VACCINES AGAINST RESPIRATORY SYNCYCIAL VIRUS (RSV), ITS METHOD OF PRODUCTION, AND ISOLATED RECOMBINA

专利摘要:
respiratory syncytial virus (rsv) vaccine, methods for vaccinating a subject against rsv, for reducing infection and/or replication of rsv, of a subject, and for producing a vaccine against respiratory syncytial virus (rsv), host cell isolated, and isolated recombinant nucleic acid. the invention provides a vaccine against respiratory syncytial virus (rsv), comprising a recombinant human adenovirus of serotype 26 which comprises nucleic acid encoding an rsv f protein or an immunologically active part thereof.
公开号:BR112014023196B1
申请号:R112014023196-6
申请日:2013-03-21
公开日:2021-09-08
发明作者:Katarina Radosevic；Jerôme H. H. V. Custers；Jort Vellinga；Myra N. Widjojoatmodjo
申请人:Janssen Vaccines & Prevention B.V.；
IPC主号:

专利说明:

[0001] The invention relates to the area of medicine. More particularly, the invention relates to a vaccine against RSV. BACKGROUND OF THE INVENTION
[0002] After the discovery of respiratory syncytial virus (RSV) in the 1950s, the virus immediately became a recognized pathogen associated with lower and upper 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 serious disease occurs particularly in premature babies, the elderly and subjects with immune problems. In children under 2 years of age, RSV is the most common respiratory tract pathogen, accounting for approximately 50% of hospitalizations for respiratory infections, and the peak of hospitalizations occurs at 2-4 months of age. It has been reported that at two years of age almost all children were infected with RSV. Repeated lifelong infection is attributed to ineffective natural immunity. The level of disease burden, mortality and morbidity from RSV in the elderly are second to those caused by non-pandemic influenza A infections.
[0003] RSV is a paramyxovirus, belonging to the pneumovirinae subfamily. Its genome encodes several proteins, including membrane proteins known as RSV glycoprotein (G) and RSV fusion protein (F) that are the main antigenic targets for neutralizing antibodies. Proteolytic cleavage of the fusion protein precursor (F0) produces two F1 and F2 polypeptides linked by disulfide bonds. Antibodies against the part that mediates the fusion of the F1 protein can prevent the virus from being absorbed into the cell and thus have a neutralizing effect. In addition to being a target for neutralizing antibodies, RSV F contains cytotoxic T cell epitopes (Pemberton et al, 1987, J. Gen.Virol. 68: 21772182).
Treatment options for RSV infection include a monoclonal antibody against RSV F protein. The high costs associated with these monoclonal antibodies and the requirement for administration in a hospital environment prevent their large-scale use in the prophylaxis of the population at risk. Thus, there is a need for an RSV vaccine, which preferably can be used in the pediatric population as well as the elderly.
[0005] Despite 50 years of research, there is still no licensed vaccine against RSV. A major obstacle to vaccine development is the legacy of the disease worsening with vaccine in a clinical trial in the 1960s with a formalin-inactivated (FI) RSV vaccine. Children vaccinated with RSV - FI were not protected against natural infection and infected children suffered from more severe illness than unvaccinated children, including two deaths. This phenomenon is known as "aggravated illness".
[0006] Since the RSV FI vaccine trial, several approaches have been followed to generate an RSV vaccine. Attempts include classical cold passage attenuated in vivo or temperature sensitive mutant RSV strains, protein subunit (chimeric) vaccines, peptide vaccines and RSV proteins expressed from recombinant viral vectors. Although some of these vaccines have shown promising preclinical data, no vaccine has been licensed 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 adenovirus-based RSV vaccine candidates that have been described.
[0008] In one approach, RSV.F has been inserted into the non-essential E3 region of replication competent adenovirus types 4, 5, and 7. Immunization of cotton rats by intranasal (in) application of 107 pfu, it was moderately immunogenic and protective of the lower respiratory tract against the threat of RSV, but not protective against the threat of the upper respiratory tract against RSV (Connors et al, 1992, Vaccine 10: 475-484; Collins, PL, Prince, GA, Camargo, E., Purcell, RH, Chanock, RM and Murphy, BR Evaluation of the protective effect of recombinant vaccine viruses and adenoviruses expressing respiratory syncytial virus glycoproteins. In: Vaccines 90: Modern Approaches to New Vaccines including prevention of AIDS ( Eds. Brown, F., Chanock, RM, Ginsberg, H. and Lerner, RA) Cold Spring Harbor Laboratory, New York, 1990, pg 79-84). Subsequent oral immunization of a chimpanzee was poorly immunogenic (Hsu et al, 1992, J Infect Dis. 66:769-775).
[0009] In other studies (Shao et al, 2009, Vaccine 27: 5460 - 71; US2011/0014220), two 5-vector recombinant replication-incompetent adenoviruses carrying the nucleic acid encoding the truncated transmembrane (rAd-F0ΔTM) were designed or the full-length version (rAd-F0) of the F protein of strain RSV-B1 and were administered intranasally to BALB/c mice. Animals were initially vaccinated i. n. with 107 pfu and boosted 28 days later with the same dose i. n. Although anti-RSV-B1 antibodies neutralized and cross-reacted with RSV-Long and RSV-A2 strains, immunization with these vectors only partially protected against replication of the RSV B1 threat. The (partial) protection with rAd-F0ΔTM was slightly better than with rAd-F0.
[00010] In another study, it was observed that BALB/c mice immunized i. n. with 1011 virus particles with replication-deficient FG-Ad adenoviruses (AdS-based) expressing wild-type RSV F (FG-Ad-F) reduced lung viral titers only by 1.5 log 10 compared with the control group ( Fu et al, 2009, Biochem. Biophys. Res. Commun. 381: 528532 .
[00011] Still in other studies, it was observed that the intranasal application of the recombinant Ad5-based replication deficient adenovector expressing the soluble F1 codon optimization fragment of the RSV A2 F protein (amino acid 155-524) (108 PFU) could reduce replication of the RSV threat in the lungs of BALB/c mice compared to control mice, but mice immunized intramuscularly (im) did not show any protection against the threat (Kim et al, 2010, Vaccine 28: 3801 -3808).
[00012] In other studies, Ad5-based adenovectors carrying the optimized full length F codon of RSV (AdV-F) or the soluble form of the F gene of RSF (AdV-Fsol) were used in order to immunize the BALB/c mice twice with a dose of 1x1010 OPU (optical particle units: a dose of 1x1010 OPU corresponds to 2x108 GTU (gene transduction unit)). These vectors strongly reduced the viral load in the lungs after immunization i. n., but only partially after subcutaneous application (s.c.) or i. m. (Kohlmann et al, 2009, J Virol 83: 1260112610; US 2010/0111989).
[00013] Still in other studies, it was observed that the intramuscular application of the recombinant Ad5-based replication-deficient adenovector expressing the sequenced cDNA F protein of the RSV A2 strain (1010 particle units) could reduce the replication of the RSV threat only partially in the lungs of BALB/c mice when compared to control mice (Krause et al, 2011, Virology Journal 8:375-386)
[00014] In addition to not being fully effective in many cases, RSV vaccines under clinical evaluation for pediatric use and most vaccines under preclinical evaluation are intranasal vaccines. The most important advantages of the intranasal strategy are the direct reinforcement of local respiratory tract immunity and the absence of aggravation of the associated disease. In fact, in general, the efficacy, for example, of adenovirus-based RSV vaccines appears to be better for intranasal administration compared to intramuscular administration. However, intranasal vaccination also raises safety concerns in children younger than 6 months. The most common adverse reactions from intranasal vaccines are itchy nose or nasal congestion at all ages. Newborns are mandatory nose breathers and therefore must breathe through their nose. Thus, nasal congestion in the first few months of a child's life can interfere with treatments, and in rare cases it can cause serious respiratory problems.
[00015] More than 50 different human adenovirus serotypes have been identified. Of these, adenovirus serotype 5 (Ad5) has historically been most extensively studied for use as a gene carrier. However, recombinant adenovirus vectors of different serotypes can give rise to different results due to the induction of immune responses and protection. For example, WO 2012/021730 describes that simian adenovirus vector serotype 7 and human adenovirus vector serotype 5 encoding the F protein confer better protection against RSV than a human adenovirus vector serotype. Furthermore, different immunogenicity was observed for vectors based on human or non-human adenovirus serotypes (Abbink et al., 2007, J Virol 81: 4654-4663; Colloca et al., 2012, Sci Transl Med 4, 115ra2) . Abbink et al. conclude that all of the rare rAd vectors of human serotypes studied were less potent than the rAd5 vectors in the absence of anti-Ad5 immunity. Furthermore, it was recently reported that while rAd5 with an Ebolavirus (EBOV) glycoprotein (gp) transgene protects 100% of non-human primates, rAd35 and rAd26 with EBOV gp transgene conferred only partial protection and was A strategy of increasing heterologous reinforcement with these vectors is necessary in order to obtain full protection against the threat of the Ebola virus (Geisbert et al, 2011, J Virol 85: 4222-4233). Thus, it is not a priori possible to predict the efficacy of a recombinant adenovirus vaccine based only on data from another adenovirus serotype.
[00016] In addition, for RSV vaccines, experiments in suitable disease models, such as the cotton rat, are required in order to determine whether a vaccine candidate is effective enough to prevent replication of RSV in the nasal and pulmonary tracts, and at the same time it is safe, that is, it does not lead to aggravation of the disease. Preferably, these vaccine candidates should be highly effective in these models, even after intramuscular administration.
[00017] For this reason, there remains a need for efficient vaccines and methods of vaccination against RSV, which do not lead to a worsening of the disease. The present invention aims to provide these vaccines and these methods of vaccination against RSV in a safe and effective way. Invention Summary
[00018] It was surprisingly found by the present inventors that recombinant adenoviruses of serotype 26 (Ad26) comprising a nucleotide sequence encoding the F protein of RSV are very effective vaccines against RSV in a well-established cotton mouse model, and have an improved efficacy when compared to the data described above for Ad5 encoding F of RSV. It has been shown that even a single administration, even intramuscularly, of Ad26 encoding F of RSV is sufficient to provide complete protection against the threat of RSV replication.
[00019] The invention provides a vaccine against respiratory syncytial virus (RSV), comprising a recombinant human adenovirus serotype 26 which comprises nucleic acid encoding an RSV F protein or fragment thereof.
[00020] In certain embodiments, the recombinant adenovirus comprises nucleic acid encoding the F protein of RSV which comprises the amino acid sequence of SEQ ID NO: 1.
[00021] In certain embodiments, the nucleic acid encoding the RSV F protein presents codon optimization for expression in human cells.
In certain embodiments, the nucleic acid encoding the RSV F protein comprises the nucleic acid sequence of SEQ ID NO:2.
[00023] In certain embodiments, the recombinant human adenovirus has a deletion in the E1 region, a deletion in the E3 region, or a deletion in both the E1 and E3 regions of the adenovirus genome.
[00024] In certain embodiments, the recombinant adenovirus has a genome comprising at its 5' terminal ends the sequence CTATCTAT.
[00025] The invention further provides a method for vaccinating a subject against RSV, the method comprising administering to the subject a vaccine according to the invention.
[00026] In certain modalities, the vaccine is administered intramuscularly.
[00027] In certain embodiments, a vaccine according to the invention is administered to the subject more than once.
In certain embodiments, the method for vaccinating a subject against RSV also comprises administering to the subject a vaccine comprising a recombinant human adenovirus of serotype 35, which comprises the nucleic acid encoding the RSV F protein or a fragment thereof.
[00029] In certain embodiments, the process of vaccinating a subject against RSV further comprises administering RSV F protein (preferably formulated as a pharmaceutical composition, thus a protein vaccine) to the subject.
[00030] In certain embodiments, the method for vaccination consists of a single administration of the vaccine to the subject.
[00031] The invention also provides a method for reducing RSV infection and/or replication, for example, in the nasal tract and lungs of a subject, comprising administering to the subject, through intramuscular injection of a composition comprising a human adenovirus recombinant serotype 26, comprising nucleic acid encoding an RSV F protein or a fragment thereof. This will reduce adverse effects resulting from RSV infection in a subject, and thus contribute to the subject's protection against those adverse effects after vaccine administration. In certain modalities, the adverse effects of RSV infection can essentially be prevented, that is, reduced to levels so low that they are not clinically relevant. The recombinant adenovirus can be in the form of a vaccine according to the invention, including the modalities described above.
The invention further provides an isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding an RSV F protein or fragment thereof.
[00033] The invention further provides a method for producing a vaccine against respiratory syncytial virus (RSV), comprising the provision of a recombinant human adenovirus serotype 26, which comprises nucleic acid encoding an RSV F protein or a fragment thereof , propagating said recombinant adenovirus in a host cell culture, isolating and purifying the recombinant adenovirus, and formulating the recombinant adenovirus into a pharmaceutically acceptable composition. The recombinant human adenovirus of this aspect can also be any of the adenoviruses described in the above embodiments.
[00034] The invention further provides an isolated recombinant nucleic acid which forms the genome of a recombinant human adenovirus of serotype 26 which comprises nucleic acid encoding an RSV F protein or fragment thereof. The adenovirus can also be any of the adenoviruses as described in the above embodiments. Brief Description of Figures
[00035] FIG. 1 shows the cellular immune response against F peptides overlaying F aa 1-252 and F peptides overlaying F aa 241-574 from rats after immunization with different doses of rAd26 (A) and rAd35 (B) based on vectors harboring the RSV F gene, 2 and 8 weeks after immunization.
[00036] FIG. 2 shows the antibody response against RSV in mice after immunization with different doses of rAd26 and rAd35 based on vectors harboring the RSV F gene, 2 and 8 weeks after immunization.
[00037] FIG. 3 shows the results of the ratio of the IgG2a vs. IgG 1 against RSV in mice after immunization with 1010 vp of rAd26 and rAd35 based on vectors harboring the F gene of RSV, 8 weeks after immunization.
[00038] FIG. 4 shows the virus neutralizing capacity against RSV Long in mice after immunization with doses of rAd26 (A) and rAd35 (B) based on vectors harboring the RSV F gene, 2 and 8 weeks after immunization.
[00039] FIG. 5 shows the cellular immune response against (A) F peptides overlaying F aa 1-252 and (B) F peptides overlaying F aa 241-574 in mice after immunization by initial vaccination - vector booster based on rAd26 and rAd35 harboring the RSV F gene, 6 and 12 weeks after primary immunization.
[00040] FIG. 6 shows the antibody response against RSV in mice after immunization by initial vaccination - boosting vectors based on rAd26 and rAd35 harboring the F gene of RSV, at different time points after the first immunization.
[00041] FIG. 7 shows the neutralizing capacity against RSV Longo in mouse serum after immunization by initial vaccination - booster with different doses of vectors based on rAd26 and rAd35 harboring the F gene of RSV, at different time points after the first immunization.
[00042] FIG. 8 shows the neutralizing capacity against RSV B1 in mice after immunization by initial vaccination - boosting with different doses of vectors based on rAd26 and rAd35 harboring the F gene of RSV, at different time points after the first immunization.
[00043] FIG. 9 shows A) pulmonary RSV titers and B) nasal RSV titers in cotton rats after immunization by initial vaccination - booster with different doses of vectors based on rAd26 and rAd35 harboring the RSV F gene, 5 days after threat .
[00044] FIG. 10 shows the induction of virus neutralizing titers after immunization by initial vaccination - boosting with different doses of vectors based on rAd26 and rAd35 harboring the RSV F gene at A) 28 days, and B) 49 days after immunization.
[00045] FIG. 11 shows the histopathological examination of the lungs of cotton rats on the day of sacrifice followed by immunization by prime-boost vaccination with different doses of vectors based on rAd26 and rAd35 harboring the RSV F gene.
[00046] FIG. 12 shows A) pulmonary RSV titers and B) nasal RSV titers in cotton rats after single dose immunization with different doses of vectors based on rAd26 and rAd35 harboring the RSV F gene, 5 days after threat , administered through different routes.
[00047] FIG. 13 shows virus neutralizing titers induced after single-dose immunization with different doses of vectors based on rAd26 and rAd35 harboring the RSV F gene at 28 and 49 days after the first immunization, administered via different routes.
[00048] FIG. 14 shows histopathological examination of cotton rat lungs on the day of sacrifice followed by immunization (i.m.) by single dose with different doses of vectors based on rAd26 and rAd35 harboring the RSV F gene on the day of sacrifice.
[00049] FIG. 15 shows plasmid maps comprising the left end of the Ad35 and Ad26 genome with the RSV F coding sequence:
[00050] A. pAdApt35BSU.RSV.F(A2)nat, and B. pAdApt26.RSV.F(A2- )nat
[00051] FIG. 16 shows A) pulmonary RSV titers and B) nasal RSV titers in cotton rats after single dose immunization on day 0 or day 28 with different doses of vectors based on rAd26 harboring the RSV F gene, 5 days after the threat. The threat occurred on the 29th.
[00052] FIG. 17 shows the induction of virus neutralizing titers after single dose immunization with different doses of rAd26 harboring the RSV F gene 49 days after immunization as described for FIG 16.
[00053] FIG. 18 shows the induction of virus neutralizing titers after single-dose immunization with different doses of rAd26 harboring the RSV F gene, over time after immunization.
[00054] FIG. 19 shows VNA titers, 49 days later, against RSV Longo and RSV B washed with serum derived from cotton rats immunized with 1010 µg Ad-RSV F or no transgene (Ad-e). PB: prime-boost vaccination.
[00055] FIG. 20 shows pulmonary RSV titers in cotton rats after single-dose immunization on day 0 with different doses of rAd26-based vectors harboring the RSV F gene, 5 days post-threat, with RSV A2 or RSV B15/97 .
[00056] FIG. 21 shows nasal RSV titers in cotton rats after single-dose immunization on day 0 with different doses of rAd26-based vectors harboring the RSV F gene, 5 days post-threat, with RSV A2 or RSV B15/97 .
[00057] FIG. 22 shows the serum VNA titers of cotton rats after single-dose immunization on day 0 with different doses of rAd26-based vectors harboring the RSV F gene, at different times after the initial vaccination.
[00058] FIG. 23 shows pulmonary RSV titers in cotton rats after single-dose immunization on day 0 with different doses of rAd26-based vectors harboring the RSV F gene, 5 days after threat, with a standard dose (105) or a high dose (5x105) of RSV A2.
[00059] FIG. 24 shows nasal RSV titers in cotton rats after single-dose immunization on day 0 with different doses of rAd26-based vectors harboring the RSV F gene, 5 days post-threat, threatened with a standard dose (105 ) or a high dose (5x105) of RSV A2.
[00060] FIG. 25 shows pulmonary RSV titers in cotton rats after immunization on day 0 and day 28 with different doses of single immunization or with prime-boost vaccination with rAd26-based vectors harboring the RSV F gene, 5 days after threat, with the threat made 210 days after the immunization.
[00061] FIG. 26 shows serum VNA titers from cotton rats after single-dose immunization and prime-boost vaccination 140 days after immunization.
[00062] FIG. 27 shows the histopathological examination of the lungs of cotton rats on the day of sacrifice followed by single-dose immunization or prime-boost vaccination with different doses of vectors based on rAd26 harboring the RSV F gene 2 days after threat. The points represent the median and the filaments the 25th and 75th percentiles.
[00063] FIG. 28 shows histopathological examination of the lungs of cotton rats on the day of sacrifice followed by single-dose immunization or prime-boost vaccination with different doses of vectors based on rAd26 harboring the RSV F gene 6 days after threat. The points represent the median and the filaments the 25th and 75th percentiles.
[00064] FIG. 29 shows induction of virus neutralizing titers after immunization with rAd26 harboring the RSV F gene (Ad26.RSV.F) followed by boosting with Ad26.RSV.F or RSV F protein adjuvant (after F).
[00065] FIG. 30 shows the induction of IgG2a and IgG1 antibodies, and the indicated ratio, after immunization with Ad26.RSV.F followed by boosting with Ad26.RSV.F or boosting with RSV F protein adjuvant (after F).
[00066] FIG. 31 shows IFN-g production by splenocytes after immunization with Ad26.RSV.F followed by boosting with Ad26.RSV.F or RSV F protein adjuvant (after F). Detailed description of the invention
[00067] The term "recombinant" for an adenovirus, as used in this document, implies that it has been modified by the hand of man, for example, it has actively cloned altered terminal ends and/or it comprises a heterologous gene, i.e., it is not a naturally occurring wild type adenovirus.
[00068] The sequences, in this document, are provided 5' to 3', as is customary in the art.
[00069] An "adenovirus capsid protein" refers to a protein in the capsid of an adenovirus that is involved in determining the serotype and/or tropism of a particular adenovirus. Adenovirus capsid proteins typically include fiber, penton and/or hexon proteins. An adenovirus of (or "based on") a particular serotype according to the invention typically comprises fiber, penton and/or hexon proteins of that particular serotype, and preferably comprises fiber, penton and/or hexon protein. of that particular serotype. These proteins are typically encoded by the recombinant adenovirus genome. A recombinant adenovirus of a particular serotype may optionally comprise and/or encode other proteins from other adenovirus serotypes. Thus, as a non-limiting example, a recombinant adenovirus comprising Ad26 hexon, penton and fiber is considered a recombinant adenovirus based on Ad26.
[00070] A recombinant adenovirus is "based on" an adenovirus, as used herein, by derivation from wild-type at least in sequence. This can be achieved through molecular cloning, using the wild-type genome or parts of it as starting material. It is also possible to use the known genome sequence of a wild-type adenovirus to generate (parts of) the genome again through DNA synthesis, which can be done using routine procedures by service companies operating in the field of DNA synthesis and/or molecular cloning (eg GeneArt, Invitrogen, GenScripts, Eurofins).
[00071] It is understood by one of skill in the art that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. It is also understood that those skilled in the art can, using routine techniques, make nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the described polynucleotides so as to reflect the codon usage of any particular host organism in which the polypeptides must be expressed. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of one another and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.
[00072] In a preferred embodiment, the nucleic acid encoding the RSV F protein or a fragment thereof exhibits codon optimization for expression in mammalian cells, such as human cells. Codon optimization methods are known and have been described previously (eg WO 96/09378). An example of a codon optimized sequence specific for the F protein of RSV is described in SEQ ID NO: 2 of EP 2102345 B1.
In one embodiment, the RSV F protein is from a strain of RSV A2, and has the amino acid sequence of SEQ ID NO: 1. In a particularly preferred embodiment, the nucleic acid encoding the RSV F protein comprises the nucleic acid sequence of SEQ ID NO: 2. It was discovered by the inventors that this modality results in stable expression and that a vaccine according to this modality confers protection for RSV replication in the nasal and pulmonary tract, even after a single dose, which was administered intramuscularly.
[00074] The term "fragment" as used herein refers to a peptide that has an amino-terminal and/or carboxy-terminal and/or internal deletion, but in which the remaining amino acid sequence is identical to the corresponding positions in the sequence of an RSV F protein, for example, the full length sequence of an RSV F protein. It will be appreciated that, for inducing an immune response and, in general, for vaccination purposes, a protein need not be full-length or have all of its wild-type functions, and that protein fragments are equally useful. Indeed, RSV F protein fragments such as F1 or soluble F have been shown to be 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 255-278 or 412-524 in active immunization induced neutralizing antibodies and some protection against the RSV threat (Sing et al, 2007, Immunol. 20, 261-275; Sing et al, 2007, Vaccine 25, 6211-6223).
[00075] 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 F protein of RSV. In certain embodiments, it comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids of the RSV F protein.
[00076] One skilled in the art will also appreciate that alterations can be made to a protein, for example, through amino acid substitutions, deletions, additions, etc., for example, using routine molecular biology procedures. Generally, conservative amino acid substitutions can be applied without loss of function or immunogenicity of a polypeptide. This can be easily verified in accordance with routine procedures well known to the person skilled in the art.
[00077] The term "vaccine" refers to an agent or composition containing an active component effective in inducing a therapeutic degree of immunity in a subject against a particular pathogen or disease. In the present invention, the vaccine comprises an effective amount of a recombinant adenovirus encoding an RSV F protein, or an antigenic fragment thereof, which results in an immune response against the RSV F protein. This provides a method for preventing severe lower respiratory tract diseases leading to hospitalization and decreasing the frequency of complications such as pneumonia and bronchiolitis due to RSV infection and replication in a subject. Thus, the invention also provides a method for preventing or reducing severe lower respiratory tract diseases, 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 of a composition comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding an RSV F protein or a fragment thereof. The term "vaccine" according to the invention implies that it is a pharmaceutical composition and thus typically includes a pharmaceutically acceptable diluent, carrier or excipient. This may or may not comprise more active ingredients. In certain embodiments this may be a combination vaccine which further comprises other components which elicit an immune response against, for example, other RSV proteins and/or against other infectious agents.
[00078] The vectors of the present invention are recombinant adenoviruses, also referred to as recombinant adenovirus vectors. The preparation of recombinant adenovirus vectors is well known in the art.
[00079] In certain embodiments, an adenovirus vector according to the invention is deficient in at least one essential gene function of the E1 Region, for example, the E1a region and/or the E1b region, of the genome of the adenovirus that it is necessary for viral replication. In certain embodiments, an adenovirus vector according to the invention is deficient in at least part of the non-essential E3 region. In certain embodiments, the vector is deficient in at least one essential gene function of the E1 region and at least part of the non-essential E3 region. The adenovirus vector can be "multiplier deficient", meaning that the adenovirus vector is deficient in one or more essential gene functions in each of the two or more regions of the adenovirus genome. For example, the aforementioned E1 deficient or E1 deficient adenovirus vectors may also be deficient in at least one essential gene from the E4 region and/or at least one essential gene from the E2 region (e.g., the E2A region and /or the E2B region).
[00080] Adenovirus vectors, methods for making them and methods for propagating them, are well known in the art and are described, for example, in US Patent Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806 , 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and 6,113,913, and in Thomas Shenk, "Adenoviridae and their Replication", MS Horwitz, "Adenoviruses", Chapters 67 and 68, respectively, in Virology, BN Fields et al. , eds., 3rd ed., Raven Press, Ltd., New York (1996), and other references mentioned herein. Typically, the construction of adenovirus vectors involves the use of standard molecular biology techniques, such as those described in, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NI (1989), Watson et al., Recombinant DNA, 2nd ed., Scientific American Books (1992), and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, NI (1995) and other cited references in this document.
[00081] According to the invention, an adenovirus is a human adenovirus of serotype 26. Vaccines according to the invention based on that serotype as well as those based on Ad35 surprisingly appear more potent than those described in the prior art which were based on Ad5, as these failed to provide complete protection against replication of the RSV threat after a single 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 present invention generally also has a low serum prevalence and/or low pre-existing neutralizing antibody titers in the human population. Recombinant adenovirus vectors of this serotype and Ad35 with different genes are evaluated in clinical trials, and so far they have been shown to have an excellent safety profile. The preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al. (2007) Virol 81(9): 4654-63. Exemplary Ad26 genomic sequences are found in GenBank Accession EF 153474 and in SEQ ID NO:1 of WO 2007/104792. The preparation of rAd35 vectors is described, for example, in U.S. Patent No. 7,270,811, WO 00/70071, and in Vogels et al. (2003) J Virol 77(15): 8263-71. Exemplary Ad35 genomic sequences are found in GenBank Accession AC_000019 and Fig. 6 of WO 00/70071.
[00082] A recombinant adenovirus according to the invention may be replication defective or replication competent.
[00083] In certain embodiments, the adenovirus is replication-deficient, for example, because it contains a deletion in the E1 region of the genome. As is known to the person skilled in the art, in case of deletions of essential regions of the adenovirus genome, the functions encoded by these regions must be conferred in trans, preferably by the producer cell, i.e. when parts or all of the E1, E2 and/or E4 regions that are deleted from the adenovirus, these must be present in the producer cell, for example integrated into its genome, or in the form of so-called helper adenoviruses or helper plasmids. Adenovirus may also have a deletion in the E3 region, which is dispensable for replication, and thus this deletion does not have to be complemented.
[00084] The producer cell (sometimes also referred to in the art and herein as "packaging cell" or "complementary cell" or "host cell") that can be used can be any producer cell, in which an adenovirus can be propagated. wanted. For example, propagation of recombinant adenovirus vectors is carried out in producer cells that complement adenovirus deficiencies. Such producer cells preferably have in their genome at least one adenovirus E1 sequence, and thus are capable of complementing the recombinant adenoviruses with a deletion in the E1 region. Any complementary E1 producing cell may be used, such as E1 immortalized human retinal cells, for example, 911 or PER.C6 cells (see US Patent 5,994,128), transformed E1 amniocytes (see EP Patent 1230354), A549 cells Transformed E1 (see e.g. WO 98/39411, US patent 5,891,690), GH329:HeLa (Gao et al, 2000, Human Gene Therapy 11: 213-219), 293, and the like. In certain embodiments, the producer cells are for example HEK293 cells, or PER.C6 cells or 911 cells, or IT293SF cells, and the like.
[00085] For adenoviruses deficient in E1 non-C subgroup, such as Ad35 (subgroup B) or Ad26 (subgroup D), it is preferable to exchange the E4-orf6 coding sequence of that non-C subgroup adenovirus with E4-orf6 of an adenovirus of subgroup C such as Ad5. This allows the propagation of these adenoviruses in well-known complementary cell lines that express the Ad5 E1 genes, such as, for example, 293 cells or PER.C6 cells (see, for example, Havenga et al, 2006, J. Gen. Virol 87: 2135-2143; WO 03/104467, incorporated in its entirety herein by reference). In certain embodiments, an adenovirus that can be used is a human adenovirus serotype 35, with a deletion in the E1 region in which the nucleic acid encoding the F protein antigen of RSV has been cloned, and with an E4 orf6 region of Ad5. In certain embodiments, the adenovirus of the vaccine composition of the invention is a human adenovirus serotype 26, with a deletion in the E1 region in which the nucleic acid encoding the RSV F protein antigen has been cloned, and with an E4 orf6 region of the Ad5.
[00086] In alternative embodiments, there is no need to place a heterologous E4orf6 region (eg Ad5) in the adenovirus vector, but instead the defective vector E1 subgroup non-C is propagated in a cell line that expresses both E1 and E4orf6 compatible , for example, the 293-0RF6 cell line that expresses both E1 and E40rf6 from Ad5 (see, for example, Brough et al, 1996, J Virol 70:6497-501 which describes the generation of 293-ORF6 cells; Abrahamsen et al, 1997, J Virol 71: 8946-51 and Nan et al, 2003, Gene Therapy 10: 326-36 each generation describing non-C subgroup deleted E1 adenovirus vectors using this cell line).
[00087] Alternatively, a complementary cell expressing E1 from the serotype that is to be propagated can be used (see for example WO 00/70071, WO 02/40665 ).
[00088] For subgroup B adenoviruses, such as Ad35, containing a deletion in the E1 region, it is preferable to retain the 3' end of the E1B 55K open reading frame in the adenovirus, for example, at 166 bp directly upstream of the E1B frame. pIX open reading frame or a fragment comprising this such as a 243 bp fragment, directly upstream of the pIX start codon (marked at the 5' end by a Bsu36I restriction site in the Ad35 genome), as this increases the stability of the adenovirus, since the promoter of the pIX gene is partially resident in that area (see, for example, Havenga et al, 2006, J. Gen. Virol. 87: 2135-2143; WO 2004/001032, incorporated herein by reference).
[00089] "Heterologous nucleic acid" (also referred to herein as "transgene") in adenoviruses of the invention is nucleic acid that is not naturally present in adenoviruses. This is introduced into adenovirus, for example, through standard molecular biology techniques. In the present invention, the heterologous nucleic acid encodes the RSV F protein or a fragment thereof. It can, for example, be cloned into an E1 or E3 deleted region of an adenovirus vector. A transgene is usually operably linked to expression control sequences. This can, for example, be done by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Additional regulatory sequences can be added. Many promoters can be used for the expression of a transgene(s), and are known to those skilled in the art. A non-limiting example of a promoter suitable for obtaining expression in eukaryotic cells is a CMV promoter (US 5,385,839), e.g. the CMV immediate early promoter, e.g. comprising nt. -735 to +95 of the CMV immediate early promoter/enhancer. A polyadenylation signal, for example the bovine growth hormone polyA signal (US 5,122,458), may be present behind the transgene(s).
[00090] In certain embodiments, the Ad26 recombinant vectors of the invention comprise as 5' terminal nucleotides the nucleotide sequence: CTATCTAT. These modalities are advantageous as such vectors exhibit improved replication in production processes, resulting in adenovirus batches with improved homogeneity, when compared to vectors having the 5' terminal sequences (generally CATCATCA) (see also Patent Applications Nos. PCT/EP2013/054846 and US 13/794,318 entitled Batches of recombinant adenovirus with altered terminal ends filed March 12, 2012 in the name of Crucell Holland BV), incorporated in their entirety by reference. Thus, the invention also provides recombinant adenovirus stocks encoding the RSV F protein or a part thereof, wherein the adenovirus is a human adenovirus serotype 26, and wherein essentially all (e.g., at least 90 %) of the adenovirus in the batch comprises a genome with terminal nucleotide sequence CTATCTAT.
[00091] According to the invention, RSV F protein can be derived from any naturally occurring or recombinant RSV strain, preferably from human RSV strains, such as A2, Longo, or B strains. In other embodiments, the sequence may be a consensus sequence based on a plurality of RSV F protein amino acid sequences. In an example of the invention, the RSV strain is RSV strain A2.
[00092] According to the invention, the RSV F protein may be the full length RSV F protein or a fragment thereof. In one embodiment of the invention, the nucleotide sequence encoding the RSV F protein encodes the entire length of the RSV F protein (F0), such as the amino acid of SEQ ID NO: 1. In an example of the invention, the sequence of the nucleotide sequence encoding the RSV F protein has the nucleotide sequence of SEQ ID NO: 2. Alternatively, the sequence encoding the RSV F protein may 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, codon-optimized sequences, such as, for example, those given in SEQ ID NO: may be used. 2, 4, 5 or 6 of WO 2012/021730.
[00093] In another embodiment of the invention, the nucleotide sequence may alternatively encode a fragment of the F protein of RSV. The fragment can result from one or both of the amino-terminal and carboxy-terminal deletions. The extent of the deletion can be determined by one skilled in the art in order, for example, to achieve a better yield of the recombinant adenovirus. The fragment will be chosen to include an immunologically active fragment of the F protein, that is, a part that will give rise to an immune response in a subject. This can be easily determined using in silico, in vitro and/or in vivo methods, all routine to one skilled in the art. In one embodiment of the present invention, the fragment is a transmembrane protein encoding the truncated region of the F protein of RSV (F0ΔTM, see for example US 20110014220). The F protein fragments can also be the F1 domain or 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).
[00094] The term "about" in numerical values as used in the present disclosure means the value of ± 10%.
[00095] In certain embodiments, the invention provides methods for producing a vaccine against respiratory syncytial virus (RSV), comprising providing a recombinant human adenovirus serotype 26, which comprises nucleic acid encoding an RSV F protein or a fragment thereof, propagating said recombinant adenovirus in a host cell culture, isolating and purifying the recombinant adenovirus, and bringing the recombinant adenovirus in a pharmaceutically acceptable composition.
[00096] Recombinant adenovirus can be propagated and prepared in host cells, according to well-known methods, which involve cell culture of the host cells that are infected with the adenovirus. The cell culture can be any type of cell culture, including adherent cell culture, for example, cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture.
[00097] Most large-scale suspension cultures are carried out as fed batch or batch processes, since these are the simplest to carry out and expand. Nowadays, continuous processes based on perfusion principles are becoming more and more common and are also suitable (see for example WO 2010/060719, and WO 2011/098592, both incorporated herein by reference, which describe suitable methods for obtaining and purifying large amounts of recombinant adenoviruses).
[00098] Producer cells are cultured in order to increase the number of cells and virus and/or virus titers. The culture of a cell is done in such a way as to allow it to metabolize, and/or grow and/or divide and/or produce viruses of interest according to the invention. This can be accomplished by methods such as those well known to those skilled in the art, and includes, but is not limited to, supplying nutrients to the cells, for example, in the appropriate culture medium. Suitable culture media are well known to the person skilled in the art and can generally be obtained from commercial sources, in large quantities, or custom made in accordance with standard protocols. The culture can be done, for example, in plates, roller bottles or in bioreactors, using continuous, batch, fed batch and the like systems. Appropriate conditions for cell culture are known (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and RI Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley -Liss Inc., 2000, ISBN 0-471-34889-9).
[00099] Typically, the adenovirus will be exposed to the appropriate producer cell in a culture, which allows for virus uptake. Usually, optimum agitation is between about 50 and 300 rpm, typically about 100-200, eg about 150, typical OD is 20-60%, eg 40%, optimum pH is between 6, 7 and 7.7, the optimum temperature between 30 and 39 °C, for example 34-37 °C, and the optimum MOI between 5 and 1000, for example around 50-300. Typically, the adenovirus infects the producer cells spontaneously, and putting the particle producer cells in contact with the rAd is sufficient for cell infection. Generally, an adenovirus seed stock is added to the culture in order to initiate the infection, and subsequently the adenovirus propagates in the producer cells. All of this is routine for the technical expert.
[000100] Upon infection with an adenovirus, the virus replicates within the cell and is thereby amplified, a process referred to herein as adenovirus propagation. Adenovirus infection ultimately results in the lysis of the cells to be infected. The lytic characteristics of adenovirus therefore allow two different modes of virus production. The first way is to harvest the virus before cell lysis, using external factors for cell lysis. The second way is to harvest the supernatant virus after (almost) complete lysis of the cells by the virus produced (see for example, U.S. patent 6,485,958, which describes harvesting adenovirus without lysis of the host cells by an external factor). It is preferable to use external factors to actively lyse cells for adenovirus harvest.
[000101] Methods that can be used for active lysis of cells are known to the person skilled in the art, and have, for example, been discussed in WO 98/22588, pg. 28-35. Methods useful in this regard are, for example, freeze-thaw, solid shear, hypertonic and/or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, combinations of the above, and the like. In one embodiment of the invention cells are lysed using at least one detergent. The use of a lysis detergent has the advantage of being an easy method, and of being easily scalable.
[000102] The detergents that can be used, and the way they are used, are generally known to the person skilled in the art. Several examples are, for example, discussed in WO 98/22588, pg. 29-33. Detergents can include anionic, cationic, zwitterionic, and nonionic detergents. The concentration of the detergent can be varied, for example, within a range of about 0.1% -5% (w/w). In one modality, the detergent used is Triton X-I00.
[000103] Nuclease can be used to remove contaminants, ie mainly from the cell producing nucleic acids. Examples of suitable nucleases for use in the present invention include Benzonase®, Pulmozyme®, or any other DNase and/or RNase commonly used in the art. In preferred embodiments, the nuclease is Benzonase®, which rapidly hydrolyzes nucleic acids through internal hydrolysis of phosphodiester bonds between specific nucleotides, thereby reducing cell lysate viscosity. Benzonase® can be commercially obtained from Merck KGaA (code W214950). The concentration at which the nuclease is used is preferably within the range of 1 to 100 units/ml. Alternatively, or in addition to nuclease treatment, it is also possible to selectively precipitate host cell DNA out of adenovirus preparations, during adenovirus purification, using selective precipitating agents such as domiphen bromide (see for example, US 7,326,555; Goerke et al., 2005, Biotechnology and bioengineering, Vol. 91: 12-21; WO 2011/045378; WO 2011/045381).
[000104] Methods for harvesting from adenovirus producer cell cultures have been extensively described in WO 2005/080556.
[000105] In certain embodiments, the harvested adenovirus undergoes further purification. Adenovirus purification can be carried out in several steps, comprising clarification, ultrafiltration, diafiltration or chromatographic separation, as described, for example, in WO 05/080556, incorporated herein by reference. Clarification can be carried out through a filtration step, removing cell debris and other impurities from the cell lysate. Ultrafiltration is used to concentrate the virus solution. Diafiltration, or buffer change, using ultrafilters is a way to remove and exchange salts, sugars and the like. The person skilled in the art knows how to find the optimal conditions for each purification step. WO 98122588, incorporated herein by reference in its entirety, also describes methods for the production and purification of adenovirus vectors. The methods comprise growing host cells, infecting the host cells with adenovirus, harvesting and lysing the host cells, concentrating the crude lysate, changing the buffer of the crude lysate, treating the lysate with nuclease, and also purifying the virus using chromatography.
[000106] Preferably, the purification uses at least one chromatography step, as for example discussed in WO 98/22588, pg. 61-70. Many processes for the further purification of adenoviruses have been described, where chromatography steps are included in the process. The person skilled in the art will be aware of these processes, and may vary the exact way in which the chromatographic steps are used in order to optimize the process. It is possible, for example, to purify adenoviruses by anion exchange chromatography steps, see for example WO 2005/080556 and Konz et al, 2005, Hum Gene Ther 16: 1346-1353. Many other methods of purifying adenoviruses have already been described and are within the purview of those skilled in the art. Other methods for the production and purification of adenoviruses are disclosed, for example, in ( WO 00/32754; WO 04/020971; US 5,837,520; US 6,261,823; WO 2006/108707; Konz et al, 2008, Methods Mol Biol 434: 13 -23; Altaras et al, 2005, Adv Biochem Eng Biotechnol 99:193-260), all incorporated herein by reference.
[000107] For administration to humans, the invention can use pharmaceutical compositions comprising rAd and a pharmaceutically acceptable carrier or excipient. In the present context, the term "Pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations used, will not cause any unwanted or harmful effects on the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds. ., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd addition, A. Kibbe, Ed., Pharmaceutical Press [2000]). Purified rAd is preferably formulated and administered as a sterile solution, although lyophilized preparations may also be used. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or placed in pharmaceutical dosage containers. The pH of the solution is generally in the pH range of 3.0 to 9.5, eg pH 5.0 to 7.5. The rAd is typically in a solution having a suitable pharmaceutically acceptable buffer, and the rAd solution may also contain a salt. Optionally, a stabilizing agent such as albumin may be present. In certain embodiments, detergent is added. In certain modalities, rAd can be formulated into an injectable preparation. These formulations contain effective amounts of rAd, or are sterile liquid solutions, liquid suspensions or lyophilized versions, and optionally contain stabilizers or excipients. An adenovirus vaccine can also be aerosolized for intranasal administration (see for example WO 2009/117134 ).
[000108] For example, adenovirus can be stored in buffer, which is also used for the Adenovirus World Standard (Hoganson et al, Development of a stable adenoviral vector formulation, Bioprocessing March 2002, pg. 43-48): 20 μm from Tris pH 8, 25 mM NaCl, 2.5% glycerol. Another useful buffer formulation suitable for human administration is 20 mM Tris, 2 mM MgCl 2 , 25 mM NaCl, 10% w/v sucrose, 0.02% w/v polysorbate-80. Obviously, many other buffers can be used, and several examples of formulations suitable for the storage and pharmaceutical administration of purified (adeno)virus preparations, for example, can be found, for example, in European patent no. 0853660, U.S. Patent 6,225,289 and International Patent Applications WO 99/41416, WO 99/12568, WO 00/29024, WO 01/66137, WO 03/049763, WO 03/078592, WO 03/061708.
[000109] In certain embodiments a composition comprising the adenoviruses also comprises one or more adjuvants. Adjuvants are known in the art to further enhance the immune response to an applied antigenic determinant, and pharmaceutical compositions comprising adenoviruses and suitable adjuvants are disclosed, for example, in WO 2007/110409, incorporated herein by reference. The terms "adjuvant" and "immune stimulant" are used interchangeably in this document and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the adenovirus vectors of the invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil-in-oil emulsion compositions (or oil-in-water compositions), including squalene-in-water emulsions such as MF59 (see, for example, WO 90/14837); saponin formulations such as, for example, QS21 and Immunostimulating Complexes (ISCOMS) (see, for example, US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG motif-containing oligonucleotides, the bacterial ADP-ribosylation toxins or mutants thereof such as the enterotoxin LTda E. heat-labile coli, cholera toxin CT, and the like. It is also possible to use vector-encoded adjuvants, for example, through the use of heterologous nucleic acid encoding a fusion of the C4-binding protein oligomerization domain (C4bp) with the antigen of interest (eg, Solabomi et al, 2008, Infect Immun 76:381723). In certain embodiments the compositions of the invention comprise aluminum as an adjuvant, for example, in the form of aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate, or combinations thereof, in concentrations of 0.05-5 mg, for example , 0.075-1.0 mg, aluminum content per serving.
[000110] In other embodiments, the compositions do not include adjuvants.
[000111] It is also possible, according to the invention, to administer other active components, in combination with the vaccines according to the invention. Such additional active components may comprise, for example, other RSV antigens or vectors comprising nucleic acid encoding them. Such vectors can be non-adenoviral or adenoviral, of which the latter can be of any serotype. An example of other RSV antigens include the RSV G protein or immunologically active parts thereof. For example, the adenovector rAd/3xG based on Ad5-deficient recombinant replication Ad5 applied intranasally, which expresses 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 RSV G is an appropriate 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 the like. Administration of additional active components can, for example, be done by separate administration or by administering the vaccine combination products of the invention and the additional active components. In certain embodiments, additional non-adenoviral antigens (in addition to RSV.F) can be encoded on the vectors of the invention. In certain embodiments, it may thus be desirable to express more than one protein from a single adenovirus, and in such cases more coding sequences, for example, may be linked to form a single transcript from a single expression cassette. , or may be present in two separate expression cassettes cloned into different parts of the adenovirus genome.
Adenovirus compositions can be administered to a subject, for example, a human. The total dose of adenovirus conferred on a subject during an administration may be varied as is known to one skilled in the art, and is generally between 1x107 viral particles (vp) and 1x1012 vp, preferably between 1x108 vp and 1x1011 vp, for example between 3x108 and 5x1010 vp, for example, between 109 and 3x1010 vp.
[000113] Administration of adenovirus compositions can be carried out using standard routes of administration. Non-limiting modalities include parenteral administration, such as for example by intradermal, intramuscular etc. injection, or by subcutaneous, transcutaneous, or mucosal administration, for example intranasal, oral and the like. Intranasal administration has generally been regarded as a preferred route for RSV vaccines. The most important advantage of the in vivo strategy is the direct stimulation of local respiratory tract immunity and the absence of aggravation of the associated disease. The only vaccines under clinical evaluation for pediatric use at present are in vivo 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, pg. 233-277). Intranasal administration represents a suitable preferred route in accordance with the present invention. However, it is particularly preferred according to the present invention to administer the vaccine intramuscularly, as it has surprisingly been 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 , unlike previously reported intramuscular RSV vaccines based on other adenovirus serotypes. The advantage of intramuscular administration is that it is simple and well established, and does not imply safety concerns for nasal application in children younger than 6 months. In one modality, a composition is administered by intramuscular injection, for example, into the deltoid muscle of the arm, or the vastus lateralis muscle of the thigh. The skilled person is aware of the various possibilities for administering a composition, for example a vaccine, in order to induce an immune response to the antigen(s) in the vaccine.
[000114] 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 being. The subject can be of any age, for example from about 1 month to 100 years of age, for example from about 2 months to about 80 years of age, for example from about 1 month to about 3 years old, from about 3 years old to about 50 years old, from about 50 years old to about 75 years old, etc.
[000115] It is also possible to give one or more booster administrations of one or more adenovirus vaccines of the invention. If a booster vaccination is performed, typically, that booster vaccination will be administered to the same subject at a time between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which it is in these cases referred to as "initial vaccination"). In alternative booster regimens, it is also possible to administer different vectors, for example, one or more adenoviruses of different serotype, or other vectors, such as MVA or DNA, or protein, to the subject after the initial vaccination. It is for example possible to administer to the subject a recombinant adenovirus vector according to the invention as initial vaccination, and to be boosted with a composition comprising RSV F protein.
[000116] In certain embodiments, administration comprises an initial administration and, at a minimum, a booster administration. In certain embodiments thereof, initial administration is with rAd35 comprising the nucleic acid encoding the RSV F protein or a fragment thereof ('rAd35-RSV.F') and booster administration is with rAd26 comprising the nucleic acid that encodes the F protein of RSV according to the invention ('rAd26-RSV.F'). In other embodiments of the same, initial administration is with rAd26-RSV.F and booster administration is with rAd35-RSV.F. In other modalities, both initial and booster administration are with rAd26.RSV.F. In certain embodiments, initial administration is with rAd26-RSV.F and booster administration is with RSV F protein. In all of these embodiments, it is possible to provide other booster administrations with the same or other vectors or protein. Modalities in which RSV F protein booster may be particularly beneficial include, for example, elderly subjects in at-risk groups (for example, having asthma or COPD) of 50 years or more, or for example, in healthy subjects of 60 years or older or 65 years or older.
[000117] In certain embodiments, the administration comprises a single administration of a recombinant adenovirus according to the invention, without additional administrations (booster). Such modalities are advantageous in view of the complexity and costs of a single single-administration regimen as compared to a prime-boost vaccination regimen. Complete protection is already seen after a single administration of the recombinant adenovirus vectors of the invention without booster administrations in the cotton rat model in the examples herein.
[000118] The invention is further explained in the following examples. The examples do not limit the invention in any way. These merely serve to clarify the invention. EXAMPLES Example 1. Preparation of adenovirus vectors Cloning of the RSV F gene in the E1 region of Ad35 and Ad26:
[000119] The RSV.F(A2)nat gene, which encodes the native RSV fusion protein (F) of the A2 strain (Genbank ACO83301.1), was optimized for human gene expression and synthesized by Geneart. A Kozak sequence (5' GCCACC 3') was included in front of the ATG start codon, and two stop codons (5' TGA TAA 3') were added at the end of the RSV.F(A2)nat coding sequence . The RSV.F(A2)nat gene was inserted into plasmid pAdApt35BSU and plasmid pAdApt26 through HindIII and XbaI sites. The resulting plasmids, pAdApt35BSU.RSV.F(A2)nat and pAdApt26.RSV.F(A2)nat are depicted in Fig. 15. The amino acid sequence of the F protein, and the optimized sequence of the codon encoding that amino acid sequence , are given in Table 1 as SEQ. ID NOs: 1 and 2, respectively. Cell culture:
[000120] PER.C6 cells (Fallaux et al., 1998, Hum Gene Ther 9:1909-1917) were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), supplemented with 10 mM MgCl2. Adenovirus generation, infections and passage:
All adenoviruses were generated in PER.C6 cells by simple homologous recombination and 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). To rescue the Ad35 vectors carrying the RSV.F(A2)nat transgene expression cassette, the plasmid pAdApt35BSU.RSV.F(A2)nat and the cosmid pWE/Ad35.pIX-rITR.dE3.5orf6 were used, while that for the Ad26 vectors carrying the RSV.F(A2)nat transgene expression cassette, plasmid pAdApt26.RSV.F(A2)nat and cosmid pWE.Ad26.dE3.5orf6 were used. Cells were harvested one day after complete CPE, frozen-thawed, centrifuged for 5 min at 3000 rpm, and stored at 20°C. Viruses were then plaque purified and amplified on PER.C6 cells grown in a single well of a 24-well tissue culture plate. Additional amplification was performed on cultured PER.C6 cells using a T25 tissue culture flask and a T175 tissue culture flask. Of the crude T175 lysate, 3 to 5 ml was used to inoculate 20*T175 triple layer tissue culture flasks containing 70% confluent layers of PER.C6 cells. Virus was purified using a two-step CsCl purification method. Finally, the virus was stored in aliquots at 85°C. Example 2. Induction of immunity against RSV F using in vivo recombinant adenovirus serotypes 26 and 35.
[000122] This is an experiment to investigate the ability of recombinant adenovirus serotype (Ad26) and recombinant adenovirus serotype 35 (Ad35) to induce immunity against RSV F glycoprotein antigen in BALB/c mice.
[000123] In this study the animals were distributed into experimental groups of 5 mice. Animals were immunized with a single dose of Ad26 or Ad35 carrying the F gene of the full length RSV (Ad26-RSV.F or Ad35-RSV.F) or without any transgene (Ad26e or Ad35e). Three 10-fold serial dilutions of rAd ranging from 1010 to 108 viral particles (vp) were administered intramuscularly. As controls, one group of 3 animals received the empty Ad26 vector and one group received the empty Ad35e vector.
[000124] The ELISPOT assay is used to determine the relative number of T cells secreting F protein-specific IFNy in the spleen, and is essentially performed as described by Radosevic et al. (Clin Vaccine Immunol. 2010;17(11):1687-94.). For the stimulation of splenocytes in the ELISPOT assay, two sets of peptides consisting of 11 amino acids overlapping 15-mer peptides covering the entire RSV F protein sequence (A2) were used. The number of spot forming units (SFU) per 106 cells was calculated.
[000125] For the determination of antibody titers an ELISA assay was used. For this, the ELISA plates (Thermo Scientific) were coated with 25 μg/ml of the totally inactivated RSV Longo antigen (Virion Serion, cat# BA113VS). Diluted serum samples were added to the plates, and IgG antibodies against RSV were determined using biotin-labeled anti-mouse IgG (DAKO, cat# E0413) using horseradish peroxidase (PO)-conjugated to streptavidin (SA) detection ). Titers were calculated by linear interpolation, using 1.5 x OD signals from naive serum diluted 50 x as cutoff. RSV-specific IgG1 and IgG2a antibody titers in mouse serum were determined using PO-labeled anti-mouse IgG1 and PO-labeled anti-mouse IgG2a (Southern Biotechnology Associates, cat#s 1070-05 and 1080-05) was used to quantify subclasses.
[000126] The virus neutralizing activity (VNA) of the antibodies was determined by microneutralization assay, carried out essentially as described by Johnson et al. (J Infect Dis. 1999 Jul;180(1):35-40.). RSV-susceptible VERO cells were seeded in 96-well cell culture plates one day before infection. On the day of infection, serially diluted sera and controls were mixed with 1200 pfu of RSV (Long or B1) and incubated 1 h at 37 °C. Subsequently, virus/antibody mixtures were transferred to 96-well plates containing VERO cell monolayers. Three days later, the monolayers were fixed with cold 80% acetone and the RSV antigen was determined with an anti-F monoclonal antibody. The neutralizing titer is expressed as the serum dilution (log2), which causes a 50% reduction in OD450 from virus-only control wells (IC50).
[000127] At week 2 and week 8 after the initial dose the animals were sacrificed and cellular and humoral responses were monitored as described above.
[000128] Fig. 1 shows that all doses of Ad26-RSV.F (Fig. 1A) and Ad35-RSV.F (Fig. 1B) were effective in inducing a good cellular immune response and that the responses were stable over time. No significant vector dose differences were observed in T cell response to either Ad26-RSV.F or Ad35-RSV.F.
[000129] Fig. 2 shows antibody titers in the same experiment as described above. Both vectors induced very clear and dose-dependent time increases in ELISA titers (Fig. 2). Anti-F titers clearly increased from 2 to 8 weeks, which was significant for the 1010 dose. At 8 weeks, there was no difference in titers between Ad26-RSV.F and Ad35-RSV.F vector.
The F-specific IgG subclass (IgG1 vs IgG2a) distribution was determined in order to assess the balance of the Th1 vs Th2 response. A skewed Th2/Th1 response predisposes animals to develop vaccine-aggravated RSV disease, as seen with formalin-inactivated RSV. As shown in Fig. 3, the IgG2a/IgG1 ratio for both Ad26-RSV.F and Ad35-RSV.F is greater than 1. This strongly indicates that Ad26-RSV.F and Ad35-RSV.F adenovectors exhibit a Th1 response type rather than a Th2 type.
[000131] Fig. 4 shows the virus neutralizing (VNA) titers of the same sera used for the antibody titers. Immunization with Ad26-RSV.F and rAd35-RSV.F led to the induction of neutralizing antibody titers. VNA titers increased strongly between two and eight weeks after boosting in mice that received 1010 vp. At eight weeks, there was no difference in titers between Ad26-RSV.F and Ad35-RSV.F vectors in mice that received 1010 vp.
[000132] From these immunization experiments it is evident that Ad35 and Ad26 vectors harboring the RSV.F transgene induce strong cellular and humoral responses against RSV.F. Example 3. Immunity against RSV.F after initial vaccination - heterologous boost using recombinant adenovirus vectors encoding RSV.F.
[000133] This study was designed to investigate the ability of prime-boost vaccination regimens based on adenovirus vectors derived from two different serotypes to induce immunity against RSV.F.
[000134] This study involved BALB/c mice distributed in experimental groups of 8 mice. Animals were immunized by intramuscular injection with 1010 vp carrying the wild-type sequence of the RSV.F gene based on/derived from RSV A2 (Ad-RSV.F or Ad35-RSV.F) or without transgene (Ad26e or Ad35e ). One group of animals was initially vaccinated at week with Ad26-RSV.F and boosted at week 4 with Ad35-RSV.F or Ad35e. Another group of animals was initially vaccinated with Ad35-RSV.F and boosted at week 4 with Ad26-RSV.F or Ad26e. A control group of mice was initially vaccinated with Ad35e and boosted at week 4 with Ad26e. At week 6 and week 12 after the initial vaccination, 8 animals were sacrificed at each time point and cellular and humoral responses were monitored with immunoassays well known to those skilled in the art and as described above.
[000135] Fig. 5 shows the cellular response at 6 and 12 weeks after the first immunization. At 6 weeks after the initial vaccination (and 2 weeks after the booster), a significant booster effect was measured by both Ad26-RSV.F and Ad35-RSV.F on T cell responses, and the magnitude of the cell response T was independent of the order of immunization with Ad26-RSV.F or Ad35-RSV.F in the prime-boost vaccination. At 12 weeks after the initial vaccination (and 8 weeks after the boost), mice immunized with Ad26-RSV.F maintained higher levels of F-specific T cells in both prime- and prime-boost-only animals, compared to with animals only with initial vaccination with rAd35-RSV.F. Overall, F-specific lymphocyte (SFU) numbers were elevated and stable for at least 12 weeks in all animals immunized with either rAd26-RSV.F or rAd35-RSV.F (prime vaccination/or prime-boost vaccination).
[000136] Fig. 6 shows the humoral response at different time points after the prime-boost vaccination with adenovirus vectors. Ad35.RSV.F and Ad26.RSV.F conferred equally good immunization, and a significant booster effect induced by either Ad26.RSV.F or rAd35.RSV.F on B cell responses was shown. B cell response to heterologous prime-boost vaccination was independent of the order of immunization with Ad35.RSV.F and Ad26.RSV.F, and after the boost ELISA titers were stable for 12 weeks.
[000137] Fig. 7 shows the titer of virus neutralizing antibodies at different time points after immunization by prime-boost vaccination. Both Ad35.RSV.F and Ad26.RSV.F vectors gave equally good immunization in order to obtain clear VNA titers, as seen in the ELISA titers. Furthermore, the increase in VNA titers after the initial heterologous booster vaccination was independent of the order of immunization with Ad35.RSV.F and Ad26.RSV.F. The effect of boosting either by Ad26.RSV.F or Ad35.RSV.F on VNA titers was significant at both time points and was already maximal at 6 weeks. Groups that were only initially vaccinated with Ad.RSV.F increased VNA titers at 12 weeks compared to 6 weeks. The RSV F sequence in the adenovirus vector constructs is derived from the isolated RSV A2. The neutralization assay described in this application is based on the RSV Longo strain, belonging to the RSV subgroup A, which demonstrates that antibodies induced by F (A2) are capable of cross-neutralizing a different subtype of A strain.
[000138] Since the F protein of RSV is well conserved among the isolated RSVs, it was tested whether the sera from animals immunized with Ad-RSV.F vectors were able to neutralize by crossing an isolated strain of RSV B, RSV B1. As shown in Fig. 8, sera from immunized mice were also able to cross-neutralize the B1 strain. The ability to cross-neutralize RSV B 1 was not dependent on the vector that was used in the prime-vaccination-only groups or on the order of immunization by prime-boost vaccination with the Ad26.RSV.F and Ad35.RSV.F vectors.
[000139] Taken together, these data show that, in a prime-boost vaccination regimen, consecutive immunizations with Ad26.RSV.F and Ad35.RSV.F induced strong humoral and cellular responses, whereas the humoral immune response includes the capacity to neutralize both isolated RSV subtypes A and B. Example 4. Induction of protection against RSV infection using recombinant adenovirus vectors in vivo in a cotton rat model.
[000140] This experiment was carried out to investigate the ability of prime-boost vaccination regimens based on adenovirus vectors derived from two different serotypes to induce protection against RSV threat replication in the cotton rat. Cotton rats (Sigmodon hispidus) are susceptible to both upper and lower respiratory tract infection with RSV, and have been found to be at least 50 times more permissive than mouse strains (Niewiesk et al, 2002, Lab. Anim 36(4):357-72). In addition, the cotton mouse has been the primary model for determining the efficacy and safety of RSV vaccine, antiviral and antibody candidates. Preclinical data generated in the cotton rat model advanced the development of two-antibody formulations (RespiGam® and Synagis®) for clinical trials, without the need for intermediate studies in non-human primates.
[000141] The study enrolled cotton rats in experimental groups of 8 cotton rats each. Animals were immunized with intramuscular injections of 109 viral particles (vp) or 1010 vp adenovirus vectors carrying the full length of the RSV F gene (A2) (Ad26.RSV.F or Ad35.RSV.F) or without transgene (Ad26e or Ad35e). The animals were boosted 28 days later with the same vp dose, either with the same vector (homologous prime-boost vaccination) or with another adenovirus serotype (heterologous prime-boost vaccination); control groups were immunized according to Ad-e vectors, except that only 1 dose (1010) was applied. Control groups consisted of 6 animals. Animals intranasally infected with RSV A2 (104 plaque-forming units (pfu)) were used as a positive control for protection against threat replication, as it is known that the major infection with the RSV virus protects against replication of secondary threat (Prince. Lab Invest 1999, 79:1385-1392). In addition, formalin-inactivated RSV (FI-RSV) served as a control for vaccine-aggravated RSV disease. Three weeks after the second immunization (boost), cotton rats were challenged intranasally with 1x105 pfu plaque purified RSV A2. As controls, one group of cotton rats was not immunized but received the threat virus, and another control group was not immunized and did not receive the threat. Cotton rats were sacrificed 5 days after infection, a point in time at which the threat virus RSV reaches maximum titers (Prince. Lab Invest 1999, 79:1385-1392), and lung and nose RSV titers were determined by virus plaque titration (Prince et al. 1978, Am J Pathology 93,711-791).
[000142] Fig. 9 shows that high titers of RSV virus were observed in lungs and nose in unimmunized controls, as well as in animals that received the transgene-free adenovirus vectors, respectively 5.3 +/- 0.13 log10 pfu/ gram and 5.4 +/- 0.35 log10 pfu. On the other hand, no threat virus could be detected in lung and nose tissue of animals that received immunization by prime-boost vaccination with Ad26.RSV.F and/or Ad35.RSV.F vectors, independent of dose or regime.
[000143] These data clearly show that both Ad35-based and Ad26-based vectors provide complete protection against replication of the RSV threat in the cotton rat model. This was surprising since Ad5-based adenovirus vectors encoding RSV F were known to not be able to induce complete protection in animal models after intramuscular administration.
[000144] During the course of the experiment, blood samples were collected before immunization (day 0), before booster immunization (day 28), on the day of threat (day 49) and on the day of sacrifice (day 54). Sera were tested in a virus neutralization assay (VNA) based on the plaque assay for the induction of systemic RSV-specific neutralizing antibodies as described by Prince (Prince et al. 1978, Am J Pathology 93,711-791). The neutralizing titer is expressed as the serum dilution (log2), which causes a 50% reduction in plaques compared to control wells with virus alone (IC50).
[000145] Fig. 10 shows that control animals do not have virus neutralizing antibodies on day 28 and day 49, while high VNA titers are induced after animals have been initially vaccinated with either Ad26.RSV.F or Ad35 vectors .RSV.F. A moderate increase in VNA titer is observed after booster immunizations. Initial infection with the RSV A2 virus resulted in very moderate VNA titers that gradually increased with time.
[000146] In order to assess whether the Ad26.RSV.F or Ad35.RSV.F vaccine can exacerbate the disease after a threat with RSV A2, histopathological analyzes of the lungs were performed 5 days after the infection. The lungs were removed, perfused with formalin, sectioned and stained with hematoxylin and eosin for histological examination. Histopathological examination scoring was performed in a blinded trial, according to criteria published by Prince (Prince et al. Lab Invest 1999, 79:1385-1392), and scored for the following parameters: peribronchiolitis, perivasculitis, interstitial pneumonitis and alveolitis. Fig. 11 shows the pulmonary pathology score from this experiment. After the threat with RSV, animals immunized with FI-RSV showed high histopathology in all histopathological parameters analyzed, compared to animals threatened with empty immunization, which was expected based on previously published studies (Prince et al. Lab Invest 1999, 79:1385-1392). Histopathological scores in immunized with Ad26.RSV.F and Ad35.RSV.F compared to animals immunized with rAd-e or empty immunization were similar, although perivasculitis in animals immunized with rAd-RSV.F appears to be somewhat smaller. Thus, Ad26.RSV.F and Ad35.RSV.F vaccines did not result in aggravated disease, unlike FI-RSV vaccines.
[000147] All vaccination strategies resulted in complete protection against replication of the RSV threat, induced strong virus neutralizing antibodies, and no aggravated pathology was observed. Example 5. Protective efficacy of rAd vectors using different routes of administration, after a single immunization
[000148] This study serves to investigate the influence of routes of administration on the protective efficacy induced by Ad26 or Ad35 vectors encoding RSV.F. The vaccine was administered both intramuscularly and intranasally.
Cotton rats received a single immunization with 1x109 or 1x1010 viral particles (vp) of Ad26 or Ad35 carrying either RSV F or transgene (Ad26.RSV.F or Ad35.RSV.F) or without transgene (Ad26- and or Ad35-e) on day 0, were threatened on day 49 with 105 RSV pfu and sacrificed on day 54.
[000150] Fig. 12 shows the results of experiments in which the lung and nose threat viruses were determined. High titers of RSV virus were detected in the lungs and nose of mice that were not immunized or were immunized with adenovirus vectors without a transgene, respectively 4.9 +/- 0.22 log10 pfu/gram and 5.4 +/- 0.16 log10 pfu. In contrast, the lungs and noses of animals that received both Ad35-RSV.F and Ad26-RSV.F were devoid of threat virus replication, regardless of route of administration and dose.
[000151] These data surprisingly demonstrated that each of the Ad26-based and Ad35-based vectors encoding the RSV F protein confer complete protection in cotton rat threat experiments, regardless of the route of administration of the vectors. This was unexpected, as none of the published adenovirus-based RSV vaccines, which were based on other serotypes, demonstrated complete protection after intramuscular vaccination.
[000152] During the experiment, blood samples were collected before immunization (day 0), 4 weeks after immunization (day 28), and on the threat day (day 49). Sera were tested in a neutralization test for the induction of antibodies specific for RSV (Fig 13). Prior to immunization no virus neutralizing antibodies were detected in any cotton rats. All adenovirus vector immunization strategies, regardless of the route of administration, clearly induced high titers of VNA, which remained stable over time. These data surprisingly demonstrated that each of the Ad26-based and Ad35-based vectors encoding the RSV F protein confer high titers of virus neutralizing antibodies in cotton rat immunization experiments, irrespective of the route of administration of the vectors.
[000153] In order to assess whether a single immunization with the Ad26.RSV.F or Ad35.RSV.F vaccine can cause a vaccine aggravated disease after a threat with RSV A2, histopathological analyzes of the lungs were performed 5 days after the infection (Fig. 14). Single immunization with rAd26.RSV.F or rAd35.RSV.F resulted in similar immunopathological scores in animals immunized with rAd26.RSV.F or rAd35.RSV.F compared to rAd-e or in animals with empty immunization, as observed in the prime-boost vaccination experiments described above. Clearly, exacerbated disease was not observed, in contrast to animals that were immunized with FI-RSV. Histopathological scores of animals immunized with rAd vectors were comparable to "empty" infected animals.
[000154] In conclusion, all single-dose vaccination strategies resulted in complete protection against replication of the RSV threat, induced strong virus neutralizing antibodies, and showed no improved pathology. Example 6. Vectors with variants such as RSV F fragments or with alternative promoters show similar immunogenicity
[000155] The previous examples were performed with vectors that express the wild type of F of RSV. Others, truncated or modified forms of F were performed in rAd35, conferring modalities of F fragments of RSV in adenovirus vectors. Such truncated or modified forms of F include a truncated form of RSV-F, in which the cytoplasmic domain and transmembrane region were missing (i.e., only the ectodomain fragment remained), and a fragment form of RSV-F with truncation of the cytoplasmic domain and the transmembrane region and an additional internal deletion in the ectodomain and addition of a trimerization domain. These vectors did not improve responses on rAd35.RSV.F with full-length F protein.
[000156] In addition, other rAd35 vectors with different alternative promoters driving wild-type expression of RSV F were constructed.
[000157] The immunogenicity of modified forms of RSV. F and promoter variants were compared in the mouse model and compared to Ad35.RSV.F which expresses wild type F. All Ad35 vectors harboring these F variants or promoter variants showed responses in the same order of magnitude as Ad35. RSV. F. Example 7. Short term protection against RSV infection after immunization by recombinant adenovirus vectors in vivo in a cotton rat model.
[000158] This experiment determines the potential for rapid onset of protection by adenovirus vectors expressing the RSV F protein in the cotton rat model. For this purpose, cotton rats were immunized with a single i.m. of 107, 108 or 109 viral particles (vp) adenovirus vectors carrying the full length of the RSV F gene (A2) (Ad26.RSV.F) or no transgene (Ad26e) on day 0 or day 21. The infected animals intranasally with RSV A2 (104 plaque-forming units (pfu)) were used as a positive control for protection against the replication of the threat, as it is known that the main infection with the RSV virus protects against the replication of the secondary threat (Prince. Lab Invest 1999, 79:1385-1392). On day 49, seven or four weeks after immunization, cotton rats were challenged intranasally with 1x105 pfu plaque purified RSV A2. Cotton rats were sacrificed 5 days after infection, a point in time at which the threat virus RSV reaches maximum titers (Prince. Lab Invest 1999, 79:1385-1392), and lung and nose RSV titers were determined by virus plaque titration (Prince et al. 1978, Am J Pathology 93,711-791). Fig. 16A and Fig. 16B show that high titers of RSV virus were observed in the lungs and nose in the animals that received the transgene-free adenovirus vectors, respectively 4.8 +/- 0.11 log10 pfu/gram and 5.1 +/- 0.32 log10 pfu. On the other hand, no threat virus could be detected in lung and nose tissue of animals that received immunization with Ad26.RSV.F vectors independent of the time between immunization and threat. This experience clearly indicates the rapid onset of protection against virus threat replication through Ad26 expressing RSV-F. Blood samples were taken from cotton rats immunized on day 0, day 28 and the day of threat (day 49). Sera were tested in a neutralization test for the induction of antibodies specific for RSV (Fig 17). Immunization with dose-dose adenovirus vectors induced dependent VNA titers. Fig. 18 shows that control animals lack virus neutralizing antibodies on day 28 and day 49, while high VNA titers are induced 28 or 49 days after immunizations with 107 to 109 Ad26.RSV.F vp. Initial infection with the RSV A2 virus resulted in very moderate VNA titers that gradually increased with time. This experience clearly indicates the rapid onset of protection against virus threat replication by Ad26 expressing RSV-F. Example 8. Protection against RSV subgroup A and subgroup B infection after immunization by recombinant adenovirus vectors in vivo in a cotton rat model.
[000159] RSV strains can be divided into two subgroups, subgroups A and B. This subgroup division is based on differences in the highly variable G-glycoprotein antigenicity. The F protein sequence is highly conserved, but it can also be classified into the same subgroups A and B. Example 3 described that sera from mice immunized with Ad-RSV.F vectors were also able to cross-neutralize the B1 strain in vitro . Fig. 19 clearly shows that cotton rat serum derived from cotton rats immunized with Ad26.RSV-FA2 shows high VNA titers at day 49 after immunization against RSV-A Long (subgroup A) and wash of B (Subgroup B, ATCC #1540). Next, in vivo protection against both subgroup A or B threats in the cotton rat was determined using low doses of adenovirus vectors in a scale of 106 to 108 vp. For this purpose the cotton rats were divided into experimental groups of 8 cotton rats each. Animals were immunized on day 0 by intramuscular injections of 106, 107 or 108 viral particles (vp) adenovirus vectors carrying the full length of the RSV F gene (A2) (Ad26.RSV.F) or without transgene (Ad26e) on day 0. On day 49 animals were threatened i. n. with either with 10A5 pfu RSV-A2 (RSV-A strain) or RSV-B 15/97 (RSV-B strain). Fig. 20 shows that high titers of RSV virus were observed in the lungs and nose in animals that received transgene-free adenovirus vectors. In contrast, no or limited threat of virus was detected in lung and nose tissue of animals that received immunization with Ad26.RSV.F. Only small differences in protection were observed when threatened with either RSV-A2 or RSV -B 15/97. Ad26.RSV.FA2 showed complete protection against lung threat replication when using doses of 108 and 107vp, and exceptionally limited advance at 106 vp Ad26.RSV.FA2. A similar trend was observed for protection against nose threatening virus replication, although partial advance was observed for all animals at 106 and 107 vp Ad26.RSV.FA2, although less than in the control groups (Fig. 21). During the experiment, blood samples were collected on the day of the threat (day 49). Sera were tested in a neutralization test for the induction of antibodies specific for RSV (Fig. 22). This example demonstrates that adenovirus vectors at low doses of 106 to 108 vp Ad26.RSV showed a dose response of VNA titers against RSV A2. Prior to immunization no virus neutralizing antibodies were detected in any cotton rats.
[000160] Ad26.RSV.F proved to be somewhat better than Ad35.RSV.F as the latter showed some advance in nasal threat experiences with a dose of 108 vp. Example 9. Protection against a high threat dose of RSV-A2 after immunization by recombinant adenovirus vectors in vivo in a cotton rat model.
[000161] This example determines protection against a high threat dose of 5x105 pfu compared to the standard dose of 1x105 pfu of RSV-A2. The study enrolled cotton rats in experimental groups of 8 cotton rats each. Animals were immunized by single intramuscular injections of 107, or 108 viral particles (vp) adenovirus vectors carrying the full length RSV F gene (A2) (Ad26.RSV.F) or no transgene (Ad26e) on day 0 Animals infected intranasally with RSV A2 (104 plaque-forming units (pfu) were used as a positive control for protection against threat replication. Cotton rats were sacrificed 5 days after infection, and RSV titers were determined of the lung and nose by titration of the virus plaque. Fig. 23 shows that a higher threat dose induces a higher pulmonary viral load in animals that received the transgene-free adenovirus vectors than with the standard threat dose. animals that received the immunization with 107 or 108 vp Ad26.RSV.F vectors were completely protected against the high titers and threat pattern by RSV in the lungs. Fig. 24 shows that the animals that received the immunization with 1 08 vp Ad26.RSV.F vectors were completely protected against high and standard nose RSV threat titers, while animals that received immunization with 107 vp Ad26.RSV.F vectors were partially protected against standard RSV threat titers. Example 10. Long-term protection against RSV-A2 and RSV-B15/97 after immunization by recombinant adenovirus vectors in vivo in a cotton rat model.
[000162] This example determines the durability of protection against RSV-A2 and RSV-B15/97 after immunization by recombinant adenovirus vectors in vivo in a cotton rat model. The study enrolled cotton rats in experimental groups of 6 cotton rats each. Animals were immunized with intramuscular injections of 108 viral particles (vp) or 1010 vp adenovirus vectors carrying the full length of the RSV F gene (A2) (Ad26.RSV.F) or without transgene (Ad26e or Ad35e). The animals were boosted 28 days later with the same vp dose, either with the same vector (Ad26.RSV.F) (homologous prime-boost vaccination) or with Ad35.RSV.F adenovirus (heterologous prime-boost vaccination); control groups were immunized according to Ad-e vectors, except that only 1 dose (1010) was applied. Some groups did not receive a booster immunization. Control groups consisted of 6 animals. Animals intranasally infected with RSV A2 and B15/97 (104 plaque-forming units (pfu) were used as a positive control for protection against threat replication.The threat was 210 days after the first immunization.
[000163] Fig. 25 shows that high titers of RSV virus were observed in the lungs and nose in animals that received transgene-free adenovirus vectors. In contrast, no threat of virus was detected in the lung tissue of animals that received immunization with Ad26.RSV.F and/or Ad35.RSV.F. No threat of RSV-A2 virus was detected in the nose tissue of animals that received immunization with Ad26.RSV.F and/or Ad35.RSV.F. The threat with RSV-B15/97 induced limited viral replication in the nasal tissues of animals that received immunization with Ad26.RSV.F and/or Ad35.RSV.F, except in animals that received an initial vaccination with Ad26.RSV.F followed by by a boost of Ad35.RSV.F with 1010 vp. Fig. 26 shows the virus neutralizing antibody titer 140 days after immunization. Only the initial adenovirus vector vaccination or immunization by initial vaccination and booster with doses of 108 and 1010 vp showed a dose response of the durable VNA titers for at least 4.5 months after immunization. In addition, the observed titers were higher than the neutralizing titers generated by immunization i. n. initial. A clear boosting effect was observed by both Ad26.RSV.F and Ad35.RSV.F on VNA titers.
[000164] In conclusion, these examples show long-lasting VNA titers after immunization with single or double doses of Ad26.RSV.F or Ad35.RSV.F, and long-term integral protection in the lung and nose against the combined homologous virus threat with long-term full protection in the lung and partial protection in the nose against heterologous virus threat. Example 11. Absence of vaccine-aggravated immunopathology after immunization by recombinant adenovirus vectors in vivo in a cotton rat model.
[000165] In order to assess whether the Ad26.RSV.F vaccine can exacerbate the disease after a threat with RSV A2, histopathological analyzes of the lungs were performed 2 and 6 days after the infection. Two days after the threat, the immediate response (including pulmonary neurophilic infiltration) peaks, while subacute changes such as lymphocyte infiltration peak at day 6 post-infection (Prince et al., J Virol, 1986 , 57:721-728). The study enrolled cotton rats in experimental groups of 12 cotton rats each. The animals were immunized with intramuscular injections of 108 viral particles (vp) or 1010 vp adenovirus vectors carrying the full length of the RSV F gene (A2) (Ad26.RSV.F) or without transgene (Ad26e). Some groups were boosted 28 days later with the same vp dose, with the same vector (Ad26.RSV.F) (homologous prime-boost vaccination); control groups were immunized according to Ad-e vectors, except that only 1 dose (1010) was applied. Control groups consisted of 12 animals. Animals intranasally infected with RSV A2 (104 plaque-forming units (pfu)) were used as a positive control for protection against threat replication. Animals immunized with FI-RSV were used as controls for aggravated disease. The lungs were removed, perfused with formalin, sectioned and stained with hematoxylin and eosin for histological examination. Histopathological examination scoring was performed in a blinded trial, according to criteria published by Prince (Prince et al. Lab Invest 1999, 79:1385-1392), and scored for the following parameters: peribronchiolitis, perivasculitis, interstitial pneumonitis and alveolitis. The lung pathology score from this experiment is represented in Fig. 27 for day 2 and in Fig. 28 for day 6. After the threat with RSV, FI-RSV immunized animals showed on day 2 and day 6 elevated histopathology in all histopathological parameters analyzed compared to threatened and empty immunized animals, which was expected based on previously published studies. Histopathological scores in all groups immunized with Ad26.RSV.F vectors were comparable to animals with empty immunization on day 2 and on day 6 post-threat always had lower scores than those threatened with empty immunization (Ad26.e). Thus, Ad26.RSV.F vaccines did not result in aggravated disease, unlike FI-RSV vaccines. Example 12. Initial vaccination with Ad26.RSVF enhanced with recombinant F protein results in a Th1 biased response in a mouse model.
[000166] In this example, it was investigated whether the immune response of the initial Ad26.RSV.F vaccination can be improved by boosting recombinant RSV F protein with adjuvant. For this purpose the mice were divided into experimental groups of 7 mice each. Animals were immunized on day 0 by intramuscular injections of 1010, viral particles (vp) adenovirus vectors carrying the full length RSV F (A2) gene (Ad26.RSV.F) or PBS. On day 28 the animals were boosted i. m. either with the same vector, at the same dose, or with RSV F protein with adjuvant (full length; post-fusion conformation: post-F) (in 2 doses: 5 μg and 0.5 μg). Fig 29 clearly shows that serum derived from mice immunized with Ad26.RSV-FA2 and boosted with RSV F with adjuvant show high VNA titers 12 weeks after immunization against Long VRS-A (subgroup A). Fig. 30 shows the IgG2a/IgG1 ratio in the sera of mice immunized with Ad26.RSV-FA2 and boosted with adjuvanted RSV F protein. A high ratio is indicative of a balanced Th1 response, while a low ratio indicates a skewed Th2 response. Clearly, animals immunized with Ad26.RSV.F, or boosted with Ad26.RSV.F or RSV F protein result in a high IgG2a/IgG1 ratio, whereas control mice immunized with FI-RSV or RSV F protein (without the context of adenovirus vectors) induce a low ratio. Since a skewed Th1 response is strongly desired in an RSV vaccine to prevent aggravated disease under threat and to induce strong T cell memory, the skewed Th2 response of a protein immunization can be directed towards a Th1 response when an initial vaccination with Ad26.RSV.F is applied. Fig. 31 shows the cellular response in spleens taken from mice immunized with Ad26.RSV-FA2 and boosted with RSV F protein with adjuvant. It can be clearly seen that RSV F protein booster, with adjuvant, will also strongly enhance the cellular response.Table 1. Sequences
[000167] SEQ ID NO: 1: RSV fusion protein amino acid sequence (Genbank ACO83301.1):
[000168] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGY LSALRTGWYTSVITIELSNIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLL MQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGV GSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLD LKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTP VSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVL AYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNA GSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQV NEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCK ARSTPVTLSKDQLSGINNIAFSN
[000169] SEQ ID NO: 2: codon optimization of the RSV.F (A2) nat gene encoding the RSV fusion protein
[000170] ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCAC CATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACAT CACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGG GCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATC ACCATCGAGCTGAGCAACATCAAAAAGAACAAGTGCAACGGCACC GACGCCAAAATCAAGCTGATCAAGCAGGAACTGGACAAGTACAAG AACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGC CACCAACAACCGGGCCAGACGGGAGCTGCCCCGGTTCATGAACT ACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGA AGCGGAAGCGGCGGTTCCTGGGCTTCCTGCTGGGCGTGGGCAG CGCCATTGCTAGCGGAGTGGCTGTGTCTAAGGTGCTGCACCTGG AAGGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAAC AAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAG CAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCC CATCGTGAACAAGCAGAGCTGCAGCATCAGCAACATCGAGACAGT GATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCC GCGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGTCCACC TACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATG CCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAG ATCGTGCGGCAGCAGAGCTACTCCATCATGTCCATCATCAAAGAA GAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGAT CGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCA CCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGAC CGGGGCTGGTACTGCGATAATGCCGGCAGCGTGTCATTCTTTCCA CAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGA CACCATGAACAGCCTGACCCTGCCCAGCGAGGTGAACCTGTGCA ACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCT CCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCC ATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAA GAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACG TGTCCAACAAGGGCGTGGACACCGTGTCCGTGGGCAACACCCTG TACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGG CGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGA CGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACC AGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAAT GTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATC ATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGC CTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTC CAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAAC

权利要求:
Claims (14)
[0001]
1. Vaccine against respiratory syncytial virus (RSV), characterized in that it comprises a recombinant human adenovirus serotype 26 which comprises nucleic acid encoding an RSV F protein comprising the amino acid sequence of SEQ ID NO: 1.
[0002]
2. Vaccine according to claim 1, characterized in that the nucleic acid encoding the RSV F protein is codon optimized for expression in human cells.
[0003]
3. Vaccine according to claim 1 or 2, characterized in that the nucleic acid encoding the RSV F protein comprises the nucleic acid sequence of SEQ ID NO: 2.
[0004]
4. Vaccine according to any one of claims 1 to 3, characterized in that the recombinant human adenovirus has a deletion in the E1 region, a deletion in the E3 region, or a deletion in both the E1 and E3 regions of the adenovirus genome .
[0005]
5. Vaccine according to any one of claims 1 to 4, characterized in that the recombinant adenovirus contains a genome comprising at its 5' terminal ends the sequence CTATCTAT.
[0006]
6. Vaccine according to any one of claims 1 to 5, characterized in that it is for use in the vaccination of a subject against RSV.
[0007]
7. Vaccine according to claim 6, characterized in that the vaccine is administered intramuscularly.
[0008]
8. Vaccine according to claim 6 or 7, characterized in that the vaccine is administered to the subject more than once.
[0009]
9. Vaccine according to any one of claims 6 to 8, characterized in that it also comprises administering to the subject a vaccine comprising a recombinant human adenovirus serotype 35, which comprises the nucleic acid encoding the F protein of RSV.
[0010]
10. Vaccine according to claim 6 or 7, characterized in that it consists of a single administration of the vaccine to the subject.
[0011]
11. Vaccine according to any one of claims 6 to 10, characterized in that it further comprises the administration of the F protein of RSV to the subject.
[0012]
12. Vaccine, characterized in that it is for use in reducing RSV infection and/or replication in a subject, by administering to the subject by intramuscular injection a composition comprising a recombinant human adenovirus serotype 26, comprising nucleic acid encoding an RSV F protein comprising the amino acid sequence of SEQ ID NO:1.
[0013]
13. Method for producing a vaccine against respiratory syncytial virus (RSV), characterized in that it comprises providing a recombinant human adenovirus serotype 26, which comprises nucleic acid encoding an RSV F protein comprising the amino acid sequence of SEQ ID NO: 1, propagating said recombinant adenovirus in a host cell culture, isolating and purifying the recombinant adenovirus, and formulating the recombinant adenovirus into a pharmaceutically acceptable composition.
[0014]
14. Isolated recombinant nucleic acid, characterized in that it forms the genome of a recombinant human adenovirus serotype 26 which comprises nucleic acid encoding an RSV F protein comprising the amino acid sequence of SEQ ID NO: 1.

类似技术:

---

公开号 | 公开日 | 专利标题

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BR112014023196B1|2021-09-08|VACCINES AGAINST RESPIRATORY SYNCYCIAL VIRUS |, ITS METHOD OF PRODUCTION, AND ISOLATED RECOMBINANT NUCLEIC ACID

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US9119813B2|2015-09-01|Vaccine against RSV

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US20200360506A1|2020-11-19|Vaccine against rsv

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BR112020004143A2|2020-09-01|method for the safe induction of immunity against respiratory syncytial virus |

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Fu et al.2009|Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

---

BR112014023195B1|2021-05-04|respiratory syncytial virus | vaccine, method for producing a respiratory syncytial virus | vaccine, and isolated recombinant nucleic acid

---

OA17940A|2018-03-12|Vaccine against RSV

---

OA17889A|2018-02-27|Vaccine against RSV.

同族专利:

---

公开号 | 公开日

---

EP2827895A1|2015-01-28|

---

AU2013237424A1|2014-10-16|

---

WO2013139916A1|2013-09-26|

---

WO2013139911A1|2013-09-26|

---

EP2827894A1|2015-01-28|

---

TW201341531A|2013-10-16|

---

SG11201405804YA|2014-10-30|

---

EA201491754A1|2014-12-30|

---

TW201343669A|2013-11-01|

---

CA2867950A1|2013-09-26|

---

MY169352A|2019-03-25|

---

MY169331A|2019-03-21|

---

AP2014007994A0|2014-10-31|

---

AU2013237424B2|2017-07-06|

---

EA026504B1|2017-04-28|

---

KR20140138765A|2014-12-04|

---

KR102050616B1|2019-12-03|

---

SG11201405803PA|2014-11-27|

---

CA2867955A1|2013-09-26|

---

AR090470A1|2014-11-12|

---

EA026620B1|2017-04-28|

---

EP2827894B1|2017-05-17|

---

TWI640534B|2018-11-11|

---

EP2827895B1|2017-08-09|

---

JP2015512380A|2015-04-27|

---

NZ630649A|2016-12-23|

---

AU2013237429B2|2015-07-23|

---

KR20140138769A|2014-12-04|

---

NZ630753A|2016-12-23|

---

AU2013237429A1|2014-10-09|

---

CN105431169B|2019-04-02|

---

CN104334188B|2016-08-24|

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JP2015519295A|2015-07-09|

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CN105431169A|2016-03-23|

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KR102023791B1|2019-09-23|

---

CN104334188A|2015-02-04|

---

AP2014007993A0|2014-10-31|

---

JP5845376B2|2016-01-20|

---

TWI624545B|2018-05-21|

---

AR090469A1|2014-11-12|

---

BR112014023196A2|2017-06-20|

---

JP5805908B2|2015-11-10|

---

EA201491752A1|2015-02-27|

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-08-21| B25D| Requested change of name of applicant approved|Owner name: JANSSEN VACCINES AND PREVENTION B.V (NL) |

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2018-09-25| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-08-06| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-03-03| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-06-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-29| B25G| Requested change of headquarter approved|Owner name: JANSSEN VACCINES AND PREVENTION B.V. (NL) |

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2021-07-20| B09X| Republication of the decision to grant [chapter 9.1.3 patent gazette]|

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2021-09-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 21/03/2013, OBSERVADAS AS CONDICOES LEGAIS. |

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EP12160682|2012-03-22|

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US61/614429|2012-03-22|

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PCT/EP2013/055935|WO2013139911A1|2012-03-22|2013-03-21|Vaccine against rsv|

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