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In this paper, we investigate the conformational behaviour of new pseudopeptide CCK2 agonists based on the common template R-Trp-NMeNle-Asp-PheNH2 where only the N-terminal substituent R varies. These compounds could be separated into two families according to their pharmacological profile: CCK2A or CCK2B. The conformational space of each compound was explored using molecular dynamics simulations and their structural properties were investigated. Our results suggest the occurrence of two families of conformations, extended and folded. Furthermore, this study brings out a possible correspondence between the CCK2 pharmacological profile and the conformational behaviour of the compounds analysed. A model of preferential conformation is proposed for each compound. This work may provide a key to understand the mechanisms underlying the heterogeneity of CCK2 receptor-associated pharmacological effects. [Copyright &y& Elsevier]

Water pollution is a one of the most contributors to aquatic biodiversity decline. Consequently, ecological risk assessment methods have been developed to investigate the effects of existing stresses on the environment, including the toxic effects of chemicals. One of the existing approaches to quantify toxic risks is called "Potentially Affected Fraction of species" (PAF), which estimates the potential loss of species within a group of species studied. In this study, the PAF method was applied to the Garonne catchment (southwest France) due to the limited information available on the involvement of water pollution in the decline of diadromous fish populations. This approach was used to quantify the potential toxic risk associated with chemical contamination of water for fish species. The objectives were to quantify this risk (1) in the Garonne and Dordogne rivers and (2) in the spawning grounds of two endangered anadromous fish species: the allis shad and the European sturgeon during the development period of their early life stages. Environmental pollution data was provided for 21 sites within the Garonne catchment between 2007 and 2022, and toxicity data was obtained specifically from freshwater toxicity tests on fish species. Then, for each site and each year, the potential toxic risk for a single substance (ssPAF) and for a mixture of substances (msPAF) was calculated and classified as high (>5 %), moderate (>1 % and < 5 %) or low (<1 %). Potential toxic risks were mostly moderate and mainly associated with: metals > other industrial pollutants and hygiene and care products > agrochemicals. In summary, this study highlights the probable involvement of water contamination on the decline, fate and restoration of diadromous fish populations in the Garonne catchment, focusing notably on the toxic effects on early life stages, a previously understudied topic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The cautious optimism following recent anti-amyloid therapeutic trials for Alzheimer's disease (AD) provides a glimmer of hope after years of disappointment. Although these encouraging results represent discernible progress, they also highlight the need to enhance further the still modest clinical efficacy of current disease-modifying immunotherapies. Here, we highlight crucial milestones essential for advancing precision medicine in AD. These include reevaluating the choice of therapeutic targets by considering the key role of both central neuroinflammation and peripheral immunity in disease pathogenesis, refining patient stratification by further defining the inflammatory component within the forthcoming ATN(I) (amyloid, tau and neurodegeneration (and inflammation)) classification of AD biomarkers and defining more accurate clinical outcomes and prognostic biomarkers that better reflect disease heterogeneity. Next-generation immunotherapies will need to go beyond the current antibody-only approach by simultaneously targeting pathological proteins together with innate neuroinflammation and/or peripheral-central immune crosstalk. Such innovative immunomodulatory combination therapy approaches should be evaluated in appropriately redesigned clinical therapeutic trials, which must carefully integrate the neuroimmune component., (© 2024. Springer Nature America, Inc.)

Introduction: Antibody production and the generation of memory B cells are regulated by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells in germinal centers. However, the precise role of Tfr cells in controlling antibody production is still unclear. We have previously shown that both Tfh and Tfr cells express the IL-1R1 agonist receptor, whereas only Tfr cells express the IL-1R2 decoy and IL-1Ra antagonist receptors. We aimed to investigate the role of IL-1 receptors in the regulation of B cell responses by Tfh and Tfr., Methods: We generated mice with IL-1 receptors inactivated in Tfh or Tfr and measured antibody production and cell activation after immunisation., Results: While IL-1β levels are increased in the draining lymph node after immunisation, antigen-specific antibody levels and cell phenotypes indicated that IL-1β can activate both Tfh and Tfr cells through IL-1R1 stimulation. Surprisingly, expression of IL-1R2 and IL-1Ra on Tfr cells does not block IL-1 activation of Tfh cells, but rather prevents IL-1/IL-1R1-mediated early activation of Tfr cells. IL-1Rs also regulate the antibody response to autoantigens and its associated pathophysiology in an experimental lupus model., Discussion: Collectively, our results show that IL-1 inhibitory receptors expressed by Tfr cells prevent their own activation and suppressive function, thus licensing IL-1-mediated activation of Tfh cells after immunisation. Further mechanistic studies should unravel these complex interactions between IL-1β and follicular helper and regulatory T cells and provide new avenues for therapeutic intervention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Belbezier, Engeroff, Fourcade, Vantomme, Vaineau, Gouritin, Bellier, Brocheriou, Tchitchek, Graff-Dubois and Klatzmann.)

Mucosal surfaces serve as the primary entry points for pathogens such as SARS- CoV-2 coronavirus or HIV in the human body. Mucosal vaccination plays a crucial role to successfully induce long-lasting systemic and local immune responses to confer sterilizing immunity. However, antigen formulations and delivery methods must be properly selected since they are decisive for the quality and the magnitude of the elicited immune responses in mucosa. We investigated the significance of using particulate antigen forms for mucosal vaccination by comparing VLP- or protein- based vaccines in a mouse model. Based on a mucosal prime-boost immunization protocol combining (i) HIV- pseudotyped recombinant VLPs (HIV-VLPs) and (ii) plasmid DNA encoding HIV- VLPs (pVLPs), we demonstrated that combination of intranasal primes and intravaginal boosts is optimal to elicit both humoral and cellular memory responses in mucosa. Interestingly, our results show that in contrast to proteins, particulate antigens induce high-quality humoral responses characterized by a high breadth, long-term neutralizing activity and cross-clade reactivity, accompanying with high T follicular helper cell (T FH ) response. These results underscore the potential of a VLP-based vaccine in effectively instigating long-lasting, HIV-specific immunity and point out the specific role of particulate antigen form in driving high-quality mucosal immune responses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

An ideal protective vaccine against SARS-CoV-2 should not only be effective in preventing disease, but also in preventing virus transmission. It should also be well accepted by the population and have a simple logistic chain. To fulfill these criteria, we developed a thermostable, orally administered vaccine that can induce a robust mucosal neutralizing immune response. We used our platform based on retrovirus-derived enveloped virus-like particles (eVLPs) harnessed with variable surface proteins (VSPs) from the intestinal parasite Giardia lamblia , affording them resistance to degradation and the triggering of robust mucosal cellular and antibody immune responses after oral administration. We made eVLPs expressing various forms of the SARS-CoV-2 Spike protein (S), with or without membrane protein (M) expression. We found that prime-boost administration of VSP-decorated eVLPs expressing a pre-fusion stabilized form of S and M triggers robust mucosal responses against SARS-CoV-2 in mice and hamsters, which translate into complete protection from a viral challenge. Moreover, they dramatically boosted the IgA mucosal response of intramuscularly injected vaccines. We conclude that our thermostable orally administered eVLP vaccine could be a valuable addition to the current arsenal against SARS-CoV-2, in a stand-alone prime-boost vaccination strategy or as a boost for existing vaccines., Competing Interests: HL and DK are inventors of a patent application claiming orally administered vaccines against coronaviruses that is owned by their public institutions. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bellier, Saura, Luján, Molina, Luján and Klatzmann.)

Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.

Retrovirus-derived virus-like particles (VLPs) are particularly interesting vaccine platforms, as they trigger efficient humoral and cellular immune responses and can be used to display heterologous antigens. In this study, we characterized the intrinsic immunogenicity of VLPs and investigated their possible adjuvantization by incorporation of Toll-like receptor (TLR) ligands. We designed a noncoding single-stranded RNA (ncRNA) that could be encapsidated by VLPs and induce TLR7/8 signaling. We found that VLPs efficiently induce in vitro dendritic cell activation, which can be improved by ncRNA encapsidation ( ncRNA VLPs). Transcriptome studies of dendritic cells harvested from the spleens of immunized mice identified antigen presentation and immune activation as the main gene expression signatures induced by VLPs, while TLR signaling and Th1 signatures characterize ncRNA VLPs. In vivo and compared with standard VLPs, ncRNA VLPs promoted Th1 responses and improved CD8 + T cell proliferation in a MyD88-dependent manner. In an HIV vaccine mouse model, HIV-pseudotyped ncRNA VLPs elicited stronger antigen-specific cellular and humoral responses than VLPs. Altogether, our findings provide molecular evidence for a strong vaccine potential of retrovirus-derived VLPs that can be further improved by harnessing TLR-mediated immune activation. IMPORTANCE We previously reported that DNA vaccines encoding antigens displayed in/on retroviral VLPs are more efficient than standard DNA vaccines at inducing cellular and humoral immune responses. We aimed to decipher the mechanisms and investigated the VLPs' immunogenicity independently of DNA vaccination. We show that VLPs have the ability to activate antigen-presenting cells directly, thus confirming their intrinsic immunostimulatory properties and their potential to be used as an antigenic platform. Notably, this immunogenicity can be further improved and/or oriented by the incorporation into VLPs of ncRNA, which provides further TLR-mediated activation and Th1-type CD4 + and CD8 + T cell response orientation. Our results highlight the versatility of retrovirus-derived VLP design and the value of using ncRNA as an intrinsic vaccine adjuvant., (Copyright © 2017 American Society for Microbiology.)

Background & Aims: Interferon-free direct-acting antiviral (DAA) therapies are effective in patients with hepatitis C virus-induced cryoglobulinemia vasculitis (HCV-CV). We analyzed blood samples from patients with HCV-CV before and after DAA therapy to determine mechanisms of these drugs and their effects on cellular immunity., Methods: We performed a prospective study of 27 consecutive patients with HCV-CV (median age, 59 y) treated with DAA therapy (21 patients received sofosbuvir plus ribavirin for 24 weeks, 4 patients received sofosbuvir plus daclatasvir for 12 weeks, and 2 patients received sofosbuvir plus simeprevir for 12 weeks) in Paris, France. Blood samples were collected from these patients before and after DAA therapy, and also from 12 healthy donors and 12 individuals with HCV infection without CV. HCV load, cryoglobulins, and cytokines were quantified by flow cytometry, cytokine multiplex assays, and enzyme-linked immunosorbent assay., Results: Twenty-four patients (88.9%) had a complete clinical response of CV to DAA therapy at week 24, defined by improvement of all the affected organs and the absence of relapse. Compared with healthy donors and patients with HCV infection without CV, patients with HCV-CV, before DAA therapy, had a lower percentage of CD4+CD25hiFoxP3+ regulatory T cells (P < .01), but higher proportions of IgM+CD21-/low memory B cells (P < .05), CD4+IFNγ+ cells (P < .01), CD4+IL17A+ cells (P < .01), and CD4+CXCR5+interleukin 21+ follicular T-helper (Tfh) cells (P < .01). In patients with HCV-CV, there was a negative correlation between numbers of IgM+CD21-/low memory B cells and T-regulatory cells (P = .03), and positive correlations with numbers of Tfh cells (P = .03) and serum levels of cryoglobulin (P = .01). DAA therapy increased patients' numbers of T-regulatory cells (1.5% ± 0.18% before therapy vs 2.1% ± 0.18% after therapy), decreased percentages of IgM+CD21-/low memory B cells (35.7% ± 6.1% before therapy vs 14.9% ± 3.8% after therapy), and decreased numbers of Tfh cells (12% ± 1.3% before therapy vs 8% ± 0.9% after therapy). Expression levels of B lymphocyte stimulator receptor 3 and programmed cell death 1 on B cells increased in patients with HCV-CV after DAA-based therapy (mean fluorescence units, 37 ± 2.4 before therapy vs 47 ± 2.6 after therapy, P < .01; and 29 ± 7.3 before therapy vs 48 ± 9.3 after therapy, P < .05, respectively)., Conclusions: In a prospective clinical trial of patients with HCV-CV, DAA-based therapy restored disturbances in peripheral B- and T-cell homeostasis., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Despite current therapy, head and neck squamous cell carcinomas (HNSCCs) arising from various mucosal sites of the upper aero-digestive tract frequently relapse in a loco-regional manner and have a poor prognosis. Our objective was to validate an innovative mucosal route of vaccination using plasmo virus-like particles (pVLPs) in a pre-clinical orthotopic model of HNSCCs. For this purpose, we used pVLP-E7, that are plasmid DNA encoding retroviral virus-like particles carrying a truncated E7 oncoprotein from HPV-16 as antigen model, to vaccinate mice bearing pre-established TC-1 tumors implanted into the buccal mucosa. pVLP-E7 were combined with clinical grade TLR agonists (Imiquimod and CpG-ODN). In this pre-clinical orthotopic model, whose tumor microenvironment resembles to those of human HNSCCs, different mucosal vaccination routes were tested for their ability to elicit efficient immune and antitumoral responses. Results showed that mucosal intra-cheek (IC) vaccinations using pVLP-E7, comparatively to intradermic vaccinations (ID), gave rise to higher mobilization of mucosal (CD49a(+)) CD8(+) specific effector T cells in both tumor draining lymph nodes (TdLNs) and tumor microenvironment resulting in better antitumor effects and in a long-term protection against tumor rechallenge. In vivo CD8(+) depletion demonstrated that antitumoral effects were fully dependent upon the presence of CD8(+) T cells. Validation of IC mucosal vaccinations with pVLPs combined with adjuvants using a pre-clinical orthotopic model of HNSCC provides valuable pre-clinical data to rapidly envision the use of such therapeutic vaccines in patients with HNSCCs, inasmuch as vaccinal components and adjuvants can be easily obtained as clinical grade reagents.

Regulatory T cells (Tregs) are pivotal for maintenance of immune self-tolerance and also regulate immune responses to exogenous Ags, including allergens. Both decreased Treg number and function have been reported in allergic patients, offering new therapeutic perspectives. We previously demonstrated that Tregs can be selectively expanded and activated by low doses of IL-2 (ld-IL-2) inducing immunoregulation without immunosuppression and established its protective effect in autoimmune diseases. In this study, we evaluated the ability of ld-IL-2 to control allergy in an experimental model of food allergy. Ld-IL-2 induced Treg expansion and activation that elicited protection against clinical manifestations of food allergy in two mouse models with OVA and peanut. This clinical effect was lost in Treg-depleted mice, demonstrating the major contribution of Tregs in ld-IL-2 efficacy. Mechanistic studies further indicated that protection from allergy could be explained by a Treg-dependent local modification of the Th1/Th2 balance and an inhibition of mast cell recruitment and activation. Preventive and therapeutic effects of ld-IL-2 were observed over a 7-mo-period, highlighting its long-term efficacy. This study demonstrated that ld-IL-2 is efficient to prevent and to treat allergic immune responses, and thus represents a promising therapeutic strategy for managing allergic diseases., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Tregs imprint an early immunotolerant tumor environment that prevents effective antitumor immune responses. Using transcriptomics of tumor tissues, we identified early upregulation of VEGF and TGF-β pathways compatible with tolerance imprinting. Silencing of VEGF or TGF-β in tumor cells induced early and pleiotropic modulation of immune-related transcriptome signatures in tumor tissues. These were surprisingly similar for both silenced tumors and related to common downstream effects on Tregs. Silencing of VEGF or TGF-β resulted in dramatically delayed tumor growth, associated with decreased Tregs and myeloid-derived suppressor cells and increased effector T cell activation in tumor infiltrates. Strikingly, co-silencing of TGF-β and VEGF led to a substantial spontaneous tumor eradication rate and the combination of their respective inhibitory drugs was synergistic. VEGF and/or TGF-β silencing also restored tumor sensitivity to tumor-specific cell therapies and markedly improved the efficacy of anti-PD-1/anti-CTLA-4 treatment. Thus, TGF-β and VEGF cooperatively control the tolerant environment of tumors and are targets for improved cancer immunotherapies.

Systems biology offers promising approaches for identifying response-specific signatures to vaccination and assessing their predictive value. Here, we designed a modelling strategy aiming to predict the quality of late T-cell responses after vaccination from early transcriptome analysis of dendritic cells. Using standardized staining with tetramer, we first quantified antigen-specific T-cell expansion 5 to 10 days after vaccination with one of a set of 41 different vaccine vectors all expressing the same antigen. Hierarchical clustering of the responses defined sets of high and low T cell response inducers. We then compared these responses with the transcriptome of splenic dendritic cells obtained 6 hours after vaccination with the same vectors and produced a random forest model capable of predicting the quality of the later antigen-specific T-cell expansion. The model also successfully predicted vector classification as low or strong T-cell response inducers of a novel set of vaccine vectors, based on the early transcriptome results obtained from spleen dendritic cells, whole spleen and even peripheral blood mononuclear cells. Finally, our model developed with mouse datasets also accurately predicted vaccine efficacy from literature-mined human datasets.

To further investigate the contribution of intercellular adhesion molecule-1 (ICAM-1) to adaptive immune responses, we analysed T-cell development and function in mice lacking full-length ICAM-1 (ICAM-1(tm1Jcgr) ). Compared with wild-type (ICAM-1(WT) ) mice, ICAM-1(tm1Jcgr) mice have impaired thymocyte development. Proportions and numbers of double negative, double positive, mature CD4(+) and CD8(+) thymocytes, as well as of regulatory T (Treg) cells were also significantly decreased. In the periphery, ICAM-1(tm1Jcgr) mice had significantly decreased proportions and numbers of naive and activated/memory CD4(+) and CD8(+) T cells, as well as of Treg cells, in lymph nodes but not in the spleen. In vitro activation of CD4(+) and CD8(+) T cells from ICAM-1(tm1Jcgr) mice with anti-CD3 antibodies and antigen-presenting cells (APCs) resulted in a significantly weaker proliferation, whereas proliferation induced with anti-CD3 and anti-CD28 antibody-coated beads was normal. In vivo immunization of ICAM-1(tm1Jcgr) mice resulted in normal generation of specific effector and memory immune responses that protect against a viral challenge. However, contrary to ICAM-1(WT) mice, immunization-induced specific effectors could not eradicate immunogen-expressing tumours. Treg cells from ICAM-1(tm1Jcgr) mice have abnormal activation and proliferation induced by anti-CD3 antibody and APCs, and have markedly decreased suppressive activity in vitro. In contrast to ICAM-1(WT) mice, they were unable to control experimentally induced colitis in vivo. Hence, our results further highlight the pleiotropic role of ICAM-1 in T-cell-dependent immune responses, with a major role in Treg cell development and suppressive function., (© 2015 John Wiley & Sons Ltd.)

In addition to CD4(+) regulatory T cells (Tregs), CD8(+) suppressor T cells are emerging as an important subset of regulatory T cells. Diverse populations of CD8(+) T cells with suppressive activities have been described. Among them, a small population of CD8(+)CD25(+)FOXP3(+) T cells is found both in mice and humans. In contrast to thymic-derived CD4(+)CD25(+)FOXP3(+) Tregs, their origin and their role in the pathophysiology of autoimmune diseases (AIDs) are less understood. We report here the number, phenotype, and function of CD8(+) Tregs cells in mice and humans, at the steady state and in response to low-dose interleukin-2 (IL-2). CD8(+) Tregs represent approximately 0.4 and 0.1% of peripheral blood T cells in healthy humans and mice, respectively. In mice, their frequencies are quite similar in lymph nodes (LNs) and the spleen, but two to threefold higher in Peyer patches and mesenteric LNs. CD8(+) Tregs express low levels of CD127. CD8(+) Tregs express more activation or proliferation markers such as CTLA-4, ICOS, and Ki-67 than other CD8(+) T cells. In vitro, they suppress effector T cell proliferation as well as or even better than CD4(+) Tregs. Owing to constitutive expression of CD25, CD8(+) Tregs are 20- to 40-fold more sensitive to in vitro IL-2 stimulation than CD8(+) effector T cells, but 2-4 times less than CD4(+) Tregs. Nevertheless, low-dose IL-2 dramatically expands and activates CD8(+) Tregs even more than CD4(+) Tregs, in mice and humans. Further studies are warranted to fully appreciate the clinical relevance of CD8(+) Tregs in AIDs and the efficacy of IL-2 treatment.

The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA) antigen to EVs: (a) by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin), which is exposed on the surface of secreted membrane vesicles; and (b) by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs). We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8(+) T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4(+) T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases.

Interleukin 2 (IL2) is the key cytokine supporting survival and function of regulatory T cells (Tregs). We recently reported that low-dose IL2 safely expands/stimulates Tregs and improves autoimmune conditions in humans. Further development of IL2 in autoimmune diseases will require chronic IL2 administration, which could affect beneficial effector immune responses regulated by Tregs. We used recombinant adeno-associated viral vector (rAAV)-mediated gene transfer to continuously release IL2 in mice and assessed its long-term effects on immune responses. A single rAAV-IL2 injection enabled sustained stimulation and expansion of Tregs without inducing Teff activation and prevented diabetes in NOD mice. After several weeks of IL2 production, mice responded normally to a viral challenge and to vaccination, and had pregnancies with offspring that developed normally. They showed no change in the occurrence and growth of chemically-induced tumors. Altogether, chronic low-dose IL2 treatment does not affect beneficial effector immune responses at doses that prevent autoimmune diabetes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Microarray analysis often leads to either too large or too small numbers of gene candidates to allow meaningful identification of functional signatures. We aimed at overcoming this hurdle by combining two algorithms: i. Independent Component Analysis to extract statistically-based potential signatures. ii. Gene Set Enrichment Analysis to produce a score of enrichment with statistical significance of each potential signature. We have applied this strategy to identify regulatory T cell (Treg) molecular signatures from two experiments in mice, with cross-validation. These signatures can detect the -1% Treg in whole spleen. These findings demonstrate the relevance of our approach as a signature discovery tool.

Human papillomavirus (HPV) is involved in the development of anogenital tumors and also in the development of oropharyngeal head and neck carcinomas, where HPV-16, expressing the E6 and E7 oncoproteins, is the most frequent serotype. Although vaccines encoding L1 and L2 capsid HPV proteins are efficient for the prevention of HPV infection, they are inadequate for treating established tumors. Hence, development of innovative vaccine therapies targeting E6/E7 is important for controlling HPV-induced cancers. We have engineered a nononcogenic mutated E7-specific plasmo-retroVLP vaccine (pVLP-E7), consisting of plasmid DNA, that is able to form recombinant retrovirus-based virus-like particles (VLPs) that display E7 antigen into murine leukemia virus Gag proteins pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G). pVLP-E7 vaccinations were studied for their ability to generate specific immune responses and for induction of protective immunity against tumor cell challenge in preventive and therapeutic models. The produced VLPs induce the maturation of human dendritic cells in vitro and mount specific E7 T cell responses. Intradermic vaccinations of mice with pVLP-E7 show their efficacy to generate antigen-specific T cell responses, to prevent and protect animals from early TC-1 tumor development compared with standard DNA or VLP immunizations. The vaccine efficacy was also evaluated for advanced tumors in mice vaccinated at various time after the injection of TC-1 cells. Data show that pVLP-E7 vaccination can cure mice with already established tumors only when combined with Toll-like receptor-7 (TLR7) and TLR9 agonists. Our findings provide evidence that pVLPs, combining the advantages of DNA and VLP vaccines, appear to be a promising strategy for the treatment of HPV-induced cancers.

While the immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood, it is now admitted that an effective vaccine against HCV will need to induce both cellular and humoral immune responses and address viral heterogeneity to prevent immune escape. We developed a vaccine platform specifically aimed at inducing such responses against HCV antigens displayed by recombinant retrovirus-based virus-like particles (VLPs) made of Gag of murine leukemia virus. Both ex vivo produced VLPs and plasmid DNA encoding VLPs can be used as vaccines. Here, we report that immunizations with plasmid DNA forming VLPs pseudotyped with HCV E1 and E2 envelope glycoproteins (HCV-specific plasmo-retroVLPs) induce strong T-cell-mediated immune responses that can be optimized by using proper DNA delivery methods and/or genetic adjuvants. Additionally, multigenotype or multi-specific T-cell responses were observed after immunization with plasmids that encode VLPs pseudotyped with E1E2 derived from numerous viral genotypes and/or displaying NS3 antigen in capsid proteins. While homologous prime-boost immunizations with HCV-specific plasmo-retroVLPs or ex vivo produced VLPs induce a low level of specific antibody responses, optimal combination of plasmo-retroVLPs and VLPs was identified for inducing HCV-specific T-cell and B-cell responses as well as neutralizing antibodies. Altogether, these results have important meanings for the development of anti-HCV preventive vaccines and exemplify the flexibility and potential of our retrovirus-based platform in inducing broad cellular and humoral immune responses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

HCV infection is a worldwide health problem and much effort is being made to develop novel therapies. New vaccines are designed for preventive and therapeutic use through induction of robust immunity, including neutralizing antibodies and T-cell-mediated immunity. Novel future vaccine approaches include virus-like particle (VLP)-based vaccines that have been successfully employed to prevent infections by hepatitis B virus or human papillomavirus. The HCV-derived VLP approach simplifies the delivery of neutralizing antibody- and core-specific T-cell epitopes in a highly immunogenic single construct resembling mature HCV virions. The size, particulate nature and dense, repetitive structure of VLPs are the basis for their innate immunogenicity. Consequently, VLPs have also been exploited as antigenic platforms. Association of HCV antigens with heterologous structural viral proteins able to form recombinant VLPs (HBsAg, HBcAg, MLV Gag, PapMV coat protein) is also a promising approach for induction of HCV-specific immune responses. This article summarizes the different VLP-based vaccine approaches that are currently under development.

Vaccines are the most effective tools to prevent infectious diseases and to minimize their impact on humans or animals. Despite the successful development of vaccines that are able to elicit potent and protective immune responses, the majority of vaccines have been so far developed empirically and mechanistic events leading to protective immune responses are often poorly understood. This hampers the development of new prophylactic as well as therapeutic vaccines for infectious diseases and cancer. Biological correlates of immune-mediated protection are currently based on standard readout such as antibody titres and ELISPOT assays. The development of successful vaccines for difficult settings, such as infectious agents leading to chronic infection (HIV, HCV...) or cancer, calls for novel 'readout systems' or 'correlates' of immune-mediated protection that would reliably predict immune responses to novel vaccines in vivo. Systems biology offers a new approach to vaccine design that is based upon understanding the molecular network mobilized by vaccination. Systems vaccinology approaches investigate more global correlates of successful vaccination, beyond the specific immune response to the antigens administered, providing new methods for measuring early vaccine efficacy and ultimately generating hypotheses for understanding the mechanisms that underlie successful immunogenicity. Using functional genomics, specific molecular signatures of individual vaccine can be identified and used as predictors of vaccination efficiency. The immune response to vaccination involves the coordinated induction of master transcription factors that leads to the development of a broad, polyfunctional and persistent immune response integrating all effector cells of the immune systems., (© 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Chronic hepatitis C virus (HCV) infection, with its cohort of life-threatening complications, affects more than 200 million persons worldwide and has a prevalence of more than 10% in certain countries. Preventive and therapeutic vaccines against HCV are thus much needed. Neutralizing antibodies (NAbs) are the foundation for successful disease prevention for most established vaccines. However, for viruses that cause chronic infection such as HIV or HCV, induction of broad NAbs from recombinant vaccines has remained elusive. We developed a vaccine platform specifically aimed at inducing NAbs based on pseudotyped virus-like particles (VLPs) made with retroviral Gag. We report that VLPs pseudotyped with E2 and/or E1 HCV envelope glycoproteins induced high-titer anti-E2 and/or anti-E1 antibodies, as well as NAbs, in both mouse and macaque. The NAbs, which were raised against HCV 1a, cross-neutralized the five other genotypes tested (1b, 2a, 2b, 4, and 5). Thus, the described VLP platform, which can be pseudotyped with a vast array of virus envelope glycoproteins, represents a new approach to viral vaccine development.

The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.

H5N1 avian influenza virus has caused widespread infection in poultry and wild birds, and has the potential to emerge as a pandemic threat to humans. The hemagglutinin (HA) is a glycoprotein on the surface of the virus envelope. Understanding its antigenic structure is essential for designing novel vaccines that can inhibit virus infection. The aim of this study was to map the amino acid substitutions that resulted in resistance to neutralization by monoclonal antibodies (MAbs) of the highly pathogenic A/crested eagle/Belgium/01/2004 (H5N1), a clade 1 virus. Two hybridomas specific to H5N1 clade 1 viruses were selected by enzyme-linked immunosorbent assay, virus neutralization test, and immunofluorescence assay. Escape mutant populations resisting neutralization by those MAbs (8C5 and 5A1) were then selected, and sequencing of these mutants allowed the prediction of the HA protein structure by molecular homology. We could detect an amino acid change in our escape mutants at position K189E corresponding to antigenic site 2 of H5 HA1 and site B of H3 HA1. Interestingly, 336 out of 350 available HA sequences from H5N1 clade 1 and clade 2.3 viruses had Lys (K) at position 189 in the HA1, whereas HA sequences analyzed from dade 2.1 and 2.2 viruses had Arg (R). This residue also interacts with the receptor-binding site, and it is thus important for the evolution of H5N1 viruses. An additional substitution K29E in HA2 subunit was also observed and identified with the use of NetChop software as a loss of a proteasomal cleavage site, which seems to be an advantage for H5N1 viruses.