Patent Application
20250195637
The present invention relates to virus strains genetically modified, comprising antigenic parts from two different respiratory viruses.
The present invention relates to genetically modified viral strains, and their use in the prophylactic or therapeutic treatment of infections with viruses, in particular respiratory viruses.
Acute infections of the lower respiratory tracts are one of the major causes of morbidity and mortality on the global scale. In children less than 5 years old in particular, they represent the second cause of mortality according to the World Health Organisation.
The majority of acute infections of the lower respiratory tracts, associated with variable pathologies ranging from a simple cold to serious pneumonia, are notably caused by viruses, such as those belonging to the following families:
The human respiratory syncytial virus (hRSV) and the human metapneumovirus (hMPV) are both responsible for acute infections of the respiratory tracts such as bronchiolitis, bronchitis or pneumonias. They mainly affect populations at risk, which are young children less than 5 years old, the older adults and immuno-compromised persons. Both viruses are present on all continents. Their annual distribution is mainly in winter and spring. Viral transmission essentially takes place by respiratory route, in particular via dispersion in the air of salivary droplets. hRSV was first identified in 1956 in a group of chimpanzees, and then documented to be a mainly human pathogen. It was isolated in 1957. It is a virus with single-stranded RNA genome of negative polarity, of “enveloped” type. Its RNA genome is linear, non-segmented and protected within a helical nucleocapsid. It comprises 10 genes encoding 11 viral proteins. It belongs to the family Pneumoviridae and to the genus Orthopneumovirus. Two hRSV subgroups, A and B, have been identified on the basis of differences of F and G genes.
hRSV is the main etiological agent of bronchiolitis and pneumonias in infants less than 1 year old, with an increased incidence below 6 months old. hRSV is responsible for 6.7% of deaths in children below 1 year of age, and causes excess mortality in the elderly at levels comparable to influenza virus.
hMPV was isolated and described for the first time in 2001 in the Netherlands (van den Hoogen et al., 2001). The following year, several strains of hMPV were isolated in patients located in North America (Peret et al., 2002). hMPV are viruses with negative single-stranded RNA, belonging to the family Pneumoviridae and to the genus Metapneumovirus.
hMPV is prevalent in bronchiolitis and pneumonias in infants, and particularly affects children between 6 months and 3 years old. The average age of children hospitalised due to complications of an infection by hMPV is 6 to 12 months, i.e., later than that caused by hRSV, which mainly occurs between 3 and 6 months.
The genomic organisations of hMPV and hRSV are close. Their genome is sub-divided into several ORFs (Open Reading Frames) encoding for N, P, M, F, M2 (M2-1 and M2-2), SH, G and L proteins. hRSV also comprises 2 additional non-structural genes NS1 and NS2, contrary to hMPV. The general genome structure is represented for the virus strain rC-8543 and its attenuated versions Delta SH in
Three types of glycoproteins are expressed at the surface of the virus particles: F, G and SH proteins.
The F fusion glycoprotein, highly conserved between different subgroups, is involved in the penetration of the virus into the target cells and the formation of syncytium (large multi-nucleated cells derived from the fusion of individual cells following their infection by the virus). The F glycoprotein, also designated as F protein in the present application, is considered as being the most antigenic protein of hRSV and hMPV viruses.
Today, few prophylactic or therapeutic options, specific for hRSV and/or hMPV infection, are available. There is no approved vaccine against either hRSV or hMPV.
For treatment, ribavirin may be used occasionally in serious cases of infections by hMPV or hRSV but this therapy presents several side effects. The usual and widely favoured clinical approach is symptomatic care of the infection: by placing patients under respiratory assistance (administration of oxygen or mechanical ventilation), by administering bronchodilators, corticosteroids and/or antibiotics for preventing or treating bacterial superinfections, and by preventing hypoxemia and electrolyte imbalance.
As a licensed prophylactic treatment, Palivizumab (recombinant humanized hRSV anti-F monoclonal antibody) can be used for hRSV infections, especially for preventing severity of the diseases in high-risk newborns. This preventive treatment is very expensive.
Scientific and clinical communities are currently deeply involved to change this situation of limitation or absence of prophylactic or therapeutic options. Initiatives are underway, such as the creation of RESCEU, a European consortium aiming to regroup all clinical data regarding hRSV infections, and to provide infrastructure to perform trials for future RSV candidate vaccines and therapeutics.
The development of a bivalent vaccine against both hRSV and hMPV thus responds not only a major public health challenge, but also a real socio-economic issue with the objective of reducing the high cost of treatments and hospitalisations associated with these infections. Furthermore, use of such vaccine would present the advantage to decrease the use of antibiotics in the context of bacterial superinfections, and thus limiting the emergence of antibiotics resistances in the population.
Vaccines based on the use of attenuated living virus strains, designated as Live-Attenuated Vaccines (LAVs), have numerous advantages.
First, attenuated living viruses are able to induce a strong immune response in vaccinated patients, because they induce a physiological response similar to a natural infection with the wild virus (although attenuated), which is not the case when an isolated recombinant antigen is administered. Furthermore, in the case of respiratory viruses, these LAVs can be administered by intra-nasal route, and thus mimic the natural entry of the pathogen.
Live-attenuated vaccines broadly stimulate innate, humoral, and cellular immunity, both systemically and locally in the respiratory tract (mucosal immunity).
Because of this strong induced immune response, use of additional adjuvant(s) is usually not necessary.
Finally, this vaccination strategy does not generate any out-of-control inflammatory reaction, as may be the case with inactivated vaccines.
Therefore, it is considered to be the optimal vaccinal strategy for prevention of respiratory viral infections in infants and young children.
The international application WO 2020/021180 discloses an attenuated hMPV strain, obtained from a clinical specimen C-85473, characterised by significant fusogenic capacities, and genetically modified in order to exhibit attenuated virulence, in particular by suppression of expression of the small hydrophobic protein (ASH) and/or the G protein (AG).
Further, the international application WO 2020/120910 discloses a method for producing this attenuated hMPV strain, with infection and culture of a specific avian immortalized cell line. Attenuated strains of hRSV have also been reported. For example, (Rostad et al., 2016) developed an attenuated viral strain: RSV-A2-dNS-ASH-BAF (DB1), with codon deoptimization of genes for non-structural proteins NS1 and NS2, deletion of the SH protein encoding gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence. The same year, an attenuated viral strain named OE4 (RSV-A2-dNS1-dNS2-DSH-dGm-Gsnull-line19F) was engineered and described in (Stobart et al., 2016). This OE4 strain expresses line 19 F protein and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. Safety and immunogenicity of an attenuated viral strain RSV/ANS2/A1313/11314L, with a deletion of the interferon antagonist NS2 gene, deletion of codon 1313 of the RSV polymerase gene and a stabilizing missense mutation 11314L, has been shown in RSV-seronegative children (Karron et al., 2020).
Among the most interesting viral antigens, F proteins, involved in the entry of the virus into the target cells and in the formation of syncytium, have been extensively studied. In particular, antibodies against F protein have been generated.
Administration of a monoclonal antibody directed against F protein of hRSV is currently used to prevent the severity of the disease. Administration to children of this humanized monoclonal antibody, Palivizumab, reduces significantly the risk of hospitalization linked to RSV infection in high-risk children.
F protein merges virus and host-cell membranes by using the difference in folding energy between two conformations:
It has been noticed that the major epitopes of F protein of hRSV are found in the prefusion state of F protein. For example, antibodies have been developed which target the antigenic site Ø, a metastable site specific to the prefusion state of the F glycoprotein. These antibodies are 10 to 100-fold more potent than palivizumab, which recognizes the antigenic site II located in pre-fusion and post-fusion state.
Soluble variants of F protein which stably expose the antigenic site Ø have been engineered (Mclellan et al., 2013). These variants consist in the extracellular domain of the F protein, stabilized in the prefusion state.
In the development of live-attenuated vaccines, another field of research is the engineering of bivalent vaccines, comprising antigens from two different pathogens.
Bivalent vaccines based on combination of hRSV and hMPV are described, for example, in the European patent application EP 3 868 874. In these recombinant viruses, based on a rearranged viral strain of hRSV, a gene encoding the F protein of another virus, in particular belonging to the family Pneumoviridae, is (i) inserted between the genes encoding the G protein and the F protein of hRSV, or (ii) replaces the gene encoding the F protein of hRSV.
Nevertheless, efforts for providing safe and efficacious bivalent vaccines against both hMPV and hRSV are still underway. The present invention relates to a novel viral strain with multiple genetic modifications, that is attenuated but still immunogenic, with significant fusogenic capacities. Advantageously, the attenuated phenotype is stable, i.e., non-reversible during the replication cycles. This attenuated viral strain is able to reproduce in vitro on cells, and can be used in vivo as a live-attenuated vaccine allowing to generate an immune response against both hMPV and hRSV in individuals to whom it is administered.
The present invention concerns a bivalent vaccine, able to generate a multiple immune response in an individual after its administration, against two viruses. Such vaccine induces a combined immune response against two viruses, which reduces the number of injections to an individual for obtaining a preventive immunization against different pathogens.
More particularly, the present invention concerns a viral strain derived from the human metapneumovirus (hMPV)C-85473 strain, having the genome sequence represented in SEQ ID NO. 1, wherein said genome sequence comprises the following genetic modifications:
In particular, the invention relates to said viral strain comprising an exogenous sequence comprising:
Another object of the invention is a genetic cassette comprising one of the following nucleotide sequences:
The present invention also concerns a viral strain derived from a human metapneumovirus (hMPV) strain comprising a genetic cassette as defined above. Preferentially, this viral strain presents an attenuated virulence.
The invention also relates to said viral strain, for use as a medicament.
The invention also relates to said viral strain, for use in preventing and/or treating infections by at least one respiratory virus, in particular at least, and more particularly two viruses of the Pneumoviridae family.
The invention also relates to a vaccine composition comprising, in a pharmaceutically acceptable vehicle, at least one viral strain such as defined above, and optionally an adjuvant.
[11C] Viral genes quantification within the pulmonary tissues of BALB/c mice prime- and boost-immunized with the bivalent Metavac®-RSV v.1 or v.3 4 days after the viral challenge with the RSV WT virus: quantification of the F RSV gene (left graph) and the N HMPV gene (right graph) is performed by RT-qPCR. The dotted line represents the threshold of detection.
Human Metapneumovirus (hMPV) Virus Strain
The present invention is based on a virus strain of human metapneumovirus, designated C-85473, isolated from a patient sample in Canada, described in the article (Hamelin et al., 2010).
The recombinant strain rC-85473, originating from this virus strain C-85473 and obtained by reverse genetic engineering, is characterised by considerable fusogenic capacities. Furthermore, rC-85473 is able to penetrate into target cells at a high frequency, i.e., a high degree of infection. Without wishing to be bound by any theory, inventors attribute these properties to a specific sequence of the F protein of rC-85473 strain, which comprises a unique peptide sequence of five amino acids that is not found among the other F proteins of other hMPV strains (Dubois et al., 2017).
This rC-85473 hMPV strain comprises the genomic sequence as shown in SEQ ID NO.1.
The first genetic modification (i) introduced into the rC-85473 strain aims to attenuate the virulence of said strain. An “attenuated virulence” corresponds to an absence of pathogenicity and a reduced inflammatory response in vivo, after administration of an efficient dose of such attenuated viral strain. (Dubois et al., 2019 and Le et al., 2019).
Advantageously, this attenuation of virulence does not affect the replication capacities of the viral strain in vitro, neither its capacities of infection of target cells.
This genetic modification consists in the inactivation of at least one endogenous gene:
This genetic modification may also be an inactivation of both genes.
In the sense of the invention, the “inactivation of a gene” designates a genetic modification inducing a loss of expression of the gene, or the expression of a non-active form of the encoded protein. This inactivation of a gene may be carried out by all techniques well known to the person skilled in the art. In particular, the inactivation of a gene may be obtained by the introduction of a point mutation into the gene, by the partial or total deletion of the coding sequences of the gene, or by modification of the gene promoter. These different genetic modifications will be carried out according to any one of the molecular biology techniques well known to the person skilled in the art.
In a specific embodiment of the invention, in the viral strain of the invention, the endogenous gene coding for the SH protein is deleted.
In the sense of the invention, “deleted gene” means that a significant part of the coding sequence of this gene has been removed, notably:
According to a preferred embodiment, in the viral strain according to the invention, the gene encoding for the SH protein is completely deleted, that is to say that all (100% of) the coding sequence for the SH protein has been removed from the original genomic sequence. In this case, the viral strain of the invention comprises the nucleotide sequence such as represented in SEQ ID NO. 2. This specific attenuated viral strain is designated in the examples section with the name “Metavac®”, also designated as “monovalent Metavac®”.
A viral strain comprising this first genetic modification (i) is designated below as the attenuated viral strain.
The second genetic modification (ii) introduced into the attenuated rC-85473 strain aims to obtain the expression of an antigen originating from a hRSV strain.
This genetic modification (ii) consists in the introduction of an exogenous coding sequence into the genome of the attenuated viral strain described above, for example comprising a genome sequence such as represented in SEQ ID NO.2.
This introduction of an exogenous coding sequence is not a replacement, but a real addition to the genome of the attenuated rC-85473 strain. In consequence, the F protein of the rC-85473 strain is still present in the genome of the strain, and is expressed, even after the introduction of an exogenous sequence.
Thus, the viral strain of the invention comprises in its genome a sequence coding for at least one extracellular domain of a F protein of the human respiratory syncytial virus (hRSV), said domain being wild-type or mutated.
In the sense of the invention, “exogenous sequence coding for” or “exogenous coding sequence” or “exogenous nucleotide sequence” means a nucleic acid sequence that has been introduced into a viral genome, that is under the control of a suitable promoter, and encodes a protein or a protein domain. In the present case, since the genome of hMPV is made of RNA, the introduced exogenous sequence will also be constituted of RNA. But it is understood that preliminary steps for introducing this exogenous sequence may use corresponding DNA sequence (reverse genetic plasmids).
In the sense of the invention, the term “F protein from hRSV” designates a glycoprotein from a hRSV subgroup A or B, preferentially from a hRSV of subgroup A.
Sequences of all known wild-type F proteins from hRSV can be found in public databases such as UniProt, for example under the access references P11209 or P03420 (precursor forms).
The person of the art knows well the biology of proteins, and can identify the three domains constituting a transmembrane protein: the cytoplasmic domain, inside the cell; the transmembrane domain, inserted into the cell membrane; and the extracellular domain, present at the surface of cell membranes.
In the sense of the invention, the phrase “coding for at least” means that the exogenous sequence codes for at least one peptidic domain, but in most cases also codes for other domains.
In the sense of the invention, the phrase “one extracellular domain of the F protein of hRSV” designates the extracellular domain (expressed at the surface of the viral particles) of any F protein from any hRSV subgroup, and includes wild-type domains and mutated domains.
As is well known by the person of the art, it is preferable to combine an extracellular domain with another peptide sequence allowing its anchoring into a viral particle, in particular at the surface of said viral particle, and/or allowing its anchoring into membranes of infected cells.
Therefore, in a preferred embodiment of the invention, an “exogenous sequence coding for at least the extracellular domain of the F protein of the human respiratory syncytial virus (hRSV), said domain being wild-type or mutated” designates an exogenous sequence coding for a mutated or wild-type extracellular domain of the F protein of hRSV, associated with a sequence coding for at least one anchoring domain, in particular coding for a cytoplasmic and/or a transmembrane domain.
In a first embodiment of the invention, the domain of the F protein is a wild-type domain, i.e., presents the peptide sequence of a domain of a natural F protein from hRSV.
The term “wild-type” designates the typical form of a protein (i.e., its typical peptide sequence) as it occurs in nature. On the contrary, the term “mutated” designates an atypical, non-standard form of the same protein.
In a particular embodiment, the exogenous nucleotide sequence comprises a sequence coding for the wild-type extracellular domain of a hRSV F protein.
In another particular embodiment, the exogenous nucleotide sequence consists in a sequence coding for the wild-type extracellular domain of a hRSV F protein.
In particular, the exogenous nucleotide sequence consists in a sequence coding for the wild-type extracellular domain of the hRSV F protein from a subgroup A virus, more particularly coding for the wild-type extracellular domain of the hRSV F protein having a peptide sequence as shown in SEQ ID NO. 3.
In a particular embodiment, the exogenous nucleotide sequence further comprises a sequence coding for the wild-type transmembrane domain of a hRSV F protein.
In a particular embodiment, the exogenous nucleotide sequence further comprises a sequence coding for the wild-type cytoplasmic domain of a hRSV F protein.
In another particular embodiment, the exogenous nucleotide sequence consists in a sequence coding for the three wild-type domains of a hRSV F protein, i.e., the extracellular, cytoplasmic and transmembrane domains constituting the whole F protein.
In particular, the exogenous nucleotide sequence consists in a sequence coding for the wild-type hRSV F protein from a subgroup A virus, more particularly coding for the wild-type hRSV F protein having a peptide sequence as shown in SEQ ID NO. 4.
In a specific embodiment of the invention, the exogenous nucleotide sequence is integrated into the genome of the attenuated hMPV viral strain at a specific site, for example:
According to one of these specific embodiments, advantageously, the exogenous nucleotide sequence consists in the three wild-type domains of a hRSV F protein, i.e., the extracellular, cytoplasmic and transmembrane domains constituting the whole F protein from hRSV.
A specific chimeric construction comprising the genome of an attenuated hMPV strain (sequence represented in SEQ ID NO. 2) and an exogenous nucleotide sequence coding for a wild-type hRSV F protein (SEQ ID NO. 4) inserted between the gene coding for the F protein and the gene coding for the M2 protein, is designated in the examples section as “Metavac®-RSV v.1”.
The full genomic sequence of this viral strain, combined with the GFP encoding gene, is shown in SEQ ID NO. 10.
In a specific embodiment, the invention concerns a viral strain derived from the human metapneumovirus (hMPV) strain having the genome sequence represented in SEQ ID NO. 1, wherein said genome sequence comprises the following genetic modifications:
In a second embodiment of the invention, the domain of the F protein is a mutated domain, i.e., presents a peptide sequence derived from a domain of a wild-type F protein from hRSV comprising at least one point mutation, that is to say the replacement of at least one residue in the wild-type peptide sequence with another residue.
In the context of the present invention, the terms “mutated domain” or “mutated sequence” or “mutated protein” all refer to peptide sequences presenting at least 80% of sequence identity with their corresponding standard, wild-type peptide sequences, and therefore presenting at most 20% of differences with their corresponding wild-type peptide sequences, after optimal alignment of both sequences. In a preferred embodiment, in said mutated domains, the main antigenic epitopes are conserved, i.e., present their wild-type sequence.
In a preferred implementation of the invention, a mutated domain presents at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97% of sequence identity with its corresponding wild-type domain. Preferentially, in said mutated domain, the main antigenic epitopes are conserved.
In a more specific embodiment of the invention, a mutated domain corresponds to a mutated domain presenting at least 97% of sequence identity with the corresponding wild-type domain. The percent identities referred to in the context of the present invention are determined on the optimal alignment of the sequences to be compared, which comprise one or more differences such as insertions, deletions, truncations and/or substitutions of amino acids.
This percent identity may be calculated by any sequence analysis method well-known to the person skilled in the art.
The percent identity may be determined after global alignment of the sequences to be compared of the sequences taken in their entirety over their entire length. In addition to manual comparison, it is possible to determine global alignment using the algorithm of Needleman and Wunsch (1970).
For peptide sequences, the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the BLOSUM62 matrix. Preferably, the percent identity is determined via the global alignment of sequences compared over their entire length.
In a particular embodiment, the exogenous nucleotide sequence comprises a sequence coding for a mutated extracellular domain of a hRSV F protein.
In another particular embodiment, the exogenous nucleotide sequence consists in a sequence coding for a mutated extracellular domain of a hRSV F protein.
More preferentially, this mutated domain corresponds to the stabilized prefusion state of the F protein, that is to say has a protein structure restrain under the prefusion state.
This mutated extracellular domain comprises at least one of the following 14 mutations: S46G, K66E, E92D, Q101P, A149C, S155C, S190F, V203L, V207L, S215P, S290C, L373R, Y458C, K465Q, such as described in (Mclellan et al., 2013).
Preferentially, the mutated extracellular domain comprises all 14 mutations listed above. These mutations are incorporated by any technique known by the person of the art, in particular by directed mutagenesis.
In a specific embodiment, this mutated extracellular domain presents a sequence as shown in SEQ ID NO. 5. This mutated domain presents 14 point-mutations over a full length of 513 residues, and therefore has 499 common residues with the corresponding wild-type extracellular domain (residues 1-513 of SEQ ID NO. 4), which corresponds to an identity percentage of 97.27% between the wild-type and the mutated extracellular domain of the F protein of hRSV.
In another embodiment, the exogenous nucleotide sequence consists in a sequence coding for the wild-type cytoplasmic domain, the wild-type transmembrane domain, and a mutated extracellular domain corresponding to the stabilized prefusion state of the F protein of hRSV.
A specific chimeric construction comprising the genome of an attenuated hMPV strain (sequence represented in SEQ ID NO. 2) and an exogenous nucleotide sequence coding for a mutated hRSV F protein, comprising a mutated extracellular domain corresponding to the stabilized prefusion state (SEQ ID NO. 5), is designated in the examples section as “Metavac®-RSV v. 2”.
In particular, the mutated F protein presents the sequence as shown in SEQ ID NO. 6. This mutated protein presents a sequence identity with the wild-type hRSV F protein of 97.5% (560 common residues over a full length of 574 residues).
The full genomic sequence of this viral strain, combined with the GFP encoding gene, is shown in SEQ ID NO. 11.
In another embodiment of the invention, the exogenous nucleotide sequence encodes a chimeric protein comprising domain(s) from F protein of hRSV and domain(s) from F protein of hMPV.
In particular, said exogenous nucleotide sequence encoding a chimeric hRSV/hMPV F protein comprises:
As mentioned earlier, in the context of the invention, the term “mutated domain” refers to domains having peptide sequences having at least 80%, preferentially at least 90%, and more preferentially at least 95%, 96% or 97% of sequence identity with peptide sequences of the corresponding wild-type domains. Preferentially, the main antigenic epitopes of said domain are conserved.
In other words, the invention concerns a viral strain as described above, wherein the exogenous nucleotide sequence encodes a chimeric hRSV/hMPV F protein comprising:
Any attenuated viral strain as described above, comprising in its genome an exogenous sequence according to any possible combination of (a) and (b), is an object of the present invention.
For example, said combinations include the following sequences coding for chimeric hRSV/hMPV F proteins consisting in:
Advantageously, a “mutated” extracellular domain corresponds to a stabilized prefusion state of the F protein, from hRSV such as described above, in particular presenting at least 97% of sequence identity with the corresponding wild-type domain.
In a specific embodiment, said attenuated viral strain from hMPV comprises an exogenous nucleotide sequence consisting in:
In a specific embodiment, the F protein of hMPV is from a subgroup strain A1.
In another specific embodiment, the F protein of hMPV is from the rC-85473 strain and presents the sequence as shown in SEQ ID NO. 7.
A specific chimeric construction, comprising the genome of an attenuated hMPV strain (sequence represented in SEQ ID NO. 2) and an exogenous nucleotide sequence coding for a chimeric protein, comprising a mutated extracellular domain corresponding to the stabilized prefusion state of F protein from hRSV (SEQ ID NO. 5) and the wild-type cytoplasmic and transmembrane domains of a F protein from hMPV, is designated in the examples section as “Metavac®-RSV v.3”.
In particular, the encoded chimeric protein presents the sequence as shown in SEQ ID NO. 8.
The full genomic sequence of this viral strain, combined with the GFP encoding gene, is shown in SEQ ID NO. 12.
In a specific embodiment, said attenuated viral strain from hMPV comprises an exogenous nucleotide sequence consisting in:
A specific chimeric construction comprising the genome of an attenuated hMPV strain (sequence represented in SEQ ID NO. 2) and an exogenous sequence coding for a chimeric protein, comprising a wild-type extracellular domain of F protein from hRSV (SEQ ID NO. 3) and the wild-type cytoplasmic and transmembrane domains from the F protein of hMPV, is designated in the examples section as “v.4”.
In particular, the encoded chimeric protein presents the sequence as shown in SEQ ID NO. 9.
In a specific embodiment, the viral strain of the invention presents the following genetic modifications:
Another aspect of the invention concerns genetic cassettes encoding chimeric proteins comprising at least one domain from F protein of hRSV and at least one domain from F protein of hMPV.
In particular, said genetic cassette comprises:
These genetic cassettes also enclose promoter sequences and all regulatory elements allowing the transcription and translation into proteins of the nucleotide sequences (a) and (b).
All possible combinations of (a) and (b) are objects of the present invention.
What is meant by a “mutated domain” has been defined previously. Advantageously, a “mutated” extracellular domain corresponds to the stabilized prefusion state of the F protein, from hRSV. In a specific embodiment, this mutated extracellular domain presents a sequence as shown in SEQ ID NO. 5.
For example, said combinations include the following nucleotide sequences coding for chimeric hRSV/hMPV F proteins consisting in:
Advantageously, a “mutated” extracellular domain corresponds to a stabilized prefusion state of the F protein, from hRSV.
In a specific embodiment, said genetic cassette comprises the following nucleotide sequences:
A specific chimeric construction according to this embodiment is designated in the examples section as “v.3”.
In particular, the encoded chimeric protein presents the sequence as shown in SEQ ID NO. 8.
In a specific embodiment, said genetic cassette comprises the following nucleotide sequences:
A specific chimeric construction according to this embodiment is designated in the examples section as “v.4”.
In particular, the encoded chimeric protein presents the sequence as shown in SEQ ID NO. 9.
The described nucleotide sequences can be under the form of DNA or RNA.
The genetic cassette described above may be expressed by any system known by the person of the art. For example, a genetic cassette according to the invention can be integrated into a plasmid, into a bacmid, into liposomes, or into any vector of expression. The encoded chimeric proteins may also be used as such.
In another aspect, the invention concerns a viral strain derived from a human metapneumovirus (hMPV) strain, comprising in its genome a genetic cassette such as described above.
Preferentially, the viral strain derived from a hMPV strain is further attenuated, i.e., its virulence is decreased compared to those of the initial viral strain. This attenuation of virulence is obtained, for example, by introducing genetic modifications into the genomic sequence of this viral strain, as is well known by the person of the art.
Viral Strains for their Use Thereof as a Medicine
The present invention also relates to any viral strain as defined above, for its use as a medicament.
Indeed, this attenuated viral strain may be used, notably, for treating and/or preventing infection by at least one respiratory virus, more specifically by at least one virus of the Pneumoviridae family.
In the sense of the invention, the term “treat” designates the fact of combatting infection by a virus in a human or animal organism. In the case of a viral infection, “treating” designates the decrease of the level of viral infection (infectious load) in the organism, and preferably the complete eradication of the virus from the organism. The term “treat” also designates the fact of attenuating the symptoms associated with the viral infection (respiratory syndrome, renal failure, fever, etc.).
In the sense of the invention, the term “prevent” designates the fact of avoiding, or at least decreasing the risk of occurrence, of an infection in an organism. In the case of a viral infection, “preventing” means that the cells of an organism become less permissive to infection, and are thus best placed not to be infected by said virus. It also means that the immune system of the organism has been prepared to react quickly and efficiently in presence of the virus, in order to resist to the infection.
More specifically, the invention relates to a viral strain such as defined above, for use in preventing and/or treating infections by at least one respiratory virus, wherein the at least one respiratory virus is from the Pneumoviridae family, in particular is a human metapneumovirus and/or is the human syncytial respiratory virus.
In particular, the invention concerns a viral strain as described above, for use in preventing infection by two respiratory viruses, a human metapneumovirus (hMPV) and a human syncytial respiratory virus (hRSV).
This viral strain will be preferably integrated in a vaccine composition comprising a pharmaceutically acceptable vehicle, suitable for suspending said viral strain and for the administration thereof.
Said vaccine composition comprises at least one viral strain according to the invention, making it possible to stimulate in a specific manner the immune system of an organism.
Thus, this vaccine composition comprises at least one live attenuated viral strain which plays the role of antigen, that is to say that is recognized and induces a specific immune response in the organism, which will retain the memory thereof.
The present invention also relates to a vaccine composition comprising, in a pharmaceutically acceptable vehicle, at least one viral strain according to the invention, and optionally an adjuvant.
In the sense of the invention, the term “pharmaceutically acceptable vehicle” designates vehicle or excipient, that is to say compound not having any specific action on the infection considered here. These vehicles or excipients are pharmaceutically acceptable, meaning that they may be administered to an individual without risk of significant deleterious effect(s) or prohibitive undesirable effect(s).
The vaccine composition according to the invention comprises at least one effective amount of the viral strain. “Effective amount” is taken to mean, in the sense of the invention, a quantity of viral strain sufficient to trigger an immune reaction in the organism to which it is administered.
The vaccine composition of the present invention is suited for oral, sublingual, inhalation, sub-cutaneous, intramuscular or intravenous administration.
According to a particular embodiment of the invention, the vaccine composition is in a galenic form intended for administration by inhalation.
Inhalation designates absorption by the respiratory tracts. It is in particular a method for absorption of compounds for therapeutic purposes, of certain substances in the form of gas, micro-droplets or powders in suspension.
The administration of pharmaceutical or veterinary compositions by inhalation, that is to say by the nasal and/or buccal passageways, is well known to the person skilled in the art.
Two types of administration by inhalation are distinguished:
The pharmaceutical form considered here is thus advantageously selected from: a powder, an aqueous suspension of droplets or a pressurised solution.
The target population of respiratory virus is mainly a paediatric population, constituted of individuals less than 18 years old, and more specifically of young children (less than 5 years old) and infants. Administration by inhalation is advantageous, since it is non-invasive.
The invention also relates to a vaccine composition such as described above, for its use for preventing and/or treating infections by at least one respiratory virus, wherein the at least one respiratory virus is from the Pneumoviridae family, in particular is a human metapneumovirus and/or is the human syncytial respiratory virus.
Such a vaccine composition could be used as a preventive vaccine, that is to say intended to stimulate a specific immune response before infection of an organism by a virus.
Such a vaccine composition could also be used as a therapeutic vaccine, that is to say intended to stimulate a specific immune response concomitantly with infection of an organism by said virus.
The present invention also relates to a method for preventing infections by at least one virus from the Pneumoviridae family, in particular hMPV and/or hRSV, comprising the administration to individuals susceptible to be infected by such viruses of a vaccine composition described above.
The present invention also relates to a method for treating an infection with a virus from the Pneumoviridae family, in particular hMPV and/or hRSV, comprising the administration to individuals infected with at least one of these viruses of a vaccine composition described above.
In particular, the individuals are children and infants.
As is shown in the examples section below, the vaccine compositions comprising the viral strains according to the invention induce the production of neutralizing antibodies against multiple strains of hMPV (HMPV A and B, see example 10) and against multiple strains of hRSV (RSV A and B, see example 11).
Four F RSV protein constructions were designed as represented in
F RSV v. 1 sequence corresponds to the native F RSV gene from the RSV A2 strain accessible to the person skilled in the art.
F RSV v.2 sequence corresponds to the F RSV v.1 gene in which 14 mutations (*) has been incorporated by directed mutagenesis. These mutations (S46G, K66E, E92D, Q101P, A149C, S155C, S190F, V203L, V207L, S215P, S290C, L373R, Y458C, K465Q) are described to stabilize the glycoprotein F in its pre-fusion metastable form (Mclellan et al., 2013 doi:10.1126/science.1243283).
F RSV v.3 sequence corresponds to the F RSV v.2 gene in which the region coding for the F RSV protein transmembrane and cytoplasmic domains (amino acid position 514-574) has been replaced by the counterpart coding sequence of the F C-85473 HMPV gene (amino acid position 482-539).
F RSV v.4 sequence corresponds to the F RSV v.1 gene in which the region coding for the F RSV protein transmembrane and cytoplasmic domains (amino acid position 514-574) has been replaced by the counterpart coding sequence of the F C-85473 HMPV gene (amino acid position 482-539).
The corresponding coding sequences were inserted into the plasmid encoding the full-length genome of the rC-85473 HMPV virus (SEQ ID NO. 1).
The insertion of the F RSV coding sequences was performed at several genomic positions, as resumed in Table 2.
Among these conditions, the insertion between the HMPV genes N and P or P and M or F and M2 allowed viral rescue after reverse genetics, following experimental protocols known by the person skilled in the art. HMPV virulence being attenuated by the deletion of the gene encoding for the SH protein (ΔSH), the construction constituted by the insertion of the F RSV coding sequences between F and M2 HMPV genes is the only construction compatible with significant rescue of recombinant viruses, efficient viral propagation and amplification.
The genetic constructions represented in
The complete sequences of these genetic constructions are presented in SEQ ID NO. 13 (GFP ΔSH-rC-85473), SEQ ID NO.10 (Bivalent Metavac® RSV v.1), SEQ ID NO.11 (Bivalent Metavac® RSV v.2) and SEQ ID NO. 12 (Bivalent Metavac® RSV v.3).
In the
The cells are observed by fluorescence microscopy at ten days post-infection.
GFP expression reveals fully functional and replicative recombinant viruses, as well as expected fusogenic phenotype (induction of cellular syncytia via efficient expression of F fusion protein), which are intrinsic characteristics of the attenuated Metavac® recombinant strain.
These results show the capacity of the Metavac® recombinant virus to accept an exogenous F RSV gene leading to the expression of fully functional F fusion proteins and production of propagative and replicative recombinant viruses (rescued by reverse genetic), especially when the corresponding coding sequence is inserted between F and M2 HMPV genes.
LLC-MK2 cells were infected separately, with a multiplicity of infection of 0.01, by the following recombinant viruses:
The cell supernatants were collected each day for 7 days, in triplicate, and viral loads were evaluated by TCID50 assays, virology techniques well known to the person skilled in the art, which represents the final viral dilution at which 50% of the cell tissue show visible cytopathic effects (50% Tissue Culture Infective Dose).
The
The recombinant virus Bivalent Metavac®-RSV v. 1 seems to have better replicative capacities than those of the viruses Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3.
In the table below (table 3) are represented the average loads of the viral stocks produced and concentrated for each recombinant virus Bivalent Metavac®-RSV v.1, Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3, illustrating that despite variable replicative kinetics, all of the three recombinant bivalent candidates lead to similar production yield.
These results show the replicative capacities of the Bivalent Metavac®-RSV v.1, Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3 viruses.
LLC-MK2 cells were infected (t=0) with the Bivalent Metavac®-RSV v.1, Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3 viruses, or with the wild-type recombinant rC-85473 HMPV strain at an MOI of 0.01. Hep-2 cells were infected (t=0) with RSV A2 strain at an MOI of 0.01. After 5 days of infection, infected cell monolayers were fixed in formaldehyde solution and specific immunostainings were performed with:
Results are presented in
Specific labelling is observed after peroxidase revelation and the representative images show that both HMPV F (after HMPV24 immunostaining) and RSV F (after Palivizumab immunostaining) fusion proteins are expressed and detected on cells infected with Bivalent Metavac®-RSV v.1, or Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3 viruses.
As expected for controls, no immunostaining with MAb HMPV24 was observed on cells infected by RSV A2 strain (which does not express HMPV F fusion protein), and no immunostaining with Palivizumab Synagis® or D25 Mab was observed on cells infected by rC-85473 HMPV (which does not express RSV F fusion protein).
With monoclonal D25 immunostaining, the pre-fusion F RSV protein form was detected on cell infected with the Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3 viruses with higher intensity than with the Bivalent Metavac®-RSV v.1, which argue in favour of stronger expression and exposition at cell surface of the stabilized pre-fusion F RSV protein, as expected.
LLC-MK2 cells were infected (t=0) with the Bivalent Metavac®-RSV v.1, Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3 viruses, or with the rC-85473 HMPV at an MOI of 0.1.
After 48 hours of infection, infected cell monolayers were trypsinized, resuspended and quantification of the F HMPV or F RSV protein expressions was performed in flow cytometry.
Infected cells were detected by GFP fluorescence and the detection of F HMPV and F RSV expression is performed with the following immunolabeling:
Results are presented in table 4 below.
The results reported in Table 4 show that more than 85% of the infected cells expose the F HMPV protein at their surface whereas 64.5%, 54.6% and 47.2% of the cells expose the F RSV proteins when they are infected with Bivalent Metavac®-RSV v.1, Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3 viruses, respectively.
These results show the ability of the three Bivalent Metavac®-RSV v.1, Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3 viruses to express and expose both the F RSV and the F HMPV proteins at the surface of infected cells, and in particular the stabilized pre-fusion F RSV form expressed by the Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3 viruses.
Viral suspensions of each Bivalent Metavac®-RSV viruses were prepared, filtered at 0.45 μm, concentrated by ultracentrifugation and then resuspended in NaCl. Viral suspensions were adsorbed on 200 Mesh coated Nickel grids and observed by transmission electron microscopy without labelling (
Results are presented in
In
In
In
These results demonstrate the effective expression of both hMPV F protein and hRSV F protein at the surface of viral particles of the three Bivalent Metavac®-RSV v.1, Bivalent Metavac®-RSV v.2 and Bivalent Metavac®-RSV v.3 viruses.
3D reconstituted human respiratory epithelia (MucilAir® HAE, Epithelix) have been cultivated at the air-liquid interface following the supplier instructions.
Epithelia were then infected at an MOI 0.5 with the monovalent Metavac® or the bivalent Metavac®-RSV v.1, v.2 or v.3 viruses.
Representative pictures of viral propagation have been taken after 3, 5 and 7 days of infection with the monovalent Metavac®, bivalent Metavac®-RSV v.1, bivalent Metavac®-RSV v.2 and bivalent Metavac®-RSV v.3 viruses.
In
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In
These results show the capacity of the three different bivalent Metavac®-RSV v. 1, v.2 and v.3 viruses to infect and propagate into ex vivo human airway epithelial tissue, and further the ability of these bivalent Metavac®-RSV viruses to express the exogenous F RSV antigen at the surface of 3D reconstituted human respiratory epithelium.
BALB/c mice were infected by intranasal instillation with: 5×105 TCID50 of recombinant bivalent Metavac®-RSV v.1, v.2 or v.3 viruses, on the basis of active immunizing dose determined for the monovalent Metavac® virus. The weight and the survival of the infected mice were monitored daily for 10 days (weight average over 5 mice±SEM).
In addition, 5 days after the infection, 2 mice per group underwent euthanasia for a measurement of pulmonary viral loads by RT-qPCR.
In
These results show the capacity of three bivalent Metavac®-RSV viruses to infect and replicate in vivo, and to express the exogenous F RSV gene at a similar level to that of N HMPV gene into the pulmonary tissue of infected mice.
BALB/c mice were infected by intranasal instillation with non-lethal doses of wild-type HMPV rC-85473 strain (1×106 TCID50) or wild-type RSV A Long strain (1×106 PFU), in order to induce a primary seroconversion of infected mice.
Three weeks after, HMPV- or RSV-primed BALB/c mice were infected by intranasal (boost) instillation with 5×105 TCID50 of the recombinant bivalent Metavac®-RSV v.1, v.2 or v.3 viruses, on the basis of active immunizing dose determined for the monovalent Metavac® virus.
The weight and the survival of the mice was monitored daily for 10 days (weight average over 8 mice±SEM).
In addition, 5 days after the intranasal boost-infection, 3 mice per group underwent euthanasia to measure pulmonary viral loads by RT-qPCR.
In
In
These results show the capacity of the three bivalent Metavac®-RSV v.1, v.2 or v. 3 viruses to induce in vivo a strong and specific neutralizing antibody response against HMPV and RSV viruses.
BALB/c mice were immunized twice, in 21-days interval, by intranasal instillation with 5×105 TCID50 of the recombinant monovalent Metavac® or the bivalent Metavac®-RSV v.3 viruses, on the basis of active immunizing dose determined for the monovalent Metavac® virus, in comparison with a group of mice mock-immunized with culture medium (mock).
Twenty-one days after the boost-infection, mice endured a hMPV lethal viral challenge by intranasal instillation of 3×106 TCID50 of the wild-type HMPV rC-85473 (an infectious dose resulting in more than 50% mortality rate for mice).
The weight and the survival of the mice was monitored daily for 14 days (weight average over 8 mice±SEM).
In
Five days after the viral challenge, 4 mice per group underwent euthanasia to measure pulmonary viral loads by RT-qPCR.
In
Finally, in
As expected, a high level of HMPV-neutralizing antibodies was measured in sera of mice immunized with 2 doses of monovalent Metavac® virus.
For mice double-immunized with the bivalent Metavac®-RSV v.3 virus, high neutralizing antibody titers against both HMPV and RSV viruses were detected 21 days after the boost-immunization and were persistent until 21 days after the HMPV viral challenge.
These results show the capacity of the bivalent Metavac®-RSV candidate (bivalent Metavac®-RSV v.3) to induce in vivo a strong specific neutralizing antibody response against both HMPV- and RSV viruses, and to fully protect mice challenged with a lethal dose of HMPV wild-type virus.
BALB/c mice were infected by intranasal instillation with 5×105 TCID50 of recombinant bivalent Metavac®-RSV v.1 or v.3 viruses, on the basis of active immunizing dose determined for the monovalent Metavac® virus.
In addition, 5 days after the infection, 3 mice per group underwent euthanasia for a lung tissue harvest. Complete lungs were fixed with formaldehyde solution for further histopathological analysis.
In
Overall, these results highlight the capacity of both bivalent Metavac®-RSV viruses to infect and replicate in vivo, to express the exogenous F RSV gene, and to induce a low inflammatory response into the pulmonary tissue of infected mice, as expected from live-attenuated vaccine candidates.
BALB/c mice were immunized twice, in 21-days interval, by intranasal instillation with 5×105 TCID50 of the recombinant monovalent Metavac® or bivalent Metavac®-RSV v.1 or v.3 viruses, on the basis of active immunizing dose determined for the monovalent Metavac® virus, in comparison with a group of mice mock-immunized with culture medium (mock) or a group of mice immunized via intramuscular route with an adjuvanted split of HMPV WT virus (HMPV split), as surrogate of a vaccination with HMPV protein vaccine.
Twenty-one days after the boost-infection, mice endured a HMPV lethal viral challenge by intranasal instillation of 2×106 TCID50 of the wild-type HMPV rC-85473 (an infectious dose resulting in more than 50% mortality rate for mice).
The weight and the survival of the mice was monitored daily for 14 days (weight average over 8 mice±SEM).
In
Five days after the viral challenge, 3 mice per group underwent euthanasia for a lung tissue harvest for further histopathological analysis and 2 or 4 mice per group underwent euthanasia to measure pulmonary viral loads by RT-qPCR.
In
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Finally, in
These results show the capacity of the bivalent Metavac®-RSV candidates (both Metavac®-RSV v.1 and v.3) to induce in vivo a strong specific neutralizing antibody response against both HMPV A and B viruses, and to fully protect mice challenged with a lethal dose of HMPV wild-type virus.
BALB/c mice were immunized twice, in 21-days interval, by intranasal instillation with 5×105 TCID50 of the bivalent Metavac®-RSV v.1 or v.3 viruses, on the basis of active immunizing dose determined for the monovalent Metavac® virus, in comparison with a group of mice mock-immunized with culture medium (mock) or a group of mice immunized by intranasal instillation with 5×105 PFU of a recombinant RSV WT virus (RSV-mCh).
Twenty-one days after the boost-infection, mice endured a RSV infectious challenge by intranasal instillation of 1×105 PFU of rRSV-Luc virus, a recombinant RSV A WT virus expressing a luminescent luciferase protein in vivo (Rameix-Welti et al., 2014).
The weight of animals was monitored daily for 14 days with no weight loss, as expected from RSV infection in mouse model.
In
Four days after the viral challenge, 4 mice per group underwent euthanasia to measure pulmonary viral loads by RT-qPCR.
In
In
As expected, an induction of RSV-neutralizing antibodies was measured in sera of mice immunized with 2 doses of bivalent Metavac®-RSV v.1 and RSV WT viruses, and higher levels of neutralizing antibodies specific to RSV A and B strains seem to be induced by the vaccination with the bivalent Metavac®-RSV v.3 candidate 21 days after the RSV viral challenge.
These results show the capacity of the bivalent Metavac®-RSV candidates (bivalent Metavac®-RSV v.1 and v.3) to induce in vivo a strong specific neutralizing antibody response against both RSV A and B viruses, and to restrain RSV replication in upper and lower respiratory tract of mice challenged with a RSV virus.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22305240.8 | Mar 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/055221 | 3/1/2023 | WO |
