Description:FIELD OF INVENTION
The present disclosure is related to development of mRNA sequences comprising at least one antigenic protein derived from rabies virus. The invention further relates to induce potent immune responses in mammalian subjects against rabies virus infection.
BACKGROUND OF INVENTION
Rabies is invariably a fatal disease causing approximately 60000 deaths, annually, worldwide. Transmission of rabies to humans, via infected dogs as vectors has posed distinct clinical complications such as hydrophobia. Mass vaccination of dogs and dog population management has improved the elimination of rabies but still in developing countries, nerve tissue vaccines are still applied for prophylaxis. Currently, inactivated and cell based vaccines are widely used for prevention of rabies in developing and developed countries, respectively. Major drawback is the requirement of repeated doses and being less immunogenic. Live vaccines are advantageous to promote mass vaccination in dogs, which can induce immunity efficiently through propagation of attenuated RV strain at injury site. Thus even smaller doses of vaccines can effectively inhibit infection at lower production costs. However, attenuated live vaccines can potentially induce rabies cases post vaccination and due to virus residual virulence, it has limited applications. New classes of vaccines have been developed but very few have been commercialized. Hence there’s a need for better immunogenic and economical rabies virus vaccines to prevent the infection. Recombinant DNA technology has significantly sped up the development of the next generation vaccines which are safe, less reactive and more immunogenic vaccines.
Rabies virus is a highly neurotropic virus of genus Lyssavirus in the family Rhabdoviridae. Rhabdoviruses contains a single stranded, non-segmented, and negative sense- RNA have two major structural components: helical ribonucleoprotein core (RNP) and an envelope surrounding it. Rabies virus genome encodes five proteins namely, nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and polymerase (L). RNP complex serves as a template for viral transcription and replication where the viral genome is encapsidated by the N in combination with L and P protein. Viral lipid envelope comprises a transmembrane glycoprotein that is tightly arranged on the surface of virus and a matrix protein being associated with envelope and RNP. G, N & M proteins are important for immunogenicity specifically G protein is the major antigen for formation of neutralizing antibodies. G protein is 524 amino acids sequence comprising 19 amino acid signal peptide at N-terminus, 21 amino acid transmembrane domain and a 44 amino acid cytoplasmic domain. Mutations in G proteins that contribute to pathogenicity are strain dependent. M proteins, consisting 202 amino acids, lies between lipid bilayer and RNP coil therefore, absence of M severely impairs the viral assembly and budding process. M directly interacts with G protein in the formation of bullet shaped virions, viral spread and pathogenicity. N protein, consisting of 450 amino acids, is responsible for encapsidation of genomic and anti-genomic RNAs. Also, N protein plays important role in evasion of innate immune responses and thereby in propagation and spread of virus in brain.
Post inoculation, the viral spread within the central nervous system (CNS) can be controlled by means of serum neutralizing antibodies. Effective vaccines are available for pre-exposure immunization as well as human rabies immune globulin (HRIG) for post exposure prophylaxis (PEP) intervention early after infection, to successfully prevent disease from progressing towards symptoms. Recombinant vaccines overcome the risk posed by the residual virulence in conventional vaccines where the genetic manipulations and reverse genetics. Modifications in viral genome has led to development of vaccines based on nucleic acids (DNA/mRNA), protein subunits vaccines, which are more potent, safer, stable and immunogenic at lower production costs. However, still there’s an unmet need to improve the effectiveness and reduce the number of doses for pre and post exposure prophylaxis. Also, the main object of this invention is to develop thermostable vaccines that do not rely on cold chain transportation thereby improving the reach of vaccines in resource limited locations.
SUMMARY OF INVENTION
The present invention provides an mRNA comprising one or more modifications, which increases the expression of the encoded protein, for medical use, wherein the modified mRNA is administered to a subject such as human and dogs. The present invention relates to design of mRNA sequence for treating or preventing in particular, the pre-exposure and post exposure rabies prophylaxis through a vaccination method as described herein.
DETAILED DESCRIPTION
As used herein, the term “vaccine” refers to a production of inactivated or dead or weakened pathogens or components of pathogens or derived antigenic determinants used to induce antibodies or immunity against the pathogen. In addition, the term “vaccine” is administered into subjects in form of a suspension or solution of the pathogen to produce immunity against the disease caused by that pathogen.
The present invention provides a modified messenger ribonucleic acid (mRNA) comprising at least one open reading frame (ORF) coding for at least one protein wherein the mRNA comprises at least one modification, which increases the expression of its encoded protein. In the meaning of the present invention, the term “redesigned mRNA” comprises any type of mRNA that is redesigned such that the amount of protein is increases in comparison with mRNA lacking the modification.
The redesigned mRNA according to the present invention may encode a protein which comprises a pathogenic antigen or a fragment, variant or derivative thereof. Such pathogenic antigens are derived from pathogens such a rabies virus which evoke an immunological reaction in a subject, in particular a mammalian subject, more particularly a human and a dog.
In a preferred embodiment, the redesigned mRNA relates to ribonucleotide sequence in particular an mRNA sequence modified for enhanced expression of mRNA encoded proteins. In present invention, the modified mRNA encodes a rabies virus protein or an antigenic fragment thereof, is a component of the vaccine injected into the dermis or muscle of a mammal may elicit immune reactions.
In one embodiment, the inventive mRNA sequence comprise a coding region, encoding at least one antigenic protein derived from glycoprotein (RABVPG), nucleoprotein (RABVPN), matrix protein (RABVPM), phosphoprotein (RABVPP) or the RNA polymerase (RABVPL) of rabies virus or a fragment, variant or derivative thereof or from any engineered rabies protein.
In one embodiment, the inventive mRNA sequence comprise a coding region, encoding at least one antigenic protein derived from glycoprotein (RABVPG) of rabies virus or a fragment, variant or derivative thereof (Figure 1).
In the preferred embodiment, the amino acid sequence encoded in the inventive mRNA is selected from the sequences with SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, and SEQ ID NO 36 or preferably an identity of at least 70%, more preferably of at least 80% and/ or most preferably of at least 90% with the nucleotide sequences according to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, and SEQ ID NO 36.
In one embodiment, the inventive mRNA sequence comprises a coding region that may occur as a mono-, di- or even multicistronic mRNA, i.e. an mRNA sequence which carries the coding sequences of one or more proteins. The coding sequences in di- or even multicistronic mRNAs may be separated by at least one internal ribosome entry site (IRES) sequence or by signal peptides/ proteins which induce cleavage of the resulting polypeptide comprising one or more proteins or peptides.
In a preferred embodiment, the full length protein of RABVPG, RABVPN, RABVPM, RABVPP or RABVPL protein is encoded by the coding region comprised in the inventive mRNA.
In a further preferred embodiment, a fragment comprising at least one epitope of the RABVPG, RABVPN, RABVPM, RABVPP or RABVPL protein is encoded by the coding region comprised in the inventive mRNA.
In further embodiment, the inventive mRNA comprises at least one of the structural elements; a 5’- and /or 3’- untranslated region element (UTR) and/ or a 5’CAP structure and/ or a poly (A) tail and /or optionally a poly (C) sequence.
In a preferred embodiment, the 5’ UTR or 3’ UTR comprises or a nucleic acid sequence are derived from a fragment, a homolog or a variant of 5’- or 3’- UTR of a gene.
In particular preferred embodiment, the inventive mRNA sequence in present invention comprises, preferably in 5’ to 3’ direction;
a) a 5’ CAP structure, preferably (m7GpppN);
b) a coding region, preferably with an increased or maximized G/C content compared with the G/C content of the coding region of the wild type Glycoprotein mRNA, encoding at least one antigenic protein derived from glycoprotein RABVPG; nuclepoprotein RABVPN; phosphopreotein RABVPP, matrix protein RABVPM or RNA polymerase RABVPL of rabies virus or fragment, variant or derivative thereof.
c) a 3’UTR element
d) a poly (A) sequence, preferably consisting of 50-250 adenosines
e) Either optionally a poly (C) sequence, preferably consisting of 30 cytosines.
In the preferred embodiment, the mRNA sequence comprises a poly (A) sequence of about 25 to about 400 adenosine nucleotides, preferably a sequence of about 50 to 400 adenosine nucleotides, more preferably a sequence of about 50 to about 300 adenosine nucleotides, even more preferably a sequence of about 50 to about 250 adenosine nucleotides, most preferably a sequence of about 60 to about 250 adenosine nucleotides.
For further improvement, the inventive mRNA is stabilized in the form of a modified nucleic acid, preferably by backbone modifications, sugar modifications and/ or base modifications, more preferred stabilized by modifications of the G/C content, preferably of the coding region thereof. One or more of these modifications may be incorporated in the inventive mRNA without impairing the translation function of the inventive mRNA to produce rabies virus derived protein in vivo.
Additionally, the inventive mRNA may be prepared using any method known in the art, including the synthetic methods such as solid phase synthesis, as well as in vitro methods, such as in vitro transcription reactions.
In preferred embodiment, the inventive mRNA consisting a coding region of at least one antigenic protein of rabies virus or a fragment or a variant or a derivative thereof may be administered naked without being associated with any further vehicle, transfection or complexation agent for increasing the transfection efficiency and or the immunostimulatory properties of the inventive mRNA or of further comprised nucleic acid.
Thereby, the inventive mRNA or any other nucleic acid comprised in the inventive composition or vaccine can also be associated with a vehicle, transfection or complexation agent for increasing the transfection efficiency and or the immunostimulatory properties of the invention mRNA or of optionally comprised further included nucleic acids.
, Claims:We claim:
1. An mRNA sequence comprising a coding region, encoding at least one antigenic protein derived from RABVP Glycoprotein (strain Lysine biotech vaccine/ LBV) of rabies virus or fragment, variant or derivative thereof.
2. The mRNA sequence as claimed in claim 1, additionally comprises of 21 amino acids signal peptide at N-terminus, 21 amino acid transmembrane domains and 43 amino acid cytoplasmic domain.
3. The mRNA sequence as claimed in claim 1, coding region encodes the full length protein of glycoprotein G of rabies virus.
4. The mRNA sequence as claimed in claim 1, comprises nucleotide sequence encoding the antigenic protein selected from sequences with SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, and SEQ ID NO 36 or preferably an identity of at least 70%, more preferably of at least 80% and/ or most preferably of at least 90% with the nucleotide sequences according to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, and SEQ ID NO 36.
5. The mRNA sequence as claimed in claim 1, comprises amino acid sequence encoding the antigenic protein selected from sequences with SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, and SEQ ID NO 72 or preferably an identity of at least 70%, more preferably of at least 80% and/ or most preferably of at least 90% with the amino acid sequences according to SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, and SEQ ID NO 72.
6. The mRNA sequence as in claims 1 to 5, further defined for stimulating an immune response in mammals, in particular, human and dogs, infected by rabies virus.