Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine.

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.

Stimulation of growth of the human gastric pathogen Helicobacter pylori by atmospheric level of oxygen under high carbon dioxide tension.

BACKGROUND: Helicobacter pylori (Hp), a human pathogen that is associated with gastritis, peptic ulcer, and gastric cancer, has been considered a microaerophile, but there is no general consensus about its specific O2 requirements. A clear understanding of Hp physiology is needed to elucidate the pathogenic mechanism(s) of Hp infection. RESULTS: We cultured Hp under a range of O2 levels with or without 10% CO2 and evaluated growth profiles, morphology, intracellular pH, and energy metabolism. We found that, in the presence of 10% CO2, the normal atmospheric level of O2 inhibited Hp growth at low density but stimulated growth at a higher density. Field emission scanning electron microscopy and fluorescence microscopy of Hp cells cultured under 20% O2 tension revealed live spiral-shaped bacteria with outer membrane vesicles on a rugged cell surface, which became smooth during the stationary phase. Fermentation products including acetate, lactate, and succinate were detected in cell culture media grown under microaerobic conditions, but not under the aerobic condition. CO2 deprivation for less than 24 h did not markedly change cytoplasmic or periplasmic pH, suggesting that cellular pH homeostasis alone cannot account for the capnophilic nature of Hp. Further, CO2 deprivation significantly increased intracellular levels of ppGpp and ATP but significantly decreased cellular mRNA levels, suggesting induction of the stringent response. CONCLUSIONS: We conclude, unlike previous reports, that H. pylori may be a capnophilic aerobe whose growth is promoted by atmospheric oxygen levels in the presence of 10% CO2. Our data also suggest that buffering of intracellular pH alone cannot account for the CO2 requirement of H. pylori and that CO2 deprivation initiates the stringent response in H. pylori. Our findings may provide new insight into the physiology of this fastidious human pathogen.

Culture media optimization for Chinese hamster ovary cell growth and expression of recombinant varicella-zoster virus glycoprotein E.

Herpes zoster (HZ) is caused by reactivation of varicella-zoster virus (VZV) latent in the sensory ganglia and causes severe pain, often leading to postherpetic neuralgia (PHN). Two prophylactic vaccines against HZ are currently licensed for human use, a live attenuated vaccine and a subunit vaccine containing recombinant VZV glycoprotein E (gE) as antigen. The latter has superior protective efficacy against HZ and PHN. During HZ subunit vaccine development, we obtained Chinese hamster ovary (CHO) cell clones expressing VZV gE. This study was performed to optimize culture media conditions for CHO cell growth and gE production. Using a high-throughput culture system, three CHO cell clones were cultured in microtiter plates containing 24 different basal media, and three basal media were selected. The clone with the highest gE expression was fed-batch cultured in each of the three basal media in combination with 13 different feed media. A pair of media, BalanCD CHO Growth A and EX-CELL Advanced CHO Feed 1, with the highest productivity was selected for gE production. Scale-up fed-batch cultures of the selected clone cultured in a wave bag bioreactor containing the optimized media yielded 2440 mg gE protein/L culture, a 11.5-fold increase compared to original culture conditions (batch culture in CD OptiCHO medium). The optimized media condition is used to produce VZV gE antigen for an HZ subunit vaccine, which is under phase I clinical trial. This study would provide valuable insights on culture media optimization for CHO cells expressing a recombinant vaccine antigen. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-021-00468-1.

The Effect of a TLR4 Agonist/Cationic Liposome Adjuvant on Varicella-Zoster Virus Glycoprotein E Vaccine Efficacy: Antigen Presentation, Uptake, and Delivery to Lymph Nodes.

Adjuvant CIA09, composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based cationic liposomes and the toll-like receptor 4 agonist de-O-acylated lipooligosaccharide (dLOS), has been shown to enhance antibody and cellular immune responses to varicella-zoster virus (VZV) glycoprotein E (gE), recombinant tuberculosis vaccine antigen, and inactivated Japanese encephalitis vaccine. In this study, we investigated its modes of action using VZV gE as a model antigen. Liposomes adsorbed gE and cooperatively with dLOS promoted endocytosis-mediated cellular uptake of gE by mouse dendritic cells in vitro. CIA09 increased the stability and cellular uptake of the antigen at the muscle site of injection, and induced immune cell recruitment and cytokine and chemokine production, which led to efficient antigen delivery to draining lymph nodes. Mouse bone marrow-derived dendritic cells, pulsed with CIA09-adjuvanted gE, efficiently presented gE to antigen-specific T cells, inducing Th1-type biased immunity, as shown by high IFN-γ production. The data indicate that liposomes and dLOS cooperate in the adjuvant activity of CIA09 by promoting antigen uptake and delivery to lymph nodes as well as antigen presentation to T cells.

Efficient induction of cell-mediated immunity to varicella-zoster virus glycoprotein E co-lyophilized with a cationic liposome-based adjuvant in mice.

Varicella zoster virus (VZV) is a neurotropic and lymphotropic alpha herpesvirus that causes varicella and herpes zoster (HZ). At a primary infection, VZV causes varicella in young children. Reactivation of latent VZV in sensory ganglia causes painful HZ in elderly people, occasionally leading to a serious complication, postherpetic neuralgia (PHN). A live attenuated VZV vaccine, the first vaccine licensed for the prevention of HZ and PHN is not very effective, while a recombinant subunit vaccine provides higher and longer protection against HZ. In the present study, we developed a new adjuvant system CIA09A, which is composed of cationic liposomes, the Toll-like receptor 4 (TLR4) agonist de-O-acylated lipooligosaccharide, and Quillaja saponin fraction QS-21. We then determined its adjuvant activity for recombinant VZV glycoprotein E (gE) in mice. Co-lyophilization of the liposomal adjuvant formulation with gE did not abolish the immune-stimulating activity. In fact, the CIA09A-adjuvanted gE vaccine was highly effective in eliciting both humoral and cellular immune responses to the recombinant gE protein and VZV in a VZV-primed mouse model. Furthermore, the frequency of gE-specific polyfunctional CD4+ T cells expressing interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2 was significantly increased in mice immunized with the adjuvanted vaccine. These data indicate that co-lyophilization of protein antigens with CIA09A enables development of a liposome-adjuvanted vaccine in a single vial to induce strong cell-mediated immunity required for vaccine efficacy. Thus, the CIA09A-adjuvanted gE vaccine warrants further development as a new prophylactic vaccine against HZ.

Analysis of growth phase-dependent proteome profiles reveals differential regulation of mRNA and protein in Helicobacter pylori.

Helicobacter pylori is a slow growing, microaerophilic bacterium that causes various gastric diseases. To understand the growth phase-dependent global regulation of protein in H. pylori, we analyzed the proteome profiles of H. pylori 26695 harvested during the course of in vitro culture. Temporal changes in protein profiles were assessed using three independent cultures harvested at 6, 12, 24, 36, 48, and 60 h. Compared with the protein spots obtained at 6 h, 151 protein spots obtained at other time points exhibited significantly altered intensity, with 57 of these protein spots identified by MALDI-TOF MS analysis. Clustering analysis showed that overall protein profile was coordinated in accordance with the growth phases of the culture. When we compared mRNA transcript levels of the identified proteins, obtained from RT-PCR analysis, with their protein levels, we observed substantial discrepancies in their patterns, suggesting that the transcriptome and proteome of H. pylori were differentially regulated during in vitro culture. Proteomic analysis also suggested that several H. pylori proteins underwent PTMs, some of which were modulated as a function of the growth phase of the culture. These findings indicate that H. pylori utilizes modulation of protein regulation and PTM as mechanisms to cope with changing growth environments. These observations should provide insight into the adaptive mechanisms employed by H. pylori within the context of growth environments.

Enhancement of the immune responses of mice to Bacillus anthracis protective antigen by CIA07 combined with alum.

Anthrax is an acute zoonotic disease caused by infection with Bacillus anthracis. B. anthracis spores are highly resistant to environmental degradation and are used as a biological weapon. In this study, we investigated the adjuvant activity of CIA07 to anthrax protective antigen (PA). A/J mice were immunized intraperitoneally once, or twice with a 4-week interval, with recombinant PA alone or combined with alum, CpG1826, or CIA07 as adjuvant, and serum anti-PA IgG antibody responses were measured 4 weeks after each immunization. All three adjuvants significantly enhanced anti-PA IgG antibody titer 4 weeks after the priming and boosting immunizations, and alum gave the highest titer. In order to evaluate the adjuvant activity of CIA07 in the presence of alum, Balb/c mice were immunized 3 times at 1-week intervals with PA in combination with alum, CIA07 or alum plus CIA07, and the immune responses were assessed 2 weeks after the third immunization. The serum anti-PA IgG antibody titer of the CIA07-treated group was 14-fold higher than the group given PA alone, and the coadministration of CIA07 with alum further increased the titer 3.5-fold (P < 0.05). The toxin neutralizing activity of the sera from the mice given the combination of CIA07 and alum was 109-times higher than the animals given PA alone. The mice given CIA07 plus alum also showed a marked increase in the number of IFN-gamma-, IL-2-, and IL-4-producing CD4(+) T cells among their splenocytes. These data suggest the potential of CIA07 in combination with alum as an adjuvant for the development of a potent anthrax vaccine.

Comparison of the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide on Japanese encephalitis vaccine in mice.

Adjuvants are essential vaccine components used to enhance, accelerate, and/or prolong adaptive immunity against specific vaccine antigens. In this study, we compared the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS), a toll-like receptor 4 agonist, on the Japanese encephalitis (JE) vaccine in mice. Mice were immunized once or twice at a two-week interval with inactivated JE vaccine in the absence or presence of adjuvant. We found that both the alum- and the liposome-based formulation induced significantly faster and higher serum IgG antibody responses as compared with the non-adjuvanted vaccine after either one or two immunizations. The antibody titers of the mouse immune sera correlated with 50% plaque reduction neutralization test (PRNT50) antibody titers. In addition, the dLOS/liposome formulation was more effective in inducing a Th1-type immune response than the dLOS/alum formulation, as suggested by a strong antigen-specific interferon (IFN)-γ response. Based on these results, we suggest that both alum- and liposome-based adjuvant formulations containing dLOS may be used for the development of JE vaccines with improved immunogenicity.

Potentiation of Th1-Type Immune Responses to Mycobacterium tuberculosis Antigens in Mice by Cationic Liposomes Combined with De-O-Acylated Lipooligosaccharide.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. Bacillus Calmette-Guérin (BCG) vaccine is the only TB vaccine currently available, but it is not sufficiently effective in preventing active pulmonary TB or adult infection. With the purpose of developing an improved vaccine against TB that can overcome the limitations of the current BCG vaccine, we investigated whether adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS) are capable of enhancing the immunogenicity and protective efficacy of TB subunit vaccine. The results revealed that dLOS/dimethyl dioctadecyl ammonium bromide (DDA) adjuvant formulation significantly increased both humoral and Th1-type cellular responses to TB subunit vaccine that are composed of three antigens, Ag85A, ESAT-6, and HspX. The adjuvanted TB vaccine also effectively induced Th1-type response in a BCG-primed mouse model, suggesting a potential as a booster vaccine. Finally, dLOS/DDA-adjuvanted TB vaccine showed protective efficacy against M. tuberculosis infection in vitro and in vivo. These data indicate that dLOS/DDA adjuvant enhances the Th1-type immunity and protective efficacy of TB subunit vaccine suggesting that it would be a promising adjuvant candidate for development of a booster vaccine.

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.

Increased Immunogenicity and Protective Efficacy of a P. aeruginosa Vaccine in Mice Using an Alum and De-O-Acylated Lipooligosaccharide Adjuvant System.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that commonly causes fatal infections in cystic fibrosis and burn patients as well as in patients who are hospitalized or have impaired immune systems. P. aeruginosa infections are difficult to treat owing to the high resistance of the pathogen to conventional antibiotics. Despite several efforts, no effective prophylactic vaccines against P. aeruginosa are currently available. In this study, we investigated the activity of the CIA06 adjuvant system, which is composed of alum and de-O-acylated lipooligosaccharide, on a P. aeruginosa outer membrane protein (OMP) antigen vaccine in mice. The results indicated that CIA06 significantly increased the antigen-specific IgG titers and opsonophagocytic activity of immune sera against P. aeruginosa. In addition, the antibodies induced by the CIA06-adjuvanted vaccine exhibited higher cross-reactivity with heterologous P. aeruginosa strains. Finally, mice immunized with the CIA06-adjuvanted vaccine were effectively protected from lethal P. aeruginosa challenge. Based on these data, we suggest that the CIA06 adjuvant system might be used to promote the immunogenicity and protective efficacy of the P. aeruginosa OMP vaccine.

A De-O-acylated Lipooligosaccharide-Based Adjuvant System Promotes Antibody and Th1-Type Immune Responses to H1N1 Pandemic Influenza Vaccine in Mice.

Vaccine adjuvants are agents that are used to promote immune responses to vaccine antigens and thereby to enhance the protective efficacy of the vaccines. In this study, we investigated the adjuvant activity of CIA06, an adjuvant system that is composed of a toll-like receptor 4 agonist de-O-acylated lipooligosaccharide (dLOS) and aluminum hydroxide, on the H1N1 pandemic influenza vaccine Greenflu-S® in mice. CIA06 significantly enhanced influenza-specific serum IgG, hemagglutination-inhibition, and virus-neutralizing antibody titers, which eliminated vaccine dose-dependency in the antibody response. Mice immunized with the CIA06-adjuvanted Greenflu-S showed Th1-type-predominant cytokine profiles, and both CD4+ and CD8+ T cell responses were induced. Immunization of mice with the CIA06-adjuvanted vaccine reduced the mortality and morbidity of mice upon lethal challenges with influenza virus, and no excessive inflammatory responses were observed in the lung tissues of the immunized mice after viral infection. These data suggest that the dLOS-based adjuvant system CIA06 can be used to promote the immune responses to influenza vaccine or to spare antigen dose without causing harmful inflammatory responses.

Characterization of the structure and immunostimulatory activity of a vaccine adjuvant, de-O-acylated lipooligosaccharide.

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. LPS elicits strong immunopathological responses during bacterial infection, and the lipid A moiety of LPS is responsible for this immunostimulatory activity. Lipid A exerts its biological activity by sending signals via TLR4 present on immune cells, and TLR4 agonists have been a target for vaccine adjuvant. Previously, we demonstrated an adjuvant activity of deacylated lipooligosaccharide (dLOS) to viral and bacterial antigens. In this study, we characterized the chemical structure of dLOS and evaluated its immunostimulatory activity on mouse and human immune cells in comparison with monophosphoryl lipid A (MPL). dLOS consists of the R3-type core, a glucosamine disaccharide with two phosphate groups, and two N-linked acyl groups [corrected], and two N-linked acyl groups. dLOS was similar to MPL in induction of cytokine production in mouse peritoneal macrophages, but was a more potent activator in human monocytes and dendritic cells (DCs). Results of an analysis of allogeneic T cell responses revealed that dLOS induces Th1, Th2, and Th17-type immune responses in a dose-dependent manner. The immunostimulatory activities of dLOS were completely abrogated in TLR4(-/-) mice, which confirms its TLR4-dependency. These results suggest that in the presence of the core oligosaccharide, O-linked acyl groups of LPS are dispensable for activating the TLR4 signaling pathway. dLOS did not cause any pathological effects or death at 0.25, 0.5, or 1 mg per kg body weight in mice in the acute toxicity tests. This result suggests that dLOS has a low toxicity. dLOS should be considered for further development as a safe and effective adjuvant for human vaccines.

Global regulation of gene expression in the human gastric pathogen Helicobacter pylori in response to aerobic oxygen tension under a high carbon dioxide level.

The human gastric pathogen Helicobacter pylori (Hp) has been considered a microaerophile. However, we recently reported that, when supplied with 10% CO2, Hp growth is stimulated by an atmospheric level of O2, suggesting that Hp is a capnophilic aerobe. In this study, we investigated the effects of aerobic O2 tension on Hp cells by comparing gene expression profiles of cultures grown under microaerobic and aerobic conditions in the presence of 10% CO2. The results showed that overall differences in gene expression in Hp cells grown under the two O2 conditions were predominantly growth-phase-dependent. At 6 h, numerous genes were down-regulated under the aerobic condition, accounting for our previous observation that Hp growth was retarded under this condition. At 36 h, however, diverse groups of genes involved in energy metabolism, cellular processes, transport, and cell envelope synthesis were highly up- or down-regulated under the aerobic condition, indicating a progression of the cultures from the log phase to the stationary phase. The expression of several oxidative stress-associated genes including tagD, katA, and rocF was induced in response to aerobic O2 level, whereas trxA, trxB, and ahpC remained unchanged. Altogether, these data demonstrate that aerobic O2 tension is not detrimental to Hp cells but stimulates Hp growth, supporting our previous finding that Hp may be an aerobic bacterium that requires a high CO2 level for its growth.

Increased long-term immunity to Bacillus anthracis protective antigen in mice immunized with a CIA06B-adjuvanted anthrax vaccine.

Anthrax is an acute infectious disease caused by Bacillus anthracis. We previously reported that the adjuvant CIA06B, which consists of TLR4 agonist CIA05 and aluminum hydroxide (alum), enhanced the immune response to anthrax protective antigen (PA) in mice. This study was carried out to determine whether CIA06B can enhance long-term immune responses to PA in mice. BALB/c mice were immunized intramuscularly three times at 2-week intervals with recombinant PA alone or PA combined with alum or CIA06B. At 8 and 24 weeks post-immunization, the immunological responses including serum anti-PA IgG antibody titer, toxin-neutralizing antibody titer, splenic cytokine secretion and the frequency of PA-specific memory B cells were assessed. Compared with mice injected with PA alone or PA plus alum, mice injected with PA plus CIA06B had higher titers of serum anti-PA IgG antibodies, and higher frequencies of PA-specific memory B cells and interferon-γ secreting cells. Furthermore, anti-PA antibodies induced by CIA06B were more effective in neutralizing anthrax toxin. These results demonstrated that CIA06B is capable of providing long-term immunity when used as an adjuvant in a PA-based anthrax vaccine.

Comparison of the immune responses to the CIA06-adjuvanted human papillomavirus L1 VLP vaccine with those against the licensed HPV vaccine Cervarix™ in mice.

CIA05 is a toll-like receptor (TLR) 4 agonist derived from an Escherichia coli lipopolysaccharide (LPS) mutant and has been shown to have potential as a vaccine adjuvant. In this study, we investigated the immunopotentiating activity of the adjuvant system CIA06, which is comprised of CIA05 and aluminum hydroxide (alum), when used with the human papillomavirus (HPV) L1 virus-like particles (VLPs) vaccine. BALB/c mice were immunized intramuscularly three times at 2-week intervals with HPV16 L1 VLPs alone or in the presence of various combinations of CIA05 and alum, and the immune responses were assessed. We found that the combination of CIA05 and alum at a ratio of 1:50 (designated CIA06B) yielded the highest immune response in terms of serum anti-HPV L1 VLP IgG antibody titers, splenocyte interferon (IFN)-γ secretion, and antigen-specific memory B cell responses. The immunogenicity of the CIA06B-adjuvanted HPV16/18 L1 VLP vaccine was compared with that of the currently licensed HPV vaccine Cervarix™. The CIA06B-adjuvanted vaccine was similar to Cervarix™ with regard to eliciting serum antigen-specific IgG antibodies and virus-neutralizing antibodies but more effective at inducing splenic cytokine production and memory B cells. We also observed that the antigen-specific IgG antibody titers, splenic IFN-γ secretion and memory B cells induced by the CIA06B-adjuvanted HPV vaccine remained high up to 24 weeks post-immunization. Based on these data, we concluded that CIA06B may have potential as an adjuvant in a potent prophylactic vaccine against HPV infection.

A combination of the TLR4 agonist CIA05 and alum promotes the immune responses to Bacillus anthracis protective antigen in mice.

Anthrax is an infectious disease caused by Bacillus anthracis. The currently licensed human anthrax vaccines contain protective antigen (PA) as a major protective component and alum as an adjuvant. In this study, we investigated whether CIA05, a TLR4 agonist, is able to promote the immune response to an anthrax vaccine adjuvanted with alum. BALB/c mice were immunized intraperitoneally three times at 2-week intervals with a recombinant B. anthracis PA alone or in combination with CIA05 in the absence or presence of alum, and immune responses were determined 2 or 3 weeks after the third immunization. The results showed that the combination of CIA05 and alum significantly increased both serum anti-PA IgG antibody and toxin-neutralizing antibody titers, and the adjuvant effects were greater when lower antigen doses were used for immunization. Both CIA05 and alum stimulated PA-specific splenocyte secretion of interleukin (IL)-4, IL-5, and IL-6. A combination of the two yielded synergistic effects on IL-4 secretion, but CIA05 tended to repress IL-5 and IL-6 secretions induced by alum. Co-administration of CIA05 and alum also increased GL7 expression in B220(+)CD24(+) splenic cells, indicating the ability to activate B cells. These data suggest that CIA05, combined with alum, could be used to achieve higher immune responses to PA, leading to the development of an effective anthrax vaccine.

Identification of anti-inflammatory target genes of Rhizoma coptidis extract in lipopolysaccharide-stimulated RAW264.7 murine macrophage-like cells.

AIM OF THE STUDY: Rhizoma coptidis is used widely in traditional Oriental medicine to treat inflammatory diseases. The aim of this study was to identify the anti-inflammatory target genes of Rhizoma coptidis extract (CEX) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage-like cells. MATERIALS AND METHODS: RAW264.7 cells were treated with CEX in the absence or presence of LPS for 6h, and changes in gene expression profiles were analyzed using oligonucleotide DNA microarrays. The results of microarray analysis were validated by semiquantitative reverse transcription-polymerase chain reaction. To confirm the anti-inflammatory activity of CEX, the concentrations of cytokines released into the media were measured by sandwich ELISA, NO production was assessed using the Griess reagent, and iNOS expression levels were determined using immunoblot analysis. RESULTS: Microarray analysis revealed that activation of RAW264.7 cells with LPS elicited marked changes in mRNA expression of numerous genes known to be associated with inflammatory responses. Treatment of the cells with CEX suppressed the expression of various cytokines/chemokines, cell surface molecules, adhesion molecules, and growth factors. An ELISA also showed a decrease in the secretion of IL-1alpha, GM-CSF, and IL-6 but not of TNF-alpha. iNOS protein expression and NO production were also reduced by CEX treatment. CONCLUSIONS: The data obtained in this study demonstrate that CEX exerts its anti-inflammatory effect by inhibiting the expression of various proinflammatory cytokines and cell surface molecules involved in inflammatory responses at the transcriptional level. These data support the traditional use of CEX as an anti-inflammatory agent and should provide useful information for the understanding of the pharmacological effects of CEX.

Comparative analysis of the anti-inflammatory activity of Huang-lian extracts in lipopolysaccharide-stimulated RAW264.7 murine macrophage-like cells using oligonucleotide microarrays.

In this study, we attempted to determine the anti-inflammatory activity of a medicinal plant huang-lian using gene expression profiles as an index. Huang-line extracts (CEXs) were prepared from seven different plant origins and compared for their chemical composition and biological activity. In order to achieve this, RAW264.7 cells were treated with CEXs in the absence or presence of LPS for 6 h, and the differential gene expression profiles were analyzed using oligonucleotide microarrays. The alkaloid content of CEXs was determined by high performance liquid chromatography analysis. Evaluation of anti-inflammatory activity of CEXs was by measuring a decrease in cytokines and nitric oxide production in LPS-stimulated RAW264.7 cells. Hierarchical clustering analysis revealed that three CEXs from Coptis chinensis formed a cluster separate from the other four CEXs in LPS-stimulated cells, and were the most effective anti-inflammatoryagents. The extract prepared from Picrorrhiza kurrooa neither induced any changes in gene expression profiles nor possessed any anti-inflammatory activity. The extract from Jeffersonia dubia, which exhibited the highest cytotoxicity among the CEXs tested, was most effective in suppressing LPS-induced nitric oxide production but was not able to inhibit proinflammatory cytokine production. The results obtained in this study demonstrate that overall gene expression profiles of the extracts correlated well with their biological activity and that CEXs prepared from plants of diverse origins vary in their biological activity. These data also suggest that gene expression profiles may serve as a good indicator for the pharmacological activities of medicinal plants arising from diverse origins.

A nontoxic derivative of lipopolysaccharide increases immune responses to Gardasil HPV vaccine in mice.

Human papillomavirus (HPV) is the causative agent of cervical cancer, the second most common cause of cancer death in women worldwide. The licensed HPV vaccine Gardasil((R)) from Merck & Co. is a quadrivalent vaccine containing virus-like particles (VLPs) of the L1 proteins from HPV types 6, 11, 16, and 18 adsorbed on aluminum salts (alum). CIA07 is an immunostimulatory agent comprised of bacterial DNA fragments (CIA02) and a nontoxic derivative of lipopolysaccharide (CIA05) that has been shown to have antitumor activity and adjuvant activity for viral and bacterial vaccine antigens. We investigated whether these CIAs are capable of promoting the immune response to Gardasil. Balb/c mice were immunized intramuscularly twice three weeks apart with 1/20 human dose of Gardasil alone or in combination with CIA02, CIA05 or both, and immune responses were assessed. The serum anti-HPV16 L1 VLP IgG antibody titer was significantly higher in mice administered CIA05 or CIA05 plus CIA02, but not in those given CIA02, compared with mice given Gardasil alone. A secreted alkaline phosphatase (SEAP)-based pseudovirus neutralization assay showed increased neutralizing antibody titers in both CIA05 and CIA05 plus CIA02 groups. Coadministration of CIA05 with Gardasil led to a marked increase in serum IgG2a antibody titer and the percentage of interferon (IFN)-gamma(+) cells in the spleen, indicating that CIA05 effectively promotes Th1-type immune responses. These data indicate that CIA05, in synergy with alum, enhances the immune response to HPV L1 VLPs and suggest its potential as an adjuvant for the development of a potent prophylactic HPV vaccine.