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Porcine astrovirus (PAstV) has been associated experimentally with diarrhea in piglets, but much more knowledge is needed about this virus. PAstV has high genetic variability, and 5 genotypes have been identified, namely PAstV1–5. To obtain information on the epidemiology of PAstV, we established a multiplex PAstV PCR assay to detect and differentiate the 5 PAstV genotypes simultaneously. The assay utilized specific primers for each genotype, producing fragments of 307, 353, 205, 253, and 467 bp, representing PAstV1–5, respectively. Our multiplex PCR assay amplified all 5 DNA fragments from single or mixed viral genomes without cross-reactions with other PAstV genotypes or other viruses in pigs. The limit of detection of the multiplex PCR assay was 5 × 102 copies/μL for PAstV1 and PAstV4, and 5 × 103 copies/μL for PAstV2, PAstV3, and PAstV5. We examined 76 pig fecal specimens with our multiplex PCR assay. PAstV was detected in 36 of 76 (47.4%) samples; ≥2 PAstVs were found in 20 of 76 (26.3%) samples. The multiplex PCR assay results were essentially the same as the results using a monoplex PAstV PCR assay, with a coincidence rate of >96%. Our multiplex PCR method provides a simple, sensitive, and specific detection tool for PAstV detection and epidemiologic surveys.

Human breastmilk is rich in T cells; however, their specificity and function are largely unknown. We compared the phenotype, diversity, and antigen specificity of T cells in the breastmilk and peripheral blood of lactating individuals who received SARS-CoV-2 mRNA vaccination. Relative to blood, breastmilk contained higher frequencies of T effector and central memory populations that expressed mucosal-homing markers. T cell receptor (TCR) sequence overlap was limited between blood and breastmilk. Overabundan t breastmilk clones were observed in all individuals, were diverse, and contained CDR3 sequences with known epitope specificity including to SARS-CoV-2 Spike. Spike-specific TCRs were more frequent in breastmilk compared to blood and expanded in breastmilk following a third mRNA vaccine dose. Our observations indicate that the lactating breast contains a distinct T cell population that can be modulated by maternal vaccination with potential implications for infant passive protection.One-Sentence SummaryThe breastmilk T cell repertoire is distinct and enriched for SARS-CoV-2 Spike-specificity after maternal mRNA vaccination.

Viral pathogens associated with diarrhea in pigs include porcine circovirus 2 (PCV2), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (RVA) and C (RVC) among others. In this study, a novel universal primer-based pentaplex PCR (UP-M-PCR) assay was developed for simultaneous detection and differentiation of these five viruses. The assay uses a short-cycle multiplex amplification by chimeric primers (CP), which are virus specific, with a tail added at the 5' end of the universal primer (UP), followed by universal amplification using UPs and a regular cycle amplification. Five universal primers with CPs (UP1-5) were designed and evaluated in an UP-based single PCR (UP-S-PCR). All five UPs were found to work efficiently and UP2 exhibited the best performance. After system optimizations, the analytical sensitivity of the UP-M-PCR, using plasmids containing the specific viral target fragments, was 5 copies/reaction for each of the five viruses irrespective of presence of a single or multiple viruses in the template. No cross-reaction was observed with other non-target viruses. When 273 fecal samples from clinically healthy pigs were tested, the assay sensitivity was 90.9-100%, the specificity was 98.0-100%, and the agreement rate with the UP-S-PCR was 98.5-99.6% with a Kappa value being 0.95-0.98. In summary, the UP-M-PCR developed here is a rapid and highly sensitive and specific detection method that can be used to demonstrate mixed infections in pigs with diarrhea.

BACKGROUNDCoronavirus disease 2019 (COVID-19) is associated with endothelial activation and coagulopathy, which may be related to pre-existing or infection-induced pro-thrombotic autoantibodies such as those targeting angiotensin II type I receptor (AT1R-Ab).METHODSWe compared prevalence and levels of AT1R-Ab in COVID-19 cases with mild or severe disease to age and sex matched negative controls.RESULTSThere were no significant differences between cases and controls. However, there were trends toward a higher proportion with AT1R-Ab positivity among severe cases versus controls (32% vs. 11%, p=0.1) and higher levels in those with mild COVID-19 compared to controls (median 9.5U/mL vs. 5.9U/mL, p=0.06).CONCLUSIONSThese findings suggest that AT1R-Ab are not consistently associated with COVID-19 but do not exclude a contribution to endothelial pathology in a subset of people.

The fate of protective immunity following mild SARS-CoV-2 infection remains ill defined. Here we characterize antibody responses in a cohort of participants recovered from mild SARS-CoV-2 infection with follow up to 6 months. We measure IgA, IgM, and IgG binding and avidity to viral antigens and assess neutralizing antibody responses over time. Further, we correlate the effect of fever, gender, age, and time since symptom onset with antibody responses. We observe that total anti-S trimer, anti-RBD, and anti-NP IgG are relatively stable over 6 months of follow-up and anti-S and anti-RBD avidity increases over time, and that fever is associated with higher levels of antibodies. However, neutralizing antibody responses rapidly decay and are strongly associated with declines in IgM levels. Thus, while total antibody against SARS-CoV-2 may persist, functional antibody, particularly IgM, is rapidly lost. These observations have implications for the duration of protective immunity following mild SARS-CoV-2 infection., Graphical Abstract, Harrington et al., report that in adults with mild COVID-19, IgG responses are maintained for over six months and are correlated with fever. Neutralization activity rapidly decays, correlating with the loss of anti-S-trimer IgM titers. Thus, neutralizing antibodies fade quickly after mild COVID-19 infection, despite the long-term maintenance of IgG.

Polymeric nanocarriers have been extensively used to improve the delivery of hydrophobic drugs, but often provide low encapsulation efficiency and percent loading for hydrophilic compounds. In particular, insufficient loading of hydrophilic antiretroviral drugs such as the integrase inhibitor raltegravir (RAL) has limited the development of sustained-release therapeutics or prevention strategies against HIV. To address this, we developed a generalizable prodrug strategy using RAL as a model where loading, release and subsequent hydrolysis can be tuned by promoiety selection. Prodrugs with large partition coefficients increased the encapsulation efficiency up to 25-fold relative to RAL, leading to significant dose reductions in antiviral activity assays. The differential hydrolysis rates of these prodrugs led to distinct patterns of RAL availability and observed antiviral activity. We also developed a method to monitor the temporal distribution of both prodrug and RAL in cells treated with free prodrug or prodrug-NPs. Results of these studies indicated that prodrug-NPs create an intracellular drug reservoir capable of sustained intracellular drug release. Overall, our results suggest that the design of prodrugs for specific polymeric nanocarrier systems could provide a more generalized strategy to formulate physicochemically diverse hydrophilic drugs with a number of biomedical applications.

High throughput serological tests that can establish the presence and functional activity of anti-SARS-COV2 antibodies are urgently needed. Here we present microsphere-based Flow Cytometry assays that quantify both anti-spike IgGs in plasma, and the ability of plasma to inhibit the binding of spike protein to angiotensin converting enzyme 2 (ACE2). First, we detected anti-spike-trimer IgGs in 22/24 and anti-spike-receptor-binding-domain (RBD) IgGs in 21/24 COVID+ subjects at a median of 36 (range 14-73) days following documented SARS-CoV-2 RNA (+) secretions. Next, we find that plasma from all 22/24 subjects with anti-trimer IgGs inhibited ACE2-trimer binding to a greater degree than controls, and that the degree of inhibition correlated with anti-trimer IgG levels. Depletion of trimer-reactive Igs from plasma reduced ACE2-trimer inhibitory capacity to a greater degree than depletion of RBD-reactive Igs, suggesting that inhibitory antibodies act by binding both within and outside of the RBD. Amongst the 24 subjects, presence of fever was associated with higher levels of anti-trimer IgG and inhibition of binding to human ACE2. This inhibition assay may be broadly useful to quantify the functional antibody response of recovered COVID19 patients or vaccine recipients in a cell-free assay system.

Monoclonal antibodies and peptides are conjugated to the surface of nanocarriers (NCs) for targeting purposes in numerous applications. However, targeting efficacy may vary with their specificity, affinity, or avidity when linked to NCs. The physicochemical properties of NCs may also affect targeting. We compared the targeting efficacy of the CD4 binding peptide BP4 and an anti-CD4 monoclonal antibody (CD4 mAb) and its fragments, when conjugated to lipid-coated poly(lactic-co-glycolic) acid nanoparticles (LCNPs). Negatively charged LCNPs with cholesteryl butyrate in the lipid layer (cbLCNPs) dramatically reduced nonspecific binding, leading to higher targeting specificity, compared to neutral or positively charged LCNPs with DOTAP (dtLCNP). cbLCNPs surface conjugated with a CD4 antibody (CD4-cbLCNPs) or its fragments (fCD4-cbLCNPs), but not BP4, showed high binding in vitro to the human T cell line 174xCEM, and preferential binding to CD3+ CD14-CD8- cells from pigtail macaque peripheral blood mononuclear cells. CD4-cbLCNPs showed 10-fold higher binding specificity for CD4+ than CD8+ T cells, while fCD4-cbLCNPs demonstrated the highest binding level overall, but only three-fold higher binding specificity. This study demonstrates the importance of ζ-potential on NC targeting and indicates that CD4 mAb and its fragments are the best candidates for delivery of therapeutic agents to CD4+ T cells. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1177-1188, 2018.

Here, we present the current challenges in women’s reproductive health and the current state-of-the-art treatment and prevention options for STI prevention, contraception, and treatment of infections. We discuss how the versatile platform of electrospun fibers can be applied to each challenge, and postulate at how these technologies could be improved. The void of approved electrospun fiber-based products yields the potential to apply this useful technology to a number of medical applications, many of which are relevant to women’s reproductive health. Given the ability to tune drug delivery characteristics and three-dimensional geometry, there are many opportunities to pursue new product designs and routes of administration for electrospun fibers. For each application, we provide an overview of the versatility of electrospun fibers as a novel dosage form and summarize their advantages in clinical applications. We also provide a perspective on why electrospun fibers are well suited for a variety of applications within women’s reproductive health, and identify areas that could greatly benefit from innovations with electrospun fiber-based approaches.

Electrospun fibers show potential as a topical delivery system for vaginal microbicides. Previous reports have demonstrated delivery of anti-HIV and anti-STI (sexually transmitted infection) agents from fibers formulated using hydrophilic, hydrophobic, or pH-responsive polymers that result in rapid, prolonged, or stimuli-responsive release, respectively. However, coaxial electrospun fibers have yet to be evaluated as a highly tunable microbicide delivery vehicle. In this research, we explored the opportunities and limitations of a model coaxial electrospun fiber system to provide broad and tunable release rates for the HIV entry inhibitor maraviroc. Specifically, we prepared ethyl cellulose (EC)-shell and polyvinylpyrrolidone (PVP)-core fibers that were capable of releasing actives over a range of hours to several days. We further demonstrated simple and effective methods for combining core-shell fibers with rapid-release formulations to provide combined instantaneous and sustained maraviroc release. In addition, we investigated the effect of varying release media on maraviroc release from core-shell fibers, and found that release was strongly influenced by media surface tension and drug ionization. Finally, in vitro cell culture studies show that our fiber formulations were not cytotoxic and that electrospun maraviroc maintained similar antiviral activity compared to neat maraviroc.

In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube. Five distinct melt peaks were obtained with different melting temperature (Tm) value corresponding to each of the five viruses. This method was highly sensitive to detect and distinguish TGEV, RVA, RVC, PEDV and PCV2 with the limits of detection ranging from 5 to 50 copies/μL. The intra-assay and inter-assay reproducibility were good with coefficient of variation of Tm and cycle threshold values less than 0.32% and 2.86%, respectively. Testing of 90 field samples by the single and multiplex real-time PCR assays demonstrated a concordance of 91.1%. Thus, the EvaGreen multiplex real-time PCR is a rapid, sensitive and low-cost diagnostic tool for differential detection and routine surveillance of TGEV, RVA, RVC, PEDV and PCV2 in pigs.

Targeted nanocarriers deliver HIV-1 latency-reversing agents to CD4+ T cells in the lymph nodes., A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified, but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety, providing sustained drug release, and simultaneously delivering multiple drugs to target tissues and cells. Here, we formulated hybrid nanocarriers that incorporate physicochemically diverse LRAs and target lymphatic CD4+ T cells. We identified one LRA combination that displayed synergistic latency reversal and low cytotoxicity in a cell model of HIV and in CD4+ T cells from virologically suppressed patients. Furthermore, our targeted nanocarriers selectively activated CD4+ T cells in nonhuman primate peripheral blood mononuclear cells as well as in murine lymph nodes, and substantially reduced local toxicity. This nanocarrier platform may enable new solutions for delivering anti-HIV agents for an HIV cure.

Porcine viral diarrhea, mainly caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine group A rotaviruses (RVA), porcine group C rotaviruses (RVC) and porcine circovirus 2 (PCV2), is a serious global problem, resulting in substantial economic losses to the swine industry. For fast and reliable diagnosis of the causative agent associated with viral diarrhea in pigs, an inexpensive and easy to perform gel-based multiplex PCR assay was developed in this study to detect and differentiate the different viruses by amplicon size. The assay was able to distinguish between all five viral agents without cross-reacting with other non-target pig viruses. The detection limits of the assay per reaction were 5 copies for PEDV, TGEV, RVC and PCV2 and 50 copies for RVA for the singleplex assays and 50 copies when all five viruses were multiplexed. Sixty-nine field samples were used to validate the developed multiplex assay. The overall prevalence of positive samples was 44.9% (31/69). PCV2 was detected in 37.7% of the samples, PEDV and RVC each in 4.3%, TGEV in 2.9%, and RVA was detected in 1.4% of the samples tested. A total of 5.8% of the samples were co-infected by two or more viruses, and the results of the multiplex assay were in agreement to those obtained by single PCR assays. These findings suggest that the developed cost-effective multiplex assay is specific, sensitive, and will serve as a valuable diagnostic tool for the rapid differential detection of these five viruses and for molecular epidemiological studies and diarrhea disease management.

A major sanctuary site for HIV infection is the gut-associated lymphoid tissue (GALT). The α4β7 integrin gut homing receptor is a promising therapeutic target for the virus reservoir because it leads to migration of infected cells to the GALT and facilitates HIV infection. Here, we developed a core-shell nanoparticle incorporating the α4β7 monoclonal antibody (mAb) as a dual-functional ligand for selectively targeting a protease inhibitor (PI) to gut-homing T cells in the GALT while simultaneously blocking HIV infection. Our nanoparticles significantly reduced cytotoxicity of the PI and enhanced its in vitro antiviral activity in combination with α4β7 mAb. We demonstrate targeting function of our nanocarriers in a human T cell line and primary cells isolated from macaque ileum, and observed higher in vivo biodistribution to the murine small intestines where they accumulate in α4β7+ cells. Our LCNP shows the potential to co-deliver ARVs and mAbs for eradicating HIV reservoirs.

Monoclonal antibodies and peptides are conjugated to the surface of nanocarriers (NCs) for targeting purposes in numerous applications. However, targeting efficacy may vary with their specificity, affinity, or avidity when linked to NCs. The physicochemical properties of NCs may also affect targeting. We compared the targeting efficacy of the CD4 binding peptide BP4 and an anti-CD4 monoclonal antibody (CD4 mAb) and its fragments, when conjugated to lipid-coated poly(lactic-co-glycolic) acid nanoparticles (LCNPs). Negatively charged LCNPs with cholesteryl butyrate in the lipid layer (cbLCNPs) dramatically reduced nonspecific binding, leading to higher targeting specificity, compared to neutral or positively charged LCNPs with DOTAP (dtLCNP). cbLCNPs surface conjugated with a CD4 antibody (CD4-cbLCNPs) or its fragments (fCD4-cbLCNPs), but not BP4, showed high binding in vitro to the human T cell line 174xCEM, and preferential binding to CD3+ CD14-CD8- cells from pigtail macaque peripheral blood mononuclear cells. CD4-cbLCNPs showed 10-fold higher binding specificity for CD4+ than CD8+ T cells, while fCD4-cbLCNPs demonstrated the highest binding level overall, but only three-fold higher binding specificity. This study demonstrates the importance of ζ-potential on NC targeting and indicates that CD4 mAb and its fragments are the best candidates for delivery of therapeutic agents to CD4+ T cells. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1177-1188, 2018.

Self-assembling peptides programed by sequence design to form predefined nanostructures are useful for a variety of biomedical applications. However, assemblies of classic ionic self-complementary peptides are unstable in neutral pH, while charged peptide hydrogels have low mechanical strength. Here, we report on the rational design of a self-assembling peptide system with optimized charge distribution and density for bioscaffold development. Our designer peptides employs a sequence pattern that undergoes salt triggered self-assembly into β-sheet rich cationic nanofibers in the full pH range (pH 0–14). Our peptides form nanofibrils in physiological condition at a minimum concentration that is significantly lower than has been reported for self-assembly of comparable peptides. The robust fiber-forming ability of our peptides results in the rapid formation of hydrogels in physiological conditions with strong mechanical strength. Moreover, fiber structure is maintained even upon dense conjugation with a model bioactive cargo OVA 257-264 peptide. Nanofibers carrying OVA 257-264 significantly enhanced CD8 + T cell activation in vitro. Subcutaneous immunization of our peptide fiber vaccine also elicited robust CD8 + T cell activation and proliferation in vivo. Our self-assembling peptides are expected to provide a versatile platform to construct diverse biomaterials. Statement of Significance This work is an attempt of rational design of materials from molecular level for targeted properties and an exploration in molecular self-assembly. Current widely studied self-assembling peptides do not have stable nanofiber structures and form weak hydrogels under physiological conditions. To address this issue, we develop charged self-assembling peptides with a novel sequence pattern for strong fiber-forming ability under physiological conditions. Our designer peptides can undergo salt-triggered self-assembly into nanofibers that are ultrastable in extreme pH (0–14) and dilute solutions, and into hydrogels with strong mechanical strength. Upon conjugation with a model bioactive cargo, our self-assembled peptides exhibit great potential as bioscaffolds for multiple applications.

Composite delivery systems where drugs are electrospun in different layers and vary the drug stacking-order are posited to affect bioavailability. We evaluated how the formulation characteristics of both burst- and sustained-release electrospun fibers containing three physicochemically diverse drugs: dapivirine (DPV), maraviroc (MVC) and tenofovir (TFV) affect in vitro and ex vivo release. We developed a poly(hydroxyethyl methacrylate) (pHEMA) hydrogel release platform for the rapid, inexpensive in vitro evaluation of burst- and sustained-release topical or dermal drug delivery systems with varying microarchitecture. We investigated properties of the hydrogel that could recapitulate ex vivo release into nonhuman primate vaginal tissue. Using a dimethyl sulfoxide extraction protocol and high-performance liquid chromatography analysis, we achieved >93% recovery from the hydrogels and >88% recovery from tissue explants for all three drugs. We found that DPV loading, but not stacking order (layers of fiber containing a single drug) or microarchitecture (layers with isolated drug compared to all drugs in the same layer) impacted the burst release in vitro and ex vivo. Our burst-release formulations showed a correlation for DPV accumulation between the hydrogel and tissue (R2= 0.80), but the correlation was not significant for MVC or TFV. For the sustained-release formulations, the PLGA/PCL content did not affect TFV release in vitro or ex vivo. Incorporation of cells into the hydrogel matrix improved the correlation between hydrogel and tissue explant release for TFV. We expect that this hydrogel-tissue mimic may be a promising preclinical model to evaluate topical or transdermal drug delivery systems with complex microarchitectures.

Concurrent infection of pigs with two or more pathogens is common in pigs under intensive rearing conditions. Porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV) and pseudorabies virus (PRV) are all associated with reproductive or respiratory disorders or both and can cause significant economic losses in pig production worldwide. An EvaGreen-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed in this study for simultaneous detection and differentiation of these six viruses in pigs. This method is able to detect and distinguish PCV2, PPV, PRRSV, CSFV, JEV and PRV with the limits of detection ranging from 100 to 500 copies/μL, high reproducibility, and intra-assay and inter-assay variation ranging from 0.11 to 3.20%. After validation, a total of 118 field samples were tested by the newly developed EG-mPCR. PCV2 was identified in 23%, PPV in 15%, PRRSV in 17% and PRV in 5% of the samples. Concurrent PCV2 and PRRSV infection was detected in 6.7%, PCV2 and PPV in 5% and PPV2 and PRRSV infection was detected in 5% of the cases. The agreement of the EG-mPCR and conventional PCR tests was 99.2%. This EG-mPCR will be a useful, rapid, reliable and cost-effective alternative for routine surveillance testing of viral infections in pigs.

Several novel porcine bocaviruses (PBoVs) have been identified in pigs in recent years and association of these viruses with respiratory signs or diarrhea has been suggested. In this study, an EvaGreen(®)-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed for simultaneous detection and grouping of novel PBoVs into the same genogroups G1, G2 and G3. Each target produced a specific amplicon with a melting peak of 81.3 ± 0.34 °C for PBoV G1, 78.2 ± 0.37 °C for PBoV G2, and 85.0 ± 0.29 °C for PBoV G3. Non-specific reactions were not observed when other pig viruses were used to assess the EG-mPCR assay. The sensitivity of the EG-mPCR assay using purified plasmid constructs containing the specific viral target fragments was 100 copies for PBoV G1, 50 for PBoV G2 and 100 for PBoV G3. The assay is able to detect and distinguish three PBoV groups with intra-assay and inter-assay variations ranging from 0.13 to 1.59%. The newly established EG-mPCR assay was validated with 227 field samples from pigs. PBoV G1, G2 and G3 was detected in 15.0%, 25.1% and 41.9% of the investigated samples and coinfections of two or three PBoV groups were also detected in 25.1% of the cases, indicating that all PBoV groups are prevalent in Chinese pigs. The agreement of the EG-mPCR assay with an EvaGreen-based singleplex real-time PCR (EG-sPCR) assay was 99.1%. This EG-mPCR will serve as a rapid, sensitive, reliable and cost effective alternative for routine surveillance testing of multiple PBoVs in pigs and will enhance our understanding of the epidemiological features and possible also pathogenetic changes associated with these viruses in pigs.

Etravirine (ETR), maraviroc (MVC) and raltegravir (RAL) are promising antiretroviral drugs being used in HIV treatment and may be interesting for prevention applications such as oral or topical pre-exposure prophylaxis. Here we describe a sensitive and accurate method for the simultaneous detection of ETR, MVC and RAL from pigtail macaque plasma, vaginal secretions, and vaginal tissue. This method is characterized by a straightforward precipitation extraction method, a limit of quantification 94% in all cases. We also show that extracted biological samples are stable after storage at room temperature or 4 °C and after three freeze/thaw cycles. This is the first analytical method capable of quantifying ETR, MVC and RAL in biological matrices relevant for pre-clinical testing of oral or topical HIV prevention methods in pigtailed macaques.

Nanocarrier-based drug delivery systems are playing an emerging role in human immunodeficiency virus (HIV) chemoprophylaxis and treatment due to their ability to alter the pharmacokinetics and improve the therapeutic index of various antiretroviral (ARV) drug compounds used alone and in combination. Although several nanocarriers have been described for combination delivery of ARV drugs, measurement of drug-drug activities facilitated by the use of these nanotechnology platforms has not been fully investigated for topical prevention. Here, we show that physicochemically diverse ARV drugs can be encapsulated within polymeric nanoparticles to deliver multidrug combinations that provide potent HIV chemoprophylaxis in relevant models of cell-free, cell-cell, and mucosal tissue infection. In contrast to existing approaches that coformulate ARV drug combinations together in a single nanocarrier, we prepared single-drug-loaded nanoparticles that were subsequently combined upon administration. ARV drug-nanoparticles were prepared using emulsion-solvent evaporation techniques to incorporate maraviroc (MVC), etravirine (ETR), and raltegravir (RAL) into poly(lactic-co-glycolic acid) (PLGA) nanoparticles. We compared the antiviral potency of the free and formulated drug combinations for all pairwise and triple drug combinations against both cell-free and cell-associated HIV-1 infection in vitro. The efficacy of ARV-drug nanoparticle combinations was also assessed in a macaque cervicovaginal explant model using a chimeric simian-human immunodeficiency virus (SHIV) containing the reverse transcriptase (RT) of HIV-1. We observed that our ARV-NPs maintained potent HIV inhibition and were more effective when used in combinations. In particular, ARV-NP combinations involving ETR-NP exhibited significantly higher antiviral potency and dose-reduction against both cell-free and cell-associated HIV-1 BaL infection in vitro. Furthermore, ARV-NP combinations that showed large dose-reduction were identified to be synergistic, whereas the equivalent free-drug combinations were observed to be strictly additive. Higher intracellular drug concentration was measured for cells dosed with the triple ARV-NP combination compared to the equivalent unformulated drugs. Finally, as a first step toward evaluating challenge studies in animal models, we also show that our ARV-NP combinations inhibit RT-SHIV virus propagation in macaque cervicovaginal tissue and block virus transmission by migratory cells emigrating from the tissue. Our results demonstrate that ARV-NP combinations control HIV-1 transmission more efficiently than free-drug combinations. These studies provide a rationale to better understand the role of nanocarrier systems in facilitating multidrug effects in relevant cells and tissues associated with HIV infection.

Nanocarriers are versatile vehicles for drug delivery, and emerging as platforms to formulate and deliver multiple classes of antiretroviral (ARV) drugs in a single system. Here we describe the fabrication of hydrogel-core and lipid-shell nanoparticles (nanolipogels) for the controlled loading and topical, vaginal delivery of maraviroc (MVC) and tenofovir disoproxil fumarate (TDF), two ARV drugs with different mechanisms of action that are used in the treatment of HIV. The nanolipogel platform was used to successfully formulate MVC and TDF, which produced ARV drug-loaded nanolipogels that were characterized for their physical properties and antiviral activity against HIV-1 BaL in cell culture. We also show that administration of these drug carriers topically to the vaginal mucosa in a murine model leads to antiviral activity against HIV-1 BaL in cervicovaginal lavages. Our results suggest that nanolipogel carriers are promising for the encapsulation and delivery of hydrophilic small molecule ARV drugs, and may expand the nanocarrier systems being investigated for HIV prevention or treatment.Topical, mucosal intervention of HIV is a leading strategy in the efforts to curb the spread of viral infection. A significant research thrust in the field has been to characterize different dosage forms for formulation of physicochemically diverse antiretroviral drugs. Nanocarriers have been used to formulate and deliver small molecule and protein drugs for a range of applications, including ARV drugs for HIV treatment. The broad significance of our work includes evaluation of lipid-shell, hydrogel-core nanoparticles for formulation and topical, vaginal delivery of two water-soluble antiretroviral drugs. We have characterized these nanocarriers for their physical properties and their biological activity against HIV-1 infection in vitro, and demonstrated the ability to deliver drug-loaded nanocarriers in vivo.

Sustained release of small molecule hydrophilic drugs at high doses remains difficult to achieve from electrospun fibers and limits their use in clinical applications. Here we investigate tunable release of several water-soluble anti-HIV drugs from electrospun fibers fabricated with blends of two biodegradable polyesters. Drug-loaded fibers were fabricated by electrospinning ratios of PCL and PLGA. Fiber morphology was imaged by SEM, and DSC was used to measure thermal properties. HPLC was used to measure drug loading and release from fibers. Cytotoxicity and antiviral activity of drug-loaded fibers were measured in an in vitro cell culture assay. We show programmable release of hydrophilic antiretroviral drugs loaded up to 40 wt%. Incremental tuning of highly-loaded drug fibers within 24 h or >30 days was achieved by controlling the ratio of PCL and PLGA. Fiber compositions containing higher PCL content yielded greater burst release whereas fibers with higher PLGA content resulted in greater sustained release kinetics. We also demonstrated that our drug-loaded fibers are safe and can sustain inhibition of HIV in vitro. These data suggest that we were able to overcome current limitations associated with sustained release of small molecule hydrophilic drugs at clinically relevant doses. We expect that our system represents an effective strategy to sustain delivery of water-soluble molecules that will benefit a variety of biomedical applications.