Non-Thermal Plasma Reduces HSV-1 Infection of and Replication in HaCaT Keratinocytes In Vitro.

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.

What's in a cure: designing a broad-spectrum HIV gene therapy.

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.

IgM N-glycosylation correlates with COVID-19 severity and rate of complement deposition.

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.

Host glycosylation of immunoglobulins impairs the immune response to acute Lyme disease.

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.

IgM N-glycosylation correlates with COVID-19 severity and rate of complement deposition.

The glycosylation of IgG plays a critical role during human SARS-CoV-2, activating immune cells and inducing cytokine production. However, the role of IgM N-glycosylation has not been studied during acute viral infection in humans. In vitro evidence suggests that the glycosylation of IgM inhibits T cell proliferation and alters complement activation rates. The analysis of IgM N-glycosylation from healthy controls and hospitalized COVID-19 patients reveals that mannosylation and sialyation levels associate with COVID-19 severity. Specifically, we find increased di- and tri-sialylated glycans and altered mannose glycans in total serum IgM in severe COVID-19 patients when compared to moderate COVID-19 patients. This is in direct contrast with the decrease of sialic acid found on the serum IgG from the same cohorts. Moreover, the degree of mannosylation and sialylation correlated significantly with markers of disease severity: D-dimer, BUN, creatinine, potassium, and early anti-COVID-19 amounts of IgG, IgA, and IgM. Further, IL-16 and IL-18 cytokines showed similar trends with the amount of mannose and sialic acid present on IgM, implicating these cytokines' potential to impact glycosyltransferase expression during IgM production. When examining PBMC mRNA transcripts, we observe a decrease in the expression of Golgi mannosidases that correlates with the overall reduction in mannose processing we detect in the IgM N-glycosylation profile. Importantly, we found that IgM contains alpha-2,3 linked sialic acids in addition to the previously reported alpha-2,6 linkage. We also report that antigen-specific IgM antibody-dependent complement deposition is elevated in severe COVID-19 patients. Taken together, this work links the immunoglobulin M N-glycosylation with COVID-19 severity and highlights the need to understand the connection between IgM glycosylation and downstream immune function during human disease.

Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9.

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.

Manipulation of Oxidative Stress Responses by Non-Thermal Plasma to Treat Herpes Simplex Virus Type 1 Infection and Disease.

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.

IgG N-glycan Signatures as Potential Diagnostic and Prognostic Biomarkers.

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.

Roles of neuropathology-associated reactive astrocytes: a systematic review.

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.

Immunomodulatory Effects of Non-Thermal Plasma in a Model for Latent HIV-1 Infection: Implications for an HIV-1-Specific Immunotherapy.

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.

Computational analysis of cas proteins unlocks new potential in HIV-1 targeted gene therapy.

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.

Examining virtual driving test performance and its relationship to individuals with HIV-associated neurocognitive disorders.

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.

Acute lyme disease IgG N-linked glycans contrast the canonical inflammatory signature.

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%.

Chronic Low Dose Morphine Does Not Alter Two In Vitro BBB Models.

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.

Off-Target Analysis in Gene Editing and Applications for Clinical Translation of CRISPR/Cas9 in HIV-1 Therapy.

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.

Differential Effect of Non-Thermal Plasma RONS on Two Human Leukemic Cell Populations.

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.

Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy.

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.

Non-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection.

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-1ß (IL-1ß) 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.

Targeting CCR5 as a Component of an HIV-1 Therapeutic Strategy.

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.

HIV-1 cure strategies: why CRISPR?

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.