Patent Grant
10350285
This application is a 371 National Phase Application of International Application No. PCT/GB2016/051358 filed May 11, 2016, which claims the benefit of and priority to United Kingdom Application No. GB 1508099.7 filed May 12, 2015; the entire contents of each of the foregoing applications are hereby incorporated by reference herein.
This invention relates to proteins, particularly immunogenic proteins, and associated viral vectors for use in a vaccine against dengue viral infection.
The work leading to this invention has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 281803.
Dengue is the most rapidly spreading mosquito-borne viral disease in the world. Incidence has increased 30-fold in the last 50 years and current WHO estimates indicate that 50 million clinical cases occur each year and approximately 2.5 billion people live in endemic countries (1) More recent estimates, however, have calculated the burden to be more than three times the current published data, reaching astonishing numbers of 390 million infections every year, of which 96 million have clinical manifestations (2) Currently, treatment remains supportive and is based on fluid management and no effective antiviral agents to treat dengue infection are available (3) Moreover, despite various vaccines are being developed, there are no licensed vaccines to prevent dengue infection.
Most DENV (dengue virus) vaccines in clinical development attempt to induce protective antibodies against the four serotypes of dengue. The most advanced is a mixture of four recombinant vectors based on the yellow-fever vaccine Y17. This induces antibodies against the pre-membrane and envelope antigen of all four dengue serotypes and is in late phase III development. However, it is unclear whether in the absence of complete protection, these antibodies will give rise to a risk of enhanced disease through immunological enhancement and this will remain unclear until very large long term follow-up trials are undertaken.
An aim of the present invention is to provide an improved vaccine for dengue viral infection.
According to a first aspect of the invention, there is provided a protein comprising a plurality of conserved peptide sequences, or variants thereof, wherein at least one of the conserved sequences is conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4, and wherein the conserved sequences comprise at least part of a sequence of one or more non-structural proteins of the dengue virus serotypes.
The invention advantageously provides a novel alternative and safer approach to vaccination whereby T cells can be induced to the internal (non-structural) antigens of the dengue virion. This poses no risk of antibody-mediated enhancement and the use of encoded conserved viral segments from the non-structural proteins can provide protection against all serotypes.
The “protein” of the invention may otherwise be termed a “polypeptide”. In one embodiment, the protein is a fusion protein. The protein may not be a wild-type protein. The protein may be synthetic/artificial, for example, the protein may not exist in nature. In one embodiment, the protein may not comprise a complete translation from a complete gene sequence. The protein may consist essentially of conserved peptide sequences. In one embodiment, the protein is a recombinant protein, such as a recombinant fusion protein.
The term “fusion protein” used herein is understood to mean a protein comprising a combination of sequences from different gene products (for example different dengue non-structural proteins) or combinations of sequences from the same gene product (for example a single dengue non-structural protein), wherein the sequences are from distinct/separate regions of the wild-type gene product. For example the fusion protein may comprise combinations of sequences which are normally separated by other sequence segments in wild-type, and the separating sequence(s) have been removed.
The term “conserved peptide sequence” used herein is defined as a sequence that is found in more than one serotype or within variant populations of the same serotype, whereby the sequence is identical or highly similar between the serotypes or variants within a serotype. The required similarity for conserved sequences may be at least 60% identity between serotypes and/or within serotype variants. In another embodiment, the required similarity for conserved sequences may be at least 70%, 80% or 90% identity between serotypes and/or within serotype variants. Conserved peptide sequences may be identified using an algorithm which uses a sliding window-based method. Conserved windows within serotypes (intra-serotype) at the same position across different numbers of serotypes (inter-serotype) are identified if the percentage identity between them is greater than 60% and used to create a normalised consensus sequence. These conserved fragments are referred to as conserved sequences. A window size of greater than 9 amino acids and less than 25 amino acids is used.
At least one of the plurality of conserved peptide sequences may comprise a known epitope or a variant of a known epitope. At least one of the plurality of conserved peptide sequences may comprise a known epitope and a known epitope variant. A known epitope variant may be inserted into at least one of the plurality of conserved peptide sequences. A known epitope and/or known epitope variant may be provided in addition to the plurality of conserved peptide sequences. The protein may not consist essentially of known epitopes and/or known epitope variants (e.g. other conserved sequences are required in the protein that are not published known epitopes or variants thereof). The conserved peptide sequences may not consist of a majority (i.e. over 50%) of known epitopes or known epitope variants (e.g. 50% or more conserved sequences are required in the protein that are not published known epitopes or variants thereof). The plurality of conserved peptide sequences may not consist of more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of known epitopes or known epitope variants. The protein may not consist of more than 1, 2, 3, 4, 5, 6, 7, or 8 known epitopes or known epitope variants. In an embodiment where the protein comprises a known epitope and/or known epitope variant, the protein may comprise a plurality of conserved peptide sequences that do not comprise a known epitope and/or known epitope variant. In an embodiment where the protein comprises a known epitope and/or known epitope variant, at least 90% of the protein sequence comprises conserved peptide sequences that do not comprise a known epitope and/or known epitope variant. In an embodiment where the protein comprises a known epitope and/or known epitope variant, at least 80% of the protein sequence comprises conserved peptide sequences that do not comprise a known epitope and/or known epitope variant. In an embodiment where the protein comprises a known epitope and/or known epitope variant, at least 70% of the protein sequence comprises conserved peptide sequences that do not comprise a known epitope and/or known epitope variant. In an embodiment where the protein comprises a known epitope and/or known epitope variant, at least 60% of the protein sequence comprises conserved peptide sequences that do not comprise a known epitope and/or known epitope variant. In an embodiment where the protein comprises a known epitope and/or known epitope variant, at least 50% of the protein sequence comprises conserved peptide sequences that do not comprise a known epitope and/or known epitope variant.
In one embodiment at least two of the conserved peptide sequences are conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. In another embodiment at least three of the conserved peptide sequences are conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. At least 4 of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. At least 5 of the conserved sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. At least 8 of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. At least 10 of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. At least 12 of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. At least 15 of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4.
In another embodiment, 100% of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. Alternatively, at least 90% of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. Further alternatively, at least 80% of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. Further alternatively, at least 60% of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. Further alternatively, at least 50% of the conserved peptide sequences may be conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4.
The plurality of conserved peptide sequences may be from distinct regions of the non-structural protein sequence relative to each other (i.e. not-naturally concurrent). For example, in the wild-type genotype the conserved sequences may be separated in the wild-type genotypes by variable/non-conserved sequences. The plurality of conserved peptide sequences may not, or may not significantly, overlap with each other. Two or more, or all, of the plurality of conserved peptide sequences may be directly joined together in the protein, for example not comprising any non-conserved residues there between. The protein sequence may not be found in nature. The protein may not comprise non-conserved sequences or residues. The conserved peptide sequences may not be distanced apart by more than 1, 2, 3, 4, or 5 residues in the protein sequence.
The protein may further comprise at least one conserved peptide sequence conserved across 2 or 3 dengue virus serotypes selected from DENV-1, DENV2, DENV-3 and DENV-4, and wherein the conserved peptide sequence comprises at least part of a sequence of a non-structural protein of the Dengue virus serotypes. The protein may further comprise at least one conserved peptide sequence conserved across 2 dengue virus serotypes selected from DENV-1, DENV2, DENV-3 and DENV-4, and wherein the conserved peptide sequence comprises at least part of a sequence of a non-structural protein of the Dengue virus serotypes. The protein may further comprise at least one conserved peptide sequence conserved across 3 dengue virus serotypes selected from DENV-1, DENV2, DENV-3 and DENV-4, and wherein the conserved peptide sequence comprises at least part of a sequence of a non-structural protein of the Dengue virus serotypes. The protein may further comprise a plurality of conserved peptide sequences conserved across 2 and/or 3 dengue virus serotypes selected from DENV-1, DENV2, DENV-3 and DENV-4, and wherein the conserved peptide sequence comprises at least part of a sequence of a non-structural protein of the Dengue virus serotypes.
The protein may comprise:
In one embodiment at least two of the conserved peptide sequences are conserved across 2 or 3 dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4. In another embodiment at least three of the conserved peptide sequences are conserved across 2 or 3 dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4. At least 4 of the conserved peptide sequences may be conserved across 2 or 3 dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4. At least 5 of the conserved sequences may be conserved across 2 or 3 dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4. At least 8 of the conserved peptide sequences may be conserved across 2 or 3 dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4. At least 10 of the conserved peptide sequences may be conserved across 2 or 3 dengue virus serotypes selected from DENV-1, DENV-2, DENV-3 and DENV-4.
The protein may further comprise at least one intra-serotype conserved peptide sequence (i.e. a conserved peptide sequence conserved within a single dengue virus serotype) of DENV-1, DENV2, DENV-3 or DENV-4, and wherein the intra-serotype conserved peptide sequence comprises at least part of a sequence of a non-structural protein of the Dengue virus serotype. The protein may further comprise a plurality of conserved peptide sequences conserved within a single dengue virus serotype selected from DENV-1, DENV2, DENV-3 and DENV-4, and wherein the conserved peptide sequence comprises at least part of a sequence of a non-structural protein of the Dengue virus serotype.
In another embodiment, the protein may comprise:
In one embodiment the protein comprises at least two intra-serotype conserved peptide sequences from DENV-1, DENV2, DENV-3 and/or DENV-4. In another embodiment the protein comprises at least three intra-serotype conserved peptide sequences from DENV-1, DENV2, DENV-3 and/or DENV-4. In another embodiment the protein comprises at least 4, 5, 6, 7 or 8 intra-serotype conserved peptide sequences from DENV-1, DENV2, DENV-3 and/or DENV-4.
The conserved peptide sequences may be selected from the group comprising SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68; SEQ ID NO: 69; SEQ ID NO: 70; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 74; SEQ ID NO: 75; SEQ ID NO: 76; SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81; SEQ ID NO: 82; SEQ ID NO: 83; SEQ ID NO: 84; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90; or combinations thereof.
The conserved peptide sequences conserved across all 4 dengue virus serotypes of DENV-1, DENV2, DENV-3 and DENV-4, may be selected from the group comprising SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; and SEQ ID NO: 22; or combinations thereof.
The conserved peptide sequences conserved across 2 or 3 dengue virus serotypes selected from DENV-1, DENV2, DENV-3 and DENV-4, may be selected from the group comprising SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; and SEQ ID NO: 52; or combinations thereof.
The conserved peptide sequences conserved within a single dengue virus serotype (i.e. intra-serotype) of DENV-1, DENV2, DENV-3 or DENV-4, may be selected from the group comprising SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68; SEQ ID NO: 69; SEQ ID NO: 70; and SEQ ID NO: 71; or combinations thereof.
The protein may further comprise at least one sequence providing an epitope of a known immunogenic epitope of Dengue virus. Additionally or alternatively, the protein may comprise variants of known epitopes (also referred to as a “known epitope variant”). A known epitope variant may differ from the known epitope by one amino acid residue. Alternatively, a variant of a known epitope may differ from the known epitope by two amino acid residues. Epitope variants are identified as being present in the same residue location with one or more amino acid change from the epitope sources (4-6). The known epitope or epitope variant may comprise at least part of a conserved peptide sequence between two or more of DENV-1, DENV2, DENV-3 and DENV-4. In an embodiment where a conserved peptide sequence comprises a known epitope or a known epitope variant, the known epitope or known epitope variant may be substituted with any other known epitope variant, for example known epitope variants described herein.
The known immunogenic epitope, or variant thereof, of Dengue virus may comprise or consist of a sequence selected from any of the group comprising SEQ ID NO: 91; SEQ ID NO: 92; SEQ ID NO: 93; SEQ ID NO: 94; SEQ ID NO: 95; SEQ ID NO: 96; SEQ ID NO: 97; SEQ ID NO: 98; SEQ ID NO: 99; SEQ ID NO: 100; SEQ ID NO: 101; SEQ ID NO: 102; SEQ ID NO: 103; SEQ ID NO: 104; SEQ ID NO: 105; SEQ ID NO: 106; SEQ ID NO: 107; SEQ ID NO: 108; SEQ ID NO: 109; SEQ ID NO: 110; SEQ ID NO: 111; SEQ ID NO: 112; SEQ ID NO: 113; and SEQ ID NO: 114; or combinations thereof.
Known epitopes or known epitope variants may comprise epitopes experimentally proven by Weiskopf et al4 or from the IEDB Database5,6. A known epitope or known epitope variant may comprise a sequence that is conserved across all four serotypes of DENV-1, DENV-2, DENV-3, and DENV-4. Alternatively, known epitopes may comprise a sequence that is conserved across 2 or 3 serotypes of DENV-1, DENV-2, DENV-3, and DENV-4. The known epitope variant may comprise a sequence that is conserved across all, 2 or 3 serotypes of DENV-1, DENV-2, DENV-3, and DENV-4, with one or two residue changes to the known epitope.
In another embodiment, the protein may comprise:
In an embodiment comprising a protein with at least one known epitope variant or a plurality of known epitope variants, the conserved peptide sequences may be selected from the group comprising SEQ ID NO: 73; SEQ ID NO: 74; SEQ ID NO: 75; SEQ ID NO: 76; SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81; SEQ ID NO: 82; SEQ ID NO: 83; SEQ ID NO: 84; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90; or combinations thereof.
The known epitope(s) may be part of the above conserved peptide sequences. The known epitope variant(s) may be insertions into the conserved peptide sequences.
The protein may comprise or consist of the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 23, SEQ ID NO: 53, or SEQ ID NO: 72; or variants thereof. In one embodiment, the protein may comprise or consist of the sequence of SEQ ID NO: 1. In another embodiment, the protein may comprise or consist of the sequence of SEQ ID NO: 2. In one embodiment, the protein may comprise or consist of the sequence of SEQ ID NO: 3. In one embodiment, the protein may comprise or consist of the sequence of SEQ ID NO: 23. In one embodiment, the protein may comprise or consist of the sequence of SEQ ID NO: 53. In one embodiment, the protein may comprise or consist of the sequence of SEQ ID NO: 72.
Variants of the protein may comprise or consist of a sequence having at least 80% identity with the protein of the invention, for example any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 23, SEQ ID NO: 53, or SEQ ID NO: 72. Alternatively, variants of the protein may comprise or consist of a sequence having at least 85% identity with the protein of the invention. Variants of the protein may comprise or consist of a sequence having at least 90% identity with the conserved sequence. Variants of the protein may comprise or consist of a sequence having at least 95% identity with the protein of the invention. Variants of the protein may comprise or consist of a sequence having at least 98% identity with the protein of the invention. Variants of the protein may comprise or consist of a sequence having at least 99% identity with the protein of the invention.
Variants of conserved peptide sequences may comprise or consist of a sequence having at least 80% identity with the conserved peptide sequence. Alternatively, variants of conserved peptide sequences may comprise or consist of a sequence having at least 85% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a sequence having at least 90% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a sequence having at least 95% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a sequence having at least 98% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a sequence having at least 99% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a truncated sequence of the conserved peptide sequences. For example the sequences of SEQ ID NOs 4 to 22, 24 to 52, 54 to 71, or 73 to 90 herein may be truncated and still provide immunogenicity in the protein. The truncated sequence may comprise at least five amino acids of the sequences of SEQ ID NOs 4 to 22, 24 to 52, 58 to 71, or 73 to 90. Alternatively, the truncated sequence may comprise at least six amino acids of the sequences of SEQ ID NOs 4 to 22, 24 to 52, 54 to 71, or 73 to 90. Alternatively, the truncated sequence may comprise at least seven amino acids of the sequences of SEQ ID NOs 4 to 22, 24 to 52, 54 to 71, or 73 to 90. Alternatively, the truncated sequence may comprise at least eight amino acids of the sequences of SEQ ID NOs 4 to 22, 24 to 52, 54 to 71, or 73 to 90.
Reference to sequence “identity” used herein may refer to the percentage identity between two aligned sequences using standard NCBI BLASTp parameters (http://blast.ncbi.nlm.nih.gov).
The plurality of conserved peptide sequences may comprise 5 or more conserved sequences. The plurality of conserved peptide sequences may comprise 8 or more conserved peptide sequences. The plurality of conserved sequences may comprise 10 or more conserved peptide sequences. The plurality of conserved peptide sequences may comprise 15 or more conserved peptide sequences. In one embodiment, the plurality of conserved peptide sequences consists of about 15 conserved peptide sequences.
At least one conserved peptide sequence may be at least about 15 amino acids in length. At least one conserved peptide sequence may be at least about 20 amino acids in length. At least one conserved peptide sequence may be no more than about 23 amino acids in length. At least one conserved peptide sequence may be no more than about 30 amino acids in length. The conserved peptide sequences may be an average length of about 18 amino acids in a population of conserved peptide sequences. The conserved peptide sequences may be an average length of between about 16 and about 20 amino acids in a population of conserved peptide sequences. The conserved peptide sequences may be an average length of between about 15 and about 23 amino acids in a population of conserved peptide sequences. The conserved peptide sequences may be an average length of between about 10 and about 30 amino acids in a population of conserved peptide sequences. The conserved peptide sequences may be an average length of between about 10 and about 40 amino acids in a population of conserved peptide sequences.
The non-structural (NS) protein may comprise NS1. The non-structural (NS) protein may comprise NS2. The non-structural (NS) protein may comprise NS3. The non-structural (NS) protein may comprise NS4. The non-structural (NS) protein may comprise NS5. The non-structural (NS) protein may be selected from NS1, NS2, NS3, NS4, or NS5. In one embodiment, the non-structural protein is selected from NS1, NS3, NS4, or NS5. In another embodiment, the non-structural protein is selected from NS3, NS4, or NS5. The peptide may comprise conserved sequences from non-structural proteins NS3, NS4, and NS5. The peptide may comprise conserved sequences from non-structural proteins NS1, NS3, NS4, and NS5. The conserved sequences may comprise sequences from non-structural proteins NS1, NS2, NS3, NS4, or NS5; or combinations thereof. The conserved sequences may comprise sequences from non-structural proteins NS1, NS3, NS4, or NS5; or combinations thereof. The conserved sequences may comprise sequences from non-structural proteins NS3, NS4, or NS5; or combinations thereof. The conserved sequence of NS1 may comprise SEQ ID NO: 117.
In one embodiment the protein is an isolated protein. In another embodiment, the protein may be encoded in nucleic acid or in a viral vector.
The protein may be immunogenic. The protein may be immunogenic in a mammal. The mammal may be human. The immune response may be a protective immune response. The protein may be capable of activating T-cell and antibody mediated immunity in a subject. The protein may be capable of activating T-cell mediated immunity in a subject. The protein may be capable of activating antibody-mediated immunity in a subject. In one embodiment the protein may comprise an NS1 sequence for inducing an antibody response in a subject. In one embodiment the protein may comprise an NS1, NS3, NS4 and NS5 sequence for inducing an antibody and T-cell response in a subject.
Combinations of proteins of the invention may be provided as a vaccine. For example, a prime and/or boost vaccine formulation may comprise nucleic acid or viral vector encoding two or more proteins of the invention.
The protein may be used in a vaccine in combination with another therapeutically or prophylactically active ingredient. The protein may be used in a vaccine in combination with an adjuvant.
According to another aspect of the invention there is provided a protein comprising the sequence of SEQ ID NO: 1; SEQ ID NO: 2; or SEQ ID NO: 3.
According to another aspect of the invention there is provided a protein comprising the sequence of SEQ ID NO: 23.
According to another aspect of the invention there is provided a protein comprising the sequence of SEQ ID NO: 53.
According to another aspect of the invention there is provided a protein comprising the sequence of SEQ ID NO: 72.
The protein may be provided in a pharmaceutically acceptable carrier.
According to another aspect of the invention there is provided a nucleic acid comprising a sequence encoding a protein according to the invention herein.
The nucleic acid may be a plasmid vector for vaccination. The nucleic acid may comprise viral vector sequences.
According to another aspect of the invention there is provided a viral vector comprising the nucleic acid according to the invention herein.
The viral vector may comprise a virus. The viral vector may comprise an adenovirus, such as a simian adenovirus. The viral vector may comprise an adenovirus when used in a prime vaccine of a prime boost regime. The viral vector may comprise ChAdOx 1 (a group E simian adenovirus, like the AdCh63 vector used safely in malaria trials). The viral vector may comprise AdCh63. The viral vector may comprise AdC3 or AdH6. The viral vector may be a human serotype. The viral vector may comprise Modified Vaccinia Ankara (MVA). The viral vector may comprise MVA when used as a vaccine boost in a prime boost regime. The viral vector may comprise Adeno-associated virus (AAV) or lentivirus. The viral vector may be an attenuated viral vector. The protein sequence of the invention may be cloned into any suitable viral vector that is known to elicit good immune response. Suitable viral vectors have been described in Dicks et al (Vaccine. 2015 Feb. 25; 33(9):1121-8. doi: 10.1016/j.vaccine.2015.01.042. Epub 2015 Jan. 25), Antrobus et al (Mol Ther. 2014 March; 22(3):668-74. doi: 10.1038/mt.2013.284. Epub 2013 Dec. 30), and (Warimwe et al. (Virol J. 2013 Dec. 5; 10:349. doi: 10.1186/1743-422X-10-349), which are incorporated herein by reference.
The viral vector may comprise or consist of a nucleic acid sequence selected from the group comprising SEQ ID NO: 118; SEQ ID NO: 119; SEQ ID NO: 120; SEQ ID NO: 121; SEQ ID NO: 122; SEQ ID NO: 123 SEQ ID NO: 124; and SEQ ID NO: 125.
According to another aspect of the invention there is provided a virus-like particle comprising the protein in accordance with the invention herein.
It is understood that a virus-like particle may provide viral structural proteins, such as envelope or capsid, but does not contain virus genetic material.
According to another aspect of the invention there is provided a composition comprising one or more of:
The composition may be immunogenic, for example in a mammal, such as a human. The composition may comprise a pharmaceutically acceptable carrier. The composition may be a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be for use in the prophylaxis or treatment of dengue viral infection.
According to another aspect of the invention there is provided a method of treatment or prophylaxis of dengue viral infection comprising the administration of:
The method of treatment or prophylaxis of dengue viral infection may be a method of vaccination.
According to another aspect of the invention there is provided an agent for use in the prophylaxis or treatment of dengue viral infection, the agent comprising or consisting of:
According to another aspect of the invention there is provided the protein according to the invention; the composition according to the invention; the nucleic acid according to the invention; the virus-like particle according to the invention; or the viral vector according to the invention; for use in, or as, a vaccine.
According to another aspect of the invention there is provided a vaccine comprising the protein of the invention comprising or consisting of:
The vaccine may be a prime vaccine. The vaccine may be a boost vaccine. Where a boost vaccine is provided following a prime vaccine, the protein may be different. For example, the protein may comprise a re-ordered sequence of conserved peptide sequences. The conserved peptide sequences may be identical, but the order in which they are provided in the protein may be changed.
Advantageously, the re-ordering of the conserved peptide sequences of the protein between prime and boost vaccines can avoid the provision of “false” epitopes formed across junctions of one conserved peptide sequence with another conserved peptide sequence. i.e. the same junction may not occur in the re-ordered protein.
According to another aspect of the invention, there is provided a protein according to the invention for use in, or as, a vaccine.
According to another aspect of the invention, there is provided a prime boost vaccination kit comprising
The prime and boost vaccinations may be different. The prime and boost vaccination may differ in the protein sequence. The prime and boost vaccination may comprise different viral vectors.
The term “immunogenic”, when applied to the protein or composition of the present invention means capable of eliciting an immune response in a human or animal body. The immune response may be protective.
The term “isolated”, when applied to the protein of the present invention means a protein: (i) encoded by nucleic acids using recombinant DNA methods; or (ii); synthesized by, for example, chemical synthetic methods; or (iii) separated from naturally-occurring biological materials, and then purified using protein analytical procedures; or (iv) associated with chemical moieties (e.g. peptides, carbohydrates, fatty acids, and the like) other than those associated with the antigenic peptide in its naturally-occurring state; or (v) that do not occur in nature. An isolated protein of the invention includes a protein expressed from a nucleotide sequence encoding the protein, or from a recombinant vector containing a nucleotide sequence encoding the protein. An isolated protein of the invention may include a protein expressed from a virus-like particle.
The term “protective” means prevention of a disease, a reduced risk of disease infection, transmission and/or progression, reduced severity of disease, a cure of a condition or disease, an alleviation of symptoms, or a reduction in severity of a disease or disease symptoms.
The term “prophylaxis” means prevention of or protective treatment for a disease. The prophylaxis may include a reduced risk of disease infection, transmission and/or progression, or reduced severity of disease.
The term “treatment”, means a cure of a condition or disease, an alleviation of symptoms, or a reduction in severity of a disease or disease symptoms.
According to another aspect of the invention, there is provided a composition comprising a peptide comprising at least part of an NS1 sequence which is conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4, and a pharmaceutically acceptable carrier.
The peptide comprising at least part of an NS1 sequence may comprise SEQ ID NO: 116. The peptide comprising at least part of an NS1 sequence may comprise or consist of SEQ ID NO: 117. The peptide comprising at least part of an NS1 sequence may comprise or consist of the NS1 (3-ladder.
The composition may not comprise sequences from NS2, NS3, NS4 and/or NS5 of dengue virus. The composition may not comprise wild-type dengue virus. The composition may not comprise structural dengue virus protein sequence.
According to another aspect of the invention, there is provided a peptide comprising at least part of an NS1 sequence which is conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4, for use as a vaccine.
The use may be with a pharmaceutically acceptable carrier. Additionally or alternatively, the use may be with an adjuvant.
According to another aspect of the invention, there is provided a virus-like particle comprising a peptide comprising at least part of an NS1 sequence which is conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4, and a pharmaceutically acceptable carrier.
The virus-like particle may not comprise sequences from NS2, NS3, NS4 and/or NS5 of dengue virus. The virus-like particle may not comprise wild-type dengue virus. The virus-like particle may not comprise non-conserved dengue virus peptide sequences. The virus-like particle may not comprise structural protein sequence of dengue virus.
According to another aspect of the invention, there is provided a nucleic acid encoding essentially or at least a peptide comprising at least part of an NS1 sequence which is conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4.
The nucleic acid encoding a peptide comprising at least part of an NS1 sequence may further encode non-dengue viral structural proteins.
According to another aspect of the invention, there is provided a viral vector encoding a peptide comprising at least part of an NS1 sequence which is conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4.
The viral vector or nucleic acid may be provided in a composition, wherein composition may comprise a pharmaceutically acceptable carrier. The viral vector or nucleic acid may not encode NS2, NS3, NS4 and/or NS5 of dengue virus. The viral vector or nucleic acid may not encode wild-type dengue virus. The viral vector or nucleic acid may not encode structural protein sequence of dengue virus. The viral vector or nucleic acid may not encode non-conserved protein/peptide sequence of dengue virus. The viral vector or nucleic acid may encode a peptide sequence comprising or consisting of SEQ ID NO: 116. The viral vector may encode a peptide sequence comprising or consisting of SEQ ID NO: 117. The viral vector or nucleic acid may comprise at least part of SEQ ID NO: 115. The viral vector or nucleic acid may encode the NS1 (3-ladder.
According to another aspect of the invention, there is provided a composition comprising or consisting of a peptide comprising at least part of an NS1 sequence which is conserved across all four dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4, for use as a vaccine.
The peptide comprising at least part of an NS1 sequence may comprise or consist of SEQ ID NO: 116. The peptide comprising at least part of an NS1 sequence may comprise or consist of SEQ ID NO: 117. The peptide comprising at least part of an NS1 sequence may comprise or consist of the NS1 (3-ladder.
According to another aspect of the invention, there is provided an isolated NS1 peptide sequence or a non-dengue viral vector encoding an NS1 sequence, for use as a vaccine. The NS1 sequence may consist of SEQ ID NO: 116 or SEQ ID NO: 117. The NS1 sequence may comprise or consist of the NS1 (3-ladder.
The above mentioned peptide or compositions of the peptide comprising at least part of an NS1 sequence (or nucleic acid or viral vectors encoding such as peptide) may also be applicable for use in the methods of treatment for the prevention or treatment of dengue virus infection.
The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention.
Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.
The invention herein uses a novel alternative and safer approach to vaccination using non-replicating vectors to induce T cells to the internal (non-structural) antigens of the dengue virus. The T cell-based vaccine against dengue should protect against all dengue serotypes and avoid the risk of deleterious antibody-mediated enhancement. A novel insert has been developed, which comprises the most conserved segments of the internal NS3-NS5 genes and this is expressed in the simian adenoviral vector ChAdOxl (a group E simian adenovirus, like the AdCh63 vector used safely in malaria trials) and in the established boosting vector MVA. Results of Immunogenicity trials in mice indicate induction of good frequencies of T cells against the dengue conserved antigen.
For the vaccine design, a bioinformatics tool was developed to assess variability and conservation of DNA or protein sequences of variable pathogens in order to identify the most conserved sequences of DENV and thus design a universal dengue vaccine. Using this data functional and immunological data such as known epitopes was incorporated to create 4 vaccine candidates. One based purely on regions conserved in all 4 serotypes (NSALL1); a second which consists of a mosaic using consensus sequences from the structural proteins that are conserved in different numbers of serotypes providing a greater coverage (NSALL2); a third consists of a mosaic using consensus sequences from the structural proteins that are conserved in different numbers of serotypes and intra-serotype conserved sequences (NSALL3). Finally to try and further promote a protective CD8 response epitopes strings containing all know epitope variants were added to NSALL3 to form NSALL4.
An in house developed engine (Rev1.0) was used to identify conserved sequences using a specific criteria. A sliding window based approach with a sequence normalisation method was used to identify windows conserved within each serotype (intra-serotype). A conserved window was classed as having a window conservation value within one quartile of the overall conservation of the whole sequence. Subsequently conservation between serotypes (inter-serotype) was assessed by, identifying windows between serotypes at the same position and conserved (i.e. within their respective serotype first quartiles) and sharing a consensus identity of greater than 60%.
The invention involved the creation of four arrays of Dengue-conserved sequences retrieved by the bioinformatics analysis. Those sequences were synthesised (Geneart™) and cloned into destination plasmid vectors: pMono 2489 and MVAp434.
To produce the pMono2489-dengue vectors, both Geneart and Empty-pMono2489 plasmids were expanded and digested with Kpnl and Notl restriction enzymes. To produce MVAp434-dengue vectors, both pMono-Dengue and Empty-MVA-p434 plasmids were expanded and digested with Kpnl and Xhol restriction enzymes. Size-specific DNA inserts and DNA backbone plasmids were purified and ligated using T7 DNA Ligase (New England Biolabs). Restriction assays (
Development of Adenoviral Vectors.
To generate the adenoviral vectored vaccine, Attl sites in pMono-containing dengue sequences and AttR sites in the adenoviral plasmid were recombined by using the Gateway LR Clonase (Life Technologies) and verified by Polymerase Chain Reaction (PCR) assay (
The resulting plasmids were further processed by the Jenner Institute Vector Core Facility to generate, expand and purify the adenoviral and MVA vaccines. In total, 4 adenoviral and 4 MVA vaccines were developed.
Prime-Boost Regimen of DNA Vaccines Containing Highly Conserved Sequences from Dengue Virus and Assessing Immunogenicity of the Vaccines by Ex Vivo IFN-γ ELISPOT Assay.
To confirm immunogenicity and to obtain preliminary results in mouse models, DNA vaccines containing highly conserved sequences from dengue virus were generated. Plasmids pMono-expressing dengue antigens were expanded and DNA purified by using the Endo-free DNA isolation Mega prep kit (Qiagen). Four groups of 6 mice were primed and boost (day 0 and day 16, respectively) with either pMono-1NSALL, pMono-2NSALL, pmono 3NSALL or pmono-Empty vaccines (
Ex vivo IFN-gamma Enzyme-Linked ImmunoSpot (ELISPOT) was utilised in order to assess cellular responses in blood drawn from previously vaccinated mice. ELISPOTs were performed two weeks after prime and two weeks after boost.
Based on the sequences retrieved by the software, a set of 180 individual 20-mer peptides overlapping by 10 were designed for T-cell stimulation and sent for synthesis (Pepscan). Lyophilised peptides were solubilised in DMSO to a stock concentration of 100 mg/ml. Peptide pools were prepared to encompass NS3 (Pool 1), NS4A-B (Pool 2) and NS5 (Pool 3) proteins, respectively. Pool peptides sequences are provided at Appendix 1 below together with an indication of which NS protein the pool peptide matches.
Immunisation and tissue collection were performed following licensed procedures for animal handling. Groups of 6 outbred CD1 mice were immunised intramuscularly with 100 ug/mouse of the DNA vaccines diluted in sterile PBS. A control, mock-vaccinated group received an empty pMono plasmid vector at a similar dose of 100 ug/mouse. At 15 days after prime immunisation and at 15 days after boost immunisation, blood was taken by tail puncture. Peripheral Blood Mononuclear Cells (PBMCs) were isolated and cultured in the presence of peptide pools in ELISPOT plates previously incubated with an anti-IFN-gamma antibody (MAbTech). Splenocytes, prepared from naïve mice, were co-cultured at 2.5×10^5 cells/well.
Peptide pool stimulation (10 ug/ml of each peptide) was performed by duplicate for each mouse. Positive controls for stimulation were included (Ionomycin, PMA or both). Upon development, plates were scanned and number of spots forming cells (SFC) per million of PBMCs were calculated. Background of wells without peptides was subtracted.
Preliminary results indicate that the construct is immunogenic in CD1 mice upon a single vaccination with the DNA vaccine expressing the conserved Dengue antigen sequences. For example,
Having demonstrated the ability of the DNA vaccines to elicit specific immune responses against the conserved regions; novel vaccine vectors based on Adenovirus (ChadOX1) and Modified Virus Ankara were made to express the sets of highly conserved dengue regions. A preclinical assessment was done in CD1 mice with Prime and Boost Regimes with different DNA and/or viral vector combinations (
Alternatively, an embodiment of only a beta-ladder domain of the NS1 that contains a highly conserved region between all 4 dengue serotypes has been synthesized and tested as a DNA vaccine. DNA vaccine coding the NS1 (3-ladder, either in a prime or a prime boost regime, elicited specific immune responses (
Peptides used in the ELISPOT assay are shown in Appendix 2.
In summary, the results provide preliminary evidence that the Dengue sequences contained in DNA vaccines are capable to enter cells, transcribe and translate into proteins. Importantly, the ELISPOT analysis demonstrates the viability of the DNA vaccines to induce specific immune responses. Those responses were greatly increased by the use of adenoviral and/or MVA vectors containing the same dengue sequences either as priming or boosting agents.
Vaccine Peptide Sequences
The sequences below are annotated with ^ and/or *. ^ represents a junction between two conserved sequences. * represents a known epitope insert.
NSALL1 Sequence.
Contains fragments that are conserved in all 4 serotypes, *Sequence* underlined=epitope identified from the EIDB5,6 or Weiskopf4 sets (See table 1 and 2) and their variant which was manually added. ^=start/end of fragment.
Conserved Peptide Sequences of NSALL1
Optional Conserved Sequence of NSALL1 in Addition to, or Alternatively to SEQ ID NO: 4
NSALL2 Fragments.
Contains Fragments Conserved in 2, 3 or 4 Serotypes. ^=Start/End of Fragment.
Conserved Peptide Sequences of NSALL2
NSALL3 Fragments.
Contains fragments conserved in 1, 2, 3 or all 4 serotypes. ^=start/end of fragment. Epitopes identified (all marked with “*” in table 3) are underlined.
ELMRRGDLPVWLSYKVASAGFQYKDREWCFDGERNNQILEENMDVEIWTK
Conserved Peptide Sequences of NSALL3
TRVVAAEMEEALKGLPIRYQTTAIKAEHTGKEIVDLMCHATFTMRLLSPV
NSALL4 Fragments:
Contained all of NSALL3, but where epitopes were matched from Weiskopf4 et al and IEDB databases5,6 all variants were also includes. Epitopes and their variants are highlighted in bold type. Underlined epitopes are epitopes which were not in Weiskopf et al or EIDB databases but variants for them which were present in the sequences.
KKDLISY*GGGWRLEGEWDEGEEVQVIAVEPGKNPKAVQTKPGLFKTPEG
RGLPIRY*QTTAIKAEHTGKEIVDLMCHATFTMRLLSPVRVPNYNLIIMD
TVLM*GLDKGWPISKMDLGVPLLALGCYSQVNPLTLTAAVLLLITHYAII
Conserved Peptide Sequences of NSALL4
Known Epitopes and Known Epitope Variants
VKKDLISY
GTSGSPIVNRE
ALKGLPIRY
IANQAAVLM
Epitope Data
Tables 2 and 3 shows which epitopes from Wesikopf et al and IEDB are present in the invention sequences and which serotypes they are present in. Table 4 shows the process by which the above epitopes (tables 2-3) are assessed to determine whether they fall within a conserved sequence. Epitopes which did not are excluded.
RYRGEDGCWYGMEIRP
LKEKEENLVNSLVTA
There is a highly conserved region that is contained in the beta ladder domain of NS1. The protein/vaccine of the invention may provide the full beta ladder domain to preserve tridimensionality. The underlined sequence is the highly conserved region.
Viral Vector Sequences
| Number | Date | Country | Kind |
|---|---|---|---|
| 1508099.7 | May 2015 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2016/051358 | 5/11/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/181147 | 11/17/2016 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2003048184 | Jun 2003 | WO |
| 2011163628 | Dec 2011 | WO |
| Entry |
|---|
| Bowie JU, Reidhaar-Olson JF, Lim WA, Sauer RT. Deciphering the message in protein sequences: tolerance to amino acid substitutions. Science. Mar. 16, 1990;247(4948):1306-10. |
| Konishi E. Issues related to recent dengue vaccine development. Trop Med Health. Dec. 2011;39(4 Suppl):63-71. Epub Aug. 6, 2011. |
| Halstead SB. Dengvaxia sensitizes seronegatives to vaccine enhanced disease regardless of age. Vaccine. Nov. 7, 2017;35(47): 6355-6358. Epub Oct. 10, 2017. |
| Flasche S, et. al. The Long-Term Safety, Public Health Impact, and Cost-Effectiveness of Routine Vaccination with a Recombinant, Live-Attenuated Dengue Vaccine (Dengvaxia): A Model Comparison Study. PLoS Med. Nov. 29, 2016;13(11):e1002181. eCollection Nov. 2016. |
| Nascimento EJ, Mailliard RB, Khan AM, Sidney J, Sette A, Guzman N, Paulaitis M, de Melo AB, Cordeiro MT, Gil LV, Lemonnier F, Rinaldo C, August JT, Marques ET Jr. Identification of conserved and HLA promiscuous DENV3 T-cell epitopes. PLoS Negl Trop Dis. Oct. 10, 2013;7(10):e2497. |
| Kim MY, Reljic R, Kilbourne J, Ceballos-Olvera I, Yang MS, Reyes-del Valle J, Mason HS. Novel vaccination approach for dengue infection based on recombinant immune complex universal platform. Vaccine. Apr. 8, 2015 8;33(15):1830-8. Epub Feb. 26, 2015. |
| Assenberg R, Mastrangelo E, Walter TS, Verma A, Milani M, Owens RJ, Stuart DI, Grimes JM, Mancini EJ. Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for polyprotein processing and viral replication. J Virol. Dec. 2009;83(24):12895-906. Epub Sep. 30, 2009. |
| “Plurality”. Merriam-Webster.com. Accessed Jul. 16, 2018. https://www.merriam-webster.com/dictionary/plurality. |
| International Preliminary Report on Patentability (IPRP) dated Nov. 23, 2017 for international application PCT/GB2016/051358. |
| Shi, et al.,Inferring Protective CD8+ T Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design, Plos One, vol. 10, No. 9, Sep. 18, 2015 (Sep. 18, 2015), p. e0138729, DOI: 10.1371/journal.pone.0138729. |
| Weiskopf, et al., Comprehensive Analysis of Dengue Virus-Specific Responses Supports an HLA-Linked Protective Role for CD8+ T Cells, Proceedings of the National Academy of Sciences, vol. 110, No. 22, May 28, 2013 (May 28, 2013 ), pp. D2046-E2053, XP055155265, ISSN: 0027-8424, DOI: 10.1073/pnas.1305227110. |
| Number | Date | Country | |
|---|---|---|---|
| 20180193446 A1 | Jul 2018 | US |
