Patent Application
20190185548
The present invention relates to the prevention and/or treatment of the Ebola virus disease.
Ebola virus disease (EVD), also known as Ebola hemorrhagic fever, is a severe and often fatal illness in humans. According to the World Health Organization, the average case fatality rate is indeed around 50% (from 25% to 90% in past outbreaks).
The virus is transmitted to humans from wild animals and then spreads in the human population via direct contact (for example with blood, secretions, other bodily fluids, or organs of infected people) or indirect contact (for example with contaminated surfaces or materials).
Mortality results from multi-organ failure and severe bleeding complications. The incubation period is from 2 to 21 days (usually 4-6 days). The symptoms comprise fever, fatigue, muscle pain, severe headache, sore throat, vomiting, diarrhea, rash, symptoms of impaired kidney and liver function and, in some cases, both internal and external bleeding. The disease is also associated with a decrease in blood cells and platelets, as well as increased liver enzymes.
There is no approved vaccine or drug able to neutralize the virus. The treatment of patients infected with Ebola virus thus essentially consists in rehydration and treatment of the specific symptoms.
Among the promising drug candidates to treat Ebola virus disease are ZMapp (a cocktail of three humanized monoclonal antibodies), TKM-Ebola (a RNA polymerase blocker using RNAi), Favipiravir (a small molecule acting as RNA polymerase inhibitor) and BCX-4430 (adenosine nucleoside analogue). Vaccine candidates include cAD3 Ebola vaccine (a recombinant Chimpanzee adenovirus serotype 3 vectored Ebola vaccine) and VSVΔG-ZEBOV (a recombinant vesicular stomatitis virus-based vaccine).
When considering antibody-based therapy, humanized monocional antibodies are used, because of their high specificity, homogeneity and limitless supply.
There is still a need of therapeutic compounds for the prevention and/or treatment of the Ebola disease.
The Inventors have surprisingly found that polyclonal antibodies may be used for the prevention and/or treatment of the Ebola virus disease, in particular in humans.
The Inventors have indeed shown that polyclonal antibodies generated against a modified Ebola virus GP glycoprotein are active against the Ebola virus and can be used effectively to treat or prevent Ebola virus disease or infection.
The present invention is thus based on passive immunotherapy. The passive transfer of antibodies confers two possible types of protective immunity: either a complete inhibition of virus replication, leading to the so-called sterile immunity, or an incomplete inhibition of virus replication. In the case of incomplete virus inhibition, the virus replicates at low level, the reduced viral load enabling the host to mount virus induced immune responses leading to the complete elimination of the virus.
One object of the invention thus relates to polyclonal antibodies specific for the Ebola virus.
Polyclonal antibodies present the advantages of being generated rapidly (for example in a few weeks), more easily and at less expense then monocional antibodies.
Besides, because they recognize multiple epitopes, polyclonal antibodies usually have a broader activity spectrum than monoclonal antibodies.
The polyclonal antibodies are preferably non-human polyclonal antibodies.
The polyclonal antibodies may be selected in the group consisting of rabbit polyclonal antibodies, mice polyclonal antibodies, rat polyclonal antibodies, guinea pig polyclonal antibodies, chicken polyclonal antibodies, goat polyclonal antibodies, horse polyclonal antibodies, cow polyclonal antibodies, sheep polyclonal antibodies and their combinations.
In a preferred embodiment, the polyclonal antibodies contain immunoglobulins that specifically bind to an Ebola virus GP glycoprotein antigen, such as a native Ebola virus GP glycoprotein, an Ebola virus GP1 subunit, an Ebola virus GP2 subunit and/or a variant thereof, for example a truncated form of a native Ebola virus GP glycoprotein, preferably a truncated form derived from the Ebola virus GP glycoprotein of sequence SEQ ID NO: 1, 12 or 13.
The polyclonal antibodies preferably contain immunoglobulins that specifically bind to an Ebola virus GP glycoprotein which comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14 and/or which is encoded by a nucleic acid comprising or consisting of a sequence at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID NO: 6.
The polyclonal antibodies are preferably obtained or obtainable from at least one biological sample of an animal immunized with (i) at least one antigen as defined above, preferably comprising or consisting of a sequence at least 80% identical to sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14 and/or (ii) at least one nucleic acid comprising or consisting of a sequence encoding said antigen and/or (iii) at least one vector comprising said nucleic acid.
In a particular embodiment, the polyclonal antibodies are obtainable by immunization of a non-human animal with an antigen composition comprising an Ebola virus GP glycoprotein antigen in trimeric form. More preferably, the composition comprises at least 50% of an Ebola virus GP glycoprotein antigen in trimeric form, even more preferably at least 60%, at least 70%, at least 80%, or at least 90%. More preferably, the Ebola virus GP glycoprotein antigen comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
In another particular embodiment, the polyclonal antibodies are obtainable by immunization of a non-human animal with an antigen composition comprising a mix of Ebola virus GP glycoprotein antigen in trimeric and monomeric forms. More preferably, the composition comprises at least 30% of an Ebola virus GP glycoprotein antigen in trimeric form and at least 20% of an Ebola virus GP glycoprotein antigen in monomeric form, more preferably at least 40% trimeric and at least 30% monomeric. More preferably, the Ebola virus GP glycoprotein antigen comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
Another object of the invention is a pharmaceutical composition comprising one or more polyclonal antibodies as defined above.
The pharmaceutical composition is preferably serum-free. Also, to further enhance the effect of the antibodies of the invention, complement may be added to the composition, or administered separately to the subject.
The pharmaceutical composition is preferably suitable for subcutaneous, intradermal, intramuscular, intraperitoneal and/or intravenous administration.
Another object of the invention is a method for producing the polyclonal antibodies as defined above, wherein said method comprises:
Another object of the invention relates to the polyclonal antibodies as defined above or to the pharmaceutical composition as defined above, for use in a method for the prevention and/or treatment of Ebola virus disease.
Said Ebola virus disease is preferably caused by at least one virus selected in the group consisting of Zaire Ebolavirus, Bundibugyo Ebolavirus, Reston Ebolavirus, Sudan Ebolavirus and Taï Forest Ebolavirus, preferably caused by Zaire Ebolavirus.
The polyclonal antibodies according to the invention may be used in combination with at least another anti-Ebola virus drug and/or at least another anti-Ebola virus vaccine.
Another object of the invention is a method for preventing and/or treating an Ebola virus disease in a subject in need thereof, said method comprising administering a polyclonal antibody as defined above or a pharmaceutical composition as defined above to said subject.
The present invention also relates to an antigen comprising or consisting of a sequence at least 80% identical to sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14, a nucleic acid comprising or consisting of a sequence encoding said antigen and a vector comprising said nucleic acid.
In a particular embodiment, the invention further relates to an antigen composition comprising an Ebola virus GP glycoprotein antigen in trimeric form. More preferably, the composition comprises at least 50% of Ebola virus GP glycoprotein antigen in trimeric form, even more preferably at least 60%, at least 70%, at least 80%, or at least 90%. In a particular embodiment, the composition comprises at least 30% of an Ebola virus GP glycoprotein antigen in trimeric form and at least 20% of an Ebola virus GP glycoprotein antigen in monomeric form. More preferably, the Ebola virus GP glycoprotein antigen comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
Ebola Virus
The Ebola virus belongs to the Filoviridae family and the Ebolavirus genus.
The Ebola virus may be of any known Ebola virus species.
Preferably, the Ebola virus may be selected in the group consisting of Zaire Ebolavirus (also called ZEBOV or EBOV), Bundibugyo Ebolavirus (also called BEBOV or BDBV), Reston Ebolavirus (also called REBOV or RESTV), Sudan Ebolavirus (also called SEBOV or SUDV) and Taï Forest Ebolavirus (also called TAFV, ICEBOV or Ivory Coast Ebolavirus).
The Ebola virus is preferably Zaire Ebolavirus or Sudan Ebolavirus, preferably Zaire Ebolavirus.
The Ebola virus consists of a non-segmented, negative single-stranded linear RNA.
Seven proteins are encoded by the Ebola virus genome: the nucleoprotein (also called NP), VP35, VP40, the GP glycoprotein (also called GP or GP protein), VP30, VP24 and the RNA-dependent RNA Polymerase protein (also called L).
NP, VP30, VP35 and L bind to the negative-sense RNA genome and form the ribonucleoprotein complex.
VP40 is the major matrix protein and VP24 is the minor matrix protein.
GP is the major membrane spike protein and is encoded by the GP gene.
As a matter of fact, the GP gene produces two proteins, which are sGP and GP.
sGP is a small polypeptide.
GP consists in a N-terminus GP1 subunit and a C-terminal GP2 subunit, both linked by disulfide bonds. GP is therefore also called GP1/GP2 heterodimer.
The GP1 subunit, also called GP1 protein or GP1, consists of a N-terminus half, a head and a C-terminus domain. The N-terminus half of the GP1 subunit is highly conserved. The head of the GP1 subunit comprises the receptor-binding domain. The C-terminus domain of the GP1 subunit comprises two highly glycosylated regions called the glycan cap and the mucin domain.
The GP2 subunit, also called GP2 protein or GP2, comprises a transmembrane domain, a fusion peptide and two heptad repeat (HR) domains that are required for virus-cell membrane fusion.
The mature GP protein exists as homo-trimers on the surface of viral particles, the trimerization being mediated by GP2.
Ebola Virus Antigen
As used herein, the term “antigen” refers to any substance that, when recognized as non-self by the adaptive immune system of an animal (human or non-human), triggers an immune response.
At least one Ebola virus antigen is preferably used to obtain the polyclonal antibodies according to the present invention.
The Ebola virus antigen is preferably selected in the group consisting of the Ebola virus GP glycoprotein, the Ebola virus GP1 subunit, the Ebola virus GP2 subunit and/or a variant thereof. The antigen may be glycosylated or not.
Many sequences of naturally occurring Ebola virus GP glycoproteins and their subunits GP1 and GP2 are known from public gene databases.
The Inventors have developed a consensus sequence for the Ebola virus GP glycoprotein from 899 sequences of the Zaire Ebola virus GP glycoprotein. This consensus sequence of 676 amino acids is referred to as sequence SEQ ID NO: 1. Although being a consensus sequence, sequence SEQ ID NO: 1 is herein considered as an Ebola virus GP glycoprotein sequence.
The Inventors have further developed consensus sequences for the Ebola virus GP glycoproteins from several sequences of the Sudan and Bundibugyo Ebola virus GP glycoproteins. These consensus sequences are referred to as sequence SEQ ID NO: 12 (Sudan) and SEQ ID NO: 13 (Bundibugyo).
The Ebola virus GP glycoprotein may thus comprise or consist of a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 1, 12 or 13. The Ebola virus GP glycoprotein preferably comprises or consists of sequence SEQ ID NO: 1, 12 or 13, of fragments thereof.
A reference sequence for the Ebola virus GP1 subunit is sequence SEQ ID NO: 8, which corresponds to amino acids 1 to 500 of sequence SEQ ID NO: 1.
The Ebola virus GP1 subunit may comprise or consist of a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 8. The Ebola virus GP1 subunit preferably comprises or consists of sequence SEQ ID NO: 8.
A reference sequence for the Ebola virus GP2 subunit is sequence SEQ ID NO: 9, which corresponds to amino acids 501 to 676 of sequence SEQ ID NO: 1.
The Ebola virus GP2 subunit may comprise or consist of a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 9. The Ebola virus GP2 subunit preferably comprises or consists of sequence SEQ ID NO: 9.
The expression “variant of a given protein” herein means a derivative of said protein, wherein said derivative shares completely or at least partially the immunogenic properties of said protein. A variant of a given protein preferably comprises at least one epitope of said protein.
A variant of a given protein is for example a biologically and/or chemically modified form of said protein, such as a fragment, a truncated form and/or a soluble form of said protein, wherein said fragment and/or truncated form and/or soluble form may comprise additional biological and/or chemical modification(s).
The biological and/or chemical modifications may increase the immunogenicity, the stability and/or the time of circulation before clearance of the variant by comparison to the protein from which it derives and/or may facilitate the purification of said variant.
Using a soluble form of a protein as an antigen is particularly advantageous for a membrane protein and/or to make the production of the antigen easier.
To facilitate purification, the variant may comprise at least one tag.
Tags for the purification of proteins are well known by the skilled person. Non-limitative examples of tag are a Strep-tag or a Histidine tag.
When present, a tag is preferably linked directly or indirectly (for example via a linker) to the C-terminal part of the protein.
A spacer may advantageously be added to the C-terminal part of the protein, for example between the protein and the at least one tag.
The spacer may be a cleavage site spacer, preferably an enzymatic cleavage site spacer, such as an enterokinase cleavage site spacer. An example of enterokinase cleavage site spacer is DDDDK (SEQ ID NO: 10).
The variant may also comprise amino acids resulting from the cleavage of a cleavage site spacer.
A preferred variant of a GP1 subunit comprises or consists of (i) at least one fragment, for example one or two fragments, of said GP1 subunit, wherein said fragment preferably lacks at least one part of the mucin domain or at least one part of the signal peptide of said GP1 subunit, and (ii), optionally, at least one tag and/or or at least one spacer.
Deleting the mucin domain advantageously allows improving the immune response by increasing the activity of the polyclonal antibodies.
A variant of a GP1 subunit may thus comprise or consist of:
In one preferred embodiment, a variant of a GP1 subunit comprises or consists of (i) two fragments of said GP1 subunit, wherein the first fragment consists of amino acids 33 to 311 of sequence SEQ ID NO: 1 and wherein the second fragment consists of amino acids 463 to 500 of sequence SEQ ID NO: 1, and (ii), optionally, at least one tag and/or or at least one spacer.
The fragment of a GP1 subunit may consist of at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids or at least 450 amino acids and/or may consist of at most 100 amino acids, at most 150 amino acids, at most 200 amino acids, at most 250 amino acids, at most 300 amino acids, at most 350 amino acids, at most 400 amino acids, at most 450 amino acids or at most 490 amino acids.
A preferred variant of a GP2 subunit comprises or consists of:
wherein said fragment preferably lacks at least one part of the transmembrane domain of said GP2 subunit.
Deleting the transmembrane domain allows facilitating the purification of GP2 subunit variant.
A variant of a GP2 subunit may thus comprise or consist of (i) a fragment of said GP2 subunit and (ii) optionally, at least one tag and/or or at least one spacer, wherein said fragment consists of a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to a sequence consisting of amino acids 501 to 632 of sequence SEQ ID NO: 1. In one preferred embodiment, a variant of a GP2 subunit comprises or consists of (i) a fragment of said GP2 subunit, wherein said fragment consists of amino acids 501 to 632 of sequence SEQ ID NO: 1, and (ii) optionally at least one tag and/or or at least one spacer. The fragment of a GP2 subunit may consist of at least 50 amino acids, at least 75 amino acids, at least 100 amino acids, at least 125 amino acids, or at least 150 amino acids and/or may consist of at most 100 amino acids, at most 125 amino acids, at most 150 amino acids or at most 170 amino acids.
By “fragment of a given protein”, it is herein meant a sequence of consecutive amino acids of said protein.
A preferred variant of an Ebola virus GP glycoprotein comprises or consists of:
A preferred variant of an Ebola virus GP glycoprotein may thus comprise or consist of:
The size of the fragments is for example as defined above.
For example, the variant of an Ebola virus GP glycoprotein may comprise or consist of (i) a first fragment of the GP1 subunit, wherein said fragment consists of amino acids 33 to 311 of sequence SEQ ID NO: 1, (ii) a second fragment of the GP1 subunit, wherein said fragment consists of amino acids 463 to 500 of sequence SEQ ID NO: 1, (iii) a fragment of the GP2 subunit, wherein said fragment consists of amino acids 501 to 632 of sequence SEQ ID NO: 1 and (iv), optionally, at least one tag and/or or at least one spacer. Such variant may comprise or consist of sequence SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11 and/or SEQ ID NO. 14.
A preferred Ebola virus antigen according to the invention thus comprises or consists of a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
Starting from the consensus sequences SEQ ID NOs: 1, 12 or 13, or from one of their derived sequences SEQ ID NO: 3, 5, 7, 8, 9, 11 or 14 advantageously allows obtaining polyclonal antibodies with a broader spectrum of activity.
As defined herein, an amino acid sequence “at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical” to a reference sequence may comprise mutations, such as deletions, insertions and/or substitutions compared to the reference sequence.
In case of substitutions, the substitution preferably corresponds to a conservative substitution as indicated in the Table 1 below. In a preferred embodiment, the sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence only differs from the reference sequence by conservative substitutions.
In another preferred embodiment, the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence corresponds to a homologous sequence derived from another Ebola virus of the same species as the reference sequence or of a different species than the reference sequence.
In a preferred embodiment, the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence differs from the reference sequence by conservative substitutions and/or corresponds to a homologous sequence derived from another Ebola virus of the same species as the reference sequence or of a different species than the reference sequence.
By “a sequence at least x % identical to a reference sequence”, it is intended that the sequence is identical to the reference sequence or differs from the reference sequence by up to 100-x amino acid alterations per each 100 amino acids of the reference sequence.
The alignment and the determination of the percentage of identity may be carried out manually or automatically using for instance the Needle program which is based on the Needleman and Wunsch algorithm, described in Needleman and Wunsch (1970) J. Mol Biol. 48:443-453, with for example the following parameters for polypeptide sequence comparison: comparison matrix: EBLOSUM62, gap open: 10, gap extend: 0.5, end gap penalty: false, end gap open penalty=10, end gap extension penalty=0.5; and the following parameters for polynucleotide sequence comparison: comparison matrix: EDNAFULL; gap open=10, gap extend=0.5, end gap penalty: false, end gap open penalty=10, end gap extension penalty=0.5.
In a particular embodiment, the Ebola virus GP glycoprotein antigen comprises or consists of a protein of SEQ ID NO: 3, 5, 7, 9, or 14.
In a further particular embodiment, an immunogen is used which comprises an Ebola virus GP glycoprotein antigen in trimeric form. Ebola virus GP naturally exists in different forms, such as monomers, dimers or trimers. Trimers are mediated by inter-GP2 domain interactions. The invention shows that when present in trimeric form, the Ebola glycoprotein generates polyclonal antibodies with high neutralization potency. Accordingly, in a preferred embodiment, the invention relates to a composition comprising an Ebola virus GP glycoprotein antigen in trimeric form. More preferably, the composition comprises at least 30% of Ebola virus GP glycoprotein antigen in trimeric form, even more preferably at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In a particular embodiment, the composition comprises at least 30% of an Ebola virus GP glycoprotein antigen in trimeric form and at least 20% of an Ebola virus GP glycoprotein antigen in monomeric form. More preferably, the antigen comprises or consists of a sequence at least 80% identical to sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
Nucleic Acid Encoding an Ebola Virus Antigen
The present invention also relates to a nucleic acid comprising or consisting of a sequence encoding an Ebola virus antigen.
The “Ebola virus antigen” is as defined above in the section of the same name.
The nucleic acid may comprise or consist of a sequence encoding an Ebola virus antigen linked to a spacer, for example a cleavage site spacer, and/or a tag.
The nucleic acid for example comprises or consists of a sequence encoding an Ebola virus GP glycoprotein, an Ebola virus GP1 subunit, an Ebola virus GP2 subunit and/or a variant thereof, said variant being preferably a soluble form of said protein(s).
The sequence encoding the Ebola virus GP glycoprotein may be at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical to, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. In one embodiment, the sequence encoding the Ebola virus GP glycoprotein is identical to sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
The nucleic acid may thus comprise or consist of a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical to, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. In one embodiment, the nucleic acid comprises or consists of sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
A nucleic sequence “at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical” to a reference sequence may comprise mutations, such as deletions, insertions and/or substitutions compared to the reference sequence.
In case of substitutions, the substitution preferably corresponds to a silent substitution or a substitution leading to a conservative substitution in the translated amino acid sequence by comparison to the reference sequence, for example as indicated in the Table 1 above.
In a preferred embodiment, the nucleic sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence only differs from the reference sequence by silent substitution(s) and/or substitution(s) leading to a conservative amino-acid substitution(s).
The nucleic acid encoding the Ebola virus antigen is preferably cloned into a vector.
The present invention thus also relates to a vector comprising at least one nucleic acid comprising or consisting of a sequence encoding an Ebola virus antigen.
The vector may comprise one and only one nucleic acid encoding an Ebola virus antigen.
Alternatively, the vector may comprise at least two nucleic acids (for example two, three or four nucleic acids) encoding the same Ebola virus antigen or at least two different Ebola virus antigens.
In a preferred embodiment, the vector comprises at least one nucleic acid encoding an Ebola virus antigen, wherein said Ebola virus antigen is an Ebola virus GP glyprotein, an Ebola virus GP1 subunit, an Ebola virus GP2 subunit or a variant thereof.
A preferred vector comprises a nucleic acid comprising or consisting of a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical to, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. In one embodiment, the vector comprises a nucleic acid comprising or consisting of sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
The “vector comprising at least one nucleic acid encoding an Ebola virus antigen” is thereafter also referred to as “vector”.
The nucleic acid is preferably an isolated nucleic acid.
The vector is preferably an isolated vector.
An “Isolated nucleic acids” or “isolated vectors” include nucleic acid molecules purified by standard purification methods.
The term “isolated” in reference to a biological component (such as a nucleic acid, a vector or a protein) refers to a biological component that has been substantially separated or purified away from other biological components in the cell of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, cells, and organelles. “Isolated nucleic acids” or “isolated vectors” include nucleic acid molecules purified by standard purification methods. These terms also encompass nucleic acids and vectors prepared by amplification and/or cloning, as well as chemically synthesized nucleic acids and vectors.
The vector preferably comprises at least one Ebola virus antigen expression cassette, i.e. a sequence encoding the Ebola virus antigen placed under the control of at least one expression signal allowing its expression.
The expression signal is particularly selected among a promoter, a terminator, an enhancer and their combinations.
The vector is preferably appropriate for an expression system distant from the human expression system.
In a preferred embodiment, the vector is appropriate for insect expression, more preferably for Drosophilia expression, or for mammalian expression. Suitable promoters, terminators and enhancers are well-known by the skilled person.
For example, the promoter is the Drosophila metallothionein gene promoter or a mammalian promoter.
For example, the terminator is SV40 PA terminator.
The vector may be a non-viral vector or a viral vector.
A non-viral vector may be selected in the group consisting of a plasmid, a liposomal nucleic acid complex and a carrier-associated nucleic acid.
By “plasmid”, it is herein meant a double-stranded circular DNA. The plasmid may include a marker gene enabling to select the cells comprising said plasmid, an origin of replication to allow the cell to replicate the plasmid and/or a multiple cloning site allowing the insertion of a DNA fragment, in particular the sequence encoding the Ebola virus antigen.
For example, the plasmid may comprise Puromycin as a marker gene.
Non-limitative examples of carrier-associated nucleic acid are polymer-carried DNA and cationic lipids.
The liposomes used in liposomal nucleic acid complexes are well-known in the art.
Said liposomes may be cationic, anionic or neutral liposomes.
Cationic lipids are also known in the art and are commonly used for gene delivery. Such lipids include Lipofectin Tm also known as DOTMA (N-[I-(2, 3-dioleyloxy) propyls N, N, N-trimethylammonium chloride), DOTAP (1, 2-bis (oleyloxy)-3 (trimethylammonio) propane), DDAB (dimethyldioctadecyl-ammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta-(N—(N′, N′-dimethyl aminomethane)carbamoyl) cholesterol). Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine).
In another embodiment, the vector comprising a nucleic acid encoding the Ebla virus antigen is a viral vector.
By “viral vector”, it is herein meant a recombinant viral vector.
The viral vector is preferably selected in the group consisting of a baculovirus vector, a retrovirus vector, a lentivirus vector, an adenovirus vector and an adeno-associated virus vector.
Baculovirus vectors are particularly suitable for insect expression.
Method for Producing an Ebola Virus Antigen
The present invention also relates to a method for producing an Ebola virus antigen, said antigen being as defined above in the section of the same name.
Methods for producing a protein, such as an antigen, are well-known by the skilled person.
The method for producing at least one Ebola virus antigen preferably comprises: (i) providing cells comprising (a) at least one nucleic acid sequence comprising or consisting of a sequence encoding said Ebola virus antigen or (b) at least one vector comprising at least one nucleic acid comprising or consisting of a sequence encoding said Ebola virus antigen, (ii) culturing the cells, in particular in conditions allowing the expression of said Ebola virus antigen, (iii) recovering said Ebola virus antigen.
The “nucleic acid” and the “vector” are as defined above in the section “Nucleic acid encoding an Ebola virus antigen”.
The cells used in step (i) are preferably eukaryotic cells, for example insect cells such as Drosophila cells, or mammalian cells, preferably distant from human cells. The cells of step (i) may be obtained by transfection of said nucleic acid or vector.
The cells comprising said nucleic acid or vector may be selected using a selectable marker, such as puromycin.
In step (ii), the cells are cultured, in particular in conditions allowing the expression of said Ebola virus antigen. The skilled person knows the conditions of culture in function of the cells used. Besides, depending on the expression system used, the expression of the antigen may be induced by adding a specific compound in the culture medium, such as for example CuSO4.
In a preferred embodiment, the cells are insect cells, such as Drosophila cells, particularly S2 cells. The invention shows the Ebola virus antigen produced with insect cells has a suitable conformation and leads to generation of high amounts of trimeric form.
The nucleic acid sequence comprising or consisting of a sequence encoding said Ebola virus antigen or the vector may be integrated or not in the genome of the cells.
In a preferred embodiment, the Ebola virus antigen is a soluble and secreted protein.
When the Ebola virus antigen is a soluble and secreted protein, step (iii) may for example comprise centrifuging the cell culture obtained in step (ii) and collecting the supernatant.
Step (iii) preferably comprises:
The steps of extracting and purifying may consist in a single step.
Methods of extracting, purifying and/or concentrating a protein are well-known by the skilled person.
For example, when the recombinant antigen comprises a Strep tag or a His tag, a Strep-Tactin resin or a Ni resin, respectively, may be used for purification.
The method may comprise a step of purifying, or separating, or enriching trimers, e.g., to obtain a composition comprising at least 30% trimers.
In a particular embodiment, the method comprises expressing a nucleic acid encoding an Ebola virus GP glycoprotein antigen in insect cells, collecting the antigen, and purifying or separating or enriching for trimers to obtain a composition comprising at least 30%, preferably at least 50%, more preferably at least 60° %, at least 70%, at least 80%, Ebola virus GP glycoprotein antigen in trimeric form. Such a step may be performed by techniques known per se in the art, such as size exclusion chromatography, conformational antibody purification, and the like.
Polyclonal Antibodies
The present invention relates to particular polyclonal antibodies that bind to an Ebola virus antigen, as well as the uses thereof.
The present invention particularly relates to polyclonal antibodies specific for the Ebola virus.
The term “antibody” herein means an immunoglobulin molecule (also called Ig) or immunologically active portion thereof that specifically binds to an antigen.
The expression “polyclonal antibodies” or “polyclonal antibody” herein means antibodies originating from different B lymphocyte clones, which are thus capable of binding to and/or reacting with at least two different specific epitopes on the same or on different antigens.
An “epitope” (also called “determinant” or “antigenic determinant”) is the portion of a molecule that is bound by an antibody.
An antibody is generally a tetramer consisting in two identical copies of both a heavy and light chains bound by disulfide and non-covalent bonds, also called herein whole IgG. The N-terminal regions of the light and heavy chains define a variable region responsible for antigen recognition. The C-terminal regions of the two heavy chains form the Fc domain.
The light chain comprises one variable region and one constant region.
The heavy chain comprises one variable region and at least three constant regions.
Each of the light and heavy chain variable region comprises three hypervariable regions, called complementary determining regions or CDRs, which form the epitope binding site.
An antibody may also be in the form a fragment, for example produced by degradation with various peptidases, such as Fab (monovalent for antigen binding), F(ab′)2 (bivalent for antigen binding) or Fc (effector domains).
The polyclonal antibodies are preferably selected from the group consisting of whole IgG, Fab, F(ab′)2 and their combinations.
Antibodies comprise different classes defined according to the amino acid sequence of the heavy chain constant region. For example, mammal classes comprise IgA, IgD, IgE, IgG and IgM; avians classes comprise IgY, IgM and IgA. Subclasses may also be defined. For example, IgG and IgA are further divided into isotypes IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 in humans.
The light chain of an antibody may also be classified as kappa or lambda, based on the amino acid sequence of the light chain constant region.
According to common knowledge, a polyclonal antibody “specific” for the Ebola virus typically refers to a polyclonal antibody that has been generated by immunization of an animal with an Ebola virus antigen, and/or which contains immunoglobulins that specifically bind an Ebola virus (i.e., and essentially do not bind a virus of a different class). In a particular embodiment, a polyclonal antibody of the invention contains at least 5% immunoglobulins that specifically bind an Ebola virus, more preferably between 5-10%. In another embodiment, the polyclonal antibody contains at least 40% immunoglobulins that specifically bind an Ebola virus, preferably between 40-60%, or even more. Such polyclonals may be prepared by clonal enrichment, e.g., by purification on affinity support. In a preferred embodiment, the polyclonal antibodies specific for an Ebola virus specifically bind to the GP glycoprotein, the GP1 subunit and/or the GP2 subunit of said Ebola virus.
The binding of the polyclonal antibodies to the Ebola virus antigen for which they are specific preferably results in blocking, completely or at least partially, the replication of the Ebola virus and/or blocking completely or at least partially, the function of the Ebola virus and/or in activating complement and/or other effector functions, thereby allowing the elimination of the Ebola virus particles.
In a preferred embodiment, the binding of the polyclonal antibodies to the Ebola virus antigen for which they are specific results in blocking, completely or at least partially, the replication of the Ebola virus. Such polyclonal antibodies are called neutralizing antibodies.
In this regard, the invention shows that polyclonal antibodies prepared according to the invention exhibit high anti-EBOLA titers and high neutralizing effect. Surprisingly, using trimeric antigen, or mix of trimeric and monomeric antigen, combined with the preferred sequence and production system of the invention generates very potent polyclonal antibodies with high neutralizing capacity, suitable for use as therapeutic/prophylactic agents.
In a particular embodiment, the polyclonal antibody of the invention has an anti-EBOLA titer of at least 10000, preferably at least 13000, and/or a neutralization effect of at least 400, preferably at least 500.
The polyclonal antibodies of the invention are preferably isolated antibodies.
The term “isolated” is as defined above.
The polyclonal antibodies are for example isolated from the blood, egg yolk, milk or colostrum of an immunized animal.
The polyclonal antibodies are preferably non-human polyclonal antibodies.
The expression “non-human polyclonal antibodies” means that the polyclonal antibodies originate from non-human animal(s). Said non-human animal is for example rabbit, mouse, rat, guinea pig, chicken, goat, horse, cow or sheep, preferably rabbit.
The non-human polyclonal antibodies may be antibodies originating from animal(s) of the same genus and/or from animals of different genus.
The polyclonal antibodies are for example selected in the group consisting of rabbit polyclonal antibodies, murine polyclonal antibodies, rat polyclonal antibodies, guinea pig polyclonal antibodies, chicken polyclonal antibodies, goat polyclonal antibodies, horse polyclonal antibodies, cow polyclonal antibodies, sheep polyclonal antibodies and their combinations.
The polyclonal antibodies according to the invention are preferably prepared by immunization of an animal, preferably a non-human animal, with at least one Ebola virus antigen as defined above and, depending on the animal, purification of the obtained polyclonal antibodies, for example from blood, egg yolk, milk or colostrum.
In one preferred embodiment, the polyclonal antibodies are obtained, in particular purified, from at least one biological sample of an animal, preferably a non-human animal, immunized with (i) at least one antigen comprising or consisting of a sequence at least 80% identical to sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 and/or (ii) at least one nucleic acid comprising or consisting of a sequence encoding said antigen and/or (ii) a vector comprising said nucleic acid.
The expression “polyclonal antibodies specific for the Ebola virus” herein means polyclonal antibodies that are specific of at least one antigen of said Ebola virus, i.e., that specifically binds to at least one antigen of the Ebola virus. The specific binding of polyclonal antibodies to a given antigen may be assessed by ELISA, in vitro neutralizing assay using a mammalian (preferably human) cell line infected by Ebola virus and/or mammalian (preferably human) cells transfected with an Ebola recombinant virus and/or in vivo assay using an animal model infected by Ebola virus.
The animal model is preferably a rodent or primate animal model.
The antigen used in the ELISA may be the antigen used for immunizing the animals and/or another Ebola virus GP glycoprotein, for example expressed in a mammalian (preferably human) or insect cell line (such as S2) transfected with a plasmid encoding said GP glycoprotein.
In the in vitro neutralizing assay, the Ebola recombinant virus used for transfecting the cells is for example a rVSV (recombinant Vesicular Stomatitis Virus) carrying Ebola virus GP glycoprotein. The Ebola recombinant virus carrying Ebola GP glycoprotein is for example incubated with the polyclonal antibodies and then with a mammalian (preferably) human cell line. Said mammalian cell line is for example HEK293 cells or VERO cells.
The “Ebola virus” and the “antigen of the Ebola virus” are as defined above in the sections of the same name.
In a preferred embodiment, the polyclonal antibodies specifically bind to the Ebola virus GP protein, the Ebola virus GP1 protein, the Ebola virus GP2 protein and/or a variant thereof, as defined above.
In a preferred embodiment, the polyclonal antibodies specifically bind to a truncated form of the Ebola virus GP glycoprotein of sequence SEQ ID NO: 1, for example to a sequence at least 80% identical to sequence SEQ ID NO: 3 and/or a sequence encoded by a sequence at least 80% identical to sequence SEQ ID NO: 2.
The polyclonal antibodies are preferably provided in a pharmaceutical composition, particularly as defined below.
Method for Producing Polyclonal Antibodies
The present invention also relates to a method, preferably an in vitro method, for producing polyclonal antibodies specific for the Ebola virus.
Methods for producing polyclonal antibodies specific for an antigen are well-known by the skilled person.
The present invention thus relates to a method for producing polyclonal antibodies specific for the Ebola virus, wherein said method comprises collecting or purifying the polyclonal antibodies from a biological sample of an animal immunized with (i) at least one Ebola virus antigen, (ii) at least one nucleic acid comprising or consisting of a sequence encoding said Ebola virus antigen and/or (iii) at least one vector comprising said nucleic acid.
The present invention particularly relates to a method for producing polyclonal antibodies specific for the Ebola virus, said method comprising:
The immunized animal is preferably non-human.
The non-human animal may be a rabbit, mouse, rat, guinea pig, chicken, goat, horse, cow or sheep, preferably a rabbit.
In a particular embodiment, the polyclonal antibodies are prepared from at least two biological samples, which may have been obtained from non-human animals immunized with the same or distinct Ebola virus antigenic compositions. In such embodiment, the at least two biological samples are preferably mixed to form a biological sample pool from which the polyclonal antibody is collected or purified.
The biological sample may be any sample from the immunized animal that comprises antibodies.
Depending on the animal, the biological sample may be blood, milk, egg yolk and/or colostrum. In a preferred embodiment, the biological sample is blood.
The pool of biological samples may comprise biological samples originating from the same animal and/or from different animals, preferably of the same genus.
The Ebola virus antigen and the nucleic acid are as defined above in the section of the same name.
In one embodiment, the animal is immunized with at least one Ebola virus antigen, for example one, two, three or at least three Ebola virus antigens. Said Ebola virus antigens may belong to the same Ebola virus species or to different Ebola virus species.
The antigen used for immunization is preferably administered with one or at least one adjuvant.
Non-limitative examples of adjuvant are Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum salts (such as Al(OH)3, AlPO4), Quil A, Iscoms, Montanide, TiterMax™ or RIBI™.
The antigen may be administered to the animal by the subcutaneous, intradermal, intramuscular, intraperitoneal and/or intravenous route.
The antigen may be administered to the animal as a single injection at one site or as multiple injections of low volumes at different sites, for example at two, three or four different sites.
The antigen may be administered to the animal several times at given intervals of time, for example two or three times.
In another embodiment, the animal is immunized with at least one nucleic acid comprising or consisting of a sequence encoding said Ebola virus antigen.
The nucleic acid may be provided in the form of a vector comprising said nucleic acid, said vector being as defined above.
The nucleic acid or vector is administered to the animal, so as to be expressed by cells of the animal and, preferably, secreted in the blood.
The Ebola virus antigen, administered as a protein or expressed by the cells from the administered nucleic acid or vector, thus allows triggering an immune response.
It is advantageous to monitor the presence of an immune response before collecting biological sample(s) from the animal(s).
In a preferred embodiment, the present invention relates to a method for producing polyclonal antibodies specific for the Ebola virus, said method comprising:
In a more preferred embodiment, the Ebola virus antigen is a variant of the Ebola virus GP glycoprotein. For example, the Ebola virus antigen comprises or consists of sequence SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 and/or is encoded by sequence SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
The present invention particularly relates to a method for producing polyclonal antibodies specific for the Ebola virus, said method comprising:
When the biological sample is blood, the purification step preferably comprises a) clotting the blood sample or pool of blood samples and b) collecting the liquid phase, in order to obtain serum or a pool of sera.
The polyclonal antibodies are preferably further purified from said serum or pool of sera.
The step of purifying the polyclonal antibodies that are specific for the Ebola virus antigen may comprise at least one step of precipitation.
The step of purification for example comprises or consists of at least one of the following steps, for example one, two or three of the following steps: precipitation in 50% ammonium sulphate, purification on protein A column, purification on ion exchange column and/or gel filtration.
In one embodiment, the step of purification comprises or consists of precipitation in 50% ammonium sulphate, purification on protein A-sepharose column, purification on ion exchange column and a gel filtration.
In one embodiment, the polyclonal antibodies are further purified, in order to remove antibodies that are not specific for the Ebola virus and/or to remove non-immunoglobulin factors initially present in the biological sample of the immunized animal(s).
The method for producing polyclonal antibodies specific for the Ebola virus preferably further comprises at least one step of viral inactivation and/or at least one step of sterilization.
The sterilization may be performed by microfiltration and/or pasteurization.
Pharmaceutical Composition
The polyclonal antibodies according to the invention are preferably formulated into a pharmaceutical composition.
The present invention thus also relates to a pharmaceutical composition comprising polyclonal antibodies as defined above.
The pharmaceutical composition is preferable serum-free.
As used herein, the term “serum-free” means that the pharmaceutical composition does not comprise or consist of serum, plasma and/or blood. A serum-free composition may only comprise some components of the serum, in particular polyclonal antibodies.
In other words, in a serum-free composition, the polyclonal antibodies have been purified from the serum of an immunized animal.
The pharmaceutical composition preferably comprises the polyclonal antibodies and a pharmaceutically acceptable vehicle.
The polyclonal antibodies are as defined above in the section of the same name.
The pharmaceutical composition may comprise polyclonal antibodies specific for different epitopes present on the same Ebola virus antigen and/or on different antigens.
Pharmaceutical compositions comprising the polyclonal antibodies according to the invention include all compositions, wherein said polyclonal antibodies are contained in an amount effective to achieve the intended purpose, in particular to achieve complete or at least partial blocking of the replication of the Ebola virus, i.e. complete or at least partial neutralization of the Ebola virus, and/or complete or at least partial blocking of the function of the Ebola virus, which is capable of preventing and/or treating Ebola virus disease.
The expression “pharmaceutically acceptable” is meant to encompass any carrier, which does not interfere with the effectiveness and/or the biological activity of the polyclonal antibodies and that is preferably not toxic to the subject to who they are administered.
Pharmaceutically acceptable vehicles may be prepared by any method known by those skilled in the art.
Suitable pharmaceutically acceptable vehicles may comprise excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Suitable pharmaceutically acceptable vehicles are described for example in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, USA, 1985), which is a standard reference text in this field. Pharmaceutically acceptable vehicles can be routinely selected in accordance with the mode of administration, solubility and stability of the polyclonal antibodies. For example, a pharmaceutical composition for intravenous administration may include a sterile aqueous solution, which may also comprise buffer(s), diluent(s) and other suitable additive(s). The use of biomaterials and other polymers for drug delivery, as well the different techniques and models to validate a specific mode of administration, are disclosed in literature.
The polyclonal antibodies according to the invention may be formulated in a liquid pharmaceutical composition (e.g. a solution or suspension) or in a solid pharmaceutical composition (for example a pulverulent or lyophilized pharmaceutical composition).
The amount of polyclonal antibodies to be used in a pharmaceutical composition depends, for example, on the mode of administration, the dosage regimen and the type of formulation.
For example, the pharmaceutical composition may comprise from 50 mg to 1 g, preferably from 50 mg to 500 mg, preferably from 50 mg to 400 mg, for example from 50 mg to 200 mg or from 200 mg to 500 mg of the polyclonal antibodies according to the invention, in particular for administration to a human patient.
The pharmaceutical composition is preferably suitable for subcutaneous, intradermal, intramuscular, intraperitoneal and/or intravenous administration.
In one embodiment, the pharmaceutical composition further comprises at least another anti-Ebola virus drug and/or at least another anti-Ebola virus vaccine
In another embodiment, the pharmaceutical composition comprises the polyclonal antibodies according to the invention as the only active ingredient.
Non-limitative examples of other anti-Ebola virus drug are a humanized monoclonal antibody, a RNA polymerase blocker or inhibitor, an adenosine nucleoside analogue.
Non-limitative examples of anti-Ebola vaccine are a recombinant virus vaccine, an inactivated virus, an antigen in combination with an adjuvant.
The pharmaceutical composition may be presented in a unit dosage form, for example to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other animals, each unit containing a pre-determined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of the liquid compositions. In such compositions, the polyclonal antibodies are usually a minor component, with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
The invention further provides kits comprising a pharmaceutical composition comprising the polyclonal antibodies according to the invention and instructions regarding the mode of administration. These instructions may e.g. indicate the medical indication, the route of administration, the dosage and/or the group of subjects to be treated.
Subject to be Treated
A subject in need of a treatment for preventing and/or treating Ebola virus disease may be a mammal, for example a human being or a non-human mammal.
A human being is also referred to as an “individual” or a “patient”.
Said human being may be of any age, for example an infant, child, adolescent, adult, elderly people, and of any sex.
A non-human mammal is preferably a mouse, rat, cat, dog, rabbit or primate.
The subject to be treated may suffer from Ebola virus disease or be likely to suffer from Ebola virus disease.
The subject likely to suffer from Ebola virus disease may be a subject who has been or is in contact with patient(s) suffering from Ebola virus disease, for example relatives and/or health personnel.
Prevention and/or Treatment of Ebola Virus Disease
The present invention thus relates to the prevention and/or treatment of the Ebola virus disease.
The “Ebola virus” is as defined as above in the section of the same name.
The Ebola virus disease is for example caused by at least one virus selected in the group consisting of Zaire Ebolavirus, Bundibugyo Ebolavirus, Reston Ebolavirus, Sudan Ebolavirus and Taï Forest Ebolavirus, preferably caused by Zaire Ebolavirus.
By the expression “treatment of Ebola virus disease”, it is herein meant to eliminate at least partially the Ebola virus particles, neutralize at least partially the Ebola virus particles and/or improve the symptoms resulting from the Ebola virus disease, in particular in a subject suffering from Ebola virus disease.
Desirable effects of treatment thus include:
By the expression “prevention of Ebola virus disease”, it is herein meant to block or limit the transmission of the Ebola virus to a subject, in particular to prevent at least partially the multiplication of the Ebola virus particles, neutralize at least partially the Ebola virus particles, eliminate at least partially the Ebola virus particles and/or prevent at least partially the symptoms resulting from the Ebola virus disease, in particular in a subject likely to suffer from Ebola virus disease.
Desirable effects of prevention thus include:
Polyclonal Antibodies for Use in the Prevention and/or Treatment of Ebola Virus Disease and Method of Prevention and/or Treatment of Ebola Virus Disease
The present invention particularly relates to the polyclonal antibodies as defined above for use in the prevention and/or treatment of Ebola virus, in particular in a subject in need thereof. The invention is particularly suited for treating or preventing Ebola virus disease or infection.
The present invention thus relates to the use of the polyclonal antibodies as defined above for the manufacture of a medicament intended for the prevention and/or treatment of Ebola virus disease or infection, in particular in a subject in need thereof.
The present invention is also directed to a method for preventing and/or treating Ebola virus disease in a subject in need thereof, said method comprising a step of administering a polyclonal antibody or a composition as defined above to said subject.
The present invention is also directed to a method for preventing and/or treating Ebola virus infection in a subject in need thereof, said method comprising a step of administering a polyclonal antibody or a composition as defined above to said subject.
The present invention is also directed to a method for reducing Ebola virus load in a subject in need thereof, said method comprising a step of administering a polyclonal antibody or a composition as defined above to said subject.
The polyclonal antibodies are preferably provided in the form of a pharmaceutical composition as defined above.
The present invention also relates to a pharmaceutical composition comprising polyclonal antibodies as defined above for use in the prevention and/or treatment of Ebola virus disease, in particular in a subject in need thereof.
The present invention also relates to a method for preventing and/or treating Ebola virus disease in a subject in need thereof, said method comprising a step of administering a pharmaceutical composition comprising the polyclonal antibodies as defined above to said subject.
The polyclonal antibodies are preferably used or administered in an effective amount.
The expression “effective amount”, also referred to as “therapeutically effective amount” is as defined above. Such effective amounts can be routinely determined by those of skilled in the art. The amount of the polyclonal antibodies actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual polyclonal antibodies administered, the age, sex, weight, and response of the individual subject, the severity of the subject's symptoms, and the like. It will also be appreciated by those of skilled in the art that the dosage may be dependent on the stability of the administered polyclonal antibodies.
The effective amount may also vary according to the drug or prodrug with which the polyclonal antibodies may be co-administered.
A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the polyclonal antibodies are outweighed by the therapeutically beneficial effects.
The polyclonal antibodies or the pharmaceutical composition may be used or administered in a single dose or in several doses. Said several doses may be used or administered at the same time, separately or sequentially.
The polyclonal antibodies or pharmaceutical composition comprising thereof are preferably used or administered by subcutaneous, intradermal, intramuscular, intraperitoneal and/or intravenous route.
A preferred route is the intravenous route, in particular in a subject infected with an Ebola virus having a high virus replication rate. The intravenous route indeed allows a quick therapeutic response.
In one embodiment of the invention, a combination of polyclonal antibodies specific for the Ebola virus GP glycoprotein, the Ebola virus GP1 subunit, the Ebola virus GP2 subunit and/or a variant thereof and of polyclonal antibodies specific for at least another Ebola virus antigen may be used. Said polyclonal antibodies specific for different Ebola virus antigens may be used or administered simultaneously, separately or sequentially, in any order.
The polyclonal antibodies may be used or administered in one dose ranging from 50 mg to 500 mg of polyclonal antibodies, preferably from 50 mg to 400 mg, for example from 50 mg to 200 mg or from 200 mg to 500 mg, in particular for intravenous administration.
The polyclonal antibodies may be used or administered in one dose or several doses, said dose being for example as defined above.
When at least two doses are used or administered, they are preferably used or administered sequentially over time, for example one dose once, twice or three times a day for one day or at least two days.
For the prevention of the Ebola virus disease, the polyclonal antibodies are advantageously used or administered in one dose, preferably as soon as possible after exposition to the Ebola virus.
The polyclonal antibodies are preferably administered at least until elimination of the viral particles and/or improvement or disappearance of the symptoms, in particular fever, fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, rash, kidney function, liver function, internal bleeding and/or external bleeding.
The polyclonal antibodies may be used or administered in combination with at least another anti-Ebola virus drug and/or at least another anti-Ebola virus vaccine.
Said other anti-Ebola virus drug and said other anti-Ebola virus vaccine are particularly as defined above.
The term “comprising” as used herein encompasses the term “consisting of”.
The present invention will be further illustrated in view of the following examples and figures.
Sequence SEQ ID NO: 1 is a consensus amino acid sequence of Ebola virus GP glycoprotein.
Sequence SEQ ID NO: 2 is a nucleic acid sequence encoding the truncated Ebola virus GP glycoprotein of sequence SEQ ID NO: 3.
Sequence SEQ ID NO: 3 is the amino acid sequence of a truncated Ebola virus GP glycoprotein consisting of amino acids 33-311 and 463-632 of sequence SEQ ID NO: 1.
Sequence SEQ ID NO: 4 is a nucleic acid sequence encoding sequence SEQ ID NO: 5.
Sequence SEQ ID NO: 5 is the amino acid sequence of the truncated Ebola virus GP glycoprotein of sequence SEQ ID NO: 3 linked to an enterokinase cleavage site and a twin strep tag.
Sequence SEQ ID NO: 6 is a nucleic acid sequence encoding sequence SEQ ID NO: 7.
Sequence SEQ ID NO: 7 is the amino acid sequence of the truncated Ebola virus GP glycoprotein of sequence SEQ ID NO: 3 linked to an enterokinase cleavage site and a his tag.
Sequence SEQ ID NO: 8 is a consensus amino acid sequence of Ebola virus GP1 subunit.
Sequence SEQ ID NO: 9 is a consensus amino acid sequence of Ebola virus GP2 subunit.
Sequence SEQ ID NO: 10 is the amino acid sequence of an enterokinase cleavage site spacer.
Sequence SEQ ID NO: 11 is the amino acid sequence SEQ ID NO: 3 linked to the amino acid sequence SEQ ID NO: 10.
Sequence SEQ ID NO: 12 is a consensus amino acid sequence of a Sudan Ebola virus serotype.
Sequence SEQ ID NO: 13 is a consensus amino acid sequence of a Bundibugyo Ebola virus serotype.
Sequence SEQ ID NO: 14 is a consensus amino acid sequence of a Sudan Ebola virus serotype devoid of signal peptide, mucin-like domain and transmembrane domain.
The recombinant antigens of sequence SEQ ID NO: 5 and SEQ ID NO: 7 are produced as follows. Two pMTpuro vectors comprising either sequence SEQ ID NO: 4 or SEQ ID NO: 6 are used for transfecting Drosophilia Schneider 2(S2) cells. A stable selection of transfected cells is then performed with 6 μg/ml puromycin. Secreted recombinant antigen expression is induced with 0.5 mM CuSO4 for 4 days. The recombinant antigens are then purified on Strep-Tactin resin (for the recombinant antigen carrying a Strep tag) or on a Ni resin (for the recombinant antigen carrying a His tag). The recombinant antigens are further purified by Superdex 200 size-exclusion chromatography in 10 mM Tris and 150 mM NaCl (pH 7,5) (1×TBS). If needed, the tag is then removed using an enterokinase acting on the cleavage site present between the truncated GP and the tag. The purified recombinant antigens comprise sequence SEQ ID NO: 11.
Rabbits are then immunized by injecting 0.1 mg of purified recombinant antigen per injection. Four injections are performed at day 10, 38, 66 and 87. Serum is collected between the injections to monitor the immunization process. The rabbits are sacrificed on day 90 and the serum is collected.
The polyclonal antibodies are purified from the serum by precipitation in 50% ammonium sulphate followed by purification on protein A-sepharose column, purification ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.
The specific binding of the polyclonal antibodies to the recombinant antigen comprising sequence SEQ ID NO: 11 is then assessed by ELISA and the neutralizing capacity of the polyclonal antibodies is assessed in infected human cells.
The recombinant Ebola virus antigen of sequence SEQ ID NO: 5 is produced in insect cells. Briefly, a pMTpuro vector comprising sequence SEQ ID NO: 4 is used for transfecting Drosophilia Schneider 2(S2) cells. A stable selection of transfected cells is then performed with 6 μg/ml puromycin. Secreted recombinant antigen expression is induced with 0.5 mM CuSO4 for 4 days. The recombinant antigen is then purified on Strep-Tactin resin.
As shown in CBB stained SDS PAGE analysis of
Fractions before (lane B) and after one of the SEC runs (lanes 1-14); P1 and P2 indicate peak 1 and 2 respectively. After pooling and concentration of the peak fractions: lane 16 (peak 1) and lane 17 (peak2). Lower panel: Loading pattern of gel.
Furthermore, as shown in
Peak 1 was further purified by SEC chromatography and analyzed by HPLC. The resulting composition comprises more than 90% antigen in trimeric form. The profile of this composition is shown in
Samples of antigen produced as above were separated on 12% SDS PAGE and blotted to nitrocellulose. The membrane was then probed with human anti-EBOLA monoclonal antibody (mAb KZ52) and revealed with mouse anti-IgGFc HRP (Thermofisher cat no A10648). The results presented in
Rabbits were immunized by injecting 0.3 mg of purified recombinant antigen prepared as disclosed in example II, per injection. In this example, three im injections were performed at day 0, 22, and 50. The rabbits are sacrificed on day 60 and the serum is collected.
The polyclonal antibodies are purified from the serum by precipitation in 50% ammonium sulphate followed by purification on protein A-sepharose column, purification ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.
The specific binding of the polyclonal antibody to the recombinant antigen comprising sequence SEQ ID NO: 5 was then assessed by ELISA, and the neutralizing capacity of the polyclonal antibody was determined in infected human cells. The results are presented in
As shown on these figures, polyclonal antibody has a high titer of anti-Ebola immunoglobulins, and can effectively neutralize Ebola virus entry into human cells.
The specific anti-GP IgG are detected by ELISA. Recombinant GP is coated onto the well and sera are titrated by serial dilution. Binding of polyclonal antibody is measured by colorimetric assay. The titer of specific IgG is calculated by defining the inverse of the dilution presenting 50% of the positive signal. Following three administrations, polyclonal antibody presents a titer of 13357±2181 (mean±SE).
Neutralization activity of the polyclonal antibody D60 is evaluated by sero-neutralization assay. Recombinant VSV-GP virus is mixed with different dilutions of polyclonal antibody and added to cultured cells. Protection is measured by counting the number of infected cells. The titer of sero-neutralization is determined by the inverse of the dilution where 50% of the cells are protected from the recombinant virus. Following three administrations, polyclonal antibody presents a SN50% of 564±154 (mean±SE).
Rabbits were immunized by injecting 0.3 mg of purified recombinant antigen prepared as disclosed in example II, per injection. In this example, four im injections were performed at day 0, 22, 36, and 50. The rabbits are sacrificed on day 68 and the serum is collected.
The polyclonal antibodies are purified from the serum by precipitation in 50% ammonium sulphate followed by purification on protein A-sepharose column, purification ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.
The specific binding of the polyclonal antibody to the recombinant antigen comprising sequence SEQ ID NO: 5 was then assessed by ELISA, and the neutralizing capacity of the polyclonal antibody was determined in infected human cells. The results are presented in
As shown on these figures, polyclonal antibody has a high titer of anti-Ebola immunoglobulins, and can effectively neutralize Ebola virus entry into human cells The specific anti-GP IgG are detected by ELISA. Recombinant GP is coated onto the well and sera are titrated by serial dilution. Binding of polyclonal antibody is measured by colorimetric assay. The titer of specific IgG is calculated by defining the inverse of the dilution presenting 50% of the positive signal. Following four administrations, polyclonal antibody presents a titer of 17177±4914 (mean±SE).
Neutralization activity of the polyclonal antibody D68 is evaluated by sero-neutralization assay. Recombinant VSV-GP virus is mixed with different dilutions of polyclonal antibody and added to cultured cells. Protection is measured by counting the number of infected cells. The titer of sero-neutralization is determined by the inverse of the dilution where 50% of the cells are protected from the recombinant virus. Following four administrations, polyclonal antibody presents a SN50% of 860±225 (mean±SE)
Cross neutralization effect of polyclonals of the invention was tested against several serotypes of Ebola virus, as well as against non-Ebola viruses. Cross neutralization activity of the polyclonal antibody is evaluated by sero-neutralization assay. Several recombinant VSV-GP virus are produced harboring different GP from EBOLA virus (Mayinga, Makona, Tai Forrest and Bundibugyo), or from Marburg or Lloviu. Recombinant virus is mixed with different dilutions of polyclonal antibody and added to cultured cells. Protection is measured by counting the number of infected cells.
The results are depicted on
As shown on
The present example discloses the production of an anti-Ebola polyclonal antibody preparations directed against Sudan Ebola virus.
The GP glycoprotein has been selected as antigen to produce polyclonal antibodies. The antigen has been modified to improve the immunogenicity. More specifically, such modified antigen comprises a consensus sequence. By aligning the sequences of several sequences of the Sudan Ebola virus GP glycoprotein, the inventors have selected most conserved positions and defined a consensus sequence which is referred to as sequence SEQ ID NO: 12.
MGGLSLLQLPRDKFRKSSFFVWVIILFQKAFSMPLGVVTNSTLEVTEIDQ
VPLHIPEGETTLPSQNSTEGRRVVNTQETITETAATIIGTNGNHMQISTI
GIRPSSSQIPSSSPTMAPSPEAQTPTTHTSGPSVMATEEPTTPPGSSPGS
TTEAPTLTTPENITTAVKTVLPQESTSNGLITSTV
TGILGSLGLRKRSRR
IPAGIGITGIIIAIIALLCVCKLLC
In a particular embodiment, the Sudan Ebola virus antigen for use in the invention comprises or consists of a sequence which is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 12. The antigen preferably comprises or consists of sequence SEQ ID NO: 12 or an epitope-containing fragment thereof.
In a further preferred embodiment, the modified antigen is devoid of a functional mucin-like domain (bold and emphasized on the sequence above).
In a further preferred embodiment, the modified antigen is devoid of a functional signal peptide (bold on the sequence above).
In a further preferred embodiment, the modified antigen is devoid of a functional transmembrane domain (emphasized on the sequence above).
In this respect, a further particular Ebova virus antigen of the invention comprises or consists of a sequence which is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 14. The antigen preferably comprises or consists of sequence SEQ ID NO: 14 or an epitope-containing fragment thereof.
In a further particular embodiment, the antigen further comprises a tag and, optionally, a spacer.
The recombinant antigen comprising sequence SEQ ID NO: 12 or 14 is produced as follows. Two pMTpuro vectors comprising the respective coding sequences are used for transfecting Drosophilia Schneider 2(S2) cells. A stable selection of transfected cells is then performed with 6 μg/ml puromycin. Secreted recombinant antigen expression is induced with 0.5 mM CuSO4 for 4 days. The recombinant antigen is then purified on Strep-Tactin resin (for the recombinant antigen carrying a Strep tag) or on a Ni resin (for the recombinant antigen carrying a His tag). The recombinant antigen is also/further purified by Superdex 200 size-exclusion chromatography in 10 mM Tris and 150 mM NaCl (pH 7,5) (1×TBS).
A polyclonal antibody preparation is obtained by administering to a rabbit the antigen as defined above. More specifically, rabbits are immunized by injecting between 0.1 and 1 mg of purified recombinant antigen per injection. Several (3 to 6) injections are performed. Serum is collected between the injections to monitor the immunization process. The rabbits are sacrificed when antibody titer is high and the serum is collected. The polyclonal antibodies are purified from the serum by precipitation in 50% caprylic acid followed by purification on protein A-sepharose column, ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.
The present example discloses the production of an anti-Ebola polyclonal antibody preparations directed against Bundibugyo Ebolavirus.
The GP glycoprotein has been selected as antigen to produce polyclonal antibodies. The antigen has been modified to improve the immunogenicity. More specifically, such modified antigen comprises a consensus sequence. By aligning the sequences of several sequences of the Bundibugyo Ebolavirus GP glycoprotein, the inventors have selected most conserved positions and defined a consensus sequence which is referred to as sequence SEQ ID NO: 13.
In a particular embodiment, the Bundibugyo Ebolavirus antigen for use in the invention comprises or consists of a sequence which is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, still more preferably at least 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 13. The antigen preferably comprises or consists of sequence SEQ ID NO: 13 or an epitope-containing fragment thereof.
In a further preferred embodiment, the modified antigen is devoid of a functional mucin-like domain.
In a further preferred embodiment, the modified antigen is devoid of a functional signal peptide.
In a further preferred embodiment, the modified antigen is devoid of a functional transmembrane domain.
In a further particular embodiment, the antigen further comprises a tag and, optionally, a spacer.
The recombinant antigen comprising sequence SEQ ID NO: 13 is produced as follows. Two pMTpuro vectors comprising the respective coding sequences are used for transfecting Drosophilia Schneider 2(S2) cells. A stable selection of transfected cells is then performed with 6 μg/ml puromycin. Secreted recombinant antigen expression is induced with 0.5 mM CuSO4 for 4 days. The recombinant antigen is then purified on Strep-Tactin resin (for the recombinant antigen carrying a Strep tag) or on a Ni resin (for the recombinant antigen carrying a His tag). The recombinant antigen is also/further purified by Superdex 200 size-exclusion chromatography in 10 mM Tris and 150 mM NaCl (pH 7,5) (1×TBS).
A polyclonal antibody preparation is obtained by administering to a rabbit the antigen as defined above. More specifically, rabbits are immunized by injecting between 0.1 and 1 mg of purified recombinant antigen per injection. Several (3 to 6) injections are performed. Serum is collected between the injections to monitor the immunization process. The rabbits are sacrificed when antibody titer is high and the serum is collected. The polyclonal antibodies are purified from the serum by precipitation in 50% caprylic acid followed by purification on protein A-sepharose column, ion exchange column, gel filtration and sterilization by microfiltration and pasteurization.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16305664.1 | Jun 2016 | EP | regional |
| 16306842.2 | Dec 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/063732 | 6/6/2017 | WO | 00 |
