Struktur virus hepatitis C
Virus Hepatitis C (hepatitis C virus, HCV) adalah virus berenvelop dan bermateri genetik RNA dan menyebabkan hepatitis C. Berdasarkan profil materi genetiknya, HCV digolongkan menjadi enam genotip yaitu 1, 2, 3, 4, 5, dan 6. Virus ini menyerang hati dan menyebabkan hepatitis C akut dan hepatitis C kronis. Strukturnya terdiri atas envelop lipid yang mengandung glikoprotein envelop E1 dan E2, protein kapsid C yang membungkus materi genetiknya, dan protein non-struktur (tidak diperlihatkan).
Genom HCV
HCV mempunyai genom RNA positif dengan ukuran 9,5 kilo basa. Genom terdiri atas daerah yang tidak ditranslasi terletak pada ujung 5’ dan 3’ (5’ dan 3’ non-translated region, NTR). Setelah 5’ NTR berlokasi gen yang mengkode protein struktur yang terdiri atas nukleokapsid C (p22), glikoprotein envelop E1 (gp35) dan E2 (gp70), gen pengkode protein non-struktur yang terdiri atas NS1 (p7), NS2 (p23), NS3 (p70), NS4 (p8), NS4B (p27), NS5a (p56/58) dan NS5B (p68). NS2 adalah suatu protein transmembran, NS3 adalah suatu metalloprotease, protease serin, RNA helikase, NS4a dan NS4b adalah kofaktor, NS5A adalah protein yang menentukan resistensi terhadap interferon, dan NS5A adalah suatu RNA polimerase. Baik protein struktur dan protein non-struktur dihasilkan sebagai poliprotein yang kemudian mengalami modifikasi pasca translasi yaitu pemotongan dengan protease.
Siklus hidup HCV
Partikel virus HCV (virion) mengenali sel yang peka terhadap infeksi HCV, terutama adalah sel hepatosit. HCV mengempel melalui GAG dan reseptornya, kemudian HCV akan diinternalisasi untuk masuk ke dalam endosom. Virion mengalami uncoating untuk melepaskan RNA virus. Karena polaritas RNA HCV adalah positif, maka RNA tersebut berfungsi sebagai mRNA dan dapat ditranslasi di dalam sitoplasma untuk menghasilkan poliprotein. Poliprotein kemudian akan dipotong dengan protease virus dan RNA polimerase, suatu RNA polimerase yang menggunakan RNA sebagai substrat (RNA-dependent RNA polymerase) mengkatalisis pembentukan RNA negatif menggunakan RNA positif sebagai cetakan dan menghasilkan RNA positif menggunakan RNA negatif yang baru dibentuk sebagai cetakan. Virion dirakir di membran retikulum endoplasma dimana protein E1 dan E2 disisipkan pada membran tersebut, protein virus lainnya dan materi genetiknya dikemas kemudian dilepaskan ke luar sel melalui retikulum endoplasma dan badan Golgi. Replikasi virus dikatalisis oleh RNA polimerase yang banyak mengintroduksi kesalahan pada saat replikasi dengan frekuensi kesalahan 1.4 – 1.9 x 103 nukleotida/tahun, sehingga HCV banyak mengalami mutasi.
Variasi genetik HCV
HCV mempunyai variasi genetik yang sangat tinggi dan sampai saat ini diketahui HCV mempunyai 6 genotipe dengan kemiripan di dalam genotipe adalah 91%. Diketahui pula, HCV mempunyai lebih dari 50 subtipe dan kemiripan diantara genotipe adalah 66-69%. Klasifikasi genotipe didasarkan pada urutan nukleotida gen NS5B dan 5’NTR. Pada individu yang terinfeksi juga terdapat variasi nukleotida yang disebut dengan ‘quasispecies’.
Reverse-transcription Polymerase Chain Reaction untuk HCV
Materi genetik HCV adalah RNA, oleh karena itu untuk mengamplifikasi genom HCV maka RNA HCV diubah dulu menjadi DNA melalui transkripsi balik yang dikatalisis oleh reverse trancritptase (RTase). DNA yang dihasilkan disebut dengan cDNA (complementary DNA) dan kemudian diamplifikasi dengan PCR. Produk PCR dapat dideteksi menggunakan pelacak yang spesifik untuk HCV. RT-PCR dapat digunakan untuk penegakan diagnosis HCV, pemantauan terapi dan indikator penyembuhan. Seorang penderita hepatitis C kronis yang telah diindikasikan untuk diterapi harus ditentukan terlebih dahulu kandungan RNA HCVnya menggunakan RT-PCR. Selama terapi, kandungan RNA HCV dipantau untuk mendapatkan informasi apakah pasien responsif terhadap pengobatan atau tidak. Breakthrough dapat diterjadi pada saat terapi dengan kenaikan RNA HCV dan ini disebabkan karena telah munculnya resistensi virus terhadap obat yang digunakan. Penentuan kandungan RNA HCV juga disarankan untuk dilakukan mengetahui terjadinya relapse yaitu peningkatan kandungan RNA HCV setelah terapi selesai
Pentingnya genotipe HCV pada terapi
Terapi hepatitis C sangat ditentukan oleh genotipe HCV. Sampai saat ini diketahui bahwa genotipe 1 adalah genotipe yang paling sulit diterapi. Sensitivitas dan ketahanan terhadap interferon ditentukan oleh NS5A. Jika interferon alfa mengenali sel yang peka terhadap interferon, maka akan dihasilkan PKR dimana PKR berikatan dengan eIF-2-P yang akan menurunkan efisiensi translasi dan sintesis protein. Namun, NS5A dari HCV genotipe 1 akan berikatan dengan PKR sehingga tidak dapat mengikat eIF-2. Penentuan genotipe dapat dilakukan dengan RT-PCR yang dilanjutkan dengan hibridisasi menggunakan pelacak spesifik genotipe.
Protein NS5A mempunyai ukuran 447 asam amino dan protein mengandung daerah penyisipan membran, situs hiperfosforilasi, pengikatan PKR yang di dalamnya mengandung daerah penentu kepekaan interferon (interferon sensitivity determining region, ISDR), sinyal lokalisasi inti dan daerah V3. Mutasi pada ISDR telah diketahui bertanggung jawab terhadap resistensi terhadap interferon dan mutasi pada daerah V3 bertanggung jawab terhadap responder.
Pustaka
1.Guillou-Guillemette H. L., S. Vallet, C. Gaudy-Graffin, C. Payan, A. Pivert, A. Goudeau F. and Lunel-Fabiani, 2007, Genetic diversity of the hepatitis Cvirus: Impact and issues in the antiviral therapy, World J Gastroenterol; 13(17): 2416-2426.
2.Kato N.,T. Nakamura, H. Dansako,K. Namba, K. Abe, A. Nozaki, K. Naka, M. Ikeda and K. Shimotohno, 2005, Genetic variation and dynamics of hepatitis C virus replicons in long-term cell culture, J. Gen. Virol, 86, 645–656
3.Halfon P., P. Trimoulet, M. Bourliere, H. Khiri, V. De Le´Dinghen, P. Couzigou, J. M. Feryn, P. Alcaraz, C. Renou, H. J. A. Fleury, and D. Ouzan, 2001, Hepatitis C Virus Genotyping Based on 5’ Noncoding Sequence Analysis (Trugene), J. Clinic microbiol. 39(5): 1771–1773.
4.Ross R. S., S. O. Viazov, C. D. Holtzer, A. Beyou, A. Monnet, C. Mazure, and M. Roggendorf, 2000, Genotyping of Hepatitis C Virus Isolates Using Clip Sequencing, J. Clin Microbiol, 38(10): 3581–3584
5.Simmonds P., 2004, Genetic Diversity and Evolution of Hepatitis C Virus – 15 years on, J. Gen. Virol. 85: 3173–3188
Friday, July 03, 2009
Basic Principles of Carbohydrate-based Therapeutics and Vaccines
In the recent years, there has been an increasing interest in the biological roles of carbohydrates in many disease progresses either in infectious or non-infectious illness. In infectious diseases, carbohydrates play roles in adhesion and survival in human body. Prevention of pathogen adhesion is an attractive strategy in the prevention of infection. Bacteria have developed a number of adhesion mechanisms commonly targeting surface carbohydrate molecules. Our understanding on human glycome and its relation to bacterial adhesion will lead to the discovery of novel substances for therapeutics and prevention. Several human pathogens display carbohydrate structure on their surface, the most important is capsule. This structure renders the pathogen to resist phagocytosis; therefore encapsulated pathogens are more virulent than non-capsulated counterparts. Capsules have been used for vaccine targets and capsular vaccines have been successfully decreased the incidence of some infectious diseases caused by encapsulated pathogens. However, certain pathogens develop a strategy to avoid human defence mechanism by producing different types of capsular polysaccharides. These diverse carbohydrates building the capsule determine the pathogen serotypes. This phenomenon makes vaccine design more troublesome; therefore a mixture of various capsular polysaccharides is needed to make effective vaccine. In this case, knowledge on predominant capsule serotype is required to select which polysaccharides suitable for vaccine development in a certain region. Carbohydrate also plays a crucial role in malignant diseases. In these diseases, carbohydrates are actually altered self antigens and for unknown reasons, the body immune response does not recognize them. The development of vaccines for cancer treatment has been more challenging than that for infectious diseases, mainly due to the difficulty to break the body’s immunological tolerance to the antigen. Therefore, methods to enhance the immunological recognition and induction of immunity in vivo are being investigated. For therapeutics purpose, carbohydrate acts as anticoagulant agents. Current anticoagulants can be either direct or indirect inhibitors of clotting enzymes. Despite their success, they suffer from the risk of serious bleeding. Current status in this field is to discover or design safer and more affective anticoagulants that act through antithrombin pathway of anticoagulation. New molecules can be classified into antithrombin and its mutants, natural polysaccharides, synthetic modified heparins and heparin-mimic, synthetic oligosaccharides and synthetic non-sugar antithrombin activators. In conclusion, carbohydrates are naturally occurring substances that function in diseases processes and the interference of its action can be used as targets therapeutic and vaccine development.
The roles of carbohydrates?
Carbohydrates have a number of key roles in our cells. They play important role in intracellular recognition and surface markers, in biomolecular processes which are involved in cell-cell contact. The cell-cell contacts are involved in cellular, bacterial and viral adhesion. In order for the bacteria and virus to establish productive infection, they have to adhere to epithelial cells otherwise they will be cleared by body defense mechanisms. Carbohydrate moeities in glycoproteins also affect protein solubility and protein folding which is required for proper activity. This presentation will focus only in intracellular recognition and surface maskers and biomolecular processes.
Carbohydrates and carbohydrate-acting enzymes in disease?
Why are carbohydrates and carbohydrates-acting enzymes important in the initiation, development and progression of a number of diseases? Many infectious diseases caused by bacteria, viruses, fungi and many others are mediated by glycosylation. The initial interaction between pathogens and host cells are mostly mediated by glycoprotein and or carbohydrate-binding proteins (lectins). Altered carbohydrate processing is also responsible for several extremely debilitating and eventually terminal diseases such as diabetes, arthritis and several organ disorders such as liver disease. Recently, altered carbohydrates displayed only in cancer cells have been reported and this is due to improper or abnormal cell surface glycosylation. A number of carbohydrate-acting enzymes such as glycosidases, glycosyltransferases and N-glycanases are important in carrying out a wide spectrum of cellular processes and also responsible for the expression of altered carbohydrate at the surface of cancer cells.
Carbohydrates and diseases?
Changes in carbohydrate chemistry at the cell surface or in metabolic pathways are key features of a wide spectrum of diseases. Here are a number of examples for infectious diseases, cancer, diseases of major organs and auto-immune disorders. Since carbohydrate to be hydrophilic in nature, most of them are located at the cell surface and they serve for molecular recognition. There are two different substances carrying carbohydrate moeities located at the cell surface namely glycolipids and glycoproteins. Cell-surface carbohydrates involve in molecular recognition for viruses, bacteria and toxins with host cells which are important in infectious diseases. They also are important for cancer cell recognition, hormone, enzyme and antibody mechanism of actions. In my presentation today, I will only focus in carbohydrate-mediated molecular recognition in virus, bacteria, and cancer cells.
Carbohydrate-based therapy
Since the mechanisms of several carbohydrate-mediated diseases have been revealed, carbohydrate-based therapy is recently being developed. The basis of this therapy are disruption of carbohydrate-lectin interactions which is crucial for the initiation or development of specific diseases, identification of unique carbohydrate antigens to a disease state for vaccine development or we can deliver an monoclonal antibody targeted to these unique antigens, inhibition of enzymes that are responsible for biosynthesis of disease-associated or altered carbohydrate, replacement of carbohydrate-processing enzymes that are absent in diseased cells and application of carbohydrate and lectin interactions to deliver drug specifically to diseased cells. Due to time constraints, I will only discuss several of this strategy.
When a bacterium enters our body, it will adhere to our cells and colonize our host tissue to establish infection. If the adhesion process is mediated by carbohydrate-carbohydrate interaction, it is possible to interfere the adhesion process by adding decoy carbohydrates. These decoy carbohydrates will bind to bacterial adhesins preventing the bacterium to access its receptor on the cell surface of our cells. The bacterium then will cleared by our defense mechanisms and cannot initiate an infection. Some bacterial pathogens utilize carbohydrate for their adhesion process to our cells namely Escherichia coli, Neisseria gonorrhoae, Mycobacterium tuberculosis, Salmonella and Staphylococci.
Carbohydrates as anti-infective substances
Decoy carbohydrates are also called anti-infective agents. Examples of anti-infective agents are presented in this slide. Some of them are isolated from human milk oligosaccharides and they are good in aborting infection of Streptococcus pneumoniae and influenzae virus. A company namely Synsorb Biotech produced a structure that targets two bacterial toxins from important strain of E. coli O157:H7 (causing hemorrhagic colitis) and Clostridium difficile (causing diarrhoea). These agents underwent phase III clinical trial, although these trials have now been discontinued.
Role of carbohydrate in viral release from host cell?
Another example is the use of carbohydrate to inhibit viral release from host cell. Influenzae A virus has neuraminidase that cleaves sialic acid at the surface of infected cells. The cleavage is necessary in order for the virus to release from infected cells. A number of neuraminidase inhibitor are now being produced by several pharmaceutical companies and used as anti-influenzae drugs.
Carbohydrate antigens in cancer
As mentioned earlier that carbohydrate antigens are important in cancer development. These carbohydrate antigens can be either glycolipids or glycoproteins. Mostly, they are unpregulated or altered and displayed on the surface of cancer cells. This self altered carbohydrate is due abnormal glycosylation. Since only cancer cells express altered carbohydrates, then these substances can be targeted for vaccination.
Carbohydrate antigens as vaccine targets for cancer
Table 1 illustrates some carbohydrate antigens as targets for vaccine development. Some of the carbohydrates are common to cancer cells such as GM2, some are produced by several cancer cells for instance globoH. Structure of monovalent vaccine Globo H and the trimer formation for Tn vaccine are depicted in Figure 1.
Carbohydrate antigens in cancer
These two figures below show carbohydrate that are specific for cancer cells and the figure below highlights a vaccine consisting of three different antigens, globo H, Lewis and Tn. Thiis vaccine is called multivalent vaccine.
Status of experimental vaccine therapeutics
This table below presents some experimental vaccine therapeutics for a number of cancers and their status as of the year of 2004.
Role of heparanase in tumor cell invasion and angiogenesis
Heparanase is a Heparan Sulfate-specific Endo-beta-D-glucuronidase and it cleaves heparan sulfate proteoglycans (HSPGs) which is expressed by mammalian cells on cell surfaces and deposited in extracellular matrices including the basement membrane. Cell surface HSPGs play a role as receptors for adhesion molecules and growth factors; therefore they are implicated in cell adhesion, migration, differentiation and proliferation. One mechanism of metastasis of cancer cells is due to cleavage of HSPG by heparanase produced by cancer cells. Heparin is a carbohydrate-based anticoagulant inhibits heparanase, however, anticoagulant activity will cause unwanted effects that is bleeding. Now, the researchers synthesize a heparin derivative that is free of anticoagulant activity.
References
Vliegenthart J., 2006, Carbohydrate based vaccines, FEBS Letters, 580(12): 2945-2950
Roy R., 2004. New trends in carbohydrate-based vaccines. Drug Discovery Today: Technologies. 1(3): 327-336.
Slovin S. F., S. J Keding and G. Ragupathi, 2005. Carbohydrate vaccines as immunotherapy for cancer. Immunology and Cell Biology 83: 418–428
A Liakatos, H Kunz. 2007. Synthetic glycopeptides for the development of cancer vaccines. Current Opinion in Molecular Therapeutics 9:35-44
The roles of carbohydrates?
Carbohydrates have a number of key roles in our cells. They play important role in intracellular recognition and surface markers, in biomolecular processes which are involved in cell-cell contact. The cell-cell contacts are involved in cellular, bacterial and viral adhesion. In order for the bacteria and virus to establish productive infection, they have to adhere to epithelial cells otherwise they will be cleared by body defense mechanisms. Carbohydrate moeities in glycoproteins also affect protein solubility and protein folding which is required for proper activity. This presentation will focus only in intracellular recognition and surface maskers and biomolecular processes.
Carbohydrates and carbohydrate-acting enzymes in disease?
Why are carbohydrates and carbohydrates-acting enzymes important in the initiation, development and progression of a number of diseases? Many infectious diseases caused by bacteria, viruses, fungi and many others are mediated by glycosylation. The initial interaction between pathogens and host cells are mostly mediated by glycoprotein and or carbohydrate-binding proteins (lectins). Altered carbohydrate processing is also responsible for several extremely debilitating and eventually terminal diseases such as diabetes, arthritis and several organ disorders such as liver disease. Recently, altered carbohydrates displayed only in cancer cells have been reported and this is due to improper or abnormal cell surface glycosylation. A number of carbohydrate-acting enzymes such as glycosidases, glycosyltransferases and N-glycanases are important in carrying out a wide spectrum of cellular processes and also responsible for the expression of altered carbohydrate at the surface of cancer cells.
Carbohydrates and diseases?
Changes in carbohydrate chemistry at the cell surface or in metabolic pathways are key features of a wide spectrum of diseases. Here are a number of examples for infectious diseases, cancer, diseases of major organs and auto-immune disorders. Since carbohydrate to be hydrophilic in nature, most of them are located at the cell surface and they serve for molecular recognition. There are two different substances carrying carbohydrate moeities located at the cell surface namely glycolipids and glycoproteins. Cell-surface carbohydrates involve in molecular recognition for viruses, bacteria and toxins with host cells which are important in infectious diseases. They also are important for cancer cell recognition, hormone, enzyme and antibody mechanism of actions. In my presentation today, I will only focus in carbohydrate-mediated molecular recognition in virus, bacteria, and cancer cells.
Carbohydrate-based therapy
Since the mechanisms of several carbohydrate-mediated diseases have been revealed, carbohydrate-based therapy is recently being developed. The basis of this therapy are disruption of carbohydrate-lectin interactions which is crucial for the initiation or development of specific diseases, identification of unique carbohydrate antigens to a disease state for vaccine development or we can deliver an monoclonal antibody targeted to these unique antigens, inhibition of enzymes that are responsible for biosynthesis of disease-associated or altered carbohydrate, replacement of carbohydrate-processing enzymes that are absent in diseased cells and application of carbohydrate and lectin interactions to deliver drug specifically to diseased cells. Due to time constraints, I will only discuss several of this strategy.
When a bacterium enters our body, it will adhere to our cells and colonize our host tissue to establish infection. If the adhesion process is mediated by carbohydrate-carbohydrate interaction, it is possible to interfere the adhesion process by adding decoy carbohydrates. These decoy carbohydrates will bind to bacterial adhesins preventing the bacterium to access its receptor on the cell surface of our cells. The bacterium then will cleared by our defense mechanisms and cannot initiate an infection. Some bacterial pathogens utilize carbohydrate for their adhesion process to our cells namely Escherichia coli, Neisseria gonorrhoae, Mycobacterium tuberculosis, Salmonella and Staphylococci.
Carbohydrates as anti-infective substances
Decoy carbohydrates are also called anti-infective agents. Examples of anti-infective agents are presented in this slide. Some of them are isolated from human milk oligosaccharides and they are good in aborting infection of Streptococcus pneumoniae and influenzae virus. A company namely Synsorb Biotech produced a structure that targets two bacterial toxins from important strain of E. coli O157:H7 (causing hemorrhagic colitis) and Clostridium difficile (causing diarrhoea). These agents underwent phase III clinical trial, although these trials have now been discontinued.
Role of carbohydrate in viral release from host cell?
Another example is the use of carbohydrate to inhibit viral release from host cell. Influenzae A virus has neuraminidase that cleaves sialic acid at the surface of infected cells. The cleavage is necessary in order for the virus to release from infected cells. A number of neuraminidase inhibitor are now being produced by several pharmaceutical companies and used as anti-influenzae drugs.
Carbohydrate antigens in cancer
As mentioned earlier that carbohydrate antigens are important in cancer development. These carbohydrate antigens can be either glycolipids or glycoproteins. Mostly, they are unpregulated or altered and displayed on the surface of cancer cells. This self altered carbohydrate is due abnormal glycosylation. Since only cancer cells express altered carbohydrates, then these substances can be targeted for vaccination.
Carbohydrate antigens as vaccine targets for cancer
Table 1 illustrates some carbohydrate antigens as targets for vaccine development. Some of the carbohydrates are common to cancer cells such as GM2, some are produced by several cancer cells for instance globoH. Structure of monovalent vaccine Globo H and the trimer formation for Tn vaccine are depicted in Figure 1.
Carbohydrate antigens in cancer
These two figures below show carbohydrate that are specific for cancer cells and the figure below highlights a vaccine consisting of three different antigens, globo H, Lewis and Tn. Thiis vaccine is called multivalent vaccine.
Status of experimental vaccine therapeutics
This table below presents some experimental vaccine therapeutics for a number of cancers and their status as of the year of 2004.
Role of heparanase in tumor cell invasion and angiogenesis
Heparanase is a Heparan Sulfate-specific Endo-beta-D-glucuronidase and it cleaves heparan sulfate proteoglycans (HSPGs) which is expressed by mammalian cells on cell surfaces and deposited in extracellular matrices including the basement membrane. Cell surface HSPGs play a role as receptors for adhesion molecules and growth factors; therefore they are implicated in cell adhesion, migration, differentiation and proliferation. One mechanism of metastasis of cancer cells is due to cleavage of HSPG by heparanase produced by cancer cells. Heparin is a carbohydrate-based anticoagulant inhibits heparanase, however, anticoagulant activity will cause unwanted effects that is bleeding. Now, the researchers synthesize a heparin derivative that is free of anticoagulant activity.
References
Vliegenthart J., 2006, Carbohydrate based vaccines, FEBS Letters, 580(12): 2945-2950
Roy R., 2004. New trends in carbohydrate-based vaccines. Drug Discovery Today: Technologies. 1(3): 327-336.
Slovin S. F., S. J Keding and G. Ragupathi, 2005. Carbohydrate vaccines as immunotherapy for cancer. Immunology and Cell Biology 83: 418–428
A Liakatos, H Kunz. 2007. Synthetic glycopeptides for the development of cancer vaccines. Current Opinion in Molecular Therapeutics 9:35-44
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