Antigen-specific IgG Titers Research Articles

Purification and assembling a fused capsid protein as an enterovirus 71 vaccine candidate from inclusion bodies to pentamer-based nanoparticles

An efficient preparation process for a novel Enterovirus 71 (EV71) vaccine was developed in this paper, which is a fused antigen by connecting the truncated capsid proteins of VP1, VP2 and VP3 into one molecule through flexible peptide linkers and expressed in E. coli as inclusion bodies. The fused protein was purified at denatured state through two-step ion exchange chromatography, with final purity above 95%, host cell proteins below 0.003% and residual DNA less than 50ng/mL. During the following refolding and assembling process through dilution, the fused antigen precipitated completely, while the precipitation was efficiently inhibited with 2M urea or 0.5M arginine as an additive. Size exclusion chromatography analysis indicated the protein formed soluble aggregates with linear or rod-like appearance in transmission electron microscopy. These aggregates transformed into pentamers with a size of 15nm at pH 8.0 after the additive removing. Moreover, most of the pentamers assembled as sphere-like particulates about 25–40nm after being induced by calcium chloride. High antigen-specific IgG titer was elicited by immunization with the nanoparticles in mouse model. Splenocytes proliferative responses and cytokines analysis indicated this particulate antigen could induce humoral and cellular immune responses. These results lay foundations for developing the fused antigen as an alternative vaccine against hand-foot-and-mouth disease (HFMD) and for the large-scale production for E.coli-based vaccines.

Injectable and Pathogen-Mimicking Hydrogels for Enhanced Protective Immunity against Emerging and Highly Pathogenic Influenza Virus.

Seasonal emerging infectious diseases such as influenza A impose substantial risk and need new translational strategies to achieve active immunomodulation. Here, a novel injectable pathogen-mimicking hydrogel (iPMH) that can enhance both cellular and humoral immune responses is suggested. By the help of poly(γ-glutamic acid) that has abundant carboxylate groups and dispersion helper function, hydrophobic immunostimulatory 3-O-desacyl-4'-monophosphoryl lipid A (MPLA) molecules and viral antigens (PR8, W150) can be successfully combined as pathogen-mimicking adjuvants. Polyelectrolyte complex between the poly(γ-glutamic acid)-based adjuvants and collagens generate in situ gel-forming hydrogel at physiological temperature. When the iPMH are immunized, they act as a pathogen-mimicking (MPLA, H1N1, H5N1) immune priming center and a depot for continuous stimulation of immune system, resulting in the induction of high levels (8.5 times higher) of antigen-specific IgG titers in the sera of mice and the increased number of IFN-γ-producing cells (7.3 times higher) compared with those in the groups immunized with antigen plus clinically used aluminum gels. Following the intranasal infection of the mouse adapted virus (emerging infectious 2009 H1N1 and highly pathogenic 2006 H5N1) at 50 times the 50% lethal dose, the mice immunized with viral antigens plus iPMH exhibit 100% protective immunity against lethal virus challenge.

The effect of different levels of crude protein on the pathophysiology of Haemonchus contortus infection in 2-year-old Boer dry does under confined conditions

The effect of dietary protein supplementation on the pathophysiology of a single Haemonchus contortus infection in 2-year-old Boer dry does under confined conditions was examined. Twenty-four does were randomly confined in individual pens and allocated into three groups balanced by their liveweight. The goats in the Control group were offered a basal diet whereas goats in Treatments 1 and 2 were fed the basal diet plus 25% and 50% dietary protein increments, respectively. Each animal was orally administrated 100 H. contortus L3 larvae/kg liveweight on Day 0 of the experiment. The goat liveweights and FAMACHA scores were recorded at weekly intervals. Packed cell volumes, eosinophil percentage, haemoglobin concentration, total serum protein, globulin, and albumin concentrations were recorded on Days 0 and 14, and then at weekly intervals during the experiments. Faecal egg counts were measured on Days 0, 21, and then at weekly intervals thereafter. Antigen-specific IgA, IgG, and IgM titres were determined from Enzyme-linked immunosorbent assays tests on Days 0, 28, and at the termination of the experiment. The results of this study showed that a single H. contortus L3 infection did not cause pathogenic effects in 2-year-old Boer dry does. Dietary protein supplementation significantly lowered faecal egg count and significantly enhanced IgG titres (P < 0.05). However, the higher protein diets did not have any significant effects on liveweight, packed cell volumes, haemoglobin concentration, biological parameters (total serum protein, albumin, and globulin concentrations), and peripheral antibodies (IgA and IgM titres) between the treatments. Further investigations are required to clarify the role of protein supplementation to control H. contortus infection in goats.

Immunogenicity of a chimeric Plasmodium falciparum merozoite surface protein vaccine in Aotus monkeys.

BackgroundThe production of properly folded, recombinant sub-unit Plasmodium falciparum malaria vaccine candidates in sufficient quantities is often a challenge. Success in vaccine immunogenicity studies in small animal models does not always predict immunogenicity in non-human primates and/or human subjects. The aim of this study was to assess the immunogenicity of a chimeric blood-stage malaria vaccine in Aotus monkeys. This vaccine candidate includes the neutralizing B cell epitopes of P. falciparum merozoite surface protein 1 (rPfMSP119) genetically linked to a highly immunogenic, well-conserved P. falciparum merozoite surface protein 8 (rPfMSP8 (ΔAsn/Asp)) partner.MethodsAotus nancymaae monkeys were immunized with purified rPfMSP1/8 or rPfMSP8 (ΔAsn/Asp) formulated with Montanide ISA 720 as adjuvant, or with adjuvant alone. Antibody responses to MSP119 and MSP8 domains were measured by ELISA following primary, secondary and tertiary immunizations. The functionality of vaccine-induced antibodies was assessed in a standard P. falciparum blood-stage in vitro growth inhibition assay. Non-parametric tests with corrections for multiple comparisons when appropriate were used to determine the significance of differences in antigen-specific IgG titres and in parasite growth inhibition.ResultsThe chimeric rPfMSP1/8 vaccine was shown to be well tolerated and highly immunogenic with boost-able antibody responses elicited to both PfMSP8 and PfMSP119 domains. Elicited antibodies were highly cross-reactive between FVO and 3D7 alleles of PfMSP119 and potently inhibited the in vitro growth of P. falciparum blood-stage parasites.ConclusionsSimilar to previous results with inbred and outbred mice and with rabbits, the PfMSP1/8 vaccine was shown to be highly effective in eliciting P. falciparum growth inhibitory antibodies upon immunization of non-human primates. The data support the further assessment of PfMSP1/8 as a component of a multivalent vaccine for use in human subjects. As important, the data indicate that rPfMSP8 (ΔAsn/Asp) can be used as a malaria specific carrier protein to: (1) drive production of antibody responses to neutralizing B cell epitopes of heterologous vaccine candidates and (2) facilitate production of properly folded, recombinant P. falciparum subunit vaccines in high yield.

Recombinant proteins from Gallibacterium anatis induces partial protection against heterologous challenge in egg-laying hens.

Gallibacterium anatis is a Gram-negative bacterium and major cause of salpingitis and peritonitis in egg-laying hens, thereby contributing to decreased egg production and increased mortality among the hens. Due to widespread drug resistance and antigenic diversity, novel prophylactic measures are urgently required. The aim of the present study was to evaluate the cross-protective capacity of three recombinant proteins recently identified as potential vaccine candidates; GtxA-N, GtxA-C, and FlfA, in an in vivo challenge model. Nine groups of birds were immunized twice with each protein, respectively, with 14 days separation. Additionally, three groups served as non-immunized controls. After 3 weeks, the birds were challenged with either of three G. anatis strains: 12656-12, 7990 or IPDH 697-78, respectively. Blood samples were taken at three different time points prior to challenge, as well as 48 h after challenge. All birds were euthanized and subjected to a post mortem procedure including scoring of lesions and sampling for bacterial growth. Moreover, ELISA assays were used to quantify antigen-specific IgG titers in serum. The results showed that all three proteins induced protection against the homologous strain 12656-12. No protein induced complete protection against strain 7990, although FlfA reduced the bacterial re-isolation rate. Moreover, immunization with GtxA-N and FlfA induced protection, while GtxA-C reduced the bacterial re-isolation, against strain IPDH 697-78. Thus although complete cross-protection against all three strains was not achieved, the results hold great promise for a new generation of immunogens in the search for novel prophylactic measures against G. anatis.

Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 and Pfs48/45.

Purified protein vaccines often require adjuvants for efficient stimulation of immune responses. There is no licensed mucosal adjuvant on the market to adequately boost the immune response to purified antigens for intranasal applications in humans. Bacterial outer membrane vesicles (OMV) are attractive candidates potentially combining antigenic and adjuvant properties in one substance. To more precisely characterize the potential of Escherichia coli OMV for intranasal vaccination with heterologous antigens, immune responses for AnAPN1 and Pfs48/45 as well as ovalbumin as a reference antigen were assessed in mice. The intranasal adjuvant cholera toxin (CT) and parenteral adjuvant MF59C.1 were used in comparison. Vaccinations were administered intranasally or subcutaneously. Antibodies (total IgG and IgM as well as subclasses IgG1, IgG2a, IgG2b, and IgG3) were measured by ELISA. T cell responses (cytotoxic T cells, Th1, Th17, and regulatory T cells) were determined by flow cytometry. When OMV were used as adjuvant for intranasal immunization, antibody and cellular responses against all three antigens could be induced, comparable to cholera toxin and MF59C.1. Antigen-specific IgG titres above 1 : 105 could be detected in all groups. This study provides the rationale for further development of OMV as a vaccination strategy in malaria and other diseases.

Immunological adjuvant effect of the peptide fraction from the larvae of Musca domestica.

BackgroundThe larvae of Musca domestica (Diptera: Muscidae) have been used traditionally for malnutritional stagnation, decubital necrosis, osteomyelitis, ecthyma and lip scald and also to treat coma and gastric cancer in the traditional Chinese medicine. Its immunomodulatory effects in naïve mice in relation to the traditional uses were also reported. However, the immunological adjuvant potentials of this insect have not yet been studied.MethodsThe peptide fraction from the larvae of Musca domestica L. (MDPF) was evaluated for its adjuvant potentials on the immune responses to ovalbumin (OVA) and avian influenza vaccine (rL–H5) by determining antigen-specific antibody titers, splenocyte proliferation, activity of natural killer (NK) cell, the secretion of cytokines from splenocytes in the immunized mice.ResultsMDPF significantly enhanced not only the concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-stimulated splenocyte proliferation, but serum antigen-specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the mice immunized with OVA and rL–H5. MDPF also remarkably promoted the killing activities of NK cells in splenocytes from the mice immunized with rL–H5. Furthermore, MDPF significantly promoted the production of Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines from splenocytes in the immunized mice.ConclusionsThe results indicated that MDPF had a potential to increase both cellular and humoral immune responses and elicit a balanced Th1/Th2 response, and that MDPF may be a safe and efficacious vaccine adjuvant candidate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0951-6) contains supplementary material, which is available to authorized users.

Maternal vaccination with a fimbrial tip adhesin and passive protection of neonatal mice against lethal human enterotoxigenic Escherichia coli challenge.

Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain H10407, which has been extensively studied in volunteers, an inoculum of 2 × 10(7) bacteria resulted in 50% lethal doses (LD50) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD50 challenge with H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.

MF59- and Al(OH)3-Adjuvanted Staphylococcus aureus (4C-Staph) Vaccines Induce Sustained Protective Humoral and Cellular Immune Responses, with a Critical Role for Effector CD4 T Cells at Low Antibody Titers.

Staphylococcus aureus (S. aureus) is an important opportunistic pathogen that may cause invasive life-threatening infections, like sepsis and pneumonia. Due to the increasing antibiotic resistance, the development of an effective vaccine against S. aureus is needed. Although a correlate of protection against staphylococcal diseases is not yet established, several findings suggest that both antibodies and CD4 T cells might contribute to optimal immunity. In this study, we show that adjuvanting a multivalent vaccine (4C-Staph) with MF59, an oil-in-water emulsion licensed in human vaccines, further potentiated antigen-specific IgG titers and CD4 T-cell responses compared to alum and conferred protection in the peritonitis model of S. aureus infection. Moreover, we showed that MF59- and alum-adjuvanted 4C-Staph vaccines induced persistent antigen-specific humoral and T-cell responses, and protected mice from infection up to 4 months after immunization. Furthermore, 4C-Staph formulated with MF59 was used to investigate which immune compartment is involved in vaccine-induced protection. Using CD4 T cell-depleted mice or B cell-deficient mice, we demonstrated that both T and B-cell responses contributed to 4C-Staph vaccine-mediated protective immunity. However, the role of CD4 T cells seemed more evident in the presence of low-antibody responses. This study provides preclinical data further supporting the use of the adjuvanted 4C-Staph vaccines against S. aureus diseases, and provides critical insights on the correlates of protective immunity necessary to combat this pathogen.

Uniform-sized water-in-oil vaccine formulations enhance immune response against Newcastle disease and avian influenza in chickens.

Water-in-oil (W/O) emulsion is one of the best "one-shot" delivery system for antigen to generate high and durable antibody response. Here a facile method using premixed Shirasu Porous Glass (SPG) membrane emulsification technique was employed to fabricate uniform-sized emulsion and mechanical stirring was employed to prepare non-uniform-sized emulsion as control. In release kinetics studies, uniform-sized emulsion showed low initial release, and during the followed 17 days, uniform-sized emulsions released antigen faster than emulsions of non-uniform size. In addition, vaccination studies against Newcastle disease virus (NDV) and avian Influenza virus (AIV) demonstrated that emulsion of uniform size induced higher HI antibodies and antigen-specific IgG titers. Furthermore, chickens vaccinated with emulsion of uniform size had a significantly greater ratio of CD8+ T cells to CD4+ T cells and a higher percentage of CD8+CD4+ T cells. Taken together, these results indicated that emulsion of uniform size induced a more potent immune response than emulsion prepared by mechanical stirring, and warranted the use of SPG membrane emulsification technique in generating new avian vaccines.

Adjuvant-active fraction from Albizia julibrissin saponins improves immune responses by inducing cytokine and chemokine at the site of injection.

The total saponin from the stem bark of Albizia julibrissin (AJSt) has previously showed the adjuvant potentials in mice. In this study, AJSt was subjected to resin column chromatography to afford four fractions (AJS30, AJS50, AJS75 and AJS95), and these fractions were further compared for the hemolytic activities and adjuvant potentials on the immune response to ovalbumin (OVA) and recombinant fowl pox virus vector-based avian influenza vaccine (rFPV). AJSt, AJS50, AJS75 and AJS95 showed a slight hemolytic effect. AJSt, AJS50 and AJS75 significantly enhanced not only the concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-stimulated splenocyte proliferation, but also serum antigen-specific IgG, IgG1, IgG2a and IgG2b antibody titers in the mice immunized with OVA and rFPV. AJSt, AJS50 and AJS75 also significantly promoted the NK cell activity and delayed-type hypersensitivity (DTH) in the OVA-immunized mice. Furthermore, the mechanisms of adjuvant action were explored by determining the effects of AJS75 on cytokines and chemokines at the site of injection using antibody array. AJS75 induced or up-regulated the protein expression of 12 cytokines (IL-12p40, IL-12p40/p70, IFN-γ, IL-13, IL-1β, IL-6, IL-10, TNF-α, sTNFR I, sTNFR III, IL-3 and IL-9) and 10 chemokines (Eotaxin, I-TAC, MIG, MIP-1α, RANTES, TECK, Fracatlkine, FasL, M-CSF and GM-CSF) in the injected muscles. The results suggested that AJS75, the most adjuvant-active fraction of AJSt, could improve antigen-specific both cellular and humoral immune responses and simultaneously elicit a Th1/Th2 response by inducing cytokine and chemokine at the site of injection.

Crucial roles of interleukin-7 in the development of T follicular helper cells and in the induction of humoral immunity.

T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses. Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

Recombinant BCG coexpressing Ag85B, ESAT-6 and Rv3620c elicits specific Th1 immune responses in C57BL/6 mice

Tuberculosis (TB) remains to be an enormous global health problem. The inconsistent protection efficacy of Bacille Calmette-Guérin (BCG) calls for new vaccines for TB. One choice to improve the efficacy of BCG vaccine is recombinant BCG (rBCG). Experimental evidences have revealed that Ag85B, ESAT-6 and Rv3620c are important immunodominant antigens of Mycobacterium tuberculosis. In this study, we have constructed a novel rBCG expressing fusion protein Ag85B-ESAT6-Rv3620c and evaluated the immunogenicity of this rBCG in C57BL/6 mice. Results show that there is a strong TB-specific CD4+ and CD8+ T lymphocytes proliferation in mice immunized with this rBCG vaccine. A single dose immunization of rBCG could induce a significantly strong Th1 immune response characterized by an increasing ratio of antigen-specific IgG2b/IgG1 as well as a high expression level of Th1 cytokines such as IFN-γ, TNF-α and IL-2. This conclusion was confirmed by a decreased secretion of Th2 cytokine IL-10. Moreover, this rBCG induced a strong humoral response in mice with an increasing antigen-specific IgG titer. Therefore, we concluded that this rBCG could significantly increase both Th1 type cellular immune response and antigen-specific humoral response compared with BCG. The above observations demonstrated that rBCG::Ag85B-ESAT6-Rv3620c is a potential candidate vaccine against M. tuberculosis for further study.

Structural analysis and cross-protective efficacy of recombinant 87 kDa outer membrane protein (Omp87) of Pasteurella multocida serogroup B:2

Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic septicaemia (HS) in cattle and buffalo especially in tropical regions of Asian and African countries, is known to possess several outer membrane proteins (OMPs) as immunogenic antigens. In the present study, omp87 gene encoding for 87 kDa OMP (Omp87) protein of P. multocida serogroup B:2 strain P52, has been amplified (∼2304 bp), cloned in to pET32a vector and over-expressed in recombinant Escherichia coli as fusion protein. The recombinant Omp87 protein (∼102 kDa) including N-terminus hexa-histidine tag was purified under denaturing condition. Immunization of mice with rOmp87 resulted in increased antigen specific IgG titres in serum and provided protection of 66.6 and 83.3% following homologous (B:2) and heterologous (A:1) challenge, respectively. A homology model of Omp87 revealed the presence of two distinct domains; N-terminal domain with four POTRA repeats in the periplasmic space and a pore forming C-terminal β-barrel domain (β1- β16) in the outer membrane of P. multocida, which belong to Omp85-TpsB transporter superfamily of OMPs. The study indicated the potential possibilities to use rOmp87 protein along with suitable adjuvant in developing subunit vaccine for haemorrhagic septicaemia and pasteurellosis in livestock.

134: Interleukin-27 as a novel antagonist of ectopic lymphoid-like structure development in early inflammatory arthritis

Evaluation of joint tissue from early inflammatory arthritis patients through ultrasound-guided synovial biopsy has enabled disease classification based on local pathology. For example, the disease can be classed as fibroblast-, myeloid- or lymphoid-driven. Lymphoid histopathology can be further divided into patients displaying either diffuse infiltrates, or ectopic lymphoid-like structures (ELS) reminiscent of the follicular compartments in secondary lymphoid organs. Approximately 25% of rheumatoid arthritis (RA) patients develop ELS and display severe symptoms associated with local immune cell priming and autoantibody production. However, cytokine control of ELS development in inflammatory arthritis is poorly defined. Interleukin (IL)-27 is a negative regulator of adaptive immune responses. To investigate the role of IL-27 in inflammatory arthritis, wild type (WT) and IL-27 receptor (IL-27R)-deficient mice were exposed to antigen-induced arthritis. IL-27R-deficient mice developed exacerbated inflammatory arthritis, displaying increased synovial infiltrate, hypertrophy and bone erosions. Heightened peripheral T H 17- and B-cell responses in IL-27R-deficiency were reflected in increased serum IL-17 levels and antigen-specific IgG titers. Strikingly, IL-27R-deficient mice developed synovial histopathology reminiscent of RA patients with ELS, comprising synovial lymphoid aggregates with distinct T-cell (CD3 + ), B-cell (B220 + ) and DC (F/480 + , CD21 + ) areas that were absent in WT mice. Development of lymphoid aggregates was associated with elevated synovial expression of homeostatic chemokines that promote ectopic lymphoneogenesis (e.g. Cxcl13, Ccl21 ) and the transcriptional regulator Bcl-6 , which is involved in germinal center organization and functional regulation. Ectopic lymphoneogenesis in IL-27R-deficient mice was not associated with an increase in synovial IL-6 or IL-21 expression. Studies in clinical RA patients reflect our experimental outcomes and reveal reduced synovial IL-27 expression in patients with joint histopathology comprising ELS compared to patients with diffuse lymphocytic infiltrates. Thus, IL-27 may negatively regulate the formation of ELS in human pathologies such as RA and represents a potential biomarker of synovial histopathology in early RA.

Engineering Biomaterial-Associated Complement Activation to Improve Vaccine Efficacy

The complement system plays an important role in innate and adaptive immunity, which suggests that complement activation could be exploited as a potential strategy for vaccine adjuvants. Here we explored the potential of chitosan-based microparticles (CS-NH2 MPs) as a vaccine adjuvant with an active surface for complement activation due to the abundance of amino groups. In vaccination studies, using recombinant anthrax protective antigen as a model antigen, compared with the control microparticles (amino-cross-linked MPs), we found that microparticles (MPs) with abundant amino groups significantly enhanced higher antigen-specific IgG titers in vivo and enhanced the production of IL-4 and IFN-γ with ex vivo restimulation. Furthermore, proliferative responses of splenocytes to ex vivo antigen restimulation were enhanced following immunization with MPs with amino groups. Overall, these results indicated that CS-NH2 MPs with a high surface density of amino groups were favorable for complement activation and immune responses. Our data provide further design principles for studies on complement-activating MPs as a vaccine platform.

Juzentaihoto Failed to Augment Antigen-Specific Immunity but Prevented Deterioration of Patients’ Conditions in Advanced Pancreatic Cancer under Personalized Peptide Vaccine

Juzentaihoto (JTT) is a well-known Japanese herbal medicine, which has been reported to modulate immune responses and enhance antitumor immunity in animal models. However, it is not clear whether JTT has similar effects on humans. In particular, there is little information on the effects of JTT in antigen-specific immunity in cancer patients. Here we conducted a randomized clinical study to investigate whether combined usage of JTT could affect antigen-specific immunity and clinical findings in advanced pancreatic cancer patients undergoing personalized peptide vaccination (PPV), in which HLA-matched vaccine antigens were selected based on the preexisting host immunity. Fifty-seven patients were randomly assigned to receive PPV with (n = 28) or without (n = 29) JTT. Unexpectedly, JTT did not significantly affect cellular or humoral immune responses specific to the vaccine antigens, which were determined by antigen-specific interferon-γ secretion in T cells and antigen-specific IgG titers in plasma, respectively. Nevertheless, JTT prevented deterioration of patients' conditions, such as anemia, lymphopenia, hypoalbuminemia, plasma IL-6 elevation, and reduction of performance status, which are frequently observed in advanced cancers. To our knowledge, this is the first clinical study that examined the immunological and clinical effects of JTT in cancer patients undergoing immunotherapy in humans.

Structure–function relationship of the saponins from the roots of Platycodon grandiflorum for hemolytic and adjuvant activity

To assess the contribution of the aglycone and sugar chain to the biological activity of saponins from Platycodon grandiflorum, seven structurally consecutive saponins, platycodin D (PD), D2 (PD2), D3 (PD3), platycoside A (PA), E (PE), deapioplatycoside E (DPE), and polygalacin D2 (PGD) were compared for their hemolytic activities and adjuvant potentials on the immune responses to Newcastle disease virus-based recombinant avian influenza vaccine (rL-H5) in mice. Among seven compounds, the order of the hemolytic activity was PGD≈PD>PD2>PA>PD3>PE>DPE. PD, PD2, PA, and PGD significantly not only promoted concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-induced splenocyte proliferation, but enhanced the NK cell activity in mice immunized with rL-H5. PD and PD2 increased the antigen specific IgG, IgG1, IgG2a, and IgG2b antibody titers, while PA and PGD only induce the IgG and IgG1 antibody responses in the immunized mice. However, the other three saponins were not observed for adjuvant activity. The results suggested that the sugar chains attached to C-3, the glycidic moiety at C-28 of aglycone, as well as aglycone affect their biological activities. Interestingly, their hemolytic and adjuvant activities increased with the retention time by reverse phase HPLC analysis. The retention time may be useful for primary estimation of fundamental adjuvanticity of saponin with the same aglycone.

Bis-(3′,5′)-cyclic dimeric adenosine monophosphate: Strong Th1/Th2/Th17 promoting mucosal adjuvant

New effective adjuvants are required to improve the performance of subunit vaccines. Here, we showed that bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP), a second messenger molecule in bacteria and archaea, exerts strong adjuvant activities when delivered by mucosal route. In vitro studies showed that c-di-AMP was able to both stimulate pre-activated murine macrophages and promote the activation and maturation of dendritic cells of murine and human origin. Co-administration of c-di-AMP with β-galactosidase (β-Gal) by intranasal route to BALB/c mice resulted in the elicitation of significantly higher serum antigen-specific IgG titres than in controls. The induction of local immune responses was shown by the production of antigen-specific secretory IgA in different mucosal territories. In addition, strong cellular immune responses were observed against both the β-Gal protein and a peptide encompassing its MHC class I-restricted epitope. The ratio of β-Gal-specific antibodies and the secreted cytokine profiles by in vitro re-stimulated splenocytes suggested that a balanced Th1/Th2/Th17 response pattern is promoted by c-di-AMP. When C57BL/6 mice were immunized with OVA and c-di-AMP, vigorous in vivo CTL responses were also observed. These results indicated that c-di-AMP exhibits a high potential as adjuvant for the development of mucosal vaccines, in particular when cellular immunity is needed.

Transgenic expression of bovine neonatal Fc receptor in mice boosts immune response and improves hybridoma production efficiency without any sign of autoimmunity

The overexpression of the bovine neonatal Fc receptor (bFcRn) in transgenic (Tg) mice boosts humoral immune response with increased numbers of antigen-specific spleen cells and a potent humoral immune response against weakly immunogenic targets. One of the interesting questions surrounding this enhanced immune response is whether these Tg mice generate higher number of antigen-specific hybridomas. To address this question, we immunized these Tg mice and wild type (wt) controls with trinitrophenylated proteins, generated hybridomas and analyzed their numbers and specificities. We observed that Tg mice generated a 3–5 fold increase in antigen-specific IgG titers and had significantly larger spleens containing higher number of antigen-specific B cells and plasma cells, analyzed by ELISA and ELISPOT assays. Fusion of the isolated splenocytes with standard mouse myeloma cells (SP2/0-Ag14) resulted in a 2–4 fold elevation of hybridization frequency for the hapten, or carrier-specific IgG positive microcultures, in Tg mice compared to controls. In addition, as augmented immune reactivity leads to autoimmunity in some genetically modified mouse strains, we analyzed autoreactive antibody levels in serum samples derived from elderly bFcRn Tg mice by a protein chip assay. In contrast to the sample from the MRL/lpr mouse suffering from autoimmunity, we did not detect autoantibodies in bFcRn Tg mice or the wt controls. Based on these and our earlier data, we propose that Tg mice that overexpress bFcRn offer major advantages in monoclonal Ab production.