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.

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.

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.

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.

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.

Nonthermal plasma as part of a novel strategy for vaccination.

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.

The Evolution of Dendritic Cell Immunotherapy against HIV-1 Infection: Improvements and Outlook.

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.

Non-Thermal Plasma-Induced Immunogenic Cell Death in Cancer: A Topical Review.

Recent advances in biomedical research in cancer immunotherapy have identified the use of an oxidative stress-based approach to treat cancers, which works by inducing immunogenic cell death (ICD) in cancer cells. Since the anti-cancer effects of non-thermal plasma (NTP) are largely attributed to the reactive oxygen and nitrogen species that are delivered to and generated inside the target cancer cells, it is reasonable to postulate that NTP would be an effective modality for ICD induction. NTP treatment of tumors has been shown to destroy cancer cells rapidly and, under specific treatment regimens, this leads to systemic tumor-specific immunity. The translational benefit of NTP for treatment of cancer relies on its ability to enhance the interactions between NTP-exposed tumor cells and local immune cells which initiates subsequent protective immune responses. This review discusses results from recent investigations of NTP application to induce immunogenic cell death in cancer cells. With further optimization of clinical devices and treatment protocols, NTP can become an essential part of the therapeutic armament against cancer.

Evidence of Divergent Amino Acid Usage in Comparative Analyses of R5- and X4-Associated HIV-1 Vpr Sequences.

Vpr is an HIV-1 accessory protein that plays numerous roles during viral replication, and some of which are cell type dependent. To test the hypothesis that HIV-1 tropism extends beyond the envelope into the vpr gene, studies were performed to identify the associations between coreceptor usage and Vpr variation in HIV-1-infected patients. Colinear HIV-1 Env-V3 and Vpr amino acid sequences were obtained from the LANL HIV-1 sequence database and from well-suppressed patients in the Drexel/Temple Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. Genotypic classification of Env-V3 sequences as X4 (CXCR4-utilizing) or R5 (CCR5-utilizing) was used to group colinear Vpr sequences. To reveal the sequences associated with a specific coreceptor usage genotype, Vpr amino acid sequences were assessed for amino acid diversity and Jensen-Shannon divergence between the two groups. Five amino acid alphabets were used to comprehensively examine the impact of amino acid substitutions involving side chains with similar physiochemical properties. Positions 36, 37, 41, 89, and 96 of Vpr were characterized by statistically significant divergence across multiple alphabets when X4 and R5 sequence groups were compared. In addition, consensus amino acid switches were found at positions 37 and 41 in comparisons of the R5 and X4 sequence populations. These results suggest an evolutionary link between Vpr and gp120 in HIV-1-infected patients.

cAMP Signaling Enhances HIV-1 Long Terminal Repeat (LTR)-directed Transcription and Viral Replication in Bone Marrow Progenitor Cells.

CD34+ hematopoietic progenitor cells have been shown to be susceptible to HIV-1 infection, possibly due to a low-level expression of CXCR4, a coreceptor for HIV-1 entry. Given these observations, we have explored the impact of forskolin on cell surface expression of CXCR4 in a cell line model (TF-1). The elevation of intracellular cyclic adenosine monophosphate (cAMP) by forskolin through adenylyl cyclase (AC) resulted in transcriptional upregulation of CXCR4 with a concomitant increase in replication of the CXCR4-utilizing HIV-1 strain IIIB. Transient expression analyses also demonstrated an increase in CXCR4-, CCR5-, and CXCR4-/CCR5-utilizing HIV-1 (LAI, YU2, and 89.6, respectively) promoter activity. Studies also implicated the protein kinase A (PKA) pathway and the downstream transcription factor CREB-1 in interfacing with cAMP response elements located in the CXCR4 and viral promoter. These observations suggest that the cAMP signaling pathway may serve as a regulator of CXCR4 levels and concomitantly of HIV-1 replication in bone marrow (BM) progenitor cells.

Specific amino acids in HIV-1 Vpr are significantly associated with differences in patient neurocognitive status.

Even in the era of combination antiretroviral therapies used to combat human immunodeficiency virus type 1 (HIV-1) infection, up to 50 % of well-suppressed HIV-1-infected patients are still diagnosed with mild neurological deficits referred to as HIV-associated neurocognitive disorders (HAND). The multifactorial nature of HAND likely involves the HIV-1 accessory protein viral protein R (Vpr) as an agent of neuropathogenesis. To investigate the effect of naturally occurring variations in Vpr on HAND in well-suppressed HIV-1-infected patients, bioinformatic analyses were used to correlate peripheral blood-derived Vpr sequences with patient neurocognitive performance, as measured by comprehensive neuropsychological assessment and the resulting Global Deficit Score (GDS). Our studies revealed unique associations between GDS and the presence of specific amino acid changes in peripheral blood-derived Vpr sequences [neuropsychological impairment Vpr (niVpr) variants]. Amino acids N41 and A55 in the Vpr sequence were associated with more pronounced neurocognitive deficits (higher GDS). In contrast, amino acids I37 and S41 were connected to measurably lower GDS. All niVpr variants were also detected in DNA isolated from HIV-1-infected brain tissues. The implication of these results is that niVpr variants alter the genesis and/or progression of HAND through differences in Vpr-mediated effects in the peripheral blood and/or the brain.

Utilization of HIV-1 envelope V3 to identify X4- and R5-specific Tat and LTR sequence signatures.

BACKGROUND: HIV-1 entry is a receptor-mediated process directed by the interaction of the viral envelope with the host cell CD4 molecule and one of two co-receptors, CCR5 or CXCR4. The amino acid sequence of the third variable (V3) loop of the HIV-1 envelope is highly predictive of co-receptor utilization preference during entry, and machine learning predictive algorithms have been developed to characterize sequences as CCR5-utilizing (R5) or CXCR4-utilizing (X4). It was hypothesized that while the V3 loop is predominantly responsible for determining co-receptor binding, additional components of the HIV-1 genome may contribute to overall viral tropism and display sequence signatures associated with co-receptor utilization. RESULTS: The accessory protein Tat and the HlV-1 long terminal repeat (LTR) were analyzed with respect to genetic diversity and compared by Jensen-Shannon divergence which resulted in a correlation with both mean genetic diversity as well as the absolute difference in genetic diversity between R5- and X4-genome specific trends. As expected, the V3 domain of the gp120 protein was enriched with statistically divergent positions. Statistically divergent positions were also identified in Tat amino acid sequences within the transactivation and TAR-binding domains, and in nucleotide positions throughout the LTR. We further analyzed LTR sequences for putative transcription factor binding sites using the JASPAR transcription factor binding profile database and found several putative differences in transcription factor binding sites between R5 and X4 HIV-1 genomes, specifically identifying the C/EBP sites I and II, and Sp site III to differ with respect to sequence configuration for R5 and X4 LTRs. CONCLUSION: These observations support the hypothesis that co-receptor utilization coincides with specific genetic signatures in HIV-1 Tat and the LTR, likely due to differing transcriptional regulatory mechanisms and selective pressures applied within specific cellular targets during the course of productive HIV-1 infection.

Defining the roles for Vpr in HIV-1-associated neuropathogenesis.

It is increasingly evident that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has a unique role in neuropathogenesis. Its ability to induce G2/M arrest coupled with its capacity to increase viral gene transcription gives it a unique role in sustaining viral replication and aiding in the establishment and maintenance of a systemic infection. The requirement of Vpr for HIV-1 infection and replication in cells of monocytic origin (a key lineage of cells involved in HIV-1 neuroinvasion) suggests an important role in establishing and sustaining infection in the central nervous system (CNS). Contributions of Vpr to neuropathogenesis can be expanded further through (i) naturally occurring HIV-1 sequence variation that results in functionally divergent Vpr variants; (ii) the dual activities of Vpr as a intracellular protein delivered and expressed during HIV-1 infection and as an extracellular protein that can act on neighboring, uninfected cells; (iii) cell type-dependent consequences of Vpr expression and exposure, including cell cycle arrest, metabolic dysregulation, and cytotoxicity; and (iv) the effects of Vpr on exosome-based intercellular communication in the CNS. Revealing that the effects of this pleiotropic viral protein is an essential part of a greater understanding of HIV-1-associated pathogenesis and potential approaches to treating and preventing disease caused by HIV-1 infection.

Critical Review: Immunomodulation by Seminal Factors and Implications for Male-to-Female HIV-1 Transmission.

The role of semen in heterosexual transmission of the HIV-1 has been marginally viewed as an inert vehicle for the delivery of virus. However, studies from the field of reproductive biology have made it clear that seminal fluid is a complex and dynamic medium containing high concentrations of factors that play key roles in modulating the local immune response in the female reproductive tract during fertilization and embryogenesis. It is therefore strongly implied that the same seminal factors responsible for guiding the immune response in reproduction also play a role in male-to-female transmission of HIV-1. To begin to understand how these factors affect male-to-female HIV-1 transmission, multiple studies have comparatively profiled the contents of seminal fluid collected from uninfected and HIV-1-infected men. This review provides an overview of these studies, as well as a discussion of the potential impact of semen on HIV-1 transmission.

Decreased cervical epithelial sensitivity to nonoxynol-9 (N-9) after four daily applications in a murine model of topical vaginal microbicide safety.

BACKGROUND: The disappointing clinical failures of five topical vaginal microbicides have provided new insights into factors that impact microbicide safety and efficacy. Specifically, the greater risk for human immunodeficiency virus type 1 (HIV-1) acquisition associated with multiple uses of a nonoxynol-9 (N-9)-containing product has highlighted the importance of application frequency as a variable during pre-clinical microbicide development, particularly in animal model studies. METHODS: To evaluate an association between application frequency and N-9 toxicity, experiments were performed using a mouse model of cervicovaginal microbicide safety. In this model system, changes in cervical and vaginal epithelial integrity, cytokine release, and immune cell infiltration were assessed after single and multiple exposures to N-9. RESULTS: After the initial application of N-9 (aqueous, 1%), considerable damage to the cervical epithelium (but not the vaginal epithelium) was observed as early as 10 min post-exposure and up to 8 h post-exposure. Subsequent daily exposures (up to 4 days) were characterized by diminished cervical toxicity relative to single exposures of like duration. Levels of pro-inflammatory cytokines released into the cervicovaginal lumen and the degree of CD14-positive immune cell infiltration proximal to the cervical epithelium were also dependent on the number of N-9 exposures. CONCLUSIONS: Rather than causing cumulative cervical epithelial damage, repeated applications of N-9 were characterized by decreased sensitivity to N-9-associated toxicity and lower levels of immune cell recruitment. These results provide new insights into the failure of N-9-based microbicides and illustrate the importance of considering multiple exposure protocols in pre-clinical microbicide development strategies.

Inhibiting early-stage events in HIV-1 replication by small-molecule targeting of the HIV-1 capsid.

The HIV-1 capsid (CA) protein plays essential roles in both early and late stages of virl replication and has emerged as a novel drug target. We report hybrid structure-based virtual screening to identify small molecules with the potential to interact with the N-terminal domain (NTD) of HIV-1 CA and disrupt early, preintegration steps of the HIV-1 replication cycle. The small molecule 4,4'-[dibenzo[b,d]furan-2,8-diylbis(5-phenyl-1H-imidazole-4,2-diyl)]dibenzoic acid (CK026), which had anti-HIV-1 activity in single- and multiple-round infections but failed to inhibit viral replication in peripheral blood mononuclear cells (PBMCs), was identified. Three analogues of CK026 with reduced size and better drug-like properties were synthesized and assessed. Compound I-XW-053 (4-(4,5-diphenyl-1H-imidazol-2-yl)benzoic acid) retained all of the antiviral activity of the parental compound and inhibited the replication of a diverse panel of primary HIV-1 isolates in PBMCs, while displaying no appreciable cytotoxicity. This antiviral activity was specific to HIV-1, as I-XW-053 displayed no effect on the replication of SIV or against a panel of nonretroviruses. Direct interaction of I-XW-053 was quantified with wild-type and mutant CA protein using surface plasmon resonance and isothermal titration calorimetry. Mutation of Ile37 and Arg173, which are required for interaction with compound I-XW-053, crippled the virus at an early, preintegration step. Using quantitative PCR, we demonstrated that treatment with I-XW-053 inhibited HIV-1 reverse transcription in multiple cell types, indirectly pointing to dysfunction in the uncoating process. In summary, we have identified a CA-specific compound that targets and inhibits a novel region in the NTD-NTD interface, affects uncoating, and possesses broad-spectrum anti-HIV-1 activity.

A nipple shield delivery system for oral drug delivery to breastfeeding infants: microbicide delivery to inactivate HIV.

A new drug delivery method for infants is presented which incorporates an active pharmaceutical ingredient (API)-loaded insert into a nipple shield delivery system (NSDS). The API is released directly into milk during breastfeeding. This study investigates the feasibility of using the NSDS to deliver the microbicide sodium dodecyl sulfate (SDS), with the goal of preventing mother-to-child transmission (MTCT) of HIV during breastfeeding in low-resource settings, when there is no safer alternative for the infant but to breastfeed. SDS has been previously shown to effectively inactivate HIV in human milk. An apparatus was developed to simulate milk flow through and drug release from a NSDS. Using this apparatus milk was pulsed through a prototype device containing a non-woven fiber insert impregnated with SDS and the microbicide was rapidly released. The total SDS release from inserts ranged from 70 to 100% of the average 0.07 g load within 50 ml (the volume of a typical breastfeed). Human milk spiked with H9/HIV(IIIB) cells was also passed through the same set-up. Greater than 99% reduction of cell-associated HIV infectivity was achieved in the first 10 ml of milk. This proof of concept study demonstrates efficient drug delivery to breastfeeding infants is achievable using the NSDS.

Infection by CXCR4-Tropic Human Immunodeficiency Virus Type 1 Is Inhibited by the Cationic Cell-Penetrating Peptide Derived from HIV-1 Tat.

Cell-penetrating peptides (CPP), which are short peptides that are capable of crossing the plasma membrane of a living cell, are under development as delivery vehicles for therapeutic agents that cannot themselves enter the cell. One well-studied CPP is the 10-amino acid peptide derived from the human immunodeficiency virus type 1 (HIV-1) Tat protein. In experiments to test the hypothesis that multiple cationic amino acids within Tat peptide confer antiviral activity against HIV-1, introduction of Tat peptide resulted in concentration-dependent inhibition of HIV-1 IIIB infection. Using Tat peptide variants containing arginine substitutions for two nonionic residues and two lysine residues, HIV-1 inhibition experiments demonstrated a direct relationship between cationic charge and antiviral potency. These studies of Tat peptide as an antiviral agent raise new questions about the role of Tat in HIV-1 replication and provide a starting point for the development of CPPs as novel HIV-1 inhibitors.

Application and removal of polyanionic microbicide compounds enhances subsequent infection by HIV-1.

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.

Antiviral breadth and combination potential of peptide triazole HIV-1 entry inhibitors.

The first stage of human immunodeficiency virus type 1 (HIV-1) infection involves the fusion of viral and host cellular membranes mediated by viral envelope glycoprotein gp120. Inhibitors that specifically target gp120 are gaining increased attention as therapeutics or preventatives to prevent the spread of HIV-1. One promising new group of inhibitors is the peptide triazoles, which bind to gp120 and simultaneously block its interaction with both CD4 and the coreceptor. In this study, we assessed the most potent peptide triazole, HNG-156, for inhibitory breadth, cytotoxicity, and efficacy, both alone and in combination with other antiviral compounds, against HIV-1. HNG-156 inhibited a panel of 16 subtype B and C isolates of HIV-1 in a single-round infection assay. Inhibition of cell infection by replication-competent clinical isolates of HIV-1 was also observed with HNG-156. We found that HNG-156 had a greater than predicted effect when combined with several other entry inhibitors or the reverse transcriptase inhibitor tenofovir. Overall, we find that HNG-156 is noncytotoxic, has a broad inhibition profile, and provides a positive combination with several inhibitors of the HIV-1 life cycle. These results support the pursuit of efficacy and toxicity analyses in more advanced cell and animal models to develop peptide triazole family inhibitors of HIV-1 into antagonists of HIV-1 infection.