The development of HIV vaccines targeting gp41 membrane-proximal external region (MPER): challenges and prospects

A human immunodeficiency virus type-1 (HIV-1) vaccine which is able to effectively prevent infection would be the most powerful method of extinguishing pandemic of the acquired immunodeficiency syndrome (AIDS). Yet, achieving such vaccine remains great challenges. The membrane-proximal external region (MPER) is a highly conserved region of the envelope glycoprotein (Env) gp41 subunit near the viral envelope surface, and it plays a key role in membrane fusion. It is also the target of some reported broadly neutralizing antibodies (bNAbs). Thus, MPER is deemed to be one of the most attractive vaccine targets. However, no one can induce these bNAbs by immunization with immunogens containing the MPER sequence(s). The few attempts at developing a vaccine have only resulted in the induction of neutralizing antibodies with quite low potency and limited breadth. Thus far, vaccine failure can be attributed to various characteristics of MPER, such as those involving structure and immunology; therefore, we will focus on these and review the recent progress in the field from the following perspectives: (1) MPER structure and its role in membrane fusion, (2) the epitopes and neutralization mechanisms of MPER-specific bNAbs, as well as the limitations in eliciting neutralizing antibodies, and (3) different strategies for MPER vaccine design and current harvests.

IL15 DNA adjuvant enhances cellular and humoral immune responses induced by DNA and adenoviral vectors encoding HIV-1 subtype B gp160 gene / 病毒学报

To enhance the immunogenicity of DNA and adenoviral vector vaccines expressing HIV-1 subtype B gp160, human interleukin 15 (hIL15) DNA adjuvant (pVR-hIL15) was constructed. BALB/c mice received DNA prime/protein boost immunization with pVR-HIVgp160/Ad5-HIVgp160 alone or combined with pVR-hIL15. Cellular and humoral immune responses were evaluated by IFN-gamma enzyme-linked immunosorbent spot assay and enzyme-linked immunosorbent assay, respectively. Compared with those immunized with vaccines alone, the mice immunized with vaccines combined with pVR-hIL15 had significantly increased specific cellular response and antibody titer (P < 0.05). It suggests that the IL15 DNA adjuvant can enhance the immune responses induced by prime-boost regimen using DNA and adenoviral vector encoding HIV-1 subtype B gp160.

Characterization of Gp41 Polymorphisms in the Fusion Peptide Domain and T-20 (Enfuvirtide) Resistance-Associated Regions in Korean HIV-1 Isolates

HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naive Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.

Expression, structure and antigenicity analysis of N51 derived from the N-terminal heptad repeat domain in gp41 of HIV-1 CRF07_BC strain / 南方医科大学学报

<p><b>OBJECTIVE</b>To express N51 derived from the N-terminal heptad repeat (NHR) domain in gp41 of the HIV-1 CRF07_BC strain and analyze its molecular structure and antigenicity.</p><p><b>METHODS</b>Overlapping PCR was used to amplify the DNA fragment encoding N51Fd gene, which was then subcloned into the vector pFUSE-hIgG1-Fc2. The construct was confirmed by DNA sequencing. The structure and antigenicity of the recombinant protein N51FdFc-BC were analyzed using bioinformatic software, circular dichroism, and Western blotting.</p><p><b>RESULTS</b>A recombinant expression vector pFUSE/N51Fd-BC was successfully constructed. N51FdFc-BC recombinant protein with a relative molecular mass of about 35 000 was effectively expressed in mammalian 293T cells and could be recognized by rabbit antibodies against HIV-1 gp41 N/C peptides as shown by Western blotting. Bioinformatic analysis showed that the recombinant protein N51FdFc-BC, with a relative molecular mass of 34 315.1 and a PI of 7.59, formed a secondary structure of random coil to allow its interactions as an antigen with antibodies. Circular dichroism measurement confirmed the random coil structure of N51FdFc-BC protein.</p><p><b>CONCLUSION</b>The recombinant protein N51FdFc-BC has a random coil structure and can be used as an immunogen for development of HIV-1 subunit vaccine.</p>

Subtype analysis of gp41 gene of HIV-1 among men who have sex with men in Zhengzhou / 中华预防医学杂志

<p><b>OBJECTIVE</b>This study aimed to investigate the subtype distribution of gp41 gene of human immunodeficiency virus-1 (HIV-1) among men who have sex with men (MSM) in Zhengzhou.</p><p><b>METHODS</b>Thirty blood samples were collected from men who have sex with men infected by HIV. The complete gp41 gene was amplified by RT-PCR and nested-PCR and sequenced. All sequences were edited by Bioedit and subtyped with HIV sequence library US Los Alamos National Laboratory and online genotyping software provided by American National Center of Biotechnology Information. Phylogenetic analysis of gp41 gene was performed using the MEGA 3.1 software, and the genic dispersion rates among subtype of gp41 gene were analyzed.</p><p><b>RESULTS</b>A total of eighteen gene sequences of HIV-1 gp41 gene were obtained from thirty men who have sex with men infected by HIV, which belonged to subtype CRF15-01B (50% (9/18)), CRF01-AE (22% (4/18)), CRF07-B (22% (4/18)) and B (6% (1/18)), respectively. The intersubtype HIV-1 strains aggregate with according reference strains. The genetic distance inter-subtype of subtype CRF15-01B, CRF01-AE and CRF07-B were 0.050 ± 0.007, 0.052 ± 0.009 and 0.082 ± 0.012, respectively.</p><p><b>CONCLUSION</b>The prevalent subtypes of HIV-1 among among MSM in Zhengzhou was complicated and recombinant HIV-1 strains were the most prevalent strains.</p>

Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion

Despite the high mutation rate of HIV-1, the amino acid sequences of the membrane-spanning domain (MSD) of HIV-1 gp41 are well conserved. Arginine residues are rarely found in single membrane-spanning domains, yet an arginine residue, R(696) (the numbering is based on that of HXB2), is highly conserved in HIV-1 gp41. To examine the role of R(696), it was mutated to K, A, I, L, D, E, N, and Q. Most of these substitutions did not affect the expression, processing or surface distribution of the envelope protein (Env). However, a syncytia formation assay showed that the substitution of R(696) with amino acid residues other than K, a naturally observed mutation in the gp41 MSD, decreased fusion activity. Substitution with hydrophobic amino acid residues (A, I, and L) resulted in a modest decrease, while substitution with D or E, potentially negatively-charged residues, almost abolished the syncytia formation. All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein (DSP) assay that scores the degree of membrane fusion based on pore formation between fusing cells. Interestingly, the D and E substitutions did show some fusion activity in the DSP assays, suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability. However, nascent pores failed to develop, due probably to impaired activity in the pore enlargement process. Our data show the importance of this conserved arginine residue for efficient membrane fusion.

Qadirvirtide

Qadirvirtide is a fusion inhibitor that may be used as prophylaxis or for the treatment of AIDS. It is a synthetic peptide that is composed of 36 amino acids. Qadirvirtide blocks the entry of HIV genome into human CD4 cells by binding to HR1 as the virus can not come close to the human cell membrane and ultimately fusion of the viral envelope with human cell membrane is prohibited

Effect of HIV-1gp41 ectodomain on Cryptococcus neoformans-induced cytoskeletal changes in human brain microvascular endothelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of HIV-1 gp41 ectodomain (gp41-I90) on the cytoskeletal changes in human brain microvascular endothelial cells (HBMECs) induced by Cryptococcus neoformans.</p><p><b>METHODS</b>HBMECs were cultured on collagen-coated chamber slide or transwell to allow the formation of cell monolayers. After pre-treatment with gp41-I90 and infection with Cryptococcus neoformans, the HBMECs were examined for the expression of actin or filamin by immunofluorescence assay. HRP permeability of the HBMECs treated with gp41-I90 was detected by ELISA. Transcytosis of Cryptococcus neoformans through the gp41-I90-treated HBMECs was detected by direct counting from a hemocytometer.</p><p><b>RESULTS</b>gp41-I90 obviously enhanced the cytoskeletal changes of the HBMECs infected by Cryptococcus neoformans, causing curved and sparse filamentous arrangement of actin and filamin. gp41-I90 treatment also resulted in obviously increased HRP permeability of the cells and transcytosis of Cryptococcus neoformans.</p><p><b>CONCLUSION</b>gp41- I90 enhances Cryptococcus neoformans binding to HBMECs, which is related to its effect in enhancing Cryptococcus neoformans-induced cytoskeletal changes of the cells.</p>

HIV entry inhibitors: progress in development and application / 药学学报

This review discusses recent progress in the development of anti-HIV agents, with emphasis on small molecule HIV-1 entry inhibitors. The entry inhibitors primarily target HIV-1 envelope glycoproteins or the cellular receptors, CD4 and chemokine receptors. Two of the entry inhibitors, enfuvirtide and maraviroc, have been approved by the US FDA for AIDS therapy. The drug resistance associated with some of the entry inhibitors will also be discussed.

The current progress in the development of HIV-1 fusion inhibitors / 药学学报

HIV-1 fusion inhibitors are a new class of anti-HIV compounds, which block the entry of HIV into target cells through preventing the fusion between viral and cell plasma membrane and thus interrupt the initial steps of viral replication. T-20 (enfuvirtide), which has been clinically approved as the first fusion inhibitor of HIV-1 by U.S. FDA in 2003, can suppress replication of HIV variants with multi-drug resistance to reverse transcriptase and protease inhibitors. Peptides and small molecules display potent anti-HIV fusion activities by targeting gp41 thus inhibit its fusogenic function. In recent years, with the development of studies on the molecular mechanism of HIV membrane fusion process and the function of gp41, many new fusion inhibitors are found and some have been in advanced clinical trials. This review discusses recent progress in the development of HIV-1 fusion inhibitors targeting the gp41.

The newest developments in anti-HIV-1 drugs / 药学学报

In the two decades since AZT was first approved for clinical use in 1987, 24 additional antiretroviral agents have been approved. They include 7 nucleoside analogs, a nucleotide analog and 4 non-nucleoside reverse transcriptase inhibitors, 10 protease inhibitors, 2 entry inhibitors and an integrase inhibitor. More than 20 investigational agents are currently being studied in clinical trials. Highly active antiretroviral therapy (HAART), which involves a combination of anti-HIV-1 drugs, is extremely effective in suppressing HIV-1 replication and increasing CD4+ number and results in substantial reductions in HIV-1-related morbidity and mortality. In last 20 years, much has been learned about resistance to antiretroviral drugs, drug interactions and metabolic complications of antiviral drug use. Drugs are now selected on the basis of resistance tests and on the risk of specific drug complications in individual patients. As a result, decisions about the therapy of HIV/AIDS have become personalized and are made on a patient-by-patient basis. With appropriate medical management, a person with HIV-1 now has the possibility of a nearly normal life expectancy.

A non-infectious and quantitative cell-based bioassay for screening HIV entry inhibitors targeting HIV envelope proteins / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors.</p><p><b>METHODS</b>HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method.</p><p><b>RESULTS</b>No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner.</p><p><b>CONCLUSION</b>We have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.</p>

Development of a rapid test kit for antibody to HIV by nano immunomagnetic lateral flow method / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a rapid test kit for antibody to HIV by nano immunomagnetic lateral flow method.</p><p><b>METHODS</b>A rapid test kit was developed by conjugation of the HIV antigen gp41 and gp36 to 200nm super paramagnetic particles by carbodiimide (EDC) and coating of the HIV antigen gp41 and gp36 to nitrocellulose membrane. Then the kit was evaluated with serials of experiments.</p><p><b>RESULTS</b>The kit was qualified with examination of national reference panel of anti-HIV antibody for colloidal gold diagnostic kit. The sensitivity was 100% by tested with 20 HIV antibody positive sera, the specificity was 98.5% by tested with 600 HIV antibody negative sera, respectively. The stability of the kit was over 12 month by storage at room temperature.</p><p><b>CONCLUSION</b>A diagnostic kit for antibody to HIV was developed with the advantages of convenience, rapid test, good stability and point of care.</p>

Association of variations of NAb 2F5 and 4E10 epitopes and disease progression in Chinese antiretroviral treatment-naïve patients infected with HIV-1 clade B' / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Studies on human immunodeficiency virus type 1 (HIV-1) vaccines have recently focused on targeting the conserved neutralizing epitopes 2F5 and 4E10, and hence it is important to understand the extent of mutations in these two viral epitopes. Here, we investigated the amino acid mutations in epitopes of 2F5 (ELDKWA, HIV-1 HXB2 env 662 - 667 aa) and 4E10 (NWFDIT, HIV-1 HXB2 env 671 - 676 aa) in the membrane proximal-external region of gp41 from clade B' HIV-1-infected individuals living in Henan province, China. We also examined the frequency of a mutation and its relation to disease progression.</p><p><b>METHODS</b>A cohort of 54 treatment-naïve HIV-1-infected individuals was recruited in this study, and 16 individuals were selected for a short-term longitudinal study on sequence evolution. The HIV-1 env gp41 gene was amplified, cloned, and sequenced, and predicted amino acid sequences were aligned for analysis.</p><p><b>RESULTS</b>The mutations E662A and K665E on the 2F5 epitope and N671S and T676S on the 4E10 epitope were seen. Simultaneous RNA sequencing showed some discrepancies with proviral DNA sequences. In our longitudinal study, mutation levels of these two neutralizing epitopes were low but diverse and persistent. The frequencies of mutations within the 4E10 peptide NWFDIT in slow progressors were noticeably lower than those in AIDS patients (P < 0.05).</p><p><b>CONCLUSIONS</b>Antigenic variation of the neutralizing epitopes 2F5 and 4E10 is limited in subtype B' infection, and that 4E10 peptide mutation is correlated with disease progression. Monitoring epitope mutations will offer useful data for development of the candidate 2F5-like and 4E10-like antibodies to prevent and treat AIDS.</p>

High level expression of 5-helix protein in HIV gp41 heptad repeat regions and its virus fusion-inhibiting activity / 生物工程学报

The artificial 5-helix can inhibit the formation of trimer-of-hairpins structure during the course of HIV-directed membrane fusion and then inhibit human immunodeficiency virus (HIV) infecting target cells. But 5-helix was apt to form inclusion body when expressed directly in prokaryotic cell and was difficult to renature, which causes inconvenience to future study. We found a proper expression vector by simulating protein structure. We simulated its proper conformation in two vectors pGEX-6P-1 and pET44b by homology modeling. The contrast of conformations showed that the energy of salvation of its fusion protein with NusA in vector pET44b was higher than its fusion protein with glutathione-S-transferase (GST) in pGEX-6P-1 and its restriction site lay on the surface of its fusion protein in vector pET44b. 5-helix gene was amplified from pGEX-6P-1-5H by PCR, and was ligated to pET44b to construct recombinant vector pET44b-PSP-5Helix after tested correctly by enzymes digestion. The recombinant vector was transformed into Escherichia coli BL21 (DE3) to express 5-helix protein at different temperatures. Aim protein was purified with Ni column and GST column, and was determined by SDS-PAGE. Then the purified 5 -Helix was used to test the inhibitive activity of pseudo HIV virus infecting GHOST-CXCR4. Results show that its fusion protein with NusA can be effectively soluble expressed and easier to be cleaved, and that the purified 5-helix can efficiently inhibit pseudo HIV virus infecting GHOST-CXCR4 and its IC50 value is (22.77 +/- 5.64) nmol/L, which lay the foundation to further discuss the application in HIV-1 infection.

Yeast surface display of HIV-1 gp41 and expression enhancement / 生物工程学报

HIV-1 gp41 has been successfully anchored on the cell surface of yeast Saccharomyces cerevisiae by yeast cell-surface display systems using His-tag for the detection of protein expression. Gp41 activity has been detected by gp41 monoclonal antibody. The vector for gp41 yeast display has been constructed as follows: the gene-encoding gp41 was amplified by PCR using pMD18T-gp41 as a template, and then inserted into shuttle vector pICAS-His by restriction enzyme digestion. Next, the vectors were introduced into Saccharomyces cerevisiae MT8-1. After cultivation, recombinant cells were immunofluorescence labelled. The bright green cells were observed by the microscopy indicating the proteins have been displayed on the cell surface successfully, flow cytometry convinced that gp41 has been folded correctly on the cell surface. Then different concentrations of initial glucose were used to enhance the expression of protein. gp41 has been expressed by 82.46% yeast cells as the concentration of glucose was 1%. Protein expression was depressed when the concentration was increased.

1,2,6-tri-O-galloyl-beta-D-glucopyranose inhibits gp41-mediated HIV envelope fusion with target cell membrane / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the inhibitory effect of 1,2,6-Tri-O-galloyl-beta-D-glucopyranose (TGGP) from Balanophora japonica Makino on human immunodeficiency virus (HIV) entry into the host cells and explore the mechanisms.</p><p><b>METHODS</b>TGGP was purified from Balanophora japonica Makino by n-hexane and ethyl acetate extraction and column chromatography. The inhibitory activity of TGGP on HIV gp41 six-helix bundle formation was measured with ELISA, N-PAGE and SE-HPLC, and the inhibitory effect of TGGP on HIV envelope grlycoprotein-induced cell-cell fusion was detected using a non-infectious cell-based assay.</p><p><b>RESULTS</b>TGGP inhibited HIV gp41 six-helix bundle formation, with an IC50 of 1.37-/+0.19 microg/ml as determined by ELISA, and this activity was further confirmed by N-PAGE and SE-HPLC. TGGP at 25 microg/ml significantly inhibited syncytium formation between the effector (CHO-WT) and the target (MT-2) cells.</p><p><b>CONCLUSION</b>The HIV transmembrane subunit gp41 mediates the entry of HIV into the target cells. TGGP can inhibit HIV fusion and entry into the target cells by inhibiting the formation of gp41 six-helix bundles, suggesting the potential of TGGP as a microbicide to prevent sexual transmission of HIV.</p>

Immune response analysis of immunized Balb/C mice with HIV-1 fusion p24 - gp41 genes as DNA vaccine candidate

The global HIV epidemic continues to expand and exceeding previous predictions. An effective vaccine represents the best hope to curtail the HIV epidemic. DNA vaccines induce humoral and cellular responses and mimic live vaccines without their pathogenic potential. The importance of CD8[+]CTL responses in controlling HIV and SIV viremia has led to production of a series of vaccines candidates that effectively induce these responses. It is now widely believed that an HIV vaccine strategy must stimulate both a strong humoral [antibody] as well as cell-mediated [CTL] immune response. The p24 and gp41 play many important roles in host-virus interaction and pathogenesis. These proteins are considered as attractive vaccine candidate in which their immunogenecity and immunomodulatory effects have been confirmed. In this study, a construct, pcDNA3.1Hygro- [p24-gp41], was evaluated as a DNA vaccine candidate in Balb/C mice for generation of effective cellular immune responses. For immunizing, we used dendrosome, a novel family of vehicles for transfection and therapy. IFN-gamma cytokine production and total antibody were detected by ELISA. Lymphoprolifration assay was performed by MTT test. ELISA and MTT assays confirmed that the cited p24-gp41 fusion gene is able to enhance immune responses in mice. The construct that was used in this research can be a good candidate for DNA vaccine against HIV-1, if the future complementary tests demonstrate the same trends of immunogenic responses shown in this study

Inhibitory effect of polysaccharides on the six-alpha-helix bundle formation of HIV gp41 protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the in vitro inhibitory effect of expolysaccharides from Streptomyces, polysaccharides of Ganoderma lucidum and rice bran on six-alpha-helix bundle formation of HIV gp41 protein.</p><p><b>METHODS</b>The amount of six-alpha-helix bundle formed in the presence of N36 and C34 was tested by ELISA in response to treatments with different doses of polysaccharides.</p><p><b>RESULTS</b>Expolysaccharides from Streptomyces potentially inhibited six-alpha-helix bundle formation with the effective concentration (IC(50)) of 145.48-/+7.25 mg /L. Polysaccharides of Ganoderma lucidum and rice bran showed no effect on the six-alpha-helix bundle formation.</p><p><b>CONCLUSION</b>Expolysaccharides from Streptomyces can inhibit the six-alpha-helix bundle formation of HIV gp41, whereas polysaccharides of Ganoderma lucidum and rice bran do not exhibit such activity.</p>

Structure-activity relationships of anti-HIV-1 peptides with disulfide linkage between D- and L-cysteine at positions i and i+3, respectively, derived from HIV-1 gp41 C-peptide

The constrained alpha-helical structure of a C-peptide is useful for enhancing anti-HIV-1 activity. The i and i+3 positions in an alpha-helical structure are located close together, therefore D-Cys (dC) and L-Cys (C) were introduced at the positions, respectively, to make a dC-C disulfide bond in 28mer C-peptides. Accordingly, this study tested whether a dC-C disulfide bond would increase the alpha-helicity and anti-HIV-1 activity of peptides. A C-peptide can be divided into three domains, the N-terminal hydrophobic domain (HPD), middle interface domain (IFD), and C-terminal hydrogen domain (HGD), based on the binding property with an N-peptide. In general, the dC-C modifications in HPD enhanced the anti-HIV-1 activity, while those in IFD and HGD resulted in no or much less activity. The modified peptides with no activity clearly showed much less alpha-helicity than the native peptides, while those with higher activity showed an almost similar or slightly increased alpha-helicity. Therefore, the present results suggest that the introduction of a dC-C bridge in the N-terminal hydrophobic domain of a C-peptide may be useful for enhancing the anti-HIV-1 activity.