Inhibition of the activity of HIV-1 protease through antibody binding and mutations probed by molecular dynamics simulations.

HIV-1 protease is an essential enzyme in the life cycle of the HIV-1 virus. The conformational dynamics of the flap region of the protease is critical for the ligand binding mechanism, as well as for the catalytic activity. The monoclonal antibody F11.2.32 raised against HIV-1 protease inhibits its activity on binding. We have studied the conformational dynamics of protease in its free, inhibitor ritonavir and antibody bound forms using molecular dynamics simulations. We find that upon Ab binding to the epitope region (residues 36-46) of protease, the overall flexibility of the protease is decreased including the flap region and the active site, which is similar to the decrease in flexibility observed by inhibitor binding to the protease. This suggests an allosteric mechanism to inhibit protease activity. Further, the protease mutants G40E and G40R are known to have decreased activity and were also subjected to MD simulations. We find that the loss of flexibility in the mutants is similar to that observed in the protease bound to the Ab/inhibitor. These insights highlight the role played by dynamics in the function of the protease and how control of flexibility through Ab binding and site specific mutations can inhibit protease activity.

Polyfunctional CD8+ T-Cell Response to Autologous Peptides from Protease and Reverse Transcriptase of HIV-1 Clade B.

BACKGROUND: The diversity of the HIV proteome influences the cellular response and development of an effective vaccine, particularly due to the generation of viral variants with mutations located within CD8+ T-cell epitopes. These mutations can affect the recognition of the epitopes, that may result in the selection of HIV variants with mutated epitopes (autologous epitopes) and different CD8+ T-cell functional profiles. OBJECTIVE: To determine the phenotype and functionality of CD8+ T-cell from HIV-infected Colombian patients in response to autologous and consensus peptides derived from HIV-1 clade B protease and reverse transcriptase (RT). METHODS: By flow cytometry, we compared the ex vivo CD8+ T-cell responses from HIV-infected patients to autologous and consensus peptides derived from HIV-1 clade B protease and RT, restricted by HLA-B*35, HLA-B*44 and HLA-B*51 alleles. RESULTS: Although autologous peptides restricted by HLA-B*35 and HLA-B*44 did not show any differences compared with consensus peptides, we observed the induction of a higher polyfunctional profile of CD8+ T-cells by autologous peptides restricted by HLA-B*51, particularly by the production of interferon-γ and macrophage inflammatory protein-1ß. The response by different memory CD8+ T-cell populations was comparable between autologous vs. consensus peptides. In addition, the magnitude of the polyfunctional response induced by the HLA-B*51-restricted QRPLVTIRI autologous epitope correlated with low viremia. CONCLUSION: Autologous peptides should be considered for the evaluation of HIV-specific CD8+ Tcell responses and to reveal some relevant epitopes that could be useful for therapeutic strategies aiming to promote polyfunctional CD8+ T-cell responses in a specific population of HIV-infected patients.

Genetic and mechanistic basis for APOBEC3H alternative splicing, retrovirus restriction, and counteraction by HIV-1 protease.

Human APOBEC3H (A3H) is a single-stranded DNA cytosine deaminase that inhibits HIV-1. Seven haplotypes (I-VII) and four splice variants (SV154/182/183/200) with differing antiviral activities and geographic distributions have been described, but the genetic and mechanistic basis for variant expression and function remains unclear. Using a combined bioinformatic/experimental analysis, we find that SV200 expression is specific to haplotype II, which is primarily found in sub-Saharan Africa. The underlying genetic mechanism for differential mRNA splicing is an ancient intronic deletion [del(ctc)] within A3H haplotype II sequence. We show that SV200 is at least fourfold more HIV-1 restrictive than other A3H splice variants. To counteract this elevated antiviral activity, HIV-1 protease cleaves SV200 into a shorter, less restrictive isoform. Our analyses indicate that, in addition to Vif-mediated degradation, HIV-1 may use protease as a  counter-defense mechanism against A3H in >80% of sub-Saharan African populations.

Conformational and dynamical basis for cross-reactivity observed between anti HIV-1 protease antibody with protease and an epitope peptide from it.

F11.2.32 is a monoclonal antibody raised against HIV-1 protease and it inhibits protease activity. While the structure of the epitope peptide in complex with the antibody is known, how protease interacts with the antibody is not known. In this study, we model the conformational features of the free and bound epitope peptide and protease-antibody interactions. We find through our simulations, that the free epitope peptide P36-46 samples conformations akin to the bound conformation of the peptide in complex with the Ab, with a ß-turn conformation sampled by the 38LPGR41 sequence highlighting the role of inherent conformational preferences of the peptide. Further, to determine the interactions present between the protease and antibody, we docked the protease in its conformation observed in the crystal structure, onto the antibody and simulated the dynamics of the complex in explicit water. We have identified the key residues involved in hydrogen-bond interactions and salt-bridges in Ag-Ab complex and examined the role of CDR flexibility in binding different conformations of the same epitope sequence in peptide and protein antigens. Thus, our results provide the basis for understanding the cross-reactivity observed between the antibody with protease and the epitope peptide from it.

Weaker HLA Footprints on HIV in the Unique and Highly Genetically Admixed Host Population of Mexico.

HIV circumvents HLA class I-restricted CD8+ T-cell responses through selection of escape mutations that leave characteristic mutational "footprints," also known as HLA-associated polymorphisms (HAPs), on HIV sequences at the population level. While many HLA footprints are universal across HIV subtypes and human populations, others can be region specific as a result of the unique immunogenetic background of each host population. Using a published probabilistic phylogenetically informed model, we compared HAPs in HIV Gag and Pol (PR-RT) in 1,612 subtype B-infected, antiretroviral treatment-naive individuals from Mexico and 1,641 individuals from Canada/United States. A total of 252 HLA class I allele subtypes were represented, including 140 observed in both cohorts, 67 unique to Mexico, and 45 unique to Canada/United States. At the predefined statistical threshold of a q value of <0.2, 358 HAPs (201 in Gag, 157 in PR-RT) were identified in Mexico, while 905 (534 in Gag and 371 in PR-RT) were identified in Canada/United States. HAPs identified in Mexico included both canonical HLA-associated escape pathways and novel associations, in particular with HLA alleles enriched in Amerindian and mestizo populations. Remarkably, HLA footprints on HIV in Mexico were not only fewer but also, on average, significantly weaker than those in Canada/United States, although some exceptions were noted. Moreover, exploratory analyses suggested that the weaker HLA footprint on HIV in Mexico may be due, at least in part, to weaker and/or less reproducible HLA-mediated immune pressures on HIV in this population. The implications of these differences for natural and vaccine-induced anti-HIV immunity merit further investigation.IMPORTANCE HLA footprints on HIV identify viral regions under intense and consistent pressure by HLA-restricted immune responses and the common mutational pathways that HIV uses to evade them. In particular, HLA footprints can identify novel immunogenic regions and/or epitopes targeted by understudied HLA alleles; moreover, comparative analyses across immunogenetically distinct populations can illuminate the extent to which HIV immunogenic regions and escape pathways are shared versus population-specific pathways, information which can in turn inform the design of universal or geographically tailored HIV vaccines. We compared HLA-associated footprints on HIV in two immunogenetically distinct North American populations, those of Mexico and Canada/United States. We identify both shared and population-specific pathways of HIV adaptation but also make the surprising observation that HLA footprints on HIV in Mexico overall are fewer and weaker than those in Canada/United States, raising the possibility that HLA-restricted antiviral immune responses in Mexico are weaker, and/or escape pathways somewhat less consistent, than those in other populations.

A novel HIV vaccine targeting the protease cleavage sites.

HIV preferentially infects activated CD4+ T cells and mutates rapidly. The classical vaccine approach aimed to generate broad immune responses to full HIV proteins largely failed to address the potential adverse impact of increased number of activated CD4+ T cells as viral targets. Learning from natural immunity observed in a group of HIV resistant Kenyan female sex workers, we are testing a novel vaccine approach. It focuses immune response to the highly conserved sequences surrounding the HIV protease cleavage sites (PCS) to disrupt viral maturation, while limiting excessive immune activation. Our pilot studies using nonhuman primate SIV infection models suggest that this approach is feasible and promising.

Modulation of apoptosis and viral latency - an axis to be well understood for successful cure of human immunodeficiency virus.

Human immunodeficiency virus (HIV) is the causative agent of the deadly disease AIDS, which is characterized by the progressive decline of CD4(+)T-cells. HIV-1-encoded proteins such as envelope gp120 (glycoprotein gp120), Tat (trans-activator of transcription), Nef (negative regulatory factor), Vpr (viral protein R), Vpu (viral protein unique) and protease are known to be effective in modulating host cell signalling pathways that lead to an alteration in apoptosis of both HIV-infected and uninfected bystander cells. Depending on the stage of the virus life cycle and host cell type, these viral proteins act as mediators of pro- or anti-apoptotic signals. HIV latency in viral reservoirs is a persistent phenomenon that has remained beyond the control of the human immune system. To cure HIV infections completely, it is crucial to reactivate latent HIV from cellular pools and to drive these apoptosis-resistant cells towards death. Several previous studies have reported the role of HIV-encoded proteins in apoptosis modulation, but the molecular basis for apoptosis evasion of some chronically HIV-infected cells and reactivated latently HIV-infected cells still needs to be elucidated. The current review summarizes our present understanding of apoptosis modulation in HIV-infected cells, uninfected bystander cells and latently infected cells, with a focus on highlighting strategies to activate the apoptotic pathway to kill latently infected cells.

[Construction and identification of HSV-1 vector vaccine carrying HIV-1 antigen].

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.

CD4+ T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes.

Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4(+) and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

Increased expression and immunogenicity of HIV-1 protease following inactivation of the enzymatic activity.

HIV-1 protease is an important target for anti-HIV therapy but has not received much attention as a vaccine antigen. To investigate the immunogenic properties of HIV-1 protease, we designed DNA plasmids encoding variants of the protease gene. Mutations resulting in enzymatic inactivation (D25N) and resistance to standard antiretroviral drugs (V82F/I84V) were introduced in order to examine the impact of the enzymatic activity on immunogenicity and the possibility to induce immune responses against drug resistant protease, respectively. The enzymatic inactivation of protease resulted in significantly increased in vitro expression as well as in vivo immunogenicity. The inactivated protease was highly immunogenic in both BALB/c and HLA-A0201 transgenic C57Bl/6 mice, and the immunogenicity was retained when the gene was delivered as a part of a multigene HIV-1 DNA vaccine. The drug resistance mutations hampered both the cellular and humoral immune responses, as the mutations also affect both CD4 and CD8 T cell epitopes. Taken together, our data demonstrates the possibility to drastically increase the immunogenicity of HIV-1 protease.

Influence of major HIV-1 protease inhibitor resistance mutations on CTL recognition.

BACKGROUND: HIV-1 protease is subjected to dual selection pressure exerted by protease inhibitors (PIs) and cytotoxic T lymphocytes (CTL). Recently, we identified KMIGGIGGF (KF9) as a HLA-B*1501-restricted CTL epitope, including several major PI resistance mutations (M46I/L, I47A/V, G48V, I50V). To assess potential interactions between KF9-specific CTL and emergence of these important resistance mutations, we studied CTL recognition of the mutations and analyzed protease sequences in an HLA-I­typed patient cohort. METHODS: CTL recognition of KF9 and resistance mutations in KF9 were studied in 38 HLA-B*1501-positive HIV-1­infected patients using variant KF9 peptides in interferon-g enzyme-linked immunospot assays. Protease sequences were analyzed in 302 HLA-I­typed HIV-1­infected patients. RESULTS: G48V abolished KF9 recognition by CTL in all patients. Furthermore, M46I, I47A, and I50V could impair or abolish CTL recognition in many patients. In contrast, M46L and I47V showed good CTL recognition in nearly all patients. HIV-1 protease sequence analysis showed no statistical correlation between the occurrence of resistance mutations in KF9 and HLA-B*1501. Viral load in patients failing therapy with KF9 mutations was significantly lower in HLA-B*1501-positive patients in comparison with HLA-B*1501-negative patients. CONCLUSIONS: PI mutations, G48V, M46I, and I47A, can abrogate CTL recognition, indicating potential interactions between development of drug resistance and CTL response. However, we could not find evidence that development of these PI mutations is influenced by KF9-specific CTL.

Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys.

The worldwide diversity of HIV-1 presents an unprecedented challenge for vaccine development. Antigens derived from natural HIV-1 sequences have elicited only a limited breadth of cellular immune responses in nonhuman primate studies and clinical trials to date. Polyvalent 'mosaic' antigens, in contrast, are designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity. Here we show that mosaic HIV-1 Gag, Pol and Env antigens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys. Polyvalent mosaic antigens therefore represent a promising strategy to expand cellular immunologic vaccine coverage for genetically diverse pathogens such as HIV-1.

Potent inhibition of drug-resistant HIV protease variants by monoclonal antibodies.

The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.

Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease.

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.

Immunological cross-reactivity against a drug mutated HIV-1 protease epitope after DNA multi-CTL epitope construct immunization.

Epitopes in HIV polymerase were analyzed by peptide binding to human leukocyte antigen (HLA) A0201 molecules, the most frequent HLA class in the Caucasian population. We found that HIV-1 protease peptides representing both the wild type and anticipated drug resistance variants of the sequence bound well to HLA-A0201. We also found that wild type as well as a double mutated variant of the epitope was strongly immunogenic in HLA-A0201 transgenic mice, either as individual peptides or encoded in DNA multi-CTL epitope constructs. Immunological cross-reactivity between different variants of the peptide could be seen, suggesting that it may be possible to induce a broad immune response by immunizing with drug resistance-mutated epitopes. This may be of advantage for HIV-1 infected patients since such a response may cause a better outcome of an anti-retroviral drug therapy.

Induction of T-cell immunity to antiretroviral drug-resistant human immunodeficiency virus type 1.

Antiretroviral drug-resistant human immunodeficiency virus type 1 (HIV-1) is a major, growing, public health problem. Immune responses targeting epitopes spanning drug resistance sites could ameliorate development of drug resistance. We studied 25 individuals harboring multidrug-resistant HIV-1 for T-cell immunity to HIV-1 proteins and peptides spanning all common drug resistance mutations. CD8 T cells targeting epitopes spanning drug-induced mutations were detected but only in the 3 individuals with robust HIV-specific T-cell activity. Novel CD8 T-cell responses were detected against the common L63P and L10I protease inhibitor fitness mutations. Induction of T-cell immunity to drug-resistant variants was demonstrated in simian human immunodeficiency virus-infected macaques, where both CD8 and CD4 T-cell immune responses to reverse transcriptase and protease antiretroviral mutations were elicited using a novel peptide-based immunotherapy. T-cell responses to antiretroviral resistance mutations were strongest in the most immunocompetent animals. This study suggests feasible strategies to further evaluate the potential of limiting antiretroviral drug resistance through induction of T-cell immunity.

Augmentation of HIV-1 subtype C vaccine constructs induced immune response in mice by CpG motif 1826-ODN.

The greatest biomedical challenge of this century is to develop a preventive vaccine against Human Immunodeficiency Virus (HIV-1). For an HIV vaccine to be effective, it appears logical to develop new strategies that enhance the level of the immune response as well as steer it towards the desirable cellular type. In view of this, there is a need for rational inclusion of biological adjuvants into the HIV-1 vaccination strategies that could potentiate the immune responses both qualitatively and quantitatively. The adjuvant may include the immunostimulatory oligonucleotides containing CpG motifs, whose immunomodulatory characters are well established and represent the basis for an effective vaccine adjuvant. In our study, we investigated the use of an immunostimulatory oligonucleotide (or CpG motif), 1826-ODN to augment the immune response elicited by plasmid DNA vaccine constructs containing Indian subtype C HIV-1 envelope gp120 and gag-protease genes in Balb/c mouse model system. A dose of 2-microg CpG motifs/mouse was found to be optimum when co-administered with the DNA vaccine constructs with the peak level of humoral and cell mediated immune responses at 6 weeks post immunization. Murine IFN-gamma ELISpot assay demonstrated that the use of 1826-ODN led to a broad based and long term recognition of the subtype C envelope and gag peptides. The use of CpG motifs has been effective in augmenting the immune responses generated by the DNA constructs. Taken together, these results are an important advancement towards the design of future preclinical and clinical trials of these vaccine constructs.

Cross-reactive cytotoxic T lymphocytes against human immunodeficiency virus type 1 protease and gamma interferon-inducible protein 30.

The gamma interferon (IFN-gamma)-inducible protein 30 (IP-30) signal peptide -11 to -3 (LLDVPTAAV) is a prominent self peptide expressed with the class I human histocompatibility leukocyte antigen A2 (HLA-A2). Stimulation of peripheral blood mononuclear cells (PBMC) from HLA-A2 human immunodeficiency virus type 1 (HIV-1)-infected individuals with an HLA-A2-restricted HIV protease (PR) peptide 76-84 (LVGPTPVNI) activated cytotoxic T lymphocytes (CTL) against the IP-30 signal peptide. Since HIV-1 PR 76-84 stimulated CD8+ T cells from these individuals to secrete IFN-gamma, we tested whether the activation of IP-30-specific CTL in vitro resulted from T-cell cross-reactivity or from up-regulation of IP-30 by IFN-gamma. Neither high levels of exogenous IFN-gamma nor incubation of PBMC with other HIV peptides triggering substantial IFN-gamma release activated IP-30-specific CTL. Although the IP-30 signal peptide did not stimulate IFN-gamma release from freshly isolated PBMC, it activated CTL in vitro against itself and HIV PR 76-84. Peptide-stimulated IFN-gamma release, cold target inhibition, and HLA-A2/immunoglobulin dimer-mediated binding and depletion of effector cells all indicated that in vitro stimulation with HIV PR 76-84 or the IP-30 signal peptide activated a comparable population of cross-reactive effector cells. Neither IP-30 nor HIV PR 76-84 activated CTL against themselves following in vitro stimulation of PBMC from non-HIV-infected HLA-A2 individuals. Peptide titrations indicated higher-avidity T-cell interactions with HIV PR 76-84 than with the IP-30 signal peptide. These data indicate that HIV PR 76-84 is a heteroclitic variant of the IP-30 signal peptide -11 to -3, which has implications for immune memory and autoimmunity.