Claude Ruffié

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

Dengue disease is an increasing global health problem that threatens one-third of the worlds 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.

A recombinant measles vaccine expressing chikungunya virus-like particles is strongly immunogenic and protects mice from lethal challenge with chikungunya virus

Chikungunya virus (CHIKV), a mosquito-transmitted alphavirus, recently reemerged in the Indian Ocean, India and Southeast Asia, causing millions of cases of severe polyarthralgia. No specific treatment to prevent disease or vaccine to limit epidemics is currently available. Here we describe a recombinant live-attenuated measles vaccine (MV) expressing CHIKV virus-like particles comprising capsid and envelope structural proteins from the recent CHIKV strain La Reunion. Immunization of mice susceptible to measles virus induced high titers of CHIKV antibodies that neutralized several primary isolates. Specific cellular immune responses were also elicited. A single immunization with this vaccine candidate protected all mice from a lethal CHIKV challenge, and passive transfer of immune sera conferred protection to naïve mice. Measles vaccine is one of the safest and most effective human vaccines. A recombinant MV-CHIKV virus could make a safe and effective vaccine against chikungunya that deserves to be further tested in human trials.

Live attenuated measles vaccine expressing HIV-1 Gag virus like particles covered with gp160ΔV1V2 is strongly immunogenic

Although a live attenuated HIV vaccine is not currently considered for safety reasons, a strategy inducing both T cells and neutralizing antibodies to native assembled HIV-1 particles expressed by a replicating virus might mimic the advantageous characteristics of live attenuated vaccine. To this aim, we generated a live attenuated recombinant measles vaccine expressing HIV-1 Gag virus-like particles (VLPs) covered with gp160DeltaV1V2 Env protein. The measles-HIV virus replicated efficiently in cell culture and induced the intense budding of HIV particles covered with Env. In mice sensitive to MV infection, this recombinant vaccine stimulated high levels of cellular and humoral immunity to both MV and HIV with neutralizing activity. The measles-HIV virus infected human professional antigen-presenting cells, such as dendritic cells and B cells, and induced efficient presentation of HIV-1 epitopes and subsequent activation of human HIV-1 Gag-specific T cell clones. This candidate vaccine will be next tested in non-human primates. As a pediatric vaccine, it might protect children and adolescents simultaneously from measles and HIV.

Persistence of bronchopulmonary hyper-reactivity and eosinophilic lung inflammation after anti-IL-5 or -IL-13 treatment in allergic BALB/c and IL-4Rα knockout mice

Background Antigen‐induced bronchopulmonary hyper‐reactivity (BHR) is generally associated with eosinophilia. It involves cytokines produced by Th2 lymphocytes, including IL‐4, IL‐5 and IL‐13, which are implicated in IgE production, eosinophil differentiation and attraction, and related events relevant to allergic inflammation, whose mechanisms remain unclear.

Interference of cetirizine with the late eosinophil accumulation induced by either PAF or compound 48/80

1 The effect of topical or systemic treatment with the histamine H1‐receptor antagonist, cetirizine, on the rat pleural eosinophil accumulation induced by PAF or compound 48/80 was investigated. The number of pleural resident eosinophils increased 6 h after the intrathoracic (i.t.) injection of PAF (1 μg/cavity), peaked within 24 h and persisted significantly augmented for at least 96 h. Compound 48/80 (25 μg/cavity) also produced a long lasting pleural eosinophilia but this was first noted only 24 h after stimulation. 2 Intraperitoneal pretreatment with cetirizine inhibited eosinophilia induced by either PAF (ED50 = 19 mg kg−1) or compound 48/80 (ED50 = 14 mg kg−1) whereas meclizine, another histamine H1‐receptor antagonist, was inactive. 3 Administered locally, cetirizine (5 and 15 μg/cavity) also dose‐dependently inhibited both PAF‐ and compound 48/80‐induced eosinophilia, providing evidence that its inhibitory effect is not due to any action upon circulating eosinophils. Consistent with this result, incubation of isolated peritoneal eosinophils with cetirizine failed to modify in vitro eosinophil migration caused by PAF. 4 Since the late eosinophilia induced by PAF may depend on the synthesis of a transferable protein mediator, cetirizine was administered to donor or recipient rats in order to determine whether it interferes with the generation or with the expression of this protein. We showed that only the treatment of recipient rats abolished the transfer of the eosinophilotactic activity, indicating that cetirizine does not modify the generation but inhibits the expression of this activity. 5 Our findings indicate that cetirizine is able to inhibit eosinophil accumulation by acting locally. The mechanism is neither related to its recognized ability to antagonize histamine H1‐receptors nor to a direct action upon circulating eosinophils.

A Role for Bid in Eosinophil Apoptosis and in Allergic Airway Reaction

Bid, a proapoptotic member of Bcl-2 family, is involved in Fas receptor signaling. Fas activation promotes human eosinophil cell death and is believed to accelerate the resolution of pulmonary Th2-driven allergic reaction in mice. We hypothesized that Bid would regulate eosinophil apoptosis and Ag-induced airway inflammation, particularly eosinophilia. C57BL/6 Bid−/− and wild-type mice were immunized and repeatedly challenged with OVA, and bronchoalveolar lavage (BAL) fluid, lung, and spleen were collected 4–240 h after the final challenge. Cultured BAL eosinophils from Bid-deficient mice showed resistance to Fas-mediated apoptotic DNA fragmentation, phosphatidylserine exposure, mitochondria depolarization, and caspase-3 activity. In addition, OVA-challenged Bid−/− mice had higher BAL eosinophilia and a lower proportion of BAL apoptotic eosinophils than Bid+/+ mice. This was accompanied by augmented BAL levels of the eosinophilotactic cytokine, IL-5, and of the eosinophil-associated mediators, TGF-β1 and fibronectin. Finally, cultured OVA-stimulated lung mononuclear cells and splenocytes from Bid-deficient mice showed increased release of the Th2-type cytokines, IL-4 and IL-5, but no change in cell number. We conclude that Bid modulates BAL eosinophilia by regulating both eosinophil apoptosis and Th2-type cytokine production.

Whole Pichia pastoris Yeast Expressing Measles Virus Nucleoprotein as a Production and Delivery System to Multimerize Plasmodium Antigens

Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening and possibly for large-scale production, distribution and delivery of a malaria vaccine in developing countries.

Cell-associated and soluble phospholipases A2 increase during carrageenan and zymosan-induced pleurisy in rat

Extra-cellular and cell-associated Ca(2+)-dependent phospholipases A2 and released thromboxane B2 were correlated to exudation and cell migration during rat pleurisy induced by carrageenan or zymosan. Extra-cellular phospholipase A2 was delayed with respect to acute inflammation, while cell-associated phospholipase A2 closely correlated with cell migration and thromboxane B2 levels. This confirms that the subcellular localization of phospholipases A2 is linked to their physiological action and, in particular, suggests that the cell-associated, rather than the extracellular enzyme, accounts for the production of eicosanoids.

Identification of a small molecule that primes the type I interferon response to cytosolic DNA

The type I interferon response plays a pivotal role in host defense against infectious agents and tumors, and promising therapeutic approaches rely on small molecules designed to boost this system. To identify such compounds, we developed a high-throughput screening assay based on HEK-293 cells expressing luciferase under the control of Interferon-Stimulated Response Elements (ISRE). An original library of 10,000 synthetic compounds was screened, and we identified a series of 1H-benzimidazole-4-carboxamide compounds inducing the ISRE promoter sequence, specific cellular Interferon-Stimulated Genes (ISGs), and the phosphorylation of Interferon Regulatory Factor (IRF) 3. ISRE induction by ChX710, a prototypical member of this chemical series, was dependent on the adaptor MAVS and IRF1, but was IRF3 independent. Although it was unable to trigger type I IFN secretion per se, ChX710 efficiently primed cellular response to transfected plasmid DNA as assessed by potent synergistic effects on IFN-β secretion and ISG expression levels. This cellular response was dependent on STING, a key adaptor involved in the sensing of cytosolic DNA and immune activation by various pathogens, stress signals and tumorigenesis. Our results demonstrate that cellular response to cytosolic DNA can be boosted with a small molecule, and potential applications in antimicrobial and cancer therapies are discussed.

OA021-02. Replicating measles-SHIV vaccine induces long term preservation of central memory CD4 cells in the gut of vaccinated macaques challenged with SHIV

Background Live attenuated vaccines are mostly appropriate for global mass immunization, controlling very efficiently global pandemics like polio or measles. Although a live attenuated HIV vaccine is not currently considered for safety reasons, a strategy based on the expression of HIV-1 particles through a live replicating viral vector might mimic the advantageous characteristics of live attenuated SIV.