Human Immunodeficiency Virus Entry Research Articles

Hybrid Virtual Screening Approach to Predict Novel Natural Compounds against HIV-1 CCR5.

Human immunodeficiency virus (HIV) infection continues to pose a major global health challenge. HIV entry into host cells via membrane fusion mediated by the viral envelope glycoprotein gp120/gp41 is a key step in the HIV life cycle. CCR5, expressed on CD4+ T cells and macrophages, acts as a coreceptor facilitating HIV-1 entry. The CCR5 antagonist maraviroc is used to treat HIV infection. However, it can cause adverse effects and has limitations such as only inhibiting CCR5-tropic viruses. There remains a need to develop alternative CCR5 inhibitors with improved safety profiles. Natural products may offer advantages over synthetic inhibitors including higher bioavailability, binding affinity, effectiveness, lower toxicity, and molecular diversity. However, screening the vast chemical space of natural compounds to identify novel CCR5 inhibitors presents challenges. This study aimed to address this gap through a hybrid ligand-based pharmacophore modeling and molecular docking approach to virtually screen large natural product databases. A reliable pharmacophore model was developed based on 311 known CCR5 antagonists and validated against an external data set. Five natural product databases containing over 306,000 compounds were filtered based on drug-likeness rules. The validated pharmacophore model screened the databases to identify 611 hits. Key residues of the CCR5 receptor crystal structure were identified for docking. The top hits were docked, and interactions were analyzed. Molecular dynamics simulations were conducted to examine complex stability. Computational prediction evaluated pharmacokinetic properties. Three compounds exhibited similar interactions and binding energies to maraviroc. MD simulations demonstrated complex stability comparable to maraviroc. One compound showed optimal predicted absorption, minimal metabolism, and a lower likelihood of interactions than maraviroc. This computational screening workflow identified three natural compounds with promising CCR5 inhibition and favorable pharmacokinetic profiles. One compound emerged as a lead based on bioavailability potential and minimal interaction risk. These findings present opportunities for developing alternative CCR5 antagonists and warrant further experimental investigation. Overall, the hybrid virtual screening approach proved effective for mining large natural product spaces to discover novel molecular entities with drug-like properties.

Glycan-Modified Peptides for Dual Inhibition of Human Immunodeficiency Virus Entry into Dendritic Cells and T Cells.

Dendritic cells (DCs) play a crucial role in HIV-1 infection of CD4+ T cells. DC-SIGN, a lectin expressed on the surface of DCs, binds to the highly mannosylated viral membrane protein gp120 to capture HIV-1 virions and then transport them to target T cells. In this study, we modified peptide C34, an HIV-1 fusion inhibitor, at different sites using different sizes of the DC-SIGN-specific carbohydrates to provide dual-targeted HIV inhibition. The dual-target binding was confirmed by mechanistic studies. Pentamannose-modified C34 inhibited virus entry into both DC-SIGN+ 293T cells (52%-71% inhibition at 500 μM) and CD4+ TZM-b1 cells (EC50 = 0.7-1.7 nM). One conjugate, NC-M5, showed an extended half-life relative to C34 in rats (T1/2: 7.8 vs 1.02 h). These improvements in antiviral activity and pharmacokinetics have potential for HIV treatment and the development of dual-target inhibitors for pathogens that require the involvement of DC-SIGN for infection.

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins.

CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

Killing two birds with one stone: CRISPR/Cas9 CCR5 knockout hematopoietic stem cells transplantation to treat patients with HIV infection and hematological malignancies concurrently.

Human immunodeficiency virus (HIV) is known to cause hematological malignancy. Hematopoietic stem cell transplantation (HPSCT) is an advanced treatment for that. Currently, there are three successful HIV-eliminated cases, and two received HPSCT from CCR5-absent donors. It is well established that the CCR5 protein on the cell surface assists human immunodeficiency virus entry. Preliminary studies have revealed that knocking out CCR5 and/or CXCR4 may inhibit the viral entry of HIV, which may prove promising in the further development of HIV treatment options. Herein, we suggest performing autologous or allogeneic HSCT with CCR5 KO hematopoietic stem cells in patients who suffer from complicated HIV conditions, particularly drug-resistant HIV or a concurrent diagnosis of HIV with lymphoma/leukemia, to achieve complete HIV remission. Nevertheless, at the clinical forefront of CRISPR-HIV technology, more efforts should be directed to advance nonhuman primate (NHP) models for studies of HIV pathogenesis and off-target assessments within this system. CRISPR-Cas9 knock out ofhost HSCT-expressing CCR5 or CXCR4 may confer HIV-resistance, which when applied to bedside therapeutics in an allogeneic or autologous manner can warrant a permanent and effective treatment outcome.

Downregulation of CCR5 on brain perivascular macrophages in simian immunodeficiency virus-infected rhesus macaques.

C-C chemokine receptor 5 (CCR5) is a major coreceptor for Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) cell entry; however, its role in brain pathogenesis is largely understudied. Thus, we sought to examine cell type-specific protein expression of CCR5 during SIV infection of the brain. We examined occipital cortical tissue from uninfected rhesus macaques and SIV-infected animals with or without encephalitis using immunohistochemistry and immunofluorescence microscopy to determine the number and distribution of CCR5-positive cells. An increase in the number of CCR5+ cells in the brain of SIV-infected animals with encephalitis was accounted for by increased CD3+CD8+ cells expressing CCR5, but not by increased CCR5+ microglia or perivascular macrophages (PVMs), and a concurrent decrease in the percentage of CCR5+ PVMs was observed. Levels of CCR5 and SIV Gag p28 protein expression were examined on a per-cell basis, and a significant, negative relationship was established indicating decreased CCR5 expression in productively infected cells. While investigating the endocytosis-mediated CCR5 internalization as a mechanism for CCR5 downregulation, we found that phospho-ERK1/2, an indicator of clathrin-mediated endocytosis, was colocalized with infected PVMs and that macrophages from infected animals showed significantly increased expression of clathrin heavy chain 1. These findings show a shift in CCR5-positive cell types in the brain during SIV pathogenesis with an increase in the number of CCR5+ CD8 T cells, and downregulated CCR5 expression on infected PVMs, likely through ERK1/2-driven, clathrin-mediated endocytosis.

Direct intranodal tonsil vaccination with modified vaccinia Ankara vaccine protects macaques from highly pathogenic SIVmac251

Human immunodeficiency virus (HIV) is a mucosally transmitted virus that causes immunodeficiency and AIDS. Developing efficacious vaccines to prevent infection is essential to control the epidemic. Protecting the vaginal and rectal mucosa, the primary routes of HIV entry has been a challenge given the significant compartmentalization between the mucosal and peripheral immune systems. We hypothesized that direct intranodal vaccination of mucosa associated lymphoid tissue (MALT) such as the readily accessible palatine tonsils could overcome this compartmentalization. Here we show that rhesus macaques primed with plasmid DNA encoding SIVmac251-env and gag genes followed by an intranodal tonsil MALT boost with MVA encoding the same genes protects from a repeated low dose intrarectal challenge with highly pathogenic SIVmac251; 43% (3/7) of vaccinated macaques remained uninfected after 9 challenges as compared to the unvaccinated control (0/6) animals. One vaccinated animal remained free of infection even after 22 challenges. Vaccination was associated with a ~2 log decrease in acute viremia that inversely correlated with anamnestic immune responses. Our results suggest that a combination of systemic and intranodal tonsil MALT vaccination could induce robust adaptive and innate immune responses leading to protection from mucosal infection with highly pathogenic HIV and rapidly control viral breakthroughs.

Characterization of the Human Immunodeficiency Virus (HIV-1) Envelope Glycoprotein Conformational States on Infectious Virus Particles.

Human immunodeficiency virus (HIV-1) entry into cells involves triggering of the viral envelope glycoprotein (Env) trimer ([gp120/gp41]3) by the primary receptor, CD4, and coreceptors, CCR5 or CXCR4. The pretriggered (State-1) conformation of the mature (cleaved) Env is targeted by broadly neutralizing antibodies (bNAbs), which are inefficiently elicited compared with poorly neutralizing antibodies (pNAbs). Here, we characterize variants of the moderately triggerable HIV-1AD8 Env on virions produced by an infectious molecular proviral clone; such virions contain more cleaved Env than pseudotyped viruses. We identified three types of cleaved wild-type AD8 Env trimers on virions: (i) State-1-like trimers preferentially recognized by bNAbs and exhibiting strong subunit association; (ii) trimers recognized by pNAbs directed against the gp120 coreceptor-binding region and exhibiting weak, detergent-sensitive subunit association; and (iii) a minor gp41-only population. The first Env population was enriched and the other Env populations reduced by introducing State-1-stabilizing changes in the AD8 Env or by treatment of the virions with crosslinker or the State-1-preferring entry inhibitor, BMS-806. These stabilized AD8 Envs were also more resistant to gp120 shedding induced by a CD4-mimetic compound or by incubation on ice. Conversely, a State-1-destabilized, CD4-independent AD8 Env variant exhibited weaker bNAb recognition and stronger pNAb recognition. Similar relationships between Env triggerability and antigenicity/shedding propensity on virions were observed for other HIV-1 strains. State-1 Envs on virions can be significantly enriched by minimizing the adventitious incorporation of uncleaved Env; stabilizing the pretriggered conformation by Env modification, crosslinking or BMS-806 treatment; strengthening Env subunit interactions; and using CD4-negative producer cells. IMPORTANCE Efforts to develop an effective HIV-1 vaccine have been frustrated by the inability to elicit broad neutralizing antibodies that recognize multiple virus strains. Such antibodies can bind a particular shape of the HIV-1 envelope glycoprotein trimer, as it exists on a viral membrane but before engaging receptors on the host cell. Here, we establish simple yet powerful assays to characterize the envelope glycoproteins in a natural context on virus particles. We find that, depending on the HIV-1 strain, some envelope glycoproteins change shape and fall apart, creating decoys that can potentially divert the host immune response. We identify requirements to keep the relevant envelope glycoprotein target for broad neutralizing antibodies intact on virus-like particles. These studies suggest strategies that should facilitate efforts to produce and use virus-like particles as vaccine immunogens.

Structural dynamics of chemokine receptors.

Membrane proteins such as G protein-coupled receptors (GPCRs) are involved in awide range of physiological and pathological cellular processes. Binding of extracellular signals to GPCRs, including hormones, neurotransmitters, peptides and proteins, can activate intracellular signaling cascades via G protein interaction. Chemokine receptors are key GPCRs implicated in cancers, immune responses, cell migration and inflammation. Specifically, the CCR5 and CXCR4 chemokine receptors serve as important therapeutic targets against Human Immunodeficiency virus (HIV) entry into human cells. Maraviroc and Vicriviroc, two clinically used HIV entry inhibitors, are antagonists of the CCR5 receptor. These drugs block HIV entry, but ultimately resistance develops, due to emergence of viruses that can utilize the CXCR4 co-receptor. Unfortunately, development of chemokine receptor antagonists as selective drugs of HIV infection has been greatly hindered as their target orthosteric site is conserved among different receptor subtypes. Accordingly, it is important to understand the structural dynamics of these receptors to develop more effective therapeutics. In this chapter, we describe the latest advances in studies of these two key chemokine receptors with respect to their structures, dynamics and function.

Investigation of the CC chemokine receptor 5 (CCR5) delta32 and gene variants in HIV infected patients

CCR5 is an important CC chemokine receptor for the normal entry of Human Immunodeficiency Virus (HIV) in host cells. Mutations in this gene have been linked with delayed HIV infection, therefore, the current case-control study was conducted to identify genetic polymorphism in CCR5 gene in Pakistani population and to evaluate its association with resistance for HIV infections. All regulations of MOOSE and Helsinki Declaration were strictly followed during the entire study. DNA was extracted from the blood samples of HIV positive and HIV negative patients. Extracted DNA was amplified for CCR5 gene by PCR. Amplified product was sequenced to screen out polymorphism mutation, including most prevalent 32 base-pairs deletion through Bioedit. Novel SNP in 5?UTR region was identified and examined by CRYP-SKIP server. None of the studied samples demonstrated any previously reported polymorphisms. CRYP-SKYP server predicted that this polymorphism has no effect on splicing or transcription of CCR5 gene. Genetic Polymorphism of delta32 mutation in CCR5 gene was found in Pakistani individuals. To the best of our knowledge, this is the first report from this region. However, large scale studies should be conducted for extensive view of the association of delta32 mutation in CCR5 gene and resistance for HIV infection.

Pharmacophore-Oriented Identification of Potential Leads as CCR5 Inhibitors to Block HIV Cellular Entry.

Cysteine-cysteine chemokine receptor 5 (CCR5) has been discovered as a co-receptor for cellular entry of human immunodeficiency virus (HIV). Moreover, the role of CCR5 in a variety of cancers and various inflammatory responses was also discovered. Despite the fact that several CCR5 antagonists have been investigated in clinical trials, only Maraviroc has been licensed for use in the treatment of HIV patients. This indicates that there is a need for novel CCR5 antagonists. Keeping this in mind, the present study was designed. The active CCR5 inhibitors with known IC50 value were selected from the literature and utilized to develop a ligand-based common feature pharmacophore model. The validated pharmacophore model was further used for virtual screening of drug-like databases obtained from the Asinex, Specs, InterBioScreen, and Eximed chemical libraries. Utilizing computational methods such as molecular docking studies, molecular dynamics simulations, and binding free energy calculation, the binding mechanism of selected inhibitors was established. The identified Hits not only showed better binding energy when compared to Maraviroc, but also formed stable interactions with the key residues and showed stable behavior throughout the 100 ns MD simulation. Our findings suggest that Hit1 and Hit2 may be potential candidates for CCR5 inhibition, and, therefore, can be considered for further CCR5 inhibition programs.

Characterization of Human Immunodeficiency Virus (HIV-1) Envelope Glycoprotein Variants Selected for Resistance to a CD4-Mimetic Compound.

Binding to the host cell receptors CD4 and CCR5/CXCR4 triggers conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer that promote virus entry. CD4 binding allows the gp120 exterior Env to bind CCR5/CXCR4 and induces a short-lived prehairpin intermediate conformation in the gp41 transmembrane Env. Small-molecule CD4-mimetic compounds (CD4mcs) bind within the conserved Phe-43 cavity of gp120, near the binding site for CD4. CD4mcs like BNM-III-170 inhibit HIV-1 infection by competing with CD4 and by prematurely activating Env, leading to irreversible inactivation. In cell culture, we selected and analyzed variants of the primary HIV-1AD8 strain resistant to BNM-III-170. Two changes (S375N and I424T) in gp120 residues that flank the Phe-43 cavity each conferred an ~5-fold resistance to BNM-III-170 with minimal fitness cost. A third change (E64G) in layer 1 of the gp120 inner domain resulted in ~100-fold resistance to BNM-III-170, ~2- to 3-fold resistance to soluble CD4-Ig, and a moderate decrease in viral fitness. The gp120 changes additively or synergistically contributed to BNM-III-170 resistance. The sensitivity of the Env variants to BNM-III-170 inhibition of virus entry correlated with their sensitivity to BNM-III-170-induced Env activation and shedding of gp120. Together, the S375N and I424T changes, but not the E64G change, conferred >100-fold and 33-fold resistance to BMS-806 and BMS-529 (temsavir), respectively, potent HIV-1 entry inhibitors that block Env conformational transitions. These studies identify pathways whereby HIV-1 can develop resistance to CD4mcs and conformational blockers, two classes of entry inhibitors that target the conserved gp120 Phe-43 cavity. IMPORTANCE CD4-mimetic compounds (CD4mcs) and conformational blockers like BMS-806 and BMS-529 (temsavir) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. Although CD4mcs and conformational blockers inhibit HIV-1 entry by different mechanisms, they both target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor CD4 and is highly conserved among HIV-1 strains. Our study identifies changes near this pocket that can confer various levels of resistance to the antiviral effects of a CD4mc and conformational blockers. We relate the antiviral potency of a CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate changes in Env conformation and to induce the shedding of the gp120 exterior Env from the spike. These findings will guide efforts to improve the potency and breadth of small-molecule HIV-1 entry inhibitors.

Targeting C-C Chemokine Receptor 5: Key to Opening the Neurorehabilitation Window After Ischemic Stroke.

Stroke is the world’s second major cause of adult death and disability, resulting in the destruction of brain tissue and long-term neurological impairment; induction of neuronal plasticity can promote recovery after stroke. C–C chemokine receptor 5 (CCR5) can direct leukocyte migration and localization and is a co-receptor that can mediate human immunodeficiency virus (HIV) entry into cells. Its role in HIV infection and immune response has been extensively studied. Furthermore, CCR5 is widely expressed in the central nervous system (CNS), is engaged in various physiological activities such as brain development, neuronal differentiation, communication, survival, and learning and memory capabilities, and is also involved in the development of numerous neurological diseases. CCR5 is differentially upregulated in neurons after stroke, and the inhibition of CCR5 in specific regions of the brain promotes motor and cognitive recovery. The mechanism by which CCR5 acts as a therapeutic target to promote neurorehabilitation after stroke has rarely been systematically reported yet. Thus, this review aims to discuss the function of CCR5 in the CNS and the mechanism of its effect on post-stroke recovery by regulating neuroplasticity and the inflammatory response to provide an effective basis for clinical rehabilitation after stroke.

Impact of Caveolin-Mediated Endocytosis on the Trafficking of HIV within the Colonic Barrier.

ABSTRACTIn the United States, most new cases of human immunodeficiency virus (HIV) belong to the at-risk group of gay and bisexual men. Developing therapies to reverse viral latency and prevent spread is paramount for the HIV cure agenda. In gay and bisexual men, a major, yet poorly characterized, route of HIV entry is via transport across the colonic epithelial barrier. While colonic tears and paracellular transport contribute to infection, we hypothesize that HIV entry through the colonic mucosa proceeds via a process known as transcytosis, involving (i) virion binding to the apical surface of the colonic epithelium, (ii) viral endocytosis, (iii) transport of virions across the cell, and (iv) HIV release from the basolateral membrane. Using Caco-2 colonic epithelial cells plated as a polarized monolayer in transwells, we characterized the mechanism of HIV transport. After exposing the monolayer to HIV apically, reverse transcription quantitative PCR (RT-qPCR) of the viral genome present in the basolateral chamber revealed that transport is dose dependent, cooperative, and inefficient, with released virus first detectable at 12 h. Inefficiency may be associated with >50% decline in detectable intracellular virus that correlates temporally with increased association of the virion with lysosomal-associated membrane protein 1 (LAMP-1+) endosomes. Microscopy revealed green fluorescent protein (GFP)-labeled HIV within the confines of the epithelial monolayer, with no virus detectable between cells, suggesting that viral transport is transcellular. Treatment of the monolayer with endocytosis inhibitors, cholesterol reducing agents, and small interfering RNA (siRNA) to caveolin showed that viral endocytosis is mediated by caveolin-coated endosomes contained in lipid rafts. These results indicate that HIV transport across the intestinal epithelial barrier via transcytosis is a viable mechanism for viral spread and a potential therapeutic target.IMPORTANCE Despite the success of combination antiretroviral therapy in suppressing HIV replication and the emergence and effectiveness of PrEP-based prevention strategies, in 2018, 37,968 people in the United States received a new HIV diagnosis, accompanied by 15,820 deaths. While the annual number of new diagnoses decreased 7% from 2014 to 2018, 14% of people with HIV did not know they were infected. Gay and bisexual men accounted for 69% of all HIV diagnoses and 83% of diagnoses among males. Due to the scope of the HIV epidemic, determining and understanding precise routes of infection and the mechanisms of viral spread are paramount to ending the epidemic. Since transcellular transport of HIV across an intact colonic epithelial barrier is poorly understood, our overall goal is to characterize the molecular events involved in HIV transcytosis across the intestinal epithelial cell.

The inhibition effects and mechanisms of sulfated chitooligosaccharides on influenza A virus in vitro and in vivo

Sulfated chitooligosaccharide was reported to possess inhibition effect on human immunodeficiency virus (HIV) entry into host cells. Herein, we prepared chitooligosaccharide COS and its sulfate derivative SCOS and explored whether the sulfation modification can enhance the anti-influenza A virus (IAV) activity of COS. Interestingly, we discovered that SCOS possessed broad-spectrum anti-IAV effects with low toxicity, while the non-sulfated chitooligosaccharide COS had very low inhibition on IAV, verifying that the sulfation modification is essential for the anti-IAV actions of chitooligosaccharide. SCOS may target virus hemagglutinin (HA) protein to block both virus adsorption and membrane fusion processes. Oral administration of SCOS significantly decreased pulmonary viral titers and improved survival rate in IAV infected mice, comparable to the effects of Oseltamivir. Therefore, our findings support further studies on the use of SCOS as a novel entry inhibitor for IAV and as a supplement to current therapeutics for influenza.

Synthesis and Evaluation of Novel Tetrahydronaphthyridine CXCR4 Antagonists with Improved Drug-like Profiles.

Our first-generation CXCR4 antagonist TIQ15 was rationally modified to improve drug-like properties. Introducing a nitrogen atom into the aromatic portion of the tetrahydroisoquinoline ring led to several heterocyclic variants including the 5,6,7,8-tetrahydro-1,6-naphthyridine series, greatly reducing the inhibition of the CYP 2D6 enzyme. Compound 12a demonstrated the best overall properties after profiling a series of isomeric tetrahydronaphthyridine analogues in a battery of biochemical assays including CXCR4 antagonism, CYP 2D6 inhibition, metabolic stability, and permeability. The butyl amine side chain of 12a was substituted with various lipophilic groups to improve the permeability. These efforts culminated in the discovery of compound 30 as a potent CXCR4 antagonist (IC50 = 24 nM) with diminished CYP 2D6 activity, improved PAMPA permeability (309 nm/s), potent inhibition of human immunodeficiency virus entry (IC50 = 7 nM), a cleaner off-target in vitro safety profile, lower human ether a-go-go-related gene channel activity, and higher oral bioavailability in mice (% FPO = 27) compared to AMD11070 and TIQ15.

TLR7 Activation of Macrophages by Imiquimod Inhibits HIV Infection through Modulation of Viral Entry Cellular Factors.

Simple SummaryThe Toll-like receptor (TLR) 7 is highly expressed by immune cells including macrophages. Agonists to TLR7 are attractive therapeutic agents as they have the potential of activating both innate and acquired immunity against viral infections. Imiquimod, a specific TLR7 agonist, has been successfully used for the topical treatment of genital/perianal warts in immunocompetent individuals. Here we examined the anti-HIV effect of imiquimod in primary human macrophages and demonstrated that TLR7 activation by imiquimod could effectively inhibit infection of the cells by different strains of HIV. Further mechanistic studies revealed that while imiquimod had little effect on interferons expression, its treatment of macrophages resulted in the increased production of the CC chemokines, the natural ligands of the HIV entry co-receptor CCR5, and decreased expression of CD4 and CCR5. These findings are clinically important and indicate that activating the intracellular antiviral immunity by imiquimod has the potential for developing TLR7 agonist-based therapy for HIV infection.The Toll-like receptor (TLR) 7 is a viral sensor for detecting single-stranded ribonucleic acid (ssRNA), the activation of which can induce intracellular innate immunity against viral infections. Imiquimod, a synthetic ligand for TLR7, has been successfully used for the topical treatment of genital/perianal warts in immunocompetent individuals. We studied the effect of imiquimod on the human immunodeficiency virus (HIV) infection of primary human macrophages and demonstrated that the treatment of cells with imiquimod effectively inhibited infection with multiple strains (Bal, YU2, and Jago) of HIV. This anti-HIV activity of imiquimod was the most potent when macrophages were treated prior to infection. Infection of macrophages with pseudotyped HIV NL4-3-ΔEnv-eGFP-Bal showed that imiquimod could block the viral entry. Further mechanistic studies revealed that while imiquimod had little effect on the interferons (IFNs) expression, its treatment of macrophages resulted in the increased production of the CC chemokines (human macrophage inflammatory protein-1 alpha (MIP-1α), MIP-1β, and upon activation regulated normal T cells expressed and secreted (RANTES)), the natural ligands of HIV entry co-receptor CCR5, and decreased the expression of CD4 and CCR5. The addition of the antibodies against the CC chemokines to macrophage cultures could block imiquimod-mediated HIV inhibition. These findings provide experimental evidence to support the notion that TLR7 participates in the intracellular immunity against HIV in macrophages, suggesting the further clinical evaluation of imiquimod for its additional benefit of treating genital/perianal warts in people infected with HIV.

Double Arylation of the Indole Side Chain of Tri- and Tetrapodal Tryptophan Derivatives Renders Highly Potent HIV-1 and EV-A71 Entry Inhibitors†.

We have recently described a new generation of potent human immunodeficiency virus (HIV) and EV-A71 entry inhibitors. The prototypes contain three or four tryptophan (Trp) residues bearing an isophthalic acid moiety at the C2 position of each side-chain indole ring. This work is now extended by both shifting the position of the isophthalic acid to C7 and synthesizing doubly arylated C2/C7 derivatives. The most potent derivative (50% effective concentration (EC50) HIV-1, 6 nM; EC50 EV-A71, 40 nM), 33 (AL-518), is a C2/C7 doubly arylated tetrapodal compound. Its superior anti-HIV potency with respect to the previous C2-arylated prototype is in consonance with its higher affinity for the viral gp120. 33 (AL-518) showed comparable antiviral activities against X4 and R5 HIV-1 strains and seems to interact with the tip and base of the gp120 V3 loop. Taken together, these findings support the interest in 33 (AL-518) as a useful new prototype for anti-HIV/EV71 drug development.

Therapeutic potential of HIV-1 entry inhibitor peptidomimetics.

Human immunodeficiency virus 1 (HIV-1) infection remains a public health concern globally. Although great strides in the management of HIV-1 have been achieved, current highly active antiretroviral therapy is limited by multidrug resistance, prolonged use-related effects, and inability to purge the HIV-1 latent pool. Even though novel therapeutic options with HIV-1 broadly neutralizing antibodies (bNAbs) are being explored, the scalability of bNAbs is limited by economic cost of production and obligatory requirement for parenteral administration. However, these limitations can be addressed by antibody mimetics/peptidomimetics of HIV-1 bNAbs. In this review we discuss the limitations of HIV-1 bNAbs as HIV-1 entry inhibitors and explore the potential therapeutic use of antibody mimetics/peptidomimetics of HIV-1 entry inhibitors as an alternative for HIV-1 bNAbs. We highlight the reduced cost of production, high specificity, and oral bioavailability of peptidomimetics compared to bNAbs to demonstrate their suitability as candidates for novel HIV-1 therapy and conclude with some perspectives on future research toward HIV-1 novel drug discovery.

A sensitive method to quantify HIV-1 antibodies in mucosal samples

Human immunodeficiency virus (HIV) remains a significant public health issue. In recent years, passive immunization with broadly neutralizing antibodies (bNabs) is being considered as a potentially efficacious approach for fighting HIV. One candidate that holds great promise is represented by the CD4-binding site targeted bNab capable of neutralizing over 90% of circulating HIV strains, VRC01. VRC01 along with its variants and clonal relatives – VRC01-LS and VRC07-523LS are currently being evaluated as vaccines in a number of clinical trials for HIV treatment and prevention. While mucosal areas of the body serve as major ports of HIV entry, reliable quantification of bNabs for pharmacokinetic and bioavailability analyses has been challenging due to low antibody concentrations in these samples. We developed an immunoassay on the Singulex platform which enables ultra-sensitive quantification of VRC01, VRC07, VRC01-LS and VRC07-523LS with a greater than 4-log linear dynamic range (LDR) and less than 120 pg/mL lower limit of quantitation (LLOQ). We implemented this assay to quantify VRC01 levels in rectal, cervical and oral mucosal samples in two passive immunization studies conducted with VRC01 – VRC 601 and VRC 602. Our assay was able to successfully quantify VRC01 levels in mucosal samples from all dosage groups (5 – –40 mg/kg) in these trials. VRC01 levels in a significant proportion of these samples (37% in oral and 25% in rectal mucosa) were below the lower limits of quantitation of other traditional immunoassays used for VRC01 quantification. We also measured VRC01 levels in sera from these trials and found that VRC01 measurements made using our assay exhibited excellent correlation (r2 = 0.9509) with measurements made previously using Enzyme-linked immunosorbent assay (ELISA). Our assay provides a reliable, sensitive and accurate method for quantification of clinically relevant bNabs and will help delineate antibody infiltration and bioavailability characteristics in complex biological matrices (CBM) such as mucosal tissues. This will in turn help determine clinical antibody threshold concentrations required to mediate protection against HIV acquisition and serve to inform dosing regimens and clinical trial design for future efficacy trials with these bNabs.

Specific synonymous mutations tightly correlate with HIV-1 co-receptor usage and differentially affect the secondary structure of HIV-1 Env RNA.

Human immunodeficiency virus (HIV) is a pathogen that infects blood cells, using CD4 molecule and two cell receptors CCR5 and CXCR4. The other major actor is gp120/gp41 viral protein complex, which interacts with receptors. Here, the presence of synonymous mutations associated with HIV-1 tropism and the related RNA secondary-structure in HIV-1 infected patients was evaluated. The analysis includes gp120-sequences from 340 HIV-1 subtype-B infected patients, all retrieved from Los Alamos database and with phenotypic HIV tropism determination based on recombinant-virus entry-assay. Frequencies of all nucleotide substitutions were calculated. Mfold and RNAfold algorithms were used to predict RNA secondary-structure of HIV-1. Nineteen codons in V2/C2, V3 and C3 domains were found to be closely related to CCR5 and CXCR4. Additionally, in X4-sequences, gp120 gca303gcu and gua222guc synonymous mutations are positively related to the gp120 S11R and T8A/I codons in V3 protein domain. Furthermore, gua222guc increases stability of the viral RNA secondary-structure. Probably, it would not be surprising if a novel escape viral strategy therapy will be related to the gp120 synonymous mutations. Moreover, in relation to the pivotal role played by gp120 in polyvalent vaccine approaches, the impact of gp120 synonymous mutations may play an important role in HIV entry into the cell. Keywords: gp120; tropism; v3; s11r; evolution; vaccine.