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Abstract Background Continued efforts are being directed toward the development of microbicides that will be used to reduce or eliminate the risk of HIV-1 sexual transmission. Unfortunately, clinical trials involving polyanion-containing microbicide formulations, including Carraguard (λ-carrageenan [LC]) and Ushercell (cellulose sulfate [CS]) demonstrated that these products were ineffective and may have, in some circumstances, increased the risk of HIV-1 infection. These findings prompted reassessments of the in vitro activities of these agents to determine whether variables that can affect agent safety and efficacy had been overlooked during preclinical testing. One such variable is product retention and loss following topical application. Results In the present studies involving an HIV-1-susceptible cell line and primary human immune cells, product loss was mimicked by introducing and then removing polyanionic compounds prior to HIV-1 infection. In these in vitro "washout" experiments, LC and CS significantly enhanced HIV-1 infection, despite potent antiviral activity when introduced simultaneously with the virus. The presence and magnitude of this effect were dependent on compound identity and concentration; target cell; interval between compound removal and virus challenge; and coreceptor usage. Levels of enhancement (relative to controls) were considerable, exceeding a 200% increase (CS) in P4-R5 MAGI cells and a 300% increase (LC) in human peripheral blood mononuclear cells. Conclusions These studies, which demonstrate significant increases in HIV-1 infection subsequent to application and removal of LC and CS, support plausible explanations for the failures of microbicides formulated from these compounds. Detailed studies are now underway to determine the mechanism responsible for this enhancement effect and to assess the potential contribution of this effect to the clinical failures of these agents.

Abstract Background Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a virion-associated regulatory protein that functions at several points within the viral life cycle and has been shown to accumulate primarily in the nucleus and at the nuclear envelope. However, most studies have investigated Vpr localization employing cell types irrelevant to HIV-1 pathogenesis. To gain a better understanding of how cellular phenotype might impact HIV-1 Vpr intracellular localization, Vpr localization was examined in several cell lines representing major cellular targets for HIV-1 infection within the peripheral blood, bone marrow, and central nervous system (CNS). Results Utilizing a green fluorescent protein-tagged Vpr, we detected Vpr mainly in foci inside the nucleus, at the nuclear envelope, and around the nucleoli, with dispersed accumulation in the cytoplasm of human endothelial kidney 293T cells. No differences were observed in Vpr localization pattern with respect to either the location of the tag (N- or C-terminus) or the presence of other viral proteins. Subsequently, the Vpr localization pattern was explored in two primary HIV-1 target cells within the peripheral blood: the CD4+ T lymphocyte (represented by the Jurkat CD4+ T-cell line) and the monocyte-macrophage (represented by the U-937 cell line). Vpr was found primarily in speckles within the cytoplasm of the Jurkat T cells, whereas it accumulated predominantly intranuclearly in U-937 monocytic cells. These patterns differ from that observed in a bone marrow progenitor cell line (TF-1), wherein Vpr localized mainly at the nuclear envelope with some intranuclear punctuate staining. Within the CNS, we examined two astroglioma cell lines and found that Vpr displayed a perinuclear and cytoplasmic distribution. Conclusions The results suggest that the pattern of Vpr localization depends on cellular phenotype, probably owing to interactions between Vpr and cell type-specific host factors. These interactions, in turn, are likely coupled to specific roles that Vpr plays in each cell type within the context of the viral life cycle. Phenotype-specific Vpr localization patterns might also provide an explanation with respect to Vpr secretion or release from HIV-1-infected cells within the peripheral blood and CNS.

Abstract Human immunodeficiency virus type 1 (HIV-1) has been shown to replicate productively in cells of the monocyte-macrophage lineage, although replication occurs to a lesser extent than in infected T cells. As cells of the monocyte-macrophage lineage become differentiated and activated and subsequently travel to a variety of end organs, they become a source of infectious virus and secreted viral proteins and cellular products that likely initiate pathological consequences in a number of organ systems. During this process, alterations in a number of signaling pathways, including the level and functional properties of many cellular transcription factors, alter the course of HIV-1 long terminal repeat (LTR)-directed gene expression. This process ultimately results in events that contribute to the pathogenesis of HIV-1 infection. First, increased transcription leads to the upregulation of infectious virus production, and the increased production of viral proteins (gp120, Tat, Nef, and Vpr), which have additional activities as extracellular proteins. Increased viral production and the presence of toxic proteins lead to enhanced deregulation of cellular functions increasing the production of toxic cellular proteins and metabolites and the resulting organ-specific pathologic consequences such as neuroAIDS. This article reviews the structural and functional features of the cis-acting elements upstream and downstream of the transcriptional start site in the retroviral LTR. It also includes a discussion of the regulation of the retroviral LTR in the monocyte-macrophage lineage during virus infection of the bone marrow, the peripheral blood, the lymphoid tissues, and end organs such as the brain. The impact of genetic variation on LTR-directed transcription during the course of retrovirus disease is also reviewed.

Abstract Background Reducing transmission of HIV-1 through breast milk is needed to help decrease the burden of pediatric HIV/AIDS in society. We have previously reported that alkyl sulfates (i.e., sodium dodecyl sulfate, SDS) are microbicidal against HIV-1 at low concentrations, are biodegradable, have little/no toxicity and are inexpensive. Therefore, they may be used for treatment of HIV-1 infected breast milk. In this report, human milk was artificially infected by adding to it HIV-1 (cell-free or cell-associated) and treated with ≤1% SDS (≤10 mg/ml). Microbicidal treatment was at 37°C or room temperature for 10 min. SDS removal was performed with a commercially available resin. Infectivity of HIV-1 and HIV-1 load in breast milk were determined after treatment. Results SDS (≥0.1%) was virucidal against cell-free and cell-associated HIV-1 in breast milk. SDS could be substantially removed from breast milk, without recovery of viral infectivity. Viral load in artificially infected milk was reduced to undetectable levels after treatment with 0.1% SDS. SDS was virucidal against HIV-1 in human milk and could be removed from breast milk if necessary. Milk was not infectious after SDS removal. Conclusion The proposed treatment concentrations are within reported safe limits for ingestion of SDS by children of 1 g/kg/day. Therefore, use of alkyl sulfate microbicides, such as SDS, to treat HIV1-infected breast milk may be a novel alternative to help prevent/reduce transmission of HIV-1 through breastfeeding.

Human immunodeficiency virus-associated neurocognitive disorders persist in the combination antiretroviral therapy era. CD4 nadir is a well-established predictor of cognition cross-sectionally, but its impact on longitudinal neurocognitive (NC) trajectories is unclear. The few studies on this topic examined trajectories of global cognition, rather than specific NC domains. The current study examined CD4 nadir in relation to domain-specific NC decline. 132 HIV + adults from the Temple/Drexel Comprehensive NeuroHIV Center, Clinical and Translational Research Support Core Cohort were administered comprehensive NC assessments longitudinally, with last visit occurring an average of 12 years after CD4 nadir. Linear mixed models were used to examine CD4 nadir in relation to longitudinal NC trajectories in three empirically identified NC domains: speed/executive function (S/EF), visuospatial memory (VM), and verbal fluency (VF). CD4 nadir was associated with change in VF (p = 0.020), but not with S/EF or VM. Specifically, those with CD4 nadir < 200 demonstrated increasing VF over time (p = .002), whereas those with CD4 nadir > 200 demonstrated stable VF (p = .568), though these differing trajectories may partly reflect regression to the mean or differential practice effect. CD4 dynamics over time were analyzed as potential mechanisms for the identified associations, with mixed findings. While low CD4 nadir has been associated with weaker neurocognition among people living with HIV, the results of this study suggest that low CD4 nadir is not associated with ongoing decline a decade later. Nadir-related deficits in VF may be stable or even improve over time, possibly reflecting the beneficial cognitive effects of long-term treatment and immune reconstitution., (© 2024. The Author(s).)

Human immunodeficiency virus type 1 (HIV-1) infection is well known as one of the most complex and difficult viral infections to cure. The difficulty in developing curative strategies arises in large part from the development of latent viral reservoirs (LVRs) within anatomical and cellular compartments of a host. The clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system shows remarkable potential for the inactivation and/or elimination of integrated proviral DNA within host cells, however, delivery of the CRISPR/Cas9 system to infected cells is still a challenge. In this review, the main factors impacting delivery, the challenges for delivery to each of the LVRs, and the current successes for delivery to each reservoir will be discussed., Competing Interests: The authors declare that the work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gurrola, Effah, Sariyer, Dampier, Nonnemacher and Wigdahl.)

Purpose of Review: The leading gene editing strategy for a human immunodeficiency virus type 1 (HIV-1) cure involves the delivery of SaCas9 and two guide RNAs (gRNAs) in an adeno-associated viral (AAV) vector. As a dual-component system, CRISPR is targeted to a genetic locus through the choice of a Cas effector and gRNA protospacer design pair. As CRISPR research has expanded in recent years, these components have been investigated for utilization in cure strategies, which will be discussed in this article., Recent Findings: Type II SpCas9 and SaCas9 have been the leading Cas effectors across gene editing therapeutics to date. Additionally, extensive research has expanded the potential to multiplex gRNAs and target them effectively to the highly genetically diverse HIV-1 provirus. More recently, the Type V family of Cas12 effectors opens a new opportunity to use a smaller Cas protein for packaging into an AAV vector with multiplexed gRNAs., Summary: In understanding the individual components of a CRISPR/Cas therapeutic cure for HIV-1, it is important to know that the currently used strategies can be improved upon. Future areas will include alternative smaller Cas effectors, multiplexed gRNAs designs, and/or alternative delivery modalities., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Herpes simplex virus type 1 (HSV-1) is a lifelong pathogen characterized by asymptomatic latent infection in the trigeminal ganglia (TG), with periodic outbreaks of cold sores caused by virus reactivation in the TG and subsequent replication in the oral mucosa. While antiviral therapies can provide relief from cold sores, they are unable to eliminate HSV-1. We provide experimental results that highlight non-thermal plasma (NTP) as a new alternative therapy for HSV-1 infection that would resolve cold sores faster and reduce the establishment of latent infection in the TG. Additionally, this study is the first to explore the use of NTP as a therapy that can both treat and prevent human viral infections. The antiviral effect of NTP was investigated using an in vitro model of HSV-1 epithelial infection that involved the application of NTP from two separate devices to cell-free HSV-1, HSV-1-infected cells, and uninfected cells. It was found that NTP reduced the infectivity of cell-free HSV-1, reduced viral replication in HSV-1-infected cells, and diminished the susceptibility of uninfected cells to HSV-1 infection. This triad of antiviral mechanisms of action suggests the potential of NTP as a therapeutic agent effective against HSV-1 infection., Competing Interests: The authors declare no conflicts of interest.

Background: Lyme disease is caused by the bacteria Borreliella burgdorferi sensu lato (Bb) transmitted to humans from the bite of an infected Ixodes tick. Current diagnostics for Lyme disease are insensitive at the early disease stage and they cannot differentiate between active infections and people with a recent history of antibiotic-treated Lyme disease., Methods: Machine learning technology was utilized to improve the prediction of acute Lyme disease and identify sialic acid and galactose sugar structures (N-glycans) on immunoglobulins associated specifically at time points during acute Lyme disease time. A plate-based approach was developed to analyze sialylated N-glycans associated with anti-Bb immunoglobulins. This multiplexed approach quantitates the abundance of Bb-specific IgG and the associated sialic acid, yielding an accuracy of 90% in a powered study., Findings: It was demonstrated that immunoglobulin sialic acid levels increase during acute Lyme disease and following antibiotic therapy and a 3-month convalescence, the sialic acid level returned to that found in healthy control subjects (p < 0.001). Furthermore, the abundance of sialic acid on Bb-specific IgG during acute Lyme disease impaired the host's ability to combat Lyme disease via lymphocytic receptor FcγRIIIa signaling. After enzymatically removing the sialic acid present on Bb-specific antibodies, the induction of cytotoxicity from acute Lyme disease patient antigen-specific IgG was significantly improved., Interpretation: Taken together, Bb-specific immunoglobulins contain increased sialylation which impairs the host immune response during acute Lyme disease. Furthermore, this Bb-specific immunoglobulin sialyation found in acute Lyme disease begins to resolve following antibiotic therapy and convalescence., Funding: Funding for this study was provided by the Coulter-Drexel Translational Research Partnership Program as well as from a Faculty Development Award from the Drexel University College of Medicine Institute for Molecular Medicine and Infectious Disease and the Department of Microbiology and Immunology., Competing Interests: Declaration of interests B.H.G. and M.A.C. hold patent App# PCT/US2022/047442 filed on October 21st 2022, and App# PCT/US23/34148 filed on September 30th 2023 detailing a glycan-based method of diagnosing Lyme disease. All other authors do not have conflicting interests to declare., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The glycosylation of IgG plays a critical role during human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, activating immune cells and inducing cytokine production. However, the role of IgM N-glycosylation has not been studied during human acute viral infection. The analysis of IgM N-glycosylation from healthy controls and hospitalized coronavirus disease 2019 (COVID-19) patients reveals increased high-mannose and sialylation that correlates with COVID-19 severity. These trends are confirmed within SARS-CoV-2-specific immunoglobulin N-glycan profiles. Moreover, the degree of total IgM mannosylation and sialylation correlate significantly with markers of disease severity. We link the changes of IgM N-glycosylation with the expression of Golgi glycosyltransferases. Lastly, we observe antigen-specific IgM antibody-dependent complement deposition is elevated in severe COVID-19 patients and modulated by exoglycosidase digestion. Taken together, this work links the IgM N-glycosylation with COVID-19 severity and highlights the need to understand IgM glycosylation and downstream immune function during human disease., (© 2024. The Author(s).)

Introduction: The human immunodeficiency virus type 1 (HIV-1) pandemic has been slowed with the advent of anti-retroviral therapy (ART). However, ART is not a cure and as such has pushed the disease into a chronic infection. One potential cure strategy that has shown promise is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing system. It has recently been shown to successfully edit and/or excise the integrated provirus from infected cells and inhibit HIV-1 in vitro , ex vivo , and in vivo . These studies have primarily been conducted with SpCas9 or SaCas9. However, additional Cas proteins are discovered regularly and modifications to these known proteins are being engineered. The alternative Cas molecules have different requirements for protospacer adjacent motifs (PAMs) which impact the possible targetable regions of HIV-1. Other modifications to the Cas protein or gRNA handle impact the tolerance for mismatches between gRNA and the target. While reducing off-target risk, this impacts the ability to fully account for HIV-1 genetic variability. Methods: This manuscript strives to examine these parameter choices using a computational approach for surveying the suitability of a Cas editor for HIV-1 gene editing. The Nominate, Diversify, Narrow, Filter (NDNF) pipeline measures the safety, broadness, and effectiveness of a pool of potential gRNAs for any PAM. This technique was used to evaluate 46 different potential Cas editors for their HIV therapeutic potential. Results: Our examination revealed that broader PAMs that improve the targeting potential of editors like SaCas9 and LbCas12a have larger pools of useful gRNAs, while broader PAMs reduced the pool of useful SpCas9 gRNAs yet increased the breadth of targetable locations. Investigation of the mismatch tolerance of Cas editors indicates a 2-missmatch tolerance is an ideal balance between on-target sensitivity and off-target specificity. Of all of the Cas editors examined, SpCas-NG and SPRY-Cas9 had the highest number of overall safe, broad, and effective gRNAs against HIV. Discussion: Currently, larger proteins and wider PAMs lead to better targeting capacity. This implies that research should either be targeted towards delivering longer payloads or towards increasing the breadth of currently available small Cas editors. With the discovery and adoption of additional Cas editors, it is important for researchers in the HIV-1 gene editing field to explore the wider world of Cas editors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dampier, Berman, Nonnemacher and Wigdahl.)

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5' portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Allen, Chung, Worrell, Nwaozo, Madrid, Mele, Dampier, Nonnemacher and Wigdahl.)

In the contexts of aging, injury, or neuroinflammation, activated microglia signaling with TNF-α, IL-1α, and C1q induces a neurotoxic astrocytic phenotype, classified as A1, A1-like, or neuroinflammatory reactive astrocytes. In contrast to typical astrocytes, which promote neuronal survival, support synapses, and maintain blood-brain barrier integrity, these reactive astrocytes downregulate supportive functions and begin to secrete neurotoxic factors, complement components like C3, and chemokines like CXCL10, which may facilitate recruitment of immune cells across the BBB into the CNS. The proportion of pro-inflammatory reactive astrocytes increases with age through associated microglia activation, and these pro-inflammatory reactive astrocytes are particularly abundant in neurodegenerative disorders. As the identification of astrocyte phenotypes progress, their molecular and cellular effects are characterized in a growing array of neuropathologies., (© 2023. The Author(s).)

IgG N-glycans are an emerging source of disease-specific biomarkers. Over the last decade, the continued development of glycomic databases and the evolution of glyco-analytic methods have resulted in increased throughput, resolution, and sensitivity. IgG N-glycans promote adaptive immune responses through antibody-dependent cellular cytotoxicity (ADCC) and complement activation to combat infection or cancer and promote autoimmunity. In addition to the functional assays, researchers are examining the ability of protein-specific glycosylation to serve as biomarkers of disease. This literature review demonstrates that IgG N-glycans can discriminate between healthy controls, autoimmune disease, infectious disease, and cancer with high sensitivity. The literature also indicates that the IgG glycosylation patterns vary across disease state, thereby supporting their role as specific biomarkers. In addition, IgG N-glycans can be collected longitudinally from patients to track treatment responses or predict disease reoccurrence. This review focuses on IgG N-glycan profiles applied as diagnostics, cohort discriminators, and prognostics. Recent successes, remaining challenges, and upcoming approaches are critically discussed.

Herpes simplex virus type 1 (HSV-1) is a contagious pathogen with a large global footprint, due to its ability to cause lifelong infection in patients. Current antiviral therapies are effective in limiting viral replication in the epithelial cells to alleviate clinical symptoms, but ineffective in eliminating latent viral reservoirs in neurons. Much of HSV-1 pathogenesis is dependent on its ability to manipulate oxidative stress responses to craft a cellular environment that favors HSV-1 replication. However, to maintain redox homeostasis and to promote antiviral immune responses, the infected cell can upregulate reactive oxygen and nitrogen species (RONS) while having a tight control on antioxidant concentrations to prevent cellular damage. Non-thermal plasma (NTP), which we propose as a potential therapy alternative directed against HSV-1 infection, is a means to deliver RONS that affect redox homeostasis in the infected cell. This review emphasizes how NTP can be an effective therapy for HSV-1 infections through the direct antiviral activity of RONS and via immunomodulatory changes in the infected cells that will stimulate anti-HSV-1 adaptive immune responses. Overall, NTP application can control HSV-1 replication and address the challenges of latency by decreasing the size of the viral reservoir in the nervous system.

In people living with HIV-1 (PLWH), antiretroviral therapy (ART) eventually becomes necessary to suppress the emergence of human immunodeficiency virus type 1 (HIV-1) replication from latent reservoirs because HIV-1-specific immune responses in PLWH are suboptimal. Immunotherapies that enhance anti-HIV-1 immune responses for better control of virus reemergence from latent reservoirs are postulated to offer ART-free control of HIV-1. Toward the goal of developing an HIV-1-specific immunotherapy based on non-thermal plasma (NTP), the early immunological responses to NTP-exposed latently infected T lymphocytes were examined. Application of NTP to the J-Lat T-lymphocyte cell line (clones 10.6 and 15.4) stimulated monocyte recruitment and macrophage maturation, which are key steps in initiation of an immune response. In contrast, CD8+ T lymphocytes in a mixed lymphocyte reaction assay were not stimulated by the presence of NTP-exposed J-Lat cells. Furthermore, co-culture of NTP-exposed J-Lat cells with mature phagocytes did not modulate their antigen presentation to primary CD8+ T lymphocytes (cross-presentation). However, reactivation from latency was stimulated in a clone-specific manner by NTP. Overall, these studies, which demonstrated that ex vivo application of NTP to latently infected lymphocytes can stimulate key immune cell responses, advance the development of an NTP-based immunotherapy that will provide ART-free control of HIV-1 reactivation in PLWH.

Significance: Existing screening tools for HIV-associated neurocognitive disorders (HAND) are often clinically impractical for detecting milder forms of impairment. The formal diagnosis of HAND requires an assessment of both cognition and impairment in activities of daily living (ADL). To address the critical need for identifying patients who may have disability associated with HAND, we implemented a low-cost screening tool, the Virtual Driving Test (VDT) platform, in a vulnerable cohort of people with HIV (PWH). The VDT presents an opportunity to cost-effectively screen for milder forms of impairment while providing practical guidance for a cognitively demanding ADL., Objectives: We aimed to: (1) evaluate whether VDT performance variables were associated with a HAND diagnosis and if so; (2) systematically identify a manageable subset of variables for use in a future screening model for HAND. As a secondary objective, we examined the relative associations of identified variables with impairment within the individual domains used to diagnose HAND., Methods: In a cross-sectional design, 62 PWH were recruited from an established HIV cohort and completed a comprehensive neuropsychological assessment (CNPA), followed by a self-directed VDT. Dichotomized diagnoses of HAND-specific impairment and impairment within each of the seven CNPA domains were ascertained. A systematic variable selection process was used to reduce the large amount of VDT data generated, to a smaller subset of VDT variables, estimated to be associated with HAND. In addition, we examined associations between the identified variables and impairment within each of the CNPA domains., Results: More than half of the participants ( N = 35) had a confirmed presence of HAND. A subset of twenty VDT performance variables was isolated and then ranked by the strength of its estimated associations with HAND. In addition, several variables within the final subset had statistically significant associations with impairment in motor function, executive function, and attention and working memory, consistent with previous research., Conclusion: We identified a subset of VDT performance variables that are associated with HAND and assess relevant functional abilities among individuals with HAND. Additional research is required to develop and validate a predictive HAND screening model incorporating this subset., Competing Interests: DG was an employee and a vesting shareholder of Diagnostic Driving, Inc. (DDI) (creator of the virtual driving test used in this study). VK was an intellectual property and financial interest in DDI and serves as its President and Chief Executive Officer. DG and VK’s potential of conflict of interest is managed by a conflict management plan from DDI whereby DG and VK had no interactions with study participants (all field data collection procedures were carried out by Drexel University CNAC staff members on Drexel University premises) and all methods and analyses were reviewed and approved by outside consultants with no intellectual or financial interest in DDI (Nicolas Skuli, PhD; a Senior Research Investigator and Director of the Stem Cell and Xenograft Core at the University of Pennsylvania and SO, PhD; an Associate Professor at Drexel University). Additionally, FW and the Children’s Hospital of Philadelphia (CHOP) have an intellectual property and financial interest in DDI. FW served as DDI’s Chief Scientific Advisor. This potential conflict of interest is managed under a conflict-of-interest management plan from CHOP and the University of Pennsylvania whereby FW had no interaction with study participants or involvement in data collection procedures and all analyses were reviewed and approved by outside consultants with no intellectual or financial interests in DDI (John Bolte, a traffic injury researcher at the Ohio State University; and Nancy Kasam-Adams, a behavioral researcher at CHOP and the University of Pennsylvania). The reviewer LF declared a shared affiliation with the authors FW and DK to the handling editor at the time of review. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Grethlein, Pirrone, Devlin, Dampier, Szep, Winston, Ontañón, Walshe, Malone, Tillman, Ances, Kandadai, Kolson and Wigdahl.)

Lyme disease (LD) infection is caused by Borrelia burgdorferi sensu lato (Bb). Due to the limited presence of this pathogen in the bloodstream in humans, diagnosis of LD relies on seroconversion. Immunoglobulins produced in response to infection are differentially glycosylated to promote or inhibit downstream inflammatory responses by the immune system. Immunoglobulin G (IgG) N-glycan responses to LD have not been characterized. In this study, we analyzed IgG N-glycans from cohorts of healthy controls, acute LD patient serum, and serum collected after acute LD patients completed a 2- to 3-week course of antibiotics and convalesced for 70-90 days. Results indicate that during the acute phase of Bb infection, IgG shifts its glycosylation profile to include structures that are not associated with the classic proinflammatory IgG N-glycan signature. This unexpected result is in direct contrast to what is reported for other inflammatory diseases. Furthermore, IgG N-glycans detected during acute LD infection discriminated between control, acute, and treated cohorts with a sensitivity of 75-100% and specificity of 94.7-100%., Competing Interests: Author AM is a founding partner in GlycoPath, Inc. and holds patents for the GlycoTyper methodology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Haslund-Gourley, Grauzam, Mehta, Wigdahl and Comunale.)

The blood-brain barrier (BBB) mediates cellular and molecular passage between the central nervous system (CNS) and peripheral circulation. Compromised BBB integrity has been linked to neurocognitive deficits in multiple diseases and various infections, including those associated with HIV-1 infection. Understanding the impact of exposure to pharmaceuticals, such as those utilized for pain management by patients suffering from CNS disease, on BBB regulation and function is clinically important. In this study, we modelled two different BBB systems; a primary human co-culture and a cell line monoculture. These systems were both exposed to three daily repeat doses of morphine and examined for alterations to BBB integrity via permeability, PBMC transmigration, and chemokine gradient changes. We did not find any significant changes to either BBB system with repeat morphine dosing, suggesting that repeat morphine exposure may not play a significant role in BBB changes.

Globally, human immunodeficiency virus type 1 (HIV-1) infection is a major health burden for which successful therapeutic options are still being investigated. Challenges facing current drugs that are part of the established life-long antiretroviral therapy (ART) include toxicity, development of drug resistant HIV-1 strains, the cost of treatment, and the inability to eradicate the provirus from infected cells. For these reasons, novel anti-HIV-1 therapeutics that can prevent or eliminate disease progression including the onset of the acquired immunodeficiency syndrome (AIDS) are needed. While development of HIV-1 vaccination has also been challenging, recent advancements demonstrate that infection of HIV-1-susceptible cells can be prevented in individuals living with HIV-1, by targeting C-C chemokine receptor type 5 (CCR5). CCR5 serves many functions in the human immune response and is a co-receptor utilized by HIV-1 for entry into immune cells. Therapeutics targeting CCR5 generally involve gene editing techniques including CRISPR, CCR5 blockade using antibodies or antagonists, or combinations of both. Here we review the efficacy of these approaches and discuss the potential of their use in the clinic as novel ART-independent therapies for HIV-1 infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mohamed, Gurrola, Berman, Collins, Sariyer, Nonnemacher and Wigdahl.)

As genome-editing nucleases move toward broader clinical applications, the need to define the limits of their specificity and efficiency increases. A variety of approaches for nuclease cleavage detection have been developed, allowing a full-genome survey of the targeting landscape and the detection of a variety of repair outcomes for nuclease-induced double-strand breaks. Each approach has advantages and disadvantages relating to the means of target-site capture, target enrichment mechanism, cellular environment, false discovery, and validation of bona fide off-target cleavage sites in cells. This review examines the strengths, limitations, and origins of the different classes of off-target cleavage detection systems including anchored primer enrichment (GUIDE-seq), in situ detection (BLISS), in vitro selection libraries (CIRCLE-seq), chromatin immunoprecipitation (ChIP) (DISCOVER-Seq), translocation sequencing (LAM PCR HTGTS), and in vitro genomic DNA digestion (Digenome-seq and SITE-Seq). Emphasis is placed on the specific modifications that give rise to the enhanced performance of contemporary techniques over their predecessors and the comparative performance of techniques for different applications. The clinical relevance of these techniques is discussed in the context of assessing the safety of novel CRISPR/Cas9 HIV-1 curative strategies. With the recent success of HIV-1 and SIV-1 viral suppression in humanized mice and non-human primates, respectively, using CRISPR/Cas9, rigorous exploration of potential off-target effects is of critical importance. Such analyses would benefit from the application of the techniques discussed in this review., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Atkins, Chung, Allen, Dampier, Gurrola, Sariyer, Nonnemacher and Wigdahl.)

Introduction: Antiretroviral therapy (ART) has transformed prognoses for HIV-1-infected individuals but requires lifelong adherence to prevent viral resurgence. Targeted elimination or permanent deactivation of the latently infected reservoir harboring integrated proviral DNA, which drives viral rebound, is a major focus of HIV-1 research., Areas Covered: This review covers the current approaches to developing curative strategies for HIV-1 that target the latent reservoir. Discussed herein are shock and kill, broadly neutralizing antibodies (bNAbs), block and lock, Chimeric antigen receptor (CAR) T cells, immune checkpoint modulation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) coreceptor ablation, and CRISPR/Cas9 proviral excision. Emphasis is placed on CRISPR/Cas9 proviral excision/inactivation. Recent advances and future directions toward discovery and translation of HIV-1 therapeutics are discussed., Expert Opinion: CRISPR/Cas9 proviral targeting fills a niche amongst HIV-1 cure strategies by directly targeting the integrated provirus without the necessity of an innate or adaptive immune response. Each strategy discussed in this review has shown promising results with the potential to yield curative or adjuvant therapies. CRISPR/Cas9 is singular among these in that it addresses the root of the problem, integrated proviral DNA, with the capacity to permanently remove or deactivate the source of HIV-1 recrudescence.

Non-thermal plasma application to cancer cells is known to induce oxidative stress, cytotoxicity and indirect immunostimulatory effects on antigen presenting cells (APCs). The purpose of this study was to evaluate the responses of two leukemic cell lines-Jurkat T lymphocytes and THP-1 monocytes-to NTP-generated reactive oxygen and nitrogen species (RONS). Both cell types depleted hydrogen peroxide, but THP-1 cells neutralized it almost immediately. Jurkat cells transiently blunted the frequency-dependent increase in nitrite concentrations in contrast to THP-1 cells, which exhibited no immediate effect. A direct relationship between frequency-dependent cytotoxicity and mitochondrial superoxide was observed only in Jurkat cells. Jurkat cells were very responsive to NTP in their display of calreticulin and heat shock proteins 70 and 90. In contrast, THP-1 cells were minimally responsive or unresponsive. Despite no NTP-dependent decrease in cell surface display of CD47 in either cell line, both cell types induced migration of and phagocytosis by APCs. Our results demonstrate that cells modulate the RONS-mediated changes in liquid chemistry, and, importantly, the resultant immunomodulatory effects of NTP can be independent of NTP-induced cytotoxicity.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based HIV-1 genome editing has shown promising outcomes in in vitro and in vivo viral infection models. However, existing HIV-1 sequence variants have been shown to reduce CRISPR-mediated efficiency and induce viral escape. Two metrics, global patient coverage and global subtype coverage, were used to identify guide RNA (gRNA) sequences that account for this viral diversity from the perspectives of cross-patient and cross-subtype gRNA design, respectively. Computational evaluation using these parameters and over 3.6 million possible 20-bp sequences resulted in nine lead gRNAs, two of which were previously published. This analysis revealed the benefit and necessity of considering all sequence variants for gRNA design. Of the other seven identified novel gRNAs, two were of note as they targeted interesting functional regions. One was a gRNA predicted to induce structural disruption in the nucleocapsid binding site (Ψ), which holds the potential to stop HIV-1 replication during the viral genome packaging process. The other was a reverse transcriptase (RT)-targeting gRNA that was predicted to cleave the subdomain responsible for dNTP incorporation. CRISPR-mediated sequence edits were predicted to occur on critical residues where HIV-1 has been shown to develop resistance against antiretroviral therapy (ART), which may provide additional evolutionary pressure at the DNA level. Given these observations, consideration of broad-spectrum gRNAs and cross-subtype diversity for gRNA design is not only required for the development of generalizable CRISPR-based HIV-1 therapy, but also helps identify optimal target sites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chung, Allen, Atkins, Link, Nonnemacher, Dampier and Wigdahl.)

Effective control of infection by human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), requires continuous and life-long use of anti-retroviral therapy (ART) by people living with HIV-1 (PLWH). In the absence of ART, HIV-1 reemergence from latently infected cells is ineffectively suppressed due to suboptimal innate and cytotoxic T lymphocyte responses. However, ART-free control of HIV-1 infection may be possible if the inherent immunological deficiencies can be reversed or restored. Herein we present a novel approach for modulating the immune response to HIV-1 that involves the use of non-thermal plasma (NTP), which is an ionized gas containing various reactive oxygen and nitrogen species (RONS). J-Lat cells were used as a model of latent HIV-1 infection to assess the effects of NTP application on viral latency and the expression of pro-phagocytic and pro-chemotactic damage-associated molecular patterns (DAMPs). Exposure of J-Lat cells to NTP resulted in stimulation of HIV-1 gene expression, indicating a role in latency reversal, a necessary first step in inducing adaptive immune responses to viral antigens. This was accompanied by the release of pro-inflammatory cytokines and chemokines including interleukin-1β (IL-1β) and interferon-γ (IFN-γ); the display of pro-phagocytic markers calreticulin (CRT), heat shock proteins (HSP) 70 and 90; and a correlated increase in macrophage phagocytosis of NTP-exposed J-Lat cells. In addition, modulation of surface molecules that promote or inhibit antigen presentation was also observed, along with an altered array of displayed peptides on MHC I, further suggesting methods by which NTP may modify recognition and targeting of cells in latent HIV-1 infection. These studies represent early progress toward an effective NTP-based ex vivo immunotherapy to resolve the dysfunctions of the immune system that enable HIV-1 persistence in PLWH., Competing Interests: The authors have declared that no competing interests exist.

Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) is a potent mediator involved in the development of HIV-1-associated neurocognitive disorders (HAND). Tat is expressed even in the presence of antiretroviral therapy (ART) and is able to enter the central nervous system (CNS) through a variety of ways, where Tat can interact with microglia, astrocytes, brain microvascular endothelial cells, and neurons. The presence of low concentrations of extracellular Tat alone has been shown to lead to dysregulated gene expression, chronic cell activation, inflammation, neurotoxicity, and structural damage in the brain. The reported effects of Tat are dependent in part on the specific HIV-1 subtype and amino acid length of Tat used. HIV-1 subtype B Tat is the most common subtype in North American and therefore, most studies have been focused on subtype B Tat; however, studies have shown many genetic, biologic, and pathologic differences between HIV subtype B and subtype C Tat. This review will focus primarily on subtype B Tat where the full-length protein is 101 amino acids, but will also consider variants of Tat, such as Tat 72 and Tat 86, that have been reported to exhibit a number of distinctive activities with respect to mediating CNS damage and neurotoxicity.

Vaccination has been one of the most effective health intervention mechanisms to reduce morbidity and mortality associated with infectious diseases. Vaccines stimulate the body's protective immune responses through controlled exposure to modified versions of pathogens that establish immunological memory. However, only a few diseases have effective vaccines. The biological effects of nonthermal plasma on cells suggest that plasma could play an important role in improving efficacy of existing vaccines and overcoming some of the limitations and challenges with current vaccination strategies. This review summarizes the opportunities for nonthermal plasma for immunization and therapeutic purposes., (© 2020 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Viral latency of human immunodeficiency virus type 1 (HIV-1) has become a major hurdle to a cure in the highly effective antiretroviral therapy (ART) era. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been demonstrated to excise or inactivate integrated HIV-1 provirus from infected cells by targeting the long terminal repeat (LTR) region. However, the guide RNAs (gRNAs) have classically avoided transcription factor binding sites (TFBSs) that are readily observed and known to be important in human promoters. Although conventionally thought unfavorable due to potential impact on human promoters, our computational pipeline identified gRNA sequences that were predicted to inactivate HIV-1 transcription by targeting the nuclear factor κB (NF-κB) binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad-spectrum activity (predicted coverage of known viral sequences). Genome-wide, unbiased identification of double strand breaks (DSBs) enabled by sequencing (GUIDE-seq) showed that the gRNAs targeting NF-κB binding sites had no detectable CRISPR-induced off-target edits in HeLa cells. 5' LTR-driven HIV-1 transcription was significantly reduced in three HIV-1 reporter cell lines. These results demonstrate a working model to specifically target well-known TFBSs in the HIV-1 LTR that are readily observed in human promoters to reduce HIV-1 transcription with a high-level safety profile and broad-spectrum activity., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Human immunodeficiency virus type-1 (HIV-1) infection has resulted in the death of upward of 39 million people since being discovered in the early 1980s. A cure strategy for HIV-1 has eluded scientists, but gene editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) offer a new approach to developing a cure for HIV infection. While the CRISPR/Cas9 system has been used successfully in a number of different types of studies, there remains a concern for off-target effects. This review details the different aspects of the Cas9 system and how they play a role in off-target events. In addition, this review describes the current technologies available for detecting off-target cleavage events and their advantages and disadvantages. While some studies have utilized whole genome sequencing (WGS), this method sacrifices depth of coverage for interrogating the whole genome. A number of different approaches have now been developed to take advantage of next generation sequencing (NGS) without sacrificing depth of coverage. This review highlights four widely used methods for detecting off-target events: (1) genome-wide unbiased identification of double-stranded break events enabled by sequencing (GUIDE-Seq), (2) discovery of in situ Cas off-targets and verification by sequencing (DISCOVER-Seq), (3) circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-Seq), and (4) breaks labeling in situ and sequencing (BLISS). Each of these technologies has advantages and disadvantages, but all center around capturing double-stranded break (DSB) events catalyzed by the Cas9 endonuclease. Being able to define off-target events is crucial for a gene therapy cure strategy for HIV-1., (Copyright © 2020 Sullivan, Allen, Atkins, Chung, Dampier, Nonnemacher and Wigdahl.)

Human immunodeficiency virus type 1 (HIV-1)- associated neurocognitive disorders (HAND) is a disease of neurologic impairment that involves mechanisms of damage similar to other degenerative neurologic diseases such as Alzheimer's disease (AD). In the current era of antiretroviral therapy (ART), HIV-1 replication is well-suppressed, and yet, HIV-1-infected patients still have high levels of chronic inflammation, indicating that factors other than viral replication are contributing to the development of neurocognitive impairment in these patients. The underlying mechanisms of HAND are still unknown, but the HIV-1 protein, Tat, has been highlighted as a potential viral product that contributes to the development of cognitive impairment. In AD, the presence of senescent cells in the CNS has been discussed as a contributing factor to the progression of cognitive decline and may be a mechanism also involved in the development of HAND. This mini-review discusses the viral protein HIV-1 Tat, and its potential to induce senescence in the CNS, contributing to the development of HAND., (Copyright © 2020 Marino, Wigdahl and Nonnemacher.)

Dendritic cells (DC) are key phagocytic cells that play crucial roles in both the innate and adaptive immune responses against the human immunodeficiency virus type 1 (HIV-1). By processing and presenting pathogen-derived antigens, dendritic cells initiate a directed response against infected cells. They activate the adaptive immune system upon recognition of pathogen-associated molecular patterns (PAMPs) on infected cells. During the course of HIV-1 infection, a successful adaptive (cytotoxic CD8 + T-cell) response is necessary for preventing the progression and spread of infection in a variety of cells. Dendritic cells have thus been recognized as a valuable tool in the development of immunotherapeutic approaches and vaccines effective against HIV-1. The advancements in dendritic cell vaccines in cancers have paved the way for applications of this form of immunotherapy to HIV-1 infection. Clinical trials with patients infected with HIV-1 who are well-suppressed by antiretroviral therapy (ART) were recently performed to assess the efficacy of DC vaccines, with the goal of mounting an HIV-1 antigen-specific T-cell response, ideally to clear infection and eliminate the need for long-term ART. This review summarizes and compares methods and efficacies of a number of DC vaccine trials utilizing autologous dendritic cells loaded with HIV-1 antigens. The potential for advancement and novel strategies of improving efficacy of this type of immunotherapy is also discussed., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this literature review., (Copyright © 2020 Hager Mohamed et al.)

The full length of HIV/R7/E - /GFP integrated in the J-Lat 10.6 cell line was sequenced in this study. The single copy of the integrated virus, including the breakpoints from the human chromosome to the provirus, was amplified by two separate PCRs. A 10,200-bp genome sequence was acquired, analyzed, and deposited in GenBank., (Copyright © 2020 Chung et al.)

Defining the variables that impact the specificity of CRISPR/Cas9 has been a major research focus. Whereas sequence complementarity between guide RNA and target DNA substantially dictates cleavage efficiency, DNA accessibility of the targeted loci has also been hypothesized to be an important factor. In this study, functional data from two genome-wide assays, genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) and circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), have been computationally analyzed in conjunction with DNA accessibility determined via DNase I-hypersensitive sequencing from the Encyclopedia of DNA Elements (ENCODE) Database and transcriptome from the Sequence Read Archive to determine whether cellular factors influence CRISPR-induced cleavage efficiency. CIRCLE-seq and GUIDE-seq datasets were selected to represent the absence and presence of cellular factors, respectively. Data analysis revealed that correlations between sequence similarity and CRISPR-induced cleavage frequency were altered by the presence of cellular factors that modulated the level of DNA accessibility. The above-mentioned correlation was abolished when cleavage sites were located in less accessible regions. Furthermore, CRISPR-mediated edits were permissive even at regions that were insufficient for most endogenous genes to be expressed. These results provide a strong basis to dissect the contribution of local chromatin modulation markers on CRISPR-induced cleavage efficiency., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The CRISPR/Cas9 system has been proposed as a cure strategy for HIV. However, few published guide RNAs (gRNAs) are predicted to cleave the majority of HIV-1 viral quasispecies (vQS) observed within and among patients. We report the design of a novel pipeline to identify gRNAs that target HIV across a large number of infected individuals. Next generation sequencing (NGS) of LTRs from 269 HIV-1-infected samples in the Drexel CARES Cohort was used to select gRNAs with predicted broad-spectrum activity. In silico, D-LTR-P4-227913 (package of the top 4 gRNAs) accounted for all detectable genetic variation within the vQS of the 269 samples and the Los Alamos National Laboratory HIV database. In silico secondary structure analyses from NGS indicated extensive TAR stem-loop malformations predicted to inactivate proviral transcription, which was confirmed by reduced viral gene expression in TZM-bl or P4R5 cells. Similarly, a high sensitivity in vitro CRISPR/Cas9 cleavage assay showed that the top-ranked gRNA was the most effective at cleaving patient-derived HIV-1 LTRs from five patients. Furthermore, the D-LTR-P4-227913 was predicted to cleave a median of 96.1% of patient-derived sequences from other HIV subtypes. These results demonstrate that the gRNAs possess broad-spectrum cutting activity and could contribute to an HIV cure.

Human immunodeficiency virus type 1 (HIV-1) encodes for Tat, a multi-functional regulatory protein involved in transcriptional enhancement and in causing neurotoxicity/central nervous system (CNS) dysfunction. This study examines Sanger sequencing of HIV-1 subtype B Tat from 2006 to 2014 within the Drexel University College of Medicine CNS AIDS Research and Eradication Study (CARES) Cohort to investigate Tat length in patients. The Los Alamos National Laboratory (LANL) database was used as a comparator. Miscoded stop codons were present in the CARES Cohort and LANL and protein variability was highly similar. Tat proteins in CARES and LANL were predominantly 101 residues. There was no observed correlation between Tat length and clinical parameters within the CARES Cohort. Unique Tat lengths found in the CARES Cohort and not in LANL were 31, 36, and 39 residues. When CARES patients were longitudinally examined, sequence lengths of 101 had a low probability of reducing to below 48, and sequences had a high probability of increasing to above 86 residues during their next visit, when below 48 residues in length. This suggests that Tat length is conserved to retain the majority of the proteins function highlighting its importance in viral replication., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human immunodeficiency virus type 1 (HIV-1) encodes a transactivator of transcription (Tat) protein, which has several functions that promote viral replication, pathogenesis, and disease. Amino acid variation within Tat has been observed to alter the functional properties of Tat and, depending on the HIV-1 subtype, may produce Tat phenotypes differing from viruses' representative of each subtype and commonly used in in vivo and in vitro experimentation. The molecular properties of Tat allow for distinctive functional activities to be determined such as the subcellular localization and other intracellular and extracellular functional aspects of this important viral protein influenced by variation within the Tat sequence. Once Tat has been transported into the nucleus and becomes engaged in transactivation of the long terminal repeat (LTR), various Tat variants may differ in their capacity to activate viral transcription. Post-translational modification patterns based on these amino acid variations may alter interactions between Tat and host factors, which may positively or negatively affect this process. In addition, the ability of HIV-1 to utilize or not utilize the transactivation response (TAR) element within the LTR, based on genetic variation and cellular phenotype, adds a layer of complexity to the processes that govern Tat-mediated proviral DNA-driven transcription and replication. In contrast, cytoplasmic or extracellular localization of Tat may cause pathogenic effects in the form of altered cell activation, apoptosis, or neurotoxicity. Tat variants have been shown to differentially induce these processes, which may have implications for long-term HIV-1-infected patient care in the antiretroviral therapy era. Future studies concerning genetic variation of Tat with respect to function should focus on variants derived from HIV-1-infected individuals to efficiently guide Tat-targeted therapies and elucidate mechanisms of pathogenesis within the global patient population.

Antiretroviral therapy has prolonged the lives of people living with human immunodeficiency virus type 1 (HIV-1), transforming the disease into one that can be controlled with lifelong therapy. The search for an HIV-1 vaccine has plagued researchers for more than three decades with little to no success from clinical trials. Due to these failures, scientists have turned to alternative methods to develop next generation therapeutics that could allow patients to live with HIV-1 without the need for daily medication. One method that has been proposed has involved the use of a number of powerful gene editing tools; Zinc Finger Nucleases (ZFN), Transcription Activator-like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 to edit the co-receptors (CCR5 or CXCR4) required for HIV-1 to infect susceptible target cells efficiently. Initial safety studies in patients have shown that editing the CCR5 locus is safe. More in depth in vitro studies have shown that editing the CCR5 locus was able to inhibit infection from CCR5-utilizing virus, but CXCR4-utilizing virus was still able to infect cells. Additional research efforts were then aimed at editing the CXCR4 locus, but this came with other safety concerns. However, in vitro studies have since confirmed that CXCR4 can be edited without killing cells and can confer resistance to CXCR4-utilizing HIV-1. Utilizing these powerful new gene editing technologies in concert could confer cellular resistance to HIV-1. While the CD4, CCR5, CXCR4 axis for cell-free infection has been the most studied, there are a plethora of reports suggesting that the cell-to-cell transmission of HIV-1 is significantly more efficient. These reports also indicated that while broadly neutralizing antibodies are well suited with respect to blocking cell-free infection, cell-to-cell transmission remains refractile to this approach. In addition to stopping cell-free infection, gene editing of the HIV-1 co-receptors could block cell-to-cell transmission. This review aims to summarize what has been shown with regard to editing the co-receptors needed for HIV-1 entry and how they could impact the future of HIV-1 therapeutic and prevention strategies.