Systems Pharmacology-based strategy to screen new adjuvant for hepatitis B vaccine from Traditional Chinese Medicine Ophiocordyceps sinensis Jingbo Wang+, Rui Liu, Baoxiu Liu, Yan Yang, Jun Xie*, Naishuo Zhu* I Laboratory of Molecular Immunology State Key Laboratory of Genetic Engineering Institute of Biomedical Science, School of Life Sciences, Fudan University, Shanghai, 200438. China *Correspondence and requests for materials should be addresses to NZ.(Email chu@fudan.edu.cn)andJX.(Email:xiejun@fudan.edu.cn) t These authors contributed equally to this work Adjuvants are common component for many vaccines but there are still few licensed for human use due to low efficiency or side effects. The present work adopted Systems Pharmacology analysis as a new strategy to screen adjuvants from traditional Chinese medicine Ophiocordyceps sinensis has been used for many years in China and other Asian countries with many biological properties, but the pharmacological mechanism has not been fully elucidated. First in this study, 190 putative targets for 17 active compounds in Ophiocordyceps sinensis were retrieved and a systems pharmacology-based approach was applied to provide new insights into the pharmacological actions of the drug. Pathway enrichment analysis found that the targets participated in several immunological processes. Based on this, we selected cordycepin as a target compound to serve as an adjuvant of the hepatitis B vaccine because the existing vaccine often fails to induce an effective immune response in
1 Systems Pharmacology-based strategy to screen new adjuvant for hepatitis B vaccine from Traditional Chinese Medicine Ophiocordyceps sinensis Jingbo Wang1+, Rui Liu1+, Baoxiu Liu1 , Yan Yang1 , Jun Xie1 *, Naishuo Zhu1 * 1 Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, Institute of Biomedical Science, School of Life Sciences, Fudan University, Shanghai, 200438, China. *Correspondence and requests for materials should be addresses to N.Z. (Email: nzhu@fudan.edu.cn) and J.X. (Email:xiejun@fudan.edu.cn) + These authors contributed equally to this work. Adjuvants are common component for many vaccines but there are still few licensed for human use due to low efficiency or side effects. The present work adopted Systems Pharmacology analysis as a new strategy to screen adjuvants from traditional Chinese medicine. Ophiocordyceps sinensis has been used for many years in China and other Asian countries with many biological properties, but the pharmacological mechanism has not been fully elucidated. First in this study, 190 putative targets for 17 active compounds in Ophiocordyceps sinensis were retrieved and a systems pharmacology-based approach was applied to provide new insights into the pharmacological actions of the drug. Pathway enrichment analysis found that the targets participated in several immunological processes. Based on this, we selected cordycepin as a target compound to serve as an adjuvant of the hepatitis B vaccine because the existing vaccine often fails to induce an effective immune response in
many subjects. Animal and cellular experiments finally validated that the new vaccine simultaneously improves the humoral and cellular immunity of BALB/c mice without side effects. All this results demonstrate that cordycepin could work as adjuvant to hepatitis b vaccine and systems-pharmacology analysis could be used as a new method to select adjuvants Introduction Adjuvants could elicit immune responses through different signaling pathways thus improve vaccine formulations for better protection, however, there are still very few adjuvants have been licensed for human because of the side effects or other problems. Even the widely used alum adjuvant could cause local reaction and IgE responses during vaccination. Therefore, there is a requirement to develop novel adjuvants for vaccines. Traditional Chinese Medicine (TCM) involves the use of natural products that have been utilized by humans for centuries for good health TCMs contain many bioactive ingredients with beneficial effects, and thus have attracted much attention in recent years. Although herbal medicines are comprised of several ingredients that target multiple organs, it is difficult to pin-point the bioactive compounds by traditional pharmacological methods. In addition, TCMs are not widely used outside of China and other Asian countries because of the lack of scientific data on their mechanisms of drug action. Thus, additional studies are needed to identify the underlying mechanisms Systems pharmacology, an emerging area of pharmacology, combines drug-like
2 many subjects. Animal and cellular experiments finally validated that the new vaccine simultaneously improves the humoral and cellular immunity of BALB/c mice without side effects. All this results demonstrate that cordycepin could work as adjuvant to hepatitis b vaccine and systems-pharmacology analysis could be used as a new method to select adjuvants. Introduction Adjuvants could elicit immune responses through different signaling pathways thus improve vaccine formulations for better protection1 , however, there are still very few adjuvants have been licensed for human because of the side effects or other problems2-4. Even the widely used alum adjuvant could cause local reaction and IgE responses during vaccination. Therefore, there is a requirement to develop novel adjuvants for vaccines. Traditional Chinese Medicine (TCM) involves the use of natural products that have been utilized by humans for centuries for good health5 . TCMs contain many bioactive ingredients with beneficial effects, and thus have attracted much attention in recent years6 . Although herbal medicines are comprised of several ingredients that target multiple organs, it is difficult to pin-point the bioactive compounds by traditional pharmacological methods. In addition, TCMs are not widely used outside of China and other Asian countries because of the lack of scientific data on their mechanisms of drug action7 . Thus, additional studies are needed to identify the underlying mechanisms. Systems pharmacology, an emerging area of pharmacology, combines drug-like
prediction, absorption, distribution, multiple drug target prediction and network analysis to analyze drugs, drug targets, pathways and drug effects". This approach, which can be used in the discovery of single bioactive ingredients, can help to identify he mechanisms of drug action 0, I Hepatitis B(HB)is an infectious disease caused by the hepatitis B virus(HBv) There are approximately 350 million individuals worldwide that carry the HBv, and China has a high incidence, with approximately 120 million carriers. Chronic HBV infection can lead to hepatitis, cirrhosis and hepatocellular carcinoma(HCC), and effective treatments are lacking. Because of the high morbidity and mortality, hepatitis b vaccination is essential for reducing the carrier rate and preventing viral infection The currently used genetically engineered hepatitis b vaccine with aluminum hydroxide as the conventional adjuvant has low efficacy, a long waiting period to immune response and large individual differences in immune effects. Approximately 5-10% of individuals with a normal immune system cannot achieve effective immunity against hepatitis b virus infection Therefore, developing a quick and effective adjuvant to enhance hepatitis b vaccine immunogenicity is of great practical Ophiocordyceps sinensis(syn. Cordyceps sinensis), placed systematically as Ophiocordycipitaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Ascomycota, is a TCM agent that has been widely used as a folk tonic for nearly one thousand years. It has an overwhelming list of pharmacological properties 8-.As one of the traditional and medical fungi, O. sinensis is currently available in China
3 prediction, absorption, distribution, multiple drug target prediction and network analysis to analyze drugs, drug targets, pathways and drug effects8,9. This approach, which can be used in the discovery of single bioactive ingredients, can help to identify the mechanisms of drug action10,11. Hepatitis B (HB) is an infectious disease caused by the hepatitis B virus (HBV). There are approximately 350 million individuals worldwide that carry the HBV, and China has a high incidence, with approximately 120 million carriers12. Chronic HBV infection can lead to hepatitis, cirrhosis and hepatocellular carcinoma (HCC), and effective treatments are lacking. Because of the high morbidity and mortality, hepatitis b vaccination is essential for reducing the carrier rate and preventing viral infection13. The currently used genetically engineered hepatitis b vaccine with aluminum hydroxide as the conventional adjuvant has low efficacy, a long waiting period to immune response and large individual differences in immune effects. Approximately 5-10% of individuals with a normal immune system cannot achieve effective immunity against hepatitis b virus infection14-16. Therefore, developing a quick and effective adjuvant to enhance hepatitis b vaccine immunogenicity is of great practical value. Ophiocordyceps sinensis (syn. Cordyceps sinensis), placed systematically as Ophiocordycipitaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Ascomycota, is a TCM agent that has been widely used as a folk tonic for nearly one thousand years17. It has an overwhelming list of pharmacological properties18-21. As one of the traditional and medical fungi, O. sinensis is currently available in China
and South East Asia22230 sinensis contains several bioactive components, and many reports have carefully analyzed its make-up of polysaccharides, sterols, nucleosides and protein 2224. Many of these components have been found to possess immunological, anti-tumorigenic, anti-oxidative, anti-inflammatory, anti-fatigue anti-fungal and anti-hypertensive properties, as well as to protect the kidney, liver and lung2526. However, there is no systematic analysis of the drug-target network of O sinensis. Therefore, the aim of the current study was to explain how the immunoregulation function was implemented and find a component that can act as an adjuvant of the hepatitis b vaccine through systems pharmacology analysis. Briefly, as seen in Figure 1, we first use a network pharmacology approach to determine the active ingredients of O. sinensis. A molecular-target network was then developed followed by enrichment analysis and functional classification. After screening,a single component was selected and validated by in vivo and in vitro experiments Results Candidate component identification. It has been difficult to identify the mechanisms of action of different TCM agents due to their complex biochemical make-up, and specific methods to identify the bioactive compounds have not been available until now. In this study, we used oral bioavailability(OB) screening and drug-likeness(DL) property evaluation or drug half-life(HL) prediction to identify the bioactive compounds".All the ingredients of O. sinensis were obtained from the Traditional Chinese Medicines Systems Pharmacology Database and Analysis
4 and South East Asia22,23. O. sinensis contains several bioactive components, and many reports have carefully analyzed its make-up of polysaccharides, sterols, nucleosides and protein21,22,24. Many of these components have been found to possess immunological, anti-tumorigenic, anti-oxidative, anti-inflammatory, anti-fatigue, anti-fungal and anti-hypertensive properties, as well as to protect the kidney, liver and lung25,26. However, there is no systematic analysis of the drug-target network of O. sinensis. Therefore, the aim of the current study was to explain how the immunoregulation function was implemented and find a component that can act as an adjuvant of the hepatitis b vaccine through systems pharmacology analysis. Briefly, as seen in Figure 1, we first use a network pharmacology approach to determine the active ingredients of O. sinensis. A molecular-target network was then developed, followed by enrichment analysis and functional classification. After screening, a single component was selected and validated by in vivo and in vitro experiments. Results Candidate component identification. It has been difficult to identify the mechanisms of action of different TCM agents due to their complex biochemical make-up, and specific methods to identify the bioactive compounds have not been available until now27. In this study, we used oral bioavailability (OB) screening and drug-likeness (DL) property evaluation or drug half-life (HL) prediction to identify the bioactive compounds7,9. All the ingredients of O. sinensis were obtained from the Traditional Chinese Medicines Systems Pharmacology Database and Analysis
Platform2 and the CancerHSP Database2 Twenty potential compounds with OB 30%, DL 20.18 or HL 4 were selected. Additionally, three compounds in the CancerhSP database with potential anti-cancer activities and ob 30% were also selected as candidate compounds for further analysis. Duplicate compounds in the two databases were eliminated, yielding 23 readily-absorbed compounds from a total of 40 compounds. The satisfied compounds are presented in Supplementary Table SI Target prediction and functional analysis. Generally, TCMs could prevent diseases through synergistic effects of different compounds and targets. Therefore, the potential therapeutic targets of the multiple compounds were important for its synergistic effects. After eliminating six additional compounds without targets, 190 targets in the database were assembled and ranked for 17 components contained in O sinensis. Detailed enrichment analysis and functional pathway classification showed that the bioactive components were involved in several cellular events, including signal transduction and cell differentiation( Figure 2a and Supplementary Table $2) Network construction. O. sinensis exerts extensive biological and pharmacological effects through multiple compound and target interactions. To understand these effects at the systemic level, a compound-target network was constructed based on the candidate components and targets. Figure 2b shows the results of the compound-target-function network, which consisted of 17 compounds, 190 candidate targets and seven functional annotations. The results displayed an average degree of 9.4 per compound and 3. 8 per target protein, respective relationships between compounds and targets, as shown in Figure 2c, the
5 Platform28 and the CancerHSP Database29. Twenty potential compounds with OB ≥ 30%, DL ≥ 0.18 or HL ≥ 4 were selected. Additionally, three compounds in the CancerHSP database with potential anti-cancer activities and OB ≥ 30% were also selected as candidate compounds for further analysis. Duplicate compounds in the two databases were eliminated, yielding 23 readily-absorbed compounds from a total of 40 compounds. The satisfied compounds are presented in Supplementary Table S1. Target prediction and functional analysis. Generally, TCMs could prevent diseases through synergistic effects of different compounds and targets. Therefore, the potential therapeutic targets of the multiple compounds were important for its synergistic effects. After eliminating six additional compounds without targets, 190 targets in the database were assembled and ranked for 17 components contained in O. sinensis. Detailed enrichment analysis and functional pathway classification showed that the bioactive components were involved in several cellular events, including signal transduction and cell differentiation (Figure 2a and Supplementary Table S2). Network construction. O. sinensis exerts extensive biological and pharmacological effects through multiple compound and target interactions. To understand these effects at the systemic level, a compound-target network was constructed based on the candidate components and targets30. Figure 2b shows the results of the compound-target-function network, which consisted of 17 compounds, 190 candidate targets and seven functional annotations. The results displayed an average degree of 9.4 per compound and 3.8 per target protein, respectively. With regard to the relationships between compounds and targets, as shown in Figure 2c, the