EP3893928A1 - Production of viral vaccines on an avian cell line - Google Patents

Abstract

The invention relates to the use of the immortalised cell line ECACC 09070703, filed on 7 July 2009 with the European Collection Of Cell Cultures (ECACC, Salisbury, United Kingdom) under the number 09070703, for the production of a viral vaccine consisting of an attenuated strain derived from a human metapneumovirus.

Description

PRODUCTION OF VIRAL VACCINES ON AN AVIAN CELL LINE

FIELD OF THE INVENTION

The present invention relates to the use of an immortalized avian cell line for the production of a pneumovirus type virus, and of viral vaccines consisting of live attenuated viral strains.

STATE OF THE ART

Pneumoviruses

Pneumoviruses are viruses responsible for acute respiratory tract infections such as bronchiolitis, bronchitis or pneumonia, mainly in the at-risk populations of young children under 5, the elderly and the immunocompromised.

The family Pneumoviridae, whose members were previously included in the family Paramyxoviridae, includes enveloped viruses with negative-polarity single-stranded RNA, including:

human respiratory syncytial virus (hVRS), representative of the orthopneumovirus subfamily, and

human metapneumovirus (hMPV), representative of the metapneumovirus subfamily (according to the Report of the International Committee on Virus Taxonomy (ICTV)).

There is currently no vaccine available on the market, nor specific and effective treatment, against these hMPV and / or hVRS viruses.

The hVRS virus is the most common cause of respiratory infections in young children. Very contagious, this virus mainly infects infants under two years of age.

The hMPV virus is also one of the major causes of pediatric bronchiolitis, responsible for 5 to 15% of hospitalizations attributable to acute lower respiratory tract infections in young children. The average age of children hospitalized as a result of hMPV infection is 6 to 12 months, later than that caused by hVRS, which mainly occurs between 0 and 3 months.

These two viruses are also the etiological agents responsible for 12 to 15% of consultations for upper and lower respiratory tract infections in non-hospitalized children. In terms of treatment, ribavirin, which is not free from adverse effects, or very expensive intravenous immunoglobulins, can be used occasionally for the treatment of severe cases of hMPV infections, as well as hVRS.

Other types of treatment are under development and / or characterization such as fusion inhibitor peptides, glycosaminoglycans sulfate, inhibitory RNAs and certain immunomodulators.

The usual clinical approach, widely favored today, consists mainly of treating the symptoms of the infection, by putting patients on respiratory assistance (administration of oxygen or mechanized ventilation) and by administering bronchodilators, corticosteroids and / or antibiotics for them. prevent and / or treat secondary bacterial infections.

Vaccination strategies developed for the prevention of pneumovirus infections

Regarding vaccination, the main populations targeted by hMPV being infants, young children and the elderly, it is crucial to have effective and safe vaccines, in order to reduce severe respiratory infections which have a dramatic impact. in these age groups.

The development of vaccines against pneumoviruses such as hMPV and / or hVRS therefore represents not only a major health issue, but also a real socio-economic issue with the objective of (i) reducing the significant costs of treatments and hospitalizations associated with these infections, and (ii) reduce the use of antibiotics in the context of secondary bacterial infections, and thus limit the emergence of resistance.

Different vaccine strategies have been developed to date (Mazur et al., 2018).

For example, the company Novavax has developed a “non-living nanoparticle” vaccine candidate. This vaccine is made up of nanoparticles with an F protein from the hVRS virus, genetically modified to increase its immunogenicity. This vaccine candidate is intended for pregnant women, it would be used to generate transient immunization of the unborn child in utero.

GSK is also developing a vaccine for pregnant women to immunize the fetus in utero, based on recombinant antigens derived from the F protein of the hVRS virus.

Another approach is to use non-pathogenic viral vectors, such as adenoviruses, by making them express pneumovirus antigens. A candidate vaccine for newborns, composed of an adenovirus encoding 3 major pneumovirus antigens (proteins F, N and M2.1) is currently being developed by the company GSK.

These vaccine approaches are based on the injection or expression of recombinant proteins in different forms. However, the disadvantage of these approaches is the low immunogenicity inherent in these proteins, which by nature induce little immune response. Adding potentially harmful adjuvants, such as aluminum salts, should therefore be considered in most cases.

Consequently, approaches based on the use of so-called “live attenuated” vaccines, made up of attenuated viral strains, are to be preferred. Indeed, live attenuated vaccines have many advantages:

they can be administered intra-nasally and mimic the natural route of entry of wild viruses, thus inducing an immune response quite similar to that physiologically observed after an hMPV and / or hVRS infection;

it is a strategy that has never been described as being associated with an exaggerated inflammatory reaction (as can be seen after administration of inactivated vaccines);

this vaccination strategy does not require the addition of adjuvants, the live attenuated vaccine being by nature sufficiently immunogenic to generate a satisfactory immune reaction.

These approaches are particularly interesting for the vaccination of children from 6 months.

Difficulties in preparing live attenuated viral vaccines on an industrial scale

These viral vaccines are produced by a step of viral replication on virus host cells, cultured in vitro. The choice of these cells is essential: they must be both:

(i) host cells of said virus, that is to say permissive with respect to the infection by the virus and the replication of said virus, and

(ii) “industrialized” cells, that is to say in accordance with the regulations in force for the production of vaccines on an industrial scale.

To date, there is no registered industrial cell line which has the capacity for permissiveness and production of live attenuated vaccine candidates against pneumovirus infections. Laboratory cell lines, in their “adherent” form in culture, such as:

the MDCK cell line, a canine cell line,

the Vero cell line, an African green monkey kidney cell line, commonly used in cell culture to test various viruses,

the PERC6 cell line, a cell line of human origin, and

the LLC-MK2 cell line, a cell line derived from rhesus monkey kidney cells, available from ATCC under the number CCL-7, and commonly used for tests of infection by various viruses,

have been used for the experimental development of certain vaccine candidates (see Tables 1 to 3); however, these adherent cell lines did not have the characteristics necessary to be used on an industrial scale.

For the production of vaccines having a replication of the viral type, “industrialized” cell lines, that is to say stable (“robust”), nonadherent, which can be cultured in the absence of serum (for example), and conform regulatory requirements for the production of viral vaccines for use in humans or animals have been established.

Two types of cell lines are notably conventionally used:

The EB66® cell line developed by VALNEVA is a line derived from duck embryonic cells; it has already enabled the development of several viral vaccines.

The AGE1.CR® cell lines distributed by ProBioGen come from several cell types, from gallinaceae or humans. In particular, the AGE1.CR.plX® line is derived from musk duck cells (Jordan et al., 2009). This very stable line allows the production, on an industrial scale, of vectors from alphavirus and paramyxovirus genomes, as well as for the growth of poxviruses.

However, to date, the production of hVRS and hMPV viruses has not been possible on these established cell lines, well known to those skilled in the art.

The present invention relates to the identification of an industrialized immortalized duck cell line allowing the replication of viral vectors derived from hMPV and / or hVRS viruses, in particular the replication of live attenuated viral vaccines derived from a specific viral strain of hMPV . STATEMENT OF THE INVENTION

Currently, there are no vaccines to prevent infections with pneumoviruses, which are responsible for acute respiratory tract infections. This is partly due to the fact that the reproduction of these viruses in vitro is difficult. In particular, these viruses have not been described as being able to multiply on known and well-established industrializable cell lines, such as the EB66® and AGE1.CR® lines.

The present invention relates to the identification of an "industrializable" cell line which allows the replication of live attenuated viral vaccines intended to prevent infections by hMPV and / or hVRS.

More specifically, the present invention relates to the use of the immortalized cell line ECACC 09070703, deposited with the European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom) under the number 09070703 on July 7, 2009, for the production of a viral vaccine consisting of an attenuated strain derived from a human metapneumovirus.

Said viral vaccine may in particular be an attenuated strain derived from a human metapneumovirus comprising the genomic sequence represented by the sequence SEQ ID NO. 1.

According to a particular implementation, said viral vaccine is an attenuated viral strain which has been genetically modified by the introduction of at least one exogenous gene, in particular a gene coding for an antigen derived from the hVRS virus, such as the fusion protein F by example.

The present invention also relates to a process for the production of a viral vaccine constituted by an attenuated strain derived from a human metapneumovirus, comprising the following steps:

a) Infection of cells in culture of the line deposited with the ECACC under the access number 09070703, with an attenuated viral strain derived from a human metapneumovirus;

b) Culture of said infected cells in step (a) for a period of between 2 and 14 days, in a suitable medium;

c) Harvesting of the viral vaccine consisting of infectious viral particles of said attenuated viral strain produced during step (b).

The present invention also relates to a viral vaccine as obtained by the method described above, as well as a pharmaceutical composition comprising said viral vaccine, and at least one pharmaceutically acceptable vehicle. According to another aspect, the present invention relates to said viral vaccine or to said pharmaceutical composition, for their use as a medicament.

More specifically, the present invention relates to said viral vaccine or to said pharmaceutical composition, for their use in the prevention or treatment of viral infections, in particular infections by pneumovirus, and more particularly by human metapneumovirus and / or human respiratory syncytial virus.

The present invention also relates to a kit for implementing the process for producing a viral vaccine, comprising at least:

The immortalized cell line ECACC 09070703; and

- An attenuated viral strain derived from a human metapneumovirus comprising the genomic sequence represented by the sequence SEQ ID NO. 1, and in particular an attenuated viral strain comprising one of the genomic sequences represented in SEQ ID NO. 2 or NO.3.

DESCRIPTION OF THE FIGURES

Figure 1. Replicative capacity of wild viral strain C-85473 in DuckCelt®-T17 cells, in comparison with the replicative capacities of wild strains CAN98-75, CAN97-82 and CAN99-81. The kinetics are stopped ( ^* ) when more than 50% of the cells are dead.

[Fig. 1 a] Monitoring of the quantity of cells according to the days post-viral infection

[Fig. 1b] Monitoring of the viral titer (TCID50 / ml) according to the days post-viral infection

Figure 2. Replicative capacities of the recombinant wild-type viruses C-85473 WT (GFP) and attenuated ASH-C-85473 (GFP) and AG-C-85473 (GFP) in DuckCelt®-T17 cells.

[Fig. 2a] Cell growth after infection. Mock cells were not infected.

[Fig. 2b] Infectivity of the recombinant viruses C-85473 WT (GFP), ASH-C-85473 (GFP) and AG- C-85473 (GFP). The percentage of infected cells is evaluated by flow cytometry (detection of the expression of GFP expressed by these recombinant viruses) during the 14 days of viral kinetics.

[Fig. 2c] Viral replication of the recombinant viruses C-85473 WT (GFP), ASH- C-

85473 (GFP) and AG-C-85473 (GFP). The viral production is measured by titration of the number of infectious particles per ml of culture medium (expressed in TCID ₅₀ / ml) from samples taken every 2 to 3 days during the 14 days of kinetics. Figure 3. Replicative capacities in LLC-MK2 cells and in a 3D model of human respiratory epithelium reconstituted and cultured at the air-liquid interface (MucilAir ™) of the recombinant viruses ASH-C-85473 (GFP) and AG-C-85473 (GFP) produced in DuckCelt®-T17 cells.

[Fig. 3a] A cell mat of LLC-MK2 cells (left) and healthy reconstituted human respiratory epithelia MucilAir ™ (right) were infected with the recombinant hMPV virus ASH-C-85473 at MOI (multiplicity of infection) 0.01 and 0.1, respectively. The photos were taken after 3, 5, 7, 12 and 17 days post-infection for the respiratory epithelium.

[Fig. 3b] A cell mat of LLC-MK2 cells (left) and healthy reconstituted human respiratory epithelia MucilAir ™ (right) were infected with the recombinant hMPV virus AG-C-85473 at MOI (multiplicity of infection). 0.1 and 0.65, respectively. The photos were taken after 3, 5, 7, 12 and 17 days post-infection for the respiratory epithelium.

[Fig. 3c] The viral secretion at the apical pole of the infected epithelia was evaluated by RT-qPCR (number of copies of the viral gene N) from washings on the apical surface carried out on days 5, 7, 12 and 17 post-infection. The viral replication of the attenuated recombinant viruses ASH-C-85473 and AG-C-85473 was compared with that of a wild-type recombinant virus C-85473 produced in DuckCelt®-T17 cells. The detection threshold by RT-qPCR is 10 copies of viral N gene.

[Fig. 3d] Viral replication within infected epithelia was evaluated by RT-qPCR (number of copies of viral gene N) from lysates of epithelium to 17 ^th day post-infection. The viral replication of the attenuated recombinant viruses ASH-C-85473 and AG-C-85473 was compared with that of a wild recombinant virus WT C-85473 produced in DuckCelt®-T17 cells. The detection threshold by

RT-qPCR is 10 ¹ copies of viral N gene.

Figure 4. The recombinant ASH-C-85473 (GFP) and AG-C-85473 (GFP) viruses produced on DuckCelt®-T17 cells retain their attenuation character in vivo.

[Fig. 4] BALB / c mice from 4 to 6 weeks of age were infected intranasally with ⁵ × 10 ⁵ TCID ₅₀ of the vaccine candidates ASH-C-85473 or AG-C-

85473 produced on DuckCelt®-T17 cells, or culture medium (mock) as a control. Daily monitoring of the weight and mortality of the infected mice was carried out for 9 days after infection and compared with the uninfected mice. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of the immortalized cell line ECACC 09070703, deposited on July 7, 2009 with the European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom) under the number 09070703, for the production of a viral vaccine consisting of an attenuated viral strain derived from a human metapneumovirus.

ECACC cell line N ° 09070703

Thus, the present invention relates to a new use of the DuckCelt®-T17 cell line, deposited with the European Collection of Cell Cultures (ECACC) under the access number 09070703, and previously described in the literature, in particular in applications WO 2007/077256, WO 2009/004016 and WO 2012/001075.

These requests relate to the preparation of immortalized avian cell lines, and to their use for the reproduction of viruses.

The term “immortalized cell line” designates cells capable of growing in culture in vitro during at least 35 subcultures (dilution of the cells in a new culture medium), without losing their functional characteristics.

Briefly, the cell lines described in WO 2012/001075, deposited with the ECACC under the numbers 09070701, 09070702 and 09070703 originate from duck embryonic cells (cairina moschata), and have been immortalized by introduction of the following nucleotide sequences:

- the nucleotide sequence of the E1A region originating from the genome of an adenovirus, coding for the 12S and 13S RNAs; and

- the gene coding for duck telomerase reverse transcriptase (dTERT).

The main destination of these cell lines is their use for the replication of viruses, such as poxviruses, adenoviruses, retroviruses, herpes viruses and influenza viruses.

Recently, Petiot and his collaborators (Petiot et al., 2018) highlighted the fact that the avian cell line DuckCelt®-T17, deposited with the ECACC under the number 09070703, could be advantageously used for the production of particles infectious influenza virus of different origins (human, avian, swine).

Attenuated viral strains of human metapneumovirus Significant research is underway to produce a viral vaccine to prevent and / or treat infections with human metapneumorvirus and / or respiratory syncytial virus.

In particular, attenuated viral strains derived from human metapneumovirus virus have been prepared and offered as vaccines.

Genetic analysis of clinical strains of hMPV made it possible to define two major groups (genotypes A and B) and four “minor” subgroups (A1, A2, B1 and B2), based mainly on the variability of the glycoprotein sequence. attachment (G) and fusion (F) surface. It was then shown that these groups could still be subdivided into sublines such as A2a, A2b and A2c (Huck et al., 2006; Nidaira et al., 2012).

Among the viral strains widely studied, there may be mentioned the strain NL 00-1, belonging to the genotype A1; CAN 97-83 strain belonging to genotype A2; and the CAN 98-75 strain, belonging to genotype B2.

In the present application, the terms "a virus" and "a viral strain" are used interchangeably to designate a particular viral strain, as identified previously.

For the purposes of the invention, the term "derived strain" means a recombinant viral strain obtained by the introduction of genetic modifications into the genome of a viral strain called "original strain". The original strain is advantageously a wild strain, for example a clinical isolate.

The virulence of a viral strain corresponds to the degree of rapidity of multiplication of a virus in a given organism, therefore its speed of invasion. By "attenuating virulence", we mean reducing the speed at which a virus invades an organism.

This attenuation may take the form of a reduction in the replication capacities of the viral strain, a reduction in its capacity for infecting target cells, or even a reduction in the pathology induced by viral infection of the virus. 'organization.

Viral strains are considered to be attenuated in vitro when they have a reduced replicative capacity compared to the wild virus (WT), and / or when these viral strains cause the formation of infectious foci, in particular of syncytia (adjacent cells fusing more to viral infection), more restricted. In vivo, attenuated viral strains replicate at a lower maximum titer and / or induce a less severe pathology (in terms of weight loss or inflammatory profile or histopathological damage) than the wild viral strain.

Thus, by “attenuated viral strain” is meant, within the meaning of the invention, a recombinant virus whose virulence is reduced compared to that of the original viral strain, that is to say less than that of the original viral strain.

To measure the virulence of a viral strain, tests in vitro, ex vivo or in vivo can be carried out, such as for example tests of replicative capacity in vitro (measured by viral titration TCID50 / ml or quantification of viral genome by quantitative PCR ), monitoring by microscopic observation of the evolution of cytopathic effects in vitro and ex vivo (in 3D model of human respiratory epithelium reconstituted and cultured at the air-liquid interface for example), or monitoring clinical signs of pathology and measurement of pulmonary viral titers in an in vivo infection model.

Tables 1, 2 and 3 below list the different approaches under development to obtain live attenuated vaccines from wild-type hMPV strains.

Table 1. List of candidate live attenuated vaccines based on a strain of hMPV virus presenting one or more mutations, developed or under development

The symbol V indicates that the cells used are susceptible / permissive to viral infection by said viral strain. The following abbreviations are used for in vivo study models: M for Mouse; H for hamster; CR for Cotton Rat; CM for Cynomolgus Macaque; AGM for African Green Monkey; Ch for Chimpanzee; Rh for Rhesus Monkey; and SCID for Severe Combinée! ImmunoDeficiency.

The abbreviations "aa" denote amino acids, "aa172" denotes the amino acid at position 172 in the protein sequence.

The point mutations are represented according to the nomenclature known to the skilled person.

Table 2. List of live attenuated vaccine candidates developed or in development, comprising a strain of hMPV virus having a complete deletion of at least one gene

All the attenuated viral strains developed and described in this table 2 are derived from the wild strain CAN97-83 of the subgroup A2.

The following abbreviations are used:

D: total deletion of the gene. 0 = no attenuation or replicative advantage. Table 3. List of candidate live attenuated vaccines developed or in development, based on a strain of chimeric hMPV virus

The following abbreviations are used:

TM = transmembrane domain; PIV = Parainfluenza virus; SeV = Sendai virus.

Other attenuated strains derived from hMPV have been described in the applications or patents summarized below.

The international application WO 2005/014626 relates to several strains of hMPV virus, designated by the following names: CAN 97-83, CAN 98-75 and HMPV 00-1. This application describes a recombinant hMPV virus strain, designated CAN 97-83, genetically modified to reduce its virulence. The proposed modifications relate in particular to the total deletion of the genes coding for the G and / or SH proteins.

US Patent 8,841,433 further describes other isolated hMPV strains and their use for the preparation of vaccines. In patent application FR1856801, attenuated strains derived from the clinical strain C-85473 of human metapneumovirus, comprising the genomic sequence represented by the sequence SEQ ID NO. 1, have been described and proposed as vaccine candidates. These attenuated strains comprise one or more genetic modifications of said sequence SEQ ID NO.1, in particular the inactivation of the gene coding for the SH protein and / or of the gene coding for the G protein of said strain of metapneumovirus.

All these attenuated viral strains derived from human metapneumovirus are candidate vaccines which are capable of being developed industrially to produce, on a large scale, vaccines intended to be administered to numerous patients, for a preventive and / or therapeutic aim.

However, to achieve this objective, it is necessary to identify a cellular support which will allow the identified attenuated viral strains to be replicated on an industrial scale.

Tests carried out on industrial cell lines, such as the lines

EB66® and AGE1.CR.plX®, did not allow sufficient replication of different viral strains derived from human metapneumovirus.

The present invention aims to meet this need, by having identified an immortalized cell line, having functional characteristics suitable for production of viruses on an industrial scale, for the replication of such attenuated strains derived from human metapneumovirus.

According to an implementation of the invention, said attenuated strain has been genetically modified by inactivation of the gene coding for the SH protein and / or of the gene coding for the G protein of metapneumovirus.

Genetic modifications designate, within the meaning of the invention, all modifications of an original nucleotide sequence such as the deletion of one or more nucleotides, the addition of one or more nucleotides, and the replacement of one or more nucleotides. These modifications include in particular all the modifications making it possible to shift the genetic reading frame, or to introduce a stop codon in the middle of a coding sequence.

Among the genetic modifications intended to attenuate the virulence of a viral strain, we know in particular the genetic modifications making it possible to inactivate or even to suppress one or more genes coding for proteins not essential for the growth of the virus in culture. In the sense of the invention, the inactivation of a gene designates the fact that this gene is modified so that the gene product is no longer expressed, or expressed in a non-active form, or expressed in an amount low that the activity of this protein is nonexistent. This inactivation of a gene can be carried out by any technique well known to those skilled in the art. In particular, the inactivation of a gene can be obtained by the introduction of a point mutation in the gene, by the partial or total deletion of the coding sequences of the gene, or by modification of the promoter of the gene. These various genetic modifications will be carried out according to any of the molecular biology techniques well known to those skilled in the art.

For example, attenuated viral strains of human metapneumoviruses have been obtained by deletion of the genes coding for the accessory proteins SH, G and / or M2-2 (see Table 2).

SH protein is a type II membrane protein, whose functions are not yet fully characterized. In the case of the hVRS virus, the deletion of the gene coding for the SH protein generates a recombinant virus capable of reproducing in vitro, and the virulence of which is attenuated in the upper respiratory tract of mice, but not in the lower part of said tract ( Bukreyev et al., 1997). In the case of the hMPV virus, the functions of the SH protein are still being evaluated.

Protein G is also a type II membrane protein, its C-terminal end being outside the cell. This protein is not essential for the assembly of viral constituents, and for replication in vitro. For the hVRS virus, the deletion of the gene encoding this protein has been shown to attenuate the virulence of the strain during infection of the respiratory tract of mice (Teng et al., 2001). For the hMPV virus, the functions of the G protein are still being evaluated.

According to an implementation of the invention, the attenuated viral strain is characterized in that the genetic modifications include the inactivation of the two genes coding for the G protein and the SH protein.

According to another particular implementation, the inactivation of the two genes corresponds to the complete deletion of one or the other or of the two genes coding for the G and SH proteins.

According to an implementation of the invention, said attenuated strain has been genetically modified by the introduction of at least one exogenous gene.

This exogenous gene could in particular be a gene coding for a viral antigen.

For the purposes of the invention, the term “viral antigen” means a protein element or of another nature, expressed by a virus, than the immunological system of an individual. recognizes as foreign and which provokes an immune response in said individual, in particular the production of specific antibodies.

The viral antigens may in particular be selected from the antigens expressed by at least one influenza virus, or by at least one virus of the pneumovirus family, such as the hVRS virus, or by at least one virus of the Paramyxoviridae family, such than the parainfluenza virus.

More particularly, said viral antigen can be chosen from all or part of the F protein of the hVRS virus, and all or part of the hemagglutinin of the influenza or parainfluenza viruses. According to another implementation, said viral antigen is the F protein of the hVRS virus, in its stabilized pre-fusion conformation as described in the article (Krarup A et al. 2015).

Such an attenuated viral strain further comprising an exogenous viral antigen will, when administered to a patient, generate a multiple immune response, both against the expressed exogenous viral antigen and against the hMPV virus. Such a strain making it possible to obtain a combined immune response against several viruses, following a single administration, is very advantageous.

HMPV virus strain C-85473

According to a preferred implementation of the invention, said attenuated strain is derived from a human metapneumovirus comprising the genomic sequence represented by the sequence SEQ ID NO. 1.

Preferably, said attenuated strain is derived from a human metapneumovirus viral strain, named C-85473, which was isolated from a patient sample in Canada. This strain belongs to the A1 subgroup of metapneumoviruses.

The complete genomic sequence of this viral strain C-85473, comprising 13394 nucleotides, was disclosed for the first time in the patent application

FR1856801. It is represented here in the list of sequences under the reference SEQ ID NO. 1.

The C-85473 strain is characterized by large fusogenic capacities, allowing it to penetrate into target cells at a high frequency and / or a high degree of infection (Dubois et al., 2017). The large fusogenic capacity of this strain makes it possible to generate syncytia, i.e. giant multinucleated cells, particularly large, made up of a very large number of cell nuclei.

According to a preferred implementation of the invention, the attenuated viral strain is derived from this strain C-85473 comprising the genomic sequence represented by the sequence SEQ ID NO. 1. The genetic modifications introduced into this strain C-85473 to obtain a so-called “derivative of” strain are intended to attenuate the virulence of said original strain, and not to modify the identity of its genome.

In particular, these genetic modifications only concern genes coding for proteins which are not essential for the growth of the virus, in other words “accessory proteins”, such as the SH and G proteins.

Advantageously, in this strain genetically modified in order to become “attenuated”, the peptide sequence of the F protein of the original rC-85473 strain is not modified, and therefore has the same peptide sequence as the original strain.

Most preferably, the attenuated viral strain is chosen from:

(i) A viral strain derived from the C-85473 strain, genetically modified by inactivation of the gene coding for the SH protein;

(ii) A viral strain derived from the C-85473 strain, genetically modified by inactivation of the gene coding for the G protein; and

(iii) A viral strain derived from the C-85473 strain, genetically modified by inactivation of the gene coding for the SH protein and of the gene coding for the protein G.

A viral strain illustrating the implementation (i) is in particular the strain used in the examples of the present application, comprising the nucleotide sequence as shown in SEQ ID NO. 2.

A viral strain illustrating implementation (ii) is in particular the strain used in the examples of the present application, comprising the nucleotide sequence as shown in SEQ ID NO. 3.

According to an implementation of the invention, the nucleotide sequence of the attenuated viral strain C-85473 can be, moreover, genetically modified by the introduction of at least one exogenous gene.

Thus, the attenuated viral strain C-85473 has a genomic sequence which comprises at least one exogenous gene. This exogenous gene could in particular be a gene coding for a viral antigen.

Said viral antigen may in particular be selected from the antigens expressed by at least one influenza virus, or by at least one virus of the family of Pneumoviridae, such as the hRSV virus, or by at least one virus of the Paramyxoviridae family, such as the parainfluenza virus.

More particularly, said viral antigen may be chosen from all or part of the protein F of the hRSV virus, and all or part of the hemagglutinin of the influenza or parainfluenza viruses.

According to another implementation, said viral antigen is the F protein of the hVRS virus, preferably in its stabilized pre-fusion conformation as described in the article (Krarup A et al., 2015).

Other characteristics of these attenuated strains derived from the strain C-85473 comprising the genomic sequence represented by the sequence SEQ ID NO. 1 are presented in patent application FR1856801 and international application PCT / FR2019 / 051759.

Origin of attenuated viral strains

The attenuated viral strains used in the implementation of the present invention can come from various origins.

The attenuated viral strain may have been isolated from a patient suffering from a viral infection by pneumovirus, in particular by an hMPV or an hVRS. In fact, certain infectious viral strains can exhibit a spontaneously attenuated character.

The attenuated viral strain may also have been genetically modified, from an unattenuated viral strain.

According to a first implementation, the attenuated viral strain can be obtained by reproduction of said virus on cells in culture. Frozen samples of the infectious viral particles thus produced may in particular be supplied by academic laboratories or hospitals.

According to a second implementation, the attenuated viral strain can be obtained from DNA sequences using reverse genetic technology, described in particular in the articles (Biacchesi et al., 2004) and (Aerts et al., 2015 ).

The principle of this technology, which allows the production of recombinant hMPV viruses, is based for example on the use of a hamster kidney cell line (BHK-21) modified to constitutively express the RNA polymerase of bacteriophage T7 (BHK cells -T7 or BSR-T7 / 5). The genomic elements are divided into five plasmid elements: a plasmid coding for the antigenome of the hMPV virus and 4 “satellite” plasmids, coding for the viral proteins of the transcription machinery (L, P, N and M2-1).

After co-transfection of the hMPV antigenomic plasmid and the four "satellite" plasmids in these cells, a RNA strand corresponding to the viral genomic strand (negative RNA strand) is transcribed by T7polymerase from its promoter.

The four proteins involved in the viral transcription of hMPV are in fact expressed by the transfected host cells to constitute an active RNA-dependent RNA polymerase (RdRP) complex. This functional viral polymerase thus transcribes genomic RNA into viral mRNA and then replicates it into new viral genomic RNA molecules, via transcription of template strands.

The translation and assembly of viral proteins with genomic RNA thus allows budding of infectious hMPV particles from the cytoplasmic membrane of transfected BHK-T7 cells. Then, the amplification of the recombinant viruses is allowed thanks to the addition in co-culture of LLC-MK2 cells (ATCC CCL-7), permissive to infection.

Thus, from the sequences described in the present application, and from these appropriate host cells, a person skilled in the art is able to create a functional recombinant enveloped virus comprising one of these sequences.

Method for producing a viral vaccine

According to another aspect, the present invention relates to a method for producing a viral vaccine as defined above, comprising the following steps:

a) Infection of cells in culture of the ECACC 09070703 line with an attenuated viral strain derived from a human metapneumovirus;

b) Culture of said cells infected in step (a) for a period of between 2 and 14 days in a suitable medium;

c) Harvesting of the viral vaccine consisting of infectious viral particles of said attenuated viral strain produced during step (b).

The attenuated viral strain used in step (a) will have been obtained in particular by one of the technologies described above.

It is understood that this process may include additional steps, optional and not indicated here. Furthermore, according to a particular implementation, this process could consist exactly of the three successive steps (a), (b) and (c) mentioned above.

The infection step (a) will be carried out under appropriate conditions, such as for example under the following conditions:

the infection medium may be the OptiPRO ™ SFM culture medium (Gibco ™) suitable for the DuckCelt®-T17 cell line and supplemented with 4mM L-Glutamine, 0.1% to 0.5% of Pluronic®, and trypsin from 0.1 pg / ml to 3pg / ml of final volume. Trypsin is supplemented in the middle during infection but also every 2 to 3 days for the duration of viral production;

- the cell density of the cells will be between 0.5 × 10 ⁶ and 5 × 10 ⁶ cells / ml; and

infection with the attenuated viral strain will be carried out at a multiplicity of infection (MOI) of between 1 and 0.0001.

Step (b) of culturing the infected cells will be carried out under conditions suitable for normal growth of the cells, well known to those skilled in the art. In particular :

the bioproduction equipment for cells in suspension may be of the TubeSpin® type or a flask of 50 ml to 200 ml on a Kühner type shaking tray incubator, or a 500 ml to 2 liter bioreactor of the miniBioReactor Applikon BioTechnology type, or of the UniVessel type SU Sartorius Stedim Biotech; and the temperature of the culture medium will be between 33 and 37 ° C; the pH between 7 and 7.4; agitation between 100 and 200rpm and the oxygen content between 40 and 60%.

The cell culture stage can last from 2 to 14 days, depending on cell growth and the replicative capacities of the virus. The culture step may in particular be carried out for a period of 3 to 12 days, or from 4 to 10 days, or again from 5 to 9 days, or from 6 to 8 days.

Stage (c) of harvesting the infectious viral particles will be carried out by any technique well known to those skilled in the art, such as clarification of the production culture medium, followed by purification, concentration and quantification stages of the viral particles .

According to another aspect, the present invention relates to a viral vaccine capable of being obtained by the method described above. Said viral vaccine consists of infectious viral particles collected in step (c) of the method described. According to another implementation, the present invention relates to a viral vaccine obtained by the method described above.

Pharmaceutical composition

According to another aspect, the present invention relates to a pharmaceutical composition comprising said viral vaccine, and at least one pharmaceutically acceptable vehicle.

For the purposes of the invention, the term “pharmaceutically acceptable vehicle” means vehicles or excipients, that is to say “inactive” compounds, having no therapeutic properties. These vehicles or excipients can be administered to an individual or an animal without risk of significant deleterious effects or unhealthy undesirable effects.

Other active compounds, usually used in pharmaceutical compositions, may be present in said composition: for example, adjuvants and / or excipients.

It is understood that the pharmaceutical composition according to the invention comprises at least one effective amount of the viral vaccine.

By "effective amount" is meant within the meaning of the invention an amount of attenuated viral strain sufficient to trigger an immune reaction in the organism to which it is administered.

The present pharmaceutical composition can also be designated as being a vaccine composition.

The pharmaceutical compositions according to the present invention are especially suitable for oral, sublingual, or inhalation administration.

According to a particular implementation, the pharmaceutical composition according to the invention is suitable for administration by inhalation, that is to say by the nasal and / or buccal routes.

Inhalation refers to absorption through the respiratory tract. It is in particular a method of absorption of compounds for therapeutic purposes, of certain substances in the form of gases, micro-droplets or powder in suspension.

There are two types of administration by inhalation:

administration by insufflation when the compositions are in the form of powders, and administration by nebulization when the compositions are in the form of aerosols (suspensions) or in the form of solutions, for example aqueous solutions, pressurized. The use of a nebulizer or a sprayer will then be recommended for administering the pharmaceutical composition.

The pharmaceutical form of the pharmaceutical composition considered here is therefore advantageously chosen from: a powder, an aqueous suspension of droplets or a solution under pressure.

According to a preferred implementation, the pharmaceutical composition according to the invention is suitable for administration by the nasal route, in particular by inhalation.

Such a composition can be used as a preventive vaccine, that is to say intended to stimulate a specific immune response before infection of an organism by a pathogenic virus.

Such a composition can also be used as a therapeutic vaccine, that is to say intended to stimulate a specific immune response concomitantly with the infection of an organism by said pathogenic virus.

Therapeutic use

The present invention also relates to the viral vaccine as described above, or to the pharmaceutical composition as described, for their use as a medicament, in other words for their therapeutic use.

Advantageously, this viral vaccine or this pharmaceutical composition will be used in therapy for the treatment and / or prevention of viral infections.

"Treatment of viral infections" means fighting a virus infection in an organism. The goal is to obtain a decrease in the rate of viral infection (infectious titer) in the body, and preferably to obtain a complete eradication of the virus from the body. The term "treatment" also refers to the action of alleviating the symptoms associated with viral infection (respiratory syndrome, kidney failure, fever, etc.).

The expression “prevention of viral infections” designates preventing, or at least reducing the risk of an infection in an organism. Thanks to this preventive action, the cells of said organism become less permissive to viral infection, and are therefore more resistant to infection by said virus. In addition, the organism will advantageously have developed specific immune cells, allowing a specific fight against the aforementioned virus, thus limiting its entry into the cells of the body.

More specifically, the invention relates to the viral vaccine or the pharmaceutical composition as described above, for their use in the prevention of viral infections, in particular infections by pneumovirus, and in particular by human metapneumovirus and / or human respiratory syncytial virus.

According to a first implementation, said viral vaccine or said pharmaceutical composition is used in the prevention of infections by pneumoviruses.

According to a second implementation, said viral vaccine or said pharmaceutical composition is used in the prevention of infections with a human metapneumovirus.

According to a third implementation, said viral vaccine or said pharmaceutical composition is used in the prevention of infections by an orthopneumovirus, in particular by the human respiratory syncytial virus.

According to another aspect, the invention relates to the viral vaccine or the pharmaceutical composition as described above, for their use in the treatment of viral infections, in particular infections by pneumovirus, and more particularly by human metapneumovirus and / or respiratory virus human syncytial.

Indeed, said viral vaccine or pharmaceutical composition comprising it may be used, in certain cases and under certain conditions, in a therapeutic approach, in individuals already infected with one of these viruses, in particular in adult individuals.

The present invention also relates to a method for preventing a viral infection in humans, in particular an infection with pneumoviruses, more particularly with a human metapneumovirus and / or with a human respiratory syncytial virus, comprising administration to individuals susceptible to be infected with such a virus with an effective amount of a viral vaccine as described above, or a pharmaceutical composition comprising it.

Said vaccine or said composition for their use in the prevention and / or treatment of viral infections are intended for all types of individuals, both newborns and elderly adults.

According to a preferred implementation of the invention, said vaccine or said composition for their use in the prevention and / or treatment of viral infections are intended for pediatric use, that is to say are intended to be administered to a pediatric population. Within the meaning of the invention, a pediatric population designates a population of individuals made up of individuals aged less than 18 years, and more specifically young children aged less than 5 years, and infants.

In fact, viruses of the Pneumoviridae family mainly infect these individuals, who tend to exhibit so-called "naive" immunity, and therefore less strong, than older individuals.

Finally, the present application also relates to a kit for implementing the process for preparing a viral vaccine, comprising:

The immortalized cell line ECACC 09070703; and

- An attenuated viral strain derived from a human metapneumovirus comprising the genomic sequence represented by the sequence SEQ ID NO. 1.

Said attenuated viral strain may in particular have been genetically modified, in particular by inactivation of the gene coding for the SH protein and / or of the gene coding for the G protein of said metapneumovirus strain.

In addition, said attenuated viral strain may have a genomic sequence which comprises at least one exogenous gene. This exogenous gene may in particular be a gene coding for a viral antigen originating from another virus, such as for example all or part of the protein F of the hRSV virus, and / or all or part of the hemagglutinin of the influenza or parainfluenza viruses .

According to a preferred aspect, said viral strain will in particular be one of the strains presented in the examples of the present application:

i) The viral strain comprising the nucleotide sequence as represented in SEQ ID NO. 2, optionally further comprising at least one exogenous gene; or

ii) The viral strain comprising the nucleotide sequence as represented in SEQ ID NO. 3, optionally further comprising at least one exogenous gene.

This kit may also include other elements, such as for example culture medium suitable for the growth of the cell line, and / or a manual specifying the ideal conditions for the preparation of the live attenuated viral vaccine. EXAMPLES

Example 1. Use of the DuckCelt®-T17 Cell Line for the Replication of Wild Viral Strains of Subgroup A1 (C-85473 and CAN99-81), of Subgroup B1 (CAN97-82) and of Subgroup B2 (CAN98-75)

DuckCelt®-T17 cells are cultured in OptiPro + L-glutamine 4mM final medium in TubeSpin 50ml in Kuhner shaker at a speed of 175 rpm, at 37 ° C with 5% C02 and 85% hygrometry, and diluted to 1 x 10 ⁶ cells / ml in 10 ml of medium. They were infected at a multiplicity of infection (MOI) of 0.01 by wild viral strains (non-GFP) of the A1 subgroup (C-85473 and CAN99-81), of the B1 subgroup (CAN97-82 ) and of subgroup B2 (CAN98-75) in the presence of trypsin (T6763 Sigma) at

0.5pg / ml.

Cells are counted in trypan blue every 2 to 3 days to assess cell growth and cell mortality during viral infection. The results are presented in Figure 1a.

Viral production is measured by titration of the number of infectious particles per ml in the culture medium (expressed in TCID50 / ml) from samples taken every 2 to 3 days up to 14 days post-infection. The results are presented in Figure 1b.

The kinetics of viral replication are stopped when cell death reaches more than 50%, that is to say 8 days post-infection for the viral strain C-85473 and 14 days post infection for the viral strains CAN99-81, CAN97-82 and CAN98 -75.

The results show that only the C-85473 virus amplifies upon infection of DuckCelt®-T17 cells with an increase in viral titer of more than 2 Iog10 in 7 days. The CAN98-75 virus demonstrates a low viral production from 4 to 9 days post-infection while the CAN99-81 and CAN97-82 viruses are detectable at levels below the initial inoculum or below the detection thresholds up to 14 days post infection.

In conclusion, the viral strain C-85473 has a replication capacity on DuckCelt®-T17 cells very significantly greater than that of other hMPV viral strains. In particular, the cell death rate observed from 8 days post-infection indicates that the infection capacities of this strain on this cell line are significant.

Example 2. Use of the DuckCelt®-T17 cell line for the replication of the wild strain C-85473 WT, which is recombinant because it expresses with the Green Fluorescent Protein (GFP), and of the recombinant viral strains ASH-C-85473 (GFP) and AG- C- 85473 (GFP). The viral strains used in this example have the following genomic sequences:

The sequence of C-85473 WT - GFP is shown in SEQ ID NO. 4;

The sequence of ASH-C-85473 - GFP is shown in SEQ ID NO. 5;

- The sequence of AG-C-85473 - GFP is represented in SEQ ID NO. 6.

DuckCelt®-T17 cells are maintained in culture in OptiPro + L-glutamine 4mM final medium in 50ml TubeSpin in Kuhner shaker at a speed of 175 rpm, at 37 ° C with 5% CO2 and 85% humidity.

Before infection, the cells are diluted to 1 × 10 ⁶ cells / ml in 10 ml of culture medium and then are infected with recombinant wild hMPV viruses (C-85473 WT), or deleted from the genomic sequence coding for the SH protein ( ASH-C-85473) or deleted from the genomic sequence coding for protein G (AG-C-85473), at a multiplicity of infection (MOI) of 0.01 in the presence of trypsin (T6763 Sigma) at 0.5pg / ml. The "Mock" cells are cells which have not been infected and constitute the negative control of the experiment.

Cells are counted in trypan blue every 2 to 3 days after infection to assess cell growth during infection. The results obtained are presented in Figure 2a.

The kinetics of viral replication are stopped when cell death reaches more than 50%, that is to say after 14 days on average.

The percentage of infected cells is evaluated by flow cytometry (detection of the expression of GFP expressed by the recombinant viruses) during the 14 days of viral kinetics. The results obtained are presented in Figure 2b.

Viral production is measured by titration of the number of infectious particles per ml of culture medium (expressed in TCID50 / ml) from samples taken every 2 to 3 days during the 14 days of kinetics. The results obtained are presented in FIG. 2C.

The results obtained represent four independent experiments.

These results show that, under these culture and viral infection conditions, the DuckCelt®-T17 (i) cells reach their maximum concentration at 7 days after infection; (ii) are infected with the wild viral strain and the modified viral strains ASH-C-85473 and AG-C-85473 more than 80%, between 9 and 11 days post-infection.

The peak of viral production for the three viral strains is 11 days post infection. In conclusion, the results indicate that the DuckCelt®-T17 line is "permissive", that it can be infected with the recombinant viruses C-85473 and in particular live attenuated viruses ASH-C-85473 and AG-C-85473, and allow the production of viral particles.

Example 3. Characterization of the viral particles obtained according to the culture method of Example 2

This example relates to the measurement of the replicative capacities of the recombinant viruses ASH-C-85473 and AG-C-85473 produced on DuckCelt®-T17 cells, in comparison with a recombinant virus C-85473 WT:

(i) in monkey kidney epithelial cells LLC-MK2 (ATCC-CCL7) and

(ii) in a 3D model of reconstituted human pulmonary epithelium (MucilAir ™,

Epithelix) and grown at the air-liquid interface.

A cell mat of LLC-MK2 cells and healthy reconstituted human respiratory epithelia MucilAir ™ were infected with:

- the recombinant hMPV virus ASH-C-85473 with MOI (multiplicity of infection) of

0.01 and 0.1, respectively;

AG-C-85473 recombinant hMPV virus with MOIs (multiplicity of infection) of 0.01 and 0.65, respectively.

The photos of the infected cells taken at 3, 5, 7, 12 and 17 days post-infection are presented in Figures 3a (ASH-C-85473) and 3b (AG-C-85473).

From 5 days of infection, viral replication within infected epithelia and viral secretion at their apical pole was evaluated by RT-qPCR (number of copies of viral gene N) from epithelium lysates and apical surface washes, respectively.

The results are presented in Figures 3c (surface washes) and 3D (epithelium lysates).

The results obtained indicate that the attenuated live viruses ASH-C-85473 and AG-C-85473 produced on DuckCelt®-T17 cells are functional and retain their capacity to infect LLC-MK2 cells and in particular the 3D model of reconstituted human pulmonary epithelium (MucilAir ™, Epithelix).

It should be noted that the attenuated strains ASH-C-85473 and AG-C-85473 replicate little, and in a less sustained manner over time, in the model of human epithelium compared to a wild C-85473 virus, as shown in Figure 3d; while their replication rate on DuckCelt®-T17 cells (and incidentally LLC-MK2) is very satisfactory.

These two characteristics make them excellent vaccine candidates, these attenuated viral strains being able to be produced easily in vitro but presenting only a very moderate risk when introduced into a living organism, in view of the results obtained in this physiologically human infection model. close to the conditions of an organization.

Example 4. The recombinant viruses ASH-C-85473 and AG-C-85473 produced on DuckCelt®-T17 cells retain their attenuated character in vivo.

BALB / c mice aged 4 to 6 weeks were infected intranasally with:

5x10 ⁵ TCID50 of vaccine candidates ASH-C-85473 or AG-C-85473 produced on DuckCelt®-T17 cells and concentrated by ultracentrifugation, or

Optimem culture medium (mock) as a control (10 mice per group).

Daily monitoring of the weight and mortality of the mice was carried out for 9 days after infection. The results are presented in FIG. 4. The results obtained indicate that the mice infected with the live attenuated viruses ASH-C-85473 or AG-C- 85473 produced on DuckCelt®-T17 cells, show no sign of pathology or mortality, thus demonstrating the attenuation character of these viruses produced on DuckCelt®-T17 cells.

Table 4. Summary of the sequences presented in the sequence listing

PATENT REFERENCES

WO 2007/077256

WO 2009/004016

WO 2012/001075

WO 2005/014626

US 8,841,433

FR1856801

SCIENTIFIC LITERATURE

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Claims

1. Use of the immortalized cell line ECACC 09070703, deposited on July 7, 2009 with the European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom) under the number 09070703, for the production of a viral vaccine consisting of a strain attenuated viral derived from a human metapneumovirus.

2. Use according to claim 1, characterized in that said attenuated strain has been genetically modified by inactivation of the gene coding for the SH protein and / or of the gene coding for the G protein of metapneumovirus.

3. Use according to claim 1 or 2, characterized in that said attenuated strain has been genetically modified by the introduction of at least one exogenous gene.

4. Use according to one of claims 1 to 3, characterized in that said attenuated strain is derived from a human metapneumovirus comprising the genomic sequence represented by the sequence SEQ ID NO. 1.

5. Method for producing a viral vaccine as defined in claims 1 to 4, comprising the following steps:

a) Infection of cells in culture of the ECACC 09070703 line with an attenuated viral strain derived from a human metapneumovirus;

b) Culture of said cells infected in step (a) for a period of between 2 and 14 days in a suitable medium;

c) Harvesting of the viral vaccine consisting of infectious viral particles of said attenuated viral strain produced during step (b).

6. Viral vaccine obtained by the method according to claim 5.

7. Pharmaceutical composition comprising the viral vaccine according to claim 6, and at least one pharmaceutically acceptable vehicle.

8. Pharmaceutical composition according to claim 7, suitable for administration by the nasal route.

9. Vaccine according to claim 6 or composition according to one of claims 7 or 8, for its therapeutic use.

10. Vaccine according to claim 6 or composition according to one of claims 7 or 8, for its use in the prevention of viral infections, in particular infections by pneumovirus, and in particular by human metapneumovirus and / or human respiratory syncytial virus.

1 1. Vaccine or composition for their use according to one of claims 9 or 10, characterized in that he / she is intended (e) for pediatric use.

12. Kit for implementing the method according to claim 5, comprising:

- The immortalized ECACC 09070703 cell line; and

- An attenuated viral strain derived from a human metapneumovirus comprising the genomic sequence represented by the sequence SEQ ID NO. 1.