD4 AlexaFluor700 mAb (Table 1) for 20mins at RT (at 1:100 dilution) and washed thrice with PBA. Similarly, to evaluate binding affinity with the latent population (CD2+ T-cells) and monocytes (CD68+ T-cells), treated PBMCs (as described above) were labeled with anti-CD68 APC mAb and anti-CD2 Pacific Blue, respectively (Table 1). In parallel, unstrained and single channel marked labeled PBMCs were considered for flow cytometry setting and gating strategy. The above marker antibody labeled treated and untreated cells were fixed for 20 min with 4% PFA at 4 • C and washed twice with PBA. The binding of Cy3-xfR5 mAb and Cy3-xfR5-D+T NPs to respective T-cell types was detected and evaluated by the BD LSRII flow cytometer instrument (BD Biosciences; San Jose, CA, USA) and Flowjo software v10 (BD, Franklin Lakes, NJ, USA), Nanomaterials 2022, 12, 1942 7 of 23 respectively. Supplementary Figure S1 details the complete gating strategies. Each experiment was performed on three healthy independent donors PBMCs (n = 3). The binding affinity was calculated based on Michaelis-Menten's non-linear fitting analysis of obtained mean ± SEM (standard errors of means) data.

Immunophenotype Study
Immunophenotype variation upon xfR5-D+T NPs treatment compared to xfR5 mAb in uninfected (mimicking pre-exposure prophylaxis, PrEP condition) and HIV-1 ADA -challenged/ infected PHA-activated PBMCs was evaluated by flow cytometry. Briefly, for immunophenotype evaluation in the absence of infection (during PrEP application), PBMCs (10 5 cells/well) were treated (day 0), respectively, with xfR5 mAb and xfR5-D+T NPs (at 20 µg/mL of xfR5 concentration) for 96 h at 37 • C and 5% CO 2 atmosphere for 4 days (detailed in Supplementary Figure S2I). As a control group and to compare activated PBMCs immunophenotype, PHA-activated PBMCs (10 5 cells/well) were maintained for 4 days [36].  Figure S2III), the cells were infected on day 0 with the HIV-1 ADA virus, as mentioned above, after washing off the PHA stimulation (day −1 ). After washing off HIV infection, cells were treated, respectively, with xfR5 mAb and xfR5-D+T NPs (20 µg/mL of xfR5 concentration) for 4 days. All cells were PAHstimulated on day −1 . On the respective days (days 0, 1, and 4), cells were washed thrice with cold PBA solution by centrifugation (400× g at 4 • C) and incubated with marker mAbs against T-lymphocytes (CD3), Table 1), for 20 min at RT (at 1:100 dilution). The cells were again washed with PBA, fixed for 20 min with 4% PFA at 4 • C, and rewashed thrice with PBA. The immunophenotype was evaluated by flow cytometry. The untreated and unstimulated were considered as the initial cellular immunophenotypic profile. Three independent studies have been performed on three healthy donors' PBMCs. The data are presented as mean ± SEM was obtained from three independent donors.

Intracellular Kinetics Experiments
The intracellular uptake and retention kinetics of D+T NPs and D+T solution were evaluated by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis following a standardized method [14,42,43]. Briefly, TZM-bl cells (10 4 cells/well) were seeded in the 24-well plate with the complete HiDMEM medium. Following O/N cell adherence, the respective cell groups were treated with D+T NPs and D+T solution at 10 µg/mL concentration of each drug, i.e., DTG and TAF. For uptake experiments, at respective time points (i.e., 1, 6, 18, and 24 h), the treated cells were washed twice with warm PBS and detached by Trypsin-EDTA (25%; Thermo Scientific, Oklahoma City, OK, USA), washed twice with PBS. One set of untreated detached cells was counted at each time point to determine cell count at the respective time point. The cells were air-dried under a biosafety cabinet. The air-dried samples were then lysed with 70% methanol and stored at −80 • C until analysis. Drug-retention experiments, the adhered TZM-bl cells were treated with xfR5-D+T NP and D+T solution, respectively, for 24 h and washed thrice with warm 1 × PBS. The washed treated cells were in fresh complete HiDMEM medium until respective time-points (i.e., 1, 6, 24, and 72 h after wash, which corresponds to 25, 30, 48, and 96 h, respectively) after treatment time points). The cells were rewashed with PBS at this time point, detached, lysed, and stored following the same method as explained above. The samples were analyzed using the LC-MS/MS method described in the section below.
For the intracellular DTG, TAF, TFV, and TFV-dp drug-kinetics evaluation by LC-MS/MS instrument, the cell lysates were centrifuged (21,952× g for 5 min at 4 • C), and the supernatant was collected. To an aliquot of 100 µL supernatant, 300 µL of internal standard spiking solution (10 ng/mL each of DTG-d4, TAF-d5, TFV-d6, and 100 nmol/mL of TFV-dp-d6 in ACN) was added and vortexed. The samples were then dried at 45 • C under the stream of nitrogen and reconstituted with 100 µL of 50% acetonitrile. The drug and metabolites were quantified using LC-MS/MS instrument from the same sample.
For TAF, TFV, and DTG estimation, the similar conditions that our group previously published were used with minor modification [43]. One µL of the processed sample was injected into LC-MS/MS and operated in positive mode. The chromatographic separation was carried out using the Restek Pinnacle DB Biph column (2.1 mm × 50 mm, 5 µm) with 0.5% formic acid in water and 0.1% formic acid in ACN (48:52 v/v) mobile phase. The calibration range for all the analytes was 0.01 to 50 ng.
For the quantification of TFV-dp, Phenomenex Kinetex C18 (75 × 4.6 mm, 2.6 µm) column was used with an isocratic mobile phase (10 mM ammonium acetate pH 10.5: ACN (70:30) at a flow rate of 0.25 mL/min. The dynamic calibration range was from 0.01 to 100 ng. The LC-MS/MS system consists of an Exion HPLC system (Applied Biosystems, Waltham, CA, USA) coupled with AB Sciex 5500 Q Trap with an electrospray ionization (ESI) source (Applied Biosystems, Waltham, CA, USA) was used in positive ionization mode. The retention time of TFV-dp was 2.1 min, and the runtime for each sample was 3.5 min. The average inter-day and intra-day variability were <15%, corresponding to the FDA bioanalytical guidelines [44].

In Vitro Cytotoxicity Experiments
The comparative in vitro cytotoxicity of D+T NP vs. D+T solution was evaluated using the TZM-bl cell line and CellTiter-Glo ® luminescent assay method, as described previously [45]. Briefly, the TZM-bl cells (10 4 cells/well) in complete DMEM medium and PBMCs (10 5 cells/well) in complete RPMI medium, were treated in triplicate, respectively, with D+T NP or D+T solution, at different concentrations (20, 10, 1, 0.1, 0.01 µg/mL each drug concentration) for 96 h. Similarly, the 5% DMSO-treated cells and 1×PBS (treatment equal volume) treated cells were the positive and negative control, respectively. The cytotoxicity was evaluated by the CellTiter-Glo ® luminescent-cell viability assay kit (Promega; Madison, WI, USA) following manufacturer protocol. The luminescence intensity was read from the Synergy II multi-mode reader with Gen5TM software (BioTek; Winooski, VT, USA). The untreated control cells were considered 100% viable. The percentage cytotoxicity (%Cytotoxicity) was calculated as % normalized viability against the untreated (negative) control group. The experiment was carried out on three independent batches of D+T NPs and D+T solution. The result represents the mean ± SEM of three independent batches of studies. The %Cytotoxicity was evaluated by following Equation (2):

In Vitro Protection Experiments
The comparative in vitro prophylaxis (PrEP), i.e., protection study between D+T NP vs. D+T solution against HIV-1 NL4-3 , was performed on TZM-bl cells and peripheral blood mononuclear cells (PBMCs) was evaluated by following standardized method [14,45]. Briefly, TZM-bl cells (10 4 cells/well) and PBMCs (10 5  For TZM-bl cells, data were collected from three independent experiments performed with three different batches of D+T NP and D+T solution (each performed in duplicate). For PBMCs, the data were obtained after treating three different healthy donors' PBMCs (each performed in triplicate) at independent time. Finally, the selectivity index (SI) was evaluated by following Equation (5): where 'CC 50 ' (cytotoxic concentration at 50%) and 'IC 50 ' (50% inhibitory concentration) were evaluated from the above-described in vitro cytotoxicity and protection study.

Statistical Analysis
All study results presented are expressed as mean ± SEM of the obtained data from at least three independent experiments. The CC 50 value was determined by non-regression curve fitting based on log (DTG or TAF concentration) vs. normalized luminescent (threeparameter logistic fits) of cytotoxicity response curves. In contrast, the IC 50 value was evaluated by fitting the non-regression inhibitory curve of log [DTG] vs. normalized TZMbl luminescence (three-parameter logistic fits) luminance values. The analysis of variance (ANOVA) method was used to determine significant differences between treated (D+T NP and D+T solution) vs. control groups at a p-value ≤ 0.05. All the statistical analysis presented was determined by GraphPad Prism 7 software (La Jolla, CA, USA).

CCR5 Targeted cARV-Loaded NPs Characterization
By using standardized water-in-oil-in-water (w-o-w) interfacial polymer deposition method [14,15], N-hydroxysuccinimide (NHS) functionalized DTG and TAF loaded NPs (D+T NP) were formulated (Figure 1). The DTG and TAF drugs were loaded at a 1:1 ratio in the modified CCR5 mAb (xfR5-D+T NP). Well-defined DTG+TAF NP (NHS-D+T NP) was formed as a result. Dynamic light scattering (DLS) size distribution analysis demonstrated D+T NPs size averaged 198.7 ± 10 nm with uniform size-distribution (poly-dispersity index, PDI: 0.153 ± 0.008) ( Table 2). The xfR5 mAb were covalently conjugated on NHS-D+T NP (xfR5-D+T NP) by replacing the NHS group. The xfR5 mAb binding on xfR5-D+T NP caused a slight increase in size (212.6 ± 20.7 nm). The shape and surface properties evaluation by scanning electron microscopy reflected that NHS-D+T NPs obtained were uniform and smooth-surfaced spherical particles (Figure 2A), and xfR5 mAbs surface conjugation did not cause changes in the particle's morphology ( Figure 2B). The xfR5 mAb binding on the D+T NP surface caused NP's surface charge suppression from −28.15 ± 1.9 mV to −17.77 ± 1.9. This surface charge reduction could be attributed to several factors, such as charge masking by the ions from the PBS buffer or xfR5 mAb surface decoration, or both. The polymeric NP encapsulation enhanced drug encapsulation efficiency (%EE) of 58.5 ± 5.2% and 55.7 ± 3.9% for DTG and TAF, respectively ( Table 2). The xfR5 mAb binding on xfR5-D+T NPs quantified by bicinchoninic acid (BCA) protein assay estimated~3.7 ± 0.52 µg of xfR5 mAb per mg xfR5-D+T NPs ( Table 2). The xfR5 mAb binding was further confirmed by FT-IR analysis, as shown in Figure 2C. The NHS ester bands at 1695-1818 cm −1 (aromatic C=O stretching) and 967 cm −1 (-CH wagging band) of PEG-PLGA-NHS polymer on NHS-Blank NPs and NHS-D+T NPs, confirmed free NHS functional group on the NP surface. The disappearance of these -NHS bands in xfR5-D+T NP proved the replacement of the NHS ester group. Additionally, the shift of primary aliphatic amine NH stretching (3350 cm −1 and 1420 cm −1 ) and -CH stretching (2947 cm −1 ), along with primary amide I and II band presence (at 1640 and 1540 cm −1 , respectively), confirmed the covalent amide (-CONH-) bond formation. Other significant bands, C-O-C stretching (1000-1300 cm −1 ) and broad O-H stretching band (3000-3500 cm −1 ) confer xfR5-D+T NPs surface, which has open PLGA & PEG surfaces along with covalently bound xfR5 mAb. Based on these results, xfR5 mAb is not densely packed on xfR5-D+T NP and has enough free space to avoid steric hindrance during targeted CCR5 receptor binding.
Overall, the enhanced electron density and BCA protein quantification validated the binding of xfR5 mAbs on the xfR5-D+T NPs. In contrast, the amide bond (NHCO) conferred a covalent bond between xfR5 mAb and PEG on the NP surface. Aside from the amide bond, the O-H stretching at 3000-3500 cm −1 confirms that the free surface on NP has predominant PEG-PLGA composition; therefore, the xfR5 mAbs are spread on the PEG-covered NP surface with a relatively good space between molecules to avoid steric hindrance.

Binding Affinity
The antibody binding affinity was estimated using flow cytometric analysis, and the data are expressed as equilibrium dissociation constant, K d . The binding affinity (K d ) of xfR5-D+T NP compared to naïve xfR5 mAb and wild-type CCR5 mAb on TZM-bl cells (Table 3) demonstrates that xfR5 mAb displayed approximately 10-fold higher affinity (0.251 ± 0.15 nM) compared to wild-type CCR5 mAb (2.023 ± 0.655 nM). The simultaneousmultiple xfR5 mAbs of xfR5-D+T NP (5.7 ± 2.7 nM) interaction with CCR5 receptors of cells improved the sensitivity by approximately 6.6 times (Supplementary Figure S1 and Table 3). Similarly, xfR5-D+T NPs showed enhanced binding with the primary CD4+ T-cells (4.31 ± 1.47 nM), as reflected from the Kd value, i.e., approximately Kd value was~5 times lower than the xfR5 mAb (20.87 ± 10.65 nM); therefore, multipoint interaction of single xfR5-D+T NP via multimeric xfR5 mAb on NP significantly improved binding affinity compared to xfR5 mAb di-valent interaction (Supplementary Figure S1).

Type
Binding Affinity (K d , nM) The binding affinity of xfR5-D+T NPs with other CCR5+ immune cell types such as cytotoxic T-cells (CTLs, CD8+), dendritic T-cells (CD2+), and monocytes (CD68+), were evaluated using a similar method (gating strategy, Supplementary Figure S3). The comparative binding affinity demonstrated that xfR5-D+T NPs had a significantly enhanced binding affinity with memory CD8+ T-cells, approximately 25 times higher affinity (p < 0.05) compared to naïve xfR5 mAb (Table 2). Additionally, xfR5-D+T NP binding with CD2+ T-cells and CD68+ T-cells displayed slightly enhanced but non-significant differences in binding affinity compared to naïve xfR5 mAb.

Intracellular Drug Kinetics
The sustained release property of xfR5-D+T NP compared to D+T solution was evaluated following the intracellular uptake/release and retention kinetics of DTG, TFV, and TFA-dp (active-metabolite) by non-compartmental analysis using Phoenix WinNonlin 8.1 software. The result demonstrated ( Table 4) that the maximum concentration (C max ) and the area under the concentration-time curve (AUC all ) of TAF after xfR5-D+T NP treatment were 12.9 and 72.4 times higher than TAF solution treatment. DTG demonstrated comparatively higher C max and AUC all , (110.9 and 254.5 times higher, respectively). These parameters showed a non-significant difference for intracellular TFV and TFV-dp between xfR5-D+T NP and D+T solution. From the C max and AUC all data of TAF and DTG, it is evident that the nanoformulation enhances cellular uptake compared to the naïve drugs in solution. In terms of retention, NP formulation demonstrated prolongation in elimination half-life (t 1/2 ) of 11.6 and 4.4 times for TAF and DTG compared to naïve drugs in solution.

Cytotoxicity and HIV Protection Study
A comparative cytotoxicity study was performed using TZM-bl cells and primary human peripheral blood mononuclear cells (hPBMCs) ( Table 5). The % viability was calculated based on untreated controls (normalized using Equation (2)). In primary hPBMCs, xfR5-D+T NP treatment demonstrated a non-significant change in CC 50 compared to xfR5 mAb (Supplementary Table S1). Overall, in vitro cytotoxicity results in TZM-bl cells and hPBMCs suggest that nano-encapsulation does not pose added toxicity and improves cell viability. The experiments revealed that all the tested nanoformulations (xfR5-D+T NP, xfR5 NP, and D+T NP) were as safe as xfR5 mAb for cellular application. These results indicated that PLGA-based CCR5 targeted ARV drugs loaded NP does not induce significant cytotoxicity. The dual protection by xfR5-D+T NP due to its multimeric xfR5 mAb blocking and prolonged ARV release was anticipated to improve the half-maximal inhibitory concentration (IC 50 ) compared to xfR5 mAb and D+T NPs alone. The protection study in TZM-bl cells demonstrated that xfR5-D+T NP improved IC 50 by 528 and 5.5 times compared to D+T NPs alone and xfR5 mAb alone, respectively (Table 5). Similarly, in primary hPBMCs, xfR5-D+T NPs lowered the IC 50 by 12, 5.5, and 3.4 times compared to naïve xfR5 mAb, xfR5 NP alone D+T NP alone, respectively (Table 5 and statistical test in Supplementary Table S2). These results support the fact that multimeric mAb-coated nanoformulation induces multivalent interaction, which may have enhanced HIV protection compared to xfR5 mAb alone (Supplementary Figure S1). Finally, using the selectivity index to evaluate the cytotoxicity versus efficacy demonstrated the potential of xfR5-D+T NPs. These results demonstrate that xfR5-D+T NP significantly (p < 0.05) improved the therapeutic index of both individual therapeutic approaches alone.

Immunophenotype during In Vitro Short-Term Prophylaxis and HIV Treatment Study
The immunological potential of targeted ARV-loaded nanoformulation was evaluated on the PBMCs isolated from healthy donors. The immune-differentiation pattern of T-cells during prophylaxis application and HIV treatment were evaluated using flow cytometry after treating uninfected hPBMCs with xfR5-D+T NP or xfR5 mAb, along with respective controls. The gating strategy for the immunophenotypic study is described in Supplementary Figure S4 The unstimulated hPBMCs was composed of 18%, 30.3%, 1.4%, 60.7%, and 36.8% of naïve, CM, TM, EM, and E T-cell subpopulations, respectively ( Figure 3A). As expected, PHA stimulation significantly (p < 0.05) changed the immunophenotype of T-cell subpopulations stimulating the naïve and E sub-population (45.7% and 66.1%, respectively). EM population was significantly (p < 0.05) decreased (27.4%) during PHA stimulation. Emergence of activated cytotoxic T-lymphocytes (aCTLs, CD8+ CD69+) from minimal (0.66%) to 41.8% could also be attributed to PHA-activation ( Figure 4). Further, the binding of xfR5-D+T NPs with different immune cell types than xfR5 NPs and xfR5 mAb (Supplementary Figure S5) persists not only with CD4+ cells but binds with all cell types expressing CCR5 receptor. compared on day 1 vs. 4, after 1 day of treatment. The differentiation pattern was evaluated following naïve, CM, TM, EM, and E sub-population as depicted in the x-axis, whereas the y-axis represents respective marker% positive cells. The data are presented as mean ± SEM of three independent experiments on three healthy donors (n = 3). The significance was represented by the asterisk (*) symbol, where '*', '**', '***', and '****' correspond to p values <0.05. <0.01, <0.001 and <0.0001, respectively.
The unstimulated hPBMCs was composed of 18%, 30.3%, 1.4%, 60.7%, and 36.8% of naïve, CM, TM, EM, and E T-cell subpopulations, respectively ( Figure 3A). As expected, PHA stimulation significantly (p < 0.05) changed the immunophenotype of T-cell subpopulations stimulating the naïve and E sub-population (45.7% and 66.1%, respectively). EM population was significantly (p < 0.05) decreased (27.4%) during PHA stimulation. Emergence of activated cytotoxic T-lymphocytes (aCTLs, CD8+ CD69+) from minimal (0.66%) to 41.8% could also be attributed to PHA-activation ( Figure 4). Further, the binding of xfR5-D+T NPs with different immune cell types than xfR5 NPs and xfR5 mAb (Supplementary Figure S5) persists not only with CD4+ cells but binds with all cell types expressing CCR5 receptor.  The short-term ex vivo HIV protection study (4 days) demonstrated that xfR5-D+T NP significantly protected cells against HIV infection (Table 5), which could be attributed to a protective immunophenotype ( Figure 3B). The xfR5-D+T NP resulted in increased CM, TM, and E sub-populations; however, compared to untreated, PHA-activated hPBMC, the xfR5-D+T NP significantly boosted TM and E sub-populations ( Figure 3B). The aCTLs and T h population demonstrated an inverse effect ( Figure 4B). The aCTL sub-population after the initial spike (day 1) followed a decline. In contrast, the T h population gradually increased over time ( Figure 4A,B).
The ex vivo HIV infection treatment and short-term (4-day) protection experiments were performed to predict the immunophenotypic differentiation pattern that the nanoformulated treatment could produce ( Figure 3C). Results demonstrated that HIV infection mainly influenced naïve and CM sub-population ( Figure 3C). In the presence of HIV infection, the naïve sub-population, after an initial increase (day 1, post-infection, PI), reverts to basal levels (day 4, after HIV infection), and the CM population showed a gradually increased population (day 4 PI). The xfR5-D+T NP treated population also showed enhanced naïve and CM sub-population during the initial active HIV infection stage (day 1 PI). It would be interesting to investigate the immunophenotypic differentiation pattern in treatment-experienced patients for long-term effects.

Discussion
Our primary aim was to investigate a potential treatment modality to investigate a strategy that could achieve 'functionally cure' HIV+ patients. These experiments demonstrated a strategy combining two different HIV-treatment approaches in a single nanoformulation with the sustained-release property. Combining anti-CCR5 HIV-entry blocking [3] and ARV-based HIV treatment [48]. We formulated xfR5-D+T NPs, a CCR5 receptor targeting ARV-loaded nanoformulation with the potential to block HIV entrance and promote cellular immunity against HIV infection, specifically in memory CD4+ T-cells as they commit themselves to provide antiviral immunity as latent HIV reservoir [34]. Alongside memory CD4+ T-cells, the myeloid lineage, such as monocytes/macrophages, are believed to be other potential HIV sanctuaries [35,36]. We hypothesize that the CCR5 mAb-targeted ARV nanoformulation combines two different strategies to ensure protection against HIV. First, CCR5 mAb docking on the CCR5+ cells blocks HIV entry and prevents new HIV infection, following a similar action mechanism as ARV maraviroc. Second, sustained intracellular ARV released from endocytosed NP degradation would provide intracellular protection against HIV to the naïve cell and latent HIV-infected cell population ( Figure 1C). In addition, prolonged intracellular ARV levels and CCR5 blocking may promote anti-HIV immunological response. Thus, prolonged HIV protection and immunity-boosting could contribute to a possible "functional cure" strategy [37].
The only effective CCR5 receptor blocker that blocks HIV entry in lymphocytes and confirmed HIV cure is the CCR5 delta32 natural mutant genotype [9,10,49]. Other strategies, including maraviroc, ZFN, ribozymes, TALENs, short hairpin RNAs, and CRISPR/Cas9 nuclease, have been shown to block HIV entry but are not yet successful in conferring HIV cure [49]. A study by Platt et al. reported that approximately 7 × 10 2 and 2 × 10 3 CCR5 receptors per cell could lead to HIV infection [50]. To block HIV infection, at least 10 3 blocker molecules are needed to occupy 7 × 10 2 unbound CRR5 receptors/cell. The most effective CCR5 blocker molecule, maraviroc in vivo, does not accumulate at a concentration around the cell to block all CCR5 receptors. Additionally, ligand/CCR5 endocytosis increases CCR5 receptor turn-over dynamics, causing increased CCR5 density as viral load increases [51]. Incomplete blockage due to an insufficient number of molecules near CD4+ cells or increased CCR5 receptor density because of CCR5 receptor regulatory dynamics [52] is a major reason behind the failure of CCR5 blocking strategies other than the CCR5 delta32 natural mutant genotype approach.
To overcome these constraints, we strategically surface decorated xfR5-D+T NP with multiple xfR5 mAb to maximize CCR5 receptor blocking and prove a hindrance to viral binding to CCR5 receptors on T-cells. The xfR5 mAb immunospecifically binds to one or more types of CCR5-receptor [32,53]. It also effectively downregulates CCR5 surface expression by promoting its internalization. The patent report claims xfR5 mAb can inhibit or abolish HIV binding, enter/fusion, and replicating ability in CCR5 expressing cells [53].
Furthermore, compared to univalent interactions such as maraviroc, each xfR5-D+T NPs could effectively block multiple CCR5 receptors due to multimeric interaction of xfR5-D+T NPs via multiple xfR5 to CCR5 receptor interaction per cell. The enhanced binding affinity of xfR5-D+T NP could be attributed to the multivalent interaction strengthening the binding affinity between the target biomolecules on the NP and the receptors on the cell surface [54]. The theoretical calculation revealed that each mAb conjugated NP using the flexible PEG spacers could occupy ten mAb/receptor complexes in T-cells [54]; however, a single naïve mAb could only block one CCR5 molecule per T-cell [55]. Similar to the theoretical assumption, the xfR5-D+T NP practically resulted in a 7-times higher binding affinity than xfR5 mAb. The CCR5 co-receptor characteristically is expressed on CD4+ T-cells in peripheral blood [56]. In addition to CD4+ T cells, the CCR5 chemokine receptor is also expressed on CTLs (CD8+) [57], dendritic cells (CD2+) [58], and monocytes/macrophages (CD68+) [59,60], which also plays critical roles in HIV infection, propagation, and latency. The binding affinity of xfR5-D+T NP on other CCR5+ expressing cell types is essential to estimate the success of this targeted nanoformulated strategy. Our studies demonstrate the multimeric binding potency of xfR5-D+T NP on primary HIV susceptible cells type, especially memory Th (CD4+) and CTLs (CD8+) T-cell populations displayed~5 and~25 times higher binding affinity compared to naïve xfR5 mAb. The enhanced CCR5 expression during infection or PHA-activation promotes migration of activated effector and memory CTLs T-cells [57].
Additionally, the assembly of NPs size (~212 nm, with a weak surface negative charge) around target cells and strong cell surface interaction together pose a substantial steric hindrance to out-compete virion particle (sized~145 nm [56]), thus minimizing their cellular binding ( Figure 1A). Hence, steric hindrance strategically also contributes toward protection against viral entry. The above strategic twofold competitive hindrance by xfR5-D+T NPs may overcome the incomplete blockage constrain of other reported CCR5 blocking strategies.
A study has shown that viral interaction with the CCR5 receptor promotes increased surface CCR5 density on the T-cell surface [57]. CCR5 receptor endocytosis and recycling rate increase compared to degradation [58]. Others have reported that blockage in HIV/CCR5 entry delayed HIV disease progression (observed in HIV+ long-term non-progressors populations) [59]. The NP interaction with the target T-cell generates strong forces following the same direction due to specific ligand-receptor binding and non-specific bonds (i.e., van der Waals, electrostatic and steric interactions). The increased membrane stiffness due to NPmultivalent receptor interaction thus possibly nullifies or overshades the receptor-mediated endocytic force as membrane tension increases [60]. Hence, NP-mediated interference with the ligand-receptor endocytosis process prolongs targeted NP retention [60]. This process could delay CCR5 receptor turn-over rate, suppressing CCR5-receptor upregulation, similar to long-term non-progressors [61]. Target-specific nanoformulation has several unique properties that block (physically and mechanically) HIV entry and delay or prolongs protection against new HIV infection.
Our strategy is to introduce internal protection against new infection or during reactivation of latently infected cells. Latent HIV+ cells, when activated, will produce new virion components (reverse transcriptase, integrase, and protease) to be packed into fresh virion particles to start a new HIV infection. Thus, the CCR5 targeted NPs were loaded with prescribed NRTI and integrase inhibitors to ensure intracellular protection [62][63][64]. The xfR5-D+T NPs upon endosomal uptake cause sustained release of ARVs in cell cytoplasm due to polymeric degradation; therefore, fresh HIV infection introducing new reverse transcriptase or integrase will immediately be inhibited by the intracellular NRTI and INSTI concentration (Figure 1, schematic diagram). The HIV protection assay results demonstrated the same possibility. The xfR5-D+T NP reduced IC50 value by 12 (hPBMCs) times compared to naïve xfR5 mAb. Further, our previous and present study confirms that nanoformulated ARV drugs promote a broad therapeutic index compared to naïve drugs [14,15,[40][41][42].
HIV infection and ART induces different immunophenotypic profile. Studies have shown that HIV infection induces naïve T-cells proliferation, whereas ART causes depletion [65], which aligns with our observed ( Figure 3C). Studies have shown that HIV-infected hPBMCs increased the naïve sub-population [66,67]. In contrast, xfR5-D+T NP induced HIV suppression and reverted the naïve sub-population to its basal level. We observed a reduced naïve T-cell population and an increased CM sub-population, indicating that targeted ARV nanoparticles promote effective memory against HIV by shifting the differentiation-induced population from the naïve ⇒ CM sub-population [68].
The memory T-cell population plays a vital role in promoting anti-HIV immunity and AIDS prognosis since these cells govern the functions of CTLs and B-cells, which regulates cellular and humoral immunity against HIV [69]. Persistent HIV viremia is known to drive the differentiation CM ⇒ EM sub-population [70,71], and the high EM sub-population may be responsible for the long-lasting and exhausted T-cell population in HIV+ patients [70]. The ART treatment partially restores the high CM sub-population and reduces the EM sub-population overtime to regain homeostasis disrupted during HIV infection [70]. Our results demonstrate that as treatment progresses, the xfR5-D+T NP treated population could significantly increase the CM sub-population and boost TM and EM sub-population (Supplementary Figure S6); however, during active HIV infection, targeted treatment using xfR5-D+T NP or naïve xfR5 mAb maintained high E and aCTL sub-population to counter HIV infection. The E and aCTLs sub-population tend to decrease upon viral suppression ( Figure 4). Studies have shown that despite persistently low antigenemia, high Th and E sub-population is essential to control HIV replication in the presence or absence of ART to achieve a "functional cure" [69,72]. Overall, the immunophenotype experiments proved the xfR5-D+T NP multivalent interaction results in enhanced protective immunophenotype compared to naive xfR5 mAb, D+T NPs, and xfR5 NPs; therefore, immunophenotypic differentiation indicated that mAb+ARV NP could also overcome the CM ⇒ TM ⇒ EM ratelimiting steps to promote high E and aCTL population maintenance during HIV; however, a more detailed study in an animal model is needed to confirm these preliminary results.
Our proof-of-concept study demonstrates that the novel CCR5 mAb decorated ARVloaded nanoformulation (xfR5-D+T NPs) could potentially be a single delivery system providing two-layered protection. The competitive binding to CCR5 on target cells provides the first layer of protection by blocking HIV entry (the primary target of HIV to invade the host cell), and the next level of protection comes from eventual ART-mediated blockage of HIV replication and integration. Our preliminary study at the immunological level indicates that prolonged CCR5 receptor blockage and ART protection may also contribute to inducing a protective anti-HIV immune response, i.e., stimulation of protective immunophenotype that could prevent and promote immune cell induce killing of fresh or latent HIV-infected cells. A dual protective mechanism may prevent HIV infection from spreading from the naive and latent CCR5+ cell population. Thus, this could be a potential strategy for achieving an "HIV-functional cure."

Conclusions
We formulated a dual-action targeted nanoformulation, i.e., xfR5-D+T NP. The ex vivo system (primary hPBMCs) experiments demonstrated xfR5-D+T NP induced CCR5 blocking and intracellular ARV drug release, which provides two levels of protection against new HIV infection or latently infected HIV+ cells. Further, xfR5-D+T NP treatment also promotes reprogramming of the immune repertoire function of memory T-cells and could help reconstitute anti-HIV immunity. Based on the proof-of-concept study, we conclude that xfR5-D+T NP combines the advantages of ART and HIV entry blockage, which augments anti-HIV immune reconstitution. Thus, the dual-action strategy could be a promising multifactorial strategy to target the HIV latent reservoir to achieve a "functional cure." Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nano12111942/s1, Figure S1: Schematic diagram explains the xfR5 mAb interaction with CCR5 receptors on T cell binding; Figure S2: Schematic diagram explaining immunophenotype study design; Figure S3: Flow cytometry gating strategy to evaluate T cell binding affinity; Figure S4: Flow cytometry gating strategy to evaluate T cell differentiation pattern upon xfR5-D+T NPs and xfR5 mAbs (free) treatment (at 66.5 nM of xfR5 mAb concentration as NP bound or free); Figure S5: A graphical presentation of comparative binding of Cy3 tagged xfR5-D+T NPs vs. different variables; Figure S6: T-cell differentiation phenotype after D+T NP vs. xfR5 NPs treatment under (A) protection against HIV challenge (HIV prophylaxis), and (B), HIV infected T-cell treatment (HIV treatment) condition; Table S1: Comparative one-way ANOVA analysis (using Tukey's multiple comparison test) of CC 50 of TZM-bl and PBMCs; Table S2: Comparative one-way ANOVA analysis (using Tukey's multiple comparisons test) of IC 50 of TZM-bl and PBMCs.