VACCINE

专利摘要:
HRV VP2 proteins useful as components of immunogenic compositions for inducing cell-mediated immunity with cross-reactivity against human rhinovirus infection; nucleic acid constructs encoding such VP2 proteins of HRV.
公开号:BE1024785B1
申请号:E2017/5714
申请日:2017-10-05
公开日:2018-07-02
发明作者:Catherine Marie Ghislaine Gerard；Sandra Giannini；Julien Thierry Massaux
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

(30) Priority data:
10/05/2016 GB 1616904.7 (73) Holder (s):
GLAXOSMITHKLINE BIOLOGICALS SA
1330, RIXENSART
Belgium (72) Inventor (s):
GERARD Catherine Marie Ghislaine
1330 RIXENSART
Belgium
GIANNINI Sandra 1330 RIXENSART Belgium
MASSAUX Julien Thierry 1330 RIXENSART Belgium (54) VACCINE (57) VP2 proteins of HRV useful as components of immunogenic compositions for the induction of cell-mediated immunity exhibiting cross-reactivity against infection by a human rhinovirus; nucleic acid constructs encoding such VP2 proteins from HRV.
Figure 1
BELGIAN INVENTION PATENT
FPS Economy, SMEs, Middle Classes & Energy
Publication number: 1024785 Deposit number: BE2017 / 5714
Intellectual Property Office International Classification: C07K 14/005 A61K 39/12 Date of issue: 02/07/2018
The Minister of the Economy,
Having regard to the Paris Convention of March 20, 1883 for the Protection of Industrial Property;
Considering the law of March 28, 1984 on patents for invention, article 22, for patent applications introduced before September 22, 2014;
Given Title 1 “Patents for invention” of Book XI of the Code of Economic Law, article XI.24, for patent applications introduced from September 22, 2014;
Having regard to the Royal Decree of 2 December 1986 relating to the request, the issue and the maintenance in force of invention patents, article 28;
Given the patent application received by the Intellectual Property Office on 05/10/2017.
Whereas for patent applications falling within the scope of Title 1, Book XI of the Code of Economic Law (hereinafter CDE), in accordance with article XI. 19, §4, paragraph 2, of the CDE, if the patent application has been the subject of a search report mentioning a lack of unity of invention within the meaning of the §ler of article XI.19 cited above and in the event that the applicant does not limit or file a divisional application in accordance with the results of the search report, the granted patent will be limited to the claims for which the search report has been drawn up.
Stopped :
First article. - It is issued to
GLAXOSMITHKLINE BIOLOGICALS SA, Rue de l'Institut 89, 1330 RIXENSART Belgium;
represented by
PRONOVEM - Office Van Malderen, Avenue Josse Goffin 158, 1082, BRUXELLES;
a Belgian invention patent with a duration of 20 years, subject to payment of the annual fees referred to in article XI.48, §1 of the Code of Economic Law, for: VACCINE.
INVENTOR (S):
GERARD Catherine Marie Ghislaine, c / o GlaxoSmithKline Biologicals SA Rue de l'Institut 89, 1330, RIXENSART;
GIANNINI Sandra, c / o GlaxoSmithKline Biologicals SA, Rue de l'Institut 89, 1330, RIXENSART;
MASSAUX Julien Thierry, c / o GlaxoSmithKline Biologicals SA, Rue de l'Institut 89, 1330, RIXENSART;
PRIORITY (S):
10/05/2016 GB 1616904.7;
DIVISION:
divided from the basic application: filing date of the basic application:
Article 2. - This patent is granted without prior examination of the patentability of the invention, without guarantee of the merit of the invention or of the accuracy of the description thereof and at the risk and peril of the applicant (s) ( s).
Brussels, 02/07/2018, By special delegation:
BE2017 / 5714
VACCINE
Context
The present invention relates to immunogenic compositions for use in the prevention or amelioration of a disease caused by a human rhinovirus.
Human rhinoviruses (HRV) are the most common infectious viral agents in humans and are the predominant cause of colds. HRV is also linked to exacerbations of chronic obstructive pulmonary disease (COPD), the development of asthma, and, more recently, severe bronchiolitis in infants and children as well as fatal pneumonia in the elderly and immunocompromised adults. Therefore, the development of a vaccine for HRV is highly recommended but efforts are hampered by the existence of more than 100 HRV serotypes, with high level sequence variability in antigenic sites. Humoral immune responses are important for the prevention of HRV infection. Infection with HRV in antibody-naive subjects is followed by the development in serum of neutralizing antibodies specific for the serotype (IgG) as well as secretory antibodies (IgA) in the respiratory tract. Human viral test studies have shown that pre-existing HRV type specific antibodies can protect against HRV infection (Alper et al., 1998). CD4-specific cellular responses develop as a result
BE2017 / 5714 of an HRV infection. CD4 cells are largely Thl 'type and their production of IFN-γ contributes to the antiviral immune response, but these CD4 cells can also facilitate the development of the humoral immune response.
The literature indicates that sensitization with the OPV protein of HRV16 adjuvanted with IFA directly impacted the extent of heterotypic neutralizing antibodies induced against infection by the rhinovirus (Glanville et al., 2013). In WO 2014/122220, based on the Glanville results, the VP4 protein was identified as having high homology among HRVs, and therefore is responsible for the cross-reactive helper CD4 T cells induced by OPV which may accelerate the production of neutralizing antibodies during natural HRV infections. In document WO 2016/134288, the peptide epitopes of CD4 + T lymphocytes were identified.
summary
HRV VP2 proteins are provided herein useful as components of immunogenic compositions for the induction of cell-mediated immunity exhibiting broad cross-reactivity against rhinovirus infection.
In some embodiments, there is provided an immunogenic composition comprising an HRV VP2 protein, particularly in combination with an adjuvant, for example, a Thl adjuvant, such as an adjuvant containing a saponin.
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In some embodiments, there is provided a nucleic acid sequence encoding a polypeptide comprising a VP2 protein from HRV. In certain embodiments, there is provided a vector comprising such a nucleic acid sequence encoding a polypeptide comprising a VP2 protein from HRV, such as an adenoviral vector. In some embodiments, there is provided a self-amplifying RNA molecule comprising the nucleic acid sequence encoding a polypeptide comprising an HRV VP2 protein, such as a SAM vector. In other embodiments, immunogenic compositions are provided comprising such vectors or nucleic acid sequences.
In certain embodiments, such immunogenic compositions are provided comprising the HRV VP2 protein in combination with an adjuvant, and / or, nucleic acid constructs encoding HRV VP2 proteins, for use in medicine , for example, for use in preventing or ameliorating a disease or symptoms of a disease caused by or associated with an HRV infection in a subject, or, for use in a subject to reduce recovery time from a subject's HRV infection and / or to lessen the severity of a disease caused by a subject's HRV infection, or, for use in a subject to reduce or prevent clinical symptoms when the subject is infected with HRV, or for use in a subject to induce an immune response with
BE2017 / 5714 cross-reactivity against at least three serotypes of HRV, as when at least one of the at least three serotypes of HRV belongs to type A of HRV and at least one of the at least three serotypes of HRV belongs to the type B of HRV or type C of HRV.
In some embodiments, the immunogenic composition is intended for use in subjects with COPD (such as the elderly) or asthma (such as infants or children).
In some embodiments, the methods of reducing recovery time from HRV infection in a subject and / or lessening the severity of disease caused by HRV infection in a subject having if necessary, include administration to said subject of an immunologically effective amount of an immunogenic composition as disclosed herein.
In some embodiments, the methods of reducing or preventing clinical symptoms upon HRV infection in a subject in need thereof, include administering to the subject subject an immunologically effective amount of an immunogenic composition such as that disclosed here.
In some embodiments, methods of inducing an immune response with cross-reactivity against at least three HRV serotypes in a subject in need thereof, include administering to the subject subject an immunologically effective amount of a immunogenic composition as disclosed herein.
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Brief description of the figures
Figure 1 - Quantification of the positive single-stranded RNA genome in fluids from LBA to D2 after the intranasal test by HRVlb - each point represents individual values of a mouse (n = 5 / group) and the results are presented with the median (horizontal lines).
Figure 2 - Differential count of leukocytes (lymphocytes, neutrophils, macrophages, eosinophils) in AML fluids. collected 2 days after the test by HRVlb. Each point represents individual values of a mouse (n = 5 / group) and the results are presented with the median (horizontal lines).
Figure 3 - The levels of inflammatory cytokines (TNF-a, IFN-γ, IL-6, IL-10 and IL-12p70) and of the chemokines (MCP-1) secreted in AML fluids were studied 2 days after test by HRVlb. Each point represents individual values of a mouse (n = 5 / group) and the results are presented with the median (horizontal lines).
Figure 4 - Response in specific CD4 + T lymphocytes following stimulation in vitro with VP2 peptides from HRV39. The data are represented for individual mice (points; n = 5 / group) with the median / group (horizontal line).
Figure 5 - Response in specific CD4 + T cells following stimulation in vitro with VP4 peptides from HRV2. The data are represented for individual mice (points; n = 5 / group) with the median / group (horizontal line).
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Figure 6 - Response in specific CD4 + T cells following stimulation in vitro with VP4 peptides from HRV39. The data are represented for individual mice (points; n = 5 / group) with the median / group (horizontal line).
Figure 7 - Response in specific CD4 + T lymphocytes following stimulation in vitro with VP2 peptides from HRV2. The data are represented for individual mice (points; n = 5 / group) with the median / group (horizontal line).
Figure 8 - Response in specific CD4 + T lymphocytes following stimulation in vitro with VP2 peptides from HRV14. The data are represented for individual mice (points; n = 5 / group) with the median / group (horizontal line).
Figure 9 - Response in specific CD4 + T cells following in vitro stimulation with UC particles of HRV25. The data are represented for individual mice (points; n = 5 / group) with the median / group (horizontal line).
Figure 10 - Response to specific CD4 + T cells following in vitro stimulation with UC peptides from HRV3. The data are represented for individual mice (points; n = 5 / group) with the median / group (horizontal line).
Figure 11 - Response in specific CD4 + T cells following in vitro stimulation with UC particles of HRV28. The data are represented for individual mice (points; n = 5 / group) with the median / group (horizontal line).
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Figure 12 - Response in specific CD8 + T cells following in vitro stimulation with UC particles of HRV25. The data are represented for individual mice (points; n = 5 / group) with the median / group (horizontal line).
Figure 13 - HRVlb specific neutralizing antibody responses detected before / after the HRVlb test. The data are represented for grouped mouse sera (5 or 7 groups of 3 mice / group) with the median / group (horizontal line).
Figure 14 - Production of IFN-γ by CD4 + CD44 + T lymphocytes in mice immunized with VP2, VP4 or OPV of HRV39; IFN-γ was triggered in mice immunized with VP2 and VPO from HRV39 in response to groups of peptides produced from VP2 of homologous (14Ά) or heterologous (14B, 14C, 14D, or 14E) HRV viruses. The data presented are individual mice (n = 6 per group) with the median indicated by a horizontal line. Moving from left to right in each figure, the columns present the data of the mice immunized with HRV39 VP2, HRV39 VP4, HRV39 OPV, the living virus HRV39, and physiological saline, respectively. Please note the differences in upper limit on the ordinate.
Figure 15 - Production of IFN-γ by CD4 + CD44 + T lymphocytes in mice immunized with VP2, VP4 or OPV of HRV39: little by little IFN was produced in response to the groups of peptides produced from types VP4 of homologous (15A) or heterologous (15B, 15C, 15D, 15E) HRV. The data presented are individual mice (n = 6 per group) with the median
BE2017 / 5714 indicated by a horizontal line. Moving from left to right in each figure, the columns present the data of the mice immunized with HRV39 VP2, HRV39 VP4, HRV39 OPV, the living virus HRV39, and physiological saline, respectively.
Figure 16A - Alignments of the amino acid sequence of the VP2 protein of the HRV types used in Example 4. An overview of the extent to which each amino acid is conserved within the types is included. The height of the bar on the "identity" line is directly related to the degree to which the amino acid is stored.
Figure 16B - Table of HRV types used in Example 4, showing the percentage of identity at the amino acid level using pairwise comparisons.
Figure 16C - Alignment of amino acids in text for the HRV types used in Example 4; HRV_39VP2 (SEQ ID NO: 1), HRV_89-VP2 (SEQ ID NO: 15), HRV_1B-VP2 (SEQ ID NO: 16), HRV_02-VP2 (SEQ ID NO: 17), and HRV_14-VP2 (SEQ ID NO : 18).
detailed description
Constructs HR2 VP2 Proteins
HRV VP2 protein constructs useful as an antigenic component of immunogenic compositions are provided for inducing a cross-reactive immune response in a subject against human rhinovirus (HRV). As used herein, the term "antigen" refers to a molecule containing one or more epitopes (for example,
BE2017 / 5714 linear, conformational or both) that will stimulate a host's immune system to produce an antigen-specific humoral and / or cellular immunological response (i.e., an immune response that specifically recognizes a naturally occurring polypeptide). An "epitope" is that part of an antigen that determines its immunological specificity. T and B cell epitopes can be identified empirically (for example, using PEPSCAN or similar methods). In the context of the present invention, the induction of a "cross-reactive immune response" means that an immune response is induced both against the type of HRV from which the HRV antigen in the immunogenic composition , for example, the HRV protein VP2 of the invention, is derived (i.e., a homologous immune response), and, against one or more types of HRV different from the type of HRV from which the HRV antigen in the immunogenic composition is derived (i.e., a heterologous immune response). In one embodiment, the immunogenic composition of the invention induces an immune response against both homologous and heterologous serotypes of human rhinovirus.
For the purpose of the present invention, the terms "construction of HR2 VP2 protein", "human rhinovirus VP2 protein" or "HRV protein VP2", or "VP2 protein" are used interchangeably and refer to any amino acid sequence corresponding to the acid sequence
BE2017 / 5714 amines of the VP2 capsid protein of any HRV serotype. Immunogenic variants of an HRV VP2 protein construct are amino acid sequences having at least or exactly 75%, 77%, 80%, 85%, 90%, 95%, 97%, or 99% of full length identity with the native HRV VP2 sequence. The protein VP2 is about 270 amino acids in length. Table 1 lists the Uniprot accession numbers of the complete polyprotein genome sequences for HRV serotypes chosen from the three clades. Generally, the protein VP2 is located between amino acids 70 and 339 of the precursor of the polyprotein. Thus, based on these sequences, a person skilled in the art can derive therefrom the wild-type sequences of the VP2 and / or VP4 proteins of HRV for the HRV serotypes, for use in the present examples and in the present invention.
As is also known to those skilled in the art, the length of the amino acid sequence of the VP2 protein may vary slightly depending on the HRV serotype. For example, the wild type protein VP2 of HRV39 corresponds to amino acids 70 to 334 of the sequence of
wild type of VP0 of HRV39 (SEQ ID NO: 4). Three species of HRV have summer identified in whichto say, HRV-A, more thanHRV-B and hundred typesHRV-C. are classified, that ' East- The species HRV-A understands in particular the
following serotypes: : HRVla, HRVlb, HRV2, HRV7, HRV8, HRV9, HRV10, HRV11, HRV12, HRV13, HRV15, HRV16, HRV18, HRV19, HRV20, HRV21, HRV22, HRV23, HRV2 4, HRV25, HRV28, HRV29, HRV30, HRV31, HRV32, HRV33, HRV34, HRV36, HRV38,
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HRV39, HRV40, HRV41, HRV43, HRV44 , HRV45, HRV46, HRV47, HRV49, HRV50, HRV51, HRV53, HRV54 , HRV55, HRV56, HRV57, HRV58, HRV59, HRV60, HRV61, HRV62 , HRV63, HRV64, HRV65, HRV66, HRV67, HRV68, HRV71, HRV73 , HRV74, HRV75, HRV76, HRV77, HRV78, HRV80, HRV81, HRV82 , HRV85, HRV88, HRV8 9, HRV90, HRV94, HRV95 , HRV96, HRV98, HRV100, HRV101, HRV102 and HRV103. L 'species HRV-B understands especially the following serotypes: HRV3, HRV4 , HRV5, HRV6, HRV14, HRV17, HRV26, HRV27, HRV35, HRV37 _r HRV42, HRV48, HRV52, HRV69, HRV70, HRV72, HRV79, HRV83 , HRV84, HRV86, HRV91, HRV92, HRV93, HRV97 and HRV99. The species HRV-C understands especially the
following serotypes: HRV-C1, HRV-C2, HRV-C3, HRV-C4,
HRV-C5, HRV-C6, HRV-C7, HRV-C8, HRV-C9, HRV-C10, HRV- Eyelash, HRV-C12, HRV-C13, HRV-C14, HRV-C15, HRV-C16, HRV- 07, HRV-C18, HRV-C19, HRV-C20, HRV-C21, HRV-C22, HRV- C23, HRV-C24, HRV-C25, HRV-C26, HRV-C27, HRV-C28, HRV- C29, HRV-C30, HRV-C31, HRV-C32, HRV-C33, HRV-C34, HRV- C35, HRV-C36, HRV-C37, HRV-C38, HRV-C39, HRV-C40, HRV- C41, HRV-C42, HRV-C43, HRV-C44, HRV-C45, HRV-C46, HRV-
C47, HRV-C48 and HRV-C49.
Table 1
Clade HRV AccessionUniprot Clade HRV AccessionOniprot AT HRV1 B9V432 AT HRV7 5 A5GZF9 AT HRV10 A5GZE7 AT HRV7 6 B9V4A3 AT HRV100 B9V496 AT HRV7 7 B9V475 AT HRV101 D2IW01 AT HRV7 8 B9V4A4 AT HRV11 A7KC06 AT HRV8 B9V434 AT HRV12 A7KC07 AT HRV 8 0 B9V477
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Clade HRV AccessionUniprot Clade HRV AccessionUniprot AT HRV13 B9V437 AT HRV81 B9V478 AT HRV15 A5GZE2 AT HRV8 2 B9V481 AT HRV16 Q82122 AT HRV85 B9V484 AT HRV18 B9V439 AT HRV88 A5GZF3 AT HRV19 B9V440 AT HRV8 9 P07210 AT HRV1B P12916 AT . HRV 9 B9V436 AT HRV2 P04936 AT HRV 90 B9V488 AT HRV20 B9V441 AT HRV 9 4 B9V4A6 AT HRV21 B9V442 AT HRV 9 5 B9V491 AT HRV 2 2 B9V443 AT HRV 9 6 B9V492 AT HRV2 3 A5GZE6 AT HRV 9 8 B9V494 AT HRV2 4 B9V4B1 B HRV 14 P03303 AT HRV25 B9V444 B HRV17 A7KC12 AT HRV2 8 A5GZF7 B HRV2 6 B9V445 AT HRV2 9 B9V446 B HRV2 7 A7KC13 AT HRV30 B9V4A0 B HRV3 A7RC14 AT HRV31 B9V447 B HRV35 B9V4A8 AT HRV32 B9V448 B HRV37 A7KC15 AT HRV33 B9V449 B HRV4 A5GZD9 AT HRV 3 4 B9V4B0 B HRV4 2 B9V451 AT HRV3 6 A5GZF4 B HRV 4 8 A5GZD7 AT HRV3 8 A5GZE4 B HRV5 B9V433 AT HRV3 9 Q5XLP5 B HRV52 B9V458 AT HRV 4 0 B9V450 B HRV 6 A5GZD5 AT HRV41 A5GZE0 B HRV 6 9 B9V472 AT HRV4 3 B9V452 B HRV7 0 A5GZD8 AT HRV4 4 A5GZE8 B HRV7 2 D6PT65 AT HRV4 5 B9V453 B HRV7 9 B9V476 AT HRV 4 6 A5GZF5 B HRV 8 3 B9V482 AT HRV4 7 B9V454 B HRV8 4 B9V483 AT HRV4 9 B9V455 B HRV8 6 B9V485 AT HRV5 0 B9V456 B HRV 91 B9V489 AT HRV51 B9V457 B HRV 9 2 B9V490 AT HRV5 3 A5GZF6 B HRV93 A7KC17 AT HRV54 B9V459 B HRV97 B9V493
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Clade HRV AccessionUniprot Clade HRV AccessionUniprot AT HRV5 5 A5GZG0 B HRV 9 9 B9V495 AT HRV5 6 B9V461 VS HRVC-11 C5HDF8 AT HRV5 7 B9V462 VS HRVC - STRAINCU072 E9LS20 AT HRV58 B9V463 VS HRVC - CU184 E9LS23 AT HRV5 9 A5GZE9 VS HRVC-15 E5D8F2 AT HRV60 B9V464 VS HRVC-24 A8S322 AT HRV 61 B9V465 VS HRVC-25 A8S330 AT HRV 6 2 B9V466 vs HRVC-26 A8S334 AT HRV63 B9V467 vs HRVC - STRAINNATO45 A7TUB2 AT HRV 6 4 B9V4A2 vs HRVC - STRAINNAT001 A7TUB1 AT HRV 6 5 B9V468 vs HRVC - STRAINNY-074 A0MHB7 AT HRV66 B9V469 vs HRVC-04 C7DUC6 AT HRV 6 7 B9V470 vs HRVC-10 C7DÜC7 AT HRV68 B9V471 vs HRVC-03 A4UHT9 AT HRV7 B9V497 vs HRVC - STRAINQCE C9DDK2 AT HRV71 B9V473 vs HRVC-54 A0A0B5HPB2 ATAT HRV7 3HRV7 4 A5GZE1A5GZE3 vs HRVC-35 H8Y6P9
The types of HRV can also be grouped according to the use of the receptor, into viruses of the minor group and viruses of the major group. Minor group viruses, such as HRV2, use the low density lipoprotein receptor family as a receptor. They are acid labile and have an absolute dependence on a low pH for decapsidation. Major group viruses, such as HRV14 and HRV16, use the intercellular adhesion molecule 1 (ICAM-1) as a receptor. They are generally acid labile
BE2017 / 5714 but, unlike the viruses of the minor group, they do not exhibit an absolute dependence on a low pH for decapsidation. As is well known to those skilled in the art, the HRVs of the minor group include 11 serotypes, including HRV1A, HRV1B, HRV2, HRV23, HRV25, HRV29, HRV30, HRV31, HRV44, HRV47, HRV49 and HRV62.
For the purpose of the invention, the VP2 protein of any of the types of HRV listed here can be used. In one embodiment, the HRV VP2 protein is the HRV VP2 protein of HRV39, HRVlb, HRV2, HRV3, HRV14, HRV25 or HRV28, or one of their immunogenic variants. In a specific embodiment, the HRV VP2 protein is the HRV39 VP2 protein (SEQ ID NO: 1) or one of its immunogenic variants having at least 90%, 95%, 97%, or 99% of identity, over the entire length, with SEQ ID NO: 1.
Identity or homology with respect to a sequence is defined here as the percentage of amino acid residues in the candidate sequence that are identical to the reference amino acid sequence after alignment of the sequences and the introduction of gaps, if necessary, to obtain the maximum percentage of sequence identity, and not considering any conservative substitution as part of the sequence identity.
Sequence identity can be determined by standard methods which are commonly used to compare the amino acid position similarity of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned for optimal match of their
BE2017 / 5714 respective amino acids (either along the full length of one or both sequences or throughout a predetermined part of one or both sequences). Programs provide a default opening penalty and a default breach penalty, and a score matrix such as PAM 250 [a classic score matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)] can be used in conjunction with the computer program. For example, the identity percentage can then be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longest sequence within the corresponding window and the number of gaps entered in the shorter sequences to align the two sequences.
When reference is made here to a sequence by a UniProt or Genbank accession code, the sequence to which reference is made is the version on the date of filing of this application.
In one embodiment, the HRV VP2 proteins described herein are appropriately isolated. An “isolated” HRV VP2 protein is one that is removed from its original environment. Similarly, the polynucleotides described here are appropriately isolated. For example, a naturally occurring protein is isolated if it is separated from some or all of the materials coexisting in the natural system. A polynucleotide is considered isolated if, for example, it is cloned into a vector that does not
BE2017 / 5714 not part of its natural environment or if it is included within a cDNA.
In one embodiment, an immunogenic variant of an HR2 VP2 protein corresponds to an HRV VP2 protein into which an amino acid sequence of up to 25, or, up to 20 amino acids can be inserted, substituted or deleted, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid (s) ). In a more specific embodiment, such insertion, substitution or deletion is localized in these parts of the amino acid sequences of the VP2 protein which correspond to highly variable regions of the VP2 protein of HRV. Regions in the HR2 VP2 protein suitable for such insertion, substitution or deletion include aal55 to 170 (i.e., the NIm-II loop), aal34 to 146, aa232 to 238 and aa72 to 75, the numbering of each of which is based on the full length sequence of VP2 of HRV39 (SEQ ID NO: 1) In a particular embodiment, an insertion, deletion and / or substitution is located at the level of aal55 to 170 (i.e., the NIm-II loop). In a specific embodiment, the HRV VP2 protein is the HRV39 VP2 protein comprising a mutation in its NIm-II loop (SEQ ID NO: 2) or one of its immunogenic variants having at least 90%, 95% , 97%, or 99% identity with SEQ ID NO: 2, over the entire length. Alternatively, an insertion, deletion and / or substitution is localized at the carboxy end of VP2.
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In another embodiment, an HRV peptide is inserted or substituted into one of said highly variable regions of the HR2 VP2 protein; the peptide is derived from one of the proteins VP1, VP2, VP3 or VP4 of the HRV capsid, and is capable of inducing an immune response exhibiting cross-reactivity and / or cross-neutralization against two or more HRV serotypes. Such peptides are chosen or derived from conserved regions of the structural proteins of human rhinoviruses. A response with cross-reactivity and / or neutralization can be obtained when the amino acid sequence of HRV is of limited length. Thus, the HRV peptide may consist of a fragment of 5 to 40 contiguous amino acids, or of 8 to 30 contiguous amino acids, derived from a full-length wild type HRV capsid protein (VP1, VP2, VP3 or VP4). Favorably, the HRV peptide consists of no more than 20 amino acids, such as 8 to 20 amino acids, for example, 16 amino acids. In any embodiment, an HRV peptide or one of its variants will have a minimum length of 8 amino acids.
The VP2 protein of the invention may include or include amino acid fragments or HRV peptides which have been described in the literature. For example, antibodies induced with recombinant amino acid fragments of HRV-14 or -89 VP1 covering amino acids 147 to 162 of HRV14 VP1 have been shown to exhibit specific activity and cross-neutralization (McCray & Werner, 1998 Nature Oct 22-28; 329 (6141): 736-8; Edlmayr et al., 2011, Eur.
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Breathe. J. 37: 44-52). It has been observed that the structure of the rhinovirus capsid is dynamic and seems to oscillate between two different structural states: one in which VP4 is deeply buried, and the other where the N-terminal end of VP4 and VP1 is accessible to proteases (Lewis et al. 1998 Proc Natl Acad Sci US A. 95 (12): 6774-8). Antibodies produced against the 30 N-terminal amino acids of VP4 but not of VP1 have been found to successfully neutralize viral infectivity in vitro (Katpally et al. 2009, J Virol. 83 (14): 7040-8. ). Antibodies produced against the N-terminal amino acids of VP4 have been found to neutralize HRV14, HRV16 and HRV29. In addition, the antibodies produced against a consensus sequence of the first 24 residues derived from rhinovirus VP4 have also shown some cross-neutralization activity (Katpally et al., 2009, J Virol. 83 (14): 7040-8.).
Other descriptions of HRV peptides and / or epitopes from the literature can be found in: Niespodziana et al. 2012 (The FASEB Journal. Vol 26, 1001-1008) in which a response against an N-terminal 20mer peptide from VP1 was not a neutralizing response, i.e., a non-protective epitope; Miao et al. 2009 (J. Clin. Microbiol. Vol 47, No 10, 3108-3113) - mAbs produced against the highly conserved N-terminal part of VP1 of enterovirus are useful in the recognition of a wide range of enteroviruses ; WO 2006/078648 regarding vaccines based on HRV peptides derived from transiently exposed regions of VP4, in
BE2017 / 5714 in particular amino acids 1 to 31 or 1 to 24 of VP4; WO 2011/050384 relating to peptides originating from the N-terminal end of VP1 comprising amino acids 1 to 8; WO 2008/057158 concerning Nim IV of rhinovirus, in particular a peptide comprising amino acids 277 to 283 or 275 to 285 originating from the carboxy-terminal region of VP1, in particular HRV14.
Other HRV peptides have been identified derived from the N-terminal sequences of VP1 and VP4, i.e., amino acid fragments of HRV comprising amino acids 32 to 45 of VP1 and fragments of amino acids of HRV comprising amino acids 1 to 16 of VP4, or their variants comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions d amino acids within the peptide sequence. When a variant of a peptide sequence has 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence , this means that the variant exhibits at least one amino acid difference compared to the reference peptide sequence, which can comprise between 0 and 4 additions or deletions of amino acids at one end and between 0 and 4 additions or deletions at l 'other end and between 0 and substitutions or additions or deletions of amino acids within the sequence.
In one embodiment, a peptide consists of at least 8 and no more than 20 amino acids
BE2017 / 5714 originating from the N-terminal end of VP4, which peptide of HRV comprises amino acids 1 to 16 of VP4 or a variant of amino acids 1 to 16 comprising 1 to 4 additions or deletions of amino acids to the either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence. In a particular embodiment, the VP4 peptide of HRV consists of amino acids 1 to 16 of VP4 or of a variant comprising one, two, three, or four additions or deletions or substitutions of amino acids. Other VP4 specific amino acid fragments of HRV include, for example, amino acids 1 to [16 to 20], amino acids 2 to [17 to 21], 3 to [18 to 22], 4 to [19 to 23], 5 to [20 to 24] where it should be understood that the numbers in square brackets include all the numbers in the range specified individually. Favorably, the HRV VP4 peptide is made up of no more than 16 contiguous amino acids from VP4. It should be understood that the numbering of the HRV VP4 peptide or of any peptide or protein (expressed recombinantly) as used herein is independent of methionine due to the starting codon.
In another embodiment, an HRV peptide consists of at least 8 and not more than 40 amino acids from the N-terminal region of VP1, which HRV peptide comprises amino acids 32 to 45 of VP1 or a variant of amino acids 32 to 45 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the
BE2017 / 5714 peptide sequence. In a particular embodiment, the amino acid fragment of VP1 of HRV consists of amino acids 32 to 45 of VP4 or of a variant comprising one, two, three, or four additions or deletions or substitutions of amino acids . VP1 peptides include, for example, amino acids [5 to 35] to 45, [6 to 35] to 46, [7 to 35] to 47, [8 to 35] to 48, [9 to 35] to 49 and similarly 32 to [45 to 72], 33 to [45 to 73], 34 to [45 to 74], 35 to [45 to 75] and 36 to [45 to 76] where the numbers in square brackets all include numbers in the individually specified range.
The HRV peptides for the purpose of the invention thus comprise:
- amino acids 147 to 162 of VP1 of HRV14 or a variant of amino acids 147 to 162 of VP1 of HRV14 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence;
- amino acids 1 to 30 of VP4 of HRV14 or a variant of amino acids 1 to 30 of VP4 of HRV14 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence;
- amino acids 1 to 24 of VP4 of HRV14 or a variant of amino acids 1 to 24 of VP4 of HRV14 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence;
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- amino acids 1 to 8 of VP1 of HRV14 or a variant of amino acids 1 to 8 of VP1 of HRV14 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence;
- amino acids 277 to 283 of VP1 of HRV14 or a variant of amino acids 277 to 283 of VP1 of HRV14 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence;
- amino acids 275 to 285 of VP1 of HRV14 or a variant of amino acids 275 to 285 of VP1 of HRV14 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence;
- amino acids 32 to 45 of VP1 or a variant of amino acids 32 to 45 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions d amino acids within the peptide sequence;
- amino acids 1 to 16 of VP4 or a variant of amino acids 1 to 16 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions d amino acids within the peptide sequence.
In a particular embodiment, the HRV peptide is derived from VP1 and has or comprises an amino acid sequence chosen from:
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HRV14 (B): 32-PILTANETGATMPV-45 (SEQ ID NO: 5)
HRV8 (A-M): 32-PALDAAETGHTSSV-45 (SEQ ID NO: 6)
HRV25 (A-m): 32-PILDAAETGHTSNV-45 (SEQ ID NO: 7)
HRV_C_026: 32-QALGAVEIGATADV-45 (SEQ ID NO: 8) or one of their variants with 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or amino acid deletions within the amino acid sequence.
In another particular embodiment, the HRV peptide is derived from VP4 and has or comprises an amino acid sequence chosen from:
HRV14 (B): 1-GAQVSTQKSGSHENQN-16 (SEQ ID NO: 9)
HRV100 (A-M): 1-GAQVSRQNVGTHSTQN-16 (SEQ ID NO: 10)
HRV_C_026: 1-GAQVSRQSVGSHETMI-16 (SEQ ID NO: 11) or one of their variants comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or amino acid deletions within the amino acid sequence. For the purpose of the present invention, the immunogenic variants consist of or comprise an amino acid sequence having at least or exactly 75%, 77%, 80%, 85%, 90%, 95%, 97%, or 99 of identity, over the entire length, with the native sequence.
In another particular embodiment, peptides derived from HR2 VP2 can be introduced into the protein VP2, where said peptide derived from HRV2 VP2 is chosen from:
SSKGWWWKLPDALKDMGIFGENMFYHYLGRS (aa 143 to 173 of HR2 VP2) (SEQ ID NO: 12), IPEHQIASALHGNVNVGYNYTHPG
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ETGREVK (aa 196 to 226 of VP2 of HRV2) (SEQ ID NO: 13), and INTIPITISISPMCAEFSGARAKRQGLPVFI (aa 306 to 336 of VP2
HRV2) (SEQ ID NO: 14). In a embodiment, the composition does understands not VP4 protein (or of polynucleotide
comprising a nucleic acid sequence encoding a VP4 protein from HRV). For the purpose of the present invention, the term "human rhinovirus VP4 protein" or "HRV protein VP4" or "protein VP4" refers to any amino acid sequence corresponding to the amino acid sequence of the protein of the VP4 capsid of any HRV serotype as well as one of its variants, where the variant is at least 90% identical to the VP4 amino acid sequence of an HRV.
HRV's VP2 protein can be synthesized chemically using standard techniques or produced by recombination.
Adjuvanted HRV VP2 protein
In one embodiment, the immunogenic composition or vaccine comprises the HRV VP2 protein as defined herein and in combination with an adjuvant, such as a Th1 adjuvant.
For the purpose of the present invention, the term "adjuvant" refers to a compound or composition which enhances the immune response against an antigen, such as the immune response against a VP2 protein of HRV in a human subject. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or
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Aluminum hydroxide), organic builders (e.g. saponins, such as QS21, or squalene), oil-based builders (e.g. full Freund's adjuvant and incomplete adjuvant Freund), cytokines (e.g. IL-1, IL-2, IL-7,
IL-12, IL-18, immunostimulatory particles
GM-CSF, and IFN-γ), adjuvants (e.g. complexes (ISCOM), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g. monophosphoryllipid A, such as monophosphoryl- 3-de-Oacylated lipid A (3D-MPL), or muramyl-peptides), synthetic adjuvants (for example, nonionic block copolymers, muramyl-peptide analogs, or synthetic lipid A), adjuvants of synthetic polynucleotides (eg, polyarginine or polylysine), and immunostimulatory oligonucleotides containing unmethylated CpG dinucleotides ("CpG").
In one embodiment, the adjuvant is an adjuvant containing a saponin. A saponin suitable for use in the present invention is Quil A and its derivatives. Quit A is a saponin preparation isolated from the South American tree Quillaja saponaria Molina and it has been described for the first time as having an adjuvant activity by Dalsgaard et al. in 1974 (Saponin adjuvants, Archiv, für die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254). Purified Quil A fragments have been isolated by HPLC, which retain adjuvant activity without the toxicity associated with Quil A (EP 0 362 278), for example QS7 and QS21 (also
BE2017 / 5714 known as QA7 and QA21). QS-21 is a natural saponin derived from the bark of Quillaja saponaria Molina, which induces CD8 + cytotoxic T lymphocytes (CTL), Thl cells and a predominant IgG2a antibody response and is a preferred saponin in the context of the present invention. In a suitable form of the present invention, the saponin adjuvant within the immunogenic composition is a Quil A derivative of Quillaja saponaria Molina, preferably an immunologically active fraction of Quil A, such as QS-7, QS17, QS -18 or QS-21, appropriately QS-21.
In one embodiment, the saponin comprises a combination of saponin fractions as disclosed in WO 1996/011711. Alternatively, (semi-) synthetic saponins are considered useful such as those described by Govind Ragupathi et al. (Expert Rev Vaccines 2011; 10 (4): 463-470).
Saponin is generally provided in its least reactogenic composition or it is neutralized by an exogenous sterol, such as cholesterol. Suitable sterols include β-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. These sterols are well known in the art, for example, cholesterol is disclosed in the Merck Index, 11th Edn., Page 341, as a naturally occurring sterol found in animal fat. There are several particular forms of less reactogenic compositions in which QS21 is neutralized with an exogenous sterol such as cholesterol. In one embodiment, the saponin / sterol is presented
BE2017 / 5714 in a liposomal formulation structure. Processes for obtaining saponin / sterol in a liposomal formulation are described in document WO 96/33739, in particular example 1.
A saponin, such as QS21, can be used in amounts between 1 and 100 µg per human dose of the adjuvant composition. QS21 can be used at a rate of approximately 50 pg, such as at least 40 pg, at least 45 pg or at least 49 pg, or, less than 100 pg, less than 80 pg, less than 60 pg, less than 55 pg or less than 51 pg. Examples of suitable ranges are between 40 and 60 pg, suitably between 45 and 55 pg or between 49 and 51 pg or 50 pg. In another embodiment, the human dose of the adjuvant composition comprises QS21 at a rate of about 25 pg, such as at least 20 pg, at least 21 pg, at least 22 pg or at least 24 pg, or , less than 30 pg, less than 29 pg, less than 28 pg, less than 27 pg or less than 26 pg Examples of lower ranges include between 20 and 30 pg, suitably between 21 and 29 pg or between 22 and 28 pg or between 23 and 27 pg or between 24 and 26 pg, or 25 pg.
When the active saponin fraction is QS21 and a sterol is included, the QS21 / sterol ratio will generally be of the order of 1/100 to 1/1 (w / w), suitably between 1/10 and 1/1 (w / w), and preferably 1/5 to 1/1 (w / w). Suitably, an excess of sterol is present, the QS21 / sterol ratio being at least 1/2 (w / w). In one embodiment, the QS21 / sterol ratio is 1/5 (w / w). In a specific embodiment, the sterol is cholesterol.
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In one embodiment, the adjuvant comprises a TLR-4 agonist (also called a TLR-4 ligand). A suitable example of a TLR-4 agonist is a lipopolysaccharide, suitably a non-toxic derivative of lipid A, particularly monophosphoryllipid A or more particularly 3-deacylated monophosphoryl lipid A (3D-MPL).
3D-MPL is sold as MPL by GlaxoSmithKline Biologicals N.A. and is referred to throughout the document MPL or 3D-MPL. See, for example, US Patents 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL mainly promotes responses in CD4 + T lymphocytes with an IFN-g (Thl) phenotype. 3D-MPL can be produced according to the methods described in document GB 2 220 211 A. From the chemical point of view, it is a mixture of 3-deacylated monophosphoryl-lipid A with 4, 5 or 6 acylated chains . In the compositions of the present invention, the small particle 3DMPL can be used to prepare the adjuvant. The small particle 3D-MPL has a particle size such that it can be sterilized by filtration through a 0.22 µm filter. Such preparations are described in document WO 94/21292.
Other TLR-4 ligands that can be used are aminoalkyl glucosaminide phosphates (AGP) such as those described in WO 98/50399 or US Patent No. 6,303,347 (methods for preparing AGP are also described), suitably RC527 or RC529 or pharmaceutically acceptable salts of AGP as described in US Patent No. 6,764,840. Other suitable AGPs are described in the document
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WO 2004/062599. Some PGAs are TLR4 agonists, and some are TLR-4 antagonists. Both are believed to be useful as an adjuvant.
Other suitable ligands for TLR-4 are as described in document WO 2003/011223 and in document WO 2003/099195, such as compound I, compound II and compound III described on pages 4 and 5 of the document WO 2003/011223 or on pages 3 and 4 of document WO 2003/099195 and in particular these compounds described in document WO 2003/011223 such as ER803022, ER803058, ER803732, ER804053, ER804057m ER804058,
ER804059, ER804442, ER804680 and ER804764. For example, a suitable TLR-4 ligand is ER804057.
Other TLR-4 ligands which can be used in the present invention include the glucopyranosyl lipid (GLA) adjuvant as described in documents WO 2008/153541 or WO 2009/143457 or the literature of Coler RN and al.
articles from (Development
Synthetic a Vaccine e! 6333.
and Characterization of Glucopyranosyl Lipid Adjuvant System as Adjuvant, PLoS ONE 6 (1):
doi: 10.1371 / journal.pone.0016333, 2011) and Arias MA et al. (Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgpl40, PLoS ONE 7 (7): e41144. Doi: 10.1371 / journal. Pone .0041144,
2012). Documents WO 2008/153541 or WO 2009/143457 are incorporated here by reference with the aim of defining ligands for TLR-4 which can be used in the present invention.
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A TLR-4 ligand such as a lipopolysaccharide, such as 3D-MPL, can be used in amounts between 1 and 100 µg per human dose of the adjuvant composition. 3D-MPL can be used at a rate of approximately 50 pg, such as at least 40 pg, at least 45 pg or at least 49 pg, or, less than 100 pg, less than 80 pg, less than 60 pg, less at 55 pg or less than 51 pg. Examples of suitable ranges are between 40 and 60 pg, suitably between 45 and 55 pg or between and 51 pg or 50 pg. In another embodiment, the human dose of the adjuvant composition comprises 3D-MPL at a rate of about 25 pg, such as at least 20 pg, at least 21 pg, at least 22 pg or at least 24 pg , or, less than 30 pg, less than 29 pg, less than 28 pg, less than 27 pg or less than 26 pg. Examples of lower ranges include between 20 and 30 pg, suitably between 21 and 29 pg or between 22 and 28 pg or between 23 and 27 pg or between 24 and 26 pg, or 25 pg.
In one embodiment, the adjuvant comprises a TLR4 agonist, such as 3D-MPL, formulated with an aluminum salt, such as aluminum hydroxide or aluminum phosphate.
In a specific embodiment, the adjuvant comprises both a saponin and a TLR4 agonist. In a specific example, the adjuvant includes QS21 and 3D-MPL. In an alternative embodiment, the adjuvant comprises QS21 and GLA.
When both a TLR4 agonist and a saponin are present in the adjuvant, then the weight ratio of the TLR4 agonist to the saponin is suitably between 1/5 and 5/1, suitably
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1/1. For example, when 3D-MPL is present in an amount of 50 pg or 25 pg, then suitably, QS21 may also be present in an amount of 50 pg or 25 pg per human dose of the adjuvant.
In one embodiment, the saponin, optionally with a TLR4 agonist, is administered in a liposomal formulation. By "liposomal formulation" it is meant that the saponin (and possibly the TLR-4 agonist) is formulated with liposomes, or, in other words, presented in a composition based on liposomes. The liposomes provided for the present invention contain a neutral lipid or consist essentially of a neutral lipid. By "neutral lipid" it should be understood that the overall net charge of the lipid is (approximately) zero. Therefore, the lipid can be generally nonionic or it can be zwitterionic. In one embodiment, the liposomes include a zwitterionic lipid. Examples of suitable lipids are phospholipids such as phosphatidylcholine species. In one embodiment, the liposomes contain phosphatidylcholine as the liposome forming lipid which is suitably non-crystalline at room temperature. Examples of such non-crystalline phosphatidylcholine lipids include egg yolk phosphatidylcholine, dioleoylphosphatidylcholine (DOPC) or dilaurylphosphatidylcholine (DLPC). In a particular embodiment, the liposomes of the present invention contain DOPC, or, consist essentially of DOPC. Liposomes can also
BE2017 / 5714 contain a limited amount of a charged lipid which increases the stability of the liposome-saponin structure for liposomes composed of saturated lipids. In these cases, the amount of loaded lipid is suitably from 1 to 20% w / w, preferably 5 to 10% w / w of the liposomal composition. Suitable examples of such charged lipids include phosphatidylglycerol and phosphatidylserine. Suitably, the neutral liposomes will contain less than 5% w / w of loaded lipid, such as less than 3% w / w or less than 1% w / w. In a particular embodiment, the liposomal formulation comprises cholesterol as a sterol.
Nucleic acid constructs encoding a polypeptide comprising an HR2 VP2 protein
In one embodiment, the immunogenic composition or the vaccine comprises the polynucleotide comprising a nucleic acid sequence coding for the VP2 protein of HRV as defined herein. In another embodiment, the nucleic acid sequence encoding the VP2 protein of HRV is placed under the control of elements allowing its expression in a cell, as in a mammalian cell.
In one embodiment, the nucleic acid sequence is incorporated into a viral vector, such as an adenoviral vector. Thus, in a specific embodiment, the composition comprises an adenoviral vector comprising a transgene coding for the protein VP2 of HRV as defined here.
Adenovirus has been widely used for gene transfer applications due to its ability to achieve highly efficient gene transfer.
BE2017 / 5714 effective in various target tissues and a high transgenic capacity. The adenoviral vectors for use in the present invention can be derived from a variety of mammalian hosts. More than 100 distinct serotypes of adenoviruses that infect various mammalian species have been isolated. These adenoviral serotypes have been classified into six subgenera (A to F; B is subdivided into B1 and B2) according to the sequence homology and the ability to agglutinate erythrocytes (Tatsis and Ertl, Molecular Therapy (2004) 10: 616-629).
In one embodiment, the adenoviral vector of the present invention is derived from a human adenovirus. Examples of such adenoviruses of human origin are Ad1, Ad2, Ad4, Ad5, Ad6, Adll, Ad 24, Ad34, Ad35, particularly Ad5, Adll and Ad35. Although Ad5-based vectors have been widely used in a number of gene therapy trials, there are limitations on the use of Ad5 and other human group C adenoviral vectors due to pre-existing immunity in the general population due to natural infection. Ad5 and other members of human group C tend to be among the most seroprevalent serotypes. In addition, immunity against existing vectors may develop as a result of exposure to the vector during treatment. These types of pre-existing or developed immunity against seroprevalent vectors can limit the effectiveness of gene therapy or vaccination efforts.
Therefore, in another embodiment, the adenoviral vector is derived from a simian adenovirus
BE2017 / 5714 non-human, also called simply simian adenovirus. Many adenoviruses have been isolated from non-human simians such as chimpanzees, bonobos, rhesus macaques and gorillas, and vectors derived from these adenoviruses induce strong immune responses against transgenes encoded by these vectors (Colloca et al . (2012) Sci. Transi. Med. 4: 1-9; Roy et al. (2004) Virol. 324: 361-372; Roy et al. (2010) J. of Gene Med. 13: 17-25) . Some advantages of vectors based on non-human simian adenoviruses include the relative lack of cross-neutralizing antibodies against these adenoviruses in the target human population. For example, the cross-reaction of certain chimpanzee adenoviruses with preexisting neutralizing antibody responses is only present in 2% of the target human population compared to 35% in the case of certain candidate human adenoviral vectors.
In specific embodiments, the adenoviral vector is derived from a non-human adenovirus, such as a simian adenovirus and in particular, a chimpanzee adenovirus such as ChAd3, ChAd63, ChAd83, ChAdl55, Pan 5, Pan 6, Pan 7 (also called C7) or Pan 9. Examples of such strains are described in documents WO 03/000283, WO 2010/086189 and
GB 1510357.5 and are also available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, and other sources. Alternatively, the adenoviral vectors can be derived from non-human simian adenoviruses isolated from bonobos, such as PanAdl, PanAd2 or PanAd3. Examples
BE2017 / 5714 such vectors described here can be found, for example, in documents WO 2005/071093 and WO 2010/086189. Adenoviral vectors can also be derived from adenoviruses isolated from gorillas as described in documents WO 2013/52799, WO 2013/52811 and WO 2013/52832.
Adenoviral vectors can be used to administer desired nucleic acid or protein sequences, for example, heterologous (gene) sequences, for expression in vivo. A vector can comprise any genetic element including naked DNA, a phage, a transposon, a cosmid, an episome, a plasmid, or a virus for administration. By "expression cassette" is meant the combination of a desired heterologous gene (transgene) and other regulatory elements necessary to direct the translation, transcription and / or expression of the gene product in a host cell.
Generally, an adenoviral vector is designed such that the expression cassette is localized in a nucleic acid molecule which contains other adenoviral sequences in the region native to a chosen adenoviral gene. The expression cassette can be inserted into an existing gene region to disrupt the function of that region, if desired. Alternatively, the expression cassette can be inserted into the site of a partially or completely deleted adenoviral gene. For example, the expression cassette can be located in the site of a mutation, an insertion or a deletion which renders at least one gene of a genomic region chosen in the
BE2017 / 5714 group consisting of EIA, E1B, E2A, E2B, E3 and E4. The term "renders non-functional" means that a sufficient amount of the gene region is eliminated or otherwise disrupted, so that the region of the gene is no longer able to produce functional gene expression products. If desired, the entire region of the gene can be eliminated (and appropriately replaced by the expression cassette). Suitably, the El genes of the adenovirus are deleted and replaced by an expression cassette consisting of the promoter of choice, a cDNA sequence of the gene of interest and a poly A signal, resulting in a recombinant virus defective for replication.
In another embodiment, the nucleic acid sequence is incorporated into a self-amplifying mRNA vector (hereinafter called SAM). SAM RNA molecules are well known in the art and can be produced using replicating elements derived, for example, from alphavirus, and substituting structural viral proteins with a nucleotide sequence encoding a protein of interest . A SAM RNA molecule is usually a ± stranded molecule that can be translated directly after administration to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from 1 'RNA administered. Thus the administered RNA leads to the production of multiple daughter molecules of RNA. These daughter molecules of RNA, as well as collinear subgenomic transcripts, can be translated themselves to provide in situ expression of an antigen.
BE2017 / 5714 encoded (i.e., a HRV VP2 protein construct), or they can be transcribed to provide other transcripts with the same meaning as the administered RNA which are translated to provide expression in antigen situ. The overall result of this transcription sequence is a huge increase in the number of RNA replicons introduced and thus the encoded antigen becomes a major polypeptide product of cells.
A suitable system for obtaining self-replication in this way is to use an alphavirus-based replicon. These replicons are stranded RNAs which lead to the translation of a replicase (or of a replicase-transcriptase) after administration to a cell. Replicase is translated as a polyprotein that self-cleaves to provide a replication complex that creates copies of the genomic strand of the administered strand ± RNA. These strand ± transcripts can themselves be transcribed to give other copies of the stranded parent RNA ± and also to give a subgenomic transcript which codes for the antigen. Translation of the subgenomic transcript thus leads to in situ expression of the antigen by the infected cell. Appropriate alphavirus replicons can use a replicase from a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus, an equine encephalitis virus from the Venezuela, etc. Mutant or wild-type viral sequences can be used, for example, the attenuated mutant TC83 of VEEV has been used in replicons, see the following reference: WO 2005/113782.
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In some embodiments, the SAM RNA molecule described herein encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the SAM RNA molecule and (ii) a VP2 protein antigen of HRV as described here. The polymerase can be an alphavirus replicase, for example, comprising one or more nsP1, nsP2, nsP3 and nsP4 proteins of alphavirus.
While natural alphaviral genomes code for structural proteins of virions in addition to the nonstructural polyprotein of replicase, in some embodiments, SAM RNA molecules do not code for structural alphaviral proteins. Thus, SAM RNA can lead to the production of copies of genomic RNA of itself in a cell, but not to the production of RNA-containing virions. The inability to produce these virions means that, unlike a wild type alphavirus, the SAM RNA molecule cannot be perpetuated in an infectious form. The alphaviral structural proteins which are necessary for perpetuation in type viruses wild-type are absent from the SAM RNAs of the present disclosure and their place is taken by one or more genes coding for the immunogen of interest, such that the subgenomic transcript codes for the immunogen rather than for the structural proteins of the virions of 'alphavirus.
Thus, a SAM RNA molecule useful with the invention can have two open reading frames. The first open reading frame (in 5 ') codes for a replicase; the second open reading frame
BE2017 / 5714 (in 3 ') code for an antigen. In some embodiments, the RNA may include additional open reading frames (eg, downstream), for example, to code for other antigens or to code for accessory polypeptides.
In some embodiments, the SAM RNA molecule disclosed herein has a 5 'cap (for example, a 7-methylguanosine). This cap can amplify the in vivo translation of RNA. In some embodiments, the 5 'sequence of the SAM RNA molecule must be chosen to ensure compatibility with the encoded replicase.
A SAM RNA molecule can have a 3 'poly-A tail. It can also include a poly-A polymerase recognition sequence (for example, AAUAAA) near its 3 'end.
SAM RNA molecules can be of various lengths, but are generally 5,000 to 25,000 nucleotides in length. SAM RNA molecules are usually single stranded. Single-stranded RNA can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and / or PKR. RNA administered in double strand form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA which is formed either during the replication of a single strand RNA or within the secondary structure of a single stranded RNA.
SAM RNA can be conveniently prepared by in vitro transcription (IVT). IVT can use a template (cDNA) created and propagated as a plasmid in bacteria, or created by synthesis (by
BE2017 / 5714 example, by engineering methods of gene synthesis and / or polymerase chain reaction (PCR)). For example, a DNA-dependent RNA polymerase (such as the RNA polymerases of bacteriophages T7, T3 or SP6) can be used to transcribe SAM RNA from a DNA template. Appropriate poly-A styling and addition reactions can be used as required (although the poly-A sequence of the replicon is usually encoded within the DNA template). These RNA polymerases may have stringent requirements for the nucleotides transcribed in 5 ′ and in certain embodiments, these requirements must correspond to the requirements of the encoded replicase,
to ensure that the RNA transcribed by IVT may operate efficiently as substrate for his self-coded replicase. A SAM RNA can include (more of all
5 'cap structure) one or more nucleotides comprising a modified nucleobase. An RNA used with the invention ideally comprises only phosphodiester bonds between the nucleosides, but in certain embodiments, it can contain phosphoramidate, phosphorothioate, and / or methylphosphonate bonds.
The SAM RNA molecule can code for a single heterologous polypeptide antigen (i.e., an HRV VP2 protein antigen as described herein) or, optionally, two or more heterologous antigens linked together so that each sequences retain their identity (e.g. linked in series) when expressed as a sequence
BE2017 / 5714 amino acids. Heterologous polypeptides produced from SAM RNA can then be produced as a fusion polypeptide or modified to produce separate polypeptide or peptide sequences.
The SAM RNA molecules described herein can be engineered to express multiple nucleotide sequences, from two or more open reading frames, thus allowing coexpression of proteins, such as one or two or more HRV antigens (e.g., a or two or more HRV antigens) together with cytokines or other immunomodulators, which can enhance the production of an immune response. Such a SAM RNA molecule may be particularly useful, for example, in the production of various gene products (for example, proteins) at the same time, for example, in the form of a bivalent or multivalent vaccine.
If desired, SAM RNA molecules can be screened or analyzed to confirm their therapeutic and prophylactic properties using various methods of in vitro or in vivo analysis which are known to those of skill in the art. For example, vaccines comprising a SAM RNA molecule can be tested for their effect on the induction of proliferation or effector function of the particular type of lymphocyte of interest, for example, B lymphocytes, T lymphocytes, T cell lines, and T cell clones. For example, spleen cells from immunized mice can be isolated and the capacity of
BE2017 / 5714 Cytotoxic T lymphocytes to lyse autologous target cells which contain a SAM RNA molecule which codes for a VP2 protein of HRV as described here. In addition, the differentiation of helper T cells can be analyzed by measuring the proliferation or induction of cytokines TH1 (IL-2 and IFN-γ) and / or TH2 (IL-4 and IL-5) by an ELISA technique or directly in CD4 + T lymphocytes by cytoplasmic staining of cytokines and flow cytometry.
SAM RNA molecules that encode an HRV antigen, e.g., an HRV peptide antigen as described herein, can also be tested for their ability to induce humoral immune responses, as demonstrated, by for example, by inducing the production by B cells of antibodies specific for an HRV antigen of interest. These analyzes can be carried out using, for example, peripheral B cells from immunized individuals. Such analysis methods are known to those skilled in the art. Other assays that can be used to characterize SAM RNA molecules may involve detection of expression of the HRV antigen encoded by the target cells. For example, the FACS technique can be used to detect the expression of the antigen on the cell surface or at the intracellular level. Another advantage of selection by FACS is that different levels of expression can be sorted; sometimes a lower expression may be desired. Another suitable method for identifying cells that express a particular antigen involves the technique
BE2017 / 5714 adhesion using monoclonal antibodies on a plate or capture using magnetic beads coated with monoclonal antibodies.
The nucleic acid vaccine may include a viral or non-viral delivery system. The delivery system (also referred to herein as the delivery vehicle) can have adjuvanting effects that enhance the immunogenicity of the encoded HRV antigen. For example, the nucleic acid molecule can be encapsulated in liposomes, non-toxic biodegradable polymer microparticles or viral replicon particles (VRP), or complexed with particles of an oil-in-water cationic emulsion. In some embodiments, the nucleic acid vaccine includes a cationic nanoemulsion delivery system (NEC) or a lipid nanoparticle delivery system (NPL). In some embodiments, the nucleic acid-based vaccine includes a non-viral delivery system, i.e., the nucleic acid-based vaccine is substantially free of viral capsid. Alternatively, the nucleic acid vaccine may include particles of viral replicon. In other embodiments, the nucleic acid vaccine may include a naked nucleic acid, such as a naked RNA (e.g., mRNA), but administration via NEC or NPL is favorite.
In some embodiments, the nucleic acid vaccine includes a cationic nanoemulsion (NEC) delivery system. NEC administration systems and methods for their
BE2017 / 5714 preparation are described in the following reference: WO 2012/006380. In an NEC delivery system, the nucleic acid molecule (eg, RNA) that codes for the antigen is complexed with a particle of an oil-in-water cationic emulsion. Cationic oil-in-water emulsions can be used to deliver negatively charged molecules, such as an RNA molecule, to cells. The emulsion particles include an oily core and a cationic lipid. The cationic lipid can interact with the negatively charged molecule, thereby anchoring the molecule to the particles of the emulsion. Further details on useful CNEs can be found in the following references: WO 2012/006380; WO 2013/006834; and WO 2013/006837 (the content of each of which is incorporated herein in its entirety).
Thus, in a nucleic acid-based vaccine of the invention, an RNA molecule encoding an HRV VP2 protein antigen can be complexed with a particle of an oil-in-water cationic emulsion. The particles generally comprise an oily core (for example, a vegetable oil or squalene) which is in the liquid phase at 25 ° C, a cationic lipid (for example, a phospholipid) and, optionally, a surfactant (for example, the trioleate sorbitan, polysorbate 80); polyethylene glycol can also be incorporated. In some embodiments, the NEC includes squalene and a cationic lipid, such as 1,2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP). In some preferred embodiments, the
BE2017 / 5714 delivery system is a non-viral delivery system, such as an NEC, and the nucleic acid-based vaccine includes SAM RNA (mRNA). This can be particularly effective in triggering humoral and cellular immune responses. Benefits also include the absence of a limiting anti-vector immune response and a lack of risk of genomic integration.
NPL delivery systems and non-toxic biodegradable polymer microparticles, and methods for their preparation are described in the following references: WO 2012/006376 (NPL and microparticle delivery systems); Geall et al. (2012) PNAS USA. Sep 4; 109 (36): 14604-9 (NPL administration system); and WO 2012/006359 (microparticle delivery systems). NPLs are non-virionic liposomal particles in which a nucleic acid molecule (eg, RNA) can be encapsulated. The particles may include some external RNA (for example, on the surface of the particles), but at least half of the RNA (and ideally all of it) is encapsulated. The liposomal particles can be formed, for example, of a mixture of zwitterionic, cationic and anionic lipids which can be saturated or unsaturated, for example; DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and / or DMG (anionic, saturated). Preferred NPLs for use with the invention include an amphiphilic lipid which can form liposomes, optionally in combination with at least one cationic lipid (such as DOTAP, DSDMA, DODMA,
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DLinDMA, DLenDMA, etc.). A mixture of DSPC, DlinDMA, PEG-DMG and cholesterol is particularly effective. Other useful NPLs are described in the following references:
WO 2012/031046 WO 2011/076807 WO 2014/136086
WO 2012/030901 WO 2012/006378 WO 2013/006825 WO 2015/095346
WO 2012/006376;
WO 2012/031043;
WO 2013/033563;
WO 2015/095340;
WO 2016/037053. In certain embodiments, the NPLs are liposomes RV01, see the following references: WO 2012/006376 and Geall et al. (2012) PNAS USA. Sep 4; 109 (36): 14604-9.
Immunogenic compositions
Also provided are compositions comprising the HRV VP2 protein or nucleic acid constructs encoding such an HRV VP2 protein. The compositions can be a pharmaceutical composition, for example, an immunogenic composition or a vaccine composition. Therefore, the composition may also include a pharmaceutically acceptable carrier.
A "pharmaceutically acceptable carrier" includes any carrier which does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). These supports are well known from a
BE2017 / 5714 person of average competence in the field. The compositions can also contain a pharmaceutically acceptable diluent, such as water, physiological saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. The physiological saline buffered by sterile pyrogen-free phosphate is a conventional support.
The pharmaceutical compositions can comprise the constructs, nucleic acid sequences, and / or polypeptide sequences described elsewhere in this document in simple sterile water (for example, water for injections "ppi") or in a buffer, for example, a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. Buffer salts will generally be included in the range of 5 to 20 mM. The pharmaceutical compositions can have a pH between 5.0 and 9.5, for example, between 6.0 and 8.0. The compositions may include sodium salts (e.g., sodium chloride) to provide the tone. A concentration of 10 ± 2 mg / ml of NaCl is conventional, for example, around 9 mg / ml. The compositions can include metal ion chelators. These can prolong the stability of RNA by removing ions which can accelerate phosphodiester hydrolysis. Thus, a composition can include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are generally present at a concentration between
BE2017 / 5714 and 500 μΜ, for example, 0.1 mM. A citrate salt, such as sodium citrate, can also act as a chelator, while also advantageously providing buffering activity. The pharmaceutical compositions can have an osmolality between 200 mOsm / kg and 400 mOsm / kg, for example, between 240 and 360 mOsm / kg, or between 290 and 310 mOsm / kg. The pharmaceutical compositions can comprise one or more preservatives, such as thiomersal or 2phenoxyethanol. Mercury-free compositions are preferred, and preservative-free vaccines can be prepared. The pharmaceutical compositions can be aseptic or sterile. The pharmaceutical compositions can be non-pyrogenic, for example, containing <1 EU (endotoxin unit, a standard measurement) per dose, and preferably <0.1 EU per dose. The pharmaceutical compositions can be free of gluten. The pharmaceutical compositions can be prepared as a unit dose. In some embodiments, a unit dose can have a volume between 0.1 and 1.0 ml, for example, about 0.5 ml.
The compositions disclosed herein will generally be administered directly to a subject. Direct administration can be accomplished by parenteral injection (for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or into the interstitial space of tissue). Variant routes of administration include rectal, oral (e.g., tablet, spray), buccal, sublingual, vaginal, topical, transdermal administration
BE2017 / 5714 or transcutaneous, intranasal, ocular, auricular, pulmonary or other mucosal. Intradermal and intramuscular administration are two preferred routes. The injection can be done through a needle (for example, a hypodermic needle), but needle-less injection can be used alternatively. A typical intramuscular dose is 0.5 ml.
A human dose or an immunologically effective amount of the protein antigen may be about or less than 50 µg of HRV VP2 protein as described herein; for example, from 1 to 50 pg, such as about 1 pg, about 2.5 pg, about 5 pg, about 7.5 pg, about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg or about 50 pg. In other embodiments, a human dose of the protein antigen may be 10 to 50 pg or 20 to 50 pg. A dose of a nucleic acid (for example, a nucleic acid-based vaccine) may vary depending on the nucleic acid vector used. A human dose or an immunologically effective amount of a nucleic acid can be suitably between 1 ng and 100 mg. For example, an appropriate amount may be 1 pg to 100 mg. An appropriate amount of the particular nucleic acid (e.g., a vector) can be readily determined by those of skill in the art. Examples of the effective amount of a nucleic acid component can be between 1 ng and 100 pg, such as between 1 ng and 1 pg (for example, 100 ng to 1 pg), or between 1 pg and 100 pg, as 10 ng, 50 ng, 100 ng, 150 ng,
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200 ng, 250 ng, 500 ng, 750 ng, or 1 gg. The effective amounts of a nucleic acid can also include from 1 gg to 500 gg, such as between 1 gg and 200 gg, as between 10 and 100 gg, for example 1 gg, 2 gg, 5 gg, 10 gg, 20 gg, 50 gg, 75 gg, 100 gg, 150 gg, or 200 gg. Alternatively, an example of an effective amount of a nucleic acid may be between 100 gg and 1 mg, such as from 100 gg to 500 gg, for example, 100 gg, 150 gg, 200 gg, 250 gg, 300 gg, 400 gg, 500 gg, 600 gg, 700 gg, 800 gg, 900 gg or 1 mg.
Generally, a human dose will be in a volume between 0.1 ml and 2 ml, generally between 0.2 and 1 ml, such as 0.5 or 0.625 ml. Thus, the composition described here can be formulated in a volume, for example, of about 0.1, 0.15, 0.2, 0.5, 1.0, 1.5 or 2.0 ml of human dose by individual or combined immunogenic component. In a particular embodiment, a human dose is contained in 0.5 ml of the composition.
For any component of the immunogenic compositions disclosed herein, the dosage may vary with the condition, sex, age and size of the target subject or population and the route of administration of the immunogenic composition or vaccine.
Human rhinoviruses are the main cause of acute upper respiratory disease in humans, known as the common cold. They are also the most common viral cause of a severe exacerbation of chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). The
BE2017 / 5714 inventors have discovered that by using compositions of the invention (that is to say comprising an antigen of protein VP2), possibly adjuvanted, an immune response can be produced, which partially or totally protects the subject against subsequent infections and / or disease by the same or other types of HRV, the immune response thus exhibiting cross-reactivity. The inventors have also discovered that the immune response produced is at least mediated by cells. In another embodiment, antibodies exhibiting cross-reactivity are also induced, which may or may not be neutralizing.
In certain embodiments, the compositions disclosed herein are intended for use in a subject for: preventing HRV infection (prophylactic use), reducing the viral load of HRV infection, reducing recovery time, and / or lessen the severity of the disease caused by an HRV in this subject. The term "recovery time" refers to the reduction of time for recovery from an HRV infection. Alternatively, the compositions are intended for use in a method for reducing or preventing disease in a subject, i.e., reducing or preventing clinical symptoms during HRV infection, for example, reducing the severity of exacerbations in a subject diagnosed with asthma or COPD.
A person of average skill in the field will understand that the prevention or prophylactic use of the compositions disclosed herein is not
BE2017 / 5714 supposed to imply 100% efficiency in any given population. Rather, there are varying degrees of prevention or prophylaxis that a person of average skill in the field recognizes as having one or more beneficial effects. In this regard, the methods of the invention can provide any level of prevention or prophylaxis. The compositions described herein and their use can simultaneously prevent or reduce HRV infection and the clinical symptoms associated with HRV such as exacerbations of asthma or COPD.
In certain embodiments, the compositions disclosed herein are intended for use in a method of inducing an immune response (exhibiting cross-reactivity) against HRVs of at least three different serotypes. The immune response produced upon administration to a subject of a composition comprising a VP2 protein derived from a type of HRV belonging to HRV-A may exhibit cross-reactivity against a test of the subject with a type of HRV belonging to HRV -A, HRV-B and / or HRVC. Similarly, the immune response produced upon administration to a subject of a composition comprising a VP2 protein derived from an HRV serotype belonging to HRV-B may exhibit cross-reactivity against the subject's challenge with a type of HRV belonging to HRV-B, HRV-A and / or HRV-C, or, the immune response produced upon administration to a subject of a composition comprising a protein VP2 derived from a type of HRV belonging to HRV -C can present
BE2017 / 5714 cross-reactivity against the test of the subject with a type of HRV belonging to HRV-A and / or HRV-B.
In other embodiments of the uses and methods disclosed herein, the target population for the uses or methods disclosed herein are human patients diagnosed with COPD or asthma. The target population may be limited to human patients with COPD.
In some embodiments, methods are provided for preventing viral infection with HRV, reducing the viral load upon infection with HRV, and / or reducing or preventing clinical symptoms of infection with HRV in a human subject in need thereof, which include administering to the subject subject an immunologically effective amount of any of the immunogenic compositions as provided herein.
In some embodiments, methods are provided for inducing a cross-reactive immune response against at least three types of HRV in a human patient in need thereof, which include administering to the subject subject an immunologically effective amount of any of the immunogenic compositions as provided herein.
In certain embodiments, the use of an HRV VP2 protein as disclosed herein is provided in the manufacture of an immunogenic composition for the prevention or reduction of the duration of an HRV infection in a subject human, and / or the reduction or prevention of clinical symptoms of an HRV infection in a human subject.
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In certain embodiments, the use of an HRV VP2 protein as disclosed herein is provided in the manufacture of an immunogenic composition inducing an immune response exhibiting cross-reactivity against at least three HRV serotypes in a human subject in need of it.
In some embodiments, the subject is a human subject. In specific embodiments, the human subject is a patient with asthma, or, the human subject is a patient with COPD.
In some embodiments, the human subject is a young subject in age such as an infant, young child or child. In other embodiments, the human subject is an infant, young child, or a child who is a patient with asthma. In some embodiments, the human subject is an elderly subject, for example, aged 50 or older, aged 60 or older, or, aged 70 or older. In other embodiments, the human subject is an elderly subject who is a patient with COPD. In these embodiments, the target population is defined accordingly.
The following examples illustrate the invention.
Examples
Example 1
The objective of the experiment was to compare the quality, diversity and extent of the neutralizing antibodies induced before / after an intranasal test with HRVlb in mice sensitized with an adjuvant combination with protein ASOlb.
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VP2 and VP4 compared to mice sensitized with VP4 proteins adjuvanted with ASOlb or ASOlb alone.
1.1 Animal model
CB6 / F1 mouse strains were chosen to study the immunogenicity of candidate vaccines for HRV since these animals are capable of mimicking both humoral and cellular immune responses induced during natural HRV infections. In addition, CB6 / F1 mice are able to summarize the virological and histological signs observed in humans (recruitment of neutrophils and production of cytokines in the lungs) during a test with HRV belonging to the minor group (replicating in mice).
1.2 Experimental design
In this study, 3 groups of CB6 / F1 mice (n = 30 / group) were immunized intramuscularly twice (in the gastrocnemius muscle) on days 0 and 14 (OJ and D14) with 5 μg of:
GROUP 1 - Combination of HR239 VP2 (SEQ ID NO: 1) + concatamer of full-length clade VP4 proteins (SEQ ID NO: 3; HRV2 type specific VP4 proteins, 8, 10, 21, 39, 60, 71 , 77, CU107 and CU150) formulated in ASOlb.
GROUP 2 - Concader of VP4 proteins of the full length clade (SEQ ID NO: 3; specific VP4 proteins of the HRV2, 8, 10, 21, 39, 61, 77, CU107 and CU150 types) formulated in ASOlb
GROUP 3 - ASOlb alone as a negative control.
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ASOlb includes 3D-MPL and QS21 in liposomes containing cholesterol. A human dose (DH) of ASOlb contains 50 µg of MPL, 50 µg of QS21, in liposomes containing cholesterol.
Four weeks later (D42), the mice were subjected to a nasal test with 10 ⁶ TCID50 units of purified HRVlb virus and the response rates in CD4 + / CD8 + T lymphocytes exhibiting cross-reactivity and the quality, diversity and the extent of neutralizing antibodies was studied in serum and spleen cells collected 14 days after the second immunization (before the test) and 14 days after the HRVlb test.
Rates of positive-strand RNA genomes, production of cytokines (MCP-1, IL-6, IL-10, IL-12p70,
TNF-a, IFN-γ) and differential blood count were studied in bronchoalveolar lavage fluids (LBA) 2 days after the test with HRVlb (D44) to ensure that the test with HRVlb a been successfully obtained. The full description for each group and the immunization schedule are reported in Table 2.
Table 2
Group NOT Sensitization Calendarimmunization Calendar of the intranasal test (10 ⁶ TCID50 units) Immunogenic(5 pg / immunogen /dose) Doseadjuvant(ASOlb) 1 30 VP2 protein fromHRV39 1/10 DH Days 0, 14 Day 42
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(SEQ ID NO: 1) +concatamer ofVP4 proteins fromclade A(SEQ ID NO: 3) 2 30 Concater ofVP4 proteins(SEQ ID NO: 3) 1/10 DH Days 0, 14 3 30 No 1/10 DH Days 0, 14
N: number of mice per group; DH = human dose; NA = not applicable.
1.3 Material - Antigens and adjuvants 5 The antigens used in this experiment were produced as follows:
The HR239 protein VP2 (SEQ ID NO: 1 with a C-terminal sequence marker His GGHHHHHH in Cterminale) was expressed in the Pichia system (yeast) and purified by centrifugation on a CsCl density gradient followed by chromatography. exclusion on Sephacryl S-500 HR / concentration on Amicon and dialysis. The protein concatamer VP4 of clade A (SEQ ID NO: 3) was expressed in E. coli (BL21). The purification was carried out using a Ni-NTA GE His trap column followed by exclusion chromatography on Superdex75 using 25 mM Bicin-4 M urea buffer (4 M urea - 25 mM Bicine - 500 mM NaCl, 1% sucrose - 0.1% pluronic
F68, pH 8.0). The material was finally dialyzed in PBS buffer supplemented with 1% Empigen.
All antigens have been formulated with the adjuvant
ASOlb (1/10 DH).
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The highly purified HRVlb viral material used for the IN test was purchased from Virapur Laboratories (VIRAPUR, San Diego, CA, USA).
Example 2 - Processes
2.1 Rhinovirus neutralization test
The quantification of the neutralizing antibodies was carried out using the following neutralization test. A suspension of 5000 Hl-HeLa cells / well was seeded in 96-well flat-bottomed plates (Nunclon Delta Surface, Nunc, Denmark) and incubated for one overnight at 37 ° C with 5% CO2.
Sera were diluted by 1/2 serial dilutions (starting at 1/10) in infection medium for HRV (MEM supplemented with 2% FCS, 30 mM MgCl2, 2 mM L -glutamine, 1% non-essential amino acids and 1% penicillin / streptomycin) in 96-well plates (Nunc, Denmark) and incubated with a construct of 100 TCID50 of virus for 2 h at 37 ° C (5% CO2). The edges of the plates were not used and one column from each plate was left without serum and was used as a negative control (no neutralization). The medium of the 96-well plates seeded with Hl-HeLa cells was decanted and the virus-antibody mixtures were then deposited on sub-confluent Hl-HeLa cells and incubated at 34 ° C with 5% CO2 for 72 hours or 120 hours (depending on the HRV strains used - see Table 3).
Table 3
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Three days of infection Five days of infection (72 h) (120 h) HRVlb, 2, 3, 8, 14, 16, HRV2 5, 2 9 28, 39
Three or five days after infection, the Hl-HeLa cells were washed and incubated at 37 ° C for 8 h (5% CO2) with a solution of WST-1 (reagent for measuring cell reliability) diluted 15x ( Roche, 1164807001, lot number 12797000) in HRV revelation medium (DMEM supplemented with 2% FCS,
0 mM MgCl2, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μΜ of β-mercaptoethanol, 1% non-essential amino acids and 1% penicillin / streptomycin). The plates are then read at a wavelength of 450 nm using the Softmaxpro software.
To calculate neutralizing antibody titers, the data sets were normalized based on the mean of the WST-1 OD in the wells of "virus-free cells" and the wells of "serum-free cells" compared to 0 and 100% cytopathic effect (CPE) respectively. The percentage of inhibition of the ECP at a dilution i was then given by:
% inhibition = (D.O.i - Average D.O. of cells without serum) / (Average D.O. of cells without virus - Average of D.O. of cells without serum)
The inverse of the dilution giving a 50% reduction in the PCE was then extrapolated using a non-linear regression.
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2.2 Measurement of rhinovirus-specific CD4 + / CD8 + T lymphocytes by intracellular staining of cytokines
The frequencies of CD4 + and CD8 + T lymphocytes specific for the antigen producing IL-2, IFN-γ and / or TNF-α were evaluated by intracellular staining of cytokines (CIC) in the spleens removed. (a) 14 days after the 2 ^nd immunization (D28 before the test), and (b) 14 days after challenge by the HRVlb (D56 after 2 ^nd immunization).
Isolation of splenocytes
The spleens were collected in RPMI 1640 medium without L-glutamine supplemented with RPMI additives (= RPMI medium) and dissociated in a suspension of single cells which was then transferred to a 100 μm cell sieve and rinsed with 5 ml. from the RPMI community. The spleen cells were then centrifuged at 335 g for 10 min (4 ° C) and the pellet was resuspended in 5 ml of RPMI medium. This previous washing step was repeated once more and the final pellet was resuspended in 5 ml of RPMI medium supplemented with 5% FCS.
The cell suspension was then diluted 20x (10 µl) in PBS buffer (190 µl) for cell counting (using a MACSQuant analyzer). After counting, the cells were again centrifuged (335 g, 10 min, RT) and the cell pellet was resuspended at ¹⁰⁷ cells / ml in RPMI medium.
Cell preparation
Splenocytes were seeded in 96-well plates with rounded bottoms to approximately 1 million
BE2017 / 5714 cells per well. The splenocytes were stimulated in vitro with 100 μΐ of:
- a group of 15-mer peptides overlapping by 11 aa covering the entire amino acid sequence of the proteins VP2, VP4 of the strains of HRV2, 14 and 39 at a working concentration of 1 pg / ml per peptide
- ultracentrifuged particles (UC) of HRV3 and 28 at 1.4 x 10 ⁷ TCID50 / ml (-MOI 1)
- UC particles of HRV25 at 1.4 x 10 ⁶ TCID50 / ml (-MOI
0.1)
- uninfected cell lysate UC or medium (as negative test controls)
- PMA - ionomycin solution at working concentrations of 0.25 pg / ml and 2.5 pg / ml respectively (as a positive control for the test).
Antibodies to CD4 49d and CD28 T lymphocytes (1 pg / ml) were added and the cells were incubated for:
- 2 h at 37 ° C followed by 4 h in the presence of brefeldine (1 pg / ml) to inhibit the secretion of cytokines for stimulation in vitro using the groups of peptides originating from the proteins VP2, VP4 of the strains of HRV2, 14 and 39
- 16 h at 37 ° C followed by 4 h in the presence of brefeldine (1 pg / ml) to inhibit the secretion of cytokines for stimulation in vitro using UC viral preparations (HRV3-25-28).
Intracellular staining of cytokines (CIC)
The cell staining was carried out as follows: the cell suspensions were placed in 96-well plates with V-bottom, centrifuged (150 g,
BE2017 / 5714 min at 4 ° C), and washed in 250 μΐ of PBS, 1% FCS. The cells were again centrifuged and resuspended in 50 μΐ of PBS, 1% of FCS containing 2% of Fc blocking reagent (1/50; CD16 / 32). After 10 min of incubation at 4 ° C, 50 μΐ of a mixture of anti-CD4-V450 T lymphocyte antibodies (1/200), CD8 perCp-cy 5.5 anti-lymphocyte 5.5 (1/100) and Live & Dead POs (1/1000) were added and incubated for 30 min in the dark at 4 ° C. After washing in PBS, 1% FCS, the cells were permeabilized in 200 μΐ of Cytofix-Cytoperm (Kit BD) and incubated for 20 min at 4 ° C.
The cells were then washed with Perm Wash (Kit BD) and resuspended with 50 μΐ of anti-IFNg APC antibody (1/200) + anti-IL-2 FITC (1/400) + anti-TNFa PE (1/700) diluted in PermWash. After 1 h of incubation at 4 ° C, the cells were washed with Perm Wash and resuspended in 220 μΐ of PBS.
Acquisition and analysis of cells
The stained cells were analyzed by flow cytometry using an LSRII and the FlowJo software. Living cells were identified with Live / Dead staining and then classified by FSC / SSC and the acquisition was performed on ~ 20,000 events (CD4 + T lymphocytes). The percentages of cells producing IFN-γ + / IL-2 + +/- TNFa were calculated on populations classified as CD4 + and CD8 + T lymphocytes.
List of reagents used (reference numbers as available at the time of deposition)
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Reagents No. ofcatalog Numbers fromlot Suppliers RPMI 1640 medium 31870-025 1683050 /1734648 Gibco RPMI additives SR120 D16R001083 Internal PBS devoid of Ca ⁺ - and MG ⁺⁺ BE17512Q 5MB165 Lonza Fetal calf seruminactivated FBS-HI-12A CP15-1084 Capricorn anti-mouse CD16 / 32 553142 5154726 Comics anti-mouse CD4 V450 560468 5065793 Comics anti-CD8 PerCp Cy 5.5 frommouse 551162 5156801 Comics Live & Dead PO L34959 1733112 MolecularProbes Cytofix / Cytoperm 51-2090K2 5075560 Comics Permwash lOx cc 554723 4198590 Comics anti-IL-2 FITC mouse 554427 4344599 Comics anti-IFN-g APC mouse 554413 4226904 Comics anti-TNF-a mouse PE 554419 3018857 Comics mouse anti-CD28 553294 83839 Comics mouse anti-CD49d 553313 4105875 Comics Acetate-myristatephorbol P8139-1MG MKBS5634V Sigma (PMA) Ionomycin I0634-1MG RNBD5728 Sigma GolgiPlug (Bréfeldine) 555029 4309737 Comics EAR LOG081D D16R001486 Internal CS&T calibration balls 94851 Comics
2.3 Differential cell counts in BLA fluids
The frequencies of the leukocytes recovered in the LBAs 2 days after the test with HRVlb were evaluated by phenotyping of the immune cells by
BE2017 / 5714 using flow cytometry. A panel of antibodies conjugated to specific fluorochromes of Ly6C-FITC, SiglecF-PE, Ly6G-PerCP, CDU-PB, CD3-APC-Cy7, CD11C PECy7 was used to easily distinguish macrophages (CDllc + / CDllb- / SiglecF +), (CDllc- / CDllb + / Ly6c + / Ly6g-), (CDllb + / CDllc- / SiglecF +), neutrophils (CDllc / CDllb + / Ly6c + / Ly6g +) and lymphocytes (CDllc- / CDllb / CD3 +).
eosinophilic monocytes
The mice were sacrificed and the lungs were washed and massaged gently 3 times with 500 μΐ of 5 mM PBS of EDTA. The recovered liquid was then centrifuged (1000 g-10 min-25 ° C), and used for CBAflex and HRVlb-specific qRT-PCR tests while the leukocyte pellet was resuspended in PBS - 2 mM EDTA (supplemented with 2% FCS) and the cells were seeded in 96-well polypropylene plates (depending on the number of cells recovered - from 7.2 x 10 ³ to 1.2 x 10 ⁵ cells / well ).
The plates were then washed with PBS + 2 mM EDTA + 2% FCS and centrifuged (1000 g-5 min 4 ° C). The supernatant was removed and the pellet was resuspended in 25 μΐ of RFc blocking reagent (mouse anti-CD16 / CD32 rat antibody (2.4 G2), (reference: 553142, lot number 4198965) prediluted to 1 / 50 in PBS + 2 mM EDTA + 2% FCS and incubated for 10 min at 4 ° C.
μΐ of a mixture of antibodies conjugated to fluorochromes diluted as follows: Ly6C-FITC (1/200) (reference: 553104, lot number: 4330779) SiglëcFPE (1/150) (reference: 552126, lot number: 3277625),
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Ly6G-PerCP (1/100) (reference: 560602, lot number: 5188651), CDllb-PB (1/300) (reference: RM2828, lot number: 1642766), CD3-APC-Cy7 (1/100) (reference: 100222, lot number: B199708), CD11C-PE-Cy7 (1/400) (reference: 558079, lot number: 4286714) were then added for 30 min at 4 ° C. The plates were then centrifuged (1000 g-5 min-4 ° C), and the pellet was resuspended in PBS, and analysis of the samples was performed by flow cytometry. Living cells were classified (FSC / SSC) and acquisition was performed on ~ 100,000 events.
2.4 Quantification of cytokines secreted in LBA fluids (CBA Flex test)
The quantification of secreted cytokines (IL-6, TNF-a, IFN-γ, IL-12p-70, IL-10, MCP-1) in LBA fluids collected 2 days after the test with HRVlb was also performed using the BD ™ Cytometric Bead Array kit (CBA, BD, United States - part number 552364; lot number 5261593) following the manufacturer's instructions on undiluted samples.
Adjustment procedures for the FACS instrument were performed using the performance verification protocol (using CS&T beads) and daily cleaning. The standards were reconstituted in the test diluent (stock concentration at 50,000 pg / ml), allowed to equilibrate at room temperature for at least 15 min, mixed and serial dilutions to 1/2 were made by starting dilution from 5000 pg / ml to 5 pg / ml.
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In the 96-well plate, 50 μΐ of mixed samples from the standard curve or undiluted were added to the appropriate test plates and 50 μΐ / well of mixed capture beads (IL-6, TNF-a, IFN- γ, IL5 12p-70, IL-10, MCP-1) were added to each well to be tested. The plates were mixed for 5 minutes using a digital shaker. The plates were then incubated for 1 hour at room temperature, protected from light.
50 μΐ / well of the mixed PE detection reagent was added to each well and mixed for 5 minutes using the digital shaker. The plates were again incubated for 1 hour at room temperature, protected from light.
The plates were centrifuged at 1000 rpm for 5 min (with braking), the supernatant was removed carefully using a multichannel pipette and the beads were resuspended in 200 μΐ of washing buffer. The samples were then acquired by FACS (Fortessa) and analyzed using the FlowJo software (FCAP Array).
2.5 Detection of positive single strand RNA genomes in LBA fluids
In order to ensure that the mice were successfully tested with the HRVlb strain, the levels of genomic single-strand positive RNA for HRVlb were studied in the LBA fluids collected 2 days after the test with HRVlb. The LBA samples were centrifuged (1000 g-10 min-25 ° C) and the supernatant was used to detect / quantify the genomic RNA (positive strand) by a qRT-PCR test. ARN
BE2017 / 5714 was purified from 100 μΐ of LBA sample (50 μΐ of LBA / 50 μΐ of RNA later) using the QIAamp Viral RNA mini kit (Qiagen) 2, 6 or 14 days after 1 ' inoculation.
Genomic RNA (positive strand) was detected as follows: reverse transcription: RNA, random primer and dNTPs were heated for 10 min at 65 ° C and then placed on ice. The cDNA was synthesized with Superscript III reverse transcriptase for 50 min at 55 ° C and then heat inactivated at 70 ° C for 15 min.
Real-time PCR was performed on 2 μΐ of cDNA with 900 nM of direct primer (RV-F1), 300 nM of reverse primer (RV-R1) and 200 nM of probes (RV-Probe) using TAQMAN Gene Expression Master Mix. The conditions of the qPCR cycles were: 2 min at 50 ° C, 10 min at 95 ° C, this followed by 45 cycles of 15 s at 95 ° C and 1 min at 60 ° C.
Example 3 - Results
3.1 Quantification of the positive single-stranded RNA genome
The following results were obtained:
- high levels of positive RNA genomes (number of copies of RNA of ~ 10 ^{7/10 8} / ml of LBA) were detected in the LBA fluids of the mice subjected to the test, attesting that the test with HRVlb by IN was successfully obtained (Figure 1)
- the ability of viral particles to replicate has not been studied because of the low rate of negative strand expressed in LBA fluids.
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3.2 Differential whole blood count The inflammatory immune response was studied by counting the number of whole blood cells (lymphocytes, neutrophils, macrophages, eosinophils) recovered in LBA fluids 2 days after the test with HRVlb. The following results were obtained:
- two to five times higher levels of neutrophils (2000 to 9000 cells / ml of LBA) were detected in some mice tested with the HRVlb strain compared to mice tested with 150 mM NaCl (exp 20140293 - <2000 cells / ml of LBA) (Figure 2), suggesting that the test with HRVlb induces
15 'the infiltration of neutrophils. However, it is important to emphasize that the levels of neutrophils detected in AML fluids after the test vary from one mouse to another.
3.3 Quantification of cytokines / chemokines secreted in AML fluids using the s test
CBA Flex
The protein levels of 6 inflammatory cytokines (TNF-cc, IFN-γ, IL-6, IL-10 & IL-12p70) and the chemokines (MCP-1) were measured.
CBAflex in LBA fluids collected 2 days after the test with HRVlb. The following results were obtained:
- high levels of MCP-1 proinflammatory chemokines (> 20 pg / ml) were detected in some of the AML fluids of certain mice subjected to the test (Figure 3). It is also important to emphasize that
BE2017 / 5714 the induction of MCP-1 chemokines was variable from one mouse to another and within the same group
- none or limited levels of cytokines of the IFN-γ, TNF-α, IL-6, IL-10, IL-12p-70 type have been detected in LBA fluids (Figure 3).
3.4 Responses to rhinovirus-specific CD4 / CD8 + T lymphocytes detected in spleen cells collected before and after the test with HRVlb
HRV-specific CD4 + / CD8 + T-cell response rates were studied in spleen cells collected 14 days after the second immunization (before the test) and 14 days after the HRVlb test.
Type-specific CD4 + / CD8 + T cell responses were studied using a group _; peptides covering the entire protein sequence VP2 (HRV39) or VP4 (serotypes HRV2 and 39) while the CD4 + / CD8 + T lymphocyte responses with cross-reactivity were studied using either a group of peptides derived from HRV2 or HRV14 covering the entire sequence of proteins VP2 or VP4, ie ultracentrifuged particles (UC) of HRV3, 25 or 28 (multiplicity of infection (MOI) from 0.1 to 1 depending on the HRV strains used). The following results have been obtained.
Type-specific CD4 + T cell response
A high frequency (0.8 to 1.7%) of HRV39 VP2 specific CD4 + T lymphocyte responses was detected before the test with HRVlb in group 1 (VP2 / VP4) but not in the other groups. Interestingly, this response was stimulated (~ 2 times
BE2017 / 5714 superior) 14 days after the test with HRVlb (1.7 to 3.2%) in group 1 (Figure 4).
None or a low frequency (0.1 to 0.5%) of HRV2 / 39 VP4-specific CD4 + T cell responses was detected in all groups. No stimulation effect was detected 14 days after the test with HRVlb (Figures 5 and 6).
CD4 + T cell response with cross-reactivity
No response in CD4 + T lymphocytes with cross-reactivity against the VP4 protein of HRV14 (clade B) was detected before / after the test with HRVlb (data not shown).
CD4 + T lymphocyte responses with cross-reactivity against VP2 of HRV2 (clade A / m) or HRV14 (clade B) were already detected before the test with HRVlb in group 1 (frequency range 0.2 - 0.8%) but not in the other groups. As for the response in specific CD4 + T lymphocytes, the responses exhibiting cross-reactivity against VP2 were also stimulated (~ 4 times greater) 14 days after the test with HRVlb (1.0 to 2.9%) (Figures 7 and 8).
CD4 + T cells showing cross-reactivity against HRV25 particles (clade A / m) were already detected before the test with HRVlb in group 1 (frequency range 0.5 to 1.5%) but not in the other groups. A response-stimulating effect was detected 14 days after the challenge with HRVlb (Figure 9).
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None or low levels of CD4 + T lymphocytes with cross-reactivity (<0.2%) against particles of HRV3 (clade B) and 28 (clade A / M) were detected before the test with HRVlb. The CD4 + T lymphocyte responses against the HRV3 (0.25 to 0.5%) or HRV28 (0.35 to 1%) strain were stimulated 14 days after the test with HRVlb in group 1 but not in the other groups (Figures 10 and 11).
CD8 + T cell responses
No CD8 + T lymphocyte response was detected before / after the HRVlb test following in vitro stimulation with peptides derived from VP2 / VP4 from HRV2 / 14/39 or UC particles from HRV3 and 28 ( data not shown).
HRV25-specific CD8 + T-cell responses were detected before the HRVlb test in group 1 and group 2. The frequency of this CD8 + T-cell response was stimulated 14 days after the HRVlb test ( 0.4 - 1%) but only in group 1 (Figure 12).
3.5 Measurements of HRV-specific neutralizing antibody responses in serum samples taken before / after the HRVlb test
The levels of neutralizing antibodies specific for HRV were studied in sera from grouped mice (5 or 7 groups of 3 mice / group) taken 14 days after the second immunization (before the test) or 14 days after the test with HRVlb. The neutralizing activity was tested against the following strains:
Clade A / m: HRVlb, 2, 25, 29
Clade A / M: HRV8, 16, 28, 39
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Clade B: HRV3 and 14
The following results were obtained:
- no neutralizing antibody (Acn) against the strains of HRV2, 3, 8, 14, 16, 25, 28, 29, 39 was detected before and after the test with HRVlb (data not shown)
- higher levels of Acn specific to the test virus serotype (HRVlb) were detected 14 days after the test in the sera of group 1 mice compared to the other groups (RMG of 13.00 between groups 1 and 2, RMG of 6.99 between groups 1 and 3) (Figure 13). This indicates that sensitization with the protein VP2 of HRV39 amplifies the production of Acn against infection with the hetero-subtype strain j (HRVlb).
Example 4 Study of Immunogenicity in Mice i
A study of immunogenicity in mice has been initiated with the recombinant protein VPO, VP2, or VP4 j j
HRV39, adjuvanted with ASOlB. The main objective) of this study was to demonstrate homologous and heterologous antigen-specific T lymphocyte responses in mice vaccinated with the (recombinant OPV, VP2 or VP4 HRV39 protein using a (HRV39 test). intracellular staining of cytokines. *
4.1 Materials and processes
Five groups of female CB6F1 mice (6 to 8 weeks old) were immunized on days 0 and 28 by intramuscular injection with either physiological saline (control) or the recombinant protein
HRV39 OPV adjuvanted with ASOlB, the protein!
I
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HRV39 VP2 adjuvanted with ASOlB, either HRV39 VP4 protein adjuvanted with ASOlB, or live HRV39 virus (Virapur).
On day 42, serum was produced from all mice for serological analysis and spleens were harvested from 6 mice per group for analysis of immunogenicity by intracellular staining of cytokines. The splenocytes were incubated overnight with groups of peptides (15-mer with overlaps of 11 amino acids) of VP2 or VP4 of five types of HRV (HRV39, HRVlb, HRV2, HRV14, and HRV89) this followed by incubation for 4 hours with brefeldine A. The cells were stained for viability, fixed, permeabilized, and then stained with fluorescently labeled antibodies directed against CD3, CD4, CD8, CD44, IFN-γ, TNF- cc, IL-2, CD107a, IL-13, IL-4, IL-17A, and IL17F. Data was acquired using a BD Fortessa flow cytometer and analyzed using FLOWJO X before being graphed in GraphPad Prism.
The remaining mice were immunized intranasally with the live HRVlb virus, with the exception of the control group treated with physiological saline, on day 56. The spleens and the serum will be collected on day 70 of the study for another analysis. immunological.
4.2 Results
In the splenocytes collected on day 42, the antigen-specific CD4 + T lymphocyte responses were detected in the mice immunized with HRV39 VP2 or OPV as described in 4.1, above, in
BE2017 / 5714 response to stimulation with a group of HR2-homologous VP2 peptides (Figure 14A) and with groups of heterologous VP2 peptides from HRV1B (Figure 14B), HRV2 (Figure 14C), HRV89 (Figure 14D) and HRV14 (Figure 14E). In Figures 14A to 14E, the data displayed represents individual mice (n = 6 per group) with the median indicated by a horizontal line. Moving from left to right in each of Figures 14A to 14E, the columns show the data from mice immunized with HRV39 VP2 (circle), HRV39 VP4 (square), HRV39 VPO (triangle), live HRV39 virus (diamond), and physiological saline (hexagon), respectively. Please note the differences in upper limit on the ordinate.
Splenocytes from mice immunized with HR439 VP4 produced little to no IFN-γ in response to stimulation with homologous (HRV39) or heterologous (HRV1B, HRV2, HRV89, or HRV14) groups of VP2 peptides (Figure 15). Few responses to antigen-specific CD8 + T cells were detected following stimulation with groups of homologous or heterologous peptides (data not shown).
Little to no IFN-γ was produced by CD4 + CD44 + T lymphocytes from the splenocytes of mice immunized with VP2, VP4 or OPV of HRV39 in response to stimulation with VP4 peptides of homologous (15A) or heterologous HRV types (15A). 15B, 15C, 15D, 15E). The data displayed represents individual mice (n = 6 per group) with the median indicated by a horizontal line. Moving from left to
BE2017 / 5714 right in each of FIGS. 15A to 15E, the columns show the data coming from the mice immunized with VP2 of HRV39 (circle), VP4 of HRV39 (square), OPV of HRV (triangle), the live HRV39 virus (diamond) , and physiological saline (hexagon), respectively.
Alignment of the amino acid sequence of VP2 of the five types of HRV used in this study was done to identify regions, showing a high degree of amino acid identity as potential regions of cross-reactivity. Figure 16A provides an overview of the extent to which each amino acid is conserved within the five types of HRV included. The height of the bar on the "identity" line is directly related to the degree to which this amino acid is stored. Multiple regions of high homology which will potentially be responsible for the phenotype of CD4 + T cells exhibiting cross-reactivity are detected. Figure 16C provides text alignment of the complete VP2 sequences of these five types of HRV (HRV_39-VP2 (SEQ ID NO: 1), HRV_89-VP2 (SEQ ID NO: 15), HRV_1B-VP2 (SEQ ID NO: 16), HRV_02-VP2 (SEQ ID NO: 17), and HRV_14-VP2 (SEQ ID NO: 18)).
As shown in Figure 16B, there is a high degree of amino acid identity when comparing sequences among HRV type A (HRV39, HRV89, HRV1B, and HRV2) as opposed to the degree of identity amino acids when comparing each of these types A to HRV14 type B (Figure 16B).
On day 70 of the study, the antigen-specific T lymphocyte responses expected against HRV
BE2017 / 5714 homologous (HRV39 and / or HRVlb) and heterologous (HRV2, HRV89 and / or HRV14) will be quantified.
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SEQ ID NO: 1 - VP2 of wild type HRV39
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FITVSI3PMF SEFSGARARP ÄAAT -2 € 4
SEQ ID NO: 2 - VP2 of the mutated HRV3 9 FT7EA3FE3 ΓΡΙΙΟΖΙΡΖΖ · STZZ ; JZTAB AWGEG7ÄPH EZTAZZASÄZ ZKPT ^ FZTSÎ
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SEQ ID NO: 3 - Full length VP4 protein concater of clade A
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Uniprot Q5XLP5
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SEQ ID NO: 6 - HRV peptide derived from VP1 of HRV8 (AM)
PAL Σ ΆΑΞ-Τ RH T S S7
SEQ ID NO: 7 - HRV peptide derived from HRV25 VP1 (A-M)
PILDAÄETGH Ï3W
SEQ ID NO: 8 - HRV peptide derived from VP1 of HRVC026 CAXGÄVEIGÄ
SEQ ID NO: 9 - HRV peptide derived from VP4 of HRV14 (B)
GAQVSTQPSG SHEXQN
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SEQ ID NO: 10 - HRV peptide derived from HRVIOO VP4 (Ά-Μ)
GÄQVSSyWG THSTQM1 5
SEQ ID NO: 11 - HRV peptide derived from VP4 of ^HRV _ ^C _ ⁰²⁶ gaqvsrqst: - shetmi ¹⁰ SEQ ID NO: 12 - HRV peptide derived from VP2 of HRV2 DÄlKDMGIFG ENMFÏ.HYLGR S
SEQ ID NO: 13 - HRV peptide derived from HR2 VP2
ZREEQIASÄL HGiü / NVZVNY EHRGEIGREV E 15
SEQ ID NO: 14 - HRV peptide derived from HR2 VP2
LEARN EZ’MCAEFSGÄ EAHRQGLP7F I
SEQ ID NO: 15 - VP2 of HRV 89
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SEQ ID NO: 17 - VP2 of HRV 02
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5 T 7EA .-ÏS _ F.II TF.-1 '3 ___ Ά. -, ΆΠ AI 'AOrUH I_iSr. £ A3AI _ . Ε3 <^ 3— c;
SEQ ID NO
- VP2 of HRV 14

PFFIPIAFLIV
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权利要求:
Claims (26)
[1]
1. Immunogenic composition comprising a human rhinovirus VP2 protein (HRV) and an adjuvant, or, a polynucleotide comprising a nucleic acid sequence encoding said HRV VP2 protein.
[2]
2. The immunogenic composition according to claim 1, in which the HRV VP2 protein is the HRV VP2 protein of HRV39, HRVlb, HRV2, HRV3, HRV14, HRV25 or HRV28.
[3]
3. The immunogenic composition according to claim 1 or 2, wherein the composition does not comprise a protein VP4 of HRV, or, of a polynucleotide comprising a nucleic acid sequence coding for a protein VP4 of HRV.
[4]
4. Immunogenic composition according to any one of claims 1 to 3, in which the HR2 VP2 protein comprises an insertion, a substitution or a deletion of up to and including 20 amino acids.
[5]
5. Immunogenic composition according to claim 4, in which the insertion, substitution or deletion is localized at the amino acid position corresponding to aal55 to 170 (NIm-II loop), aal34 to 146, aa232 to 238 , or aa72 to 75 of VP2 of HRV 39 (SEQ ID NO: 1).
[6]
6. Immunogenic composition according to claim 4 or 5, in which the HR2 VP2 protein comprises an insertion or a substitution in its amino acid sequence of an HRV peptide capable of inducing an immune response exhibiting cross-reactivity and / or cross neutralization.
[7]
7. Immunogenic composition according to any one of claims 1 to 6, in which the protein VP2
BE2017 / 5714 of HRV comprises an insertion or a substitution in its amino acid sequence of a peptide chosen from (a) peptides corresponding to amino acids 32 to 45 of VP1, (b) a variant of amino acids 32 to 45 of VP1 comprising 1 to 4 additions or deletions of amino acids at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence, (c) a peptide comprising a sequence chosen from SEQ ID NO: 5, 6, 7, and 8, (d) peptides corresponding to amino acids 1 to 16 of VP4, (e) a variant of amino acids 1 to 16 of VP4 comprising 1 to 4 additions or amino acid deletions at either end and / or 1 or 2 substitutions or additions or deletions of amino acids within the peptide sequence, or (f) a peptide comprising a sequence chosen from SEQ ID NO: 9, 10 and 11.
[8]
8. Immunogenic composition according to any one of claims 1 to 7, in which at least 5 amino acids are deleted from the NIm-II region of the protein VP2.
[9]
9. Immunogenic composition according to any one of claims 1 to 8, comprising a VP2 protein of human rhinovirus (HRV) and an adjuvant comprising a saponin.
[10]
10. The immunogenic composition according to claim 10, in which the adjuvant further comprises a lipopolysaccharide A.
[11]
11. The immunogenic composition according to claim 11, in which the saponin is QS21 and / or the lipopolysaccharide A is 3D-MPL.
BE2017 / 5714
[12]
12. Immunogenic composition according to any one of claims 9 to 11, in which the adjuvant further comprises a sterol, such as cholesterol and / or a cholesterol derivative.
[13]
13. Immunogenic composition according to any one of claims 9 to 11, in which the adjuvant further comprises liposomes.
[14]
14. Immunogenic composition according to any one of claims 1 to 8, wherein the composition comprises the polynucleotide.
[15]
15. The immunogenic composition according to claim 9, in which the nucleic acid sequence coding for the protein VP2 of HRV is placed under the control of the elements necessary for its expression in a mammalian cell.
[16]
16. Immunogenic composition according to any one of the preceding claims, in which the nucleic acid sequence is administered in a viral vector, such as an adenoviral vector.
[17]
17. The immunogenic composition according to claim 16, in which the polynucleotide is a self-amplifying mRNA vector (SAM vector).
18. Composition immunogenic according to 1 'a any of claims previous, for a use in prevention or improvement of a disease or of symptoms of a caused disease from where associated with a infection with HRV in a subject • 19. Composition immunogenic according to 1 'a any of claims previous, for a use
in a subject to reduce recovery time from a subject's HRV infection and / or for
BE2017 / 5714 lessen the severity of the disease caused by a subject's HRV infection.
[18]
20. The immunogenic composition according to any one of the preceding claims, for use in a subject to reduce or prevent clinical symptoms during an HRV infection of the subject.
[19]
21. Immunogenic composition according to any one of the preceding claims, for use in a subject for inducing an immune response exhibiting cross-reactivity against at least three serotypes of HRV, as when at least one of the at least three serotypes of HRV belongs to HRV type A and at least one of the at least three serotypes of HRV belongs to HRV type B or HRV type C.
[20]
22. Immunogenic composition for use according to any one of the preceding claims, in which the subject suffers from COPD or asthma.
[21]
23. The immunogenic composition according to any one of claims 21 or 22, wherein the cross-reactive immune response is a cell-mediated immune response.
[22]
24. The immunogenic composition according to any one of claims 21 to 23, wherein the cross-reactive immune response is further characterized by the production of cross-reactive antibodies.
[23]
25. The immunogenic composition of any one of claims 21 to 24, wherein the immune response is stimulated after subsequent exposure to an HRV.
BE2017 / 5714
[24]
26. Method for reducing the recovery time from an HRV infection in a subject in need thereof and / or for reducing the severity of a disease caused by an HRV infection in a subject in need thereof , which comprises administering to said subject an immunologically effective amount of an immunogenic composition according to any one of claims 1 to 17.
[25]
27. A method of reducing or preventing clinical symptoms during an HRV infection in a subject in need thereof, which comprises administering to said subject an immunologically effective amount of an immunogenic composition according to any one of claims 1 to 17.
[26]
28. A method of inducing an immune response exhibiting cross-reactivity against at least three serotypes of HRV in a subject in need thereof, which comprises administering to the said subject an immunologically effective amount of an immunogenic composition according to the invention. 'Any one of claims 1 to 17.
BE2017 / 5714

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法律状态:
2018-08-29| FG| Patent granted|Effective date: 20180702 |

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2020-08-13| MM| Lapsed because of non-payment of the annual fee|Effective date: 20191031 |

优先权:

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申请号 | 申请日 | 专利标题

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GB1616904.7|2016-10-05|

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GBGB1616904.7A|GB201616904D0|2016-10-05|2016-10-05|Vaccine|

---