Intranasal administration of octavalent next-generation influenza vaccine elicits protective immune responses against seasonal and pre-pandemic viruses.

Development of next-generation influenza virus vaccines is crucial to improve protection against circulating and emerging viruses. Current vaccine formulations have to be updated annually due to mutations in seasonal strains and do not offer protection against strains with pandemic potential. Computationally optimized broadly reactive antigen (COBRA) methodology has been utilized by our group to generate broadly reactive immunogens for individual influenza subtypes, which elicit protective immune responses against a broad range of strains over numerous seasons. Octavalent mixtures of COBRA hemagglutinin (HA) (H1, H2, H3, H5, H7, and influenza B virus) plus neuraminidase (NA) (N1 and N2) recombinant proteins mixed with c-di-AMP adjuvant were administered intranasally to naive or pre-immune ferrets in prime-boost fashion. Four weeks after final vaccination, collected sera were analyzed for breadth of antibody response, and the animals were challenged with seasonal or pre-pandemic strains. The octavalent COBRA vaccine elicited antibodies that recognized a broad panel of strains representing different subtypes, and these vaccinated animals were protected against influenza virus challenges. Overall, this study demonstrated that the mixture of eight COBRA HA/NA proteins mixed with an intranasal adjuvant is a promising candidate for a universal influenza vaccine. IMPORTANCE: Influenza is a respiratory virus which infects around a billion people globally every year, with millions experiencing severe illness. Commercial vaccine efficacy varies year to year and can be low due to mismatch of circulating virus strains. Thus, the formulation of current vaccines has to be adapted accordingly every year. The development of a broadly reactive influenza vaccine would lessen the global economic and public health burden caused by the different types of influenza viruses. The significance of our research is producing a promising universal vaccine candidate which provides protection against a wider range of virus strains over a wider range of time.

Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. All three itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.

Correction: Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

[This corrects the article DOI: 10.1371/journal.ppat.1010219.].

Diversification of a Novel α-Galactosyl Ceramide Hotspot Boosts the Adjuvant Properties in Parenteral and Mucosal Vaccines.

The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α-galactosyl ceramide (α-GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B-cell activation. Herein, we introduce a novel derivatization hotspot at the α-GalCer skeleton, namely the N-substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self-adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen-specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α-GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α-GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.

Coatsome-replicon vehicles: Self-replicating RNA vaccines against infectious diseases.

Herein, we provide the first description of a synthetic delivery method for self-replicating replicon RNAs (RepRNA) derived from classical swine fever virus (CSFV) using a Coatsome-replicon vehicle based on Coatsome® SS technologies. This results in an unprecedented efficacy when compared to well-established polyplexes, with up to ∼65 fold-increase of the synthesis of RepRNA-encoded gene of interest (GOI). We demonstrated the efficacy of such Coatsome-replicon vehicles for RepRNA-mediated induction of CD8 T-cell responses in mice. Moreover, we provide new insights on physical properties of the RepRNA, showing that the removal of all CSFV structural protein genes has a positive effect on the translation of the GOI. Finally, we successfully engineered RepRNA constructs encoding a porcine reproductive and respiratory syndrome virus (PRRSV) antigen, providing an example of antigen expression with potential application to combat viral diseases. The versatility and simplicity of modifying and manufacturing these Coatsome-replicon vehicle formulations represents a major asset to tackle foreseeable emerging pandemics.

Interdependencies between Toll-like receptors in Leishmania infection.

Multiple pathogen-associated molecular patterns (PAMPs) on a pathogen's surface imply their simultaneous recognition by the host cell membrane-located multiple PAMP-specific Toll-like receptors (TLRs). The TLRs on endosomes recognize internalized pathogen-derived nucleic acids and trigger anti-pathogen immune responses aimed at eliminating the intracellular pathogen. Whether the TLRs influence each other's expression and effector responses-termed TLR interdependency-remains unknown. Herein, we first probed the existence of TLR interdependencies and next determined how targeting TLR interdependencies might determine the outcome of Leishmania infection. We observed that TLRs selectively altered expression of their own and of other TLRs revealing novel TLR interdependencies. Leishmania major-an intra-macrophage parasite inflicting the disease cutaneous leishmaniasis in 88 countries-altered this TLR interdependency unfolding a unique immune evasion mechanism. We targeted this TLR interdependency by selective silencing of rationally chosen TLRs and by stimulation with selective TLR ligands working out a novel phase-specific treatment regimen. Targeting the TLR interdependency elicited a host-protective anti-leishmanial immune response and reduced parasite burden. To test whether this observation could be used as a scientific rationale for treating a potentially fatal L. donovani infection, which causes visceral leishmaniasis, we targeted the inter-TLR dependency adopting the same treatment regimen. We observed reduced splenic Leishman-Donovan units accompanied by host-protective immune response in susceptible BALB/c mice. The TLR interdependency optimizes TLR-induced immune response by a novel immunoregulatory framework and scientifically rationalizes targeting TLRs in tandem and in sequence for redirecting immune responses against an intracellular pathogen.

Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge.

Cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.

Role of Autophagy in Von Willebrand Factor Secretion by Endothelial Cells and in the In Vivo Thrombin-Antithrombin Complex Formation Promoted by the HIV-1 Matrix Protein p17.

Although the advent of combined antiretroviral therapy has substantially improved the survival of HIV-1-infected individuals, non-AIDS-related diseases are becoming increasingly prevalent in HIV-1-infected patients. Persistent abnormalities in coagulation appear to contribute to excess risk for a broad spectrum of non-AIDS defining complications. Alterations in coagulation biology in the context of HIV infection seem to be largely a consequence of a chronically inflammatory microenvironment leading to endothelial cell (EC) dysfunction. A possible direct role of HIV-1 proteins in sustaining EC dysfunction has been postulated but not yet investigated. The HIV-1 matrix protein p17 (p17) is secreted from HIV-1-infected cells and is known to sustain inflammatory processes by activating ECs. The aim of this study was to investigate the possibility that p17-driven stimulation of human ECs is associated with increased production of critical coagulation factors. Here we show the involvement of autophagy in the p17-induced accumulation and secretion of von Willebrand factor (vWF) by ECs. In vivo experiments confirmed the capability of p17 to exert a potent pro-coagulant activity soon after its intravenous administration.

New Horizons in the Development of Novel Needle-Free Immunization Strategies to Increase Vaccination Efficacy.

The young twenty-first century has already brought several medical advances, such as a functional artificial human liver created from stem cells, improved antiviral (e.g., against HIV) and cancer (e.g., against breast cancer) therapies, interventions controlling cardiovascular diseases, and development of new and optimized vaccines (e.g., HPV vaccine). However, despite this substantial progress and the achievements of the last century, humans still suffer considerably from diseases, especially from infectious diseases. Thus, almost one-fourth of all deaths worldwide are caused directly or indirectly by infectious agents. Although vaccination has led to the control of many diseases, including smallpox, diphtheria, and tetanus, emerging diseases are still not completely contained. Furthermore, pathogens such as Bordetella pertussis undergo alterations making adaptation of the respective vaccine necessary. Moreover, insufficient implementation of vaccination campaigns leads to re-emergence of diseases which were believed to be already under control (e.g., poliomyelitis). Therefore, novel vaccination strategies need to be developed in order to meet the current challenges including lack of compliance, safety issues, and logistic constraints. In this context, mucosal and transdermal approaches constitute promising noninvasive vaccination strategies able to match these demands.

Intranasal vaccination with an adjuvanted polyphosphazenes nanoparticle-based vaccine formulation stimulates protective immune responses in mice.

The most promising strategy to sustainably prevent infectious diseases is vaccination. However, emerging as well as re-emerging diseases still constitute a considerable threat. Furthermore, lack of compliance and logistic constrains often result in the failure of vaccination campaigns. To overcome these hurdles, novel vaccination strategies need to be developed, which fulfill maximal safety requirements, show maximal efficiency and are easy to administer. Mucosal vaccines constitute promising non-invasive approaches able to match these demands. Here we demonstrate that nanoparticle (polyphosphazenes)-based vaccine formulations including c-di-AMP as adjuvant, cationic innate defense regulator peptides (IDR) and ovalbumin (OVA) as model antigen were able to stimulate strong humoral and cellular immune responses, which conferred protection against the OVA expressing influenza strain A/WSN/OVAI (H1N1). The presented results confirm the potency of nanoparticle-based vaccine formulations to deliver antigens across the mucosal barrier, but also demonstrate the necessity to include adjuvants to stimulate efficient antigen-specific immune responses.

Bivalent mucosal peptide vaccines administered using the LCP carrier system stimulate protective immune responses against Streptococcus pyogenes infection.

Despite the broad knowledge about the pathogenicity of Streptococcus pyogenes there is still a controversy about the correlate of protection in GAS infections. We aimed in further improving the immune responses stimulated against GAS comparing different vaccine formulations including bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and BPPCysMPEG, a derivative of the macrophage-activating lipopeptide (MALP-2), as adjuvants, respectively, to be administered with and without the universal T helper cell epitope P25 along with the optimized B cell epitope J14 of the M protein and B and T cell epitopes of SfbI. Lipopeptide based nano carrier systems (LCP) were used for efficient antigen delivery across the mucosal barrier. The stimulated immune responses were efficient in protecting mice against a respiratory challenge with a lethal dose of a heterologous S. pyogenes strain. Moreover, combination of the LCP based peptide vaccine with c-di-AMP allowed reduction of antigen dose at the same time maintaining vaccine efficacy.

Pegylated bisacycloxypropylcysteine, a diacylated lipopeptide ligand of TLR6, plays a host-protective role against experimental Leishmania major infection.

TLRs recognize pathogen-expressed Ags and elicit host-protective immune response. Although TLR2 forms heterodimers with TLR1 or TLR6, recognizing different ligands, differences in the functions of these heterodimers remain unknown. In this study, we report that in Leishmania major-infected macrophages, the expression of TLR1 and TLR2, but not TLR6, increased; TLR2-TLR2 association increased, but TLR2-TLR6 association diminished. Lentivirus-expressed TLR1-short hairpin RNA (shRNA) or TLR2-shRNA administration reduced, but TLR6-shRNA increased L. major infection in BALB/c mice. Corroboratively, Pam3CSK4 (TLR1-TLR2 ligand) and peptidoglycan (TLR2 ligand) increased L. major infection but reduced TLR9 expression, whereas pegylated bisacycloxypropylcysteine (BPPcysMPEG; TLR2-TLR6 ligand) reduced L. major number in L. major-infected macrophages, accompanied by increased TLR9 expression, higher IL-12 production, and inducible NO synthase expression. Whereas MyD88, Toll/IL-1R adaptor protein, and TNFR-α-associated factor 6 recruitments to TLR2 were not different in Pam3CSK4-, peptidoglycan-, or BPPcysMPEG-treated macrophages, only BPPcysMPEG enhanced p38MAPK and activating transcription factor 2 activation. BPPcysMPEG conferred antileishmanial functions to L. major-infected BALB/c-derived T cells in a macrophage-T cell coculture and in BALB/c mice; the protection was TLR6 dependent and IL-12 dependent, and it was accompanied by reduced regulatory T cell number. BPPcysMPEG administration during the priming with fixed L. major protected BALB/c mice against challenge L. major infection; the protection was accompanied by low IL-4 and IL-10, but high IFN-γ productions and reduced regulatory T cells. Thus, BPPcysMPEG, a novel diacylated lipopeptide ligand for TLR2-TLR6 heterodimer, induces IL-12-dependent, inducible NO synthase-dependent, T-reg-sensitive antileishmanial protection. The data reveal a novel dimerization partner-dependent duality in TLR2 function.

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines.

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14 kb); their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines. FROM THE CLINICAL EDITOR: The use of self-amplifying replicon RNA (RepRNA) to increase vaccine antigen payloads can potentially be useful in effective vaccine design. Nonetheless, its use is limited by the degradation during the uptake process. Here, the authors attempted to solve this problem by packaging RepRNA using polyethylenimine (PEI)-polyplex delivery vehicles. The efficacy was confirmed in vivo by the appropriate humoral and cellular immune responses. This novel delivery method may prove to be very useful for future vaccine design.

Efficient nanoparticle-mediated needle-free transcutaneous vaccination via hair follicles requires adjuvantation.

Trans-follicular (TF) vaccination has recently been studied as a unique route for non-invasive transcutaneous vaccination. The present study aims to extensively characterize the immune responses triggered by TF vaccination using ovalbumin loaded chitosan-PLGA (poly lactic-co-glycolic acid) nanoparticles without skin pre-treatment to preserve skin integrity. The impact of formulation composition i.e. antigenic solution or antigen-loaded nanoparticles with or without adjuvant [bis-(3',5')-cyclic dimeric adenosine monophosphate] on immune response quality following TF immunization was analyzed and compared with immune responses obtained after tape stripping the skin. The results presented in this study confirm the ability of nanoparticle based vaccine formulations to deliver antigen across the intact skin via the follicular route, but at the same time demonstrate the necessity to include adjuvants to generate efficient antigen-specific humoral and cellular immune responses.

New Generation Self-Replicating RNA Vaccines Derived from Pestivirus Genome.

While mRNA vaccines have shown their worth, they have the same failing as inactivated vaccines, namely they have limited half-life, are non-replicating, and therefore limited to the size of the vaccine payload for the amount of material translated. New advances averting these problems are combining replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (typically 12-15 kb) derived from viral genomes defective in at least one essential structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitations with RepRNA are RNase-sensitivity and inefficient uptake by dendritic cells (DCs), which need to be overcome for efficacious RNA-based vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Condensing RepRNA with polyethylenimine (PEI) and encapsulating RepRNA into novel Coatsome-replicon vehicles are two approaches that have proven effective for delivery to DCs and induction of immune responses in vivo.

Development and characterization of attenuated metabolic mutants of Bordetella bronchiseptica for applications in vaccinology.

Bordetella bronchiseptica is an important pathogen causing a number of veterinary respiratory syndromes in agriculturally important and food-producing confinement-reared animals, resulting in great economic losses annually amounting to billions of euros worldwide. Currently available live vaccines are incompletely satisfactory in terms of efficacy and safety. An efficient vaccine for livestock animals would allow reducing the application of antibiotics, thereby preventing the massive release of pharmaceuticals into the environment. Here, we describe two new potential vaccine strains based on the BB7865 strain. Two independent attenuating mutations were incorporated by homologous recombination in order to make negligible the risk of recombination and subsequent reversion to the virulent phenotype. The mutations are critical for bacterial metabolism, resistance to oxidative stress, intracellular survival and in vivo persistence. The resulting double mutants BB7865 risA aroA and BB7865 risA dapE were characterized as promising vaccine candidates, which are able to confer protection against colonization of the lower respiratory tract after sublethal challenge with the wild-type strain.

Protective Efficacy of a Mucosal Influenza Vaccine Formulation Based on the Recombinant Nucleoprotein Co-Administered with a TLR2/6 Agonist BPPcysMPEG.

Current influenza vaccines target highly variable surface glycoproteins; thus, mismatches between vaccine strains and circulating strains often diminish vaccine protection. For this reason, there is still a critical need to develop effective influenza vaccines able to protect also against the drift and shift of different variants of influenza viruses. It has been demonstrated that influenza nucleoprotein (NP) is a strong candidate for a universal vaccine, which contributes to providing cross-protection in animal models. In this study, we developed an adjuvanted mucosal vaccine using the recombinant NP (rNP) and the TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl-poly-ethylene-glycol (BPPcysMPEG). The vaccine efficacy was compared with that observed following parenteral vaccination of mice with the same formulation. Mice vaccinated with 2 doses of rNP alone or co-administered with BPPcysMPEG by the intranasal (i.n.) route showed enhanced antigen-specific humoral and cellular responses. Moreover, NP-specific humoral immune responses, characterized by significant NP-specific IgG and IgG subclass titers in sera and NP-specific IgA titers in mucosal territories, were remarkably increased in mice vaccinated with the adjuvanted formulation as compared with those of the non-adjuvanted vaccination group. The addition of BPPcysMPEG also improved NP-specific cellular responses in vaccinated mice, characterized by robust lymphoproliferation and mixed Th1/Th2/Th17 immune profiles. Finally, it is notable that the immune responses elicited by the novel formulation administered by the i.n. route were able to confer protection against the influenza H1N1 A/Puerto Rico/8/1934 virus.

Pulmonary Application of Novel Antigen-Loaded Chitosan Nano-Particles Co-Administered with the Mucosal Adjuvant C-Di-AMP Resulted in Enhanced Immune Stimulation and Dose Sparing Capacity.

The most successful medical intervention for preventing infectious diseases is still vaccination. This effective strategy has resulted in decreased mortality and extended life expectancy. However, there is still a critical need for novel vaccination strategies and vaccines. Antigen cargo delivery by nanoparticle-based carriers could promote superior protection against constantly emerging viruses and subsequent diseases. This should be sustained by the induction of vigorous cellular and humoral immunity, capable of acting both at the systemic and mucosal levels. Induction of antigen-specific responses at the portal of entry of pathogens is considered an important scientific challenge. Chitosan, which is widely regarded as a biodegradable, biocompatible and non-toxic material for functionalized nanocarriers, as well as having adjuvant activity, enables antigen administration via less-invasive mucosal routes such as sublingual or pulmonic application route. In this proof of principle study, we evaluate the efficacy of chitosan nanocarriers loaded with the model antigen Ovalbumin (OVA) co-administrated with the STING agonist bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) given by pulmonary route. Here, BALB/c mice were immunized with four doses of the formulation that stimulates enhanced antigen-specific IgG titers in sera. In addition, this vaccine formulation also promotes a strong Th1/Th17 response characterized by high secretion of IFN-γ, IL-2 and IL-17, as well as induction of CD8+ T cells. Furthermore, the novel formulation exhibited strong dose-sparing capacity, enabling a 90% reduction of the antigen concentration. Altogether, our results suggest that chitosan nanocarriers, in combination with the mucosal adjuvant c-di-AMP, are a promising technology platform for the development of innovative mucosal vaccines against respiratory pathogens (e.g., Influenza or RSV) or for therapeutic vaccines.

Immune responses following neonatal vaccination with conserved F4 fragment of VtaA proteins from virulent Glaesserella parasuis adjuvanted with CAF®01 or CDA.

Glaesserella parasuis is a Gram-negative bacterium that colonizes the upper airways of swine, capable of causing a systemic infection called Glässer's disease. This disease is more frequent in young post-weaning piglets. Current treatments against G. parasuis infection are based on the use of antimicrobials or inactivated vaccines, which promote limited cross-protection against different serovars. For this reason, there is an interest in developing novel subunit vaccines with the capacity to confer effective protection against different virulent strains. Herein, we characterize the immunogenicity and the potential benefits of neonatal immunization with two different vaccine formulations based on the F4 polypeptide, a conserved immunogenic protein fragment from the virulence-associated trimeric autotransporters of virulent G. parasuis strains. With this purpose, we immunized two groups of piglets with F4 combined with cationic adjuvant CAF®01 or cyclic dinucleotide CDA. Piglets immunized with a commercial bacterin and non-immunized animals served as control groups. The vaccinated piglets received two doses of vaccine, at 14 days old and 21 days later. The immune response induced against the F4 polypeptide varied depending on the adjuvant used. Piglets vaccinated with the F4+CDA vaccine developed specific anti-F4 IgGs, biased towards the induction of IgG1 responses, whereas no anti-F4 IgGs were de novo induced after immunization with the CAF®01 vaccine. Piglets immunized with both formulations displayed balanced memory T-cell responses, evidenced upon in vitro re-stimulation of peripheral blood mononuclear cells with F4. Interestingly, pigs immunized with F4+CAF®01 controlled more efficiently a natural nasal colonization by a virulent serovar 4 G. parasuis that spontaneously occurred during the experimental procedure. According to the results, the immunogenicity and the protection afforded by F4 depend on the adjuvant used. F4 may represent a candidate to consider for a Glässer's disease vaccine and could contribute to a better understanding of the mechanisms involved in protection against virulent G. parasuis colonization.

Toll-like receptor-2 agonist-allergen coupling efficiently redirects Th2 cell responses and inhibits allergic airway eosinophilia.

Toll-like receptor (TLR) agonists beneficially modulate allergic airway inflammation. However, the efficiency of TLR agonists varies considerably, and their exact cellular mechanisms (especially of TLR 2/6 agonists) are incompletely understood. We investigated at a cellular level whether the administration of the pharmacologically improved TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPP) conjugated to antigenic peptide (BPP-OVA) could divert an existing Th2 response and influence airway eosinophilia. The effects of BPP-OVA on airway inflammation were assessed in a classic murine sensitization/challenge model and an adoptive transfer model, which involved the adoptive transfer of in vitro differentiated ovalbumin (OVA)-specific Th2 cells. Functional T-cell stimulation by lung dendritic cells (DCs) was determined both in vitro and in vivo, combined with a cytokine secretion analysis. A single mucosal application of BPP-OVA efficiently delivered antigen, led to TLR2-mediated DC activation, and resulted in OVA-specific T-cell proliferation via lung DCs in vivo. In alternative models of allergic airway disease, a single administration of BPP-OVA before OVA challenge (but not BPP alone) significantly reduced airway eosinophilia, most likely through altered antigen-specific T-cell stimulation via DCs. Analyses of adoptively transferred Th2-biased cells after BPP-OVA administration in vivo suggested that BPP-OVA guides antigen-specific Th2 cells to produce significantly higher amounts of IFN-γ upon allergen challenge. In conclusion, our data show for the first time that a single mucosal administration of a TLR 2/6 agonist-allergen conjugate can provoke IFN-γ responses in Th2-biased cells and alleviate allergic airway inflammation.