Characterization of human immunodeficiency virus type 1 CRF01_AE env genes derived from recently infected Thai individuals

Transmitted/founder virus is responsible for the establishment of human immunodeficiency virus type 1 (HIV-1) infection and induces primary anti-HIV-1 immune responses; therefore, it is important to study the viral population to understand the early events of HIV-1 infection. We amplified HIV-1 env genes from sera derived from recently infected Thai individuals, and established envelope glycoproteins (Env)-recombinant viruses. Generated Env-recombinant viruses were tested for their neutralization susceptibility to neutralizing human monoclonal antibodies (NHMAbs) and entry inhibitors, as well as being subjected to genotypic analysis. Most recombinant viruses were susceptible to neutralization by NHMAbs to Env gp41, whereas approximately one-third of the recombinant viruses were susceptible to a NHMAb against the CD4 binding site of gp120. In addition, all env genes were classified into CRF01_AE genes and showed low genetic divergence. Taken together with our previous studies on CRF01_AE env genes derived from chronically infected Thai individuals, these results suggested that the immunological and genetic characteristics of CRF01_AE Env derived from recently infected Thai individuals were different from those derived from chronically infected individuals.     

Laos is Affected by HIV CRF01_AE and the Newly Identified CRF97_01B

Virologica Sinica - Laos is the only landlocked country in Southeast Asia and borders Thailand, Myanmar and Cambodia, the three countries in this region that have been hardest hit by human...     

CRF01-AE亚型人类免疫缺陷病毒的基因序列特征研究

从确诊的HIV-1感染者的全血样本中提取基因组DNA,经套式聚合酶链反应(PCR)扩增其gag蛋白P17/P24交界区基因片断后,将扩增产物进行纯化和测序,分析其氨基酸序列。进而了解所检出的病毒基因变异和分子流行病学特征。结果发现,HIV-1 CRF01-AE亚型病毒分别与3株不同来源的国际参考毒株具有紧密的亲缘关系,表明这些毒株可能分别由不同的传播路线进入我国大陆境内。     

Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.     

Exploring the drug resistance mechanism of active site,non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations

Human immunodeficiency virus type 1 protease is essential for virus replication and maturation and has been considered as one of the important drug target for the antiretroviral treatment of HIV infection. The majority of HIV infections are caused due to non-B subtypes in developing countries. Subtype AE is spreading rapidly and infecting huge population worldwide. Understanding the interdependence of active and non-active site mutations in conferring drug resistance is crucial for the development effective inhibitors in subtype AE protease. In this work, we have investigated the mechanism of resistance against indinavir (IDV) due to therapy selected active site mutation V82F, non-active site mutations PF82V and their cooperative effects PV82F in subtype AE-protease using molecular dynamics simulations and binding free energy calculations. The simulations suggested all the three complexes lead to decrease in binding affinity of IDV, whereas the PF82V complex resulted in an enhanced binding affinity compared to V82F and PV82F complexes. Large positional deviation of IDV was observed in V82F complex. The preservation of hydrogen bonds of IDV with active site Asp25/Asp25′ and flap residue Ile50/50′ via a water molecule is crucial for effective binding. Owing to the close contact of 80s loop with Ile50′ and Asp25, the alteration between residues Thr80 and Val82, further induces conformational change thereby resulting in loss of interactions between IDV and the residues in the active site cavity, leading to drug resistance. Our present study shed light on the effect of active, non-active site mutations and their cooperative effects in AE protease.

Communicated by Ramaswamy H. Sarma     

The development of CD4 binding site antibodies during HIV-1 infection

Broadly neutralizing antibodies to the CD4 binding site (CD4bs) of gp120 are generated by some HIV-1-infected individuals, but little is known about the prevalence and evolution of this antibody response during the course of HIV-1 infection. We analyzed the sera of 113 HIV-1 seroconverters from three cohorts for binding to a panel of gp120 core proteins and their corresponding CD4bs knockout mutants. Among sera collected between 99 and 258 weeks post-HIV-1 infection, 88% contained antibodies to the CD4bs and 47% contained antibodies to resurfaced stabilized core (RSC) probes that react preferentially with broadly neutralizing CD4bs antibodies (BNCD4), such as monoclonal antibodies (MAbs) VRC01 and VRC-CH31. Analysis of longitudinal serum samples from a subset of 18 subjects revealed that CD4bs antibodies to gp120 arose within the first 4 to 16 weeks of infection, while the development of RSC-reactive antibodies was more varied, occurring between 10 and 152 weeks post-HIV-1 infection. Despite the presence of these antibodies, serum neutralization mediated by RSC-reactive antibodies was detected in sera from only a few donors infected for more than 3 years. Thus, CD4bs antibodies that bind a VRC01-like epitope are often induced during HIV-1 infection, but the level and potency required to mediate serum neutralization may take years to develop. An improved understanding of the immunological factors associated with the development and maturation of neutralizing CD4bs antibodies during HIV-1 infection may provide insights into the requirements for eliciting this response by vaccination.     

Biochemically defined HIV-1 envelope glycoprotein variant immunogens display differential binding and neutralizing specificities to the CD4-binding site

HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.     

Lipid binding properties of 4E10, 2F5, and WR304 monoclonal antibodies that neutralize HIV-1

Two human mAbs (2F5 and 4E10), originally derived from HIV-1-infected patients, are important, but rare, mAbs that exhibit broad cross-clade neutralizing activities against HIV-1. In addition to peptide sequences on the gp41 envelope protein, both antibodies reportedly also bound specifically to several phospholipid antigens. However, the phospholipid binding property of 2F5 has been disputed and, because of uncertainly regarding phospholipid binding, the modeling of neutralizing mechanisms has been difficult. To explore this issue, we examined the binding of 4E10 and 2F5 to a broad range of lipid antigens by ELISA. 4E10 and 2F5 both bound to a variety of purified phospholipids, and 4E10 bound, but 2F5 did not bind, to cardiolipin. Both mAbs also bound to a sulfated glycolipid, sulfogalactosyl ceramide (sulfatide), and to two neutral glycolipids, galactosyl ceramide and glucosyl ceramide, but not to other galactosyl glycolipids. 4E10, but not 2F5, also bound to cholesterol, although both mAbs bound to squalene. Interestingly, 4E10, but not 2F5, exhibited striking binding to lipid A, the lipid moiety of Gram-negative bacterial lipopolysaccharide. The binding properties of 4E10 to phospholipids, sulfatide, cholesterol, squalene, and lipid A were similar to those of a neutralizing murine mAb (WR304) induced by liposomes containing phosphatidylinositol phosphate and lipid A, although WR304 did not bind to neutral glycolipids. The discovery of a binding specificity of 4E10 for lipid A, a widely used vaccine adjuvant, suggests that innate immunity stimulated by lipid A could have played a role for induction of multispecific antibodies that simultaneously recognize both HIV-1 protein and lipid antigens.     

Active site binding modes of the beta-diketoacids: a multi-active site approach in HIV-1 integrase inhibitor design

Predicting a bioactive conformation of a ligand is of paramount importance in rational drug design. The task becomes very difficult when the receptor site possesses a region with unusual conformational flexibility. Significant conformational differences are present in the active site regions in the available crystal structures of the core domains of HIV-1 integrase (IN). Among all reported IN inhibitors, the β-diketoacid class of compounds has proved to be of most promise and indeed S-1360 was the first IN inhibitor to enter clinical studies. With an aim to predict the bioactive (active site bound) conformation of S-1360, we performed extensive docking studies using three different reported crystal structures where the active site or partial active site region was resolved. For comparison we extended our studies to include 5CITEP (the first compound cocrystallized with IN core domain) and a bis-diketoacid (BDKA). We found that the conformation of S-1360 when bound in one of the active sites matches that of the experimentally observed results of IN escape mutants resistant to S-1360. Therefore, we propose that this active site conformation is the biologically relevant conformation and can be used for the future structure-based drug design studies selectively targeting IN.     

The Effect of HLA Polymorphisms on the Recognition of Gag Epitopes in HIV-1 CRF01_AE Infection

Introduction

The design of a globally effective vaccine rests on the identification of epitopes capable of eliciting effective cytotoxic T lymphocyte (CTL) responses across multiple HIV clades in different populations. This study aims to discern the effect of HLA polymorphisms and the cross-clade reactivity or clade-specificity of epitopes in Thailand where HIV-1 CRF01_AE is circulating.

Materials and Methods

14 peptides based on consensus HIV-1 CRF01_AE amino acid sequences were designed for use in IFN-γ ELISpot assays and ⁵¹Cr release assays among 66 HIV-1 CRF01_AE-infected Thai patients. For ELISpot responders carrying HLA alleles currently unknown to restrict CRF01_AE epitopes, in silico epitope-HLA prediction was performed.

Results

29/66 (43.9%) patients recognized at least one peptide. In total 79 responses were seen against all 14 peptides. 28/79 (35.4%) of the responses were in patients with HLA alleles previously reported to restrict CRF01_AE epitopes, 24/79 (30.4%) responses were in individuals with HLA alleles previously reported to restrict epitopes of HIV clades other than CRF01_AE, and the remaining 27/79 (34.2%) responses were not associated with HLA alleles previously known to restrict HIV epitopes. In silico epitope prediction detected 19 novel, epitope-HLA combinations, and 11/19 (57.9%) were associated with HLA-C alleles. We further confirmed a novel HLA restriction of a previously identified HIV-1 Gag epitope [p24_122–130: PPIPVGDIY (PY9)] by HLA-B*40:01 with a standard ⁵¹Cr release assay.

Discussion

CTL recognition sites in HIV-1 Gag were similar among different clades but the HLA restriction differed in Thai patients. This disparity in HLA restriction along different populations illustrated the importance of clade- and population-specific HLA analysis prior to CTL vaccine design.     

Increased rate of CD4+ T-cell decline and faster time to antiretroviral therapy in HIV-1 subtype CRF01_AE infected seroconverters in Singapore

Background

It remains controversial as to whether HIV-1 subtypes influence disease progression. Singapore offers a unique opportunity to address this issue due to the presence of co-circulating subtypes. We compared subtype CRF01_AE and non-CRF01_AE infected patients, with regards to estimated annual rate of CD4+ T-cell loss and time from estimated data of seroconversion (EDS) to antiretroviral therapy (ART).

Methods

We recruited ART-naive patients with known dates of seroconversion between October 2002 and December 2007 at the Singapore Communicable Disease Centre, the national reference treatment centre. Multilevel mixed-effects models were used to analyse the rate of CD4+ T-cell decline. Time from EDS to ART was analyzed with the Kaplan-Meier survival method and compared with Cox proportional hazards models.

Results

54 patients with previously assigned HIV-1 subtypes (24 CRF01_AE, 17 B, 8 B'', 1 CRF33_01B, 3 CRF34_01B and 1 G) were observed for 89 patient-years. Subtype CRF01_AE and non-CRF01_AE infected patients did not differ in age, gender, risk factor, rate of symptomatic seroconversion, baseline CD4+ T-cell count, log₁₀ viral load or haemoglobin concentration. The estimated annual rate of CD4+ T-cell loss was 58 cells/mm³/year (95% CI: 7 to 109; P = 0.027) greater in subtype CRF01_AE infected patients compared to non-CRF01_AE patients, after adjusting for age, baseline CD4+ T-cell count and baseline log₁₀ viral load. The median time from EDS to ART was 1.8 years faster comparing CRF01_AE to non-CRF01_AE infected patient with a 2.5 times (95% CI: 1.2-5.0; P = 0.013) higher hazard for ART initiation, after controlling for age, baseline CD4+ T-cell count and baseline log₁₀ viral load.

Conclusions

Infecting subtype significantly impacted the rate of CD4+ T-cell loss and time to treatment in this cohort. Studies to understand the biological basis for this difference could further our understanding of HIV pathogenesis.     

Prediction of HIV-1 entry inhibitors neomycin-arginine conjugates interaction with the CD4-gp120 binding site by molecular modeling and multistep docking procedure

Developing of multi-target HIV-1 entry inhibitors represents an important avenue of drug therapy. Two such inhibitors are hexa-arginine-neomycin-conjugate (NeoR6) and nona-d-arginine-neomycin-conjugate (Neo-r9). Our findings that NeoR6-resistant mutations appear in the gp120 constant regions; and NeoR6 is not CCR5 antagonist, but inhibits CXCR4 and CCR5 HIV-1 using isolates, led us to suggest that NeoR6 may inhibit HIV-1 entry by interfering with the CD4-gp120 binding. To support this notion, we constructed a homology model of unliganded HIV-1_IIIB gp120 and docked NeoR6 and Neo-r9 to it, using a multistep docking procedure: geometric-electrostatic docking by MolFit; flexible ligand docking by Autodock3 and final refinement of the obtained complexes by Discover3. Binding free energies were calculated by MM-PBSA methodology. The model predicts competitive inhibition of CD4-gp120 binding by NeoR6 and Neo-r9. We determined plausible binding sites between constructed CD4-bound gp120 trimer and homology modeled membranal CXCR4, and tested NeoR6 and Neo-r9 interfering with this interaction. These models support our notion that another mechanism of anti-HIV-1 activity of NeoR6 is inhibition of gp120-CXCR4 binding. These structural models and interaction of NeoR6 and Neo-r9 with gp120 and CXCR4 provide a powerful approach for structural based drug design for selective targeting of HIV-1 entry and/or for inhibition of other retroviruses with similar mechanism of entry.     

Identification of a novel HIV-1 intra-circulating recombinant form 01_AE in China: a descendant of the previously identified CRF01_AE transmission clusters 1 and 6

<正>Dear Editors,Here,we report a novel HIV-1 intra-circulating recombinant form 01_AE(intra-CRF01_AE:CRF01-1AE/CRF01-6AE)composed of CRF01_AE transmission clusters 1 and 6,which was identified in a heterosexual male from Fujian,with one breakpoint observed in the vif gene.The CRF01_AE region I(HXB2:868–5184)of the recombinant clustered with CRF01_AE transmission cluster 1,which is     

Molecular mechanism of HIV-1 gp120 mutations that reduce CD4 binding affinity

The interaction of the HIV-1 fusion protein gp120 with its cellular receptor CD4 represents a crucial step of the viral infection process, thus rendering gp120 a promising target for the intervention with anti-HIV drugs. Naturally occurring mutations of gp120, however, can decrease its affinity for anti-infective ligands like therapeutic antibodies or soluble CD4. To understand this phenomenon on a structural level, we performed molecular dynamics simulations of two gp120 variants (termed gp120_3-2 and gp120_2-1), which exhibit a significantly decreased binding of soluble CD4. In both variants, the exchange of a nonpolar residue byglutamate was identified as an important determinant for reduced binding. However, those glutamates are located at different sequence positions and affect different steps of the recognition process: E471 in gp120_3-2 predominantly affects the CD4-bound conformation, whereas E372 in gp120_2-1 mainly modulates the conformational sampling of free gp120. Despite these differences, there exists an interesting similarity between the two variants: both glutamates exert their function by modulating the conformation and interactions of glycine-rich motifs (G366–G367, G471–G473) resulting in an accumulation of binding incompetent gp120 conformations or a loss of intermolecular gp120–CD4 hydrogen bonds. Thus, the present data suggests that interference with the structure and dynamics of glycine-rich stretches might represent a more widespread mechanism, by which gp120 mutations reduce binding affinity. This knowledge should be helpful to predict the resistance of novel gp120 mutations or to design gp120–ligands with improved binding properties.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:41     

Candidate multi-epitope vaccines in aluminium adjuvant induce high levels of antibodies with predefined multi-epitope specificity against HIV-1

Some neutralizing epitopes on HIV-1 envelope proteins were identified to induce antibodies which could effectively inhibit the infection of different strains in vitro. But only very low levels of these antibodies were determined in the HIV-1 infected individuals. To increase the levels of protective antibodies in vivo, we suggested multi-epitope vaccine as a new strategy to induce high level of neutralization antibodies with predefined multi-epitope specificity. A synthesized epitope peptide MP (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) containing three neutralizing epitopes (GPGRAFY, ELDKWA, RILAVERYLKD) was conjugated to carrier protein KLH, and then used for immunization in mouse together with aluminium adjuvant or Freund's adjuvant (FA). The candidate MP-KLH multi-epitope vaccine in aluminium adjuvant could induce antibody response very strongly to the epitope peptide C-(RILAVERYLKD-G)2 and the immunosuppressive peptide (P1) (LQARILAVERYLKDQQL) (antibody titer: 1:51200), strongly to the epitope peptide C-(ELDKWA-G)4 and the C-domain peptide (P2) (1:12800), and moderately to the epitope peptide C-(GPGRAFY)4 and the V3 loop peptide (1:1600). The immunoblotting analysis demonstrated that the antibodies in sera could recognize P1, P2, V3 loop peptides and rsgp41 (aa 539-684). These results are similar with that in the case of PI-BSA in FA, and suggest that the multi-epitope vaccine in aluminium could induce high levels of antibodies of predefined multi-epitope specificity, which provides experimental evidence for the new strategy to develop an effective neutralizing antibody-based multi-epitope vaccine against HIV-1.     

Differential profile of CRF receptor distribution in the rat stomach and duodenum assessed by newly developed CRF receptor antibodies

Peripheral corticotropin-releasing factor (CRF) receptor ligands inhibit gastric acid secretion and emptying while stimulating gastric mucosal blood flow in rats. Endogenous CRF ligands are expressed in the upper gastrointestinal (GI) tissues pointing to local expression of CRF receptors. We mapped the distribution of CRF receptor type 1 (CRF1) and 2 (CRF2) in the rat upper GI. Polyclonal antisera directed against the C-terminus of the CRF receptor protein were generated in rabbits and characterized by western blotting and immunofluorescence using CRF1- and CRF2-transfected cell lines and in primary cultured neurons from rat brain cortex. A selective anti-CRF1 antiserum (4467a-CRF1) was identified and used in parallel with another antiserum recognizing both CRF1 and CRF2 (4392a-CRF1&2) to immunostain gastric tissue sections. Antiserum 4467a-CRF1 demonstrated specific immunostaining in a narrow zone in the upper oxyntic gland within the stomach corpus. Conversely, 4392a-CRF1&2 labeled cells throughout the oxyntic gland and submucosal blood vessels. Pre-absorption with the specific antigen peptide blocked immunostaining in all experiments. Doublestaining showed co-localization of 4392a-CRF1&2 but not 4467a-CRF1 immunoreactivity with H/K-ATPase and somatostatin immunostaining in parietal and endocrine cells of the oxyntic gland. No specific staining was observed in the antrum with either antisera, whereas only antiserum 4392a-CRF1&2 showed modest immunoreactivity in the duodenal mucosa. Finally, co-localization of CRF2 and urocortin immunoreactivity was found in the gastric glands. These results indicate that both CRF receptor subtypes are expressed in the rat upper GI tissues with a distinct pattern and regional differences suggesting differential function.