WO2011092253A1 - Modified tuberculosis antigens - Google Patents
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- WO2011092253A1 WO2011092253A1 PCT/EP2011/051158 EP2011051158W WO2011092253A1 WO 2011092253 A1 WO2011092253 A1 WO 2011092253A1 EP 2011051158 W EP2011051158 W EP 2011051158W WO 2011092253 A1 WO2011092253 A1 WO 2011092253A1
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- modified rv3616c
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/06—Antibacterial agents for tuberculosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to modified Mycobacterium tuberculosis Rv3616c proteins, associated polynucleotides and the use of such proteins and polynucleotides in the treatment or prevention of tuberculosis, in particular use in the treatment or prevention of latent tuberculosis and in the prevention or delay of reactivation of tuberculosis.
- Tuberculosis is a chronic infectious disease caused by infection with
- Mycobacterium tuberculosis and other Mycobacterium species It is a major disease in developing countries, as well as an increasing problem in developed areas of the world. More than 2 billion people are believed to be infected with TB bacilli, with about 9.2 million new cases of TB and 1 .7 million deaths each year. 10% of those infected with TB bacilli will develop active TB, each person with active TB infecting an average of 10 to 15 others per year. While annual incidence rates have peaked globally, the number of deaths and cases is still rising due to population growth (World Health Organisation Tuberculosis Facts 2008).
- Mycobacterium tuberculosis infects individuals through the respiratory route. Alveolar macrophages engulf the bacterium, but it is able to survive and proliferate by inhibiting phagosome fusion with acidic lysosomes. A complex immune response involving CD4+ and CD8+ T cells ensues, ultimately resulting in the formation of a granuloma. Central to the success of Mycobacterium tuberculosis as a pathogen is the fact that the isolated, but not eradicated, bacterium may persist for long periods, leaving an individual vulnerable to the later development of active TB.
- an infection may be asymptomatic for a considerable period of time, the active disease is most commonly manifested as an acute inflammation of the lungs, resulting in tiredness, weight loss, fever and a persistent cough. If untreated, serious complications and death typically result.
- Tuberculosis can generally be controlled using extended antibiotic therapy, although such treatment is not sufficient to prevent the spread of the disease. Actively infected individuals may be largely asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behaviour is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistance.
- Multidrug-resistant TB (MDR-TB) is a form which fails to respond to first line medications. 5% of all TB cases are MDR-TB, with an estimated 490,000 new MDR-TB cases occurring each year. Extensively drug-resistant TB (XDR-TB) occurs when resistance to second line medications develops on top of MDR-TB. It is estimated that 40,000 new cases of the virtually untreatable XDR-TB arise annually (World Health Organisation Tuberculosis Facts 2008).
- BCG Bacillus Calmette- Guerin
- M. bovis an avirulent strain of M. bovis which was first developed over 60 years ago.
- BCG Bacillus Calmette- Guerin
- the safety and efficacy of BCG is a source of controversy - while protecting against severe disease manifestation in children, BCG does not prevent the establishment of latent TB or the reactivation of pulmonary disease in adult life.
- some countries, such as the United States do not vaccinate the general public with this agent. Almost all new generation TB vaccines which are currently in clinical development have been designed as pre-exposure vaccines.
- subunit vaccines which have been particularly effective in boosting immunity induced by prior BCG vaccination
- advanced live mycobacterial vaccines which aim to replace BCG with more efficient and/or safer strains.
- these vaccines aim to improve resistance to infection, they are likely to be less effective as post-exposure or therapeutic vaccines in latent TB cases (Lin MY et al Endocrine, Metabolic & Immune Disorders - Drug Targets 2008 8:15-29).
- Example 2 of US20080269151 discloses the cloning, construction and expression of certain modified Rv3616c proteins, including: ⁇ -1 , an Rv3616c polypeptide wherein residues 150 to 160 have been deleted (SEQ ID No: 22 of US20080269151 ); ⁇ -2, an Rv3616c polypeptide wherein residues 101 to 203 have been deleted (SEQ ID No: 24 of
- the present invention relates generally to the use of modified Rv3616c polypeptides, or polynucleotides encoding them, in the field of latent Mycobacterial infections. Additionally, the present invention relates to particular modified Rv3616c proteins. The inventors have surprisingly discovered that disrupting the hydrophobicity of a particular region of a Rv3616c sequence can lead to improved expression without detrimental impact to immunogenic properties.
- the modified Rv3616c proteins are of use as TB antigens, in particular as latent TB antigens.
- the present invention provides a modified Rv3616c protein in which the hydrophobicity of the amino acid residues corresponding to residues 134-183 of the H37Rv sequence has been disrupted, suitably a modified Rv3616c protein in which the hydrophobicity of the amino acid residues corresponding to residues 135-154 of the H37Rv sequence is disrupted.
- a modified Rv3616c protein said modified Rv3616c protein comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the C-terminus of the modified Rv3616c protein relative to the second polypeptide, and wherein:
- the first polypeptide is a sequence having at least 90% identity to residues 1 -
- the second polypeptide is a sequence having at least 90% identity to residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly or indirectly linked.
- modified Rv3616c protein comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the C-terminus of the modified Rv3616c protein relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -133 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 155 amino acids within residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly or indirectly linked.
- a modified Rv3616c protein comprising or, alternatively, consisting essentially or consisting of, a Rv3616c sequence in which at least one amino acid (e.g. at least 2) has been deleted from the region corresponding to residues 134-183 in SEQ ID No:1 .
- a fourth aspect of the invention provides a modified Rv3616c protein, said protein comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the N- terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -133 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 155 amino acids within residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly or indirectly linked via a third
- a fifth aspect of the invention provides modified Rv3616c proteins comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the N- terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -134 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 175 amino acids within residues 155-392 of SEQ ID No: 1 ;
- first and second polypeptides are either directly linked or indirectly linked via a third polypeptide, wherein said third polypeptide corresponds to residues 135-154 in SEQ ID No:1 in which at least 1 amino acid (e.g. at least 2) has been deleted.
- a sixth aspect of the invention provides a modified Rv3616c protein, said protein comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the N- terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a sequence having at least 90% identity to residues 1 -
- the second polypeptide is a sequence having at least 90% identity to residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly linked or indirectly linked via a third polypeptide, said third polypeptide having at least 90% identity to a sequence corresponding to residues 134-183 in SEQ ID No:1 in which a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted.
- a seventh aspect of the invention provides modified Rv3616c proteins comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the N- terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a sequence having at least 90% identity to residues 1 - 134 of SEQ ID No: 1 ;
- the second polypeptide is a sequence having at least 90% identity to residues 155-392 of SEQ ID No: 1 ;
- first and second polypeptides are either directly linked or indirectly linked via a third polypeptide, said third polypeptide having at least 80% identity to a sequence
- a modified Rv3616c protein comprising a Rv3616c sequence in which a contiguous portion of at least 3 amino acids (e.g. at least 4) from the region corresponding to residues 134-183 in SEQ ID No: 1 has been substituted with hydrophilic residues.
- Modified Rv3616c proteins may be based on a wild-type Rv3616c protein sequence from any strain of M. tuberculosis.
- any one of SEQ ID Nos: 3-7, in particular any one of SEQ ID Nos: 3-6 may be substituted for SEQ ID No:1 in the foregoing embodiments.
- Exemplary modified Rv3616c proteins according to the present invention are those comprising the amino acid sequences provided in SEQ ID Nos: 161 -169, 179 and 180 (such as those consisting of the amino acid sequences provided in SEQ ID Nos: 161 -169, 179 and 180).
- a further aspect of the invention relates to a method for inducing an immune response in a subject, comprising the administration of a modified Rv3616c protein.
- a further aspect of the invention relates to a method for the treatment, amelioration or prevention of TB comprising the administration of an effective amount of a modified Rv3616c protein to a subject in need thereof, wherein said polypeptide induces an immune response.
- the method further comprises inducing an immune response against Mycobacterium tuberculosis.
- the present invention provides a polynucleotide comprising a nucleic acid sequence encoding a modified Rv3616c protein.
- exemplary polynucleotides comprising a nucleic acid sequence encoding modified Rv3616c proteins are those comprising the nucleotide sequences provided in SEQ ID Nos: 170-178, such as those consisting of the nucleotide sequences provided in SEQ ID Nos: 170-178.
- Other exemplary polynucleotides comprising a nucleic acid sequence encoding modified Rv3616c proteins are those comprising (e.g. consisting of) a nucleotide sequence encoding an amino acid sequences provided in SEQ ID Nos: 161 -169, 179 or 180, such as SEQ ID Nos: 161 , 163-169, 179 or 180.
- polynucleotide comprising a nucleic acid sequence encoding a modified Rv3616c protein for use as a medicament.
- a further aspect of the invention relates to a method for inducing an immune response in a subject, comprising the administration of a polynucleotide comprising a nucleic acid sequence encoding a modified Rv3616c protein.
- a further aspect of the invention relates to a method for the treatment, amelioration, delaying or prevention of tuberculosis reactivation comprising the administration of an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a modified Rv3616c protein to a subject in need thereof, wherein said polypeptide induces and immune response.
- the method further comprises inducing an immune response against
- polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising a modified Rv3616c protein in the manufacture of a medicament for the treatment, amelioration or prevention of TB, represents another aspect of the invention.
- composition comprising:
- an immunogenic composition comprising:
- a non-specific immune response enhancer (c) a non-specific immune response enhancer. Also provided is an expression vector comprising a nucleic acid sequence encoding a modified Rv3616c protein.
- Host cells transformed with said expression vector, form a further aspect of the invention. Additionally provided is a host cell which recombinantly expresses a modified Rv3616c protein.
- said method comprising the step of recombinantly expressing said polypeptide within a host cell.
- diagnostic kits comprising:
- apparatus sufficient to contact said modified Rv3616c protein with a sample (e.g. whole blood or more suitably PBMC) from an individual; and (c) means to quantify the T cell response of the sample.
- a sample e.g. whole blood or more suitably PBMC
- Another aspect of the invention relates to a diagnostic kit comprising:
- a further aspect of the invention relates to a method for detecting Mycobacterium tuberculosis infection in a subject comprising:
- the invention also provides a diagnostic kit comprising:
- the subject receiving a modified Rv3616c protein, polynucleotide or composition according the invention may have active tuberculosis (e.g. active infection by M. tuberculosis).
- the subject may have latent tuberculosis (e.g. dormant infection by M. tuberculosis).
- the subject may be free from tuberculosis (e.g. free from infection by M. tuberculosis).
- a subject receiving a modified Rv3616c protein, polynucleotide or composition according to the invention may have previously been vaccinated for tuberculosis (e.g. vaccinated against infection by M. tuberculosis), such as having been vaccinated with a Bacillus Calmette-Guerin (BCG).
- a subject receiving a polypeptide, polynucleotide or composition of the invention may not have previously been vaccinated for tuberculosis (e.g. not vaccinated against infection by M. tuberculosis), such as not having been vaccinated with a Bacillus Calmette-Guerin (BCG).
- a modified Rv3616c protein, polynucleotide or composition according the invention may be provided for the purpose of:
- tuberculosis especially the delay of TB reactivation, for example by a period of months, years or even indefinitely.
- a polypeptide of the present invention in the manufacture of a diagnostic kit for the identification of TB (e.g. latent TB) in a test subject.
- Figure 1 Rv3616c peptide alignment with full length sequence.
- Figure 2 PBMC responses to Rv3616c peptides. Percentage of CD4 and CD8 cells from immunised CB6F1 mice expressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 21 (i.e. 7 days post second immunisation).
- Cytokine profile at day 21 i.e. 7 days post second immunisation of the antigen specific CD4 response in immunised CB6F1 mice.
- Cytokine profile at day 21 i.e. 7 days post second immunisation of the antigen specific CD8 response in immunised CB6F1 mice.
- CD4 and CD8 cells Percentage of CD4 and CD8 cells from immunised CB6F1 mice expressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 35 (i.e. 7 days post third immunisation).
- Cytokine profile at day 35 i.e. 7 days post third immunisation of the antigen specific CD4 response in immunised CB6F1 mice.
- Cytokine profile at day 35 i.e. 7 days post third immunisation of the antigen specific CD8 response in immunised CB6F1 mice.
- CD4 and CD8 cells Percentage of CD4 and CD8 cells from immunised C57BL/6 mice expressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 21 (i.e. 7 days post second immunisation).
- Cytokine profile at day 21 i.e. 7 days post second immunisation of the antigen specific CD4 response in immunised C57BL/6 mice.
- CD4 and CD8 cells Percentage of CD4 and CD8 cells from immunised C57BL/6 mice expressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 35 (i.e. 7 days post third immunisation).
- Cytokine profile at day 35 i.e. 7 days post third immunisation of the antigen specific CD4 response in immunised C57BL/6 mice.
- Cytokine profile at day 35 i.e. 7 days post third immunisation of the antigen specific CD8 response in immunised C57BL/6 mice.
- Figure 22 Cytokine profile of the Rv3616 specific CD4 T cell response at 7 days post third immunisation with Rv3616A138-145.
- Figure 23 Percentage of CD8 cells from immunised mice expressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at 7 days post second and 7 days post third immunisations with Rv3616A138-145.
- Figure 24 Cytokine profile of the Rv3616 specific CD8 T cell response at 7 days post second immunisation with Rv3616A138-145.
- Figure 25 Cytokine profile of the Rv3616 specific CD8 T cell response at 7 days post third immunisation with Rv3616A138-145.
- SEQ ID No: 1 polypeptide sequence of Rv3616c from M. tuberculosis H37Rv strain
- SEQ ID No: 2 polynucleotide sequence of Rv3616c from M. tuberculosis H37Rv strain
- SEQ ID No: 3 polypeptide sequence of Rv3616c from M. tuberculosis CDC1551 strain
- SEQ ID No: 4 polypeptide sequence of Rv3616c from M. tuberculosis F1 1 strain
- SEQ ID No: 5 polypeptide sequence of Rv3616c from M. tuberculosis Haarlem A
- SEQ ID No: 6 polypeptide sequence of
- SEQ ID No: 7 polypeptide sequence of
- SEQ ID No: 8 polypeptide sequence of
- SEQ ID No: 9 polypeptide sequence of
- SEQ ID No: 10 polypeptide sequence of
- SEQ ID No: 1 1 polypeptide sequence of
- SEQ ID No: 12 polypeptide sequence of
- SEQ ID No: 13 polypeptide sequence of
- SEQ ID No: 14 polypeptide sequence of
- SEQ ID No: 15 polypeptide sequence of
- SEQ ID No: 16 polypeptide sequence of
- SEQ ID No: 17 polypeptide sequence of
- SEQ ID No: 18 polypeptide sequence of
- SEQ ID No: 19 polypeptide sequence of
- SEQ ID No: 20 polypeptide sequence of SEQ ID No: 21 polypept de sequence of Ser/Ala mutated mature Mtb32A.
- SEQ ID No: 26 polypept de sequence of M92 fusion.
- SEQ ID No: 27 polypept de sequence of M103 fusion.
- SEQ ID No: 28 polypept de sequence of M1 14 fusion.
- SEQ ID No: 56 tative human CD8 ce ep tope 9.
- SEQ D No: 57 putative human CD8 eel epitope 10.
- SEQ D No: 58 putative human CD8 eel epitope 1 1 .
- SEQ D No: 59 putative human CD8 eel epitope 12.
- SEQ D No: 60 putative human CD8 eel epitope 13.
- SEQ D No: 61 putative human CD8 eel epitope 14.
- SEQ D No: 62 putative human CD8 eel epitope 15.
- SEQ D No: 63 putative human CD8 eel I epitope 16.
- SEQ D No: 64 putative human CD8 eel I epitope 17.
- SEQ D No: 65 putative human CD8 eel I epitope 18.
- SEQ D No: 66 putative human CD8 eel I epitope 19.
- SEQ D No: 67 putative human CD8 eel I epitope 20.
- SEQ D No: 68 putative human CD8 eel I epitope 21 .
- SEQ D No: 69 putative human CD8 eel I epitope 22.
- SEQ ID No: 70 putative human CD8 eel I epitope 23
- SEQ ID No: 71 putative human CD8 eel I epitope 24
- SEQ ID No: 72 putative human CD8 eel I epitope 25
- SEQ ID No: 73 putative human CD8 eel I epitope 26
- SEQ ID No: 74 putative human CD8 ce I epitope 27
- SEQ ID No: 75 putative human CD8 ce I epitope 28
- SEQ ID No: 77 putative human CD8 eel I epitope 30
- SEQ ID No: 78 putative human CD8 ce I epitope 31
- SEQ ID No: 80 putative human CD8 ce I epitope 33
- SEQ ID No: 81 putative human CD8 ce I epitope 34
- SEQ ID No: 82 putative human CD8 ce I epitope 35
- SEQ ID No: 84 putative human CD8 ce I epitope 37
- SEQ ID No: 85 putative human CD8 ce I epitope 38
- SEQ ID No: 88 putative human CD8 ce I epitope 41
- SEQ ID No: 90 putative human CD8 ce I epitope 43
- SEQ ID No: 92 putative human CD8 ce I epitope 45
- SEQ D No: 93 putative human CD8 cell epitope 46.
- SEQ D No: 94 putative human CD8 cell epitope 47.
- SEQ D No: 95 putative human CD8 cell epitope 48.
- SEQ D No: 96 putative human CD8 cell epitope 49.
- SEQ D No: 97 putative human CD8 cell epitope 50.
- SEQ D No: 98 putative human CD8 cell epitope 51 .
- SEQ D No: 99 putative human CD8 cell epitope 52.
- SEQ D No: 100 putative human CD8 cell epitope 53.
- SEQ D No: 101 putative human CD8 cell epitope 54.
- SEQ ID No: 102 putative human CD8 cell epitope 55.
- SEQ ID No: 103 putative human CD8 cell epitope 56.
- SEQ ID No: 104 putative human CD8 cell epitope 57.
- SEQ ID No: 105 putative human CD8 cell epitope 58.
- SEQ ID No: 1 18 putative human CD8 cell epitope 71 SEQ ID No: 1 18 putative human CD8 cell epitope 71 .
- SEQ ID No: 120 putative human CD8 cell epitope 73 SEQ ID No: 120 putative human CD8 cell epitope 73.
- tuberculosis H37Rv strain H37Rv strain.
- SEQ ID No: 161 polypeptide sequence of Rv3616cA136-183 derived from M.
- SEQ ID No: 162 polypeptide sequence of Rv3616cA150-160 derived from M. tuberculosis H37Rv strain.
- SEQ ID No: 163 polypeptide sequence of Rv3616cA136-154 derived from M.
- tuberculosis H37Rv strain H37Rv strain.
- SEQ ID No: 164 polypeptide sequence of Rv3616cA166-182 derived from M.
- tuberculosis H37Rv strain H37Rv strain.
- SEQ ID No: 165 polypeptide sequence of Rv3616cA135-139 derived from M.
- tuberculosis H37Rv strain H37Rv strain.
- SEQ ID No: 166 polypeptide sequence of Rv3616cA142-145 derived from M.
- tuberculosis H37Rv strain H37Rv strain.
- SEQ ID No: 167 polypeptide sequence of Rv3616cA138-145 derived from M.
- tuberculosis H37Rv strain H37Rv strain.
- SEQ ID No: 168 polypeptide sequence of Rv3616cA145-152 derived from M.
- tuberculosis H37Rv strain H37Rv strain.
- SEQ ID No: 169 polypeptide sequence of Rv3616cA149-154 derived from M.
- tuberculosis H37Rv strain H37Rv strain.
- SEQ ID No: 170 £ coli codon optimised polynucleotide sequence encoding
- Rv3616cA136-183 derived from M. tuberculosis H37Rv strain.
- SEQ ID No: 171 £ coli codon optimised polynucleotide sequence encoding
- Rv3616cA136-154 derived from M. tuberculosis H37Rv strain.
- SEQ ID No: 173 £. coli codon optimised polynucleotide sequence encoding
- Rv3616cA166-182 derived from M. tuberculosis H37Rv strain.
- Rv3616cA135-139 derived from M. tuberculosis H37Rv strain.
- SEQ ID No: 175 £ coli codon optimised polynucleotide sequence encoding
- Rv3616cA142-145 derived from M. tuberculosis H37Rv strain.
- SEQ ID No: 176 £ coli codon optimised polynucleotide sequence encoding
- SEQ ID No: 177 £ coli codon optimised polynucleotide sequence encoding
- Rv3616cA145-152 derived from M. tuberculosis H37Rv strain.
- SEQ ID No: 178 £ coli codon optimised polynucleotide sequence encoding
- Rv3616cA149-154 derived from M. tuberculosis H37Rv strain.
- SEQ ID No: 179 polypeptide sequence of modified Rv3616c protein based on separating and rearrangement around the residues 137-139 from M. tuberculosis H37Rv strain, including deletion of Cys138.
- SEQ ID No: 180 polypeptide sequence of modified Rv3616c protein based on separating and rearrangement around the residues 152-153 from M. tuberculosis
- the present invention relates generally to the use of modified Rv3616c polypeptides, or polynucleotides encoding them, in the field of latent Mycobacterial infections. Additionally, the present invention relates to particular modified Rv3616c proteins. The inventors have surprisingly discovered that disrupting the hydrophobicity of a particular region of a Rv3616c protein sequence can lead to improved expression without substantial detrimental impact to immunogenic properties.
- the modified Rv3616c proteins are of use as TB antigens, in particular as latent TB antigens.
- late stage antigens could be used to complement and improve BCG vaccination (either by boosting the BCG response or through the development of advanced recombinant BCG strains).
- macrophages have been shown to act as the principal effectors of Mycobacterium immunity
- T cells are the predominant inducers of such immunity.
- the essential role of T cells in protection against tuberculosis is illustrated by the increased rates of TB reactivation in human immunodeficiency virus infected individuals, due to the associated depletion of CD4+ T cells.
- adoptive transfer of CD4+ T cells taken at the height of the primary immune response to M. tuberculosis has been shown to confer protection against M.
- Mycobacterium-reactwe CD4+ T cells have been shown to be potent producers of ⁇ -interferon (IFN- ⁇ ), which, in turn, has been shown to trigger the anti-mycobacterial effects of IFN- ⁇
- Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot always be easily distinguished from infected individuals (this is particularly important in light of the fact that BCG does not protect against latent infection).
- individuals who have received BCG but are not infected by M. tuberculosis show a PPD reaction below 10 mm in diameter whereas people who have a PPD reaction above 10 mm in diameter are considered to have been infected by M. tuberculosis.
- M. tuberculosis vaccine candidates have been proposed based on a bioinformatics analysis of the whole genome M. tuberculosis genome (Zvi et al. BMC Medical Genetics 2008 1 :18) and on the testing of differentially expressed proteins in actively and latently infected individuals (Schuck SD et al. PLoS ONE 2009 4(5):e5590).
- Rv3616c also known as Mtb40, HTCC1 and EspA, is involved in the Mycobacterium tuberculosis ESX-1 secretion system (Woodsworth et al. Infection and Immunity 2008
- Rv3616c has previously been implicated in the immune responses associated with tuberculosis (see, for example, WO98/53075). Al-Attiyah et al. Clin. Exp. Immunol. 2004 138:139-144 have shown that Rv3616c is well recognised (through PMBC proliferation and IFN-gamma production) by pulmonary tuberculosis patients. Mustafa et al. Infect. Immun. 2006 74(8):4566-4572 have investigated the recognition of Rv3616c by M. bovis infected and BCG vaccinated cattle.
- Vaccine antigens are desirably produced having their wild-type sequence, thus ensuring that the immunological responses solicited by the vaccine correspond closely to those required to counter infection by a pathogen. Nevertheless, efficient production of antigens is an important factor in reducing the costs associated with vaccine manufacture. Consequently, modified antigens which are conveniently expressed at high levels but which avoid any detrimental impact on immunogenicity could provide a substantial benefit.
- the present invention seeks to provide modified Rv3616c antigens which address this and other issues.
- amino acid residues 134-183 of the Mycobacterium are amino acids residues 134-183 of the Mycobacterium.
- tuberculosis H37Rv strain Rv3616c are thought to correspond to a potential transmembrane region, a low complexity region and a coiled-coil. The disruption of one, two or all three of these structural elements enables the resultant modified Rv3616c protein sequence to be expressed at improved levels.
- the present invention provides a modified Rv3616c protein in which the hydrophobicity of the amino acid residues corresponding to residues 134- 183 of the H37Rv sequence has been disrupted, suitably a modified Rv3616c protein in which the hydrophobicity of the amino acid residues corresponding to residues 135-154 of the H37Rv sequence is disrupted.
- 'disrupting the hydrophobicity' is meant a sequence modification which results in a sufficiently reduced hydrophobicity such that the modified Rv3616c protein sequence may be expressed more efficiently.
- a 'direct peptide linkage' is a peptide linkage in which two peptides are linked via peptide bonds directly to each other and without an intervening amino acid sequence.
- An 'indirect peptide linkage' is a peptide linkage in which two peptides are linked via peptide bonds to a third, intervening peptide.
- Separating hydrophobic residues may be achieved by splitting an Rv3616c protein sequence at a location between the amino acids corresponding to residues 133 to 184 of SEQ ID No: 1 into an N-terminal and a C-terminal fragment, followed by rearranging such portions such that the N-terminal fragment is located in the C-terminal region of the modified Rv3616c protein and the C-terminal fragment is located in the N-terminal region of the modified Rv3616c protein.
- modified Rv3616c protein comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the C-terminus of the modified Rv3616c protein relative to the second polypeptide, and wherein:
- the first polypeptide is a sequence having at least 90% identity to residues 1 - 133 of SEQ ID No: 1 ;
- the second polypeptide is a sequence having at least 90% identity to residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly or indirectly linked.
- the modified Rv3616c protein consists essentially of, or alternatively consists of, a first polypeptide and a second polypeptide, the first polypeptide being located towards the C-terminus of the modified Rv3616c protein relative to the second polypeptide, and wherein:
- the first polypeptide is a sequence having at least 90% identity to residues 1 - 133 of SEQ ID No: 1 ;
- the second polypeptide is a sequence having at least 90% identity to residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly or indirectly linked.
- the first polypeptide may be a sequence having at least 95% identity to residues 1 -133 of SEQ ID No: 1 , such as at least 97% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the second polypeptide may be a sequence having at least 95% identity to residues 184-392 of SEQ ID No: 1 , such as at least 97% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the first polypeptide may be a sequence having at least 90% identity to residues 1 - 134 of SEQ ID No: 1 , in particular at least 95% identity, such as at least 97% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the second polypeptide may be a sequence having at least 90% identity to residues 155-392 of SEQ ID No: 1 , in particular at least 95% identity, such as at least 97% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the modified Rv3616c protein of the first aspect does not comprise a sequence having at least 90% identity to full length SEQ ID No: 1 .
- the modified Rv3616c protein of the first aspect is less than 500 amino acids long, such as less than 450 amino acids long, in particular less than 400 amino acids long.
- the peptide linkage may be direct.
- the peptide linkage may alternatively be indirect.
- modified Rv3616c protein comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the C-terminus of the modified Rv3616c protein relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -133 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 155 amino acids within residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly or indirectly linked.
- the modified Rv3616c protein consists essentially of, or alternatively consists of, a first polypeptide and a second polypeptide, the first polypeptide being located towards the C-terminus of the modified Rv3616c protein relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -133 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 155 amino acids within residues 184-392 of SEQ ID No: 1 ;
- the first polypeptide may be a contiguous sequence of at least 1 10 amino acids within residues 1 -133 of SEQ ID No: 1 , such as at least 120 amino acids or at least 130 amino acids,for example residues 1 -133.
- the second polypeptide may be a contiguous sequence of at least 180 amino acids within residues 184-392 of SEQ ID No: 1 , such as at least 190 amino acids or at least 200 amino acids, for example residues 184-392.
- the first polypeptide may be a contiguous sequence of at least 100 amino acids within residues 1 -134 of SEQ ID No: 1 , in particular at least 1 10 amino acids, such as at least 120 amino acids or at least 130 amino acids, for example residues 1 -134.
- the second polypeptide may be a contiguous sequence of at least 175 amino acids within residues 155-392 of SEQ ID No: 1 , in particular at least 200 amino acids such as at least 210 amino acids or at least 220 amino acids, for example residues 155-392.
- Embodiments wherein the second polypeptide is a contiguous sequence of at least 235 amino acids within residues 155-392 of SEQ ID No: 1 are also of interest.
- the modified Rv3616c protein of the second aspect does not comprise a contiguous sequence of more than 259 amino acids from SEQ ID No: 1 .
- the modified Rv3616c protein of the second aspect does not comprise a contiguous sequence of more than 259 amino acids from SEQ ID No: 1 .
- Rv3616c protein of the second aspect does not comprise a contiguous sequence of more than 257 amino acids, a contiguous sequence of more than 255 amino acids or a contiguous sequence of more than 253 amino acids.
- the modified Rv3616c protein of the second aspect is less than 500 amino acids long, such as less than 450 amino acids long, in particular less than 400 amino acids long.
- the peptide linkage may either be a direct or indirect linkage.
- first and second aspects include modified Rv3616c proteins wherein the first and second polypeptide correspond to the N-terminal and C-terminal fragments resulting from splitting an Rv3616c sequence at a location between the amino acids corresponding to residues 135-154 in SEQ ID No: 1 , e.g. the residues 138-139 or 152-153, e.g. the residues 138-139 or 152-153 where the peptide linkage is direct.
- the start methionine is left at the N-terminus of the modified Rv3616c protein. See for example SEQ ID Nos: 179 and 180 which illustrate this type of arrangement.
- Deleting hydrophobic residues may be achieved through the removal of at least one amino acid corresponding to residues 134 to 183 of SEQ ID No: 1 .
- Deleted residues may be non- contiguous, and/or contiguous.
- deleting hydrophobic residues may be achieved through the removal of at least two amino acids corresponding to residues 134 to 183 of SEQ ID No: 1 .
- Deleting hydrophobic residues may also be achieved through the removal of at least three amino acids
- Deleted residues may be non-contiguous, and/or contiguous.
- wild-type Rv3616c sequences contain a Cys residue at location 138.
- this Cys residue is deleted or replaced (e.g. C138Q).
- a modified Rv3616c protein comprising or, alternatively, consisting essentially or consisting of, a Rv3616c sequence in which at least one amino acid (e.g. at least 2) has been deleted from the region corresponding to residues 134-183 in SEQ ID No:1 .
- the modified Rv3616c protein may comprise or, alternatively, consists essentially or consist of, a Rv3616c sequence in which a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted from the region corresponding to residues 134-183 in SEQ ID No:1.
- modified Rv3616c proteins comprising a Rv3616c sequence in which at least 1 amino acid (e.g. at least 2) has been deleted from the region corresponding to residues 135-154 in SEQ ID No:1.
- modified Rv3616c proteins comprising a Rv3616c sequence in which a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted from the region corresponding to residues 135-154 in SEQ ID No:1 .
- the deleted contiguous portion may be at least 5 amino acids (e.g. 5 to 30, such as 5 to 20 or 5 to 15), especially at least 6 amino acids (e.g. 6 to 30, such as 6 to 20 or 6 to 15), in particular at least 7 amino acids (e.g. 7 to 30, such as 7 to 20 or 7 to 15), such as at least 8 amino acids (e.g. 8 to 30, such as 8 to 20 or 8 to 15), or at least 10 amino acids (e.g. 10 to 30, such as 10 to 20 or 10 to 15).
- amino acids e.g. 5 to 30, such as 5 to 20 or 5 to 15
- at least 6 amino acids e.g. 6 to 30, such as 6 to 20 or 6 to 15
- at least 7 amino acids e.g. 7 to 30, such as 7 to 20 or 7 to 15
- at least 8 amino acids e.g. 8 to 30, such as 8 to 20 or 8 to 15
- at least 10 amino acids e.g. 10 to 30, such as 10 to 20 or 10 to 15
- the deleted contiguous portion may be 3 to 10 amino acid residues, such as 4 to 10, for example 4 to 8.
- the particular number of deleted amino acids may be 3, 4, 5, 6, 7, 8, 9 or 10, especially 4, 5, 6 or 8.
- the deleted portion may be those corresponding to residues 135-138 in SEQ ID No: 1 , residues 136-138 in SEQ ID No: 1 , residues 137-138 in SEQ ID No: 1 , residues 138-140 in SEQ ID No: 1 , residues 138-141 in SEQ ID No: 1 , residues 152-154 in SEQ ID No: 1 or the deletion of residues 149-151 in SEQ ID No: 1 .
- a fourth aspect of the invention provides a modified Rv3616c protein, said protein comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the N- terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -133 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 155 amino acids within residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly or indirectly linked via a third
- the modified Rv3616c protein consists essentially of, or alternatively consists of, a first polypeptide and a second polypeptide, the first polypeptide being located towards the N-terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -133 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 155 amino acids within residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly or indirectly linked via a third
- polypeptide said third polypeptide corresponding to residues 134-183 in SEQ ID No:1 in which at least 1 amino acid (e.g. at least 2) has been deleted.
- proteins comprising, or alternatively consisting essentially or consisting of, a first polypeptide and a second polypeptide, the first polypeptide being located towards the N-terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -133 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 155 amino acids within residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly or indirectly linked via a third
- polypeptide said third polypeptide corresponding to residues 134-183 in SEQ ID No:1 in which at least a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted.
- the first polypeptide may be a contiguous sequence of at least 1 10 amino acids within residues 1 -133 of SEQ ID No: 1 , such as at least 120 amino acids or at least 130 amino acids (for example residues 1 -133).
- the second polypeptide may be a contiguous sequence of at least 180 amino acids within residues 184-392 of SEQ ID No: 1 , such as at least 190 amino acids or at least 200 amino acids (for example residues 184-392).
- the deleted contiguous portion from the residues corresponding to 134-183 in SEQ ID No:1 may be at least 5 amino acids (e.g. 5 to 30, such as 5 to 20 or 5 to 15), especially at least 6 amino acids (e.g. 6 to 30, such as 6 to 20 or 6 to 15), in particular at least 7 amino acids (e.g. 7 to 30, such as 7 to 20 or 7 to 15), such as at least 8 amino acids (e.g. 8 to 30, such as 8 to 20 or 8 to 15), or at least 10 amino acids (e.g. 10 to 30, such as 10 to 20 or 10 to 15).
- the deleted contiguous portion from the residues corresponding to 134-183 in SEQ ID No:1 may be:
- - 1 1 amino acids such as those corresponding to residues 150-160 in SEQ ID No:1 ; - 17 amino acids, such as those corresponding to residues 166-182 in SEQ ID No:1 ; - 19 amino acids, such as those corresponding to residues 136-154 in SEQ ID No:1 ;
- the deleted contiguous portion from the residues corresponding to 134- 183 in SEQ ID No:1 may be may be 3 to 10 amino acid residues, such as 4 to 10, for example 4 to 8.
- the particular number of deleted amino acids may be 3, 4, 5, 6, 7, 8, 9 or 10, especially 4, 5, 6 or 8.
- the deleted contiguous portion from the residues corresponding to 134- 183 in SEQ ID No:1 may be those corresponding to residues 135-138 in SEQ ID No: 1 , residues 136-138 in SEQ ID No: 1 , residues 137-138 in SEQ ID No: 1 , residues 138-140 in SEQ ID No: 1 , residues 138-141 in SEQ ID No: 1 , residues 152-154 in SEQ ID No: 1 or the deletion of residues 149-151 in SEQ ID No: 1.
- the first polypeptide and second polypeptide will in some embodiments be directly linked. In other embodiments the first polypeptide and second polypeptide will be indirectly linked via a third polypeptide.
- the third polypeptide may correspond to residues 134-183 in SEQ ID No: 1 wherein deletion has occurred at a single contiguous portion of at least 3 amino acids (e.g. at least 4). Additionally, the third polypeptide may correspond to residues 134-183 in SEQ ID No: 1 wherein deletions have occurred at a plurality of distinct locations (e.g. 1 -10, such as 1 -5, in particular 1 or 2 locations), each deletion being of 1 -10, such as 1 -5 amino acid residues.
- the third polypeptide is 48 amino acids or fewer (e.g. 10-48, such as 20-48 or 30-48 residues), such as 46 amino acids or fewer (e.g. 10-46, such as 20-46 or 30-46 residues), 44 amino acids or fewer (e.g. 10-44, such as 20-44 or 30-44 residues), or 42 amino acids or fewer (e.g. 10-42, such as 20-42 or 30-42 residues).
- a fifth aspect of the invention provides modified Rv3616c proteins comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the N- terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -134 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 175 amino acids within residues 155-392 of SEQ ID No: 1 ;
- first and second polypeptides are either directly linked or indirectly linked via a third polypeptide, wherein said third polypeptide corresponds to residues 135-154 in SEQ ID No:1 in which at least 1 amino acid (e.g. at least 2) has been deleted.
- the modified Rv3616c protein consists essentially of, or alternatively consists of, a first polypeptide and a second polypeptide, the first polypeptide being located towards the N-terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -134 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 175 amino acids within residues 155-392 of SEQ ID No: 1 ;
- first and second polypeptides are either directly linked or indirectly linked via a third polypeptide, wherein said third polypeptide corresponds to residues 135-154 in SEQ ID No:1 in which at least 1 amino acid (e.g. at least 2) has been deleted.
- proteins comprising, or alternatively consisting essentially or consisting of, a first polypeptide and a second polypeptide, the first polypeptide being located towards the N-terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a contiguous sequence of at least 100 amino acids within residues 1 -134 of SEQ ID No: 1 ;
- the second polypeptide is a contiguous sequence of at least 175 amino acids within residues 155-392 of SEQ ID No: 1 ;
- first and second polypeptides are either directly linked or indirectly linked via a third polypeptide, wherein said third polypeptide corresponds to residues 135-154 in SEQ ID No:1 in which at least a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted.
- the first polypeptide may also be a contiguous sequence of at least 1 10 amino acids within residues 1 -134 of SEQ ID No: 1 , such as at least 120 amino acids or at least 130 amino acids, for example residues 1 -134.
- the second polypeptide may also be a contiguous sequence of at least 200 amino acids within residues 155-392 of SEQ ID No: 1 , such as at least 210 amino acids or at least 220 amino acids, for example residues 155-392.
- the second polypeptide is a contiguous sequence of at least 235 amino acids within residues 155-392 of SEQ ID No: 1 are also of interest.
- the deleted contiguous portion from the residues corresponding to 135-154 in SEQ ID No: 1 may be at least 5 amino acids (e.g. 5 to 20, such as 5 to 15 or 5 to 10), especially at least 6 amino acids (e.g. 6 to 20, such as 6 to 15 or 6 to 10), in particular at least 7 amino acids (e.g. 7 to 20, such as 7 to 15 or 7 to 10), such as at least 8 amino acids (e.g. 8 to 20, such as 8 to 15 or 8 to 10), or at least 10 amino acids (e.g. 10 to 20, such as 10 to 15).
- amino acids e.g. 5 to 20, such as 5 to 15 or 5 to 10
- at least 6 amino acids e.g. 6 to 20, such as 6 to 15 or 6 to 10
- 7 amino acids e.g. 7 to 20, such as 7 to 15 or 7 to 10
- at least 8 amino acids e.g. 8 to 20, such as 8 to 15 or 8 to 10
- at least 10 amino acids e.g. 10 to 20, such as 10 to 15
- the deleted contiguous portion from the residues corresponding to 135-154 in SEQ ID No: 1 may be:
- the deleted contiguous portion from the residues corresponding to 135- 154 may be 3 to 10 amino acid residues, such as 4 to 10, for example 4 to 8.
- the particular number of deleted amino acids may be 3, 4, 5, 6, 7, 8, 9 or 10, especially 4, 5, 6 or 8.
- the deleted contiguous portion from the residues corresponding to 135- 154 in SEQ ID No: 1 may be those corresponding to residues 135-138 in SEQ ID No: 1 , residues 136-138 in SEQ ID No: 1 , residues 137-138 in SEQ ID No: 1 , residues 138-140 in SEQ ID No: 1 , residues 138-141 in SEQ ID No: 1 , residues 152-154 in SEQ ID No: 1 or the deletion of residues 149-151 in SEQ ID No: 1.
- the first polypeptide and second polypeptide may in some embodiments be directly linked. In other embodiments the first polypeptide and second polypeptide may be indirectly linked via a third polypeptide.
- the third polypeptide may correspond to residues 135-154 in SEQ ID No: 1 wherein deletion has occurred at a single contiguous portion of at least 3 amino acids (e.g. at least 4). Additionally, the third polypeptide may correspond to residues 135-154 in SEQ ID No: 1 wherein deletions have occurred at a plurality of distinct locations (e.g. 1 -10, such as 1 -5, in particular 1 or 2 locations), each deletion being of 1 -10, such as 1 -5 amino acid residues.
- a plurality of distinct locations e.g. 1 -10, such as 1 -5, in particular 1 or 2 locations
- the third polypeptide is 20 amino acids or fewer (e.g. 5-20, such as 10-20 residues), such as 18 amino acids or fewer (e.g. 5-18, such as 10-18 residues), 16 amino acids or fewer (e.g. 5-16, such as 10-16 residues), or 14 amino acids or fewer (e.g. 5-14, such as 10-14 residues).
- a sixth aspect of the invention provides a modified Rv3616c protein, said protein comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the N- terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a sequence having at least 90% identity to residues 1 - 133 of SEQ ID No: 1 ;
- the second polypeptide is a sequence having at least 90% identity to residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly linked or indirectly linked via a third polypeptide, said third polypeptide having at least 90% identity to a sequence corresponding to residues 134-183 in SEQ ID No:1 in which a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted.
- the modified Rv3616c protein consists essentially of, or alternatively consists of, a first polypeptide and a second polypeptide, the first polypeptide being located towards the N-terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a sequence having at least 90% identity to residues 1 - 133 of SEQ ID No: 1 ;
- the second polypeptide is a sequence having at least 90% identity to residues 184-392 of SEQ ID No: 1 ;
- first and second polypeptides are directly linked or indirectly linked via a third polypeptide, said third polypeptide having at least 90% identity to a sequence corresponding to residues 134-183 in SEQ ID No:1 in which a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted.
- the first polypeptide may be a sequence having at least 95% identity to residues 1 -133 of SEQ ID No: 1 , such as at least 97% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the second polypeptide may be a sequence having at least 95% identity to residues 184-392 of SEQ ID No: 1 , such as at least 97% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the first polypeptide and second polypeptide may in some embodiments be directly linked. In other embodiments the first polypeptide and second polypeptide will be indirectly linked via a third polypeptide.
- the third polypeptide may be a sequence having at least 95% identity to a sequence corresponding to residues 134-183 in SEQ ID No:1 in which a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted, such as at least 97% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the contiguous portion deleted from the residues corresponding to 134-183 in SEQ ID No: 1 may be at least 5 amino acids (e.g. 5 to 30, such as 5 to 20 or 5 to 15), especially at least 6 amino acids (e.g. 6 to 30, such as 6 to 20 or 6 to 15), in particular at least 7 amino acids (e.g. 7 to 30, such as 7 to 20 or 7 to 15), such as at least 8 amino acids (e.g. 8 to 30, such as 8 to 20 or 8 to 15), or at least 10 amino acids (e.g. 10 to 30, such as 10 to 20 or 10 to 15).
- amino acids e.g. 5 to 30, such as 5 to 20 or 5 to 15
- 6 amino acids e.g. 6 to 30, such as 6 to 20 or 6 to 15
- 7 amino acids e.g. 7 to 30, such as 7 to 20 or 7 to 15
- at least 8 amino acids e.g. 8 to 30, such as 8 to 20 or 8 to 15
- at least 10 amino acids e.g. 10 to 30, such as 10
- the contiguous portion deleted from the residues corresponding to 134-183 in SEQ ID No: 1 may be:
- the deleted contiguous portion may be 3 to 10 amino acid residues, such as 4 to 10, for example 4 to 8.
- the particular number of deleted amino acids may be 3, 4, 5, 6, 7, 8, 9 or 10, especially 4, 5, 6 or 8.
- the contiguous portion deleted from the residues corresponding to 134- 183 in SEQ ID No: 1 may be those corresponding to residues 135-138 in SEQ ID No: 1 , residues 136-138 in SEQ ID No: 1 , residues 137-138 in SEQ ID No: 1 , residues 138-140 in SEQ ID No: 1 , residues 138-141 in SEQ ID No: 1 , residues 152-154 in SEQ ID No: 1 or the deletion of residues 149-151 in SEQ ID No: 1.
- the third polypeptide is 48 amino acids or fewer (e.g. 10-48, such as 20-48 or 30-48 residues), such as 46 amino acids or fewer (e.g. 10-46, such as 20-46 or 30-46 residues), 44 amino acids or fewer (e.g. 10-44, such as 20-44 or 30-44 residues), or 42 amino acids or fewer (e.g. 10-42, such as 20-42 or 30-42 residues).
- 10-48 such as 20-48 or 30-48 residues
- 46 amino acids or fewer e.g. 10-46, such as 20-46 or 30-46 residues
- 44 amino acids or fewer e.g. 10-44, such as 20-44 or 30-44 residues
- 42 amino acids or fewer e.g. 10-42, such as 20-42 or 30-42 residues.
- a seventh aspect of the invention provides modified Rv3616c proteins comprising a first polypeptide and a second polypeptide, the first polypeptide being located towards the N- terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a sequence having at least 90% identity to residues 1 - 134 of SEQ ID No: 1 ;
- the second polypeptide is a sequence having at least 90% identity to residues 155-392 of SEQ ID No: 1 ;
- first and second polypeptides are either directly linked or indirectly linked via a third polypeptide, said third polypeptide having at least 80% identity to a sequence
- the modified Rv3616c protein consists essentially of, or alternatively consists of, a first polypeptide and a second polypeptide, the first polypeptide being located towards the N-terminus relative to the second polypeptide, and wherein:
- the first polypeptide is a sequence having at least 90% identity to residues 1 - 134 of SEQ ID No: 1 ;
- the second polypeptide is a sequence having at least 90% identity to residues 155-392 of SEQ ID No: 1 ;
- first and second polypeptides are either directly linked or indirectly linked via a third polypeptide, said third polypeptide having at least 80% identity to a sequence corresponding to residues 135-154 in SEQ ID No:1 in which a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted.
- the first polypeptide may be a sequence having at least 95% identity to residues 1 -134 of SEQ ID No: 1 , such as at least 97% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the second polypeptide may be a sequence having at least 95% identity to residues 155-392 of SEQ ID No: 1 , such as at least 97% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the first polypeptide and second polypeptide will in some embodiments be directly linked. In other embodiments the first polypeptide and second polypeptide will be indirectly linked via a third polypeptide.
- the third polypeptide may be a sequence having at least 90% identity to a sequence corresponding to residues 135-154 in SEQ ID No:1 in which a contiguous portion of at least 3 amino acids (e.g. at least 4) has been deleted, such as at least 95% identity, at least 98% identity, at least 99% identity or even 100% identical.
- the contiguous portion deleted contiguous portion from the residues corresponding to 135-154 in SEQ ID No: 1 may be at least 5 amino acids (e.g. 5 to 20, such as 5 to 15 or 5 to 10), especially at least 6 amino acids (e.g. 6 to 20, such as 6 to 15 or 6 to 10), in particular at least 7 amino acids (e.g. 7 to 20, such as 7 to 15 or 7 to 10), such as at least 8 amino acids (e.g. 8 to 20, such as 8 to 15 or 8 to 10), or at least 10 amino acids (e.g. 10 to 20, such as 10 to 15).
- the contiguous portion deleted contiguous portion from the residues corresponding to 135-154 in SEQ ID No: 1 may be:
- the deleted contiguous portion may be 3 to 10 amino acid residues, such as 4 to 10, for example 4 to 8.
- the particular number of deleted amino acids may be 3, 4, 5, 6, 7, 8, 9 or 10, especially 4, 5, 6 or 8.
- the contiguous portion deleted contiguous portion from the residues corresponding to 135-154 in SEQ ID No: 1 may be those corresponding to residues 135-138 in SEQ ID No: 1 , residues 136-138 in SEQ ID No: 1 , residues 137-138 in SEQ ID No: 1 , residues 138-140 in SEQ ID No: 1 , residues 138-141 in SEQ ID No: 1 , residues 152-154 in SEQ ID No: 1 or the deletion of residues 149-151 in SEQ ID No: 1.
- the third polypeptide is 20 amino acids or fewer (e.g. 5-20, such as 10-20 residues), such as 18 amino acids or fewer (e.g. 5-18, such as 10-18 residues), 16 amino acids or fewer (e.g. 5-16, such as 10-16 residues), or 14 amino acids or fewer (e.g. 5-14, such as 10-14 residues).
- Substituting hydrophobic residues may be achieved through the replacement of at least one (e.g. at least 2) amino acid corresponding to residues 134 to 183 of SEQ ID No: 1 with a hydrophilic residue.
- suitable hydrophilic residues will typically be Gin (Q), Asp (D), Glu (E), Asn (N), His (H), Lys (K), Arg (R), Ser (S) or Thr (T).
- suitable hydrophilic residues will typically be Gin (Q), Asp (D), Glu (E), Asn (N), His (H), Lys (K), Arg (R), Ser (S) or Thr (T).
- Substituted residues may be non-contiguous, although are suitably contiguous.
- a modified Rv3616c protein said protein comprising a Rv3616c sequence in which a contiguous portion of at least 3 amino acids (e.g. at least 4) from the region corresponding to residues 134-183 in SEQ ID No: 1 has been substituted with hydrophilic residues.
- the modified Rv3616c protein consists essentially of, or alternatively consists of a Rv3616c sequence in which a contiguous portion of at least 3 amino acids (e.g.
- Rv3616c proteins comprising an Rv3616c sequence in which a contiguous portion of at least 3 amino acids (e.g. at least 4) from the region corresponding to residues 135-154 in SEQ ID No: 1 has been substituted with hydrophilic residues.
- the substituted contiguous portion may be at least 5 amino acids (e.g. 5 to 30, such as 5 to 20 or 5 to 15), especially at least 6 amino acids (e.g. 6 to 30, such as 6 to 20 or 6 to 15), in particular at least 7 amino acids (e.g. 7 to 30, such as 7 to 20 or 7 to 15), such as at least 8 amino acids (e.g. 8 to 30, such as 8 to 20 or 8 to 15), or at least 10 amino acids (e.g. 10 to 30, such as 10 to 20 or 10 to 15).
- the substituted contiguous portion may be:
- - 1 1 amino acids such as those corresponding to residues 150-160 in SEQ ID No:1 ; - 17 amino acids, such as those corresponding to residues 166-182 in SEQ ID No:1 ; - 19 amino acids, such as those corresponding to residues 136-154 in SEQ ID No:1 ;
- the substituted contiguous portion may be 3 to 10 amino acid residues, such as 4 to 10, for example 4 to 8.
- the particular number of substituted amino acids may be 3, 4, 5, 6, 7, 8, 9 or 10, especially 4, 5, 6 or 8.
- the substituted portion may be those corresponding to residues 135-138 in SEQ ID No: 1 , residues 136-138 in SEQ ID No: 1 , residues 137-138 in SEQ ID No: 1 , residues 138-140 in SEQ ID No: 1 , residues 138-141 in SEQ ID No: 1 , residues 152-154 in SEQ ID No: 1 or the deletion of residues 149-151 in SEQ ID No: 1.
- Disrupting the hydrophobicity may also be achieved by adding hydrophilic residues, e.g. the addition of at least one hydrophilic amino acid residue (e.g. at least 2, such as 2-10) at a location between those residues corresponding to residues 133 to 184 of SEQ ID No: 1.
- hydrophilic residues e.g. the addition of at least one hydrophilic amino acid residue (e.g. at least 2, such as 2-10) at a location between those residues corresponding to residues 133 to 184 of SEQ ID No: 1.
- At least 3 hydrophilic residues may be added (e.g. 3 to 20, such as 3 to 15, especially 3 to 10), such as at least 4 residues (e.g. 4 to 20, such as 4 to 15, especially 4 to 10), in particular at least 5 residues (e.g. 5 to 20, such as 5 to 15, especially 5 to 10), optionally at least 6 residues (e.g. 6 to 20, such as 6 to 15, especially 6 to 10).
- suitable hydrophilic residues will typically be Gin (Q), Asp (D), Glu (E), Asn (N), His (H), Lys (K), Arg (R), Ser (S) or Thr (T).
- the additional hydrophilic residues will typically be located between those residues
- the additional hydrophilic residues may be distributed at different positions between those residues corresponding to residues 133 to 184 of SEQ ID No: 1 (e.g. 1 -10 locations, such as 1 - 5, in particular 1 or 2 locations), each location having 1 -10 additional hydrophilic residues, such as 1 -5 additional residues.
- the additional hydrophilic residues will suitably be located in one contiguous group.
- the modified Rv3616c protein is not SEQ ID No: 162 (Rv3616cA150-160). In other embodiments the modified Rv3616c protein does not comprise SEQ ID No: 162
- Modified Rv3616c proteins may be based on a wild-type Rv3616c protein sequence from any strain of M. tuberculosis. For example, any one of SEQ ID Nos: 3-7, in particular any one of SEQ ID Nos: 3-6, may be substituted for SEQ ID No:1 in the foregoing embodiments. Proteins of the various aspects discussed above are collectively referred to herein as modified Rv3616c proteins. Also provided are such modified Rv3616c proteins for use as medicaments, such as a medicament for the treatment or prevention of TB.
- a further aspect of the invention relates to a method for inducing an immune response in a subject, comprising the administration of a modified Rv3616c protein.
- a further aspect of the invention relates to a method for the treatment, amelioration or prevention of TB comprising the administration of a safe and effective amount of a modified Rv3616c protein to a subject in need thereof, wherein said polypeptide induces an immune response.
- the method further comprises inducing an immune response against Mycobacterium tuberculosis.
- a further aspect of the invention relates to a method for the treatment, amelioration, delaying or prevention of tuberculosis reactivation comprising the administration of an effective amount of a modified Rv3616c protein to a subject in need thereof, wherein said polypeptide induces and immune response.
- the method further comprises inducing an immune response against Mycobacterium tuberculosis.
- the present invention provides a polynucleotide comprising a nucleic acid sequence encoding a modified Rv3616c protein. Also provided is a polynucleotide comprising a nucleic acid sequence encoding a modified Rv3616c protein for use as a medicament, such as a medicament for the treatment, amelioration or prevention of TB.
- a further aspect of the invention relates to a method for inducing an immune response in a subject, comprising the administration of a polynucleotide comprising a nucleic acid sequence encoding a modified Rv3616c protein.
- a further aspect of the invention relates to a method for the treatment, amelioration or prevention of TB comprising the administration of a safe and effective amount of a
- polynucleotide comprising a nucleic acid sequence encoding a modified Rv3616c protein to a subject in need thereof, wherein said polynucleotide induces an immune response.
- the present invention provides a method for inducing an immune response against Mycobacterium tuberculosis.
- a further aspect of the invention relates to a method for the treatment, amelioration, delaying or prevention of tuberculosis reactivation comprising the administration of an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a modified Rv3616c protein to a subject in need thereof, wherein said polypeptide induces and immune response.
- the method further comprises inducing an immune response against
- a polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising a modified Rv3616c protein in the manufacture of a medicament for the treatment, amelioration or prevention of TB, represents another aspect of the invention. Additionally, there is provided a pharmaceutical composition comprising:
- an immunogenic composition comprising:
- an expression vector comprising a nucleic acid sequence encoding a modified Rv3616c protein.
- Host cells transformed with said expression vector, form a further aspect of the invention. Additionally provided is a host cell which recombinantly expresses a modified Rv3616c protein.
- said method comprising the step of recombinantly expressing said polypeptide within a host cell.
- diagnostic kits comprising:
- apparatus sufficient to contact said modified Rv3616c protein with a sample (e.g. whole blood or more suitably PBMC) from an individual; and (c) means to quantify the T cell response of the sample.
- a sample e.g. whole blood or more suitably PBMC
- Another aspect of the invention relates to a diagnostic kit comprising:
- a further aspect of the invention relates to a method for detecting Mycobacterium tuberculosis infection in a subject comprising:
- the invention also provides a diagnostic kit comprising:
- the subject receiving a modified Rv3616c protein, polynucleotide or composition according the invention may have active tuberculosis (e.g. active infection by M. tuberculosis).
- the subject may have latent tuberculosis (e.g. dormant infection by M. tuberculosis).
- the subject may be free from tuberculosis (e.g. free from infection by M. tuberculosis).
- a subject receiving a modified Rv3616c protein, polynucleotide or composition according to the invention may have previously been vaccinated for tuberculosis (e.g. vaccinated against infection by M. tuberculosis), such as having been vaccinated with a Bacillus Calmette-Guerin (BCG).
- a subject receiving a polypeptide, polynucleotide or composition of the invention may have not been previously vaccinated for tuberculosis (e.g. not vaccinated against infection by M. tuberculosis), such as not having been vaccinated with a Bacillus Calmette-Guerin (BCG).
- a modified Rv3616c protein, polynucleotide or composition according the invention may be provided for the purpose of:
- active tuberculosis such as by administering to a subject who is uninfected, or alternatively a subject who has latent infection
- tuberculosis especially the delay of TB reactivation, for example by a period of months, years or even indefinitely.
- a method for the treatment of latent TB comprising the steps: (i) identifying a subject as having a latent TB infection (e.g. by PPD or T cell based assays); and
- a polypeptide of the present invention in the manufacture of a diagnostic kit for the identification of TB (e.g. latent TB) in a test subject.
- TB e.g. latent TB
- Mycobacterium species of the tuberculosis complex includes those species traditionally considered as causing the disease tuberculosis, as well as Mycobacterium environmental and opportunistic species that cause tuberculosis and lung disease in immune compromised patients, such as patients with AIDS, e.g., M. tuberculosis, M. bovis, or
- the present invention is particularly directed to infection with M. tuberculosis.
- active infection refers to an infection (e.g. infection by M. tuberculosis) with manifested disease symptoms and/or lesions (suitably with manifested disease symptoms).
- inactive infection refers to an infection (e.g. infection by M. tuberculosis) without manifested disease symptoms and/or lesions (suitably without manifested disease symptoms).
- a subject with latent infection will suitably be one which tests positive for infection (e.g. by PPD or T cell based assays) but which has not demonstrated the disease symptoms and/or lesions which are associated with an active infection.
- primary tuberculosis refers to clinical illness (e.g., manifestation of disease symptoms) directly following infection (e.g. infection by M. tuberculosis). See, Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005).
- second tuberculosis or “postprimary tuberculosis” refer to the reactivation of a dormant, inactive or latent infection (e.g. infection by M. tuberculosis). See, Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005).
- tuberculosis reactivation refers to the later manifestation of disease symptoms in an individual that tests positive for infection (e.g. in a tuberculin skin test, suitably in an in vitro T cell based assay) test but does not have apparent disease symptoms. Suitably the individual will not have been re-exposed to infection.
- the positive diagnostic test indicates that the individual is infected, however, the individual may or may not have previously manifested active disease symptoms that had been treated sufficiently to bring the tuberculosis into an inactive or latent state. It will be recognised that methods for the prevention, delay or treatment of tuberculosis reactivation can be initiated in an individual manifesting active symptoms of disease.
- tuberculosis refers to an infection (e.g. infection by M. tuberculosis) wherein the infecting strain is not held static or killed (i.e. is resistant to) one or more of so- called “front-line” chemotherapeutic agents effective in treating tuberculosis (e.g., isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide).
- front-line chemotherapeutic agents effective in treating tuberculosis
- multi-drug resistant tuberculosis refers to an infection (e.g. infection by M.
- tuberculosis wherein the infecting strain is resistant to two or more of "front-line"
- chemotherapeutic agents effective in treating tuberculosis.
- a “chemotherapeutic agent” refers to a pharmacological agent known and used in the art to treat tuberculosis (e.g. infection by M. tuberculosis).
- Exemplified pharmacological agents used to treat tuberculosis include, but are not limited to amikacin, aminosalicylic acid, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, kanamycin, pyrazinamide, rifamycins (i.e., rifampin, rifapentine and rifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin and fluoroquinolones.
- "First-line” or “Front-line” chemotherapeutic agents used to treat tuberculosis that is not drug resistant include isoniazid, rifampin, ethambutol,
- “Second-line” chemotherapeutic agents used to treat tuberculosis that has demonstrated drug resistance to one or more "first-line” drugs include ofloxacin, ciprofloxacin, ethionamide, aminosalicylic acid, cycloserine, amikacin, kanamycin and capreomycin. Such pharmacological agents are reviewed in Chapter 48 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, Hardman and Limbird eds., 2001 .
- the terms "polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
- Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, v- carboxyglutamate, and O-phosphoserine.
- a polypeptide according to the present invention will consist only of naturally occurring amino acid residues, especially those amino acids encoded by the genetic code.
- Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
- the term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single- letter codes.
- Rv3616c protein sequence' as used herein is meant the Rv3616c polypeptide sequence provided in SEQ ID No: 1 or a homologue thereof from a Mycobacterium species of the tuberculosis complex, e.g., a species such as M. tuberculosis, M. bovis, or M. africanum, or a Mycobacterium species that is environmental or opportunistic and that causes opportunistic infections such as lung infections in immune compromised hosts (e.g., patients with AIDS), e.g., BCG, M. avium, M. intracellulare, M. celatum, M. genavense, M. haemophilum, M.
- immune compromised hosts e.g., patients with AIDS
- BCG immune compromised hosts
- M. avium M. intracellulare
- M. celatum M. genavense
- M. haemophilum M.
- the Rv3616c protein is derived from M. tuberculosis H37Rv (i.e. the polypeptide sequence provided in SEQ ID No: 1 ) or a homologue thereof from another M. tuberculosis strain (such as CDC1551 , F1 1 , Haarlem A and C strains).
- M. tuberculosis H37Rv i.e. the polypeptide sequence provided in SEQ ID No: 1
- a homologue thereof such as CDC1551 , F1 1 , Haarlem A and C strains.
- Strains of M. tuberculosis which are associated with drug resistance are a particularly valuable basis for the wild-type Rv3616c protein sequence. Strains of interest include:
- the Rv3616c protein is selected from the polypeptide sequences provided in SEQ ID Nos: 1 and 3-7, in particular SEQ ID Nos: 1 and 3-6, such as SEQ ID No: 1.
- SEQ ID Nos: 1 and 3-7 An alignment of SEQ ID Nos: 1 and 3-7 is provided in Figure 15.
- Modified Rv3616c proteins of particular interest are those comprising (e.g. consisting of) SEQ ID Nos: 161 -169.
- Polynucleotides of particular interest are those derived from the wild-type sequences corresponding to the M. tuberculosis strains discussed above, such as those derived from SEQ ID No: 2 or its related E. coli codon optimised SEQ ID No: 160.
- a sequence containing the modified Rv3616c proteins (or associated polynucleotides) of the present invention can further comprise other components designed to enhance their immunogenicity or to improve these antigens in other respects.
- improved isolation of the polypeptide antigens may be facilitated through the addition of a stretch of histidine residues (commonly known as a his-tag) towards one end of the antigen.
- his-tag refers to a string of histidine residues, typically six residues, that are inserted within the reference sequence.
- a his-tag is typically inserted at the N-terminus, usually immediately after the initiating methionine residue, or else at the C-terminus. They are usually heterologous to the native sequence but are incorporated since they facilitate isolation by improving the protein binding to immobilised metal affinity chromatography resins (I MAC).
- I MAC immobilised metal affinity chromatography resins
- polypeptides (and nucleic acids encoding them) can be prepared which comprise multiple modified Rv3616c sequences and/or additional heterologous polypeptides or the
- polynucleotides encoding them from Mycobacterium species in particular M. tuberculosis.
- all antigen components are provided as polypeptides (e.g. within a single fusion protein).
- all antigen components are provided as polynucleotides (e.g. a single polynucleotide, such as one encoding a single fusion protein).
- heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
- the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
- a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
- Fusion polypeptide refers to a protein having at least two heterologous polypeptides (e.g. at least two Mycobacterium sp. polypeptides) covalently linked, either directly or via an amino acid linker.
- the polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N- terminus to N-terminus, or N-terminus to C-terminus.
- the polypeptides of the fusion protein can be in any order. This term also refers to conservatively modified variants, polymorphic variants, alleles, mutants, immunogenic fragments, and interspecies homologs of the antigens that make up the fusion protein.
- Mycobacterium tuberculosis antigens are described in Cole et al., Nature 393:537 (1998), which discloses the entire Mycobacterium tuberculosis genome.
- Antigens from other Mycobacterium species that correspond to M. tuberculosis antigens can be identified, e.g., using sequence comparison algorithms, as described herein, or other methods known to those of skill in the art, e.g., hybridisation assays and antibody binding assays.
- the term "fused” refers to the covalent linkage between two polypeptides in a fusion protein. The polypeptides are typically joined via a peptide bond, either directly to each other or via an amino acid linker.
- peptides can be joined via non-peptide covalent linkages known to those of skill in the art.
- exemplary M. tuberculosis antigens which may be combined with a modified Rv3616c sequence include one or more of (e.g. 1 to 5, such as 1 to 3, in particular 1 ) the following:
- Mtb8.4 also known as DPV and Rv1 174c
- the polypeptide sequence of which is described in SEQ ID No: 102 of WO97/09428 (cDNA in SEQ ID No: 101 ) and in Coler et al Journal of Immunology 1998 161 :2356-2364.
- SEQ ID No: 102 of WO97/09428 cDNA in SEQ ID No: 101
- Coler et al Journal of Immunology 1998 161 :2356-2364 Of particular interest is the mature
- Mtb8.4 sequence which is absent the leading signal peptide (i.e. amino acid residues 15-96 from SEQ ID No: 102 of WO97/09428).
- the full-length polypeptide sequence of Mtb8.4 is shown in SEQ ID No: 8; (ii) Mtb9.8 (also known as MSL and Rv0287), the polypeptide sequence of which is described in SEQ ID No: 109 of WO98/53075 (fragments of MSL are disclosed in SEQ ID Nos: 1 10-124 of WO98/53075, SEQ ID Nos: 1 19 and 120 being of particular interest) and also in Coler et al Vaccine 2009 27:223-233 (in particular the reactive fragments shown in Figure 2 therein).
- Mtb9.8 The full-length polypeptide sequence for Mtb9.8 is shown in SEQ ID No: 9; (iii) Mtb9.9 (also known as Mtb9.9A, MTI, MTI-A and Rv1793) the polypeptide sequence of which is described in SEQ ID No: 19 of WO98/53075 and in Alderson et al Journal of Experimental Medicine 2000 7:551 -559 (fragments of MTI are disclosed in SEQ ID Nos: 17 and 51 -66 of WO98/53075, SEQ ID Nos: 17, 51 , 52, 53, 56 and 62-65 being of particular interest). A number of polypeptide variants of MTI are described in
- Ra12 also known as Mtb32A C-terminal antigen
- Mtb32A C-terminal antigen the polypeptide sequence of which is described in SEQ ID No: 10 of WO01/98460 and in Skeiky et al Journal of Immunology 2004 172:7618-7682.
- the full-length polypeptide sequence for Ra12 is shown in SEQ ID No: 1 1 ;
- Ra35 also known as Mtb32A N-terminal antigen
- the full-length polypeptide sequence for Ra35 is shown in SEQ ID No: 12;
- TbH9 also known as Mtb39, Mtb39A, TbH9FL and Rv1 196 the polypeptide sequence of which is described in SEQ ID No: 107 of WO97/09428, and also in Dillon et al Infection and Immunity 1999 67(6):2941 -2950 and Skeiky et al Journal of
- Mtb41 also known as MTCC2 and Rv0915c
- the polypeptide sequence of which is described in SEQ ID No: 142 of WO98/53075 (cDNA in SEQ ID No: 140) and in Skeiky et al Journal of Immunology 2000 165:7140-7149.
- the full-length polypeptide sequence for Mtb41 is shown in SEQ ID No: 14;
- ESAT-6 also known as esxA and Rv3875
- the polypeptide sequence of which is described in SEQ ID No: 103 of WO97/09428 (cDNA in SEQ ID No: 104) and in Sorensen et al Infection and Immunity 1995 63(5):1710-1717.
- the full-length polypeptide sequence for ESAT-6 is shown in SEQ ID No: 15;
- Ag85 complex antigens e.g.
- Ag85A also known as fbpA and Rv3804c; or Ag85B, also known as fbpB and Rv1886c
- SEQ ID No: 16 the mature protein of residues 43-338, i.e. lacking the signal peptide, being of particular interest
- SEQ ID No: 17 the mature protein of residues 41 -325, i.e. lacking the signal peptide, being of particular interest
- Alpha-crystallin also known as hspX and Rv2031 c
- hspX and Rv2031 c Alpha-crystallin
- Mpt64 (also known as Rv1980c) which is described in Roche et al Scandinavian Journal of Immunology 1996 43:662-670.
- the full-length polypeptide sequence for MPT64 is shown in SEQ ID No: 19 (the mature protein of residues 24-228, i.e. lacking the signal peptide, being of particular interest):
- Mtb32A the polypeptide sequence of which is described in SEQ ID No: 2 (full- length) and residues 8-330 of SEQ ID No: 4 (mature) of WO01/98460, especially variants having at least one of the catalytic triad mutated (e.g. the catalytic serine residue, which may for example be mutated to alanine).
- the full-length polypeptide sequence for Mtb32A is shown in SEQ ID No: 20.
- the mature form of Mtb32A having a Ser/Ala mutation is shown in SEQ ID No: 21 ;
- Rv1753c the full-length polypeptide sequence for Rv1753c from Mycobacterium tuberculosis H37Rv is shown in SEQ ID No: 157;
- Rv2386c the full-length polypeptide sequence for Rv2386c from Mycobacterium tuberculosis H37Rv is shown in SEQ ID No: 158; and/or (xvi) Rv2707c, the full-length polypeptide sequence for Rv2707c from Mycobacterium tuberculosis H37Rv is shown in SEQ ID No: 159. inations thereof, such as:
- Mtb72f a combination of Ra12, TbH9 and Ra35 components, for example in the form of a fusion protein, such as Mtb72f.
- the polypeptide sequence of Mtb72f is described in SEQ ID No: 6 of WO2006/1 17240 (cDNA in SEQ ID No: 5) and in Skeiky et al Journal of Immunology 2004 172:7618-7682 (where it incorporates an optional His-tag to aid purification, when utilised in the present invention suitably Mtb72f is absent the optional histidine residues).
- the polypeptide sequence for Mtb72f is shown in SEQ ID No: 23;
- M72 may also incorporate a double histidine though suitably M72 is absent the optional double histidine (i.e. residues 4-725 from SEQ ID No: 4 of WO2006/1 17240 are of particular interest).
- the polypeptide sequence for M72 is shown in SEQ ID No: 24;
- Mtb71f a combination of Mtb8.4, Mtb9.8, Mtb9.9 and Mtb41 components, for example in the form of a fusion protein, such as Mtb71f.
- the polypeptide sequence of Mtb71f is described in SEQ ID No: 16 of WO99/051748 (cDNA in SEQ ID No: 15), where it incorporates an optional His-tag to aid purification, when utilised in the present invention suitably Mtb71f corresponds to amino acid residues 9-710 of SEQ ID NO: 16 from WO99/051748.
- the polypeptide sequence for Mtb71f is shown in SEQ ID No: 25;
- Mtb72f or M72 suitable without optional histidine residues to aid expression
- Mtb9.8 and Mtb9.9 for example in a fusion protein.
- the polypeptide sequence for an M72-Mtb9.9-Mtb9.8 fusion is shown in SEQ ID No: 26 (M92 fusion), when used in the present invention, the M72-Mtb9.9-Mtb9.8 fusion may optionally incorporate a double histidine following the initiating methionine residue to aid manufacture;
- a combination of Mtb72f or M72 suitableably without optional histidine residues to aid expression
- Ag85B for example in a fusion protein, such Mtb103f.
- Mtb103f The polypeptide sequence of Mtb103f is described in SEQ ID No: 18 of WO03/070187 (cDNA in SEQ ID No: 10), where it incorporates an optional His-tag to aid purification, when utilised in the present invention suitably Mtb103f corresponds to amino acid residues 8-1016 of SEQ ID No: 18 from WO03/070187. Also of particular interest is M103, i.e. Mtb103f incorporating a Ser/Ala mutation in the Ra35 component, when utilised in the present invention suitably M103 corresponds to amino acid residues 8- 1016 of SEQ ID No: 18 from WO03/070187 wherein the Ser residue at position 710 has been replaced with Ala.
- the polypeptide sequence for M103 is shown in SEQ ID No:
- the M72-Mtb9.9-Mtb9.8 fusion may optionally incorporate a double histidine following the initiating methionine residue to aid manufacture;
- Mtb1 14f a combination of Mtb72f or M72 (suitably without optional histidine residues to aid expression) with Mtb41 , for example in a fusion protein, such Mtb1 14f.
- the polypeptide sequence of Mtb1 14f is described in SEQ ID No: 16 of WO03/070187 (cDNA in SEQ ID No: 9), where it incorporates an optional His-tag to aid purification, when utilised in the present invention suitably Mtb1 14f corresponds to amino acid residues 8-1 154 of SEQ ID No: 16 from WO03/070187.
- Mtb1 14f incorporating a Ser/Ala mutation in the Ra35 component, when utilised in the present invention suitably M1 14 corresponds to amino acid residues 8-1 154 of SEQ ID No: 16 from WO03/070187 wherein the Ser residue at position 710 has been replaced with Ala.
- the polypeptide sequence for M1 14 is shown in SEQ ID No: 28, when used in the present invention, the M72-Mtb9.9-Mtb9.8 fusion may optionally incorporate a double histidine following the initiating methionine residue to aid manufacture;
- Combinations of a modified Rv3616c sequence component and an Rv1753c component are of particular interest. Obviously such combinations could optionally contain other additional antigen components (e.g. an M72 component).
- Another combination of interest comprises a modified Rv3616c sequence component and an M72 component.
- a further combination of interest comprises a modified Rv3616c sequence component and an Rv2386c component.
- An additional combination of interest comprises a modified Rv3616c sequence component and an alpha-crystallin component.
- compositions, polypeptides, and nucleic acids of the invention can also comprise additional polypeptides from other sources.
- the compositions and fusion proteins of the invention can include polypeptides or nucleic acids encoding polypeptides, wherein the polypeptide enhances expression of the antigen, e.g., NS1 , an influenza virus protein (see, e.g. WO99/40188 and WO93/04175).
- the nucleic acids of the invention can be engineered based on codon preference in a species of choice, e.g., humans (in the case of in vivo expression) or a particular bacterium (in the case of polypeptide production).
- SEQ ID No: 160 for example, provides a codon optimised polynucleotide for the expression of Rv3616c from H37Rv in E. coli.
- the modified Rv3616c sequence component may also be administered with one or more chemotherapeutic agents effective against tuberculosis (e.g. M. tuberculosis infection).
- chemotherapeutic agents effective against tuberculosis e.g. M. tuberculosis infection.
- chemotherapeutic agents include, but are not limited to, amikacin, aminosalicylic acid, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, kanamycin, pyrazinamide, rifamycins (i.e., rifampin, rifapentine and rifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin and fluoroquinolones.
- rifamycins i.e., rifampin, rifapentine and rifabutin
- streptomycin ofloxacin
- ciprofloxacin clarithromycin
- azithromycin azithromycin and fluoroquinolones.
- First-line chemotherapeutic agents used to treat tuberculosis include isoniazid, rifampin, ethambutol, streptomycin and
- chemotherapeutic agents used to treat tuberculosis include ofloxacin, ciprofloxacin, ethionamide, aminosalicylic acid, cycloserine, amikacin, kanamycin and capreomycin.
- chemotherapeutic agents are generally administered over a relatively long period (ca. 9 months).
- Combination of conventional chemotherapeutic agents with the administration of a modified Rv3616c sequence component according to the present invention may enable the chemotherapeutic treatment period to be reduced (e.g. to 8 months, 7 months, 6 months, 5 months, 4 months, 3 months or less) without a decrease in efficacy.
- modified Rv3616c sequence component in conjunction with Bacillus Calmette-Guerin (BCG).
- BCG Bacillus Calmette-Guerin
- the modified Rv3616c sequence component may be used to enhance the response of a subject to BCG vaccination, either by co-administration or by boosting a previous BCG vaccination.
- the modified Rv3616c sequence component may obviously be provided in the form of a polypeptide or a polynucleotide
- polypeptides, polynucleotides and compositions of the present invention will usually be administered to humans, but may be expected to be effective in other mammals including domestic mammals (e.g., dogs, cats, rabbits, rats, mice, guinea pigs, hamsters, chinchillas) and agricultural mammals (e.g., cows, pigs, sheep, goats, horses).
- domestic mammals e.g., dogs, cats, rabbits, rats, mice, guinea pigs, hamsters, chinchillas
- agricultural mammals e.g., cows, pigs, sheep, goats, horses.
- T cell epitopes are short contiguous stretches of amino acids which are recognised by T cells (e.g. CD4+ or CD8+ T cells). Identification of T cell epitopes may be achieved through epitope mapping experiments which are well known to the person skilled in the art (see, for example, Paul, Fundamental Immunology, 3rd ed., 243-247 (1993); Beipbarth et al Bioinformatics 2005 21 (Suppl. 1 ):i29-i37). Alternatively, epitopes may be predicted or mapped using the approaches discussed in the Examples.
- an optimal modified Rv3616c protein is one which contains the majority (or suitably all) T cell epitopes intact.
- “Variants” or “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
- nucleic acid sequence As a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations lead to "silent” or “degenerate” variants, which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
- each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
- Non-silent variations are those which result in a change in the encoded amino acid sequence (either though the substitution, deletion or addition of amino acid residues). Those skilled in the art will recognise that a particular polynucleotide sequence may contain both silent and non-silent conservative variations.
- polymorphic variants interspecies homologs, and alleles of the invention.
- substitutions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen.
- Protein variants may also include those wherein additional amino acids are inserted compared to the reference sequence. Suitably such insertions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen.
- insertions includes a short stretch of histidine residues (e.g. 2-6 residues) to aid expression and/or purification of the antigen in question.
- Protein variants include those wherein amino acids have been deleted compared to the reference sequence. Suitably such deletions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen.
- nucleic acids or polypeptide sequences refer to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence.
- the identity exists over a region that is at least about 25 to about 50 amino acids or nucleotides in length, or optionally over a region that is 75-100 amino acids or nucleotides in length.
- the comparison is performed over a window corresponding to the entire length of the reference sequence (as opposed to the variant sequence).
- sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
- test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
- sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
- a "comparison window" references to a segment in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art.
- Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981 ), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci.
- PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 -360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151 -153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
- the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
- the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
- PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984).
- Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively.
- Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov/).
- This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
- T is referred to as the neighborhood word score threshold (Altschul et al., supra).
- M forward score for a pair of matching residues; always > 0
- N penalty score for mismatching residues; always ⁇ 0).
- a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the
- the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
- the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
- One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
- P(N) the smallest sum probability
- a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001.
- variants of a polypeptide sequence will have essentially the same activity as the reference sequence (in the case of polynucleotides, variant polynucleotide sequences will encode a polypeptide which has essentially the same activity as the reference sequence).
- essentially the same activity is meant at least 50%, suitably at least 75% and especially at least 90% activity of the reference sequence in an in vitro restimulation assay of PBMC or whole blood with specific antigens (e.g.
- essentially the same activity is meant at least 50%, suitably at least 75% and especially at least 90% activity of the reference sequence in a T cell proliferation and/or IFN- gamma production assay.
- polynucleotides of use in this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins,
- polypeptides may be naturally isolated, or modified synthetically by the hand of man.
- polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
- RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
- Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Mycobacterium antigen or a portion thereof) or may comprise a variant, or a biological or functional equivalent of such a sequence.
- Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, such that the immunogenicity of the encoded polypeptide is not diminished relative to the reference protein.
- Polynucleotides may be identified, prepared and/or manipulated using any of a variety of well established techniques. For example, a polynucleotide may be identified, as described in more detail below, by screening a microarray of cDNAs. Such screens may be performed, for example, using a Synteni microarray (Palo Alto, CA) according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA
- polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as M. tuberculosis cells. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesised.
- PCR polymerase chain reaction
- An amplified portion of a polynucleotide may be used to isolate a full length gene from a suitable library (e.g., a M. tuberculosis cDNA library) using well known techniques.
- a library cDNA or genomic
- a library is screened using one or more polynucleotide probes or primers suitable for amplification.
- a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequences.
- a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32 P) using well known techniques.
- a bacterial or bacteriophage library is then generally screened by hybridising filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual (2000)).
- Hybridising colonies or plaques are selected and expanded, and the DNA is isolated for further analysis.
- cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones.
- the complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones. The resulting overlapping sequences can then be assembled into a single contiguous sequence.
- a full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
- Primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68°C to 72°C.
- the amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.
- One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 16:8186 (1988)), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularised by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region.
- sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region.
- the amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region.
- a variation on this procedure which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591.
- Another such technique is known as "rapid amplification of cDNA ends" or RACE. This technique involves the use of an internal primer and an external primer, which hybridises to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence.
- Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 1 :1 1 1 -19 (1991 )) and walking PCR (Parker et al., Nucl. Acids. Res. 19:3055-60 (1991 )). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.
- EST expressed sequence tag
- Full length DNA sequences may also be obtained by analysis of genomic fragments.
- Polynucleotide sequences or fragments thereof which encode polypeptides, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.
- polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
- the polynucleotide sequences can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
- site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.
- Natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein.
- a heterologous sequence For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognised by a commercially available antibody.
- a fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.
- Sequences encoding a desired polypeptide may be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al., Nucl. Acids Res. Symp. Ser. pp. 215-223 (1980), Horn et al., Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)).
- the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof.
- peptide synthesis can be performed using various solid-phase techniques (Roberge et al., Science 269:202-204 (1995)) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer, Palo Alto, CA).
- a newly synthesised peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, Proteins, Structures and Molecular Principles (1983)) or other comparable techniques available in the art.
- composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
- the nucleotide sequences encoding the polypeptide, or functional equivalents may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
- an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
- Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (2000), and Ausube/ et al., Current Protocols in Molecular Biology (updated annually).
- a variety of expression vector/host systems may be utilised to contain and express
- polynucleotide sequences include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
- yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
- plant cell systems transformed with virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
- virus expression vectors e.g., cauliflower
- control elements or "regulatory sequences" present in an expression vector are those non-translated regions of the vector-enhancers, promoters, 5' and 3' untranslated regions- which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) and the like may be used.
- inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) and the
- promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.
- a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, for example for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used.
- Such vectors include, but are not limited to, the multifunctional £. co// cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke &Schuster, J. Biol. Chem.
- pGEX Vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
- GST glutathione S-transferase
- fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
- Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
- a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
- Other vectors containing constitutive or inducible promoters include GAP, PGK, GAL and ADH.
- viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 3:1671 -1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991 )).
- plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671 -1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991 )).
- These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
- An insect system may also be used to express a polypeptide of interest.
- Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
- the sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
- the recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard et ai, Proc. Natl. Acad. Sci. U.S.A. 91 :3224-3227 (1994)).
- S. frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard et ai, Proc. Natl. Acad. Sci. U.S.A. 91 :3224-3227 (1994)).
- a number of viral-based expression systems are generally available.
- sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A. 81 :3655-3659 (1984)).
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
- RSV Rous sarcoma virus
- adenovirus vectors Methods and protocols for working with adenovirus vectors are reviewed in Wold, Adenovirus Methods and Protocols, 1998. Additional references regarding use of adenovirus vectors can be found in Adenovirus: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References, 2004.
- Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
- Enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf. et al., Results Probl. Cell Differ. 20:125-162 (1994)).
- a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
- modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
- Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function.
- Different host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.
- stable expression is generally preferred.
- cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1 -2 days in an enriched media before they are switched to selective media.
- the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
- Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
- any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 1 1 :223-32 (1977)) and adenine phosphonbosyltransferase (Lowy et al., Cell 22:817-23 (1990)) genes which can be employed in tk.sup.- or aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci.
- npt which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1 -14 (1981 )); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilise indole in place of tryptophan, or hisD, which allows cells to utilise histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad.
- marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a polypeptide-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
- host cells which contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridisations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
- a variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescence activated cell sorting
- a two-site, monoclonal-based immunoassay utilising monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed. These and other assays are described, among other places, in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:121 1 -1216 (1983).
- Means for producing labelled hybridisation or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labelling or PCR amplification using a labelled nucleotide.
- the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe.
- Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
- reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
- the protein produced by a recombinant cell may be secreted or contained intracellular ⁇ depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane.
- recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
- purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
- the inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen.
- One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
- the histidine residues facilitate purification on IMIAC (immobilised metal ion affinity chromatography) as described in Porath et al., Prot. Exp. Purif. 3:263-281 (1992) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein.
- IMIAC immobilised metal ion affinity chromatography
- genetic constructs comprising one or more of the polynucleotides of the invention are introduced into cells in vivo. This may be achieved using any of a variety or well known approaches, several of which are outlined below for the purpose of illustration. 1 .
- ADENOVIRUS ADENOVIRUS
- adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein in a sense or antisense orientation.
- expression does not require that the gene product be synthesised.
- the expression vector comprises a genetically engineered form of an adenovirus.
- retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
- adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
- Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titre, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are c/ ' s elements necessary for viral DNA replication and packaging.
- ITRs inverted repeats
- the E1 region (E1 A and E1 B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
- the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
- MLP major late promoter
- TPL 5'-tripartite leader
- adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kB of DNA. Combined with the approximately 5.5 kB of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kB, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the E1 -deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).
- MOI multiplicities of infection
- Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
- the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.
- the currently preferred helper cell line is 293.
- Racher et al. (1995) have disclosed improved methods for culturing 293 cells and propagating adenovirus.
- natural cell aggregates are grown by inoculating individual cells into 1 litre siliconised spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
- Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml
- the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
- Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain a conditional replication-defective adenovirus vector for use in the present invention, since Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
- the typical vector according to the present invention is replication defective and will not have an adenovirus E1 region.
- the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
- the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al. (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.
- Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titres, e.g., 10 9 -10 11 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971 ), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
- Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991 ;
- Adenovirus vectors may originate from human adenovirus. Alternatively they may originate from adenovirus of other species e.g.
- Adenovirus type 35 which is relatively uncommon and therefore there are low levels of preexisting immunity to the vector itself, has been used as a delivery system in certain
- Adenovirus type 35 may also be of particular value in the present invention as a delivery vector.
- the retroviruses are a group of single-stranded RNA viruses characterised by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular
- the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
- a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
- Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
- a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
- a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983).
- Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).
- AAV (Ridgeway, 1988; Hermonat & Muzycska, 1984) is a parovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replication is dependent on the presence of a helper virus, such as adenovirus. Five serotypes have been isolated, of which AAV-2 is the best characterised.
- AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VP1 , VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter (Muzyczka & McLaughlin, 1988).
- the AAV DNA is approximately 4.7 kilobases long. It contains two open reading frames and is flanked by two ITRs.
- rep and cap There are two major genes in the AAV genome: rep and cap. The rep gene codes for proteins responsible for viral replications, whereas cap codes for capsid protein VP1 -3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential c/ ' s components of the AAV for chromosomal integration.
- the AAV can be used as a vector with all viral coding sequences removed and replaced by the cassette of genes for delivery.
- Three viral promoters have been identified and named p5, p19, and p40, according to their map position. Transcription from p5 and p19 results in production of rep proteins, and transcription from p40 produces the capsid proteins (Hermonat & Muzyczka, 1984).
- rAAV rAAV as an expression vector.
- AAV is also a good choice of delivery vehicles due to its safety. There is a relatively complicated rescue mechanism: not only wild type adenovirus but also AAV genes are required to mobilise rAAV. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimises immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response.
- viral vectors may be employed as expression constructs in the present invention for the delivery of oligonucleotide or polynucleotide sequences to a host cell.
- Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Coupar ei a/., 1988), lentiviruses, polio viruses and herpes viruses may be employed.
- Other poxvirus derived vectors such as fowl- pox derived vectors, may also be expected to be of use. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Coupar ei a/., 1988; Horwich ef a/., 1990).
- VLPs virus like particles
- the expression construct In order to effect expression of the oligonucleotide or polynucleotide sequences of the present invention, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. As described above, one preferred mechanism for delivery is via viral infection where the expression construct is encapsulated in an infectious viral particle.
- the nucleic acid encoding the desired oligonucleotide or polynucleotide sequences may be positioned and expressed at different sites.
- the nucleic acid encoding the construct may be stably integrated into the genome of the cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
- the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronisation with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
- the expression construct comprising one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed, for example, by any method which physically or chemically permeabilises the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984)
- Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
- Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991 ). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated.
- Bacteria may also be utilised as a delivery method (e.g. listeria, see WO2004/1 1048) and in particular BCG.
- Polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by DNA sequences as described above may be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells, such as mammalian cells and plant cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.
- Supernatants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.
- Shorter polypeptides may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesised using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146 (1963). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.
- a polypeptide may be a fusion protein that comprises multiple modified Rv3616c proteins as described herein, or that comprises at least one modified Rv3616c proteins as described herein and an unrelated sequence such as those described in (i) to (xvi) and (a) to (g) above.
- a fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognised by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein.
- Certain preferred fusion partners are both immunological and expression enhancing fusion partners.
- Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments.
- Still further fusion partners include affinity tags, which facilitate purification of the protein.
- Fusion proteins may generally be prepared using standard techniques, including chemical conjugation.
- a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system.
- DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector.
- the 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.
- a peptide linker sequence may be employed to separate the fusion partners by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
- Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art.
- Suitable peptide linker sequences may be chosen based on the following factors: (1 ) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
- Preferred peptide linker sequences contain Gly, Asn and Ser residues.
- linker sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262 (1986); U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751 ,180.
- the linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
- an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926).
- a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-1 10 amino acids), and a protein D derivative may be lipidated.
- the first 109 residues of a lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer).
- the lipid tail ensures optimal presentation of the antigen to antigen presenting cells.
- Other fusion partners include the non-structural protein from influenzae virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
- the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion).
- LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292 (1986)).
- LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
- the C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E.
- coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798 (1992)). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.
- polynucleotide or polypeptide compositions disclosed herein may be formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
- Compositions may be presented in powder form (e.g. freeze-dried) for reconstitution shortly before use, such dry compositions generally are more stable during storage.
- compositions may comprise a fusion protein or a polynucleotide encoding a fusion protein, in combination with a pharmaceutically-acceptable carrier or excipient.
- the nucleic acid segment e.g., RNA or DNA
- the nucleic acid segment that expresses a polypeptide as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents, including chemotherapeutic agents effective against a M. tuberculosis infection.
- agents such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents, including chemotherapeutic agents effective against a M. tuberculosis infection.
- chemotherapeutic agents effective against a M. tuberculosis infection.
- the compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesised as described herein.
- compositions may further comprise substituted or derivatised RNA or DNA compositions.
- Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and
- formulations comprising a therapeutically effective amount deliver about 0.01 ug to about 1000 ug of modified Rv3616c polypeptide per administration, more typically about 0.1 ug to about 100 ug of polypeptide per administration (e.g. 0.5 to 50 ug).
- these typically deliver about 10 ug to about 20 mg of the inventive polynucleotide per administration, more typically about 0.1 mg to about 10 mg of the inventive polynucleotide per administration
- the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
- Factors such as solubility, bioavailability, biological half-life, route of
- compositions disclosed herein may be delivered via oral administration to an animal.
- these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
- the active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et ai, 1997; Hwang et ai, 1998; U. S. Patent 5,641 ,515; U. S.
- the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavouring agent, such as peppermint, oil of wintergreen, or cherry flavouring.
- a binder as gum tragacanth, acacia, cornstarch, or gelatin
- excipients such as dicalcium phosphate
- a disintegrating agent such as corn starch, potato starch, alginic acid and the like
- a lubricant such as magnesium stearate
- a sweetening agent such as sucrose, lactose or saccharin may be added or a flavour
- the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both.
- a syrup of elixir may contain the active component, sucrose as a sweetening agent methyl and propylparabens as
- any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- the active components may be incorporated into sustained-release preparation and formulations.
- compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation.
- a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
- the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
- the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth. 2. INJECTABLE DELIVERY
- compositions disclosed herein parenterally, intravenously, intramuscularly, intradermally, or even intraperitoneally as described in U. S. Patent 5,543,158; U. S. Patent 5,641 ,515 and U. S. Patent 5,399,363 (each specifically incorporated herein by reference in its entirety).
- Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
- Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U. S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
- the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
- polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
- suitable mixtures thereof e.g., vegetable oils
- vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
- suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
- vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
- Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
- isotonic agents for example, sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- aqueous solution for parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
- a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
- one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp.
- Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- compositions disclosed herein may be formulated in a neutral or salt form.
- Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
- solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
- the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
- carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
- solvents dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
- compositions is contemplated.
- Supplementary active ingredients can also be incorporated into the compositions.
- pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
- the preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art.
- such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
- the preparation can also be emulsified.
- the pharmaceutical compositions may be delivered by intranasal sprays, buccal sprays, inhalation, and/or other aerosol delivery vehicles.
- Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs eg via nasal and buccal aerosol sprays has been described e.g., in U. S. Patent 5,756,353 and U. S. Patent 5,804,212 (each specifically incorporated herein by reference in its entirety).
- U. S. Patent 5,756,353 and U. S. Patent 5,804,212 each specifically incorporated herein by reference in its entirety.
- the delivery of drugs using intranasal microparticle resins Takenaga et al., 1998) and
- lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871 , specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Patent 5,780,045 (specifically incorporated herein by reference in its entirety). 4. LIPOSOME-, NANOCAPSULE-, AND MICROPARTICLE-MEDIATED DELIVERY
- the inventors contemplate the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the present invention into suitable host cells.
- the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
- Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of the nucleic acids or constructs disclosed herein.
- liposomes are generally known to those of skill in the art (see for example, Couvreur et al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases).
- liposomes were developed with improved serum stability and circulation half-times (Gabizon & Papahadjopoulos, 1988; Allen and Choun, 1987; U. S. Patent 5,741 ,516, specifically incorporated herein by reference in its entirety).
- Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al., 1990; Muller et al., 1990). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems.
- Liposomes have been used effectively to introduce genes, drugs (Heath & Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989; Fresta & Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses (Faller & Baltimore, 1984), transcription factors and allosteric effectors (Nicolau & Gersonde, 1979) into a variety of cultured cell lines and animals.
- Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 ⁇ . Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
- SUVs small unilamellar vesicles
- Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e. in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.
- Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure.
- the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability.
- phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less- ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs. In addition to temperature, exposure to proteins can alter the permeability of liposomes.
- soluble proteins such as cytochrome c
- Cholesterol inhibits this penetration of proteins, apparently by packing the phospholipids more tightly. It is contemplated that the most useful liposome formations for antibiotic and inhibitor delivery will contain cholesterol.
- SUVs unilamellar vesicles
- LUVs large unilamellar vesicles
- Polar compounds are trapped in the aqueous spaces and nonpolar compounds bind to the lipid bilayer of the vesicle. Polar compounds are released through permeation or when the bilayer is broken, but nonpolar compounds remain affiliated with the bilayer unless it is disrupted by temperature or exposure to lipoproteins. Both types show maximum efflux rates at the phase transition temperature.
- Liposomes interact with cells via four different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
- liposomes The fate and disposition of intravenously injected liposomes depend on their physical properties, such as size, fluidity, and surface charge. They may persist in tissues for h or days, depending on their composition, and half lives in the blood range from min to several h. Larger liposomes, such as MLVs and LUVs, are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the capillary endothelium, such as the sinusoids of the liver or spleen. Thus, these organs are the predominate site of uptake.
- MLVs and LUVs are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the ca
- SUVs show a broader tissue distribution but still are sequestered highly in the liver and spleen. In general, this in vivo behavior limits the potential targeting of liposomes to only those organs and tissues accessible to their large size. These include the blood, liver, spleen, bone marrow, and lymphoid organs.
- Antibodies may be used to bind to the liposome surface and to direct the antibody and its drug contents to specific antigenic receptors located on a particular cell-type surface.
- determinants may also be used as recognition sites as they have potential in directing liposomes to particular cell types. Usually, it is contemplated that intravenous injection of liposomal preparations would be used, but other routes of
- the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention.
- Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987).
- ultrafine particles sized around 0.1 ⁇
- Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention.
- Such particles may be are easily made, as described (Couvreur et al., 1980; 1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and U. S. Patent 5,145,684, specifically incorporated herein by reference in its entirety).
- Skin patches may also be utilised for transcutaneous delivery.
- immunogenic compositions are provided.
- the immunogenic compositions will comprise one or more modified Rv3616c sequences (polypeptides or polynucleotides) as those discussed above, in combination with an immunostimulant.
- Immunogenic compositions may also comprise a fusion protein or a polynucleotide encoding a fusion protein, in combination with a pharmaceutically-acceptable carrier or excipient.
- An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen.
- immunostimulants include adjuvants.
- compositions and immunogenic compositions within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.
- one or more immunogenic portions of other M. tuberculosis antigens may be present, either incorporated into a fusion polypeptide or as a separate component, within the pharmaceutical or immunogenic composition.
- Illustrative immunogenic compositions may contain a polynucleotide (e.g. DNA) encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ (thereby eliciting an immune response).
- a polynucleotide e.g. DNA
- the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198 (1998), and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
- Bacterial delivery systems involve the administration of a bacterium host cell (for example, a Mycobacterium, Bacillus or Lactobacillus strain, including Bacillus-Calmette-Guerrin or Lactococcus lactis) that expresses the polypeptide (e.g. on its cell surface or secretes the polypeptide) (see, for example, Ferreira, et al., An Acad Bras Cienc (2005) 77:1 13-124; and Raha, et al., AppI Microbiol Biotechnol (2005) PubMedID 15635459).
- a bacterium host cell for example, a Mycobacterium, Bacillus or Lactobacillus strain, including Bacillus-Calmette-Guerrin or Lactococcus lactis
- the polypeptide e.g. on its cell surface or secretes the polypeptide
- the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus.
- a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
- vaccinia or other pox virus e.g., vaccinia or other pox virus, retrovirus, or adenovirus
- an immunogenic composition may comprise both a polynucleotide and a polypeptide component. Such immunogenic composition may provide for an enhanced immune response. It will be apparent that an immunogenic composition may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein. Such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts). While any suitable carrier known to those of ordinary skill in the art may be employed in the immunogenic compositions of this invention, the type of carrier will vary depending on the mode of administration.
- compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.
- the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
- any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
- Biodegradable microspheres e.g., polylactate polyglycolate
- suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,81 1 ,128;
- compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
- buffers e.g., neutral buffered saline or phosphate buffered saline
- carbohydrates e.g., glucose, mannose, sucrose or dextrans
- mannitol proteins
- proteins polypeptides or amino acids
- proteins e.glycine
- antioxidants e.g., g., bacteriostats,
- Compounds may also be encapsulated within liposomes using well known technology. Any of a variety of immunostimulants may be employed in the immunogenic compositions of this invention.
- an adjuvant may be included.
- An adjuvant refers to the components in a vaccine or therapeutic composition that increase the specific immune response to the antigen (see, e.g., Edelman, AIDS Res. Hum Retroviruses 8:1409-141 1 (1992)). Adjuvants induce immune responses of the Th1 -type and Th-2 type response.
- Th1 - type cytokines e.g., IFN- ⁇ , IL-2, and IL-12
- Th-2 type cytokines e.g., IL-4, IL-5, 11-6, IL-10 tend to favour the induction of humoral immune responses.
- the adjuvant composition is preferably designed to induce an immune response predominantly of the Th1 type.
- Suitable adjuvant compositions include oil in water emulsions.
- oil in water emulsions which comprises 0.5 - 10 mg metabolisable oil (e.g. squalene), 0.5 - 1 1 mg tocol (e.g. alpha-tocopherol) and 0.1 - 4 mg emulsifying agent (e.g. polyoxyethylene sorbitan monooleate) per human dose.
- metabolisable oil e.g. squalene
- 0.5 - 1 1 mg tocol e.g. alpha-tocopherol
- emulsifying agent e.g. polyoxyethylene sorbitan monooleate
- An alternative adjuvant comprises an immunologically active saponin fraction derived from the bark of Quillaja Saponaria Molina (such as the HPLC purified fraction known as QS21 , as described in US5,057,540) presented in the form of a liposome and a lipopolysaccharide (such as 3-de-O-acylated monophosphoryl lipid A).
- These compositions may further comprise a sterol (e.g. cholesterol), such as wherein the ratio of saponin:sterol is from 1 :1 to 1 :100 w/w (e.g. the ratio of saponin:sterol is from 1 :1 to 1 :10 w/w).
- adjuvant systems of interest include those based on aluminium salts in conjunction with the lipopolysaccharide 3-de-O-acylated monophosphoryl lipid A.
- the antigen and 3-de-O- acylated monophosphoryl lipid A may be co-adsorbed to the same metallic salt particles or may be adsorbed to distinct metallic salt particles. See, for example, WO00/23105,
- Any immunogenic composition provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient (as necessary).
- Any of a variety of delivery vehicles may be employed within pharmaceutical compositions and immunogenic compositions to facilitate production of an antigen-specific immune response.
- Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells,
- APCs may generally be isolated from any of a variety of biological fluids and organs, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
- Dendritic cells are highly potent APCs (Banchereau & Steinman, Nature 392:245-251 (1998)) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic immunity (see Timmerman & Levy, Ann. Rev. Med. 50:507-529 (1999)).
- dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses.
- Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention.
- secreted vesicles antigen-loaded dendritic cells called exosomes
- exosomes secreted vesicles antigen-loaded dendritic cells
- Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid.
- dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM- CSF, IL-4, IL-13 and/or TNFa to cultures of monocytes harvested from peripheral blood.
- CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFa, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
- Dendritic cells are conveniently categorised as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterised phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation.
- Immature dendritic cells are characterised as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fey receptor and mannose receptor.
- the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD1 1 ) and
- costimulatory molecules e.g., CD40, CD80, CD86 and 4-1 BB.
- APCs may generally be transfected with a polynucleotide encoding a protein (or portion or other variant thereof) such that the polypeptide, is expressed on the cell surface. Such transfection may take place ex vivo, and a pharmaceutical composition or immunogenic composition comprising such transfected cells may then be used, as described herein.
- a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo.
- In vivo and ex vivo transfection of dendritic cells may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and Cell Biology 75:456-460 (1997).
- Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).
- the polypeptide Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule).
- a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
- Immunogenic compositions and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use.
- formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles.
- an immunogenic composition or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
- a "priming" or first administration of a modified Rv3616c protein (including fusion proteins), or polynucleotide encoding said protein is followed by one or more "boosting" or subsequent administrations of a modified Rv3616c protein (including fusion proteins) or polynucleotide encoding said protein ("prime and boost" method).
- a first administration with a modified Rv3616c polypeptide (including fusion proteins) or polynucleotide encoding said protein is followed by one or more subsequent administrations of a modified Rv3616c polypeptide (including fusion proteins) or polynucleotide encoding said polypeptide.
- a first administration with a modified Rv3616c protein or polynucleotide is followed by one or more subsequent administrations of a modified Rv3616c protein.
- a first administration with a modified Rv3616c protein or polynucleotide is followed by one or more subsequent administrations of a modified Rv3616c polynucleotide.
- first or “priming” administration and the second or “boosting” administration are given about 2-12 weeks apart, or up to 4-6 months apart. Subsequent "booster"
- administrations are given about 6 months apart, or as long as 1 , 2, 3, 4 or 5 years apart.
- Conventional booster treatment e.g., a protein priming administration followed by a protein boosting administration
- tuberculosis e.g.
- this invention provides methods for using one or more of the modified
- Rv3616c proteins described above to diagnose tuberculosis such as latent tuberculosis (for example using T cell response based assays or antibody based assays of conventional format).
- a method for determining latent M. tuberculosis infection in an individual comprising:
- the sample may for example be whole blood or purified cells.
- the sample will contain peripheral blood mononucleated cells (PBMC).
- PBMC peripheral blood mononucleated cells
- the individual will be seropositive.
- the individual will be seronegative.
- the individual will not previously have been vaccinated against M. tuberculosis infection (e.g. suitably the individual will not previously have been vaccinated with a BCG).
- the sample response may be quantified by a range of means known to those skilled in the art, including the monitoring of lymphocyte proliferation or the production of specific cytokines or antibodies.
- T-cell ELISPOT may be used to monitor cytokines such as interferon gamma (IFNv), interleukin 2 (IL2) and interleukin 5 (IL5).
- B-cell ELLISPOT may be used to monitor the stimulation of M. tuberculosis specific antigens.
- the cellular response may also be characterised by the use of by intra- and extra-cellular staining and analysis by a flow cytometer.
- Methods of quantifying a sample proliferation response include:
- Quantifying a sample cytokine response includes in particular the monitoring of interferon gamma production.
- a positive response to an antigen may be defined by a signal to noise ratio (S/N ratio) of at least 2:1 (for example, at least 3:1 or at least 5:1 ).
- a skin test is any assay performed directly on a patient in which a delayed-type hypersensitivity (DTH) reaction (such as swelling, reddening or dermatitis) is measured following intradermal injection of a modified Rv153c protein as described above.
- DTH delayed-type hypersensitivity
- Such injection may be achieved using any suitable device sufficient to contact the antigen combinations with dermal cells of the patient, such as a tuberculin syringe or 1 mL syringe.
- the reaction is measured after a period of time, for example at least 48 hours after injection, especially 48-72 hours.
- the DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to the test antigen.
- the response may be measured visually, using a ruler.
- a response that is greater than about 0.5 cm in diameter, especially greater than about 1.0 cm in diameter is a positive response, indicative of prior M. tuberculosis infection, which may or may not be manifested as an active disease.
- the modified Rv3616c protein component is suitably formulated as a pharmaceutical composition containing a physiologically acceptable carrier.
- a physiologically acceptable carrier for use in a skin test, the modified Rv3616c protein component is suitably formulated as a pharmaceutical composition containing a physiologically acceptable carrier.
- the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 80TM.
- kits for use within any of the above diagnostic methods.
- Such kits typically comprise two or more components necessary for performing a diagnostic assay.
- Components may be compounds, reagents, containers and/or equipment.
- one container within a kit may contain a modified Rv3616c protein.
- Such protein may be provided attached to a support material.
- kits may also, contain a detection reagent that contains a reporter group suitable for direct or indirect detection of antibody binding.
- kits include those designed for the detection of cell mediated responses (which may, for example, be of use in the diagnostic methods of the present invention). Such kits will typically comprise:
- (iii) means for detecting or quantifying the cellular response to stimulation.
- Suitable means for quantifying the cellular response include a B-cell ELISPOT kit or alternatively a T-cell ELISPOT kit, which are known to those skilled in the art.
- One possible kit comprises:
- a diagnostic kit comprising:
- a diagnostic kit comprising:
- (c) means to quantify the T cell response (e.g. proliferation or IFN-gamma
- the gene Rv3616c encodes for a conserved hypothetical alanine and glycine rich protein.
- Rv3616c was selected based on a genome-wide analysis of Mycobacterium tuberculosis genes associated with dormancy phase maintenance and infectivity as in Murphy and Brown BMC. Infect. Dis. 2007 7:84-99. Potential dormancy phase gene targets in Mycobacterium tuberculosis were prioritised through a bioinformatics meta-analysis of published genome-wide DNA microarray datasets of bacterial gene expression under simulated dormancy conditions. Subcellular localisation of M. tuberculosis proteins encoded by genes, was subsequently carried out on the entire genome to identify vaccine targets.
- Table 1 Sources, experimental models, and scoring criteria for M. tuberculosis DNA microarray gene expression and genome-wide gene knock-out (growth phase essentiality).
- Step 2 In applying the second criterion, the rank order of gene expression, gene scores from each dataset were ordered from highest to lowest based on expression ratio (fold expression in the experimental condition versus cells in log-phase liquid culture). The highest scoring gene received the maximum score for that particular dataset (listed in column 3 of Table 1 . (e.g. 5, 4 1 point)). The score was decreased by 0.005 points for each gene in order until zero, or the end of the data set was reached. Thus when the maximum score was 4 points, the 100th ranked gene would receive a score of 3.500. For a maximum score of 5 points, 1000 genes or 25% of the M. tuberculosis genome received a score. For experiments where data from multiple time points were collected, the maximum score across all time points was used as the final score.
- Step 3 scores for each gene in each of the experimental conditions were collected into a Microsoft Access database. Reference fields were added to facilitate prioritisation, such as the Refseq ID, Genbank function, Genbank note, Tuberculist classification, and KEGG and Sanger Center links.
- Step 4 a prioritised list of therapeutic targets was derived utilising the top 400 scoring genes (-10% of the genome) supplemented by expert computational and manual analysis of biochemical pathways, enzymology, drug tractability, homology to human genes and other prior knowledge. The great majority of the high scoring genes come from the subset where two or three of the groups intersect.
- Step 5 the identification of subcellular localisation of M. tuberculosis proteins encoded by genes, was carried out on the entire genome.
- the heuristic used for membrane protein prediction is described in Chalker et al. J. Bacteriol. 2001 183:1259-1268.
- Average hydropathy profiles (H) (von Heijne G J. Mol. Biol. 1992 225:487-494) were generated using GES hydropathy values (Engelman DM et al. Annu. Rev. Biophys. Biophys. Chem. 1986 15:321 - 353) weighted using a trapezoid window.
- GES hydropathy values Engelman DM et al. Annu. Rev. Biophys. Biophys. Chem. 1986 15:321 - 353
- helical transmembrane segments (TMS) were predicted for each peptide sequence by selecting 19 amino acids centered on the highest H value (MaxH), masking these from further
- Anchored proteins were defined as having exactly two TMS, one starting before amino acid (aa) 35 and one having a H of >1.15 with the other having a H not lower than 0.5.
- SignalP with Gram positive settings was specifically used for M. bacterium to identify secreted proteins amongst those classified as either cytoplasmic or "unknown" in the heuristic analysis (Nielsen H et al. Protein Eng. 1997 10:1 -6).
- Rv3616c ranked very high as a vaccine antigen according to several criteria:
- Rv3616c is consistently up-regulated across all models of dormancy. Among the entire suite of 3999 genes scored in the meta-analysis, Rv3616c was ranked in the top quartile of overexpressed genes across all dormancy models. The up-regulated score for Rv3616c was 6.52 which favourably compared with the top gene score of 22.28.
- Rv3616c can a elicit protective response against initial tuberculosis challenge.
- Rv3616c is broadly recognised as an antigen.
- CD4 and Multipred website antigen. i2r.a-star.edu. sg/multipred/
- CD4 ProPred website www.imtech.res.in/raghava/propred/
- Tepitope2 In house program based on :
- PredTAP website antigen. i2r.a-star.edu. sg/predTAP/
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BR112012018669A BR112012018669A2 (en) | 2010-01-27 | 2011-01-27 | MODIFIED RV3616C PROTEIN, USE OF A MODIFIED RV3616C PROTEIN, POLYNUCLEOTIDE, USE OF A POLYNUCLEOTIDE, PHARMACEUTICAL COMPOSITION, IMMUNOGENIC COMPOSITION, FUSION PROTEIN, AND, POLYPEPTIDE. |
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Citations (65)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4603112A (en) | 1981-12-24 | 1986-07-29 | Health Research, Incorporated | Modified vaccinia virus |
US4751180A (en) | 1985-03-28 | 1988-06-14 | Chiron Corporation | Expression using fused genes providing for protein product |
GB2200651A (en) | 1987-02-07 | 1988-08-10 | Al Sumidaie Ayad Mohamed Khala | A method of obtaining a retrovirus-containing fraction from retrovirus-containing cells |
US4769330A (en) | 1981-12-24 | 1988-09-06 | Health Research, Incorporated | Modified vaccinia virus and methods for making and using the same |
US4777127A (en) | 1985-09-30 | 1988-10-11 | Labsystems Oy | Human retrovirus-related products and methods of diagnosing and treating conditions associated with said retrovirus |
WO1989001973A2 (en) | 1987-09-02 | 1989-03-09 | Applied Biotechnology, Inc. | Recombinant pox virus for immunization against tumor-associated antigens |
EP0345242A2 (en) | 1988-06-03 | 1989-12-06 | Smithkline Biologicals S.A. | Expression of gag proteins from retroviruses in eucaryotic cells |
US4897268A (en) | 1987-08-03 | 1990-01-30 | Southern Research Institute | Drug delivery system and method of making the same |
US4935233A (en) | 1985-12-02 | 1990-06-19 | G. D. Searle And Company | Covalently linked polypeptide cell modulators |
WO1991002805A2 (en) | 1989-08-18 | 1991-03-07 | Viagene, Inc. | Recombinant retroviruses delivering vector constructs to target cells |
US5017487A (en) | 1985-04-04 | 1991-05-21 | Hoffmann-La Roche Inc. | Vaccinia DNA |
WO1991012325A1 (en) * | 1990-02-09 | 1991-08-22 | E.I. Du Pont De Nemours And Company | Htlv envelope antigenic segment |
US5057540A (en) | 1987-05-29 | 1991-10-15 | Cambridge Biotech Corporation | Saponin adjuvant |
WO1991018926A1 (en) | 1990-05-31 | 1991-12-12 | Arne Forsgren | PROTEIN D - AN IgD-BINDING PROTEIN OF HAEMOPHILUS INFLUENZAE |
US5075109A (en) | 1986-10-24 | 1991-12-24 | Southern Research Institute | Method of potentiating an immune response |
US5145684A (en) | 1991-01-25 | 1992-09-08 | Sterling Drug Inc. | Surface modified drug nanoparticles |
WO1993004175A1 (en) | 1991-08-15 | 1993-03-04 | Smithkline Beecham Biologicals (S.A.) | Osp a proteins of borrelia burgdorferi subgroups, encoding genes and vaccines |
US5399363A (en) | 1991-01-25 | 1995-03-21 | Eastman Kodak Company | Surface modified anticancer nanoparticles |
US5466468A (en) | 1990-04-03 | 1995-11-14 | Ciba-Geigy Corporation | Parenterally administrable liposome formulation comprising synthetic lipids |
US5543158A (en) | 1993-07-23 | 1996-08-06 | Massachusetts Institute Of Technology | Biodegradable injectable nanoparticles |
US5552157A (en) | 1990-08-27 | 1996-09-03 | Kabushiki Kaisha Vitamin Kenkyusya | Liposome for entrapping gene, liposomal preparation and process for the manufacture of the preparation |
US5565213A (en) | 1990-07-26 | 1996-10-15 | Taisho Pharmaceutical Co., Ltd. | Stable liposome aqueous suspension |
US5567434A (en) | 1989-03-31 | 1996-10-22 | The Regents Of The University Of California | Preparation of liposome and lipid complex compositions |
US5580579A (en) | 1995-02-15 | 1996-12-03 | Nano Systems L.L.C. | Site-specific adhesion within the GI tract using nanoparticles stabilized by high molecular weight, linear poly (ethylene oxide) polymers |
WO1996038591A1 (en) | 1995-06-02 | 1996-12-05 | Incyte Pharmaceuticals, Inc. | IMPROVED METHOD FOR OBTAINING FULL-LENGTH cDNA SEQUENCES |
WO1997009429A2 (en) | 1995-09-01 | 1997-03-13 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
WO1997009428A2 (en) | 1995-09-01 | 1997-03-13 | Corixa Corporation | Compounds and methods for immunotherapy and diagnosis of tuberculosis |
US5641515A (en) | 1995-04-04 | 1997-06-24 | Elan Corporation, Plc | Controlled release biodegradable nanoparticles containing insulin |
WO1997024447A1 (en) | 1996-01-02 | 1997-07-10 | Chiron Corporation | Immunostimulation mediated by gene-modified dendritic cells |
US5655698A (en) | 1994-07-13 | 1997-08-12 | Yoon; Inbae | Surgical stapler with curved jaws |
US5725871A (en) | 1989-08-18 | 1998-03-10 | Danbiosyst Uk Limited | Drug delivery compositions comprising lysophosphoglycerolipid |
US5738868A (en) | 1995-07-18 | 1998-04-14 | Lipogenics Ltd. | Liposome compositions and kits therefor |
US5741516A (en) | 1994-06-20 | 1998-04-21 | Inex Pharmaceuticals Corporation | Sphingosomes for enhanced drug delivery |
WO1998016645A2 (en) | 1996-10-11 | 1998-04-23 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
WO1998016646A2 (en) | 1996-10-11 | 1998-04-23 | Corixa Corporation | Compounds and methods for immunotherapy and diagnosis of tuberculosis |
US5756353A (en) | 1991-12-17 | 1998-05-26 | The Regents Of The University Of California | Expression of cloned genes in the lung by aerosol-and liposome-based delivery |
US5780045A (en) | 1992-05-18 | 1998-07-14 | Minnesota Mining And Manufacturing Company | Transmucosal drug delivery device |
US5792451A (en) | 1994-03-02 | 1998-08-11 | Emisphere Technologies, Inc. | Oral drug delivery compositions and methods |
US5795587A (en) | 1995-01-23 | 1998-08-18 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
US5804212A (en) | 1989-11-04 | 1998-09-08 | Danbiosyst Uk Limited | Small particle compositions for intranasal drug delivery |
US5811128A (en) | 1986-10-24 | 1998-09-22 | Southern Research Institute | Method for oral or rectal delivery of microencapsulated vaccines and compositions therefor |
WO1998053075A2 (en) | 1997-05-20 | 1998-11-26 | Corixa Corporation | Compounds for immunotherapy and diagnosis of tuberculosis and methods of their use |
US5928647A (en) | 1993-01-11 | 1999-07-27 | Dana-Farber Cancer Institute | Inducing cytotoxic T lymphocyte responses |
WO1999040188A2 (en) | 1998-02-05 | 1999-08-12 | Smithkline Beecham Biologicals S.A. | Tumor-associated antigen derivatives from the mage family, and nucleic acid sequences encoding them, used for the preparation of fusion proteins and of compositions for vaccinations |
WO1999051748A2 (en) | 1998-04-07 | 1999-10-14 | Corixa Corporation | Fusion proteins of mycobacterium tuberculosis antigens and their uses |
WO2001098460A2 (en) | 2000-06-20 | 2001-12-27 | Corixa Corporation | Fusion proteins of mycobacterium tuberculosis |
WO2003070187A2 (en) | 2002-02-15 | 2003-08-28 | Corixa Corporation | Fusion proteins of mycobacterium tuberculosis |
WO2004011048A1 (en) | 2002-07-26 | 2004-02-05 | Kimberly-Clark Worldwide, Inc. | Absorbent binder desiccant composition and articles incorporating it |
WO2006117240A2 (en) | 2005-04-29 | 2006-11-09 | Glaxosmithkline Biologicals S.A. | Novel method for preventing or treating m tuberculosis infection |
WO2007068907A2 (en) | 2005-12-13 | 2007-06-21 | Glaxosmithkline Biologicals Sa | Vaccine compositions comprising a saponin adjuvant |
US7259698B2 (en) | 2005-06-01 | 2007-08-21 | Olympus Imaging Corp. | Data encoding apparatus, data encoding method, and recording medium recorded with program |
US7296798B2 (en) | 2006-01-31 | 2007-11-20 | Matt Overfield | Gameboard, games played on board and methods of play requiring strategy and luck |
US7301098B2 (en) | 2005-01-19 | 2007-11-27 | Panduit Corp. | Communication channels with suppression cores |
US7300998B2 (en) | 2002-02-14 | 2007-11-27 | Mitsui Chemicals, Inc. | Polyester resin and catalyst for polyester production, process for producing polyester resin with the catalyst, polyester resin obtained with the catalyst, and hollow molded container comprising the polyester resins |
WO2008007942A1 (en) * | 2006-07-10 | 2008-01-17 | Het Nederlands Kanker Instituut | Means and methods for manipulating sequential phagolysomal-cytosolic translocation of mycobacteria, and uses thereof |
US7357936B1 (en) | 1998-10-16 | 2008-04-15 | Smithkline Beecham Biologicals, Sa | Adjuvant systems and vaccines |
WO2008043774A1 (en) | 2006-10-12 | 2008-04-17 | Glaxosmithkline Biologicals S.A. | Vaccine comprising an oil in water emulsion adjuvant |
US20080269151A1 (en) | 1999-10-07 | 2008-10-30 | Corixa Corporation | Fusion proteins of mycobacterium tuberculosis |
WO2010010177A1 (en) | 2008-07-25 | 2010-01-28 | Glaxosmithkline Biologicals S.A. | Novel compositions and methods |
WO2010121618A1 (en) | 2009-04-24 | 2010-10-28 | Statens Serum Institut | A tuberculosis tb vaccine to prevent reactivation |
US9810407B2 (en) | 2009-07-21 | 2017-11-07 | Cooper Technologies Company | Interfacing a light emitting diode (LED) module to a heat sink |
US9810514B2 (en) | 2015-09-08 | 2017-11-07 | Deufol Sunman Inc. | Ammunition carrier consumer package |
US9903265B2 (en) | 2013-10-28 | 2018-02-27 | Ford Global Technologies, Llc | Method for estimating charge air cooler condensation storage with an intake oxygen sensor |
US9903268B2 (en) | 2015-04-02 | 2018-02-27 | Ford Global Technologies, Llc | Internal combustion engine with two-stage supercharging capability and with exhaust-gas aftertreatment arrangement, and method for operating an internal combustion engine |
US9907717B2 (en) | 2011-10-08 | 2018-03-06 | Michael Dennis | Mattress overlay system with positionally adjustable, lateral ramp-wedge bolster structure |
Family Cites Families (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US1786412A (en) * | 1929-11-20 | 1930-12-23 | American Telephone & Telegraph | Shielding arrangement |
AT402926B (en) | 1994-12-05 | 1997-09-25 | Hafslund Nycomed Pharma | HETEROCYCLIC AMIDES, METHOD FOR THEIR PRODUCTION AND USE |
US6458366B1 (en) | 1995-09-01 | 2002-10-01 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
US6338852B1 (en) | 1995-09-01 | 2002-01-15 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
US6592877B1 (en) | 1995-09-01 | 2003-07-15 | Corixa Corporation | Compounds and methods for immunotherapy and diagnosis of tuberculosis |
US6544522B1 (en) | 1998-12-30 | 2003-04-08 | Corixa Corporation | Fusion proteins of mycobacterium tuberculosis antigens and their uses |
US6627198B2 (en) | 1997-03-13 | 2003-09-30 | Corixa Corporation | Fusion proteins of Mycobacterium tuberculosis antigens and their uses |
US6350456B1 (en) | 1997-03-13 | 2002-02-26 | Corixa Corporation | Compositions and methods for the prevention and treatment of M. tuberculosis infection |
US6555653B2 (en) | 1997-05-20 | 2003-04-29 | Corixa Corporation | Compounds for diagnosis of tuberculosis and methods for their use |
AU2001241738A1 (en) * | 2000-02-25 | 2001-09-03 | Corixa Corporation | Compounds and methods for diagnosis and immunotherapy of tuberculosis |
AU2002302919A1 (en) | 2001-02-22 | 2002-10-03 | Institut Pasteur | Comparative mycobacterial geneomics as a tool for identifying targets for the diagnosis, prophylaxis or treatment of mycobacterioses |
AU2002303135A1 (en) * | 2001-03-13 | 2002-09-24 | Corixa Corporation | Heterologous fusion protein constructs comprising a leishmania antigen |
EP1511768B1 (en) * | 2002-06-11 | 2007-04-11 | GlaxoSmithKline Biologicals S.A. | Immunogenic compositions |
GB0323965D0 (en) * | 2003-10-13 | 2003-11-19 | Glaxosmithkline Biolog Sa | Immunogenic compositions |
CA2606962A1 (en) * | 2005-04-29 | 2006-11-09 | Fusion Antibodies Limited | Assays for diagnosis of tuberculosis and uses thereof |
PT1880008E (en) | 2005-05-03 | 2015-08-27 | Inserm Inst Nat De La Santé Et De La Rech Médicale | Expression of mitochondrial protein by an enhanced allotopic approach |
EP2368568A1 (en) * | 2006-11-01 | 2011-09-28 | Immport Therapeutics, INC. | Compositions and methods for immunodominant antigens |
GB0622282D0 (en) * | 2006-11-08 | 2006-12-20 | Novartis Ag | Quality control methods |
MX2009009342A (en) * | 2007-03-02 | 2009-09-11 | Glaxosmithkline Biolog Sa | Novel method and compositions. |
GB0716070D0 (en) * | 2007-08-17 | 2007-09-26 | Fusion Antibodies Ltd | Diagnostic method and kit |
US7670609B2 (en) * | 2007-11-27 | 2010-03-02 | Aeras Global Tb Vaccine Foundation | Recombinant BCG tuberculosis vaccine designed to elicit immune responses to Mycobacterium tuberculosis in all physiological stages of infection and disease |
FR2933552B1 (en) | 2008-07-04 | 2014-10-10 | Centre Nat Rech Scient | AMPLIFICATION CIRCUIT OF A SIGNAL REPRESENTING A RESISTANCE VARIATION OF A VARIABLE RESISTANCE AND CORRESPONDING SENSOR |
KR20120129927A (en) * | 2010-01-27 | 2012-11-28 | 글락소 그룹 리미티드 | Modified tuberculosis antigens |
-
2011
- 2011-01-27 KR KR1020127022290A patent/KR20120129927A/en not_active Application Discontinuation
- 2011-01-27 DK DK11700859.9T patent/DK2528621T3/en active
- 2011-01-27 EP EP11700859.9A patent/EP2528621B1/en active Active
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-
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-
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- 2016-12-01 HR HRP20161608TT patent/HRP20161608T1/en unknown
- 2016-12-06 CY CY20161101254T patent/CY1119518T1/en unknown
Patent Citations (71)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4603112A (en) | 1981-12-24 | 1986-07-29 | Health Research, Incorporated | Modified vaccinia virus |
US4769330A (en) | 1981-12-24 | 1988-09-06 | Health Research, Incorporated | Modified vaccinia virus and methods for making and using the same |
US4751180A (en) | 1985-03-28 | 1988-06-14 | Chiron Corporation | Expression using fused genes providing for protein product |
US5017487A (en) | 1985-04-04 | 1991-05-21 | Hoffmann-La Roche Inc. | Vaccinia DNA |
US4777127A (en) | 1985-09-30 | 1988-10-11 | Labsystems Oy | Human retrovirus-related products and methods of diagnosing and treating conditions associated with said retrovirus |
US4935233A (en) | 1985-12-02 | 1990-06-19 | G. D. Searle And Company | Covalently linked polypeptide cell modulators |
US5853763A (en) | 1986-10-24 | 1998-12-29 | Southern Research Institute | Method for delivering bioactive agents into and through the mucosally-associated lymphoid tissue and controlling their release |
US5820883A (en) | 1986-10-24 | 1998-10-13 | Southern Research Institute | Method for delivering bioactive agents into and through the mucosally-associated lymphoid tissues and controlling their release |
US5814344A (en) | 1986-10-24 | 1998-09-29 | Southern Research Institute | Method for delivering bioactive agents into and through the mucosally associated lymphoid tissues and controlling their release |
US5811128A (en) | 1986-10-24 | 1998-09-22 | Southern Research Institute | Method for oral or rectal delivery of microencapsulated vaccines and compositions therefor |
US5075109A (en) | 1986-10-24 | 1991-12-24 | Southern Research Institute | Method of potentiating an immune response |
US5942252A (en) | 1986-10-24 | 1999-08-24 | Southern Research Institute | Method for delivering bioactive agents into and through the mucosally-associated lymphoid tissues and controlling their release |
GB2200651A (en) | 1987-02-07 | 1988-08-10 | Al Sumidaie Ayad Mohamed Khala | A method of obtaining a retrovirus-containing fraction from retrovirus-containing cells |
US5057540A (en) | 1987-05-29 | 1991-10-15 | Cambridge Biotech Corporation | Saponin adjuvant |
US4897268A (en) | 1987-08-03 | 1990-01-30 | Southern Research Institute | Drug delivery system and method of making the same |
WO1989001973A2 (en) | 1987-09-02 | 1989-03-09 | Applied Biotechnology, Inc. | Recombinant pox virus for immunization against tumor-associated antigens |
EP0345242A2 (en) | 1988-06-03 | 1989-12-06 | Smithkline Biologicals S.A. | Expression of gag proteins from retroviruses in eucaryotic cells |
US5567434A (en) | 1989-03-31 | 1996-10-22 | The Regents Of The University Of California | Preparation of liposome and lipid complex compositions |
US5725871A (en) | 1989-08-18 | 1998-03-10 | Danbiosyst Uk Limited | Drug delivery compositions comprising lysophosphoglycerolipid |
WO1991002805A2 (en) | 1989-08-18 | 1991-03-07 | Viagene, Inc. | Recombinant retroviruses delivering vector constructs to target cells |
US5804212A (en) | 1989-11-04 | 1998-09-08 | Danbiosyst Uk Limited | Small particle compositions for intranasal drug delivery |
WO1991012325A1 (en) * | 1990-02-09 | 1991-08-22 | E.I. Du Pont De Nemours And Company | Htlv envelope antigenic segment |
US5466468A (en) | 1990-04-03 | 1995-11-14 | Ciba-Geigy Corporation | Parenterally administrable liposome formulation comprising synthetic lipids |
WO1991018926A1 (en) | 1990-05-31 | 1991-12-12 | Arne Forsgren | PROTEIN D - AN IgD-BINDING PROTEIN OF HAEMOPHILUS INFLUENZAE |
US5565213A (en) | 1990-07-26 | 1996-10-15 | Taisho Pharmaceutical Co., Ltd. | Stable liposome aqueous suspension |
US5552157A (en) | 1990-08-27 | 1996-09-03 | Kabushiki Kaisha Vitamin Kenkyusya | Liposome for entrapping gene, liposomal preparation and process for the manufacture of the preparation |
US5399363A (en) | 1991-01-25 | 1995-03-21 | Eastman Kodak Company | Surface modified anticancer nanoparticles |
US5145684A (en) | 1991-01-25 | 1992-09-08 | Sterling Drug Inc. | Surface modified drug nanoparticles |
WO1993004175A1 (en) | 1991-08-15 | 1993-03-04 | Smithkline Beecham Biologicals (S.A.) | Osp a proteins of borrelia burgdorferi subgroups, encoding genes and vaccines |
US5756353A (en) | 1991-12-17 | 1998-05-26 | The Regents Of The University Of California | Expression of cloned genes in the lung by aerosol-and liposome-based delivery |
US5780045A (en) | 1992-05-18 | 1998-07-14 | Minnesota Mining And Manufacturing Company | Transmucosal drug delivery device |
US5928647A (en) | 1993-01-11 | 1999-07-27 | Dana-Farber Cancer Institute | Inducing cytotoxic T lymphocyte responses |
US5543158A (en) | 1993-07-23 | 1996-08-06 | Massachusetts Institute Of Technology | Biodegradable injectable nanoparticles |
US5792451A (en) | 1994-03-02 | 1998-08-11 | Emisphere Technologies, Inc. | Oral drug delivery compositions and methods |
US5741516A (en) | 1994-06-20 | 1998-04-21 | Inex Pharmaceuticals Corporation | Sphingosomes for enhanced drug delivery |
US5655698A (en) | 1994-07-13 | 1997-08-12 | Yoon; Inbae | Surgical stapler with curved jaws |
US5795587A (en) | 1995-01-23 | 1998-08-18 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
US5580579A (en) | 1995-02-15 | 1996-12-03 | Nano Systems L.L.C. | Site-specific adhesion within the GI tract using nanoparticles stabilized by high molecular weight, linear poly (ethylene oxide) polymers |
US5641515A (en) | 1995-04-04 | 1997-06-24 | Elan Corporation, Plc | Controlled release biodegradable nanoparticles containing insulin |
WO1996038591A1 (en) | 1995-06-02 | 1996-12-05 | Incyte Pharmaceuticals, Inc. | IMPROVED METHOD FOR OBTAINING FULL-LENGTH cDNA SEQUENCES |
US5738868A (en) | 1995-07-18 | 1998-04-14 | Lipogenics Ltd. | Liposome compositions and kits therefor |
WO1997009429A2 (en) | 1995-09-01 | 1997-03-13 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
WO1997009428A2 (en) | 1995-09-01 | 1997-03-13 | Corixa Corporation | Compounds and methods for immunotherapy and diagnosis of tuberculosis |
WO1997024447A1 (en) | 1996-01-02 | 1997-07-10 | Chiron Corporation | Immunostimulation mediated by gene-modified dendritic cells |
WO1998016645A2 (en) | 1996-10-11 | 1998-04-23 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
WO1998016646A2 (en) | 1996-10-11 | 1998-04-23 | Corixa Corporation | Compounds and methods for immunotherapy and diagnosis of tuberculosis |
WO1998053075A2 (en) | 1997-05-20 | 1998-11-26 | Corixa Corporation | Compounds for immunotherapy and diagnosis of tuberculosis and methods of their use |
WO1999040188A2 (en) | 1998-02-05 | 1999-08-12 | Smithkline Beecham Biologicals S.A. | Tumor-associated antigen derivatives from the mage family, and nucleic acid sequences encoding them, used for the preparation of fusion proteins and of compositions for vaccinations |
WO1999051748A2 (en) | 1998-04-07 | 1999-10-14 | Corixa Corporation | Fusion proteins of mycobacterium tuberculosis antigens and their uses |
US20080226672A1 (en) | 1998-10-16 | 2008-09-18 | Smithkline Beecham Biologicals, S.A. | Adjuvant Systems and Vaccines |
US7357936B1 (en) | 1998-10-16 | 2008-04-15 | Smithkline Beecham Biologicals, Sa | Adjuvant systems and vaccines |
US20080269151A1 (en) | 1999-10-07 | 2008-10-30 | Corixa Corporation | Fusion proteins of mycobacterium tuberculosis |
WO2001098460A2 (en) | 2000-06-20 | 2001-12-27 | Corixa Corporation | Fusion proteins of mycobacterium tuberculosis |
US7300998B2 (en) | 2002-02-14 | 2007-11-27 | Mitsui Chemicals, Inc. | Polyester resin and catalyst for polyester production, process for producing polyester resin with the catalyst, polyester resin obtained with the catalyst, and hollow molded container comprising the polyester resins |
WO2003070187A2 (en) | 2002-02-15 | 2003-08-28 | Corixa Corporation | Fusion proteins of mycobacterium tuberculosis |
WO2004011048A1 (en) | 2002-07-26 | 2004-02-05 | Kimberly-Clark Worldwide, Inc. | Absorbent binder desiccant composition and articles incorporating it |
US7301098B2 (en) | 2005-01-19 | 2007-11-27 | Panduit Corp. | Communication channels with suppression cores |
WO2006117240A2 (en) | 2005-04-29 | 2006-11-09 | Glaxosmithkline Biologicals S.A. | Novel method for preventing or treating m tuberculosis infection |
US7259698B2 (en) | 2005-06-01 | 2007-08-21 | Olympus Imaging Corp. | Data encoding apparatus, data encoding method, and recording medium recorded with program |
WO2007068907A2 (en) | 2005-12-13 | 2007-06-21 | Glaxosmithkline Biologicals Sa | Vaccine compositions comprising a saponin adjuvant |
US20080279926A1 (en) | 2005-12-13 | 2008-11-13 | Pierre Vandepapeliere | Vaccine Compositions Comprising a Saponin Adjuvant |
US7296798B2 (en) | 2006-01-31 | 2007-11-20 | Matt Overfield | Gameboard, games played on board and methods of play requiring strategy and luck |
WO2008007942A1 (en) * | 2006-07-10 | 2008-01-17 | Het Nederlands Kanker Instituut | Means and methods for manipulating sequential phagolysomal-cytosolic translocation of mycobacteria, and uses thereof |
WO2008043774A1 (en) | 2006-10-12 | 2008-04-17 | Glaxosmithkline Biologicals S.A. | Vaccine comprising an oil in water emulsion adjuvant |
WO2010010177A1 (en) | 2008-07-25 | 2010-01-28 | Glaxosmithkline Biologicals S.A. | Novel compositions and methods |
WO2010121618A1 (en) | 2009-04-24 | 2010-10-28 | Statens Serum Institut | A tuberculosis tb vaccine to prevent reactivation |
US9810407B2 (en) | 2009-07-21 | 2017-11-07 | Cooper Technologies Company | Interfacing a light emitting diode (LED) module to a heat sink |
US9907717B2 (en) | 2011-10-08 | 2018-03-06 | Michael Dennis | Mattress overlay system with positionally adjustable, lateral ramp-wedge bolster structure |
US9903265B2 (en) | 2013-10-28 | 2018-02-27 | Ford Global Technologies, Llc | Method for estimating charge air cooler condensation storage with an intake oxygen sensor |
US9903268B2 (en) | 2015-04-02 | 2018-02-27 | Ford Global Technologies, Llc | Internal combustion engine with two-stage supercharging capability and with exhaust-gas aftertreatment arrangement, and method for operating an internal combustion engine |
US9810514B2 (en) | 2015-09-08 | 2017-11-07 | Deufol Sunman Inc. | Ammunition carrier consumer package |
Non-Patent Citations (128)
Title |
---|
"Adenovirus: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References", 2004 |
"Current Protocols in Molecular Biology", 1995 |
"Goodman and Gilman's The Pharmacological Basis of Therapeutics", 2001 |
"Harrison's Principles of Internal Medicine", 2005, pages: 953 - 966 |
"Remington's Pharmaceutical Sciences", pages: 1035 - 1038,157 |
"Tuberculosis", 2003 |
AI-ATTIYAH ET AL., CLIN. EXP. IMMUNOL., vol. 138, 2004, pages 139 - 144 |
ALDERSON ET AL., JOURNAL OF EXPERIMENTAL MEDICINE, vol. 7, 2000, pages 551 - 559 |
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1997, pages 403 - 410 |
ALTSCHUL ET AL., NUC. ACIDS RES., vol. 25, 1977, pages 3389 - 3402 |
ANDERSON P, TRENDS IN MICROBIOLOGY, vol. 15, no. 1, 2007, pages 7 - 13 |
AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY |
BANCHEREAU, STEINMAN, NATURE, vol. 392, 1998, pages 245 - 251 |
BEIPBARTH ET AL., BIOINFORMATICS, vol. 21, no. 1, 2005, pages I29 - I37 |
BERKNER, BIOTECHNIQUES, vol. 6, 1988, pages 616 - 627 |
BIOTECHNOLOGY, vol. 10, 1992, pages 795 - 798 |
BOYD D ET AL., PROTEIN SCI., vol. 7, 1998, pages 201 - 205 |
BROGLIE ET AL., SCIENCE, vol. 224, 1984, pages 838 - 843 |
CAMUS, MICROBIOLOGY, vol. 148, 2002, pages 2967 - 2973 |
CARDANA P-J, INFECTION, vol. 37, no. 2, 2009, pages 80 - 86 |
CARDANA P-J, INFLAMMATION & ALLERGY - DRUG TARGETS, vol. 6, 2006, pages 27 - 39 |
CARUTHERS, M. H. ET AL., NUCL. ACIDS RES. SYMP. SER., 1980, pages 215 - 223 |
CHALKER ET AL., J. BACTERIOL., vol. 183, 2001, pages 1259 - 1268 |
CHAN, KAUFMANN: "Tuberculosis: Pathogenesis, Protection and Control", 1994 |
CLAROS MG ET AL., COMPUT. APPL. BIOSCI., vol. 10, 1994, pages 685 - 686 |
COHEN, SCIENCE, vol. 259, 1993, pages 1691 - 1692 |
COLBERE-GARAPIN ET AL., J. MOL. BIOL., vol. 150, 1981, pages 1 - 14 |
COLE ET AL., NATURE, vol. 393, 1998, pages 537 |
COLE ET AL., NATURE, vol. 393, 1998, pages 537 - 544 |
COLER ET AL., JOURNAL OF IMMUNOLOGY, vol. 161, 1998, pages 2356 - 2364 |
COLER ET AL., VACCINE, vol. 27, 2009, pages 223 - 233 |
CONTENT ET AL., INFECTION AND IMMUNITY, vol. 59, 1997, pages 3205 - 3212 |
CORUZZI ET AL., EMBO J., vol. 3, 1984, pages 1671 - 1680 |
CREIGHTON, PROTEINS, 1984 |
DEVEREAUX ET AL., NUC. ACIDS RES., vol. 12, 1984, pages 387 - 395 |
DIETRICH ET AL., JOURNAL OF IMMUNOLOGY, vol. 174, no. 10, 2005, pages 6332 - 6339 |
DILLON ET AL., INFECTION AND IMMUNITY, vol. 67, no. 6, 1998, pages 2941 - 2950 |
DOHERTY ET AL., JOURNAL OF INFECTIOUS DISEASES, vol. 190, 2004, pages 2146 - 2153 |
EDELMAN, AIDS RES. HUM RETROVIRUSES, vol. 8, 1992, pages 1409 - 1411 |
EHLERS S, INFECTION, vol. 37, no. 2, 2009, pages 87 - 95 |
ENGELHARD ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 91, 1994, pages 3224 - 3227 |
ENGELMAN DM ET AL., ANNU. REV. BIOPHYS. BIOPHYS. CHEM., vol. 15, 1986, pages 321 - 353 |
FENG, DOOLITTLE, J. MOL. EVOL., vol. 35, 1987, pages 351 - 360 |
FERREIRA ET AL., AN ACAD BRAS CIENC, vol. 77, 2005, pages 113 - 124 |
FISHER-HOCH ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 317 - 321 |
FLEXNER ET AL., ANN. N. Y. ACAD. SCI., vol. 569, 1989, pages 86 - 103 |
FLEXNER ET AL., VACCINE, vol. 8, 1990, pages 17 - 21 |
FLYNN ET AL., J. EXP. MED., vol. 178, 1993, pages 2249 - 2254 |
FRIEDMAN ET AL., J. INFECT. DIS., vol. 176, no. 2, 1998, pages 478 - 84 |
FRISCIA ET AL., CLINICAL AND EXPERIMENTAL IMMUNOLOGY, vol. 102, 1995, pages 53 - 57 |
GAGNEUX ET AL., PNAS, vol. 103, no. 8, 2006, pages 2869 - 2873 |
GENE, vol. 43, 1986, pages 265 - 292 |
GRANT ET AL., METHODS ENZYMOL., vol. 153, 1987, pages 516 - 544 |
GUZMAN ET AL., CIR. RES., vol. 73, 1993, pages 1202 - 1207 |
GUZMAN ET AL., CIRCULATION, vol. 88, 1993, pages 2838 - 2848 |
HAMPTON ET AL.: "Serological Methods, a Laboratory Manual", 1990 |
HANAHAN D.: "DNA cloning", 1985, IRL PRESS, article "Plasmid transformation by Simanis", pages: 109 - 135 |
HARTMAN, MULLIGAN, PROC. NATL. ACAD. SCI. U.S.A., vol. 85, 1988, pages 8047 - 51 |
HELLER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 94, 1998, pages 2150 - 2155 |
HENIKOFF, HENIKOFF, PROC. NATL. ACAD. SCI. USA, vol. 89, 1997, pages 10915 |
HERBERT ET AL., INFECT. IMMUN., vol. 75, no. 12, 2007, pages 5798 - 5805 |
HIGGINS, SHARP, CABIOS, vol. 5, 1989, pages 151 - 153 |
HIRSH ET AL., PNAS, vol. 101, 2004, pages 4871 - 4876 |
HOBBS: "Yearbook of Science and Technology", 1992, MCGRAW HILL, pages: 191 - 196 |
HODGKINS, PD ET AL., J. EXP. MED., vol. 184, 1996, pages 277 - 281 |
HORN ET AL., NUCL. ACIDS RES. SYMP. SER., 1980, pages 225 - 232 |
HUYGEN ET AL., NATURE MEDICINE, vol. 2, no. 8, 1996, pages 893 - 898 |
KARLIN, ALTSCHUL, PROC. NAT'1. ACAD. SCI. USA, vol. 90, 1997, pages 5873 - 5787 |
KASS-EISLER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 11498 - 11502 |
KOLLS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 215 - 219 |
KROLL ET AL., DNA CELL BIOL., vol. 12, 1993, pages 441 - 453 |
LAGERSTROM ET AL., PCR METHODS APPLIC., vol. 1, 1991, pages 111 - 19 |
LALVANI A ET AL., J. INFECT. DIS., vol. 180, 1999, pages 1656 - 1664 |
LIN MY ET AL., ENDOCRINE, METABOLIC & IMMUNE DISORDERS - DRUG TARGETS, vol. 8, 2008, pages 15 - 29 |
LOCHT ET AL., EXPERT OPIN. BIOL. THER., vol. 7, no. 11, 2007, pages 1665 - 1677 |
LOGAN, SHENK, PROC. NATL. ACAD. SCI. U.S.A., vol. 81, 1984, pages 3655 - 3659 |
LOWY ET AL., CELL, vol. 22, 1990, pages 817 - 23 |
MADDOX ET AL., J. EXP. MED., vol. 158, 1983, pages 1211 - 1216 |
MAECKER HT ET AL., J. IMMUNOL. METHODS, vol. 255, no. 1-2, 2001, pages 27 - 40 |
MAHVI ET AL., IMMUNOLOGY AND CELL BIOLOGY, vol. 75, 1997, pages 456 - 460 |
MARATEA ET AL., GENE, vol. 40, 1963, pages 39 - 46 |
MERRIFIELD, J. AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2146 |
MURPHY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 8258 - 8262 |
MURPHY, BROWN, BMC.INFECT. DIS., vol. 7, 2007, pages 84 - 99 |
MUSTAFA ET AL., INFECT. IMMUN., vol. 74, no. 8, 2006, pages 4566 - 4572 |
NEEDLEMAN, WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 |
NIELSEN H ET AL., PROTEIN ENG., vol. 10, 1997, pages 1 - 6 |
ORME ET AL., J. EXP. MED., vol. 158, 1983, pages 74 - 83 |
PAI M ET AL., EXPERT REV. MOL. DIAGN., vol. 6, no. 3, 2006, pages 413 - 422 |
PARKER ET AL., NUCL. ACIDS. RES., vol. 19, 1991, pages 3055 - 60 |
PAUL: "Fundamental Immunology", 1993, pages: 243 - 247 |
PEARSON, LIPMAN, PROC. NAT'L. ACAD. SCI. USA, vol. 85, 1988, pages 2444 |
PORATH ET AL., PROTO EXP. PURIF., vol. 3, 1992, pages 263 - 281 |
RADOSEVIC ET AL., INFECTION AND IMMUNITY, vol. 75, no. 8, 2007, pages 4105 - 4115 |
RAHA ET AL., APPL MICROBIOL BIOTECHNOL, 2005 |
RHODES ET AL., METHODS MOL. BIOL., vol. 55, 1995, pages 121 - 131 |
ROBERGE ET AL., SCIENCE, vol. 269, 1995, pages 202 - 204 |
ROLLAND, CRIT. REV. THERAP. DRUG CARRIER SYSTEMS, vol. 15, 1998, pages 143 - 198 |
ROMAS ET AL., YEAST, vol. 8, 1987, pages 423 - 88 |
ROSENFELD ET AL., SCIENCE, vol. 252, 1991, pages 431 - 434 |
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", 2000 |
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2000 |
SCANDINAVIAN, JOURNAL OF IMMUNOLOGY, vol. 43, 1996, pages 662 - 670 |
SCHARF. ET AL., RESULTS PROBL. CELL DIFFER., vol. 20, 1994, pages 125 - 162 |
SCHENA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 10614 - 10619 |
SCHUCK SD ET AL., PLOS ONE, vol. 4, no. 5, 2009, pages E5590 |
SKEIKY ET AL., JOURNAL OF IMMUNOLOGY, vol. 165, 2000, pages 7140 - 7149 |
SKEIKY ET AL., JOURNAL OF IMMUNOLOGY, vol. 172, 2004, pages 7618 - 7682 |
SMITH, WATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482 |
SORENSEN ET AL., INFECTION AND IMMUNITY, vol. 63, no. 5, 1997, pages 1710 - 1717 |
TAKAMATSU, EMBO J., vol. 6, 1987, pages 307 - 311 |
TATSIS N ET AL., GENE THERAPY, vol. 13, 2006, pages 421 - 429 |
TIMMERMAN, LEVY, ANN. REV. MED., vol. 50, 1999, pages 507 - 529 |
TRIGLIA ET AL., NUCL. ACIDS RES., vol. 16, 1988, pages 8186 |
TUBERCULOSIS FACTS, 2008 |
ULMER ET AL., SCIENCE, vol. 259, 1993, pages 1745 - 1749 |
VAN HEEKE, SCHUSTER, J. BIOL. CHEM., vol. 264, 1989, pages 5503 - 5509 |
VERBON ET AL., JOURNAL OF BACTERIOLOGY, vol. 174, 1992, pages 1352 - 1359 |
VON ESCHEN ET AL., HUM. VACCIN., vol. 5, no. 7, 2009 |
VON HEIJNE G, J. MOL. BIOL., vol. 225, 1992, pages 487 - 494 |
WALZER T ET AL., CELL IMMUNOL., vol. 206, no. 1, 2000, pages 16 - 25 |
WIGLER ET AL., CELL, vol. 11, 1977, pages 223 - 32 |
WIGLER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 77, 1980, pages 3567 - 70 |
WINTER ET AL., RESULTS PROBL. CELL DIFFER., vol. 17, 1991, pages 85 - 105 |
WOLD, ADENOVIRUS METHODS AND PROTOCOLS, 1998 |
WOODSWORTH ET AL., INFECTION AND IMMUNITY, vol. 76, no. 9, 2008, pages 4199 - 4205 |
ZITVOGEL ET AL., NATURE MED., vol. 4, 1998, pages 594 - 600 |
ZVI ET AL., BMC MEDICAL GENETICS, vol. 1, 2008, pages 18 |
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