Chemokine receptors as fusion cofactors for human immunodeficiency virus type 1 (HIV-1)

CD4 is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1), but is not sufficient for entry of HIV-1 into cells. After a decade-long search, the cellular coreceptors that HIV-1 requires in conjunction with CD4 have been identified as members of the chemokine receptor family of seven-transmembrane G-protein coupled receptors. The discovery of distinct chemokine receptors that support entry of T-cell tropic (CXCR-4) and macrophage tropic HIV-1 strains (CCR-5) explains the differences in cell tropism between viral strains, the inability of HIV-1 to infect most nonprimate cells, and the resistance of a small percentage of the population to HIV-1 infection. Further understanding of the role of chemokine receptors in viral entry may also help explain the evolution of more pathogenic forms of the virus, viral transmission, and HIV-induced pathogenesis. These recent discoveries will aid the development of strategies for combating HIV-1 transmission and spread, the understanding of HIV-1 fusion mechanisms, and the possible development of small animal models for HIV-1 drug and vaccine testing.     

含中国流行株HIV—1Gag—gp120融合蛋白的重组鸡痘病毒的构建

目的用重组鸡痘病毒表达中国流行株HIV-1Gag-gp120融合蛋白.方法在以鸡痘病毒282E4株为基础构建的重组表达质粒pUTAL的ATI-P7.5复合启动子下游,插入编码中国流行株HIV-1Gag-gp120嵌合基因,构建了重组表达质粒pUTALGP.重组质粒与FPV共转染CEF细胞,进行了同源重组.通过PUdR加压筛选,X-gal染色,获得重组鸡痘病毒vUTALGP.结果经Westernblot检测表明,重组病毒表达了Gap-gp120融合蛋白,其分子量分别为110×103、69×103、48×103、24×103.电镜观察可见Gag蛋白在CEF细胞中形成的反转录病毒样粒子.重组病毒免疫小鼠,能够诱导HIV-1特异的血清抗体反应.结论重组鸡痘病毒成功的表达了Gag-gp120融合蛋白,并进行了正确的加工.     

Immunisation with recombinant modified vaccinia virus Ankara expressing HIV-1 gag in HIV-1-infected subjects stimulates broad functional CD4+ T cell responses

Virus-specific CD4+ T cells with IL-2-secreting and/or proliferative capacity are detected readily in HIV-1-infected long-term nonprogressors and rarely in persons with untreated progressive infection. The contribution of these cells to viraemia control is uncertain, but this question might be addressed in clinical therapeutic vaccination studies. However, the quality of T helper responses induced by currently available HIV-1 vaccine candidates has not been explored in depth. We determined the effect of vaccination with modified vaccinia virus Ankara (MVA) expressing HIV-1 gag p24/p17 (MVA.HIVA) on HIV-1-specific CD4+ T cell responses in 16 chronically infected, highly active antiretroviral therapy (HAART)-treated subjects using CD8-depleted IFN-gamma ELISPOT assays, intracellular cytokine staining assays for IL-2 and IFN-gamma, and a CFSE-based proliferation assay. Gag-specific CD4+ T cell responses were significantly increased in magnitude and breadth after vaccination and targeted both known and new epitopes, several of which were also recognised by healthy HIV-uninfected volunteers immunised with the same vaccines. The frequencies of CD4+ T cells expressing IL-2 or IFN-gamma, alone or simultaneously, were also augmented. These findings indicate that functional virus-specific T helper cells can be boosted by vaccination in chronic HIV-1 infection. Further evaluation of their role in viraemia control is warranted.     

Bet v 1 homologues in strawberry identified as IgE-binding proteins and presumptive allergens

BACKGROUND: No strawberry allergen has so far been identified and characterized. METHODS: Serum samples were collected from patients with a suggestive case history of adverse reactions to strawberry and other fruits. Extracts from fresh and frozen strawberries were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and mass spectrometry. Patient blood samples were analysed for inhibition of IgE binding and basophil degranulation. RESULTS: Several IgE-binding proteins could be detected. In more than half of the patient sera, a 20/18-kDa doublet band was observed in Western blotting. These two bands were excised and analysed by mass spectrometry showing the presence of proteins belonging to the Bet v 1 family of allergens. Inhibition of the IgE binding to the 20/18-kDa doublet was obtained by addition of two recombinantly expressed allergens belonging to the Bet v 1 family (Bet v 1 and Mal d 1) and strawberry protein extract. In a cell-based assay of patient blood samples, basophil degranulation could be induced by strawberry protein extract and by Bet v 1 and Mal d 1. CONCLUSIONS: We conclude that strawberry homologues to Bet v 1 may be allergens of importance for adverse reactions to strawberry.     

A novel HIV-1 reporter virus with a membrane-bound Gaussia princeps luciferase

HIV-1 reporter viruses are a critical tool for investigating HIV-1 infection. By having a reporter gene incorporated into the HIV-1 genome, the expressed reporter protein acts as a specific tag, thus enabling specific detection of HIV-1 infected cells. Currently existing HIV-1 reporter viruses utilize reporters for the detection of HIV-1 infected cells by a single assay. A reporter virus enabling the detection of viral particles as well as HIV-1 infected cells by two assays can be more versatile for many applications. In this report, a novel reporter HIV-1 was generated by introducing a membrane-anchored form of the Gaussia princeps luciferase gene (mGluc) upstream of the nef gene in the HIV-1(NL4-3) genome using a picornaviral 2A-like sequence. The resulting HIV-1(NL4-3mGluc) virus expresses G. princeps luciferase efficiently on viral membrane and the cell surface of infected human T cell lines and primary peripheral blood mononuclear cells. This HIV-1 reporter is replication competent and the reporter gene mGluc is expressed during multiple rounds of infection. Importantly, viral particles can be detected by bioluminescence and infected cells can be detected simultaneously by bioluminescence and flow cytometric assays. With the versatility of two sensitive detection methods, this novel luciferase reporter has many applications such as cell-based screening for anti-HIV-1 agents or studies of HIV-1 pathogenicity.     

An O-linked carbohydrate neutralization epitope of HIV-1 gp 120 is expressed by HIV-1env gene recombinant vaccinia virus

Summary Previous studies have disagreed about the presence of O-linked carbohydrate epitopes on gp 120 of HIV, although antibodies against short-chain O-linked glycans neutralize HIV infection and block syncytium formation in vitro. To settle this question, we analysed the O-linked glycans of gp 120 by chemical methods using purified HIV-1 gp 120 from cells infected with recombinant vaccinia virus solely expressing gp 160 or gp 120. Alkaline borohydride degradation of recombinant gp 120 released monosaccharides and also slightly larger structures (di/trisaccharides) by a -elimination, confirming the presence of simple O-linked oligosaccharides. The functional activity as neutralisation epitopes of the O-linked oligosaccharides expressed on recombinant gp 120 was preserved, since fusion between uninfected CD 4⁺ cells and cells infected with recombinant vaccinia was blocked by monoclonal antibodies to the O-linked oligosaccharides of gp 120. Although the mechanism for HIV induction of O-linked oligosaccharide neoantigens is unknown, these results indicate that the O-linked neutralization epitopes are inherent to the glycoprotein itself, and that the unusual appearance of simple O-linked oligosaccharides on gp 120 is independent of any interaction between the host cell and retroviral genes other thanenv.     

Epitope-vaccine as a new strategy against HIV-1 mutation

An effective vaccine is urgently needed to stop AIDS-epidemic. Up to now none of the candidate HIV-vaccines has been developed to prevent HIV-1 infection. A few neutralizing antibodies against HIV-1 enveloping proteins proved to be highly effective to neutralize different strains in vitro. Unfortunately, these antibodies are rare in infected humans, and have never yet been raised by a vaccine. The multiple sequential and antigenic variability of HIV-1 led to unprecedented difficulties in development of effective vaccines and anti-viral drugs. More and more experimental evidences indicated that HIV-1 mutants resulted in immune evasion may be a grave challenge for conventional strategy to prepare effective vaccines. We suggested that epitope-vaccine could be a new strategy to induce high levels of neutralizing antibodies with predefined epitope-specificity against HIV-1. Several candidate epitope-vaccines including mono-epitope-vaccine, multi-epitope-vaccine, epitope-vaccines in combination, were prepared and systematically studied in animal experiments. These studies provided experimental evidences that epitope-vaccine could be a new strategy to develop effective vaccines for prevention and immunotherapy against viral infection of HIV-l or other viruses.     

Adenoviral gene delivery for HIV-1 vaccination

The AIDS epidemic continues to spread throughout nations of Africa and Asia and is by now threatening to undermine the already frail infrastructure of developing countries in Sub-Saharan Africa that are hit the hardest. The only option to stem this epidemic is through inexpensive and efficacious vaccines that prevent or at least blunt HIV-1 infections. Despite decades of pre-clinical and clinical research such vaccines remain elusive. Most anti-viral vaccines act by inducing protective levels of virus-neutralizing antibodies. The envelope protein of HIV-1, the sole target of neutralizing antibodies, is constantly changing due to mutations, B cell epitopes are masked by heavy glycosylation and the protein's structural unfolding upon binding to its CD4 receptor and chemokine co-receptors. Efforts to induce broadly cross-reactive virus-neutralizing antibodies able to induce sterilizing or near sterilizing immunity to HIV-1 have thus failed. Studies have indicated that cell-mediated immune responses and in particular CD8+ T cell responses to internal viral proteins may control HIV-1 infections without necessarily preventing them. Adenoviral vectors expressing antigens of HIV-1 are eminently suited to stimulate potent CD8+ T cell responses against transgene products, such as antigens of HIV-1. They performed well in pre-clinical studies in rodents and nonhuman primates and are currently in human clinical trials. This review summarizes the published literature on adenoviral vectors as vaccine carriers for HIV-1 and discusses advantages and disadvantages of this vaccine modality.     

Altered IgE epitope presentation: A model for hypoallergenic activity revealed for Bet v 1 trimer

In order to reduce side effects in the course of allergen specific immunotherapy hypoallergenic allergen derivatives with reduced IgE reactivity have been made by genetic engineering. In contrast to other recombinant hypoallergenic allergen derivatives which showed reduced IgE reactivity, a recombinant trimer of the major birch pollen allergen Bet v 1 showed reduced allergenic activity despite preserved IgE reactivity. We studied rBet v 1 trimer by SDS-PAGE, mass spectrometry, circular dichroism and gel filtration. Furthermore we investigated IgE and IgG reactivity of the rBet v 1 trimer in solid and liquid phase assays and compared its allergenic activity with that of rBet v 1 wildtype using basophil activation assays. In solid phase immunoassays rBet v 1 trimer exhibited even stronger IgE reactivity than the rBet v 1 wildtype, whereas both proteins were equally well recognized by Bet v 1-specific IgG antibody probes. In fluid phase IgE experiments rBet v 1 trimer inhibited IgE reactivity to rBet v 1 wildtype but showed a more than 10-fold reduced allergenic activity compared to the rBet v 1 monomer. By analytical gel filtration it was demonstrated that, despite its monomeric appearance in SDS-PAGE the trimer occurred in fluid phase in the form of defined high molecular weight (>600 kDa) aggregates whereas rBet v 1 wildtype strictly appeared as monomeric protein. The results indicate that the hypoallergenic nature of the rBet v 1 trimer is due to formation of defined high molecular weight aggregates which may be responsible for an altered presentation of IgE epitopes in a form with reduced capacity to crosslink effector-cell bound IgE. We thus provide evidence for a novel mechanism for hypoallergenic activity.     

Use of rubella virus E1 fusion proteins for detection of rubella virus antibodies.

Two glutathione S-transferase fusion proteins containing 44 (p1503) and 75 (p1509) amino acid residues of the rubella virus E1 glycoprotein were expressed in Escherichia coli with the aim of producing a recombinant rubella virus antigen for use in serological assays. p1503 contained three neutralizing and hemagglutinating epitopes (G. M. Terry, L. M. Ho-Terry, P. Londesborough, and K. R. Rees, Arch. Virol. 98:189-197, 1988); p1509 contained the putative neutralization domain described by Mitchell et al. (L. A. Mitchell, T. Zhang, M. Ho, D. Decarie, A. Tingle, M. Zrein, and M. Lacroix, J. Clin. Microbiol. 30:1841-1847, 1992) in addition to the three epitopes present in p1503. Both fusion proteins were soluble and affinity purified on glutathione-Sepharose 4B. In Western blots (immunoblots), p1503 and p1509 reacted with human sera containing rubella virus-specific immunoglobulin G. When used as antigens in indirect enzyme immunoassays to detect rubella virus-specific immunoglobulin G, p1503 correctly identified the rubella virus antibody status of 43 (76.8%) and p1509 correctly identified that of 48 (85.7%) of 56 serum samples received for routine rubella virus antibody screening. The results obtained with p1509 compare well with those obtained with a latex agglutination assay.     

Development of a novel allo-independent HIV-1 virus preparation for use in immunoassays

The study of the immunologic response to whole human immunodeficiency virus type 1 (HIV-1) antigen is limited by the presence of highly immunogenic human leukocyte antigen (HLA) alloantigens on the envelope of wild type virus. This paper outlines the production of HIV-1 infectious virions free of HLA for use as whole viral antigens in immunoassays. An infectious molecular clone of HIV-1 was transfected into the K-562 cell line, which does not express HLA on the cell surface. After a 30-day selection period, to ensure stable transfection, cells and culture supernatants were analyzed for productive HIV-1 infection and virion infectivity. An enzyme-linked immunosorbent assay (ELISA) confirmed the presence of p24 in the culture supernatants. Molecular confirmation of HIV-1 transfection was achieved by gene amplification. Flow cytometric analysis was used to identify gp120 on the surface of the infected cells. Viral supernatants were tested for HIV infectivity in peripheral blood mononuclear cells (PBMCs). The usefulness of this viral preparation as whole virus antigens was validated using PBMCs from HIV-infected individuals. These results indicate the successful production of HIV-1 infectious virions, which do not have HLA molecules on their viral envelope, and demonstrate their utility for immunoassays.     

Monitoring of HIV-1 envelope-mediated membrane fusion using modified split green fluorescent proteins

A simple, cell-based, membrane fusion assay system that uses split green fluorescent proteins (spGFPs) as an indicator was developed. The attachment of the pleckstrin homology (PH) domain to the N-termini of each spGFP not only localized the reporter signal to the plasma membrane but also helped the stable expression of the smaller spGFP of seventeen amino acid residues. It was shown that this system allowed real-time monitoring of membrane fusion by HIV-1 envelope protein (Env) without the addition of external substrates. This method can be adapted to the analyses of other viral membrane fusion.     

Bet v 1142-156 is the dominant T-cell epitope of the major birch pollen allergen and important for cross-reactivity with Bet v 1-related food allergens

BACKGROUND: Individuals with birch pollen allergy frequently experience hypersensitivity reactions to certain foods, primarily because of IgE antibodies specific for the major birch pollen allergen Bet v 1 that cross-react with homologous food allergens. OBJECTIVE: We sought to characterize the major T-cell epitopes of Bet v 1 and to investigate their involvement in the cellular cross-reactivity with homologous food allergens. METHODS: T-cell epitope mapping of Bet v 1 was performed by testing Bet v 1-specific T-cell lines derived from 57 individuals with birch pollen allergy, with overlapping peptides representing the entire allergen. T-cell lines and T-cell clones were stimulated with Bet v 1-related major allergens from apple (Mal d 1), cherry (Pru av 1), hazelnut (Cor a 1), celery (Api g 1), carrot (Dau c 1), and soybean (Gly m 4) and with peptides deduced from the C-terminal amino acid sequences of these molecules. Results Bet v 1 142-156 , positioned in the highly conserved C-terminal region of Bet v 1, was identified as the major T-cell epitope recognized by 61% of individuals. Most T lymphocytes specific for Bet v 1 142-156 were activated by one or more homologous food proteins or the respective peptides, as indicated by proliferation and cytokine production. CONCLUSION: The major T-cell epitope of Bet v 1, Bet v 1 142-156 , plays an important role in the cellular cross-reactivity between this respiratory allergen and related food allergens. Thus T lymphocytes specific for Bet v 1 142-156 might be activated by various Bet v 1-related food allergens in vivo, even out of the pollen season.     

Prospects of HIV-1 entry inhibitors as novel therapeutics

A great deal of progress has been made in understanding the mechanism of human immunodeficiency virus entry into target cells. Landmark discoveries such as the identification of viral coreceptors and the structure of a portion of the viral envelope protein (Env) bound to its receptor provided important insight into how Env mediates fusion of the viral and cellular membranes. This knowledge has been successfully applied to the development of inhibitors that target discrete steps of the entry process. Some of these compounds efficiently block HIV-1 replication in vitro and are currently being evaluated in clinical trials. In this review, we will introduce the challenges of antiviral therapy and highlight the need for novel therapeutics, such as entry inhibitors, to complement current antiviral regimens. The mechanism by which Env mediates HIV-1 entry and the therapeutic potential of small molecule inhibitors of this dynamic process will be discussed in detail.     

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)--II. Synthetic peptides linked to HIV-1 carrier proteins gag and nef.

Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.     

Recombinant vesicular stomatitis virus as an HIV-1 vaccine vector

Recombinant vesicular stomatitis virus (rVSV) is currently under evaluation as a human immunodeficiency virus (HIV)-1 vaccine vector. The most compelling reasons to develop rVSV as a vaccine vector include a very low seroprevalence in humans, the ability to infect and robustly express foreign antigens in a broad range of cells, and vigorous growth in continuous cell lines used for vaccine manufacture. Numerous preclinical studies with rVSV vectors expressing antigens from a variety of human pathogens have demonstrated the versatility, flexibility, and potential efficacy of the rVSV vaccine platform. When administered to nonhuman primates (NHPs), rVSV vectors expressing HIV-1 Gag and Env elicited robust HIV-1-specific cellular and humoral immune responses, and animals immunized with rVSV vectors expressing simian immunodeficiency virus (SIV) Gag and HIV Env were protected from AIDS after challenge with a pathogenic SIV/HIV recombinant. However, results from an exploratory neurovirulence study in NHPs indicated that these prototypic rVSV vectors might not be adequately attenuated for widespread use in human populations. To address this safety concern, a variety of different attenuation strategies, designed to produce a range of further attenuated rVSV vectors, are currently under investigation. Additional modifications of further attenuated rVSV vectors to upregulate expression of HIV-1 antigens and coexpress molecular adjuvants are also being developed in an effort to balance immunogenicity and attenuation.