MX2014011340A - Vaccine against rsv. - Google Patents

Abstract

The invention provides a vaccine against respiratory syncytial virus (RSV), comprising a recombinant human adenovirus of serotype that comprises nucleic acid encoding a RSV F protein or immunologically active part thereof.

Description

VACCINE AGAINST SYNTHETIC RESPIRATORY VIRUS (RSV) The invention relates to the field of medicine. More particularly, the invention relates to vaccines against respiratory syncytial virus (RSV).

BACKGROUND OF THE INVENTION After the discovery of respiratory syncytial virus (RSV) in the 1950s, the virus soon became a recognized pathogen associated with upper and lower respiratory tract infections in humans. Worldwide, an estimated 64 million RSV infections occur each year resulting in 160,000 deaths (WHO Acute Respiratory Infections Update September 2009). The most severe disease occurs particularly in premature infants, in the elderly and in immunocompromised individuals. In children younger than 2 years, RSV is the most common respiratory tract pathogen, accounting for approximately 50% of hospitalizations due to respiratory infections, and the peak of hospitalizations occurs between 2 and 4 months of age. It has been reported that almost all children have been infected with RSV at the age of two. Repeated infection during life is attributed to a non-effective natural immunity. The level of disease burden due to RSV, mortality and morbidity in the Elderly people are the seconds after those caused by non-pandemic influenza A infections.

RSV is a paramyxovirus, which belongs to the subfamily of pneumovirinae. Its genome codes for various proteins, including the membrane proteins that are known as RSV glycoprotein (G) and VSR fusion protein (F) which are the most important antigenic targets for neutralizing antibodies. The proteolytic cleavage of the fusion protein precursor (FO) yields two F1 and F2 polypeptides linked through a disulfide bridge. The antibodies against the part that mediates the fusion of the F1 protein can prevent the uptake of the virus in the cell and therefore have a neutralizing effect. In addition to being a target for neutralizing antibodies, RSV F contains epitopes for cytotoxic T cells (Pemberton et al., 1987, J. Gen.Virol.68: 2177-2182).

Treatment options for RSV infection include a monoclonal antibody against the RSV F protein. The high costs associated with these monoclonal antibodies and the requirement for their administration in a hospital space preclude their use for prophylaxis in the population at risk on a large scale. Therefore there is a need for a vaccine for RSV, which can preferably be used for the pediatric population as well as for the elderly Despite fifty years of research, there is still no certified vaccine against RSV. A major obstacle to the development of the vaccine is the legacy of disease accentuated by vaccine in a clinical trial in the 1960s with an inactivated formalin (IF) vaccine for RSV. Children vaccinated with FI-RSV were not protected against natural infection and infected children experienced a more severe illness than unvaccinated children, including two deaths. This phenomenon is called "accentuated disease".

Since the trial with the IFV vaccine for RSV, different strategies have been tried to generate a vaccine for RSV. Attempts include classical cold-attenuated live strains or temperature-sensitive mutants of RSV, protein (chimeric) subunit vaccines, peptide vaccines, and VSR proteins expressed from recombinant viral vectors. Although some of these vaccines showed promising pre-clinical data, no vaccine has been authorized for human use due to safety concerns or lack of efficacy.

Adenovirus vectors are used for the preparation of vaccines for a variety of diseases, including disease associated with RSV infections. The following paragraphs provide examples of candidate vaccines for adenovirus-based RSV that have been described.

In one strategy, VSR.F has been inserted in the non-essential region E3 of adenoviruses competent for type 4, 5, and 7 replication. Immunization in cotton rats, by intranasal (in) application of 107 pfu, was moderately immunogenic, and protective against VSR challenge in the lower respiratory tract, but was not protective against VSR challenge in the upper respiratory tract (Connors et al., 1992, Vaccine 10: 475-484; Collins, PL, Prince, GA, Camargo, E. , Purcell, RH, Chanock, RM Murphy, BR Evaluation of the protective efficacy of recombinant vaccinia viruses and adenoviruses that express respiratory syncytial virus glycoproteins In: Vaccines 90: Modern Approaches to New Vaccines including prevention of AIDS (Eds. Brown, F. , Chanock, RM, Ginsberg, H. Lerner, RA) Coid Spring Harbor Laboratory, New York, 1990, pages 79-84). The subsequent oral immunization of a chimpanzee was poorly immunogenic (Hsu et al., 1992, J Infect Dis.66: 769-775).

In other studies (Shao et al., 2009, Vaccine 27: 5460-71; US2011 / 0014220), two recombinant adenovirus 5 vectors incompetent for replication were manufactured carrying a nucleic acid encoding the truncated transmembrane version (rAd-F0áTM) or full length (rAd-FO) of the F protein of strain VSR-Bl, and BALB / c mice were given by intranasal route. The animals were sensitized i.n. with 107 pfu and were boosted 28 days later with the same dose via i.n. Although the anti-RSV-Bl antibodies were neutralizing and reacted cross-reactive with the strains RSV-Long and RSV-A2, immunization with these vectors only partially protected against replication by challenge with VSR Bl. The (partial) protection with rAd-FOATM was slightly higher than with rAd-F0.

In another study, it was observed that immunization via i.n. of BALB / c mice with 1011 viral particles with the replication-defective adenovirus FG-Ad (based on Ad5) expressing the natural RSV F (FG-Ad-F) reduced the viral titers in lung by only 1.5 log 10 in comparison with the control group (Fu et al., 2009, Biochem. Biophys. Res. Com un.381: 528-532.

In still other studies, it was observed that the deficient replication adenovector based on recombinant Ad5 applied intranasally expressing the soluble F1 fragment optimized by codons of the F protein of RSV A2 (amino acids 155-524) (108 pfu) could reduce the replication challenge with RSV in the lungs of BALB / c mice compared to control mice, but mice that immunized by intramuscular route (i.m.) did not exhibit any protection for the challenge (Kim et al., 2010, Vaccine 28: 3801-3808).

In other studies, adenovectors based on Ad5 carrying the full-length RSV F (AdV-F) optimized by codons or the soluble form of the RSF F gene (AdV-Fsol) were used to immunize BALB / c mice twice with a dose of 1x1010 OPU (units of optical particles: a dose of 1x1010 OPU corresponds to 2x108 GTU (unit of gene transduction)). These vectors strongly reduced viral loads in the lungs after i.n. immunization, but only partially after subcutaneous (s.c.) or i.m. (Kohlmann et al., 2009, J Virol 83: 12601-12610; US 2010/0111989).

In still other studies, it was observed that the deficient replication adenovector based on recombinant Ad5 applied intramuscularly expressing the sequenced cDNA of the F protein of the strain RSV A2 (1010 particle units) could reduce replication by challenge with RSV alone partially in the lungs of BALB / c mice compared to control mice (Krause et al., 2011, Virology Journal 8: 375-386) Apart from not being totally effective in many cases, vaccines for RSV that are under clinical evaluation for use Pediatric and most vaccines under preclinical evaluation, are intranasal vaccines. The most important advantages of the intranasal strategy are the direct stimulation of the local respiratory tract immunity and the lack of associated potentiation of the disease. In factIn general, the efficacy of for example candidate vaccines against adenovirus-based VSR seems better for intranasal administration compared to intramuscular administration. However, intranasal vaccination also gives rise to safety implications in children under 6 months of age. The most common adverse reactions of intranasal vaccines are watery nose or nasal congestion at all ages. Newborn babies are forced nasal respirators and therefore must breathe through the nose. Therefore, nasal congestion in children a few months of age can interfere with care, and in rare cases can cause serious breathing problems.

Human adenoviruses of more than 50 different serotypes have been identified. Of these, serotype 5 adenoviruses (Ad5) have historically been the most studied as vehicles for genes. The recombinant adenoviral vectors of different serotypes, however, may give different results with respect to the induction of immune responses and protection. For example, WO 2012/021730 discloses that the serotype 7 simian adenoviral vector and the serotype 5 human adenoviral vector encoding protein F provide better protection against RSV than a serotype 28 human adenoviral vector. different immunogenicity for vectors based on human or non-human adenovirus serotypes (Abbink et al., 2007, J Virol 81: 4654-4663, Colloca et al., 2012, Sci Transí Med 4, 115ra2). Abbink et al. conclude that all the rare serotypes of human rAd vectors studied were less potent than the rAd5 vectors in the absence of immunogenicity by anti-Ad5. Furthermore, it has recently been reported that, while rAd5 with a transbole of Ebolavirus glycoprotein (gp) (EBOV) protected 100% non-human primates, rAd35 and rAd26 with the transgene gp of EBOV provided only partial protection and was required a heterologous strategy of sensitization and reinforcement with these vectors to obtain complete protection against challenge with Ebola virus (Geisbert et al., 2011, J Virol 85: 4222-4233). Therefore, a priori it is not possible to predict the efficacy of a recombinant adenoviral vaccine, only on the basis of the data of other adenovirus serotypes.

Moreover, for RSV vaccines, experiments in appropriate disease models such as cotton rat to determine if a candidate vaccine is effective enough to prevent the replication of RSV in the nasal tract and lungs and at the same time it is safe, that is, it does not lead to severe disease. Preferably said candidate vaccines should be highly effective in said models, even before muscular administration.

Therefore, a need remains for efficient vaccines and methods of vaccination against RSV, which do not lead to marked disease. The present invention has the objective of providing said vaccines and vaccination methods against RSV in a safe and effective manner.

SUMMARY OF THE INVENTION Surprisingly, the present inventors found that recombinant serotype 35 (Ad35) adenoviruses comprising a nucleotide sequence coding for RSV protein F are highly effective vaccines against RSV in a well-established model of cotton rat, and that they have better efficacy in comparison with the data previously described for Ad5 that codes for RSV F. It is shown that even a single administration, even by intramuscular route, of Ad35 coding for RSV F is sufficient to provide complete protection against challenge replication with RSV.

The invention provides a respiratory syncytial virus (RSV) vaccine, comprising a human serotype 35 recombinant adenovirus comprising a nucleic acid encoding a VSR F protein or a fragment thereof.

In certain embodiments, the recombinant adenovirus comprises nucleic acid encoding the RSV F protein comprising the amino acid sequence of SEQ ID NO: 1.

In certain embodiments, the nucleic acid encoding the VSR F protein is optimized by codons for expression in human cells.

In certain embodiments, the nucleic acid encoding the VSR F protein comprises the nucleic acid sequence of SEQ ID NO: 2.

In certain embodiments, the recombinant human adenovirus has a deletion in the El region, a deletion in the E3 region, or a deletion in both the El region and the E3 region of the adenoviral genome.

In certain embodiments, the recombinant adenovirus has a genome comprising at its 5 'ends the CTATCTAT sequence.

The invention further provides a method for vaccinating a subject against RSV, wherein the method comprises administering to the subject a vaccine according to the invention.

In certain embodiments, the vaccine is administered intramuscularly.

In certain embodiments, a vaccine according to the invention is administered to the subject more than once.

In certain embodiments, the method of vaccination consists of a single administration of the vaccine to the subject.

In certain embodiments, the method for vaccinating a subject against RSV further comprises administering to the subject a vaccine comprising a recombinant human adenovirus of « serotype 26 comprising nucleic acid encoding a VSR F protein or a fragment thereof.

In certain embodiments, the method of vaccination of a subject against RSV further comprises administering VSR protein F (preferably formulated as a pharmaceutical composition, hence a protein vaccine) to the subject.

The invention also provides a method for reducing infection and / or replication of RSV in, for example, the nasal tract and lungs of a subject, which comprises administering to the subject by intramuscular injection a composition comprising a recombinant human adenovirus of serotype 35. comprising nucleic acid encoding a protein F of VSR or a fragment of it. This will reduce the adverse effects that result from RSV infection in a subject, and will therefore contribute to the protection of the subject against said adverse effects upon administration of the vaccine. In certain embodiments, essentially the adverse effects of RSV infection can be prevented, that is to say reducing those low levels that are not clinically relevant. The recombinant adenovirus may be in the form of a vaccine according to the invention, including the embodiments that were previously described.

The invention also provides an isolated host cell comprising a recombinant human adenovirus of serotype 35 comprising nucleic acid encoding a VSR F protein or a fragment thereof.

The invention further provides a method for making a respiratory syncytial virus (RSV) vaccine, which comprises providing a serotype 35 recombinant human adenovirus comprising nucleic acid encoding a VSR F protein or a fragment thereof, propagating said adenovirus recombinant in a culture of host cells, isolate and purify the recombinant adenovirus, and formulate the recombinant adenovirus in a pharmaceutically acceptable composition. The recombinant human adenovirus of this aspect can also be any of the adenoviruses that are described in the previous embodiments.

The invention also provides an isolated recombinant nucleic acid forming the genome of a serotype 35 recombinant human adenovirus comprising nucleic acid encoding a VSR F protein or a fragment thereof. The adenovirus can also be any of the adenoviruses as described in the previous embodiments.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the cellular immune response against F-peptides that overlap aa 1-252 of F and F-peptides that overlap aa 241-574 of F of mice before immunization with different doses of vectors based on rAd26 (A) and rAd35 (B) carrying the RSV F gene at 2 and 8 weeks after immunization.

Figure 2 shows the response of antibodies against RSV in mice before immunization with different doses of vectors based on rAd26 and rAd35 carrying the F gene of RSV at 2 and 8 weeks after immunization.

Figure 3 shows the results of the proportion of the IgG2a antibody response vs. IgG 1 against RSV in mice before immunization with 1010 pv vector based in rAd26 and rAd35 carrying the RSV F gene at 8 weeks after immunization.

Figure 4 shows the capacity of virus neutralization against Long VSR in mice before immunization with different doses of vectors based on rAd26 (A) and rAd35 (B) carrying the F gene of RSV at 2 and 8 weeks after immunization .

Figure 5 shows the cellular immune response against (A) F peptides that overlap aa 1-252 of F and (B) F peptides that overlap aa 241-574 of F of mice before immunization by sensitization and vector reinforcement based on rAd26 and rAd35 carrying the RSV F gene at 6 and 12 weeks after the primary immunization.

Figure 6 shows the response of antibodies against RSV in mice before immunization by sensitization and reinforcement with vectors based on rAd26 and rAd35 carrying the F gene of RSV at different time poiafter the first immunization.

Figure 7 shows the capacity of virus neutralization against Long VSR in sera of mice before immunization by sensitization and reinforcement with different doses of vectors based on rAd26 and rAd35 carrying the F gene of RSV at different time poiafter the first immunization.

Figure 8 shows the neutralization capacity of virus against RSV B1 in mice before immunization by sensitization and reinforcement with different doses of vectors based on rAd26 and rAd35 carrying the F gene of RSV at different time poiafter the first immunization.

Figure 9 shows the A) lung titers of RSV and B) nasal RSV titres in cotton rats after immunization by sensitization and reinforcement with different doses of vectors based on rAd26 and rAd35 carrying the VSR F gene 5 days later of the challenge.

Figure 10 shows the induction of virus neutralizing titers after immunization by sensitization and reinforcement with different doses of vectors based on rAd26 and rAd35 carrying the F gene of RSV to A) 28 days, and B) 49 days after the first immunization.

Figure 11 shows the histopathological examination of the cotton rat lungs on the day of sacrifice after immunization by sensitization and reinforcement with different doses of vectors based on rAd26 and rAd35 carrying the F gene of RSV.

Figure 12 shows A) the lung RSV titers and B) the nasal RSV titers in cotton rats after single dose immunization with different doses of vectors based on rAd26 and rAd35 that carry the F gene of VSR 5 days after the challenge, administered by different routes.

Figure 13 shows virus-neutralizing titers induced after single-dose immunization with different doses of vectors based on rAd26 and rAd35 carrying the RSV F gene at 28 and 49 days after the first immunization, administered by different routes.

Figure 14 shows the histopathological examination of the cotton rat lungs on the day of sacrifice after single dose immunization (i.m.) with different doses of vectors based on rAd26 and rAd35 carrying the F gene of RSV on the day of sacrifice.

Figure 15 shows the plasmid maps comprising the left end of the Ad35 and Ad26 genome with the sequence coding for RSV F: A. pAdApt35BSU.VSR.F (A2) nat and B. pAdApt26.VSR.F (A2) nat Figure 16 shows A) the lung RSV titers and B) the nasal RSV titers in cotton rats after single dose immunization on day 0 or day 21 with different doses of rAd35-based vectors carrying the gene VSR F The challenge was on day 49 and sacrifice on day 54.

Figure 17 shows the induction of titles virus neutralizers after single dose immunization with different doses of rAd35 carrying the RSV F gene at 49 days after immunization as described for Figure 16.

Figure 18 shows the induction of virus neutralizing titers after single dose immunization with different doses of rAd35 carrying the RSV F gene over time after immunization.

Figure 19 shows the VNA titres 49 days later against VSR Long and VSR Bwash with serum derived from cotton rats immunized with a single dose of 1010 Ad-35VSR F or no transgene (Ad-e). PB: sensitization and reinforcement.

Figure 20 shows lung RSV titres in cotton rats after single dose immunization on day 0 with different doses of rAd35-based vectors carrying the RSV F gene at 5 days after challenge with RSV A2 or VSR B15 / 97.

Figure 21 shows the nasal RSV titres in cotton rats after single-dose immunization on day 0 with different doses of rAd26-based vectors carrying the RSV F gene at 5 days after challenge with RSV A2 or VSR B15 / 97.

Figure 22 shows the titers of VNA in the serum of cotton rats after immunization by single dose in on day O with different doses of rAd35-based vectors carrying the RSV F gene for a period after immunization.

Figure 23 shows the lung RSV titres in cotton rats after single dose immunization on day 0 with different doses of rAd35-based vectors carrying the RSV F gene at 5 days after challenge with a standard dose (105 ) or a high dose (5x105) of RSV A2.

Figure 24 shows the nasal RSV titres in cotton rats after single-dose immunization on day 0 with different doses of rAd35-based vectors carrying the RSV F gene at 5 days after challenge with a standard dose (105 ) or a high dose (5x105) of RSV A2.

Figure 25 shows the induction of virus neutralizing titers after immunization with rAd26 carrying the RSV F gene (Ad26.VSR.F) followed by reinforcement with Ad26.VSR.F or with F protein of RSV with adjuvant (post -F).

Figure 26 shows the induction of IgG2a and IgG1 antibodies, and the proportion thereof, after immunization with Ad26.VSR.F followed by reinforcement with Ad26.VSR.F or by reinforcement with F protein of RSV with adjuvant (post-F).

Figure 27 shows the production of IFN-g by splenocytes after immunization with Ad26.VSR.F followed by reinforcement with Ad26.VSR.F or with F protein of RSV with adjuvant (post-F).

DETAILED DESCRIPTION OF THE INVENTION The term "recombinant", as used herein in relation to adenoviruses, refers to the result of a modification by man, for example, to an alteration in the ends based on an active cloning and or the introduction of a heterologous gene, whereby said adenoviruses are not natural adenoviruses.

In the present, the sequences are described in the 5 'to 3' direction, which is usual in the art.

The term "capsid protein of an adenovirus" refers to a protein that is found in the capsid of an adenovirus and that participates in the determination of the serotype and / or tropism of a particular adenovirus. Adenovirus capsid proteins typically encompass proteins that are related to the fibers, to pentones and / or to hexones. An adenovirus of a serotype determined in accordance with the invention (or an adenovirus that is "based" on said serotype) typically comprises proteins related to the fibers, pentones and / or hexones of the serotype in question, and preferably comprises proteins related to the fibers, the pentons and the hexones of said serotype. These proteins are typically encoded by the genome of recombinant adenoviruses. Recombinant adenoviruses of a given serotype may optionally comprise and / or encode adenovirus proteins of other serotypes. Accordingly, as a non-limiting example, a recombinant adenovirus comprising hexon, penton and Ad35 fiber is considered a recombinant adenovirus based on Ad35.

As used herein, a recombinant adenovirus "is based on" a natural adenovirus at least as far as the sequence is concerned. This result can be obtained on the basis of a molecular cloning, by using the natural genome or parts of it as starting material. It is also possible to use known sequences of natural adenovirus genomes to generate new genomes or parts thereof, through a DNA synthesis method, which can be practiced according to routine methods, by companies related to the area of DNA synthesis and / or molecular cloning (eg, GeneArt, GenScripts, Invitrogen, Eurofins).

A person skilled in the art will understand that several different polynucleotides and nucleic acids can code for the same polypeptide as a result of the degeneracy of the genetic code. It will also be understood that those skilled in the art can, using routine techniques, make substitutions of nucleotides that do not affect the polypeptide sequence that is encoded by the polynucleotides that are described to reflect the codon usage of any particular host organism in which they are going to Express the polypeptides. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNA may include introns.

In a preferred embodiment, the nucleic acid encoding the VSR F protein or a fragment thereof is codon optimized for expression in mammalian cells, such as human cells. Methods for codon optimization are known and have been previously described (for example in WO 96/09378). An example of a specific sequence optimized by VSR F protein codons is described in SEQ ID NO: 2 of EP 2102345 Bl.

In one embodiment, the RSV F protein is from an RSV A2 strain, and has the amino acid sequence of SEQ ID NO: 1. In a particularly preferred embodiment, the nucleic acid encoding the F protein of RSV comprises the nucleic acid sequence of SEQ ID NO: 2. The inventors found that this embodiment results in stable expression and that a vaccine according to this embodiment provides protection against the replication of RSV in the nasal tract and in the lungs even after a single dose that was administered intramuscularly. .

The term "fragment" as used herein refers to a peptide having an amino-terminal and / or carboxyl-terminal and / or internal elimination, but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence of a VSR F protein, e.g., the full length sequence of an RSV F protein. It will be appreciated that to induce an immune response and in general for vaccination purposes, a protein does not need to have its full length or have all of its innate functions, and that the protein fragments are equally useful. In fact, it has been demonstrated that fragments of soluble type F1 or F type VSR F proteins are effective in inducing immune responses such as full length F (Shao et al., 2009, Vaccine 27: 5460-71, Kohlmann et al., 2009, J Virol 83: 12601-12610). The incorporation of F protein fragments corresponding to amino acids 255 to 278 or 412 to 524 in active immunization induces neutralizing antibodies and some protection against challenge with RSV (Sing et al., 2007, Virol.Immunol.20, 261-275; Sing et al., 2007, Vaccine 25, 6211-6223).

A fragment according to the invention is an immunologically active fragment, and typically comprises at least 15 amino acids, or at least 30 amino acids, of the VSR F protein. In certain embodiments, it comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids, of the VSR F protein.

The person skilled in the art will also appreciate that changes can be made to a protein, for example by amino acid substitutions, deletions, additions, etc., for example using routine molecular biology procedures. In general, conservative amino acid substitutions can be applied without loss of function or immunogenicity of a polypeptide. This can easily be verified according to routine procedures well known to a person skilled in the art.

The term "vaccine" refers to an agent or composition that contains an active component that is capable of inducing a therapeutic degree of immunity in a subject against a certain pathogen or disease. In the present invention, the vaccine comprises an effective amount of a recombinant adenovirus that encodes a VSR F protein, or a fragment antigenic thereof, which results in an immune response against the RSV F protein. It provides a method for preventing serious lower respiratory tract disease leading to hospitalization and decreasing the frequency of complications such as pneumonia and bronchiolitis due to RSV infection and replication in a subject. Accordingly, the invention also provides a method for preventing or reducing a serious lower respiratory tract disease, preventing or reducing (e.g., shortening) hospitalization, and / or reducing the frequency and / or severity of pneumonia or bronchiolitis caused by RSV in a subject, comprising administering to the subject by intramuscular injection a composition comprising a recombinant human adenovirus of serotype 35 comprising nucleic acid encoding a VSR F protein or a fragment thereof. The term "vaccine" according to the invention implies that it is a pharmaceutical composition, and therefore typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise other active ingredients. In certain embodiments, it may be a combination vaccine which also comprises other components that induce an immune response, for example against other RSV proteins and / or against other infectious agents.

The vectors of the present invention are recombinant adenoviruses, so it is also possible to call them recombinant adenovirus-based vectors. The preparation of recombinant adenovirus-based vectors is well known in the art.

In certain embodiments, a vector based on an adenovirus according to the invention exhibits a deficiency in the function of at least one essential gene of the El region of the adenovirus genome, for example, of the Ela region and / or of the Elb region, which is essential for the replication of the virus. In certain embodiments, a vector based on an adenovirus according to the invention exhibits a deficiency in at least a portion of the non-essential region E3. In certain embodiments, the vector exhibits a deficiency in the function of at least one essential gene of the El region and in at least a portion of the non-essential region E3. The vector based on an adenovirus may present "multiple deficiencies", that is, it may present a deficiency in the function of one or more essential genes, which may be present in two or more regions of its genome. By way of example, adenovirus-based vectors exhibiting deficiencies in the El region or in the El and E3 regions may also exhibit deficiencies in at least one other essential gene, as is the case of the E4 region and / or the E2 region (e.g., the E2A region and / or the E2B region).

Adenovirus-based vectors, methods for constructing them, and methods for propagating them are well known in the art and are described, for example, in U.S. Pat. UU No. 5559099, 5837511, 5846782, 5851806, 5994106, 5994128, 5965541, 5981225, 6040174, 6020191 and 6113913 and in Thomas Shenk, "Adenoviridae and their Replication", and MS Horwitz, "Adenovirus", chapters 67 and 68, respectively, from Virology, BN Fields et al. , editors, 3rd edition., Raven Press, Ltd., New York (1996), as well as other references cited herein. Typically, the construction of adenovirus-based vectors is based on the use of standard molecular biology protocols, such as those described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd edition, Coid Spring Harbor Press, Coid Spring Harbor, NY (1989); Watson et al., Recombinant DNA, 2nd edition, Scientific American Books (1992); and Ausubel et al., Current Protocols in Molecular Biology, Wilcy Interscience Publishers, NY (1995), as well as other references cited herein.

According to the invention, an adenovirus is a human adenovirus of serotype 35. Vaccines according to the invention that are based on this serotype as well as those based on Ad26 surprisingly seem more potent than those described in the prior art that were based on Ad5, because the latter failed to provide complete protection against replication by challenge with RSV after a simple intramuscular administration (Kim et al., 2010, Vaccine 28: 3801-3808; Kohlmann et al., 2009, J Virol 83: 12601-12610; Krause et al., 2011, Virology Journal 8: 375). The serotype of the invention also generally has a low seroprevalence and / or low pre-existing titres of neutralizing antibodies in the human population. The recombinant adenoviral vectors of this serotype and of Ad26 with different transgenes are evaluated in clinical trials, and also demonstrate an excellent safety profile. The preparation of rAd26 vectors is described, for example, in WO 2007/104792 and Abbink et al., (2007) Virol 81 (9): 4654-63. Examples of sequences of the genomes of the Ad26 vectors can be found in the access GenBank EF 153474 and in SEQ ID NO 1 of WO 2007/104792. The preparation of rAd35 vectors is described, for example, in U.S. Pat. UU No. 7270811, in WO 00/70071 and in Vogels et al., (2003) J. Virol., 77 (15): 8263-71. Examples of sequences of the genomes of Ad35 vectors can be found in the access GenBank AC_000019 and in Figure 6 The recombinant adenoviruses according to the invention may exhibit replication deficiencies or may be capable of replication.

In certain embodiments, the adenoviruses exhibit replication deficiencies, for example, because they contain a deletion in the El region of the genome.

Those versed in the art should be aware that the deletion of the essential regions of the genome of an adenovirus can result in the loss of the functions that are encoded by the regions in question, which can normally operate in trans, preferably in the cell that it is used for production. Therefore, when part or all of the El, E2 and / or E4 regions of the adenoviruses are eliminated, it is imperative that they be present in the cell used for production, for example, that they are integrated into their cell. genome or take the form of the so-called assistant adenoviruses or attendant plasmids. The adenoviruses can also present a deletion in the E3 region, which is dispensable for replication, so it is not necessary to complement a deletion of this type.

For production, any cell may be used (which, in the art and in the present, may also be known as "packaging cell", "host cell" or "cell" "In this context, vectors based on recombinant adenoviruses can be propagated in cells that are appropriate to complement the deficiencies that adenoviruses may present." In the genome of the cells that are used for the production, preferably there will be at least one sequence of the adenovirus El, which will allow the complementation of those recombinant adenoviruses that have a deletion in the El region. To implement the production, any cell that is appropriate to complement can be used. a deletion in the El region, for example, an immortalized human retinal cell comprising the El region, as is the case with the 911 or PER.C6 cells (see U.S. Patent No. 5994128), an amino cell that has been transformed with the El region (see Patent EP 1230354), an A549 cell that has been transformed with the El region (see, for example, WO 98/39411 and U.S. Pat. UU No. 5891690), a GH329: HeLa cell (Gao et al., 2000, Human Gene Therapy, 11: 213-219), 293 and a similar cell. In certain embodiments, the cells that are used for production may be, for example, HEK293 cells, PER.C6 cells, 911 cells, IT293SF cells and the like cells.

For adenoviruses deficient in El that are not subgroup C, such as Ad35 (subgroup B) or Ad26 (subgroup D), it is preferred to exchange the coding sequence of E4-orf6 of these adenoviruses which are not subgroup C by E4-orf6 of a subgroup C adenovirus such as Ad5. In this way, adenoviruses can be propagated in lines of well-known complementary cells where genes encoding the El region of Ad5 adenovirus are expressed, as is the case with 293 cells or PER.C6 cells (see, for example, Havenga et al., 2006, J. Gen. Virol., 87: 2135-2143, and WO 03/104467, which are incorporated herein by reference in their entirety). In certain embodiments, the adenovirus of the vaccine composition is a human adenovirus of serotype 35, with a deletion in the El region in which the nucleic acid encoding the VSR protein F antigen has been cloned, and with an E4 region orf6 of Ad5. In certain embodiments, the adenovirus that can be used is a human adenovirus of serotype 26, with a deletion in the El region in which the nucleic acid encoding the antigen of the VSR protein F has been cloned, and with an E4 region orf6 of Ad5.

In alternative embodiments, it will not be necessary to introduce a heterologous region encoding the ORF 6 of the E4 region (which, for example, comes from an Ad5 adenovirus) into the vector based on an adenovirus. Instead of this the vector that has a deficiency in the region El and that is not related to subgroups C may be propagated in a cell line in which both the El region and an ORF 6 of the E4 region are expressed, for example, in the 293-ORF6 cell line, in which the El region and the ORF 6 of the E4 region of the Ad5 adenovirus are expressed (see, for example, Brough et al., 1996, J. Virol., 70: 6497- 501, where the generation of 293-ORF6 cells is described, Abrahamsen et al., 1997, J. Virol., 71: 8946-51; and Nan et al., 2003, Gene Therapy 10: 326-36, which describes the generation of adenovirus-based vectors that do not belong to subgroup C from which the El region has been deleted from the cell line that is Has mentioned) .

As an alternative, a line of complementary cells in which an El region which is specific to the serotype to be propagated may be used (see, for example, WO 00/70071 and WO 02/40665).

In the case of adenoviruses of subgroup B, as is the case of Ad35, comprising a deletion in the El region, it is preferable that the 3 'end of the open reading frame of E1B 55K be retained in the adenovirus, for example, the 166 base pairs that are just before the open reading frame of pIX, or that you retain a fragment that understands them, which may be a fragment of 243 base pairs that is just before the start codon of pIX (comprising a restriction site for Bsu36I at the 5 'end of the Ad35 genome), for the purpose of that its stability be increased, since the promoter of the pIX gene resides in part in the aforementioned area (see, e.g., Havenga et al., 2006, J. Gen. Virol., 87: 2135-2143, and WO 2004/001032, which are incorporated herein by reference).

The term "heterologous nucleic acid" (also referred to herein as "transgene") applied to an adenovirus according to the invention refers to a nucleic acid that is not naturally present in the adenovirus. It is introduced into the adenovirus, for example, on the basis of conventional molecular biology procedures. In the present invention, the heterologous nucleic acid encodes a VSR F protein or a fragment thereof. By way of example, it is possible to clone it at the location where the El or E3 region of a vector based on an adenovirus has been deleted. A transgene is generally operably linked to sequences that are appropriate for controlling expression. This can be achieved, for example, by placing a nucleic acid encoding one or more transgenes under the control of a promoter.

It is possible to add more regulatory sequences. To express the transgenes, numerous promoters known to those skilled in the art can be used. A non-limiting example of an appropriate promoter for carrying out expression in eukaryotic cells is the CMV promoter (US 5385839), which for example, can be the immediate early promoter of CMV, which may comprise, for example, nucleotides -735 to +95 of the immediate early enhancer / promoter of the corresponding CMV gene. Behind the transgenes, a polyadenylation signal may be present, for example, the polyadenylation signal of bovine growth hormone (US 5122458).

In certain embodiments, the recombinant Ad26 or Ad35 vectors of the invention comprise the nucleotide sequence: CTATCTAT as terminal 5 'nucleotides. These embodiments are advantageous because said vectors show better replication in production processes, resulting in batches of adenovirus with better homogeneity, as compared to vectors having the original 5 'terminal sequences (in general CATCATCA) (also see Patent Applications No. PCT / EP2013 / 054846 and US Pat. No. 13 / 794,318, entitled "Batches of Recoding Adenovirus with Altered Terminal Ends" filed on March 12, 2012 in the name of Crucell Holland BV), which It is incorporated in its entirety as a reference to the present. The invention therefore also provides batches of recombinant adenoviruses that code for the RSV F protein or for a portion thereof, wherein the adenovirus is a human adenovirus serotype 35, and where essentially all (eg, at least 90%) the adenovirus in the lot comprises a genome with terminal nucleotide sequence CTATCTAT.

According to the invention, the RSV F protein can be derived from any RSV strain of natural or recombinant origin, preferably from strains of human RSV, such as A2, Long, or B strains. In other embodiments, the sequence can be be a consensus sequence based on a plurality of amino acid sequences of RSV protein F. In one example of the invention, the RSV strain is the VSR-A2 strain.

According to the invention, the RSV F protein can be the full length RSV F protein, or a fragment thereof. In one embodiment of the invention, the nucleotide sequence encoding the VSR F protein encodes the full-length RSV F protein (F0), such as the amino acid sequence of SEQ ID NO: 1.

In one example of the invention, the nucleotide sequence coding for the RSV F protein has the sequence of nucleotides of SEQ ID NO: 2. Alternatively, the sequence encoding the RSV F protein can be any sequence that is at least about 80%, preferably more than about 90%, more preferably at least about 95%, identical to the nucleotide sequence of SEQ ID NO: 2. In other embodiments, sequences optimized by codons can be used such as for example those provided in SEQ ID NO: 2, 4, 5 or 6 of WO 2012/021730.

In another embodiment of the invention, the nucleotide sequence can alternatively code for a fragment of the RSV protein F. The fragment can be the result of either an amino-terminal or a carboxyl-terminal deletion or both. The extent of the elimination can be determined by a person skilled in the art to, for example, achieve better performance of the recombinant adenovirus. The fragment will be chosen to comprise an immunologically active fragment of the F protein, that is, a part that originates an immune response in a subject. This can be easily determined using in silico, in vitro and / or in vivo methods, all routinely for a person skilled in the art. In one embodiment of the present invention, the fragment is a RSV F protein truncated in the coding region of transmembrane (FOATM, see for example US 20110014220). The F protein fragments can also be the Fl domain or the F2 domain of the F protein. The F fragments can also be fragments containing neutralizing epitopes and T cell epitopes (Sing et al., 2007, Virol. Immunol. 20, 261-275 / Sing et al., 2007, Vaccine 25, 6211-6223).

The term "approximately" for numerical values as used in the present disclosure means a value of ± 10%.

In certain embodiments, the invention provides methods for making a respiratory syncytial virus (RSV) vaccine, which comprises providing a serotype 35 recombinant human adenovirus comprising nucleic acid encoding a VSR F protein or a fragment thereof. , propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and placing the recombinant adenovirus in a pharmaceutically acceptable composition.

Recombinant adenoviruses can be prepared and propagated in host cells, according to well-known methods, including cell culture of host cells that are infected with the adenovirus. The cell culture can be any type of cell culture, including culture of adherent cells, for example cells that are attached to the surface of a culture vessel or to microcarriers, as well as suspension culture.

Most large-scale suspension crops are operated as single or consecutive batches, which are the simplest to operate and adaptable at any scale. At present, continuous processes that are based on the principle of perfusion are becoming more common, and may also be appropriate in the context of the present invention (see, for example, WO 2010/060719 and WO 2011/098592, which are incorporated herein by reference, where appropriate methods are described for obtaining and purifying large quantities of recombinant adenoviruses).

The cells that are used for production are grown to increase the number of cells and viruses and / or the titration of the viruses. The cells are cultured to enable metabolism and or growth and / or division and / or generation of the viruses of interest according to the invention. This can be carried out according to methods which are to be known to those skilled in the art, and includes, without limitation, the supply of nutrients to the cells, for example, through an appropriate culture medium. The appropriate culture media they should be known to those versed in the art, and generally can be obtained in large quantities from commercial suppliers or can be made to measure, according to conventional protocols. The cultivation can be carried out, for example, in plates, in stirred bottles or in bioreactors, by the use of systems in individual, consecutive, continuous batches and similar systems. The appropriate conditions for culturing the cells should be known (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, eds. (1973), and RI Freshncy, Culture of animal cells: A manual of basic technique, fourth edition ( Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

Typically, the adenoviruses will be exposed to the cells that are appropriate to carry out the production, which will be found in a culture, so that they can capture it. Usually, the optimum agitation is between about 50 and 300 rpm, typically about 100-200, for example, it can be about 150. The typical OD is 20-60%, for example, it can be 40%. The optimum pH is between 6.7 and 7.7. The optimum temperature is between 30 ° C and 39 ° C, for example, it can be 34-37 ° C. The optimal MOI is between 5 and 1000, by example, it can be approximately 50-300. Typically, the adenoviruses infect the cells that are used for production spontaneously, and usually the contact between said cells and the rAd particles is sufficient for infection of the cells to take place. In general, a stock solution comprising the adenoviruses is added to the culture to initiate the infection, and subsequently, the adenoviruses are propagated in the cells that are used for production. All these procedures are of usual use for those versed in the technique.

Once the infection with the adenoviruses takes place, the viruses replicate in the cells, whereby their amount can be increased. This process is known herein as the propagation of adenoviruses. Eventually, infection of the cells with the adenoviruses results in their lysis. On the basis of the lytic characteristics of the adenoviruses, two different virus elaboration modalities can be practiced. The first modality is based on the harvesting of the viruses before the lysis of the cells, and includes the use of external factors to effect said lysis. The second mode is based on the harvest of the supernatant comprising the viruses produced after the (almost) complete lysis of the cells (see, for example, US Patent UU 6485958, where the adenovirus harvest is described without lysis of the host cells by the use of an external factor). It is preferable to employ external factors to actively lyse the cells and harvest the adenoviruses.

Those versed in the art have to know various methods to lyse cells actively. They are described, for example, in WO 98/22588, p.28-35. Methods useful in this context include, for example, freeze-thaw cycles, agitation on a solid phase, hypertonic and / or hypotonic lysis, agitation in a liquid phase, sonication, extrusion at elevated pressure, lysis with detergents, combinations of these methods and similar methods. In one embodiment of the invention, the cells are lysed using at least one detergent. The use of a detergent for lysis is advantageous, since it is a simple method, whose scale can be easily modified.

The detergents that can be used and the method of their use have to be known in general to those skilled in the art. By way of example, reference can be made to WO 98/22588, p.29-33. The detergents can be anionic, cationic, zwitterionic or nonionic. The concentration of the detergents can vary, and for example, can be found in the approximate range of 0, 1% -5% (w / w). In one embodiment, the detergent used is Triton X-100.

Nucleases can be used to remove contaminants, which, especially in the context of cells used for production, encompass nucleic acids. Examples of nucleases that are suitable for use in the present invention include Benzonase®, Pulmozyme® or any other DNase or RNase commonly used in the art. In preferred embodiments, the nuclease is Benzonase®, which can rapidly hydrolyze the nucleic acids by cleaving the internal phosphodiester bonds between specific nucleotides, whereby the viscosity of the cell lysate can be reduced. Benzonase® can be obtained from the commercial supplier Merck KGaA (code W214950). The concentration at which the nuclease is used is preferably in the range of 1-100 units / ml. As an alternative to a treatment with a nuclease, or additionally, it is also possible to separate the DNA from the host cells of the adenovirus preparations during the purification of the adenoviruses, on the basis of a selective precipitation, for which precipitation agents can be used. selective agents such as domiphene bromide (see, for example, US 7326555, Goerke et al., 2005, Biotechnology and bioengineering, Vol. 91: 12-21, WO Methods for harvesting the adenoviruses from the cultures comprising the cells that are used for production are described in detail in WO 2005/080556.

In certain embodiments, the harvested adenoviruses are also subjected to a purification. Purification of the adenoviruses can be carried out in several steps, which may include purification, ultrafiltration, diafiltration or separation based on chromatography, as described, for example, in WO 05/080556, which is incorporated herein by reference. The depuration can be based on a filtration step, in which cell debris and other impurities from the cell lysate can be removed. Ultrafiltration is used to concentrate the solution comprising the virus. The diafiltration or the exchange of shock absorbers using an ultrafiltration device is an appropriate method to remove and change salts, sugars and other similar elements. Those versed in the technique must know how to determine the optimal conditions for each step of purification. In WO 98/22588, which is incorporated herein by reference in its entirety, methods for producing and purifying adenovirus-based vectors are described. These methods comprise culture the host cells, infect them with adenovirus, harvest them and remove them, concentrate the crude solution, change the buffer in said lysate, treat it with a nuclease and subject the virus to further purification through chromatography.

Preferably, at least one chromatography step is used in the purification, as described, for example, in WO 98/22588, p.61-70. Numerous processes have been described to carry out the step of further purification of adenoviruses, which usually comprise chromatography procedures. Those versed in the technician have to know these processes and have to be able to make modifications in the chromatography procedures to optimize them. By way of example, it is possible to purify the adenoviruses through various steps based on anion exchange chromatography methods, as described, for example, in WO 2005/080556 and in Konz et al., 2005, Hum Gene Ther 16: 1346-1353. A significant number of different methods have been described for purifying the adenoviruses, which are to be known to those skilled in the art. Other methods for producing and purifying adenoviruses are described, for example, in WO 00/32754, WO 04/020971, US 5837520, US 6261823, WO 2006/108707; Konz et al., 2008, Methods Mol Biol 434: 13-23; Altaras et al., 2005, Adv Biochem Eng Biotechnol 99: 193-260), which are incorporated herein by reference.

In the context of administration in humans, pharmaceutical compositions comprising a rAd and a pharmaceutically acceptable carrier or excipient can be employed in the present invention. Thus, the term "pharmaceutically acceptable" denotes that the vehicle or excipient, in the doses and concentrations that are employed, do not result in undesirable or harmful effects in the subjects to whom they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, editor, Mack Publishing Company (1990), Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard , editors, Taylor and Francis (2000), and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, editor, Pharmaceutical Press (2000)). The purified rAd is preferably formulated and administered in the form of a sterile solution, although it is also possible to use lyophilized preparations. Sterile solutions are made on the basis of filtration under sterile conditions or with methods known in the art.

Subsequently, the solutions are lyophilized or placed in suitable containers for packaging pharmaceutical doses. The pH of these solutions is generally in the range of 3.0 to 9.5, for example, between 5.0 and 7.5. RAd is typically found in a solution comprising a suitable pharmaceutically acceptable buffer, and which may also contain a salt. Optionally, a stabilizing agent, such as albumin, may be present. In certain embodiments, a detergent is added. In certain embodiments, rAd can be formulated into an injectable preparation. Formulations of this type comprise effective amounts of rAd, and are liquid solutions, liquid suspensions or sterile lyophilized versions, and optionally contain stabilizers or excipients. An adenovirus-based vaccine can also be administered intranasally, in the form of an aerosol (see, for example, WO 2009/117134).

By way of example, the adenoviruses can be stored in the same buffer used to store the global adenovirus references (Hoganson et al., Development of a stable vector adenoviral formulation, Bioprocessing, March 2002, pp. 43-48), comprising 20 mM Tris, pH 8, 25 mM NaCl and 2.5% glycerol. The formulation of another suitable buffer that is useful for administering it to humans comprises 20 mM Tris, Mg122 mM, 25 NaCl mM, 10% w / v sucrose and 0.02% w / v polysorbate-80. Obviously, it should be possible to employ other buffers, and various examples of suitable formulations can be found for storing purified (adeno) virus preparations and for carrying out their pharmaceutical administration in, for example, European Patent No. 0853660, in the US Patent UU.6225289 and in the international patent applications WO 99/41416, WO 99/12568, WO 00/29024, WO 01/66137, WO 03/049763, WO 03/078592 and WO 03/061708.

In certain embodiments, a composition comprising adenovirus also comprises one or more adjuvants. It is known in the art that adjuvants are useful to further increase the immune response against the antigenic determinant that is administered. By way of example, WO 2007/110409, which is incorporated herein by reference, describes pharmaceutical compositions comprising adenoviruses and appropriate adjuvants. The terms "adjuvant" and "immunostimulant" are used interchangeably herein, and encompass those substances that are useful for stimulating the immune system. In this context, an adjuvant is employed to increase the immune response that is generated with the adenovirus-based vectors according to the invention. Examples of appropriate adjuvants they include aluminum salts, such as aluminum hydroxide and / or aluminum phosphate, oil-in-water emulsion compositions (oil-in-water compositions), which may be squalene and water emulsions, such as MF59 (see, for example, example, WO 90/14837), saponin-based formulations, such as QS21 and immunostimulatory complexes (ISCOMS; see, for example, US 5057540, WO 90/03184, WO 96/11711, WO 2004/004762 and WO 2005 / 002620), bacterial or microbial derivatives, such as monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), oligonucleotides containing CpG motifs, bacterial toxins that can ribosulate ADP or mutants of these, as is the case of LT enterotoxin of heat-labile E. coli, CT toxin of cholera, and the like. It is also possible to use adjuvants encoded by vectors, for example, by the use of a heterologous nucleic acid encoding a fusion between the oligomerization domain of the protein that can bind C4 (C4bp) and the antigen of interest (eg, Solabomi). et al., 2008, Infect. Im un., 76: 3817-23). In certain embodiments, the compositions according to the invention comprise aluminum as an adjuvant, for example, in the form of aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate or a combination of these, in a concentration between 0.05 and 5 mg, for example, between 0.075 and 1.0 mg per dose.

In other embodiments, the compositions do not comprise adjuvants.

It is also possible according to the invention to administer other active components, in combination with the vaccines according to the invention. Said other active components may comprise, for example, other RSV antigens or vectors comprising nucleic acids encoding these. Said vectors can be non-adenoviral or adenoviral, of which the latter can be of any serotype. An example of other RSV antigens includes the VSR G protein or immunologically active portions thereof. For example, the recombinant adenovector rAd / 3xG deficient replication based on Ad5 applied intranasally, expressing the soluble core domain of glycoprotein G (amino acids 130 to 230) was protective in a murine model (Yu et al., 2008 , J Virol 82: 2350-2357), and although it was not protective when applied intramuscularly, it is clear from these data that the VSR G protein is a suitable antigen to induce protective responses. Other active components can also comprise non-RSV antigens, for example from other pathogens such as viruses, bacteria, parasites, and Similar. The administration of other active components for example can be done by separate administration or by administration of combination products of the vaccines of the invention and the other active components. In certain embodiments, there may be other non-adenoviral antigens (in addition to VSR.F), encoded in the vectors of the invention. In certain embodiments, therefore it may be desired to express more than one protein from an individual adenovirus, and in such cases more coding sequences may be connected eg to form an individual transcript from an individual expression cassette or They can be present in two separate expression cassettes that are cloned in different parts of the adenoviral genome.

The adenovirus compositions can be administered to subjects that can be human subjects. The total dose of the adenoviruses administered to the subjects in each administration may vary, which should be evident to those skilled in the art. In general, the dose is between 1 x 107 viral particles (pv) and 1 x 1012 pv, preferably it is between 1 x 108 pv and 1 x 1011 pv, and for example, it can be between 3 x 108 and 5 x 1010 pv, such as between 109 and 3 x 1010 pv.

The administration of the adenovirus compositions it can be carried out through conventional administration routes. Non-limiting embodiments encompass parenteral administration, for example, by means of an injection, which can be applied intradermally, intramuscularly, among others, or through a subcutaneous route, a transcutaneous route or a route through a mucosa, such as the intranasal, oral and other routes. Intranasal administration has generally been seen as a preferred route for RSV vaccines. The most important advantage of the live intranasal strategy is the direct stimulation of the local respiratory tract immunity and the lack of associated potentiation of the disease. The only vaccines under clinical evaluation for pediatric use in the present are live intranasal vaccines (Collins and Murphy. Vaccines against human respiratory syncytial virus). In: Perspectives in Medical Virology 14: Respiratory Syncytial Virus (Ed. Cane, P.), Elsevier, Amsterdam, The Netherlands, p. 233-277). Intranasal administration is a suitable preferred route according to the present invention as well. However, it is particularly preferred according to the present invention to administer the vaccine intramuscularly, because surprisingly it was found that intramuscular administration of the vaccine according to the invention resulted in protection against RSV replication in the nose and lungs of cotton rats, in contrast to intramuscular vaccines previously reported for RSV based on other adenovirus serotypes. The advantage of intramuscular administration is that it is simple and well established, and that it does not cause safety problems for intranasal application in children under 6 months. In one embodiment, a composition is administered by means of an intramuscular injection, for example, in the deltoid muscle of the arm or in the vastus lateralis muscle of the thigh. Those skilled in the art must know the various possible modalities for administering a composition, for example, a vaccine, for the purpose of inducing an immune response against the one or more antigens it comprises.

A subject as used herein is preferably a mammal, for example a rodent, for example a mouse, a cotton rat, or a non-human primate, or a human. Preferably, the subject is a human subject. The subject can be of any age, for example between about 1 month and 100 years of age, for example between about 2 months and about 80 years of age, for example between about 1 month and about 3 years of age, between about 3 years and approximately 50 years old, between approximately 50 years and approximately 75 years of age, etc.

It is also possible to perform one or more booster administrations with one or more vaccines of the invention based on adenovirus. If a booster vaccination is used, it is typically administered to the same subject between one week and one year after the first administration of the composition (which in these cases is known as the "initial vaccination"), preferably between two weeks and four months after the first administration. In alternative booster regimens, subjects may also be administered various vectors, eg, one or more adenoviruses of different serotypes, or vectors of different types, which may comprise MVA, DNA or proteins, as initial vaccinations. For example it is possible to administer to the subject a recombinant adenoviral vector according to the invention as sensitization, and to do the boost with a composition comprising the VSR F protein.

In certain embodiments, the administration comprises a sensitization and at least one reinforcement administration. In certain embodiments thereof, the administration of sensitization is with a nucleic acid comprising rAd35 which codes for the RSV F protein according to the invention ('rAd35-VSR.F') and the administration of reinforcement is with a nucleic acid comprising rAd26 which codes for the RSV F protein ('rAd26-VSR.F'). In other embodiments thereof, the administration of sensitization is with rAd26-VSR.F and the administration of reinforcement is with rAd35-VSR.F. In other embodiments, both the sensitization and the reinforcement administration are with rAd35.VSR.F. In certain embodiments, the administration of sensitization is with rAd35-VSR.F and the administration of reinforcement is with the F protein of RSV. In all these embodiments, it is possible to provide additional administration of boosters with the same or with other vectors or proteins. Embodiments where the reinforcement with RSV F protein can be particularly beneficial include, for example, subjects of age in risk groups (for example having COPD or asthma) of 50 years or older, or for example in subjects healthy people 60 years of age or older or 65 years or older.

In certain embodiments, the administration comprises a simple administration of a recombinant adenovirus according to the invention, without other administrations (reinforcement). Such embodiments are advantageous in view of the lower complexity and costs of a simple administration regimen compared to a sensitization and booster regimen. The complete protection it is already observed after the simple administration of the recombinant adenoviral vectors of the invention without administration of reinforcement in the cotton rat model in the examples herein.

The invention is described in greater detail in the following examples, which are not intended to limit the invention, but are only provided to facilitate its understanding.

EXAMPLES EXAMPLE 1. Preparation of adenoviral vectors Cloning of the RSV F gene in the El Ad35 region and Ad26: The VSR.F (A2) nat gene, which codes for the native RSV fusion protein (F) of strain A2 (Genbank ACO83301.1), was optimized for expression in humans and synthesized in Geneart. A Kozak sequence (5 'GCCACC 3') directly in front of the ATG start codon was included, and two stop codons (5 'TGA TAA 3') were added at the end of the VSR.F coding sequence (A2 nat. The VSR.F (A2) nat gene was inserted into the plasmid pAdApt35BSU and the plasmid pAdApt26 via the HindIII and Xbal sites. The resulting plasmids, pAdApt35BSU.VSR.F (A2) nat and pAdApt26.VSR.F (A2) nat are shown in Figure 15. The amino acid sequence of the F protein, and the sequence optimized by codons coding for the amino acid sequence, are provided in Table 1 as SEQ. ID. NOs: 1 and 2, respectively.

Cell culture: PER.C6 cells were maintained (Fallaux et al., 1998, Hum Gene Ther 9: 1909-1917) in the middle of Eagle modified by Dulbecco (DMEM) with 10% fetal bovine serum (FBS), supplemented with MgC1210 mM.

Generation of adenoviruses, infections and passage: All adenoviruses were generated in PER.C6 cells by simple homologous recombination and were produced as previously described (for rAd35: Havenga et al., 2006, J. Gen. Virol. 87: 2135-2143; for rAd26: Abbink et al., 2007, J. Virol. 81: 4654-4663). Briefly, PER.C6 cells were transfected with Ad vector plasmids, using Lipofectamine according to the instructions provided by the manufacturer (Life Technologies). For the rescue of the Ad35 vectors carrying the transgene expression cassette VSR.F (A2) nat, the plasmid pAdApt35BSU.VSR.F (A2) nat and the cosmid pWE / Ad35.pIX-rITR.dE3.5orf6 were used. , whereas for the Ad26 vectors carrying the transgene expression cassette VSR.F (A2) nat, the plasmid pAdApt26.VSR.F (A2) nat and the cosmid pWE.Ad26.dE3.5orf6 were used. The cells were harvested one day after the full CPE, they were frozen and thawed, centrifuged for 5 minutes at 3,000 rpm, and stored at 20 ° C. The viruses were then purified by plaque and amplified in PER.C6 grown in an individual well of a 24-well multi-well tissue culture plate. Further amplification was carried out in cultured PER.C6 using a tissue culture flask T25 and a T175 tissue culture flask. From the crude U17 of T175, between 3 and 5 ml were used to inoculate 20 vials of T175 triple-layer tissue culture with 70% confluent layers of PER.C6 cells. The virus was purified using a two step purification method in CsCl. Finally, the virus was stored in aliquots at 85 ° C.

Example 2. Induction of immunity against RSV F using recombinant adenoviruses of serotype 26 and 35 in vivo.

This is an experiment to investigate the ability of the recombinant adenovirus serotype (Ad26) and the recombinant adenovirus serotype 35 (Ad35) to induce immunity against the antigen of VSR F glycoprotein in BALB / c mice.

In this study the animals were distributed in experimental groups of 5 mice. The animals were immunized with a single dose of Ad26 or Ad35 carrying the full-length RSV F gene (Ad26-VSR.F or Ad35-VSR.F) or without transgene (Ad26e or Ad35e). Three serial dilutions were given to the tenth of rAd in the range between 1010 and 108 particles of virus (pv) by intramuscular route. As controls, a group of 3 animals received the empty vector Ad26e and one group received the empty vector Ad35e.

The ELISPOT assay is used to determine the relative number of T cells secreting protein-specific IFNy in the spleen, and is essentially done as described in Radosevic et al. (Clin Vaccine Immunol., 2010; 17 (11): 1687-94.). For the stimulation of the splenocytes in the ELISPOT assay, two peptide groups consisting of 15 amino acid peptides overlapping in 11 amino acids spanning the complete sequence of the RSV F protein (A2) were used. Number of point-forming units (SFU) were calculated for every 106 cells.

For the determination of antibody titers, an ELISA assay was used. For this, ELISA plates (Thermo Scientific) were coated with 25 μg / ml of complete inactivated antigen VSR Long (Virion Serion, catalog # BA113VS). Diluted serum samples were added to the plates, and IgG antibodies against RSV were determined using biotin-labeled anti-mouse IgG (DAKO, catalog # E0413), using streptavidin (SA) detection conjugated with horseradish peroxidase (PO) . The titers were calculated by linear interpolation, using the 1.5x of the DO signal of the virgin serum diluted 50x as a cut. HE determined the titres of specific RSV IgG1 and IgG2a antibodies in mouse serum using anti-mouse IgGl antibody labeled with PO and anti-mouse IgG2a antibody labeled with PO (Southern Biotechnology Associates, catalog # 1070-05 and 1080-05) to quantify the subclasses.

The virus neutralizing activity (VNA) of the antibodies was determined by microneutralization assay, done essentially as described in Johnson et al. (J Infect Dis. 1999 July; 180 (1): 35-40.). VERO cells susceptible to RSV were seeded in 96-well cell culture plates one day before infection. On the day of infection, sera and controls were mixed in serial dilution with 1200 pfu of RSV (Long or Bl) and incubated 1 hour at 37 ° C. Subsequently, the virus / antibody mixtures were transferred to 96-well plates with monolayers of VERO cells. Three days later the monolayers were fixed with 80% acetone cooled on ice and the RSV antigen was determined with an anti-F monoclonal antibody. The neutralizing titre is expressed as the serum dilution (log2) that causes 50% reduction in the OD450 compared to the single virus control wells (IC50).

At week 2 and week 8 after sensitization, animals were sacrificed and cellular and humoral responses were monitored as described previously.

Figure 1 shows that all doses of Ad26-VSR.F (Figure 1A) and Ad35-VSR.F (Figure IB) were effective in inducing a good cellular immune response and that the responses were stable over time. No significant differences were observed with the vector dose in the T cell response either with Ad26-VSR.F or with Ad35-VSR.F.

Figure 2 shows the antibody titers in the same experiment as previously described. Both vectors induced an increase very clearly dependent on time and dose in the ELISA titers (Figure 2). The anti-F titers clearly increased between 2 and 8 weeks, which was significant for the 1010 dose. At 8 weeks there was no difference in the titers between Ad26-VSR.F or Ad35-VSR.F vectors.

The distribution of subclasses (IgGl vs IgG2a) of IgG specific for F was determined to evaluate the balance of the Thl vs. Th2 response. A skewed Th2 / Thl response predisposes animals to develop a vaccine-enhanced RSV disease as seen with formalin-inactivated RSV. As shown in Figure 3, the IgG2a / IgG1 ratio for both Ad26-VSR.F and Ad35-VSR.F is greater than 1. This is a strong indication that the Adenovectors Ad26-VSR.F and Ad35-VSR.F exhibit a more Th1-type response than Th2-type.

Figure 4 shows the virus neutralizing titers (VNA) of the same sera that were used for the antibody titers. Immunization with Ad26-VSR.F and rAd35-VSR.F led to the induction of antibody neutralizing titers. VNA titres increased strongly between two and eight weeks after sensitization in mice receiving 1010 pv. At eight weeks there was no difference in the titers between Ad26-VSR.F and Ad35-VSR.F vectors in mice that received 1010 pv.

From these immunization experiments it is evident that Ad35 and Ad26 vectors carrying the VSR.F transgene induce strong cellular and humoral responses against VSR.F.

Example 3. Immunity against VSR.F after sensitization and heterologous reinforcement using recombinant adenoviral vectors encoding VSR.F.

This study was designed to investigate the ability of sensitization and reinforcement regimens based on adenoviral vectors derived from two different serotypes to induce immunity against RSV.

This study involved BALB / c mice that were distributed in experimental groups of 8 mice. HE immunized the animals by intramuscular injection with 1010 pv carrying the wild type sequence of the VSR.F gene based on / RSV A2 derivative (Ad-VSR.F or Ad35-VSR.F) or without transgene (Ad26e or Ad35e). A group of animals was sensitized during the week with Ad26-VSR.F and reinforced in week 4 with Ad35-VSR.F or Ad35e. Another group of animals was sensitized with Ad35-VSR.F and reinforced at week 4 with Ad26-VSR.F or Ad26e. A control group of mice was sensitized with Ad35e and strengthened at week 4 with Ad26e. At week 6 and week 12 after sensitization, 8 animals were sacrificed at each time point and the cellular and humoral responses were monitored with immunological assays well known to those skilled in the art and as previously described.

Figure 5 shows the cellular response at 6 and 12 weeks after the first immunization. At 6 weeks after sensitization (and 2 weeks after reinforcement), a significant reinforcing effect was measured by both Ad26-VSR.F and Ad35-VSR.F on T-cell responses, and the magnitude of the T cell response was independent of the order of immunization with Ad26-VSR.F or with Ad35-VSR.F in the sensitization and reinforcement. At 12 weeks after sensitization (8 weeks after booster), mice sensitized with Ad26-VSR.F speak maintained high levels of F-specific T cells either in animals only sensitized or in sensitized and reinforced animals, in comparison with animals sensitized with rAd35-VSR.F. Overall, the numbers of lymphocytes specific for F (SFU) were high and stable for at least 12 weeks in all animals immunized either with rAd26-RSR.F or with rAd35-VSR.F (sensitization / or sensitization and reinforcement) .

Figure 6 shows the humoral response at different time points after vaccination by sensitization and reinforcement with the adenoviral vectors. Ad35.VSR.F and Ad26.VSR.F sensitized equally well, and a significant reinforcing effect induced either by Ad26.VSR.F or by rAd35.VSR.F on the B cell responses was shown. Furthermore, the magnitude of the B cell responses by sensitization and heterologous reinforcement was independent of the order of immunization with Ad35.VSR.F and Ad26.VSR.F, and after reinforcement the ELISA titers remained stable for 12 weeks.

Figure 7 shows virus neutralizing antibody titers at different time points after immunization by sensitization and reinforcement. Both Ad35.VSR.F and Ad26.VSR.F vectors sensitized equally well to achieve clear VNA titres, as observed for the ELISA titles. Also, the increase in VNA titers after sensitization and heterologous reinforcement was independent of the order of immunization with Ad35.VSR.F and Ad26.VSR.F. The effect of the reinforcement with either Ad26.VSR.F or Ad35.VSR.F on the VNA titers was significant at both time points and was maximum already at 6 weeks. Groups that were only sensitized with Ad.VSR.F had higher VNA titers at 12 weeks compared to 6 weeks. The F sequence of RSV in the adenoviral vector constructions is derived from the RSV A2 isolate. The neutralizing assay described in this application is based on the VSR Long strain, which belongs to RSV subgroup A, demonstrating that F (A2) induced antibodies are able to cross-neutralize a different VSR strain subtype TO.

Because the RSV F protein is well conserved among the RSV isolates, it was tested whether the sera of animals immunized with Ad-VSR.F vectors were capable of cross-neutralizing a prototypical isolate of strain VSR B, VSR Bl. As shown in Figure 8, sera from immunized mice were also able to cross-neutralize strain Bl. The ability to cross-neutralize RSV Bl was not dependent on the vector used in the single-sensitization groups, or the order of immunization by sensitization and reinforcement with the vectors Ad26.VSR.F and Ad35.VSR.F.

Collectively, these data show that in a regimen of sensitization and reinforcement, consecutive immunizations with Ad26.VSR.F and Ad35.VSR.F induce strong humoral and cellular responses, and that the humoral immune response includes the ability to neutralize isolates from both the VSR A subtypes as B.

Example 4. Induction of protection against RSV infection using recombinant adenoviral vectors in vivo in a cotton rat model. This experiment was carried out to investigate the capacity of the sensitization and reinforcement regimes based on adenoviral vectors derived from two different serotypes to induce protection against replication by challenge with RSV in the cotton rat. Cotton rats (Sigmodon hispidus) are susceptible to upper and lower respiratory tract infection with RSV and were found to be at least 50 times more permissive than mouse strains (Niewiesk et al., 2002, Lab. Anim.36 (4): 357-72). Furthermore, the cotton rat has been the primary model for evaluating the efficacy and safety of candidate vaccines, antivirals and VSR. The preclinical data generated in the cotton rat model advanced the development of two formulations of antibody (RespiGam® and Synagis®) to clinical trials without the need for intermediate studies in non-human primates.

The study included cotton rats in experimental groups of 8 cotton rats each. Animals were immunized by intramuscular injections of 109 virus particles (pv) or 1010 pv of adenoviral vectors carrying the full length gene of RSV F (A2) (Ad26.VSR.F or Ad35.VSR.F) or without transgene (Ad26e or Ad35e). The animals were boosted 28 days later with the same dose of pv, either with the same vector (sensitization and homologous reinforcement) or with another adenoviral serotype (sensitization and heterologous reinforcement); the control groups were therefore immunized with Ad-e vectors, except that only one dose was applied (1010). The control groups consisted of 6 animals. Animals infected intranasally with RSV A2 (104 plaque-forming units (pfu)) were used as a positive control for protection against replication by challenge, since it is known that primary infection with RSV virus protects against replication by secondary challenge ( Prince, Lab Invest 1999, 79: 1385-1392). Furthermore, formalin-inactivated RSV (FI-RSV) was used as a control for histopathological disease potentiated by vaccine. Three weeks after the second immunization (booster), cotton rats were challenged intranasally with 1x105 pfu of purified RSV A2 per plate. As controls, a group of cotton rats was not immunized but challenged with virus, and another control group was not immunized and challenged. The cotton rats were sacrificed 5 days after the injection, a time point at which the RSV challenge virus reaches peak titers (Prince, Lab Invest 1999, 79: 1385-1392), and the RSV titers were determined. in lung and nose by plaque virus titration (Prince et al 1978, Am J Pathology 93,711-791).

Figure 9 shows that high titers of RSV virus were observed in lungs and nose in non-immunized controls as well as in the animals that received adenoviral vectors without transgene, respectively 5.3 +/- 0.13 loglO pfu / gram and 5, 4 +/- 0.35 loglO pfu. Conversely, challenge virus could not be detected in lung and nose tissue of the animals that received immunization by sensitization and reinforcement with Ad26.VSR.F and / or Ad35.VSR.F vectors, regardless of the dose or regimen. .

These data clearly demonstrate that vectors based on both Ad35 and Ad26 give complete protection against replication by challenge with RSV in the cotton rat model. This was surprising, since it was known that adenoviral vectors based on Ad5 that encode for RSV F they were not able to induce complete protection in animal models after intramuscular administration.

In the course of the experiment, blood samples were taken before the immunization (day 0), before the booster immunization (day 28), on the day of the challenge (day 49) and on the day of sacrifice (day 54). The sera were tested in a virus neutralization assay based on plaque assay (VNA) for the induction of neutralizing antibodies specific for systemic RSV as described in Prince (Prince et al., 1978, Am J Pathology 93,711-791). The neutralizing titer is expressed as the dilution of serum (log2) that causes 50% reduction of plaques compared to the control wells of only virus (IC50).

Figure 10 shows that the control animals do not have virus neutralizing antibodies on day 28 and day 49, while high titers of VNA are induced after the animals were sensitized with Ad26.VSR.F or Ad35.VSR vectors .F. A moderate increase in the VNA titer is observed after the booster immunizations. Primary infection with RSV A2 virus resulted in rather moderate titers of VNA that increased gradually over time.

To assess whether the Ad26.VSR.F or Ad35.VSR.F vaccine can exacerbate the disease after a challenge with RSV A2, histopathological analysis of the lungs was carried out 5 days after the injection. The lungs were removed, perfused with formalin, sectioned, and stained with hematoxylin and eosin for histological examination. The histopathological qualification was double-blind, according to the criteria published by Prince (Prince et al., Lab Invest 1999, 79: 1385-1392), and was qualified for the following parameters: peribronchiolitis, perivasculitis, interstitial pneumonitis, and alveolitis. Figure 11 shows the lung pathology score for this experiment. After challenge with RSV, the animals immunized with FI-RSV showed high histopathology in all the histopathological parameters that were examined, compared to challenged animals immunized with simulation, which was expected according to previously published studies (Prince et al., Lab Invest 1999, 79: 1385-1392). Histopathology scores in animals immunized with Ad26.VSR, F and Ad35.VSR.F compared to animals immunized with rAd-e or simulation, were similar, although perivasculitis in animals immunized with rAd-RSV .F seemed slightly inferior. Therefore, Ad26.VSR.F and Ad35.VSR.F vaccines did not result in severe disease, contrary to FI-RSV vaccines.

All vaccination strategies resulted in a complete protection against replication by challenge with RSV, induced strong virus neutralizing antibodies, and no enhanced pathology was observed.

Example 5. Protective efficacy of rAd vectors using different routes of administration after simple immunization This study is to investigate the influence of administration routes on the protective efficacy induced by Ad26 or Ad35 vectors that code for VSR.F. The vaccine was administered either intramuscularly or intranasally.

Cotton rats that had received a simple immunization with 1 × 10 9 or 1 × 10 10 Ad26 or Ad35 virus particles (pv) carrying either the RSV F as a transgene (Ad26.VSR.F or Ad35.VSR.F) or without transgene (Ad26-e or Ad35-e) on day 0, on day 49 with 105 pfu RSV and sacrificed on day 54.

Figure 12 shows the results of the experiments where the lung and nasal challenge viruses were determined. High titers of RSV virus were detected in lungs and noses of rats that were not immunized or immunized with adenoviral vectors without a transgene, respectively 4.9 +/- 0.22 loglO pfu / gram and 5.4 +/- 0.16 loglO pfu. On the contrary, the lungs and noses of the animals that received either Ad35-VSR.F or Ad26-VSR.F were devoid of challenge viruses in replication, regardless of the route of administration and dose.

These data surprisingly demonstrate that each of the vectors based on Ad26 and Ad35 coding for the RSV F protein provides complete protection in challenge experiments in cotton rats, regardless of the route of administration of the vectors. This was unexpected, because none of the published vaccines based on adenovirus for RSV, which were based on other serotypes, had shown complete protection after intramuscular vaccination.

During the experiment, blood samples were taken before immunization (day 0), 4 weeks after immunization (day 28), and on the day of challenge (day 49). The sera were tested in a neutralization test for the induction of specific RSV antibodies (Figure 13). No virus neutralizing antibodies were detected in any cotton rat prior to immunization. All strategies of immunization with adenoviral vector, independently of the route of administration, clearly induced high VNA titers, which remained stable over time. This data surprisingly show that each of the vectors based on Ad26 and Ad35 coding for the RSV F protein provides high titers of virus neutralizing antibodies in cotton rat immunization experiments, regardless of the route of administration of the vectors.

To assess whether a single immunization of Ad26.VSR.F or Ad35.VSR.F vaccine can cause accentuated disease by vaccine after challenge with RSV A2, histopathological analyzes of the lungs were carried out 5 days after injection (Figure 14). Single immunization with rAd26.VSR.F or rAd35.VSR.F resulted in similar immunopathology scores in animals immunized with rAd26.VSR.F or rAd35.VSR.F compared to animals immunized with rAd-e or simulation, as observed in the immunization experiments by sensitization and reinforcement that were previously described. Clearly, no exacerbated disease was observed, in contrast to the animals that were sensitized with IF-RSV. Immunopathology scores of animals immunized with rAd vectors were comparable to animals infected by simulation.

In conclusion, all single-dose vaccination strategies resulted in complete protection against replication by challenge with RSV, induced strong virus-neutralizing antibodies and did not show potentiated pathology.

Example 6. Vectors with variants such as Fragments of RSV or with alternative promoters showed similar immunogenicity The previous examples were carried out with vectors expressing wild type RSV F. Other truncated or modified forms of F have been constructed in rAd35, and provided embodiments of Fragments of RSV in adenoviral vectors. These truncated or modified forms of F include a truncated form of RSV-F where the cytoplasmic domain and the transmembrane region were absent (ie only the ectodomain fragment remained), and a fragment form of RSV-F with truncated cytoplasmic domain and transmembrane region and additional internal elimination in the ectodomain and the addition of a trimerization domain. These vectors did not improve the responses by over rAd35.VSR.F with full-length F protein.

In addition, other rAd35 vectors have been constructed with different alternative promoters that drive wild-type RSV F expression.

The immunogenicity of the modified forms of RSV was compared. F and the promoter variants in the mouse model and compared to Ad35.VSR.F expressing wild-type F.

All Ad35 vectors carrying these F variants or promoter variants showed responses in the same order of magnitude as those of Ad35.VSR.F.

Example 7. Short-term protection against RSV infection after immunization with recombinant adenoviral vectors in vivo in a cotton rat model.

This experiment determines the potential for rapid onset of protection by adenoviral vectors expressing RSV F protein in the cotton rat model. For this purpose, cotton rats were immunized in experimental groups of 8 cotton rats each with an i.m. simple 107, 108 or 109 virus particles (pv) of adenoviral vectors carrying the full length F gene of RSV (A2) (Ad35.VSR.F) or without transgene (Ad26e) on day 0 or day 21. Intranasally infected animals were used with RSV A2 (104 plaque forming units (pfu)) as a positive control for protection against challenge replication, since it is known that primary infection with RSV virus protects against replication by challenge secondary (Prince, Lab Invest 1999, 79: 1385-1392). On day 49, seven or four weeks after immunization, the cotton rats were challenge intranasally with 1x105 pfu of purified RSV A2 per plate. The cotton rats were sacrificed 5 days after the injection, a point in time in which the RSV challenge virus reaches the peak titers (Prince, Lab Invest 1999, 79: 1385-1392), and the RSV titers in the lung and nose were determined by plaque virus titre ( Prince et al.1978, Am J Pathology 93,711-791). Figure 16 shows that high titers of RSV virus were observed in lungs and nose in animals receiving adenoviral vectors without transgene, respectively 4.8 +/- 0.11 loglO pfu / gram and 5.1 +/- 0.32 loglO pfu / gram. In contrast, all animals that received high dose immunization (109 vp) of Ad35.VSR.F vectors were fully protected against RSV titers in the lungs and nose 7 weeks after immunization and almost completely protected 4 weeks after of immunization. Minor doses of Ad35.VSR.F vectors confer complete protection against RSV titers in the lungs and partial protection against nose titers at 4 and 7 weeks after immunization. The blood samples were taken on the day of challenge (day 49). The sera were tested in a neutralization test for the induction of specific antibodies against RSV (Figure 17). Immunization with Ad vectors induced VNA titers dependent on the dose. Figure 18 shows that the control animals do not have virus-neutralizing antibodies during the experiments, while they are induced high VNA titres in animals 28 or 49 days after immunization with between 107 and 109 Ad35.VSR.F vp. No virus neutralizing antibodies were detected in any cotton rat prior to immunization. Immunization with adenoviral vectors induced dose-dependent VNA titers that were greater or comparable to the neutralizing titers generated by i.n. This experiment clearly indicates the rapid onset of protection against virus replication by challenge with the RSV-F expressing Ad35.

Example 8. Protection against RSV infection subgroup A and subgroup B after immunization with recombinant adenoviral vectors in vivo in a cotton rat model.

The strains of RSV can be divided into two subgroups, subgroups A and B. This subtyping is due to differences in the antigenicity of highly variable G glycoprotein. The sequence of the F protein is highly conserved but can also be classified in the same subgroups A and B. Patent application 0200 EO POO described that sera from mice immunized with Ad-VSR.F vectors were also able to neutralize in a cross strain B1 in vitro. Figure 19 clearly shows that the cotton rat serum derived from cotton rats immunized with Ad35.VSR-FA2 shows high VNA titers on day 49 after immunization against VSR Long (subgroup A) and Bwash (subgroup B, ATCC # 1540). In vivo protection against challenge was determined with either subgroup A or B in the cotton rat using low doses of adenoviral vector at 107 and 108 pv. For this purpose, cotton rats were divided into experimental groups of 8 cotton rats each. Animals were immunized on day 0 by intramuscular injections of 107, or 108 virus particles (pv) of adenoviral vectors carrying the full length gene of RSV F (A2) (Ad35.VSR.F) or without transgene (Ad26e ) on day 0. Animals infected intranasally with A2 RSV (104 plaque forming units (pfu)) were used as a positive control for protection against challenge replication. On day 49 i.n. animals with 105 pfu RSV-A2, a strain of subgroup RSV-A or VSR-B 15/97, a VSR-B.

Figure 20 shows that high titers of RSV virus were observed in lungs in animals that received adenoviral vectors without transgene. In contrast, challenge virus could not be detected in lung and nose tissue of the animals that received immunization with Ad35.VSR.F. No differences in protection were observed when challenged with RSV-A2 or RSV-B 15/97. Ad35.VSR.FA2 showed complete protection against replication in lungs by challenge at 107 and 108 vp. Figure 21 shows that high titers of RSV were observed in the nose in animals that received adenoviral vectors without transgene. The Ad35.VSR.FA2 showed partial protection against virus replication in the nose by challenge at 108 vp. No differences in protection were observed when challenged with RSV-A2 or RSV-B 15/97. During this experiment, blood samples were taken on day 28 and day of challenge (day 49). The sera were tested in a neutralization test for the induction of specific antibodies against RSV. Figure 22 shows the virus neutralization titers during the course of the experiment and shows that the control animals do not have neutralizing antibodies against the virus during the experiments. High titers of VNA were induced in animals 28 days after immunization with 108 or 107 of Ad35.VSR.F vp.

Example 9. Protection against a high dose of RSV-A2 challenge after immunization with recombinant adenoviral vectors in vivo in a cotton rat model.

This example determines the protection against a high challenge dose of 5 x 0.05 pfu compared to the standard dose of 1 x 0.05 pfu of RSV-A2. The study included cotton rats in experimental groups of 8 cotton rats each. Animals were immunized by intramuscular injections of low doses of 107 or 108 virus particles (pv) of adenoviral vectors carrying the full length F gene of RSV (A2) (Ad35.VSR.F) or without transgene (Ad26e) in on day 0. Infected animals were used intranasally with RSV A2 (104 plaque-forming units (pfu)) as a positive control for protection against replication by challenge. The cotton rats were sacrificed 5 days after the injection, and the RSV titers in lung and nose were determined by plaque virus titration. Figure 23 shows that a higher challenge dose induces a higher viral load in the lung in the animals that received adenoviral vectors without transgene than with the standard challenge dose. Animals that received immunization with 108 pV Ad35.VSR.F vectors were completely protected in the lung against high and standard RSV challenge titers. Figure 24 shows that the animals that received immunization with 108 or 107 pv. Ad35.VSR.F vectors were partially protected in the nose against high and standard VSR challenge titles.

Example 10. Sensitization and reinforcement with Ad26.VSR.F with recombinant F protein results in a Thl response in a mouse model.

In this example, it was investigated whether the immune response to sensitization with Ad26.VSR.F can be enhanced by reinforcement with recombinant RSV protein F with adjuvant. For this purpose mice were divided into experimental groups of 7 mice each. Animals were immunized on day 0 by intramuscular injections of 1010 virus particles (pv) of adenoviral vectors carrying the full length F gene of RSV (A2) (Ad26.VSR.F) or PBS. On day 28 the animals were reinforced by i.m. either with the same vector in the same dose, or with F protein of RSV with adjuvant (full length; conformation of post fusion: post-F) (in 2 doses: 5 mg and 0.5 pg). Figure 25 clearly shows that serum derived from mice that were immunized with Ad26.VSR-FA2 and boosted with RSV F with adjuvant shows high VNA titers at 12 weeks after immunization against VSR-A Long (subgroup) TO). Figure 26 shows the IgG2a / IgGl ratio in the serum of mice immunized with Ad26.VSR-FA2 and reinforced with F protein of RSV with adjuvant. A high proportion is indicative of balanced Thl responses, while a low proportion indicates a response with Th2 bias. Clearly, animals that were immunized with Ad26.VSR.F, and boosted either with Ad26.VSR.F or with RSV F protein result in a high proportion of IgG2a / IgGl, while that control mice that were immunized with FI-VSR or with RSV F protein (without the context of adenoviral vectors) induce a low ratio. Because a response with Thl bias in a RSV vaccine is strongly desired to avoid severe disease in the face of challenge, the Th2 bias response of a protein immunization can be directed to a Thl response when an Ad26.VSR sensitization is applied. F. Figure 27 shows cell responses in spleens derived from mice that were immunized with Ad26.VSR-FA2 and boosted with RSV F protein with adjuvant. Clearly it can be seen that reinforcement with F protein of RSV with adjuvant will also strongly increase the cellular response. Based on the above experiments described in the examples above, it is expected that Ad26 substitution by Ad35 will generate similar results.

Table 1. sequences SEQ ID NO: 1: VSR fusion protein (Genbank ACO83301.1) amino acid sequence: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSV ITIELSNIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNY TLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAV VSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSV NAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV VQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQ SNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKC TASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVF PSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAV GLLLYCKARSTPVTLSKDQLSGINNIAFSN SEQ ID NO: 2: gene VSR.F (A2) nat optimized by codon coding for the fusion protein of RSV ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGAC CTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCC GTGTCCAAGGGCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATCACCATCG AGCTGAGCAACATCAAAAAGAACAAGTGCAACGGCACCGACGCCAAAATCAAGCTGATCAA GCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACC CCCGCCACCAACAACCGGGCCAGACGGGAGCTGCCCCGGTTCATGAACTACACCCTGAACA ACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTCCT GCTGGGCGTGGGCAGCGCCATTGCTAGCGGAGTGGCTGTGTCTAAGGTGCTGCACCTGGAA GGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGA GCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCA GCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCAGCAACATCGAGACAGTGATCGAG TTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCCGGCG TGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGA CATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAG CAGAGCTACTCCATCATGTCCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGC CCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCAC CAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGAT AATGCCGGCAGCGTGTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGG TGTTCTGCGACACCATGAACAGCCTGACCCTGCCCAGCGAGGTGAACCTGTGCAACGTGGA CATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCC GTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCA ACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGG CGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGC CTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACG AGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAG AAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATC ACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGC TGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAA CAATATCGCCTTCTCCAAC

Claims (16)

NOVELTY OF THE INVENTION Having described the present invention as above, it is considered as a novelty and, therefore, the content of the following is claimed as property: CLAIMS

1. A respiratory syncytial virus (RSV) vaccine, comprising a recombinant human serotype 35 adenovirus comprising nucleic acid encoding a VSR F protein or a fragment thereof.

2. A vaccine according to claim 1, wherein the recombinant adenovirus comprises nucleic acid encoding the RSV protein F comprising the amino acid sequence of SEQ ID NO: 1.

3. A vaccine according to any one of the preceding claims, wherein the nucleic acid encoding the VSR F protein is optimized by codons for expression in human cells.

4. A vaccine according to any one of the preceding claims, wherein the nucleic acid encoding the VSR protein F comprises the sequence of nucleic acid of SEQ ID NO: 2.

5. A vaccine according to any of the preceding claims, wherein the recombinant human adenovirus has a deletion in the El region, a deletion in the E3 region, or a deletion in both the El region and the E3 region of the adenoviral genome.

6. A vaccine according to any of the preceding claims, wherein the recombinant adenovirus has a genome comprising at its 5 'ends the CTATCTAT sequence.

7. A method for vaccinating a subject against RSV, wherein the method comprises administering to the subject a vaccine according to any of the preceding claims.

8. A method according to claim 7, wherein the vaccine is administered intramuscularly.

9. A method according to claim 7 or 8, wherein a vaccine according to any of claims 1 to 6 is administered to the subject more than once.

10. A method according to any of claims 7 to 9, further comprising administering to the subject a vaccine comprising a recombinant human adenovirus serotype 26 comprising nucleic acid encoding a VSR F protein or a fragment of the same

11. A method according to claim 7 or 8, which consists of an individual administration of the vaccine to the subject.

12. A method according to any of claims 7 to 11, further comprising administering RSV F protein to the subject.

13. A method for reducing infection and / or replication of RSV in a subject, comprising administering to the subject by intramuscular injection a composition comprising a human serotype 35 recombinant adenovirus comprising nucleic acid encoding a VSR F protein or fragment Of the same.

14. An isolated host cell comprising a human serotype 35 recombinant adenovirus comprising nucleic acid encoding a VSR F protein or a fragment thereof.

15. A method for making a respiratory syncytial virus (RSV) vaccine, comprising providing a recombinant human serotype 35 adenovirus comprising nucleic acid encoding a VSR F protein or a fragment thereof, propagating said recombinant adenovirus in a culture of host cells, isolate and purify the recombinant adenovirus, and formulate the adenovirus recombinant in a pharmaceutically acceptable composition.

16. An isolated recombinant nucleic acid forming the genome of a serotype 35 recombinant human adenovirus comprising nucleic acid encoding a VSR F protein or a fragment thereof.