Stabilized trimeric peptide immunogens of the complete HIV-1 gp41 N-heptad repeat and their use as HIV-1 vaccine candidates.

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.

Antiviral activity and in vitro mutation development pathways of MK-6186, a novel nonnucleoside reverse transcriptase inhibitor.

MK-6186 is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) which displays subnanomolar potency against wild-type (WT) virus and the two most prevalent NNRTI-resistant RT mutants (K103N and Y181C) in biochemical assays. In addition, it showed excellent antiviral potency against K103N and Y181C mutant viruses, with fold changes (FCs) of less than 2 and 5, respectively. When a panel of 12 common NNRTI-associated mutant viruses was tested with MK-6186, only 2 relatively rare mutants (Y188L and V106I/Y188L) were highly resistant, with FCs of >100, and the remaining viruses showed FCs of <10. Furthermore, a panel of 96 clinical virus isolates with NNRTI resistance mutations was evaluated for susceptibility to NNRTIs. The majority (70%) of viruses tested displayed resistance to efavirenz (EFV), with FCs of >10, whereas only 29% of the mutant viruses displayed greater than 10-fold resistance to MK-6186. To determine whether MK-6186 selects for novel resistance mutations, in vitro resistance selections were conducted with one isolate each from subtypes A, B, and C under low-multiplicity-of-infection (MOI) conditions. The results showed a unique mutation development pattern in which L234I was the first mutation to emerge in the majority of the experiments. In resistance selection under high-MOI conditions with subtype B virus, V106A was the dominant mutation detected in the breakthrough viruses. More importantly, mutant viruses selected by MK-6186 showed FCs of <10 against EFV or etravirine (ETR), and the mutant viruses containing mutations selected by EFV or ETR were sensitive to MK-6186 (FCs of <10).

Small molecule mimetics of an HIV-1 gp41 fusion intermediate as vaccine leads.

We describe here a novel platform technology for the discovery of small molecule mimetics of conformational epitopes on protein antigens. As a model system, we selected mimetics of a conserved hydrophobic pocket within the N-heptad repeat region of the HIV-1 envelope protein, gp41. The human monoclonal antibody, D5, binds to this target and exhibits broadly neutralizing activity against HIV-1. We exploited the antigen-binding property of D5 to select complementary small molecules using a high throughput screen of a diverse chemical collection. The resulting small molecule leads were rendered immunogenic by linking them to a carrier protein and were shown to elicit N-heptad repeat-binding antibodies in a fraction of immunized mice. Plasma from HIV-1-infected subjects shown previously to contain broadly neutralizing antibodies was found to contain antibodies capable of binding to haptens represented in the benzylpiperidine leads identified as a result of the high throughput screen, further validating these molecules as vaccine leads. Our results suggest a new paradigm for vaccine discovery using a medicinal chemistry approach to identify lead molecules that, when optimized, could become vaccine candidates for infectious diseases that have been refractory to conventional vaccine development.

Distinct mutation pathways of non-subtype B HIV-1 during in vitro resistance selection with nonnucleoside reverse transcriptase inhibitors.

Studies were conducted to investigate mutation pathways among subtypes A, B, and C of human immunodeficiency virus type 1 (HIV-1) during resistance selection with nonnucleoside reverse transcriptase inhibitors (NNRTIs) in cell culture under low-multiplicity of infection (MOI) conditions. The results showed that distinct pathways were selected by different virus subtypes under increasing selective pressure of NNRTIs. F227C and Y181C were the major mutations selected by MK-4965 in subtype A and C viruses during resistance selection. With efavirenz (EFV), F227C and V106M were the major mutations responsible for viral breakthrough in subtype A viruses, whereas a single pathway (G190A/V106M) accounted for mutation development in subtype C viruses. Y181C was the dominant mutation in the resistance selection with etravirine (ETV) in subtype A, and E138K/H221Y were the mutations detected in the breakthrough viruses from subtype C viruses with ETV. In subtype B viruses, on the other hand, known NNRTI-associated mutations (e.g., Y181C, P236L, L100I, V179D, and K103N) were selected by the NNRTIs. The susceptibility of the subtype A and B mutant viruses to NNRTIs was determined in order to gain insight into the potential mechanisms of mutation development. Collectively, these results suggest that minor differences may exist in conformation of the residues within the NNRTI binding pocket (NNRTIBP) of reverse transcriptase (RT) among the three subtypes of viruses. Thus, the interactions between NNRTIs and the residues in the NNRTIBPs of different subtypes may not be identical, leading to distinct mutation pathways during resistance selection in cell culture.

Potent and selective HIV-1 ribonuclease H inhibitors based on a 1-hydroxy-1,8-naphthyridin-2(1H)-one scaffold.

Optimization studies using an HIV RNase H active site inhibitor containing a 1-hydroxy-1,8-naphthyridin-2(1H)-one core identified 4-position substituents that provided several potent and selective inhibitors. The best compound was potent and selective in biochemical assays (IC(50)=0.045 µM, HIV RT RNase H; 13 µM, HIV RT-polymerase; 24 µM, HIV integrase) and showed antiviral efficacy in a single-cycle viral replication assay in P4-2 cells (IC(50)=0.19 µM) with a modest window with respect to cytotoxicity (CC(50)=3.3 µM).

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates.

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.

Assessment of the susceptibility of mutant HIV-1 to antiviral agents.

Traditional phenotypic assays used to assess the susceptibility of mutant human immunodeficiency virus type-1 (HIV-1) obtained from infected patients or from resistance selection to antiviral agents in cell culture are rather tedious and time consuming. To improve the efficiency of this process, a novel method was developed in which mutant viruses are captured with magnetic nano-beads and used to infect gag-GFP reporter cells to evaluate the extent of resistance conferred by the mutant viruses against antiviral agents. The optimal timing for measuring the inhibitory potencies of antiviral agents was found to be day 3 post-infection for integrase strand transfer inhibitors and protease inhibitors and day 4 for non-nucleoside reverse transcriptase inhibitors. Comparable EC(50) values were obtained when bead-captured breakthrough virus from in vitro resistance selection experiments and its matched site-directed mutagenesis virus were tested side by side in this assay. This assay protocol was also employed to evaluate the inhibitor susceptibility of breakthrough viruses collected from resistance selections that were conducted in the presence of increasing concentrations of an HIV-1 protease inhibitor. Taken together, these findings suggest that a rapid, sensitive, non-invasive, and homogeneous phenotypic assay has been developed for assessing the antiviral agent susceptibility of mutant viruses that emerge from in vitro resistance selection studies.

Addition of a cholesterol group to an HIV-1 peptide fusion inhibitor dramatically increases its antiviral potency.

Peptides derived from the heptad repeat 2 (HR2) region of the HIV fusogenic protein gp41 are potent inhibitors of viral infection, and one of them, enfuvirtide, is used for the treatment of therapy-experienced AIDS patients. The mechanism of action of these peptides is binding to a critical intermediate along the virus-cell fusion pathway, and accordingly, increasing the affinity for the intermediate yields more potent inhibitors. We took a different approach, namely to increase the potency of the HR2 peptide inhibitor C34 by targeting it to the cell compartment where fusion occurs, and we show here that a simple, yet powerful way to accomplish this is attachment of a cholesterol group. C34 derivatized with cholesterol (C34-Chol) shows dramatically increased antiviral potency on a panel of primary isolates, with IC(90) values 15- to 300-fold lower than enfuvirtide and the second-generation inhibitor T1249, making C34-Chol the most potent HIV fusion inhibitor to date. Consistent with its anticipated mechanism of action, the antiviral activity of C34-Chol is unusually persistent: washing target cells after incubation with C34-Chol, but before triggering fusion, increases IC(50) only 7-fold, relative to a 400-fold increase observed for C34. Moreover, derivatization with cholesterol extends the half-life of the peptide in vivo. In the mouse, s.c. administration of 3.5 mg/kg C34-Chol yields a plasma concentration 24 h after injection >300-fold higher than the measured IC(90) values. Because the fusion machinery targeted by C34-Chol is similar in several other enveloped viruses, we believe that these findings may be of general utility.

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41.

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.

Short communication: In vitro synergy between peptides or neutralizing antibodies targeting the N- and C-terminal heptad repeats of HIV Type 1 gp41.

Class 1 and class 2 fusion peptides bind to the trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of HIV-1 envelope glycoprotein gp41, respectively, and block its intramolecular folding required for Env-mediated viral and host cell membrane fusion and subsequent viral entry. Using a combination of T-20 (class 1) and (CCIZN17)(3) (class 2), we provide evidence that these classes of fusion peptides work synergistically in an in vitro infectivity assay in inhibiting the entry of primary HIV-1 isolate 89.6 with combination indexes reaching 0.37 and 0.32 at IC(50) and IC(90), respectively. We further demonstrate a similar degree of neutralization synergy between a monoclonal antibody (MAb), D5, targeting the hydrophobic pocket region of the NHR, and 2F5, a well-characterized MAb that targets the C-terminal end of CHR and the membrane-proximal external region (MPER), providing a rational basis for developing combination vaccines targeting these two highly conserved regions of gp41.

An oligosaccharide-based HIV-1 2G12 mimotope vaccine induces carbohydrate-specific antibodies that fail to neutralize HIV-1 virions.

The conserved oligomannose epitope, Man(9)GlcNAc(2), recognized by the broadly neutralizing human mAb 2G12 is an attractive prophylactic vaccine candidate for the prevention of HIV-1 infection. We recently reported total chemical synthesis of a series of glycopeptides incorporating one to three copies of Man(9)GlcNAc(2) coupled to a cyclic peptide scaffold. Surface plasmon resonance studies showed that divalent and trivalent, but not monovalent, compounds were capable of binding 2G12. To test the efficacy of the divalent glycopeptide as an immunogen capable of inducing a 2G12-like neutralizing antibody response, we covalently coupled the molecule to a powerful immune-stimulating protein carrier and evaluated immunogenicity of the conjugate in two animal species. We used a differential immunoassay to demonstrate induction of high levels of carbohydrate-specific antibodies; however, these antibodies showed poor recognition of recombinant gp160 and failed to neutralize a panel of viral isolates in entry-based neutralization assays. To ascertain whether antibodies produced during natural infection could recognize the mimetics, we screened a panel of HIV-1-positive and -negative sera for binding to gp120 and the synthetic antigens. We present evidence from both direct and competitive binding assays that no significant recognition of the glycopeptides was observed, although certain sera did contain antibodies that could compete with 2G12 for binding to recombinant gp120.

Automated high-throughput purification of antibody fragments to facilitate evaluation in functional and kinetic based assays.

Screening antibodies from phage displayed in vitro libraries and from affinity maturation of lead antibodies requires testing of antibody fragments (scFvs and Fabs) for function and binding affinities. Crude scFv or Fab periplasmic preparations from Escherichia coli are often not pure and/or concentrated enough for use in functional and affinity assays. We have developed an automated high-throughput approach for small and large-scale expression and purification of His-tagged scFvs and Fabs using the Qiagen BioRobot 3000 LS with optimized application software. This automated procedure enabled us to rapidly evaluate antibody fragments in functional and surface plasmon resonance (SPR) assays. We have used these procedures to make thousands of purified scFv/Fabs for several antibody maturation campaigns and significantly decreased the time needed to select the best candidates. The assay results from these purified samples were used to prioritize candidates before converting them to IgG. This protocol can process up to 300 small-scale and up to 72 large-scale scFvs or Fabs per week per full-time employee (FTE).

Structural basis for HIV-1 neutralization by a gp41 fusion intermediate-directed antibody.

Elicitation of potent and broadly neutralizing antibodies is an important goal in designing an effective human immunodeficiency virus-1 (HIV-1) vaccine. The HIV-1 gp41 inner-core trimer represents a functionally and structurally conserved target for therapeutics. Here we report the 2.0-A-resolution crystal structure of the complex between the antigen-binding fragment of D5, an HIV-1 cross-neutralizing antibody, and 5-helix, a gp41 inner-core mimetic. Both binding and neutralization depend on residues in the D5 CDR H2 loop protruding into the conserved gp41 hydrophobic pocket, as well as a large pocket in D5 surrounding core gp41 residues. Kinetic analysis of D5 mutants with perturbed D5-gp41 interactions suggests that D5 persistence at the fusion intermediate is crucial for neutralization. Thus, our data validate the gp41 N-peptide trimer fusion intermediate as a target for neutralizing antibodies and provide a template for identification of more potent and broadly neutralizing molecules.

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope.

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a "hot spot" for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.

Covalent stabilization of coiled coils of the HIV gp41 N region yields extremely potent and broad inhibitors of viral infection.

Peptides from the N-heptad repeat region of the HIV gp41 protein can inhibit viral fusion, but their potency is limited by a low tendency to form a trimeric coiled-coil. Accordingly, stabilization of N peptides by fusion with the stable coiled-coil IZ yields nanomolar inhibitors [Eckert, D. M. & Kim, P. S. (2001) Proc. Natl. Acad. Sci. USA 98, 11187-11192]. Because the antiviral potency of IZN17 is limited by self-association equilibrium, we covalently stabilized the peptide by using interchain disulfide bonds. The resulting covalent trimer, (CCIZN17)3, has an extraordinary thermodynamic stability that translates into unprecedented antiviral potency: (CCIZN17)3 (i) inhibits fusion in a cell-cell fusion assay (IC50 = 260 pM); (ii) is the most potent fusion inhibitor described to date (IC50 = 40-380 pM) in a single-cycle infectivity assay against HIV(HXB2), HIV(NL4-3), and HIV(MN-1); (iii) efficiently neutralizes acute viral infection in peripheral blood mononuclear cells; and (iv) displays a broad antiviral profile, being able to neutralize 100% of a large panel of HIV isolates, including R5, X4, and R5/X4 strains. In all of these assays, the potency of N-peptide inhibitor (CCIZN17)3 was equal to or more than the C-peptide inhibitor in clinical use, DP178 (also known as Enfuvirtide and Fuzeon). More importantly, we show that the two inhibitors, which have different targets in gp41, synergize when used in combination. These features make (CCIZN17)3 an attractive lead to develop as an antiviral drug, alone or in combination with DP178, as well as a promising immunogen to elicit a fusion-blocking neutralizing antibody response.

Potent 1,3,4-trisubstituted pyrrolidine CCR5 receptor antagonists: effects of fused heterocycles on antiviral activity and pharmacokinetic properties.

A series of 1,3,4-trisubstituted pyrrolidine CCR5 receptor antagonists containing a variety of fused heterocycles at the 4-position of the piperidine side chain has been discovered, which are orally bioavailable with potent anti-HIV activity.

Synthesis and biological evaluation of 5R- and 5S-methyl substituted D- and L-configuration 1,3-dioxolane nucleoside analogs.

1,3-Dioxolane and 1,3-oxathiolane nucleoside analogs play an important role in anti-viral and anti-neoplastic chemotherapy. We report here the synthesis of 2-hydroxymethyl-5-methyl-1,3-dioxolanylpurine nucleosides from 4-acetoxy-2-(benzyloxymethyl)-5-methyldioxolane. Dioxolanes of alpha-D-, beta-D-, alpha-L-, and beta-L-configuration were prepared, that included 5-methyl derivatives of both 5R and 5S configuration. Molecular mechanics calculations indicate that the 5S and 5R diastereoisomeric 1,3-dioxolanes possess distinct conformational bias, suggesting that methyl substitution may alter the conformational preference of 1,3-dioxolanes. The ability of the 1,3-dioxolanes to inhibit HCV RNA replication was evaluated in a cell-based, subgenomic replicon assay. In addition, activity against vaccinia and HIV was evaluated in cell-based assays. The 2-hydroxymethyl-5-methyl-1,3-dioxolanes were found to be inactive.