Growth of macrophage-tropic and primary human immunodeficiency virus type 1 (HIV-1) isolates in a unique CD4+ T-cell clone (PM1): failure to downregulate CD4 and to interfere with cell-line-tropic HIV-1.

Human immunodeficiency virus type 1 (HIV-1) isolates derived directly from clinical samples are usually unable to grow in cytokine-independent continuous cell lines, thus hindering the study of their biological features and their sensitivity to humoral and cellular protective immunity. To overcome these limitations, we have derived from the Hut78 T-cell line a CD4+ clone (PM1) characterized by a unique susceptibility to a wide range of HIV-1 isolates, including primary and biologically pure macrophage (M phi)-tropic isolates (e.g., HIV-1BaL), which are unable to infect other human T- or promonocytic cell lines. Both primary and M phi-tropic HIV-1 establish persistent infection in PM1, with sustained levels of virus replication for prolonged periods. Experiments with chimeric viruses containing envelope fragments of HIV-1BAL inserted into the genetic framework of HXB2, a molecular clone derived from the cell-line-tropic isolate HIV-1IIIB, showed the third hypervariable domain (V3) of gp120 to be a critical determinant of the cell line tropism of HIV-1. Nevertheless, the V3 loop of HIV-1BaL was not sufficient to confer on the chimeras a bona fide M phi tropism. The biological characteristics of HIV-1BaL and of a primary isolate (HIV-1(573)) were investigated by using the PM1 clone. Infection of PM1 by HIV-1BaL was critically dependent on the CD4 receptor, as shown by competition experiments with an anti-CD4 monoclonal antibody (OKT4a) or with soluble CD4. However, the amount of soluble CD4 required for inhibition of HIV-1BaL was approximately 100-fold higher than for HIV-1IIIB, suggesting that the affinity of HIV-1BaL for CD4 is significantly lower. Infection of PM1 with either HIV-1BaL or HIV-1(573) failed to induce downregulation of surface CD4 expression and syncytium formation. Analogous results were obtained with a chimeric virus (HXB2[BaL PvuII-BamHI]) encompassing a large portion of gp120 and gp41 of HIV-1BaL, indicating that the env genes contain critical determinants for CD4 downregulation and syncytium formation. Consistent with the lack of CD4 downregulation, persistent infection of PM1 by HIV-1BaL or HIV-1(573) failed to interfere with HIV-1IIIB superinfection, as revealed by the expression of a type-specific V3 loop epitope (M77) and by the induction of extensive syncytium formation. This lack of interference suggests that a direct viral interaction may occur in vivo between biologically diverse HIV-1 strains.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relative replicative fitness of human immunodeficiency virus type 1 mutants resistant to enfuvirtide (T-20)

Resistance to enfuvirtide (ENF; T-20), a fusion inhibitor of human immunodeficiency virus type 1 (HIV-1), is conferred by mutations in the first heptad repeat of the gp41 ectodomain. The replicative fitness of recombinant viruses carrying ENF resistance mutations was studied in growth competition assays. ENF resistance mutations, selected in vitro or in vivo, were introduced into the env gene of HIV-1(NL4-3) by site-directed mutagenesis and expressed in HIV-1 recombinants carrying sequence tags in nef. The doubling time of ENF-resistant viruses was highly correlated with decreasing ENF susceptibility (R(2) = 0.859; P < 0.001). Initial fitness experiments focused on mutants identified by in vitro selection in the presence of ENF (L. T. Rimsky, D. C. Shugars, and T. J. Matthews, J. Virol. 72:986-993, 1998). In the absence of drug, these mutants displayed reduced fitness compared to wild-type virus with a relative order of fitness of wild type > I37T > V38 M > D36S/V38 M; this order was reversed in the presence of ENF. Likewise, recombinant viruses carrying ENF resistance mutations selected in vivo displayed reduced fitness in the absence of ENF with a relative order of wild type > N42T > V38A > N42T/N43K approximately N42T/N43S > V38A/N42D approximately V38A/N42T. Fitness and ENF susceptibility were inversely correlated (r = -0.988; P < 0.001). Similar results were obtained with recombinants expressing molecularly cloned full-length env genes obtained from patient-derived HIV-1 isolates before and after ENF treatment. Further studies are needed to determine whether the reduced fitness of ENF-resistant viruses alters their pathogenicity in vivo.     

CD4-induced T-20 binding to human immunodeficiency virus type 1 gp120 blocks interaction with the CXCR4 coreceptor

The synthetic peptide T-20, which corresponds to a sequence within the C-terminal heptad repeat region (HR2) of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope glycoprotein, potently inhibits viral membrane fusion and entry. Although T-20 is thought to bind the N-terminal heptad repeat region (HR1) of gp41 and interfere with gp41 conformational changes required for membrane fusion, coreceptor specificity determined by the V3 loop of gp120 strongly influences the sensitivity of HIV-1 variants to T-20. Here, we show that T-20 binds to the gp120 glycoproteins of HIV-1 isolates that utilize CXCR4 as a coreceptor in a manner determined by the sequences of the gp120 V3 loop. T-20 binding to gp120 was enhanced in the presence of soluble CD4. Analysis of T-20 binding to gp120 mutants with variable loop deletions and the reciprocal competition of T-20 and particular anti-gp120 antibodies suggested that T-20 interacts with a gp120 region near the base of the V3 loop. Consistent with the involvement of this region in coreceptor binding, T-20 was able to block the interaction of gp120-CD4 complexes with the CXCR4 coreceptor. These results help to explain the increased sensitivity of CXCR4-specific HIV-1 isolates to the T-20 peptide. Interactions between the gp41 HR2 region and coreceptor-binding regions of gp120 may also play a role in the function of the HIV-1 envelope glycoproteins.     

Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor

T-20 is a synthetic peptide that corresponds to 36 amino acids within the C-terminal heptad repeat region (HR2) of human immunodeficiency virus type 1 (HIV-1) gp41. T-20 has been shown to potently inhibit viral replication of HIV-1 both in vitro and in vivo and is currently being evaluated in a Phase III clinical trial. T-649 is an inhibitory peptide that also corresponds to 36 amino acids within HR2. This sequence overlaps the T-20 sequence but is shifted 10 residues toward the N terminus of gp41. Both inhibitors are thought to exert their antiviral activity by interfering with the conformational changes that occur within gp41 to promote membrane fusion following gp120 interactions with CD4 and coreceptor molecules. We have shown previously that coreceptor specificity defined by the V3 loop of gp120 modulates sensitivity to T-20 and that a critical region within the N-terminal heptad repeat (HR1) of gp41 is the major determinant of sensitivity (C. A. Derdeyn et al., J. Virol. 74:8358-8367, 2000). This report shows that (i) regions within gp41 distinct from those associated with T-20 sensitivity govern the baseline sensitivity to T-649 and (ii) T-649 sensitivity of chimeric viruses that contain sequences derived from CXCR4- and CCR5-specific envelopes is also modulated by coreceptor specificity. Moreover, the pattern of sensitivity of CCR5-specific chimeras with only minor differences in their V3 loop was consistent for both inhibitors, suggesting that the individual affinity for coreceptor may influence accessibility of these inhibitors to their target sequence. Finally, an analysis of the sensitivity of 55 primary, inhibitor-naive HIV-1 isolates found that higher concentrations of T-20 (P < 0.001) and T-649 (P = 0.016) were required to inhibit CCR5-specific viruses compared to viruses that utilize CXCR4. The results presented here implicate gp120-coreceptor interactions in driving the complex conformational changes that occur in gp41 to promote fusion and entry and suggest that sensitivity to different HR1-directed fusion inhibitors is governed by distinct regions of gp41 but is consistently modulated by coreceptor specificity.     

Mutations in gp120 Contribute to the Resistance of Human Immunodeficiency Virus Type 1 to Membrane-Anchored C-Peptide maC46

Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41.The entry process of the human immunodeficiency virus type 1 (HIV-1) has become a major target for new antiviral drugs. Viral entry is initiated by binding of the HIV-1 envelope glycoprotein subunit gp120 to the CD4 receptor and a chemokine coreceptor, generally CCR5 or CXCR4. Upon coreceptor binding, the viral transmembrane subunit gp41 undergoes conformational changes that eventually lead to the formation of the six-helix bundle (6HB) and membrane fusion. The 6HB is composed of a central trimeric coiled-coil structure formed by the N-terminal heptad repeat (NHR) domains of three gp41 molecules and the corresponding C-terminal heptad repeats (CHRs) that pack into the longitudinal grooves on the surface of the NHR coiled-coil in an antiparallel orientation (23). C-peptide fusion inhibitors (CFI) derived from the CHR of gp41 compete with the viral CHR for binding to the NHR trimer, thus blocking 6HB formation and viral entry (18).T-20 (enfuvirtide) is the first clinically approved CFI with high antiviral activity and a low-toxicity profile. However, as with many anti-HIV-1 drugs, resistance can emerge rapidly (13). The majority of the resistance mutations are found in the NHR of gp41 among the amino acids 544 to 553 (32, 35) (numbering refers to gp160 of the HIV-1 HXB2 strain throughout the article). Most of these mutations cause resistance by reducing the affinity of the NHR target region to inhibitory C peptides (13). Additionally, viral entry kinetics were found to correlate with the baseline susceptibility of different HIV strains to CFI. Determinants for viral entry kinetics are found in gp41 as well as in gp120 (1, 14, 35). Here, the influence of coreceptor affinity on virus entry kinetics and CFI susceptibility has been studied extensively (28, 30, 31). Recently, a statistical approach was used that highlighted positions in gp120 that underwent mutations in patients under enfuvirtide treatment (38). However, to our knowledge, selected CFI resistance mutations outside of gp41 have never been confirmed experimentally.Previously, we developed a retroviral vector expressing a membrane-anchored antiviral C peptide (maC46) that efficiently inhibits a broad range of different HIV-1 isolates. Enfuvirtide-resistant HIV-1 strains with mutations in the GIV motif of NHR were fully susceptible to maC46 (10). In the present study, we selected an HIV-1 variant with reduced sensitivity to maC46 by passaging an enfuvirtide-resistant BaL strain of HIV-1 on cells expressing increasing concentrations of maC46. Mutations in gp120 and gp41 were found to contribute to maC46 resistance.     

Cooperative involvement of the S1 and S2 subunits of the murine coronavirus spike protein in receptor binding and extended host range

To study the process of spike (S)-receptor interaction during coronavirus entry, we evaluated the contributions of mutations in different regions of the murine hepatitis virus (MHV) S protein to natural receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (CEACAM1a) dependence and to the acquisition of extended host range. Extended-host-range variants of MHV strain A59 were previously obtained from persistently infected cells (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9504, 1997). These variant viruses contain several mutations in the S protein that confer to the viruses the ability to enter cells in a heparan sulfate-dependent manner (C. A. de Haan, Z. Li, E. te Lintelo, B. J. Bosch, B. J. Haijema, and P. J. M. Rottier, J. Virol. 79:14451-14456, 2005). While the parental MHV-A59 is fully dependent on murine CEACAM1a for its entry, viruses carrying the variant mutations in the amino-terminal part of their S protein had become dependent on both CEACAM1a and heparan sulfate. Substitutions in a restricted, downstream part of the S protein encompassing heptad repeat region 1 (HR1) and putative fusion peptide (FP) did not alter the CEACAM1a dependence. However, when the mutations in both parts of the S protein were combined, the resulting viruses became independent of CEACAM1a and acquired the extended host range. In addition, these viruses showed a decreased binding to and inhibition by soluble CEACAM1a. The observations suggest that the amino-terminal region of the S protein, including the receptor-binding domain, and a region in the central part of the S protein containing HR1 and FP, i.e., regions far apart in the linear sequence, communicate and may even interact physically in the higher-order structure of the spike.     

Why are HIV-1 fusion inhibitors not effective against SARS-CoV? Biophysical evaluation of molecular interactions

The envelope spike (S) glycoprotein of the severe acute respiratory syndrome associated coronavirus (SARS-CoV) mediates the entry of the virus into target cells. Recent studies point out to a cell entry mechanism of this virus similar to other enveloped viruses, such as HIV-1. As it happens with other viruses peptidic fusion inhibitors, SARS-CoV S protein HR2-derived peptides are potential therapeutic drugs against the virus. It is believed that HR2 peptides block the six-helix bundle formation, a key structure in the viral fusion, by interacting with the HR1 region. It is a matter of discussion if the HIV-1 gp41 HR2-derived peptide T20 (enfuvirtide) could be a possible SARS-CoV inhibitor given the similarities between the two viruses. We tested the possibility of interaction between both T20 (HIV-1 gp41 HR2-derived peptide) and T-1249 with S protein HR1- and HR2-derived peptides. Our biophysical data show a significant interaction between a SARS-CoV HR1-derived peptide and T20. However, the interaction is only moderate (K(B)=(1.1+/-0.3)x10(5) M(-1)). This finding shows that the reasoning behind the hypothesis that T20, already approved for clinical application in AIDS treatment, could inhibit the fusion of SARS-CoV with target cells is correct but the effect may not be strong enough for application.     

A simple, rapid, and sensitive system for the evaluation of anti-viral drugs in rats

The lack of small animal models for the evaluation of anti-human immunodeficiency virus type 1 (HIV-1) agents hampers drug development. Here, we describe the establishment of a simple and rapid evaluation system in a rat model without animal infection facilities. After intraperitoneal administration of test drugs to rats, antiviral activity in the sera was examined by the MAGI assay. Recently developed inhibitors for HIV-1 entry, two CXCR4 antagonists, TF14016 and FC131, and four fusion inhibitors, T-20, T-20EK, SC29EK, and TRI-1144, were evaluated using HIV-1(IIIB) and HIV-1(BaL) as representative CXCR4- and CCR5-tropic HIV-1 strains, respectively. CXCR4 antagonists were shown to only possess anti-HIV-1(IIIB) activity, whereas fusion inhibitors showed both anti-HIV-1(IIIB) and anti-HIV-1(BaL) activities in rat sera. These results indicate that test drugs were successfully processed into the rat sera and could be detected by the MAGI assay. In this system, TRI-1144 showed the most potent and sustained antiviral activity. Sera from animals not administered drugs showed substantial anti-HIV-1 activity, indicating that relatively high dose or activity of the test drugs might be needed. In conclusion, the novel rat system established here, "phenotypic drug evaluation", may be applicable for the evaluation of various antiviral drugs in vivo.     

Loss of the Protease Dimerization Inhibition Activity of Tipranavir (TPV) and Its Association with the Acquisition of Resistance to TPV by HIV-1

Tipranavir (TPV), a protease inhibitor (PI) inhibiting the enzymatic activity and dimerization of HIV-1 protease, exerts potent activity against multi-PI-resistant HIV-1 isolates. When a mixture of 11 multi-PI-resistant (but TPV-sensitive) clinical isolates (HIV_11MIX), which included HIV_B and HIV_C, was selected against TPV, HIV_11MIX rapidly (by 10 passages [HIV_11MIX^P10]) acquired high-level TPV resistance and replicated at high concentrations of TPV. HIV_11MIX^P10 contained various amino acid substitutions, including I54V and V82T. The intermolecular FRET-based HIV-1 expression assay revealed that TPV''s dimerization inhibition activity against cloned HIV_B (cHIV_B) was substantially compromised. The introduction of I54V/V82T into wild-type cHIV_NL4-3 (cHIV_{NL4-3^I54V/V82T}) did not block TPV''s dimerization inhibition or confer TPV resistance. However, the introduction of I54V/V82T into cHIV_B (cHIV_B^I54V/V82T) compromised TPV''s dimerization inhibition and cHIV_B^I54V/V82T proved to be significantly TPV resistant. L24M was responsible for TPV resistance with the cHIV_C genetic background. The introduction of L24M into cHIV_NL4-3 (cHIV_NL4-3^L24M) interfered with TPV''s dimerization inhibition, while L24M increased HIV-1''s susceptibility to TPV with the HIV_NL4-3 genetic background. When selected with TPV, cHIV_{NL4-3^I54V/V82T} most readily developed TPV resistance and acquired E34D, which compromised TPV''s dimerization inhibition with the HIV_NL4-3 genetic background. The present data demonstrate that certain amino acid substitutions compromise TPV''s dimerization inhibition and confer TPV resistance, although the loss of TPV''s dimerization inhibition is not always associated with significantly increased TPV resistance. The findings that TPV''s dimerization inhibition is compromised with one or two amino acid substitutions may explain at least in part why the genetic barrier of TPV against HIV-1''s development of TPV resistance is relatively low compared to that of darunavir.     

HbAHP-25, an In-Silico Designed Peptide,Inhibits HIV-1 Entry by Blocking gp120 Binding to CD4 Receptor

Human Immunodeficiency Virus (HIV-1) poses a serious threat to the developing world and sexual transmission continues to be the major source of new infections. Therefore, the development of molecules, which prevent new HIV-1 infections, is highly warranted. In the present study, a panel of human hemoglobin (Hb)-α subunit derived peptides and their analogues, with an ability to bind gp120, were designed in-silico and their anti-HIV-1 activity was evaluated. Of these peptides, HbAHP-25, an analogue of Hb-α derived peptide, demonstrated significant anti-HIV-1 activity. HbAHP-25 was found to be active against CCR5-tropic HIV-1 strains (ADA5 and BaL) and CXCR4-tropic HIV-1 strains (IIIB and NL4-3). Surface plasmon resonance (SPR) and ELISA revealed direct interaction between HbAHP-25 and HIV-1 envelope protein, gp120. The peptide prevented binding of CD4 to gp120 and blocked subsequent steps leading to entry and/or fusion or both. Anti-HIV activity of HbAHP-25 appeared to be specific as it failed to inhibit the entry of HIV-1 pseudotyped virus (HIV-1 VSV). Further, HbAHP-25 was found to be non-cytotoxic to TZM-bl cells, VK2/E6E7 cells, CEM-GFP cells and PBMCs, even at higher concentrations. Moreover, HbAHP-25 retained its anti-HIV activity in presence of seminal plasma and vaginal fluid. In brief, the study identified HbAHP-25, a novel anti-HIV peptide, which directly interacts with gp120 and thus has a potential to inhibit early stages of HIV-1 infection.     

Albumin-conjugated C34 peptide HIV-1 fusion inhibitor: equipotent to C34 and T-20 in vitro with sustained activity in SCID-hu Thy/Liv mice

Entry inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been the focus of much recent research. C34, a potent fusion inhibitor derived from the HR2 region of gp41, was engineered into a 1:1 human serum albumin conjugate through stable covalent attachment of a maleimido-C34 analog onto cysteine 34 of albumin. This bioconjugate, PC-1505, was designed to require less frequent dosing and less peptide than T-20 and was assessed for its antifusogenic activity both in vitro and in vivo in the SCID-hu Thy/Liv mouse model. PC-1505 was essentially equipotent to the original C34 peptide and to T-20 in vitro. In HIV-1-infected SCID-hu Thy/Liv mice, T-20 lost activity with infrequent dosing, whereas the antiviral potency of PC-1505 was sustained, and PC-1505 was active against T-20-resistant ("DIV") virus with a G36D substitution in gp41. The in vivo results are the direct result of a significantly improved pharmacokinetic profile for the C34 peptide following albumin conjugation. Contrary to previous reports that the gp41 NHR trimer is poorly accessible to C34 fused to protein cargoes of increasing size (Hamburger, A. E., Kim, S., Welch, B. D., and Kay, M. S. (2005) J. Biol. Chem. 280, 12567-12572), these results are the first demonstration of the capacity for a large, endogenous serum protein to gain unobstructed access to the transient gp41 intermediates that exist during the HIV fusion process, and it supports further development of albumin conjugation as a promising approach to inhibit HIV-1 entry.     

Introducing metallocene into a triazole peptide conjugate reduces its off-rate and enhances its affinity and antiviral potency for HIV-1 gp120

In this work, we identified a high affinity and potency metallocene-containing triazole peptide conjugate that suppresses the interactions of HIV-1 envelope gp120 at both its CD4 and co-receptor binding sites. The ferrocene-peptide conjugate, HNG-156, was formed by an on-resin copper-catalysed [2+3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl-containing triazole conjugate HNG-105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV-1 gp120 due mainly to the decreased dissociation rate of the conjugate-gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037-08) and clade B (YU-2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV-1YU-2 gp120 at both the CD4 binding site and CD4-induced binding sites. HNG-156 showed a close-to nanomolar IC50 for inhibiting cell infection by HIV-1BaL whole virus. The dual receptor site antagonist activity and potency of HNG-156 make it a promising viral envelope inhibitor lead for developing anti-HIV-1 treatments.     

Resistance profiles of novel electrostatically constrained HIV-1 fusion inhibitors

Human immunodeficiency virus (HIV) gp41 plays a key role in viral fusion; the N- and C-terminal heptad repeats (N-HR and C-HR) of gp41 form a stable 6-helical conformation for fusion. Therefore, HR-derived peptides, such as enfuvirtide (T-20), inhibit HIV-1 fusion by acting as decoys, and have been used for the treatment of HIV-1 infection. However, the efficacy of T-20 is attenuated by resistance mutations in gp41, including V38A and N43D. To suppress the resistant variants, we previously developed electrostatically constrained peptides, SC34 and SC34EK, and showed that both exhibited potent anti-HIV-1 activity against wild-type and T-20-resistant variants. In this study, to clarify the resistance mechanism to this next generation of fusion inhibitors, we selected variants with resistance to SC34 and SC34EK in vitro. The resistant variants had multiple mutations in gp41. All of these mutations individually caused less than 6-fold resistance to SC34 and SC34EK, indicating that there is a significant genetic barrier for high-level resistance. Cross-resistance to SC34 and SC34EK was reduced by a simple difference in the polarity of two intramolecular electrostatic pairs. Furthermore, the selected mutations enhanced the physicochemical interactions with N-HR variants and restored activities of the parental peptide, C34, even to resistant variants. These results demonstrate that our approach of designing gp41-binding inhibitors using electrostatic constraints and information derived from resistance studies produces inhibitors with enhanced activity, high genetic barrier, and distinct resistance profile from T-20 and other inhibitors. Hence, this is a promising approach for the design of future generation peptide fusion inhibitors.     

Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization

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.     

The LLSGIV stretch of the N-terminal region of HIV-1 gp41 is critical for binding to a model peptide,T20

A number of peptides and peptide analogs derived from the membrane proximal region of gp41 ectodomain are found to be effective inhibitors of human immunodeficiency virus type 1 (HIV-1)-mediated fusion events. One of them, T20 (aa 638-673), was found disordered and sparingly soluble in water, but became soluble upon mixing with selected, structured peptides from the amino terminal heptad repeat (HR1) region of gp41 using a simple and sensitive method of reduction in the scattering of T20 suspension. From the results on mapping the locus of interaction with T20 by employing partially overlapping peptides derived from HR1, it was concluded that the LLSGIV segment was a critical docking site for the C-terminal peptide of gp41 in its putative inhibitory action consistent with a previous fluorescence study. It was also found that peptides capable of solubilizing T20 dispersion have a high content of helix, as well as beta-strand, conformation in aqueous solution. Specificity of T20/HR1-derived peptide binding was ascertained by using a scrambled sequence of a T20-active peptide and a plateau in scattering reduction of T20 suspension with variation in the concentration of a T20-active HR1 peptide. Implications on the mechanism of T20 inhibition and the sequence of folding of the gp41 core structure are discussed.     

Immunogenicity of constrained monoclonal antibody A32-human immunodeficiency virus (HIV) Env gp120 complexes compared to that of recombinant HIV type 1 gp120 envelope glycoproteins

One strategy for the generation of broadly reactive neutralizing antibodies (NA) against human immunodeficiency virus type 1 (HIV-1) primary isolates is to use immunogens that have constrained HIV-1 envelope gp120 conformations reflective of triggered envelope on the surface of virions. A major change in gp120 following binding to CD4 is the enhanced exposure of the CCR5 binding site. One inducer of CCR5 binding site epitopes on gp120 is the human anti-gp120 monoclonal antibody, A32. We have made cross-linked A32-rgp120(89.6) and A32-rgp120(BaL) complexes and have compared their immunogenicities to those of uncomplexed recombinant gp120(BaL) (rgp120(BaL)) and rgp120(89.6). A32-rgp120(89.6) and A32-rgp120(BaL) complexes had stable induced CCR5 binding site expression compared to that of uncomplexed rgp120s. However, the A32-rgp120 complexes had similar capacities in guinea pigs for induction of NA against HIV-1 primary isolates versus that of rgp120 alone. A32-rgp120(89.6) induced antibodies that neutralized 6 out of 11 HIV-1 isolates, while rgp120(89.6) alone induced antibodies that neutralized 4 out of 11 HIV-1 isolates. A32-rgp120(BaL) complexes induced antibodies that neutralized 4 out of 14 HIV-1 isolates while, surprisingly, non-cross-linked rgp120(BaL) induced antibodies that neutralized 9 out of 14 (64%) HIV-1 isolates. Thus, stable enhanced expression of the coreceptor binding site on constrained gp120 is not sufficient for inducing broadly neutralizing anti-HIV-1 NA. Moreover, the ability of HIV-1 rgp120(BaL) to induce antibodies that neutralized approximately 60% of subtype B HIV-1 isolates warrants consideration of using HIV-1 BaL as a starting point for immunogen design for subtype B HIV-1 experimental immunogens.