ANTIGENIC CONSTRUCTS OF ZIKA VIRUS

专利摘要:
Compounds useful as components of immunogenic compositions for inducing an immunogenic response in a subject against viral infection, methods for their use in a treatment, and methods for their manufacture are provided herein. The compounds comprise a nucleic acid construct comprising a sequence that encodes a Zika virus antigen.
公开号:BE1024796B1
申请号:E2017/5392
申请日:2017-06-01
公开日:2018-07-10
发明作者:Mayuri Sharma；Dong Yu
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

(73) Holder (s):
GLAXOSMITHKLINE BIOLOGICALS SA
1330, RIXENSART
Belgium (72) Inventor (s):
SHARMA Mayuri 02139 CAMBRIDGE United States
YU Dong
20850 ROCKVILLE United States (54) ANTIGENIC CONSTRUCTIONS OF ZIKA VIRUS (57) Compounds useful herein are provided as components of immunogenic compositions for inducing an immunogenic response in a subject against viral infection, methods for their use in a treatment, and methods for their manufacture. The compounds include a nucleic acid construct comprising a sequence which codes for a Zika virus antigen.
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BELGIAN INVENTION PATENT
FPS Economy, SMEs, Middle Classes & Energy
Publication number: 1024796 Deposit number: BE2017 / 5392
Intellectual Property Office International Classification: A61K 39/12 A61K 39/39 C07K 14/18 C12N 15/86 A61K 39/00 Date of issue: 07/10/2018
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 01/06/2017.
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: ANTIGENIC VIRUS CONSTRUCTIONS
ZIKA.
INVENTOR (S):
SHARMA Mayuri, 40 Landsdowne Street, 02139, CAMBRIDGE;
YU Dong, 14200 Shady Grove Road, 20850, ROCKVILLE;
PRIORITY (S):
06/02/2016 US 62344417;
09/15/2016 US 62394769;
04/13/2017 US 62485081;
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, 07/10/2018, By special delegation:
BE2017 / 5392
ANTIGENIC CONSTRUCTIONS OF ZIKA VIRUS
FIELD OF THE INVENTION
This invention is in the field of treatment and prevention of viral infections. In particular, the present invention relates to vaccine constructs based on nucleic acids encoding Zika virus antigens and the use of Zika virus antigens for the treatment and prevention of infections due to Zika virus.
TECHNOLOGICAL BACKGROUND OF THE INVENTION
Zika virus was first identified in Uganda in 1947 in Rhesus monkeys through a yellow fever surveillance network. It was then identified in humans in 1952 in Uganda and the United Republic of Tanzania. . Outbreaks of Zika virus disease have been recorded in Africa, the Americas, Asia and the Pacific. The Zika virus belongs to the genus flavivirus. Its reservoir is unknown.
Zika virus is a positive strand RNA virus belonging to the family of Flaviviridae. Disease
BE2017 / 5392 due to the Zika virus is caused by a virus transmitted mainly by Aedes mosquitoes. People suffering from Zika virus disease may have symptoms which may include a mild fever, rash, conjunctivitis, muscle and joint pain, feeling sick or headache. These symptoms normally last for 2 to 7 days.
The Zika virus is known to circulate in Africa, the Americas, Asia and the Pacific. Transmitted by Aedes mosquitoes, the virus has been known to cause either an asymptomatic infection (in the majority of infected people) or a spontaneously resolving disease with a decrease in rash, conjunctivitis and mild fever. However, during the current Zika virus epidemic in the Americas, there has been an alarming increase in the number of babies born with microcephaly, as well as an increase in the incidence of Guillain-Barré syndrome. In addition to microcephaly, other fetal malformations and neurological disorders have been described.
Dowd et al. (Science, Vol. 354 Issue 6309, pp. 23740 (2016) recently reported that DNA vaccines expressing the premembrane and Zika virus envelope proteins were immunogenic in mice and non-human primates when administered by electroporation or injection without a needle; and this protection against viraemia after a test with the Zika virus correlated with the neutralizing activity of the serum.
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Chahal et al. (Scientific Reports, 7: 252, pp. 1-9 (2017)) describe an alphavirus RNA vector encoding structural antigens of the Zika virus. When formulated with modified dendrimer material and administered to mice intramuscularly, the vaccine has been found to be immunogenic.
Richner et al. (Cell, 168, pp. 1-12, (2017)) describe a modified mRNA vaccine encoding wild-type or mutant Zika virus structural proteins. When encapsulated in lipid nanoparticles and intramuscularly in mice, the vaccine elicited high titers of neutralizing antibodies and protection against viral testing.
Given the worrying burden of the disease and the potential for rapid spread, there is an urgent need for the development of components for use in an immunogenic or vaccine composition against Zika virus.
administered via mRNA a
SUMMARY OF THE INVENTION
The present inventors provide constructs useful as components of immunogenic compositions for inducing an immune response in a subject against infection with Zika virus, methods for their use in treatment, and methods for their manufacture.
In some embodiments, there is provided a nucleic acid-based vaccine construct encoding a polypeptide comprising a preBE2017 / 5392 antigen
Full-length M-E (prME) of the Zika virus, or one of its immunogenic fragments.
In some embodiments, a vector is provided comprising the construct as described.
In some embodiments, there is provided a self-replicating RNA molecule (also referred to herein as a self-amplifying mRNA molecule, or SAM) comprising the construct as described.
In certain embodiments, there is provided a composition comprising an immunologically effective amount of one or more of the self-replicating RNA constructs, vectors, or molecules as described above.
In certain embodiments, there is provided a composition as described above in which the composition comprises an RNA-based vaccine.
In certain embodiments, there is provided a composition as described above in which the composition comprises one or more self-replicating RNA constructs, vectors, or molecules as described above complexed with a particle of an oil-in-water cationic emulsion.
In certain embodiments, there is provided a composition as described above for use in inducing an immune response against Zika virus infection in a subject in need thereof.
In some embodiments, there is provided a self-replicating RNA construct, vector, and / or molecule as described herein for use in therapy or medicine. In some modes
BE2017 / 5392 embodiment, the compositions disclosed here are intended for use in therapy or medicine. In a preferred embodiment, the therapy is vaccine therapy. Preferably, the therapy is a vaccine to prevent infection with the Zika virus.
In some embodiments, there is provided a self-replicating RNA construct, vector, and / or molecule as described herein for use in the prevention or treatment of Zika virus infection in a subject in need. In certain embodiments, one of the compositions disclosed herein is intended for use in the prevention or treatment of Zika virus infection in a subject in need thereof.
In certain embodiments, there is provided a method for inducing an immune response against Zika virus infection in a subject in need thereof, which comprises administering to said subject an immunologically effective amount composition comprising one or more self-replicating RNA constructs, vectors, or molecules as described above.
In certain embodiments, there is provided a method for inducing an immune response sufficient to prevent or treat a Zika virus infection in a subject, which comprises administering to the subject a composition comprising a ( e) or more of the self-replicating RNA constructs, vectors, or molecule as described above in an amount sufficient to prevent or treat infection with the Zika virus.
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In some embodiments, there is provided a method as described above in which the composition comprises one or more self-replicating RNA constructs, vectors, or molecules as described above complexed with a particle of an oil-in-water cationic emulsion.
In certain embodiments, there is provided a process for the production of an RNA-based vaccine comprising a step of transcribing a vector or a DNA molecule encoding a self-RNA molecule replicating as described above to produce an RNA comprising a coding region for the antigen.
In certain embodiments, there is provided a process for preparing a composition as described above in which the process comprises 1) preparing a cationic oil-in-water emulsion; 2) the preparation of one or more self-replicating RNA constructs, vectors, or molecules as described above; and 3) adding one or more constructs, vectors, or RNA molecules that self-replicate to the cationic oil-in-water emulsion such that the construct, vector, or molecule of self-replicating RNA complexes with the emulsion.
In some embodiments, there is provided a composition produced by the method described above.
In some embodiments, use is made of the construct, the vector, the self-replicating RNA molecule, or the composition described above for inducing a response.
BE2017 / 5392 immune against infection by the Zika virus in a subject.
In certain embodiments, there is provided a use of the construct, vector, self-replicating RNA molecule, or composition described above in the manufacture of a medicament which induces an immune response against a Zika virus infection in a subject.
DESCRIPTION OF THE FIGURES
Figure 1 - Organization of the Flavivirus genome, showing that the polyprotein is cleaved into structural and non-structural proteins by a combination of viral and cellular proteases.
Figure 2 - Formation of virions and subviral particles of Flavivirus. (1) In natural infections, flavivirus proteins are produced by the processing of a polyprotein translated from viral genomic RNA and inserted cotraductionally into the membrane of the endoplasmic reticulum (ER). The horizontal arrows indicate the cleavage of the polyprotein by the peptidase signal and the arrowheads indicate the cleavage by the viral protease NS2B-3. The empty arrow indicates signalase cleavage which is ineffective unless cleavage of the cytoplasmic capsid (C) has occurred. (2) The minimum requirement for the production of sub-viral particles is the precursor proteins of the membrane (prM) and of the envelope (E). (3) The particles of flavivirus are formed by budding on the membrane of the ER directed by the proteins prM and E
BE2017 / 5392 independent of protein C or preformed nucleocapsides. Viral infection mainly results in the formation of virions. (4) Pseudoviral particles devoid of nucleocapsides are efficiently produced by recombinant expression of the proteins prM and E and are a by-product of infection by a flavivirus. (5) Virions and pseudoviral particles follow the exocytic pathway for secretion from infected / transfected cells. "Cy" indicates the cytoplasmic side of the ER membrane.
Figures 3A to D - Alignment of multiple CLUSTAL 0 (1.2.1) sequences of Zika virus CprME proteins. See the sequences here and SEQ ID NO: 2 and SEQ ID NO: 15 to 23.
Table 1
Zika virus strain, year, and Genbank reference
STRAIN YEAR GENBANK NUMBER Uganda NC 012532 Micronesia 2007 EU545988.1 Natal (Brazil) 2016 KU527068 Salvador (Brazil) 2016 KU707826.1 Sao Paulo (Brazil) 2016 KU321639 French Polynesia 2013 KJ776791
Figures 4A to C - Multiple CLUSTAL 0 sequence alignment (1.2.1) of CprME proteins from Brazilian strains of the Zika virus: Natal (SEQ ID NO: 2);
Salvador (SEQ ID NO: 15); No. of accession Genbank KU365777 (SEQ ID NO: 20); No. of accession Genbank KU365778 (SEQ ID NO: 21); No. of accession Genbank
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KU365779 (SEQ ID NO: 22); Genbank accession number KU365780 (SEQ ID NO: 23); and Sao Paulo (“Sao”) (SEQ ID NO: 16). See Table 1 for Zika virus strains, year, and Genbank reference.
Figure 5 - SAM-Zika construction. The context of self-amplifying mRNA (SAM) consists of the VEE TC-83 replicon encoding non-structural viral proteins 1 to 4 (nsP1 to 4), followed by the subgenomic promoter, and either the prME of Zika is the CprME of Zika. The empty vector is represented by SEQ ID NO: 24; The insert starts immediately after nucleotide 7561.
Figure 6 - Design of a Zika-SAM construct coding for prME viral proteins. The diagram shows the region of the prM signal sequence of the prM gene, which contains basic residues in the NH2-terminal region (region n), a hydrophobic nucleus uninterrupted by charged or polar residues (region h), and a motif of amino acids -3, -1 in the COOH-terminal cleavage region (region c) suitable for the recognition of signalase. The first construct is a prME which has been optimized for codons for expression in mammalian cells. An unusual feature of the flavivirus prM signal peptide is the lack of residue
polar in the region vs. Previously, he been shown that replacement of Gly, Phe and Ala at level of positions -5, -4 and -2 per Pro, Gin and Gin, respectively (mutation PQAQA), increases
significantly the extent of prM signalase cleavage in vitro without the requirement for prior cleavage from C. Stocks, et al. 1998. Signal peptidase cleavage at
BE2017 / 5392 the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM. J. Virol. 72: 2141-2149.
The second construct - ESS.l, includes the PQAQA mutation. The third construct - ESS.2, has the native prM signal sequence replaced by the IgG signal peptide, which was previously used for the expression and secretion of IgG and Fab proteins from mammalian cells. Ciferri et al. (2015) Antigenic Characterization of the HCMV gH / gL / gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies, PLoS Pathog. Oct 20; 11 (10): el005230.
Figure 7 - Zika-SAM construction design coding for the capsid viral proteins and prME. The Zika virus capsid protein (C) is incorporated into constructs to test whether the presence of a cleavable capsid protein increases the efficiency of PSV production. CprME.1 is a nucleic acid sequence optimized for codons encoding the native capsid proteins, prM and E of the Zika virus, with the native cleavage sites for NS2B-3 and signalase. CprME.2 is identical to CprME.1, but contains the mutation -PQ-Q in the signal peptide region of C. Finally, CprME.3 is identical to CprME.1, except that a P2A sequence is inserted at the level of cleavage site for NS2B-3.
Figure 8 - Analysis of the expression and secretion of protein E from Zika-SAM constructs. Protein E expression has been detected
BE2017 / 5392 by immunoblot in cell lysates for all the Zika-SAM constructs tested (see Table 2: wild-type prME ("WT"; construction No. 1), prME optimized for codons ("CO", construction No . 2), amplified -PQ-Q signal sequence ("ESS.l", construct No. 3), IgG signal sequence ("ESS.2", construct No. 4), and the three capsid constructs. : CprME.1 (construction No. 5), CprME.2 (construction No. 6) and CprME.3 (construction No. 7). As a positive control, a SAM construction which expresses protein E of another flavivirus , yellow fever virus, was included ("SAM-YFV"). Negative controls included a SAM-respiratory syncytial virus ("SAM-RSV") construct and a pseudotransfection ("Nickname").
Secretion of protein E in cell supernatants has also been detected by immunoblotting for at least construction No. 1 ("WT"), construction No. 2 ("CO"), construction No. 4 (ESS.2 ), and construction No. 7 (CprME.3).
Figure 9 - Immunoblots of wild-type cell culture supernatants ("WT") and optimized for codons ("CO") after concentration with a cutoff of 100 kDa show that protein E of the Zika virus is present in a high molecular weight which is retained in the column but not in the fraction not retained. This result suggests that the secreted protein E forms complexes of a higher order than the monomers or dimers, which is consistent with the hypothesis of PSV.
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Figure 10 - Neutralizing antibody responses. It was discovered that the mice have significant neutralizing antibodies against the Zika virus two weeks after a single vaccination with the constructs Zika-SAM No. 1 or No. 2 (CO.prM-E and WT.prM-E, respectively) , or with the construct of positive control Zika virus DNA No. 5283, as measured by analysis of neutralization of reporter viral particles (PVR). The neutralizing antibody titers increased further two weeks after a second vaccination with the same ZikaSAM construct or the positive control. A dose-response effect was observed for the constructions of SAM No. 1 and No. 2, with 15 μg of RNA triggering more neutralizing antibodies than 1.5 μg of RNA.
Figure 11 - Protection against a test with the Zika virus. Mice vaccinated on days 0 and 21 were tested with live Zika virus on day 49. The viral load was measured 3 days after the test. Vaccinations with the constructs of SAM No. 1 and No. 2 (at doses of 1.5 pg and 15 pg), as well as the positive control (Zika virus DNA No. 5283) were protective against the viremia of the virus Zika, compared to unvaccinated mice. The dotted line indicates the limit of quantification (LOQ) of the analysis.
BE2017 / 5392
DETAILED DESCRIPTION OF THE INVENTION
Antigens; variants; fragments; and constructions
The present inventors provide constructs useful as components of immunogenic compositions for inducing an immune response in a subject against infection with Zika virus, constructs useful for expression of antigens, methods for their use in a treatment, and methods for their manufacture. By "construction" is meant a nucleic acid which codes for the polypeptide sequences described herein, and may include DNA, RNA, or non-naturally occurring nucleic acid monomers. The nucleic acid components of the constructs are described more fully in the Nucleic acids section here.
In some embodiments, the constructs disclosed herein encode wild type polypeptide sequences from a Zika virus, or one of their variants, or fragments. The constructs may further code for a polypeptide sequence heterologous to the polypeptide sequences of a Zika virus. In certain embodiments, the constructs code for wild type polypeptide sequences of a Brazilian strain Zika virus, or one of their variants, or fragments. By "Zika virus of Brazilian strain" is meant any strain of Zika virus indicated as "Brazilian" in Table 1. Unless otherwise indicated, the descriptions of the prME wild-type antigen are made with reference to the strain of Natal (Brazil), number
BE2017 / 5392
GenBank KU527068.1, as represented by SEQ ID NO: 1 (nucleic acid) and SEQ ID NO: 2 (polypeptide), and as illustrated in Figures 3A to D, and Figures 4A to C.
A "variant" of a polypeptide sequence comprises amino acid sequences comprising one or more substitutions, insertions and / or deletions of amino acids compared to the reference sequence. The variant may include an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at at least 97%, at least 98%, or at least 99% to a full-length wild-type polypeptide, for example, to a polypeptide according to SEQ ID NO: 2. Alternatively, or additionally, a fragment of a polypeptide can comprising an immunogenic fragment (i.e., an epitope-containing fragment) of the full-length polypeptide which may include a contiguous amino acid sequence of at least 8, at least 9, at least 10 , at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at minus 19 or more amino acids that is the same as a contiguous amino acid sequence of the full-length polypeptide.
A fragment of a polypeptide can comprise N- and / or C-terminal deletions compared to a full-length polypeptide, for example SEQ ID NO: 2, in which the fragment comprises a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acids from the N-terminal end, the Cterminal end, or both from the N-terminal end and from
BE2017 / 5392 the C-terminus of the full-length sequence. It can be specified that the deletions are consecutive amino acids.
As used herein, the term "antigen" refers to a molecule containing one or more epitopes (eg, linear, conformational or both) which will stimulate the immune system of a host to produce a specific immunological response to the humoral and / or cellular antigen (i.e., an immune response which specifically recognizes an antigenic 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). See the following references: Geysen et al. (1984) PNAS USA 81: 3998-4002; Carter (1994) Methods Mol Biol 36: 207-23. They can be predicted (for example, using the Jameson-Wolf antigenic index (see Jameson et al. (1988) CABIOS 4 (1): 181-186)), matrix-based approaches (see Raddrizzani & Hammer ( 2000) Brief Bioinform 1 (2): 179-89), TEPITOPE (see De Lalla et al. (1999) J. Immunol. 163: 172529), neural networks (see Brusic et al.
(1998) Bioinformatics 14 (2): 121-30), OptiMer and EpiMer (see Meister et al. (1995) Vaccine 13 (6): 581-91; see Roberts et al. (1996) AIDS Res Hum Retroviruses 12 ( 7): 593-610), ADEPT (see Maksyutov & Zagrebelnaya (1993) Comput Appl Biosci 9 (3): 291-7), Tsites (see Feller & de la Cruz (1991) Nature 349 (6311): 720-1 ),
Hydrophilicity (see Hopp (1993) Peptide Research 6:
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183-190), the antigenic index (see Welling et al. (1985) FEBS Lett. 188: 215-218) or the methods disclosed in the reference Davenport et al. (1995) Immunogenetics 42: 392-297, etc.).
In some embodiments, the constructs herein code for a prME antigen of the Zika virus. By "prME Zika virus antigen" is meant the amino acid sequence, or a nucleotide sequence coding for the amino acid sequence, of a prME wild-type Zika virus structural protein, of one of its variants, or fragments. Figure 3 and Figure 4 identify the amino acid sequence of several variants of the wild-type prME structural protein of the Zika virus. The sequence identification numbers for each are presented in the Sequences section and the Sequence List here. See SEQ ID NO: 2 and 15 to 23.
Thus, when a Zika virus prME antigen is a variant of a wild-type prME polypeptide, the variant may include an amino acid sequence that is at least 70%, at least 75%, at least 80% identical , at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a full-length wild-type polypeptide, for example, to a polypeptide according to SEQ ID NO: 2 and 15 to 23. Alternatively, or in addition, a fragment of a polypeptide may comprise an immunogenic fragment (i.e., a fragment containing an epitope) of the full-length polypeptide which may comprise a sequence of contiguous amino acids of at least 8, at least 9, at least 10, at least 11, at least 12,
BE2017 / 5392 of at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 amino acids or more which is identical to a contiguous amino acid sequence of the full-length polypeptide.
A fragment of a prika polypeptide of the Zika virus can comprise N- and / or C-terminal deletions compared to a full-length polypeptide, for example SEQ ID NO: 2 and 15 to 23, in which the fragment comprises a deletion of up to 'to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acids from the Nterminal end, the C-terminal end, or both N-terminus and C-terminus of the full length sequence. It can be specified that the deletions are consecutive amino acids. In certain embodiments, the prME polypeptide of the Zika virus comprises a fragment chosen from the group consisting of amino acids 1 to 692 of SEQ ID NO: 2 and amino acids 21 to 692 of SEQ ID NO: 2.
In some embodiments, an immunogenic fragment of a prME antigen includes the full length of the Zika virus M antigen. By "Zika virus M antigen" is meant the amino acid sequence, or a nucleotide sequence coding for the amino acid sequence, of SEQ ID NO: 28. When a Zika virus M antigen is a variant of a wild-type polypeptide M, the variant may comprise an amino acid sequence which is identical to at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a type polypeptide
BE2017 / 5392 wild full-length, for example, to a polypeptide according to SEQ ID NO: 28.
A fragment of an antigen M of the Zika virus can comprise N- and / or C-terminal deletions compared to a full-length polypeptide, for example SEQ ID NO: 28, in which the fragment comprises a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acids from the N-terminal end, the C-terminal end, or both from the N-terminal end and from the C-terminus of the full length sequence. It can be specified that the deletions are consecutive amino acids.
In some embodiments, an immunogenic fragment of a prME antigen includes the full length of the Zika virus E antigen. By "Zika virus E antigen" is meant the amino acid sequence, or a nucleotide sequence coding for the amino acid sequence, of SEQ ID NO: 29. When a Zika virus E antigen is a variant of a wild-type polypeptide E, the variant may comprise an amino acid sequence which is identical to at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a full-length wild-type polypeptide, for example, to a polypeptide according to SEQ ID NO: 29.
A fragment of a Zika virus E antigen may comprise N- and / or C-terminal deletions compared to a full-length polypeptide, for example SEQ ID NO: 29, in which the fragment comprises a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
BE2017 / 5392 or 20 amino acids from the Nterminal end, the C-terminal end, or both the N-terminal end and the C-terminal end of the full length sequence. In one embodiment, a Zika virus E antigen comprises amino acids 1 to 520 of SEQ ID NO: 29. It can be specified that the deletions are consecutive amino acids.
As noted elsewhere in this document, Zika virus RNA is translated as a polyprotein comprising a prM signal sequence. The prM signal sequence is located in the N-terminal position relative to the prM antigen sequence. The cleavage takes place in the ER lumen by a cellular peptidase signal and produces the N-terminal end of the prM. When the polyprotein comprises a wild-type amino acid sequence, the polyprotein comprises a native prM signal sequence, SEQ ID NO: 5. By “native prM signal sequence”, the amino acid sequence is meant , or a nucleotide sequence coding for the amino acid sequence, of a signal sequence of a wild-type viral prME, SEQ ID NO: 5. FIGS. 3A and B and FIG. 4A identify the amino acid sequence of several variants of the native signal sequence of full-length prM from various strains of Zika virus.
In some embodiments, the constructs code for a native prM signal sequence. When the prM signal sequence is a variant of a native prM signal sequence, the variant may comprise an amino acid sequence which
BE2017 / 5392 is identical to at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% , or at least 99% to the full-length polypeptide according to SEQ ID NO: 5. Alternatively, or in addition, a fragment of a polypeptide may comprise a functional fragment (that is to say, containing the recognized and cleaved sequence by protease) of the full-length polypeptide which may include a contiguous amino acid sequence of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 amino acids of SEQ ID NO: 5 which is the same as a contiguous amino acid sequence of full-length polypeptide.
In some embodiments, the construction codes for a mutated prM signal sequence for amplified prM cleavage. By "mutated prM signal sequence for amplified cleavage of prM" is meant a prM signal sequence in which the native amino acid sequence is modified such that residues are added, replaced or deleted to increase the extent of signalase cleavage. In one embodiment, the amino acid sequence of the native prM signal sequence is modified by replacing Gly, Phe, and Ala at positions -5, -4, and -2 from the site of signalase cleavage by Pro, Gin, and Gin, respectively. See Figure 6. The signalase cleavage site is located at the junction of the prM signal sequence and the prME antigen. This is illustrated in Figure 3A and Figure 4A for several strains of Zika virus.
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In some embodiments, the mutated prM signal sequence for amplified cleavage of prM to the amino acid sequence shown in Figure 6 (ESS.l) (SEQ ID NO: 8) or may be one of its variants or fragments. A variant may include an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a full-length wild-type polypeptide, for example, to a polypeptide according to SEQ ID NO: 8. Alternatively, or in addition, a fragment of a polypeptide can comprise a functional fragment (i.e., containing the sequence recognized and cleaved by the protease) of the full-length polypeptide which may comprise a sequence of contiguous amino acids of at least 9, at least 10, d '' at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 amino acids, which is identical to a sequence d contiguous amino acids of the full-length polypeptide.
In some embodiments, the construction codes for a non-Zika heterologous signal sequence. In some embodiments, the construct codes for an IgG signal sequence, one of its variants or fragments, in place of the prM signal sequence of the Zika virus. By "signal sequence of IgG" is meant the amino acid sequence as shown in Figure 6 (ESS.2) (SEQ ID NO: 10). A variant may include an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
BE2017 / 5392 minus 96%, at least 97%, at least 98%, or at least 99% to a full-length wild-type polypeptide, for example, to a polypeptide according to SEQ ID NO: 10. Alternatively, or in addition , a fragment of a polypeptide can comprise a functional fragment (that is to say, containing the sequence recognized and cleaved by the protease) of the full-length polypeptide which can comprise a sequence of contiguous amino acids of at least 9 , at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 amino acids , which is identical to a contiguous amino acid sequence of the full-length polypeptide.
In some embodiments, the construction codes for a polypeptide comprising a cleavable capsid protein. By "capsid sequence" is meant any of the amino acid sequences designated by "capsid (C)" as shown in Figure 3 (for example, amino acids 1 to 104 of SEQ ID NO : 12). A capsid sequence may further comprise the cleavage sites of the native proteins C and prM (corresponding to the cleavage sites by NS2B-3 and the signalase illustrated in FIG. 7). When the capsid sequence is a variant of a wild-type capsid sequence, the variant may comprise an amino acid sequence which is at least 70%, at least 75%, at least 80% identical to at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a full-length wild type capsid polypeptide, e.g. amino acids 1 to 104 of SEQ ID NO: 12. Alternatively, or in addition, a fragment
BE2017 / 5392 of a polypeptide can comprise a functional fragment (that is to say, containing the sequence recognized and cleaved by the protease) of the full-length polypeptide which can comprise a sequence of contiguous amino acids of at least 8 , at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, d '' at least 17, at least 18, at least 19, at least 25, at least 50 and at least 75 amino acids, which is identical to a contiguous amino acid sequence of the polypeptide full length capsid.
A fragment of a Zika virus capsid protein can comprise N- and / or C-terminal deletions compared to a full-length polypeptide, for example amino acids 1 to 104 of SEQ ID NO: 12, in which the fragment includes a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acids from the N-terminus, the Cterminal, or both from the N-terminus and from the C-terminus of the full length sequence.
In some embodiments, the construction codes for a polypeptide comprising a cleavage sequence of the porcine teschovirus-1 protein 2A. By “cleavage sequence of the porcine teschovirus1 protein 2A”, is meant the amino acid sequence as represented in FIG. 7 (P2A sequence) (SEQ ID NO: 14).
In certain embodiments, the construction codes for a polypeptide comprising from the Cterminal part to the N-terminal part: a prME antigen, one of its immunogenic variants or fragments; one or
BE2017 / 5392 several components chosen from the group consisting of a prM signal sequence, one of their variants or fragments; a mutated prM signal sequence for amplified cleavage of prM, one of its variants or fragments; an IgG signal sequence, one of its variants or fragments; a variant of the cleavage sequence of the porcine teschovirus-1 protein 2A, one of its variants or fragments; and a capsid sequence, one of its variants or fragments.
In certain embodiments, a construction codes for each component of the polypeptide, if present, juxtaposed immediately next to the adjacent component, that is to say, without any intervening amino acid. In some embodiments, a linker group of 1, 2, 3, 4, or 5 amino acids is present between one or more of the components.
In certain embodiments, the construction codes for a polypeptide having a sequence chosen from the group consisting of SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 46 ,
SEQ ID NO: 52, and SEQ ID NO: 58. In certain embodiments, the construction codes for a polypeptide which is at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in a sequence chosen from the group consisting of 26, SEQ ID NO: 31, SEQ ID NO: 36,
41, SEQ ID NO: 46, SEQ ID NO: 52, and 58. In certain embodiments, the construction codes for a polypeptide which comprises a fragment of a full-length sequence chosen from the
SEQ ID NO SEQ ID NO SEQ ID NO
BE2017 / 5392 group consisting of SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 46,
SEQ ID NO: 52, and SEQ ID NO: 58, wherein the fragment comprises a contiguous sequence of the amino acid sequence of the full length sequence of up to 1, 2, 3, 4, 5, 6, 7 , 8, 9, or 10 amino acids shorter than the full length sequence.
In certain embodiments, the construction comprises a DNA sequence chosen from the group
constituted of SEQ ID NO : 25, SEQ ID NO : 30, SEQ ID NO : 35, SEQ ID NO : 40, SEQ ID NO : 45, SEQ ID NO : 51, and SEQ ID NO : 57. In some modes of achievement, the construction includes a sequence DNA that is the same as at least 70 %, at less 75%, at least 80 OGold at minus 85%, at least 90 Oθ r at less 95%, at least 96% , at least 97%, at less 98 οθ r or to minus 99 OO at a sequence chosen in the group constituted of SEQ ID NO : 25, SEQ ID NO : 30, SEQ ID NO : 35, SEQ ID NO : 40, SEQ ID NO : 45, SEQ ID NO : 51, and SEQ ID NO : 57. In some modes
incorporated SEQ ID NO:
of
35, and of embodiment, the construction comprises a DNA sequence which comprises a fragment of a full-length sequence chosen from the group
SEQ ID NO: 25, SEQ ID NO: 30,
SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 51,
SEQ ID NO: 57, in which the fragment comprises a contiguous sequence of the DNA sequence of the full length sequence of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, or 30 nucleic acids shorter than the full length sequence.
BE2017 / 5392
In includes length of realization, of a sequence the group the construction of full DNA made up of certain modes a fragment chosen in
SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 51, and SEQ ID NO: 57, wherein the fragment includes a deletion from up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleic acids from the 5 'end, from the 3 end ', or both 5' and 3 'ends of the full length sequence. In some embodiments, the construction includes nucleic acids 1 to 2076 of a selected sequence
in the group made of SEQ ID NO : 25, SEQ ID NO : 30, SEQ ID NO: 35, SEQ ID NO : 40, SEQ ID NO : 45, SEQ ID NO: 51, and SEQ ID NO : 57 • In some embodiments, construction understands a selected RNA sequence in the group constituted of SEQ ID NO: 77, SEQ ID NO : 78, SEQ ID NO : 79, SEQ ID NO: 80, SEQ ID NO : 81, SEQ ID NO : 82, and SEQ ID NO: 83. In some modes of realization, the construction includes a sequence of RNA that i is at least 70% identical, at less 75%, at least 80%, at least 85%, at least 90 %, at less 95%, at least 96%, at least 97%, at least 98%, or to minus 99 % to one sequence chosen in the group constituted of SEQ ID NO: 77, SEQ ID NO : 78, SEQ ID NO : 79, SEQ ID NO: 80, SEQ ID NO : 81, SEQ ID NO : 82, and SEQ ID NO: 83. In some modes
of embodiment, the construction comprises an RNA sequence which comprises a fragment of a full-length sequence chosen from the group consisting of
BE2017 / 5392
SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83, in which the fragment comprises a sequence contiguous RNA sequence of the full length sequence of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleic acids shorter than the full length sequence.
In certain embodiments, the construction comprises a fragment of a full-length RNA sequence chosen from the group consisting of SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83, wherein the fragment comprises a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleic acids from the 5 'end, the 3' end, or both the 5 'and 3' ends of the full length sequence. In some embodiments, the construction includes
the acids nucleic 1 to 2076 of a sequence chosen in the constituted group of SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83.
Polypeptides
In some embodiments, a polypeptide herein is a non-naturally occurring form (for example, a recombinant or modified form).
For example, the polypeptides (e.g., antigens) disclosed herein can be prepared by chemical synthesis (in whole or in part), by digestion of longer polypeptides using
BE2017 / 5392
Synthesis proteases, by translation from RNA, by purification from a cell culture (for example, from a recombinant expression), from the organism itself, etc. An example of a process for the production of peptides <40 amino acids in length involves chemical synthesis in vitro, see the following references: Bodanszky (1993) Principles of Peptide Synthesis (ISBN: 0387564314); and Fields et al. (1997) Meth Enzymol 289:
Solid-Phase Peptide Synthesis. ISBN: 0121821900. Techniques for the synthesis of peptides on a solid phase, such as methods based on tBoc or Fmoc chemistry are known in the art, see the following reference: Chan & White (2000) Fmoc Solid Phase Peptide Synthesis. ISBN: 0199637245. An enzymatic synthesis can also be used in part or in whole, see the following reference: Kullmann (1987) Enzymatic Peptide ISBN: 0849368413. As a variant of chemical synthesis, biological synthesis can be used, for example, polypeptides can be produced by translation. This can be done in vitro or in vivo. Biological processes are generally restricted to the production of L-amino acid polypeptides, but manipulation of translation machinery (for example, aminoacyl-tRNA molecules) can be used to allow the introduction of D- acids. amino (or other unnatural amino acids, such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.), see the following reference: Kullmann (1987) Enzymatic Peptide Synthesis. ISBN: 0849368413. However, when based on the
BE2017 / 5392 D-amino acids are included, it is preferred to use chemical synthesis. The polypeptides of the disclosure may have covalent modifications at the C-terminal and / or N-terminal. They can also take various forms (for example, native, fusions, glycosylated, non-glycosylated, lipidized, non-lipidized, phosphorylated, non phosphorylated, myristoylated, non myristoylated, monomer, multimeric, particulate, denatured, etc.). The polypeptides can be glycosylated naturally or unnaturally (i.e., the polypeptide can exhibit a glycosylation profile which differs from the glycosylation profile found in the corresponding naturally occurring polypeptide).
Non-naturally occurring forms of the polypeptides herein may include one or more heterologous amino acid sequences (e.g., another antigen sequence, another signal sequence, a detectable marker, or the like) in addition of a sequence of the prME antigen of the Zika virus. For example, a polypeptide here can be a fusion protein. As a variant, or in addition, the amino acid sequence or the chemical structure of the polypeptide can be modified (for example, by one or more non-natural amino acids, by covalent modification, and / or by having a different glycosylation profile, for example, by removing or adding one or more glycosylated groups) compared to a naturally occurring polypeptide sequence.
The polypeptides (e.g. antigens) disclosed herein are preferably provided in a form
BE2017 / 5392 purified or substantially purified, i.e., substantially free of other polypeptides (for example, free of naturally occurring polypeptides), particularly other polypeptides of the Zika virus or the host cell ; for example, at least about 50% pure (by weight), at least about 60% pure (by weight), at least about 70% pure (by weight), at least about 80% pure (by weight) , or at least 90% pure (by weight), etc. Alternatively, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% of a composition is consisting of other expressed polypeptides.
Nucleic acids
The present inventors here disclose nucleic acid molecules comprising a sequence which codes for a prME antigen of the Zika virus. The nucleic acids as disclosed herein can take various forms (e.g., single strand, double strand, vectors, etc.). Nucleic acids can be circular or branched, but they will generally be linear.
The nucleic acids used herein are preferably provided in a purified or substantially purified form, i.e., substantially free of other nucleic acids (e.g., free of naturally occurring nucleic acids), particularly other nucleic acids of the Zika virus or of the host cell, generally being at least about 50% pure (by weight), at least about 60% pure (by weight), at least
BE2017 / 5392 about 70% less pure (by weight), at least about 80% pure (by weight), and usually at least about 90% pure.
Nucleic acids can be prepared in many ways, for example, by chemical synthesis (for example, DNA synthesis by phosphoramidites) in whole or in part, by digestion of longer nucleic acids using nucleases (for example , restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g., using ligases or polymerases), from genomic or cDNA libraries, etc.
The term "nucleic acid" generally means a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and / or their analogs. It includes DNA, RNA, DNA / RNA hybrids. It also includes DNA or RNA analogs, such as those containing modified backbones (eg, peptide nucleic acids phosphorothioates) or nucleic acid bases of the disclosure includes mRNA, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, etc. When the nucleic acid takes the form of RNA, it may or may not have a 5 'cap.
The nucleic acids herein include a sequence which codes for at least one prME antigen of the Zika virus. Generally, the nucleic acids of the invention will be in recombinant form, that is to say, a form which does not exist in nature. For example, des (PNA) or modified acid. So,
BE2017 / 5392 nucleic acid can comprise one or more heterologous nucleic acid sequences (for example, a sequence coding for another antigen and / or a control sequence such as a promoter or an internal ribosome entry site) in addition of the sequence coding for at least one prME antigen of the Zika virus. The nucleic acid can be part of a vector, that is, part of a nucleic acid construct designed for the translation / transfection of one or more cell types. The vectors may be, for example, "expression vectors" which are designed to express a nucleotide sequence in a host cell, or "viral vectors" which are designed to cause the production of a recombinant virus or a pseudoviral particle.
Alternatively, or in addition, the sequence or chemical structure of the nucleic acid may be modified compared to a naturally occurring sequence which codes for a prME antigen of the Zika virus. The sequence of the nucleic acid molecule can be modified, for example, to increase the efficiency of expression or replication of the nucleic acid, or to provide additional stability or resistance to degradation.
The nucleic acid encoding the polypeptides described above can be optimized for codons. By "optimized for codons" is meant a modification with respect to the use of codons which can increase the efficiency of translation and / or half-life of the nucleic acid. A poly A tail (e.g., about 30 or more adenosine residues) can be
BE2017 / 5392 attached to the 3 'end of the RNA to increase its half-life. The 5 'end of the RNA can be capped by a ribonucleotide modified with the structure m7G (5') ppp (5 ') N (cap structure 0) or one of its derivatives, which can be incorporated during the synthesis of RNA or can be created by enzymes after RNA transcription (for example, using the vaccinia virus capping enzyme (VCE) consisting of mRNA triphosphatase, guanylyl transferase and guanine- 7methyltransferase, which catalyzes the construction of N7-monomethylated cap structures). The cap structure 0 plays an important role in maintaining the stability and efficiency of translation of the RNA molecule. The 5 'cap of the RNA molecule can be further modified by a 2'-0methyltransferase which results in the production of a cap structure 1 (m7Gppp [m2'-O] N), which can further increase the efficiency of translation.
The nucleic acids may include one or more modified nucleotide analogs or nucleotides. As used herein, "nucleotide analogue" or "modified nucleotide" refers to a nucleotide that contains one or more chemical modifications (eg, substitutions) in or on the nitrogenous basis of the nucleoside (eg, cytosine (C) , thymidine (T) (or uracil (U)), adenine (A) or guanine (G)). A nucleotide analog may contain other chemical changes in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analog, or open chain sugar analog), or
BE2017 / 5392 of phosphate. The preparation of nucleotides and modified nucleotides and nucleosides is well known in the art, see the following references: U.S. patents numbers 4,373,071, 4,458,066, 4,500,707, 4,668,777,
973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530,
700,642. Many modified nucleosides and modified nucleotides are commercially available.
Modified nucleobases that can be incorporated into modified nucleosides and nucleotides and be present in RNA molecules include: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2- thiouridine), Um (2'-O-methyluridine), mlA (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6- (cis-hydroxyisopentenyl) adenosine); ms2io6A (2-methylthio-N6- (cishydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyl (N6-hydroxynorvalylcarbamoylhn6A adenosine) adenosine) ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine); Ar (p) (2'-O-ribosyladenosine (phosphate)); I (inosine); millet (1-methylinosine); m'im (1,2'-O-dimethylinosine); m3C (3-methylcytidine);
Cm (2T-O-methylcytidine); (N4-acetylcytidine); 5FC (5,2-O-dimethylcytidine);
s2C (2-thiocytidine); ac4C (5-formylcytidine); m5Cm ac4Cm (N4-acetyl-2T-Omethylcytidine) k2C (lysidine) mlG (1-methylBE2017 / 5392 guanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2, N2dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2, N2,2'-O-trimethylguanosine); Gr (p) (2'-Oribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW * (sub-modified hydroxywybutosine); imG (wyosin); mimG (methylguanosine); Q (queuosin); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G * (archaeosine); D (dihydrouridine); m5Um (5,2'-Οdimethyluridine); s4U (4-thiouridine); m5s2U (5methyl-2-thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3- (3-amino-3-carboxypropyl) uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine-5-oxyacetic acid); mcmo5U (methyl ester of uridine-5-oxyacetic acid); chm5U (5 (carboxyhydroxymethyl) uridine)); mchm5U (5- (carboxyhydroxymethyl) uridine methyl ester); mcm5U (5-methoxycarbonyl-methyluridine); mcm5Um (5methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-seleno-uridine); ncm5U (5-carbamoylmethyl-uridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-L-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6, N6BE2017 / 5392 dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6, T-O-dimethyladenosine); m62Am (N6, N6, O-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2, N2,7trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); £ 5Cm (5-formyl-2'-0methylcytidine); mlGm (1,2'-O-dimethylguanosine); m'Am (1,2-O-dimethyladenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine); iniG-14 (4demethyl-guanosine); imG2 (isoguanosine); ac6A (N6acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C1-C6 alkyl) -uracil, 5 -methyluracil, 5- (C2 to Ce alkenyl) -uracil, 5- (C2 to Ce alkynyl) -uracil, 5- (hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5bromouracil, 5-hydroxycytosine, 5- (C1 to Ce alkyl) ~ cytosine, 5-methylcytosine, 5- (C2 to Ce alkenyl) ~ cytosine, 5- (C2 to Ce alkynyl) -cytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-substituted 7-deazaguanine, 7-deaza-7- (C2-C6 alkynyl) ~ guanine, 7-deaza-guanine 8-substituted, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8azapurine, 7-deazapurine substituted, 7-deaza-purine 7substituted, 7-deaza-purine 8-substituted, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methylU. Many of these modified nucleobases and
BE2017 / 5392 their corresponding ribonucleosides are available from commercial suppliers.
Nucleic acid vaccines
The present inventors disclose compositions comprising a nucleic acid sequence which codes for a polypeptide comprising a Zika virus antigen, one of its variants or fragments. Such compositions can be a nucleic acid-based vaccine. Another composition comprising a nucleic acid sequence which codes for one or more (for example, a second, third, fourth, fifth or sixth) additional Zika virus antigens can also be provided in the form of a vaccine based on 'nucleic acid. In some embodiments, a composition includes a nucleic acid sequence coding for a Zika virus prME antigen from a first strain of Zika virus and an additional nucleic acid sequence coding for an additional Zika virus prME antigen one or more other strains of Zika virus. In some embodiments, a composition comprises a nucleic acid sequence encoding a prika Zika virus antigen and one or more (for example, a second, third, fourth, fifth or sixth) additional Zika virus antigens. Alternatively, one or more additional non-Zika virus antigens can be encoded.
The nucleic acid can be, for example, RNA (i.e., an RNA-based vaccine) or DNA (i.e., a d-based vaccine DNA, such as a vaccine
BE2017 / 5392 based on plasmid DNA). In some embodiments, the nucleic acid vaccine is an RNA vaccine. In some embodiments, the RNA-based vaccine includes a self-replicating RNA molecule, also referred to herein as a self-amplifying mRNA molecule (SAM). The self-replicating RNA molecule can be an alphavirus-derived RNA replicon.
Self-replicating RNA molecules are well known in the art and can be produced by using replicating elements derived, for example, from alphavirus, and by substituting viral structural proteins with a nucleotide sequence encoding a protein of interest. A self-replicating RNA molecule is usually a stranded molecule which 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 administered RNA. Thus, the administered RNA leads to the production of multiple daughter RNA molecules. These daughter RNA molecules, as well as the collinear subgenomic transcripts, can be translated themselves to provide in situ expression of a coded antigen (i.e., a prM-F Zika virus antigen), or can be transcribed to provide other transcripts with the same meaning as the administered RNA which are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is a huge increase in the number
BE2017 / 5392 of the RNAs of replicons introduced and thus the encoded antigen becomes a major polypeptide product of the cells.
A suitable system for obtaining self-replication in this way is to use an alphavirus-based replicon. These replicons are strand + RNAs (positive sense strand) which lead to the translation of a replicase (or a replicase-transcriptase) after administration to a cell. Replicase is translated into the form of a polyprotein which self-activates to provide a replication complex which creates copies of genomic strands of the strand + administered RNA. These negative strand transcripts (strand -) can even be transcribed to give d other copies of the parent + strand RNA and also to give a subgenomic transcript which codes for the antigen. The translation of the subgenomic transcript thus leads to the 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 Venezuela, etc. The 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, the context of which is incorporated by reference.
In some embodiments, the self-replicating RNA molecule described here encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule and ( ii) a Zika virus prME antigen. The polymerase can
BE2017 / 5392 be an alphavirus replicase, for example, comprising one or more nsP1, nsP2, NsP3 and nsP4 proteins of alphavirus.
While natural alphavirus genomes code for viral structural proteins in addition to the non-structural polyprotein of replicase, in some embodiments, the self-replicating RNA molecules do not code for structural proteins of d 'alphavirus. Thus, self-replicating RNA can lead to the production of genomic RNA copies 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 self-replicating RNA molecule cannot be perpetuated in an infectious form. The structural proteins of alphaviruses which are necessary for perpetuation in wild type viruses are absent from the self-replicating 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 encodes the immunogen rather than the structural proteins of alphavirus virions.
Thus, a self-replicating RNA molecule useful with the invention can have two open reading frames. The first open reading frame (5 ') codes for a replicase; the second open reading frame (3 ') codes for an antigen. In some embodiments, the RNA may include additional open reading frames (e.g., downstream), for example
BE2017 / 5392 example, to code for other antigens or to code for accessory polypeptides.
In some embodiments, the self-replicating 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 self-replicating RNA molecule must be chosen to ensure compatibility with the encoded replicase.
A self-replicating RNA molecule can have a 3 'poly-A tail. It may also include a recognition sequence for poly-A polymerase (for example, AAUAAA) near its 3 'end.
Self-replicating RNA molecules can have various lengths, but they are generally 5,000 to 25,000 nucleotides in length. Self-replicating RNA molecules will generally be single-stranded. Single-stranded RNAs can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and / or PKR. RNA administered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNAs which are formed either during the replication of a single-stranded RNA or within the secondary structure of a single stranded RNA.
Self-replicating RNA can be conveniently prepared by in vitro transcription (IVT). IVT can use a template (cDNA) created and propagated in plasmid form in bacteria, or created by synthesis (for example, by the synthesis of genes and / or methods of creation by polymerase chain reaction (PCR)) . For example, an ADNBE2017 / 5392 RNA polymerase dependent (such as the RNA polymerases of bacteriophages T7, T3 or SP6) can be used to transcribe the self-replicating RNA from a DNA template. Appropriate styling and poly-A addition reactions can be used as necessary (although the poly-A of the replicon is usually encoded within the template DNA). These RNA polymerases may have stringent requirements for the nucleotide (s) transcribed in 5 'and in some embodiments, these requirements must correspond to the requirements of the encoded replicase, in order to ensure that the RNA transcribed by TIV can function effectively as as a substrate for its self-coded replicase.
A self-replicating RNA can comprise (in addition to any 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 self-replicating RNA molecule can code for a single heterologous polypeptide antigen (i.e., a prika Zika antigen) or, optionally, two or more heterologous polypeptide antigens linked together in such a way that each of the sequences retain their identity (for example, linked in series) when expressed as an amino acid sequence. Heterologous polypeptides produced from self-replicating RNA can then be
BE2017 / 5392 produced in the form of a fusion polypeptide or modified so as to produce distinct polypeptide or peptide sequences.
The self-replicating RNA molecules described herein can be engineered to express multiple nucleotide sequences, from two or more open reading frames, thereby allowing the coexpression of proteins, such as one, two or more antigens, Zika virus (eg, one, two or more prME antigens of Zika virus) together with cytokines or other immunomodulators, which can enhance the production of an immune response. Such a self-replicating 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, the self-replicating 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 self-replicating 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 cells, T cell lines, and T cell clones. For example, spleen cells from immunized mice can be isolated and the ability of cytotoxic T cells to lyse cells
BE2017 / 5392 autologous targets which contain a self-replicating RNA molecule which codes for a prME antigen of Zika virus. In addition, the differentiation of helper T lymphocytes can be analyzed by measuring the proliferation or production 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 cells by cytoplasmic staining of cytokines and flow cytometry.
Self-replicating RNA molecules that code for a prika Zika virus antigen can also be tested for their ability to induce humoral immune responses, as evidenced, for example, by induction by lymphocytes B of antibodies specific for a Zika virus prME 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 self-replicating RNA molecules may involve detection of the expression of the Zika virus prME antigen encoded by the target cells. For example, the FACS technique can be used to detect the expression of antigens on the cell surface or at the intracellular level. Another advantage of selection by the FACS technique 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 immunoadhesion using monoclonal antibodies
BE2017 / 5392 on a plate or capture using magnetic beads coated with monoclonal antibodies.
In some embodiments, the self-replicating RNA molecules include a selected sequence
in the group constituted of SEQ ID NO : 70, SEQ ID NO : 71, SEQ ID NO: 72 r SEQ ID NO : 73, SEQ ID NO : 74, SEQ ID NO: 75, and SEQ ID NO: 76. In some modes of production, the molecules RNA
self-replicating include a sequence that is at least 70%, at least 75%, at least 80%,
at less 85 Oθ r at least 90%, at least 95%, at least 96 %, at less 97%, at less 98%, or at least 99 o d. a sequence chosen in the group formed of SEQ ID NO : 70, SEQ ID NO : 71, SEQ ID NO: 72, SEQ ID NO : 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO : 76. In some embodiments, the
SEQ ID NO: 71, SEQ ID NO: 74, 76 in which the
SEQ ID NO: 65, self-replicating RNA molecule comprises a fragment of a full-length sequence chosen from the group consisting of SEQ ID NO: 70,
SEQ ID NO: 72, SEQ ID NO: 73,
SEQ ID NO: 75, and SEQ ID NO fragment includes a contiguous sequence of the nucleic acid sequence of the full length sequence of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 15, 20, 25, or nucleic acids shorter than the full length sequence.
In certain embodiments, a DNA sequence coding for a self-replicating RNA molecule is provided, said DNA sequence being chosen from the group consisting of SEQ ID NO: 63,
SEQ ID NO: 66,
SEQ ID NO: 64, SEQ ID NO: 67,
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SEQ ID NO: 68, and SEQ ID NO: 69. In some embodiments, the DNA sequence encoding a self-replicating RNA molecule includes a sequence that is at least 70% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in a sequence chosen from the group made of
ID NO: 63, SEQ ID NO : 64, SEQ ID NO: 65, ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and ID NO: 69. In some modes of achievement, the
DNA sequence encoding a self-replicating RNA molecule comprises a fragment of a full-length sequence selected from the group consisting of
SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69 in which the fragment comprises a contiguous sequence of the full-length nucleic acid sequence of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleic acids shorter than the full length sequence.
The nucleic acid vaccine may include a viral or non-viral delivery system. The delivery system (also referred to herein as the delivery vehicle) may have adjuvanting effects which enhance the immunogenicity of the prME antigen of the encoded Zika virus. For example, the nucleic acid molecule can be encapsulated in liposomes, non-toxic biodegradable polymer microparticles or viral-type replicon particles (PRV), or complexed with particles of an oil-in-water cationic emulsion. In some embodiments, the
BE2017 / 5392 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-like virions. 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. The administration systems of NEC and the methods for their 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 oil 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. Other useful NEC details can be found
BE2017 / 5392 find in the following references: WO 2012/006380; WO 2013/006834; and WO 2013/006837 (the content of which is incorporated herein in its entirety).
Thus, in a nucleic acid-based vaccine of the invention, an RNA molecule coding for a prME antigen of Zika virus can be complexed with a particle of an oil-in-water cationic emulsion. The particles generally comprise an oil core (for example, a vegetable oil or a squalene) which is in the liquid phase at 25 ° C., a cationic lipid (for example, a phospholipid) and, optionally, a surfactant (for example, sorbitan trioleate, polysorbate 80); polyethylene glycol can also be included. 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 delivery system is a non-viral delivery system, such as an NEC, and the nucleic acid vaccine includes self-replicating RNA (mRNA). This can be particularly effective in triggering humoral and cellular immune responses. The benefits also include the absence of a limiting anti-vector immune response and the lack of risk of genomic integration.
In some embodiments, an RNA molecule encoding a Zika virus prME antigen can be complexed with a cationic oil-in-submicron water emulsion. In some embodiments, the oil-in-water cationic emulsion is characterized by an average particle size of about 80 nm at
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180 nm in diameter (or alternatively about 80 to about 150 nm; about 80 to 130 nm; or about 100 nm). In some embodiments, the concentration of DOTAP in said emulsion, before complexation of the RNA, is at least about 2.5 mM, or from about 2.5 mM to about 8 mM. In a particular embodiment, the concentration of DOTAP in said emulsion is approximately 4 mg / ml (5.73 mM). The oil can be squalene or squalane.
In some embodiments, an RNA molecule encoding a Zika virus prME antigen is complexed to an oil-in-water cationic emulsion comprising DOTAP, squalene, sorbitan trioleate and polysorbate 80 in citrate buffer. Cationic oil-in-water emulsions suitable for the administration of an RNA molecule encoding a prME antigen of Zika virus may contain approximately 2 mg / ml to 7 mg / ml of DOTAP, approximately 3 mg / ml to 6 mg / ml Span 85; about 3 mg / ml to 6 mg / ml of Tween 80; and about 30 mg / ml to 50 mg / ml of squalene. In certain embodiments, the cationic oil-in-water emulsion, before complexing with the RNA, contains approximately 4.3% of squalene w / v, 0.5% of Tween 80, 0.5% of SPAN 85, and 4 mg / ml of DOTAP.
Also provided is a method of preparing a composition comprising an RNA molecule encoding a ZME virus prME antigen complexed with an oil-in-water cationic emulsion, the method comprising: (i) providing an emulsion oil in water as described here; (ii) providing an aqueous solution comprising the RNA molecule; and
BE2017 / 5392 (iii) the combination of the aqueous solution of (ii) and the oil-in-water emulsion of (i), thereby preparing the composition. If desired, the aqueous solution comprising the RNA molecule can be a buffer. The buffer can include one or more salts, buffers, saccharides, or polymers. In a preferred embodiment, the buffer comprises 560 mM sucrose, 20 mM NaCl, and 10 mM citrate, which can be mixed with an oil-in-water cationic emulsion described herein to produce a final aqueous phase which comprises 280 mM sucrose, 10 mM NaCl and 10 mM citrate.
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 system). NPLs are non-virion 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
BE2017 / 5392 comprise an amphiphilic lipid which can form liposomes, optionally in combination with at least one cationic lipid (such as DOTAP, DSDMA, DODMA, 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 2012/006376; WO 2012/030901;
WO 2012/031046; WO 2012/031043; WO 2012/006378; WO 2013/033563;
WO 2015/095340;
WO 2016/037053. In some embodiments, the NPLs are liposomes RV01, see references WO 2012/006376 and Geall ei al
WO 2011/076807 WO 2014/136086
WO 2013/006825 WO 2015/095346 survivors (2012) PNAS
USA. Sep 4; 109 (36): 14604-9.
Pharmaceutical compositions; immunogenic compositions
The disclosure provides compositions comprising a nucleic acid comprising a sequence which codes for a Zika virus polypeptide, for example, a prika Zika virus antigen. The composition can be a pharmaceutical composition, for example, an immunogenic composition or a vaccine composition. Therefore, the composition may also include a pharmaceutically acceptable carrier. In certain embodiments, the Zika virus is a Zika virus of Brazilian strain.
A "pharmaceutically acceptable carrier" includes any carrier which does not itself induce the production of antibodies harmful to the individual receiving the composition. The suitable carriers are generally large metabolized macromolecules
BE2017 / 5392 slowly such as proteins, polysaccharides, poly lactic acids, poly glycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as droplets oil or liposomes). Such supports are well known to a person with average skills 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 sterile pyrogen-free phosphate buffered saline is a typical carrier.
Pharmaceutical compositions may include the constructs, amino acid sequences, and / or polypeptide sequences described elsewhere in this document in simple water (eg, "eppi") or in a buffer, for example, a buffer phosphate, Tris buffer, borate buffer, succinate buffer, histidine buffer, or 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 NaCl is typical, for example, around 9 mg / ml. The compositions can include metal ion chelators. These can prolong the stability of RNA by
BE2017 / 5392 eliminating the ions which can accelerate the hydrolysis of phosphodiesters. Thus, a composition can include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are generally present at a concentration between 10 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 2-phenoxyethanol. 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 in the form of unit doses. In some embodiments, a unit dose can have a volume between 0.1 and 1.0 ml, for example, about 0.5 ml.
In some embodiments, the compositions disclosed herein are immunogenic compositions which, when administered to a subject, induce a humoral and / or cellular immune response specific for the antigen (i.e., a
BE2017 / 5392 immune response which specifically recognizes a naturally occurring Zika virus polypeptide). For example, an immunogenic composition can induce a population of memory T and / or B lymphocytes relative to a subject not treated following an infection with the Zika virus, particularly in these embodiments where the composition comprises an acid. nucleic acid comprising a sequence which codes for a prika Zika virus antigen or comprises a Zika virus antigen. In some embodiments, the subject is a vertebrate, such as a mammal, for example, a human or a veterinary mammal.
The compositions of the invention can be formulated as vaccine compositions. The vaccine will include an immunologically effective amount of antigen. By "an immunologically effective amount" it is meant that administration of this amount to a subject, either in a single dose or as part of a series, is effective in inducing a measurable immune response against the Zika virus in the subject . This amount varies according to the state of health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (for example, human, non-human primate, etc.), the ability of the the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the composition or vaccine, the evaluation of the attending physician of the medical situation, the severity of the disease, the potency of the compound administered, the method of administration, and other relevant factors. We
BE2017 / 5392 expects the amount to be within a relatively wide range which can be determined by routine testing. In one embodiment, an immunologically effective amount of a Zika virus antigen is an amount sufficient to prevent or treat infection with the Zika virus. The vaccines as disclosed herein can be either prophylactic (i.e., to prevent an infection) or therapeutic (i.e., to treat an infection), but they will generally be prophylactic In some modes of In one embodiment, the vaccine compositions disclosed herein can induce an effective immune response against Zika virus infection, i.e., a response sufficient for the treatment or prevention of Zika virus infection.
In some embodiments, the composition further comprises an additional antigen. In some embodiments, the composition is administered to a subject in combination with another composition which includes an additional antigen.
A composition of the present disclosure may also include, or be administered in conjunction with, one or more adjuvants (e.g., vaccine adjuvants), particularly where the composition comprises an immunologically effective amount of a nucleic acid encoding a prME antigen of Zika virus or a Zika virus prME antigen. By "adjuvant", it is meant that it is capable of increasing an immune response against an antigen compared to the administration of said antigen alone. In some
BE2017 / 5392 Aspects, the adjuvant compositions as described herein further include one or more immunostimulants, for example, a saponin such as QS21.
The adjuvants which can be used in compositions of the invention include, but are not limited to: (A) compositions containing minerals, for example, aluminum and calcium salts, such as phosphates aluminum. (B) Oily emulsions, for example, squalene emulsions in water, such as MF59 or AS03. The complete Freund's adjuvant (CFA) and the incomplete Freund's adjuvant (IFA) can also be used. (C) Formulations of saponins. (D) Virosomes and pseudoviral particles (PPV). (E) Bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), derivatives of lipid A, immunostimulatory oligonucleotides and ADP-ribosylating toxins and their detoxified derivatives. (F) Human immunomodulators, for example, cytokines, such as interleukins, interferons, macrophage colony stimulating factor, and tumor necrosis factor. (G) Bioadhesives and mucoadhesives, such as esterified hyaluronic acid microspheres, crosslinked derivatives of poly (acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethylcellulose. (H) Microparticles, for example, particles from ~ 100 nm to ~ 150 µm in diameter, preferably ~ 200 nm to ~ 30 µm in diameter, and most preferably ~ 500 nm to ~ 10 µm in diameter
BE2017 / 5392 diameter, formed from materials which are biodegradable and non-toxic (for example, a poly (α-hydroxylated acid), a hydroxybutyric polyacid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with a poly ( lactide-coglycolide) are preferred, optionally treated to have a negatively charged surface (for example, with SDS) or a positively charged surface (for example, with a cationic detergent, such as CTAB). (I) Liposomes. (J) Formulations of polyoxyethylene ethers and polyoxyethylene esters. (K) A polyphosphazene (PCPP). (L) Muramyl peptides. (M) Imidazoquinolone compounds, for example, Imiquimod and its counterparts.
Combinations of one or more of the adjuvants identified above can also be used with the invention.
Operating procedures / uses
In certain embodiments, methods are provided for inducing an immune response against Zika virus infection in a subject in need thereof comprising a step of administering an immunologically effective amount of a construct or composition as disclosed here. In certain embodiments, there is provided the use of the constructs or compositions disclosed herein to induce an immune response against a prME Zika virus antigen in a subject in need thereof. In some embodiments, the use of constructions or
BE2017 / 5392 compositions disclosed herein to induce an immune response against Zika virus infection in a subject. In certain embodiments, there is provided the use of the construct or composition as disclosed herein in the manufacture of a medicament which induces an immune response against Zika virus infection in a subject. By "subject" is meant a vertebrate, such as a mammal, for example, a human or a veterinary mammal. In some embodiments, the subject is human. By "immune response" is meant a humoral and / or cellular immunological response specific for the antigen (ie, an immune response which specifically recognizes an antigenic polypeptide) which can be demonstrated to neutralize the Zika virus in vitro or controlling / reducing / eliminating Zika virus infection in vivo.
In certain embodiments, the immune response is characterized by an immunological memory against the Zika virus and / or an effective population of T lymphocytes with memory sensitive to the Zika virus.
In certain embodiments, the composition comprises an RNA molecule encoding a polypeptide chosen from the group consisting of SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 58. In some embodiments, the composition comprises an RNA molecule encoding a polypeptide which is at least 70%, at least 75%, at least identical 80%,
BE2017 / 5392
at me ns 85 oθ r at least 90%, at least 95%, at least ins 96 O0 f at least 97%, at least 98%, or at least 99 Her•her a sequence chosen in the group ; constituted of SEQ ID NO: 23, SEQ ID NO : 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO : 46, SEQ ID NO: 52, SEQ ID NO: 58. In some modes of production, the composition includes an RNA molecule coding for a polypeptide which includes a fragment of a sequence full length chosen in the group formed of SEQ ID NO: 26, SEQ ID NO : 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO : 46, SEQ ID NO: 52, SEQ ID NO: 58, in which the fragment includes a after contiguous of the amino acid sequence of the
full length sequence of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids shorter than the full length sequence.
In some embodiments, there is provided a construct, a vector, a self-replicating RNA and / or a molecule as described herein for use in therapy or medicine. In some embodiments, the compositions disclosed herein are intended for use in therapy or medicine. In a preferred embodiment, the therapy is vaccine therapy. Preferably, the therapy is a vaccine to prevent infection with the Zika virus.
In certain embodiments, there is provided a construct, a vector, a self-replicating RNA and / or a molecule as described herein for use in the prevention or treatment of a Zika virus infection in a subject in need
BE2017 / 5392
In some
In some modes modes
of production, the are intended to one or the treatment of a a subject having need of production, the are intended to one a immune response
against a Zika virus infection in a subject in need.
In certain embodiments, there is provided a construct, a vector, a self-replicating RNA and / or a molecule, and / or a composition as described herein for use in a method of inducing a response. immune against Zika virus infection in a subject in need.
In some embodiments, methods are provided for preventing or shortening Zika virus infection and / or reducing or preventing clinical symptoms during Zika virus infection in a subject having need, which includes administering to the subject a immunologically effective amount of an immunogenic composition as provided herein.
In certain embodiments, there is provided the use of a construct or composition disclosed herein in the manufacture of an immunogenic composition for preventing or shortening Zika virus infection in a subject and / or the reduction or prevention of clinical symptoms during a Zika virus infection in a subject.
BE2017 / 5392
In certain modes of administration, methods are provided for preventing or reducing the transmission of Zika virus infection from one subject to another. In specific embodiments, methods are provided for preventing or reducing the transmission of Zika virus infection to a fetus across the placental barrier. In certain embodiments, a composition as described herein is administered to a woman in an amount effective to prevent the transmission of Zika virus infection across the placental barrier.
In certain embodiments, there are provided methods for inducing an immune response sufficient to prevent or treat Zika virus infection in a subject, which comprises administering to the subject a composition comprising a ( e) or more of the self-replicating RNA constructs, vectors, or molecules as described above in an amount sufficient to prevent or treat infection with the Zika virus. In certain embodiments, there is provided the use of a construct or composition disclosed herein in the manufacture of an immunogenic composition for preventing or reducing the transmission of Zika virus infection to a fetus across the placental barrier.
In certain embodiments, there is provided a construct, a vector, a self-replicating RNA molecule, and / or a composition as described herein for use in a prevention or
BE2017 / 5392 reduction of the transmission of a Zika virus infection to a fetus across the placental barrier.
In some embodiments, the subject is a human subject. In specific embodiments, the human subject has been exposed, or is at risk of being exposed, to infection with the Zika virus.
Routes of administration / dosages
The compositions disclosed herein will generally be administered directly to a subject. Direct administration can be accomplished by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or into the interstitial space of tissue) Variants of the routes of administration include rectal, oral (e.g. tablet, spray), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, auricular, pulmonary or other mucosal administration. Intradermal and intramuscular administration represent the preferred routes of administration. The injection can be done using a needle (for example, a hypodermic needle), but needle-less injection can be used alternatively. A typical human intramuscular dose volume is 0.5 ml.
A dose of a nucleic acid (for example, a nucleic acid-based vaccine, such as an SAM-based vaccine against Zika virus) can range from about 50 µg to about 100 µg of nucleic acid. In one embodiment, a dose of SAM-based vaccine against
BE2017 / 5392 Zika virus contains 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 pg of RNA. In other embodiments, a dose of an SAM-based vaccine against the Zika virus may include <10 pg of nucleic acid, for example, from 1 to 10 pg, such as about 1 pg, 2.5 pg , 5 pg, 7.5 pg or 10 pg, but expression can be seen at much lower rates; for example, using <1 pg / dose, <100 ng / dose, <10 ng / dose, <1 ng / dose, etc. Similarly, a dose of a protein antigen may include <10 pg of protein; for example, from 1 to 10 pg, like about 1 pg, 2.5 pg, 5 pg,
7.5 pg or 10 pg.
In preferred embodiments, a SAM vaccine or vaccine composition against the Zika virus is administered to a subject at an effective dose, which means a dose sufficient to achieve a desired immune response, such as induction of neutralizing antibodies against Zika virus and / or protection against Zika virus infection.
In some embodiments, an SAM-based vaccine against the Zika virus described herein has an effective dose which is less than or equal to 50%, 40%, 30%, 20% or 10% of the effective dose of a vaccine or a vaccine composition based on DNA coding for the same antigen. In some embodiments, an SAM-based vaccine against the Zika virus described herein has an effective dose that is one-third or less than the effective dose of a DNA-encoding vaccine or vaccine composition for the same antigen.
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Manufacturing / formulation processes
Methods for making self-replicating RNA are provided here. In certain embodiments, the method for manufacturing a self-replicating RNA comprises an in vitro transcription step (IVT) as described elsewhere in this document. In some embodiments, the method of making a self-replicating RNA includes a step of TIV to produce an RNA, and further includes a step of combining the RNA with a non-viral delivery system such than described elsewhere in this document. In some embodiments, the method of making a self-replicating RNA includes a step of TIV to produce an RNA, and further includes a step of combining the RNA with an NEC delivery system such than described elsewhere in this document.
Sequence identity
Identity or homology to an amino acid 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 introducing gaps, if necessary, to obtain the maximum percent sequence identity, and not considering any of the conservative substitutions as part of the sequence identity. Identity or homology to a nucleic acid sequence is defined here as the percentage of nucleotides in the candidate sequence that are identical to the nucleic acid sequence of
BE2017 / 5392 reference after alignment of sequences and introduction of gaps, if necessary, to obtain the maximum percentage of 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, two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences or throughout a predetermined portion of the '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 ei 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 match range and the number of gaps introduced in the shorter sequences finally to align the two sequences. The same methods used to compare polypeptides can also be used to calculate the percent identity of two polynucleotide sequences.
When this disclosure relates to a sequence by reference to a UniProt or Genbank accession code, the sequence to which reference is made
BE2017 / 5392 is the current version on the date of filing of this application.
General
Unless otherwise explained, all technical and scientific terms used here have the same meaning as that commonly understood by a person of average skill in the field to which this disclosure belongs. The singular terms "un", "une", "le" and "la" include articles in the plural unless the context clearly indicates otherwise. Similarly, the word "or" is meant to include "and" unless the context clearly indicates otherwise. The term "plurality" refers to two or more. In addition, the numerical limitations given with respect to concentrations or rates of a substance, such as concentrations or ratios of solution components thereof, and reaction conditions such as temperatures, pressures and times cycles are expected to be approximate. The term "approximately" used here is intended to mean the amount ± 10%.
The term "includes" means "includes". Thus, unless the context dictates otherwise, the word "includes", and variations such as "understand" and "comprising" will be understood to imply the inclusion of a indicated compound or composition (for example, a nucleic acid, a polypeptide, an antigen) or a step, or a group of compounds or steps, but not the exclusion of any other compound, composition, step or group thereof. The modes of
BE2017 / 5392 embodiment described as including certain components are intended to include embodiments made up of the indicated components.
The abbreviation “e.g.” is derived from the Latin exempli gratia, and is used here to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example”.
The invention will be further described with reference to the following non-limiting figures and examples.
EXAMPLES
Example 1 - Project summary
The present inventors have undertaken work on a vaccine against the Zika virus using the SAM - synthetic self-amplifying mRNA (SAM) platform derived from the alphavirus genome, expressing antigens of interest. The SAM constructs are evaluated for rigorous production and antigenicity of the antigens and further tested for their immunogenicity and efficacy using in vivo models.
Processes
The SAM VEE TC-83 vector was used as the context construct for cloning in the examples. See SEQ ID NO: 24.
Example 2 - Selection of the antigen
The Flavivirus genome consists of single stranded RNA of positive polarity approximately 11.3 kb in length (Figure 1). The quarter
BE2017 / 5392 proximal 5 ′ of the genome codes for the structural proteins of the capsid (c), premembrane (prM), and of the envelope (E). The non-structural proteins NSI, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 are involved in the replication of viral RNA. The coding region is flanked by 5 'and 3' untranslated regions (5 'and 3' UTR) of approximately 100 and 600 nucleotides in length, respectively. Translation of the viral genome produces a unique polypeptide which is processed into individual proteins by a combination of cellular proteases and a viral protease consisting of a catalytic subunit, NS3, and its cofactor, NS2B.
The structural proteins prM and E are co-translated into the membrane of the endoplasmic reticulum (ER) and processed by peptidase signals, producing proteins which encapsulate protein C together with viral RNA, by budding in the ER lumen ( figure 2). At a later stage in viral maturation, the prM protein on these particles is cleaved into mature M protein by a cellular furin protease before release from the cell. This cleavage of the prM protein is necessary for the infectivity of the released virions. In addition to infectious virions, cells infected with fiavivirus release subviral particles (PSV) (Figure 2). These particles are smaller than virions, but contain the antigenically important protein E and prM / Μ protein, which is essential for proper folding and incorporation of protein E in PSVs and viral particles. However, unlike
BE2017 / 5392 virions, PSVs contain neither protein C nor the viral genome, and are therefore non-infectious. PSVs can be produced in various systems by the coexpression of prM and E proteins, and PSVs share properties with wild-type viruses, such as fusogenic activity and the induction of a neutralizing immune response, and has been repeatedly demonstrated to stimulate protective immune responses against a number of flavivirus diseases. The present inventors chose the structural proteins of the Zika virus, namely, prM and E, and in certain cases, C, for another experiment.
Example 3 - Selection of the strain
The amino acid sequences of C-prME proteins from Zika virus strains (available from NCBI / Genbank) from Zika epidemics worldwide since 2007 have been aligned to look for similarities and differences (Figure 3 ). These included the strain of the African line of origin from Uganda, Micronesia (2007), French Polynesia (2013), the Brazilian strains since 2016 (Figure 3). In addition, seven strains of Zika virus from various regions of Brazil were also compared for differences in amino acids in the CprME region (Figure 4). High conservation was observed among strains from different epidemics, with Brazilian strains almost identical in the CprME region. Natal's strain, Bahia (KU527068) was chosen as the representative strain. KU527068 was
BE2017 / 5392 one of the first strains to be isolated from the brain of a fetus with microcephaly.
Example 4 - Design of constructions
The design of the Zika-SAM constructs in FIG. 5 includes the cloning of the sequence coding for the premembrane structural proteins (prM) and of the envelope (E) of the Zika virus (strain from Natal, Brazil) [with or without the capsid (C)], under the subgenomic promoter in a SAM vector. A series of modifications was carried out on the SAMprME constructs (Table 1, Figure 6 and Figure 7). These include:
i. Optimization of codons in the coding sequence for the antigen (CO-prME or CO-CprME).
ii. Genetic changes in the native prM signal peptide (Figure 6). In addition to proteolytic processing with peptidase signals, the viral protease NS3 / NS2B is also involved in the processing of structural proteins. The junction of region C and prM undergoes two proteolytic cleavage events during maturation. Cleavage releases protein C from its transmembrane anchor sequence and is dependent on NS2B / NS3 activity. A second cleavage occurs at the end of the protein C anchor sequence in the ER lumen by a cellular peptidase signal and produces the N-terminal end of prM. Previous studies conclude that treatment with the viral protease, regardless of the presence of the signal peptidase cleavage site, is necessary for efficient secretion of the viral particles. However,
BE2017 / 5392 in some flaviviruses, when this obligatory cleavage sequence was decoupled in a mutant virus, there was a greatly reduced incorporation of virions in the budding membranes and an increase in the release of subviral particles (Lobigs et al (2004) ) Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes, J Virol. 2004 Jan; 78 (1): 178-86).
iii. Replacement of the native signal peptide of prM by a heterogeneous signal peptide to amplify the production of PSV. The present inventors used the signal peptide of the IgGl previously used for the cleavage and the secretion of the IgG or Fab proteins (Ciferri et al. (2015) Antigenic Characterization of the HCMV gH / gL / gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies, PLoS Pathog. Oct 20; 11 (10): el005230).
iv. The Zika virus capsid protein (C) is incorporated into other constructs to test whether the presence of a cleavable capsid protein increases the efficiency of PSV production (Figure 7). This includes cleavage mediated by porcine teschovirus-1 protein 2A (P2A) of protein C in the absence of the viral protease.
Table 2
SAM-Zika constructions.
No. SAM insert Description 1 WT-prME Wild-type prM and E sequences 2 CO-prME PrM and E sequences optimized forcodons
BE2017 / 5392
3 CO-prME-ESS. 1 Same as CO-prME but with aamplified signal sequence (ESS) -PQAQA mutation in region C ofprM signal peptide to promotePPV 4 CO-prME-ESS. 2 Same as CO-prME but with an ESS -the prM signal peptide sequenceis replaced by the peptide sequenceIgG signal to favor PPV 5 CO-CprME.1 Same as CO-prME but expressingalso the capsid protein of theZika virus with cleavage sitesnative to proteins C and prM 6 CO-CprME.2 Same as CO-prME but expressingalso the capsid protein of theZika virus with the PQAQA mutation inthe C region of the signal peptide 7 CO-CprME.3 Same as CO-prME but expressingalso the capsid protein of theZika virus with a P2A site insertedafter the native cleavage site of theNS2B-3 Legend: WT - wild type; CO - optimized forcodons. Constructions are in the SAM vectordescribed elsewhere. Zika sequences are derived from theNatal strain (Brazil) - KU527068.1, except where notedopposite.
Study evaluation / design
The constructs are evaluated in mammalian cells following electroporation of Zika-SAM RNA in BHK cells using the following methods:
BE2017 / 5392
at. The replication-power of SAM RNA from SAM-Zika constructs is tested using antibodies directed against dsRNA and by FACS.
b. The expression of the antigens is determined by immunoblots and immunofluorescence tests, to study the prM and E protein cleaved in the cell lysates and the cell supernatant.
vs. PSV production is tested in mammalian cells using established procedures for the isolation of PSV from a cell supernatant.
Following the identification of the most effective candidate constructs, the formulation in NPL / NEC-based delivery systems is carried out and the analysis for antigenicity and immunogenicity is carried out in vivo.
Example 5 Expression and Secretion of Zika-SAM Constructions
The capacity of the cells to express and secrete protein E of the Zika virus from the ZikaSAM constructs described above was evaluated according to the following methods.
On day 0, BHK cells were plated at 8 x 10 ⁶ in T225 flasks in growth medium. For trypsination, the medium was removed and the cells were washed with 5 ml of PBS. PBS wash liquid was removed, and 5 ml of preheated trypsin was added and distributed carefully over the entire plate. Trypsin was removed and the plates were kept at 37 degrees C
BE2017 / 5392 for 1 to 2 min. The cells were then resuspended in 10 ml of growth medium (5% FBS). The cells were counted and plated at the required concentration in a new vial. The cells were incubated at 37 degrees C, 5% CCg for about 20 hours.
On day 1, the plates were prepared by adding 2 ml of DMEM + 1% FBS + P / S (growth medium) in each well of a 6-well plate (one well by electroporation). The plates were kept warm in an incubator at 37 degrees C. The electroporator was prepared to deliver 120 V, 25 ms pulse, 0.0 pulse interval, 1 pulse for a 2 mm cuvette. The bowls were marked and kept on ice. The cells in growth phase were harvested as normal in BHK medium (growth) and counted using a hemocytometer. The cells were trypsinized following the same trypsinization protocol as above. Electroporations of the standards and of the negative control were also prepared.
The cells were centrifuged at 1500 rpm (462 x g) for 5 min. The medium was aspirated, and the cells were washed once with 20 ml of cold Opti-MEM medium. The cells were again centrifuged at 1500 rpm (462 x g) for 5 min. The medium was aspirated, and the cells were resuspended in Opti-MEM medium to 0.25 ml by electroporation.
For each sample, 4000 ng of RNA were mixed with 250 μΐ of cells, and the mixture was
BE2017 / 5392 gently pipetted 4 to 5 times. The cells and the RNA mixture were transferred to 2 mm cuvettes and subjected to an electroporation pulse using the parameters described above. The cells were left to stand at room temperature for 10 min. Cells from a cuvette were added to one well of a preheated 6-well plate, and the plate was tilted back and forth and then side to side at an angle of 45 ° to distribute the cells evenly.
On day 2 (30 h post-electroporation), the supernatant was collected. A 75 μΐ aliquot was taken for the Western blot analysis, 25 μΐ of NuPAGE 4X buffer were added to the aliquot (no reducing agent), and the aliquot was stored at -20 ° C. The rest of the supernatant was stored at -80 degrees C.
The cells were washed once with ice-cold PBS, and then scraped in 200 μΐ of RIPA buffer containing a cocktail of protease inhibitors (1 tablet in 10 ml) while keeping the plate on ice. The cell-containing buffer was collected in microcentrifuge tubes, and subjected to two freeze-thaw cycles on dry ice. The samples were briefly vortexed, and centrifuged at 8000 rpm for 5 min. The pellets were discarded and the supernatants were retained. 25 μΐ of NuPAGE 4X buffer were added to a 75 μΐ aliquot of the lysates for the Western blot analysis. The aliquots were stored at -20 degrees C. The rest of the lysates were stored at -80 degrees C.
BE2017 / 5392
Concentration and filtration of the protein E species of the Zika virus from the cell supernatant
Cell supernatants from simple transfections (1 million cells, 4000 ng of RNA, 30 h post-electroporation) were loaded, 500 μΐ each, into the upper chamber of Amicon Ultra-0.5 centrifugal filter devices, 100 K (LMMN of 100,000). The centrifuge filter devices were centrifuged at 14,000 x g for 10 minutes each. After each centrifugation, the fraction not retained was collected in a separate tube. After centrifugation, the samples remaining in the upper chamber were further washed by mixing with 500 μΐ of 20 mM HEPES buffer, pH = 7.4, and centrifugation again at 14,000 x g for 10 minutes each.
The final samples remaining in the upper chamber, approximately 35 to 40 μΐ, were centrifuged in a fresh Eppendorf tube according to the manufacturer's instructions. Five μΐ of this sample was mixed with 5 μΐ of NuPAGE 4X buffer for the immunoblot, and the remaining samples were stored at -80 degrees C. In addition, 75 μΐ of the non-retained fraction were mixed with 25 μΐ of NuPAGE 4X buffer for the immunoblot, and the remaining unrecorded fraction was saved at 80 degrees C.
Immunoblot μΐ of cell culture supernatants and 15 μΐ of cell lysates (or 10 μΐ of concentrated supernatants and 15 μΐ of the fraction not retained) were
BE2017 / 5392 passed over a 4 to 12% SDS-PAGE gel (Bis-Tris) in the MOPS IX migration buffer. The separate samples were transferred to nitrocellulose membranes. The membranes were blocked for 2 to 3 hours in PBS-Tween 20 + 5% milk. The primary antibody 4G2 of flavivirus was added at a 1/120 dilution in PBS-T-milk and the membranes were incubated overnight at 4 degrees C. The membranes were then washed 3 times for 10 minutes at each time in PBS-T. A secondary anti-mouse antibody (Odyssey® anti-Mouse 800CW-green (LI-COR, Inc., Lincoln, NE) at 1/5000) in LI-COR blocking buffer was then added, and the membranes were incubated for 1 hour The membranes were washed three times for 2 minutes each, and then read on a LI-COR Odyssey® imager (LI-COR, Inc., Lincoln, NE) at channel 800, medium intensity.
Results
The expression of protein E of the Zika virus was detectable by immunoblotting in all the lysates originating from cells electroporated with the ZikaSAM constructs or with the positive control SAM-YFV (FIG. 8). The expression of protein E of the Zika virus was not detected in the SAM-RSV construct or the pseudo-infected negative controls. However, the secretion of protein E of the Zika virus was detectable by immunoblot only in the supernatants of construction No. 1 (wild type, WT), of construction No. 2 (codon optimized, CO), of construction No. 4 (optimized for codons with IgG signal peptide, CO-prMEBE2017 / 5392
ESS.2), and of construction No. 7 (optimized for codons with the capsid protein of the Zika virus and the P2A site, CO-CprME.3).
Filtration of the supernatant through filters with a cutoff of 100 kD and the immunoblot showed that almost all of the protein E was retained in the filter and that there was no detectable protein E in the fraction not retained (Figure 9). This indicated that the protein E detected in the supernatant may be part of a higher molecular weight molecular structure, presumably PSV.
EXAMPLE 6 Oil-in-Water Cationic Emulsions
Cationic nanoemulsions (NEC) were prepared essentially according to the methods described in Brito et al., Molecular Therapy, Vol. 22, No. 12, pp. 2118-29 (2014) and the international patent publication WO 2013006834.
In short, squalene (Sigma, St. Louis, MO) was heated to 37 ° C, and DOTAP (Lipoid, Ludwigshafen Germany) was dissolved directly in squalene in the presence of sorbitan trioleate (SPAN 85; Sigma, St. Louis, MO). The resulting oily phase was then combined with the aqueous phase (Tween 80; Sigma, St. Louis, MO, in citrate buffer) and immediately homogenized for 2 min using a T25 homogenizer (IKA, Wilmington, NC) at 24K rpm. / min to produce a primary emulsion. The primary emulsions were passed three to five times through an M-110S microfluidizer or an M-110P microfluidizer
BE2017 / 5392 (Microfluidics, Newton, MA) with a cooling coil in an ice bath at a homogenization pressure of approximately 15K to 20K PSI (1000 to 1400 bar). Lot samples were taken from the unit and stored at 4 ° C. The NEC formulation used in the present examples contains 4 mg / ml of DOTAP; 4.7 mg / ml Span 85; 4.7 mg / ml Tween 80; and 39 mg / ml squalene.
Example 7 Preparation of the RNA-NEC Complexes
1. Synthesis of RNA
The Zika-SAM constructs contain a bacteriophage T7 promoter located upstream of the alphavirus cDNA to facilitate the synthesis of RNA of the replicon in vitro. SAM-Zika RNA for construct No. 1 (coding for wild-type prM and E protein sequences of Zika virus (WTprME)) and construct No. 2 (coding for optimized prM and E protein sequences for codons (CO-prME)), was synthesized using standard molecular biology techniques. In short, the plasmid DNAs coding for the ZikaSAM constructs were linearized by digestion with endonucleases at a single site located at the 3 'end of the replicon sequence. The linearized DNA was then transcribed into RNA by synthesis in vitro using a T7 RNA polymerase in the presence of template DNA and nucleoside triphosphates (ATP, CTP, GTP and UTP). Following transcription, the template DNA was digested with DNase, and the RNA transcripts were purified by precipitation with LiCl and
BE2017 / 5392 reconstituted in nuclease-free water. The RNA was then capped using the vaccinia capping system (New England BioLabs, Ipswich, MA) and purified by precipitation with LiCl. The concentration of RNA in each reaction was determined by spectrophotometry. Before RNA complexation, the RNA was diluted to a concentration of 300 pg / ml in citrate buffer (10 mM citrate pH 6.2, 20 mM NaCl, 560 mM sucrose).
2. RNA complexation
Zika-SAM RNA has been complexed with cationic nanoemulsion (NEC) particles essentially as described in Brito et al., Molecular Therapy, Vol. 22, No. 12, pp. 2118-29 (2014). Briefly, the Zika-SAM RNA (300 pg / ml in citrate buffer) was added an equal volume of the NEC produced in Example 6, mixed, and allowed to complex on ice for 30 minutes at 2 hours. The final concentration of NEC complexed RNA was 150 pg / ml.
The ratio of RNA to cationic lipid can be expressed in N / P ratio, defined as the amount (moles) of phosphates (P) on RNA. DOTAP, for example, has a nitrogen that can be protonated by molecule. The concentration of RNA was used to calculate the amount of phosphate in solution using an estimate of 3 nmol of phosphate per microgram of RNA. The formulations of NEC described above have an N / P ratio of 6.3 / 1.
Example 8 Immunogenicity and In Vivo Protection of NEC Formulations of Zika-SAM
BE2017 / 5392
Female BALB / c mice (6 to 12 weeks old; The Jackson Laboratory), were housed and fed at the pet store of the Vaccine Research Center, NIAID, NIH, Bethesda, MD. All animal experiments have been described and approved by the VRC, NIAID, NIH Animal Care and Use Committee. All animals were housed and cared for in accordance with local, state, federal, and constitutional rules in an establishment accredited by the American Association for Accreditation of Laboratory Animal Care at NIH.
The mice were immunized twice according to the study design presented in Table 2. Briefly, it was administered to groups of 10 mice each of the NEC formulations containing the RNA constructs of Zika-SAM No. 1 or No. 2. As a positive control, another group of mice received 50 µg of a DNA-based vaccine against the Zika virus (construct No. 5283, as described in Dowd et al., Science , Vol. 354 Issue 6309, pp. 237-40 (2016)) by intramuscular electroporation. All mice were tested by intraperitoneal injection (i.p.) of live Zika virus on day 49.
Table 2
Study design for mice
Group not Administration Construction ImmunizationDay 0 Immunizationday 21 TestDay 49 1 10 CNE56 / RNA CO.prME 15 pg 15 pg 100 PFU,IP
BE2017 / 5392
2 10 CNE56 / ARN CO.prME 1.5 pg 1.5 pg 100IP PFU, 3 10 CNE56 / ARN WT.prME 15 pg 15 pg 100IP PFU, 4 10 CNE56 / ARN WT.prME 1.5 pg 1.5 pg 100IP PFU, 5 10 Electroporation /DNA 5283 50 pg 50 pg 100IP PFU,
Blood sera were collected on day 0, as well as 2 weeks after the first immunization, 2 weeks after the second immunization, and three days after the Zika virus challenge.
The neutralizing antibody titers for the Zika virus were measured by the viral reporter particles (PVR) neutralization test according to methods described in Dowd, KA et al. Cell Rep. 16 (6): 1485-9 (2016). The results are shown in Figure 10. Two weeks after the first immunization with Zika-SAM constructs No. 1 or No. 2 or the construct of positive control Zika virus DNA, there were significant levels of neutralizing antibodies for the Zika virus detected in the sera of immunized mice. The levels of neutralizing antibodies for the Zika virus were higher two weeks after the second immunization with the same Zika-SAM construct or the positive control.
A dose-dependent effect was observed, with the 15 pg dose of the Zika-SAM Constructions No. 1 and No. 2 producing higher levels of neutralizing antibodies compared to the dose of 1.5 pg. In particular, the 15 pg dose of the Zika-SAM No. 1 and
BE2017 / 5392
No. 2 produced a neutralizing antibody response which was comparable to the 50 pg dose of the Zika virus DNA vaccine construct (DNA No. 5283). These results indicate that the Zika-SAM No. 1 and No. 2 constructs are capable of inducing a significant response in neutralizing antibodies against the Zika virus.
On day 49 of the study, the mice were subjected to a test with intraperitoneal injections of live Zika virus (strain PRVABC57) at a dose of 100 plaque-forming units (PFU). Serum samples were taken three days after the test, and viral loads were determined by quantitative real-time PCR (qPCR) of the Zika virus capsid gene.
As shown in Figure 11, mice vaccinated with the Zika-SAM No. 1 or No. 2 constructs (1.5 or 15 pg doses) or the positive control construct (DNA No. 5283) showed so marked a reduction in Zika virus detected in serum compared to unvaccinated animals. These results indicate that the constructs Zika-SAM No. 1 and No. 2 are capable of producing a protective immune response against infection by the Zika virus.
BE2017 / 5392

权利要求:
Claims (39)
[1]
1. Construction of a nucleic acid-based vaccine encoding a polypeptide comprising a full-length prME antigen of the Zika virus, or one of its immunogenic fragments or variants.
[2]
2. Construction according to claim 1, in which said nucleic acid is an RNA comprising the coding region for the antigen.
[3]
3. Construction according to any one of claims 1 or 2, in which the construction is optimized for codons.
[4]
4. Construction according to any one of claims 1 to 3 comprising a prM signal sequence.
[5]
5. Construction according to any one of claims 1 to 4, further comprising a mutated signal sequence of prM for amplified cleavage of prM.
[6]
6. Construction according to any one of claims 1 to 5, further comprising an IgG signal sequence.
[7]
7. Construction according to any one of claims 1 to 6, further comprising a sequence of the capsid optimized for codons.
[8]
8. Construction according to any one of claims 1 to 7, further comprising a cleavage sequence of the 2A protein of porcine teschovirus-1.
[9]
9. Construction according to any one of claims 1 to 8, wherein said construction
BE2017 / 5392 comprises a nucleic acid sequence chosen from the group consisting of:
(a) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 26; SEQ ID NO: 31; SEQ ID NO: 36; SEQ ID NO: 41; SEQ ID NO: 46; SEQ ID NO: 52; and SEQ ID NO: 58;
(b) a nucleic acid sequence comprising the DNA sequence of SEQ ID NO: 25; SEQ ID NO: 30;
SEQ ID NO: 35; SEQ ID NO: 40; SEQ ID NO: 45;
SEQ ID NO: 51; and SEQ ID NO: 57;
(c) a nucleic acid sequence comprising the RNA sequence corresponding to the DNA sequence of SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81; SEQ ID NO: 82; and SEQ ID NO: 83; and (d) a variant or fragment of (a) to (c).
[10]
10. Vector comprising the construction according to any one of claims 1 to 9.
[11]
11. A self-replicating RNA molecule comprising the construct according to any one of claims 1 to 9.
[12]
12. Self-replicating RNA molecule encoding an antigen comprising a nucleic acid sequence chosen from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76.
[13]
13. DNA molecule coding for the self-replicating RNA molecule according to claims 11 or 12 comprising a nucleic acid sequence chosen from the group consisting of SEQ ID NO: 63, SEQ ID NO: 64,
BE2017 / 5392
SEQ ID NO: 65, SEQ ID NO: 66,
SEQ ID NO: 68, and SEQ ID NO: 69.
[14]
14. Composition including
SEQ ID NO: 67, an immunologically effective amount of one or more of the constructs of claims 1 to claim 10 according to any of to 9; of the vector according to the; or the self-replicating RNA molecule according to claims 11 or 12.
[15]
15. Composition according to comprising an RNA-based vaccine.
[16]
16. The composition of claim 14 claim 15 according to the comprising a self-replicating RNA molecule.
[17]
17. The composition as claimed in claim 16, in which the self-replicating RNA molecule comprises a sequence chosen from the group consisting of SEQ ID NO: 71, SEQ ID NO: 72,
SEQ ID NO: 74, SEQ ID NO: 75, and
ID NO: 70, ID NO: 73, ID NO: 76.
[18]
18. Composition according to any one of claims 14 to 17, in which the composition comprises a non-viral administration material, such as a cationic oil in submicron water emulsion; a liposome; or a system for administering biodegradable polymer microparticles.
[19]
19. The composition of claim 18, wherein the cationic oil-in-submicron water emulsion comprises an oily core, a cationic lipid, and a surfactant.
[20]
20. Composition according to any one of claims 14 to 19 in which the composition
BE2017 / 5392 further comprises one or more nucleic acid sequences which code for one or more additional antigens and / or the composition further comprises one or more additional antigens.
[21]
21. A composition according to any of claims 14 to 20 wherein the composition is pharmaceutically acceptable for administration to a subject in combination with another composition which comprises a nucleic acid comprising a sequence which codes for an additional antigen; and / or the composition is pharmaceutically acceptable for administration to the subject in combination with another composition which comprises an additional antigen.
[22]
22. Composition according to any one of claims 14 to 21 in which the composition comprises one or more adjuvants.
[23]
23. A method of inducing an immune response against infection by the Zika virus in a subject in need thereof, which comprises administering to the said subject an immunologically effective amount of a composition comprising one or more a construction according to claims 1 to 9; the vector of claim 10; the self-replicating RNA molecule according to claims 11 or 12; or of a composition according to any one of claims 14 to 22.
[24]
24. The method of claim 23 wherein the immune response is characterized by an immunological memory against the Zika virus and / or an effective population of T cells with memory sensitive to the Zika virus.
BE2017 / 5392
[25]
25. Method according to any one of claims 23 or 24 in which the subject is human.
[26]
26. A method of producing an RNA-based vaccine comprising a step of transcribing the vector according to claim 10 or the DNA according to claim 13 to produce an RNA comprising a region coding for the antigen.
[27]
27. The method of claim 26, wherein said transcription is in vitro.
[28]
28. The method of claim 26, wherein said transcription is in vivo.
[29]
29. The method according to any one of claims 26 to 28, further comprising a step of formulating the RNA comprising the coding region for the antigen with a delivery system.
[30]
30. The method of claim 29, wherein the delivery system is a non-viral delivery material.
[31]
31. The method of claim 30, wherein the delivery system is selected from the group consisting of: a cationic oil-in-submicron water emulsion; a liposome; and a delivery system for biodegradable polymer microparticles.
[32]
32. The method according to any one of claims 26 to 31, further comprising a step of combining the RNA comprising the coding region for the antigen with an additional composition comprising an adjuvant.
[33]
33. The method of claim 32, wherein said adjuvant comprises an immunostimulant.
BE2017 / 5392
[34]
34. Composition produced by the method according to any one of claims 26 to 33.
[35]
35. Use of the construction according to claims 1 to 9; the vector of claim 10; the self-replicating RNA molecule according to claims 11 or 12; or a composition according to any one of claims 14 to 22 for the induction of an immune response against a Zika virus infection in a subject.
[36]
36. Use of the construction according to claims 1 to 9; the vector of claim 10; the self-replicating RNA molecule according to claims 11 or 12; or a composition according to any of claims 14 to 22 in the manufacture of a medicament inducing an immune response against infection by the Zika virus in a subject.
[37]
37. Construction according to claims 1 to 9; vector according to claim 10; self-replicating RNA molecule according to claims 11 or 12; or composition according to any of claims 14 to 22 or 34 for use in therapy or medicine.
[38]
38. Construction according to claims 1 to 9; vector according to claim 10; self-replicating RNA molecule according to claims 11 or 12; or composition according to any of claims 14 to 22 or 34 for use in vaccine therapy.
[39]
39. Construction according to claims 1 to 9; vector according to claim 10; self-replicating RNA molecule according to claims 11 or 12; or
BE2017 / 5392 composition according to any one of claims to 22 or 34 for use in the prevention or treatment of a Zika virus infection in a subject in need thereof.
the one
Genome
NCR.
, _in g> __— 1 | (ni · struct! .rail /
B E2017 / 5392
Polyprotein prM
NS1

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法律状态:
2018-08-31| FG| Patent granted|Effective date: 20180710 |

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2020-03-27| MM| Lapsed because of non-payment of the annual fee|Effective date: 20190630 |

优先权:

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申请号 | 申请日 | 专利标题

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US201662344417P| true| 2016-06-02|2016-06-02|

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US62344417|2016-06-02|

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US201662394769P| true| 2016-09-15|2016-09-15|

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US201762485081P| true| 2017-04-13|2017-04-13|

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