Targeted immuno-antiretroviral HIV therapeutic approach to provide dual protection and boosts cellular immunity: A proof-of-concept study

Human immunodeficiency virus (HIV)-infected active and latent CCR5 expressing long-lived T-cells are the primary barrier to HIV/AIDS eradication. Broadly neutralizing antibodies and latency-reversing agents are the two most promising strategies emerging to achieve ‘functional cure’ against HIV infection. Antiretrovirals (ARVs) have shown to suppress plasma viral loads to non-detectable levels and above strategies have demonstrated a ‘functional cure’ against HIV infection is achievable. Both the above strategies are effective at inducing direct or immune-mediated cell death of latent HIV+ T-cells but have shown respective limitations. In this study, we designed a novel targeted ARVs-loaded nanoformulation that combines the CCR5 monoclonal antibody and antiretroviral drugs (ARV) as a dual protection strategy to promote HIV ‘functional cure’. The modified CCR5 monoclonal antibody (xfR5 mAb) surface-coated dolutegravir (DTG) and tenofovir alafenamide (TAF) loaded nanoformulation (xfR5-D+T NPs) were uniformly sized <250 nm, with 6.5 times enhanced antigen-binding affinity compared to naïve xfR5 mAb, and provided prolonged DTG and TAF intracellular retention (t1/2). The multivalent and sustained drug release properties of xfR5-D+T NPs enhance the protection efficiency against HIV by approximately 12, 3, and 5 times compared to naïve xfR5 mAb, D+T NP alone, and xfR5 NPs, respectively. Further, the nanoformulation demonstrated high binding-affinity to CCR5 expressing CD4+ cells, monocytes, and other HIV prone/latent T-cells by 25, 2, and 2 times, respectively. Further, the xfR5-D+T NPs during short-term pre-exposure prophylaxis induced a protective immunophenotype, i.e., boosted T-helper (Th), temporary memory (TM), and effector (E) sub-population. Moreover, treatment with xfR5-D+T NPs to HIV-infected T-cells induced a defensive/activated immunophenotype i.e., boosted naïve, Th, boosted central memory, TM, EM, E, and activated cytotoxic T-cells population. Therefore, this dual-action targeted mAb-ARV loaded nanoformulation could potentially become a multifactorial/multilayered solution to achieve a “functional cure.”


50
Currently, antiretroviral therapy (ART) is the prime treatment strategy for human 51 immunodeficiency virus (HIV) infection. ART improves HIV patient's life-expectancy by effectively 52 controlling plasma viral load (pVL), but is unable to eradicate the virus, thus patients have to 53 commit to continuing life-long ART. Additionally, ART withdrawal could reactivate latent virus. 54 Therefore, alternative ways are under investigation to search for potential candidates to 55 'functionally-cure' HIV infection (1). One of the essential targets of HIV research is the C-C motif 56 chemokine receptor 5 (CCR5), a co-receptor that is predominant expressed on CD4+ T-cells, 57 latently HIV-1 infected cells, dendritic cells (DC) and macrophages, and is responsible for HIV-1 58 entrance into the targeted cells (2).

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Maraviroc, a CCR5 antagonist that blocks HIV entry and infection by docking on the CCR5 60 receptor on CCR5+ cells, is the first approved CCR5-antagonist drug for HIV-1 treatment (3). The 61 other promising approach is genome editing that disrupts CCR5 alleles in CD4+ T-cells due to 62 the infusion of an engineered zinc finger nuclease (ZFN) (4). However, the only gene-therapy 63 approach to date that had conferred HIV cure is the use of CCR5 delta32 natural mutant genotype. 64 It resists HIV-1 entry in three patients, i.e., the "Berlin Patient", "London Patient", and the 65 "Düsseldorf patient" upon transplantation of stem cells from a CCR5 delta32 genotype donor (5).

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Even though the sustained ART-free remission is a well-accepted and effective strategy to 67 control HIV infection, the main focus of HIV research is to develop strategies that prolong 68 protection and target latent HIV+ cells. Recent studies have demonstrated designing a long-acting 69 (LA) ARV delivery system would boost the HIV prevention and treatment strategy (6-9). The LA 70 ARV delivery system has shown to enhance drug-solubility, stability, biodistribution, 71 pharmacokinetics, efficiency, and concurrent drug safety, due to reduced ARV associated side-72 effects, such as mitochondrial toxicity, renal abnormality, and reduced bone mineral density (10-73 12). Even though LA ARV nanoparticles (NPs) are promising to be a successful injectable LA 74 antiretroviral candidate, these systems still need to show a good tolerability profile. In the LATTE-2 75 trial, the cabotegravir plus rilpivirine LA injection group has reported grade 3-4 adverse events 76 higher compared to the oral comparative treatment group (13). The ECLAIR prevention study of 77 LA ARVs (e.g., cabotegravir LA plus rilpivirine LA injection group) (14, 15) revealed a significantly 78 prolonged sub-therapeutic tail of residual drugs which places patients at high-risk to contracting 79 HIV and the possibility of developing resistance (16). The emergence of ARV resistance would 80 limit future treatment options in those patients treated with LA ARV NPs. However, 'targeting' HIV 81 prone cells is another strategy that is still under investigation.

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The broadly neutralizing antibodies (bNAbs) against HIV have emerged as a promising 83 strategy to protect or to treat circulating HIV (17). However, bNAbs for HIV is still a naïve field, as 84 the search is on-going to find Abs that have good neutralization potency (18). Eliminating the 85 circulating HIV is not sufficient to achieve "functional cure" against HIV, due to the presence of 86 latently HIV-infected cells. Furthermore, the reported bNAbs still need optimization in terms of 87 their effector function and plasma half-life (19)(20)(21). Therefore, the bNAbs strategy reveals that 88 antibody-based HIV therapy has two limiting factors, i.e., plasma concentration and neutralization 89 potency. Recently, studies have already confirmed the existence of resistant HIV-1 strains against 90 bNAbs (22,23), challenging the bNAbs clinical potency.

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Furthermore, combination ARV (cARV) therapy could suppress plasma viral load but has been 92 unsuccessful in immune restoration. Recovery from the viral infection needs reestablishment of 93 strong immunity. Therefore, research against HIV-1, faces challenges related to immune 94 reconstitution failure. Studies have revealed that the HIV infection induce terminal differentiation 95 of effector (E) CD8+ T cells to the memory phenotypes that causes progressive E population 96 reduction, immune exhaustion, and promote activation-induced cell death (24). HIV-infection 97 strongly compromises the immune system and immune impairment results in its inability to 98 respond to HIV and other pathogens; consequently, rapid progression to acquired 99 immunodeficiency syndrome (AIDS).

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However, ideally, the immune reconstitution could be promoted by stimulating both humoral 101 immune responses and cellular immune responses to prevent and control HIV infection, 102 respectively. The adaptive immune system plays the most critical role in protection against HIV-103 1 infection. Studies have shown that promoting T-cell-based immunity, specifically cytotoxic T 104 lymphocyte (CTL) stimulation (25,26), would promote effective production of neutralizing 105 antibodies, and would affect synergistically to protection against active and latent HIV-1 infection.

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Our hypothesis is a combination of HIV therapeutic strategies, i.e., ART and antibody-107 mediated HIV-entry blockage, would be an effective strategy to suppress HIV viremia, and 108 promote protective immune-response. The cumulative effect could potentially induce a "functional 109 cure." In this study, we propose an innovative approach to combine ART and CCR5 mAb in a 110 novel single nano-module to achieve dual protection against HIV infection. To achieve this, a 111 novel LA ARV loaded and anti-CCR5 mAbs surface decorated nanoparticles (NPs) have been 112 formulated ( Figure 1). The first level of protection comes from CCR5 mAb (decorated on the ARV 113 NP surface), that blocks HIV-1 entry in the CD4+ cells surface (i.e., CCR5+ CD4+ cells) similar to 114 the CCR5 antagonist. Wherein, internalization of CCR5/ CCR5 mAb-ARVs NP complex causes 115 release and maintaining intracellular ARV, providing the second level of protection again HIV 116 infection ( Figure 1). Our study shows the novel CCR5 mAb decorated ARV nanoformulation act 117 as a single delivery-system with dual-layers of defense that could prevent primary HIV-infection 118 as well as potentially induce a protective immunophenotype, to target and suppress HIV latency. 119 Therefore, a dual protection mechanism will prevent new HIV infection of the naive and latent 120 CCR5+ cell population, potentially achieving a "functional cure."

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The binding affinity of xfR5-D+T NPs with other CCR5+ immune cell types and HIV-prone 177 cells such as cytotoxic T-cells (CTLs, CD8+), dendritic T-cells (CD2+) and monocytes (CD68+), 178 were evaluated in similar a method (gating strategy, supplementary Figure 2). The comparative 179 binding affinity study demonstrated xfR5-D+T NPs had an enhanced binding affinity with memory 180 CD8+ T-cells, approximately 25 times higher affinity compared to naïve xfR5 mAb (Table 2). 181 Whereas xfR5-D+T NP binding with CD2+ T-cells and CD68+ T-cells illustrated slightly enhanced 182 but non-significant difference in binding affinity compared to naïve xfR5 mAb.

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The sustained release property of xfR5-D+T NP compared to D+T in solution was evaluated 185 by evaluating the intracellular uptake/release and retention kinetics based on the non-186 compartmental analysis using Phoenix WinNonlin 8.1 software (Table 3).

NP Solution Parameter
Units TAF TFV DTG TFVdp TAF TFV DTG TFVdp showed a non-significant difference for intracellular TFV and TFV-dp, between xfR5-D+T NP and 195 D+T solution treatment. The rationale behind this non-significant difference in the active-drug, 196 TFV and TFV-dp could be due to its dependence on cellular enzyme kinetics. The conversion of 197 TAF TFVTFV-dp intracellularly, is restricted due to acquired steady state. Thus, in the 198 absence of intracellular drug utilization, the cellular TFV and TFV-dp steady-state concentration 199 are maintained in both case of xfR5-D+T NP and D+T solution treatments. From the C max and 200 AUC all data of TAF and DTG, it is evident that the nanoformulation enhanced cellular uptake of 201 ARV compared to the same drugs in solution. In terms of retention, NP demonstrated 11.6 and 202 4.4 times higher TAF and DTG elimination half-life (t 1/2 ), than naïve drugs in solution, which is 203 indicative of improved retention kinetics.
208 nM) treatment demonstrated non-significant CC 50 differences (Table 4). The in vitro results on 217 TZM-bl cells and PBMCs suggest that nano-encapsulation reduces the toxic effect of DTG and 218 TAF as well as promotes cell viability.

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Therefore, the selectivity index (SI) evaluation significantly reflected the potential of xfR5-D+T 232 NPs (Table 4). In TZM-bl cells (all cells uniformly CCR5+ CD4+ cells), the xfR5-D+T NP improves 233 SI value by 5.5 and 4.5 times higher than individualized treatment by xfR5 NP and D+T NPs. 234 However, in primary PBMCs, xfR5-D+T NP demonstrated 2.7, 2.4, and 11 times higher SI value 235 compared to xfR5 NP, D+T NP, and naïve xfR5 mAb, respectively. These results showed xfR5 236 decorated D+T NP, significantly improved the therapeutic index of both individual therapeutic 237 approaches, i.e., xfR5 mAb and D+T NPs.

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Immunophenotype during in vitro short-term PrEP and HIV-1 treatment study

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Immunophenotype performed on PBMCs isolated from healthy donors to evaluate the 240 immunological potential of targeted ARV-loaded nanoformulation. The immune-differentiation 241 pattern of T-cells during PrEP and HIV-1 treatment was evaluated by flow cytometry after treating 242 uninfected PBMCs with xfR5-D+T NP or xfR5 mAb, along with respective controls. ). The immunophenotype of the T-cell subpopulations were determined over different time-points 249 and conditions, day before PHA activation (unstimulated T-cell population); PHA-activation (i.e., 250 one day after PHA-stimulated); HIV infection (i.e., one day after HIV infection); as well as one and 251 four days after xfR5-D+T NP or xfR5 mAb treatment ( Figure 3).

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The short-term PrEP (4 days) ex-vivo study, demonstrated xfR5-D+T NP significant protection 260 against HIV infection (Table 4), which in part could be attributed to a protective immunophenotype 261 ( Figure 3B). The xfR5-D+T NP resulted in increased CM, TM, and E sub-populations. However, 262 compared to PHA-activated (untreated), the xfR5-D+T NP demonstrated a significant boost in TM 263 and E sub-populations ( Figure 3B). The aCTLs and T h population demonstrated a reciprocal effect 264 ( Figure 4B). The aCTL sub-population after initial spike (day 1) followed a decline; in contrast, T h 265 population showed a gradual increase in overtime (day 4) ( Figure 4A, B).

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The ex-vivo HIV-1 infection and short-term treatment (4 days) study were performed to predict 267 the immunophenotypic differentiation pattern that the novel treatment could present ( Figure 3C).

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The study showed the presence of HIV infection, mainly influencing the naïve and CM sub-269 population ( Figure 3C). In the presence of HIV infection, the naïve sub-population after initial 270 increased (day 1, post-infection, PI) reverts to basal levels (day 4, after HIV infection), and the 271 CM population showed gradually increased population (day 4 PI). The xfR5-D+T NP treated 272 population also showed enhanced naïve and CM sub-population during the initial active HIV 273 infection stage (day 1 PI). In the long-term, however, the xfR5-D+T NP treated population 274 demonstrated a reverse effect in the CM and TM sub-population. Prolong xfR5-D+T NP treatment 275 (day 4, after treatment) of HIV-infected PBMCs (day 5 PI) displayed 2.4 times higher CM sub-276 population, as well as 6-times higher TM sub-population boost, compared to initial HIV infection. 277 Moreover, xfR5-D+T NP treatment also significantly induced increases in EM sub-population. The 278 xfR5-D+T NP treatment displayed significantly higher CM, EM, E, and T h sub-population 279 compared to xfR5 mAb and untreated controls. Therefore, these results demonstrate the 280 multimeric interaction of xfR5-D+T NP could potentially improve the CM TM  EM rate-limiting 281 steps that would help in maintaining a high EM population during possible HIV challenge.

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In this study, we have demonstrated a strategy to combine two different HIV treatments in a 284 single nanoformulation with sustained release property. To achieve this we formulated CCR5 285 targeting ARV loaded nanoformulation with the potential to block HIV entrance and promote 286 cellular immunity against HIV infection, specifically in memory CD4+ T-cells as they commit 287 themselves to provide antiviral immunity as latent HIV-1 reservoir (34). Alongside memory CD4+ 288 T-cells, the myeloid lineage, such as monocytes/macrophages, are believed to be other potential 289 HIV-1 sanctuaries (35, 36). Our hypothesis is the novel CCR5 targeted ARV nanoformulation 290 approach could combine different strategies to ensure dual protection against HIV to the CCR5+ 291 cell types. First, CCR5 receptor docking on the CCR5+ cells will block HIV entrance to prevent 292 HIV infection; and second, intracellular ARV released from endocytosed NP will providing 293 protection against HIV-1 intracellularly to the naïve cell or latent HIV-infected cell population 294 ( Figure 1). The novel nanoformulation will also boost anti-HIV immunity to contribute to the 295 possible "functional cure" strategies (37).

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To target and block CCR5 receptors, the nanoformulation was surface decorated with high-297 affinity CCR5 mAb (xfR5 mAb) and D+T ARV were encapsulated within the nanoformulation. The 298 w-o-w emulsion method produced uniform size xfR5-D+T NPs with a weak surface negative 299 charge. The enhanced electron density ( Figure 2B) and BCA protein quantification (Table 1) 300 validated covalent binding of xfR5 mAbs were attached to the xfR5-D+T NPs ( Figure 2C). 301 Besides, the presence of amide bond ( Figure 2C), and O-H stretching (3000-3500 cm-1) 302 conferred xfR5 mAbs are surface decorated with an ample amount of free surface on NP with 303 predominant PEG-PLGA composition. It is known that the onset of steric crowding compromises 304 the binding avidity during multimeric interaction (38). Therefore, the spare xfR5 mAb coverage 305 reduces the possibility of steric hindrance during multimeric xfR5 to CCR5-binding on the T-cells.

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The observed enhanced binding affinity of xfR5-D+T NP (  (Supplementary figure 1). 312 Therefore, based on the above theoretical assumption, the xfR5-D+T NP was expected to 313 elucidate 10-times higher affinity than the naive xfR5 mAb. However, practically xfR5-D+T NP 314 resulted in 7-times higher binding affinity compared to xfR5 mAb on TZM-bl cells.

335
The pronounced HIV protection efficacy of xfR5-D+T NP, as observed in the short-term PrEP 336 study (Table 4), could be attributed to the enhanced binding affinity (Table 2) as well as sustained 337 maintenance of intracellular ARV within xfR5-D+T NP treatment (Table 3). Further, the xfR5-D+T 338 NP dual protection, i.e., multimeric xfR5 induced CCR5 blocking and intracellular ARV mediated 339 HIV inhibition, boosted IC 50 value by 526 (TZM-bl cells) and 12 (PBMCs) times compared to xfR5 340 mAb (Table 4). The combination or dual protection of xfR5-D+T NP demonstrates more rigorous 341 protection compared to only blocking CCR5 receptors on T-cells by multimeric CCR5 T-cell 342 blocking, i.e., 5.5 times higher; and ARV induced HIV protection by treating with D+T NP alone, 343 i.e., 3.5 times compared to ARV by D+T NP alone. Further, it is well established (9, 10, 30, 31) 344 and our study also reflects (Table 4), that reduced cytotoxic effect and the increased protection 345 efficacy against HIV, widens the therapeutic index of xfR5-D+T NPs against HIV-1 virus. The 346 enhanced high therapeutic index, therefore, advocates the potency of xfR5-D+T NPs as an 347 advanced HIV therapeutic.

348
In general terms, the T-cell differentiation progresses in the following path: Naïve cells  CM over the entire study period ( Figure 3B). Therefore, the immunophenotype suggests that xfR5-354 D+T NP for PrEP application will keep the immune-system ready to promote fast clearance of the 355 virions upon HIV challenge.  (Table 4) reverted the naïve sub-population to its basal level (similar to untreated+unfected+non-362 activated condition, Figure 3A). Moreover, the declined naïve T-cell population and increased CM 363 sub-population could be due to differentiation induced population shifting from naïve  CM sub-364 population.

365
The memory T-cell population plays a vital role not only in promoting anti-HIV immunity but 366 also in AIDS prognosis since these cells govern the functions of CTLs and B-cells, which in turn 367 regulates cellular and humoral immunity against HIV (50). Persistent HIV-1 viremia is known to 368 drive the differentiation CM  EM sub-population (51, 52), and the high EM sub-population may 369 be responsible for the long-lasting and exhausted T-cell population in HIV+ patients (52). The 370 ART treatment effectively partially restores high CM sub-population and declines EM sub-371 population over time to rebalance the homeostasis disrupted during HIV infection (52). This study 372 shows as treatment progresses, xfR5-D+T NP treated population demonstrated significant 373 increased CM sub-population (day 4, after treatment), as well as atypical to ART treatment, also 374 significantly boosted TM and EM sub-population ( Figure 3C). During active infection (Day 1 PI), 375 targeted specific treatment (xfR5-D+T NP and naïve xfR5 mAb) maintained high E and aCTL sub-376 population, to counter HIV infection. Upon viral suppression (Table 4), E and aCTLs sub-377 population tend to decrease ( Figure 4B). Studies have shown that despite persistently low 378 antigenemia, high T h and E sub-population is essential to control HIV replication in the presence 379 or absence of ART to achieve "functional cure" (50, 53). However, long-term patients under ART-380 treatment results in the loss of effector functions. We observed HIV targeted treatment prolongs 381 high E ( Figure 3C) and T h ( Figure 4B) sub-population. Overall, the immunophenotypic study 382 displayed the xfR5-D+T NP multivalent interaction results in enhanced and improves protective 383 and defensive immunophenotype compared to naive xfR5 mAb treatment. Additionally, the 384 multivalent phenomenon also contributes to maintaining high TM and EM subpopulation. 385 Therefore, the immunophenotypic differentiation study indicated that targeted cARV NP could 386 also overcome the CM  TM  EM rate-limiting steps to promote high E and aCTL population 387 maintenance during the HIV challenge.

388
In summary, we formulated a dual-action targeted nanoformulation, i.e., xfR5-D+T NP. The 389 clinically relevant ex-vivo system (primary PBMCs) study showed xfR5-D+T NP induced CCR5 390 blocking and intracellular ARV drug release which provides two-levels of protection against fresh 391 HIV-1 infection or in latently infected HIV+ cells. Further, xfR5-D+T NP treatment also promotes 392 reprogramming of the immune repertoire function of memory T-cells and could help to reconstitute 393 anti-HIV immunity. This novel targeted ARV nanoformulation promotes protective immune 394 differentiation in T-cells. However, further investigations are essential to confirm the anti-HIV 395 immune efficacy of xfR5-D+T NP, especially with HIV+ patients. However, based on this study, 396 we conclude that xfR5-D+T NP combines the advantages of ART and augmentation of anti-HIV 397 immunity reconstitution could be a promising multifactorial strategy to target the complex HIV 398 latent reservoir to achieve HIV "functional cure." Materials and methods.

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The TZM-bl cell line obtained from the National Institutes of Health (NIH) acquired 419 immunodeficiency syndrome (AIDS) reagent program is a JC53-bl (clone 13)/HeLa cell line that 420 are phenotypic similar to HIV-1 infecting cell type (stably overexpresses CD4 and CCR5 receptor).

446
The targeted nanoformulation was fabricated by following multiple steps. First, NHS 447 functionalized D+T loaded nanoformulation was obtained by following the modified oil-in-water 448 emulsions phase inversion method (9, 10). Briefly, in the DCM organic phase PLGA, PLGA-PEG-449 NHS, PEG-PLGA, PF127 along with TAF and DTG have dissolved at 1:1:2:2:4:4 ratios, TAF (at 450 comparative ratio 2) in PBS were added dropwise under constant stirring condition. The water-in-451 oil (w-o) emulsion was sonicated as described below and then added dropwise to the three times 452 higher volume of 1 % PVA solution (aqueous phase) under high-speed stirring conditions. The 453 above w-o-w emulsion was immediately probe-sonicated for 5 mins on ice (setting: 90% 454 Amplitude; pulse 0.9 cycle/bursts) with the help of UP100H ultrasonic processor (Hielscher Inc. 455 Mount Holly, NJ, USA). The organic phase from the o-w emulsion was completely evaporated 456 overnight (O/N). The NHS functionalized D+T NPs were desiccated by lyophilization using 457 Millrock LD85 lyophilizer (Kingston, NY, USA) to eliminate the aqueous phase. The complete 458 formulation method was carried out under the hood to maintain sterility during fabrication.

459
The NHS functionalized D+T NPs, xfR5 mAb was conjugated using its amine group to were analyzed. By scanning electron microscopy, the morphology and shape of the D+T NPs 486 were evaluated (59). Briefly, D+T NPs were deposited on Whatman® Nuclepore Track-Etch 487 Membrane (~50 nm pore size) and air-dried for one day at RT under a chemical hood. The air-

488
dried NPs membrane was sputter-coated with a thin layer (~3-5 nm thick) of chromium and 489 imaged under a Hitachi S-4700 field-emission SEM (New York, NY, USA).

490
The % drug entrapment efficiency (%EE) of DTG and TAF in D+T NPs and xfR5-D+T NPs 491 were evaluated by high-performance liquid chromatography (HPLC) instrument by following 492 published methodology (30, 31, 60). Briefly, 1 mg of D+T NPs dissociated in 50 µL DMSO and 493 mobile phase (25mM KH 2 PO 4 45%: ACN 55%) added to get 10% DMSO final concentration in 494 the injection volume (20 µL). For the standard curve evaluation, the same procedure was followed 495 to prepare the D+T standard solutions (with each drug concentration from 0.5 to 0.0019 mg/mL). 496 The chromatography separation was performed under a HPLC instrument (Shimadzu Scientific 497 Instruments; MD, USA) equipped with SIL-20AC auto-sampler, LC-20AB pumps, and SPD-20A 498 UV/Visible detector, using Phenomenex® C-18 (150×4.6 mm, particle size 5 μm) column 499 (Torrance, CA, USA), under isocratic elution process with 0.5 mL/min mobile phase flow rate, 500 temperature: 25°C; and detection at 260 nm (retention time of 4 mins for TAF and 6.3 mins for 501 DTG). The quantification of the drug was determined by evaluating the peak area under the curve 502 (AUC) analysis at their respective retention time. The amount of TAF and DTG loaded in the D+T 503 NPs was analyzed based on the standard curve construction (linear correlation, r 2 ≥0.99) 504 respective from TAF, and DTG standard concentration ranges from 0.5 mg/mL to 0.0019 mg/mL. 505 The HPLC instrument illustrated inter-day and intra-day variability of <10%. The % encapsulation 506 efficiency (%EE) of each drug in the D+T NPs batch was estimated by equation 1, respectively. 507 The data presented as mean± standard error of the mean (SEM) of three D+T NPs batches (n=3). 508

510
Antibody binding and binding affinity evaluation

511
To establish and estimate the binding affinity of isolated xfR5 mAb and xfR5-D+T NPs in 512 comparison to wild-type CCR5 mAb, Cy3 conjugated to xfR5 mAb by Cy3 NHS Ester Mono-513 Reactive CyDye (GE Healthcare; PA, USA), following manufacturer's protocol. The Cy3 dye to 514 xfR5 mAb binding ratio was evaluated based on regression analysis of respective standards (i.e., 515 0.5 to 0.00625 mg/mL) data obtained from UV/vis spectroscopy and BCA assay. The 3:1 dye to 516 xfR5 mAb ratio Cy3 conjugated xfR5 mAb (Cy3-xfR5 mAb) batches considered for further studies. 517 To study binding affinity by flow-cytometry, Cy3-xfR5 mAb conjugated D+T NPs (Cy3-xfR5-D+T 518 NPs) formulation were fabricated and characterized, similarly as described for xfR5-D+T NPs. By 519 using the standardized formulation, three independent batches were obtained and evaluated for 520 further studies.
533 Table 5. Detailed information about different T lymphocyte phenotypic markers used for the 534 immunophenotypic study. Similarly, to evaluate binding affinity with the latent population (CD2+ T-cells) and monocytes 537 (CD68+ T-cells), PBMCs were treated as mentioned above. The above-treated cells incubated 538

T-lymphocyte target
for 20 mins with anti-CD68 APC mAb and anti-CD2 Pacific Blue (Table 5). The above marker 539 antibody bound treated cells were fixed for 20 min with 4% PFA at 4ºC and washed again twice 540 with PBA. The binding of Cy3-xfR5 mAb and Cy3-xfR5-D+T NPs to respective T-cell type was 541 detected and evaluated respectively by the BD LSRII flow cytometer instrument (BD Biosciences; 542 San Jose, CA, USA) and Flowjo software v10 (BD, Franklin Lakes, NJ, USA). The supplementary 543 figure 1 details the complete gating strategies. Each study mentioned above was performed on 544 three healthy independent donors PBMCs. The binding affinity was calculated based on 545 Michaelis-Menten's non-linear fitting analysis of mean ± SEM (standard errors of means).

547
Immunophenotype variation upon xfR5-D+T NPs treatment compared to xfR5 mAb in 548 uninfected (mimicking PrEP condition) and HIV-1 ADA -infected PHA-activated PBMCs was 549 evaluated by flow cytometry. Briefly, PBMCs (10 5 cells/well) were treated respectively with xfR5 550 mAb and xfR5-D+T NPs (at 20 µg/mL of xfR5 concentration) for 96 h at 37°C and 5% CO 2 551 atmosphere. As the control and to compare activated PBMCs immunophenotype, PHA-activated 552 PBMCs ( (Table 5), for 20 mins at RT (at 1:100 dilution). The marker-557 bound treated cells were washed with PBA, fixed for 20 min with 4% PFA at 4ºC, and rewashed 558 thrice with PBA. The immunophenotype of the markers bound treated cells were evaluated by 559 flow cytometry. Three independent studies have been performed on three healthy donor's 560 PBMCs. The data presented as mean ± SEM obtained from three independent donors.

561
Intracellular kinetics study

562
The intracellular uptake and retention kinetics of D+T NPs and D+T solution were 563 evaluated by LC-MS/MS analysis following a standardized method (9,30,62 the same method as explained above. The samples were analyzed using the LC-MS/MS method 578 described in the section below.

579
For the intracellular DTG, TAF, TFV, and TFV-dp drug-kinetics evaluation by LC-MS/MS 580 instrument, the respective cell lysates were centrifuged (14000 rpm for 5 mins at 4°C) and the 581 supernatant was collected. To an aliquot of 100 µL supernatant, 300 µL of internal standard 582 spiking solution (10 ng/mL each of DTG-d4, TAF-d5, TFV-d6, and 100 n/mL of TFV-dp-d6 in ACN) 583 was added, and vortexed. The samples were then dried at 45°C under the stream of nitrogen and 584 reconstituted with 100 µL 50% acetonitrile. The drug and metabolites were quantified from the 585 same sample using LC-MS/MS instrument.

586
For TAF, TFV, and DTG estimation, the similar conditions that were previously published 587 by our group were used with minor modification (62). One µL of the processed sample was 588 injected on to LC-MS/MS operated in positive mode. The chromatographic separation was 589 carried-out using the Restek Pinnacle DB Biph column (2.1 mm × 50 mm, 5 µm) with 0.5% formic 590 acid in water and 0.1% formic acid in ACN (48:52 v/v) mobile phase. The calibration range for all 591 the analytes was 0.01 to 50 ng.

592
For the quantification of TFV-dp, Phenomenex Kinetex C18 (75×4.6 mm, 2.6µm) column 593 was used with an isocratic mobile phase (10mM ammonium acetate pH 10.5: ACN (70:30) at a 594 flow rate of 0.25 mL/min. The dynamic calibration range was from 0.01 to 100 ng. The LC-MS/MS 595 system consisting of an Exion HPLC system (Applied Biosystems, CA, USA) coupled with AB

596
Sciex 5500 Q Trap with an electrospray ionization (ESI) source (Applied Biosystems, CA, USA) 597 was used in positive ionization mode. The retention time of TFV-dp was 2.1 min, and the runtime 598 for each sample was 3.5 min. The average inter-day and intra-day variability were < 15%, which 599 corresponds to the FDA bioanalytical guidelines (63).

600
In vitro cytotoxicity Study

601
The comparative in vitro cytotoxicity of D+T NP vs. D+T solution was evaluated on the 602 TZM-bl cell line using CellTiter-Glo® luminescent assay method, as described previously (64). 603 Briefly, the TZM-bl cells (10 4 cells/well) in complete HiDMEM medium and PBMCs (10 5  Where, 'CC 50 ' (cytotoxic concentration at 50%) and 'IC 50 ' (50% inhibitory concentration), 641 was evaluated from the above described in vitro cytotoxicity and protection study.  latently HIV+ cells, free intracellular high-affinity DTG (INSTI), will bind with fresh integrase 856 enzymes produced during the reactivation stage, resulting in a non-functional INSTI/integrase 857 complex. Therefore fresh virons thus produced will be with non-functional INSTIs. Therefore, fresh 858 viron will not be able to integrate viral DNA in the host genome. shows NH-band shifting from 1450 cm -1 to 1420 cm -1 indicating COO-NC bond conversion to 867 amide bond (-CONH-); and presence of Amide I and II at 1540 cm -1 and 1640 cm -1 respectively. on aCTL (left graph) and T h (right graph) on day 1, and day 4. Each data set is representing mean 882 ± SEM of three independent experiments on PBMCs obtained from 3 healthy donors (n=3). The 883

868
gating strategy for this study has been explained in Supplementary Figure 3. The data 884 represented in the graph were mean ± SEM of three independent studies on three healthy donors 885 (n=3). The significance represented as the asterisk (*) symbol, where, '*', and '****' corresponding 886 to P values <0.05. and <0.0001, respectively.