Abstract: Sexually transmitted infections and unplanned pregnancies present a great risk to the reproductive health of women. Therefore, female-controlled vaginal products directed toward disease prevention and contraception are needed urgently. In the present study, efforts were made to evaluate the contraceptive potency of dermaseptin DS4, an antimicrobial peptide derived from frog skin. To assess the structure-activity relationship between the native DS4 and its derivatives, a set of chemically modified peptides was synthesized and evaluated. Normal human semen samples were used to detect the spermicidal activity of the new compounds. HeLa cultures were used to determine the safety of compounds toward their toxicity. Fluorescent-binding assays were performed to evaluate the rapidity and the irreversibility of the sperm-immobilizing activity of peptides. All DS4 derivatives elicited concentration-dependent spermicidal activity at microgram concentrations (EC(100) values: 25 microg/ml-l mg/ml). The order was K4S4=S4a>S4>K4S4(1-16)a>S4(6-28). In cytotoxicity assay, some compounds were found to be significantly safer than nonoxynol-9, the most widely used spermicide, and their activity was not accompanied by total loss of plasma membrane integrity as detected by fluorescent microscopy. Our data also show that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the spermicidal effect. This study indicates that dermaseptins are spermicidal molecules that deserve to be tested as topical contraceptive with useful activities that can add to their prophylaxis, safety, and effectiveness.

Abstract: Despite conventional medical and surgical treatments, malignant pleural mesothelioma (MPM) remains incurable. Oncovirotherapy (i.e., the use of replication-competent virus for cancer treatment) is currently explored in clinical trials. In this study, we investigated the antineoplastic potential of a new oncolytic viral agent, a live-attenuated measles virus (MV) strain derived from the Edmonston vaccine lineage (Schwarz strain). We evaluated both oncolytic activity and immunoadjuvant properties of the MV vaccine strain on mesothelioma tumor cells. Infectivity, syncytium formation, and cytolytic activity of MV were studied on a panel of mesothelioma cells derived from pleural effusions of MPM patients. We observed that MV infected preferentially MPM cell lines in comparison with nontransformed mesothelial cells, leading to an efficient killing of a significant fraction of tumor cells. A cytoreductive activity was also evidenced through formation of multinuclear cellular aggregates (syncytia). The susceptibility of MPM cell lines to measles infection was assessed by the analysis of cell surface expression of the MV vaccine receptor (CD46). We also evaluated whether MV infection of mesothelioma cells could elicit an autologous antitumor immune response. We showed that MV Schwarz strain induced apoptotic cell death of infected mesothelioma cells, which were efficiently phagocytosed by dendritic cells (DC). Loading of DCs with MV-infected MPM cells induced DC spontaneous maturation, as evidenced by the increased expression of MHC and costimulatory molecules along with the production of proinflammatory cytokines. Priming of autologous T cells by DCs loaded with MV-infected MPM cells led to a significant proliferation of tumor-specific CD8 T cells. Altogether, these data strongly support the potential of oncolytic MV as an efficient therapeutic agent for mesothelioma cancer.

Abstract: OBJECTIVES: Sexually transmitted infections present a great risk to the reproductive health of women. Therefore, female-controlled vaginal products directed toward disease prevention are needed urgently. In the present study, efforts were made to evaluate the antimicrobial potency of dermaseptin DS4, an antimicrobial peptide derived from frog skin. To assess the structure-activity relationship between the native DS4 and their derivatives, a set of chemically modified peptides was synthesized and evaluated. METHODS: Different strains of bacteria and fungi were used to detect the antimicrobial activity of the new compounds. HeLa cultures were used to determine the safety of compounds towards their toxicity. RESULTS: All DS4 derivatives elicited concentration-dependent antimicrobial activity at micrograms concentrations (MIC values: 4 microg/mL-l mg/mL). K4S4(1-28) and K4S4(1-16)a were the most active peptides whereas S4(6-28) was the less one. The order was K4S4(1-28)>K4S4(1-16)a>S4a>S4>S4(6-28). In cytotoxicity assay, some compounds were found to be significantly safer than current antibiotics. Our data also show that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the antimicrobial effect. CONCLUSIONS: This study indicates that dermaseptins are antimicrobial molecules that deserve to be tested as topical microbicide with useful associated activities that can add to their prophylaxis, safety and effectiveness.

Abstract: The measles vaccine is one of the best vaccines currently available. Over the last 30 years, it has been administered to hundreds of millions of children and has proved to be both effective and safe. This attenuated live virus induces life-long immunity after only one or two injections. It is produced on a large scale, with ease, in many countries and is distributed at low cost. These excellent characteristics led us to consider its use as a pediatric live vector, to simultaneously immunize children or adolescents against measles and other viral infections, such as human immunodeficiency virus (HIV) or flavivirus infections. For this purpose, we have developed a vector derived from the live attenuated Schwarz strain of the measles virus (MV). We have demonstrated the capacity of this vector to strongly and stably express genes encoding proteins from HIV and to induce specific humoral and cellular immune responses in vivo. Importantly, we observed that, at least in animal models, the vector can bypass measles vaccine pre-existing immunity when two doses of recombinant vaccine are administered. Clinical trials are in progress to confirm this observation in immunized adults. We also produced MV vectors expressing proteins from West Nile virus and other flaviviruses, which in the case of West Nile virus, protected against experimental challenge. Recombinant live attenuated MV might be used as bivalent vaccination vector to mass immunize children and adolescents against both measles and flaviviral diseases such as Dengue or Japanese Encephalitis in the developing world.

Abstract: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes in humans an acute infection characterized by polyarthralgia, fever, myalgia and headache. Since 2005, this virus has been responsible for an epidemic outbreak of unprecedented magnitude. Up to now, no treatment of the disease has been shown to be effective. By analogy with other alphaviruses, it is thought that cellular proteases are able to process the viral precursor protein E3E2 to produce the receptor-binding E2 protein that associates as a heterodimer with E1. Destabilization of the heterodimer by exposure to low pH allows viral fusion and infection. We show that among a large panel of proprotein convertases, membranous Furin but also PC5B can process E3E2 from African CHIKV strains at the HRQRR64ST site, whereas a CHIKV strain of Asian origin is cleaved at RRQRR64SI by membranous and soluble Furin, PC5A, PC5B, and PACE4 but not by PC7 or SKI-1. Using intramolecularly quenched fluorogenic model peptides and recombinant convertases, we measured in vitro the kinetic parameters and observed that the Asian strain E3E2 model peptide is cleaved most efficiently by Furin and PC5A. This cleavage was also observed in CHIKV infected cells and could be blocked by Furin inhibitor dec-RVKR-cmk. This inhibitor was compared to chloroquine for its ability to inhibit CHIKV spreading in myoblast cell cultures, a cell-type previously described as a natural target of this virus. Our results demonstrate the role of Furin-like proteases in the processing of CHIKV particles and point out new approaches to inhibit this infection.

Abstract: DNA complementarity is expressed by way of three hydrogen bonds for a G:C base pair and two for A:T. As a result, careful control of the denaturation temperature of PCR allows selective amplification of AT-rich alleles. Yet for the same reason, the converse is not possible, selective amplification of GC-rich alleles. Inosine (I) hydrogen bonds to cytosine by two hydrogen bonds while diaminopurine (D) forms three hydrogen bonds with thymine. By substituting dATP by dDTP and dGTP by dITP in a PCR reaction, DNA is obtained in which the natural hydrogen bonding rule is inversed. When PCR is performed at limiting denaturation temperatures, it is possible to recover GC-rich viral genomes and inverted Alu elements embedded in cellular mRNAs resulting from editing by dsRNA dependent host cell adenosine deaminases. The editing of Alu elements in cellular mRNAs was strongly enhanced by type I interferon induction indicating a novel link mRNA metabolism and innate immunity.

Abstract: Dengue disease is an increasing global health problem that threatens one-third of the world's population. Despite decades of efforts, no licensed vaccine against dengue is available. With the aim to develop an affordable vaccine that could be used in young populations living in tropical areas, we evaluated a new strategy based on the expression of a minimal dengue antigen by a vector derived from pediatric live-attenuated Schwarz measles vaccine (MV). As a proof-of-concept, we inserted into the MV vector a sequence encoding a minimal combined dengue antigen composed of the envelope domain III (EDIII) fused to the ectodomain of the membrane protein (ectoM) from DV serotype-1. Immunization of mice susceptible to MV resulted in a long-term production of DV1 serotype-specific neutralizing antibodies. The presence of ectoM was critical to the immunogenicity of inserted EDIII. The adjuvant capacity of ectoM correlated with its ability to promote the maturation of dendritic cells and the secretion of proinflammatory and antiviral cytokines and chemokines involved in adaptive immunity. The protective efficacy of this vaccine should be studied in non-human primates. A combined measles-dengue vaccine might provide a one-shot approach to immunize children against both diseases where they co-exist.

Abstract: Viruses have evolved various strategies to escape the antiviral activity of type I interferons (IFN-alpha/beta). For measles virus, this function is carried by the polycistronic gene P that encodes, by an unusual editing strategy, for the phosphoprotein P and the virulence factor V (MV-V). MV-V prevents STAT1 nuclear translocation by either sequestration or phosphorylation inhibition, thereby blocking IFN-alpha/beta pathway. We show that both the N- and C-terminal domains of MV-V (PNT and VCT) contribute to the inhibition of IFN-alpha/beta signaling. Using the two-hybrid system and co-affinity purification experiments, we identified STAT1 and Jak1 as interactors of MV-V and demonstrate that MV-V can block the direct phosphorylation of STAT1 by Jak1. A deleterious mutation within the PNT domain of MV-V (Y110H) impaired its ability to interact and block STAT1 phosphorylation. Thus, MV-V interacts with at least two components of IFN-alpha/beta receptor complex to block downstream signaling.

Abstract: We evaluated the antimicrobial effect of antimicrobial peptides from frog skin belonging to the dermaseptin family against reference and clinical Neisseria gonorrhoeae strains, including penicillin-resistant strains. Dermaseptin S4 exhibited anti-N. gonorrhoeae activity against all strains with MICs ranging between 10 and 100 microg/mL. We then used derivatives of DS4 and determined the anti-N. gonorrhoeae activity of each of analogs. All the derivatives showed antimicrobial activity. Among the different molecules tested, we found that dermaseptins K4S4 (1-16)a and K4S4 (1-28) were the more potent to inhibit N. gonorrhoeae growth with MIC of 10 microg/mL against all strains.

Abstract: BACKGROUND: The Expanded Program on Immunization (EPI) is the most cost-effective measures to control vaccine-preventable diseases. Currently, the EPI schedule is similar for HIV-infected children; the introduction of antiretroviral therapy (ART) should considerably prolong their life expectancy. METHODS AND PRINCIPAL FINDINGS: To evaluate the persistence of antibodies to the EPI vaccines in HIV-infected and HIV-exposed uninfected children who previously received these vaccines in routine clinical practice, we conducted a cross-sectional study of children, aged 18 to 36 months, born to HIV-infected mothers and living in Central Africa. We tested blood samples for antibodies to the combined diphtheria, tetanus, and whole-cell pertussis (DTwP), the measles and the oral polio (OPV) vaccines. We enrolled 51 HIV-infected children of whom 33 were receiving ART, and 78 HIV-uninfected children born to HIV-infected women. A lower proportion of HIV-infected children than uninfected children had antibodies to the tested antigens with the exception of the OPV types 1 and 2. This difference was substantial for the measles vaccine (20% of the HIV-infected children and 56% of the HIV-exposed uninfected children, p<0.0001). We observed a high risk of low antibody levels for all EPI vaccines, except OPV types 1 and 2, in HIV-infected children with severe immunodeficiency (CD4(+) T cells <25%). CONCLUSIONS AND SIGNIFICANCE: Children were examined at a time when their antibody concentrations to EPI vaccines would have still not undergone significant decay. However, we showed that the antibody concentrations were lowered in HIV-infected children. Moreover, antibody concentration after a single dose of the measles vaccine was substantially lower than expected, particularly low in HIV-infected children with low CD4(+) T cell counts. This study supports the need for a second dose of the measles vaccine and for a booster dose of the DTwP and OPV vaccines to maintain the antibody concentrations in HIV-infected and HIV-exposed uninfected children.

Abstract: Human T cell lymphotropic virus (HTLV) type I is the agent of adult T cell leukemia and HTLV-I-associated myelopathy. Because its pathogenesis is not well understood, a mouse model of HTLV-I infection would be valuable. We report the infection of adult BALB/c, C3H/He, 129Sv, and 129Sv IFNAR(-/-) mice with an infectious chimeric HTLV-I provirus bearing the Moloney-murine leukemia virus (Mo-MuLV) envelope glycoprotein truncated for the C-terminal R peptide. Mice were persistently infected (500-800 proviral DNA copies/10(5) splenocytes) for at least 20 weeks after inoculation. The chimeric virus disseminated to several organs, such as spleen, thymus, lung, brain, and spinal cord. The amplification of proviral integration sites showed an oligoclonal integration resembling that reported in HTLV-I-infected humans. Infected mice developed lasting humoral and cellular immune responses. This DeltaR HTLV-I/Mo-MuLV chimeric virus, with the Mo-MuLV Env tropism and HTLV-I replication characteristics, could provide a mouse model of HTLV-I infection.

Abstract: The Schwarz strain of measles virus (MV), a live attenuated RNA virus, is one of the safest and most effective human vaccines available. Immunization with MV vaccine expressing heterologous antigen is an attractive strategy to prevent emerging viral diseases. West Nile virus (WNV), which recently emerged in North America, is an important mosquito-borne flavivirus that causes numerous cases of human encephalitis, thus urging the development of a vaccine. To evaluate the efficacy of recombinant MV for the prevention of WNV encephalitis, we constructed a live attenuated Schwarz MV (MVSchw-sE(WNV)) expressing the secreted form of the envelope glycoprotein from the virulent IS-98-ST1 strain of WNV. Inoculation of MV-susceptible mice with MVSchw-sE(WNV) induced both high levels of specific anti-WNV neutralizing antibodies and protection from a lethal challenge with WNV. Passive administration with antisera to MVSchw-sE(WNV) prevented WNV encephalitis in BALB/c mice challenged with a high dose of WNV. The present study is the first to report that a recombinant live attenuated vector based on an approved and widely used MV vaccine can protect against a heterologous, medically important pathogen.

Abstract: Most of HIV-1 infections are acquired through sexual contact. In the absence of a preventive vaccine, the development of topical microbicides that can block infection at the mucosal tissues is needed. Dermaseptin S4 (DS4) is an antimicrobial peptide derived from amphibian skin, which displays a broad spectrum of activity against bacteria, yeast, filamentous fungi, and herpes simplex virus type 1. We show here that DS4 inhibits cell-free and cell-associated HIV-1 infection of P4-CCR5 indicator cells and human primary T lymphocytes. The peptide is effective against R5 and X4 primary isolates and laboratory-adapted strains of HIV-1. Its activity is directed against HIV-1 particles by disrupting the virion integrity. Increasing the number of DS4-positive charges reduced cytotoxicity without affecting the antiviral activity. The modified DS4 inhibited HIV-1 capture by dendritic cells and subsequent transmission to CD4(+) T cells, as well as HIV-1 binding on HEC-1 endometrial cells and transcytosis through a tight epithelial monolayer.

Abstract: Live attenuated measles vaccine (MV) could provide a safe and efficient pediatric vaccination vector to immunize children simultaneously against measles and human immunodeficiency virus type 1 (HIV-1). To evaluate the capacity of a vector derived from the certified Schwarz measles vaccine (MVSchw) to prime effective cytotoxic T cells (CTL) and broad neutralizing antibodies against HIV-1 conserved epitopes, we generated recombinant MVSchw viruses expressing HIV-1 antigens. We demonstrate that a recombinant MVSchw virus expressing an HIV-1-derived CTL polyepitope primes effective HLA-A0201-restricted CTLs against multiple conserved HIV-1 epitopes in mice susceptible to measles and humanized for the major histocompatibility complex (MHC) class-I molecule HLA-A0201. Additionally, we show that a recombinant MVSchw virus expressing an HIV-1(89.6) gp140 glycoprotein whose hyper variable V1, V2 and V3 loops were deleted (DeltaV1V2V3gp140), induces broadly neutralizing antibodies against HIV-1 primary isolates. These results show that the MVSchw pediatric vaccination vector induces efficient cellular and humoral HIV-specific immune responses.

Abstract: Live attenuated RNA viruses make highly efficient vaccines. Among them, measles virus (MV) vaccine has been given to a very large number of children and shown to be highly effective and safe. MV vaccine induces a life-long immunity after a single or two low-dose injections. It is easily produced on a large scale in most countries and can be distributed at low cost. Reversion to pathogenicity has never been observed with this vaccine. Because of all these characteristics, MV vaccine might be a very promising vector to immunise children against both measles and other infectious agents, such as HIV or flaviviruses, in the developing world. In this article, we describe recent data that we obtained showing the capacity of recombinant Schwarz MVs to express proteins from human immunodeficiency or West Nile viruses, and to induce specific immune responses able, in the case of West Nile virus, to protect from an experimental challenge.

Abstract: Live attenuated RNA viruses make highly efficient vaccines. Among them is the live attenuated measles virus (MV) vaccine that has been given to a very large number of children and has been shown to be highly efficacious and safe. MV vaccine induces a life-long immunity after a single injection or two low-dose injections. It is easily produced on a large scale in most countries and can be distributed at low cost. Reversion to pathogenicity has never been observed with this vaccine. For all of these characteristics, developing of MV vaccine vector as a multivalent vaccine to immunize children against both measles and other infectious agents such as human immunodeficiency virus (HIV), flaviviruses, or malaria might be very promising for worldwide use. As MV vaccine is inexpensive to produce, the generation of recombinant vaccines may remain affordable and attractive for the developing word. In this article, we describe the development of MV vector and present some recent data showing the capacity of recombinant MV vaccine to express various proteins from HIV and West Nile virus. In addition, the ability of recombinant MV to induce specific immune responses against these different pathogens are presented and discussed.

Abstract: The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8+ and CD4+ cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.

Abstract: One of the most exciting recent developments in the field of retroviruses is the finding that their Gag proteins hijack cellular proteins from the mutivesicular body (MVB) pathway during the budding process. The Gag proteins of oncoretroviruses possess a PPxY motif that recruits a ubiquitin ligase from the Nedd4 family, whereas those of the human immunodeficiency virus interact through a PTAP motif with Tsg101, a protein of the ESCRT-1 complex. It is currently assumed that Nedd4 and Tsg101 represent equivalent entry gates towards the same cellular process leading to budding, and that both partners are recruited to the plasma membrane where viral budding occurs. However, we report here that the budding of the human oncoretrovirus HTLV-1, the Gag proteins of which possess tandem PPPY/PTAP motifs, requires both Nedd4 and Tsg101. We show that Nedd4.1, but not Nedd4.2, is recruited by the PPPY motif of Gag and subsequently catalyzes Gag ubiquitination. We also demonstrate that Gag interacts first with Nedd4.1 at the plasma membrane and then with Tsg101 in late endosomes/MVBs. Consistently, we found that HTLV-1 particles mutated in the PPPY motif remain underneath the plasma membrane, blocked at an early step of the budding process, whereas PTAP-mutated viruses accumulate in intracellular vesicles, blocked at a later step. Our findings indicate that Nedd4.1 and Tsg101 act successively in the assembly process of HTLV-1 to ensure proper Gag trafficking through the endocytic pathway up to late endosomes where the late steps of retroviral release occur.

Abstract: Live attenuated RNA viruses make highly efficient vaccines. Among them, measles virus (MV) vaccine has been given to a very large number of children and has been shown to be highly efficacious and safe. Therefore, this vaccine might be a very promising vector to immunize children against both measles and other infectious agents, such as human immunodeficiency virus. A vector was previously derived from the Edmonston B strain of MV, a vaccine strain abandoned 25 years ago. Sequence analysis revealed that the genome of this vector diverges from Edmonston B by 10 amino acid substitutions not related to any Edmonston subgroup. Here we describe an infectious cDNA for the Schwarz/Moraten strain, a widely used MV vaccine. This cDNA was constructed from a batch of commercial vaccine. The extremities of the cDNA were engineered in order to maximize virus yield during rescue. A previously described helper cell-based rescue system was adapted by cocultivating transfected cells on primary chicken embryo fibroblasts, the cells used to produce the Schwarz/Moraten vaccine. After two passages the sequence of the rescued virus was identical to that of the cDNA and of the published Schwarz/Moraten sequence. Two additional transcription units were introduced in the cDNA for cloning foreign genetic material. The immunogenicity of rescued virus was studied in macaques and in mice transgenic for the CD46 MV receptor. Antibody titers and T-cell responses (ELISpot) in animals inoculated with low doses of rescued virus were identical to those obtained with commercial Schwarz MV vaccine. In contrast, the immunogenicity of the previously described Edmonston B strain-derived MV clone was much lower. This new molecular clone will allow for the production of MV vaccine without having to rely on seed stocks. The additional transcription units allow expressing heterologous antigens, thereby providing polyvalent vaccines based on an approved, safe, and efficient MV vaccine strain that is used worldwide.

Abstract: We constructed a chimeric human T-cell lymphotropic virus type 1 (HTLV-1) provirus in which the original envelope precursor sequence was replaced by that of ecotropic Moloney murine leukemia virus (Mo-MuLV). Chimeric particles produced by transient transfection of this chimeric provirus were infectious for murine cells, such as NIH 3T3 fibroblasts, lymphoid EL4 cells, and primary CD4(+) T lymphocytes, whereas HTLV-1 particles were not. The infectivity of chimeric particles increased 10 times when the R peptide located at the carboxy terminus of the MuLV envelope glycoprotein was deleted. Primary murine CD4(+) T lymphocytes, infected by the Delta R chimeric virus, released particles that could spread the infection to other naive murine lymphoid cells. This chimeric virus, with the Mo-MuLV envelope glycoprotein and the replication characteristics of HTLV-1, should be useful in studying the pathogenesis of HTLV-1 in a mouse model.

Abstract: The aim of this study was to investigate the distribution of human T-cell leukemia virus type 1 (HTLV-1) in various organs of serially sacrificed squirrel monkeys (Saimiri sciureus) in order to localize the reservoir of the virus and to evaluate the relationship between viral expression and the humoral or cellular immune response during infection. Six squirrel monkeys infected with HTLV-1 were sacrificed 6, 12, and 35 days and 3, 6, and 26 months after inoculation, and 20 organs and tissues were collected from each animal. PCR and reverse transcription-PCR (RT-PCR) were performed with gag and tax primers. Proviral DNA was detected by PCR in peripheral blood mononuclear cells (PBMCs) of monkeys sacrificed 6 days after inoculation and in PBMCs, spleens, and lymph nodes of monkeys sacrificed 12 and 35 days and 3, 6, and 26 months after inoculation. Furthermore, tax/rex mRNA was detected by RT-PCR in the PBMCs of two monkeys 8 to 12 days after inoculation and in the spleens and lymph nodes of the monkey sacrificed on day 12. In this animal, scattered HTLV-1 tax/rex mRNA-positive lymphocytes were detected by in situ hybridization in frozen sections of the spleen, around the germinal centers and close to the arterial capillaries. Anti-HTLV-1 cell-mediated immunity was evaluated at various times after inoculation. Anti-p40(Tax) and anti-Env cytolytic T-cell responses were detected 2 months after infection and remained detectable thereafter. When Tax peptides were used, this response appeared to be directed against various Tax epitopes. Our results indicate that squirrel monkeys represent a promising animal model for studying the early events of HTLV-1 infection and for evaluating candidate vaccines against HTLV-1.

Abstract: Human T cell leukemia virus type I (HTLV-I) sequences were sought in labial salivary glands of patients with HTLV-I-associated myelopathy or tropical spastic paraparesis and of seropositive neurologically healthy carriers. HTLV-I proviral DNA was found by polymerase chain reaction amplification in DNA extracted from lip biopsies of every patient. Viral RNA was found by in situ hybridization in the acini epithelium, as well as in lymphocytic infiltrates. This observation suggests that HTLV-I expression in labial salivary glands could participate in the inflammatory lesions observed in these patients. Some seronegative patients with Sjögren's syndrome or dryness syndrome were also positive for viral transactivator tax DNA (41% in Martinique and 16% in non-HTLV-I-endemic region). Despite histologic signs of lymphocytic infiltration, no viral expression was found in the labial salivary glands of these patients.

Abstract: The infection by human T lymphotropic virus type I is associated with adult T cell leukemia and several inflammatory degenerative disorders, including tropical spastic paraparesis. To investigate the role of the Tax protein in the development of diseases linked to human T lymphotropic virus type I infection, we generated two lines of transgenic mice carrying the tax gene under the control of the viral promoter. The expression of the transgene was low in these mice and was restricted to the central nervous system and testis. Mice from both lines developed various types of tumors, including fibrosarcomas and adenocarcinomas. Tax was expressed at a high level in fibrosarcomas and in cell lines derived from these tumors. In tumor-derived cells, the expression of Tax led to an increased degradation of IkappaB alpha and IkappaB beta and caused stable nuclear translocation of nuclear factor-kappaB. This translocation was essential for cell proliferation, as shown by expressing a nondegradable form of IkappaBbeta in these cells. Therefore, Tax-induced cell transformation in mice correlates with the degradation of IkappaB alpha and IkappaB beta and with the constitutive activation of NF-kappaB.

Abstract: We searched for the presence of human T-cell leukemia virus type I (HTLV-I) sequences in central nervous system and muscle lesions of 3 patients with tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) and 3 patients with HTLV-I-associated polymyositis. Proviral DNA coding for the Tax protein was found by polymerase chain reaction amplification in DNA extracted from lesions of every patient with TSP/HAM or HTLV-I-associated polymyositis. In contrast, viral RNA was found occasionally by in situ hybridization in muscle lesions of some patients with polymyositis, but was never found in central nervous system lesions of TSP/HAM patients.

Abstract: Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either an acute encephalitis or a persistent demyelinating disease. Following intracranial inoculation, the demyelinating strains infect sequentially the grey matter of the brain, the grey matter of the spinal cord, and finally the white matter of the spinal cord, where they persist and cause chronic demyelination. The neurovirulent strains cause a generally fatal encephalitis with lytic infection of neurons. The study of chimeric Theiler's viruses, obtained by recombining the genomes of demyelinating and neurovirulent strains, has shown that the viral capsid contains determinants for persistence and demyelination. In this article we describe the recombinant virus R5, in which the capsid protein VP1 and a small portion of protein 2A come from the neurovirulent GDVII strain and the rest of the genome comes from the persistent DA strain. The capsid of virus R5 also contains one mutation at amino acid 34 of VP3 (Asn-->His). Virus R5 does not persist in the central nervous system (CNS) of immunocompetent SJL/J or BALB/c mice. However, it replicates efficiently and persists in the CNS of BALB/c nu/nu mice, showing that its growth in the CNS is not impaired. In BALB/c nu/nu mice, whereas virus DA causes mortality with large amounts of viral antigens in the white matter of the spinal cord, virus R5 does not kill the animals, persists in the neurons of the grey matter of the brain, and never reaches the white matter of the spinal cord. This phenotype is due to the chimerism of the capsid and/or to the mutation in VP3. These results indicate that the capsid plays an important role in the characteristic migration of Theiler's virus within the CNS.

Abstract: The DA strain of Theiler's virus persists in the central nervous systems of mice and causes chronic inflammation and demyelination. The GDVII strain, on the other hand, causes an acute encephalitis that kills the host in a matter of days. We constructed a series of recombinants between two infectious cDNA clones of the genomes of DA and GDVII viruses. Analysis of the phenotypes of the recombinant viruses yielded the following results. (i) Determinants of persistence and demyelination are found only in the VP1 capsid protein of DA virus. (ii) Whereas the VP1 capsid protein of DA virus is able to fully attenuate the neurovirulence of GDVII virus and to allow the chimeric virus to persist and demyelinate, the VP1 capsid protein of GDVII virus is unable to render DA virus neurovirulent. (iii) The mere attenuation of the neurovirulence of GDVII virus does not allow it to persist and demyelinate.

Abstract: Theiler's virus, a murine picornavirus, is responsible for two different types of disease: strains DA, BeAn, and WW persist for more than a year in the white matter of the central nervous system and cause primary demyelination; strains GDVII and FA, on the other hand, cause an acute encephalitis that kills the host in a matter of days. To map the regions of the viral genome responsible for persistence and demyelination, cDNA clones of the entire genomes of the DA and GDVII strains were constructed and cloned into Bluescript plasmid (A. McAllister, F. Tangy, C. Aubert, and M. Brahic, Microb. Pathogen. 7:381-388, 1989; F. Tangy, A. McAllister, and M. Brahic, J. Virol. 63:1101-1106, 1989). We constructed chimeric viruses obtained by exchanging regions between the cDNA clones. Analysis of the disease phenotypes produced by the chimeric viruses allowed us to map persistence and demyelination to a genome segment coding for the VP1 capsid protein and 27 amino acids of protein 2A.

Abstract: We constructed a complete cDNA clone of the genome of Theiler's virus strain DA in a Bluescript plasmid. This recombinant plasmid, called pTMDA, was used to synthesize full length RNA transcripts of the viral insert. The RNA was infectious for BHK cells. Virus R1-DA, obtained from transfected BHK cells, caused the biphasic disease classically observed with this strain of Theiler's virus. SJL/J mice did not show clinical symptoms during the first week following intracranial inoculation, although viral antigens were found in a few neurons of brain and spinal cord. By 45 days post-inoculation, the mice had developed a chronic demyelinating disease and viral RNA and antigens could be found only in spinal cord white matter in areas surrounded by inflammatory infiltrates. At this stage no RNA or antigens were found in neurons. Therefore the phenotype of R1-DA was indistinguishable from that of genuine DA Theiler's virus.

Abstract: We constructed a complete cDNA clone of strain GDVII of Theiler's virus in Bluescript plasmid. This recombinant plasmid, called pTMGDVII, was used to synthesize full-length RNA transcripts of the viral insert. This RNA was infectious for BHK cells. Virus R1-GDVII, obtained from transfected BHK cells, caused rapidly fatal encephalomyelitis in BALB/c mice. High amounts of viral antigens were present in neurons. No antigens were found in white matter. Therefore, the phenotype of R1-GDVII was indistinguishable from that of genuine GDVII Theiler's virus.

Abstract: Gentamicin C2 interacts cooperatively with ribosomes from a sensitive Escherichia coli strain in a multiphasic way with several classes of sites. It is shown that this binding is highly-dependent on Mg++ and natural endogenous polyamine concentrations. The differences observed between ribosomes from sensitive and resistant strains may be explained by the absence of specific cooperative gentamicin interactions with resistant ribosomes. The effects of other aminoglycoside antibiotics are discussed in terms of structure-activity relationships.

Abstract: Theiler's virus causes a persistent demyelinating infection of the mouse central nervous system. Our study of the molecular mechanism of persistence led us to sequence 1925 nucleotides located at the 3' end of the viral genome. We observed extensive homologies between this region and the corresponding region of encephalomyocarditis virus, the prototype cardiovirus, and only some homologies with the 3' ends of foot-and-mouth disease virus, rhinovirus, and poliovirus genomes.

Abstract: The binding of gentamicin (Gm) to Escherichia coli ribosomes and ribosomal subunits has been studied. By means of equilibrium dialysis and of statistical interpretation of the data it was found that [3H]gentamicin C2 and 6'-N-[3H]methylgentamicin C1a interact with three classes of sites on tight-coupled 70-S species: a first class concerning the tight and non-cooperative interaction with one drug molecule (Kd = 0.6 microM), a second class in which about five Gm molecules bind cooperatively (mean Kd = 10 microM), and a third class of very high capacity in which up to 70 drug molecules may interact. The extreme cooperativity of the third class of sites induces such an increase in the affinity for Gm that it may allow the shift of molecules already bound from high-affinity sites towards lower-affinity sites. The alteration of a ribosomal protein, L6, in a gentamicin-resistant mutant of E. coli abolished the multiclass and the cooperative aspects of ribosomes--gentamicin interaction. The large ribosomal subunits from E. coli MRE 600 strain interact cooperatively with Gm, whereas 50-S particles from the resistant mutant bind the drug in a diffuse way with high capacity and low affinity. The small subunits from both strains behave identically towards Gm. A good correlation is observed in comparing the gentamicin concentrations capable of saturating the different ribosomal classes of sites with concentrations inducing its multiphasic effects on protein synthesis.

Abstract: An [3H]azidobenzyl derivative of tobramycin, a 4,6-disubstituted 2-deoxystreptamine aminoglycoside, has been synthesized, and its ability to label Escherichia coli 70-S ribosomes under photoactivation has been studied. Two concentrations of the photolabel, corresponding to the saturation of the two classes of tobramycin sites on the ribosomes, were used. The results show that, at high antibiotic concentrations which induce maximal misreading during protein synthesis, most of the ribosomal proteins are labelled. At low antibiotic concentration, which results in the saturation of the first-class sites, a few proteins of both subunits are labelled, including L6, S4, S5, and, to a lesser extent, L2, L13 and S18. The 30-S subunit is, on the whole, labelled more efficiently than the 50-S subunit.

Abstract: (3H) Tobramycin was used as a probe to determine the relationship between the structure of aminoglycoside antibiotics and their ability to remove this drug from its higher affinity binding site on the ribosome. The dissacharide moieties (neamine, tobramine, gentamine) appeared to have a common binding site, whereas the kanosamine, garosamine and ribose moieties determined the specificity of this binding. Amikacin and butikacin behaved in an anomalous manner in spite of their close structural relationship to tobramycin.

Abstract: 6'-N-[14C]Acetyl-tobramycin and [3H]tobramycin were synthesized and their binding to Escherichia coli ribosomes and ribosomal subunits studied using equilibrium dialysis. THE 70-S ribosome, as well as its 50-S and 30-S subunits, bound tightly to 6'-N-acetyl-tobramycin. The binding of [3H]tobramycin to ribosomes was quite different. The 70-S ribosome was observed to possess several classes of binding sites; of these, one was determined to be of higher affinity and lower capacity, the 6'-N-[14C]acetyl-tobramycin site. The isotopic dilution method was used to define the specificity of the interaction. The selective binding of 6'-N-[14C]acetyl-tobramycin was highly reversible by tobramycin, kanamycins A, B, C and neomycin, but not by streptomycin or erythromycin. Gentamicin C1a was a poor inhibitor. This suggested that either the kanosamin or garosamin rings might be determinant in the binding of these molecules, as well as the 6'-amino group.

Abstract: A sample of [3H] tobramycin (5,000 Ci/Mole) has been synthetized and incubated with the bacterial ribosome and its subunits. The results obtained show that this antibiotic has two types of binding sites. The primary one is probably responsible for the inhibition of protein synthesis whereas the secondary one is probably related to the misreading and reading through of the messenger RNA.