KR20190107182A - Vaccine against rsv - Google Patents

Abstract

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

Description

Vaccine against RSV {VACCINE AGAINST RSV}

The present invention relates to the field of medicine. In particular, the present invention relates to a vaccine against RSV.

After the discovery of respiratory syncytial virus (RSV) in the 1950s, the virus was soon recognized in humans as a pathogen associated with upper respiratory tract infections. Worldwide, it is estimated that 64 million cases of RSV infection occur each year, killing 160,000 people (WHO Acute Respiratory Infection Update September 2009). The most serious diseases occur especially in premature infants, the elderly and individuals with immune dysfunction. In infants under 2 years of age, RSV is the most common respiratory pathogen, accounting for approximately 50% of hospitalizations due to respiratory infections, with hospitalization peaking at 2 to 4 months of age. Almost all children have been reported to be infected with RSV at the age of 2 years. Repeated infection throughout life is due to ineffective natural immunity. The degree of RSV disease burden, mortality and morbidity in the elderly is comparable to those resulting from nonpandemic influenza A infection. RSV is a paramyxovirus belonging to the subfamily of pneumovirinae. Its genome encodes a variety of proteins, including membrane proteins known as RSV glycoprotein (G) and RSV fusion (F) protein, which are major antigenic targets for neutralizing antibodies. Proteolytic cleavage of the fusion protein precursor (F0) results in the release of two polypeptides, F1 and F2, linked via a disulfide bridge. Antibodies to the fusion-mediated portion of the F1 protein can have a neutralizing effect by preventing virus uptake in cells. In addition to being a target for neutralizing antibodies, RSV F contains cytotoxic T cell epitopes (Pemberton et al., 1987, J. Gen. Virol . 68: 2177-2182).

Treatment options for RSV infection include monoclonal antibodies to the F protein of RSV. The high cost associated with such monoclonal antibodies and the requirements for administration in hospital settings preclude their use for large-scale prophylaxis in large populations at risk. There is a need for a RSV vaccine that can be preferably used for the elderly as well as the pediatric population.

Despite 50 years of research, there is still no approved vaccine for RSV. One major obstacle to vaccine development is the legacy of vaccine-enhanced disease in clinical trials of formalin-inactivated (FI) RSV vaccines in the 1960s. FI-RSV vaccinated children were not protected against spontaneous infection, and infected children experienced more serious illness than non-vaccinated children, including two deaths. This phenomenon is referred to as'enhanced disease'.

Since testing with FI-RSV vaccines, various approaches have been pursued to generate RSV vaccines. These attempts include classic live attenuated cold passaged or temperature sensitive mutant strains of RSV, (chimeric) protein subunit vaccines, peptide vaccines and RSV protein expressed from recombinant viral vectors. These are included. Some of these vaccines have shown promising pre-clinical data, but no vaccines have been approved for human use due to safety concerns or lack of efficacy.

Adenovirus vectors are used in the manufacture of vaccines against a variety of diseases, including diseases associated with RSV infections. The following paragraphs provide examples of the described adenovirus-based RSV candidate vaccines.

In one approach, RSV.F was inserted into the non-essential E3 region of replicable adenovirus types 4, 5 and 7. Immunization in cotton rats with an intranasal (in) application of 10 ⁷ pfu showed mild immunity and protected the lower respiratory tract against RSV attack, but did not protect against RSV attack in the upper respiratory tract (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 efficacy of recombinant vaccinia viruses and adenoviruses that express respiratory syncytial virus glycoproteins. 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, pp 79-84). Oral immunization of the ensuing chimpanzee immune insufficient (Hsu, etc., 1992, J Infect Dis 66: . 769 - 775).

In other studies (Shao et al. , 2009, Vaccine 27: 5460-71; US2011/0014220), transmembrane truncated (rAd-F0ΔTM) or full-length (rAd-F0) versions of the F protein of the RSV-B1 strain were used. Two recombinant replication-deficient adenovirus 5 vectors carrying the encoding nucleic acid were engineered and given to BALB/c mice via an intranasal route. Animals were ^{initially administered 10 7} pfu intranasally, followed by additional intranasal administration at the same dose 28 days later. Anti-RSV-B1 antibodies neutralized and cross-reacted with RSV-Long and RSV-A2 strains, but immunization with these vectors only partially protected against RSV B1 attack replication. The (partial) protection with rAd-F0ΔTM was slightly higher than that of rAd-F0.

In another study, intranasal immunization of BALB/c mice ^{with 10 11} viral particles using replication-deficient (Ad5-based) FG-Ad adenovirus expressing wild-type RSV F (FG-Ad-F), control It was observed to reduce lung virus titers by only 1.5 log 10 compared to (Fu et al., 2009, Biochem. Biophys. Res. Commun. 381: 528-532).

In other studies, a recombinant Ad5-based replication-deficient adenovector applied intranasally expressing a codon optimized soluble F1 fragment of the F protein of RSV A2 (amino acids 155-524) (10 ^{8 PFU) was compared to control mice.} Thus, it was possible to reduce the replication of RSV attack in the lungs of BALB/c mice, but it was observed that mice immunized by the intramuscular (im) route did not show any protection from attack (Kim et al. , 2010, Vaccine 28: 3801. -3808).

In other studies, 1 x 10 ¹⁰ OPU (optical particle units (optical particle units ( optical particle units): BALB/c mice were immunized twice with a dose of ^{1 x 10 10 OPU, which corresponds to a 2 x 10 8 GTU (gene transduction unit).} These vectors strongly reduced the viral loads in the lungs after intranasal immunization, but only partially after subcutaneous (sc) or intramuscular application (Kohlmann et al., 2009, J Virol 83: 12601- 12610; US 2010/0111989).

In other studies, a recombinant Ad5-based replication-deficient adenovector applied intramuscularly expressing the sequenced F protein cDNA of the RSV A2 strain (10 ^{10 particle units) was compared to control mice in the lungs of BALB/c mice.} It has been observed that replication of RSV attacks can be reduced only partially within (Krause et al. , 2011, Virology Journal 8:375-386).

Most of the pediatric clinically evaluated RSV vaccines and preclinical evaluation vaccines are intranasal vaccines, except in many cases not being sufficiently effective. The most important advantages of the intranasal strategy are the lack of direct stimulation of local airway immunity and associated disease strengthening. In fact, in general, the efficacy of, for example, adenovirus-based RSV candidate vaccines appears to be superior to intranasal administration compared to intramuscular administration. However, intranasal vaccination also raises safety concerns in infants younger than 6 months. The most common adverse reactions of intranasal vaccines are runny nose or stuffy nose at all ages. Since newborns are absolute nasal breathers, they must breathe through the nose. Therefore, in the first few months of an infant's birth, stuffy nose can interfere with lactation and, in very rare cases, can cause serious breathing problems.

More than 50 different human adenovirus serotypes have been identified. Of these, adenovirus serotype 5 (Ad5) has historically been the most extensively studied for its use as a gene carrier. However, recombinant adenovirus vectors of different serotypes can lead to different consequences for the induction and protection of immune responses. For example, WO 2012/021730 describes that simian adenovirus vector serotype 7 and human adenovirus vector serotype 5 encoding F protein provide better protection against RSV than human adenovirus vectors of serotype 28. have. In addition, differential 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 . concluded that all rare serotype human rAd vectors studied had lower titers than rAd5 vectors in the absence of anti-Ad5 immunity. In addition, rAd5 with Ebolavirus (EBOV) glycoprotein (gp) transgene protected 100% of non-human primates, and rAd35 and rAd26 with EBOV glycoprotein transgene provided only partial protection, and complete protection against Ebola virus attack. It was recently described that a heterogeneous prime-boost strategy using these vectors is required to obtain protection (Geisbert et al., 2011, J Virol 85: 4222-4233). Therefore, it is a priori impossible to predict the efficacy of a recombinant adenovirus vaccine based solely on data from different adenovirus serotypes.

In addition, for RSV vaccines, cotton is used to determine whether the vaccine candidate is efficacious enough to prevent replication of RSV in the nasal tract and lungs, and at the same time is safe, i.e., does not lead to enhanced disease. Testing is required in appropriate disease models such as rats. Preferably, such candidate vaccines should be highly efficacious in such models, even in intramuscular administration.

Therefore, there is still a need for efficient vaccines and vaccination methods for RSV that do not lead to enhanced disease. The present invention aims to provide such vaccines and vaccination methods against RSV in a safe and efficacious manner.

We found that recombinant adenoviruses of serotype 26 (Ad26), containing the nucleotide sequence encoding the RSV F protein, are highly effective vaccines against RSV in the established cotton rat model, and previously against Ad5 encoding RSV F. It has been surprisingly found by the inventors to have an improved efficacy compared to the data described. It has been demonstrated that a single intramuscular administration of Ad26, which encodes RSV F, is sufficient to provide complete protection against aggressive RSV replication.

The present invention provides a vaccine against RSV comprising a recombinant human adenovirus of serotype 26 comprising a nucleic acid encoding a respiratory syncytial virus (RSV) F protein or a fragment thereof.

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

In certain embodiments, the nucleic acid encoding the RSV F protein is a codon optimized 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.

In certain embodiments, the recombinant human adenovirus has a deletion in the E1 region, in the E3 region, or in both the E1 and E3 regions of the adenovirus genome.

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

The present invention further provides a method of vaccinating a subject against RSV, comprising the step of inoculating the subject with the vaccine according to the present invention.

In certain embodiments, the vaccine is administered intramuscularly.

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

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

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

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

In addition, the present invention comprises the step of administering to the subject by intramuscular injection of a composition comprising a recombinant human adenovirus of serotype 35 comprising a nucleic acid encoding RSV F protein or a fragment thereof, in a subject, for example, A method of reducing the infection and/or replication of RSV in the nose and lungs of a subject is provided. This will reduce adverse effects due to RSV infection in the subject, thus contributing to the subject's protection against such adverse effects upon administration of the vaccine. In certain embodiments, adverse effects of RSV infection can be essentially prevented, ie, reduced to low levels where they are not clinically problematic. The recombinant adenovirus may be in the form of a vaccine according to the present invention, including the embodiments described above.

In addition, the present invention provides an isolated host cell comprising a recombinant human adenovirus of serotype 35, comprising a nucleic acid encoding a respiratory syncytial virus (RSV) F protein or a fragment thereof.

The present invention provides a recombinant human adenovirus of serotype 35 comprising a nucleic acid encoding RSV F protein or fragment thereof, propagating the recombinant adenovirus in a culture of host cells, isolating the recombinant adenovirus It further provides a method for preparing a vaccine against respiratory syncytial virus (RSV), comprising the steps of purifying and purifying, and formulating a recombinant adenovirus in a pharmaceutically acceptable composition. In addition, the recombinant human adenovirus of this aspect may be any one of the adenoviruses described in the above embodiments.

In addition, the present invention provides an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 35 comprising a nucleic acid encoding the RSV F protein or fragment thereof. Further, the adenovirus may be any one of the adenoviruses as described in the above embodiments.

Figure 1 shows F peptides overlapping with aa 1-252 of F of mice 2 and 8 weeks after immunization when immunized with different doses of rAd26 (a) and rAd35 (b) based vectors carrying the RSV F gene and F Shows the cellular immune response to F peptides overlapping with aa 241-574 of.
Figure 2 shows the antibody responses to RSV in mice 2 and 8 weeks after immunization when immunized with different doses of rAd26 and rAd35 based vectors carrying the RSV F gene.
3 shows the results of the ratio of IgG2a to IgG 1 antibody response to RSV in mice 2 and 8 weeks after immunization when immunized with ^{10 10} vp of rAd26 and rAd35 based vectors carrying the RSV F gene.
Figure 4 shows the viral neutralizing dose for RSV Long in mice 2 and 8 weeks after immunization when immunized with different doses of rAd26 (A) and rAd35 (B) based vectors carrying the RSV F gene.
FIG. 5 shows (a) F peptides overlapping with aa 1-252 of F of mice at 6 and 12 weeks after primary immunization when immunizing with rAd26 and rAd35-based vectors carrying RSV F gene for the first time. And (b) F peptides overlapping with aa 241-574 of F.
Figure 6 shows the antibody response to RSV of mice at different time points after the first immunization when the first additional immunization with rAd26 and rAd35 based vectors carrying the RSV F gene.
Figure 7 shows the virus neutralizing dose of serum of mice to RSV Long at different time points after the first immunization when first boosting with different doses of rAd26 and rAd35 based vectors carrying the RSV F gene.
Figure 8 shows the viral neutralizing dose of mice to RSV B1 at different time points after the first immunization when first boosting with different doses of rAd26 and rAd35 based vectors carrying the RSV F gene.
Figure 9 shows A) RSV lung titers and B) RSV nasal titers of cotton mice following initial further immunization with different doses of rAd26 and rAd35 based vectors carrying the RSV F gene 5 days after challenge.
FIG. 10 shows the induction of viral neutralizing titers following initial further immunization with different doses of rAd26 and rAd35 based vectors bearing the RSV F gene on days A) 28 and B) 49 after the first immunization.
FIG. 11 shows histopathological examination of cotton rat lungs on the day of sacrifice of cotton rats following initial booster immunization with different doses of rAd26 and rAd35 family vectors carrying the RSV F gene.
Figure 12 shows A) RSV lung titers and B) RSV nasal titers of cotton mice following a single dose immunization with different doses of rAd26 and rAd35 based vectors carrying the RSV F gene 5 days after challenge, administered via different routes. Shows.
Figure 13 shows the induced viral neutralization titers following a single dose immunization with different doses of rAd26 and rAd35 based vectors bearing the RSV F gene on days 28 and 49 after the first immunization, administered via different routes.
14 shows histopathological examination of cotton rat lungs on the day of sacrifice of cotton rats following single-dose immunization (intramuscular) with different doses of rAd26 and rAd35 based vectors carrying the RSV F gene on the day of sacrifice.
Figure 15 is a map of plasmids comprising the left end of the genome of Ad35 and Ad26 with sequences encoding RSV F: A. pAdApt35BSU.RSV.F(A2)nat, and B. pAdApt26.RSV.F(A2)nat Show them.
Figure 16 shows A) RSV lung titers and B) RSV nasal titers of cotton mice following single dose immunization on day 0 or 21 with different doses of rAd35 based vectors carrying the RSV F gene. The attack was on day 49, and the sacrifice was on day 54.
FIG. 17 depicts the induction of viral neutralizing titers following single dose immunization with different doses of rAd35 bearing the RSV F gene on day 49 post immunization as described for FIG. 16.
Figure 18 shows the induction of viral neutralizing titers following single dose immunization with different doses of rAd35 bearing the RSV F gene during the post-immunization period.
Figure 19 shows VNA titers after 49 days for RSV Long and RSV Bwash with sera derived from cotton mice immunized with Ad-35RSV F 10 ^{10 or without a transgene (Ad-e).} PB: First addition.
FIG. 20 shows RSV lung titers of cotton mice following single dose immunization at day 0 with different doses of rAd35 family vectors carrying the RSV F gene 5 days after challenge with RSV A2 or RSV B15/97.
Figure 21 shows RSV nasal titers of cotton mice after single dose immunization on day 0 with different doses of rAd26 family vectors bearing the RSV F gene on day 5 after challenge with RSV A2 or RSV B15/97.
Figure 22 shows the VNA titers of cotton mice after single dose immunization on day 0 with different doses of rAd35 based vectors bearing the RSV F gene during the time post immunization.
Figure 23 shows cotton mice following single-dose immunization at day 0 with different doses of rAd35-based vectors bearing the RSV F gene on day 5 after challenge with standard dose (10 ⁵ ) or high dose (5 x 10 ^{5) RSV A2.} RSV lung titers are shown.
Figure 24 shows cotton mice following single-dose immunization at day 0 with different doses of rAd35-based vectors carrying the RSV F gene on day 5 after challenge with standard dose (10 ⁵ ) or high dose (5 x 10 ^{5) RSV A2.} RSV nasal titers are shown.
Figure 25 shows the induction of viral neutralizing titers after immunization with rAd26 carrying the RSV F gene (Ad26.RSV.F) followed by further administration with Ad26.RSV.F or with the supplemented RSV F protein (post-F).
FIG. 26 shows the induction of IgG2a and IgG1 antibodies and their ratio after further administration with Ad26.RSV.F after immunization with Ad26.RSV.F or after further administration with supplemented RSV F protein (post-F).
Figure 27 shows the generation of IFN-g by splenocytes after additional administration with Ad26.RSV.F after immunization with Ad26.RSV.F or additional administration with supplemented RSV F protein (post-F) do.

As used herein, the term'recombinant' for adenovirus means that the adenovirus is modified by the human hand, e.g., the adenovirus has altered terminal ends actively cloned herein and/or heterologous. gene), that is, it is not a naturally occurring wild-type adenovirus.

Sequences herein are provided in the direction of 5'to 3', as is art practice.

“Adenovirus capsid protein” refers to a protein on 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. Adenoviruses of a particular serotype according to the invention (or'based on it') usually comprise fibers of that particular serotype, Fenton and/or hexon proteins, preferably fibers of that particular serotype, Fenton and Contains hexon protein. These proteins are typically encoded by the genome of a recombinant adenovirus. Recombinant adenoviruses of a particular serotype may selectively contain and/or encode other proteins from other adenovirus serotypes. Thus, as a non-limiting example, a recombinant adenovirus comprising hexon, fenton and fiber of Ad35 is considered a recombinant adenovirus based on Ad35.

Recombinant adenoviruses are'based' on adenoviruses as used herein at least in sequence by induction from wild type. This can be achieved by molecular cloning, using the wild-type genome or portions thereof as starting material. Genome (parts of) from scratch by DNA synthesis, which can be performed using conventional procedures by service companies (e.g., GeneArt, GenScripts, Invitrogen, Eurofins) doing business in the field of DNA synthesis and/or molecular cloning. It is also possible to use known sequences of the wild-type adenovirus genome to generate.

One of skill in the art knows that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of degeneracy of the genetic code. Those skilled in the art can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described in the conventional techniques to reflect the codon utilization of any particular host organism in which the polypeptides will be expressed. I also know that there is. Therefore, unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” is a degenerate version of each other and includes all nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding proteins and RNA can include introns.

In one preferred embodiment, the nucleic acid encoding the RSV F protein or fragment thereof is a codon optimized for expression in mammalian cells such as human cells. Codon-optimization methods are known and have been described previously (eg WO 96/09378). Examples of specific codon-optimized sequences of RSV F protein are described in SEQ ID NO: 2 of EP 2102345 B1.

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

As used herein, the term "fragment" has an amino-terminal and/or carboxy-terminal and/or internal deletion, but the remaining amino acid sequence is the sequence of the RSV F protein, for example the corresponding position in the full length sequence of the RSV F protein. Refers to the same peptides as those. It will be appreciated that for eliciting an immune response, and for vaccination purposes in general, the protein does not have to be full-length, nor does it need to have all its wild-type functions, and the fragments of the protein are equally useful. In fact, fragments of RSV F protein, such as soluble F1 or F, have been shown to be effective in inducing immune responses like full-length F (Shao et al. , 2009, Vaccine 27: 5460-71, Kohlmann et al., 2009, J Virol 83: 12601-12610). Incorporation of F-protein fragments corresponding to amino acids 255-278 or amino acids 412-524 by active immunization induces neutralizing antibodies and some protection against RSV attack (Sing et al., 2007, Virol. Immunol . 20, 261-275; Sing et al. , 2007, Vaccine 25, 6211-6223).

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

In addition, one of ordinary skill in the art will appreciate that changes can be made to proteins, for example by amino acid substitutions, deletions, additions, etc. using routine molecular biological procedures. In general, conservative amino acid substitutions can be applied without loss of function or immunogenicity of the polypeptide. This can be easily checked according to routine procedures well known to those skilled in the art.

The term “vaccine” refers to an agent or composition containing an active ingredient effective to induce a therapeutic degree of immunity in a subject against a particular pathogen or disease. In the present invention, the vaccine contains an effective amount of a recombinant adenovirus encoding the RSV F protein or antigenic fragment thereof, which causes an immune response against the F protein of RSV. This provides a way to prevent serious lower respiratory tract disease that leads to hospitalization and reduces the frequency of complications such as pneumonia and bronchiolitis due to RSV infection and replication in the subject. Accordingly, the present invention includes administering a composition comprising a recombinant human adenovirus of serotype 35 comprising a nucleic acid encoding RSV F protein or a fragment thereof, by intramuscular injection, to prevent serious lower respiratory tract diseases. Methods are also provided for reducing or reducing, preventing or reducing (eg, shortening) hospital admission, and/or reducing the frequency and/or severity of pneumonia or bronchiolitis caused by RSV in a subject. The term "vaccine" according to the present invention implies that it is a pharmaceutical composition, and thus includes usually pharmaceutically acceptable diluents, carriers or excipients. The vaccine may or may not contain additional active ingredients. In certain embodiments, it may be a combination vaccine further comprising other components that elicit an immune response against, for example, other proteins of RSV and/or other infectious agents.

The vectors of the present invention are recombinant adenoviruses, also called recombinant adenovirus vectors. The preparation of recombinant adenovirus vectors is well known in the art.

In certain embodiments, the adenovirus vector according to the invention lacks at least one essential gene function of the E1 region of the adenovirus genome, such as the E1a region and/or the E1b region, required for viral replication. In certain embodiments, the adenovirus vector according to the invention lacks at least a portion of the non-essential E3 region. In certain embodiments, the vector lacks at least one essential gene function of the E1 region and at least a portion of the non-essential E3 region. Adenovirus vectors may be "multiplely deficient," meaning that the adenovirus vector lacks one or more essential gene functions in each of two or more regions of the adenovirus genome. For example, the aforementioned E1-deficient or E1-, E3-deficient adenovirus vectors are at least one essential gene of the E4 region and/or at least one of the E2 region (e.g., E2A region and/or E2B region). The essential gene of may be more deficient.

Adenovirus vectors, their construction methods and their propagation methods are well known in the art, for example, U.S. 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 And Virology , BN Fields et al ., eds ., 3rd edition, Raven Press, Ltd., New York (1996), chapter 67 and 68, respectively, Thomas Shenk, "Adenoviridae and their Replication", MS Horwitz, "Adenoviruses", and the present application. It is described in other references mentioned in. Typically, the composition of adenovirus vectors is, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual , 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), Watson, etc. , Recombinant DNA , 2nd edition, Scientific American Books (1992), and the use of standard molecular biological techniques such as those described in Ausubel et al. , Current Protocols in Molecular Biology , Wiley Interscience Publishers, NY (1995) and other references cited herein.

According to the present invention, the adenovirus is a human adenovirus of serotype 35. The vaccines according to the invention based on this serotype as well as the Ad26 based vaccines surprisingly appear to be more potent than those described in the prior art based on Ad5, which provides complete protection against RSV attack replication 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 serotypes of the invention also generally have low seroprevalence and/or low existing neutralizing antibody titers in the human population. Recombinant adenovirus vectors of Ad35 with this serotype and different transgenes have been evaluated in clinical trials and appear to some extent to have a good stability profile. The preparation of rAd26 vectors is described, for example, in WO 2007/104792 and Abbink et al ., (2007) Virol 81(9): 4654-63. Exemplary genomic sequences of Ad26 can be found in Gene Bank Accession No. EF 153474 and SEQ ID NO: 1 of WO 2007/104792. The preparation of rAd35 vectors is described, for example, in US Pat. No. 7,270,811, WO 00/70071 and Vogels et al., (2003) J Virol 77(15): 8263-71. Exemplary genomic sequences of Ad35 can be found in Figure 6 of Gene Bank Accession No. AC_000019 and WO 00/70071.

The recombinant adenovirus according to the present invention may be replication-competent or replication-deficient.

In certain embodiments, the adenovirus is replication deficient, eg, because it contains deletions in the E1 region of the genome. As known to those skilled in the art, when deleting essential regions from the adenovirus genome, the functions encoded by these regions should preferably be provided in trans form by the producer cell, i.e. E1, E2 and/ Alternatively, if some or all of the E4 regions are deleted from adenovirus, they must be present in the producer cell, for example, integrated into their genome or in the form of so-called helper adenoviruses or helper plasmids. Adenoviruses may also have deletions in the E3 region that are not required for replication, and thus such deletions do not need to be supplemented.

Producer cells that can be used (sometimes referred to in the industry and herein as'packaging cells'or'complementingcells'or'hostcells') Any producer cell in which the desired adenovirus can be propagated. Can be For example, propagation of recombinant adenovirus vectors is carried out in producer cells that compensate for deficiencies in adenovirus. Such producer cells preferably have at least one adenovirus E1 sequence in their genome, thereby being able to supplement recombinant adenoviruses with deletions in the E1 region. Human retinal cells immortalized by E1, such as 911 or PER.C6 cells (see US Pat. No. 5,994,128), E1-transformed amniotic cells (see European Pat. No. 1230354), E1-transform Any E1-supplementary producer such as converted A549 cells (see, e.g., WO 98/39411, U.S. Patent No. 5,891,690), GH329:HeLa (Gao et al., 2000, Human Gene Therapy 11: 213-219), 293, etc. Cells can be used. In certain embodiments, the producer cells are, for example, HEK293 cells or PER.C6 cells or 911 cells or IT293SF cells, and the like.

For non-subgroup C E1-deficient adenoviruses such as Ad35 (subgroup B) or Ad26 (subgroup D), the E4-orf6 coding sequence of these non-subgroup C adenoviruses was changed to Ad5 and It is preferred to exchange for E4-orf6 of adenoviruses of the same subgroup C. This allows the propagation of such adenoviruses in known supplemental cell lines that express the E1 gene of Ad5, such as, for example, 293 cells or PER.C6 cells (e.g., incorporated herein by reference in its entirety. Havenga et al., 2006, J. Gen. Virol. 87: 2135-2143; WO 03/104467). In certain embodiments, the adenovirus in the vaccine composition is a human adenovirus of serotype 35 with a deletion in the E1 region into which the nucleic acid encoding the RSV F protein antigen is cloned, and with the E4 orf6 region of Ad5. In certain embodiments, the adenovirus that can be used is a human adenovirus of serotype 26 with a deletion in the E1 region into which the nucleic acid encoding the RSV F protein antigen is cloned, and with the E4 orf6 region of Ad5.

In alternative embodiments, it is not necessary to locate the heterologous E4orf6 region (e.g., Ad5) within the adenovirus vector, but instead, the E1-deficient non-subgroup C or E vector is both E1 and compatible E4orf6. Cell lines expressing both, such as, from Ad5, are proliferated in 293-ORF6 cell lines that express both E1 and E4orf6 (eg, Brough et al., 1996, J Virol 70: 6497-501, describing 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, which describe the generation of E1 deleted non-subgroup C adenovirus vectors using such cell lines, respectively).

Alternatively, supplementary cells expressing E1 from a serotype to be proliferated can be used (see, eg, WO 00/70071, WO 02/40665).

For subgroup B adenoviruses such as Ad35, which have deletions within the E1 region, the 3'end of the E1B 55K open reading frame in the adenovirus, e.g., 166 base pairs directly upstream of the pIX open reading frame, or ( It is desirable to maintain a segment containing it, such as the 243 base pair segment directly upstream of the pIX start codon (marked at the 5'end by the Bsu36I restriction site in the Ad35 genome), which means that the promoter of the pIX gene is partially within this region. It is because it increases the stability of adenovirus because it is present in (see, eg, Havenga et al., 2006, J. Gen. Virol . 87: 2135-2143; WO 2004/001032, incorporated herein by reference).

In the adenoviruses of the present invention, a "heterologous nucleic acid" (also referred to herein as a "transplant gene") is a nucleic acid that does not naturally exist in the adenovirus. It is introduced into adenoviruses, for example by standard molecular biology techniques. In the present invention, the heterologous nucleic acid encodes the RSV F protein or a fragment thereof. Heterologous nucleic acids can be cloned, for example, into the deleted E1 or E3 regions of an adenovirus vector. Transgenes are generally operably linked to expression control sequences. This can be done, for example, by positioning 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 expression of the transgene(s) and are known to those of skill in the art. Non-limiting examples of suitable promoters for obtaining expression in eukaryotic cells are from CMV-promoter (US Pat. No. 5,385,839), such as CMV immediate early gene enhancer/promoter, for example, It is a CMV immediate early promoter comprising -735 to +95 nucleotides. A polyadenylation signal, such as a bovine growth hormone polyA signal (US Pat. No. 5,122,458), may be present after the transgene(s). A polyadenylation signal, such as a bovine growth hormone polyA signal (US Pat. No. 5,122,458), may be present after the transgene(s).

In certain embodiments, recombinant Ad26 or Ad35 vectors of the invention comprise the nucleotide sequence: CTATCTAT as 5'terminal nucleotides. These embodiments are advantageous, as such vectors show improved replication in the production processes compared to vectors with original 5'terminal sequences (generally CATCATCA), which are incorporated herein by reference in their entirety, This is because it generates batches of adenoviruses with improved homogeneity (a patent application titled'Batch of Recombinant Adenovirus with Changed Terminal End' filed on March 12, 2012 in the name of Crucell Holland BV. See also numbers PCT/EP2013/054846 and US 13/794,318). Accordingly, the present invention is a batch of recombinant adenoviruses encoding RSV F protein or a portion thereof, wherein the adenovirus is a human adenovirus serotype 35, and essentially all of the adenoviruses in the batch (e.g., at least 90%) are terminal nucleotides. A batch of recombinant adenoviruses comprising a genome with sequence CTATCTAT is also provided.

According to the invention, the F protein of RSV can be derived from any strains of naturally occurring RSV or recombinant RSV, preferably from human RSV strains such as A2, Long 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 present invention, the RSV strain is an RSV-A2 strain.

According to the present invention, the F protein of RSV may be the full length of the F protein of RSV or a fragment thereof. In one embodiment of the present invention, the nucleotide sequence encoding the F protein of RSV encodes the full length (F0) of the F protein of RSV, such as the amino acid of SEQ ID NO: 1. In an example of the present invention, the nucleotide sequence encoding the F protein of RSV has the nucleotide sequence of SEQ ID NO: 2. Alternatively, the sequence encoding the F protein of RSV can be any sequence that is at least 80%, preferably more than about 90%, more preferably at least about 95% identical to the nucleotide sequence of SEQ ID NO: 2 have. In other embodiments, codon-optimized sequences such as those provided in, for example, SEQ ID NO: 2, 4, 5 or 6 of WO 2012/021730 can be used.

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 either or both amino-terminal and carboxy-terminal deletions. The degree of deletion can be determined by one of skill in the art, for example, to achieve a better yield of recombinant adenovirus. The segment will be selected to contain an immunologically active segment of the F protein, ie, the portion that will cause an immune response in the subject. This can be readily determined using routine in silico, in vitro and/or in vivo methods, all of which are routine to those skilled in the art. In one embodiment of the invention, the fragment is a transmembrane coding region-truncated F protein of RSV (F0ΔTM, such as US 20110014220). The fragments of the F protein may be the F1 domain or the F2 domain of the F protein. The fragments of F may also be those containing neutralizing epitopes and T cell epitopes (Sing et al., 2007, Virol. Immunol . 20, 261-275; Sing et al., 2007, Vaccine 25, 6211-6223).

For numerical values used in this disclosure, the term "about" means a numerical value ± 10%.

In certain embodiments, the present invention provides a recombinant human adenovirus of serotype 35 comprising a nucleic acid encoding a respiratory syncytial virus (RSV) F protein or a fragment thereof, in a culture of host cells the Proliferating a recombinant adenovirus, isolating and purifying the recombinant adenovirus, and incorporating the step of putting the recombinant adenovirus in a pharmaceutically acceptable composition, it provides a method for producing a vaccine against RSV.

Recombinant adenovirus can be produced and propagated in host cells according to well known methods involving cell culture of host cells infected with adenovirus. Cell cultures can be suspension cultures as well as adherent cell cultures, such as any type of cell culture, including cells attached to the surface of a culture vessel or to microcarriers.

Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the easiest to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and also suitable (e.g., describing suitable methods for obtaining and purifying large quantities of recombinant adenoviruses, both of which are incorporated herein by reference. See WO 2010/060719 and WO 2011/098592).

Producer cells are cultured to increase cell and virus numbers and/or viral titers. Cell culture is carried out to allow the cells to metabolize, grow, and/or differentiate and/or produce a virus of interest according to the invention. This can be accomplished by methods well known to those of skill in the art, including, but not limited to, providing nutrients to the cells in an appropriate culture medium, for example. Suitable culture media are well known to those of skill in the art and can generally be obtained from large quantities of commercial sources or can be customized according to standard protocols. The cultivation can be carried out in dishes, roller bottles or in bioreactors using, for example, batch, fed-batch, continuous systems, and the like. Suitable conditions for cell culture are known (e.g., Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and RI Freshney, Culture of animal cells: A manual of basic technique, 4th edition (Wiley-Liss Inc. , 2000, ISBN 0-471-34889-9).

Typically, adenoviruses will be exposed to appropriate producer cells in culture to allow uptake of the virus. In general, the optimum agitation is between about 50 to 300 rpm, typically about 100 to 200 rpm, such as about 150 rpm, with a typical DO of 20 to 60%, such as 40%, with an optimal pH of 6.7 to 7.7 Between, and the optimum temperature is between 30 and 39° C., such as 34 to 37° C., and the optimum MOI is between 5 and 1000, such as about 50 and 300. Typically, adenovirus spontaneously infects producer cells, and the step of contacting the producer cells with rAd particles is sufficient to infect the cells. Typically, the adenovirus seed stock is added to the culture to initiate infection, and the adenovirus then proliferates in the producer cells. This is all routine for a person skilled in the art.

After infection with adenovirus, the virus replicates inside the cell and is thereby amplified, a process referred to herein as propagation of adenovirus. Adenovirus infection eventually results in the lysis of the infected cells. Therefore, the dissolution properties of adenovirus enable two different modes of virus production. The first mode is to harvest the virus prior to cell lysis using foreign factors that lyse the cells. The second mode is to harvest the viral supernatant after (almost) complete cell lysis by the generated virus (e.g., U.S. Patent No. 6,485,958, which describes the harvesting of adenovirus without lysing host cells by external factors. Reference). It is desirable to use external factors that actively lyse cells to harvest adenovirus.

Methods that can be used for vigorous cell lysis are known to those skilled in the art and are discussed, for example, in WO 98/22588, pages 28 to 35. Methods useful in this respect include, for example, freeze-thaw, solid shear, hypertonic and/or hypotonic dissolution, liquid shear, sonication, high pressure extrusion, detergent lysis, such as Such as combinations of. In one embodiment of the invention, the cells are lysed using at least one detergent. The use for dissolution of the detergent is an easy method, and has the advantage that it can be easily expanded and contracted.

The detergents that can be used and the manner in which they are used are generally known to those skilled in the art. Several examples are discussed in, for example, WO 98/22588, pages 29 to 33. Detergents can include anionic, cationic, amphoteric and nonionic detergents. The concentration of the detergent may vary within a range of, for example, about 0.1% to 5% (w/w). In one embodiment, the detergent used is Triton X-100.

Nucleases can be used to contaminate, ie, to remove nucleic acids from most producer cells. Exemplary nucleases suitable for use in the present invention include Benzonase®, Pulmozyme®, or any other DNA degrading enzyme and/or RNA degrading enzyme commonly used in the industry. In preferred embodiments, the nuclease is Benzonase®, which rapidly hydrolyzes nucleic acids by hydrolyzing internal phosphodiester bonds between specific nucleotides, thereby reducing the viscosity of cell lysates. Benzonase® can be obtained commercially from Merck KGaA (code W214950). The concentration at which the nuclease is used is preferably in 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 from the adenovirus preparation during adenovirus purification using selective precipitating agents such as domiphene bromide (e.g., U.S. Patent No. 7,326,555; See Goerke et al., 2005, Biotechnology and bioengineering, Vol. 91: 12-21; WO 2011/045378; WO 2011/045381).

Methods for harvesting adenoviruses from cultures of producer cells are extensively described in WO 2005/080556.

In certain embodiments, the adenovirus harvested is further purified. Purification of adenovirus can be carried out in several steps including purification, ultrafiltration, diafiltration or separation with chromatography, for example as described in WO 05/080556, incorporated herein by reference. Purification can be carried out by means of a filtration step to remove cell debris and other impurities from the cell lysate. Ultrafiltration is used to concentrate the virus solution. Diafiltration or buffer exchange using ultrafiltration membranes is a method of removing and exchanging salts, sugars, and the like. The skilled person knows how to find the optimal conditions for each purification step. In addition, methods of production and purification of adenovirus vectors are described in WO 98/22588, which is incorporated herein by reference in its entirety. These methods include growing host cells, infecting host cells with adenovirus, harvesting and lysing host cells, concentrating the crude lysate, exchanging the buffer of the crude lysate, and It includes treating the lysate with a nuclease and further purifying the virus using chromatography.

Preferably, purification uses at least one chromatographic step, as discussed in, for example, WO 98/22588, pages 61 to 70. Many processes have been described for further purification of adenoviruses, and chromatographic steps are included in this process. Those of skill in the art will be aware of these processes and can vary the exact way to use the chromatography steps to optimize the process. For example, adenoviruses can be purified by anion exchange chromatography steps, see, for example, WO 2005/080556 and Konz et al. , 2005, Hum Gene Ther 16: 1346-1353. Many other adenovirus purification methods have been described and are within the competence of one of skill in the art. Other methods of producing and purifying adenoviruses are, for example, (WO 00/32754; WO 04/020971; US 5,837,520; US 6,261,823; WO 2006/108707; Konz et al. , 2008, which are all incorporated herein by reference. Methods Mol Biol 434: 13-23; Altaras et al., 2005, Adv Biochem Eng Biotechnol 99: 193-260).

For administration to humans, the present invention can use pharmaceutical compositions comprising rAd and a pharmaceutically acceptable carrier or excipient. In this context, the term "pharmaceutically acceptable" means that the carrier or excipient will, at the dosages and concentrations used, will never cause any undesired or deleterious effects in the subjects to which they are being administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (Remington's Pharmaceutical Sciences, 18th ed., 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 ed., A. Kibbe, Ed., Pharmaceutical Press [2000]). Purified rAd is preferably formulated and administered as a sterile solution, although lyophilized formulations can be utilized. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution is generally in the range of pH 3.0 to 9.5, such as pH 5.0 to 7.5. rAd is usually in a solution with a suitable pharmaceutically acceptable buffer, and the solution of rAd may also contain salts. Optionally, stabilizers such as albumin may be present. In certain embodiments, detergent is added. In certain embodiments, rAd can be formulated as an injectable formulation. These formulations contain an effective amount of rAd and are either sterile liquid solutions, liquid suspensions or lyophilized versions, and optionally contain stabilizers or excipients. Adenovirus vaccines may also be aerosolized for intranasal administration (see, eg, WO 2009/117134).

For example, adenovirus can be stored in a buffer that is also used for adenovirus world standards (Hoganson et al., Development of a stable adenoviral vector formulation, Bioprocessing March 2002, p. 43-48): 20 mM Tris pH 8 , 25 mM NaCl, 2.5% glycerol. Other useful formulation buffers suitable for administration to humans are 20 mM Tris, 2 mM MgCl ₂ , 25 mM NaCl, 10% w/v sucrose, 0.02% w/v polysorbate-80. Obviously, many different buffers can be used, and some examples of formulations suitable for storage and pharmaceutical administration of purified (adeno)viral preparations are, for example, European Patent No. 0835660, US Patent No. 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.

In certain embodiments, the composition comprising adenovirus further comprises one or more adjuvants. Adjuvants that further increase the immune response to the applied antigenic determinant are known in the art, and pharmaceutical compositions comprising adenovirus and suitable adjuvants are disclosed, for example, in WO 2007/110409, which is incorporated herein by reference. have. The terms “adjuvant” and “immune stimulant” are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to the adenovirus vectors of the present invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; Oil-emulsion compositions (or oil-in-water compositions) including squalene-water emulsions such as MF59 (see, eg, WO 90/14837); Saponin formulations such as, for example, QS21 and immunostimulatory complexes (ISCOMS) (see, eg, US Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); As bacterial or microbial derivatives, examples thereof include monophosphoryl lipid A (MPL), 3-O-diacylated MPL (such as E. coli) heat labile enterotoxin LT, cholera toxin CT, etc. 3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylated bacterial toxins or mutants thereof, bacterial or microbial derivatives. For example, a vector-encoded adjuvant may be used by using a heterologous nucleic acid encoding the fusion of the oligomerization domain of a C4-binding protein (C4bp) as an antigen of interest (e.g., Solabomi et al., 2008, Infect Immun 76: 3817. -23). In certain embodiments, the compositions of the present invention contain aluminum as an adjuvant in the form of, for example, aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate or combinations thereof, with an aluminum dose of 0.05 to 5 mg per dose, such as 0.075 to 1.0 mg. Included in the concentration of.

In other embodiments, the compositions do not contain adjuvants.

It is also possible according to the invention to administer other active ingredients in combination with the vaccines according to the invention. Such other active ingredients may include, for example, vectors containing other RSV antigens or nucleic acids encoding them. Such vectors may be non-adenoviral or adenoviral, the latter of which may be of any serotype. Examples of other RSV antigens include the RSV G protein or immunologically active portions thereof. For example, a recombinant replication-deficient Ad5-based adenovector rAd/3xG applied intranasally expressing the soluble central domain of G glycoprotein (amino acids 130-230) was protective in a rat model (Yu et al. , 2008). , J Virol 82: 2350-2357), said that there was no protection when applied intramuscularly, but from this data it is clear that RSV G is a suitable antigen for inducing protective responses. Other active ingredients may also contain non-RSV antigens from other pathogens such as, for example, viruses, bacteria, parasites, and the like. Administration of the other active ingredients can be carried out, for example, by separate administration or by administering the vaccines of the present invention and combination products of the other active ingredients. In certain embodiments, other non-adenovirus antigens (except RSV.F) can be encoded in the vectors of the invention. In certain embodiments, therefore, it may be desirable to express more than one protein from a single adenovirus, in such cases, more coding sequences are linked, for example, to a single translocation from a single expression cassette. It can form a cadaver, or it can exist in two separate expression cassettes that have been cloned within different portions of the adenovirus genome.

Adenovirus compositions can be administered to a subject, such as a human subject. The total dose of adenovirus provided to a subject upon single administration can be varied as known to those skilled in the art, and generally, between 1 x 10 ⁷ viral particles (vp) to 1 x 10 ¹² vp, preferably 1 x Between 10 ⁸ vp and 1 x 10 ¹¹ vp, for example between 3 x 10 ⁸ and 5 x 10 ¹⁰ vp, for example between 10 ⁹ and 3 x 10 ¹⁰ vp.

Administration of adenovirus compositions can be carried out using standard routes of administration. Non-limiting embodiments include parenteral administration by injection, such as, for example, endothelial, intramuscular, etc., or subcutaneous or transdermal administration, or mucosal administration such as, for example, intranasal, oral, etc. Intranasal administration is generally considered the preferred route for vaccines against RSV. The most important advantage of the live intranasal strategy is the lack of direct stimulation of local airway immunity and associated disease enhancement. The only vaccines currently under clinical evaluation for pediatric use are attenuated 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, pages 233-277). Intranasal administration is also a suitable and preferred route according to the invention. However, according to the present invention it is particularly preferred to administer the vaccine intramuscularly, which is different from previously reported intramuscular RSV vaccines based on other adenovirus serotypes due to the intramuscular administration of the vaccine according to the present invention, cotton rats This is because they have surprisingly found that it results in protection against RSV replication in the nose and lungs of the animals. The advantage of intramuscular administration is that it is simple and established, and there are no safety concerns for intranasal application in infants younger than 6 months. In one embodiment, the composition is administered by intramuscular injection, such as to the deltoid muscle of the arm or the lateral femoral muscle of the thigh. Those of skill in the art are aware of the various possibilities of administering a composition, such as a vaccine to elicit an immune response to the antigen(s) in the vaccine.

As used herein, the subject is preferably a mammal, such as a rodent, such as a mouse, a cotton rat or a non-human primate or a human. Preferably, the subject is a human subject. Subjects are of any age, such as from about 1 month to 100 years, such as from about 2 months to about 80 years, such as from about 1 month to about 3 years, from about 3 years to about 50 years, from about 50 years to about 75 years Etc.

It is also possible to provide one or more booster administrations of one or more adenovirus vaccines of the present invention. If a booster vaccination is performed, typically, such booster vaccinations are performed in the same subject from 1 week to 1 year after the composition was first administered to the subject (in such cases, referred to as'priming vaccination'). It will be administered at any point in between, preferably between 2 weeks and 4 months. In alternative booster methods, different vectors, such as one or more adenoviruses of different serotypes, or other vectors such as MVA, or DNA, or proteins may be administered to the subject after the initial vaccination. For example, it is possible to administer the recombinant adenovirus vector according to the present invention to a subject as an initial vaccination, and administer it as an additional vaccination with a composition containing the RSV F protein.

In certain embodiments, administration includes an initial administration and at least one additional administration. In certain embodiments thereof, the first administration is with rAd35 ('rAd35-RSV.F') comprising a nucleic acid encoding the RSV F protein or a fragment thereof, and the further administration encodes the RSV F protein according to the present invention. It is to use rAd26 ('rAd26-RSV.F') containing a nucleic acid. In other embodiments thereof, the first administration is with rAd26-RSV.F and the further administration is with rAd35-RSV.F. In other embodiments, the first and additional administration is with rAd26.RSV.F. In certain embodiments, the first administration is with rAd35-RSV.F and the further administration is with the RSV F protein. In all these embodiments, it is possible to provide different additional administrations with the same or different vectors or proteins. Embodiments in which further administration with RSV F protein may be particularly beneficial are, for example, in elderly subjects in risk groups 50 years of age or older (e.g., with COPD or asthma), or healthy subjects 60 years of age or older or 65 years of age or older. It includes embodiments in.

In certain embodiments, administration comprises a single administration of the recombinant adenovirus according to the invention without other (additional) administration. Such embodiments are advantageous considering the reduced complexity and cost of a single dose regimen compared to an initial-additional regimen. Complete protection is already observed after a single administration of the recombinant adenovirus vectors of the invention without further administration in the cotton rat model in the examples herein.

The invention is further illustrated in the following examples. The examples in no way limit the invention. These only serve to clarify the invention.

Example

Example 1. Preparation of adenovirus vectors

Cloning of the RSV F gene into the E1 region of Ad35 and Ad26:

The RSV.F(A2)nat gene encoding the native RSV fusion (F) protein of the A2 strain (gene bank ACO83301.1) was a gene optimized and synthesized for human expression by Geneart. The Kozak sequence (5' GCCACC 3') was included just before 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 the pAdApt35BSU plasmid and pAdApt26 plasmid through the HindIII and XbaI sites. The obtained plasmids pAdApt35BSU.RSV.F(A2)nat and pAdApt26.RSV.F(A2)nat are shown in FIG. 15. The amino acid sequence of the F protein and the codon-optimized sequence encoding the amino acid sequence are respectively shown in SEQ. ID. NO: 1 and SEQ. ID. NO: 2 is provided.

Cell culture:

PER.C6 cells (Fallaux et al., 1998, Hum Gene Ther 9: 1909-1917) Dulbecco? with 10% fetal calf serum (FBS) supplemented with 10 _{mM MgCl 2} The modified Eagle? It was maintained in medium (DMEM).

Adenovirus outbreak, infection and passage:

All adenoviruses were generated in PER.C6 cells by single homologous recombination and were generated as previously described (for rAd35: Havenga et al., 2006, J. Gen. Virol . 87: 2135-143; for rAd26: Abbink et al., 2007, J. Virol . 81: 4654-4663). Briefly, PER.C6 cells were transfected with Ad vector plasmids using Lipofectamine according to the instructions provided by the manufacturer (Life Technologies). For the rescue of Ad35 vectors carrying the RSV.F(A2)nat transgene expression cassette, the pAdApt35BSU.RSV.F(A2)nat plasmid and pWE/Ad35.pIX-rITR.dE3.5orf6 cosmid were used. On the other hand, for Ad26 vectors carrying the RSV.F(A2)nat transgene expression cassette, pAdApt26.RSV.F(A2)nat plasmid and pWE.Ad26.dE3.5orf6.cosmid were used. Cells were harvested 1 day after complete CPE, freeze-thaw, centrifuged at 3,000 rpm for 5 minutes, and stored at -20°C. Next, the viruses were plaque purified and amplified in PER.C6 cultured on a single well of a multiwell 24 tissue culture plate. Further amplification was performed in PER.C6 cultured using T25 tissue culture flasks and T175 tissue culture flasks. In the T175 crude lysate, 3 to 5 ml were used to inoculate 20 x T175 3 multilayer tissue culture flasks containing 70% confluent layers of PER.C6 cells. Viruses were purified using a two-step CsCl purification method. Finally, the virus was aliquoted and stored at -85°C.

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

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

In this study, animals were distributed into experimental groups of 5 mice. Animals were immunized with a single dose of Ad26 or Ad35 (Ad26-RSV.F or Ad35-RSV.F) carrying the full length RSV F gene or without a transgene (Ad26e or Ad35e). Three 10-fold serial dilutions of rAd up to 10 ¹⁰ to 10 ^{8 viral particles (vp) were given intramuscularly.} As controls, one group of 3 animals received an empty vector Ad26e, and one group received an empty vector Ad35e.

The ELISPOT assay is used to determine the relative number of F protein-specific IFNγ-secreting T cells in the spleen and is essential as described by Radosevic et al. (Clin Vaccine Immunol. 2010;17(11):1687-94.) It is done with. For stimulation of splenocytes in the ELISPOT assay, two peptide pools of 11 amino acids overlapping with 15-mer peptides extending over the entire sequence of the RSV F(A2) protein were used. The number of spot-forming units (SFU) per 10 ^{6 cells was calculated.}

For the determination of antibody titers, an ELISA assay was used. To this end, ELISA plates (Thermo Scientific) were coated with 25 μg/ml RSV Long total inactivated antigen (Virion Serion, cat# BA113VS). Diluted serum samples were added to the plates, using biotin-labeled anti-mouse IgG (DAKO, cat# E0413) using detection by horseradish peroxidase (PO)-conjugated streptavidin (SA). Thus, IgG antibodies against RSV were measured. Titers were calculated by linear interpolation using a 1.5 x OD signal from 50-fold diluted naive serum as cut-off. Titers of RSV-specific IgG1 and IgG2a antibodies in the serum of mice were PO-labeled anti-mouse IgG1 and PO-labeled anti-mouse IgG2a (Southern Biotechnology Associates, cat#s 1070-) used to quantify subclasses. 05 and 1080-05).

The virus neutralizing activity (VNA) of the antibodies was determined by Johnson et al . (J Infect Dis. 1999 Jul; 180(1):35-40.) It was measured by a microneutralization assay, which is essentially performed as described. RSV-sensitive VERO cells were sprayed into 96-well cell-culture plates 1 day prior to infection. On the day of infection, serially diluted sera and controls were mixed with 1200 pfu of RSV (Long or B1) and incubated for 1 hour at 37°C. The virus/antibody mixes were then transferred to 96-well plates containing VERO cell monolayers. After 3 days, the monolayers were fixed with 80% ice-cooled acetone and RSV antigen was measured using an anti-F monoclonal antibody. Neutralizing titers are expressed as serum dilutions (log ₂ ) that resulted in a 50% reduction (IC _{50) in OD450 from virus-only control wells.}

At 2 and 8 weeks after the initial dosing, animals were sacrificed and cellular and humoral responses were monitored as described above.

Figure 1 shows that all doses of Ad26-RSV.F (Figure 1A) and Ad35-RSV.F (Figure 1B) are effective in inducing a good cellular immune response, and the responses are stable over time. I'm doing it. No significant differences in vector dose were observed for T cell responses with either Ad26-RSV.F or Ad35-RSV.F.

2 depicts antibody titers in the same experiment as described above. Both vectors induced a very sharp time and dose-dependent increase in ELISA titers (FIG. 2 ). Anti-F titers apparently increased from week 2 to week 8, significant for ^{10 10 doses.} There was no difference in titer between the Ad26-RSV.F or Ad35-RSV.F vectors at 8 weeks.

The subclass distribution of F-specific IgG (IgG1 vs. IgG2a) was measured to assess the balance of Th1 vs. Th2 responses. The skewed Th2/Th1 response causes animals to develop vaccine-enhanced RSV disease as seen in formalin-inactivated RSV. As shown in Fig. 3, the IgG2a/IgG1 ratio in both Ad26-RSV.F and Ad35-RSV.F exceeds 1. This strongly suggests that the adenoviruses Ad26-RSV.F and Ad35-RSV.F have a Th1 type rather than a Th2 type of response.

Figure 4 shows the virus neutralizing titers (VNA) of the same sera used for antibody titers. Immunization with Ad26-RSV.F and rAd35-RSV.F led to induction of neutralizing antibody titers. VNA titers increased strongly between 2 and 8 weeks after initial administration in mice given ^{at 10 10 vp.} There was no difference in titer between the Ad26-RSV.F and Ad35-RSV.F vectors in mice given ^{at 10 10} vp at 8 weeks.

It is clear from these immunization experiments that Ad35 and Ad26 vectors carrying the RSV.F transgene induce strong cellular and humoral responses to RSV.F.

Example 3. RSV.F Immunization against RSV.F after heterologous first-additional administration with encoding recombinant adenovirus vectors

This study was designed to investigate the ability of initial-additional schemes based on adenovirus vectors derived from two different serotypes to induce immunity to RSV.F.

This study included BALB/c mice distributed in experimental populations of 8 mice. Animals were subjected to intramuscular injection at ^{10 10} vp with the wild-type sequence of the RSV.F gene based/derived from RSV A2 (Ad-RSV.F or Ad35-RSV.F) or without a transgene (Ad26e or Ad35e). Was immunized. One group of animals was initially administered Ad26-RSV.F at week 1, followed by Ad35-RSV.F or Ad35e at 4 weeks. Animals of another group of animals were initially dosed with Ad35-RSV.F, followed by additional doses of Ad26-RSV.F or Ad26e at 4 weeks. Among the mice, the control group was initially administered with Ad35e, and additionally administered with Ad26e at 4 weeks. At 6 and 12 weeks after the initial administration, 8 animals were sacrificed at each time point, and cellular and humoral responses were well known to those of skill in the art and monitored using immunological assays as described above.

5 depicts cellular responses at 6 and 12 weeks after the first immunization. At 6 weeks after the first administration (and 2 weeks after the additional administration), a significant additional administration effect on T cell responses was measured by Ad26-RSV.F and Ad35-RSV.F, and the magnitude of the T cell response was initially determined. -Independent of the order of immunization with Ad26-RSV.F or Ad35-RSV.F in further administration. At 12 weeks after initial administration (8 weeks after additional administration), mice initially dosed with Ad26-RSV.F were compared to animals initially dosed with rAd35-RSV.F. The level of F-specific T cells was maintained higher in either of the animals. Overall, the number of F-specific lymphocytes (SFU) was high and stable for at least 12 weeks in all animals immunized with either rAd26-RSV.F or rAd35-RSV.F (first dose or first-addition). Was.

Figure 6 shows the humoral response at different time points after initial-boost vaccination with adenovirus vectors. A significant additional dosing effect on B cell responses induced by either Ad26.RSV.F or rAd35.RSV.F initially administered equally well to Ad35.RSV.F and Ad26.RSV.F is shown. have. In addition, the magnitude of the B cell responses in the xenogeneic first-additional administration was independent of the order of Ad35.RSV.F and Ad26.RSV.F immunization, and the ELISA titers remained stable for 12 weeks after the additional administration.

7 shows virus neutralizing antibody titers at different time points after first-boost dose immunization. Both Ad35.RSV.F and Ad26.RSV.F vectors were initially administered equally well, achieving apparent VNA titers, as observed for ELISA titers. In addition, the increase in VNA titers after xenogeneic first-additional administration was independent of the order of Ad35.RSV.F and Ad26.RSV.F immunization. The effect of further administration on VNA titers by either Ad26.RSV.F or Ad35.RSV.F was remarkable at both time points and was already maximal at 6 weeks. The groups initially dosed with Ad.RSV.F alone increased VNA titers at 12 weeks compared to 6 weeks. The RSV F sequence in adenovirus vector constructs is derived from the RSV A2 isolate. The neutralization assay described in this application is based on the RSV Long strain belonging to RSV subgroup A, showing that antibodies induced by F(A2) can cross-neutralize different RSV A strain subtypes.

Since the RSV F protein is well conserved between RSV isolates, sera obtained from animals immunized with Ad-RSV.F vectors were not able to cross-neutralize the original RSV B strain isolate, RSV B1. Was tested. As shown in Figure 8, the sera of immunized mice were also able to cross-neutralize the B1 strain. The ability to cross-neutralize RSV B1 does not depend on which vector was used in the first dose only groups, or the order of first-additional immunization with Ad26.RSV.F and Ad35.RSV.F vectors.

Taken together, these data show that, in the initial-additional regimen, successive immunizations with Ad26.RSV.F and Ad35.RSV.F induce strong humoral and cellular responses, and humoral immune responses in RSV A and B It has been shown to include the ability to neutralize the isolates of both subtypes.

Example 4. Induction of protection against RSV infection using recombinant adenovirus vectors in vivo in a cotton mouse model

This experiment was conducted to investigate the ability of initial-additional schemes based on adenovirus vectors derived from two different serotypes to induce protection against RSV attack replication in cotton mice. It was found that the cotton rat (Sig sows Heath blood Douce (Sigmodon hispidus)) is that the by RSV road and also are susceptible to both infection, at least 50 times more permissive than the mouse strain (Niewiesk etc., 2002, Lab .. Anim 36 (4): 357-72). In addition, cotton rats were the primary model to evaluate the efficacy and safety of RSV candidate vaccines, antivirals and antibodies. The preclinical data generated in the cotton rat model facilitated the development of two antibody formulations (RespiGam® and Synagis®) into clinical trials without the need for intermediate studies in non-human primates.

The study placed cotton rats into experimental groups of 8 cotton rats each. Animals carry the full length RSV F (A2) gene (Ad26.RSV.F or Ad35.RSV.F) or without the transgene (Ad26e or Ad35e) 10 ⁹ viral particles (vp) or 10 ¹⁰ vp adenovirus Immunized by intramuscular injection of vectors. Animals were further dosed 28 days later at the same vp dose with either the same vector (allogeneic first-addition) or a different adenovirus serotype (heterologous first-additional administration); Controls were accordingly immunized with Ad-e vectors, except that only 1 dose was applied (10 ^10). Controls consisted of 6 animals. Animals infected intranasally with RSV A2 (10 ⁴ plaque forming units (pfu)) were positive controls for protection against attack replication, as the primary infection with RSV virus is known to provide protection against secondary attack replication. (Prince. Lab Invest 1999, 79:1385-1392). In addition, formalin-inactivated RSV (FI-RSV) served as a control for vaccine-enhanced histoimmune diseases. Three weeks after the second (additional) immunization, cotton rats were challenged intranasally with ^{1 x 10 5 pfu of plaque-purified RSV A2.} As controls, one group of cotton rats was not immunized, but received attack virus, and the other control group was neither immunized nor attacked. Cotton rats were sacrificed 5 days post infection, when RSV attack virus reached peak titers (Prince. Lab Invest 1999 , 79:1385-1392), and lung and nasal RSV titers were measured by viral plaque titration. (Prince et al. 1978, Am J Pathology 93,711-791).

Figure 9 shows that the high RSV virus titers in the lungs and nose are not transgene-free _{animals receiving adenovirus vectors of 5.3 +/- 0.13 log 10} pfu/gram and 5.4 +/- 0.35 log ₁₀ pfu, respectively, as well as non- It is shown that it was observed in the immunized controls. In contrast, irrespective of dose or schedule, no attacking virus could be detected in lung and nasal tissues from animals that were first-additionally immunized with Ad26.RSV.F and/or Ad35.RSV.F vectors.

These data clearly show that both Ad35- and Ad26-based vectors provide complete protection against RSV attack replication in the cotton rat model. This was surprising because Ad5 based adenovirus vectors encoding RSV F are known to be unable to induce full protection in animal models after intramuscular administration.

In the course of the experiment, blood samples were taken before immunization (day 0), before further immunization (day 28), on the day of challenge (day 49) and on the day of sacrifice (day 54). Serums were tested in a plaque assay-based virus neutralization assay (VNA) for induction of systemic RSV specific neutralizing antibodies as described by Prince (Prince et al. 1978, Am J Pathology 93,711-791). Neutralizing titers are expressed as serum dilutions (log ₂ ) that resulted in 50% plaque reduction (IC _{50) compared from virus-only control wells.}

10 shows that control animals do not have virus neutralizing antibodies on days 28 and 49, whereas high VNA titers are induced after the animals are first administered with Ad26.RSV.F or Ad35.RSV.F vectors. do. A modest increase in VNA titer is observed after further immunizations. Primary infection with RSV A2 virus produced slightly moderate VNA titers that gradually increased over time.

To evaluate whether the Ad26.RSV.F or Ad35.RSV.F vaccine can exacerbate the disease after attack with RSV A2, histopathological analyzes of the lungs were performed 5 days after infection. Lungs were harvested, perfused with formalin, sectioned, and stained with hematoxylin and eosin for histological examination. The histopathology score was performed blinded according to the criteria published by Prince (Prince et al . Lab Invest 1999 , 79:1385-1392), and the following parameters: peribronchial gastritis, perangiitis, interstitial pneumonia and alveoli It was scored for salt. 11 shows the scoring of the lung pathology of this experiment. After RSV challenge, FI-RSV immunized animals compared to mock-immunized challenge animals expected based on previously published studies (Prince et al . Lab Invest 1999, 79:1385-1392). , Showed improved histopathology for all histoimmunological parameters examined. Perivascular gastritis appeared to be slightly lower in rAd-RSV.F immunized animals, but histopathological scores in immunized Ad26.RSV.F and Ad35.RSV.F compared to rAd-e or sham immunized animals. It was similar. Thus, Ad26.RSV.F and Ad35.RSV.F vaccines did not lead to enhanced disease unlike FI-RSV vaccines.

All vaccination strategies resulted in complete protection against RSV attack replication, induced strong virus neutralizing antibodies, and no enhanced pathologic changes were observed.

Example 5. Protective efficacy of rAd vectors using different routes of administration after single immunization

This study investigates the effect of routes of administration on the protective efficacy induced by Ad26 or Ad35 vectors encoding RSV.F. The vaccine was administered either intramuscularly or intranasally.

A transgenic day 0 (Ad26.RSV.F or Ad35.RSV.F) RSV F or hold the transgene member (Ad26-Ad35-e or e) a 1 x 10 ⁹ Ad26 or one or 1x10 ¹⁰ of the Ad35 Cotton mice single immunized with canine virus particles (vp) were challenged on ^{day 49 with 10 5} RSV pfu and sacrificed on day 54.

12 shows the results of experiments in which lung and nasal attack viruses were measured. High RSV virus titers were detected in the lungs and nose of non-immunized or immunized mice _{without transgene, with adenoviruses of 4.9 +/- 0.22 log 10} pfu/gram and 5.4 +/- 0.16 log _{10 pfu, respectively.} In contrast, the lungs and noses of animals that received either Ad35-RSV.F or Ad26-RSV.F were free of replication attack virus regardless of the route and dose of administration.

This data surprisingly shows that each of the Ad26- and Ad35-based vectors encoding the RSV F protein provides complete protection in cotton rat challenge experiments, regardless of the route of administration of the vectors. This was unexpected because none of the published adenovirus-based RSV vaccines based on different serotypes showed complete protection after intramuscular vaccination.

During the experiment, blood samples were taken before immunization (day 0), 4 weeks after immunization (day 28) and on the day of challenge (day 49). Serums were tested in a neutralization test for induction of RSV specific antibodies (FIG. 13 ). Prior to immunization, no virus neutralizing antibodies were detected in cotton mice. Regardless of the route of administration, all adenovirus vector immunization strategies clearly induced high VNA titers that remained stable over time. This data surprisingly shows that each of the Ad26- and Ad35-based vectors encoding the RSV F protein provides high titers of virus neutralizing antibodies in cotton rat immunization experiments, regardless of the route of administration of the vectors.

To assess whether a single immunization of the Ad26.RSV.F or Ad35.RSV.F vaccine can cause vaccine-enhanced disease after attack with RSV A2, histopathological analyzes of the lungs 5 days after infection were performed. Was done (Fig. 14). A single immunization with rAd26.RSV.F or rAd35.RSV.F immunized rAd26.RSV.F or rAd35. compared to rAd-e or mock immunized animals as observed in the first-additional immunization experiments described above. It led to similar immunopathological scores in RSV.F. Obviously, no worsening disease was observed compared to animals initially dosed with FI-RSV. The histopathological scores of animals immunized with rAd vectors were comparable to that of mock infected animals.

In conclusion, all single dose vaccination strategies showed complete protection against RSV challenge replication, induced strong virus neutralizing antibodies, and did not show enhanced pathology.

Example 6. Vectors with the same variant as fragments of RSV F or with alternative promoters show similar immunogenicity.

The above examples were performed using vectors expressing wild type RSV F. Other truncated or modified forms of F are constructed in rAd35, providing embodiments of fragments of RSV F in adenovirus vectors. These truncated or modified forms of F have a truncated form of RSV-F without a cytoplasmic domain and a transmembrane region (i.e., leaving only an ectodomain fragment left) and a cleavage of the cytoplasmic and transmembrane regions, with additional internal deletions to the ectodomain. And contains a fragmented form of RSV-F with the addition of a trimerization domain. These vectors did not improve responses to rAd35.RSV.F with full-length F protein.

In addition, other rAd35 vectors were constructed with different alternative promoters driving the expression of wild-type RSV F.

RSV. Immunogenic and promoter variants of the modified forms of F were compared in a mouse model and compared to Ad35.RSV.F expressing wild type F. All Ad35 vectors with these F variants or promoter variants showed responses in the order of the same size as Ad35.RSV.F.

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

This experiment measures the potential of rapid onset of protection by adenovirus vectors expressing the RSV-F protein in a cotton rat model. For this purpose, cotton rats in each experimental group of 8 cotton rats carry the full length RSV F (A2) gene (Ad35.RSV.F) or no transgene (Ad26e) on day 0 or 21. Immunized with a single intramuscular injection of an adenovirus vector of 10 ⁷ , 10 ⁸ or 10 ^{9 viral particles (vp).} As it is known that primary infection with RSV virus protects against secondary attack replication, ^{animals infected intranasally with RSV A2 (10 4} plaque forming units (pfu)) were used as positive controls for protection against attack replication. ( Prince.Lab Invest 1999 , 79:1385-1392). At 7 weeks or 49 days after 4 weeks of immunization, cotton rats were challenged intranasally with ^{1 x 10 5 pfu of plaque-purified RSV A2.} Cotton rats were sacrificed 5 days post infection, when RSV attack virus reached peak titers (Prince. Lab Invest 1999 , 79:1385-1392), and lung and nasal RSV titers were measured by viral plaque titration. (Prince et al. 1978, Am J Pathology 93,711-791). Figure 16 shows that high RSV virus titers in the lungs and nose were _{observed in animals receiving 4.8 +/- 0.11 log 10} pfu/gram and 5.1 +/- 0.32 log ₁₀ pfu/gram of adenovirus, respectively, without transgene. Shows that. In contrast, all animals immunized with Ad35.RSV.F vectors at high doses (10 ⁹ vp) were completely protected against RSV titers in the lungs and nose at 7 weeks post immunization, and almost completely at 4 weeks post immunization. Was protected. Lower doses of Ad35.RSV.F vectors conferred complete protection at RSV titers in the lungs and partial protection at nasal titers at 4 and 7 weeks after immunization. Blood samples were taken on the day of challenge (49 days). Serums were tested in a neutralization test for induction of RSV specific antibodies (FIG. 17 ). Immunization with Ad vectors induced dose dependent VNA titers. Figure 18 shows that control animals do not have virus neutralizing antibodies during the experiment, whereas high VNA titers are induced in animals at 28 or 49 days after immunization with ^{10 7} to 10 ^{9 Ad35.RSV.F vp.} . Prior to immunization, no virus neutralizing antibodies were detected in cotton mice. Immunization with adenovirus vectors induced dose-dependent VNA titers higher than or comparable to neutralizing titers caused by primary intranasal RSV infection. This experiment clearly suggests the rapid onset of protection against attack viral replication by Ad35 expressing RSV-F.

Example 8. Protection against RSV subgroup A and subgroup B infection after immunization of in vivo recombinant adenovirus vectors in a cotton mouse model

RSV strains can be divided into two subgroups, A and B subgroups. This subtype distinction is based on differences in antigenicity of the highly variable G glycoprotein. The sequence of the F protein is highly conserved, but can also be classified into the same A and B subgroups. Patent application 0200 EO POO described that sera of Ad-RSV.F vector immunized mice may cross-neutralize the B1 strain in vitro. Figure 19 shows high VNA titer 49 days after immunization against RSV Long (subgroup A) and Bwash (subgroup B, ATCC #1540) derived from cotton mice immunized with _{Ad35.RSV-F A2.} It clearly shows that they are visible. In vivo protection against either subgroup A or B attack was measured in cotton mice using low adenovirus vector doses at ^{10 7} and 10 ^{8 vp.} For this purpose, cotton rats were divided into experimental groups of 8 cotton rats each. Animals are muscle of adenovirus vectors of ^{10 7} or 10 ⁸ viral particles (vp) with full length RSV F (A2) gene (Ad35.RSV.F) or without transgene (Ad26e) on day 0. I was immunized by my injections. Animals infected intranasally with RSV A2 (10 ⁴ plaque forming units (pfu)) were used as positive controls for protection against challenge replication. On day 49, animals were challenged intranasally ^{with 10 5} pfu of either RSV-A subgroup RSV-A2 or RSV-B strain RSV-B 15/97.

Figure 20 shows that high RSV viral titers in the lungs were observed in animals receiving adenovirus vectors without the transgene. In contrast, no attacking virus could be detected in lung and nasal tissues from animals immunized with Ad35.RSV.F vectors. No differences were observed regarding protection when attacked with either RSV-A2 or RSV-B 15/97. Ad35.RSV.F _A2 showed complete protection against lung attack replication at 10 ⁷ and 10 ^{8 vp.} Figure 21 shows that high RSV virus titers in the nose were observed in animals receiving transgene-free adenovirus vectors. Ad35.RSV.F _A2 showed partial protection against nasal attack virus replication at 10 ^{8 vp.} No difference was observed regarding protection when attacked with either RSV-A2 or RSV-B 15/97. During the experiment, blood samples were taken on day 28 and the day of challenge (49 days). Serums were tested in a neutralization test for induction of RSV specific antibodies. Figure 22 shows virus neutralizing titers during the run of the experiment, and that the control animals do not have virus neutralizing antibodies during the run. High VNA titers were induced in animals 28 days after immunization with 10 ⁸ or 10 ^{7 Ad35.RSV.F vp.}

Example 9. Protection against high attack dose of RSV-A2 after immunization of in vivo recombinant adenovirus vectors in cotton rat model

This example measures protection against a high attack dose of ^{5 x 10 5} pfu compared to a standard dose of 1 x 10 ^{5 pfu RSV-A2.} The study placed cotton rats into experimental groups of 8 cotton rats each. ^{Animals with low dose adenovirus of 10 7} or 10 ⁸ viral particles (vp) with full length RSV F (A2) gene (Ad35.RSV.F) or without transgene (Ad26e) on day 0 Immunized by intramuscular injections of vectors. Animals infected intranasally with RSV A2 (10 ⁴ plaque forming units (pfu)) were used as positive controls for protection against challenge replication. Cotton rats were sacrificed 5 days after infection, and lung and nasal RSV titers were measured by viral plaque titration. Figure 23 shows that higher challenge dose induces higher lung virus load in animals receiving transgene-free adenovirus vectors than that of standard challenge dose. Animals immunized with 10 ⁸ vp Ad35.RSV.F vectors were completely protected against standard and high RSV challenge titers in the lungs. Figure 24 shows ^{that animals immunized with 10 8} or 10 ⁷ vp Ad35.RSV.F vectors were partially protected against standard and high RSV challenge titers in the nose.

Example 10. Ad26.RSV.F, first-additionally administered with recombinant F protein, leads to a Th1 sloped response in a mouse model.

In this example, it was investigated whether the immune response upon the initial administration of Ad26.RSV.F could be enhanced by further administration with the supplemented recombinant RSV F protein. For this purpose, mice were divided into experimental groups of 7 mice each. Animals were immunized on day 0 by intramuscular injection of ^{10 10} viral particles (vp) adenovirus vectors bearing the full length RSV F (A2) gene (Ad26.RSV.F) or PBS. On day 28, animals were further administered intramuscularly with the same vector at the same dose or with the supplemented RSV F protein (full length; postfusion conformation: post-F) (2 doses: 5 μg and 0.5 μg). . Figure 25 clearly shows _{that sera derived from mice supplemented with Ad26.RSV-F A2} immunized and supplemented RSV F show high VNA titers 12 weeks after immunization against RSV-A Long (subgroup A). do. FIG. 26 depicts the IgG2a/IgG1 ratio in sera of mice supplemented with _{Ad26.RSV-F A2 immunized and supplemented RSV F protein.} Higher proportions represent Th1 balanced responses, while lower proportions represent Th2 sloped responses. Obviously, Ad26.RSV.F immunized animals supplemented with Ad26.RSV.F or RSV F protein resulted in a high IgG2a/IgG1 ratio, whereas (without the context of adenovirus vectors) FI-RSV or RSV F protein Control mice immunized with induce a low rate. Because the Th1 sloped response is strongly required in RSV vaccines to avoid enhanced disease and induce strong T cell memory upon attack, the Th2 sloped response of protein immunization is the case when the first dose of Ad26.RSV.F is applied. It can be guided by the Th1 reaction. FIG. 31 depicts cellular responses in spleens derived from mice immunized with _{Ad26.RSV-F A2 and further administered with supplemented RSV F protein.} It can also be clearly observed that further administration with the assisted RSV F protein will strongly increase the cellular response. Based on the previously performed experiments described in the above examples, it is predicted that similar results will occur if Ad26 is substituted with Ad35.

Sequences

SEQ ID NO: 1: RSV fusion protein (gene bank number ACO83301.1) amino acid sequence:

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKIKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

SEQ ID NO: 2: codon optimized RSV.F(A2)nat gene encoding RSV fusion protein

ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGCGGACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCAACATCAAAAAGAACAAGTGCAACGGCACCGACGCCAAAATCAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCCAGACGGGAGCTGCCCCGGTTCATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTCCTGCTGGGCGTGGGCAGCGCCATTGCTAGCGGAGTGGCTGTGTCTAAGGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGTCCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCAGCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGTCCATCATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCC TGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCAGCGTGTCATTCTTTCCACAAGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCAGCGAGGTGAACCTGTGCAACGTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGCGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCATCATCGTGATCATCGTCATCCTGCTGTCCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCCGGTCCACCCCTGTGACCCTGTCCAAGGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAAC

SEQUENCE LISTING <110> Crucell Holland B.V. Radosevic, Katarina Widjojoatmodjo, Myra Custers, Jerome Vellinga, Jort <120> Vaccine against RSV <130> 0200 EP P00 PRI <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 574 <212> PRT <213> respiratory syncytial virus <220> <221> RSV F protein (A2 strain) <222> (1)..(574) <400> 1 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Lys Asn Lys Cys Asn Gly Thr Asp Ala Lys Ile Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr 115 120 125 Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn 195 200 205 Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val 370 375 380 Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Ala Val Lys Ser Thr Thr Asn Ile Met Ile Thr 515 520 525 Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val 530 535 540 Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser 545 550 555 560 Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn 565 570 <210> 2 <211> 1722 <212> DNA <213> respiratory syncytial virus <220> <221> codon optimized sequence encoding RSV F protein (A2 strain) <222> (1)..(1722) <400> 2 atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gaattctacc agagcacctg tagcgccgtg 120 tccaagggct acctgagcgc cctgcggacc ggctggtaca ccagcgtgat caccatcgag 180 ctgagcaaca tcaaaaagaa caagtgcaac ggcaccgacg ccaaaatcaa gctgatcaag 240 caggaactgg acaagtacaa gaacgccgtg accgagctgc agctgctgat gcagagcacc 300 cccgccacca acaaccgggc cagacgggag ctgccccggt tcatgaacta caccctgaac 360 aacgccaaaa agaccaacgt gaccctgagc aagaagcgga agcggcggtt cctgggcttc 420 ctgctgggcg tgggcagcgc cattgctagc ggagtggctg tgtctaaggt gctgcacctg 480 gaaggcgaag tgaacaagat caagtccgcc ctgctgagca ccaacaaggc cgtggtgtcc 540 ctgagcaacg gcgtgtccgt gctgaccagc aaggtgctgg atctgaagaa ctacatcgac 600 aagcagctgc tgcccatcgt gaacaagcag agctgcagca tcagcaacat cgagacagtg 660 atcgagttcc agcagaagaa caaccggctg ctggaaatca cccgcgagtt cagcgtgaac 720 gccggcgtga ccacccccgt gtccacctac atgctgacca acagcgagct gctgagcctg 780 atcaacgaca tgcccatcac caacgaccag aaaaagctga tgagcaacaa cgtgcagatc 840 gtgcggcagc agagctactc catcatgtcc atcatcaaag aagaggtgct ggcctacgtg 900 gtgcagctgc ccctgtacgg cgtgatcgac accccctgct ggaagctgca caccagcccc 960 ctgtgcacca ccaacaccaa agagggcagc aacatctgcc tgacccggac cgaccggggc 1020 tggtactgcg ataatgccgg cagcgtgtca ttctttccac aagccgagac atgcaaggtg 1080 cagagcaacc gggtgttctg cgacaccatg aacagcctga ccctgcccag cgaggtgaac 1140 ctgtgcaacg tggacatctt caaccctaag tacgactgca agatcatgac ctccaagacc 1200 gacgtgtcca gctccgtgat cacctccctg ggcgccatcg tgtcctgcta cggcaagacc 1260 aagtgcaccg ccagcaacaa gaaccggggc atcatcaaga ccttcagcaa cggctgcgac 1320 tacgtgtcca acaagggcgt ggacaccgtg tccgtgggca acaccctgta ctacgtgaac 1380 aaacaggaag gcaagagcct gtacgtgaag ggcgagccca tcatcaactt ctacgacccc 1440 ctggtgttcc ccagcgacga gttcgacgcc agcatcagcc aggtcaacga gaagatcaac 1500 cagagcctgg ccttcatcag aaagagcgac gagctgctgc acaatgtgaa tgccgtgaag 1560 tccaccacca atatcatgat caccacaatc atcatcgtga tcatcgtcat cctgctgtcc 1620 ctgatcgccg tgggcctgct gctgtactgc aaggcccggt ccacccctgt gaccctgtcc 1680 aaggaccagc tgagcggcat caacaatatc gccttctcca ac 1722

Claims (1)

Vaccine against RSV as disclosed in the specification and figures.

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