Induction Of IgG Research Articles

Distinct Effects of Monophosphoryl Lipid A, Oligodeoxynucleotide CpG, and Combination Adjuvants on Modulating Innate and Adaptive Immune Responses to Influenza Vaccination.

Monophosphoryl lipid A (MPL) and oligodeoxynucleotide CpG are toll-like receptor (TLR) 4 and 9 agonist, respectively. Here, we investigated the effects of MPL, CpG, and combination adjuvants on stimulating in vitro dendritic cells (DCs), in vivo innate and adaptive immune responses, and protective efficacy of influenza vaccination. Combination of MPL and CpG was found to exhibit distinct effects on stimulating DCs in vitro to secrete IL-12p70 and tumor necrosis factor (TNF)-α and proliferate allogeneic CD8 T cells. Prime immunization of mice with inactivated split influenza vaccine in the presence of low dose MPL+CpG adjuvants increased the induction of virus-specific IgG and IgG2a isotype antibodies. MPL and CpG adjuvants contribute to improving the efficacy of prime influenza vaccination against lethal influenza challenge as determined by body weight monitoring, lung function, viral titers, and histology. A combination of MPL and CpG adjuvants was effective in improving vaccine efficacy as well as in reducing inflammatory immune responses locally and in inducing cellular immune responses upon lethal influenza virus challenge. This study demonstrates unique adjuvant effects of MPL, CpG, and combination adjuvants on modulating innate and adaptive immune responses to influenza prime vaccination.

Intradermal Delivery of Antigens Enhances Specific IgG and Diminishes IgE Production: Potential Use for Vaccination and Allergy Immunotherapy.

Skin is protected by a tough but flexible multilayered barrier and is a front line for immune responses against invading particles. For many years now, skin has been a tissue where certain vaccines are injected for the prevention of infectious disease, however, the detailed mechanisms of the skin immune response are not yet well understood. Using thin and small injection needles, we carefully injected OVA into a restricted region of mouse skin, i.e., intradermal (ID), and examined the antibody response in comparison with subcutaneous (SC) injection or epicutaneous patch administration of OVA. Epicutaneous patches induced a high IgE response against OVA, but IgG production was low. High IgG production was induced by both ID and SC injection, moreover, ID injection induced higher IgG production without any adjutants. Furthermore, OVA-specific IgE production was diminished by ID injection. We found that ID injection could efficiently stimulate skin resident DCs, drive Th1-biased conditions and diminish IgE production. The ID injection response was regulated by Langerin+ dermal DCs, because OVA was taken up mainly by these cells and, after transiently deleting them, the IgE response was no longer diminished and IgG1 production was enhanced. We also tested whether ID injection might be an effective allergy treatment by attempting to inhibit ongoing IgE production in mice with experimentally induced high serum IgE levels. Multiple ID injections of OVA were shown to prevent elevation of serum OVA-specific IgE after repeated allergen challenge. In contrast, SC OVA injection could only transiently inhibit the OVA-specific IgE production. These findings indicated that ID injection results in higher induction of antigen-specific IgG, and thus may be useful for vaccine delivery with little or no adjuvant components. Moreover, the observed diminishment of IgE and induction of Th1-biased immune responses suggest that ID may be a useful injection route for allergy immunotherapy.

Antibody Production in Response to Staphylococcal MS-1 Phage Cocktail in Patients Undergoing Phage Therapy.

In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties toward applied phages (K rate). Among 20 examined patients receiving the MS-1 phage cocktail orally and/or locally, the majority did not show a noticeably higher level of antiphage antibodies in their sera during phage administration. Even in those individual cases with an increased immune response, mostly by induction of IgG and IgM, the presence of antiphage antibodies did not translate into unsatisfactory clinical results of phage therapy. On the other hand, a negative outcome of the treatment occurred in some patients who showed relatively weak production of antiphage antibodies before and during treatment. This may imply that possible induction of antiphage antibodies is not an obstacle to the implementation of phage therapy and support our assumption that the outcome of the phage treatment does not primarily depend on the appearance of antiphage antibodies in sera of patients during therapy. These conclusions are in line with our previous findings. The confirmation of this thesis is of great interest as regards the efficacy of phage therapy in humans.

Norovirus (NoV) specific protective immune responses induced by recombinant P dimer vaccine are enhanced by the mucosal adjuvant FlaB.

BackgroundNoroviruses (NoVs) are a major cause of childhood gastroenteritis and foodborne diseases worldwide. Lack of appropriate animal models or cell-based culture systems makes the development and evaluation of NoV-specific vaccines a daunting task. VP1 is the major capsid protein of the NoVs that acts as a binding motif to human histo-blood group antigens (HBGAs) through its protruding 2 (P2) domain and can serve as a protective antigen candidate for vaccine development.MethodsRecombinantly produced NoV specific P domain (Pd) vaccine was inoculated into groups of mice either alone or in conjugation with mucosal adjuvant FlaB, the flagellar protein from Vibrio vulnificus. Antigen specific humoral and cell mediated immune responses were assessed by enzyme linked immunosorbent assay (ELISA) or fluorescent activated cell sorting (FACS). A comparative analysis of various routes of vaccination viz. intranasal, sublingual and subcutaneous, was also done.ResultsIn this study, we show that a recombinant Pd-vaccine administered through intranasal route induced a robust TH2-dependent humoral immune response and that the combination of vaccine with FlaB significantly enhanced the antibody response. Interestingly, FlaB induced a mixed TH1/TH2 type of immune response with a significant induction of IgG1 as well as IgG2a antibodies. FlaB also induced strong IgA responses in serum and feces. FlaB mediated antibody responses were toll like receptor 5 (TLR5) dependent, since the FlaB adjuvanticity was lost in TLR5−/− mice. Further, though the Pd-vaccine by itself failed to induce a cell mediated immune response, the Pd-FlaB combination stimulated a robust CD4+IFNγ+ and CD8+IFNγ+ T cell response in spleen and mesenteric lymph nodes. We also compared the adjuvant effects of FlaB with that of alum and complete Freund’s adjuvant (CFA). We found that subcutaneously inoculated FlaB induced more significant levels of IgG and IgA in both serum and feces compared to alum or CFA in respective samples.ConclusionWe validate the use of TLR5 agonist as a strong mucosal adjuvant that would facilitate the development of NoV specific vaccines for humans and veterinary use. This study also highlights the importance of route of immunization in inducing the appropriate immune responses in mucosal compartments.

IgG subclasses determine pathways of anaphylaxis in mice

Animal models have demonstrated that allergen-specific IgG confers sensitivity to systemic anaphylaxis that relies on IgG Fc receptors (FcγRs). Mouse IgG2a and IgG2b bind activating FcγRI, FcγRIII, and FcγRIV and inhibitory FcγRIIB; mouse IgG1 binds only FcγRIII and FcγRIIB. Although these interactions are of strikingly different affinities, these 3 IgG subclasses have been shown to enable induction of systemic anaphylaxis. We sought to determine which pathways control the induction of IgG1-, IgG2a-, and IgG2b-dependentpassive systemic anaphylaxis. Mice were sensitized with IgG1, IgG2a, or IgG2b anti-trinitrophenyl mAbs and challenged with trinitrophenyl-BSA intravenously to induce systemic anaphylaxis that was monitored by using rectal temperature. Anaphylaxis was evaluated in mice deficient for FcγRs injected with mediator antagonists or in which basophils, monocytes/macrophages, or neutrophils had been depleted. FcγR expression was evaluated on these cells before and after anaphylaxis. Activating FcγRIII is the receptor primarily responsible for all 3 models of anaphylaxis, and subsequent downregulation of this receptor was observed. These models differentially relied on histamine release and the contribution of mast cells, basophils, macrophages, and neutrophils. Strikingly, basophil contribution and histamine predominance in mice with IgG1- and IgG2b-induced anaphylaxis correlated with the ability of inhibitory FcγRIIB to negatively regulate these models of anaphylaxis. We propose that the differential expression of inhibitory FcγRIIB on myeloid cells and its differential binding of IgG subclasses controls the contributions of mast cells, basophils, neutrophils, and macrophages to IgG subclass-dependent anaphylaxis. Collectively, our results unravel novel complexities in the involvement and regulation of cell populations in IgG-dependent reactions invivo.

Proof of concept study with an HER-2 mimotope anticancer vaccine deduced from a novel AAV-mimotope library platform

ABSTRACTBackground: Anticancer vaccines could represent a valuable complementary strategy to established therapies, especially in settings of early stage and minimal residual disease. HER-2 is an important target for immunotherapy and addressed by the monoclonal antibody trastuzumab. We have previously generated HER-2 mimotope peptides from phage display libraries. The synthesized peptides were coupled to carriers and applied for epitope-specific induction of trastuzumab-like IgG. For simplification and to avoid methodological limitations of synthesis and coupling chemistry, we herewith present a novel and optimized approach by using adeno-associated viruses (AAV) as effective and high-density mimotope-display system, which can be directly used for vaccination. Methods: An AAV capsid display library was constructed by genetically incorporating random peptides in a plasmid encoding the wild-type AAV2 capsid protein. AAV clones, expressing peptides specifically reactive to trastuzumab, were employed to immunize BALB/c mice. Antibody titers against human HER-2 were determined, and the isotype composition and functional properties of these were tested. Finally, prophylactically immunized mice were challenged with human HER-2 transfected mouse D2F2/E2 cells. Results: HER-2 mimotope AAV-vaccines induced antibodies specific to human HER-2. Two clones were selected for immunization of mice, which were subsequently grafted D2F2/E2 cells. Both mimotope AAV clones delayed the growth of tumors significantly, as compared to controls. Conclusion: In this study, a novel mimotope AAV-based platform was created allowing the isolation of mimotopes, which can be directly used as anticancer vaccines. The example of trastuzumab AAV-mimotopes demonstrates that this vaccine strategy could help to establish active immunotherapy for breast-cancer patients.

S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen.

BackgroundPorcine epidemic diarrhea virus (PEDV) is a highly contagious virus infecting pigs of all ages with high morbidity and mortality among newborn piglets. Currently, there is no effective vaccine available to protect the pigs from PEDV. The N-terminal subunit of spike protein (S1) is responsible for virus binding to the cellular receptor and contains a number of neutralizing antibody epitopes. Thus, we expressed and produced recombinant S1 protein to protect newborn piglets by immunization of sows.MethodsAffinity tagged PEDV S1 protein was expressed in a secretory form in yeast, insect and mammalian cells to identify the most suitable production system. Purified recombinant protein was analysed by SDS-PAGE, Western blot and deglycosylation assay. A pregnant sow was intramuscularly immunized three times with adjuvanted recombinant protein prior to farrowing. PEDV-specific immune responses in sera and colostrum of the sow and piglets were assayed by ELISA and virus neutralization assays. Piglets were challenged orally with PEDV, and clinical parameters were monitored for 6 days post-challenge.Results and conclusionOf three eukaryotic expression systems tested (yeast, insect-cell, and mammalian), expression by HEK-293 T cells gave the highest yield of protein that was N-glycosylated and was the most appropriate candidate for vaccination. Administration of the subunit vaccine in a sow resulted in induction of S1-specific IgG and IgA that were passively transferred to the suckling piglets. Also, high virus neutralization titres were observed in the serum of the vaccinated sow and its piglets. After PEDV challenge, piglets born to the vaccinated sow exhibited less severe signs of disease and significantly lower mortality compared to the piglets of a control sow. However, there were no significant differences in diarrhea, body weight and virus shedding. Thus, vaccination with S1 subunit vaccine failed to provide complete protection to suckling piglets after challenge exposure, and further improvements are needed for the development of a subunit vaccine that fully protects against PEDV infection.

Lupus Nephritis IgG Induction of Calcium/Calmodulin-Dependent Protein Kinase IV Expression in Podocytes and Alteration of Their Function.

Kidney podocytes and their slit diaphragms prevent urinary protein loss. T cells from patients with systemic lupus erythematosus display increased expression of calcium/calmodulin-dependent protein kinase IV (CaMKIV). The present study was undertaken to investigate the role of CaMKIV in podocyte function in lupus nephritis (LN). We treated kidney podocytes with IgG derived from healthy individuals or patients with LN and then analyzed gene expression using a DNA microarray. The localization of IgG in podocytes was analyzed by immunofluorescence staining, with or without silencing of neonatal Fc receptor (FcRn). In addition, we silenced CAMK4 in podocytes and analyzed the expression of selected genes. We also examined the expression of CD86 in kidney podocytes from MRL/lpr, MRL/lpr.camkiv(-/-), and MRL/MPJ mice by in situ hybridization. We found that exposure of podocytes to IgG resulted in entry of IgG into the cytoplasm. IgG entered podocytes via the FcRn because less IgG was found in the cytoplasm of podocytes treated with FcRn small interfering RNA. DNA microarray studies of podocytes exposed to LN-derived IgG revealed up-regulation of genes related to the activation of immune cells or podocyte damage. Interestingly, CD86 expression decreased after silencing CAMK4 in podocytes. Also, in situ hybridization experiments showed that the expression of CD86 was reduced in podocytes from MRL/lpr.camkiv(-/-) mice. LN-derived IgG enters podocytes and up-regulates CAMK4, which is followed by increased expression of genes known to be linked to podocyte damage and T cell activation. Targeted inhibition of CAMK4 in podocytes may prove to be clinically useful in patients with LN.

Chronic cat allergen exposure induces a TH2 cell–dependent IgG4 response related to low sensitization

In human subjects, allergen tolerance has been observed after high-dose allergen exposure or after completed allergen immunotherapy, which is related to the accumulation of anti-inflammatory IgG4. However, the specific T-cell response that leads to IgG4 induction during chronic allergen exposure remains poorly understood. We sought to evaluate the relationship between cat allergen-specific T-cell frequency, cat allergen-specific IgE and IgG4 titers, and clinical status in adults with cat allergy with and without cat ownership and the cellular mechanism by which IgG4 is produced. Fel d 1-, Fel d 4-, Fel d 7-, and Fel d 8-specific T-cell responses were characterized by CD154 expression after antigen stimulation. In allergic subjects without cat ownership, the frequency of cat allergen (Fel d 1 and Fel d 4)-specific TH2 (sTH2) cells correlates with higher IgE levels and is linked to asthma. Paradoxically, we observed that subjects with cat allergy and chronic cat exposure maintain a high frequency of sTH2 cells, which correlates with higher IgG4 levels and low sensitization. B cells from allergic, but not nonallergic subjects, are able to produce IgG4 after cognate interactions with sTH2 clones and Fel d 1 peptide or the Fel d 1 recombinant protein. These experiments suggest that (1) allergen-experienced B cells with the capacity to produce IgG4 are present in allergic subjects and (2) cat allergen exposure induces an IgG4 response in a TH2 cell-dependent manner. Thus IgG4 accumulation could be mediated by chronic activation of the TH2 response, which in turn drives desensitization.

Synthesis of a conjugate of 3´-sialyllactoside with recombinant flagellin as a carrier protein and assessment of its immunological activity in comparison with that of a similar hemocyanin-based conjugate

In the framework of a project directed to the design of synthetic cancer vaccines based on carbohydrate chains of tumor-associated gangliosides, a new prototype cancer vaccine, a polyvalent carbohydrate-protein conjugate 1 with ∼11 3´-sialyllactoside ligands conjugated to one molecule of recombinant flagellin was synthesized. Dendritic cell vaccines were designed using the conjugate 1 and a neoglycoconjugate 2 consisting of ∼400 3´-sialyllactose residues linked to hemocyanin from a snail Megathura crenulata (KLH). Comparative ELISA study was conducted to assess the induction of carbohydrate-specific immunoglobulins IgM and IgG after immunization of mice with conjugates 1 and 2 and dendritic cells loaded with 1 and 2. Synthetic polyacrylamide carrier conjugated to 3´-sialyllactose ligands and B16F0 melanoma cells were used as coating antigens.

Pretreatment IgE sensitization patterns determine the molecular profile of the IgG4 response during updosing of subcutaneous immunotherapy with timothy grass pollen extract

Allergen immunotherapy is an effective treatment of allergic rhinoconjunctivitis. Clinical efficacy is associated with improvement of basophil sensitivity and an increase in allergen-specific immunoglobulin concentration. We sought to determine whether changes in allergen component-specific serum IgE and IgG4 levels during the updosing phase of subcutaneous immunotherapy (SCIT) are biomarkers of the immunologic changes that can lead to treatment efficacy. Twenty-four subjects with grass pollen-induced allergic rhinoconjunctivitis were randomized 3:1 to receive SCIT (Alutard SQ) or to an open control group. IgE and IgG4 concentrations were determined for the major allergens Phl p 1 or Phl p 5 by using ImmunoCAP and for 8 grass pollen molecules by using Immuno Solid-phase Allergy Chip (ISAC) before treatment and after updosing. Levels of specific IgE against the dominant major allergens Phl p 1 and Phl p 5 increased from a mean of 23.0 to 48.8 kU/L (P=.01, n=18) during the updosing phase in ImmunoCAP measurements but decreased from a median of 4.6 ISAC specific units (ISU) to 2.14 ISU (P<.0001, n=102) when measured by using ISAC against 8 grass allergen components. The updosing phase induced a specific IgG4 level increase from a median of 0 ISU before treatment to 0.83 ISU after 12weeks (P<.0001, n=102) but only for allergen molecules to which pretreatment-specific IgE antibodies were detected (Spearman σ=0.72, P<.0001, n=102). Pretreatment allergen component-specific IgE appears to determine the induction of IgG4 in the updosing phase. Induced IgG4 seems to suppress IgE levels on ISAC, resulting in a marked decrease in ISAC-measured specific IgE levels after updosing of SCIT. Thus this decrease in ISAC IgE levels can be used to monitor the blocking effect of allergen-specific immunotherapy-induced non-IgE antibodies.

Intraperitoneal Immunization with Cry1Ac Protoxin from Bacillus thuringiensis Provokes Upregulation of Fc-Gamma-II/and Fc-Gamma-III Receptors Associated with IgG in the Intestinal Epithelium of Mice.

In humans, intestinal epithelial FcRn is expressed throughout life and mediates the bidirectional transport of IgG, but in mice, it is markedly expressed in neonatal intestine. In adults, its expression is only faintly upregulated after intestinal IgG induction such as that elicited by i.p. immunization with Cry1Ac protoxin (pCry1Ac) Bacillus thuringiensis. This led us to suggest that additional Fcγ receptors (Fcγ-R) may be participating in epithelial IgG uptake. So, first we determined whether CD16/32 [an epitope shared by Fcγ-RII (CD32) and Fcγ-RIII (CD16)] was expressed in the intestinal epithelia of mice. Using confocal microscopy and flow cytometry, we detected co-localization of IgG and CD16/32 in epithelial cells, whose frequency was increased by immunization with pCry1Ac. Western blot and cross-immunoprecipitation results with anti-CD16/32 and IgG antibodies in epithelial cell extracts suggested that epithelial cells bear both Fcγ-RII and Fcγ-RIII and contained IgG associated with Fcγ-RII/RIII. Using anti-CD32 and anti-CD16 antibodies, we confirmed by Western blot, confocal microscopy and flow cytometry that both Fcγ-RII and Fcγ-RIII were expressed and suggested that upregulation occurred upon immunization in intestinal epithelia. Finally, we examined the in vitro effect of anti-CD16/32, anti-CD16 and anti-CD32 antibodies on IgG uptake and transport by intestinal epithelial cells and found that it was partially reduced. Although further studies are still required, our results suggest that Fcγ-RII and Fcγ-RIII might participate in the uptake and/or transport of IgG through the intestinal epithelia of adult mice.

Co-immunization of cattle with a vaccine against babesiosis and Lactobacillus casei increases specific IgG1 levels to Babesia bovis and B. bigemina

The effect of Lactobacillus casei administered along with a live attenuated vaccine vs. bovine babesiosis (VAC) on induction of IgG1 and IgG2 antibodies to Babesia bovis and Babesia bigemina was assessed by the indirect fluorescent antibody test (IFAT) in bovines of an endemic babesiosis area before (day 0) and after vaccination (days 15 and 30). We previously reported that L. casei increases the efficiency of VAC under controlled conditions and under extreme conditions in the field; however, the levels of IgG1 and IgG2 antibodies to B. bovis and B. bigemina are not known in vaccinated animals. Twenty-one dairy cows were allocated into three groups (seven animals per group): unvaccinated, vaccinated with VAC and vaccinated simultaneously with VAC and L. casei (VAC–LC). All animals were kept in a babesiosis endemic area at Tlalixcoyan, Veracruz. At days 15 and 30 after vaccination, the average levels of IgG1 to B. bovis and to B. bigemina were significantly higher in VAC–LC group than levels observed in VAC and control groups (P<0.01). Levels of IgG2 were similar in VAC and VAC–LC groups but higher than in the control group (P<0.01). When tested in in vitro cultures of B. bovis, sera from VAC–LC group significantly inhibited parasite growth as compared with the sera of the other two groups. It is suggested that the efficiency improvement of VAC, in part, is due to the L. casei boost of IgG1 over IgG2 antibodies to B. bovis and B. bigemina when the bacteria is co-inoculated with this vaccine.

Peptide mimotopes of malondialdehyde-epitopes for clinical applications in cardiovascular disease

IntroductionAutoantibodies specific for malondialdehyde modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, the generation of MDA-LDL results in the formation of many different epitopes with high variability. Because it is not known, which MDA epitope is biological important, the aim of this study was to identify and characterize peptide mimotopes of MDA-LDL that could be used as antigens to improve the reproducible detection of MDA-specific autoantibodies. Methods and ResultsPeptide phage display libraries were screened for phages binding to the MDA-LDL specific natural IgM antibody LR04. After biopanning two consensus sequences (P1 & P2) of binding phages were synthesized. P1 and P2 were specifically bound by LR04, as the binding of LR04 to coated peptides was fully competed by MDA-LDL but not native LDL. P1 and P2 were also bound by other MDA specific murine (EN1) and human (IK17) antibodies. Furthermore, the binding of LR04 to late apoptotic cells was completely inhibited by both peptides, identifying them as mimotopes of naturally occurring epitopes on dying cells. Immunization of C57BL/6 mice with P2 conjugated to BSA, but not BSA alone, resulted in the robust induction of IgG1 and IgM antibodies against MDA-LDL. Moreover, serum IgG of immunized mice specifically stained epitopes in atherosclerotic lesion of rabbits and humans. Finally, we measured anti-mimotope antibody titers in serum samples previously collected from healthy subjects (n=17) and from patients (n=140) with stable angina pectoris undergoing percutaneous coronary intervention (Tsimikas, 2004). In patients a significant positive correlation was observed between anti-MDA-LDL and anti-mimotope IgM (P1, r=0.8; P2, r=0.6; p<0.0001) and IgG (P1, r=0.4; P2, r=0.3; p<0.0001) antibodies. A similar correlation was also found in sera of healthy subjects with IgM (P1, r=0.6; P2, r=0.4; p<0.0004) and IgG (P1, r=0.7; P2, r=0.5; p<0.0001) antibodies. ConclusionsThus, we have identified specific mimotopes of MDA-LDL that serve as highly reproducible antigens to assess autoantibody titers in patients with cardiovascular disease. Future studies will reveal their usefulness for therapeutic vaccination approaches against atherosclerosis.

Biochemical composition of haemagglutinin‐based influenza virus‐like particle vaccine produced by transient expression in tobacco plants

Influenza virus-like particles (VLPs) are noninfectious particles resembling the influenza virus representing a promising vaccine alternative to inactivated influenza virions as antigens. Medicago inc. has developed a plant-based VLP manufacturing platform allowing the large-scale production of GMP-grade influenza VLPs. In this article, we report on the biochemical compositions of these plant-based influenza candidate vaccines, more particularly the characterization of the N-glycan profiles of the viral haemagglutinins H1 and H5 proteins as well as the tobacco-derived lipid content and residual impurities. Mass spectrometry analyses showed that all N-glycosylation sites of the extracellular domain of the recombinant haemagglutinins carry plant-specific complex-type N-glycans having core α(1,3)-fucose, core β(1,2)-xylose epitopes and Lewis(a) extensions. Previous phases I and II clinical studies have demonstrated that no hypersensibility nor induction of IgG or IgE directed against these glycans was observed. In addition, this article showed that the plant-made influenza vaccines are highly pure VLPs preparations while detecting no protein contaminants coming either from Agrobacterium or from the enzymes used for the enzyme-assisted extraction process. In contrast, VLPs contain few host cell proteins and glucosylceramides associated with plant lipid rafts. Identification of such raft markers, together with the type of host cell impurity identified, confirmed that the mechanism of VLP formation in planta is similar to the natural process of influenza virus assembly in mammals.

Mechanisms of peptide immunotherapy in allergic airways disease.

Allergen immunotherapy with whole proteins is clinically efficacious but requires a protracted treatment period because of frequent allergic adverse events. A combination of duration of treatment and adverse events leads to poor compliance. Short synthetic peptides containing the major immunodominant T cell epitopes of allergenic proteins have been shown to reduce IgE cross-linking ability, thereby leading to fewer allergic adverse events following their administration to patients with allergies. Peptide immunotherapy has been shown to result in clinically meaningful efficacy in several Phase II, randomized, double-blind, placebo-controlled clinical trials. Exactly how peptide immunotherapy achieves its efficacy remains incompletely understood, but the mechanisms are thought to include immune deviation and induction of regulatory T cells capable of suppressing allergen-specific immune responses. Limited data are available on the effects of peptide therapy on humoral immune responses. Induction of allergen-specific IgG has been observed after peptide therapy, but the levels of antibody induced were much lower than generally seen with the utilization of whole allergen approaches. Thus, the immunological mechanisms of peptide immunotherapy appear to overlap, although not completely, with those seen in whole allergen therapy. Further studies are required to fully elucidate mechanisms of action.

Transdermal immunization using solid-in-oil nanodispersion with CpG oligodeoxynucleotide adjuvants.

Simple and noninvasive vaccine administration alternatives to injections are desired. A solid-in-oil (S/O) nanodispersion system was able to overcome skin barriers and induce an immune response; however, antibody levels remained low. We applied an immune potentiator, CpG oligodeoxynucleotide (ODN), to enhance the immune response by controlling the T helper 1 (Th1)/T helper 2 (Th2) balance. S/O nanodispersions containing ovalbumin (OVA) and CpG ODN (CpG-A or CpG-B) were characterized by size distribution analysis and a protein release test. The skin permeation of fluorescence-labeled OVA was observed by fluorescence microscopy. Antigen-specific IgG, IgG1, and IgG2a responses were measured by enzyme-linked immunosorbent assay. Co-encapsulation of CpG ODNs in S/O nanodispersions enhanced induction of OVA-specific IgG. S/O nanodispersion containing OVA and CpG-A had a smaller mean particle size and permeated the skin more efficiently. In contrast, CpG-B showed the highest protein release and induction of OVA-specific IgG. IgG subclass analysis revealed that OVA induced a Th2-dominant immune response, while the S/O nanodispersion containing CpG-A skewed the immune response toward a Th1-bias. In combination with CpG ODN, the S/O nanodispersion system efficiently induced an antigen-specific antibody response. The Th1/Th2 immune balance could be controlled by the selection of CpG ODN type.

A single, low dose oral antigen exposure in newborn piglets primes mucosal immunity if administered with CpG oligodeoxynucleotides and polyphosphazene adjuvants

By definition, soluble antigens ingested orally trigger mucosal tolerance such that any subsequent re-exposure by a systemic route results in suppression of immunity. We propose that antigens introduced in extreme early life can readily traverse the gut wall and therefore circumvent induction of mucosal tolerance and instead induce immunity. Piglets were drenched with low-doses of ovalbumin (OVA; 5mg or 0.05mg) alone, OVA plus adjuvants (CpG oligodeoxynucleotides and PCEP polyphosphazene) or saline within 6h of birth. At 28 days of age, they were administered 10mg OVA plus 1:1 Montanide adjuvant (or saline) via the intraperitoneal (i.p.) route or via the oral route. Serum was obtained on day 28 and day 49 to measure OVA-specific antibodies titres. All piglets boosted orally with OVA plus Montanide, regardless of prior OVA exposure, failed to induce immunity. As expected, piglets drenched with saline but boosted via the i.p. route with OVA plus Montanide showed significant induction of anti-OVA IgA, IgG, IgG1 and IgG2 relative to saline control piglets. Newborn animals drenched with 5mg or 0.05mg OVA failed to induce oral immunity. A second intramuscular injection in adulthood triggered immunity in the piglets that were drenched with 0.05mg OVA and boosted initially by the i.p. route suggesting that some systemic lymphocytes were primed despite initial lack of induction of humoral immunity. In contrast, piglets orally immunized with 5mg or 0.05mg OVA plus adjuvants resulted in significant induction of anti-OVA IgA (5mg only), IgM, IgG, IgG1 and IgG2 in serum relative to saline control piglets as well as significant induction of anti-OVA IgA, IgM (5mg only) IgG, IgG1 (5mg only) or IgG2 relative to piglets drenched with OVA alone. These data clearly show that the response was sensitive to the oral vaccine components and was not simply a response to the i.p. immunization at day 28. This work demonstrates that newborn piglets respond to oral antigens with immunity if re-exposure to the antigen occurs via a systemic route and if adjuvants are included with the oral vaccine administered at birth. These results should be further explored to establish whether early life oral vaccination can be exploited to protect this susceptible population against infectious diseases.

Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins.

Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4+ and CD8+ T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania infantum proteins, cysteine peptidase A (CPA), histone H1, KMP-11, and Leishmania eukaryotic initiation factor (LeIF) were analyzed for the prediction of binding epitopes to H2d MHC class I and II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in complete Freund’s adjuvant/incomplete Freund’s adjuvant. CPA_p2, CPA_p3, H1_p1, and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3, and LeIF_p6 induced IFN-γ-producing CD4+ T cells indicating a TH1-type response. In addition, CPA_p2, CPA_p3, and H1_p1 induced also the induction of CD8+ T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3, and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.

Ovalbumin-coated pH-sensitive microneedle arrays effectively induce ovalbumin-specific antibody and T-cell responses in mice

The aim of this work was to study the applicability of antigen-coated pH-sensitive microneedle arrays for effective vaccination strategies. Therefore, a model antigen (ovalbumin) was coated onto pH-sensitive (pyridine-modified) microneedle arrays to test pH-triggered antigen release by applying the coated arrays onto ex vivo human skin, and by conducting a dermal immunization study in mice.The release of antigen into ex vivo human skin from the coated microneedles was determined by using radioactively labeled ovalbumin. To investigate the induction of antigen-specific IgG, and CD4+ and CD8+ T-cell responses, BALB/c mice were immunized with antigen-coated pH-sensitive microneedles by the ‘coat and poke’ approach. These responses were compared to responses induced by the ‘poke and patch’ approach, and subcutaneous and intradermal vaccination with classic hypodermic needles.The pH-sensitive microneedle arrays were efficiently coated with ovalbumin (95% coating efficiency) and upon application of six microneedle arrays 4.27 of 7μg ovalbumin was delivered into the skin, showing a release efficiency of 70%. In contrast, the ‘poke and patch’ approach led to a delivery of only 6.91 of 100μg ovalbumin (7% delivery efficiency). Immunization by means of ovalbumin-coated microneedles resulted in robust CD4+ and CD8+ T-cell responses comparable to those obtained after subcutaneous or intradermal immunization with conventional needles. Moreover, it effectively induced IgG responses; however, it required prime-boost immunizations before antibodies were produced.In conclusion, antigen delivery into ex vivo human skin by antigen-coated pH-sensitive microneedle arrays is more efficient than the ‘poke-and-patch’ approach and in vivo vaccination studies show the applicability of pH-sensitive microneedles for the induction of both T cell and B cell responses.