STADI DELL’INFEZIONE VIRALE

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2 STADI DELL’INFEZIONE VIRALE
Interazione con la superficie cellulare Ingresso nella cellula

3 Clathrin-mediated endocytosis, for example, adenovirus
Clathrin-mediated endocytosis, for example, adenovirus. Endocytosis by caveolae can also occur, for example, SV40. b | Fusion at the cell membrane, for example, HIV.Fusion can also occur from inside an endosome, for example, influenza

4 STADI DELL’INFEZIONE VIRALE
Interazione con la superficie cellulare Ingresso nella cellula Uncoating Espressione dei geni virali e replicazione Assemblaggio delle particelle virali e fuoruscita dalla cellula

5 VIRUS DIVERSI UTILIZZANO STRATEGIE
DIVERSE PER LA REPLICAZIONE DEL GENOMA Virus a RNA “positive strand” (o “sense strand”) Virus a RNA “negative strand” (o “antisense strand”) Virus a RNA a doppio filamento segmentato Virus a DNA

6 CICLO REPLICATIVO DELL’HERPES VIRUS
Herpesvirus replication cycle.   a | Attachment and entry. Viral membrane proteins on virus particles bind to cellular receptors on the plasma membrane of the cell, which initiates fusion of the two membranes. Nucleocapsids containing the viral genome (red hexagons) are liberated into the cytoplasm and transported to nuclear pores. Viral DNA is released into the nucleus and circularizes. b | Transcription. Three classes of viral genes are transcribed and translated into proteins. Immediate-early proteins (yellow) participate in further transcription. c | Replication. Early proteins (green) synthesize new viral DNA molecules using circularized input DNA as a template. d | Assembly, encapsidation and nuclear egress. Late proteins (blue) assemble into capsids, which incorporate newly replicated viral DNA. Nucleocapsids leave the nucleus by budding through the inner nuclear membrane (a process termed 'envelopment') into the perinuclear space. Through a complex process of de- and re-envelopment, mature virus particles reach exocytic vesicles, which fuse with the plasma membrane and release new virus particles into the extracellular space.

7 FARMACI PER IL TRATTAMENTO DI INFEZIONI DA VIRUS A DNA
Analoghi nucleosidici Effetti collaterali: Tossicità GI Neurotossicità Tossicità ematologica Possibili effetti teratogeni e oncogenici

8 TIMIDINA CHINASI VIRALE
ATTIVAZIONE DEI NUCLEOSIDI ACICLICI TIMIDINA CHINASI VIRALE CHINASI CELLULARE CHINASI CELLULARE

9 MECCANISMO D’AZIONE DELL’ACICLOVIR
MECCANISMO D’AZIONE DEI NUCLEOSIDI ACICLICI

10 FARMACI PER IL TRATTAMENTO DI INFEZIONI DA VIRUS A DNA
Analoghi nucleosidici VALACICLOVIR FAMCICLOVIR VALGANCICLOVIR

11 The mechanism of action of acyclic nucleoside analogues
The mechanism of action of acyclic nucleoside analogues.  Examples of these compounds include acyclovir and its oral prodrug valaciclovir, penciclovir and its oral prodrug famciclovir, and ganciclovir and its oral prodrug valganciclovir. These acyclic nucleoside analogues require phosphorylation by the viral thymidine kinase (TK) to exert their antiviral activity for HSV and VZV (valaciclovir and famciclovir) and a protein kinase (PK; UL97) for CMV (valganciclovir).

12 EFFETTI COLLATERALI DEI NUCLEOSIDI ACICLICI
Aciclovir: Effetti GI e cutanei Nefrotossicità Neurotossicità Penciclovir: Effetti GI e cutanei Possibile effetto mutageno Ganciclovir: Mielosoppressione Neurotossicità Possibili effetti teratogeni ed embriotossici

13 FARMACI PER IL TRATTAMENTO DI INFEZIONI DA VIRUS A DNA
Nucleosidi aciclici fosfonati (analoghi nucleotidici) CIDOFOVIR ( )

14 PROPRIETÀ DEI NUCLEOSIDICI ACICLICI FOSFONATI
Richiedono solo due passaggi per la conversione a metabolita attivo sono attivi anche contro virus privi di TK specifiche vengono incorporati stabilmente negli acidi nucleici hanno un’emivita intracellulare prolungata non inducono facilmente resistenza non interagiscono metabolicamente con altri farmaci

15 FARMACI PER IL TRATTAMENTO DI INFEZIONI
DA VIRUS A DNA Composti non-nucleosidici

16 EPATITE VIRALE Hepatitis A virus (HAV): virus a ssRNA, trasmesso quasi esclusivamente per via oro-fecale; non dà luogo a forme croniche Hepatitis B virus (HBV): virus a DNA, trasmesso per via percutanea, sessuale e perinatale. Può dare luogo a forme croniche Hepatitis C virus (HCV): virus a ssRNA, trasmesso prevalentemente per via percutanea. Dà spesso luogo a forme croniche Hepatitis D virus (HDV): virus difettivo a ssRNA , richiede la funzione helper di HBV. È trasmesso per via percutanea, sessuale e perinatale e da spesso luogo a forme croniche Hepatitis E virus (HEV): virus a ssRNA, trasmesso quasi esclusivamente per via oro-fecale, non da luogo a forme croniche.

17 EPATITE VIRALE: prospettiva storica
trasmessa per via enterica “infettiva” A E epatite virale “NANB” C Trasmessa per via parenterale [SLIDE 2] Viral Hepatitis: Historical Perspective Before the discovery of hepatitis A virus (HAV) and hepatitis B virus (HBV) during the 1960s and 1970s, patients with viral hepatitis were classified based on epidemiologic studies as having either infectious (transmitted person to person by the fecal-oral route) or serum (transmitted by transfusion of blood products) hepatitis. When diagnostic tests for hepatitis A virus (HAV) and hepatitis B virus (HBV) infection were developed, HAV was found to be the major cause of infectious hepatitis and HBV was found to be the major cause of serum hepatitis. Hepatitis Delta virus (HDV), discovered in 1977, is a defective virus requiring the presence of HBV in order to replicate. However, some patients with typical signs and symptoms of viral hepatitis did not have serologic markers of HAV, HBV or HDV infection and were categorized based on epidemiologic characteristics as having either parenterally transmitted non-A, non-B hepatitis or enterically transmitted non-A, non-B hepatitis. During the past decade, two additional viruses have been discovered: hepatitis C virus (HCV) and hepatitis E virus (HEV). HCV is the major cause of parenterally transmitted non-A, non-B hepatitis and HEV is the major cause of enterically transmitted non-A, non-B hepatitis. In addition, some patients with typical signs and symptoms of acute viral hepatitis do not have serologic markers of any of these types of viral hepatitis and can be classified as having non-ABCDE hepatitis. Recently, new viruses have been discovered in patients with non-ABCDE hepatitis. Characterization of the epidemiology and clinical features of these and other possible agents of viral hepatitis will await the development of diagnostic assays. B D “da siero” altro

18 Tipi di epatite virale A B C D E Sorgente del virus
Sangue/fluidi organici di der. ematica Sangue/fluidi organici di der. ematica Sangue/fluidi organici di der. ematica feci feci Via di trasmissione percutanea permucosale percutanea permucosale percutanea permucosale oro-fecale oro-fecale Infezione cronica no sì sì sì no immunizazione prima/dopo l’esposizione; modificazione comportamenti a rischio uso di acqua potabile di origine “sicura” Prevenzione immunizazione prima della esposizione immunizazione prima/dopo l’esposizione screening donatori di sangue; modificazione comportamenti a rischio 3

19 Hepatitis B Virus Hepatitis B virus (HBV) is composed of an inner protein core and an outer protein capsule. The outer capsule contains the hepatitis B surface antigen (HBsAg). The inner core contains HBV core antigen (HBcAg) and hepatitis B e-antigen (HBeAg). This cell also contains polymerase, which catalyzes the formation of the cell's DNA. HBV is the only hepatitis-causing virus that has DNA, instead of RNA. 28

20 Distribuzione geografica dell’infezione cronica da HBV
Prevalenza di HBsAg ³8% - elevata 2-7% - intermedia <2% - bassa [SLIDE 41, SLIDE 42] Global Patterns of Chronic HBV Infection, Geographic Distribution of Chronic HBV infection* Approximately 45% of the global population live in areas with a high prevalence of chronic HBV infection (> 8% of the population is HBsAg‑positive); 43% in areas with a moderate prevalence (2%‑7% of the population is HBsAg‑positive); and 12% in areas with a low prevalence (< 2% of the population is HBsAg‑positive). In high prevalence areas, the lifetime risk of HBV infection is >60%, and most infections are acquired at birth or during early childhood when the risk of developing chronic infection is greatest. In these areas, because most infections in children are asymptomatic, very little acute disease related to HBV occurs, but rates of chronic liver disease and liver cancer in adults are very high. In moderate prevalence areas, the lifetime risk of being infected is 20%‑60% and infections occur in all age groups. Acute disease related to HBV is common in these areas because many infections occur in adolescents and adults; however, the high rates of chronic infection are maintained mostly by infections occurring in infants and children. In low prevalence areas, the lifetime risk of infection is <20%. Most HBV infections in these areas occur in adults in relatively well defined risk groups. *(Note: The map of HBsAg prevalence generalizes available data and patterns may vary within countries.)

21 Infezione cronica da HBV
Prevalenza elevata (>8%): 45% della popolazione globale rischio di infezione >60% infezioni comuni nella prima infanzia Prevalenza intermedia (2%-7%): 43% della popolazione globale rischio di infezione 20%-60% l’infezione si presenta in tutte le fasce di età Prevalenza bassa (<2%): 12% della popolazione globale rischio di infezione <20% la maggior parte delle infezioni si verifica in gruppi di adulti a rischio [SLIDE 41, SLIDE 42] Global Patterns of Chronic HBV Infection, Geographic Distribution of Chronic HBV infection* Approximately 45% of the global population live in areas with a high prevalence of chronic HBV infection (> 8% of the population is HBsAg‑positive); 43% in areas with a moderate prevalence (2%‑7% of the population is HBsAg‑positive); and 12% in areas with a low prevalence (< 2% of the population is HBsAg‑positive). In high prevalence areas, the lifetime risk of HBV infection is >60%, and most infections are acquired at birth or during early childhood when the risk of developing chronic infection is greatest. In these areas, because most infections in children are asymptomatic, very little acute disease related to HBV occurs, but rates of chronic liver disease and liver cancer in adults are very high. In moderate prevalence areas, the lifetime risk of being infected is 20%‑60% and infections occur in all age groups. Acute disease related to HBV is common in these areas because many infections occur in adolescents and adults; however, the high rates of chronic infection are maintained mostly by infections occurring in infants and children. In low prevalence areas, the lifetime risk of infection is <20%. Most HBV infections in these areas occur in adults in relatively well defined risk groups. *(Note: The map of HBsAg prevalence generalizes available data and patterns may vary within countries.)

22 POSSIBILI ESITI DELL’INFEZIONE DA HBV

23 settimane dall’esposizione
Infezione Acuta da HBV Andamento tipico dei markers nel siero settimane dall’esposizione Sintomi HBeAg anti-HBe anti-HBc totali IgM anti-HBc anti-HBs HBsAg 4 8 12 16 20 24 28 32 36 52 100 Titolo [SLIDE 34] Acute Hepatitis B Virus Infection with Recovery: Typical Serologic Course Serologic markers of HBV infection vary depending on whether the infection is acute or chronic. The first serologic marker to appear following acute infection is HBsAg, which can be detected as early as 1 or 2 weeks and as late as 11 or 12 weeks (mode, 30‑60 days) after exposure to HBV. In persons who recover, HBsAg is no longer detectable in serum after an average period of about 3 months. HBeAg is generally detectable in patients with acute infection; the presence of HBeAg in serum correlates with higher titers of HBV and greater infectivity. A diagnosis of acute HBV infection can be made on the basis of the detection of IgM class antibody to hepatitis B core antigen (IgM anti‑HBc) in serum; IgM anti‑HBc is generally detectable at the time of clinical onset and declines to subdetectable levels within 6 months. IgG anti‑HBc persists indefinitely as a marker of past infection. Anti‑HBs becomes detectable during convalescence after the disappearance of HBsAg in patients who do not progress to chronic infection. The presence of anti‑HBs following acute infection generally indicates recovery and immunity from reinfection.

24 Cronicizzazione dell’infezione da HBV
Andamento tipico dei markers nel siero IgM anti-HBc anti-HBc totali HBsAg Acuta (6 mesi) HBeAg Cronica (anni) anti-HBe 4 8 12 16 20 24 28 32 36 52 settimane dall’esposizione Titolo [SLIDE 35] Progression to Chronic Hepatitis B Virus Infection: Typical Serologic Course In patients with chronic HBV infection, both HBsAg and IgG anti‑HBc remain persistently detectable, generally for life. HBeAg is variably present in these patients. The presence of HBsAg for 6 months or more is generally indicative of chronic infection. In addition, a negative test for IgM anti‑HBc together with a positive test for HBsAg in a single serum specimen usually indicates that an individual has chronic HBV infection.

25 Modalità di trasmissione di HBV
Sessuale Parenterale Perinatale 2 2 2

26 Concentrazione di HBV nei liquidi organici
Bassa/Non rilevabile Elevata Moderata sangue sperma urina siero secreti vaginali feci essudati saliva sudore lacrime latte 1 1 1

27 FARMACI UTILIZZATI NELLE INFEZIONI DA HBV
INTERFERONE  (IFN) LAMIVUDINA LAMIVUDINA ADEFOVIR

28 MECCANISMO D’AZIONE DELL’ADEFOVIR

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30 Prevalence of HCV Infection Among Blood Donors
[SLIDE 60] Prevalence of HCV Infection among Blood Donors* Knowledge of the geographic distribution of HCV infection is based primarily on seroprevalence studies among blood donors because few population‑based seroprevalence studies have been conducted worldwide. An extremely low anti‑HCV prevalence (<0.1%) has been reported among blood donors in the United Kingdom and Scandinavia; a slightly higher prevalence (0.2‑1%) has been reported in other Western European countries, Australia, and North America; an intermediate prevalence (1.1%‑5%) has been reported in South America, Eastern Europe, Mediterranean countries, South Africa and Asia; and the highest prevalence (as high as 20% in Egypt) has been reported in the Middle East. * (Note: The map of anti‑HCV prevalence is based on data available for studies in which first‑ or second‑generation anti‑HCV tests and supplemental testing was used. This map generalizes available data and patterns may vary within countries.)

31 Hepatitis C [SLIDE 1] Hepatitis C Virus
HCV is an enveloped, single‑stranded RNA virus, approximately 50 nm in diameter, that has been classified as a separate genus in the Flaviviridae family. Before HCV was visualized, the virus genome was cloned and sequenced. The 5' end of the genome codes for core and envelope proteins, followed by nonstructural proteins, which extend to the 3' end. The ability of HCV to undergo rapid mutation in a hypervariable region(s) of the genome coding for envelope protein allows it to escape immune surveillance by the host; thus, most persons infected with HCV develop chronic infection.

32 Storia naturale dell’infezione da HCV
100 individui Tempo 15% 85% Risoluzione (15) Infezione cronica (85) 80% Malattia stabile (68) 20% Cirrosi (17) 75% 25% Malattia stabile (13) Mortalità (4) Indicazione principale per il trapianto di fegato

33 Andamento dell’infezione acuta da HCV
HCV RNA Sintomi +/- tempo dall’esposizione Titolo Anti-HCV ALT Normale 1 2 3 4 5 6 anni Mesi [SLIDE 52] Acute HCV Infections with Recovery: Typical Serologic Course The incubation period for acute hepatitis C has been reported to average 6 – 7 weeks, but may range 2 – 26 weeks. Children and adults with acute hepatitis C are typically either asymptomatic or exhibit mild clinical illness. In adults with acute HCV infection, studies have shown up to 40% had some symptomatic illness and 15 –30% had jaundice. The course of acute hepatitis C is variable, although its most characteristic feature is fluctuating alanine aminotransferase (ALT) patterns. Normalization of ALT may occur and suggest full recovery although symptomless ALT elevations can follow, indicating chronic infection. Fulminant hepatic failure following acute hepatitis C is rare.

34 Andamento dell’infezione acuta da HCV seguita da cronicizzazione
Sintomi +/- Tempo dall’esposizione Titolo ALT Normale 1 2 3 4 5 6 Anni Mesi HCV RNA Anti-HCV [SLIDE 53] Progression to Chronic HCV Infection: Typical Serologic Course Persistent HCV infection develops after the onset of acute hepatitis C in most persons (>85%). Chronic hepatitis C develops in 60 – 70% of HCV-infected persons, and 10 – 20% of these individuals may develop cirrhosis over the course of 20 – 30 years. Chronic hepatitis C progresses at a slow rate without symptoms in the majority of patients during the first two decades subsequent to infection. Usually chronic hepatitis C is not diagnosed until symptoms, such as fatigue appear with advanced liver disease.

35 Fattori che favoriscono la progressione o la gravità dell’epatite C cronica
Elevata assunzione di alcool Età > 40 anni al momento dell’infezione co-infezione da HIV Altri Sesso maschile co-infezione cronica da HBV

36 FATTORI DI RISCHIO ASSOCIATI CON LA
TRASMISSIONE DI HCV Uso illegale di farmaci per via parenterale Trasfusione o trapianto da donatore infetto Esposizione professionale al sangue principalmente punture accidentali con aghi infetti Trasmissione iatrogena Nascita da madre infetta da HCV Esposizione sessuale/domestica a soggetti positivi al test anti-HCV Partner sessuali multipli [SLIDE 54] Risk Factors Associated with Transmission of HCV Percutaneous exposures, including transfusion and transplantation from an infectious donor and injecting drug use, are the most efficient modes of HCV transmission. The overall prevalence of anti-HCV among persons with these exposures generally exceeds 60%. Other types of percutaneous exposures, including hemodialysis and needlestick injuries, have also been associated with HCV transmission. The risk of HCV transmission following a needlestick exposure to an anti-HCV‑positive patient is approximately 5% to 10%. Other risk factors that have been associated with HCV transmission include sexual or household exposure to an anti-HCV‑positive contact, having multiple sex partners, and being an infant of an HCV-infected mother. However, the magnitude of the risk associated with these exposures has not been well defined.

37 FARMACI UTILIZZATI NELL’INFEZIONE DA HCV
INTERFERONE  (IFN) RIBAVIRINA et al.

38 MECCANISMO D’AZIONE DELLA RIBAVIRINA
The mechanism of action of ribavirin and mycophenolic acid, the active component of mycophenolate mofetil.   Both these compounds block RNA synthesis by inhibiting the action of inosine 5'-monophosphate (IMP) dehydrogenase—this blocks the conversion of IMP to XMP (xanthosine 5'-monophosphate) and thereby stops GTP and, consequently, RNA synthesis. Diminuzione del pool di GTP Inibizione della RNA polimerasi virale Aumento del tasso di mutazione nel genoma virale

39 INDUZIONE DELLA PRODUZIONE DI INTERFERONI
Figure 1 | Mammalian Toll-like receptors (TLrs) and their ligands. TLR1, TLR2, TLR4, TLR5 and TLR6 are located on the cell surface. Their extracellular domains (depicted as rods) bind specific microbial products that act as ligands and the intracellular domains (depicted as spheres) signal via specific cytoplasmic signalling proteins. TLRs function as homodimers or heterodimers. The ligands specific for several such dimers are listed at the top of the figure. Several other TLRs, such as TLR3, TLR7/8 and TLR9, recognize specific nucleic acids that are often produced by viruses. They span the endosomal membrane with the ligand-binding domains inside the lumen and the signalling domains in the cytoplasm. They also function as dimers and recognize double-stranded (ds) RNA, single-stranded (ss) RNA or dsDNA containing CpG sequences. GPI, glycosylphosphatidylinisotol;LPS, lipopolysaccharide. TLRs = Toll-like receptors

40 GLI INTERFERONI E I LORO RECETTORI
Figure 3 | receptor activation or ligand–receptor complex assembled by type i, type ii or type iii interferons. Type I interferons (IFNs) (α, β ω, κ, ε, δ (pigs), τ (ruminants)) interact with IFN (α, β and ω) receptor 1 (IFNAR1) and IFNAR2; type II IFNγ with IFNγ receptor 1 (IFNGR1) and IFNGR2; and type III IFN-λs with IFNλ receptor 1 (IFNLR1; also known as IL28RA) and interleukin 10 receptor 2 (IL10R2; also known as IL10RB). Type II IFNγ is an antiparallel homodimer exhibiting a two-fold axis of symmetry. It binds two IFNGR1 receptor chains, assembling a complex that is stabilized by two IFNGR2 chains. These receptors are associated with two kinases from the JAK family: JAK1 and TYK2 for type I and III IFNs; JAK1 and JAK2 for type II IFN. All IFN receptor chains belong to the class 2 helical cytokine receptor family, which is defined by the structure of the extracellular domains of their members: approximately 200 amino acids structured in two subdomains of 100 amino acids (fibronectin type III modules), themselves structured by seven β-strands arranged in a β-sandwich. The 200 amino-acids domain usually contain the ligand binding site. IFNAR2, IFNLR1, IL10R2, IFNGR1 and IFNGR2 are classical representatives of this family, while IFNAR1 is atypical as its extracellular domain is duplicated. GAS, IFNγ-activated site; IRF9, IFN regulatory factor 9; ISGF3, IFN-stimulated gene factor 3, refers to the STAT1–STAT2–IRF9 complex; ISRE, IFN-stimulated response element; P, phosphate; STAT1/2, signal transducers and activators of transcription 1/2.

41 MECCANISMI DELL’AZIONE ANTIVIRALE DEGLI INTERFERONI

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43 VANTAGGI DELLA CONIUGAZIONE
A PEG o ALBUMINA aumento della solubilità diminuzione della clearance renale e del sequestro recettore-mediato da parte del sistema reticolo-endoteliale prolungamento dell’emivita plasmatica  diminuzione della frequenza di somministrazione PEG è comunemente utilizzato come eccipiente farmaceutico ed è noto per essere non-tossico e non immunogenico Ha una catena flessibile e altamente idrosolubile che dà luogo a un raggio idrodinamico circa 5-10 volte maggiore rispetto a una proteina globulare di peso molcolare equivalente, la catena polimerica ha in effetti un guscio d’acqua che aiuta a mascherare la proteina cui il PEG è legato. (R. Duncan, Nat Rev Cancer 2006)

44 Reduced dosing frequency and sustained exposure
Reduced dosing frequency and sustained exposure. The observed therapeutic index of alb-IFN versus unmodified IFN is used as an example to illustrate the sustained exposure and reduced dosing frequency associated with the albumin-fusion technology platform. Unmodified IFN reaches peak levels shortly after administration, followed by a rapid decline to undetectable levels at the end of each dosing interval (TIW (three times weekly) dosing, on Monday, Wednesday and Friday). By contrast, alb-IFN administered Q2w (every two weeks) provides sustained drug exposure that lies within the range of therapeutic efficacy throughout each dosing interval. Sustained drug exposure accompanied by infrequent dosing might improve tolerability.

45 EFFETTI COLLATERALI DEL TRATTAMENTO CON INTERFERONI
SINDROME INFLUENZALE ACUTA (febbre; brividi; cefalea; dolori muscolari e articolari; nausea, vomito, diarrea): regredisce entro 12h MIELOSOPPRESSIONE (granulocitopenia e trombocitopenia) NEUROTOSSICITA’ (sonnolenza; confusione; disturbi comportamentali; crisi epilettiche (rare) NEURASTENIA Affaticamento e perdita di peso DISORDINI AUTOIMMUNI COMPLICANZE CARDIO-VASCOLARI (rare)

46 MODULAZIONE FARMACOLOGICA DEL SISTEMA DEGLI INTERFERONI
Figure 5 | Potential drug targets in the interferon (iFN) system. Examples of potential or developmental drugstargeted at different steps in the pathways are presented. IPS1, IFN-β promoter stimulator 1 (also known as VISA);ISG, IFN-stimulated gene; JAK, Janus kinase; RNASEL, ribonulcease L; SOCS1, suppressor of cytokine signalling 1;TRAIL, tumour necrosis factor-related apoptosis-inducing ligand (also known as APO2L); TRIF, TLR adapter molecule 1.

47 Sustained virological response rates with treatments for hepatitis C.
DA, direct antiviral; IFN, interferon; PEG, pegylated interferon; RBV, ribavirin

48 The proportion of patients achieving a sustained virological response (SVR) has increased with advances in the treatment of hepatitis C virus (HCV) infection, from interferon (IFN) monotherapy to the current standard of care. The numbers above the columns, and the paler shaded area of the columns, represent the ranges of SVR reported in the literature for each treatment or patient population. PegIFN, pegylated-interferon.

49 HCV The 9.6-kb positive-strand RNA genome is schematically depicted at the top. Simplified RNA secondary structures in the 5'- and 3'-non-coding regions (NCRs) and the core gene, as well as the NS5B stem-loop 3 cis-acting replication element (5B-SL3) are shown. Internal ribosome entry site (IRES)-mediated translation yields a polyprotein precursor that is processed into the mature structural and non-structural proteins. Amino-acid numbers are shown above each protein (HCV H strain; genotype 1a; GenBank accession number AF009606). Solid diamonds denote cleavage sites of the HCV polyprotein precursor by the endoplasmic reticulum signal peptidase. The open diamond indicates further C-terminal processing of the core protein by signal peptide peptidase. Arrows indicate cleavages by the HCV NS2–3 and NS3–4A proteases. Dots in E1 and E2 indicate the glycosylation of the envelope proteins (4 and 11 N-linked glycans, respectively, in the HCV H strain). Note that polyprotein processing, illustrated here as a separate step for simplicity, occurs co- and post-translationally. Genetic organization and polyprotein processing of hepatitis C virus (HCV). Solid diamonds denote cleavage sites of the HCV polyprotein precursor by the endoplasmic reticulum signal peptidase. Amino acid positions are shown above each protein. The open diamond indicates further processing of the core protein by signal peptide peptidase. Arrows indicate cleavages by HCV NS2–3 and NS3 proteases. Asterisks in the E1 and E2 region indicate glycosylation of the envelope proteins. Abbreviation: NCR, noncoding region.

50 Subgenomic replicon RNAs are generated from an appropriate plasmid by in vitro transcription using T7 RNA polymerase. These RNAs are introduced into cells of the human hepatoma cell line Huh-7 by transfection, and cells are kept in culture medium that contains G418. Cells that did not take up the RNA (shown in white) and cells in which the replicon RNA does not replicate (orange cells) will die because of the toxic effect of G418. Only cells in which the replicon self-amplifies will carry a sufficient copy number of the gene encoding neomcyin phosphotransferase, which inactivates G418, to become resistant to G418. These cells survive and form a colony that can be isolated from the plate and expanded to establish a cell clone that carries a stably replicating hepatitis C virus (HCV) replicon.

51

52 Hepatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flaviviridae family75. a | Genomic organization of proteins encoded by HCV, comprising the structural proteins core (C), envelope 1 (E1), envelope 2 (E2), and P7 (presumed to be an ion channel) and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), which are mainly enzymes essential to the viral life cycle. b | The nucleocapsid of the HCV genome is surrounded by an envelope that facilitates attachment and penetration into host cells. Upon enty into the host cell by endocytosis, the virus undergoes a fusion and uncoating step. Its RNA genome is translated into a polyprotein of approximately 3,000 amino acids5, that is processed by cellular and viral proteases (including NS3) to yield four structural and six non-structural proteins50. The non-structural protein NS5B, a RNA-dependent RNA polymerase, catalyses the replication of the viral genome; negative-strand RNA intermediates are formed, which, in turn, serve as templates for the synthesis of new positive-strand RNAs. These are either encapsulated to form new viruses or used as mRNA for viral protein synthesis. The newly formed viral particles are released by exocytosis97. Each HCV structure represents a potential antiviral target for drug and vaccine development46, 98. For example, protease inhibitors target the NS3/4 protease, which is essential for viral polyprotein processing; polymerase inhibitors target the NS5B RNA-dependent RNA polymerase, which is essential for viral RNA replication; cyclophilin inhibitors block cyclophilin-induced stimulation of RNA-binding activity of NS5B; and -glucosidase inhibitors block the action of a host enzyme required for viral assembly, release and infectivity. Examples of drugs that are or have been in clinical development are included

53 Circulating HCV particles can be associated with low- and very-low-density lipoproteins (LP). Virus binding to the cell surface and entry may involve the low density lipoprotein receptor (LDLR), glycosaminoglycans (GAG), scavenger receptor class B type I (SR-BI), the tetraspanin protein CD81 and claudin-1 (CLDN1). CLDN1 functions at a late stage of cell entry, possibly at tight junctions of polarized hepatocytes. Internalization depends on clathrin-mediated endocytosis. Acidification of the endosome induces HCV glycoprotein membrane fusion. Little is known about the uncoating process, which results in genome release into the cytosol.

54 HCV The 9.6-kb positive-strand RNA genome is schematically depicted at the top. Simplified RNA secondary structures in the 5'- and 3'-non-coding regions (NCRs) and the core gene, as well as the NS5B stem-loop 3 cis-acting replication element (5B-SL3) are shown. Internal ribosome entry site (IRES)-mediated translation yields a polyprotein precursor that is processed into the mature structural and non-structural proteins. Amino-acid numbers are shown above each protein (HCV H strain; genotype 1a; GenBank accession number AF009606). Solid diamonds denote cleavage sites of the HCV polyprotein precursor by the endoplasmic reticulum signal peptidase. The open diamond indicates further C-terminal processing of the core protein by signal peptide peptidase. Arrows indicate cleavages by the HCV NS2–3 and NS3–4A proteases. Dots in E1 and E2 indicate the glycosylation of the envelope proteins (4 and 11 N-linked glycans, respectively, in the HCV H strain). Note that polyprotein processing, illustrated here as a separate step for simplicity, occurs co- and post-translationally. Genetic organization and polyprotein processing of hepatitis C virus (HCV). Solid diamonds denote cleavage sites of the HCV polyprotein precursor by the endoplasmic reticulum signal peptidase. Amino acid positions are shown above each protein. The open diamond indicates further processing of the core protein by signal peptide peptidase. Arrows indicate cleavages by HCV NS2–3 and NS3 proteases. Asterisks in the E1 and E2 region indicate glycosylation of the envelope proteins. Abbreviation: NCR, noncoding region.

55 Meccanismo d’azione degli inibitori nucleosidici
della RNA replicasi (NRRI) a | Mechanism of action of nucleoside RNA replicase inhibitors (NRRIs) as exemplified for valopicitabine (2'-C-methylcytidine) and 4'-azidocytidine. Three phosphorylation steps convert the nucleoside analogues (that is, 2'-C-methylcytidine or 4'-azidocytidine) to their 5'-triphosphates, which then act as non-obligate chain terminators (in competition with the natural substrate CTP) of the HCV NS5B RNA-dependent RNA polymerase (RdRp) as they interfere with further elongation through steric hindrance. b | Structures of NRRIs.

56 Struttura di alcuni inibitori nucleosidici della RNA replicasi (NRRI)

57 Siti di legame degli inibitori della RNA replicasi
NNRRI NRRI a | X-ray crystallographic structure of the NS5B RNA replicase. The thumb, palm and finger domains are coloured in blue, green and red, respectively. The two catalytic aspartates (residues 318 and 319) are shown in stick and ball style in the active site. Residues that are responsible for resistance to the various inhibitors are coloured according to the resistance pattern: magenta (benzimidazoles/indoles), light blue (thiophenes), brown (benzothiadiazines), yellow (dihydroxypyrimidines) and orange (2'-C-Me nucleosides)63. Figure reproduced with permission from Ref. 63 © (2005) International Medical Press. b | Binding sites for non-nucleoside RNA replicase inhibitors (NNRRIs) at the NS5B polymerase. Whereas there is only one binding site (S282) for the nucleoside reverse replicase inhibitors (NRRIs), there are at least four binding sites for the NNRRIs (NNI sites 1, 2, 3 and 4 for benzimidazole, thiophene carboxylic acid, benzothiadiazine and benzofuran, respectively)147, 148, 149 (and A. Y. M. Howe, personal communication). Yellow spheres represent amino acid residues at the NNRRI binding sites; turquoise dots represent Mg2+ ions at the catalytic aspartates. Figure is courtesy of Inge Vliegen and Weidong Zhong.

58 Struttura di alcuni inibitori non nucleosidici
della RNA replicasi (NNRRI) c | Structures of NNRRIs.

59 New antivirals for the treatment of HCV and present stage of development.

60 INIBITORI DELLA PROTEASI NS3
Chemical structure of a macrocyclic inhibitor of HCV NS3 protease.   This compound is a modified tripeptide carboxylic-acid inhibitor of the hepatitis C virus (HCV) non-structural protein 3 (NS3) serine protease.

61 Proposed replicative cycle of HCV and potential sites of therapeutic intervention.   The life cycle of the hepatitis C virus (HCV) has several specific steps, many of which are targets for antiviral drugs: a | attachment; b | endocytosis; c | virion– membrane fusion; d | uncoating; e | translation and polyprotein processing; f | replicase assembly; g | RNA replication; h | viral assembly and ER budding; i | vesicle transport and glycoprotein maturation; j | vesicle fusion and virion release. ER, endoplasmic reticulum; IRES, internal ribosome-entry site; LDLR, low-density lipoprotein receptor; NS, non-structural protein; siRNA, small interfering RNA.

62 Origine delle pandemie di influenza
The two mechanisms by which pandemic influenza originates. De Clercq Nature Reviews Drug Discovery 5, 1015–1025 (December 2006) In 1918, the 'Spanish influenza' H1N1 virus, closely related to an avian virus, adapted to replicate efficiently in humans. In 1957 and 1968, reassortment events led to, respectively, the 'Asian influenza' H2N2 virus and the 'Hong Kong influenza' H3N2 virus. The 'Asian influenza' H2N2 virus acquired three genetic segments from an avian species (a haemagglutinin (H), a neuraminidase (N) and a polymerase (PB1) gene). The 'Hong Kong influenza' H3N2 virus acquired two genetic segments from an avian species (H and PB1). Future pandemic strains could arise through either mechanism. Figure adapted, with permission, from Ref. 7 © (2005) Massachusetts Medical Society.

63 CICLO REPLICATIVO DEL VIRUS DELL’INFLUENZA
Inhibition of the influenza-virus replication cycle by antiviral agents. After binding to sialic-acid receptors, influenza virions are internalized by receptor-mediated endocytosis. The low pH in the endosome triggers the fusion of viral and endosomal membranes, and the influx of H+ ions through the M2 channel releases the viral RNA genes in the cytoplasm. Adamantan(amin)e derivatives block this uncoating step. RNA replication and transcription occur in the nucleus. This process can be blocked by inhibitors of inosine 5'-monophosphate (IMP) dehydrogenase (a cellular enzyme) or viral RNA polymerase. The stability of the viral mRNA and its translation to viral protein might be prevented by small interfering RNAs (siRNAs). Packaging and budding of virions occur at the cytoplasmic membrane. Neuraminidase (N) inhibitors block the release of the newly formed virions from the infected cells. Figure adapted with permission from Ref. 8 © (2004) Macmillan Magazines Ltd. H, haemagglutinin.

64 FARMACI PER IL TRATTAMENTO DELL’INFLUENZA
1) Inibitori della proteina M2 (A)

65 Uncoating of influenza virus and effect of ion channel blockers
Uncoating of influenza virus and effect of ion channel blockers. Influenza virus enters host cells by receptor-mediated endocytosis (not shown). The early endosome contains a H+ATPase that acidifies the endosome by pumping protons from the cytosol into the endosome. A low pH-dependent conformational change in the viral envelope hemagglutinin (HA) protein triggers fusion of the viral membrane. HA binding alone is not sufficient to cause viral uncoating, however. In addition, protons from the low-pH endosome must enter the virus through M2, a proton channel in the viral envelope that opens in response to acidification. The entry of protons through the viral envelope causes dissociation of matrix proteins from the influenza virus ribonucleoprotein (RNP) releasing the genetic material of the virus into the host cell cytosol. The viral genome is subsequently transcribed into mRNAs. AMantadine and rimantadine block M2 and thereby inhibit viral acidification, dissociation of matrix protein, and uncoating. NA, neuraminidase.

66 3) Inibitori della neuraminidasi (A e B)

67 Il virus influenzale riduce l’affinità di legame dell’AcCh per il recettore M2 attraverso la rimozione dei residui di acido sialico recettoriali per azione della neuraminidasi. Poiché i recettori M2 fungono da autorecettori inibitori, la loro ridotta attività provoca una maggior liberazione di ACh dalle terminazioni colinergiche. Si comprende così come le resistenze bronchiali possano aumentare durante l’influenza.

68

69 3) Inibitori della neuraminidasi (A e B)

70 VANTAGGI DELL’USO DEGLI INIBITORI DELLA NEURAMINIDASI
Riduzione di 1-3 giorni della durata della malattia Riduzione del rischio di trasmissione del virus Riduzione dell’incidenza e della gravità delle complicazioni Riduzione dell’uso di antibiotici Prevenzione delle influenze stagionali