Relation of HTLV-4 to simian and human immunodeficiency-associated viruses

Human immunodeficiency virus type 1 (HIV-1) is the aetiologic agent of AIDS (acquired immune deficiency syndrome) in most countries and probably originated in Central Africa like the AIDS epidemic itself. Evidence for a second major group of human immunodeficiency-associated retroviruses came from a report that West African human populations like wild-caught African green monkeys had serum antibodies that reacted more strongly with a simian immunodeficiency virus (STLV-3Mac) (ref.6) than with HIV-1. Novel T-lymphotropic retroviruses were reported to have been isolated from healthy Senegalese West Africans (HTLV-4) (ref. 4) and from African green monkeys (STLV-3AGM) (ref. 7), and a different retrovirus (HIV-2) was identified in other West African AIDS patients. Genomic analysis of HIV-2 clearly distinguished it from STLV-3 (ref. 9), but restriction enzyme site-mapping of three different HTLV-4 isolates and six different STLV-3AGM isolates showed them to be essentially indistinguishable. In this report we clone, restriction map, and partially sequence three isolates of HTLV-4 (PK82, PK289, PK190) (ref. 4). We find that these viruses differ in nucleotide sequence from each other and from three isolates of STLV-3AGM (K78, K6W, K1) (ref. 7) by 1% or less. We also report the isolation of a T-lymphotropic retrovirus from the peripheral blood of a healthy Senegalese woman which hybridizes preferentially to HIV-2 specific DNA probes. We conclude that HTLV-4 (ref. 4) and STLV-3AGM (ref. 7) are not independent virus isolates and that HIV-2 is present in Senegal as it is in other West African countries.     

Inhibition of phosphorylcholine binding to antibodies using synthetic peptides

The amino-acid sequence Phe-Tyr-Met-Glu is unique to phosphorylcholine (PC)-binding antibodies. It occurs in the first complementarity-determining region (CDR1) of the immunoglobulin heavy chains in 89% of all the anti-PC myeloma and hybridoma proteins but is not present in 490 other immunoglobulin heavy chains, 854 light chains or in 2,260 other unrelated proteins. This unique tetrapeptide therefore seems to be involved in PC binding. Here we compare the effectiveness of Phe-Tyr-Met-Glu and other structurally related peptides in inhibiting the binding of PC to PC-binding proteins McPC603 and HOPC8. We also test a surface-simulation peptide that was constructed to mimic the combining site of McPC603. Our data suggest that all these peptides inhibit the binding of PC to PC-binding proteins non-specifically and we show by computer modelling that the surface-simulation peptide does not duplicate the combining site of McPC603.     

The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4

CD4, a cell-surface glycoprotein expressed on a subset of T-cells and macrophages, serves as the receptor for the human immunodeficiency virus (HIV) (reviewed in ref. 1), binding to the HIV envelope glycoprotein, gp120 with high affinity. Attempts to block infection in vivo by raising antibodies against gp120 have failed, probably because these antibodies have insufficient neutralizing activity. In addition, because of the extensive polymorphism of gp120 in different isolates of HIV, antibodies raised against one HIV isolate are only weakly effective against others. Because interaction with CD4 is essential for infectivity by all isolates of HIV, an agent that could mimic CD4 in its ability to bind to gp120, such as a peptide or monoclonal antibody, might block infection by a wide spectrum of isolates. To aid the identification of such a ligand we have defined regions of CD4 that are required for binding to gp120. Although human CD4 is similar to mouse CD4 in amino-acid sequence (55% identity, ref. 6) and structure, we have found that the murine protein fails to bind detectably to gp120 and have exploited this finding to study binding of gp120 to mouse-human chimaeric CD4 molecules. These studies show that amino-acid residues within the amino-terminal immunoglobulin-like domain of human CD4 are involved in binding to gp120 as well as to many anti-CD4 monoclonal antibodies.     

HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.     

Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope, and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity. GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope. All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies, 9D8 and 2D7, could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays. In the flow cytometry analysis, the mAbs 9D8 and 2D7 could bind to HIV-Env CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide, which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane. However, in syncytium assays, none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion. The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes. The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.     

Induction of multi-epitopespecific antibodies against HIV-1 by multi-epitopevaccines

Some neutralizing antibodies against HIV-1 envelope proteins were highly effective to inhibit the infection of different strainsin vitro, and existed in the infected individuals with very low levels. We suggested multi-epitope-vaccine as a new strategy to increase levels of neutralizing antibodies and the abilities against HIV mutationin vivo. Two candidate multi-epitope-vaccines induced antibodies with predefined multi-epitope-specificity in rhesus macaque. These antibodies recognized corresponding neutralizing epitopes on epitope-peptides, gp41 peptides, V3 loop peptide, rsgp41 and rgp120. Besides, three candidate epitope-vaccines in combination (another kind of multi-epitopevaccines) showed similar potency to induce predefined multiple immune responses in rabbits. These results suggest that multi-epitope-vaccines may be a new strategy to induce multi-antiviral activities against HIV-1 infection and mutations.     

MHC class II region encoding proteins related to the multidrug resistance family of transmembrane transporters

The T-cell immune response is directed against antigenic peptide fragments generated in intracellular compartments, the cytosol or the endocytic system. Peptides derived from cytosolic proteins, usually of biosynthetic origin, are presented efficiently to T-cell receptors by major histocompatibility complex (MHC) class I molecules, with which they assemble, probably in the endoplasmic reticulum (ER). In the absence of recognizable N-terminal signal sequences, such cytosolic peptides must be translocated across the ER membrane by a novel mechanism. Genes apparently involved in the normal assembly and transport of class I molecules may themselves be encoded in the MHC. Here we show that one of these, the rat cim gene, maps to a highly polymorphic part of the MHC class II region encoding two novel members of the family of transmembrane transporters related to multidrug resistance. Other members of this family of transporter proteins are known to be capable of transporting proteins and peptides across membranes independently of the classical secretory pathway. Such molecules are credible candidates for peptide pumps that move fragments of antigenic proteins from the cytosol into the ER.     

Interleukin-2 production used to detect antigenic peptide recognition by T-helper lymphocytes from asymptomatic HIV-seropositive individuals

T lymphocytes from mice and healthy humans immunized against the human immunodeficiency virus (HIV) envelope have recently been shown to recognize two antigenic regions of the gp160 HIV-envelope protein which have been located on the basis of amphipathicity. In HIV-infected humans, T-cell proliferative responses are lost soon after infection. Here we demonstrate that interleukin-2 production is often retained even when proliferative activity is absent, and that it can be used to monitor T-helper cell responses by HIV-seropositive donors. We use this approach to investigate the T-helper cell response of 42 asymptomatic HIV-seropositive patients to four synthetic gp160 peptides and to influenza A virus, an antigen requiring intact CD4 T-helper cell function. As many as 67% of the HIV-seropositive donors who retain responsiveness to influenza A virus respond to a single peptide, and 85-90% responded to at least one of the peptides.     

Direct binding of influenza peptides to class I HLA molecules

Activation of T lymphocytes requires the intracellular fragmentation of foreign antigens and their presentation by class I or class II major histocompatibility complex (MHC) glycoproteins. The direct binding of peptides to class II molecules has been demonstrated using equilibrium dialysis, gel filtration and fluorescence energy transfer at planar membranes, and its specificity compared to that of T-cell activation. In contrast, direct binding of peptides to class I molecules has been difficult to detect; although peptide sensitization experiments and the crystallographic structure of HLA-A2 (ref. 9) persuasively argue for its occurrence and importance. Here we describe a gel filtration assay from which we derive direct evidence for selective binding of an influenza matrix peptide to HLA-A2 and for binding of an influenza nucleoprotein peptide to HLA-B37. These two peptides have previously been shown to act respectively as targets for certain HLA-A2 or HLA-B37 restricted influenza-specific cytotoxic T lymphocytes (CTL). In addition we demonstrate binding to some, but not all, HLA allospecificities that cannot present these peptides to CTL. We estimate that less than 0.3% of the HLA molecules present in any given purified preparation were able to bind the added peptides.     

T细胞功能学与结构免疫学结合方法鉴定HLA-A2限制性的细胞毒性T淋巴细胞表位富集区

 T细胞表位在抗病毒的T细胞免疫中发挥核心作用,目前已在多种病原微生物的蛋白序列上发现存在T细胞表位的聚集现象。本文建立了一套功能学与结构学结合的策略鉴定病原体上细胞毒性T细胞(Cytotoxic T Lymphocyte,CTL)表位富集区的方法,并以严重急性呼吸道综合征(SARS)相关冠状病毒(SARS-CoV)的M蛋白为例,成功地鉴定了一个HLA-A2限制性的表位富集区。首先通过生物信息学的方法预测并合成M蛋白跨膜区的HLA-A2潜在结合多肽,通过体外复性实验和T2细胞结合实验验证多肽与HLA-A2的结合力;然后在HLA-A2.1/Kb转基因小鼠中检测这些多肽的免疫原性;最后通过X射线衍射技术,成功解析了其中一条多肽与HLA-A^*0201的复合物结构,其结构显示该多肽具有典型的HLA-A^*0201表位的结构特点,但却呈现出与以往鉴定多肽不同的构象和锚定残基。本文对于理解机体对SARS-CoV等病原体产生的T细胞免疫反应,以及为更广泛的人群设计T细胞疫苗具有重要意义。     

Sequence analysis on biological active peptides using electrospray ionizationFourier transform ion cyclotron resonance mass spectrometer

This paper describes experience with the sequencing of synthetic segment peptides of gp41 which are used in trimeric peptides as potential peptide-based vaccines and as inhibitors to mimic the fusion state of gp41 in HIV infection using ESI/CID-FT-ICR MS. All b and y““ series ions are found in the CID spectra and the mass difference between the calculated and observed results is very slight. FT-ICR MS again shows its power in peptide sequencing, successfully helping us obtain the sequence of an unknown peptide.     

New human and simian HIV-related retroviruses possess functional transactivator (tat) gene

New human retroviruses antigenically related to HIV and even more closely to STLV-III have been recently isolated from individuals from some West African countries. One of these viruses, HTLV-IVP, was reportedly isolated from lymphocytes of a healthy female prostitute. Another isolate, LAV-2FG, was obtained from an AIDS patient and third, SBL-6669, from an individual with lymphadenopathy. Current epidemiological studies indicate that some of these virus isolates cause immune deficiency whereas others may not or may be less efficient at inducing immune deficiency. Similarly, STLV-III apparently does not cause immune deficiency in its natural host, African green monkey. A novel feature of HIV is the possession of a gene termed tat, which is implicated in its pathobiology. We report here that, like HIV, HTLV-IVP, LAV-2FG (HIV-2) and SBL-6669, as well as STLV-IIIAGM possess the putative tat gene, irrespective of their pathogenic potential in vivo. Interestingly, HTLV-IVP/LAV-2FG long terminal repeat (LTR) is equally well transactivated by the HTLV-IVP/LAV-2FG and HTLV-IIIB tat function, HTLV-IIIB LTR responds better to its own tat function.     

A novel active pentapeptide from chicken brain identified by antibodies to FMRFamide

The tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and peptides structurally related to it, have been isolated from molluscan ganglia. They have widespread actions on both invertebrate and vertebrate tissues and there is increasing evidence that they are an important group of invertebrate peptide neurotransmitters. It is of interest that the primary amino acid sequence of FMRFamide forms the C-terminal tetrapeptide of an enkephalin-like heptapeptide (Met-enkephalin-ArgPhe) isolated from bovine adrenal medulla and striatum. Antisera to FMRFamide have been shown to react in radioimmunoassay and immunohistochemistry with material in the central nervous system of various vertebrate species, but the identity of this material, and in particular its relationship to the opioid heptapeptide, remains uncertain. We have used antibodies specific for the C-terminus of FMRFamide in radioimmunoassays to monitor purification of the material in chicken brain. We describe here the sequence of one of the peptides obtained. It is a biologically active peptide which does not seem to be related to other known vertebrate neuropeptides.     

Characterization of Antibody Responses Against the 2F5 Epitope ELDKWA sing HIV-1 Env-Mediated Membrane Fusion and Neutralization Assays

The epitope ELDKWA, which is located in the membrane-proximal external region (MPER) of HIV-1 gp41, is an important neutralizing epitope. The human monoclonal antibody (mAb) 2F5 against this epitope shows broad neutralizing activity toward many HIV strains. However, several reports have shown that the epitope-specific mAbs induced by peptides containing MPER did not exhibit the same neutralizing activities as human mAb 2F5. In this study, four ELDKWA epitope specific mAbs (9E7, 7E10, 6B5, and 2B4) induced by immunization with the ELDKWA epitope in varied molecular contexts, all showed inhibitory activities with different potencies in HIV-1 Env-mediated membrane fusion assays and pseudovirus neutralization assays. This result indicates that though these antibodies recognize the epitope ELDKWA, their characterizations differ from that of neutralizing antibodies, implying that the neutralizing mAbs can be induced but also need to be screened, and the protective ability of a related vaccine antigen depends on the concentration of the neutralizing mAbs in the induced polyclonal antibodies.     

Biological function of H1V-1 transmembrane protein gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539–684) and gp41 immunosuppressive peptide (aa583–599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583–599 and 641–675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class I, II and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type I interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type I interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Biological function of HIV-1 transmembrane protein gp41──A study on a putative cellular receptor of gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539-684) and gp41 immunosuppressive peptide (aa583-599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583-599 and 641-675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class Ⅰ, Ⅱ and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type Ⅰ interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type Ⅰ interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases

Alpha atrial natriuretic peptide (alpha-ANP) and brain natriuretic peptide are homologous polypeptide hormones involved in the regulation of fluid and electrolyte homeostasis. These two natriuretic peptides apparently share common receptors and stimulate the intracellular production of cyclic GMP as a second messenger. Molecular cloning has defined two types of natriuretic peptide receptors: the ANP-C receptor of relative molecular mass (Mr) 60-70,000 (60-70 K), which is not coupled to cGMP production and may function in the clearance of ANP and the ANP-A receptor of Mr 120-140 K, which is a membrane form of guanylate cyclase in which ligand binding to the extracellular domain activates the cytoplasmic domain of the enzyme. Here we report the cloning and expression of a second human natriuretic peptide-receptor guanylate cyclase, the ANP-B receptor. The ANP-B receptor is preferentially activated by porcine brain natriuretic peptide rather than human alpha-ANP, whereas the ANP-A receptor responds similarly to both natriuretic peptides. These observations may have important implications for our understanding of the central and peripheral control of cardiovascular homeostasis.     

A malaria T-cell epitope recognized in association with most mouse and human MHC class II molecules

An ideal vaccine should elicit a long lasting immune response against the natural parasite, both at the T- and B-cell level. The immune response should occur in all individuals and be directed against determinants that do not vary in the natural parasite population. A major problem in designing synthetic peptide vaccines is that T cells generally recognize peptide antigens only in association with one or a few of the many variants of major histocompatibility complex (MHC) antigens. During the characterization of epitopes of the malaria parasite Plasmodium falciparum that are recognized by human T cells, we analysed a sequence of the circumsporozoite protein, and found that synthetic peptides corresponding to this sequence are recognized by T cells in association with many different MHC class II molecules, both in mouse and in man. This region of the circumsporozoite protein is invariant in different parasite isolates. Peptides derived from this region should be capable of inducing T-cell responses in individuals of most HLA-DR types, and may represent good candidates for inclusion in an effective anti-malaria peptide vaccine.     

The interaction between the membrane-proximal external region and the N-trimer region of HIV-1 gp41: Involvement in viral fusion

The membrane proximal external region (MPER) of gp41 is extremely conserved among diverse HIV-1 variants, implying its important role in viral infection. Interestingly, two of the most broadly neutralizing antibodies, 2F5 and 4E10, specifically recognize this region. Our previous study demonstrated that the antigenicity and immunogenicity of 4E10 epitope are affected by remodeling gp41 fusion core, suggesting that the MPER may be associated with gp41 core and involved in gp41-mediated membrane fusion. Here we measured the binding activity of 4E10 epitope peptide (D4E10P) with various gp41 core-derived peptides and found that the N-trimer region in a construct designated N-trimer-6HB interacted significantly with D4E10P. Using N-trimer-6HB to screen a phage library, we identified a motif (WF) located in 4E10 epitope that may play a certain role in the interaction of gp41 MPER with the N-trimer in gp41 fusion core and, we thus speculated upon the potential involvement of MPER in the fusion process between viral envelope and target cell membrane. Supported by National Key Basic Research and Development Program of China (Grant No. 2007CB914402)