Aditya N. Bade

Worldwide implementation of antiretroviral therapy (ART) for pregnant women living with human immunodeficiency virus type-1 (HIV-1) has markedly reduced mother-to-child HIV-1 transmission rate to less than one percent. Despite this benefit, adverse pregnancy outcomes remain a major concern. Placental deficits induced by antiretroviral drugs (ARVs) are increasingly recognized as a potential causative factor. Additionally, comorbid effects of substance use on placenta cannot be overstated. Addictive substances are commonly used by people living with HIV-1 (PLWH). However, globally, nicotine exposure through smoking or tobacco use remains a major comorbidity in PLWH. Yet, to date, whether and how, HIV-1, ARVs, and nicotine together exacerbate adverse effects on placenta and affect birth outcomes is unknown. Due to ethical concerns and impracticality of obtaining placenta tissues at different stages of pregnancy, appropriate experimental models that closely recapitulate in vivo placenta physiology are essential to define how HIV-1, ART, and nicotine independently or collectively affect placental development and functions. Herein, we provide a comprehensive review of the experimental placenta models that have been or are yet to be utilized to study placental comorbid pathobiology induced by HIV-1 infection, ART, and/or nicotine exposure. We emphasize how each model is used (or underused) to interrogate infection biology, pharmacokinetic profile, and toxico-pharmacologic mechanisms. Moreover, key limitations associated with each placenta model and necessary improvements for investigating HIV-1 and associated comorbidity are highlighted. Overall, this review seeks to educate both clinical and basic scientists on advances made, and remaining challenges in understanding comorbid effects of HIV-1-ART-nicotine on pregnancy outcomes using experimental placenta models.

Advances in antiretroviral drugs (ARVs) have achieved remarkable success in preventing gestational human immunodeficiency virus type 1 (HIV-1) transmission from mother to fetus. This is reflected in the rising numbers of HIV-1-exposed uninfected (HEU) children. Worldwide, the number of HEU children exceeds sixteen million, with more than one million children joining this group each year. Although HEU children remain uninfected, they are at an increased risk of neurodevelopmental deficits. Notably, in utero exposure to HIV-1 and ARVs is a causative factor. Both are linked to adverse neurodevelopment, warranting close clinical monitoring and therapeutic intervention. We now demonstrate that chemical exchange saturation transfer (CEST) MRI can be used to successfully monitor in utero ARV-exposure-associated embryo brain metabolomic and macromolecular dysregulations in a mouse model. CEST hyperintensities at −3.5 ppm (nuclear Overhauser effect) and 3.5 ppm (amide/amine protons) are measured in the brains of mouse embryos exposed to dolutegravir (DTG). These reflect DTG-induced alterations in cellular membrane lipids, mobile proteins or peptides, and glutamate levels. All demonstrate impaired neuronal development. Non-targeted metabolomics confirms the CEST results. These support the observations of DTG-induced differential expression of lipids and metabolites that reflect deficits in energy production, cell metabolism, post-translational protein modifications, and transport pathways. Furthermore, CEST MRI demonstrated the therapeutic benefits of long-acting nanoformulation delivery of DTG in mitigating neurodevelopmental impairments. These data, taken together, support the utility of CEST MRI as a non-invasive imaging biomarker for detecting neurodevelopmental deficits.

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.

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 ofin uteroantiretroviral 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.

Children born to mothers, with or at risk, of human immunodeficiency virus type-1 (HIV-1) infection are on the rise due to affordable access of antiretroviral therapy (ART) to pregnant women or those of childbearing age. Each year, up to 1.3 million HIV-1-infected women on ART have given birth with recorded mother-to-child HIV-1 transmission rates of less than 1%. Despite this benefit, the outcomes of children exposed to antiretroviral drugs during pregnancy, especially pre- and post- natal neurodevelopment remain incompletely understood. This is due, in part, to the fact that pregnant women are underrepresented in clinical trials. This is underscored by any potential risks of neural tube defects (NTDs) linked, in measure, to periconceptional usage of dolutegravir (DTG). A potential association between DTG and NTDs was first described in Botswana in 2018. Incidence studies of neurodevelopmental outcomes associated with DTG, and other integrase strand transfer inhibitors (INSTIs) are limited as widespread use of INSTIs has begun only recently in pregnant women. Therefore, any associations between INSTI use during pregnancy, and neurodevelopmental abnormalities remain to be explored. Herein, United States Food and Drug Administration approved ARVs and their use during pregnancy are discussed. We provide updates on INSTI pharmacokinetics and adverse events during pregnancy together with underlying mechanisms which could affect fetal neurodevelopment. Overall, this review seeks to educate both clinical and basic scientists on potential consequences of INSTIs on fetal outcomes as a foundation for future scientific investigations.

Dolutegravir (DTG) is a first-line antiretroviral drug (ARV) used in combination therapy for the treatment of human immunodeficiency virus type-1 (HIV-1) infection. The drug is effective, safe, and well tolerated. Nonetheless, concerns have recently emerged for its usage in pregnant women or those of child-bearing age. Notably, DTG-based ARV regimens have been linked to birth defects seen as a consequence of periconceptional usages. To this end, uncovering an underlying mechanism for DTG-associated adverse fetal development outcomes has gained clinical and basic research interest. We now report that DTG inhibits matrix metalloproteinases (MMPs) activities that could affect fetal neurodevelopment. DTG is a broad-spectrum MMPs inhibitor and binds to Zn⁺⁺ at the enzyme’s catalytic domain. Studies performed in pregnant mice show that DTG readily reaches the fetal central nervous system during gestation and inhibits MMP activity. Postnatal screenings of brain health in mice pups identified neuroinflammation and neuronal impairment. These abnormalities persist as a consequence of in utero DTG exposure. We conclude that DTG inhibition of MMPs activities during gestation has the potential to affect prenatal and postnatal neurodevelopment.

Strain specific mouse brain magnetic resonance imaging (MRI) atlases provide coordinate space linked anatomical registration. This allows longitudinal quantitative analyses of neuroanatomical volumes and imaging metrics for assessing the role played by aging and disease to the central nervous system. As NOD/scid-IL-2Rγ _c ^null (NSG) mice allow human cell transplantation to study human disease, these animals are used to assess brain morphology. Manganese enhanced MRI (MEMRI) improves contrasts amongst brain components and as such can greatly help identifying a broad number of structures on MRI. To this end, NSG adult mouse brains were imaged in vivo on a 7.0 Tesla MR scanner at an isotropic resolution of 100 μm. A population averaged brain of 19 mice was generated using an iterative alignment algorithm. MEMRI provided sufficient contrast permitting 41 brain structures to be manually labeled. Volumes of 7 humanized mice brain structures were measured by atlas-based segmentation and compared against non-humanized controls. The humanized NSG mice brain volumes were smaller than controls (p < 0.001). Many brain structures of humanized mice were significantly smaller than controls. We posit that the irradiation and cell grafting involved in the creation of humanized mice were responsible for the morphological differences. Six NSG mice without MnCl₂ administration were scanned with high resolution T₂-weighted MRI and segmented to test broad utility of the atlas.