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We have previously reported that the CBD1 peptide (SLEQIWNNMTWMQWDK), corresponding to the consensus caveolin-1 binding domain in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, elicits peptide-specific antibodies. Here, we have investigated the cellular immune response and the protective efficacy against a simian/human immunodeficiency virus (SHIV(162P3)) challenge. In addition to the CBD1 peptide, peptides overlapping the caveolin-binding-motif (CBM) ((622)IWNNMTWMQW(631) or (622)IWNNMTW(628)) were fused to a Gag-p24 T helper epitope for vaccination. All immunized cynomolgus macaques responded to a cocktail peptide immunization by inducing specific T cells and the production of high-titer CBD1/CBM peptide-specific antibodies. Six months after the fourth vaccine boost, six control and five vaccinated animals were challenged weekly by repeated exposure to SHIV(162P3) via the mucosal rectal route. All control animals were infected after 1 to 3 challenges with SHIV, while among the five vaccinated monkeys, three became infected after a delay compared to control; one was infected after the eighth viral challenge, and one remained uninfected even after the ninth SHIV challenge. Immunized animals maintained a CD4 T cell count, and their central memory CD4 T cells were less depleted than in the control group. Furthermore, SHIV challenge stimulates antigen-specific memory T cell response in vaccinated macaques. Our results indicate that peptides derived from the CBM region can be immunogenic and provide protection against SHIV infection in cynomolgus monkeys. IMPORTANCE In HIV-1-producing cells, gp41 exists in a complexed form with caveolin-1, an interaction most probably mediated by the caveolin-1 binding motif. This sequence is highly conserved in every single HIV-1 isolate, thus suggesting that there is constant selective pressure to preserve this sequence for a specific function in the HIV infectious cycle. Consequently, the CBM sequence may represent the “Achilles' heel” of HIV-1 in the development of an efficient vaccine. Our results demonstrate that macaques immunized with the CBM-based peptides displayed a delay in the onset of viral infection and CD4 depletion, as well as a significant induction of antigen-specific memory T cell response, which is essential for the control of HIV/SIV infections. Finally, as HIV-infected individuals lack anti-CBM immune responses, CBM-based vaccines could have applications as a therapeutic vaccine in AIDS patients. more...

Follicular T helper (Tfh) cells, a subset of CD4 T lymphocytes, are essential for memory B cell activation, survival, and differentiation and assist B cells in the production of antigen-specific antibodies. Work performed in recent years pointed out the importance of Tfh cells in the context of HIV and SIV infections. The importance of tissue distribution of Tfh is also an important point since their frequency differs between peripheral blood and lymph nodes compared to the spleen, the primary organ for B cell activation, and differentiation. Our recent observations indicated an early and profound loss of splenic Tfh cells. The role of transcriptional activator and repressor factors that control Tfh differentiation is also discussed in the context of HIV/SIV infection. Because Tfh cells are important for B cell differentiation and antibody production, accelerating the Tfh responses early during HIV/SIV infection could be promising as novel immunotherapeutic approach or alternative vaccine strategies. However, because Tfh cells are infected during the HIV/SIV infection and represent a reservoir, this may interfere with HIV vaccine strategy. Thus, Tfh represent the good and bad guys during HIV infection., JE from the Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS) and from The Canadian HIV Cure Enterprise Team Grant HIG-13305 from the Canadian Institutes of Health Research (CIHR) in partnership with CANFAR and IAS. FM is supported by a fellowship from Fondation du CHU de Québec. CB and YF are supported by fellowships from ANRS. JE acknowledges the support of the Canada Research Chair program. RS is supported by FCT—Fundaçao para a Ciência e a Tecnologia/MEC—Ministério da Educaçao e Ciência através de fundos nacionais e quando aplicavel cofinanciado pelo FEDER, no âmbito do Acordo de Parceria PT2020 referente à unidade de investigaçao n°4293. RS is supported by the Fundaçao para a Ciência e a Tecnologia (FCT) (IF/00021/2014), info:eu-repo/semantics/publishedVersion more...

International audience; CBD1 peptide (SLEQIWNNMTWMQWDK), corresponding to the consensus caveolin-1 binding domain in HIV-1 envelope glycoprotein gp41, elicits the production of antibodies that inhibit infection of primary CD4(+) T lymphocytes by various primary HIV-1 isolates. Here the immunogenicity of the CBD1 peptide was investigated in cynomolgus macaques using adjuvants that are acceptable for human use. In the first set of studies, macaques were immunized with the CBD1 peptide in association with muramyl dipeptide derivative MDP-Lys(L18) combined with the oil-in-water emulsion, MF-59. After five immunizations at 4 weeks interval, the antibody titer against the CBD1 peptide was found to be either medium, poor, weak or none, thus suggesting that the CBD1 immune response might be restricted by the major histocompatibility complex (MHC) class II molecules. In the second set of studies therefore, macaques were immunized with the CBD1 peptide in association with the 'promiscuous' T cell epitope from the tetanus toxin, either as free peptides or covalently linked with the dilysine linker using CpG ODN and Montanide ISA 51 as adjuvants. This latter immunization procedure boosted markedly the anti-CBD1 antibody response, since even the non-responders generated high-titered peptide-specific antibodies. Moreover, co-immunization of the CBD1 and the T helper epitope as free peptides seemed to be favorable for the production of neutralizing antibodies, with 50% inhibition of HIV-1 infection occurring at 300-400-fold dilution of the immune sera. Finally, neutralizing and non-neutralizing immune macaque sera could be differentiated by the profile of cross-reactivity with overlapping CBD1-related peptides in ELISA. Taken together, our results demonstrate that the CBD1 peptide is immunogenic in macaques and that an eventual MHC-restriction could be overcome by the administration with an appropriate T helper epitope. more...

Follicular T helper cells (Tfh), a subset of CD4 T lymphocytes, provide crucial help to B cells in the production of antigen-specific antibodies. Although several studies have analyzed the dynamics of Tfh cells in peripheral blood and lymph nodes (LNs) during Aids, none has yet addressed the impact of SIV infection on the dynamics of Tfh cells in the spleen, the primary organ of B cell activation. We show here a significant decrease in splenic Tfh cells in SIVmac251-infected rhesus macaques (RMs) during the acute phase of infection, which persists thereafter. This profound loss is associated with lack of sustained expression of the Tfh-defining transcription factors, Bcl-6 and c-Maf but with higher expression of the repressors KLF2 and Foxo1. In this context of Tfh abortive differentiation and loss, we found decreased percentages of memory B cell subsets and lower titers of SIV-specific IgG. We further demonstrate a drastic remodeling of the lymphoid architecture of the spleen and LNs, which disrupts the crucial cell-cell interactions necessary to maintain memory B cells and Tfh cells. Finally, our data demonstrated the early infection of Tfh cells. Paradoxically, the frequencies of SIV DNA were higher in splenic Tfh cells of RMs progressing more slowly suggesting sanctuaries for SIV in the spleen. Our findings provide important information regarding the impact of HIV/SIV infection on Tfh cells, and provide new clues for future vaccine strategies., This work was supported by CHIR (Canada) and ANRS grants (France). JE thanks the Canada Research Chair program for financial assistance. VR was supported by a doctoral fellowship from FCT (Fundacao para a Ciencia e Tecnologia); code SFRH/BD/64064/2009. We would like to thank the Nonhuman Primate Reagent Resource for kindly providing CXCR5 antibodies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. more...

Caveolin-1 is a scaffolding protein that organizes and concentrates specific ligands within the caveolae membranes. We identified a conserved caveolin-1 binding motif in the HIV-1 transmembrane envelope glycoprotein gp41 and designed several synthetic peptides, referred to as CBD1, corresponding to the consensus caveolin-1 binding domain in gp41. In rabbits, these peptides elicit the production of antibodies that inhibit infection of primary CD4+ T lymphocytes by various primary HIV-1 isolates. Interestingly, gp41 exists as a stable complex with caveolin-1 in HIV-infected cells. Anti-CBD1 peptide antibodies, therefore, might be functional by inhibiting the potential interaction of gp41 with caveolin-1. Because of their capacity to elicit antibodies that inhibit the different clades of HIV-1, CBD1-based peptides may represent a novel synthetic universal B cell epitope vaccine candidate for HIV/AIDS. Moreover, such peptides could also have an application as a therapeutic vaccine since CBD1-specific antibodies are rare in HIV-infected individuals from several geographic origins. more...

The cross-linking of HIV on permissive cells results aggregation of HIV particles with surface nucleolin, CD4, and CXCR4, but without affecting the organization of CD45. In addition, HIV particles and nucleolin coaggregate with glycolipid-enriched membrane microdomains (GEMs) containing ganglioside, and glycosylphosphatidylinositol-linked proteins CD90 and CD59, pointing out that HIV anchorage induces lateral assemblies of specific membrane components into lipid rafts in which surface nucleolin is also incorporated. Consequently, equilibrium density fractionation of extracts from infected cells revealed that HIV proteins and nucleolin copurify with Triton X-100-resistant GEM-associated proteins. After HIV entry, nucleolin is recovered also in fractions containing HIV DNA, viral matrix, and reverse transcriptase, thus suggesting that it could accompany viral entry. We show that surface nucleolin is markedly down-regulated a few hours following HIV entry into permissive cells; an effect that appears to be the consequence of its translocation into the cytoplasm. Our findings demonstrate that anchorage of HIV particles on permissive cells induces aggegation of surface nucleolin and its association with detergent-insoluble lipid raft components. Moreover, they support the suggestion that surface nucleolin and lipid rafts are implicated in early events in the HIV entry process. more...

The HB-19 pseudopeptide 5[Kψ(CH 2 N)PR]-TASP, ψ(CH 2 N) for reduced peptide bond, is a specific inhibitor of HIV infection in different CD4 + cell lines and in primary T-lymphocytes and macrophages. It blocks virus-particle attachment to permissive cells by binding and forming a stable complex with nucleolin expressed on the cell surface. Here, we have investigated the tissue distribution of the tritiated HB-19 by using β-radio imager whole-body mapping in rats. A rapid, selective, and stable distribution and accumulation of the systematically administered HB-19 was demonstrated within the spleen, liver, bone, and kidney as soon as 5 min following its administration. No apparent uptake of HB-19 occurred in the brain and the muscle tissue. Interestingly and despite its rapid clearance from the blood, at 24 h postexposure a significant proportion of HB-19 was still recovered from target organs, of which 16–37% could be acounted for intact pseudopeptide. The elimination of HB-19 mainly occurred by renal glomerular filtration and most of the excreted radioactivity appeared to be HB-19 metabolites. Finally, injection of the biotin-labeled HB-19 pseudopeptide but not its control counterpart allowed the recovery of the HB-19–nucleolin complex from the liver, spleen, thymus, and bone marrow, thus indicating that the in vivo molecular target of HB-19 is surface nucleolin. Our results demonstrate the preferential uptake and stability of HB-19 in lymphoid organs that are the site of HIV propagation. more...

The growth factor midkine (MK) has been reported to bind heparan sulfate and nucleolin, two components of the cell surface implicated in the attachment of HIV-1 particles. Here we show that synthetic and recombinant preparations of MK inhibit in a dose-dependent manner infection of cells by T-lymphocyte- and macrophage-tropic HIV-1 isolates. The binding of labeled MK to cells is prevented by excess unlabeled MK or by the anti-HIV pseudopeptide HB-19 that blocks HIV entry by forming a stable complex with the cell-surface-expressed nucleolin. MK mRNA is systematically expressed in adult peripheral blood lymphocytes from healthy donors, while its expression becomes markedly but transiently increased upon in vitro treatment of lymphocytes with IL-2 or IFN-gamma and activation of T-lymphocytes by PHA or antibodies specific to CD3/CD28. In MK-producing lymphocytes, MK is detectable at the cell surface where it colocalizes with the surface-expressed nucleolin. Finally, by using MK-producing CD4(+) and CD4(-) cell clones we show that HIV infection in cell cultures could be inhibited in both an autocrine and a paracrine manner. The potent and distinct anti-HIV action of MK along with its enhanced expression in lymphocytes by various physiological stimuli suggests that MK is a cytokine that could be implicated in HIV-induced pathogenesis. more...

The membrane-expressed HIV-1 envelope glycoprotein complex, gp120 and gp41, has been shown to be responsible for the initiation of cell killing by apoptosis in CD4+T cells. By using two experimental approaches we demonstrate here that CD26, also known as dipeptidyl peptidase IV (DPP IV), appears to be implicated in this function of the gp120/gp41 complex to initiate apoptosis. In the first experimental model, we used persistently HIV-1-infected H9/IIIB cells expressing the membrane-associated gp120/gp41 complex as effector cells to induce apoptosis in Jurkat CD4+T cells: parental or transfected in order to express high levels of recombinant CD26, either wild-type or mutated at its Ser-630 which inactivates the DPP IV activity of CD26. Parental Jurkat cells and transfected control cell clones express low but reproducibly detectable levels of endogenous CD26. In coculturing experiments using H9/IIIB and Jurkat cells, the occurrence of apoptosis was found to be retarded by at least 24 hr in Jurkat cells expressing low levels of endogenous CD26, compared to cell clones expressing high levels of either wild-type or mutated catalytically inactive transfected CD26. In the second experimental model, the different Jurkat cell lines were infected with vaccinia recombinant viruses expressing HIV-1envgene, either wild-type to generate a functional gp120/gp41 complex or mutated to generate an uncleavable membrane-expressed precursor of the envelope glycoprotein gp160. At 18 hr postinfection with such vaccinia recombinant viruses, apoptosis was observed only in Jurkat cells with enhanced levels of CD26 and expressing the gp120/gp41 complex. Apoptosis was not detected in the different Jurkat cell lines expressing the uncleavable precursor gp160. In both of the experimental models used, no significant differences were observed between the transfected cells expressing either the wild-type or the mutated form of CD26, thus suggesting that the DPP IV activity of CD26 is not essential for its function as a cofactor of CD4 in the mechanism of initiation of apoptosis by the HIV envelope gp120/gp41 complex. Taken together, these results indicate that CD26 in CD4+T cells may determine the rate of initiation of apoptosis by the mature HIV-1 envelope glycoproteins, i.e., CD26 is being involved as a cofactor of CD4 in the mechanism of triggering apoptosis by the gp120/gp41 complex. As signaling through CD26 could lead to T cell activation, we propose that this latter might be modified following the binding of the gp120/gp41 complex to CD4 and thus leading to apoptosis. more...

The T-cell activation antigen CD26, is a type II membrane glycoprotein with intrinsic dipeptidyl-peptidase IV (DPP IV) activity, characterized by its capacity to cleave off N-terminal dipeptides containing proline as the penultimate residue. Independent of its catalytic activity, CD26 has also been characterized as adenosine deaminase binding protein. By using CD26 negative human C8166 cells, here we describe the existence of another cell-surface protein which manifests CD26-like DPP IV activity. For convenience, this protein will be referred to as DPP IV-beta. Consistent with the cell-surface expression of DPP IV-beta, intact C8166 cells manifested a high level of DPP IV, whereas, they manifested poor activity against substrates of DPP II known to have an intracellular localization. A partially purified preparation of CD26 from human MOLT4 cells, and the DPP IV-beta expressed on intact cells were found to possess similar catalytic activity and pH optimum. In addition, cell-surface CD26 and DPP IV-beta on intact MOLT4 and C8166 cells, respectively, resisted digestion by proteolytic enzymes such as trypsin and proteinase K. However, adenosine deaminase activity was not detectable on the surface of C8166 cells in contrast to CD26 positive MOLT4 cells. In accord with this, 125I-labeled adenosine deaminase which binds CD26 was found not to bind DPP IV-beta. Gel-filtration experiments using 0.5% Triton X-100 extracts from C8166 and MOLT4 cells, revealed that the apparent molecular mass of DPP IV-beta is 82 kDa, whereas that of CD26 is 110 kDa as expected. Taken together, our results suggest that DPP IV-beta is a CD26-like protein which could be characterized by distinct properties. more...

The mechanism of action of different types of interferons (IFN-alpha, -beta, and -gamma) against human immunodeficiency virus (HIV)-1 infection was investigated in chronically infected monocytoid U937 cells and during an acute infection of the T lymphoblastoid CEM cells. Two chronically infected U937 cell populations, obtained independently (referred to as type A and B cells), were analyzed for their response to IFNs. In type A cells, IFNs mainly inhibited virus particle release, whereas in type B cells, the anti-HIV effect of IFNs cells was found to be largely due to a specific inhibition of viral protein synthesis without any apparent effect on total cellular protein synthesis. Interestingly, such a differential inhibition of HIV protein synthesis could also be demonstrated in acutely infected CEM cells in response to treatment with IFN-alpha. Both in chronically infected U937 type B and acutely infected CEM cells, equivalent amounts of nuclear and cytoplasmic HIV-1 mRNA were detected in control and IFN-treated cells in spite of at least 80% inhibition of HIV protein synthesis. Analysis of the distribution of cellular and viral mRNAs on polysomes in HIV-1-infected cells demonstrated that IFN treatment induces a specific block on viral mRNA translation. These results indicate that the antiviral mechanism of IFN on later stages of HIV replication cycle may be partly due to the inhibition of HIV mRNA translation, besides an effect on virus budding or release. more...

Background Nucleolin expressed at the cell surface is a binding protein for a variety of ligands implicated in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal RGG domain of nucleolin, the HB-19 pseudopeptide, we recently reported that targeting surface nucleolin with HB-19 suppresses progression of established human breast tumor cells in the athymic nude mice, and delays development of spontaneous melanoma in the RET transgenic mice. Methods By the capacity of HB-19 to bind stably surface nucleolin, we purified and identified nucleolin partners at the cell surface. HB-19 and related multivalent Nucant pseudopeptides, that present pentavalently or hexavalently the tripeptide Lysψ(CH2N)-Pro-Arg, were then used to show that targeting surface nucleolin results in distinct inhibitory mechanisms on breast, prostate, colon carcinoma and leukemia cells. Results Surface nucleolin exists in a 500-kDa protein complex including several other proteins, which we identified by microsequencing as two Wnt related proteins, Ku86 autoantigen, signal recognition particle subunits SRP68/72, the receptor for complement component gC1q-R, and ribosomal proteins S4/S6. Interestingly, some of the surface-nucleolin associated proteins are implicated in cell signaling, tumor cell adhesion, migration, invasion, cell death, autoimmunity, and bacterial infections. Surface nucleolin in the 500-kDa complex is highly stable. Surface nucleolin antagonists, HB-19 and related multivalent Nucant pseudopeptides, exert distinct inhibitory mechanisms depending on the malignant tumor cell type. For example, in epithelial tumor cells they inhibit cell adhesion or spreading and induce reversion of the malignant phenotype (BMC cancer 2010, 10:325) while in leukemia cells they trigger a rapid cell death associated with DNA fragmentation. The fact that these pseudopeptides do not cause cell death in epithelial tumor cells indicates that cell death in leukemia cells is triggered by a specific signaling mechanism, rather than nonspecific cellular injury. Conclusions Our results suggest that targeting surface nucleolin could change the organization of the 500-kDa complex to interfere with the proper functioning of surface nucleolin and the associated proteins, and thus lead to distinct inhibitory mechanisms. Consequently, HB-19 and related Nucant pseudopeptides provide novel therapeutic opportunities in treatment of a wide variety of cancers and related malignancies. more...

Polyadenylic-polyuridylic acid referred to as poly (A)·poly(U), is a synthetic double-stranded RNA that has been shown to manifest both antitumoral and immunodulatory activities. Previously, we have reported that poly(A)·poly(U) inhibits HIV infection in cell cultures. Here we provide direct evidence to demonstrate that the inhibitory action of poly(A)·poly(U) is through its capacity to prevent entry of HIV particles into CD4-positive T lymphocytes. Such inhibition of HIV entry is also observed in the case of other polyanions such as heparin, dextran sulfate, and poly(I)·poly(C). The mechanism of inhibition appears to occur postbinding of HIV particles to the CD4 receptor molecules, because the binding of the external envelope glycoprotein of HIV-1 (gp120) is not affected significantly in the presence of poly(A)·poly(U) or other polyanions. These results confirm the potential of poly(A)·poly(U) as an antiviral drug against HIV infection. more...

The cytopathic effect of HIV-1 and HIV-2 in CD4+ lymphocytes has been shown to be associated with apoptosis or programmed cell death. Using different experimental conditions, we demonstrate here that apoptosis is triggered by cell membrane expression of the mature HIV envelope glycoproteins, gp120-gp41 complex, and their interaction with CD4 receptor molecules. Viral entry alone did not induce apoptosis but virus replication was required in order to produce the gp120-gp41 complex. Indeed, expression of the HIV env gene alone in the CD4+ T cell line (CEM) was sufficient for the induction of apoptosis. In general, syncytium formation and apoptosis induction were closely associated as both events require functional envelope glycoproteins and CD4 molecules. Nevertheless, apoptosis but not syncytium formation was suppressed by a monoclonal antibody against CD4 that does not affect gp120 binding. Furthermore, single-cell killing by apoptosis was observed in infected cell cultures treated with a monoclonal antibody against gp41, which completely abolishes the formation of syncytia. These results indicate that apoptosis is not the consequence of toxic effects induced by the formation of syncytia but is triggered by the HIV envelope glycoproteins. Therefore, cell death during HIV infection in CD4+ lymphocyte cultures is due to a specific event triggered by the gp120-gp41 heterodimer complex programming death in metabolically active cells. more...

Several studies have indicated that the cell-surface expressed nucleolin is implicated in tumorigenesis and angiogenesis, and represents an important target for cancer therapy. Here we show that treatment of rhabdoid tumor derived G401 cells with a nucleolin antagonist, the HB-19 pseudopeptide, could restore contact inhibition, impair anchorage-independent growth, and suppress tumor development in nude mice. G401 cells grow without contact inhibition, which is an in vitro characteristic property of malignant tumor cells. At concentrations of HB-19 that does not affect cell viability and multiplication index, there is restoration of contact inhibition thus suggesting that HB-19 treatment causes reversion of the malignant phenotype. Accordingly, HB-19 pretreated G401 cells lose the capacity to form colonies in soft agar. When assayed for tumorigenicity in nude mice, only 50% of mice injected with HB-19 pretreated G401 cells developed tumors with the mean tumor weight of 0.32 g, compared to 100% of mice injected with control G401 cells with the mean tumor weight of 2.36 g. Interestingly, the restoration of contact inhibition in HB-19 treated G401 cells is concomitant with marked reduction of transcripts coding the Wilms’ tumor 1 gene, matrix metalloproteinase-2, epithelial isoform of CD44, and vascular endothelial growth factor, whereas no apparent modification is detected for transcripts coding the proto-oncogene c - Myc , anti-apoptotic Bcl-2, pro-apoptotic Bax, tissue inhibitor of metalloproteinase TIMP-1, angiogenesis inhibitor TSP-1, and growth factor Midkine. These findings indicate that the molecular mechanism of action of HB-19 on such highly malignant rhabdoid tumor cells is associated with a selective inhibitory effect on the expression of genes implicated in tumorigenesis and angiogenesis. more...

Polyadenylic-polyuridylic acid referred to as poly(A).poly(U) is a synthetic double-stranded RNA which has been shown to manifest both antitumoral and immunomodulatory activities. Here we used this agent to demonstrate its antiviral activity against the human immunodeficiency virus (HIV-1 and HIV-2). Treatment of cells with poly(A).poly(U) resulted in a significant delay in the development of the HIV-specific cytopathic effect characterized by the formation of syncytia and cell lysis. Furthermore, the production of virus measured by the concentration of the HIV major core protein was reduced by 90-95%. Under these experimental conditions, the synthesis of HIV proteins was reduced at least tenfold whereas the metabolism and proliferation of cells apparently were not affected. The inhibitory action of poly(A).poly(U) seems to be at the level of viral entry into cells. Combined treatment of infected cells with poly(A).poly(U) and azidothymidine (AZT) resulted in a 4-5-fold synergistic inhibitory effect. Previously, no toxicity has been observed in cancer patients with long-term treatment with poly(A).poly(U). In view of this and the significant anti-HIV effect, poly(A).poly(U) provides a potential candidate as a therapeutic drug in AIDS disease. more...

The CBD1 peptide (SLEQI W NNMT W MQ W DK), corresponding to the consensus caveolin-1 binding domain in HIV-1 envelope glycoprotein gp41 (CBD1), elicits the production of antibodies that inhibit infection of primary CD4 + T lymphocytes by various primary HIV-1 isolates. Here we show that HIV-neutralizing antibodies against CBD1 react with multiple conformational epitopes that overlap the highly conserved caveolin-1 binding motif (CBM) with the N-terminal conserved isoleucine residue. The CBM-based peptides I W NNMT W MQ W and I W NNMT W when fused to a T helper epitope are immunogenic by inducing high titer CBM-specific antibodies capable of neutralizing HIV-1 infection in primary T lymphocyte cultures. Interestingly, neutralizing immune sera raised against a given peptide do not cross-react with related CBM-derived peptides, thus suggesting the existence of distinct neutralizing epitopes that probably reflect the dynamic conformational features of CBD1. In accord with this, the mixture of neutralizing immune sera raised against several CBM-derived peptides exerts a synergistic neutralizing activity against HIV-1 infection. Finally, the existence of several distinct overlapping epitopes in CBD1 is confirmed by murine monoclonal antibodies that we generated against the CBM-derived chimeric peptides. Our results indicate that CBD1- and CBM-based peptides mimic distinct dynamic conformations of CBD1, and thus such peptides could provide specific immunogens for an efficient vaccine preparation against HIV/AIDS infection. more...

BACKGROUND: Emerging evidences suggest that nucleolin expressed on the cell surface is implicated in growth of tumor cells and angiogenesis. Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, here we show that the growth of tumor cells and angiogenesis are suppressed in various in vitro and in vivo experimental models. HB-19 inhibited colony formation in soft agar of tumor cell lines, impaired migration of endothelial cells and formation of capillary-like structures in collagen gel, and reduced blood vessel branching in the chick embryo chorioallantoic membrane. In athymic nude mice, HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in nude mice, and in some cases eliminated measurable tumors while displaying no toxicity to normal tissue. This potent antitumoral effect is attributed to the direct inhibitory action of HB-19 on both tumor and endothelial cells by blocking and down regulating surface nucleolin, but without any apparent effect on nucleolar nucleolin. CONCLUSION/SIGNIFICANCE: Our results illustrate the dual inhibitory action of HB-19 on the tumor development and the neovascularization process, thus validating the cell-surface expressed nucleolin as a strategic target for an effective cancer drug. Consequently, the HB-19 pseudopeptide provides a unique candidate to consider for innovative cancer therapy. more...

The growth factor pleiotrophin (PTN) has been reported to bind heparan sulfate and nucleolin, two components of the cell surface implicated in the attachment of HIV-1 particles to cells. Here we show that PTN inhibits HIV-1 infection by its capacity to inhibit HIV-1 particle attachment to the surface of permissive cells. The beta-sheet domains of PTN appear to be implicated in this inhibitory effect on the HIV infection, in particular the domain containing amino acids 60-110. PTN binding to the cell surface is mediated by high and low affinity binding sites. Other inhibitors of HIV attachment known to bind specifically surface expressed nucleolin, such as the pseudopeptide HB-19 and the cytokine midkine prevent the binding of PTN to its low affinity-binding site. Confocal immunofluorescence laser microscopy revealed that the cross-linking of surface-bound PTN with a specific antibody results in the clustering of cell surface-expressed nucleolin and the colocalization of both PTN and nucleolin signals. Following its binding to surface-nucleolin, PTN is internalized by a temperature sensitive mechanism, a process which is inhibited by HB-19 and is independent of heparan and chondroitin sulfate proteoglycans. Nevertheless, proteoglycans might play a role in the concentration of PTN on the cell surface for a more efficient interaction with nucleolin. Our results demonstrate for the first time that PTN inhibits HIV infection and suggest that the cell surface-expressed nucleolin is a low affinity receptor for PTN binding to cells and it is also implicated in PTN entry into cells by an active process. more...

The growth factor midkine (MK) is a cytokine that inhibits the attachment of human immunodeficiency virus particles by a mechanism similar to the nucleolin binding HB-19 pseudopeptide. Here we show that the binding of MK to cells occurs specifically at a high and a low affinity binding site. HB-19 prevents the binding of MK to the low affinity binding site only. Confocal immunofluorescence laser microscopy revealed the colocalization of MK and the cell-surface-expressed nucleolin at distinct spots. The use of various deletion constructs of nucleolin then indicated that the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, is the domain that binds MK. The specific binding of MK to cells is independent of heparan sulfate and chondroitin sulfate expression. After binding to cells, MK enters cells by an active process. Interestingly, the cross-linking of surface-bound MK with a specific antibody results in the clustering of surface nucleolin along with glycosylphosphatidylinositol-linked proteins CD90 and CD59, thus, pointing out that MK binding induces lateral assemblies of nucleolin with specific membrane components of lipid rafts. Our results suggest that the cell surface-expressed nucleolin serves as a low affinity receptor for MK and could be implicated in its entry process. more...

The HB-19 pseudopeptide 5[Kpsi(CH2N)PR]-TASP[psi(CH2N) indicating a reduced peptide bond], which binds the cell surface-expressed nucleolin, is a potent inhibitor of HIV infection. Here, by using primary T lymphocyte cultures and an experimental cell model to monitor HIV entry, we show that HB-19 inhibits in a dose-dependent manner both T lymphocyte- and macrophage-tropic HIV isolates. Similar positively charged control pseudopeptides have no effect on HIV infection even at high concentrations. These observations, and the fact that HB-19 has no effect on SIV-mac and HIV-1 pseudotyped with VSV envelope glycoproteins, confirm the specific nature of this inhibitor against the entry process mediated by the HIV envelope glycoproteins. Finally, association of low doses of HB-19 with beta-chemokines or AZT results in an increased inhibitory effect on HIV infection. HB-19 has no inhibitory effect when added to cells a few hours after HIV entry. On the other hand, in HB-19-pretreated cells, the inhibitory effect persists for several hours, even after washing cells to remove away the unbound pseudopeptide. Under such conditions, the attachment of HIV particles to cells is inhibited as efficiently as by neutralizing monoclonal antibodies directed against the V3 loop. In view of its specific mode of action on various HIV isolates, HB-19 represents a potential anti-HIV drug. more...

The HB-19 pseudopeptide 5[Kpsi(CH(2)N)PR]-TASP, psi(CH(2)N) for reduced peptide bond, is a specific inhibitor of human immunodeficiency virus (HIV) infection in different CD4(+) cell lines and in primary T-lymphocytes and macrophages. Here, by using an experimental CD4(+) cell model to monitor HIV entry and infection, we demonstrate that HB-19 binds the cell surface and inhibits attachment of HIV particles to permissive cells. At concentrations that inhibit HIV attachment, HB-19 binds cells irreversibly, becomes complexed with the cell-surface-expressed nucleolin, and eventually results in its degradation. Accordingly, by confocal immunofluorescence microscopy, we demonstrate the drastic reduction of the cell-surface-expressed nucleolin following treatment of cells with HB-19. HIV particles can prevent the binding of HB-19 to cells and inhibit complex formation with nucleolin. Such a competition between viral particles and HB-19 is consistent with the implication of nucleolin in the process of HIV attachment to target cells. We show that another inhibitor of HIV infection, the fibroblast growth factor-2 (FGF-2) that uses cell-surface-expressed heparan sulfate proteoglycans as low affinity receptors, binds cells and blocks attachment of HIV to permissive cells. FGF-2 does not prevent the binding of HB-19 to cells and to nucleolin, and similarly HB-19 has no apparent effect on the binding of FGF-2 to the cell surface. The lack of competition between these two anti-HIV agents rules out the potential involvement of heparan sulfate proteoglycans in the mechanism of anti-HIV effect of HB-19, thus pointing out that nucleolin is its main target. more...

By using a CD26 negative human lymphoblastoid cell line (C8166), here we describe the characterization of a cell-surface protein which manifests CD26-like dipeptidyl peptidase IV (DPP IV) activity. This protein, referred to as DPP IV-β, shows a higher KM value for Gly-Pro-pNA than CD26 (0,31 mM compared to 0,11 mM, respectively). In addition, DPP IV-β was found not to bind 125I-labeled adenosine deaminase (a property of human CD26). Gel filtration experiments using extracts from C8166 and MOLT4 (a CD26 positive human T cell line) cells, revealed that the apparent molecular mass of DPP IV-β is 82 kDa, whereas that of CD26 is 110 kDa. In order to conveniently differentiate both activities, a new family of inhibitors, that selectively blocks peptidase activity associated to CD26, has been developed. more...

We have reported that CD26 could serve as a cofactor of CD4 in HIV entry. Recently, more evidence has been provided for the implication of CD26 in HIV entry, replication and cytopathic effect. Along with, we have demonstrated that the level of CD26 may determine the rate of HIV-envelope induced-apoptosis. The role of CD26 in HIV entry was further investigated using CEM T-cell line. Clones were established by transfection, expressing different levels of CD26. Entry, infection and cytopathic effect were monitored in several independent clones, and were found to be delayed in clones CD26Low and CD26-SuperHigh compared to clones CD26-High. The delay was most significant in clones CD26-AntiSense, without any apparent cytopathic effect. These results demonstrate that relatively enhanced levels of CD26 contribute to an increased virus infection. Furthermore, they illustrate that CD26-SuperHigh clones manifest a phenotype similar to CD26-Low clones. This point out the critical role of CD26 in the rate of HIV entry and its cytopathic effect, two events which are initiated by the interaction of HIV envelope glycoproteins with cell-surface CD4. more...

Adenosine deaminase (ADA) and the HIV-1 transactivator protein Tat have been reported to bind to human CD26, also known as dipeptidyl peptidase IV (DPP IV). In order to demonstrate the specificity of such binding under native conditions of CD26, i.e., when expressed on the cell surface, we established murine cell lines expressing transfected human CD26, either wild-type or mutated at its serine-630, which inactivates the DPP IV activity. This experimental system is advantageous since murine ADA does not bind human CD26, whereas human and bovine ADA bind. Consequently, murine cell clones expressing either the wild-type or mutated form of human CD26 were found to bind specifically bovine125I-labeled ADA with a high affinity (KD= 12 ± 2 nM and 11 ± 4 nM,respectively). No specific binding of125I-labeled ADA was observed to murine clones not expressing human CD26. The binding of125I-labeled ADA to CD26 was further characterized by the use of monoclonal antibodies specific to human CD26. The results obtained were in accord with those reported previously using other experimental models. These observations indicated that the murine cells expressing human CD26 provide a highly suitable model to investigate the potential binding of HIV-1 Tat to CD26. In contrast to previously published results, however, we could not demonstrate a specific interaction between Tat and human CD26. The125I-labeled ADA-specific binding to human CD26 was not affected by Tat, even at concentrations which induced cell death. Similarly, the binding of several monoclonal antibodies to human CD26 was not modified by the addition of Tat. More significantly, Tat binding to different murine cell clones (human CD26 negative or positive) was found not to be correlated with the expression of human CD26. Finally, the toxic effect of Tat on the growth of different murine cell clones was independent of human CD26 expression. Taken together, these observations further confirm the specific binding of ADA to human CD26 and point out that CD26 is not the target of HIV-1 Tat protein. more...

The CD4 molecule is essential for binding HIV particles, but is not sufficient for efficient viral entry and infection. The cofactor was shown to be dipeptidyl peptidase IV (DPP IV), also known as CD26. This serine protease cleaves its substrates at specific motifs; such motifs area also highly conserved in the V3 loops of HIV-1, HIV-2, and related simian isolates. Entry of HIV-1 or HIV-2 into T lymphoblastoid and monocytoid cell lines was inhibited by a specific monoclonal antibody against DPP IV or specific peptide inhibitors of this protease. Coexpression of human CD4 and CD26 in murine NIH 3T3 cells rendered them permissive to infection by HIV-1 and HIV-2. These observations could provide the basis for developing simple and specific inhibitors of HIV and open a possibility for vaccine development. more...

Interferon-treated mouse cells show an enhanced level of protein kinase activity which is manifested by the phosphorylation of an endogenous 67,000-molecular weight protein (p67K kinase). This kinase activity can be assayed efficiently after its partial purification on poly(I) · poly(C)-Sepharose. We have previously shown that the p67K kinase is present in the liver, spleen and plasma (heparinized) of mice with high levels of circulating interferon. Here we confirm these results by treatment of mice with interferon and furthermore show that besides the liver and spleen, the level of p67K kinase is enhanced in several other tissues such as thymus, brain, pancreas, heart, and lung. The action of interferon in mice was further monitored by the assay of pppA(2′p5′A)n synthetase (2–5A synthetase) in different tissues. The level of 2-5A synthetase was enhanced several fold in the following tissues: heart, pancreas, thymus, liver, and spleen. The detection of 2–5A synthetase and p67K kinase activities in the different tissues of mice provides suitable markers for the response of each individual tissue toward treatment with interferon. The phosphorylated 67K protein (pp67K) from control and interferon-treated mouse L-929 cells and from the plasma and different tissues of control and interferontreated mice was characterized by two-dimensional gel electrophoresis. The isoelectric point ( pl ) of pp67K from the different tissues and L-929 cells was 8 to 8.5. On the other hand the p I of pp67K from the plasma had a range of 7.5 to 8. These results indicated that the presence of p67K kinase in the plasma of mice is not due to lysis of tissue cells. more...

Treatment of patients with interferon or inducers of interferon results in an enhanced level of a protein kinase activity found in platelets (1,3). The kinase activity is responsible for the phosphorylation of a 70–72,000 molecular weight protein (72K protein) found in blood plasma. By the means of a technique based on the precipitation of this protein kinase system (the protein kinase and its substrate), we show here that the 72K protein is the α-chain of fibrinogen. During the coagulation process induced by thrombin, the 32P-labelled 72K protein is recovered in the clot. After incubation in the presence of thrombin, the 72K protein looses a small polypeptide of 2–3000 in molecular weight resulting a shift in its isoelectric point (pI) from 6.8–7.0 to 7.5. At the end of the coagulation process, the 32P-labelled 72K protein becomes undetectable since it gives rise to a covalently linked α-polymer of a high molecular weight. In accord with these results, the 72K protein could be precipitated by antibodies against human fibrinogen. more...

Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous 67,000 and 72,000 Mr proteins, respectively. Enhanced levels of such kinase activity are also detectable in the plasma of patients treated with interferon and in the plasma and tissues of interferon-treated mice. A rapid and efficient method of assay for these protein kinase activities is described. The samples are first incubated with heparin (100 units/ml), which results in the inhibition of different protein kinase activities, but not the one mediated by interferon. The latter one is then assayed after partial purification on poly(rI):(rC)-Sepharose or poly(rG)-Sepharose. The protein kinase from human and mouse cells in culture and from the different tissues of mice binds specifically to poly(rI):(rC)-Sepharose. On the other hand, the protein kinase activity from both mouse and human plasma shows a higher affinity toward poly(rG)-Sepharose. These methods are successfully applied for the determination of the interferon-mediated protein kinase activity from tissue extracts and plasma. more...

A new isolate of the human immunodeficiency virus (HIV) related to the HIV-2 strain was isolated from peripheral blood lymphocytes of an Ivory Coast patient with AIDS. This isolate referred to as HIV-2 EHO could be differentiated by its envelope precursor and external glycoprotein which are 20-kDa smaller than those of HIV-2 ROD isolate. Furthermore, the apparent molecular weight of the major core protein of HIV-2 EHO is 27 kDa instead of 26 kDa as in HIV-2 ROD isolate. In addition, the product of the vpx gene which is a characteristic feature of the HIV-2 strain, is 14 kDa in HIV-2 EHO compared with 16 kDa in HIV-2 ROD. In contrast to these, the envelope precursor of HIV-2 EHO forms a transient dimer during its maturation as is the case for HIV-2 ROD. In both cases the transmembrane proteins are 36 kDa and exist as homodimers of 80 kDa. Endoglycosidase H digestion experiments indicated that the 20-kDa difference between the two HIV-2 isolates is not due to a difference in the number of N-linked oligosaccharide chains per polypeptide, since deglycosylated envelope precursors of HIV-2 ROD and EHO have an apparent molecular weight of 80 and 60 kDa, respectively. Partial proteolysis of the envelope precursors from the two isolates with Staphylococcus aureus V8 protease gave a distinct pattern of polypeptides. These results suggest that the differences between the external envelope proteins of the two HIV-2 isolates are due to their amino acid composition. Accordingly, polyclonal antibodies raised against HIV-2 ROD envelope do not recognize the corresponding envelope proteins of HIV-2 EHO by immunoblotting and immunoprecipitation assays. These data illustrate that analysis of viral proteins could be useful for a rapid characterization of new viral isolates and show the heterogeneity of HIV-2 isolates in West Africa. more...

Extracts from interferon-treated human cells show an enhanced level of a double-stranded RNA-dependent protein kinase activity that is manifested by the phosphorylation of an endogenous Mr 69,000-72,000 protein in its phosphate-saturated state. By using a highly purified protein kinase fraction from interferon-treated human Daudi cells, we can now describe the preparation of murine monoclonal antibodies directed against this phosphoprotein, the Mr of which in its native state is found to be 68,000. These monoclonal antibodies (class IgG1) can identify the electrophoresed protein (p68) in polyacrylamide gels by the electrophoretic transfer blotting technique. Immunoprecipitates formed after incubation of extracts from interferon-treated human cells with the monoclonal antibodies can be conveniently recovered by protein A-Sepharose. Such immune complex preparations have associated protein kinase activity--i.e., addition of [gamma-32P]ATP results in the phosphorylation of p68 and added substrates, calf thymus histone, and eukaryotic initiation factor 2. Immune complex preparations from [35S]methionine-labeled extracts show the specific immunoprecipitation of p68. In addition, several other [35S]methionine-labeled proteins are bound unspecifically in these immune complexes prepared under similar experimental conditions as for the assay of protein kinase activity. These unspecifically bound proteins can be washed out by using a buffer containing detergents or high concentrations of KCl and magnesium acetate. Immune complex preparations washed similarly with these buffers still retain p68 but lose their capacity to phosphorylate p68 or exogenous substrates. These results indicate that p68 by itself has no protein kinase activity. The induction of [35S]methionine-labeled p68 in Daudi cells occurs with as little as 1 unit of human alpha interferon, with maximal synthesis between 6 to 9 hr after the addition of interferon. Actinomycin D blocks this induction. more...

A mouse hybridoma cell line has been isolated, which secretes a monoclonal antibody specific of human leukocyte (alpha) interferon. The antibody secreted by the hybridoma belongs to the IgG1 class. It neutralizes biological activities (cellular and antiviral) of alpha interferon. Mass production of the antibody in ascitic fluid has been obtained. A convenient method of purification of the IgG from the ascitic fluid on DEAE-Trisacryl M is described. more...

Background The importance of cell-surface nucleolin in cancer biology was recently highlighted by studies showing that ligands of nucleolin play critical role in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, we recently reported that HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in the athymic nude mice without apparent toxicity. Methods The in vivo antitumoral action of HB-19 treatment was assessed on the spontaneous development of melanoma in the RET transgenic mouse model. Ten days old RET mice were treated with HB-19 in a prophylactic setting that extended 300 days. In parallel, the molecular basis for the action of HB-19 was investigated on a melanoma cell line (called TIII) derived from a cutaneous nodule of a RET mouse. Results HB-19 treatment of RET mice caused a significant delay in the onset of cutaneous tumors, several-months delay in the incidence of large tumors, a lower frequency of cutaneous nodules, and a reduction of visceral metastatic nodules while displaying no toxicity to normal tissue. Moreover, microvessel density was significantly reduced in tumors recovered from HB-19 treated mice compared to corresponding controls. Studies on the melanoma-derived tumor cells demonstrated that HB-19 treatment of TIII cells could restore contact inhibition, impair anchorage-independent growth, and reduce their tumorigenic potential in mice. Moreover, HB-19 treatment caused selective down regulation of transcripts coding matrix metalloproteinase 2 and 9, and tumor necrosis factor-α in the TIII cells and in melanoma tumors of RET mice. Conclusions Although HB-19 treatment failed to prevent the development of spontaneous melanoma in the RET mice, it delayed for several months the onset and frequency of cutaneous tumors, and exerted a significant inhibitory effect on visceral metastasis. Consequently, HB-19 could provide a novel therapeutic agent by itself or as an adjuvant therapy in association with current therapeutic interventions on a virulent cancer like melanoma. more...