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WO2008002165A2 - Procédé, dosage et coffret pour la prédiction et le traitement d'un sujet humain infecté par un virus - Google Patents

Procédé, dosage et coffret pour la prédiction et le traitement d'un sujet humain infecté par un virus Download PDF

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WO2008002165A2
WO2008002165A2 PCT/PL2007/000044 PL2007000044W WO2008002165A2 WO 2008002165 A2 WO2008002165 A2 WO 2008002165A2 PL 2007000044 W PL2007000044 W PL 2007000044W WO 2008002165 A2 WO2008002165 A2 WO 2008002165A2
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treatment
viral
virus
human subject
polynucleotides
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PCT/PL2007/000044
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WO2008002165A3 (fr
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Krzysztof Kucharczyk
Radoslaw Kaczanowski
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Krzysztof Kucharczyk
Radoslaw Kaczanowski
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis

Definitions

  • This invention relates to the diagnosis and treatment of RNA virus infections in humans.
  • the diagnosis comprises the determination whether the number of different viral variants and quasispecies diversity before and after commencing the treatment is reduced or not. While reduction is an indication of treatment effectiveness.
  • RNA viruses could replicate in wide spectrum of organisms like humans, animals and plants. Often there are pathogens responsible for severe diseases like AIDS, SARS or heppatais carcinoma. There are two most common way of virus infection development in human body: first is acute (with symptoms duration for a few hours or days) and chronic or persistent infections (with or without apparent symptoms but with detectable virus for weeks, months, years or decades). Examples of acute human infections due to RNA viruses are: flu, measles, common cold, poliomyelitis, different kinds of viral encephalitis and hemorrhagic fevers and those associated with the Hepatitis A virus. The second basic method is chronic viral infection which could be represented by Hepatitis C and HIV infection.
  • RNA viruses The genetic material of RNA viruses is RNA (this is the material that replicates inside infected cells and is responsible for the biological properties and the virulence of a virus). This contains information or a genetic message in the form of a polynucleotide produced by the polymerization of four nucleotides (adenosine monophosphate (AMP), Cytosine monophosphate (CMP), Guanosine monophosphate (GMP) and Uridine monophosphate (UMP). Polymers of these four monomers constitute the genetic material of RNA viruses.
  • AMP adenosine monophosphate
  • CMP Cytosine monophosphate
  • GMP Guanosine monophosphate
  • UMP Uridine monophosphate
  • Polymers of these four monomers constitute the genetic material of RNA viruses.
  • infectious process multiplication of a virus in cells or organisms
  • viral populations often form groups with a high number of infectious particles that can reach from 10. sup.5 to 10.
  • the genetic message (represented in the nucleotide sequence of genomic viral RNA) is not identical in the individual genomes that make up the viral population and many individual genomes differ from the others in one or more positions. These comprise a group of very closely related but not identical sequences and this group of sequences is called a viral quasispecies.
  • a quality factor was defined that represents the fraction of the copying process that produces an exact copy of the template.
  • the accuracy of the copy is not equal to 1 (the maximum possible value) the copy of the main sequence, also called the master sequence, will produce some mistaken molecules that will have a certain probability of distribution. Therefore, depending on the accuracy of the copy there will be a different abundance of genomes with one, two, three or more differences with respect to the master sequence.
  • HCV hepatocellular carcinoma
  • HCV infection commonly occurs after direct parental exposure. Recipients of blood or blood products, intravenous drug users, renal dialysis patients and needle-stick victims all represent high risk groups for infection. Viremia generally occurs within 1 week after transfusion of contaminated blood (Farci et al, 1991. N Engl J Med. .325(2):98-104). The acute phase of infection can last several months during which patients are generally viremic. Increase in serum aminotransferases; as a result of release from damaged hepatocytes, can occur within a few weeks of infection but generally ends 5 to 10 weeks post-infection.
  • HCV is a spherical enveloped virus, approximately 50 nm in diameter. It belongs to the Flaviviridae family and is the only member of the Hepacivirus genus. This family includes two other genera, Flavivirus and Pestivirus and an unclassified virus GBV-B. They all share the same genomic organization and perhaps also have similar structural characteristics. They are enveloped viruses and their genome is composed of a single-stranded RNA of positive polarity.
  • the viral life cycle is initiated by binding of the virus to a receptor, and after or during penetration, the viral RNA is uncoated and translated into the viral proteins by the cellular machinery.
  • the viral RNA first replicates into the negative-strand RNA, followed by positive- strand RNA, using the nonstructural (NS) proteins.
  • the newly synthesized positive-strand RNA is encapsidated together with the core protein. Viral encapsidation most likely occurs in the ER-Golgi compartments. It is not known whether the viral particles are released through vesicles or are released after cell lysis.
  • the HCV genome contains a long open reading frame (ORF) of approximately 9000 nucleotides, flanked UTRs at its 5' and 3' ends (Choo et al., 1989. Science. 44(4902):359- 362).
  • the 5' UTR is 341 bp long has a complex structure (stem-loops and pseudoknots) and contains an internal ribosomal entry site (IRES) which mediates the cap-independent translation of the ORF. It also contains elements implicated in genome replication. These structures are much conserved and interact with multiple cellular factors.
  • the IRES contains four stem-loops which recruit translation factors such as the eukaryotic initiation factor 3 (eIF3), the eIF2-GTP-initiator tRNA complex, the 40s ribosome subunit and the other noncanonical factors.
  • the 3'-UTR is 200 to 235 bp long and can be divided into three regions. First (from the 5' end) is a region of variable sequence of 27 to 70 bp that has been shown not to be required for viral replication in chimpanzees after intrahepatic inoculation with an HCV RNA transcript.
  • This region is followed by a poly-U/UC stretch which interacts with several cellular proteins such as the La antigen, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and the polypyrimidine tract binding protein (PTB) and is essential for replication in vivo.
  • GPDH glyceraldehyde-3-phosphate-dehydrogenase
  • PTB polypyrimidine tract binding protein
  • the ORF encodes a long polyprotein of 3022 amino acids. It is cleaved by cellular and viral proteases into ten different products. Two more proteins are encoded in the 3'-terminal part of the ORF by translational frameshifting. It is also possible that additional small proteins are encoded by this mechanism in other parts of the ORF.
  • the N-terminal part encodes three to four structural proteins whereas the others are the NS proteins. Their organization and functions are presented in Figure 1.
  • the first structural protein from the N-terminus of the polyprotein is the core protein. It constitutes the virion nucleocapsid and most likely interacts with the viral RNA.
  • the full length core protein has been shown to localize in the cytoplasm of the external membrane of the endoplasmic reticulum (ER).
  • An internal signal sequence located between the core protein and El mediates targeting of the nascent polyprotein chain to the ER membrane.
  • Processing of the core protein comprises sequential cleavage of the signal sequence first by cell signal peptidase and secondly by the cell signal-peptide peptidase, an intra-membranous aspartic protease. The resulting mature core protein is released from the ER membrane.
  • the core protein appears to play multiple roles in various cellular signaling pathways and potentially in oncogenesis. It can also activate virus promoters. Some of its functions can be shared or mediated by two proteins, F and 1.5 kDa protein encoded by ribosomal frameshifting triggered by a frameshift sequence within the core protein coding sequence. The next two proteins are the envelope glycoproteins El and E2. They are believed to associate as non- covalent heterodimers and are exposed on the virion surface. E2 mediates viral binding to the cells, as shown by a decrease of activity by incubation of the virus with the anti-E2 antibodies but the HCV receptor has not yet been identified. Among the potential candidates are CD81, a ubiquitous molecule, and the LDL receptor (LDLR).
  • LDLR LDL receptor
  • El and E2 are intracellulary exclusively localized in the ER. Cell surface expression of the El and E2 is very limited, which may partially explain why infected cells can escape immune recognition. Their ER localization along with the presence of the core protein in the ER strongly suggests that as for other members of the F ⁇ aviviridae family, HCV assembles at the ER membrane. E2 has also been shown to modulate the interferon- ⁇ response.
  • the next protein on the polyprotein is p7. It is not known whether p7 is structural or nonstructural protein. Studies of HCV subgenomic replicons showed that p7 is not critical for RNA replication, but its actual role in the virus life cycle has not been determined. P7 has characteristics similar to those of a group of proteins called viroporins. These proteins form ion channels that might be important for virus assembly and/or release. Furthermore it was demonstrated that recombinant HCV p7 could form ion channels in artificial lipid membranes, suggesting that this protein could be responsible for ion transport from the ER to the cytoplasm of HCV-infected host cells.
  • part of p7 can function as a signal sequence that most likely promotes the translocation of NS2 into the ER lumen for appropriate cleavage by host signal peptidases.
  • Full-length HCV chimeras, engineered with deletions in the p7 protein gene are replication deficient and noninfectious.
  • specific replacement of p7 in clone Ia by p7 from the infectious genotype 2a clone results in loss of replication ability, suggesting a genotype specific interaction between the p7 envelope protein and the other genomic regions. P7 is most likely required for virus assembly and release.
  • the nonstructural (NS) proteins (NS2, NS3, NS4A NS4B, NS5A and NS5B) perform various functions involved in viral RNA replication or proteolytic processing of the polyprotein.
  • NS2 (metallo/thiol protease) and NS3 are the two viral proteases responsible for the cleavage of all the NS proteins.
  • NS3 has helicase and NTPase activity; it also plays a role in RNA replication.
  • NS3 forms a heterodimer with NS4A and can interact with NS4B and NS5B to form a complex modulating recognition of the template by NS5B.
  • the role of NS 5 A in the life cycle of the virus is not known. Recent studies of HCV RNA replication have shown that many adaptive mutations that enhance viral replication are located in NS5A, suggesting that this protein is involved in replication and resistance to interferon- ⁇ .
  • NS5B is the viral RNA-dependent RNA polymerase (RdRp). It does not show rigid template specificity in vitro but can copy full-length HCV genomic RNA. NS5B possesses a hydrophobic domain at its C-terminus allowing its insertion into membranes. Liver is considered as the primary site of infection, as HCV is associated with hepatitis. However HCV infection is not limited to the liver: several studies have shown that HCV can replicate in peripheral blood mononuclear cells (PBMC). Moreover the possibility that HCV infects other cells than hepatocytes comes from the observation that transplanted livers in HCV patients are usually very rapidly re-infected by HCV. PL2007/000044
  • RdRp including the HCV RNA polymerase
  • they do not possess a 3'-5' proofreading function
  • each new genome differs from the parental template by up to ten nucleotides due to RdRp infidelity that introduces errors at ca. 1 x 10 "5 mutations/RNA base synthesized.
  • This mutation frequency results in a mutation rate as high as approximately 1.5 mutations per genome per round of replication. This mutation rate is similar to that of other RNA viruses, and is 10 3 to 10 6 fold greater than the mutation rate of DNA viruses.
  • HCV The small size of the HCV genome, its high mutation rate and tolerance to genetic diversity favor the establishment of complex mixtures of related viruses in the same patient.
  • HCV is never present in vivo as a homogeneous population of identical RNA genomes but rather as a mixture of divergent, closely related genomes, exhibiting a distribution that follows the model referred as quasispecies.
  • Genetic variability is one of most important features of the HCV genome.
  • the genetic heterogeneity of HCV is not uniform throughout the genome. The most highly conserved regions are located in parts of the 5'-UTR and the terminal 3'-UTR region.
  • the capsid protein is the most highly conserved region of the ORF, followed by conserved sequences in NS3 and NS5B.
  • the most heterogeneous regions of the genome are the genes encoding two envelope proteins, El and E2.
  • the N terminus of the E2 gene contains a domain of 31 amino acids, which constitutes the most variable regions of the entire HCV genome and has been called the first hypervariable region (HVRl).
  • HVRl A second hypervariable region located just 3' of HVRl has been described and named HVR2, but it seems to be limited only to strains of genotype Ib.
  • the HVR2 is only seven amino acids long and its significance is not well understood.
  • the genetic heterogeneity of HCV is complex. HCV heterogeneity has been classified in four hierarchical strata: genotypes, subtypes, isolates and quasispecies.
  • HCV isolates obtained from south-west Asia. These isolates were proposed as new major genotypes designated as 7 through 11. However more detailed phylogenetic analyses of such variants rather suggest that with the exception of genotype 10, these isolates should be classified as divergent subtypes of type 6 and isolates designated as genotype 10 should instead be classified as divergent subtypes of genotype 3. These genetically diverse subtypes are indications that HCV heterogeneity may in reality be a continuum. Nevertheless the hierarchical classification of HCV remains of practical use for studies of the molecular epidemiology and transmission of hepatitis C worldwide.
  • RNA viruses may represent a fundamental strategy of adaptation to the environment.
  • the viral variants present in quasispecies do not reflect the entire spectrum of mutants generated during replication but only those that survive as a result of Darwinian selection. It is estimated that approx. 10 12 virions/day are produced within an infected individual and 1.5 mutations occur for every replication cycle. This results in 1.5 x 10 12 mutations/day produced in HCV quasispecies.
  • An ensemble of viral RNAs rather than an individual particle is the actor of selection and evolution (i.e. there may be a complementation of gene functions among individual components of the molecular spectrum). Genomes displaying a higher ability to produce progeny are represented in larger numbers in quasispecies.
  • quasispecies consist of mutant distributions centered around a master genome -mutant clouds. It is assumed that quasispecies represent the best fitting population that has established a status of equilibrium. This equilibrium can be lost as a result of changes in the host environment i.e. loss of dominance in a previous master genome and appearance of a new master genome; there are reports of major changes in viral strain composition after superinfection with different HCV strains which suggests competition between viral strains as predicted by the quasispecies theory. Those perturbations in quasispecies composition could reflect virus adaptation to the changing environment, i.e. antiviral therapy, and are potential variables that could be used as an indicator of therapy effectiveness.
  • HCV infection results in chronic infection in 80% of the cases.
  • a virus that persists in the host must evolve successful strategies to survive by avoiding recognition by the immune system.
  • Genetic variation is an important strategy of HCV to avoid specific cellular and humoral responses.
  • HCV T- cell receptor
  • HVRl functions as TCR antagonist. This role of HVRl was demonstrated for CD8+ T cells or for CD4+ T helper cells. A strong inhibition of cell proliferation and cytokine production was observed in vitro when agonist and antagonist were presented by the same antigen presenting cells (APCs).
  • T cell antagonism may be another important strategy by which HCV eludes a protective response, because sustained helper T cells response directed against several HCV antigens are associated with a self-limited course of infection.
  • Those results may provide an explanation for association between the evolution of quasispecies complexity and the course of infection observed by Farci et al., (2000. Science. 228(5464):339-345). It has been proposed that the antigenic variability, specifically the quasispecies nature of HCV, may allow the virus to circumvent the immune response, leading to the chronic infection.
  • HCV quasispecies diversity may be a mechanism of escape from immune system come from the study of immune deficient patients infected with RNA viruses. If host immunity is deficient or suppressed, there is a trend toward a more homogeneous viral population, suggesting that viral diversity is driven by host immune pressure. A dramatic change in HCV quasispecies evolution was observed as a result of bone marrow transplantation.
  • HCV genotype has been associated with response to HCV anti-viral therapy (Fried et al., 2002. New Engl Med. 347(13):975-982)
  • the observations of different responses in patients with the same genotype and titer of viremia suggest that other viral factors may be responsible for these differences.
  • the heterogeneity within virus quasispecies is routinely evaluated at two levels: genetic complexity defined as the total number of viral variants identified in the sample, and genetic diversity, the average genetic distance between the viral variants (Farci et al., 2002. Proc Natl. Acad. Sc. 99(5):3081-3086).
  • genetic complexity defined as the total number of viral variants identified in the sample
  • genetic diversity the average genetic distance between the viral variants
  • Farci (Farci et al., 2002. Proc Natl. Acad. Sci. 99(5):3081-3086) evaluated the evolution of the HCV quasispecies tracked by sequence analyses in serial serum samples obtained from 24 patients with chronic hepatitis C selected according to their different patterns of response to interferon therapy. The results showed a significant decrease in complexity and diversity of the HCV quasispecies within 2 weeks of starting the therapy in long-term responders but not in patients who had a breakthrough or relapse after treatment, suggesting that early evolution of the HCV quasispecies may predict a sustained virological response defined as loss of detectable HCV RNA 6 months or more after completion of the treatment.
  • interferon-sensitivity-determining region because it was associated with the sensitivity of HCV genotype Ib to interferon treatment.
  • PLR RNA-activated protein kinase R
  • interferon As a crucial mediator of innate antiviral immune response, interferon was a natural choice for HCV infected human treatment. Unfortunately therapy with interferon alone had very limited success. A 6-month treatment led to sustained response rates of 6-12%, and extending the treatment to 12 months raised this rate to 16-20% (Di Bisceglie et al., 2002. Hepatology. 36(5 suppl 1):S121-S127). A major advance came from the addition of the broad spectrum antiviral agent ribavirin to the interferon treatment: this more than doubled the sustained response level, bringing it to 35-40%.
  • SVR sustained virological response
  • end-of treatment response and relapse end-of treatment response and relapse
  • non-response SVR is defined as the loss of detectable HCV RNA during treatment and 6 months after stopping the therapy.
  • a transient response with relapse occurs in 10-25% of the patients. These patients show little evidence of a long-term benefit.
  • Non-response to treatment occurs in about one-third of the patients. These patients never become HCV RNA negative.
  • SVR typically has a very rapid initial decrease (phase one) - believed to reflect the effect of suppression of replication, followed by a second slower phase of decline - due to clearance of virus-infected cells, until undetectable levels of circulating virus are achieved.
  • virus genotype - SVR rates range from 42-46% in patients infected with genotype 1, which accounts for ca. 70% of the cases in the USA and approx. 80% of the cases in Tru to 76-80% for the less common genotypes 2 and 3.
  • interferon - ⁇ has antiviral activity but does not act directly on the virus or replication complex. It acts rather by inducing interferon stimulated genes (ISGs) that establish a non- virus antiviral state within the cell. Microarray analyses show that hundreds of genes are induced by type-1 interferon. Additionally, some studies suggest that interferon inhibits translation of viral proteins.
  • interferon In addition to its direct antiviral actions interferon strongly interacts with the adaptive and innate immune responses, interferon promotes multiple actions including memory T-cell proliferation, prevention of T-cell apoptosis, stimulation of natural- killer-cell activation and dendritic cell maturation, and it also up regulates the production of the major histocompatibility (complex (MHC) class-I and class-II peptides.
  • MHC major histocompatibility
  • Ribavirin a guanosine analogue was synthesized in 1970 and immediately shown to be active against several RNA and DNA viruses. It does not influence HCV titers when used in monotherapy, but when used with interferon it improves the number of patients that cleared the virus and decrease the relapse probability. Currently it is approved for use in chronic hepatitis but only in combination with interferon. Three mechanism of ribavirin action are proposed.
  • Ribavirin has a direct inhibitory effect on replication of HCV RNA.
  • ribavirin As a guanosine analogue ribavirin is phosporylated intracellularly to form the monophosphate (RMP), diphosphate (RDP) and triphosphate (RTP).
  • RMP monophosphate
  • RDP diphosphate
  • RTP triphosphate
  • the miscorporation of RTP by the RNA polymerase leads to chain termination and inhibition of replication in all six HCV genotypes.
  • this effect requires ribavirin concentrations of 50-100 ⁇ M compared to lO ⁇ M, the concentration reached for therapeutic purposes.
  • ribavirin might have a small direct effect on HCV- RNA replication through polymerase inhibition, this is unlikely to be a major mechanism of action against hepatitis C.
  • RMP is a competitive inhibitor of inosine monophosphate dehydrogenase (IMPD), which leads to shortage in the GTP supply necessary for viral RNA synthesis.
  • IMPD inosine monophosphate dehydrogenase
  • ribavirin only partly inhibits HCV replication. This finding is consistent with the minimal effects of ribavirin monotherapy on serum levels of HCV RNA and indicates that IMPD inhibition and GTP depletion might contribute to, but are not likely to be major determinants of, the effects of ribavirin therapy in hepatitis C.
  • the third concept of ribavirin action introduced by Crotty Crotty et al, 2000. Nature Med.
  • Peg-interferon and ribavirin treatment of hepatitis C virus infection is efficient in approximately 56% of the patients and its side effects are common and significant. They include flu-like side effects: fever, malaise, tachycardia, chills, headaches, arthralgias, myalgias, apathy and cognitive changes. Between 10% and 15% of the patients find the chronic side effects intolerable and discontinue the treatment. Neuropsychiatric side effects can be the most troublesome and unpredictable; they include: fatigue, asthenia, drowsiness, lack of initiative, irritability, confusion, and apathy; behavioral, mood, and cognitive changes are relatively frequent. Serve depression may occur and suicidal inclinations have been described.
  • thyroid disorders have been reported in 2.5-20% of the patients and may be irreversible after stopping therapy unless therapy is stopped early.
  • Cardiovascular, renal, hepatic, dermatologic, hormonal and metabolic side effects and myelosuppression have also been reported.
  • Peg-interferon plus ribavirin therapy predictions are based on the early virological response - (EVR) defined as a 2-log decrease from base-line HCV RNA levels or undetectable RNA by week 12 of the therapy. Of those who did not have SVR at 12 th week of the therapy (14% for treated with Peg-interferon plus ribavirin therapy), 97% did not have a sustained virologic response (Fried et al., 2002. New Engl Med. 347(13):975-982). Moreover, no vaccine is available for HCV infection.
  • EMR early virological response -
  • PBMC peripheral blood mononuclear cells
  • mutant distributions This can be expressed either as the number of mutations that distinguishes each genome (genetic distance between components of the mutant spectrum) or as mutation frequency calculated by comparing the nucleotide sequence of individual genomes sampled from the mutant spectrum with the consensus sequence derived from this mutant spectrum.
  • Virus titers are routinely measured using real time PCR based on the amplification of the conserved virus genome regions (e.g. HCV; Cobas TaqMan, Roche). The information concerning the level of viral replication before therapy and its kinetics after implementing the therapy provides important and clinically relevant information (Fried et al., 2002. New Engl Med. 347(13):975-982).
  • HMA heteroduplex mobility assay
  • HTA heteroduplex tracking assay
  • USPTO Al 20030180717 nucleic acids hybridysation assay
  • HTA and HMA were used to access quasispecies heterogeneity of HCV.
  • HMA analysis single stranded PCR products representing single clones are mixed to form double-stranded heterozygotes.
  • the mobility ratio of these products is measured by means of agarose gel electrophoresis, and on the basis of relative differences of their mobility ratios, an assumption can be made concerning the genetic distance between the samples measured.
  • the limitation of this technique is that it requires single clones to be analyzed. Estimates of genetic distance between virus strains can also be obtained without isolation of individual clones, by tracking heteroduplexes formed between entire pools of viral genetic variants present within individuals, the HTA method.
  • PCR products representing viral variants present in an infected individual is hybridized with trace amounts of radioactively labeled PCR products representing the quasispecies composition of another individual.
  • radio labeled DNA strands form heteroduplexes with unlabeled strands and the electrophoretic mobility of the radio labeled tracer reflects the genetic distance between the two quasispecies.
  • the major limitations of these techniques are (as with SSCP) the lack of information concerning specific residues in the viral genome. They are also more work intensive than SSCP and hence are far less commonly used for viral quasispecies analyses than SSCP.
  • Another classical test amplifies the viral gene that is the target of the anti-viral agent.
  • the viral gene from a given patient is amplified and then recombined into a biologically active proviral clone of HIV.
  • This pro viral clone is transfected into human cells to generate a viral stock of known moi which can then be used to infect a target indicator cell line.
  • this class of test still requires the culturing of virus to determine drug resistance, and is thus difficult, lengthy and costly and requires the laboratory investigator to handle hazardous viral cultures. Furthermore, given the attendant variation of the virus itself during the culture process, the results may be correspondingly inaccurate.
  • a second class of test attempts to provide specific information on the genotype of the patient's HIV, with the ultimate goal of correlating this genotypic information with the virus' drug resistant phenotype.
  • specific amino acid substitutions within viral genes such as reverse transcriptase and protease genes have been shown to correspond to specific levels of viral resistance to reverse transcriptase and protease inhibitors, respectively (Larder et al., 1994. J. Gen Virol. 75, 951-957).
  • a major shortcoming associated with such an analysis is that it is indirect and can be obfuscated by secondary mutations which have been shown to add to or counter the effects of the first mutation.
  • a third class of test a recently developed bacterial-based assay makes use of a molecularly cloned viral gene (specifically, the reverse transcriptase gene) which has been inserted into a bacterial expression vector.
  • the reverse transcriptase gene Upon transformation of special strains of E. coli which are deficient in the bacterial DNA polymerase I, the cloned reverse transcriptase gene can rescue the growth of the bacteria under selected growth conditions.
  • E. coli dependent upon reverse transcriptase for their growth one can ascertain the effects of certain reverse transcriptase inhibitors on the activity of the viral gene (PCT Application No. WO 95/22622).
  • nucleoside analog also blocks bacterial growth for reasons other than its effects on reverse transcriptase, it can not be adequately tested in this system.
  • the present invention provides methods and compositions and a kit for determining whether a virus present in human body is affected by a treatment.
  • the present invention is based on the discovery that in human subject infected with the RNA virus, there is a significant association between change of number of different viral quasispecies and their genetic and polypeptide diversity and the infected human subject response to treatment in the first weeks of treatment. Specifically, individuals in whom the number of different viral subspecies and their polypeptide diversity is reduced in the first weeks of treatment, will have an increased likelihood of sustained virologic response to treatment comprising interferon. Conversely, individuals in whom in the first weeks of treatment the number of different viral subspecies and their polypeptide diversity is not reduced, will have an increased likelihood of no sustained virologic response to treatment comprising interferon.
  • the viral quasispecies genetic diversity and complexity reduction is determined at highly variable regions of the virus genome which are coding viral structural capsid proteins which are most often interact with human body and host immunological system. That suggest that the reduction in the diversity and complexity of the viral quasispecies in the first week of treatment is reflecting not only the direct influence of the drug on the virus replication or development in the human subject but also might reflect the human subject anti viral activity, like immunological response and other capability to fight the virus infection.
  • the method described in this invention permits, among other applications, to detect and identify minority viral genomes, to study viral quasispecies responsible for viral drug resistance, or host immune defense /escape and thus could facilitate individualized therapeutic regimes
  • the present invention provides a method for predicting response of a human subject infected with an virus to treatment comprising a) providing polynucleotide from viral quasispecies from said human subject comprising a portion that includes polynucleotide being from the highly variable region of said viral quasispecies, before and after commencement of the treatment, b) determining the sequence of the polynucleotides, and c) determining whether the number of different polynucleotides or translated polypeptide variants is, the diversity of 44
  • said polynucleotides or translated polypeptide is, or both are, reduced after commencement of the treatment as compared to before commencement of the treatment, wherein the reduction indicates an increased likelihood of response to the treatment.
  • the present invention provides a method for determining whether a hepatitis C virus (HCV) has an altered susceptibility to a treatment, comprising a) providing polynucleotide from viral quasispecies from said human subject comprising a portion that includes polynucleotide being from the highly variable region of said viral quasispecies, before and after commencement of the treatment, b) determining the sequence of the polynucleotides, and c) determining whether the number of different polynucleotides or translated polypeptide variants is, the diversity of said polynucleotides or translated polypeptide is, or both are, reduced after commencement of the treatment as compared to before commencement of the treatment, wherein the reduction indicates an increased likelihood of sustained viro logical response to applied treatment.
  • HCV hepatitis C virus
  • the present invention provides a method for determining whether a hepatitis C virus (HCV) is susceptible to applied treatment in the first, second, there, four, five, six, seven, eight, nine or ten week of treatment.
  • HCV hepatitis C virus
  • Available methods for predicting the response to antiviral treatment are based on the determination of one or more viral sequence mutations that provide drug-resistant viral variants or determination of the total number of virus copies present in the human body.
  • Another approach is based on the determination of human subject organism capabilities in the prevention of virus infection or clearance after infection or both. Hover both of that above approaches considers neither changes of the viral population diversity and complexity during the course of infection nor human subject body individual capabilities to defend particular viral infection at particular moment.
  • natural human body response to the viral infection depends on several parameters like: immunological system status, past and present resolved infections with pathogens (bacterial, viruses, fungi etc) allergic conditions, metabolisms, food amount and quality, environment etc.
  • the present invention solves some of those obstacles by analyzing at the begging of the treatment the viral quasispecies population reaction to the applied treatment. That measure reflects at last three parameters: viral quasispecies population changes, human organism anti viral activity and applied treatment effectiveness. In case of the positive response to the applied treatment, we can observe elimination of the viral quasispecies population diversity and lowering the number of different viral quasispecies. However at that some moment the total number of viral copies present in the human bodies is not lowering by at last two orders of magnitude - which is the accepted measure of the HCV virus treatment respond prediction.
  • the present invention provides an assay for determination of the number of different said polynucleotide or translated polypeptide variants and their diversity in the sample obtained form the human subject comprising a) providing the population of viral polynucleotides from said sample and, b) amplification of polynucleotides which covers highly variable regions of said viruses, and c) separation of genetically different amplified polynucleotide, and d) isolation of separated polynucleotide, and e) re-amplification and sequencing of said isolated polynucleotide, and f) translating of said polynucleotide sequences into polypeptide sequences, and g) determining the number of different said polynucleotide or translated polypeptide variants and their diversity in the sample.
  • the present invention provides the method for separation of genetically different amplified polynucleotide in which single stranded DNA is separated in native polyacrylamide gels under different temperature conditions that are applied subsequently during one electrophoresis.
  • MSSCP Multitemperature SSCP
  • MSSCP method is significantly more sensitive in the detection of point mutations in polynucleic fragments, when compared to the classical one temperature SSCP.
  • dedicated DNA Pointer System that allows the electrophoresis to be performed at high voltage and temperature of samples controlled with 0,1-0,2 0 C accuracy. This further improves the reproducibility of electrophoresis and speed of genetic diversity analysis. This technique, which is more sensitive than SSCP is applied to analyze the genetic heterogeneity of HCV.
  • the method which is subject of the present inventions solves some of that problems by determining the dynamic changes of the viral quasispecies population during the first weeks of treatment in said human subject, which are strongly correlated with the long term respond to the applied anti viral treatment and which are reflecting: virus pathological capabilities, human subject capabilities to fait the viral infection and effectiveness of applied treatment.
  • viral quasispecies refer to a population of that same virus genomes present in that same host in that same time, comprised of dynamic distributions of mutant and recombinant genomes that are not identical but are closely related, that undergo a continuous process of genetic variation, competition and selection and that operate as a selection unit.
  • Domingo As defined by Domingo (Domingo et al., 1996. FASEB J. 10(8):859-64. ): "Viral quasispecies are reserves of genetic variants (and phenotypic; i.e. a variant of biological behavior) that have a certain probability of being selected in response to a selection applied from outside the organism or endogenous selection from the infected organism itself '). Even more among the variants that make up the mutant spectrum of a viral quasispecies there are mutants with a reduced sensitivity to inhibitors used in the treatment of viral diseases. The presence of minority variants with different level of drug resistance is one of the factors that contribute to therapeutic failure in the treatment of viral infections.
  • viral quasispecies my comprise the whole spectrum of mutants with reduced sensitivity to antibodies or to cytotoxic T cells (CTLs).
  • viral quasispecies may comprise antigenic variants or other types of variants with altered biological properties influence the progression of a viral disease in vivo.
  • most pathogenic viruses circulate as different genomes that have been divided into types, subtypes, genotypes or biotypes and can require specific reagents for diagnosis. Such subdivisions is used in human immunodeficiency virus, hepatitis C virus, the flu virus, human and animal rotaviruses, poliomyelitis virus, foot-and-mouth virus and many others (see, for example European patent application EP 0 984 067 A2). 44
  • the term "diversity" as used herein refers to refer to the measure of the difference on the nucleic acids or polypeptide level of two identical in size polynucleotide or polypeptides.
  • the mean Hamming distance which is the average of the values taken for all sequence pairs derived from a single sample, was separately calculated for the HVRl region and the entire area sequenced.
  • mutation refers to a change introduced into a parental or wild type nucleic acids sequence that changes the amino acid sequence encoded by the nucleic acids, including, but not limited to, substitutions, insertions, deletions or truncations.
  • substitutions substitutions, insertions, deletions or truncations.
  • the consequences of a mutation include, but are not limited to, the creation of a new character, property, function, or trait not found in the protein encoded by the parental nucleic acids.
  • polynucleotide template or “target polynucleotide template” refers to a polynucleotide containing an amplified region.
  • the "amplified region,” as used herein, is a region of a polynucleotide that is to be, for example, synthesized by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • an amplified region of a polynucleotide template resides between two sequences, to which two PCR primers are complementary. It is not intended that the present invention be limited by the source of a nucleic acid, nucleotide, polynucleotide or oligonucleotide.
  • a nucleic acid, nucleotide, polynucleotide or oligonucleotide can be from a human or non-human mammal, or any other organism, or derived from any recombinant source, synthesized in vitro or by chemical synthesis.
  • a nucleic acid, nucleotide, polynucleotide or oligonucleotide may be DNA, RNA, cDNA, DNA- RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), a hybrid or any mixture of the same, and may exist in a double-stranded, single-stranded or partially double-stranded form.
  • the nucleic acids of the invention include both nucleic acids and fragments thereof, in purified or unpurified forms, including genes, chromosomes, plasmids, the genomes of biological material such as microorganisms, e.g., bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals, humans, and the like.
  • primer refers to a single stranded DNA or RNA molecule that can hybridize to a polynucleotide template and prime enzymatic synthesis of a second polynucleotide strand.
  • “Complementary” refers to the broad concept of sequence complementarity between regions of two polynucleotide strands or between two nucleotides through base- pairing. It is known that an adenine nucleotide is capable of forming specific hydrogen bonds ("base pairing") with a nucleotide which is thymine or uracil. Similarly, it is known that a cytosine nucleotide is capable of base pairing with a guanine nucleotide.
  • sequencing means to determine one by one the positions of nucleotides at a given nucleic acid fragment or amino acids at given polypeptide At the DNA fragment at each position could located one of the four nucleotides: A 5 G 5 C 5 T and for RNA there are A 5 C 5 G or U. For polypeptide building there are at last 24 amino acids well know in the art.
  • DNA sequencing means of the DNA sequencing methods, first devised in 1975, based on the production of a nested set of labeled DNA fragments from a template strand by replicating the template strand to be sequenced and interrupting the replication at one of the four bases.
  • SSCP single strand conformation polymorphism method used for the detection of mutations in fragments of nucleic acids based on their difference in electrophoreses mobility of single strand nucleic acids conformers during their native electrophoresis.
  • MSSCP means multitemperature single strand conformation polymorphism method based on the mobility changes of different nucleic acids fragments caused by at least two temperature changes during the separation of said single stranded nucleic acids in native conditions
  • PMES means method consists in four steps: PCR 5 MSSCP, Electroelution, and Sequencing. Briefly, after PCR or RT-PCR amplification of genetic material of e.g. virus origin (or any other source) is separated into major populations of DNA strands by MSSCP method, representing different sequences in native polyacrylamide gels. After elution from the gel and re-amplification by PCR these populations are used as templates for sequencing. This technique was estimated to be an alternative to molecular cloning for the detection of genetic heterogeneity in the samples.
  • TGGE temperature gradient gel electrophoresis based on changes of nucleic acids separation mobility during the separation under gradient of temperature perpendicular to the direction of separated molecules migration.
  • DGGE denaturing gradient gel electrophoresis based on changes during separation mobility of nucleic acids under gradient of concentration of chemical substance (e.g. urea) and substance in the concentration forms gradient parallel to the migration direction of analyzed nucleic acids.
  • DPLC Denaturing High Performance Liquid Chromatography is an analytical method based on separation heteroduplex molecules from are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands.
  • PCR means polymerase chain reaction based on replication of specific DNA fragments .
  • polyacrylamide is used herein and refers to an acrylate polymer (-CH2CHCONH2-) formed from acrylamide subunits that is readily cross-linked used in electroforetic gels preparation.
  • label refers to molecule or any atom which can provide a signal when attached to protein or a nucleic acid. Labels have to provide the signal which is measurable and/or in quant table manner. The label may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Convenient labels for the present invention include those that facilitate detection of the electrophoretic mobility an oligonucleotide fragment.
  • a "label” is a fluorescent dye.
  • Fluorescent labels may include dyes that are negatively charged, such as dyes of the fluorescein family including e.g., HEX, TET, FAM, JOE, NAN and ZOE., Or dyes that are neutral in charge, such as dyes of the rhodamine family which include ROX, Texas Red, RIlO, R6G, and TAMRA. Or dyes that are positively charged, such as dyes of the cyanine family, which include Cy2, Cy3, Cy5, and Cy7.
  • agarose is used herein and refers to polysaccharide used as a matrix for electrophoretic separation of e.g. nuclei acids or proteins.
  • chromatography refers to analytical method of separating mixtures of biomolecules based on their size or their affinity to the solid phase of chromatographic column.
  • capillary electrophoresis refers to electrophoretic separation of biomoleclues while separation is performed inside capillary which dimension could be in the range of 1-100 micrometers .
  • interferon is used herein and refers to the family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune response.
  • pegylated interferon alpha means polyethylene glycol modified conjugates of interferon, preferably interferon alpha-2a and alpha-2b. Typical suitable pegylated interferon alpha include, but are not limited to, Pegasys.RTM. and Peg- Intron.RTM.
  • therapeutic treatment is a treatment administered to a subject in need, for the purpose of eliminating signs or symptoms of pathology, disease, or disorder.
  • therapeutic activity is an activity of a substance, or composition thereof that eliminates or diminishes signs or symptoms of pathology, disease or disorder, when administered to a subject suffering from such signs or symptoms.
  • response to treatment with interferon is herein used interchangeably and refers to the absence of detectable HCV RNA in the sample taken of an infected human subject by RT-PCR.
  • the samples might be taken at several time points, usually the are taken before the treatment (baseline), during at the week 12, 24, 48, at the end of treatment or 24 weeks after the end of treatment.
  • Virtual Non-Response to treatment with interferon is herein used interchangeably and refers to the presence of detectable HCV RNA in the sample of an infected subject by RT-PCR throughout treatment and at the end of treatment. Interferon therapy can help normalize liver function through its antiviral activity.
  • Elevated ALT and aspartate aminotransferase are known to occur in uncontrolled hepatitis C, and a complete response to treatment is generally defined as the normalization of these serum enzymes, particularly ALT. See Davis (Davis et al., 1989. New Eng. J. Med. 321 -.1501-1506. ALT is an enzyme released when liver cells are destroyed and is symptomatic of HCV infection. Interferon induces synthesis of the enzyme 2',5'-oligoadenylate synthetase (2'5'OAS), which in turn, causes the degradation of the viral mRNA. Increases in serum levels of the 2'5'OAS coincide with decreases in ALT levels.
  • RNA viruses is used herein and refers to viruses which possess ribonucleic acid as their genetic material and according the term “DNA viruses” is used herein and refers to viruses which possess deoxy ribonucleic acid as their genetic material
  • viral genome variable region refers to a region that shows high mutation frequency. Usually such regions are located at the genes which codes for structural or viral proteins which directly interact with host body. For example in the HCV virus the most variable region cold hyper variable regions located at the glycoprotein El and E2 which codes for viral capsid protein and being direct interaction of the human subject immunological system components.
  • RNA virus infection refers to a patient who is infected with an RNA virus.
  • a salient characteristic of viruses with RNA genomes is that they have relatively high rates of spontaneous mutation reportedly on the order of 10.sup.-3 to 10. sup. -4 per incorporated nucleotide. See Fields & Knipe 1986. "Fundamental Virology” (Raven Press, NY). Since heterogeneity and fluidity of genotype are inherent in RNA viruses, there are multiple types/subtypes, within the species which may be virulent or avirulent. For example, the propagation, identification, detection, and isolation of various HCV types or isolates are documented in the literature.
  • HCV-I genome and amino acid residues sequences are as described in (Choo et al. 1990. Brit. Med. Bull. 46:423-441). These patients having chronic hepatitis C include those who are infected with multiple HCV genotypes including type 1 as well as those infected with HCV genotype 2 and/or 3. Hepatitis C Virus infections may be detected by measuring the patient's anti-HCV antibody response and the diagnosis is usually verified using polymerase chain reaction amplification of viral RNA.
  • HBV Hepatitis B virus
  • HBV is one of a family of animal viruses, hepadnaviruses, and is classified as hepadnavirus type 1. Similar viruses infect certain species of woodchucks, ground and tree squirrels, and Pekin ducks.
  • All hepadnaviruses including HBV, share the following characteristics: 1) three distinctive morphological forms exist, 2) all members have proteins that are functional and structural counterparts to the envelope and nucleocapsid antigens of HBV, 3) they replicate within the liver but can also exist in extrahepatic sites, 4) they contain an endogenous DNA polymerase with both RNA- and DNA-dependent DNA polymerase activities, 5) their genomes are partially double stranded circular DNA molecules, 6) they are associated with acute and chronic hepatitis and hepatocellular carcinoma and 7) replication of their genome goes through an RNA intermediate which is reverse transcribed into DNA using the virus's endogenous RNA-dependent DNA polymerase activity in a manner analogous to that seen in retroviruses.
  • the partially double stranded DNA is converted to a covalently closed circular double stranded DNA (cccdsDNA) by the DNA-dependent DNA polymerase.
  • cccdsDNA covalently closed circular double stranded DNA
  • Transcription of the viral DNA is accomplished by a host RNA polymerase and gives rise to several RNA transcripts that differ in their initiation sites but all terminate at a common polyadenylation signal. The longest of these RNAs acts as the pregenome for the virus as well as the message for the some of the viral proteins.
  • HIV Human Immunodeficiency Virus
  • HIV-I and HIV-2 are enveloped retroviruses with a diploid genome having two identical RNA molecules.
  • the molecular organization of HIV is (5 1 ) U3-R-U5-gag-pol-env-U3-R-U5 (3')
  • HIV isolates have been identified for nucleoside and non-nucleoside reverse transcriptase inhibitors and for protease inhibitors.
  • the emergence of HIV isolates resistant to AZT is not surprising since AZT and other reverse transcriptase inhibitors only reduce virus replication by about 90%. High rates of virus replication in the presence of the selective PL2007/000044
  • isatoribine is used herein and refers to a selective agonist of Tool like receptor no 7 (TLR7).
  • TLRs Toll-like receptors
  • Isatoribine a guanosine analog
  • isatoribine is structurally related to many direct-acting antiviral nucleoside analogs
  • the TLR7-activating mechanism of isatoribine is entirely different, eliciting antiviral activity through stimulation of a host receptor rather than through inhibition of a viral receptor. See Horsmans (Horsmans et al., 2005. Hepatology 42 (3), 724-731).
  • viramidine is used herein and refers to an anti-viral drug. It is a prodrug of ribavirin, active against a number of DNA and RNA viruses. Viramidine has better liver- targeting than ribavirin, and has a shorter life in the body due to less penetration and storage in red blood cells. It is expected eventually to be the drug of choice for viral hepatitis syndromes in which ribavirin is active. These include hepatitis C and perhaps also hepatitis B and yellow fever.
  • Lamivudine is used herein and refers to 2',3'-dideoxy-3'-thiacytidine, 3TC a potent reverse transcriptase inhibitor of the class nucleoside analog reverse transcriptase inhibitor (NARTI).
  • Lamivudine is an analogue of cytidine. It can inhibit both types (1 and 2) of HIV reverse transcriptase and also the reverse transcriptase of hepatitis B. It needs to be phosphorylated to its triphosphate form before it is active. 3TC-triphosphate also inhibits cellular DNA polymerase. It has been used for treatment of chronic HBV at a lower dose than for treatment of HIV.
  • the methods of the present invention are used for predicting the efficiency of treating infections caused by RNA viruses.
  • the methods of the present invention are especially suited for predicting the efficiency of treating infections arising from RNA viruses having a positive stranded genome and the various associated types.
  • the phrase "positive stranded genome" of a virus is one in which the genome, whether RNA or DNA, is single-stranded and which encodes a viral polypeptide(s).
  • positive stranded RNA viruses include the virus families Flaviviridae, Togaviridae, Coronaviridae, Retroviridae, Picornaviridae, and Caliciviridae. See Fields & Knipe 1986. "Fundamental Virology" (Raven Press, NY).
  • any anti-viral drug known in the art could be used in the combination with interferon with the invention.
  • anti-viral agents that can be used include, but are not limited to, those that target flaviviral proteins (e.g., protease/helicase, RDRP) or nucleic acids, or that otherwise inhibit any process or aspect of the viral life cycle, including, e.g., binding of or entry into cells, genome replication or processing, protein translation, modification or processing, proteolysis, capsid assembly, or viral maturation.
  • the flavivirus is HCV.
  • the anti-viral agent is ribavirin.
  • the anti-viral agent targets viral NSSB or NS 3 protein.
  • the anti-viral agent is an anti-sense nucleic acid.
  • the anti-viral agent is a ribozyme that targets specific sequences in the viral RNA.
  • An anti-viral drug or compound is said to "target” an HCV nucleic acid or protein if the drug or compound exerts its anti-viral effects directly or indirectly on the nucleic acid or protein.
  • interferon's complex anti-viral effect is mediated by a short sequence in the HCV NS5A protein termed the interferon sensitivity determining region (ISDR; Witherell et al., 2001. J. Med. Virol. 63:8-16), suggesting that the ISDR is a target of interferon.
  • An anti-viral drug or compound can directly or indirectly target an HCV nucleic acid or protein.
  • the anti-viral compound ribavirin although it is a nucleoside analog, is thought to not exert its anti-HCV effects through direct contact with or incorporation by the HCV NS5B polymerase. Thus, it might indirectly inhibit HCV through a direct interaction with one or more host cell factors.
  • the antiviral treatment is a delivery to the human body in the pharmaceutically accepted form and dose anti-viral drug.
  • the antiviral drug is a drug that targets one, two or more viral proteins encoded by the HCV-derived nucleic acid, including, but not limited to, C, El, E2, NS2, NS3, NS4A, NS4B, NS5A or NS5B.
  • the compound is an anti-hepatitis C compound or a biomolecule.
  • the biomolecule is a protein, nucleic acid, RNA or DNA
  • the present invention also provides a method for screening for viral mutants that are resistant to an anti-viral compound.
  • a virus derived nucleic acid sequence determined by the assay of the invention has a decreased susceptibility for the anti-viral compound, then it is collected or analyzed to determine one or more of its mutations.
  • a library of viral mutants having resistance to anti- viral compounds can thus be assembled enabling the screening of candidate anti-virals, alone or in combination, using the diagnostic method as described herein. This will enable one of ordinary skill to identify effective anti-virals and design effective therapeutic regimens.
  • the present invention also provides the MSSCP -based technique comprising: MSSCP separation of ssDNA derived PCR products representing highly variable region of selected virus (e.g. HCV), isolation of ssDNA bands from the MSSCP gel and sequencing. That makes possible to generate more information about HCV quasispecies and to investigate the genetic heterogeneity of the HCV at a better resolution when compared with the evaluation of HCV quasispecies based on the number of MSSCP or SSCP gel profiles alone.
  • This technique is applied here to analyze the evolution of the HCV quasispecies in the HVRl region in two groups of patients receiving Peg-interferon plus ribavirin therapy. These groups were constructed based on the EVR response at the 12th week.
  • HCV RNA viruses Typing Methodologies
  • the nucleic acid amplification products that have been generated by the previously most sensitive and specific exponential target-specific nucleic acid amplification reactions such as e.g. PCR (U.S. Pat. No. 4,683,202 or EP-B-O 202 362), RT-PCR, SDA, NASBA (EP-A-O 329 822) or TAM (WO 91/01384) were single or double-stranded nucleic acid amplification products produced by target sequence-dependent thermocyclic or isothermal enzymatic elongation of primers running in opposite directions that are sequence-specific for the nucleic acid to be detected and bind to the ends of the nucleic acid amplification unit (amplicon) of the deoxyribonucleic acids or ribonucleic acids to be detected or to complements thereof and thus restrict the nucleic acid amplification products. All four base specificities are incorporated in these elongation reactions.
  • HCV virus two mutations have been suggest to be responsible directly for interferon treatment failure (USPTO Al 20050260567) in case of the HIV the whole library of mutants have been collected and correlated with the specific drug resistance and the level of that resistance. Based on that knowledge dedicated assay and kit has been designed and offered to the healthcare service. Hover there is not offered by the inventors /applicant diagnostic tools based on the HCV single point interferon resistant mutant and the kit offered for prediction of the HIV virus drug resistance is only partially accurate, so not very widely used.
  • Cloning followed by sequencing makes it possible to acquire the sequence of single viral particles that are present within the viral quasispecies.
  • this approach is time consuming. It usually requires at least 2-3 days to obtain sequences and is expensive. Most of the costs are consumed by sequencing, and often the sequences delivered represent the same major population of viral particles present in the quasispecies — results are highly redundant.
  • Random picking up of clones for sequencing reflects the ratios of variants present in the quasispecies. When there is a one major variant within the quasispecies most of the clones have the same sequence, the sequence of this major variant.
  • SSCP PCR products representing a chosen region of the genome are analyzed in native polyacrylamide gels for the mobility of their single-stranded conformers. This results in an electrophoretical pattern that is unique for a given quasispecies composition.
  • the number of bands in the pattern reflects the number of different major sequences present in the sample and thus reflects the heterogeneity of the quasispecies.
  • the band pattern reflects only the major variants present in the quasispecies, since minor variants present in low molecular ratios in quasispecies are not visible.
  • the SSCP technique is robust, cheap and easy to perform. Its main limitation is that no information can be obtained concerning the sequences of the variants.
  • the genetic diversity (genetic distance) of the quasispecies can not be deducted directly from the band pattern.
  • the pattern reflects the number of viral variants within the viral quasispecies, but provides no information of their sequences.
  • MSSCP technique with higher mutation detection capacity than SSCP and was used with combination with the elution and sequencing of the isoforms present in the gel.
  • This method consists in the following steps: PCR amplification of a chosen region, MSSCP analysis of the PCR product, electroelution of bands isolated from MSSCP containing different ssDNA conformers and most probably different viral sequences, and sequencing of the eluted forms of HCV quasispecies. This approach is referred to as PMES.
  • MSSCP instead of SSCP results in better separation of the different viral variants present within quasispecies since MSSCP was shown to have a higher detection capacity for point mutations in PCR products.
  • MSSCP usually produces more complex band patterns than SSCP, and this should be beneficial when subsequent isolation and re-amplification of the bands are to be carried out.
  • Initial application of MSSCP or SSCP would result in the separation of major viral variants.
  • subsequent electroelution and sequencing of the DNA from those bands will allow more variable sequences to be achieved.
  • Viral variants should be genetically more diverse than those that are detected by molecular cloning and subsequent sequencing; initially they are separated by electrophoresis and not randomly chosen. Using PMES we expect to obtain a higher proportion of minor genetic variants compared to the cloning and sequencing approach.
  • SSCP is one of the methods used to separate different polynucleotide to determine the presence of different quasispecies in the host.
  • the SSCP analysis was used evaluate the genetic heterogeneity of HCV. The results depend on many electrophoresis parameters, the temperature of the samples being among the most important. SSCP electrophoresis of the HCV HVRl region has usually been performed without temperature control during electrophoresis at "room temperature" (Lopez- Labrador et al, 1999. Hepatol. 29(3):897-903, McKechnie et al., 2001. J Virol Met., 92(2):131-139, Koizumi et al., 1995. Hepatol. 22(l):30-35) or with temperature control at various temperatures ranging from 9 to 25 0 C (Pawlowsky et al., 1998).
  • MSSCP creates higher complexity patterns than SSCP. Not surprisingly SSCP and MSSCP electrophoresis of the PCR products from the 5'- UTR, results in different patterns for patients harboring the same virus subtype.
  • MSSCP and SSCP can be efficiently used for the separation of viral variants in the PMES technique. This separation allows more diverse populations of viral variants to be isolated for sequencing as compared to the molecular approach.
  • PMES takes advantage of the initial separation of the PCR products by MSSCP and is more efficient than molecular cloning to detect minor variants in the sample without the need of sequencing a large number of samples as is required with molecular cloning (i.e. 10 sequences in PMES versus 300 sequences when molecular cloning is used to detect a minor variant representing 0,3% of the quasispecies population). Sequencing is an expensive technique and substantial amounts of the cost of identifying minor variants in a heterogeneous sample comes from sequencing, PMES therefore is a cost effective alternative to molecular cloning for the identification of minor variants in a sample.
  • the PMES strategy evaluated versus direct cloning appears to better detect genetic heterogeneity in PCR products, measured as both genetic diversity (Hamming distance) (8,13 vs. 5,55) and number of identified variants (9 vs. 7). This results from initial separation of PCR products in the form of ssDNA conformers by MSSCP. In contrast, molecular cloning, resting on the isolation of clones in a repetitively random process, results in sequence redundancy and reflects the proportions between predominant viral variants in the quasispecies mixture. PMES is also more robust and therefore is a rational alternative to molecular cloning for the detection of heterogeneity.
  • the PMES method is be used but is not limited to, as an assay to determine the heterogeneity of HCV isolates in samples derived from HCV positive patients undergoing antiviral therapy.
  • the present invention addresses the need for individual scheme of combined therapy comprising the interferon which exhibit enhanced antiviral and/or immunomodulatory efficacy.
  • the invention provides novel method of assessing the effectiveness of antiviral treatment and novel therapeutic method based on the individual antiviral treatment, particularly for viral infections and diseases and conditions associated with viral infections.
  • the present invention fulfills these and other needs.
  • Serum from HCV positive patients was obtained from the Hospital for Infectious Diseases, Warsaw. Serum of patients used to optimize the quasispecies isolation method technique was obtained from patients diagnosed as infected with HCV genotypes: Ia, Ib 5 3a, and co-infected with HCV genotypes 4c/4d and lb/4a.
  • primer specific primer: see Table 1 or random hexamer primer
  • the reaction was incubated at 42 0 C for 1 hr, and after 5 min of deactivation at 95 0 C, DEPC-treated H2O was added to reach 100 ⁇ l.
  • the cDNA was stored at -8O 0 C for further analysis. Primers used for synthesis of first strand DNA.
  • HVRl HVRE2 Primer sequences are presented in Table IA.
  • HVRE2 TGGGAGTGAAGCAATATACCG
  • PCR amplification of the HVRl, 5'-UTR and NS5B regions of HCV were performed in PE 9600 Apparatus. Amplifications were performed with specific primers (Table IA). The reaction mixture contained 20 pmol of each primer, 0,2 mM dNTPs (Sigma Aldrich), 1 x Taq polymerase buffer (Kucharczyk TE), IU of Taq Polymerase (Kucharczyk TE), and cDNA matrix. Details are presented in Table 1 B. After denaturation at 94 0 C for 2 min, 35 or fewer amplification cycles were performed followed by 7 min at 72°C for final elongation. Table 1.
  • Taq polymerase was lu/25 1, and the Taq polymerase buffer was Ix.
  • Concentration of dNTPs was 0.2 mM and the concentration of each primer was 20 pmol .
  • Nested PCR amplification of the HVRl region was performed with specific primers.
  • Reaction mixture contained 20 pmol of each primer, 0,2 mM dNTPs, 1 x Taq polymerase buffer, IU of Taq Polymerase, 0.5 ⁇ l PCR amplification product. Details are presented in Table IB. After initial denaturation at 94 0 C for 2 min, 35 or fewer amplification cycles were performed followed by 7 min at 72 0 C for final elongation.
  • SSCP electrophoresis was performed at 35 0 C, 15 0 C or 5 0 C in 0.5 x TBE buffer. Initially 100V were applied for 10 min followed by 75 min of electrophoresis at 40 W resulting in 600-900 V.
  • electrophoresis was performed at temperatures 35-15-5 0 C for 25 min at each temperature and at 40 W.
  • a pre-electrophoresis was carried out to calibrate the gel for 100 Vh at 35°C and at 40 W.
  • the reaction mixture - (20 ⁇ l) contained: 20 pmol of each primer (NSIl and NSI2), 0.2 mM dNTP (Sigma Aldrich), 1 x Taq buffer (Kucharczyk TE), 1 U Taq polymerase (Kucharczyk), 1 ⁇ l of eluted ssDNA as template for reaction and 0,5 x SybrGreen I (Invitrogene).
  • the amplification procedure was as follows: denaturation at 94 0 C for 60 sec, 50 cycles at 94 0 C for 10 sec, 5O 0 C for 10 sec and 72 0 C for 12 sec. Final elongation was at 72 0 C for 7 min.
  • Electroelution was performed according to manufacturer's recommendations with minor modifications. Briefly, cartridges after calibration with washing mix were placed in a BGM system and electrophoresis buffer added to the cell to cover the cartridges. Fragments of polyacrylamide gel containing DNA were placed in the cartridges and electroelution was performed at 300 V for 30 min. The cartridges were washed with washing mix containing C 2 H 3 OH and the DNA was eluted with TE. After precipitation with isopropanole the DNA was diluted in distilled H 2 O.
  • PCR product amplified from the E1/E2 region with primers NSI1/NSI2 was cloned into pGEM-T vector systems (Promega), and transformed into Escherichia coli strain Jl 09.
  • the bacteria were grown on L-amp plates with X-GaI (40 ⁇ /ml) and IPTG (300 ⁇ g/ml).
  • plasmid DNA was extracted from 30 white clones with Plasmid Miniprep Kit (Kucharczyk TE, Warszawa) according to manufacturer's recommendations and plasmid size was inspected by submarine electrophoresis on an agarose gel containing EtBr. Ten colonies with plasmid containing the insert were selected and the plasmid DNA was sequenced using the T7 primer.
  • the genetic diversity was calculated by analysis of DNA sequences amplified from the E1/E2 gene of the HCV genome and the sequences of predicted amino acids including the 27 amino acids of HVRl.
  • the genetic diversity was calculated by the Hamming distance, which is defined as the number of nucleotide or amino acid differences between two sequences (Hamming et al, 1986. "Coding and info ⁇ nation Theory", Pretiee-Hall, Englewood Cliffs, NJ.,2-nd ed., Greneshan et al., 1997. J. Virol. 71(l),663-677).
  • the mean Hamming distance which is the average of the values taken for all sequence pairs derived from a single sample, was separately calculated for the HVRl region and the entire area sequenced. Analysis of genetic diversity and number of viral variants was performed after exclusion of defective or unreadable sequences on the 160 sequences (mean of 10 sequences for each sample).
  • MSSCP electrophoresis was performed at 3 temperatures: 35 0 C followed by 15 0 C followed by 5 0 C using polyacrylamide gels with or without a glycerol. This temperature profile is usually used for MSSCP electrophoresis and appears to be highly efficient for the detection of point mutations in PCR fragments. Glycerol is known to alter ssDNA conformations in polyacrylamide gels and influence the SSCP and MSSCP patterns.
  • the NS5B region of HCV is more variable than the 5'-UTR. Electrophoretical conditions for NS5B have been optimized for heterogeneity analysis. This includes maximizing the number of ssDNA bands and differentiating the electrophoretical mobility of the ssDNA isoforms.
  • the evaluation of SSCP and MSSCP include performing electrophoresis with different gel matrix compositions (addition of glycerol).
  • MSSCP electrophoresis of NS5B derived from genotypes Ia, Ib, 3a, lb/4a and 4c/4d in a gel without glycerol produces higher numbers of ssDNA forms visible on the gel compared to results of SSCP performed at temperatures 5°C, 15°C and 35 0 C.
  • SSCP electrophoresis performed at 35 0 C results in better separation between ssDNA fractions than SSCP at other temperatures and than MSSCP electrophoresis.
  • MSSCP electrophoresis results in electrophoretical patterns similar to those of SSCP performed at 15°C.
  • MSSCP electrophoresis is more efficient than SSCP since MSSCP results in a more complex pattern.
  • MSSCP more ssDNA isoforms can be differentiated and isolated from the gel for subsequent analysis.
  • HVRl SSCP and MSSCP electrophoresis of PCR products amplified from the most variable region of the HCV genome, HVRl, results in a large number of bands corresponding to different ssDNA conformers.
  • the electrophoretical pattern of HVRl is different for every sample. This reflects the high variability of the virus in this region of genome. Electrophoresis with MSSCP in a gel without glycerol results in better separation between ssDNA isoforms and more diverse electr ⁇ phoretical patterns than SSCP at 35, 15 or 5 0 C in gels with or without glycerol, and thus will allow to separate more bands corresponding to different variants.
  • MSSCP is a method of choice for sample heterogeneity detection. Since the ssDNA isoforms (electrophoretical bands) must be isolated from the gel, MSSCP better suites the PMES approach than SSCP for heterogeneity analysis of HVRl andNS5B.
  • ssDNA isoforms representing different viral variants are isolated from the polyacrylamide gel, and the DNA from these fragments eluted. Elution from polyacrylamide gels is more problematic than elution from agarose gels. Polyacrylamide can not be melted either by temperature or chemically. Usually elution is made by diffusion of the DNA from the gel as a result of Brownian movement of the DNA particles within the gel. However this technique is inefficient, most of the DNA particles staying within the gel matrix. To improve elution efficiency in the PMES approach the DNA was eluted by electroelution. The viral DNA variants are present in the isolated slices of the gel in very low amounts and to ensure that re-amplification of those variants will be possible, an efficient method of elution should be applied. Electroelution is more efficient than passive diffusion and is the preferred method.
  • Electroelution was performed using the Blue Gene machine (BGM) System and DNA electroelution Kits (Kucharczyk TE Inc). Since kits were optimized for elution of double stranded DNA, optimal conditions for elution of ssDNA fragments from polyacrylamide gels was needed. After optimization electroelution at 300V for 30 min was chosen as being more efficient than the standard procedure for elution of dsDNA - 100V for 30 min. The construction of a BGM was modified by introducing separate cells for each electroelution tube, to avoid contamination during elution procedure.
  • BGM Blue Gene machine
  • the PMES procedure was evaluated to estimate the minimal level of viral variant that can be detected in a sample.
  • a region of HCV NS5B was amplified in two samples representing two different HCV genotypes Ib and 3a as described earlier.
  • the PCR products were mixed in 1 :1, 1:10, 1:30, 1:100 and 1 :300 ratios.
  • the samples were analyzed by MSSCP in the DNA Pointer System.
  • DNA electroelution was performed at 300V for 30 min. Re-amplification was as described above and the products of PCR were analyzed on an agarose gel. PCR amplification results in clear specific products: no contamination was observed in samples that represent samples electroeluted from parts of the MSSCP gel where no DNA was loaded. No amplification was present in sample 10 that was electroeluted from an empty tube without gel, control of electroelution procedure.
  • PMES allows for the identification of minor viral variants without requiring extensive sequencing of viral clones.
  • the viral variants are initially separated, then different DNA isoforms are eluted and sequenced. This procedure greatly improves the probability of detecting minor viral variants without performing many sequencing reactions.
  • Viral RNA was isolated from the serum of a patient infected with HCV genotype Ib. A fragment of the viral genome representing the HVRl region was amplified. The quality of the PCR product was checked on an agarose gel and the PCR product was analyzed by MSSCP electrophoresis: 10 bands were isolated from the gel. The DNA was electroeluted from the gel slices, re-amplified and sequenced. For molecular cloning the PCR product was cloned into the pGem-T vector and 10 clones containing the HVRl insert were sequenced.
  • HVRl Genetic diversity within HVRl measured as the normalized mean Hamming distance was: 8.13 for PMES and 5.55 for the cloning and sequencing approach. Outside HVRl, genetic diversity was also higher when using PMES, 3.40 compared to 2.46 when using molecular cloning approach (Table T). Table 2
  • Region E1/E2 of HCV including the HVRl region was amplified with primers HVR E1/E2 and then with nested PCR using primers HVRIA/HVRIB.
  • the PCR products were then analyzed by MSSCP in a 9% polyacrylamide gel; 10 bands containing ssDNA isoforms for each sample was isolated, and DNA from the gel was eluted.
  • Ip O.001 for the change between time points a and b by paired t test.
  • the first time point "a" corresponds to the baseline, the second "b” to the 2nd week of the therapy and the third "c" to the 12th week of therapy.
  • Patterns of responses to interferon therapy were essentially associated with genetic variation within HVRl.
  • a weaker immunological response in group B that can be observed as more viral variants remain after two weeks of treatment, accounts for the diverse quasispecies population with higher numbers of variants able to escape the immunological system. Since no specific sequence classes are associated with resistance to treatment it can be speculated that those changes in genetic diversity and complexity within HCV quasispecies reflects fitness of the immunological system and its ability to eradicate the virus in the presence of antiviral drugs.
  • the sequence of the viral RNA within the HVRl region is not associated with response pattern in contrast to sequence of the 5'-UTR and NS5B regions, which determine the genotype of the virus and are strongly associated with resistance to antiviral therapy.
  • HIT Hepatitis Interventional Therapy
  • Domingo E Escarmis C, Sevilla N, Moya A, Maria SF, Quer J, Novewlla IS, Holland JJ.
  • Farci P. Shimoda A., Coiana A., et al.
  • the outcome of acute hepatitis C predicted by the evolution of the viral quasispecies, Science 2000; 228(5464):339-345
  • Farci P Shimoda A, Wong D, et al, Prevention of hepatitis C infection in Chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein, Proc Natl
  • Popovic M Sarngadharan MG, Read E, Gallo RCDetection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS

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Abstract

L'invention concerne des procédés et des réactifs pour déterminer la diversité de quasi-espèces virales et le nombre des différents variants viraux présents dans l'échantillon, lesquels procédés et réactifs sont utiles pour prédire la réponse à un traitement chez l'humain. La présente invention se fonde sur la découverte du fait que, chez un sujet humain infecté par un virus, on constate une association significative entre le changement du nombre de quasi-espèces virales différentes et leur diversité génétique et polypeptidique, et la réponse du sujet humain infecté au traitement dans les premières semaines de traitement. De façon spécifique, la probabilité d'une réponse virologique prolongée à un traitement comportant un interféron est supérieure chez les sujets présentant un nombre de sous-espèces virales différentes et une diversité polypeptidique moindre.
PCT/PL2007/000044 2006-06-30 2007-06-30 Procédé, dosage et coffret pour la prédiction et le traitement d'un sujet humain infecté par un virus WO2008002165A2 (fr)

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WO2013184553A1 (fr) * 2012-06-04 2013-12-12 Gen-Probe Incorporated Compositions et procédés d'amplification et de caractérisation d'un acide nucléique du vhc
WO2014108475A1 (fr) 2013-01-11 2014-07-17 Österreichische Akademie der Wissenschaften Peptides pour le traitement du cancer
WO2024223887A1 (fr) 2023-04-26 2024-10-31 Karl-Franzens-Universität Graz Peptides pour thérapie antimicrobienne
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010040756A1 (fr) * 2008-10-06 2010-04-15 Virco Bvba Procédé pour déterminer des mutations de résistance aux médicaments dans l'une quelconque des régions protéiques non structurales ns3 à ns5b du virus de l'hépatite c (vhc) pour les génotypes 1 à 6
US20110189684A1 (en) * 2008-10-06 2011-08-04 Lieven Jozef Stuyver Method for determining drug resistance mutations in any of the non-structural protein regions ns3 to ns5b of hepatitis c virus (hcv) for genotypes 1 to 6
US8945833B2 (en) 2008-10-06 2015-02-03 Lieven Jozef Stuyver Method for determining drug resistance mutations in any of the non-structural protein regions NS3 to NS5B of hepatitis C virus (HCV) for genotypes 1 to 6
WO2013184553A1 (fr) * 2012-06-04 2013-12-12 Gen-Probe Incorporated Compositions et procédés d'amplification et de caractérisation d'un acide nucléique du vhc
US20150152513A1 (en) * 2012-06-04 2015-06-04 Gen-Probe Incorporated Compositions and methods for amplifying and characterizing hcv nucleic acid
US10190180B2 (en) 2012-06-04 2019-01-29 Gen-Probe Incorporated Compositions and methods for amplifying and characterizing HCV nucleic acid
US11118237B2 (en) 2012-06-04 2021-09-14 Gen-Probe Incorporated Compositions and methods for amplifying and characterizing HCV nucleic acid
WO2014108475A1 (fr) 2013-01-11 2014-07-17 Österreichische Akademie der Wissenschaften Peptides pour le traitement du cancer
WO2024223887A1 (fr) 2023-04-26 2024-10-31 Karl-Franzens-Universität Graz Peptides pour thérapie antimicrobienne
WO2024223883A1 (fr) 2023-04-26 2024-10-31 Karl-Franzens-Universität Graz Peptides pour le traitement du cancer

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