Jacqueline D. Reeves

Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics

HIV entry inhibitors include coreceptor antagonists and the fusion inhibitor T-20. T-20 binds the first helical region (HR1) in the gp41 subunit of the viral envelope (Env) protein and prevents conformational changes required for membrane fusion. HR1 appears to become accessible to T-20 after Env binds CD4, whereas coreceptor binding is thought to induce the final conformational changes that lead to membrane fusion. Thus, T-20 binds to a structural intermediate of the fusion process. Primary viruses exhibit considerable variability in T-20 sensitivity, and determinants outside of HR1 can affect sensitivity by unknown mechanisms. We studied chimeric Env proteins containing different V3 loop sequences and found that gp120/coreceptor affinity correlated with T-20 and coreceptor antagonist sensitivity, with greater affinity resulting in increased resistance to both classes of entry inhibitors. Enhanced affinity resulted in more rapid fusion kinetics, reducing the time during which Env is sensitive to T-20. Reduced coreceptor expression levels also delayed fusion kinetics and enhanced virus sensitivity to T-20, whereas increased coreceptor levels had the opposite effect. A single amino acid change (K421D) in the bridging sheet region of the primary virus strain YU2 reduced affinity for CCR5 and increased T-20 sensitivity by about 30-fold. Thus, mutations in Env that affect receptor engagement and membrane fusion rates can alter entry inhibitor sensitivity. Because coreceptor expression levels are typically limiting in vivo, individuals who express lower coreceptor levels may respond more favorably to entry inhibitors such as T-20, whose effectiveness we show depends in part on fusion kinetics.

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell–cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell–cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell–cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.

Maraviroc versus Efavirenz, Both in Combination with Zidovudine-Lamivudine, for the Treatment of Antiretroviral-Naive Subjects with CCR5-tropic HIV-1 Infection

BACKGROUND The MERIT (Maraviroc versus Efavirenz in Treatment-Naive Patients) study compared maraviroc and efavirenz, both with zidovudine-lamivudine, in antiretroviral-naive patients with R5 human immunodeficiency virus type 1 (HIV-1) infection. METHODS Patients screened for R5 HIV-1 were randomized to receive efavirenz (600 mg once daily) or maraviroc (300 mg once or twice daily) with zidovudine-lamivudine. Coprimary end points were proportions of patients with a viral load <400 and <50 copies/mL at week 48; the noninferiority of maraviroc was assessed. RESULTS The once-daily maraviroc arm was discontinued for not meeting prespecified noninferiority criteria. In the primary 48-week analysis (n = 721), maraviroc was noninferior for <400 copies/mL (70.6% for maraviroc vs 73.1% for efavirenz) but not for <50 copies/mL (65.3% vs 69.3%) at a threshold of -10%. More maraviroc patients discontinued for lack of efficacy (11.9% vs 4.2%), but fewer discontinued for adverse events (4.2% vs 13.6%). In a post hoc reanalysis excluding 107 patients (15%) with non-R5 screening virus by the current, more sensitive tropism assay, the lower bound of the 1-sided 97.5% confidence interval for the difference between treatment groups was above -10% for each end point. CONCLUSIONS Twice-daily maraviroc was not noninferior to efavirenz at <50 copies/mL in the primary analysis. However, 15% of patients would have been ineligible for inclusion by a more sensitive screening assay. Their retrospective exclusion resulted in similar response rates in both arms Trial registration. ClinicalTrials.gov identifier: (NCT00098293) .

Use of a small molecule CCR5 inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian-human immunodeficiency virus infection

Human immunodeficiency virus type 1 (HIV-1) fuses with cells after sequential interactions between its envelope glycoproteins, CD4 and a coreceptor, usually CC chemokine receptor 5 (CCR5) or CXC receptor 4 (CXCR4). CMPD 167 is a CCR5-specific small molecule with potent antiviral activity in vitro. We show that CMPD 167 caused a rapid and substantial (4–200-fold) decrease in plasma viremia in six rhesus macaques chronically infected with simian immunodeficiency virus (SIV) strains SIVmac251 or SIVB670, but not in an animal infected with the X4 simian–human immunodeficiency virus (SHIV), SHIV-89.6P. In three of the SIV-infected animals, viremia reduction was sustained. In one, there was a rapid, but partial, rebound and in another, there was a rapid and complete rebound. There was a substantial delay (>21 d) between the end of therapy and the onset of full viremia rebound in two animals. We also evaluated whether vaginal administration of gel-formulated CMPD 167 could prevent vaginal transmission of the R5 virus, SHIV-162P4. Complete protection occurred in only 2 of 11 animals, but early viral replication was significantly less in the 11 CMPD 167-recipients than in 9 controls receiving carrier gel. These findings support the development of small molecule CCR5 inhibitors as antiviral therapies, and possibly as components of a topical microbicide to prevent HIV-1 sexual transmission.

Truncation of the Cytoplasmic Domain Induces Exposure of Conserved Regions in the Ectodomain of Human Immunodeficiency Virus Type 1 Envelope Protein

ABSTRACT We have described a CD4-independent variant of HXBc2, termed 8x, that binds directly to CXCR4 and mediates CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4-C4 domains together with a single nucleotide deletion in the transmembrane domain which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of 27 amino acids. We demonstrate here that while introduction of the 8x FS mutation into heterologous R5, X4, or R5X4 Env proteins did not impart CD4 independence, it did affect the conformation of the gp120 surface subunit, exposing highly conserved domains involved in both coreceptor and CD4 binding. In addition, antigenic changes in the gp41 ectodomain were also observed, consistent with the idea that the effects of cytoplasmic domain truncation must in some way be transmitted to the external gp120 subunit. Truncation of gp41 also resulted in the marked neutralization sensitivity of all Env proteins tested to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may prove useful for vaccine development.

Enfuvirtide Resistance Mutations: Impact on Human Immunodeficiency Virus Envelope Function, Entry Inhibitor Sensitivity, and Virus Neutralization

ABSTRACT Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed.

Co-receptor use by HIV and inhibition of HIV infection by chemokine receptor ligands

Human and simian immunodeficiency viruses (HIV and SIV) require a seven transmembrane chemokine (7TM) receptor in addition to CD4 for efficient entry into cells. CCR5 and CXCR4 act as major co-receptors for non-syncytium-inducing and syncytium-inducing strains respectively. We have examined the co-receptor requirement for HIV-1 infection of cells of macrophage lineage. Both CCR5 and CXCR4 can operate as functional co-receptors for infection in these cell types. Other co-receptors utilised by multi-co-receptor-using strains of HIV-1, including CCR3 and STRL33, were not used for macrophage infection. HIV-2 and SIV strains, however, can replicate in both peripheral blood mononuclear cells (PBMCs) and other primary cell types such as fibroblasts independently of CCR5 or CXCR4. HIV co-receptors, particularly CCR5, will be major targets for new therapeutics in this decade. We have therefore investigated different chemokines and derivatives that bind co-receptors for their capacity to inhibit HIV infection. These included derivatives of a CCR5 ligand, RANTES, with modified N-termini as well as Kaposis sarcoma-associated herpesvirus-encoded chemokines that bind a wide range of co-receptors, including CCR5, CXCR4, CCR3 and CCR8, as well as the orphan 7TM receptors GPR1 and STRL33. One compound, aminooxypentane or AOP-RANTES, was a particularly potent inhibitor of HIV infection on PBMCs, macrophages and CCR5+ cell lines and demonstrated the great promise of therapeutic strategies aimed at CCR5.

Emerging Drug Targets for Antiretroviral Therapy

Current targets for antiretroviral therapy (ART) include the viral enzymes reverse transcriptase and protease. The use of a combination of inhibitors targeting these enzymes can reduce viral load for a prolonged period and delay disease progression. However, complications of ART, including the emergence of viruses resistant to current drugs, are driving the development of new antiretroviral agents targeting not only the reverse transcriptase and protease enzymes but novel targets as well. Indeed, enfuvirtide, an inhibitor targeting the viral envelope protein (Env) was recently approved for use in combination therapy in individuals not responding to current antiretroviral regimens.Emerging drug targets for ART include: (i) inhibitors that directly or indirectly target Env; (ii) the HIV enzyme integrase; and (iii) inhibitors of maturation that target the substrate of the protease enzyme. Env mediates entry of HIV into target cells via a multistep process that presents three distinct targets for inhibition by viral and cellular-specific agents. First, attachment of virions to the cell surface via nonspecific interactions and CD4 binding can be blocked by inhibitors that include cyanovirin-N, cyclotriazadisulfonamide analogues, PRO 2000, TNX 355 and PRO 542. In addition, BMS 806 can block CD4-induced conformational changes. Secondly, Env interactions with the co-receptor molecules can be targeted by CCR5 antagonists including SCH-D, maraviroc (UK 427857) and aplaviroc GW 873140), and the CXCR4 antagonist AMD 070. Thirdly, fusion of viral and cellular membranes can be inhibited by peptides such as enfuvirtide and tifuvirtide (T 1249). The development of entry inhibitors has been rapid, with an increasing number entering clinical trials. Moreover, some entry inhibitors are also being evaluated as candidate microbicides to prevent mucosal transmission of HIV.The integrase enzyme facilitates the integration of viral DNA into the host cell genome. The uniqueness and specificity of this reaction makes integrase an attractive drug target. However, integrase inhibitors have been slow to reach clinical development, although recent contenders, including L 870810, show promise. Inhibitors that target viral maturation via a unique mode of action, such as PA 457, also have potential. In addition, recent advances in our understanding of cellular pathways involved in the life cycle of HIV have also identified novel targets that may have potential for future antiretroviral intervention, including interactions between the cellular proteins APOBEC3G and TSG101, and the viral proteins Vif and p6, respectively.In summary, a number of antiretroviral agents in development make HIV entry, integration and maturation emerging drug targets. A multifaceted approach to ART, using combinations of inhibitors that target different steps of the viral life cycle, has the best potential for long-term control of HIV infection. Furthermore, the development of microbicides targeting HIV holds promise for reducing HIV transmission events.

Virally Induced CD4 + T Cell Depletion Is Not Sufficient to Induce AIDS in a Natural Host

Peripheral blood CD4+ T cell counts are a key measure for assessing disease progression and need for antiretroviral therapy in HIV-infected patients. More recently, studies have demonstrated a dramatic depletion of mucosal CD4+ T cells during acute infection that is maintained during chronic pathogenic HIV as well as SIV infection. A different clinical disease course is observed during the infection of natural hosts of SIV infection, such as sooty mangabeys (Cercocebus atys), which typically do not progress to AIDS. Previous studies have determined that SIV+ mangabeys generally maintain healthy levels of CD4+ T cells despite having viral replication comparable to HIV-infected patients. In this study, we identify the emergence of a multitropic (R5/X4/R8-using) SIV infection after 43 or 71 wk postinfection in two mangabeys that is associated with an extreme, persistent (>5.5 years), and generalized loss of CD4+ T cells (5–80 cells/μl of blood) in the absence of clinical signs of AIDS. This study demonstrates that generalized CD4+ T cell depletion from the blood and mucosal tissues is not sufficient to induce AIDS in this natural host species. Rather, AIDS pathogenesis appears to be the cumulative result of multiple aberrant immunologic parameters that include CD4+ T cell depletion, generalized immune activation, and depletion/dysfunction of non-CD4+ T cells. Therefore, these data provide a rationale for investigating multifaceted therapeutic strategies to prevent progression to AIDS, even following dramatic CD4 depletion, such that HIV+ humans can survive normal life spans analogous to what occurs naturally in SIV+ mangabeys.

Impact of Mutations in the Coreceptor Binding Site on Human Immunodeficiency Virus Type 1 Fusion, Infection, and Entry Inhibitor Sensitivity

ABSTRACT An increasingly large number of antiviral agents that prevent entry of human immunodeficiency virus (HIV) into cells are in preclinical and clinical development. The envelope (Env) protein of HIV is the major viral determinant that affects sensitivity to these compounds. To understand how changes in Env can impact entry inhibitor sensitivity, we introduced six mutations into the conserved coreceptor binding site of the R5 HIV-1 strain YU-2 and measured the effect of these changes on CD4 and coreceptor binding, membrane fusion levels and rates, virus infection, and sensitivity to the fusion inhibitors enfuvirtide (T-20) and T-1249, the CCR5 inhibitor TAK-779, and an antibody to CD4. The mutations had little effect on CD4 binding but reduced CCR5 binding to various extents. In general, reductions in coreceptor binding efficiency resulted in slower fusion kinetics and increased sensitivity to TAK-779 and enfuvirtide. In addition, low CCR5 binding usually reduced overall fusion and infection levels. However, one mutation adjacent to the bridging sheet β21 strand, P438A, had little effect on fusion activity, fusion rate, infectivity, or sensitivity to enfuvirtide or T-1249 despite causing a marked reduction in CCR5 binding and a significant increase in TAK-779 sensitivity. Thus, our findings indicate that changes in the coreceptor binding site of Env can modulate its fusion activity, infectivity, and entry inhibitor sensitivity by multiple mechanisms and suggest that reductions in coreceptor binding do not always result in prolonged fusion kinetics and increased sensitivity to enfuvirtide.