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 the 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:BE1025121B1
申请号:E2017/5835
申请日:2017-11-15
公开日:2018-11-05
发明作者:Kimberley DOWD；Barney S. Graham；Sung-Youl Ko；Wing-Pui Kong；John Mascola；Theodore Pierson；Mayuri Sharma；Dong Yu
申请人:Glaxosmithkline Biologicals Sa；The United States Of America, As Represented By The Department Of Health And Human Services；
IPC主号:

专利说明:

(30) Priority data:
11/17/2016 US 62423398
04/13/2017 US 62485090
10/05/2017 US 62568559 (73) Holder (s):
Human Services GLAXOSMITHKLINE BIOLOGICALS SA1330, RIXENSARTBelgiumThe United States of America, As Represented by the Department of Health and20892-7660, ROCKVILLEUnited States (72) Inventor (s): DOWD Kimberley20852-3804 ROCKVILLEUnited StatesGRAHAM Barney S.20852-3804 ROCKVILLE United StatesKO Sung-Youl20852-3804 ROCKVILLEUnited StatesKONG Wing-Pui20852-3804 ROCKVILLEUnited States
MASCOLAJohn
20852-3804 ROCKVILLE
United States
PIERSON Theodore
20852-3804 ROCKVILLE
United States
SHARMA Mayuri
02139 CAMBRIDGE
United States
YU Dong
20850 ROCKVILLE
USA (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 processing, and methods for their manufacture. The compounds include a nucleic acid construct comprising a sequence which codes for a Zika virus antigen.
Genome S ‘coiife —Signal peptidase from host JL Viral protease (NS2B-NS3)
Host's furin Unknown protease
BELGIAN INVENTION PATENT
FPS Economy, SMEs, Middle Classes & Energy Publication Number: 1025121Filing number: BE2017 / 5835 Intellectual Property Office International Classification: A61K 39/12 A61K 39/00 Date of issue: 05/11/2018
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 November 15, 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'arstitut 89, 1330 RIXENSART Belgium;
The United States of America, As Represented by the Department of Health and Human Services, 6011 Executive Boulevard, Suite 325, MSC 7660, 20892-7660 ROCKVILLE United States;
represented by
PRONOVEM - Office Van Malderen, Avenue Josse Goffin 158, 1082, BRUXELLES;
a 20-year Belgian invention patent, subject to payment of the annual fees referred to in article XI.48, §1 of the Code of Economic Law, for: ANTIGENIC CONSTRUCTIONS OF ZIKA VIRUS.
INVENTOR (S):
DOWD Kimberley, 6011 Executive Boulevard, Suite 325, 20852-3804, ROCKVILLE;
GRAHAM BarneyS., 6011 Executive Boulevard, Suite 325, 20852-3804, ROCKVILLE;
KO Sung-Youl, 6011 Executive Boulevard, Suite 325, 20852-3804, ROCKVILLE;
KONG Wing-Pui, 6011 Executive Boulevard, Suite 325, 20852-3804, ROCKVILLE;
MASCOLAJohn, 6011 Executive Boulevard, Suite 325, 20852-3804, ROCKVILLE;
PIERSON Theodore, 6011 Executive Boulevard, Suite 325, 20852-3804, ROCKVILLE;
SHARMA Mayuri, 40 Landsdowne Street 75-3050T, 02139, CAMBRIDGE;
YU Dong, 14200 Shady Grove Road, 20850, ROCKVILLE;
PRIORITY (S):
11/17/2016 US 62423398;
04/13/2017 US 62485090;
10/05/2017 US 62568559;
DIVISION:
divided from the basic request:
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, 05/11/2018,
By special delegation:
B E2017 / 5835
ANTIGENIC CONSTRUCTIONS OF ZIKA VIRUS
Declaration of the interest of the American government
This invention was developed in the bet out of ^T urt agreement for research and development. collaboration with the National Institutes of Health, an agency of the Department of Health and Human Services.
The United States government has certain rights in this invention.
Field of the invention
This invention is in the field of treatment and prevention of viral infections.
In particular, the present invention relates to nucleic acid vaccine constructs encoding Zika virus antigens and the use of Zika virus antigens for the treatment and prevention of Zika virus infections.
Context of the invention
The Zika virus was first identified in Uganda in 1947 in: Rhesus monkeys through a selvatiqae 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. His tank is. unknown.
Zika virus is a positive strand RNA virus belonging to the flaviviridae family. Disease
B E2017 / 5835 due to the Zika virus is caused by a virus transmitted mainly by Aedes mosquitoes. People with Zika virus disease may have symptoms which may include mild fever, rash, conjunctivitis, muscle and joint pain, malaise or headache. These symptoms normally last for 2 to 7 days.
The Zika virus is known to circulate in Africa, 10 in the Americas, Asia and the Pacific.
Transmitted by Aedes mosquitoes, the virus is known to cause either an asymptomatic infection (in most people: infected) or a spontaneously resolving disease with a decrease of 15 .1 rash, conjunctivitis and a slight fever.
However, during the continuing outbreak of the Zika virus 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. 3.54 Issue 6309, pp, 23740 (2016) recently reported that DNA vaccines expressing the premembrane and envelope proteins of the Zika virus were immunogenic in mice and non-human primates when administered by electroporation or injection without a needle; and that the protection against viremia after the test with the Zika virus was correlated with the neutralizing activity of the serum.
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Chahai et al.
(Scientific (2.017)) de c r i v e n t for antigens is formulated with a structural vector of the virus s h a vira 1 coda n t nanomaterial of modified dendrimers has been found that the vaccine is
Richner et al.
Zika virus.
aanopart icu.1 e s (Cell, immunogenic.
Modified mRNA encoding e st in lipids and administered
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MRNA has and protection against viral testing.
Given the worrying burden due to the disease, there is an urgent need to develop components for use in an immunogenic or vaccine composition against the Zika virus.
The present inventors provide as components of immunogenic compositions for the induction of an immune response in a subject against infection by the Zika virus, methods for their use in treatment, and methods for their manufacturing.
In some embodiments, there is provided an acid-based, nucleic acid vaccine construct for a polypeptide comprising a pre25 antigen
B E2017 / 5835
M-E (prME) full length of. Zika virus, or one of its immunogenic fragments.
In some embodiments, there is provided a vector comprising the: construction as described.
In some embodiments, there is provided a self-replicating RNA molecule (also referred to herein as self-amplifying mRNA, or SAM molecule) comprising the construct as described.
a
In some embodiments, there is provided a composition comprising an iramunologically effective amount of one or more of the self-replicating RNAs as described
In some embodiments of a composition as described above, the composition includes a RNA-based vaccine.
In some embodiments, it. is a composition, as described above provided or where provided, the composition comprises one or more constructs, vectors, or molecules of self-replicating RNA such as oil-in-water cationic emulsion.
In some modes of.
embodiment, there is provided a composition as described above for use in inducing an immune response against infection with Zika virus in a subject and in need thereof
In some embodiments, there is provided a method for inducing an immune response.
B E2017 / 5835 of an immunologically effective amount of a composition comprising uti. (E) or more of the self-replicating RNA constructs, vectors, or molecules as described above.
In some embodiments, there is provided a method as described above in which the composition comprises unite) or multiple constructs, vectors, or self-replicating RNA molecules as described above complexed with a particle of an oil-in-water cationic emulsion.
In some embodiments, there is provided a process for the production of an RNA-based vaccine comprising a step of transcribing a vector pu of a DNA molecule encoding a 15 s RNA molecule self-replicating described above to produce an RNA comprising a coding region for the antigen.
In certain embodiments, there is provided a process for the preparation of a composition as described above in which the process comprises 1) the preparation of an oil-in-water cationic emulsion;
2) the preparation of one or more constructs, vectors: or molecules of self-replicating RNA as described above; and 3) the addition of the united) or more constructs, vectors, or RNA molecules self-replicating to the cationic emulsion: oil in water such that the construct, vector, or molecule of self-replicating RNA. complex with the emulsion.
In some embodiments, there is provided a composition produced by the method described above.
B E2017 / 5835. In certain embodiments, use is made of the construct, the vector, of the self-replicating RNA molecule, or of the composition described above for the induction of an immune response against Zika virus infection in a subject.
In some embodiments, there is provided a use of the construct, the vector, the self-replicating RNA molecule, or the composition described above in the manufacture of a drug which induces a response. immune to Zika virus infection in a subject.
In some embodiments, it. provided is a construct, vector, self-replicating RNA molecule, or composition described above for use in therapy.
In some embodiments, there is provided a construct, vector, self-replicating RNA molecule, or composition described above for use in a method of inducing an immune response against infection by Zika virus in a subject.
Description of s sins / figures
Figure 1 - Organization of the Flavivirus genome, showing the polyprotein which is cleaved into structural and non-structural proteins by a combination of viral and cellular proteases.
Figure 2 - Formation of Flavivirus virions and subviral particles. (1) In natural infections, flavivirus proteins are produced
B E2017 / 5835 by the treatment of a polyprotein translated from the genomic RNA of the virus and. Inserted in a cotraductional manner in the membrane of the. reticulum etcioplasmi qi (RE). · The horizontal arrows indicate 5 cleavages of the polyprotein by the signal peptidase and the arrow heads indicate, cleavage by the viral protease NS2B-3. The empty arrow indicates a signalase cleavage which is ineffective unless the cleavage of the cytoplasmic capsid (C) is. occurred.
(2) The minimum requirement for the production of subviral particles consists of the membrane precursor (prM) and envelope (E) proteins. (3) The flavivirus particles are formed by budding, on the membrane of the ER directed by the proteins prM and E 15 independent of the protein 0 or of the preformed nucleocapsides. Viral infection mainly results in the formation of virions. (4) Pseudoviral particles devoid of nucleocapsid are efficiently produced by recombinant expression of the proteins prM and E and are a byproduct of flavivirus infection. (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-D - Alignment of multiple CLUSTAL 0 (1.2.1) sequences of CprME proteins from: virus strains
Zika: Uganda (Uganda) (SEQ ID NO: 10); Micronesia (Micronesia) (SEQ ID NO: 11.); Natal (SEQ ID NO: 2);
Salvador (SEQ ID NO: 8); No. of accession. Genbank KU 3 65 7 7 7 (SEQ ID NO: 13); No. of accession Genbank KÜ 3 65 7 78 (SEQ ID NO: 14); No. of accession Genbank
B E2017 / 5835
K. U365779 (SEQ ID NO: 15); Genbank accession number KU365780 (SEQ ID NO; 16); French Polynesia. ("French >>) (SEQ ID NO: 12); and Sao Paolo (. “Sao”) (SEQ ID NO: 9). An "*" (asterisk) indicates the 7 positions which have a unique residue, totally preserved.
":" (Colon) indicates conservation between groups of strongly similar properties. A “,” (dot) indicates conservation between groups of weakly similar properties.
θ 1
Zika virus strain, year, and Genbank reference
Strain Year Genbank number UgandaNC 012532 Micponesia 2007 EUS 45988.1 Natal (Brazil) 2016 KU527068 Salvador (Brazil) 2016 KU707826.1 Sao Paulo (Brazil) 2016 KU321639 French Polynesia 2013 KJ’77 6791
Figures 4A-C - Multiple sequence alignment
CLUSTAL 0 (1.2.1) of protein CprME of strains brazilian virus Zika : Natal. (SEQ ID: NO: 2); Salvador (SEQ ID NO: 8); No. of accession Genbank KU365777 (SEQ ID NO: 13); No. accession Genbank KU365778 (SEQ ID NO: 14); No. of accession Genbank KU365779 (SEQ ID NO: 15); No. d ^! accession Genbank KU365780 (SEQ ID NO: 16); and Sao Paolo ("Sao >>)
(SEQ ID NO: 9). See Table 1 for Zika virus strains, years, and Genbank references.
Figure 5 - A SAM-Zika construction. The context of self-amplifying mRNA (SAM) consists of
B E2017 / 5835 repllcoh TC ~ 83 of the VEE virus coding for non-structural proteins, viral 1 to. 4 (nsPl to 4), followed by the subgenomic promoter, and an insert, coding for an anti-gene of the Zika virus. The empty vector is represented by SEQ: ID. NO: 17; 1 insert starts immediately after 1 n. 1. é and i d e 7,561.
Figure 6 '- Design of antigen inserts for Zika-SAM constructions.
The antigen insert # 5283, shown in (A), 10 includes a signal sequence of the Japanese encephalitis virus (JEV) followed by the prMEs of. Zika virus, including the starting sequence, of prM type: wild type (“AEVTR”). The full length amino acid sequence of the antigen insert # 5283 is represented by SEQ ID NO: : 19, including the signal sequence of JEV (SEQ LD NO: 5), followed by the pr region of Zika virus (SEQ LD NO: 20), protein M (SEQ ID NO: 21) and protein E ( SEQ ID NO: 22), The DNA and RNA sequences encoding the antigen insert shown in (A) are represented by SEQ ID NO: 18 and 39, respectively.
The antigen insert # 5288, presented in (B), is identical to the insert of (A), except that the last 98 amino acids of protein E of the Zika virus are replaced by the last 98 acids amines from the C-terminus of JEV protein E (Genbank Accession No. AFV52311.1; SEQ ID NO: 7). Chang et al. (2003) Virol. 306: 170-180. The full length amino acid sequence of antigen insert # 5288 is represented by SEQ ID NO: 24, including the signal sequence of JEV (SEQ ID NO: 5), followed by the pr region and the Zika virus M protein
B E2017 / 5835 (SEQ ID NO: 25 and 26, respectively), and a hybrid protein E of the zika-JEV virus (SEQ ID NO: 27), The DNA and RNA sequences coding for the insert of antigen presented in. (B) are represented by 5 SEQ ID NO. : 23 and 40, respectively.
Figure 7 ~ Analysis of the expression and secretion of protein E from .de: Zika-SAM constructs.
Protein: E expression and secretion were detected by immunoblot in cell lysates for self-amplifying mRNA constructs encoding antigen inserts # 5283 (signal from JEV t prME Zika virus) and # 5288 (signal of JEV + prME hybrid Zika-JEV virus). The expression and secretion of protein E were also detected for a positive control antigen construct (# 8111) but not for a negative control construct (# 4974) or pseudo-transfer ("Mock"). See 1 example 5.
Figure 8 - Responses xi antic caps neut ra 11 jumped 20 mice. It was found that the mice exhibited significant neutralizing antibodies against the Zika virus two weeks after a single vaccination with the Zika-SAM construct # 5283 (JEV L prMe signal from the Zika virus), or with the positive control construct d DNA from the Zika virus # 5283, it is measured by the neutralizing reporter virus particles test (RVPj. The neutralizing antibody titers were further increased two weeks after a second vaccination with the same Zika-SAM construct, or on 30 positive control "The antibodies neutralizing the Zika virus were below the limit of quantification before
B E2017 / 5835 la. vaccination (Day 0), and after vaccination with the negative control construct (# 4974, assayed, dice 1.5 pg) or with # 5288 (signal of JEV + hybrid prME of the ZikaJEV virus, dose of 1.5 pg) . A dose-response effect was observed for the construction SAM # 5283., With 15 pg of RNA triggering more neutralizing antibodies than 1.5 pg of RNA.
Figure 9 - Protection of mice against the test with Zika virus: mice vaccinated on days 0 and 10 21 were subjected to a test with live zika virus on day 49. The viral load was measured 3 days after viral test. Vaccination with the SAM construct # 5283 (at doses of 1.5 pg and 15 pg), as well as the positive control (DNA # 5283) was protective against viremia by the Zika virus. Unvaccinated mice and mice vaccinated with SAM construct # 5288 (1.5 pg dose) or negative control construct # 4974 were not protected in the test with Zika virus. The dotted line indicates the limit of -quantification (LOQ). of the test.
Figure 10 - Neutralizing antibody responses in non-human primates (PNH). Rhesus macaques were immunized at weeks 0 and 4 with construct Zika-SAM # 5283 (75 pg / dose) or construct. SAM with optimized codons coding for the native prME antigen pg / dgse). Has PNH placebo or they have. was Zika virus DNA # neutralizing the Zika virus virus (Go.prME SAM; controls, it was administered an immunized with a construct) 283 (4 mg./dose). . .Zika antibodies were significantly elevated four weeks after first immunization
B E2017 / 5835 with the construct Zika-SAM # 5283 or the DNA of the Zika virus # 5283 compared to placebo, with titers further increased 4 weeks after Here second immunization. The Zik.a ~ SAM Co.prME SAM construct produced significantly less neutralizing antibodies compared to DNA # 5283 after a single dose, but similar titers after two injections. The dotted line indicates the limit of quantification (LOQ) of the test.
Figure 11 - Protection of PNB against the test with the Zika virus: rhesus macaques vaccinated as described in Figure 10 were then subjected to a test with the live Zika virus, at week 8. Viral load was measured daily 3-7 days after the viral test. The placebo animals developed elevated viremia as early as 3 days after the viral challenge (A). Vaccination with the Zika-SAM construct: # 5283 (B), Zika-SAM Co · »prME (C) _r and DNA # 5283 (D) was protective against viremia by the Zika virus, with the Zika construct -SAM # 5283 with complete protection against viremia by the Zika virus. The dotted line indicates the limit of quantification (LOQ) of the test.
Figure 12 ~ Serology after viral test in PNB. The neutralizing antibody titers were determined 8, 10 and 12 weeks after the Zika virus test, and are expressed as a factor of variation from the rates before the viral test. The placebo animals showed an abrupt increase in neutralizing antibodies after: the 'Zika virus test, suggesting Zika virus infection in these animals. Two animals from the SAM Co.prME group, and one animal from DNA group # 5283, showed high neutralizing antibody titers after the viral test, indicating that the protection was not sterilizing in these animals. On the contrary, the animals in the group
Zika-SAM # 5283 did not show elevated neutralizing antibodies after the Zika virus test, indicating that sterilizing protection was obtained in all subjects. The dotted line in {B}, (C) and (P) indicates a variation factor of 4 of the neutralizing titers, which is considered to be non-sterilizing in the field of flaviviruses.
Detailed weighing 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 production. use in treatment, and methods for their manufacture. By construction, a nucleic acid is meant which codes for the polypeptide sequences described herein, and may include DNA, RNA, to non-naturally occurring nucleic acid monomers. The nucleic acid components of the constructs are described more fully in the Nucleic acids section of this document.
In some embodiments, the constructs disclosed herein encode sequences
B E2017 / 5835
B E2017 / 5835 wild-type polypeptides of a Zika virus, or one of their variants, or one of their fragments. The constructs may further code for a polypeptide sequence heterologous to the polypeptide sequences of a Zika virus. In some embodiments, the constructs encode wild-type polypeptide sequences of a Brazilian strain of the Zika virus, or one of their variants, or one of their fragments. By "Brazilian strain of Zika virus" is meant any strain of Zika virus indicated as "Brazilian. In Table 1, Unless otherwise indicated, the descriptions of the wild-type prME antigen are made with reference to the strain Natal (Brazil), GenBank number 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 G.
A "variant" of a polypeptide sequence includes amino acid sequences having one or more amino acid substitutions, insertions and / or deletions compared to the reference sequence. The variant may include an amino acid sequence which is at least 7'0%, at least 75 '%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least%, or at least 9.9% 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 may comprise an immunogenic fragment (i.e., an epitope-containing fragment) of the full-length polypeptide which may include a
B E2017 / 5835 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 least 19, amino acids or 5. plus which is identical to 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, 10 where 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-terminus, the 0-terminus terminated it, or both
The N-terminal end and the C-specified end of its consecutive amino acids,
As it is used, the term "antigen" is used. ea a molecule containing u η or more if eur s (for example, linear, conformational or of 1 which will stimulate the immune system of one to produce a specific immunological response 'humoral- and / or cellular antigen (i.e. say, two) an immune response that specifically recognizes an antigenic polypeptide).
An "epitope" part of an antigen that has immuno1og i that.
The epitopes of the T lymphocytes and empirically identified (for example, may be using one of the following similar methods). See references: Geysen et al. (1984) PNAS USÄ 81: 39984002; Carter (1994) Methods Mol Biol 36: 207-23. They
B E2017 / 5835 can be predicted (for example, using the damesoh-Wolf antigenic index (see Jameson et al. (1988) CABIOS 4 (1): 181-18-6 '), matrix-based approaches (see Raddrizzani & Hammer (2000) Brief Bioinform 1 (2):
179-89), TEPITOPE (see De Laila 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): 581-9.1; 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 Crus (1991) Nature 349 (6311): 720-1),
1 hydrophilicity (see Hopp (1993) Peptide Research 6: 18: 3-1.90), the antigenic index (see Welling et al.
(1985) FEB S Lett. 188:. 215-218) or the methods disclosed in the reference Davenport et al. (19.95) Immunogenetics 42: 392-297, etc.).
In some embodiments, the constructs herein code for a prika Zika virus antigen. By "prMe antigen of the Zika Z virus, it is: signified the amino acid sequence, or a nucleotide sequence coding for the amino acid sequence, of a wild type prika structural protein of the Zika virus, one of its variants, or one of its fragments. Figure 3 and Figure 4 identify the amino acid sequence of several variants of the full-length wild-type prME structural protein of the Zika virus. The sequence identifier numbers for each are presented in the Sequence List here. See 30 SSQ ID NO: 2 and 8 to 16.
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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 8 to 16. 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 include 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, at least 17, at least 18, at least 19, or more amino acids that is the same as 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 8 to 16, where the fragment comprises a deletion of up to '' at 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 the N end -terminal and C-terminal end 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
BE2017 / 5835 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 ig antigen. By "Zika virus ène antigen" is meant the amino acid sequence, or a nucleotide sequence coding for the amino acid sequence, of SEQ ID NO: 21. When a Zika virus ène antigen is a variant of a wild-type Μ polypeptide, 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: 21.
A fragment of a Zika virus antig antigen can comprise N- and / or C-terminal deletions compared to a full-length polypeptide, for example, SEQ ID NO: 21, where 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-terminus, the C-terminus, or both the Nterminal and l 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 encoding the
BE2017 / 5835 amino acid sequence, of SEQ ID NO: 22. When a Zika virus E antigen is a variant of a wild-type E polypeptide, the variant may include 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: 22. In certain embodiments, a Zika virus variant E antigen may be a hybrid E antigen comprising an N-terminal fragment of a Zika virus E protein fused to a Cterminal fragment of a heterologous viral E protein. In some embodiments, the heterologous viral protein E is derived from a heterologous flavivirus, such as the Japanese encephalitis virus (JEV).
A fragment of a Zika virus E antigen can comprise N- and / or C-terminal deletions compared to a full-length polypeptide, for example, SEQ ID NO: 22, where 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-terminus, the C-terminus, or both the Nterminal and l C-terminus of the full length sequence. It can be specified that the deletions are consecutive amino acids.
In some embodiments, a prika variant antigen of the Zika virus comprises a hybrid E protein of the Zika-JEV virus. A hybrid Zika-JEV E antigen may comprise an N-terminal fragment of a Zika virus E protein comprising at least the acids
BE2017 / 5835 amino (1 + x) to (422 ± y) of SEQ ID NO: 22, where x is an integer chosen from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and y is an integer chosen from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In one embodiment, the N-terminal fragment of a protein E of the Zika virus comprises amino acids 1 to 422 of SEQ ID NO: 22.
A hybrid E antigen can comprise a C-terminal fragment of a JEV E protein comprising at least amino acids (205 ± x) to (302 ± y) of SEQ ID NO: 7, where x is an integer chosen from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and y is an integer chosen from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In one embodiment, the Cterminal fragment of a JEV protein E comprises amino acids 205 to 302 of SEQ ID NO: 7.
In one embodiment, a hybrid E antigen comprises the amino acid sequence of SEQ ID NO: 27, or one of its fragments or variants. 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 the polypeptide according to SEQ ID NO: 27. 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, such as a contiguous amino acid sequence of at least 50, at least 100, at least 150, at least 200, at least
BE2017 / 5835
250, at least 300, at least 350, at least 400, at least 450, at least 500 amino acids or more which is identical to a sequence of contiguous amino acids of
SEQ ID NO: 27.
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 Nterminal position relative to the prM antigen sequence. Cleavage occurs in the ER lumen by a cellular peptidase signal and produces the N-terminus of prM. When the polyprotein comprises a wild-type amino acid sequence, the polyprotein comprises a native prM signal sequence, SEQ ID NO: 3. By "native prM signal sequence" is meant the amino acid sequence, or a sequence nucleotide encoding the amino acid sequence of a signal sequence of a wild-type viral prME, SEQ ID NO: 3. Figures 3A and B and Figure 4A identify the amino acid sequence of several variants of the native full length prM signal sequences from various strains of the Zika virus.
In certain 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 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% to the full-length polypeptide according to SEQ ID NO: 3. Alternatively, or additionally,
BE2017 / 5835 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 amino acids contiguous with minus 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: 3 which is identical to a contiguous amino acid sequence of the full-length polypeptide.
In some embodiments, the construction codes for a non-Zika heterologous signal sequence. In certain embodiments, the construction codes for a signal sequence of the Japanese encephalitis virus (JEV), one of its variants, or one of its fragments, in place of the prM signal sequence of the virus. Zika. By “signal sequence of JEV” is meant the amino acid sequence as presented in FIG. 6: MGKRSAGSIMWLASLAVVIACAGA (SEQ ID NO: 5). A variant may comprise an amino acid sequence which is identical to at or at least 99 a full-length wild type polypeptide, for example, to a polypeptide according to
SEQ ID NO: 5.
Alternatively, or in addition, a fragment of a polypeptide may comprise a functional fragment (i.e., containing the recognized and cleaved sequence of the full-length polypeptide which may comprise a sequence of contiguous amino acids of at least at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
BE2017 / 5835 amino acids, which is identical to a contiguous amino acid sequence of the full-length polypeptide.
In certain embodiments, the construction codes for a polypeptide comprising from the Nterminal part to the C-terminal part: a signal sequence of JEV and a prME antigen of the Zika virus, one of its variants, or one of its immunogenic fragments. In certain embodiments, the construction codes for a polypeptide comprising from the N-terminal part to the C-terminal part: a JEV signal sequence, a prM antigen of the Zika virus, and a hybrid E antigen comprising an N-terminal fragment of a Zika virus E protein fused to a C-terminal fragment of a JEV E protein.
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 some embodiments, the construction codes for a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 24. In some embodiments, the construction codes for a polypeptide 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% at a sequence chosen from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 24. In certain embodiments, the construction codes
BE2017 / 5835 for a polypeptide which comprises a fragment of a full-length sequence chosen from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 24, where the fragment comprises a contiguous sequence of the amino acid sequence of the full length sequence up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids shorter than the full length sequence.
In some embodiments, the construct includes a DNA sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 23. In some embodiments, the construct includes a DNA 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 sequence chosen from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 23. In certain embodiments, the construction comprises a DNA sequence which comprises a fragment of a full-length sequence chosen from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 23 where the fragment comprises a contiguous sequence of the DNA sequence of the full length sequence 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 some embodiments, the construct includes an RNA sequence selected from the group consisting of SEQ ID NO: 39 and SEQ ID NO: 40. In some embodiments, the construct includes an RNA sequence that 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
BE2017 / 5835 group consisting of
In some modes% to a sequence
SEQ ID NO: 39 and realization, chosen in the
SEQ ID NO: 40.
the construction comprises an RNA sequence which comprises a fragment of a full-length sequence chosen from the group consisting of SEQ ID
NO: 39 and SEQ ID NO: 40 where the fragment comprises a contiguous sequence of the RNA sequence of the full length sequence up to 1, 2, 3, 4, 5, 6,
25, or 30 nucleic acids shorter than the full length sequence.
In some embodiments, the construct includes a fragment of a full-length RNA sequence selected from the group consisting of
SEQ ID NO: 39 and SEQ ID NO
0, where the fragment includes a deletion of up to
1, 2, 3,
4, 5, 6, 7,
9, 10, 15, 20, 25, or 30 nucleic acids from the 5 'end, the 3' end, or both 5 'and 3' ends of the full length sequence.
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 proteases, by translation from RNA, by purification at from a cell culture (for example, from a recombinant expression), from the organism itself, etc. An example of a process for the production of long peptides
BE2017 / 5835 <40 amino acids in vitro, see implies a chemical synthesis following references: Bodanszky of Peptide Synthesis (ISBN:
0387564314); and Fields et al. (1997) Meth Enzymol 289:
Solid-Phase Peptide Synthesis. ISBN: 0121821900. Techniques for synthesizing peptides on a solid phase, such as methods based on tBoc or Fmoc chemistry, are known in the state of the art, see the following reference: Chan & White (2000) Fmoc Solid Peptide Synthesis phase. ISBN: 0199637245. An enzymatic synthesis can also be used in part or in whole, see the following reference: Kullmann (1987) Enzymatic
Peptide Synthesis. 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-based polypeptides, but manipulation of the translation machinery (e.g. aminoacyl-tRNA molecules) can be used to allow introduction D-amino acids (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 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
BE2017 / 5835 take various forms (for example, native, fusions, glycosylated, non-glycosylated, lipid, non-lipid, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomer, multimeric, particulate, denatured, etc.). The polypeptides can be glycosylated naturally or not naturally (ie the polypeptide can have a glycosylation profile which differs from the glycosylation profile found in the polypeptide existing in the corresponding natural state).
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 a prME antigen sequence of the Zika virus or a chimeric prME sequence of the Zika virus. For example, a polypeptide here can be a fusion protein. Alternatively, or in addition, the amino acid sequence or chemical structure of the polypeptide can be modified (for example, with one or more unnatural amino acids, by covalent modification, and / or by having a different glycosylation profile , for example, by removing or adding a naturally occurring polypeptide sequence (s).
The polypeptides (e.g., antigens) disclosed herein are preferably provided in a purified or substantially purified form, i.e., substantially free of other polypeptides (e.g., free of existing polypeptides
BE2017 / 5835 natural), particularly other polypeptides of the Zika virus or of 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, etc. Alternatively, less than about
50%, less than about 40%, less about 30 %, less about 20%, less than about 10 %, or less about 5% of a composition are incorporated others expressed polypeptides. Nucleic acids The present inventors disclose here some
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 (for example, free of naturally occurring nucleic acids), particularly d other nucleic acids from the Zika virus or the host cell, generally being 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), and usually at least about 90% pure (by weight).
BE2017 / 5835
Nucleic acids can be prepared in many ways, for example, by chemical synthesis (for example, synthesis with DNA 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 contains deoxyribonucleotides, ribonucleotides, and / or their analogs. It includes DNA, RNA, DNA / RNA hybrids. It can also include DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or phosphorothioates) or modified bases. Thus, the nucleic acid 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, the nucleic acid may comprise one or more heterologous nucleic acid sequences (for example, a sequence encoding another antigen and / or a sequence
BE2017 / 5835 control (such as a promoter or an internal ribosome entry site) in addition to 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 result in the production of a virus recombinant 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 here 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 (for example, about 30 or more adenosine residues) can be attached to the 3 'end of the RNA to increase its half-life. The 5 'end of the RNA can be capped with a ribonucleotide modified with the structure m7G (5') ppp
BE2017 / 5835 (5 ') N (cap structure 0) or one of its derivatives, which can be incorporated during RNA synthesis or which can be modified by enzymes after RNA transcription ( for example, using the vaccinia virus styling enzyme (VCE) consisting of mRNA triphosphatase, guanylyltransferase and guanine-7-methytransferase, which catalyzes the construction of N7monomethylated cap structures). The structure of cap 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'-O-methyltransferase which results in the production of a cap structure 1 (m7Gppp [m2'-0] N), which can also increase the efficiency of the translation.
The nucleic acids may include one or more nucleotide analogs or modified nucleotides. As used herein, "nucleotide analog" 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), thymine (T) or uracil (U)), adenine (A) or guanine (G)). A nucleotide analog may contain other chemical modifications 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 phosphate. The preparation of nucleotides and modified nucleotides and nucleosides is well known in the art, see the
BE2017 / 5835
references following: US patents numbers 4 373 071, 4,458,066, 4,500,707, 4,668 777, 4,973 679, 5 047 524, 5 132 418, 5,153,319, 5,262 530, 5 700 642. Of numerous nucleosides modified and nucleotides modified are
commercially available.
Modified nucleobases that can be incorporated into nucleosides and modified 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) adenosine) adenosine);
ms2t6A (2-methylthio-N6-threonylcarbamoylm6t6A (N6-methyl-N6-threonylcarbamoylhn6A (N6-hydroxynorvalylcarbamoyladenosine) ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyosenosine);
Ar (p) (2'-O-ribosyladenosine (phosphate))
I (inosine) mil (1-methylinosine);
mlm (1,2'-O-dimethylinosine) m3C (3-methylcytidine);
Cm (2T-O-methylcytidine) s2C (2-thiocytidine); ac4C (N4-acetylcytidine)
5FC (5-formylcytidine); m5Cm (5,2-O-dimethylcytidine);
ac4Cm (N4-acetyl-2TOmethylcytidine);
k2C (lysidine); mlG (1methylguanosine) m2G (N2-methylguanosine); m7G (7methylguanosine)
Gm (2'-O-methylguanosine); m22G
BE2017 / 5835 (N2, N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2, N2,2'-O-trimethylguanosine);
Gr (p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW * (sub-modified hydroxywybutosine); imG (wyosin); mimG (methylguanosine); Q (queuosin); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine);
manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G * (archaeosine); D (dihydrouridine); m5Um (5,2'-Odimethyluridine); 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 5-oxyacetic uridine acid); chm5U (5 (carboxyhydroxymethyl) uridine)); mchm5U (5- (carboxyhydroxymethyl) uridine methyl ester); mcm5U (5-methoxycarbonyl-methyluridine); mcm5Um (Smethoxycarbonylmethyl-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-methyl-uridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6, N6dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C
BE2017 / 5835 (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-methyldihydro-uridine); £ 5Cm (5-formyl-2'-0methylcytidine); mlGm (1,2'-O-dimethylguanosine); m'Am (1,2-O-dimethyl-adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); iniG-14 (4demethyl-guanosine); imG2 (isoguanosine); ac6A (N6acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, their 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-alkyl (C1 to C6) uracil, 5- methyluracil, 5-alkenyl (C2 to C6) uracil, 5-alkynyl (C2 to Ce) - uracil, 5 (hydroxymethyl) uracil, 5-chlorouracil, 5fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5alkyl (C1 to C6) Cs) cytosine, 5-methylcytosine, 5-alkenyl (C2 to Cs) cytosine, 5-alkynyl (C2 to Cs) - cytosine, 5chlorocytosine, 5-fluorocytosine, 5-bromo-cytosine, N2dimethylguanine, 7-deazaguanine, 8 -azaguanine, 7-substituted 7-deazaguanine, 7-deaza-7-alkynyl (C2 to Ce) guanine, 7-deaza-guanine 8-substituted, 8hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2aminopurine, 2-amino- 6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-purine 7-substituted, 7-deaza-purine 8-substituted, hydrogen (abasic residue), m5C , m5U, m6A, s2U, W, or 2'-O-methyl-U . Many of these modified nucleobases and their ribonucleosides
BE2017 / 5835 correspondents 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 additional antigens (for example, a second, third, fourth, fifth or sixth) of the Zika virus can also be provided in the form of a vaccine based on 'nucleic acid. In some embodiments, a composition includes a nucleic acid sequence encoding a Zika virus prME antigen from a first strain of Zika virus and an additional nucleic acid sequence encoding an additional Zika prME antigen from one or more other strains of Zika virus. In some embodiments, a composition includes a nucleic acid sequence encoding a Zika virus prME antigen and one or more additional antigens (e.g., a second, third, fourth, fifth, or sixth) of the Zika virus. Alternatively, one or more additional Zika nonvirus antigens can be encoded.
The nucleic acid may be, for example, RNA (i.e., an RNA-based vaccine) or DNA (i.e., a d-based vaccine DNA such as a plasmid DNA vaccine).
In some modes of
BE2017 / 5835 realization, the nucleic acid-based vaccine is an RNA-based 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 using replicating elements derived, for example, from alphavirus, and substituting structural proteins of the virus with a nucleotide sequence encoding a protein of interest. A self-replicating RNA molecule is usually a strand + molecule that can be translated directly after administration to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from administered RNA. Thus, the administered RNA leads to the production of multiple daughter molecules of RNA. These RNA daughter molecules, as well as the collinear subgenomic transcripts, can be translated themselves to provide in situ expression of a coded gene (i.e., a prM-F antigen of the Zika virus), or they 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 transcription sequence is a vast amplification of the number of RNA replicons introduced and thus
BE2017 / 5835 the coded antigen is found to be a major polypeptide product of cells.
A suitable system for obtaining self-replication in this way is to use an alphavirus-based replicon. These replicons are 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 that self-activates to provide a replication complex that creates genomic strand copies of the administered strand + RNA. These negative strand (-) transcripts can themselves be transcribed to give other copies of the parent strand RNA + and also to give a subgenomic transcript which codes for the antigen. Translation of the subgenomic transcript thus leads to in situ expression of the antigen by the infected cell. Appropriate alphavirus replicons can use a replicase from a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus, an equine encephalitis virus from the Venezuela, etc. The mutant or wild-type virus 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 / 5835 be an alphavirus replicase, for example, comprising one or more nsP1, nsP2, nsP3 and nsP4 proteins of alphavirus.
While natural alphaviral genomes code for structural virion proteins in addition to the non-structural replicase polyprotein, in some embodiments, the self-replicating RNA molecules do not code for structural alphavirus proteins . Thus, self-replicating RNA can lead to the production of copies of genomic RNA of itself in a cell, but not to the production of RNA-containing virions. The inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule cannot be perpetuated in infectious form. The structural proteins of alphaviruses which are necessary for the perpetuation of 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 in the context of the invention can have two open reading frames. The first open reading frame (in 5 ') codes for a replicase; the second open reading frame (in 3 ') codes for an antigen. In some embodiments, the RNA may have additional open reading frames (for example,
BE2017 / 5835 downstream), for 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 can also include a poly-A polymerase recognition sequence (for example, AAUAAA) near its 3 'end. Self-replicating RNA molecules can have various lengths, but are generally 5,000 to 25,000 nucleotides in length. The 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 strand form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA which is formed either during the replication of a single strand RNA or within the secondary structure of a single stranded RNA.
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 methods of creation by gene synthesis and / or polymerase chain reaction (PCR)) . For example, a DNA40 RNA polymerase
BE2017 / 5835 dependent (like the RNA polymerases of bacteriophages T7, T3 or SP6) can be used to transcribe the self-replicating RNA from a template DNA. Appropriate styling and polyA addition reactions can be used as required (although the poly-A sequence 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, to ensure that the RNA transcribed by IVT can function effectively as substrate for its self-encoded 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 in the context of the invention ideally comprises only phosphodiester bonds between the nucleosides, but in certain embodiments, it may contain phosphoramidate, phosphorothioate, and / or methylphosphonate bonds.
The self-replicating RNA molecule can encode 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 produced as a fusion polypeptide or modified to
BE2017 / 5835 result in separate polypeptide or peptide sequences.
The self-replicating RNA molecules described here can be modified to express multiple nucleotide sequences, from two or more open reading frames, thereby allowing coexpression of proteins, such as one or two Zika virus antigens or more (for example, one or two or more ZME virus prME antigens) 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 (eg 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 autologous target cells that contain an RNA molecule self42
BE2017 / 5835 replicator which codes for a prME antigen of the Zika virus can be analyzed. In addition, the differentiation of helper T lymphocytes can be analyzed by measuring the proliferation or production of cytokines TH1 (IL2 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 Zika virus prME antigen can also be tested for the ability to induce humoral immune responses, as evidenced, for example, by induction of production by the B lymphocytes of antibodies specific for a prME antigen of the Zika virus 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, FACS analysis can be used to detect expression of antigens on the cell surface or at the intracellular level. Another advantage of selection by FACS is that different expression rates can be sorted; sometimes a lower expression may be desired. Another suitable method for identifying cells which express a particular antigen involves specific adhesion using monoclonal antibodies on a plate or capture using magnetic beads coated with monoclonal antibodies.
In some embodiments, the molecules
BE2017 / 5835 of self-replicating RNAs comprise a sequence chosen from the group consisting of
SEQ ID NO: 36 and
SEQ ID NO: 37. In some embodiments, the self-replicating RNA molecules include a sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at 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 SEQ ID NO: 36 and SEQ ID NO: 37. In certain embodiments, the self-replicating RNA molecule comprises a fragment of a full-length sequence chosen from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 37 where the fragment comprises a contiguous sequence of the sequence full-length nucleic acid 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 some embodiments, there is provided a DNA sequence encoding a self-replicating RNA molecule, said DNA sequence selected from the group consisting of SEQ ID NO: 33 and SEQ ID NO: 34. In some embodiments, the DNA sequence encoding a self-replicating RNA molecule includes a 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 sequence chosen from the group consisting of SEQ ID NO: 33 and SEQ ID NO: 34. In
BE2017 / 5835 certain embodiments, the DNA sequence coding for a self-replicating RNA molecule comprises a fragment of a full-length sequence chosen from the group consisting of SEQ ID NO: 33 and SEQ ID NO: 34 where the fragment comprises a contiguous sequence of the nucleic acid sequence of the full length sequence 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 replicon particles (VRP), or complexed with particles of an oil-in-water cationic emulsion. In some embodiments, the nucleic acid vaccine includes a cationic nanoemulsion (CNE) delivery system or a lipid nanoparticle delivery system (LNP). 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 replicons. In other embodiments, the nucleic acid vaccine may include a naked nucleic acid, such as a naked RNA (e.g., mRNA), but administration via CNE or
BE2017 / 5835
LNP is preferred.
In certain embodiments, the nucleic acid-based vaccine comprises a cationic nanoemulsion delivery system CNE delivery systems and the methods for their preparation are described in the following reference:
For administration of CNE, the nucleic acid molecule (eg, RNA) which codes for the antigen is complexed with a particle of an oil-in-water cationic emulsion. Cationic oil in emulsions can be used to deliver negatively charged molecules, such as an RNA molecule to
The emulsion particles include cells.
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 CNE details can be found in the following references: WO 2012/006380;
WO 2013/006834; and WO 2013/006837 (the content of each of which is incorporated herein in its entirety).
Thus, in a nucleic acid-based vaccine of the invention, an RNA molecule coding for a prME antigen of the 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 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,
BE2017 / 5835 polysorbate 80); polyethylene glycol can also be included. In some embodiments, the CNE comprises 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 a CNE, and the nucleic acid vaccine comprises self-replicating RNA (mRNA). This is particularly effective in triggering humoral and cellular immune responses. The benefits also include the absence of a limiting anti-vector immune response and a 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 from about 80 nm to 180 nm in diameter (or alternatively from about 80 to about 150 nm; from about 80 at 130 nm; or approximately 100 nm). In certain 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.
BE2017 / 5835 comprising DOTAP, squalene, sorbitan trioleate and polysorbate 80 in citrate buffer. The cationic oil-in-water emulsions suitable for the administration of an RNA molecule encoding a prME antigen of the Zika virus can contain approximately 2 mg / ml to 7 mg / ml of DOTAP; about 3 mg / ml to 6 mg / ml of 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 emulsion in
Water, before complexation with RNA, contains about 4.3 % w / v of squalene, 0 .5% of Tween 80, 0.5% from SPAN 85, and 4 mg / ml DOTAP. II East also provided a process of preparation
of a composition comprising an RNA molecule encoding a prME antigen of the Zika virus complexed with an oil-in-water cationic emulsion, the method comprising: (i) providing an oil-in-water emulsion as described here ; (ii) providing an aqueous solution comprising the RNA molecule; and (iii) combining 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 salt, buffer, saccharide, or polymer. 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.
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LNP delivery systems and non-toxic biodegradable polymeric microparticles, and methods for their preparation are described in the following references: WO 2012/006376 (LNP and microparticle delivery systems); Geall et al. (2012) PNAS USA. Sep 4; 109 (36): 14604-9 (LNP delivery system); and WO 2012/006359 (microparticle delivery systems). LNPs are non-virion liposomal particles in which a nucleic acid molecule (e.g., 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 LNPs for use in the context of the invention 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 LNPs are described in the following references: WO 2012/006376; WO 2012/030901;
WO 2012/031046; WO 2012/031043; WO 2012/006378;
WO 2011/076807; WO 2013/033563; WO 2013/006825;
WO 2014/136086; WO 2015/095340; WO 2015/095346;
WO 2016/037053. In some embodiments, the
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LNP are liposomes RV01, see the following references: WO 2012/006376 and Geall et al. (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 some embodiments, the Zika virus is a Brazilian strain of the Zika virus.
A "pharmaceutically acceptable carrier" includes any carrier which does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, poly lactic acids, poly glycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and aggregates lipids (such as oil droplets or liposomes). Such supports are well known to those skilled in the art. The compositions can also contain a pharmaceutically acceptable diluent, such as water, physiological saline, glycerol, etc. In addition, auxiliary substances, such as wetting or emulsifying agents, buffering substances
BE2017 / 5835 pH, and the like, may be present. The physiological saline buffered by sterile pyrogen-free phosphate is a conventional support.
The pharmaceutical compositions can comprise the constructs, the nucleic acid sequences, and / or the polypeptide sequences described elsewhere in this document in still water (for example, “ppi” water) or in a buffer, for example. for example, a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. Buffer salts will generally be included in the range of 5 to 20 mM. The pharmaceutical compositions can have a pH between 5.0 and 9.5, for example, between 6.0 and 8.0. The compositions may include sodium salts (e.g., sodium chloride) to provide the tone. A concentration of 10 ± 2 mg / ml of NaCl is conventional, for example, around 9 mg / ml. The compositions can include metal ion chelators. These can prolong the stability of RNA by removing ions that 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 essays
BE2017 / 5835 pharmaceuticals can include 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 as a unit dose. In some embodiments, a unit dose may have a volume between 0.1 and 1.0 ml, for example, approximately
0.5 ml.
In certain embodiments, the compositions disclosed herein are immunogenic compositions which, when administered to a subject, induce an immune response specific for the humoral and / or cellular antigen (i.e., a response immune system that specifically recognizes an existing Zika virus polypeptide
For example, an immunogenic composition can induce a population of T lymphocytes and / or
B with memory with respect to a subject not treated following an infection by the Zika virus, particularly in these embodiments where the composition comprises a nucleic acid comprising a sequence which codes for a prME antigen of the Zika virus or comprises a Zika virus antigen. In some embodiments, the subject
BE2017 / 5835 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 quantity varies according to the individual's health and physical condition, age, taxonomic group of the individual to be treated, to be treated (for example, capacity of the human being, non-human primate, immune system of the individual. to synthesize antibodies, the desired degree of protection, the formulation of the composition or of the vaccine,
The medical practitioner's assessment of the medical situation, the severity of the disease, the potency of the compound administered, the mode of administration, and other relevant factors. It is expected that the amount will fall within a relatively wide range which can be determined by routine testing.
The vaccines as disclosed herein may 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 embodiments, the vaccine compositions disclosed herein can induce an effective immune response against infection with the Zika virus, i.e., a sufficient response
BE2017 / 5835 for the treatment or prevention of infection with the Zika virus.
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 the 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 certain aspects, the adjuvant compositions as disclosed herein further include one or more immunostimulants, for example, a saponin such as QS21.
The adjuvants which can be used in the 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 particles
BE2017 / 5835 pseudovirals (VLP). (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 poly (acrylic acid) derivatives, polyvinyl alcohols, polyvinylpyrrolidone, polysaccharides and carboxymethylcellulose. (H) Microparticles, for example, particles with a diameter of -100 nm to -150 μm, more preferably a diameter of -200 nm to -30 μm, and most preferably of a diameter from -500 nm to -10 μm) formed from materials which are biodegradable and non-toxic (for example, a poly (a-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) mmuramyl-peptides.
(M) Imidazoquinolone compounds, for example, imiquimod and its counterparts.
Combinations of one or more of the adjuvants identified above can also be used in the context of the invention.
Operating procedures / uses
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In certain embodiments, there are provided methods 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 of a composition as disclosed here. In certain embodiments, there is provided the use of the constructs or compositions disclosed herein for inducing an immune response against a virus prME antigen
Zika in a subject in need. In certain embodiments, the use of the constructions or compositions disclosed herein is provided to
Induction of an immune response against virus infection
Zika 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 an immunological response specific to the humoral and / or cellular antigen (i.e., an immune response which specifically recognizes an antigenic polypeptide) which may be
BE2017 / 5835 demonstrated to neutralize the Zika virus in vitro or to control / reduce / eliminate infection with the Zika virus in vivo.
In certain embodiments, the immune response is characterized by an immunological memory against the Zika virus and / or a population of efficient T lymphocytes with memory and sensitive to the Zika virus.
In some embodiments, the composition includes an RNA molecule encoding a polypeptide selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 24. In some embodiments, the composition includes an RNA molecule encoding a polypeptide 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 sequence chosen from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 24. In certain embodiments, the composition comprises an RNA molecule coding for a polypeptide which comprises a fragment of a full length sequence chosen from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 24, wherein the fragment comprises a contiguous sequence of the amino acid sequence of the full length sequence 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 self-replicating construct, vector, RNA and / or molecule as described herein for use in therapy or medicine. In some modes
BE2017 / 5835 of realization, the compositions disclosed here are 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 construct, vector, RNA and / or molecule as described herein for use in the prevention or treatment of Zika virus infection in a subject needing it.
In certain embodiments, the compositions disclosed herein are for use in the prevention or treatment of Zika virus infection in a subject in need thereof.
In certain embodiments, the compositions disclosed herein are 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 construct, vector, RNA molecule, and / or composition as described herein for use in a method of inducing an immune response against infection by the Zika virus 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 subject an immunologically effective amount of a composition
BE2017 / 5835 immunogen as provided here.
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.
In some embodiments, 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 virus infection
Zika has a fetus across the placenta. In certain embodiments, a composition as described herein is administered to a woman in an amount effective to prevent transmission of Zika virus infection across the placental barrier.
In certain embodiments is provided the use of a construct or composition disclosed herein in the manufacture of an immunogenic composition for the prevention or reduction of the transmission of virus infection
Zika has a fetus across the placenta.
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 / 5835 reduction in the transmission of virus infection
Zika has a fetus across the placenta.
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 (for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or into the interstitial space of tissue). Variant routes of administration include rectal, oral (e.g., tablet, spray), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, auricular, pulmonary or other mucosal administration. Intradermal and intramuscular administration are two preferred routes. The injection can be done through a needle (for example, a hypodermic needle), but needle-less injection can be used alternatively. A conventional 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 the Zika virus contains 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 µg of RNA. In other embodiments,
BE2017 / 5835 a dose of a SAM-based vaccine against the Zika virus may contain <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 observed at much lower rates; for example, using <1 pg / dose, <100 ng / dose, <10 ng / dose, <1 ng / dose, etc. Similarly, a dose of protein antigen may include <10 pg of protein; for example, from 1 to 10 pg, such as about 1 pg, 2.5 pg, 5 pg, 7.5 pg or 10 pg.
In preferred embodiments, an SAM-based vaccine against the Zika virus or a vaccine composition is administered to a subject at an effective dose, meaning a dose sufficient to achieve a desired immune response, such as the 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 based on DNA or a vaccine composition 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-based vaccine or a vaccine composition encoding for the same antigen.
Manufacturing / formulation processes
Methods for making self-replicating RNA are provided herein. In some modes of
BE2017 / 5835 realization, the process 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 IVT to produce an RNA, and further includes a step of combining the RNA with a non-viral delivery system as described elsewhere in this document. In some embodiments, the method of making a self-replicating RNA includes an IVT step to produce an RNA, and further includes a step of combining the RNA with a CNE delivery system as 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 percentage of sequence identity, and not considering any conservative substitution 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 reference nucleic acid sequence after alignment of the sequences and introduction breaches, if necessary, to obtain the maximum percentage of sequence identity.
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Sequence identity can be determined by conventional methods which are commonly used to compare the similarity in position of the amino acids 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 et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)] can be used in conjunction with the computer program. For example, the identity percentage can then be calculated by: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longest sequence within the scope of the match and the number breaches introduced in the shortest sequence 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 with reference to a UniProt or Genbank accession code, the sequence to which reference is made is the current version on the date of filing of this application.
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General
Unless explained otherwise, all the technical and scientific terms used here have the same meaning as that commonly understood by those skilled in the art 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 the concentrations or rates of a substance, such as concentrations of solution components or their ratios, and reaction conditions such as temperatures, pressures, and cycle times are expected to be approximate. . The term "approximately" used here is intended to mean ± 10%.
The term "includes" means "includes". Thus, unless the context otherwise requires, the word "includes", and variations such as "include" and "comprising" will be understood to imply the inclusion of a indicated compound or composition ( (e.g., nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not excluding any other compound, composition, step, or group thereof . The embodiments described as including certain components are intended to include the embodiments made up of the indicated components.
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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 nonlimiting figures and examples which follow.
Examples
Example 1- Project summary
The present inventors began to 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 robust production of antigens and antigenicity and further tested for their immunogenicity and efficacy using in vivo models.
Processes
The VEE virus SAM TC-83 vector was used as the context construct for cloning in the examples. See SEQ ID NO: 17.
Example 2 - Selection of the antigen
The Flavivirus genome consists of a single stranded RNA of positive polarity with a length of approximately 11.3 kb (Figure 1). The 5 ′ proximal quarter of the genome encodes the structural proteins of the capsid (C), premembrane (prM), and envelope (E). The non-structural proteins NSI, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 are involved in
BE2017 / 5835 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 that encapsulate C together with the viral RNA, by budding in the ER lumen (Figure 2 ). At a later stage in viral maturation, prM on these particles is cleaved into mature Μ protein by a cellular protease, furin, before release from the cell. This cleavage of prM is necessary for the infectivity of the released virions. In addition to infectious virions, cells infected with the flavivirus release subviral particles (SVP) (Figure 2). These particles are smaller than virions, but contain the antigenically important protein E and the protein prM / Μ, which is essential for correct folding and incorporation of protein E in SVP and particles viral. However, unlike virions, SVPs do not contain protein C or the viral genome, and are therefore non-infectious. SVPs can be produced in various systems by coexpression of prM and E proteins, and SVPs share properties with wild-type viruses, such as
BE2017 / 5835 fusogenic activity and the induction of a neutralizing immune response and it has been repeatedly shown to stimulate protective immune responses against a number of flavivirus diseases. The present inventors chose structural proteins of the Zika virus, namely prM and E, for another experiment.
Example 3 - Selection of strains
The amino acid sequences of the C-prME proteins of Zika virus strains (available from NCBI / Genbank) originating from Zika virus outbreaks worldwide since 2007 were aligned to look for similarities and differences (Figure 3) . These included the original African line strain from Uganda, Micronesia (2007), French Polynesia (2013), and 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 C-prME region (Figure 4). High conservation was observed in all strains from different flares, with Brazilian strains almost identical in the CprME region. Natal strain, Bahia (KU527068) was chosen as the representative strain. KU527068 was one of the first strains to be isolated from the brain of a fetus with microcephaly.
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Example 4 - Design of constructions
The design of the Zika-SAM constructions of FIG. 6 includes the cloning of the sequence coding for the premembrane structural proteins (prM) and of the envelope (E) (strain Natal, Brazil) under the subgenomic promoter in a SAM vector. A series of modifications to the SAM-prME constructions were made (Table 1, Figure 6). These include:
i. Substitution of the native prM signal peptide by the JEV signal peptide (Figures 6A and B, antigen inserts # 5283 and # 5288).
ii. Substitution of the native C-terminal end of protein E of the Zika virus by the terminal end of protein E of JEV (FIG. 6B, antigen insert # 5288).
iii. Truncation of the prM starting sequence of the wild-type Zika virus to alter the expression and secretion of the prME antigen (antigen insert # 4974). Antigen insert # 4974 was used as a negative control. The amino acid sequence of antigen insert # 4974 is represented by SEQ ID NO: 29, including the signal sequence of JEV (SEQ ID NO: 5), followed by a pruncated region of the Zika virus ( SEQ ID NO: 30), protein M of the Zika virus (SEQ ID NO: 31) and protein E of the Zika virus (SEQ ID NO: 32). The DNA and RNA sequences encoding the antigen insert # 4974 are represented by SEQ ID NO: 28 and 41, respectively.
BE2017 / 5835 iv. A Zika non-virus antigen construct, # 8111, was designed and used as a positive control.
Study evaluation / design
The constructs are evaluated in mammalian cells following the electroporation of the Zika-SAM RNA in BHK cells using the following methods:
at. The replication-power of 1 'SAM RNA of SAM-Zika constructions is tested in using of antibody directed against dsRNA and a FACS technique. b. The expression of antigens East determined by
immunoblots and immunofluorescence tests, to study the prM and E proteins cleaved in cell lysates and the cell supernatant.
vs. The production of SVP is tested in mammalian cells using established procedures for the isolation of SVP from the cell supernatant.
Following the identification of the most effective candidate constructs, formulation in LNP / CNE-based delivery systems is performed and analysis for antigenicity and immunogenicity is performed in vivo.
Example 5 Expression and Secretion of Zika-SAM Constructions
The ability of cells to express and secrete protein Zika virus E and protein hybrid Zika-JEV virus from Zika-SAM constructs
BE2017 / 5835 described above was evaluated according to the following methods.
On day 0, BHK cells were deposited 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 then removed, and 5 ml of preheated trypsin was added and spread evenly across the plate. The trypsin was removed and the plates were held at 37 degrees C for 1 to 2 min. The cells were then resuspended in 10 ml of growth medium (5% FBS). The cells were counted and deposited at the required concentration in a new vial. The cells were then incubated at 37 degrees C, 5% CO2 for about 20 hours.
On day 1, the plates were prepared by the addition of 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. Cells in the 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 standards and negative controls were also prepared.
BE2017 / 5835
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 0.25 ml Opti-MEM medium by electroporation.
For each sample, 4000 ng of RNA were mixed with 250 μΐ of cells, and the mixture was gently pipetted 4 to 5 times. The mixture of cells and RNA was 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 flipped back and forth and then side to side at an angle of 45 ° to distribute the cells evenly.
On day 2 (30 h after electroporation), the supernatant was collected. A 75 μΐ aliquot was taken for a 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 buffer containing the cells was
BE2017 / 5835 collected in microcentrifuge tubes, and subjected to two series of freezing / thawing 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.
Immunoblot μΐ of the cell culture supernatants and 15 μΐ of the cell lysates were 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
BE2017 / 5835
The expression and the secretion of protein E of the Zika virus are detectable by immunoblot in the lysates of cells electroporated with the constructs Zika-SAM # 5283 (signal of JEV + prME of the wild-type Zika virus), # 5288 (signal of the JEV + prME hybrid virus Zika-JEV) and # 8111 (positive control antigen). (Figure 7). The expression and secretion of protein E of the Zika virus were not detected in the truncated construction at the N-terminal end (# 4974) or the Mock controls.
EXAMPLE 6 Oil-in-Water Cationic Emulsions
Cationic nanoemulsions (CNE) 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.
Briefly, 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 24,000 rpm to produce a primary emulsion. Primary emulsions were passed three to five times through a microfluidizer
M-110S or an M-110P microfluidizer (Microfluidics,
Newton, MA) with one pressure ice bath cooling coil
BE2017 / 5835 homogenization of approximately 15,000 to 20,000 psi. Lot samples were removed from the unit and stored at 4 ° C. The CNE 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-CNE 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 the RNA replicon in vitro. The SAM-Zika RNA for constructs # 5283 (coding for the signal of JEV + prME of the Zika virus) and # 5288 (coding for the signal of the JEV + prME hybrid of the Zika-JEV virus), as well as the negative control construct # 4974, was synthesized using standard molecular biology techniques. Briefly, the plasmid DNA encoding the Zika-SAM constructs was linearized by digestion with endonucleases from 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 LiCl precipitation and reconstituted in nuclease-free water. The RNA was then styled using the vaccinia styling system (New England BioLabs, Ipswich, MA) and purified by
BE2017 / 5835 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 (CNE) particles essentially as described in Brito et al., Molecular Therapy, Vol. 22, No. 12, pp. 211829 (2014). Briefly, the Zika SAM RNA (300 μg / ml in citrate buffer) was added in a volume equal to the CNE produced in Example 6, mixed, and allowed to complex on ice for 30 minutes to 2 hours . The final concentration of RNA complexed with CNE was 150 pg / ml.
The ratio of RNA to cationic lipid can be expressed in N / P ratio, defined as the quantity (moles) of protonatable nitrogen atoms (N) in the cationic lipid divided by the quantity (moles) of phosphates ( P) on the 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 CNE formulations described above have an N / P ratio of 6.3 / 1.
Example 8 Immunogenicity in Vivo and Protection of CNE Formulations of Zika SAM
The immunogenicity and protection of CNE formulations of Zika SAM were examined in mice and non-human primates (PNH).
I. Study in mice
Female BALB / c mice (6 to 12 weeks old; The Jackson Laboratory), were housed and
BE2017 / 5835 bred in the Vaccine animal establishment
Research Center, NIAID, NIH,
Bethesda, MD. All animal experiments have been reviewed and approved by
1'Animal Care and Use
VRC Committee,
NIAID, NIH.
All animals have been housed and cared for in accordance with local, state, federal, and institutional policies 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.
In short, to groups of 10 mice, it was administered to each of the formulations of
CNE containing the Zika RNA constructs or the negative control RNA construct. Another group of mice received 50 µg of a DNA-based vaccine coding for intramuscular construction as a positive control. See Dowd et al., Science, Vol. 354
Issue 6309,
All mice were tested with live Zika virus on day 49.
Table 2
Study design in mice
B E2017 / 5835
Groupe not Administration Constructionot ImmunizationnotDay 0 ImmunizationDay 21 Day Trial49 1 10 CNE56 / RNA 4974 1.5 pg 1.5 pg 100-PFU,IP 2 10 CNES6 / ARN 5288 1.5 og 1.5 pg 100PFU,IP 3 10 CNE56 / RNA 5283 15 pg 15 pg 100PFU,IP 4 10 CNES6 / ARN 5283 1.5 pg 1.5 pg 100PFU,.IP 5 10 Electroporation/ DNA 5283 50 pg 50 pg 100PFU,! P
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.
Zika virus neutralizing antibody titers were measured by the reporter viral particle neutralization (PVR) test according to methods described in Dowd, KA et al. Cell Rep. 16 (6): 1485-9 (2016). The results are presented in FIG. 8. Two weeks after the first immunization with the Zika SAM # 5283 construct, or the construct of positive control DNA, significant levels of antibodies neutralizing the Zika virus were detected in the sera of immunized mice. Zika virus neutralizing antibody levels were even higher two weeks after
B E2017 / 5835 the second immunization with the construction SAM # 5283 or the construction of positive control DNA.
A dose-dependent effect was observed, with the 15 pg dose of the Zika SAM # 5283 construct producing higher levels of neutralizing antibodies compared to the 1.5 pg dose. In particular, the 15 pg dose of the Zika SAM # 5283 construct produced a neutralizing antibody response which was comparable to the 50 pg dose of the DNA format of the same vaccine construct (DNA # 5283). These results indicate that the Zika SAM # 5283 construct is 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 the 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 FIG. 9, the mice vaccinated with the Zika SAM construct (doses of
1.5 or 15 µg) or the positive control construct (DNA # 5283) showed little or no detectable Zika virus in the serum. On the contrary, a significant load in Zika virus was detected in unvaccinated animals, as well as in animals vaccinated with the Zika SAM 5288 construct or the negative control construct. These results indicate that the Zika SAM # 5283 construct is capable of producing a protective immune response against infection by the Zika virus.
B E2017 / 5835
II · Study in non-human primates (PNH)
The immunogenicity of the Zika SAM constructs was evaluated in non-human primates (PNH). Rhesus macaques were immunized twice according to the study design presented in Table 3. Briefly, CNE-RNA SAM-Zika formulations were prepared as described in Example 7. A-des groups of 8 PNH, each was administered either placebo (phosphate buffer solution) or a CNE formulation containing a SAM construct optimized for codons, Co.prME, coding for the native prME antigen of the Zika virus (group 2 , 75 pg x 2; as described in document PCT / IB2017 / 053242, filed on June 1, 2017); or the construction Zika SAM # 5283 (group 3; 75 pg x 2}. Another group of PNH (group 4; n = 8) received
two immunizations (4 mg each) d ¹ a vaccine based of DNA encoding the construction # 5283 by track intramuscularly by a device injection without needle (PharmaJet) , in as a positive control. See
Dowd et al., Science, Vol. 354 Issue 6309, pp. 237-40 (2016). All animals were challenged with an intraperitoneal injection (i.p.) of live Zika virus (1000 PFU) 8 weeks after the first immunization.
Table 3
Group Vaccine Group size VaccinationWeek 0 ReminderWeek 4 TestWeek 8 1 Placebo 8 ΡΒΞ PBS 1000 PFU ZIKV 2 CO, prME SAM 8 7 5 pg 75 pg 1000 PFU ZIKV 3 SAM # 5283 8 75 pg 75 pg 1000 PFU ZIKV 4 DNA # 5283 8 4 mg 4 mg 1000 PFU ZIKV
B E2017 / 5835
Blood serum samples were collected on day 0, as well as 4 and 8 weeks after the first immunization; on each day 3 to 7 after the test with the Zika virus; and at weeks 8, 10 and 12 after the Zika virus test. Zika virus neutralizing antibody titers were measured by the reporter viral particle neutralization (PVR) test according to methods described in Dowd, KA et al. Cell Rep. 16 (6): 1485-9 (2016).
The immunogenicity results presented in FIG. 10 demonstrate that the antibodies neutralizing the Zika virus were significantly elevated four weeks after the first immunization with * the Zika SAM # 5283 construct compared to placebo, with titers further increased 4 weeks after the second immunization. DNA from Zika virus # 5283 triggered similar titers of neutralizing antibodies at 4 and 8 weeks. The ZikaSAM Co.prME SAM construct produced significantly fewer neutralizing antibodies compared to DNA # 5283 after a single dose, but similar titers after two injections.
In week 8 of the study, NHPs were tested with intraperitoneal injections of live Zika virus (strain PRVABC57) at a dose of 1000 plaque forming units (PFU). The viral loads after 1 ¹ test were determined by quantitative real time PCR (qPCR) of the gene. the capsid of the Zika virus.
As shown in Figure 11, the animals that received the placebo showed high viraemia
B E2017 / 5835 as early as 3 days after the event (A). Vaccination with the construct Zika-SAM # 5283 (B), as well as the positive controls Zika-SAM Co.prME (C) and DNA # 5283 (D), was protective against viremia by the Zika virus, with the construct Zika-SAM # 5283 with complete protection against viremia by the Zika virus.
In accordance with the results of the viraemia, the animals which received the placebo showed an abrupt increase in neutralizing antibodies after the test with the Zika virus, further confirming an infection by the Zika virus in these animals (FIG. 12). Two animals in the SAM Co.prME group, and one animal in the DNA group # 5283, also showed high titers of neutralizing antibodies after the test, indicating that the protection was not sterilizing in these animals. On the contrary, the animals in the Zika SAM # 5283 group did not show high neutralizing antibodies after the test with the Zika virus, indicating that sterilizing protection was obtained in all the subjects.
BE2017 / 5835

权利要求:
Claims (15)
[1]
1. Construction of a nucleic acid-based vaccine coding for a polypeptide comprising a 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, wherein the construction codes for a signal sequence.
[4]
4. Construction according to claim 3, in which the signal sequence is a heterologous flavivirus signal sequence.
[5]
5 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 claims 14 to 22 for use in therapy.
5. Construction according to claim 4, in which the heterologous flavivirus signal sequence is a JEV signal sequence.
[6]
6. Construction according to any one of claims 3 to 5, in which the signal sequence comprises SEQ ID NO: 5.
[7]
7. Construction according to any one of claims 1 to 6, wherein the prME antigen of the Zika virus comprises a protein E hybrid of the ZikaJEV virus.
[8]
8. Construction according to claim 7, in which the hybrid protein E of the Zika-JEV virus comprises the amino acid sequence of SEQ ID NO: 27.
[9]
9. Construction according to any one of claims 1 to 8, wherein said construction comprises
BE2017 / 5835 a nucleic acid sequence chosen from the group consisting of:
(a) a nucleic acid sequence coding for a polypeptide comprising the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 24;
(b) a nucleic acid sequence comprising the DNA sequence of SEQ ID NO: 18 or SEQ ID NO: 23;
(c) a nucleic acid sequence comprising the RNA sequence of SEQ ID NO: 39 or SEQ ID NO: 40; and (d) a variant or fragment of (a) to (c).
[10]
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 claims 14 to 22 for use in a method of inducing a response
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: 36 and SEQ ID NO: 37.
[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: 33 and SEQ ID NO: 34.
[14]
14. Composition comprising an immunologically effective amount of one or more of the constructs according to any one of claims 1 to 9; the vector of claim 10; or the self-replicating RNA molecule according to claims 11 or 12.
15. The composition of claim 14 comprising an RNA-based vaccine.
BE2017 / 5835
16. The composition of claim 15 comprising a self-replicating RNA molecule.
17. Composition according to claim 16, in which the self-replicating RNA molecule comprises a sequence chosen from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 37.
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. The composition of claim 18, wherein the cationic oil in submicron water emulsion comprises an oil core, a cationic lipid, and a surfactant.
20. A composition according to any one of claims 14 to 19 wherein the composition 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. 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.
BE2017 / 5835
22. Composition according to any one of claims 14 to 21 wherein the composition comprises one or more adjuvants.
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 a composition according to claims 14 to 22.
24. The method of claim 23, wherein the immune response is characterized by an immunological memory against the Zika virus and / or a population
T cells effective memory and sensitive at Zika virus. 25. Process according to one any of claims 23 or 24, in which the subject is human.
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. The method of claim 26, wherein said transcription is in vitro.
28. The method of claim 26, wherein said transcription is in vivo.
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.
BE2017 / 5835
30. The method of claim
29, wherein the delivery system is a non-viral delivery material.
31. Method according to 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. Method according to any one of claims 26
31, further comprising a step of combining the RNA comprising the coding region for the antigen with an additional composition comprising an adjuvant.
33. The method of claim 32, wherein said adjuvant comprises an immunostimulant.
34. Composition produced by the method according to any one of claims 26 to 33.
35. Use of the construction according to the claims 1 at 9 r of the vector according to the claim 10 r of the RNA molecule self- replicating according the claims 11 or 12; or of a composition according to the claims 14 at 22 for the induction of a reply immune against a infection with Zika virus in a subject.36. Use of the construction according to the claims 1 at 9 r of the vector according to the
claim 10; the self-replicating RNA molecule according to claims 11 or 12; or a
BE2017 / 5835 composition according to claims 14 to 22 in the manufacture of a medicament which induces an immune response against an infection by the Zika virus in a subject.
[15]
15 immune to Zika virus infection in a subject.

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法律状态:
2018-12-07| FG| Patent granted|Effective date: 20181105 |

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2020-08-21| MM| Lapsed because of non-payment of the annual fee|Effective date: 20191130 |

优先权:

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申请号 | 申请日 | 专利标题

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US201662423398P| true| 2016-11-17|2016-11-17|

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US62423398|2016-11-17|

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US201762485090P| true| 2017-04-13|2017-04-13|

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US201762568559P| true| 2017-10-05|2017-10-05|

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