CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice.

Treatment of HIV-1ADA-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4+ T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.

Diagnostics for SARS-CoV-2 infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every corner of the globe, causing societal instability. The resultant coronavirus disease 2019 (COVID-19) leads to fever, sore throat, cough, chest and muscle pain, dyspnoea, confusion, anosmia, ageusia and headache. These can progress to life-threatening respiratory insufficiency, also affecting the heart, kidney, liver and nervous systems. The diagnosis of SARS-CoV-2 infection is often confused with that of influenza and seasonal upper respiratory tract viral infections. Due to available treatment strategies and required containments, rapid diagnosis is mandated. This Review brings clarity to the rapidly growing body of available and in-development diagnostic tests, including nanomaterial-based tools. It serves as a resource guide for scientists, physicians, students and the public at large.

HIV-Tat Exacerbates the Actions of Atazanavir, Efavirenz, and Ritonavir on Cardiac Ryanodine Receptor (RyR2).

The incidence of sudden cardiac death (SCD) in people living with HIV infection (PLWH), especially those with inadequate viral suppression, is high and the reasons for this remain incompletely characterized. The timely opening and closing of type 2 ryanodine receptor (RyR2) is critical for ensuring rhythmic cardiac contraction-relaxation cycles, and the disruption of these processes can elicit Ca2+ waves, ventricular arrhythmias, and SCD. Herein, we show that the HIV protein Tat (HIV-Tat: 0-52 ng/mL) and therapeutic levels of the antiretroviral drugs atazanavir (ATV: 0-25,344 ng/mL), efavirenz (EFV: 0-11,376 ng/mL), and ritonavir (RTV: 0-25,956 ng/mL) bind to and modulate the opening and closing of RyR2. Abacavir (0-14,315 ng/mL), bictegravir (0-22,469 ng/mL), Rilpivirine (0-14,360 ng/mL), and tenofovir disoproxil fumarate (0-18,321 ng/mL) did not alter [3H]ryanodine binding to RyR2. Pretreating RyR2 with low HIV-Tat (14 ng/mL) potentiated the abilities of ATV and RTV to bind to open RyR2 and enhanced their ability to bind to EFV to close RyR2. In silico molecular docking using a Schrodinger Prime protein-protein docking algorithm identified three thermodynamically favored interacting sites for HIV-Tat on RyR2. The most favored site resides between amino acids (AA) 1702-1963; the second favored site resides between AA 467-1465, and the third site resides between AA 201-1816. Collectively, these new data show that HIV-Tat, ATV, EFV, and RTV can bind to and modulate the activity of RyR2 and that HIV-Tat can exacerbate the actions of ATV, EFV, and RTV on RyR2. Whether the modulation of RyR2 by these agents increases the risk of arrhythmias and SCD remains to be explored.

Humanized Mice for Infectious and Neurodegenerative disorders.

Humanized mice model human disease and as such are used commonly for research studies of infectious, degenerative and cancer disorders. Recent models also reflect hematopoiesis, natural immunity, neurobiology, and molecular pathways that influence disease pathobiology. A spectrum of immunodeficient mouse strains permit long-lived human progenitor cell engraftments. The presence of both innate and adaptive immunity enables high levels of human hematolymphoid reconstitution with cell susceptibility to a broad range of microbial infections. These mice also facilitate investigations of human pathobiology, natural disease processes and therapeutic efficacy in a broad spectrum of human disorders. However, a bridge between humans and mice requires a complete understanding of pathogen dose, co-morbidities, disease progression, environment, and genetics which can be mirrored in these mice. These must be considered for understanding of microbial susceptibility, prevention, and disease progression. With known common limitations for access to human tissues, evaluation of metabolic and physiological changes and limitations in large animal numbers, studies in mice prove important in planning human clinical trials. To these ends, this review serves to outline how humanized mice can be used in viral and pharmacologic research emphasizing both current and future studies of viral and neurodegenerative diseases. In all, humanized mouse provides cost-effective, high throughput studies of infection or degeneration in natural pathogen host cells, and the ability to test transmission and eradication of disease.

A year-long extended release nanoformulated cabotegravir prodrug.

Long-acting cabotegravir (CAB) extends antiretroviral drug administration from daily to monthly. However, dosing volumes, injection site reactions and health-care oversight are obstacles towards a broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18 and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics and biodistribution. Pharmacokinetics studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. NM2CAB, compared with NMCAB and NM3CAB, demonstrated a prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg per kg body weight intramuscular injection. These prodrug modifications could substantially improve CAB's effectiveness.

EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment.

Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication.

Synthesis and Characterization of Long-Acting Darunavir Prodrugs.

Antiretroviral therapy (ART) has improved the quality of life in patients infected with HIV-1. However, complete viral suppression within anatomical compartments remains unattainable. This is complicated by adverse side effects and poor adherence to lifelong therapy leading to the emergence of viral drug resistance. Thus, there is an immediate need for cellular and tissue-targeted long-acting (LA) ART formulations. Herein, we describe two LA prodrug formulations of darunavir (DRV), a potent antiretroviral protease inhibitor. Two classes of DRV prodrugs, M1DRV and M2DRV, were synthesized as lipophilic and hydrophobic prodrugs and stabilized into aqueous suspensions designated NM1DRV and NM2DRV. The formulations demonstrated enhanced intracellular prodrug levels with sustained drug retention and antiretroviral activities for 15 and 30 days compared to native DRV formulation in human monocyte-derived macrophages. Pharmacokinetics tests of NM1DRV and NM2DRV administered to mice demonstrated sustained drug levels in blood and tissues for 30 days. These data, taken together, support the idea that LA DRV with sustained antiretroviral responses through prodrug nanoformulations is achievable.

A long-acting 3TC ProTide nanoformulation suppresses HBV replication in humanized mice.

Nowadays, there is a strong request for the treatment of chronic HBV-infection with direct acting antivirals. Furthermore, prevalent human immunodeficiency virus (HIV-1) and hepatitis B (HBV) co-infections highlight an immediate need for dual long-acting and easily administered antivirals. To this end, we modified lamivudine (3TC), a nucleoside analog inhibitor of both viruses, into a lipophilic monophosphorylated prodrug (M23TC). Prior work demonstrated that nanoformulation of M23TC (NM23TC) enhanced drug stability, controlled dissolution and improved access to sites of viral replication. The present study evaluated the efficacy of a NM23TC in HBV-infected chimeric liver humanized mice. Levels of HBV DNA and HBsAg in plasma were monitored up to 8 weeks posttreatment. A single intramuscular dose of 75 mg/kg 3TC equivalents of nanoformulated NM23TC provided sustained drug levels and suppressed HBV replication in humanized mice for 4 weeks. The results support further development of this long-acting 3TC nanoformulation for HBV treatment and prevention.

Pharmacokinetics of a Long-Acting Nanoformulated Dolutegravir Prodrug in Rhesus Macaques.

A nanoformulated myristoylated dolutegravir prodrug (NMDTG) was prepared using good laboratory practice protocols. Intramuscular injection of NMDTG (118 ± 8 mg/ml, 25.5 mg of DTG equivalents/kg of body weight) to three rhesus macaques led to plasma DTG levels of 86 ± 12 and 28 ± 1 ng/ml on days 35 and 91, respectively. The NMDTG platform showed no significant adverse events. Further modification may further extend the drug's apparent half-life for human use.

A mature macrophage is a principal HIV-1 cellular reservoir in humanized mice after treatment with long acting antiretroviral therapy.

BACKGROUND: Despite improved clinical outcomes seen following antiretroviral therapy (ART), resting CD4+ T cells continue to harbor latent human immunodeficiency virus type one (HIV-1). However, such cells are not likely the solitary viral reservoir and as such defining where and how others harbor virus is imperative for eradication measures. To such ends, we used HIV-1ADA-infected NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice reconstituted with a human immune system to explore two long-acting ART regimens investigating their abilities to affect viral cell infection and latency. At 6 weeks of infection animals were divided into four groups. One received long-acting (LA) cabotegravir (CAB) and rilpivirine (RVP) (2ART), a second received LA CAB, lamivudine, abacavir and RVP (4ART), a third were left untreated and a fourth served as an uninfected control. After 4 weeks of LA ART treatment, blood, spleen and bone marrow (BM) cells were collected then phenotypically characterized. CD4+ T cell subsets, macrophages and hematopoietic progenitor cells were analyzed for HIV-1 nucleic acids by droplet digital PCR. RESULTS: Plasma viral loads were reduced by two log10 or to undetectable levels in the 2 and 4ART regimens, respectively. Numbers and distributions of CD4+ memory and regulatory T cells, macrophages and hematopoietic progenitor cells were significantly altered by HIV-1 infection and by both ART regimens. ART reduced viral DNA and RNA in all cell and tissue compartments. While memory cells were the dominant T cell reservoir, integrated HIV-1 DNA was also detected in the BM and spleen macrophages in both regimen-treated mice. CONCLUSION: Despite vigorous ART regimens, HIV-1 DNA and RNA were easily detected in mature macrophages supporting their potential role as an infectious viral reservoir.

Long-acting antituberculous therapeutic nanoparticles target macrophage endosomes.

Eradication of Mycobacterium tuberculosis (MTB) infection requires daily administration of combinations of rifampin (RIF), isoniazid [isonicotinylhydrazine (INH)], pyrazinamide, and ethambutol, among other drug therapies. To facilitate and optimize MTB therapeutic selections, a mononuclear phagocyte (MP; monocyte, macrophage, and dendritic cell)-targeted drug delivery strategy was developed. Long-acting nanoformulations of RIF and an INH derivative, pentenyl-INH (INHP), were prepared, and their physicochemical properties were evaluated. This included the evaluation of MP particle uptake and retention, cell viability, and antimicrobial efficacy. Drug levels reached 6 µg/10(6) cells in human monocyte-derived macrophages (MDMs) for nanoparticle treatments compared with 0.1 µg/10(6) cells for native drugs. High RIF and INHP levels were retained in MDM for >15 d following nanoparticle loading. Rapid loss of native drugs was observed in cells and culture fluids within 24 h. Antimicrobial activities were determined against Mycobacterium smegmatis (M. smegmatis). Coadministration of nanoformulated RIF and INHP provided a 6-fold increase in therapeutic efficacy compared with equivalent concentrations of native drugs. Notably, nanoformulated RIF and INHP were found to be localized in recycling and late MDM endosomal compartments. These were the same compartments that contained the pathogen. Our results demonstrate the potential of antimicrobial nanomedicines to simplify MTB drug regimens.

Polymer Delivery Systems for Long-Acting Antiretroviral Drugs.

The success of long-acting (LA) drug delivery systems (DDSs) is linked to their biocompatible polymers. These are used for extended therapeutic release. For treatment or prevention of human immune deficiency virus type one (HIV-1) infection, LA DDSs hold promise for improved regimen adherence and reduced toxicities. Current examples include Cabenuva, Apretude, and Sunlenca. Each is safe and effective. Alternative promising DDSs include implants, prodrugs, vaginal rings, and microarray patches. Each can further meet patients' needs. We posit that the physicochemical properties of the formulation chemical design can optimize drug release profiles. We posit that the strategic design of LA DDS polymers will further improve controlled drug release to simplify dosing schedules and improve regimen adherence.

Synthesis of azide derivative and discovery of glyoxalase pathway inhibitor against pathogenic bacteria.

A glyoxalase inhibitor was synthesized and tested against Staphylococcus aureus for first time and showed MIC90 of 20 µg/ml. Henceforth, we synthesized unnatural azide derivative of the same inhibitor to improve the biological activity. In that order, an azide carboxylate was synthesized from dimethyl tartrate by tosylation and azide substitution. The synthesized, azide compound was coupled with glutathione derivative in high yield and tested against S. aureus and showed improved MIC90 of 5 µg/ml. In general, it can be also easily converted to unnatural ß-amino acid in good yield. The shown methodology will be extended to study induced suicide in Burkholderia mallei, Francisella tularensis and Mycobacterium tuberculosis in future.

Macrophage folate receptor-targeted antiretroviral therapy facilitates drug entry, retention, antiretroviral activities and biodistribution for reduction of human immunodeficiency virus infections.

Macrophages serve as vehicles for the carriage and delivery of polymer-coated nanoformulated antiretroviral therapy (nanoART). Although superior to native drug, high drug concentrations are required for viral inhibition. Herein, folate-modified ritonavir-boosted atazanavir (ATV/r)-encased polymers facilitated macrophage receptor targeting for optimizing drug dosing. Folate coating of nanoART ATV/r significantly enhanced cell uptake, retention and antiretroviral activities without altering cell viability. Enhanced retentions of folate-coated nanoART within recycling endosomes provided a stable subcellular drug depot. Importantly, up to a five-fold enhanced plasma and tissue drug levels followed folate-coated formulation injection in mice. Folate polymer encased ATV/r improves nanoART pharmacokinetics bringing the technology one step closer to human use. FROM THE CLINICAL EDITOR: This team of authors describes a novel method for macrophage folate receptor-targeted antiretroviral therapy. Atazanvir entry, retention, and antiretroviral activities were superior using the presented method, and so was its biodistribution, enabling a more efficient way to address human immunodeficiency virus infections, with a hoped for clinical application in the near future.

Inhibition of matrix metalloproteinases by HIV-1 integrase strand transfer inhibitors.

More than fifteen million women with the human immunodeficiency virus type-1 (HIV-1) infection are of childbearing age world-wide. Due to improved and affordable access to antiretroviral therapy (ART), the number of in utero antiretroviral drug (ARV)-exposed children has exceeded a million and continues to grow. While most recommended ART taken during pregnancy suppresses mother to child viral transmission, the knowledge of drug safety linked to fetal neurodevelopment remains an area of active investigation. For example, few studies have suggested that ARV use can be associated with neural tube defects (NTDs) and most notably with the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). After risk benefit assessments, the World Health Organization (WHO) made recommendations for DTG usage as a first and second-line preferred treatment for infected populations including pregnant women and those of childbearing age. Nonetheless, long-term safety concerns remain for fetal health. This has led to a number of recent studies underscoring the need for biomarkers to elucidate potential mechanisms underlying long-term neurodevelopmental adverse events. With this goal in mind, we now report the inhibition of matrix metalloproteinases (MMPs) activities by INSTIs as an ARV class effect. Balanced MMPs activities play a crucial role in fetal neurodevelopment. Inhibition of MMPs activities by INSTIs during neurodevelopment could be a potential mechanism for adverse events. Thus, comprehensive molecular docking testing of the INSTIs, DTG, bictegravir (BIC), and cabotegravir (CAB), against twenty-three human MMPs showed broad-spectrum inhibition. With a metal chelating chemical property, each of the INSTI were shown to bind Zn++ at the MMP's catalytic domain leading to MMP inhibition but to variable binding energies. These results were validated in myeloid cell culture experiments demonstrating MMP-2 and 9 inhibitions by DTG, BIC and CAB and even at higher degree than doxycycline (DOX). Altogether, these data provide a potential mechanism for how INSTIs could affect fetal neurodevelopment.

Advances in long-acting slow effective release antiretroviral therapies for treatment and prevention of HIV infection.

Adherence to daily oral antiretroviral therapy (ART) is a barrier to both treatment and prevention of human immunodeficiency virus (HIV) infection. To overcome limitations of life-long daily regimen adherence, long-acting (LA) injectable antiretroviral (ARV) drugs, nanoformulations, implants, vaginal rings, microarray patches, and ultra-long-acting (ULA) prodrugs are now available or in development. These medicines enable persons who are or at risk for HIV infection to be treated with simplified ART regimens. First-generation LA cabotegravir, rilpivirine, and lenacapavir injectables and a dapivirine vaginal ring are now in use. However, each remains limited by existing dosing intervals, ease of administration, or difficulties in finding drug partners. ULA ART regimens provide an answer, but to date, such next-generation formulations remain in development. Establishing the niche will require affirmation of extended dosing, improved access, reduced injection volumes, improved pharmacokinetic profiles, selections of combination treatments, and synchronization of healthcare support. Based on such needs, this review highlights recent pharmacological advances and a future treatment perspective. While first-generation LA ARTs are available for HIV care, they remain far from ideal in meeting patient needs. ULA medicines, now in advanced preclinical development, may close gaps toward broader usage and treatment options.

Chronic Hepatitis B Infection: New Approaches towards Cure.

Chronic hepatitis B virus (HBV) infection leads to the development of cirrhosis and hepatocellular carcinoma. Lifelong treatment with nucleotides/nucleoside antiviral agents is effective at suppressing HBV replication, however, adherence to daily therapy can be challenging. This review discusses recent advances in the development of long-acting formulations for HBV treatment and prevention, which could potentially improve adherence. Promising new compounds that target distinct steps of the virus life cycle are summarized. In addition to treatments that suppress viral replication, curative strategies are focused on the elimination of covalently closed circular DNA and the inactivation of the integrated viral DNA from infected hepatocytes. We highlight promising long-acting antivirals and genome editing strategies for the elimination or deactivation of persistent viral DNA products in development.

Long-acting dolutegravir formulations prevent neurodevelopmental impairments in a mouse model.

The World Health Organization has recommended dolutegravir (DTG) as a preferred first-line treatment for treatment naive and experienced people living with human immunodeficiency virus type one (PLWHIV). Based on these recommendations 15 million PLWHIV worldwide are expected to be treated with DTG regimens on or before 2025. This includes pregnant women. Current widespread use of DTG is linked to the drug's high potency, barrier to resistance, and cost-effectiveness. Despite such benefits, potential risks of DTG-linked fetal neurodevelopmental toxicity remain a concern. To this end, novel formulation strategies are urgently needed in order to maximize DTG's therapeutic potentials while limiting adverse events. In regard to potential maternal fetal toxicities, we hypothesized that injectable long-acting nanoformulated DTG (NDTG) could provide improved safety by reducing drug fetal exposures compared to orally administered native drug. To test this notion, we treated pregnant C3H/HeJ mice with daily oral native DTG at a human equivalent dosage (5 mg/kg; n = 6) or vehicle (control; n = 8). These were compared against pregnant mice injected with intramuscular (IM) NDTG formulations given at 45 (n = 3) or 25 (n = 4) mg/kg at one or two doses, respectively. Treatment began at gestation day (GD) 0.5. Magnetic resonance imaging scanning of live dams at GD 17.5 was performed to obtain T1 maps of the embryo brain to assess T1 relaxation times of drug-induced oxidative stress. Significantly lower T1 values were noted in daily oral native DTG-treated mice, whereas comparative T1 values were noted between control and NDTG-treated mice. This data reflected prevention of DTG-induced oxidative stress when delivered as NDTG. Proteomic profiling of embryo brain tissues harvested at GD 17.5 demonstrated reductions in oxidative stress, mitochondrial impairments, and amelioration of impaired neurogenesis and synaptogenesis in NDTG-treated mice. Pharmacokinetic (PK) tests determined that both daily oral native DTG and parenteral NDTG achieved clinically equivalent therapeutic plasma DTG levels in dams (4,000-6,500 ng/mL). Importantly, NDTG led to five-fold lower DTG concentrations in embryo brain tissues compared to daily oral administration. Altogether, our preliminary work suggests that long-acting drug delivery can limit DTG-linked neurodevelopmental deficits.