The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells

The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti–human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1–specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system

Pediatric Measles Vaccine Expressing a Dengue Antigen Induces Durable Serotype-specific Neutralizing Antibodies to Dengue Virus

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

Etude du mécanisme d'inhibition de la fusion des flavivirus par les anticorps et mise au point d'un candidat vaccin contre la dengue basé sur l'expression d'un antigène d'enveloppe par un vecteur dérivé du vaccin contre la rougeole

La dengue est une maladie ré-émergente qui menace le tiers de la population mondiale, et pour laquelle aucun vaccin n'est disponible. Dans ce travail, nous avons évalué une nouvelle stratégie de vaccination basée sur l'expression d'un antigène combiné du virus de la dengue par un vecteur dérivé du vaccin vivant atténué pédiatrique contre la rougeole. La compréhension des mécanismes de neutralisation des flavivirus par les anticorps est essentielle pour la conception d approches vaccinales innovantes contre la dengue. C est pourquoi, dans un premier temps, nous avons étudié le mécanisme de l inhibition de fusion par des anticorps neutralisants dirigés contre les trois domaines de la protéine d enveloppe (E). Le virus de l encéphalite à tiques (TBEV) a été utilisé comme modèle dans des tests de fusion, de neutralisation et d association aux membranes lipidiques. Les résultats montrent, que les anticorps peuvent interférer avec les étapes précoces ou tardives du processus de fusion. Cette étude montre aussi que les anticorps dirigés contre le domaine III de la protéine E peuvent bloquer à la fois l entrée et la fusion virale. Pour faire la preuve de concept de notre stratégie de vaccination, nous avons donc inséré dans le vecteur rougeole le domaine III de la glycoprotéine E (EDIII) qui contient le site de liaison au récepteur ainsi que des épitopes neutralisants sérotype-spécifiques. Pour renforcer son immunogénicité, le EDIII a été fusionné à l'ectodomaine pro-apoptotique de la protéine de membrane (ectoM) du VDEN-1. Testé chez des souris sensibles à la rougeole, ce candidat vaccin s est montré immunogène et capable d induire des anticorps neutralisants sérotype-spécifiques à long terme. La présence de l'ectoM a été déterminante pour l'immunogénicité du EDIII. Sa capacité adjuvante a été corrélée avec sa capacité à induire la maturation des cellules dendritiques et à favoriser la sécrétion de cytokines pro inflammatoires et antivirales ainsi que des chimiokines impliquées dans l'établissement de l'immunité adaptative. Un candidat rougeole-dengue tétravalent a été ensuite construit dans le but d induire la même immunité contre les 4 sérotypes du virus de la dengue. Cette stratégie de vaccination combinée rougeole-dengue permettrait d offrir des vaccins pédiatriques accessibles particulièrement attrayants pour immuniser les enfants simultanément contre la rougeole et la dengue dans des régions du monde où les deux maladies co-existentDengue fever is a reemerging disease that threatens one third of the world's population, for which no vaccine is available. In this work, we evaluated a new vaccination strategy based on the expression of a combined dengue antigen by a vector derived from the pediatric live attenuated measles vaccine. Understanding the mechanisms of flavivirus neutralization by antibodies is essential for the design of innovative vaccine approaches against dengue. Therefore, as a first step, we studied the mechanism of inhibition of fusion by neutralizing antibodies directed against the three domains of the envelope protein (E). The Tick-borne encephalitis virus (TBEV) was used as a model in in vitro fusion tests, and coflottation assays with lipid membranes. The results showed that the neutralizing antibodies can interfere with the early or late stages of the fusion process. This study also shows that antibodies against domain III of the E protein can block both the viral entry and fusion. As a proof-of-concept of our vaccination strategy, we inserted into measles vector the domain III of the glycoprotein E of dengue virus (EDIII), which contains the putative receptor binding site and serotype-specific neutralizing epitopes. To strengthen its immunogenicity, EDIII was fused with the pro-apoptotic ectodomain of the membrane protein (ectoM) of VDEN-1. Tested in mice susceptible to measles, this vaccine candidate was immunogenic and able to induce long term serotype-specific neutralizing antibodies. The presence of the ectoM proved crucial for the immunogenicity of EDIII. Its adjuvant capacity correlated with its ability to mature dendritic cells and to enhance the secretion of proinflammatory and antiviral cytokines, as well as chemokines involved in the development of adaptive immunity. A tetravalent measles-dengue candidate was then generated in order to induce the same immunity against the 4 serotypes of dengue virus. This vaccination strategy combining measles and dengue might offer an affordable pediatric vaccine particularly attractive to immunize children both against measles and dengue fever in areas of the world where the two diseases co-existAIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Recombinant vector derived from live attenuated measles virus: potential for flavivirus vaccines.

International audienceThe 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

Vaccines in Development against West Nile Virus

West Nile encephalitis emerged in 1999 in the United States, then rapidly spread through the North American continent causing severe disease in human and horses. Since then, outbreaks appeared in Europe, and in 2012, the United States experienced a new severe outbreak reporting a total of 5,387 cases of West Nile virus (WNV) disease in humans, including 243 deaths. So far, no human vaccine is available to control new WNV outbreaks and to avoid worldwide spreading. In this review, we discuss the state-of-the-art of West Nile vaccine development and the potential of a novel safe and effective approach based on recombinant live attenuated measles virus (MV) vaccine. MV vaccine is a live attenuated negative-stranded RNA virus proven as one of the safest, most stable and effective human vaccines. We previously described a vector derived from the Schwarz MV vaccine strain that stably expresses antigens from emerging arboviruses, such as dengue, West Nile or chikungunya viruses, and is strongly immunogenic in animal models, even in the presence of MV pre-existing immunity. A single administration of a recombinant MV vaccine expressing the secreted form of WNV envelope glycoprotein elicited protective immunity in mice and non-human primates as early as two weeks after immunization, indicating its potential as a human vaccine

Probing the Flavivirus Membrane Fusion Mechanism by Using Monoclonal Antibodies▿

In this study, we investigated in a flavivirus model (tick-borne encephalitis virus) the mechanisms of fusion inhibition by monoclonal antibodies directed to the different domains of the fusion protein (E) and to different sites within each of the domains by using in vitro fusion assays. Our data indicate that, depending on the location of their binding sites, the monoclonal antibodies impaired early or late stages of the fusion process, by blocking the initial interaction with the target membrane or by interfering with the proper formation of the postfusion structure of E, respectively. These data provide new insights into the mechanisms of flavivirus fusion inhibition by antibodies and their possible contribution to virus neutralization

Measles virus induces oncolysis of mesothelioma cells and allows dendritic cells to cross-prime tumor-specific CD8 response.

International audienceDespite 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

Pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses.

International audienceDengue disease is an increasing global health problem that threatens one-third of the world's population. To control this emerging arbovirus, an efficient preventive vaccine is still needed. Because four serotypes of dengue virus (DV) coexist and antibody-dependent enhanced infection may occur, most strategies developed so far rely on the administration of tetravalent formulations of four live attenuated or chimeric viruses. Here, we evaluated a new strategy based on the expression of a single minimal tetravalent DV antigen by a single replicating viral vector derived from pediatric live-attenuated measles vaccine (MV). We generated a recombinant MV vector expressing a DV construct composed of the four envelope domain III (EDIII) from the four DV serotypes fused with the ectodomain of the membrane protein (ectoM). After two injections in mice susceptible to MV infection, the recombinant vector induced neutralizing antibodies against the four serotypes of dengue virus. When immunized mice were further inoculated with live DV from each serotype, a strong memory neutralizing response was raised against all four serotypes. A combined measles-dengue vaccine might be attractive to immunize infants against both diseases where they co-exist

Preclinical Studies of a Modified Vaccinia Virus Ankara-Based HIV Candidate Vaccine: Antigen Presentation and Antiviral Effect▿

Poxvirus-based human immunodeficiency virus (HIV) vaccine candidates are currently under evaluation in preclinical and clinical trials. Modified vaccinia virus Ankara (MVA) vectors have excellent safety and immunogenicity records, but their behavior in human cell cultures remains only partly characterized. We studied here various virological and immunological aspects of the interactions of MVA-HIV, a vaccine candidate developed by the French National Agency for AIDS Research (ANRS), with primary human cells. We report that MVA-HIV infects and drives Gag expression in primary macrophages, dendritic cells (DCs), and epithelial and muscle cells. MVA-HIV-infected DCs matured, efficiently presented Gag, Pol, and Nef antigens, and activated HIV-specific cytotoxic T lymphocytes (CTLs). As expected with this type of vector, infection was cytopathic and led to DC apoptosis. Coculture of MVA-HIV-infected epithelial cells or myotubes with DCs promoted efficient Gag antigen major histocompatibility complex class I (MHC-I) cross-presentation without inducing direct infection and death of DCs. Antigen-presenting cells (APCs) infected with MVA-HIV also activated HIV-specific CD4+ T cells. Moreover, exposure of DCs to MVA-HIV or to MVA-HIV-infected myotubes induced type I interferon (IFN) production and inhibited subsequent HIV replication and transfer to lymphocytes. Altogether, these results show that MVA-HIV promotes efficient MHC-I and MHC-II presentation of HIV antigens by APCs without facilitating HIV replication. Deciphering the immune responses to MVA in culture experiments will help in the design of innovative vaccine strategies