Title of Invention

A POLYNUCLEOTIDE VACCINE

Abstract The present invention relates to methods and composition useful in the treatment and prevention of Hepatitis C virus (RCV) infections and the symptoms and diseases associated therewith. In particular the present invention relates to DNA vaccines that encodes the HCV. Core protein and a polynucleotide sequence that encodes at least one other HCV protein, wherein the vaccine caused expression of the proteins within th same cell and the sequence of the polynucleotide sequence encoding the core protein has been mutated or positioned relative to the polynucleotide sequence encoding the at least one other HCV protein such that the negative effect of expression of the Core protein upon the expression of the said at least one other HCV protein is reduced.
Full Text The present invention relates to methods and compositions useful in the treatment and
prevention of Hepatitis C virus (HCV) infections and the symptoms and diseases associated
5 therewith. In particular the present invention relates to DNA vaccines comprising
polynucleotide sequences encoding the HCV core protein and at least one additional HCV
protein, and methods of treatment of individuals infected with HCV comprising
administration of the vaccines of the present invention.
HCV was identified recently as the leading causative agent of post-transfusion and
10 community acquired non A, nun B hepatitis. Approximately 170m people are chronically
infected with HCV, with prevalence between 1-10%. The health care cost in the US, where
the prevalence is 1.8%, is estimated to be $2 billion. Between 40-60% of liver disease is due
to HCV and 30% UK transplants arc for ECV infections. Although HCV is initially a sub-
clinical infection more than 90% of patients develop chronic disease. The disease process
15 typically develops from chronic active hepatitis (70%), fibrosis, cirrhosis (40%) to liepato-
cellular carcinoma (60%). Infection to cirrhosis has a median time of 20 years and that for
hepato-cellujar carcinoma of 20 years (Lauer G.and Walker B. 2001, N. Engl J. Med 345, 41,
Cohen J. 2001, Science 285 (5424) 2G).
There is a great need for the unproved treatment of HCV. The current gold standard
20 of ribavhin and PEGyhtted interferon represents the mainstay for treating HCV infection.
However the ability of the current regimens to achieve sustained response remains sub-
optimal (overall 50% response rate for up to 6 months, however, for genotype lb the
response rate is lower (27%). This treatment is also associated with unpleasant side effects.
This results in high fall out rale, especially after first 6 months of treatment.
25 Several studies have shown that the individual HCV proteins are immunogenic in
normal mice, including following immunisation with DNA. Several HCV vaccines are
currently in clinical trial for either prophylaxis or therapy. The most advanced are currently in
Phase 2 by Chiron and. Innogenetics using El or E2 envelope proteins. An epitope vaccine by
Transvax is also in Phase 2. Several vaccines are in preclinical development which use
30 sequences from core and non-structural antigens using a variety of delivery systems including
DNA.
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WO 2004/046175 PCT/EP2003/012793
HCV is a positive strand RNA virus of the flaviviradae femily, whose genome is
9.4ko in length, with one open reading frame. The HCV genome is translated as a single
polyprotein, which is then processed by host and viral proteases to produce structural proteins
(core,, envelope El aad E2, and p7) and six non-structural proteins with, various enzymatic
5 activities. The genome of the HCV J4L6 isolate, which is an example of the lb genotype, is
found as accession number AFD54247 (Yanagi,M., St Claire.M., Shapiro, M, Emerson.S. U,
Purcell, R H. and Bukh,J. "Transcripts of a chimeric cDNA clone of hepatitis C virus
genotype lb are infectious in vivo". Virology 244 (I), ] 61-172 (1998)), and is shown in
Figure l.
10 The envelope proteins ate responsible for recognition, binding and entry of virus onto
target cells. The major non-structural proteins involved in viral replication include N S2 (Zn
dependent netaloprateinase), NS3 (serinc protease / belicase), NS4A (protease co-factor),
NS4B, NS5A and MS5B (RHA polymerase)( Bartenscnlager B and Lohmatin V. 2000.
Replication of hepatitis C virus- 1. Gen Virol 81,1531).
15 The structure of the HCV polyprotein can be represented as follows (the figures refer
to the position of the first amino acid of each protein; the full polypiotein of the J4L6 isolate
is 3010 amino acids in length)
Core1-191 El E2 P7 NS2 NS31027-1657 NS4A NS4B1712-1972 NS5A NS5B2420-3010
The virus has a high mutation rate and at least six major genotypes have been defined
20 based in the nucleotide sequence of conserved and non-conserved regions. However there is
additional heterogeneity as HCV isolated from a single patient is always presented as a
mixture of closely related genomes, or quasi-species.
The HCV genome shows a high degree of genetic variation, which has been classified
into 6 major genotypes (la, lb. 2,3, 4, 5, and 6). Genotypes la, lb, 2 and 3 are the most
25 prevalent in Europe, North and South America, Asia, China, Japan said Australia. Genotypes
4 and 5 are predominant in Africa and genotype 6 S.E Asia.
There is a great need for improved treatments of HCV infection and also to provide
treatments that are diverse in the ability to treat a number of HCV genotypes,
HCV vaccines comprising polynucleotides encoding one or more HCV proteins have
30 been described. Vaccines comprisbg plasmid DNA or Semliki Forest Virus vectors encoding
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WO 2004/046175 PCT/EP2003/012793
NS3 were described by Brinster et al. (2002, Journal of General Virology,, 83,369-381).
Polynucleotide vaccines encodirigNS5B are disclosed in "WO 99/51781, Codon optimised
genes, and vaccines comprising them, encoding HCV El, E1+E2 fusions, NS5A and NS5B
proteins are described in WO 97/47358. WO 03/04149 discloses polypeptides or
5 polynudeotides encoding mosaics of HCV epitopes, derived from within Core, NS3, NS4 or
NS5A. Fusion protein, and DMA encoding such fusion proteins, comprising NS3, NS4,,
NS5A and NS5B, that are useful in vaccines are described in WO 01/30812; optionally the
fusion proteins ane said to comprise fragments of the Core protein. WO 03/031586 describes
an adenovirus vector, that is suitable for use as a vaccine, which encodes the HCV proteins
10 NS3-NS4A-NS4B-NS5A-NS5B.
Vaccin.es comprising polypeptides comprising "unprocessed" core protein and a non-
structural protein are described in WO 96/37606.
It is desirable to include in a polynucleotide vaccine, a gene that encodes the Core
protein and at least one other HCV protein. However, it ia known that the co-expression of
15 Core and other HCV proteins within the same cell can lead to a decrease in the level of
production of the other HCV protein in comparison with thai produced in a cell where the
Cote protein is not co-expressed. For this reason the art is relatively silent about the use of the
Core protein ia polynacleotide vaccines.
The present invention provides a solution to this problem, and provides a
20 polynucleotide vaccine comprising a polynucleotide sequence that encodes the HCV Core
protein and a polynucleotide sequence that encodes at least one other HCV protein, wherein
the vaccine causes expression of the proteins within the same cell, and wherein the sequence
of the polynucleotide encoding the core protein has been mutated or is positioned relative to
the polynudootide sequence encoding the at least one other HCV protein in such a way that
25 the negative effect of expression of the Core protein upon the expression o f the said at least
one other HCV protein is reduced, or abrogated.
It has been found that the reduction or prevention of the down regulation of
expression of other HCV proteins by the expression of the core protein, leads to the increase
in the magnitude of the immune response raised against the other HCV proteins. Preferably
30 the increase in magnitude of immune response against the non-core HCV protein is two fold
or greater, as measured by ELISPOT measuring the numbers of EL-2 producing splenocytes
after vaccination and restimilation in vitro with antigen,
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WO 2004/046175 PCT/EP2003/012793
The vaccines of the present invention are designed in such a way that the down
regulation effect of Core upon the expression levels of the other HCV proteins is reduced or
abrogated. It is preferred that the polynucleotide vaccines of the present invention cause the
production of the non-core HCV protein in a cell, at a quantity that is not less than 50% of the
5 quantity that is produced by transfection of the tells with an equivalent amount of a similar
vaccine that does not cause expression of the Core protein within the same cell More
preferably, the polynucleotides cause the production of the non-core HCV protein in a cell at
a level that is not less than 60%, more preferably not less than 70%, more preferably not less
than 80%, more preferably not less than 90%, and most preferably not less than 95% of the
10 levels that are produced by transfection of the cells with an equivalent amount of a similar
vaccine that does not cause expression of the Core protein within ibe same cell. Most
preferably the levels of protein production are measured using Western Blot techniques
revealed by real-time chemiluminescent technology
Most preferably the vaccine is designed such that the core protein is present in an
15 expression cassette that is downstream of an expression cassette that encodes the other HCV
protein, or alternatively the amino acid sequence of the core protein is mutated.
The at least one other HCV antigen encoded by the polynucleotide vaccines of the
invention may be any of the non-Core ECV proteins, such as E1, E2, NS3, NS4A, NS4B,
KS5A, NS5B or p7. Preferably, however,the other HCV protein are selected from NS3;
20 NS4B and NS5B. Preferably, the polynucleotide vaccines of the present invention do not
vaccines of the present invention encode the Core protein or mutated Core protein (mCore)
and NS3, NS4B and NS5B HCV proteins, and no other HCV protein. The present invention
also provides the use of a polynucleotide vaccine encoding these antigens in medicine, and in
25 the manufacture of A medicament for the treatment, or prevention, of an HCV infection.
The polynucleotide sequences used in the vaccines of the present invention are
preferably DNA sequences.
The polynudeotides encoding the HCV proteins maybe in many combinations or
configurations. For example, the proteins may be expressed as individual proteins, or as
30 fusion proteins. An example of a fusion, which could either be at the DNA or protein level,
would be a double fusion which consists of a single polypeptide or polynucleotide containing
or encoding the amino acid sequences of NS4B and NS5B (NS4B-NS5B), a triple fusion
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WO 2004/046175 PCT/EP2003/012793
containing or encoding the amino acid sequences of NS3-NS4B-NS5B, or a fusion of all four
antigens of the present invention (mCora-NS3-NS4B-NS5B)
Preferred fusions of the present invention are polynucleotides that encode the double
fusion between NS4B and NS5B (NS4B-NS5B or NS5B-NS4B); and between Core or
5 mCore and NS3 (HS3-mCore or mCore-NS3). Preferred triple fusions are polynucleotides
that encode the amino acid sequences of NS3-NS4B-NS5B.
Preferably the polynuclootides encoding each antigen are present in the same
expression vector or plasmid such that expression of the HCV proteins occurs in the same
cell. In this context the polynucleotide encoding the HCV proteins may be in a single
10 expression cassette, or in multiple in series expression cassettes within the same
polynucleotide vector.
The biological functions of HCV core protein are complex and do nor correlate with
discrete point mutations (McLauchlan 1.2000. Properties of the hepatitis C virus core
protein: a structural protein that modulates cellular processes, I of Viral Hepatitis 7, 2-4).
15 There is evidence that core directly interacts with the lymphotoxin β receptor, and can also
interfere with NFKB and PKR pathways and can influence cell survival and apoptosis. A
recombinant vaccinia construct expressing core was found to inhibit cellular responses to
vaccinia making it more virulent in vivo.
During an infection, the Core protein is cleaved at two sites from the viral polypiotein
20 by host cell proteases. The first cleavage is at 191 which generates the N-terminal end of El.
The residue at which the second cleavage takes place has not been precisely located and lies
between amino acids 174 and 191 thereby liberating a short Core peptide sequence of
approximately 17 amino acids in length (McLauchlan J, (2000) J. Viral Hepatitis. 7,2-14;
YasuiK, Lan IYR Mizokami M, et al, J. Virol 1998. 72 604S-6055),
25 The Core polypeptides encoded in the vaccines of the preset invention are either full
length or in a inncated form.
In order to optimise the expression of the other HCV proteins, the polynucleotide
encoding the HCV Core protein of mCore protein is preferably present in an expression
cassette that is downstream of an expression cassette that contains the polynuclcotide that
30 encodes al least one of the other HCV proteins. Preferably the HCV Core protein is
i preferably present in an expression cassette that is downatream of an expression cassette that

WO 2004/046175 PCT/EP2003/012793
contains the polymcleotide that encodes NS5B. ln this context is it possible for Core protein
to be expnessed in fusion with the HCV NS3 protein.
In outer to minimise the negative effect of Core upon the production of other HCV
proteins in the same cell the Core potein used is a truncated protein. This aspect of the
5 present invention is particularly preferred if the core protein is not encoded by a
polynucleotide present in an expression cassette that is downstream of an expression cassette
that contains the polynucleotide that encodes the other HCV protein. Also, this aspect of the
present invention is preferred if the Core protein is to be present as part of a fusion protein,
comprising Core and the other HCV protein sequence. In this aspect of the present invention
10 it is preferred that the Core protein that is encoded is truncated from the carboxy terminal end
in a sufficient amount to reduce the inhibitory effect of Core upon the expression of other
HCV proteins. Most preferably the Core protein is truncated from the carboxy teerminal end
such that the sequence of the protein produced lacks the naturally liberated C-terninal
peptide sequence arising from the second cleavage of Core; more preferably the protein lacks
15 at least the last 10 amino acids, preferably lacks at least the last 15 amino acids, more
preferably lacks the 20 amino acids, more preferably lacks the last 26 amino acids and
most preferably lacks the last 40 amino acids. The most preferred polynucldeotides encoding
Core that are suitable far use in the present invention are those that encode a truncated core
containing the amino acids 1-l71,1-165, 1-151. Most preferably the polynucleotide encoding
20 Core that is suitable for use in the present invention is that which encodes a truncated Core
protein between amino acids I -151. One or more consensus mutations as set forth in
example I may be present.
The other non-core HCV polypeptides encoded, by the oligonuclootide vaccines of the
present invention may comprise the full length amino acid sequence or alternatively the
25 polypeptides may be shorter than the full length protein in that they comprise a sufficient
proportion of the full length polynucleotide sequence to enable the expression product of the
shortened gene to generate an immune response which cross reacts with the full length
protein. For example, a polynucleoride of the invention may encode a fragment of a HCV
protein which is a trucated HCV protein in which regions of the original sequence have been
30 deleted, the final fragment comprisng less than 90% of the original full length amino acid
sequence, and maybe less than 70% or less than 50% of the original sequence. Alternatively
speaking, a poiynucleotide which encodes a fragment of at least 3, for example 8-10 amino
6

WO 2004/046175 PCT/EP2003/012793
acids or up to 20, 50, 60, 70, 80, l00, 150 or 200 amino acids in length is considered to fall
within scope of the invention as long as the encoded oligo or polypeptide demonstrates
HCV antigericity. In particular, but not exclusively, this aspect of the invention encompasses
the situation when the polynucleotide encodes a fragment of a complete HCV protein
5 sequence and may represent one or more discrete epitopes of that protein.
In preferred vaccines of the present invention at least one, and preferably all, of the
HCV polypeptides are inactivated by truncation or mutation. For example the helicase and
protease activity of NS3 is preferably reduced or abolished by mutation of the gene,
Preferably NS5B polymetaso activity of the expressed polypeptide is reduced or abolished by
10 mutation. Preferably N54B activity of the expressed polypeptide is reduced of abolished by
mutation Preferably activity of the Core protein of the expressed potypeptide is reduced or
abolished by truncation or mutation. Mutation in this sense could comprise an addition,
deletion, substitution or rearragement event to polynucleotide encoding the polypeptide.
Allernatively the full length sequence may be expressed in two or more separate parts,
15 The functional structure and enzymatic function of the HCV polypeptides NS3 and
NS5B are described in the art.
NS5B has heen described as an ENA-dependent RNA polymerase Qin et al,, 200lt
Hepatology, 33, pp 72S-737; Lohman et at, 2000, Journal of Viral Hepatitis; Lohmann et
al., 1997, Nov., Journal of Virology, 84l6-8428; De Francesco et al, 2000, Seminars in
20 Liver Disease, 20(1), 69-83. The N85B polypeptide has been described as having four
functional motifs A, B, C and D.
Preferably the NS5B polypcptide sequence encoded by polynucleotidc vaccines of the
present invention is mutated to reduce or remove RNA-dependent RNA polymerase activity.
Preferably the polypeptide is mutated to disrupt motif A of NS5B, for example a substitution
25 of the Aspartic acid (D) in position 2639 to Glycine (G); or a substitution of Aspartic acid (D)
2644 to Glycine G). Preferably, the NS5B polypeptide encoded by the vaccine
polynucleotide contains both of these Aspartic acid mutations.
Preferably, the encoded NS5B contains a disruption in. its motif C. For example,
Mutstion of D2737, an invariant aspartic acid residue, to H, N or E leads to the complete
30 inactivation of NS5B.
Preferably the NS5B encoded by the PNA vaccines of the present invention comprise
a motif A mission which may optionally comprorise a motif X mutaion. Preferred motations
7

WO 2004/046175 PCT/EP2003/012793
in motif A include Aspastic add (D) 2639 to Glycine awl aspartic acid (D) 2644 Glycine.
Preferably both mutations are present. Additional further consensus mutations may be
present, as set forth below in example 1.
NS3 has been described as having both protease and belicase activity.The NS3
5 polypeptides encoded by the DNA vaccines of the present invention are preferably mutated to
disrupt both the protease and helicase activities of NS3. It is known that the protease activity
of NS3 is linked to the linked to the “catalytic triad” of H-1083, D-l107 and S-l165. Preferably the NS3
encoded by the vaccines of the present invention comprises a mutation in the Catalytic triad
residues, and most preferably the N33 comprises single point mutation of Serine 1165 to
10 voline (De Francesco, R., Pessi, a and Steinkuhler C.1993. The hepatitis C Virus NS3
proteinase : structure and function of a zinc containing proteinase. Anti- Viral Therapy 3, 1-
18.)
The structure and function of NSE can be represented as:
Protease Helicase
Catalytic triad: Established functional motifs:
H-1083: I. II III IV
D-1107 GK5 DECH TAT QRrGRtGK
S-1165
15
Four critical motifs for the helicase activity of NS3 have been identified , I , II, III and
IV. Preferably the NS3 encoded by the DNA vaccines of the present invention comprise
disruptin mutations to at least one of the these motifs. Most preferably, there is a substitution of
the Aspartic acid 1316 to ghutarnine (Paolini, C, Lahn A, De Francesco R and Gallinari P
20 2000, Mutational htbesse activity of ^S3 bav& been iitantifi$d, \ &, m and
IV. Preferably tbe NS3 encoded by the DNA vaccines of the present invention comprise
disruptive mutalions to at least one of these motifs. Most preferably, there is a substitution of
the Aspaitic acid 1316 to ghitamine (Paolim, C, Labmt A, De Francesco R. and Gatlinari ?
20 2000 Mutations analysis of hepatitis C virus NS3 associated belicase. J.Gen Virol. 81,
1649)- Neither of these most preferred NS3 matations, S1165 or D1316Q, lie within known
or predicted T cell epitopes.
Most preferably the NS3 polypeptide encoded by the DNA vaccines of the present
invention comprise Serine (S) to Valine (V) and an Aspartic acid (D) 1316 to
25 Glutamine (Q) mutation. Additionally one or more of the consensus mutations as set forth in
exaple 1 may be present.
The preferred NS4B polypetide encoded by the polynucleotides of the present
invention contain an N-terminal truncation to remove a region that is hypervariable between
HCV isolates and genotypes, Preferably the NS4B polypetide contains a deletion of between
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WO 2004/046175 PCT/EP2003/012793
30- 100 amino acids from the N-terminus, more preferably between 40-80 amino acids, and
most preferably adeletion of fhe first N-tenriinaJ 48 amino acids (in tbe context of the J4 L6
isolate this corresponds to a. truncation to amino acid 176Q, which is a loss of the first 48
amino acids of NS4B; equivalent truncations in other HCV isolates also from part of the
5 present invention). Additionally, the NS4B sequence may be divided into two or more
fragments and expressed in a potypeptide having the sequence of NS4B arranged in a
different order to that found in the wild-type molecule.
The polynucleotides which are present in the vaccines of tbe present invention may
comprise the natural nucleotidc .sequence as found in the HCV virus however it is preferred
10 that the nucleotide sequence is codon optimised for expression in mammalian cells.
In addition to codon optimisation, it is preferred that the codon usage in the
polynucleotides of tbe present invention encoding HCV Core, NS3, NS4B and NS5B is
altered such that rare codons do not appear in concentrated clusters, and are on the contrary
either relatively evenly spaced throughout the polynucleotide sequence, or are excluded from
15 the codon optimised gene.
The DNA code bas 4 letters {A, T,C and G) and uses these to spell three letter
"codoos" which represent the amino adds of the proteins encoded in. an organism's genes.
The linear sequence of cadon along the DNA molecule is translated into the linear sequence
of amino acids in the protein(s) encoded by those genes. The code is highly degenerate, with
20 61 condons coding for the 20 natural amino acids and 3 codons representing "stop" signlas,
Thus, most amino acids are coded for by more than one codon - in fact several arc coded for
by four or more different codons.
Where mow than one codon is availabl to cods for given amino acid, it has been
observed that the codon usage patterns of organisms are highly nor-random. Different
25 species show a different bias in their codon selection and, furthermore, utilisation of codons
may be markedly different in a single species between genes which are expressed at high and
low levels. This bias is different in viruses, plants, bacteria and mammalian cells, and some
species show a stronger bias away from random codon selection than others. For example,
humans and other mammals are less strongly biased than certain bacteria or viruses. For these-
30 reasons, there is a significant probability that a mammalian gene expressed in E,coli or a viral
gene expressed in mammalian cells will have an inappropriate distribution of colons far
efficient expression. However, a gene with a codon usage pattern suitable for E.coli
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WO 2004/046175 PCT/EP2003/012793
expression may also be efficiently expressed in humans. It is believed that the presence in a
heterologous DNA sequence of dusters of codons which are rardy observed in the host in
which expression is to occur, is predictive of low heterologous expression levels in that host.
There are several examples where changing codons from those which are rare in the
5 host to those which are host-preferred ("codon optimisation") has enhanced heterologous
expression levels, for example the BPV {bovine papilloma virus) late genes I,I and I.2 have
been codon optimised for mammalian condon usage patterns and this has been shown to give
increased expression levels over the wild-type HPV sequences in mammlian (Cos-1) cell
culture (Zhou et. al. j. Virol 1993. 73, 4972-4982). In this work, every BPV codon which
10 occured more thatn twice as frequency in BPV than in mammals (ratio of usage>2), and
most codons with a usage ration of >1.5 were conservatively replaced by the preferentially
used mammalian codon. In WO97/31115, WO97/48370 and WO98/3460 (Merck & Co.
Inc.) codon optimisation of HIV genes or segments thereof has been shown to result in
increased protein expression and improved imnunogcnicity when the codon optimised
15 sequences are used as DNA vaccines in the host mammal for which the optimisation was
tailored. In these documnents, the sequences consist entirely of optimised codons (except
where this would introduce an undesired restriction site; intron splice site etc,} because cash
viral codon is conservativety replaced with the optimal codon for the intended host.
The term "codon usage pattern" refers to the average frequenciese for all codons in the
20 nucleotide sequence, gene or class of genes under discussion (e.g. highly expressed
mammalian genes), Codon usage patterns for mammals, including humans can be found in
the literature (sec e.g. Nakatmura et.aL Nucleic Acids Resesrth 1996,24:214-215).
In the polynutlcotides of the present invention, the todon usagc pattern is preferably
altered from that typical of HCV to mote closely represent the codon bias of the target
25 organism, e.g. E.coli or a mammal, especially a human. The "cedon usage coefficient" or
codon adaptation index (Sharp PM Li WH. Nucleic Acids Research. 15 (3): 1281-95,1987 )
is a measure of how closely the codon usage pattern of a given polynucleotide sequence
resembles that of a target species, The codon frequencies for each of the 61 codons
expressed as the number of occurrences per 1000 codons of five selected class of genes) are
30 normalised for each of hte twenty natural amino acids, so that the value for the most
freqeuntly used codon for each amino acid is set to 1 and the frequencies for the less common
codons are scaled proportionaly to lie between zero and 1. Thus each of the 61 codons is
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WO 2004/046175 PCT/EP2003/012793
assigned a value of 1 or lower for the highly expressed genes of the target species. This is
referred to as the preference value (W). In order to calculate a codon usage coefficient for a
specific polynucleotide, relative to the highly expressed genes of that species, the scaled
value for each codon of the specific polynucleotidc are noted and the geometric mean of all
5 these values is taken (by dividing the sum of the natural logs of these values by the total
number of oodons and take the anti-log). The coefficient will have a value between zero and
1 and the higher the coefficient the more codons in the polynucleotide are frequently used
codons. If a polynucleotide sequence has a codon usage coefficient of 1, all of the codons are
"most frequent" codons for highly- expressed genes of the target species,
10 The present invention provides polynuclcotide sequences which encode HCV Core,
NS3, NS4B or NS5B amino acid sequences, wherein thecodon usage pattern of the
polyuncleotide sequence resembles that of highly expressed mammalian genes. Preferably the
polynucleotide sequence is a DNA sequence. Desirably the codon usage pattern of the
polyuncleotide sequence resembles that of highly expressed human genes.
15 The codon optimise polyunleotide sequence encoding HCV core (1-191) is shown
in Figure 2. The codon optimised polynuleotide sequence encoding HCV NS3, comprising
the SI 165V and D1316Q polypeptide mutation, is shown in Figure 3, The codon optimised
polynucleotide sequence encoding HCV HS4B, comprising the N terminal 1-48 truncation of
the polypeptide, is shown in Figure 4. The codon optimised polynucleotide sequence
20 encoding HCV NCS5B, comprising the D2639G aad D2644G polypeptide mutation, is shown
in Figme5.
Accordingly, there is provided a synthetic gene comprising a plurality of codons
together encoding HCV Core, NS3, NS4Bor NS5B amino acid sequences to form, vaccines
of the present invention wherein the selection of the possible codons used for encoding the
35 amino acid sequence has been changed to resemble the optimal mammalian codon usage such
that the frequency of codon usage inthe synthetic gene more closely resembles that of highly
expressed mammalian genes than that of Hepatitis C virus genes. Preferably rlie codon usage
pattern is substantially the same as that for highly expressed human genes. The "natural"
HCV core, NS3, N34B and NS5B sequences have been analysed for codon usage. The
30 Codon usage coefficient for the HCV prateins are Core (0.4S7), N S3 (0.482), NS4B (0.481)
and HS5B (0.459), A polynunclotide of the present invention will generally have a codon
usage coefficient (as defined above) for highly expressed human genes of greater thatn 0.5,
11

WO 2004/046175 PCT/EP2003/012793
preferably greater than 0.6, most preferably greater than 0.7 but less than 1. Desirably the
polynucleotide will also have a codon usage coefficient for highly expressed E.coli genes of
greater than 0.5, preferably greater than 0.6, most preferably greater than 0.7,
In addition to Codon optimisation the synthetic genes are also mutated so as to
5 exclude the appearance of clusters of rare codons. This can be achieved in one of two ways.
The prefereed way of achieving this is to exclude tare codons from, the gene sequence. One
method to define rare codons would te codons representing particular amino acid and preferably highly expressed genes of the target organism . Alternatively rare codons may be defienes as
10 codons with a relative synonymous codon usage (RSCU) value of highly expressed genes of the target organism. An RSCU value is the observed number of
codons divided by the number expected if all codons for that amino acid were used equall
frequently. An propriate definition of a rare codon would be apparent to a person stalled in
the art.
15 Alternativley the HCV core, NS3, NS4B and NS5B polyuucleotides are optimised to
prevent clustening of rare, non-optimal, codons being present in concentrated areas. The
polynucleotides, therefore, are optimised such that individual rare to codons, such as those with
an RSCU of polynucleotides.
20 The vaccines of the present invention may comprise a vector that directs individual
expression of the HCV polypeptides, alternatively the HCV polynucleotides may be expressed
as one or more fusion proteins.
Preferred vaccines of the present invention comprise tetra-fusions either at the protein
or polynucleotide level, including:
25
HCV combination :
Moore 1 NS3^ NS4B NS5B
HCV combination E:
NS3 INS4B mCore
30 HCV combination C:
12

WO 2004/046175 PCT/EP2003/012793
NS4B NS5B mCore NS3
HCV combination D;
NS5B mCore NS3 NS4B
Other preferred vaccines of the present invention are given below and comprise
5 polynucleotide double and triple fusions being present in different expression cassettes within
the same plasmid, each cassette being under the independent control of a promoter unit (e.g
HGMV IE), (indicated by arrow).
Such dual promoter constructs drive the experssion of the four protein antigens as two
separate proteins {as indicated below) in the same cell.
10

13

WO 2004/046175 PCT/EP2003/012793
For HCV combinations E-L above, it is intended that the terminology used, eg.
(CoreNS3) + (NS4B5B), read to disclose a polynucleotide vector comprising two
expression cassettes each independently controlled by a individual promoter, and in the tase
5 of this example one expression cassette encoding a CoreNS3 double fusion protein and the
other encoding a NS4B-NS5B double fusion protein. Each HCV combination E-L should be
interpreted accordingly.
The above HCV combinations A-L disclose the relative orientations of the HCV
proteins, polyprotein fusions, or ppolynucleotide . It is also specifically disclosed herein that
10 all of the above HCV combinations A-L are also disclosed with each of the preferred
mutation or truncations to remove the activity of the component proteins. For example, the
preferred variants of the combinations A-L (unless otherwise indicated to the contrary)
comprise the nucleotide sequence for Core (1-191 (the complete sequence in its correct
order or divided into two or more fragment to disable biological activity) or preferably Core
15 being present in its truncated forms 1-151 or 1-165 or 1-171); S3 1027-1657 (mutations to
inactivate helicase (Aspartic acid 1316 to Glutamine) and protease (serine 1165 to valine)
activity; NS5B 2420-3010 (mutation at Aspartic acid 2633 to Glycine and Aspartic acid 2644
to Glycine Motif A to inactivate polymerase activity): and NS4B 1712-L972 (optionally
turmeated to 1760-1972 remove N-terminal highly variable fragment)
20 The present invention provides the novel DNA vaccines and polynucleotide as
described above. Also provided by the present invention are analogues of the described
polypeptides and DNA vaccines comprising them.
The terns " analogue" refers to a polynucleotide which encodes the same amino acid.
sequence as another polymeleotide of the present invention but which, through the
25 redundancy of the genetic code, has a different nucleotide sequence whilst maintaining the
same codon usage pattern, for example having the same codon usage coefficient or a codon
usage coefficient within 0.1, preferably within 0.05 of that of the other polynucleotide .
The HCV polynucleotide sequences may be derived from any of the various HCV
genotypes, strains or isolates. HCV isolates can be classified into the following six major
30 genotypes comprising one or more subtypes:HCV l(la lb or lc),HCV 2(2a, 2b or 3c)
HCV 3 (3a, 3b, 10a), HCV 4 (4a), HCV 5 (5a) and HCV 6 (6a, 6b, 7b, 8b 9a and 11a);
Simmonds, J. Gen. Virol., 2001, 693-712. in the context of the present invention each HCV
14

WO 2004/046175 PCT/EP2003/012793
protein may be derived from the polynucleotide sequence of the same HCV genotype or
subtype, or altenatively any combinatiuon of HCV genotype or subtype, and HCV protein
may be used. Preferably, the genss are derived from a type lb genotype such as the infectious
clone J4L.6 (Accession No AF0542478 - see figure 1).
5 Specific strains that have been sequent include HCV-J {Kato et al1990, PNAS,
USA, 87;9724-528) and BK (Takamizawa et al, 1991, J.Virol. 65: 1105-1113).
The polynucleotides according to the invention have utility in the production by
expression of lie encoded proteins, which expession may tate place in vitro, in vivo or ex
vivo. The nucleotides may therefore be involved in recombimant protein) synthesis for
10 example to increase yields, or indeed my find use as therpeutic agents in their own right,
utilised in DNA vaccination techniques. Where the polynucleotides of present invention
are used in the production of the encoded proteins in vitro or ex vivo, cells, for example in
cell culture, will be modified to include the polynucleotide to be expressed. Such cells
include transient, or preferably stable mammalian cell lines. Particular examples of cells
15 which may be modified by insertion of vectors encoding for a polyproteins according to the
invention includes mammalian HEK293T, CHO, HaLa, 293 and COS cells. Preferably the
cell line selected will be one which is not only stable, but also allows for mature
glycosylation and cell surface expression of a polyprotein. Expression may be achieved in
transformed cocytes, A polypeptide may be expressed from a polynucleotide of the present
20 invention, in cells of a transgenic non-human animal, preferably a mouse. A transgenic non-
human animal expressing a polypeptide than a polynucleotide of the invention is included
within the scope of the invention.
The present invention includes expression. vectors that comprise the nucleotide
sequence of the invention. Such expression vectors are routinely constructed in the art of
25 molecular biology and may for example involve the use of plasmid DNA and appropriate
initiators, promoters, enhancers and other elements, inch as for example polyadenylation
signals which njay be necessary, and which are positioned in the correct orientation, in order
to allow for protein expression. Other suitable vectors would be apparent to persons skilled in
the art. By way of furrthar example, in this, regard we refer to Sambrook et al Molecular;
30 Cloning: a Laboratory Manual. 2nd Edition CSH Laboratory Press. (1989).
Preferably, a polynucleotide of the invention, or for use in the invention in a vector, is
operrably linked to a control sequence which is capable of providing for the expression of the
15
WO 2004/046175 PCT/EP2003/012793
coding sequence by the host cell, i.e, the vector is an expression vector. The term "openably
linked refers to a juxtaposition wherein the components described are in a relationship
permitting them to fraction in their intended manner. A regulatory sequence, such as a
promoter, "operably linked" to a coding sequence is positioned in such a way that expression
5 of the coding sequence is achieved under conditions compatible with the regulatory sequence.
An expression cassette is an assembly which is capable of directing the expression of
the sequence or gene of interest. The expression cassette comprises control elements, such as
a promoter which is operably linked to the gene of interest.
The vectors may be, for example, plasmids, artificial chromosomes (e.g , BAC, PAC,
10 YAC), virus or phage vectors provided with. an origin of replication, optionally a promoter
for the expression of the polynucleotide and optionally a regulator of the promoter. The
vectors may contain one or more selectable marker genes, for example an ampicilin or
kanamycin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal
vector. Vedors may be used in vitro, for example for the production of DNA or RNA or
15 used to transfect or transform a host cell, for example, a mammalian host cell e.g. for the
production of protein encoded by the vector. The vectors may also be adapted to be used in
vivo, for example in a method of DNA vaccination or of genes therapy.
Promoters and other expression regulation signals may be selected to be compatible
with the host cell for which expression is designed. For example, mammalian promoters
20 include the metallothionein promoter, which can be induced in response to heavy metals such
as cadmium, and. the β-actin promoter. Viral promoters such, as the SV40 large T antigen.
promoter, human cytomegalovirus (CMV) inimediate early (IE) promoter, rous sarcoma virus
LTR promoter, adenovirus promoter, or an HPV promoter, particularly the HPV upstream
regulatory region (URK) may also be used. All these promoters are well described and
25 readily available in the art.
Examples of suitable viral vectors include herpes simplex viral vectors, vaccinia or
alpha-vims vectors and retrovirus, including lentivirus, adenoviruses and adeno-
associated viruses. Gene transfer techniques using these viruses are known to those skilled in
the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide of
30 the invention into the host genome, although such recombination is not preferred.
Replication-defective adenovirus vectors by contrast remain episomal and therefore allow
transient expression. Vectors capable of driving expression on in insect cells (for example
16

WO 2004/046175 PCT/EP2003/012793
becelovrius vectors), in human cells or in bacteria may be emloyed in order to produce
quantities of the HCV protein encoded by the polynucleotide of the present invention, for
example for use as subumit vaccines or in immunoassays..
In a further aspect, the present invention provides a pharmaceutical composition
5 comprising a polynucleotide sequence as described herein. Preferably the composition
comprises as a DNA vector according to the second aspect of the present invention. In preferred
embodiments the composition comprises a plurality of particles, preferably gold particles,
coated with DNA comprising a vector encoding a polynucleotide sequence which encode an
HCV-amino acid sequence, wherein the codon usage pattern of the polynucleotide sequence
10 resembles that of highly expressed mammalian genes particularly human genes. In
alternative embodiments, the composition comprises a pharmaceutically acceptable excipient
and a DNA vector according to the second aspect of the present invention. The composition
may also include and adjuvant..
DNA vaccines may be delivered by interstitial administration of liquid vaccines into
15 the muscle (WO90/11092) or by mechanism other than intrs-muscular injection. For
example, delivay into the skin takes advantage ol the fact that immune mechanisms are
highly active in tissues that are barriers to infection such as skin and mucous membrances.
Delivery into skin could be via injection, via jet injector (which forces a liquid into the skin,
or underlying tissues including muscles, underpressure) or via particle bombardment, in
20 which the DNA may be coated onto particles of sufficient density to penetrate the epithelium
(US Patent No. 5371015). For example, the nucleotide sequences may be incorporated into a
plasmid which is coated on to gold beads which are then administered under high pressure
into the epidermis, such us, for example, as described in Haynes et al j. Biotechnology 44:
37-42 (19%). Projection of these particles into the skin results in direct transfection of both
25 epidermal cells and epidermal Langerhan cells, Langerhan cells are antigen presenting cells
(APC) which take up the DNA, express the encoded peptides, and process these for display
an cell surface MHC proteins. Transfected Langedhan cells migrate to the lymph, nodes where
they present the display antigen fragments to lymphocytes, evoking an immune response.
Very small amounts of DNA (less than 1μg. often less than 0.5μg) are required to induce an
30 immune response via particle mediated delivery into ski9n and theis contrast with the
miligram quantities of DNA known to be required to generate immune responses subsequent
to direct intramuscular injection.
17

WO 2004/046175 PCT/EP2003/012793
Where the polynucleotide of the present invention find use as therapeutic agents, e.g
in DNA vaccination, the nucleic acid will be administered the mammal e.g. human to be
vaccinated. The nucleic acid, such as RNA or DNA, preferably DNA, is provided in the form
of a vector, such as those described above, which may be expressed in the cells of the
5 mammal. The polynucleotides may be administered by any available technique. For
example, the nucleic acid may be introduced by needle injection, preferably intradermall y,
subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly
into the skin using a nucleic acid delivery such as particle mediated DNA delivery
(PMDD). In this method, men particles (such as gold beads) are coated with a nucletic acid.
10 and are accelerated at speeds sufficient to enable them to penetrate a surface of a recipient
(e.g. akin), for example by means of discharge under high pressure from a projecting device.
(Partices coated with a nucleic acid molecule of the present invention are within the scope of
the present invention, as are delivery devices loaded with such particles). The composition
desirably comprises gold particles having an average diameter of 0.5-5 μn, preferably about 2
15 μn. In preferred embodiments, the coated gold beads are loaded into tubing to serve as
cartridges such that each cartridge contains 0.1-1 mg, preferably 0.5mg gold coated with 0.1
5 μg, preferably about 0.5 μg DNA/cartridge.
According to another aspect of the invention there is provided a host cell comprising a
polynucleotide sequence as described herein. The host cell may be bactrial, e.g. E. colo.
26 mammlain, e.g. human, or may be an insect cell. Mammalian cell comprising a vector
accordiing to the present invention may be cultured cells transfected in vitro or may be
transfected in vivo by administration of the vector to the mammal.
In a further aspect, the present invention provides a method of making a
parmaceutical composition as described above, including the step of altering the codon
25 usage pattern of a wild-type HC V nucleotide sequence, or creating a polynucleotide sequence
synthetically,to produce a sequence having a codon usage pattern resembling that of highly
expressed mammlian genes and encoding a wild-type HCV amino aci sequence or a
mutated HCV amino acid sequence comprising the wild-type sequence with amino acid
changes sufficient to inactivate one or more of the natural functions of the polypeptide.
30 Also provided are the use of a polynucleotids or vaccine as described herein in the
treatment or prophylaxis of an HCV infection,
18

WO 2004/046175 PCT/EP2003/012793
Suitable techniques for introducing the naked polynucleotide or vector into a patient
include topical application with an appropriate vehicle. The nucleic aciti maybe administered..
topically to the skin, or to mucosal surfaces for example by intrsnasal, oral, intravaginal or
intrarectal administration, The naked polynucleotide or vector may be present together with a
5 pharmaceutically acceptable excipient, such asp hosphate buffered saline (PBS), DNA uptake
may be further facilitated by use of facilitating agents such as bupivacaine, either separately
or included in the DNA formulation. Otter methods of administering the nucleic acid directly
to a recipient include ultrasound,, electrical stimulation, electoporation and microseeding
which is described in US-5,697,901.
10 Uptake of nucleic acid constructs may be enhanced by several Known transfection
techniques, for example those including the use of transfection agents. Examples of these
agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and
lipofectants, for examples, lipofectam and trasfectam. The dosage of the nucleic acid to be
administered can be altered. Typically the nucleic acid is administered in an amount in the
15 ra nge of lpg to lmg, preferably lpg to 10μg nucleic acid for particle mediated gene delivery
add 10μg to Img for other routes..
A nucleic acid sequence of the present invention may also be administered by means
of specialised delivery vectors useful in gene therapy. Gene therapy approaches are discussed
for example by Verme et al. Nature 1997, 389:239-242. Both viral and non-viral vector
20 systems can be used. Viral based systems include retroviral, lentiviral, adenoviral adeno-
associated viral, herpes viral, Canarypox nnd vaccinia-viral based systems. Preferred
adenoriral vectors are those derived from non-human primates. In particular Pan 9 (C68) as
described in US patent 60K3716, Pan5, 6 or 7 as described in WO03/046:24.
Non-viral based system include direct administration of nucleic acids, microspher
25 encapsulation technology (pol(lactido-co-glycolide) and, liposome-based systems. Viral and
non-viral delivery systems may be combined where it is desirable to provide booster
injections after an initial vaccination, for example an initial "prime" DNA vaccination using a
non-virall vector such as a plasmid followed by one or more "boost" vaccinations using a viral
vector or non-viral based system. Prime boost protocols may also take advantage of priming
30 with protein in adjuvant and boosting with DNA or a viral vector encoding the polynucleotide
of the invention. Alternatively the protein based vaccine may be used as a booster. It is
19

WO 2004/046175 PCT/EP2003/012793
preferred that ther protein vaccine will contain all the antigens that the DNA/viral vectored
vaccine contain. The protein however, may be presented individually or as a polyprotein.
A nucleic acid sequence of the present invention may also be administrated by means
of transforemd cells. Such cells include cells harvested from a subject. The naked
5 polynucleotide or vector of the present invention can be introduced into such cells in vitro
and the trnsformed cells can later be returned to the subject. The polynucleotide of the
invention may integrate into nucleic acid already present in a cell by homologous
recomination events. A transformd cell may, if desired, be grown up in vitro and one or
more of the resulted cell may be used in the present invention. Cells can be provided at an
appropriate site in patient by known surgical or microsurgical techniques (e.g. grafting.
micro-injection, etc.)
Suitable cells include antigen-presenting cells (APCs), such as dendritic cells,
macrophages, B cells, monocytes and other cells that may be engineered to be efficient
APCs. Such cell may, but need not, be genetically modified to increase the capacity for
15 presenting the antigen, to improve activation and/or to be immunologically comatible with the
receiver (i. e., matched HLA haplotype). APCs my generally be isolated from any of a
variety of biological fluids anf organs, including tumour and peri-turmoural tissue, and may
be autologous, allogeneic, syngeneic or xenogeneic cells.
20 Certain preferred embodiments of the present inventin use dendritic cells or
progenitors thereof as antigen presenting cells, either for transformation in vitro and return to
the patient or as the in vivo target of nucleotide delivered in the vaccine, for example by
particle mediated DNA delivery. Dendritic cells are highly potent APCs (Bancherenu and
Steiman, Nature 392:245-251, 1998). and have been shown to be effective as a physiological
25 adujuvant for eliciting ptophylactiv or therapeuttic antitumour immunity (see Timmerman and
Levy, Ann. Rev. Med. 50:507-529, 1999). In general, dendritic cell may be identified based
on their typical shape (stallate in situ, wth marked cytoplasmic processes (denodrited) visible
in vitro), their ability to take up, process and present antigens with hgih effeciency and their
ability to achivate naive T cell response. Dendritic cell may, of course be engineered to
30 express specific cell-surface receptors or ligands that are not commonly found on dendritic
cell in vivo or ex vivo, for example the antigens(s) encoded in the constructs of the invention,
and such modified dendritic cells are contemplated by tghe present invention
20

WO 2004/046175 PCT/EP2003/012793
Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow,
tumour-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes,, spleen, skin,
umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be
differentiated er vivo by adding a combination of cytokines such as GM-CSF IL-4; IL-13
5 and/or TNF to cultures of monocytes harvested from peripheral blood- Alternatively, 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 combination of GM-CSF,
CL-3, TNF, CD40 ligand, Iipopolysaccharide LPS, flt3 ligand (a cytokine important in the
generation of professional antigen presenting cells, particularly dendritic cells) and/or other
10 compound(s) that induce differentiation, maturation adn proliferation of dentritic cell.
APCs may generally be transfected with a polynucleotide encoding an antigentic HCV
amino acid sequcnce, such as a codon-optimised polynucleotide as envisaged in the present
invention. Such transfection may then be used for therepeutic purpose, as described herein.
such transfected cells may then be used for therapeutic purposes, as described herein.
15 Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell
rnay be administered to a patient, resulting in transfection that occurs in vivo In vivo and ex
vivo transfection of dendritic cells, for example, may generally be performed using any
methods known in the art, such as those described in WO97/24447, or the particle mediated
approach described by Mahvi et al., Immunology and cell Biology 75A56-46O, 1997.
20 The Vaccines and pharmaceutical compositions of the invention may be used in
conjunction with antiviral agents such as α-interferon, preferably PEGylated α-interferon
and a ribavirin Vaccines 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. In general, formulations
25 may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles,
Alternatively, a vaccine or pharmaceutical composition may be stored in a freeze-dried
condition requiring only the addition of a sterile liquid carrier immediately prior to use.
Vaccines comprising nucleotide sequences intended for administration via particle mediated
delivery may be presented as cartridges suitable for use with a compressed gas delivery
30 instrument, in which case the cartridges may consist of hollow tubes the inner surface of
which is coated with particles bearing the vaccine nucleotide sequence; optionally in the
presence- of other pharmeceutically acceptable ingredients.
21

WO 2004/046175 PCT/EP2003/012793
The pharmaceautical compositions of the present invention may include adjuvant
compound, or other substance which may serve to modulate or increase the immune
response induced by fee protein which is encoded by the DNA. These maybe encoded by the
DNA, either separately from or as a fusion with the antigen, or may be included as non-DNA
5 elements of the formulation. Examples of adjuvant-type substances which may be included in
the formulations of the present invention include obiquitim, lysosomal associated membrane
protein (LAMP), hepatitis B virus core antigen, flt3-ligand and other cytokines such as IFN-y
and GMCSF,
Other suitable adjuvants are commercially, available such as, for example, Freund's
10 Incomplete Adjuvant and Complete Adjuvant Difco Laboratories, Detroit, MI); Imiquimod
(3M, St Paul, MN); Resimiquimod (3M, St, Paul, MN); Merk Adjuvant 65 (Merck and
Company, Inc, Rahway, N1); aluminium salts such as aluminium hydroxide gel (alum) or
aluminium phosphate; salts of calcium, iron or zinc; and insolible suspension of acylated
tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides:
15 polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A
Cytokines, such as GM-CFS or interlenkin- 2,-7, or -12 may also be used as adjuvants.
In the formulations of the inventions it is preferred that the adjuvant composition
induces and immune response predominantly of the Th 1 type. Thus the adjuvant may server to
modulate the immunce response generated in response to the DNA encoded antigens from a
20 predominantly Th2 to a prodominantly Th1 type response. High level of the Th1-type cytokines
(e.g IFN TNF, IK-2 and IL-12) tend to favour the induction of cell mediated immune
responses to an administered antigen. Within a preferred embodiment, in which a response is
-CSF or iLberleukin-2, -1, or -12, may also be used as adjuvants.
ID the formulations of the ioventionit is preferred thai the adjuvant composition
induces ao immune response predominantly of the Thl type. Thus (he gdjuvani may serve to
m 20 predominantly Th2 to a prsdomJDantly Th 1 type response. High levels of Th 1 ^ype cytokines
(e.g.t IFN-, TIS3F, EU2 and IL-12) tend to favour the induction af cell mediated imnrune
responses to an administered antigen. Within a preferred embodiment, in which a response is
predominantly Th1-type cytokines. The levels of these cytokines may be readily assessed using
25 standard assays, For a review of the families of cytokines, sec Mosmann anf Coffiman, Ann-
Rev. Immol. 7:145-173, 1989.
According, suitable adjuvants for use in eliciting a predominantly Th1-type
response include, for example, a combination of monophosphory lipid A, preferably 3-de-O-
acylated monophosphoryl lipid A(3D-MPL) together with an aluminium salt. Other know
30 adjuvants which preferentially induce a TH1 type immune response include CpG containing
oligonucleotides. The oligonucleotides are characterised in that CpG dinucleotide is
unmethylated. Such oligonucleotides are well known and are described in, for example


WO 2004/046175 PCT/EP2003/012793
WO96/02555. Immunostimulatory DNA sequences are also described, for example, by Sato
et al,, Science 273:352, 1996. CpG-containing oligonucleotides maybe encoded separately
from the HCV antigens) in the same or a different polynucleotide construct, or may be
immediately adjacent thereto, e.g. as a fusion therewith. Alternatively the CpG-containing
5 oligonucleotides may be administered separately i,e. not as part of the composition which
includes the encoded antigen. CpG oligonucleotides may be used alone or in combination
with other adjuvants. For example, an enhanced system involves the combination of a CpG~
containing oligonucleotides and a saponin derivative particularly the combination of CpG and
QS21 as disclosed in WO 00/09159 and WO 00/62800. Preferably the formulation
10 additionally comprises an oil in water emulsion and/or tocopherol.
Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals
Inc., Framingham, MA), which may be used alone or in combination with other adjuvants.
For example, an enhanced system involves the combination of a monopbosphoryl lipid A and
saporin derivative, such as the combination of QS21 and 3D-MPL as described in WO
15 34700153 or a less reactogenic composition where the QS21 is quenched with cholesterol, as
described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion
and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL and
locopherol in sm oil-in-water emulsion is described in WO 95/17210.
Other preferred adjuvants include Motjtanide ISA 720 (Seppic, France), SAF (Chiron,
20 California, United Slates), ISCOMS (CSi-), MF-59 (Chiron), Detox (Ribi, Hamilton, MT)
RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosarminide 4-phosphates (AGPs).
Where the vaccine includes an adjuvant, the vaccine formulation may be administered
in two parts. For example, the part of the formulation containing the nucleotide construct
which encodes the antigen may be administered first, e.g, by subcutaneous or intramuscular
25 injection, or by intradermal particle-mediated delivery, then the part of the formulation
containing the adjuvant may be administered subsequently, either immediately or after a
suitable time period which will be apparent to lite physician skilled in the vaccines arts.
Udder these circumstances the adjuvant may be administered by the same route as the
antigenic formulation or by an alternate route. In ether embodiments the adjuvant part of the
30 formulation will be administered before the antigenic part. In one emobodiment, the adjuvant
is administered as a topical formulation, applied to skin at the site of particle mediated
23

WO 2004/046175 PCT/EP2003/012793
delivery of &e nucleotid sequences which, encode the antigen (s), either before or after the
particle mediated driver thereof.
Preferably the DNA vaccines of the present invention stimulate an effective immune
response, typically CD4+ snd CD8+ immunity against the HCV antigens . Preferably against
5 a broad range of epitopes. It is preferred in a therapeutic setting that liver fibrosis and/or
inflammation be reduced following vaccination.
As used herein the term comprising is intended to be used in its noo-limiting sense
such. that the presense of other elements is net excluded. However, it is also intended that the
word "cornprsing" could also be undeerstood in its exclusive sense, being commensurat with
10 "consisting" of "consisting". The present invention is illustrated by, but not limited to the
following examples..
Example 1, Mutations introduced into antigen panel:-
15
1). Consensus mutations
A comparison of the full genome sequences of all known HCV isolates was carried
out. Certain positions within the J4L6 polyprotien were identified as unusual/ deviating from
the majority of other HCV isolates, With particular importance were those positions found to
20 deviate from a more consensus residue across related lb-group isolates, extending across
groups la, 2, 3a and others, where one or two alternative amino acid residues otherwise
dominated in the equivalent position. None of the chosen consensus mutations interferes with
a known CD4 or CD8 epitope. Two change within NS 3 actually testore an immunodominant
HLA-B35-restricted CDS epitope [Isolencine (1) 13 65 to Valine (V) and Glycine (G) 1366 to
25 Alanine (A)] .
The first 48 amino acids of NS4B have been removed due to useful variability.
Core
Alanine (A) 52 to Tyreonine (T)
NS3
30 Valine (V0 1040 to Leucine (L)
Leucine (L)( 1106 to Glutamine (Q)
Serine (S) 1124 to Threonine (T)
24

WO 2004/046175 PCT/EP2003/012793
Valine (V) 1179 to Isoleudcie (I)
Threonine (T) 1215 to Serine (S)
Glycine (G) 1289 to Alanime (A)
Serine (S) 1290 to Proline (P)
5 Isoleucine (I) 1365 to Valine (V)
Glyrine{G) 1366 to Alanine (A)
Threonine (T) 1408 to Serine (S)
Praline (P) 1428 to Threonine (T)
Isoleucine (I) 1429 to Serine (S)
10 Isoleucine (I) 163 6 to Threonine (T)
NS4B
Start ORF at Phenylalaminc (F) 1760
15 NS5B
laolcucine (I) 2824 to Valine (V)
Threonine (T) 2392 to Serine (S)
Threonine (T) 2918 to Valine (V)
20 N.B. Numbering is according to position in polyprotein for 14L6 isolate.
Example 2, Construction of plasmid DNA vaccines
Polynucleotide sequences encoding HCV Core NS3, truncated NS4B, and NS5B,
were codon optimised for mammalian codon usags using SynGene 2e software. The codon
25 usage coefficient was improved to greater man 0.7 for each polynucleotide.
The sense and anti-sense strands of each new polynuclcotide sequence, incorporating codon
optimisation, enzymatic knockout mutations, and consensus mutations, were divided into
regions of 40-60 nuclcotides, with a 20 nucleotide overlap. These regions were synthesised
commercially and the polynucleotide generated by an oligo assembly PCR method.
30 The outer forward and reverse PCR primers for each potynucleotide, illustrating
unique restriction endonuclease sites used for cloning, are outlined below:
25

01 -04-2004 EP03127
VB60547P
HCV Cora
Forward primer (SEQ ID NO. 1)
5
GAATTCGCGGCCGCCATGAGCACCAACCCCAAGCCCCAGCGCAAGACCAAGCGGAACACA-3
Nett translation
5 start carton
Reverse primer (SEQ ID NO. 2)
S- GAATTCGGATCCTCATGCGCTAGCGGGGATGGTGAGGCAGCTCAGCAGCGCCAGCAGGA-3'
BamHI Stop
10 codon
HCV MN3
Forwar primar (SEQ ID NO. 3)
5- GAATTCGCGGCCGCCATGGCCCCCATCACCGCCTACAGCCAGCAGACCGGGGGAC-3'
15 Noll translation
slan codon
Reverse primer (SEQ 10 NO. A)
20 BamHi stop
codon
KCV NS4B
25 Forward primer (SEQ ID NO. 5}
5 -GAATTCGCGGCCGCCAGTGTTTTGGGCAAGATATGTGGAACMTTCA-3'
Noll traslatian
start codon
30 Revers primer (SEQ ID NO-6)
5-GAATTCGGATCCTCAGGAAgGGGTGGAGCAGTCCTCGTTGATCCAC-3'
BamHL Stop
35 HCV NS5B
Forward primer (SEQ 10 NO, 7)
5-GAATTCGCGGCCGCCATGTCCATGTCCTACACCTGGACCGGOCGCCCTGA-3'
NoLl TranslatIon
start codon
40 Reverse primer (SEQ 10 NO. 8)
5-GAATCGGATCCTCAGCGGTTGGGGCAGCAGGTAGATGCCGACTCCGACG-3'
BAmHt Stop
codon
45 All polynucleotides, encoding single antigens, were cloned into mammalian expression
vector p7313ie via Not I and BamHl unique cloning sites (see figure 7),
The polyproteins that were encoded were as follows (including mutations and codon
optimisations):
5 0 HCV Core translation (SEQ ID NO. 9):
MSTNPKPQRKTKRNTNRRQDVKFPGGGQIVCKJVYLLPRRGPRLGVRATEKTSERS
QPRGRRQPIPKJUIRPEGRAWAQPGYPWPLYGNEGLGWAGWIXSPRGSRPSWGPTDP
26
AMENDED SHEET

1-04-2004 EP03127
VB60547P
RRRSRNUGKVIDTLTCGFADLMGVTPLVGAPLGGAARALAHGVRVLEDGVNYATGN
LPGCSFSIFLLALISCLTIPASA
5 HCV NS3 translation (SEQ ID NO. 10):
MAPlTAYSQQTRGLLGCIITSLTGRDKNQVEGEVQWSTATQSFLATClNGVCWTVY
HGAGSKTLAGPKGPITQMYTNVBQDLVGWQAPPGARSMTPCTCGSSDLYLVTRHA
DVIPVRRRGDSRGSLLSPRPVSYLKGSVGGPLLCPSGHVVGIFRAAVCTRGVAKAVD
10 FlPVESMETTMRSPVFTDNSSPPAVPQTFQVAHUHAPTGSGKSTKVPAAYAAQGYKV
LVlJIPSVAATLGFGAYWSKJUIGIDPNIRTGVRTITTGAPITYSTYGKFLADGGCSGGA
YDIIICQECHSTDSTTILGTGTV LDQAETAGARLWLATATPPGS VTVPHPNTEEVALSN
NGEIPFYGKAIPIEAIKGGRHLIFCHSKKKCDELAAKLSGLGINAVAYYRGLDVSVIPT
SGDVVVVATDAILHTGFTGDFDSVIDCNTCVTQTVDFSDPTFTIETTTVPQDAVSRS
15 QRRGRTGRGRSGIYRFPVTPGERPSGMFDSSVLCECYDAGCAWYELTPAETSVKLRAY
LNTPGLPVCQDHLEFTWESVFTGLTHIDAHDLSQTKQAGDNFTYLVAYQATVCARAQ
APPPSWPQMQKCLIRLKPTLHGPTPLLYRLGAVQMEVTLTHPITYIMACMSADLEV
VT
20
HCV NS4B translation (SEQ ID NO. 11):
MFWAKHMWNFISGIQYLAGSTLPGNPAIASIMAFASITSPLTTQNTLLFNILGGWV
25 AAQLAPPSAASAFVGAGIAGAAVGSIGLGKVLVDILAGYGAGVAGALVAFKVMSGE
VPSTEDLVNLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASKGNH
VSPTHYVPESDAAARVTQLLSSLTITQLLKRLHQWINEDCSTPC
30 HCV MS5B translation (SEQ ID NO, 12):
MSMSYTWTG ALTTPC AAEESKUINPLSNSLLRHHKMYYATTSRSASLRQKKYTFDR
LQVLDPHYRDVLKEMKAKASTVKAKLLSIEACKLTPPHSAKSKFGYGAKDVRNLS
SRAVNHIRSVWEDLLEDTETPIDTTIMAKSEVFCVQPEKGGRRPARLIVFPDLGVRVC
35 EKMALYDWSTLPQAVMGSSYGFQYSPKQRVEFLVNTWKSKKCPMGFSYGTRCFG
STVTESDIRVEESIVQCCDUKPEARQAIRSLTERLYTGGPLlNSKGQKCGYKRCRASG
VLTTSCGNTLTCYLKATAACRAAKLQDCTMLVNGDDLVVLCESAGTQEDAAALRAF
TEAWTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDASGKKVYYLTRDPTTPLARAA
WETARHTPVNSWLGNIIMYAPTLWARMILAMTHFFSILLAQEQLEKALDCQIYGACYS
40 IEPLDLPQIIERLHGLSAFSLHSYSPGEINRVASCLRKLGVPPLRVWRHRARSVRAKLL
SQGGRAATCGRYLFNWAVRTKLKLTPIPAASQLDLSGWFVAGYSGGDITYHSLSRAR
PRWFPLCLLLLSVGVGIYLLPNR
45 Example 3 Immune response assays
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WO 2004/046175 PCT/EP2003/012793
C57BL or BALB/c mice were immonised with either WI or codon optimised +
mutated versions, of the four HCV antigens expressed individually in a p7313 vector. Miice
were immunised by PMID with a standard dose of I.0 μg/cartridge and boosted and day 21
(boost 1), and again at day 49 (boost 2) Spleen tells were harvested from individual mice
5 and restimulated in EL1SPOT with different HCV antigen preparations. Both IL2 and IFNy
responses were measured, The reagents used to measure immune responses were purified
HCV core, NS3, NS4 and NS5B (genotype lb) proteins from Miktogen, Vaccinia-Core and
Vaccimia NS3-5 (genotype lb in house)
HCV Core
10 C57BL Mice immunised with WT full length (FL-1 -191) or truncated (TR 1-115)
core were Testimulated with HCV cote protein and good responses were observed with.
purified core protein (figure %) ^
HCV M S3
15 Mice were immunised with p7313 WT and codon optimised NS3 usising PMID, Good
responses to MS3 following immunisation and & single boost were demonstrated inC57B1
mice using both NS3 protein and Vacctnia 3-5 to read oat the response by ELISFOT. Both
ILa and IFNy responses were detected. No significantt differences between wild type and
codon optimised (co + m) versions of the constracts were observed in this experiment (figure
20 9). However differences in in vitro expression following transient transfection were observed
betvrean wild type and codon optimised construct. Experiments to compare constructs at
lower DNA dose of in the primary response may reveal differences in the potency of the
plasmids,
25 HCV NS4B
Responses to full length WT p7313 NS4B were observed following PMID
immunisation of BALB/c mice. Both IL2 and IFNy EL1SPOT responses were observed
following in vitro restimulatlan with either NS4E protein and Vaccinia 3-5 (figure 10).
The NS4B protein was truncated at the W-terminus to remove a highly variable
30 region, however expression of this protein could not be detected following in vitro tranfection
studies because the available anti-sera had been raised against the N-terminal region. In order
to confirm expression of this region it was fused with the NS5B protein. Recent experiments
28

WO 2004/046175 PCT/EP2003/012793
have confirmed that immune responses can be detected against the truncated NS4B protein,
cither alone or as a fusion wtth NS5B, using the NS4B protein and NS3-5 vaccinia. Good
responses were observed to WT and codoo optimised NS4B,
5 HCV NSSB
The immune response to NSSB following PMID was investigated following
immunisation withWTand codon optimised (co+ M) sequence. Good responses to HS5B
following immunisation and a single boost were demonstrated in C57BL mice using both
NS3 protein and vaccinia 3-5 to read out the response by ELTSPOT. As with NS3 no
10 differences in the immune response were observed between WT and co +m versions of the
constructs in this experiment (figure 11).
Example 4, Expression qf HCV polyproteins
The four selected HCV antigens Care, NS3, NS4B and NSSB were formatted in
15 p73l3ie ID express 33 a single fusion polyprotein. The antigens were expressed in a different
order in the different constructs as shown below. The construct panel encoding the expression
of single polyproteins was designed so the amino -terminal position was taken by each of the
four antigens in turn, to monitor whether the level of expression was significantly improved
oneduccd more by the presence of one antigen than another in this important position. In
20 addition, two construct were generated in which the Core protein was re-arranged via 2
fragments ie Core 66-l9l>l-65 and 105-151>1-104.
HCV 500
Core NS3 NS4B NS5B
25 HCV 510
NS3 NS4B NS5B Core
HCVHCV 520
NS4B NS5B Core NS3
KCV 530
29

WO 2004/046175 PCT/EP2003/012793
NS5B Core NS3 NS4B
HCV 501
Core (66-l9l)-(1-65) | NS3 NS4B NS5B
HCV 502
Core (105-191) -(1- 104) NS3 NS4B NS5B
A standardised amount of DNA. was transfected into HEK 293T cells using
Lipofectamine 2000 transfection reagent (Invitrogen/Life Technologies}, following the
standard manufactures protocol. Cells were harvested 24 hours past transfection, and
polyacrylamide gel electrophoresis carried out using NuPAGE 4-12% Bis-Tris pre-formed
10 gels with either MOPS or MES ready-made buffers (Invitrogen/Life Technologies}. The
separated proteins were blotted onto FVDF membrane and protein expression monitored
using rabbit antiserum raised against NS5B whole protein. The secondary probe was an anti-
rabbit immunoglobulin antiserum conjugated to horseradish peroxidase (hrp), followed by
chemi-luminescent detection using ECL reagents (Amersham Bioseinces).
15 The results of this expression study ate shown in FIG. 12. The results show that all the
polyproteins are expressed to similar extent although at lower levels than that seen to single
antigen expressing NS5B.The slightly lower molecular weight of HCV500 is due to cleavage
of HCV core from the N-terminal position. HCV502 was not detected in this experiment due
to a cloning error. In a repeat experiment with another clone the level of expression of
20 HCV502 was similar to the other polyproteins.
Example 5, Detection of Immune response to HCV polyproieitis
C57BL mice were immunised by PMID with DNA (1μg) encoding each of the
polyproteins, followed by boosting 3 weeks later as described in example 4. Immune
25 responses were monitored 7 days post boost using ELISPOT or intracellular cytokine
production to the HCV antigens.
ELKPOT assays far T cell responses to HCV gene products
30

WO 2004/046175 PCT/EP2003/012793
Preparation of splenocytes
Spleens were obtained from immunised animals at 7 days post boost- Spleens were
processed by grinding between glass slides to produce a cell suspension. Red blood cells
were lysed by ammonium chloride treatment and debris was removed to leave a fine
5 suspension of splenocytes. Cells were resuspended at a concentration of 4x106 /ml in RPMI
complete media for use in ELISPOT assays where mice had received only a primary
immunisation and 2xl06/ml where mice had been boosted,
EUSPOT assay
10 Plates were coated with 15 μg/ml (in PBS) rat anti mouse IFNy or rat anti mouse IL-2
(Phamingen). Plates were coated overnight at +40C. Before use the plates were washed
three times with PBS. Splenoeytes were added to the plates at 4x 105 cells/well. Recombinant
HCV antigens were obtained from Mitrogcn and used at lμg/ml. Peptide was used in assays
at a final concentration of 1 -10ΜM to measure CD4 or CDS responses. These peptides were
15 obtained from Genemed Synthesis, Total volume in each well was 200μl. Plates containing
antigen stimulated cells were incubated for 16 hours is a humidified 37°C incubator. In some
experiments cells infected with recombinant Vaccinia expressing NS3-5 or Vaccinia Wild
type were used as antigens in EOSPOT assay.
20 Development of ELISPOT assay plates,
Cells were removed from the plates by washing once with water (with. 1 minute soak
to ensure lysis of cells) and three times with PBS. Biotin conjugated rat anti mouse IFN-y or
IL-2 (Phamingen) was added at 1μg/ml in PBS, Plates were incubated with snaking for 2
hours at room temperature. Plates were then washed three times with PBS before addition of
25 Streptavidin alkaline phosphatase (Caltag) at 1 /1000 dilution. Following three washes in PBS
spots were revealed by incubation with BCICP substrate (Biorad) for 15-45 mins. Substrate
was washed off using water and plates were allovred to dry. Spots were enumerated using an
image analysis system
30 Flow cytometry to detect IFNy and 112 production from T cells in response to peptide
stimulation.
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WO 2004/046175 PCT/EP2003/012793
Approximately 3x106 splenocytes were aliquoted per test tube, and spun to pellet.
The supernatant was removed and samples vortexed to break up the pellet. 0.5 μg of anti-
CD28 +0.5μg of anti-CD49d (Pharmingen) were added to each tube, and left to incubate at
room temperature for 10 minutes. 1 ml of medium, was added to appropriate tubes, which
5 contained either medium alone, or medium with HCV antigens. Samples were then
incubated for an hour at 37°C in a heated water bath. 10μg/ml Brefeldin A was added to each
tube and the incubation at 37°C continued for a further 5 hours. The programmed water bath
then returned to 60C, and was maintained at that temperature overnight.
Samples were then stained with anti-mouse CD4-CyChrame (Phamingen) and anti-
10 mouse CD3 biotin (imniunotcch). Samples were washed, and stained with streptavidin-ECD.
Samples were washed and 100μl of Fixative was added from the "Intraprep Petmeabilization
Reagent" kit (Immunotec) for 15 minutes at room temperature. After washing, 100μl of
permaeabilization reagent from the Infcraprep tit was added to each sample with anti-IFN-7-PE
+ antML-^-FTTC Samples were incubated at room temperature for 15 minutes, and washed.
15 Samples, were resuspended. in 0.5ml buffer, and analysed on the Flow Cytometer.
A total of 500,000 cells were collected per sample and subsequently CD4 and CD8
cells were gated to determine the populations of cells secreting IFNy and/or IL-2 is response
to stimulus.
The results show that all the polyproteis encodins Core NS3, NS4B and NS5B in
20 different aiders are able to stimulate immune responses to NS3 (ie HCV 500,510,520,530).
The results are shown in FIG. 13. Responses to NS3 protein were similar between each of the
HCV polyproteins (HCV 500,510,520 and 530), When monitored by IL2 (FIG 13 A.) and
IFNy (FIG 13B) ELISPOT.
The phenotype of the responding cells was analysed in more detail by 1CS. A good
25 CD4+ T cell response was elicited to aa immunodominant NS3 CD4 specific peptide which
was similar between HCV 500, 510, 520, 530.
Table J Frequency of NSS specific CD4 and CDS T ceils producing IPNyfollowing
immunisation with HCV polyroteins.
Construct |nil NS3 protein NS3 CD4 peptide NS3 CD Peptide
32

WO 2004/046175 PCT/EP2003/012793
NS3 single 0.05 0.29 0.24 4.4
HCV 500 0.09 0.27 0.38 5.54
HCV 510 0.1 0.17 0.29 3.95
HCV520 0.1 0.14 0.28 3.32
HCV530 0.07 0.15 0.21 4.89
HCV 501 0.1 0.05 0.08 0.16
IFNyspecific T cell responses were detected following of stimulation of spienocyt sin
presence or absence of antigen for 6 hours, in presence of Brefedin A for last 4hours. IFNg
was detected by gating on CD4 or CDS T celts and staining -with IFNyFJTC.
5 A strong CD 8 response to the immunodominant NS3 specific peptide was also
generated following immunisation with HCV 500,510,520 and 530, reaching frequencies of
between 2.5-6% of CD8+ cells.
Immunisation with HCV 500, 510,520 and 530 also resulted in detection of CD4 and
CDS responses to both NS4B and NS5B antigens, although the CDS responses were weaker
10 to the polyproteins than following immounisation with the single antigen.
Table 2, Frequency of NSSB CD4 or CDS specific T cells producing IFNy following
immunisation with HCV potyproleins.
Plasmid nil NSSB protein NSSB CD4 NSSB CDS peptide
peptide
NS5B single 0.05 0.1 0.26 1.67
HCV 500 0.09 0.14 0.43 0.35
HCV 510 0.11 0.1 0.29 0.11
HCV 520 0.11 0.09 0.18 0.08
HCV 530 0.07 0.06 0.7 0.12
HCV 501 0.1 0.03 0.13 0.09
15 IFNy specific T cell responses were detected following of stimulation of spienocytes in
presence or absence of antigen for 6 hours, in presence of Brefeidin A for last 4hours. IFNg
was detected by gating on CD4 or CDS T cells and staining with IFNyFJTC
33

01-G4-2Q04 EP03127
VB6G547P
Table 3 Frequency of NS4B CD4 or CDS specific T cell producing IFNyfollowing
immunisation with HCV polyproteins.
Plasmid nil NS4B protein NS4BCD4 pep tide NS4B CDS peptide
NS4B 0.05 0.17 0.18 2.04
HCV500 009 0.09 0.1 0.6
KCV510 0.05 0.09 0.09 0.34
HCV520 0.06 0.08 0.05 0.33
HCV 530 0.1 0.17 0.1 0.37
HCV501 0.04 0.09 0.06 0.13
S IFNyspecific T cell responses were detected following of stimulation of splenocytes in
presence or absence of antigen for 6 hours, in presence of Erefeldin A for last 4honrs. IFNg
was delected by gating on CD4 or CDS T cells and staining with IFNyFlTC
The peptides used have following sequence:
Protein Peptides
NS3 C57B1)CD4 FRFGKAIPlEAIKGG (SEQ ID NO. 13)CD8 YRLGAVQNEVILTHP (SEQ ID NO. 14)
NS5 (CS7BI76).CD4 SMSYTWTGALTPCA (SEQ ID NO. 15)CP8 AAALRAFTEAMTRYS (SEQ 10 NO. 16)
NS4B (Batb/c)CD4 IQYLAGLSTLPGNOA (SEQ ID NO. 17)CD8 FWAKHMWNFlSGWY(SEQ ID NO. 18)

10
Recognition of endogenouly processed antigen
In order to determine if PMID immunisation with the HCV polyproteins induced a
response that could recognise endogenously processed antigen, targets cells infected with
Vaccinia recombinant virus expressing NS3-5 were used as stimulators in the ELISPOT
34
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WO 2004/046175 PCT/EP2003/012793
assay. The results show that good IL2 and IFNy ELISPOT responses were, detected following
immunisation with 500,510,520 and 530 (FlG 14).
Immunisation with HCV polyproteins induces functional CTL activity.
5 C57BL mice were immunised with 0.01μg DNA encoding NS3 alone, HCV 500, 510
and 520 Following a prime and a single boost, spleen cells from each group were re-
stimulated in vitro with the NS3 CD8 peptide and IL2 for 5 days, CTL activity was measured
against EL4 cells pulsed with the same peptide. Mice Immunised with all constructs showed
similar levels of killing in this assay.
10 This shows that PMID immunisation with HCV polyproteins can induce fuctional
CDS responses. The results are shown in FIG- 15.
Example 6, Delivery of HCV antigens via dual promoter construct.
15 Dual promoter constructs were generated using the following method. A fragment carrying
expression cassette 1 (including Iowa-length CMV promoter, Exon 1, gene encoding
protein/fusion protein of interest, plus rabbit g Job in poIy-A signal) was excised from its host
vector, qamely p7313ie, by unique restriction andonuclease sites CM and XmnI Xmnl
generates a blunt end at the 3-prime end of the excised fragment.
20 The recipient plasmid vector was p7313ie containing expression cassette 2, This was
prepared by digest with unique restriction endonuclease Sse83K7l followed by incubation
with T4 DMA polymerase to remove the created 3-prime overhangs, resulting in blunt ends
both 5-prime and 3-prime to the linear molecule. This was cut with unique restriction
endonuclease Clal which removes a 259 bp fragment
25 Expression cassette 1 was cloned into p7313 it/Expression cassette 2via Clal/blunt
compatible ends, generating p73Bis/Expression cassette 1 + Expression cassette 2, where
cassette 1 is upstream of cassette 2.
p7313ie Plasmids comprising the following were generated

35

WO 2004/046175 PCT/EP2003/012793

Footnote:
Arrow = Human Cytomegalovirus IE gese promoter (HCMV IE)
NS4B = truncated NS4B containing amino acids 49-260-as outlined above.
5 Core = the Core protein containg amino acids 1-191.
The construct panel shown above is complete and has been monitored for experssion
from transient transfection in 293T cells by Western blot . The results of the Western blot
analysis are shown in FIG. 16 Lane key:
10 1: p73l3ie/Core S. p7313ie/CoreNS3+NS4B5B
2.p7313ie/NS3 9.p73l3ie/ NS4B5B+CoreNS3
3.p73l3ie/NS5B 10.p7313ie/NS3Core+S4B5B
4. p7313ie/CoreNS3 11. p7313ie/HS4B5B+KS3Core
5. p7313ie/NS4B5B 12. p7313ie/Core+NS34B5B
15 6.p7313ie/NS3Core I3.p7313ie/NS34B5B+Core
7. p7313ie/NS34B5B
Each pair of construct caries two independent experession cassettes. It was not
expect that the onder in which the cassettes were insetted into the vector would have an
2D effectt upon the expression from either cassette These results indicate, however, 3 significant
disadvantage to the expression of NS4BSB or NS34B5B fusion proteins when their
respective expression cassettes are positioned downstream of the Core, NS3Core, or
CoreNS3 cassette.
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WO 2004/046175 PCT/EP2003/012793
Expression level is not as positive as for the single antigen constructs, however some
reduction is to be expected due to the significant increase in size (175-228%), translating into
a reduction in copy number of plasmid delivered to the cell by-50% for the same mass of
DNA.
5
In vivo immunogetticity induced by dual promoter constructs,
Three dual promoter constructs were selected for immunogenicirty studies, which
showed the greatest expression of all four antigens. These were p7313ic NS4B/NS5B +
Core/NS3, p73l3ieNS4B/NS5B + NSSCore and p73l3ie NS3/MS4B/NS5B + Core. C57BL
10 mice were immunised with 1μg DNA by PMID end responses detennined 7 days later to the
dominant NS3 CD8 T cell epitope, using ELISPOT for II.2. The results (shown in FIG. 17)
show that responses were observed to ell there dual promoter constructs,coostivicte, after single
immunisation (Splenocytes stimulated with CD$ and CD8 NS3 T cell specific peptides)..
15 Example 7, Deletion mutation of Core.
A. number of genes encodingg the ORF of Core, progressivly deleted by a region
spanning 20 amino acids per time from the 3' end, were generated and fully sequenced.

Core component Nomenclature
1S-I911-191I-17I1-1511-1311-1111-911-711-51 Core Δl5Cere 191Core 17] Core 151Core 131Core 111Core 91Core 71Core 51
FIG, 18 depicts a DNA agarose gel showing the range of gents encoding fragments of
20 Core. These consitucts were tested for expression, combined with their effect upon the
expression level of NS4B5B fusion (p73l3ie/NS4B5B), by co-transfection in 293T cells. The
results are shown in FIG. 19.19. The lanes being loaded as follows:
37

WO 2004/046175 PCT/EP2003/012793
Lane Loaded with (each comprising 0.5 μg DNA)
1 P73l3ie/NS4B5B p7313ie
2 p73l3ie/NS4B5B Core 191
3 p73l3ie/S4B5B Core Δ15
4 p7313ie/NS4B5B Core 171
5 p73l3ie/S4B5B Core 151
6 p7313ie/NS4B5B Core 131
7 p7313ie/NS4B5B Core 111
8 p7315ie/NS4B5B Core 91
9 p73l3ie/NS4B5B Core 71
10 p7313ie/NS4B5B Core 51
The expression of Core 191, Core Δ15, Core171, Core 151, and Corel 31 are clearly detected
when the Western blot is probed with sntj-Cbre, after anti-NS5B detection of the expression
5 of NS4B5B. Further truncated forms of Core are not detected. possibly due to size capture
restrictions of the gel system used.
The result demonstrates a significant reduction in expression level of NS4B5B in the
presence of Corel 91 and Δ15, which recovers with Corel 71, and again with Core 151, despite
the strong expression of both Core species. This observation has been repeated twice with
10 HS4B5B, and once with NS3 and NS5B.
Example S, Effect of Core and Core 151 upon expression of NS3, NS5B. an NS4B-NS5B
fusion and an NS3-NS4B-NS5B triple fusion
15 Experiment I Expression in Trans formal
An experiment was performed to monitor the effect of expression of Core 191 vs Corel 51
upon the Expression of the non-structural antigens, when Core is expressed in trans, or
encoded on a separate plasmid. The experimental protocol was the same as that described in
Example 7. Briefly, 0.5 μg each of two DNA plasmid vectors, outlined in ths table below,
20 were co-transfected into HEK 293T tells using Lipofectamine 2000 transfection reagent in a
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WO 2004/046175 PCT/EP2003/012793
standard protocol (Invitrogsn/Life Technologies). (Transfection and Western blot method as
Example 4)
The results are shown in FIG 20 where the lanes were loaded as described in the
following table, and Western blot analysis was performed to detect the expression of non-
5 structural proteins primarily, using anti-NS3 and anti-NS5B antisera, and that of Core by a
secondary probe of the same blot wilh anti-Core.
Lane Non-structural element Core element
1 NS3 Empty vector
2 NS3 Core 191
3 NS3 Core 151
4 NS5B Empty vector
5 NS5B Core 191
6 NS5B Core 151
7 NS4B-NS5B Empty vector
8 NS4B-NS5B Core 191
9 NS4B-NS5B Core 151
10 NS3-NS4B-NS5B Empty vector
11 NS3-NS4B-NS5B Core 191
12 NS3-NS4B-NS5B Core 151
In all cases, the amount of non-structural protein or fusion (NS3, NS5B, NS4B-5B)
10 when produced in traits with Core 151 has been demonstrated to be significantly increased in
comparison with the Level produced when expressed in trans with Core 191.
Experiment 2 - Expression in Cis format
An experiment was performed to monitor the effect of expression of Corel 91 vs
15 Corel 51 upon the expression of the non-structural antigens, when Core is expressed in cis, or
encoded on the same plasmid in fusion with the non-structural elements. In each case.
Core 151 was substituted for Core 91 in carboxy-terminal fusion with the non-structural
region specified.
39

WO 2004/046175 PCT/EP2003/012793
1μg of DNA plasmid vector, outlined in the table below, was transfected into HEK
293T cells using Lipofectamine 2000 transfection reagent in a standard protocol
(Invitrogen/Life Technologies). (Transfection and Western blot method as Example 4)
The results are shown, in FIG 21, Western blot analysis was, performed to detect the
5 expression of non-structural components primarily, using anti-NS3 aad anti-NS5B antisera,
and that of Core by a secondary probe of the same blot with anti-Core, in Gel A The lanes
were loaded as described in the following table:
Lane Non-Structural element Core element
1 - Corel 191
3 NS5B -
4 NS3 Core 191
5 NS3 Core 151
6 NS5B Core 191
7 NS5B Core 151
Core 191
9 NS4E-NS5B Core 151
10 NS3-NS4B-US5B (HCV 510) Core 191
11 NS3-NS4B-M S5B (BC V 510c) Core 151
10 The results indicate that in a Cis format, where the antigens are in 6 polyprotein
fusion the truncation of Core increase the expression of the fusion protein.
Comparison of effect of Core l 91 and Cora 151 on immune responses to NS3.
C57BL mice were immunised wilh l.5μg x 2 shots total DNA by PMID. The groups
15 immunised included empty vector p7313ie alone, co coasting of gold beads with p73l3ieNS3
p731l3ieNS5B ond p73I3ieCore 131 or p7313ieNS3, p73l3ieNS5B and P73l3ieCorel5l.
Co-coating was used as this should deliver all plasmids to the same cell that should mimic the
in vitro co-transfection studies described above. Immune responses to the dominant CD8 and
CD4 T cell ephopes from NS3 were determined 14 days post primary immunisstion using
20 intracellular cytokine staining to measure IFNy and IL2 antigen -specific responses. The
40

WO 2004/046175 PCT/EP2003/012793
results (shown in FIG. 22) show that both CD4 and CDS NS3 responses were approximately
2 fold higher in the presence of Corel51 compared to Core 191.
In another experiment C57BL mice were immunised with gold beads co-coated
with plasmids expressing p7313ieNS3/NS4B/NS5B triple fusion together with either Core
5 191 or core 151, Animals were further boosted with the same constructs and responses to
NS3 were monitored 7 days post-boost, using intracellular cytokine staining to measure
responses. The results shown in FIG. 23, show that both NS3 antigen specific CD4 and CDS
responses were approximately 2 fold hi,gh in the presence of Core 151 compared to Core 191.
Overall the in vivo studies comparing the response to NS3 in the presence of Core
10 support the in vitro expression data that co-delivery of FL core and non-structural protein can
reduce expression of the non-structural antigens and this reduces the immunogenicity of the
constructs. This effect can at least partiarly be overcome by co-coating with truncated, core
from which the C terminal 40 amino acids have heen removed.
41

04-11-2004 EP0127
Claims
1. A polynucleotide vaccine comprising a polynucleotide sequence that encodes the
HCV Core protein and a polynucleotide sequence that encodes at least one other HCV
protein, wherein the vaccine causes expression of the proteins within, the same cell
wherein the Core protein and the at least one other HCV protein art encoded in more than
one expression cassette characterised in that the expression cassette encoding the Core
ptotetn is in a eis location downstream of the expression cassette which encodes at least
one of the other HCV proteins.
2. A polynucleotide vaccine comprising a polynucleotide sequence that encodes the
HCV Core protein and a polynucleotide sequence that encodes at least one other HCV
protein, wherein the vaccine causes expression of the proteins within the same cell and
the sequence of the polynucleotide sequence encoding the core protein has ben mutated
such that the negaqtive effect of expression of the Core protein, upon the expression of the
said at least one other HCV protein is reduced, wherein the HCV proteins, are encoded by
the polynucleotide vaccine in more than one expression cassettes.
3 A polynucleotide vaccine as claimed in claim 1 or 2, wherein polynucleotide
encodes a core protein that is touncated from the carboxy terminal end in a sufficient.
amount to reduce the inhibitory effect of Core upon the expression of other HCV
proteins.
4 A polynucleotide vaccine as claimed in claim 3 wherein the polynucleotide
encodes the mature form of HCV core protein after the second naturally occuring
cleavage during normal HCV infection.
5. A polynucleotide vaccine as claimed in 3 wherein the truncated core protein has a
deletion of at least the C-terminal 10 amino acids.
6. A polynucleotide vaccine as claimed in claim 3 wherein the truncated core protein
consists of the Core 1-151 sequence.
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04-11-2004 EP03127
7. A polynucleotide vaccine as claimed in claim 3 Wherein the truncated core protein
consists of the Core 1-165 science.
S, A polynucleotide vaccine as claimed in claim I or claim 2 wherein the expression
cassette encoding the Core protein is downstream of an expression cassette that encodes
the NS5B protein.
9. A polynucleotide vaccine as claimed in claim 8 wherein the expression cassette
encoding the Core protein encodes for Core protein in fusion with the HKCV NS3 protein.
10. An HCV vaccine as claimed in claim 8, wherein one expression cassette encodes
the double fusion protein NS3-Core nd the other encoding a NS4B-NS5B double fusion
protein.
11, An HCV vaccins as claimed in claim 10 whereto the Core element of the NS3-
Core double fusion protein is selected from the group consisting of Corel-I71,Core 1-
165 and Core 1-150.
12. An HCV vaccince claimed in claim 11, wherein the Core clement of the NS3-
Core double fusion protein is Core 1-165.
13. A polynucleotide vaccine as claimed in claim 1 or claim 2, wherein the at least
one other HCV protein comprises the HCV proteins: NS3,NS4B and NS5B
14. A polynucleotide vaccine as claimed in claim 13, wherein the polynucleotide
encodes no other HCV protein.
15. A polynucleotide vaccine as claimed in anyone of claims I to 14 wherein the
polynucleotide sequence is in the form of a plasmid.
16. Apolynucleotide vaccine as claimed in any one of claim 1 to 14 wherein the
polynucleotide are codon optimised for expression in mammalian cells.
17 A polynucleotide vaccine comprising a polynucleotide sequence that encodes the
HCV Core protein and a polynucleotide sequence that encodes at least one other HCV
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04-11-2004 EP03127
protein, wherein the vaccine causes expression of the proteins within the same cell and
the sequence of the polynucleotide sequence encoding the core protein has been mutated
or positioned relative tothe polynucleotide sequence encoding the at least one other HCGV
protein such that the negative effect of expression of the Core protein upon the expression
of the said at least oce other HCV protein is reduced, characterised in that the Core
protein encoded by the polynucleotide vaccine consist of one of the following group of
sequences: Core 1-115, Core 1-165 and Core 1-171.
18. A. method of preventing of treating an HCV injection in A mammal comprising
administering a vaccine as claimed in any one of claims1 to 17 to a mammal,
19 A method of vaccination of an individual comprising takinng a polynucleotide
vaccine as claimed in any one of claims 1 to 17, coating polynucleotide onto gold
beads and delviering the gold beads into the skin.
20, Use of a polynucleotide vaccine as claimed in any one of claims 1 to 17 in the
manufacture of a medicament, for the treatment of HCV,
44
AMEMDEO SHEET

The present invention relates to methods and composition useful in the treatment and prevention of Hepatitis C virus
(RCV) infections and the symptoms and diseases associated therewith. In particular the present invention relates to DNA vaccines
that encodes the HCV. Core protein and a polynucleotide sequence that encodes at least one other HCV protein, wherein the vaccine
caused expression of the proteins within th same cell and the sequence of the polynucleotide sequence encoding the core protein
has been mutated or positioned relative to the polynucleotide sequence encoding the at least one other HCV protein such that the
negative effect of expression of the Core protein upon the expression of the said at least one other HCV protein is reduced.

Documents:


Patent Number 216043
Indian Patent Application Number 00724/KOLNP/2005
PG Journal Number 10/2008
Publication Date 07-Mar-2008
Grant Date 06-Mar-2008
Date of Filing 25-Apr-2005
Name of Patentee GLAXO GROUP LIMITED
Applicant Address GLAXO WELLCOME HOUSE, BERKELEY AVENUE, GREENFORD, MIDDLESEX UB6 ONN. GREAT BRITAIN.
Inventors:
# Inventor's Name Inventor's Address
1 HAMBLIN PAUL ANDREW GLAXOSMITHKLINE, GUNNELS WOOD ROAD, STEVENAGE, HERTFORDSHIRE SGI 2NY, GREAT BRITAIN.
2 OGILVIE LOUISE GLAXOSMITHKLINE, GUNNELS WOOD ROAD, STEVENAGE. HERTFORDSHIRE SGI 2NY, GREAT BRITAIN.
3 BRETT SARA GLAXOSMITHKUNE, GUNNELS WOOD ROAD, STEVENAGE, HERTFORDSHIRE SGI 2NY, GREAT BRITAIN.
PCT International Classification Number C07K 14/18, 16/10
PCT International Application Number PCT/EP2003/012793
PCT International Filing date 2003-11-13
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0226722.7 2002-11-15 U.K.