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Influenza viruses continuously circulate in the human population and escape recognition by virus neutralizing antibodies induced by prior infection or vaccination through accumulation of mutations in the surface proteins hemagglutinin (HA) and neuraminidase (NA). Various strategies to develop a vaccine that provides broad protection against different influenza A viruses are under investigation, including use of recombinant (r) viral vectors and adjuvants. The replication-deficient modified vaccinia virus Ankara (MVA) is a promising vaccine vector that efficiently induces B and T cell responses specific for the antigen of interest. It is assumed that live vaccine vectors do not require an adjuvant to be immunogenic as the vector already mediates recruitment and activation of immune cells. To address this topic, BALB/c mice were vaccinated with either protein- or rMVA-based HA influenza vaccines, formulated with or without the saponin-based Matrix-M™ adjuvant. Co-formulation with Matrix-M significantly increased HA vaccine immunogenicity, resulting in antigen-specific humoral and cellular immune responses comparable to those induced by unadjuvanted rMVA-HA. Of special interest, rMVA-HA immunogenicity was also enhanced by addition of Matrix-M, demonstrated by enhanced HA inhibition antibody titres and cellular immune responses. Matrix-M added to either protein- or rMVA-based HA vaccines mediated recruitment and activation of antigen-presenting cells and lymphocytes to the draining lymph node 24 and 48 h post-vaccination. Taken together, these results suggest that adjuvants can be used not only with protein-based vaccines but also in combination with rMVA to increase vaccine immunogenicity, which may be a step forward to generate new and more effective influenza vaccines. Electronic supplementary material The online version of this article (10.1007/s12026-018-8991-x) contains supplementary material, which is available to authorized users.

Summary Because of the high variability of seasonal influenza viruses and the eminent threat of influenza viruses with pandemic potential, there is great interest in the development of vaccines that induce broadly protective immunity. Most probably, broadly protective influenza vaccines are based on conserved proteins, such as nucleoprotein (NP). NP is a vaccine target of interest as it has been shown to induce cross-reactive antibody and T cell responses. Here we tested and compared various NP-based vaccine preparations for their capacity to induce humoral and cellular immune responses to influenza virus NP. The immunogenicity of protein-based vaccine preparations with Matrix-M™ adjuvant as well as recombinant viral vaccine vector modified Vaccinia virus Ankara (MVA) expressing the influenza virus NP gene, with or without modifications that aim at optimization of CD8+ T cell responses, was addressed in BALB/c mice. Addition of Matrix-M™ adjuvant to NP wild-type protein-based vaccines significantly improved T cell responses. Furthermore, recombinant MVA expressing the influenza virus NP induced strong antibody and CD8+ T cell responses, which could not be improved further by modifications of NP to increase antigen processing and presentation.

Cytotoxic cells of the immune system may kill infected or transformed host cells via the perforin/granzyme or the Fas ligand (FasL) pathways. The purpose of this study was to determine mRNA expression of perforin, granzyme A and FasL in Eimeria tenella infected tissues at primary infection and infection of immune chickens as an indirect measure of cytotoxic cell activity. Chickens were rendered immune by repeated E. tenella infections, which was manifested as an absence of clinical signs or pathological lesions and significantly reduced oocyst production upon challenge infection. During primary E. tenella infection perforin, granzyme A and FasL mRNA expression in caecal tissue was significantly increased at 10 days after infection, compared to uninfected birds. In contrast at infection of immune birds perforin and granzyme A mRNA expression in caecal tissue was significantly increased during the early stages of E. tenella challenge infection, days 1-4, which coincided with a substantial reduction of parasite replication in these birds. These results indicate the activation of cytotoxic pathways in immune birds and support a role for cytotoxic T-cells in the protection against Eimeria infections.

Background: Basophils are inflammatory cells associated with allergy and parasite infections. Investigation of their true biological function has long been hampered by the difficulty in obtaining sufficient amounts of pure basophils and by the lack of phenotypic markers. Moreover, it has been very difficult to clone and identify basophil-specific granule proteins, partially because of an almost complete lack of mRNA in mature circulating basophils. Methods: To obtain transcriptionally active immature basophils, umbilical cord blood cells were cultured in the presence of interleukin (IL)-3. The cells were analysed by flow cytometry and by histological staining. Results: The continuous presence of IL-3 in cord blood cultures resulted in the expansion of basophil precursors co-expressing FcɛRI and the recently described mast cell/basophil marker, 97A6 (CD203c). Several nonbasophil markers (i.e. CD3, CD14, CD15, CD16, CD19 and CD21) were absent on the cultured basophils. However, we show that in early cultures, almost 60% of the CD203c+ cells co-express human leukocyte antigen (HLA)-DR, a marker that is absent on mature circulating basophils. The presence of HLA-DR on basophil precursors may explain the low recovery (24 ± 5.2%) obtained after isolation of cultured basophils, when using a conventional basophil isolation kit that removes HLA-DR+ cells. A novel purification method was developed, including a two-step cocktail of antibodies against selected markers, which resulted in both high purity (95 ± 0.5%) and recovery (59 ± 1.5%) of cultured basophils. Conclusions: We here establish cord blood cultures as a source from which transcriptionally active basophil precursors can be isolated in reasonable quantities for thorough biochemical characterization.

ABSTRACT West Nile virus (WNV), an emerging mosquito-borne and zoonotic flavivirus, continues to spread worldwide and represents a major problem for human and veterinary medicine. In recent years, severe outbreaks were observed in the USA and Europe with neighboring countries, and the virus is considered to be endemic in an increasing number of areas. Although most infections remain asymptomatic, WNV can cause severe, even fatal, neurological disease, which affects mostly the elderly and immunocompromised individuals. Several vaccines have been licensed in the veterinary sector, but no human vaccine is available today. This review summarizes recent strategies that are being followed to develop WNV vaccines with emphasis on technologies suitable for the use in humans.

Given the critical role of Fc gamma receptors (FcγR) as primary targets for autoantibody-mediated effects an important issue is how the FcγR pathway is affected in autoimmune disorders. Here we investigated the FcγR function in monocytes from rheumatoid arthritis (RA) patients in relation to immunoglobulin levels and disease activity. Peripheral blood was obtained from 30 RA patients with clinical acute joint synovitis (active RA), 28 RA patients with no clinical signs of acute joint synovitis (non-active RA) and 34 healthy controls. Prior the functional studies the monocytes were characterized of their FcγRI (CD64), II (CD32), IIb (CD32b) and III (CD16) expression as well as their cell surface bound IgG. The monocytic FcγR function was assessed by binding of human IgG1 and IgG3 immune complexes (IC) and TNF secretion in vitro. IgG anti-citrullinated peptide antibodies (ACPA) were analyzed in the plasma. We found that monocytes from active RA patients had increased levels of FcγRI, II and cell surface IgG concurrently with impaired FcγR function. This was evident by reduced IgG1-IC binding and decreased TNF secretion in response to IgG3-IC. In contrast, monocytes from non-active RA patients displayed a normal FcγR function and had increased FcγRIIb expression together with elevated FcγRI, II and cell surface IgG. The ACPA levels did not differ in active and non-active RA patients but correlated with the monocytic FcγRIII expression in the patients. In conclusion, active RA patients display a dysregulated FcγR function that may represent a novel phenotypic and likely pathogenetic marker for active RA. A disease and FcγR function controlling effect is suggested by the increased inhibitory FcγRIIb in non-active RA.

The mosquito-borne West Nile virus (WNV) causes human and animal disease with outbreaks in several parts of the world including North America, the Mediterranean countries, Central and East Europe, the Middle East, and Africa. Particularly in elderly people and individuals with an impaired immune system, infection with WNV can progress into a serious neuroinvasive disease. Currently, no treatment or vaccine is available to protect humans against infection or disease. The goal of this study was to develop a WNV-vaccine that is safe to use in these high-risk human target populations. We performed a vaccine efficacy study in non-human primates using the contemporary, pathogenic European WNV genotype 1a challenge strain, WNV-Ita09. Two vaccine strategies were evaluated in rhesus macaques (Macaca mulatta) using recombinant soluble WNV envelope (E) ectodomain adjuvanted with Matrix-M, either with or without DNA priming. The DNA priming immunization was performed with WNV-DermaVir nanoparticles. Both vaccination strategies successfully induced humoral and cellular immune responses that completely protected the macaques against the development of viremia. In addition, the vaccine was well tolerated by all animals. Overall, The WNV E protein adjuvanted with Matrix-M is a promising vaccine candidate for a non-infectious WNV vaccine for use in humans, including at-risk populations.

Adjuvants belong to a diverse collection of substances or formulations that, in one way or another, increase or improve immune responses to antigen(s). Classical adjuvants target the physical prese...

The novel saponin based adjuvant Matrix-M™ was recently used in a Phase I study of seasonal influenza in elderly. The present study is a pre-clinical evaluation of the efficacy and mode-of-action of Matrix-M™ formulated influenza vaccine in mice. A manuscript on safety profile and immunogenicity in elderly humans is under preparation. We have previously shown that subcutaneous injections of Matrix-M™, without coformulated antigen, results in a dose-dependent recruitment of leukocytes to draining lymph nodes (dLNs). Herein we compared the mode of action of Matrix-M™ with Alum, FCA and AS03 alone or formulated with influenza split virion antigen injected intramuscularly. The elicited responses in dLNs and spleen were investigated 48h later. Matrix-M™ was particularly efficient in activation of central innate immune cells such as neutrophils, DCs and macrophages compared to the other adjuvants analyzed. Moreover, the adjuvant influence on the recall immune response to influenza antigen was studied by in vitro re-stimulation of splenocytes from mice immunized with influenza antigen adjuvanted with Matrix-M™, Alum or AS03. Splenocytes from mice immunized with influenza antigen and Matrix-M™ produced both Th1 and Th2 cytokines upon re-stimulation. This response was significantly stronger than that induced by the other adjuvants studied. Interestingly, increased levels of the neutrophil chemoattractant KC were produced by antigen stimulated splenocytes from mice immunized with Matrix-M™ adjuvanted vaccine, which is in agreement with the increase of neutrophils into dLNs and spleen after Matrix-M™ injection. Furthermore, influenza antigen adjuvanted with Matrix-M™ induced significantly higher antigen-specific IgG1 and IgG2a responses compared to antigen alone. In conclusion, adjuvant Matrix-M™ activates the innate immune system without antigen present. This activation may explain the enhanced immunity to influenza seen with Matrix-M™ adjuvant. Despite this potent immune activation mediated by Matrix-M™, GLP-toxicity studies and clinical data suggest that Matrix-M™ adjuvant has a mild to moderate safety profile.

Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL)-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95%) and expression of surface markers FcεRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cell-specific transcripts, i.e. β-tryptase, serglycin and FcεRI α-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP), charcot leyden crystal (CLC), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and basophils are closely linked during human hematopoietic development. The dual phenotype also indicates that other cytokines than IL-3 or cell surface interactions are needed to obtain the full basophil specific phenotype in vivo.

Saponin-based adjuvants are widely used to enhance humoral and cellular immune responses towards vaccine antigens, although it is not yet completely known how they mediate their stimulatory effects. The aim of this study was to elucidate the mechanism of action of adjuvant Matrix-M™ without antigen and Alum was used as reference adjuvant. Adjuvant Matrix-M™ is comprised of 40 nm nanoparticles composed of Quillaja saponins, cholesterol and phospholipid. BALB/c mice were subcutaneously injected once with, 3, 12 or 30 µg of Matrix-M™, resulting in recruitment of leukocytes to draining lymph nodes (dLNs) and spleen 48 h post treatment. Flow cytometry analysis identified CD11b(+) Gr-1(high) granulocytes as the cell population increasing most in dLNs and spleen. Additionally, dendritic cells, F4/80(int) cells, T-, B- and NK-cells were recruited to dLNs and in spleen the number of F4/80(int) cells, and to some extent, B cells and dendritic cells, increased. Elevated levels of early activation marker CD69 were detected on T-, B- and NK-cells, CD11b(+) Gr-1(high) cells, F4/80(int) cells and dendritic cells in dLNs. In spleen CD69 was mainly up-regulated on NK cells. B cells and dendritic cells in dLNs and spleen showed an increased expression of the co-stimulatory molecule CD86 and dendritic cells in dLNs expressed elevated levels of MHC class II. The high-dose (30 µg) of Matrix-M™ induced detectable serum levels of IL-6 and MIP-1β 4 h post administration, most likely representing spillover of locally produced cytokines. A lesser increase of IL-6 in serum after administration of 12 µg Matrix-M™ was also observed. In conclusion, early immunostimulatory properties were demonstrated by Matrix-M™ alone, as therapeutic doses resulted in a local transient immune response with recruitment and activation of central immune cells to dLNs. These effects may play a role in enhancing uptake and presentation of vaccine antigens to elicit a competent immune response.

Occupancy of Fc gamma receptors (FcgammaR) by immune complexes (IC) induces secretion of various inflammatory mediators and cytokines. Therefore, knowledge of the FcR function is fundamental for understanding inflammatory processes. Here, we report an alteration in the FcR function in collagen-induced arthritis (CIA). The FcgammaR-binding activity of peritoneal macrophages from arthritis-susceptible DBA/1 mice following collagen type II (CII)/CFA immunization was assessed by Fc rosetting of SRBC opsonized with different IgG subclasses. A progressive reduction of IgG1 IC-binding was observed after immunization, and by the time of arthritis onset, the IgG1 IC-binding was abolished. Binding of IgG2a or IgG2b IC, however, was not affected. The blocked IgG1 IC-binding was reversed by a prior mild acid wash of the CIA macrophages, indicating receptor occupancy as the cause of the blocked binding. The impaired IgG1 IC-binding was associated with arthritis development, as macrophages from CII/CFA-immunized, arthritis-resistant SWR mice or DBA/1 mice, immunized with CFA alone, did not show this effect. Normal DBA/1 macrophages, blocked with a monoclonal antibody to FcgammaRIIB/FcgammaRIII, and macrophages from FcgammaRIII-deficient mice did not bind IgG1 IC, indicating FcgammaRIII as responsible for IgG1 IC-binding. Our data suggest that an increased degree of saturation of FcgammaRIII precedes the development of CIA, which is reflected by a reduced IgG1 IC-binding in macrophages of CII/CFA-immunized DBA/1 mice.

Activating Fc gamma receptors (FcγRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcγRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcγRIIb isoform. FcγRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcγRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcγRI, FcγRII and FcγRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcγRII, FcγRIII but not FcγRI, all activating FcγRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcγRIIb and the activating FcγRs. Anti-inflammatory treatment with glucocorticoids reduced FcγR expression in arthritic joints, particularly that of FcγRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcγRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcγRIIb and activating FcγRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcγRs may represent valuable therapeutics in this disease.