Ginseng stem‐leaf saponins in combination with selenium enhance immune responses to an attenuated pseudorabies virus vaccine

Pseudorabies, a herpesvirus infection, is mainly controlled by using attenuated live vaccines. In this study, the effect of ginseng stem and leaf saponins (GSLS) in combination with selenium (Se; in the form of sodium selenite) on vaccination against attenuated pseudorabies virus (aPrV) was evaluated. It was found that GSLS and Se have an adjuvant effect and that a combination of GSLS and Se stimulates significantly enhanced immune responses than does GSLS or Se alone. Following oral administration of GSLS, mice immunized with an attenuated PrV vaccine diluted in Se‐containing physiological saline solution (PSS) provoked a significantly stronger gB‐specific serum antibodies response (IgG, IgG1 and IgG2a), enhanced lymphocyte proliferation and cytolytic activity of NK cells, along with higher production of cytokines (IFN‐γ, IL‐12, IL‐5 and IL‐10) by splenocytes. Notably, the combination of GSLS and Se conferred a much higher resistance to fPrV challenge after immunization of the mice with aPrV vaccine. This study offers convincing experimental evidence that an injection of Se with oral GSLS is a promising adjuvant combination that improves the efficacy of vaccination against PrV and deserves further study regarding improvement of responses to other animal vaccines.     

Enhancing effects of the chemical adjuvant levamisole on the DNA vaccine pVIR‐P12A‐IL18‐3C

DNA‐based vaccination is an attractive alternative for overcoming the disadvantages of inactivated virus vaccines; however, DNA vaccines alone often generate only weak immune responses. In this study, the efficacy of LMS as a chemical adjuvant on a DNA vaccine (pVIR‐P12A‐IL18‐3C) encoding the P1‐2A and 3C genes of the FMDV and swine IL‐18, which provides protection against FMDV challenge, was tested. All test pigs were administered booster vaccinations 28 days after the initial inoculation, and were challenged with 1000 ID₅₀ FMDV O/NY00 20 days after the booster vaccination. Positive and negative control groups were inoculated with inactivated virus vaccine and PBS respectively. The DNA vaccine plus LMS induced greater humoral and cell‐mediated responses than the DNA vaccine alone, as evidenced by higher concentrations of neutralizing and specific anti‐FMDV antibodies, and by higher concentrations of T‐lymphocyte proliferation and IFN‐γ production, respectively. FMDV challenge revealed that the DNA vaccine plus LMS provided higher protection than the DNA vaccine alone. This study demonstrates that LMS may be useful as an adjuvant for improving the protective efficiency of DNA vaccination against FMDV in pigs.     

Platycodin D2 Improves Specific Cellular and Humoral Responses to Hepatitis B Surface Antigen in Mice

Platycodin D2 ( 1 ), a less hemolytic saponin from the root of Platycodon grandiflorum than platycodin D ( 2 ), was evaluated for the potential to enhance specific cellular and humoral immune responses to hepatitis B surface antigen (HBsAg) in mice. It significantly increased the concanavalin A (Con A)‐, lipopolysaccharide (LPS)‐, and HBsAg‐induced splenocyte proliferation in HBsAg‐immunized mice (P<0.05, P<0.01, and P<0.001, resp.). HBsAg‐specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the serum were also markedly enhanced by 1 compared to the HBsAg control group (P<0.01 or P<0.001). Moreover, 1 significantly promoted the production of Th1 (IL‐2 and IFN‐γ) and Th2 (IL‐4 and IL‐10) cytokines from splenocytes in the HBsAg‐immunized mice (P<0.001). The adjuvant potential of 1 on splenocyte proliferation, serum HBsAg‐specific IgG2a and IgG2b antibody response, as well as Th1‐cytokine secretion from splenocytes in the HBsAg‐immunized mice was higher than that of Alum. The results suggest that 1 could improve both cellular and humoral immune responses to HBsAg in mice. Hence, 1 might be a promising adjuvant for hepatitis B vaccine with dual Th1‐ and Th2‐potentiating activity.     

Resiquimod and polyinosinic–polycytidylic acid formulation with aluminum hydroxide as an adjuvant for foot-and-mouth disease vaccine

Background

Toll-like receptor (TLR) agonists reportedly have potent antiviral and antitumor activities and may be a new kind of adjuvant for enhancing immune efficacy. Resiquimod (R848) is an imidazoquinoline compound with potent antiviral activity and functions through the TLR7/TLR8 MyD88-dependent signaling pathway. Polyinosinic-polycytidylic acid [poly(I:C)] is a synthetic analog of double-stranded RNA that induces the production of pro-inflammatory cytokines by the activation of NF-κB through TLR3. This study investigated the potential of R848 and poly(I:C) as an adjuvant 146S foot-and-mouth disease virus (FMDV) vaccine formulated with aluminum hydroxide (Al(OH)₃).

Results

Antibody titers to FMDV and CD8⁺ T cells were markedly enhanced in mice immunized to 146S FMDV?+?Al(OH)₃?+?R848?+?poly(I:C) compared with mice immunized to FMDV?+?ISA206. IFN-γ secretion substantially increased compared with IL-4 secretion by splenic T cells stimulated with FMDV antigens in vitro, suggesting that R848, poly(I:C), and with Al(OH)₃ together biased the immune response toward a Th1-type direction.

Conclusions

These results indicated that the R848 and poly(I:C) together with Al(OH)₃ enhanced humoral and cellular immune responses to immunization with 146S FMDV antigens. Thus, this new vaccine formulation can be used for FMDV prevention.     

Oral immunization of mice with Saccharomyces cerevisiae expressing a neutralizing epitope of ApxIIA exotoxin from Actinobacillus pleuropneumoniae induces systemic and mucosal immune responses

An oral delivery system based on ApxIIA#5‐expressed on Saccharomyces cerevisiae was studied for its potential to induce immune responses in mice. Murine bone marrow‐derived dendritic cells (DCs) stimulated in vitro with ApxIIA#5‐expressed on S. cerevisiae upregulated the expression of maturation and activation markers, leading to production of tumor necrosis factor‐α, interleukin (IL)‐1β, IL‐12p70 and IL‐10. Presentation of these activated DCs to cluster of differentiation CD4+ T cells collected from mice that had been orally immunized with the ApxIIA#5‐expressed on S. cerevisiae elicited specific T‐cell proliferation. In addition, the orally immunized mice had stronger antigen‐specific serum IgG and IgA antibody responses and larger numbers of antigen‐specific IgG and IgA antibody‐secreting cells in their spleens, Peyer's patches and lamina propria than did those immunized with vector‐only S. cerevisiae or those not immunized. Furthermore, oral immunization induced T helper 1‐type immune responses mediated via increased serum concentrations of IgG2a and an increase predominantly of IFN‐γ‐producing cells in their spleens and lamina propria. Our findings suggest that surface‐displayed ApxIIA#5‐expressed on S. cerevisiae may be a promising candidate for an oral vaccine delivery system for eliciting systemic and mucosal immunity.     

Lipopolysaccharide accelerates collagen‐induced arthritis in association with rapid and continuous production of inflammatory mediators and anti‐type II collagen antibody

Collagen‐induced arthritis (CIA) is an animal model for rheumatoid arthritis (RA). Lipopolysaccharide (LPS) is known to accelerate CIA; however, the pathogenetic mechanisms are not yet fully understood. In this study, type II collagen (CII)‐immunized mice were found to have marked increases in degree of expression of mRNA of inflammatory mediators such as tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐1β, and macrophage inflammatory protein‐2 (MIP‐2) in their arthritic paws and of serum anti‐CII antibody concentration before the onset of arthritis induced by LPS injection. The gene expression was rapid and continuous after direct activation of nuclear factor κB. The amounts of mRNA of TNF‐α, IL‐1β, and MIP‐2, as well as of matrix metalloproteinases and the receptor activator of nuclear factor κB ligand, increased with the development of arthritis, correlated positively with clinical severity and corresponded with histopathological changes. Moreover, anti‐TNF‐α neutralizing antibody inhibited the development of LPS‐accelerated CIA and a single injection of recombinant mouse TNF‐α induced increases in anti‐CII antibody concentrations, suggesting TNF‐α may contribute to the development of arthritis by both initiation of inflammation and production of autoantibodies. These data suggest that exacerbation of RA by LPS is associated with rapid and continuous production of inflammatory mediators and autoantibodies.     

Adjuvant effect of CIA07, a combination of Escherichia coli DNA fragments and modified lipopolysaccharides, on the immune response to hepatitis B virus surface antigen

CIA07 is an immunostimulatory agent composed of bacterial DNA fragments and modified lipopolysaccharide, which has antitumor activity against bladder cancer in mice. In this study, the adjuvant activity of CIA07 was evaluated using hepatitis B virus surface antigen (HBsAg) as the immunogen. Mice were immunized intramuscularly three times at 1-week intervals with HBsAg alone or in combination with alum, bacterial DNA fragments, modified lipopolysaccharide, CIA07 or CpG1826, and immune responses were assessed. At 1 week after the final injection, the HBsAg-specific total serum IgG antibody titer in CIA07-treated mice was 14 times higher than that in animals administered antigen alone, six times higher than in mice given alum or bacterial DNA fragments and twice as high as those treated with modified lipopolysaccharide or CpG1826, and remained maximal until 8 weeks postimmunization. Animals receiving antigen alone or plus alum displayed barely detectable HBsAg-specific serum IgG2a antibody responses. However, coadministration of CIA07 with antigen led to markedly enhanced serum IgG2a antibody titer and IFN-gamma(+) production in splenocytes, indicating that CIA07 effectively induces Th1-type immune responses. In addition, the number of HBsAg-specific CD8(+) T cells in peripheral blood mononuclear cells was elevated in CIA07-treated mice. These data clearly demonstrate that CIA07 is able to induce both cellular and humoral immune responses to HBsAg, and confirm its potential as an adjuvant in therapeutic vaccines for hepatitis B virus infections.     

人参多糖对新甲型H1N1流感病毒灭活疫苗的免疫增强作用

在流感灭活疫苗中添加佐剂可以提高疫苗的免疫原性,节约抗原用量。一些天然中草药多糖具有潜在的佐剂效应。本文探讨了人参多糖（ginseng polysaccharide,GPS）在新甲型H1N1流感病毒裂解型灭活疫苗中的佐剂效应。将不同剂量GPS与新甲型H1N1流感病毒灭活疫苗混合,共同免疫小鼠一次,通过检测免疫后在小鼠体内诱导产生的疫苗特异性IgM、IgG、IgG1和IgG2a抗体情况来评价GPS作为流感病毒灭活疫苗佐剂的免疫增强效果,并与不添加佐剂的疫苗和加有铝佐剂的疫苗的免疫效果作比较。结果显示,GPS与铝佐剂一样能显著提高和维持疫苗特异性IgG抗体滴度,同时提高IgM抗体水平,其中800μgGPS的佐剂效果最好。因此我们认为GPS可以作为流感病毒灭活疫苗的一种候选佐剂。     

A Novel Mucosal Vaccine Against Foot-and-Mouth Disease Virus Induces Protection in Mice and Swine

Epitopes of a foot-and-mouth disease virus (FMDV) capsid protein VP1 complex and a chimera of 6×His-tagged cholera toxin B subunit (hCTB) were expressed in Hansenula polymorpha and used together as a mucosal vaccine. Antibody and cytokine responses to VP1–hCTB vaccine and protection against FMDV were evaluated by ELISA and a virus challenge test in mice, respectively. VP1–hCTB directly enhanced the expression of interleukin-5 (IL-5) both in serum and supernatants of cultured spleen cells. After challenging suckling mice with 10⁵ FMDV (=50% lethal dosage per mouse) a greater protection was seen after intraperitoneal and intranasal vaccinations than after oral vaccination. In swine immunized with VP1–hCTB, immune responses were achieved after three administrations, and the vaccine protected swine (80%) when challenged with 10^6.5 FMDV (=50% infectious dosage per swine). These results demonstrated the possibility of using CTB as a mucosal adjuvant to elicit protective immune responses against FMDV. Houhui Song, Zhiliang Wang and Dongxia Zheng contributed equally to this work.     

Plant‐derived recombinant immune complexes as self‐adjuvanting TB immunogens for mucosal boosting of BCG

Progress with protein‐based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency‐associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self‐polymerization of the complexes. TB‐RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen‐presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB‐RICs developed antigen‐specific serum IgG antibody responses with mean end‐point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN‐γ, IL‐2 and TNF‐α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG‐immunized mice with TB‐RICs led to a reduced M. tuberculosis infection in their lungs from log₁₀ mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant‐expressed TB‐RICs represent a novel molecular platform for developing self‐adjuvanting mucosal vaccines.     

Interleukin-1 family cytokines as mucosal vaccine adjuvants for induction of protective immunity against influenza virus

A safe and potent adjuvant is needed for development of mucosal vaccines against etiological agents, such as influenza virus, that enter the host at mucosal surfaces. Cytokines are potential adjuvants for mucosal vaccines because they can enhance primary and memory immune responses enough to protect against some infectious agents. For this study, we tested 26 interleukin (IL) cytokines as mucosal vaccine adjuvants and compared their abilities to induce antigen (Ag)-specific immune responses against influenza virus. In mice intranasally immunized with recombinant influenza virus hemagglutinin (rHA) plus one of the IL cytokines, IL-1 family cytokines (i.e., IL-1α, IL-1β, IL-18, and IL-33) were found to increase Ag-specific immunoglobulin G (IgG) in plasma and IgA in mucosal secretions compared to those after immunization with rHA alone. In addition, high levels of both Th1- and Th2-type cytokines were observed in mice immunized with rHA plus an IL-1 family cytokine. Furthermore, mice intranasally immunized with rHA plus an IL-1 family cytokine had significant protection against a lethal influenza virus infection. Interestingly, the adjuvant effects of IL-18 and IL-33 were significantly decreased in mast cell-deficient W/W(v) mice, indicating that mast cells have an important role in induction of Ag-specific mucosal immune responses induced by IL-1 family cytokines. In summary, our results demonstrate that IL-1 family cytokines are potential mucosal vaccine adjuvants and can induce Ag-specific immune responses for protection against pathogens like influenza virus.     

RKIP mediates autoimmune inflammation by positively regulating IL‐17R signaling

Th17 cells contribute to the development of autoimmune diseases by secreting interleukin‐17 (IL‐17), which activates its receptor (IL‐17R) that is expressed on epithelial cells, macrophages, microglia, and resident neuroectodermal cells. However, the mechanisms through which IL‐17R‐mediated signaling contributes to the development of autoimmune disease have not been completely elucidated. Here, we demonstrate that Raf‐1 kinase inhibitor protein (RKIP) deficiency in mice ameliorates the symptoms of experimental autoimmune encephalomyelitis (EAE). Adoptive T‐cell‐transfer experiments demonstrate that RKIP plays a predominant role in Th17‐mediated, but not in Th1‐mediated immune responses. RKIP deficiency has no effect on Th17‐cell differentiation ex vivo, nor does it affect Th17‐cell differentiation in EAE mice. However, RKIP significantly promotes IL‐17R‐induced proinflammatory cytokine and chemokine production. Mechanistically, RKIP directly interacts with IL‐17RA and Act1 to promote the formation of an IL‐17R‐Act1 complex, resulting in enhanced MAPK‐ and P65‐mediated NF‐κB activation and downstream cytokine production. Together, these findings indicate that RKIP functions as an essential modulator of the IL‐17R‐Act1 axis in IL‐17R signaling, which promotes IL‐17‐induced inflammation and autoimmune neuroinflammation.     

Long‐term regulation of local cytokine production following immunization in mice

Vaccines based on pathogen components require adjuvants to enhance the antigen‐specific adaptive immune response. Intramuscular injection of adjuvanted‐vaccines induces inflammatory cytokines and inflammatory nodules at the injection site within 48 hr after injection (Vaccine 2014; 32 : 3393–401). In the present study, long‐term regulation of cytokine production was investigated at 3, 6, 24, and 48 hr, 5 and 7 days, and 2 and 4 weeks after immunization with human papilloma virus (HPV), diphtheria and tetanus toxoids combined with acellular pertussis (DTaP), Haemophilus influenza type B (Hib), and pneumococcal conjugated (PCV) vaccines in mouse models. The second dose was given 4 weeks later, and cytokine profiles were investigated 2, 5, and 7 days after re‐immunization. IL‐1β, IL‐6, granulocyte‐colony stimulating factor (G‐CSF), and MCP‐1 were produced from 3 hr and peaked at 48 hr after immunization with Cervarix in mice. IL‐4, MCP‐1, and TNF‐α peaked at 5 or 7 days after immunization with Gardasil. These cytokines decreased 7 days after immunization with Cervarix and Gardasil. After the second dose, similar responses were observed. Both vaccines induced neutrophil extracellular traps (NET) in inflammatory nodules. The peak amount of IL‐1β, IL‐6, G‐CSF, and MCP‐1 was observed on day 5 of immunization and that of IL‐4 on days 5‐7 of immunization with DTaP, but no increase in IL‐6 and G‐CSF was observed after re‐immunization. A similar response was noted after immunization with PCV13. An inflammatory response is essential for the development of adaptive immunity through the production of inflammatory cytokines.

     

IL‐6 Trans‐Signaling Plays Important Protective Roles in Acute Liver Injury Induced by Acetaminophen in Mice

Our study was undertaken to evaluate the important role of interleukin‐6 (IL‐6) trans‐signaling in acetaminophen (AAP)‐induced liver injury. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL‐6 trans‐signaling, whereas an IL‐6/soluble IL‐6 receptor (sIL‐6R) fusion protein (hyper‐IL‐6) mimics IL‐6 trans‐signaling. Using these tools, we investigated the role of IL‐6 trans‐signaling in AAP‐induced liver injury. Blockade of IL‐6 trans‐signaling during AAP‐induced liver injury remarkably increased the levels of serum aspartate aminotransferase and alanine aminotransferase; lowered the level of serum sIL‐6R; aggravated liver injury; inhibited the expression of phosphorylation of STAT3 (pSTAT3), proliferating cell nuclear antigen, vascular endothelial growth factor, and glycogen synthesis; and induced the expression of Caspase3, cytochrome P450 2E1 (CYP2E1), and hepatocyte apoptosis in the liver of mice. In summary, our study suggested that IL‐6 trans‐signaling plays important protective roles by regulating the hepatocyte proliferation and apoptosis, angiogenesis, CYP2E1 expression, and glycogen metabolism during AAP‐induced liver injury in mice.     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体(pmSEA),对HBVDNA疫苗诱导Balbc小鼠(H2d)免疫应答的调节作用。肌内注射空载体pcDNA3、HBVDNA疫苗加pmSEA佐剂(pHBVS2S+pmSEA)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs;ELISPOT检测分泌IFNγ的脾淋巴细胞;4h51Cr释放法检测小鼠脾细胞CTL活性。HBVDNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBVDNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFNγ的分泌量是不加佐剂组2～3倍。CTL细胞杀伤活性(E:T=100)佐剂组与不加佐剂组分别为:69.77%±7.5%、42.81%±7.7%,差异显著(P<0.05)。HBVDNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA疫苗的免疫应答,有望成为DNA疫苗的免疫佐剂。     

Peptidoglycan‐induced T helper 2 immune response in mice involves interleukin‐10 secretion from Langerhans cells

Patients with atopic dermatitis (AD) have superficial skin colonization with Staphylococcus aureus and an increased number of T helper (Th)2 cells in their peripheral blood. The purpose of this study was to clarify the involvement of interleukin (IL)‐10 secretion from Langerhans cells (LCs) in staphylococcal peptidoglycan (PEG)‐induced Th2 immune responses in mice. Mice were primed with LCs pulsed with PEG (or LPS) and ovalbumin (OVA) and then given a booster OVA injection 2 days later in the hind footpad. Five days after the OVA injection, cytokine responses in the draining popliteal lymph nodes were investigated by RT‐PCR and ELISA. Production of both IL‐10 and IL‐12 by cultured LCs was detected by ELISA. Administration of PEG‐ or LPS‐stimulated LCs into the hind footpads of the mice induced Th2‐prone and Th1‐prone immune responses, respectively, as represented by expression of IL‐4 and interferon ‐γ . In vitro experiments showed that PEG induced greater production of IL‐12 p40 from LCs than did LPS, whereas LPS induced greater production of IL‐12 p70 from LCs than did PEG. Furthermore, it was found that PEG‐stimulated LCs induced greater production of IL‐10 than did LPS‐stimulated LCs, and that neutralization of IL‐10 augmented IL‐12 p70 production and inhibited Th2 development by PEG‐stimulated LCs. These results suggest that PEG can induce Th2 development through down‐regulation of IL‐12 p70 production by LCs in an IL‐10 production‐dependent manner and would explain the role of S. aureus colonization in patients with AD.