EA043031B1 - RSV VACCINE - Google Patents

Description

Respiratory syncytial virus (RSV) is a highly contagious pathogen that causes respiratory tract infections in children and is thought to be responsible for ~200,000 childhood deaths annually. In children under 2 years of age, RSV accounts for approximately 50% of hospitalizations due to respiratory tract infections, with the highest number of hospitalizations occurring between 2 and 4 months of age. Almost all children have been reported to have had an RSV infection by the age of two years, with re-infection throughout life associated with reduced innate immunity. In the elderly, the severity of disease due to RSV is similar to that due to infections caused by the non-pandemic influenza A virus. There is currently no vaccine against RSV, but it is necessary due to the high severity of the disease.

RSV is a paramyxovirus belonging to the Pneumovirinae subfamily. Its genome encodes various proteins, including membrane proteins known as RSV glycoprotein (G) and RSV fusion protein (F), which are major antigenic targets for neutralizing antibodies.

In contrast to the G protein, the RSV F protein is conserved among RSV strains, making it a promising vaccine candidate capable of inducing the production of a broad spectrum of neutralizing antibodies. The F protein is a transmembrane protein, and during virus budding it is incorporated into the virion membrane from the cell membrane. The RSV F protein promotes infection by fusing the membranes of the virus and the host cell. During the fusion process, the F protein undergoes an irreversible refolding from a labile pre-fusion conformation to a stable post-fusion conformation. The F0 precursor protein requires cleavage during intracellular maturation by a furin-like protease. There are two sites for furins, the cleavage of which results in the removal of the p27 peptide and the formation of two domains: an N-terminal F2 domain and a C-terminal F1 domain (Fig. 1). The F2 and F1 domains are linked by two cysteine bridges. Antibodies to the fusion protein can prevent the uptake of the virus by the cell and thus have a neutralizing effect. In addition to being a target for neutralizing antibodies, F RSV contains epitopes for cytotoxic T cells (Pemberton et al., 1987, J. Gen. Virol. 68: 2177-2182).

Despite 50 years of research, there is still no approved RSV vaccine. One of the major obstacles to vaccine development is the experience of vaccine-enhanced disease in clinical trials in the 1960s. when using a formalin-inactivated (FI) RSV vaccine. Children vaccinated with FI-RSV were not protected from natural infection, and infected children showed more severe illness than unvaccinated children, including two deaths. This phenomenon is called enhanced disease.

Since the trial of the FI-RSV vaccine, various approaches have been taken to develop an RSV vaccine. Efforts have included classical live cold attenuated passable or temperature sensitive mutant strains of RSV, vaccines based on subunit (chimeric) proteins, peptide vaccines, and RSV proteins expressed with recombinant viral vectors, including adenovirus vectors. Although some of these vaccines have shown promising preclinical data, none of the vaccines have been approved for use in humans due to safety concerns or lack of efficacy.

Therefore, there remains a need for effective vaccines and vaccination methods against RSV, in particular vaccines that do not lead to increased disease. It is an object of the present invention to provide such vaccines and methods for vaccinating against RSV in a safe and effective manner.

Brief description of the invention

The present invention provides novel nucleic acid molecules encoding stable pre-fusion RSV F proteins, wherein the pre-fusion RSV F proteins comprise the amino acid sequence of SEQ ID NO: 1 or 2.

In certain embodiments, nucleic acid molecules encoding pre-fusion RSV F proteins are codon-optimized for expression in human cells.

In certain embodiments, the nucleic acid molecules encoding RSV F proteins prior to fusion comprise the nucleic acid sequence of SEQ ID NO: 3 or 4.

The present invention further provides vectors containing nucleic acid molecules encoding pre-fusion RSV F proteins, wherein the RSV F protein contains the amino acid sequence of SEQ ID NO: 1 or 2.

In certain embodiments, the vector is a recombinant human adenovirus.

In certain embodiments, the recombinant adenovirus is adenovirus serotype 26 or 35.

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

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In certain embodiments, the implementation of the recombinant adenovirus has a genome containing in its 5'-terminal regions of the sequence CTATCTAT.

The present invention also provides compositions, for example respiratory syncytial virus (RSV) vaccines, containing nucleic acid molecules or a vector of the present invention.

The present invention further provides a method of vaccinating a subject against RSV, the method comprising administering to the subject a composition according to the present invention.

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

The present invention also provides a method of reducing the severity of RSV infection and/or replication, for example, in the nasopharynx and lungs of a subject, comprising administering to the subject a composition comprising a nucleic acid or vector in accordance with the present invention. This will reduce the adverse effects experienced by the subject as a result of an RSV infection and thus, by administering the composition, help protect the subject from such adverse effects. In certain embodiments, the adverse effects of an RSV infection may actually be prevented, i. reduced to such low levels that they would not be clinically significant.

The present invention also provides an isolated host cell comprising a nucleic acid molecule encoding a pre-fusion RSV F protein, wherein the pre-fusion RSV F protein comprises the amino acid sequence of SEQ ID NO: 1 or 2. In certain embodiments, the nucleic acid molecule, encoding the RSV F protein prior to fusion contains the nucleic acid sequence of SEQ ID NO: 3 or 4.

The present invention further provides a method for producing a respiratory syncytial virus (RSV) vaccine, comprising providing a recombinant human adenovirus that contains a nucleic acid encoding an RSV F protein before fusion or a fragment thereof, propagating said recombinant adenovirus in a host cell culture, isolating and purifying the recombinant adenovirus and formulating the recombinant adenovirus into a pharmaceutically acceptable composition, wherein the RSV F protein prior to fusion comprises the amino acid sequence of SEQ ID NO: 1 or 2. In certain embodiments, the recombinant adenovirus is adenovirus serotype 26 or 35.

Brief description of graphic materials

Fig. 1 is a schematic representation of the RSV F protein. The precursor protein includes the F2 and F1 domains and the p27 peptide, which is removed in mature proteins by cleavage with furin-like proteases. Cleavage sites are indicated by arrows. The numbers above the boxes indicate the positions of the amino acids in the full-length protein, excluding the signal peptide. F1 shows the structural elements: fusion peptide (FP), refolding region 1 (RR1) including heptad repeat A (HRA), and refolding region 2 (RR2) including heptad repeat B (HRB).

Fig. 2 - relative expression of F-protein variants on the surface. Full length F protein variants were expressed in HEK293T cells. Cells were stained with anti-F RSV antibody (CR9503) and analyzed by flow cytometry (FACS). Mean fluorescence intensity (MFI) values were calculated and normalized to MFI for a control sample of cells transfected with wild-type F protein (Fwt). The MFI for Fwt was set to 1. The bars represent the mean values, the error bars represent the range of values.

Fig. 3 - F-protein fraction before fusion on the cell surface. Full length F protein variants were expressed in HEK293T cells. Cells were stained with anti-F RSV antibody CR9503 and anti-F RSV antibody to CR9501 confluence and analyzed by flow cytometry. Mean fluorescence intensity (MFI) values were calculated and MFI measured by CR9501 was normalized to MFI measured by CR9503. Normalized MFI values indicate the pre-F fraction on the cell surface. Bars represent averages, error bars represent ranges.

Fig. 4 - relative expression of F-protein variants on the surface. Soluble variants with a transmembrane region and a cytoplasmic region (Fsl) removed, as well as full-length variants of the F protein, were expressed in HEK293T cells. Soluble protein expression level was measured in the culture supernatant with Octet and full length variants were tested by cell staining with anti-F RSV antibody (CR9503) and analyzed by flow cytometry. Mean fluorescence intensity (MFI) values were calculated and normalized to MFI for a control sample of cells transfected with wild-type F-protein (Fwt). The MFI for Fwt was set to 1. The bars represent the mean values, the error bars represent the range of values.

Fig. 5 - temperature stability of F-protein variants. Full length F protein variants were expressed in HEK293T cells. After heat shock, the cells were stained with an antibody to F

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RSV (CR9501 - solid lines and CR9503 - dashed lines) and analyzed by flow cytometry. The percentage of positively stained cells was determined. Symbols represent averages, error bars represent ranges. (A) The percentage of positively stained cells was determined. (B) Mean fluorescence intensity (MFI) values were calculated and normalized to MFI for a cell sample at 37°C. The MFI for the sample at 37°C was set as 1. The dotted lines correspond to background staining at 60°C.

Fig. 6 - stability of F-proteins. PreF is more stable than the FA2 protein in the pre-fusion conformation on the cell surface in the heat stress assay. A549 cells were infected with Ad26 and Ad35 containing the FA2 insert (F RSV dt, gray boxes) or pre-fusion stabilized F insert (preF2.2, black boxes) at the indicated MOI. Cells were exposed to temperature at the indicated temperature for 15 min before staining. Top: Percentage of cells with F present on their surface before confluence (detected with CR9501 antibody); bottom: percentage of cells with any form of F-protein present before and after fusion (detected with CR9503 antibody). Values were normalized to samples at 37°C. All bars represent a single measurement.

Fig. 7 - Ad26.RSV.preF2.1 and F2.2 induce a cellular immune response in mice after a single injection. The horizontal bars depict the geometric mean of the answer in the group. Baseline is calculated as the 95% percentile of spot forming units (PFU) detected in unstimulated splenocytes and is indicated by a dotted line.

Fig. 8 - Ad26.RSV.preF2.1 and F2.2 induction of increased levels of virus-neutralizing antibodies compared to Ad26.RSV.FA2 in mice after a single immunization. Balb/c mice (n=4 per group) were immunized with the indicated dose of 108 to 1010 viral particles (vp) of Ad26.RSV.FA2 or Ad26.RSV.preF2.1 or Ad26.RSV.preF2.2 or buffer for compilation, and then analyzed the blood serum isolated 8 weeks after immunization, regarding the humoral immune response. (A) Virus neutralizing antibodies were determined against RSV A Long using a microneutralization assay followed by ELISA reading. Titers are given as log2 value from IC ₅₀ . (B) Pre-fusion or post-fusion anti-F antibody titers were determined by ELISA, calculating the ratio of anti-F pre-fusion and post-fusion antibodies for all samples showing pre-fusion and post-fusion anti-F antibody titers above the lower limit of quantitation (LLoQ). ). (C) ELISA to determine subclasses was performed using RSV F A2 after fusion as a coating reagent and the ratio of IgG2a/IgG1 (log10) was plotted. The ratio found for Th1 (serum obtained from animals immunized with adenoviral vectors based on F RSV expression) and Th2 (serum obtained from animals immunized with FI-RSV) reference samples are indicated by dotted lines. LLoQ is indicated by dotted lines (panels A) and the horizontal bars represent the mean of responses per group.

Fig. 9 - call through Ad26.RSV.preF2.2 immune response, which neutralizes a wide range of RSV isolates. Serum from Balb/c mice immunized with 1010 viral particles (vp) Ad26.RSV.FA2 (n=3) or Ad26.RSV.preF (n=4) or formulation buffer (n=2), were used in virus neutralization assays (microneutralization assay with ELISA readout) with the indicated RSV strains A (upper panels) and B (lower panels). Titers are given as log2 IC ₅₀ values and the horizontal bars represent the mean response per group. LLoQ is indicated by a broken line.

Fig. 10 Protection of cotton hamsters with a single immunization with Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2 at low doses against challenge with homologous RSV A2. Cotton hamsters (Sigmodon hispidus) (n=7-9 per group) were immunized at the indicated doses (in vp/animal) with Ad26.RSV.preF2.2, Ad26.RSV.FA2 (left panels), Ad35.RSV.preF2.2 or Ad35.RSV.FA2 (right panels) by single intramuscular injection. Control immunizations were performed using formulation buffer, FI-RSV, or intranasal low dose RSV A2. Seven weeks after immunization, animals were challenged intranasally with 105 PFU RSV A2. Virus titers in the lungs (A-B) and in the nasal cavity (C-D) were determined by plaque assay 5 days post infection. (E-F) Serum samples taken just before challenge were used for virus neutralization assay using RSV A Long strain (microneutralization assay with ELISA readout). The dotted line represents the lower limit of quantification (LLoQ). The horizontal bars represent the mean titer value per group.

Fig. 11 - No increase in alveolitis histopathological scores as a result of immunization of cotton hamsters with Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2 after challenge with RSV A2. Cotton hamsters (Sigmodon hispidus) (n=7-9 per group) were immunized with the indicated doses (in vp/animal) of Ad26.RSV.preF2.2, Ad26.RSV.FA2 (upper panel), Ad35.RSV.preF2.2 or Ad35.RSV.FA2 (bottom panel) by single intramuscular injection. Control immunizations were performed using formulation buffer, FI-RSV or intranasal application.

- 3 043031 low dose RSV A2. Seven weeks after immunization, animals were challenged intranasally with 105 PFU RSV A2. Alveolitis was scored by histopathological assessment of one lobe of the lung 5 days post-challenge on a non-linear scale from 0 to 4. The horizontal dotted line indicates the maximum score for control animals that were pre-exposed to RSV-A2 prior to challenge to mimic natural exposure to RSV, which does not lead to ERD.

Detailed description of the invention

RSV vaccines containing a human adenovirus that contains a nucleic acid encoding an RSV F protein have previously been described in WO2013/139911 and WO2013/139916. They showed that recombinant adenoviruses of serotypes 26 or 35, which contain a nucleic acid encoding the RSV F protein, are highly effective vaccines against RSV in the conventional cotton rat model and have improved performance when compared with data previously described for Ad5 encoding F- RSV protein. It has been demonstrated that a single administration, even intramuscularly, of RSV F-encoding Ad26 or Ad35 is sufficient to provide complete protection against RSV infection and further replication.

However, due to the instability of the RSV F protein, the RSV F protein is prone to premature refolding to its more stable conformation after fusion. This phenomenon is a natural property of the protein both in solution and on the surface of virions. In human serum, most RSV-neutralizing antibodies, however, are directed against RSV F in the pre-fusion conformation. Therefore, the studies that led to the present invention attempted to identify modifications that stabilize the RSV F protein in its pre-fusion conformation into the full length protein.

A number of possible mutations stabilizing the RSV F protein in a pre-fusion conformation have previously been described in WO2014/174018 and WO2014/202570. The nucleic acid molecules of the present invention encode RSV F proteins containing a unique and specific set of mutations. According to the present invention, it has been shown that this unique combination of mutations of the present invention results in increased expression levels of the RSV F protein and stability of the RSV F protein in the pre-fusion conformation. In addition, the nucleic acid molecules of the present invention have been shown to encode an RSV F protein that is stabilized in the prefusion conformation and that induces higher neutralizing antibody titers compared to the wild-type RSV F protein (as disclosed in WO2013/139911 and WO2013/139916).

Thus, in a first aspect, the present invention provides novel nucleic acid molecules encoding a respiratory syncytial virus F protein before fusion (RSV F protein before fusion, or RSV pre-F protein), wherein the RSV F protein before fusion comprises amino acid sequence under SEQ ID NO: 1 or 2.

In certain embodiments, the nucleic acid molecules encode RSV pre-F proteins of SEQ ID NO: 1 or SEQ ID NO: 2.

As used herein, the terms nucleic acid, nucleic acid molecule, nucleic acid or nucleotide sequence, and polynucleotide are used interchangeably and all refer to linear biopolymers (strands) derived from nucleotides, including DNA and RNA.

One skilled in the art will appreciate that several different nucleic acid molecules can encode the same polypeptide as a result of the degeneracy of the genetic code. It is also understood that those skilled in the art can, using conventional techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides will be expressed. Therefore, unless specifically stated otherwise, the expression nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that are degenerate variants of each other and that encode the same amino acid sequence. Nucleotide sequences that code for proteins and RNA may include introns. In this document, sequences are presented in the 5' to 3' direction, as is customary in the art.

In certain embodiments, the nucleic acid molecule encodes a pre-fusion F RSV fragment containing the amino acid sequence of SEQ ID NO: 1 or 2.

In certain embodiments, the nucleic acid molecules encoding the pre-fusion F protein of RSV, or a fragment thereof, are codon-optimized for expression in mammalian cells, such as human cells. Methods for optimizing codons are known and have been described previously (eg WO 96/09378).

In certain embodiments, the nucleic acid molecule encoding the RSV F protein prior to fusion comprises the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the nucleic acid encoding the RSV F protein comprises the nucleic acid sequence of SEQ ID NO: 4 .

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In certain embodiments, the nucleic acid encoding the RSV F protein consists of the nucleic acid sequence of SEQ ID NO: 3 or 4.

As used herein, the term fragment refers to a peptide that has an amino-terminal and/or carboxy-terminal and/or internal deletion, but in which the remainder of the amino acid sequence is identical to the corresponding positions in the sequence of a full-length RSV F protein, e.g., the RSV F protein under SEQ ID NO 1 or 2. It will be understood that in order to induce an immune response and for vaccination purposes in general, the protein does not need to be full-length and does not need to have all the functions of a wild-type protein, but fragments of the protein are equally applicable. One of skill in the art will also appreciate that changes can be made to a protein, for example, by substitutions, deletions, additions of amino acids, etc., for example, using conventional molecular biology techniques. In general, conservative amino acid substitutions can be used without loss of function or immunogenicity of the polypeptide. This can be easily checked based on conventional procedures well known to the person skilled in the art.

The present invention also relates to vectors containing a nucleic acid molecule encoding a pre-fusion RSV F protein, comprising the amino acid sequence of SEQ ID NO: 1 or 2, or a fragment thereof.

According to the present invention, the vector may be any vector that can be easily subjected to procedures using recombinant DNA and which can cause expression of the nucleic acid molecule of the present invention. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced.

In certain embodiments, the vector is a recombinant human adenovirus, also referred to as recombinant adenovirus vectors. The production of recombinant adenoviral vectors is well known in the art. As used herein, in relation to an adenovirus, recombinant implies that it has been modified by a human, for example, it has altered terminals actively cloned into it, and/or it contains a heterologous gene, i.e. it is not a naturally occurring wild-type adenovirus .

In certain embodiments, the adenoviral vector of the present invention is defective in at least one essential functional E1 region gene, e.g., the E1a region and/or the E1b region of the adenoviral genome, which is required for viral replication. In certain embodiments, the implementation of the adenoviral vector in accordance with the present invention is defective in at least part of the E3 region, which is not essential. In certain embodiments, the vector is defective in at least one essential functional E1 region gene and in at least a non-essential portion of the E3 region.

The adenoviral vector may be multi-defective, which means that the adenoviral vector is defective in one or more essential functional genes in each of two or more regions of the adenoviral genome. For example, the aforementioned E1 or E1 deficient, E3 deficient adenoviral vectors may additionally be deficient in at least one essential E4 region gene and/or at least one essential E2 region gene (e.g., E2L region and/or section E2B).

Adenovirus vectors, methods for constructing them, and methods for propagating them are well known in the art and are described, for example, in US Pat. Nos. 5,559,099; 5,837,511; and 6113913 and Thomas Shenk , Adenoviridae and their Replication, M. S. Horwitz, Adenoviruses, Chapters 67 and 68, respectively, in Virology, B. N. Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996), and elsewhere mentioned in this document. Typically, construction of adenoviral vectors involves the use of standard molecular biology techniques such as those described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), Watson et al., Recombinant DNA, 2d ed., Scientific American Books (1992) and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, NY (1995), and other references cited in this document.

In certain embodiments, the adenovirus is human adenovirus serotypes 26 or 35. The vaccines of the present invention based on these serotypes proved to be more effective than the prior art vaccines based on Ad5 because they could not provide complete protection against RSV replication upon challenge after a single intramuscular injection (Kim et al., 2010 , Vaccine 28: 3801-3808; Kohlmann et al., 2009, J Virol 83: 12601-12610; Krause et al., 2011, Virology Journal 8:375). The serotype of the present invention as a whole is further characterized by low serotype dominance and/or low titers of pre-existing neutralizing antibodies in the human population. Recombinant adenoviral vectors of these serotypes with different transgenes are being evaluated in clinical trials and have been shown to have an excellent safety profile to date. The production of vectors based on rAd26 is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplary sequences of the Ad26 genome are found in GenBank under accession number EF 153474 and under SEQ ID NO: 1 in WO 2007/104792. The production of rAd35 vectors is described, for example, in US Pat. No. 7,270,811, in WO 00/70071 and in Vogels et al., (2003) J Virol 77(15): 8263-71. Exemplary Ad35 genome sequences are found in GenBank accession number AC_000019 and FIG. 6 of WO 00/70071.

The recombinant adenovirus of the present invention may be replication competent or replication defective. In certain embodiments, the adenovirus is replication defective, for example, because it contains a deletion in the E1 region of the genome. As the skilled person knows, in the case of deletions of essential regions from the adenovirus genome, the functional elements encoded by these regions must be provided in the trans position, preferably by the producer cell, i.e. if parts or whole regions of E1, E2 and/or E4 removed from the adenovirus, they must be present in the producer cell, for example, be built into its genome or be in the form of the so-called auxiliary adenovirus or auxiliary plasmids. The adenovirus may also contain a deletion in the E3 region that is not essential for replication and therefore such a deletion should not be complemented.

A producer cell (also sometimes referred to in the art and herein as a packaging cell or complementary cell or host cell) that can be used can be any producer cell in which the desired adenovirus can be propagated. For example, propagation of vectors based on recombinant adenovirus is carried out in producer cells that complement defects in the adenovirus. Preferably, such producer cells contain in their genome at least the E1 sequence of an adenovirus, and thus they are capable of complementing recombinant adenoviruses with a deletion in the E1 region. Any E1-complementing producer cell can be used, e.g. human retinal cells immortalized with E1, e.g. 911 or PER.C6 cells (see US Pat. ), E1 transformed A549 cells (see, for example, WO 98/39411, US patent No. 5891690), GH329: HeLa (Gao et al., 2000, Human Gene Therapy 11: 213-219), 293, etc. B 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-defective adenoviruses such as Ad35 (subgroup B) or Ad26 (subgroup D), it is preferable to replace the E4-orf6 coding sequence of these non-subgroup C adenoviruses with the E4-orf6 adenovirus of subgroup C , such as Ad5. This allows such adenoviruses to propagate in well-known complementary cell lines that express E1 genes from Ad5, such as 293 cells or PER.C6 cells (see, for example, Havenga et al., 2006, J. Gen. Virol. 87: 2135-2143; WO 03/104467, incorporated herein in its entirety by reference). In certain embodiments, the adenovirus that can be used is human adenovirus serotype 35 with a deletion in the E1 region into which the nucleic acid encoding the RSV F protein antigen has been cloned and with the E4 orf6 region of Ad5. In certain embodiments, the adenovirus in the vaccine composition of the present invention is a human serotype 26 adenovirus with a deletion at the E1 region into which the nucleic acid encoding the RSV F protein antigen has been cloned and with the E4 orf6 region of Ad5.

In alternative embodiments, it is not necessary to place the heterologous E4orf6 region (e.g., from Ad5) in an adenoviral vector, but instead, the E1-defective non-subgroup C vector is propagated into cell lines that express both E1 and compatible E4orf6, e.g. , in the 293-ORF6 cell line that expresses both E1 and E4orf6 from Ad5 (see, for example, Brough et al. al., 1997, J Virol 71: 8946-51 and Nan et al., 2003, Gene Therapy 10: 326-36, each of which describes the production of non-subgroup C adenoviral vectors with E1 deleted).

Alternatively, a complementary cell can be used that expresses E1 from the serotype to be propagated (see, for example, WO 00/70071, WO 02/40665).

For subgroup B adenoviruses, such as Ad35, with a deletion in the E1 region, it is preferable to retain the 3' end of the E1B 55K open reading frame in the adenovirus, for example, 166 bp immediately upstream of the pIX open reading frame, or a fragment including them , for example, a 243 bp fragment immediately upstream of the pIX start codon (terminated at the 5' end by the Bsu36I restriction site in the Ad35 genome), as this increases the stability of the adenovirus because the pIX gene promoter is partially located in this region (See, for example, Havenga et al., 2006, J. Gen. 87: 2135-2143; WO 2004/001032, incorporated herein by reference).

A heterologous nucleic acid (also referred to herein as a transgene) in the adenoviruses of the present invention is a nucleic acid that is not naturally present in an adenovirus. It is introduced into adenovirus using, for example, a stan

- 6 043031 gift methods of molecular biology. In the present invention, the heterologous nucleic acid encodes an RSV pre-F protein (or fragment thereof) comprising the nucleic acid sequence of SEQ ID NO: 1 or 2. For example, it can be cloned into the remote E1 or E3 region of an adenoviral vector. The transgene is usually operably linked to expression control sequences. This can be done, for example, by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Additional regulatory sequences may be added. A variety of promoters can be used to express the transgene(s) and are known to those skilled in the art. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is the CMV promoter (US 5385839), for example, the CMV immediate early response gene promoter, for example, containing nucleotides -735 to +95 from the CMV immediate early response gene enhancer/promoter. The polyadenylation signal, for example, the polyA signal of the bovine growth hormone gene (US 5122458), can be located behind the transgene(s).

In certain embodiments, the implementation of the recombinant adenoviral vectors of the present invention contain as 5'-terminal nucleotides the nucleotide sequence: CTATCTAT. These embodiments are advantageous because such vectors exhibit improved replication in production methods, resulting in adenovirus batches with improved homogeneity compared to vectors having the original 5' end sequences (typically CATCATCA) (see also patent applications nos. PCT/EP2013/054846 and US 13/794318, under the heading Batches of recombinant adenovirus with altered terminal ends' filed March 12, 2012 on behalf of Crucell Holland B.V., incorporated herein by reference in their entirety. Thus, the present invention also provides batches of recombinant adenovirus encoding an RSV F protein or a portion thereof, wherein the RSV F protein comprises the amino acid sequence of SEQ ID NO: 1 or 2, and where virtually all (e.g., at least 90% ) adenoviruses in the batch contain a genome with a terminal nucleotide sequence CTATCTAT.

In certain embodiments, the nucleic acid molecule may encode a F protein fragment of RSV prior to fusion. The fragment may be the result of any one or both of the amino-terminal and carboxy-terminal deletions. The size of the deletion can be determined by a person skilled in the art, for example, to achieve a better yield of recombinant adenovirus. A fragment will be selected that includes an immunologically active fragment of the F protein, ie. the part that will elicit an immune response in the subject. It can be readily determined using in silico, in vitro and/or in vivo methods, all of which are well known to the skilled person.

In addition, the present invention provides compositions containing a nucleic acid molecule encoding a pre-fusion RSV F protein, comprising the amino acid sequence of SEQ ID NO: 1 or 2.

Also provided in the present invention are compositions containing the vector described herein.

In certain embodiments, compositions comprising a nucleic acid molecule and/or a vector are for use in reducing the severity of RSV infection and/or replication in a subject. In certain preferred embodiments, the compositions are for use as an RSV vaccine. The term vaccine refers to an agent or composition containing an active ingredient effective to induce in a subject, at a therapeutic level, immunity against a specified pathogen or disease. In the present invention, the vaccine comprises an effective amount of a nucleic acid molecule that encodes a pre-fusion RSV F protein, comprising SEQ ID NO: 1 or 2, or an antigenic fragment thereof, that elicits an immune response to the RSV F protein. This provides a way of preventing the onset of severe lower respiratory disease leading to hospitalization and reduces the incidence of complications such as pneumonia and bronchiolitis due to RSV infection and replication in a subject. Thus, the present invention also provides a method for preventing or reducing severe lower respiratory disease, preventing or reducing (e.g., shortening) hospitalization, and/or reducing the frequency and/or severity of pneumonia or bronchiolitis caused by RSV in a subject, comprising administering to the subject a composition comprising a nucleic acid molecule encoding an RSV pre-F protein or a fragment thereof, wherein the RSV F protein comprises the amino acid sequence of SEQ SEQ ID NO: 1 or 2. The term vaccine according to the present invention implies that it is a pharmaceutical composition and thus generally includes a pharmaceutically acceptable diluent, carrier, or excipient. It may or may not contain additional active ingredients. In certain embodiments, it may be a combination vaccine that additionally contains other components that induce an immune response, for example against other RSV proteins and/or against other infectious agents.

In certain embodiments, compositions containing the nucleic acid molecule or vector further comprise or are administered with one or more adjuvants. Hell

- 7 043031 juvants known in the art to further increase the immune response to the antigenic determinant used, and pharmaceutical compositions containing adenovirus and suitable adjuvants are disclosed, for example, in WO 2007/110409, incorporated herein by reference. The terms adjuvant and immunostimulant are used interchangeably herein and are defined as one or more substances that stimulate the immune system. In this context, an adjuvant is used to enhance the immune response to RSV F proteins prior to the fusion of the present invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil emulsion compositions (or oil-input compositions), including emulsions of squalene in water, for example MF59 (see, for example, WO 90/14837); saponin-based formulations such as, for example, QS21 and immunostimulatory complexes (ISCOM) (see, for example, US 5057540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); derivatives of bacteria or microorganisms, examples of which are monophosphoryl lipid A (MPL), 3-Odeacetylated MPL (3dMPL), oligonucleotides containing the CpG motif, ADP-ribosylated bacterial toxins or mutants thereof, such as E. coli heat-labile enterotoxin LT, cholera toxin CT and similar to them. It is also possible to use a vector-encoded adjuvant, for example, by using a heterologous nucleic acid that encodes a C4 binding protein (C4bp) oligomerization domain fusion to the 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, for example, in the form of aluminum hydroxide, aluminum phosphate, aluminum-potassium phosphate, or combinations thereof, at concentrations of 0.05-5 mg, for example, 0.075-1.0 mg of aluminum per dose.

In other embodiments, the compositions do not contain adjuvants.

According to the present invention it is also possible to introduce additional active ingredients in combination with compositions, for example vaccines, according to the present invention. Such additional active ingredients may include, for example, other RSV antigens or vectors containing the nucleic acid encoding them. Such vectors may, again, be non-adenoviral or may be adenoviral, of which the latter may be of any serotype. An example of other RSV antigens includes the RSV F or G protein or immunologically active portions thereof. For example, an intranasally administered recombinant replication-deficient Ad5-based adenoviral vector rAd/3xG expressing the soluble core domain of glycoprotein G (amino acids 130-230) conferred protection in a mouse model (Yu et al., 2008, J Virol 82: 2350-2357 ), and although it did not provide protection when administered intramuscularly, it is clear from these data that RSV G is a suitable antigen to induce protective responses. Additional active ingredients may also include non-RSV antigens, eg derived from other pathogens such as viruses, bacteria, parasites and the like. The administration of additional active ingredients can, for example, be carried out by administration alone or by administration of a combination of products from the vaccines of the present invention and additional active ingredients. In certain embodiments, additional non-adenoviral antigens (other than RSV.F) may be encoded in the vectors of the present invention. In certain embodiments, expression of more than one protein from a single adenovirus may be required, and in such cases, for example, more coding sequences may be linked to form a single transcript from a single expression cassette, or they may be present in two separate expression cassettes cloned into different parts of the adenovirus. genome.

Compositions of the present invention, eg adenovirus compositions, can be administered to a subject, eg a human subject. The total dose of adenovirus provided to a subject in a single administration may vary, as known to the practitioner, and is typically from 1x10 ⁷ viral particles (vp) to 1x10 ¹² vp, preferably from 1x108 vp to 1x1011 vp, for example, from 3x108 to 5x10 ¹⁰ vp, e.g. 109 to 3x10 ¹⁰ vp.

The introduction of the compositions can be carried out using standard routes of administration. Non-limiting embodiments include parenteral administration, such as injection, eg, intradermal, intramuscular, etc., or subcutaneous, transdermal, or mucosal administration, eg, intranasal, oral, and the like. In general, it has been noted that intranasal administration is the preferred route for RSV vaccines. The most important advantage of the intranasal strategy with live viruses is the direct stimulation of local immunity in the respiratory tract and the absence of an associated increase in the disease. The only pediatric vaccines currently in clinical trials are live intranasal vaccines (Collins and Murphy. Vaccines against human respiratory syncytial virus). B: Perspectives in Medical Virology 14: Respiratory Syncytial Virus (Ed. Cane, P.), Elsevier, Amsterdam, the Netherlands, pp233-277). Intranasal administration is also a suitable preferred route according to the present invention. The advantage of intramuscular administration is that it is simple and reliable and does not pose a safety risk when administered intranasally in infants less than 6 months of age. In one embodiment, the composition is administered by intramuscular injection, for example, into the deltoid muscle of the arm or the vastus lateralis muscle of the thigh. The person skilled in the art is aware of various options for administering a composition, such as a vaccine, to induce an immune response to the antigen(s) present(s) in the vaccine.

The subject, as used herein, is preferably a mammal, such as a rodent, such as a mouse, a cotton rat, or a non-human or human primate. Preferably the subject is a human subject. The subject may be of any age, for example, from about 1 month to about 100 years, for example, from about 2 months to about 80 years, for example, 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 compositions, eg vaccines, of the present invention. In a booster vaccination, typically such a booster vaccine will be administered to the same subject at intervals of one week to one year, preferably two weeks to four months, following the administration of the composition to the subject for the first time (referred to here as a primer vaccination). ). In alternative booster regimens, different vectors can also be administered to the subject after priming, for example one or more adenoviruses of different serotypes, or other vectors such as MVA or DNA or protein. For example, a recombinant adenoviral vector containing a nucleic acid sequence encoding an RSV F protein prior to fusion can be administered to a subject as a priming antigen and boosted using a composition containing the RSV F protein. In certain embodiments, the RSV F protein is also stabilized in a pre-fusion conformation.

In certain embodiments, the administration includes a primer and at least one booster. In certain embodiments, the composition is administered as a priming composition and/or as a booster composition according to a priming-booster schedule. In certain appropriate embodiments, the priming is performed using rAd35 comprising a nucleic acid encoding an RSV pre-F protein or fragment thereof ('rAd35-RSV.preF'), wherein the RSV pre-F protein contains the amino acid sequence of SEQ ID NO : 1 or 2, and booster introduction is carried out using rAd26 containing a nucleic acid encoding the RSV F protein ('rAd26-RSV.pre-F'), where the RSV pre-F protein contains the amino acid sequence under SEQ SEQ ID NO: 1 or 2. In appropriate other embodiments, the priming is performed using rAd26-RSV.pre-F and the booster is performed using rAd35-RSV.pre-F. In other embodiments, both the primer and booster are performed using rAd26.RSV.pre-F. In certain embodiments, the priming is performed using rAd26-RSV.pre-F or rAd35-RSV.pre-F and the booster is performed using RSV F protein. In all of these embodiments, additional boosters can be provided with the same or different vectors or protein. Embodiments in which boosting with the RSV F protein may be particularly useful include, for example, elderly subjects at risk (eg, with COPD or asthma) 50 years of age or older, or, for example, healthy subjects 60 years of age or older or 65 years of age or older.

In certain embodiments, the implementation of the introduction provides for a single introduction of the composition according to the present invention without additional (booster) introductions. Such embodiments are advantageous in terms of reducing the complexity and cost of a single dose regimen compared to a primer-booster regimen. Complete protection is observed after a single administration of the recombinant adenoviral vectors of the present invention without booster administrations in the cotton hamster model of the examples in this document.

In an additional aspect, the present invention provides methods for producing a respiratory syncytial virus (RSV) vaccine, comprising providing a recombinant human adenovirus that contains a nucleic acid encoding an RSV F protein or a fragment thereof, propagating said recombinant adenovirus in a host cell culture, isolating and purifying the recombinant adenovirus and formulating the recombinant adenovirus into a pharmaceutically acceptable composition, wherein the RSV F protein contains the amino acid sequence of SEQ ID NO: 1 or 2.

Recombinant adenovirus can be generated and propagated in host cells according to well known methods which involve cell culture of host cells that are infected with adenovirus. The cell culture can be any type of cell culture, including adherent cell culture, such as cells attached to the surface of a culture flask or microcarriers, and suspension culture.

Suspension cultures are handled on the largest scale in a batch or fed-batch process because they are the easiest to manage and

- 9 043031 zoom. Nowadays, continuous processes based on perfusion principles are becoming more common, and they are also suitable (see, for example, WO 2010/060719 and

WO 2011/098592, both of which are incorporated herein by reference, which describe suitable methods for the preparation and purification of large quantities of recombinant adenoviruses).

Producer cells are cultured to increase cell and virus numbers and/or virus titers. Cultivation of the cell is performed to enable it to metabolize and/or grow and/or divide and/or produce the virus of interest in accordance with the present invention. This can be achieved using methods that are generally well known to those skilled in the art, and this includes, without limitation, providing nutrients to the cell, for example, in appropriate nutrient media. Suitable growth media are well known to those skilled in the art and can generally be obtained in large quantities from commercial sources or manufactured to order according to standard protocols. Culture can be carried out, for example, in dishes, roller bottles, or in bioreactors using batch, fed-batch, continuous systems, etc. Suitable cell culture conditions are known (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9).

Typically, the adenovirus will be exposed to the appropriate producer cell in culture, allowing uptake of the virus. Typically optimal mixing is between about 50 to 300 rpm, typically about 100-200, e.g. about 150, typically DO is 20-60%, e.g. 40%, optimum pH is 6.7 to 7.7, the optimum temperature is 30 to 39°C, eg 34-37°C, the optimum MOI is 5 to 1000, eg approximately 50-300. Typically, adenovirus infects producer cells spontaneously, and bringing the producer cells into contact with rAd particles is sufficient to infect the cells. Typically, adenovirus inoculum is added to the culture to initiate infection, and the adenovirus subsequently replicates in the producer cells. All this is customary for a person skilled in the art.

After infection with an adenovirus, the virus replicates within the cell and is thus amplified, a process referred to herein as adenovirus propagation. As a result, adenovirus infection leads to lysis of the infected cell. Therefore, the lytic characteristics of the adenovirus make it possible to obtain the virus in two different ways. The first method is to harvest the virus prior to cell lysis using external factors to lyse the cell. The second method is harvesting the viral supernatant with (substantially) complete lysis of the cell by the produced virus (see, for example, US Pat. No. 6,485,958 which describes the harvesting of adenovirus without lysis of host cells with an external agent). It is preferred to use external factors to actively lyse the cells to harvest the adenovirus.

Methods that can be used for active cell lysis are known to the person skilled in the art and are discussed, for example, in WO 98/22588, p. 28-35. Applicable methods in this regard are, for example, freeze-thaw, disruption of cells with abrasive materials, hypertonic and/or hypotonic lysis, fluid shear, ultrasonic disruption, high pressure extrusion, detergent lysis, combinations of the above, and the like. In one embodiment of the present invention, the cells are lysed using at least one detergent. The advantage of using a lysis detergent is that the method is simple and easy to scale up.

Detergents that can be used and how they are used are generally known to those skilled in the art. For example, several examples are discussed in WO 98/22588, p. 29-33. Detergents may include anionic, cationic, zwitterionic and nonionic detergents. The detergent concentration can be varied, for example in the range of about 0.1-5% (w/w). In one embodiment, the detergent used is Triton X-100.

Nucleases can be used to remove contaminants, i. mainly from the cell producer, nucleic acids. Exemplary nucleases suitable for use in the present invention include Benzonase®, Pulmozyme®, or any other DNase and/or RNase commonly used in the art. In preferred embodiments, the nuclease is a Benzonase® that rapidly hydrolyzes nucleic acids by hydrolyzing internal phosphodiester bonds between certain nucleotides, thereby reducing the viscosity of the cell lysate. Benzonase® is commercially available from Merck KGaA (code W214950). The concentration at which the nuclease is used is preferably in the range of 1-100 U/ml. As an alternative or in addition to nuclease treatment, it is also possible to selectively precipitate host cell DNA from adenovirus preparations during adenovirus purification using selective precipitants such as domiphen bromide (see, for example, US 7326555; Goerke et al., 2005, Biotechnology and bioengineering , Vol 91: 12-21; WO 2011/045378; WO 2011/045381).

Methods for harvesting adenovirus from producer cell cultures are described in detail in WO 2005/080556.

- 10 043031

In certain embodiments, the harvested adenovirus is further purified. Purification of adenovirus can be carried out in several stages, involving clarification, ultrafiltration, diafiltration or separation using chromatography, as described, for example, in WO 05/080556, incorporated herein by reference. Clarification can be accomplished with 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 ultrafilters is a method of removing or replacing salts, sugars and the like. One skilled in the art knows how to select the optimal conditions for each purification step. Also, WO 98/22588, incorporated herein by reference in its entirety, describes methods for the preparation and purification of adenoviral vectors. The methods include growing host cells, infecting host cells with adenoviruses, harvesting and lysing host cells, concentrating the crude lysate, replacing the buffer in the crude lysate, treating the lysate with nuclease, and further purifying the virus using chromatography.

Preferably, at least one chromatographic step is used in the purification, as for example discussed in WO 98/22588, p. 61-70. Many methods for further purification of adenoviruses have been described, where chromatography steps are included in the method. One of skill in the art will be aware of these methods and may make changes to the correct way of using the chromatographic steps to optimize the method. For example, it is possible to purify adenoviruses using anion exchange chromatography steps, see eg WO 2005/080556 and Konz et al., 2005, Hum Gene Ther 16: 1346-1353. Many other methods for purifying adenovirus have been described and are available to those skilled in the art. Additional methods for the preparation and purification of adenoviruses are disclosed, for example, in (WO 00/32754; WO 04/020971; US 5837520; US 6261823; WO 2006/108707; Konz et al., 2008, Methods Mol Biol 434: 13-23; Altaras et al., 2005, Adv Biochem Eng Biotechnol 99: 193-260), all of which are incorporated herein by reference.

For administration to humans, pharmaceutical compositions containing rAd and a pharmaceutically acceptable carrier or excipient may be used in the present invention. In the present context, the term pharmaceutically acceptable means that the carrier or excipient, at the dosages and concentrations used, will not cause any undesirable or adverse effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. 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 edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The purified rAd is preferably formulated and administered as a sterile solution, although lyophilized preparations may also be used. Sterile solutions are obtained by sterilizing filtration or by other methods known per se in this field. The solutions are then lyophilized or filled into dosage form containers. The pH of the solution is typically in the range of pH 3.0 to 9.5, for example pH 5.0 to 7.5. Typically, rAd is in solution with a suitable pharmaceutically acceptable buffer, while the rAd solution may also contain a salt. Optionally, a stabilizing agent such as albumin may be present. In certain embodiments, a detergent is added. In certain embodiments, rAd can be formulated as an injectable. These formulations containing effective amounts of rAd are either sterile liquid solutions, liquid suspensions, or lyophilized versions and optionally contain stabilizers or excipients. Also, the adenovirus vaccine may be in the form of an aerosol for intranasal administration (see, for example, WO 2009/117134).

For example, adenovirus can be stored in a buffer also used for the Adenovirus World Standard (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% glycerin. Another useful formulation buffer suitable for administration to humans is 20 mM Tris, 2 mM MgCl ₂ , 25 mM NaCl, 10% w/v. sucrose, 0.02% w/v polysorbate-80. Many other buffers can of course be used, and some examples of suitable formulations for storage and for pharmaceutical administration of purified (adeno)virus preparations can be found, for example, in EP 0853660, US Pat. No. 6225289 and in international patent applications WO 99/41416, WO 99/12568, WO 00/29024, WO 01/66137, WO 03/049763, WO 03/078592, WO 03/061708.

The present invention is further explained in the following examples. The examples do not limit the invention in any way. They serve only to explain the present invention.

Examples

Example 1 Stabilization of the RSV F protein in its conformation prior to fusion.

Plasmids encoding the basic F RSV sequences were synthesized and amino acid substitutions were introduced into the protein by site-directed mutagenesis. Protein variants were transiently expressed in HEK293 cells. The relative expression of the protein on the cell surface was assessed using flow cytometry. The stability of the F proteins in the pre-fusion conformation was assessed in a thermal stability assay.

- 11 043031

Protein sequence information used for RSV A2 F protein variants was obtained from GenBank under accession number ACO83301.1. Amino acid substitutions were introduced into the sequence using site-directed mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit, Agilent technologies). Mutagenesis primers were designed using the online PrimerX program. HEK293T (CRL-11268) cells were purchased from the American Tissue Culture Collection and cultured under standard cell culture conditions (37° C., 10% CO ₂ ).

Fully humanized anti-RSV F protein IgG1 antibodies CR9501 and CR9503 were constructed by cloning the heavy (VH) and light (VL) variable regions into an IgG1 expression vector. PER.C6® cells (Crucell) were transfected with IgG1 expression constructs and expressed antibodies were purified from culture supernatant using POROS Mabcapture A chromatography (Applied Biosystems) followed by buffer exchange with 50 mM NaAc, 50 mM NaCl, pH 5, 5. The antibody concentration was measured by optical absorbance at 280 nm. The amount of antibody was also confirmed using size exclusion chromatography (SEC), SDS-PAGE and isoelectric focusing. The CR9501 antibody contains the VH and VL regions of 58C5 (described in WO2011/020079) that specifically binds to the RSV F protein in its pre-fusion conformation but not in its post-fusion conformation. CR9503 contains the VH and VL regions from motavizumab, which recognizes F RSV in both pre-fusion and post-fusion conformations.

Protein expression and heat treatment.

Plasmids were transiently transfected into adherent HEK293T cells using 293fectine transfection reagents (p/n 12347-019) (Life Technologies) as recommended by the supplier. 48 h after transfection, cells were harvested by detaching them with EDTA-containing FACS buffer (without trypsin, see next section) and the cell suspension was exposed to heat for 10 min either in a water bath or in a PCR block to perform experiments for thermal stability. After heat treatment, the cells were prepared for analysis using flow cytometry.

In order to analyze the expression of F-proteins in adenoviruses, A549 cells were infected with Ad26 virus at MOI 10000 or 5000 and Ad35 virus at MOI 5000, 2500 or 1250. After 48 h, the cells were peeled off and subjected to heat treatment for 15 min at 37°C, 50°C. C and 56°C. During heat treatment, cells were stained using CR9501-Alexa647 or CR9503-Alexa647 and propidium iodide (PI). After staining, cells were fixed and analyzed using a BD FACS CANTO II cell analyzer.

Analysis based on flow cytometry.

For each staining group, the following controls were included: 1) a negative control sample, i.e. cells that have not been subjected to any treatment and not stained with any antibody labeled with a fluorophore; 2) positive control samples, i.e. cells that were stained with only one fluorophore (one of which was used for the experiment).

Cells were resuspended in flow cytometry buffer (Buffer FC, 5 mM EDTA, 1% FBS in PBS) and dispensed in volumes of 50 µl of cell suspension per well in a 96 well capped plate (U- or V-shaped well plates). ). Two-step or one-step protocols were used for staining.

In the case of a two-step protocol, 50 µl of the first Ab (or buffer for negative controls) was added to the wells and incubated at rt. within 30 min. Biotinylated CR9501 and CR9503 were used at 2 μg/ml (final well concentration 1 μg/ml). After incubation, cells were washed 2 times with FC buffer. Then 50 µl of streptavidin-APC (Molecular Probes cat. no. SA1005, 0.1 mg/ml used at 1:100) or negative control buffer was added to the wells and incubated at rt. within 30 min. Cells were again washed 2 times with FC buffer. After the last wash, the cells were resuspended in 100 µl FC buffer +/- staining for the presence of live and dead cells (PI from Invitrogen, cat. no. P1304MP, used at 2 µg/ml) and incubated at rt. within 15 min. The cells were centrifuged at 200g (1000 rpm) for 5 min, the PI buffer was removed and the cells were resuspended in 150 µl of FC buffer.

For the one-step protocol, CR9501 and CR9503 antibodies were labeled with the Alexa647 fluorescent probe (Molecular Probes, Cat # A-20186) according to the manufacturer's instructions. Cells were stained according to the protocol above, excluding the streptavidin-APC step.

In a living cell population, the percentage of cells positive for binding to the CR9501/CR9503 antibody was determined. Cells positive for CR9503 binding expressed the RSV F protein on their surface. Cells positive for CR9501 binding expressed the RSV F protein to confluence on their surface.

The intensity of antibody staining (mean fluorescence intensity - MFI) is proportional to the amount of F-protein on the cell surface. MFI was calculated for a population of living cells expressing the F-protein.

- 12 043031

Results.

Expression of full-length F-protein variants on the cell surface.

A previously identified set of mutations that increase the expression or stability of the RSV F protein ectodomain in a pre-fusion conformation were introduced into the full-length F RSV A2 wild-type sequence (Genbank accession number ACO83301). Mutations were introduced singly or in multiple combinations and evaluated for their effect on protein expression and stability.

Protein expression level was measured as mean fluorescence intensity (MFI) by flow cytometry after staining with the CR9503 antibody, which recognizes the F protein both before and after fusion. The combination of two amino acid substitutions that have previously been described to stabilize the soluble RSV pre-F protein (i.e., N67I and S215P) also increased the expression level of the full-length RSV F-protein by 2.3-fold compared to the full-length wild-type F RSV. (Fig. 2).

The most significant increase in expression was observed for variants with combined 3 amino acid substitutions. Notably, a combination of more than three mutations in one variant did not further increase protein expression. The reason may be the limited ability of the cell membrane to accommodate multiple copies of the F-protein.

The amount of pre-fusion F on the cell surface was assessed by staining with the CR9501 antibody specific for the pre-fusion protein (FIG. 3). Transfection of cells with all F variants resulted in a more or less similar amount of pre-fusion F protein at the cell surface. The presence of the transmembrane domain stabilizes the full-length protein to a certain extent and therefore differences in stability in pre-fusion conformation are not apparent under environmental conditions between full-length F proteins. Thus, a thermal stability assay was designed to better determine the difference in stability between the full length variants, as described below.

The A2 strain that was used as the parent sequence for the previously described F-protein variants (WO2014/174018 and WO2014/202570) is a cell line-adapted laboratory strain that assembles two unique and rare mutations (i.e., lysine 66 and isoleucine 76). In the present invention, these two residues were mutated to match natural clinical isolates (K66E, I76V). The K66E and I76V mutations were included in the selected protein constructs. Compared to the Lys66 and Ile76 variants, the variants with glutamate at position 66 (K66E) tend to be slightly overexpressed. The addition of valine to the residue at position 76 (double substitution of K66E and I76V) did not affect the level of expression compared to variants containing only the substitution of K66E (Fig. 4).

Stability of full-length F-protein variants on the cell surface.

Under environmental conditions, over a short period of time, there was no significant difference in pre-fusion conformational stability between full-length F variants compared to different combinations of stabilizing mutations. Elevated temperature is known to be an effective in vitro trigger for the refolding of the RSV F protein from a pre-fusion conformation to a post-fusion conformation. Therefore, the heat shock assay has been adapted and used to assess the stability of membrane-bound full-length proteins. Briefly, HEK293T cells were transfected with F-protein constructs and used for analysis 48 h after transfection. The cells were removed from the cell culture dishes and the cell suspension was heat treated at increasing temperatures for 10 minutes. After heat treatment, the cells were stained with antibodies to F RSV and analyzed by flow cytometry. Flow cytometry data were analyzed in two different ways. The percentage of cells positive for anti-F staining was analyzed and the mean fluorescence intensity (MFI) of the positive cells was calculated (FIG. 5).

Both stains with CR9501 (antibody recognizing F-protein only before fusion) and CR9503 (antibody recognizing F-protein both before and after fusion) were used in flow cytometric assays. The CR9503 antibody served as a positive control. In the event that the F protein loses its pre-fusion conformation but is still present on the cell surface, the protein is still detected using the CR9503 antibody. Loss of staining ability by both antibodies indicates that the protein is not available on the cell surface to bind to the antibody, for example due to aggregation.

Full-length proteins with three or more amino acid substitutions were tested in the assay and compared to wild-type F RSV. The expression of these variants was the highest, and thus these variants were preferred candidates. All proteins contained the N67I and S215P substitutions, and one or two additional stabilizing mutations were added.

The unmodified wild-type protein showed rather stable staining with the CR9503 antibody. The MFI for CR9503 staining increased at higher temperatures, but the scatter was also very high. This indicates that there was no protein aggregation after heat shock. Half of the pre-fusion conformation was lost after cell incubation at about 55°C, after incubation at 60°C, all of the pre-fusion conformation was lost, as was

- 13 043031 demonstrated by reducing the binding of CR9501 to wild-type F samples after heat shock at increasing temperatures.

All pre-fusion F protein variants tested were more stable than wild-type F RSV, with most of the CR9501 staining also retained after treatment at higher temperatures (FIGS. 5 and 6). Proteins with the K498R amino acid substitution were less stable than the others. The introduction of the K66E mutation additionally stabilized the proteins, as well as the variants with the K498R amino acid substitution became as stable as the others, and no loss of pre-fusion conformation at 60°C was noted. Only selected combinations of stabilizing mutations were tested with a combination of K66E and I76V. All four proteins tested were stable when the percentage of positive cells were analyzed, however, in the MFI analysis, the K498R variant showed a clear decrease in CR9501 binding after heat treatment at 60°C, indicating that this variant is less stable when assessed in the heat analysis. stress.

Finally, a combination of three stabilizing mutations (including N67I and S215P) was considered sufficient for high levels of expression and stability. Mutations S46G or D486N were chosen as the third stabilizing mutation because of their position in the protein structure. K66E and I76V were included because they did not adversely affect protein expression and stability, but made the sequence more similar to that found in nature.

Thus, RSV F protein before fusion with K66E, N67I, I76V, S215P and D486N (F2.2) (SEQ ID NO: 2) mutations and RSV F protein before fusion with K66E, N67I, I76V, S215P and S46G mutations (F2.1) (SEQ ID NO: 1) was chosen for the construction of adenoviral vectors. These proteins were shown to be stable in pre-fusion conformation in a thermal stability assay at temperatures up to 60° C. and expressed at high levels.

Example 2. Obtaining adenovirus vectors.

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

Nucleic acid sequences encoding the pre-fusion F proteins of the present invention were gene-optimized for human expression and synthesized using 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.pre-F coding sequence. The RSV.pre-F genes were inserted into the pAdApt35BSU plasmid and into the pAdApt26 plasmid at the HindIII and XbaI sites.

Cell culture.

PER.C6 cells (Fallaux et al., 1998, Hum Gene Ther 9: 1909-1917) were maintained on Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) supplemented with 10 mM MgCl ₂ .

Obtaining adenovirus, infection and reseeding.

All adenoviruses were generated in PER.C6® cells by single homologous recombination and grown as previously described (for rAd35: Havenga et al., 2006, J. Gen. Virol. 87: 2135-2143; for rAd26: Abbink et al., 2007, J. Virol 81: 4654-4663). Briefly, PER.C6 cells were transfected with plasmids based on Ad vectors using Lipofectamine according to the instructions provided by the manufacturer (Life Technologies). To rescue, for example, the Ad35 vectors carrying the cassette for the expression of the RSV.pre-F transgenes, the pAdApt35BSU.RSV.pre-F plasmid and the pWE/Ad35.pIX-rITR.dE3.5orf6 cosmid were used, while for the Ad26 vectors carrying the cassette to express the RSV.pre-F transgene, pAdApt26.RSV.pre-F plasmid and pWE.Ad26.dE3.5orf6 cosmid were used. Cells were harvested one day after complete CPE, freeze-thawed, centrifuged for 5 min. at 3000 rpm and stored at -20°C. The viruses were then purified by plaque assay and amplified in PER.C6 cultured in one well of a 24-well tissue culture plate. Further amplification was performed in PER.C6 cultured using a T25 tissue culture flask and a T175 tissue culture flask. From the T175 flask with crude lysates, 3-5 ml were used to inoculate 20xT175 trilayer tissue culture flasks containing layers of PER.C6 cells at 70% confluence. The virus was purified using a two-step purification method using CsCl. Finally, the virus was stored in aliquots at -85°C.

Example 3 In vivo induction of immunity against F RSV using recombinant adenovirus serotypes 26 and 35

The immunogenicity of Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 was evaluated in mice comparing cellular and humoral immune responses induced by the same doses of Ad26.RSV.FA2 (ie, expressing wild type RSV F protein). Balb/c mice (n=4 per group) were immunized with the indicated doses of 108 to 10 ¹⁰ virus particles (vp) of Ad26.RSV.FA2 or Ad26.RSV.preF2.1 or Ad26.RSV.preF2. 2, or compiling buffer. 8 weeks after priming, the number of RSV F A2-specific IFNγ-spot forming units (PFU) per 106 splenocytes

- 14 043031 was determined using ELISpot. It was shown that Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 induced an increase in the humoral immune response in mice compared to Ad26.RSV.FA2 with a pronounced neutralizing ability and persisting cellular responses. A single intramuscular immunization with Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 elicited a cellular response (Figure 7) characterized by the induction of CD8+ T cells positive for IFNy, IL2, and/or TNFa (data not shown). ).

The number and quality of cellular responses were comparable between Ad26.RSV.preF2.1, Ad26.RSV.preF.2.2 and Ad26.RSV.FA2. In contrast, Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 induced significantly higher titers of RSV-neutralizing antibodies than did Ad26.RSV.FA2. A more detailed analysis of antibody responses demonstrated that Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 induced higher levels of anti-F antibodies before fusion, while anti-F antibody titers after fusion remained comparable to Ad26.RSV.FA2, resulting in significantly increased ratios of preF/postF antibodies. In addition, the ratio of IgG2a/IgG1 antibody responses remained unchanged, showing a similar shift towards Th1 in the humoral response as previously demonstrated for Ad26.RSV.FA2 (FIG. 8).

In addition, with respect to Ad26.RSV.preF2.2, it was demonstrated that the resulting antibodies were capable of neutralizing different strains of RSV A and B, laboratory strains, as well as clinical isolates, similar to what was observed in the case of Ad26.RSV. FA2 (Fig. 9).

Then, the efficacy and immunogenicity of the vector constructs Ad26.RSV.preF2.2 and Ad35.RSV.preF2.2 were evaluated in the cotton hamster model. These animals are susceptible to human RSV replication, with maximal RSV lung titers on days 4 and 5. Control groups in the experiments included groups that were intranasally infected with a low dose of RSV A2, thereby mimicking natural infection, and groups immunized with FI -RSV using the original batch of 100 which induced Enhanced Respiratory Disease (ERD) in clinical studies at a dilution shown to induce ERD in cotton rats.

A single intramuscular immunization of animals with Ad26.RSV.preF2.2 at doses ranging from 105 to 10 ⁸ vp/animal or Ad35.RSV.preF2.2 at doses ranging from 106 to 109 vp/animal resulted in complete protection lungs from infection with vaccine homologous strain RSV A2, except for 3 animals immunized with 105 vp Ad26.RSV.preF2.2 (FIGS. 10A and 10B). Dose-dependent protection against RSV replication in the nasal cavity was noted for both vectors. It ranged from complete protection at 10 ⁸ vp/animal to partial protection at 105 vp in the case of Ad26.RSV.preF2.2, while for Ad35.RSV.preF2.2 the nasal cavities of animals immunized with 109 vp were completely protected against RSV. A2, with 106 vp resulting in partial protection (FIGS. 1°C and 10D). The nasal cavities of animals immunized with Ad26.RSV.preF2.2 and Ad35.RSV.preF2.2 were better protected against RSV A2 infection than when immunized with their wild-type Ad26.RSV.FA2 and Ad35.RSV.FA2 F equivalents when analyzing by dose (p=0.0003 and p=0.0001). Protection against RSV infection was accompanied by a dose-dependent induction of virus-neutralizing antibody titers against RSV A Long, which appeared already at the lowest doses of Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2 (FIGS. 10E and 10F). Dose-based statistical comparison of VNA titers to A Long showed that Ad26.RSV.preF2.2 is more immunogenic than Ad26.RSV.FA2 (p=0.0414), while the occurrence of VNA titers was not significantly different between Ad35.RSV .preF2.2 and Ad35.RSV.FA2.

Additionally, it has been demonstrated that Ad26.RSV.preF and Ad35.RSV.preF do not induce the appearance of histopathological signs of enhanced respiratory disease (ERD) after infection with RSV A2 at any tested concentrations. The cotton hamster is the most commonly used and most studied model for ERD observation. In this animal model, vaccination with FI-RSV stably induces ERD after challenge with RSV, which is visually observed on histopathological analysis of infected lung sections for criteria such as alveolitis consisting mainly of neutrophilic infiltrates and peribronchiolitis consisting mainly of lymphocytic infiltrates. In cotton rats, scoring against these criteria for FI-RSV-induced disease can be performed from day 1 post RSV infection with a maximum of days 4-5 post RSV infection.

ERD was analyzed 5 days post challenge using RSV A2 by scoring 4 criteria for changes in lung inflammation (peribronchiolitis, perivasculitis, interstitial pneumonia, alveolitis). Immunization with FI-RSV resulted in increased scores for most of the histopathological markers, which were especially pronounced for alveolitis (FIG. 11), the marker that was previously shown to be most specific for ERD. An increase in score for alveolitis or any other histopathological marker for ERD was not observed in animals immunized with either Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2 in a priming-only regimen after RSV challenge, even at low vaccine doses , which can induce low affinity/or low antibody levels (FIG. 11). It confirms

- 15 043031 previous results of the authors obtained with vectors Ad26.RSV.FA2 and Ad35.RSV.FA2.

Thus, according to the present invention, it was shown that Ad26.RSV.preF and Ad35.RSV.preF are highly active adenoviral vectors expressing F RSV A2, which is stabilized in the pre-fusion conformation. These vectors induce strong humoral and cellular immune responses. The elicited immune response is protective against RSV A2 challenge and provides a wide range of in vitro neutralization of the virus against clinical and laboratory RSV isolates. Induction of ERD was not detected in cotton rats after exposure to RSV in vaccinated animals and therefore confirmed the data obtained for Ad26 and Ad35 encoding wild-type RSV F A2 antigen. Neither mice nor cotton hamsters showed significant signs of reactogenicity after injection of either Ad26.RSV.preF or Ad35.RSV.preF.

Table 1 Pre-fusion RSV F protein amino acid sequences encoded by the nucleic acid molecules of the present invention (mutations underlined) SEQ ID NO: 1: RSV preF2.1 amino acid sequence:

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITI

ELSNIKEIKCNGTDAKVKLIKOELDKYKNAVTELOLLMQSTPATNNRARRELPRFMNYTLNNAK

KTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAWSLSNGVSV LTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYm nTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYWQLPLYGVIDTPCWK LHTSPLC TTNTKEGSNICLTRTDRGWYCflHKGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEV NLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVS NKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIR KSDELLHNVNAVKS TTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIA FSN SEQ ID NO: 2: RSV preF2.2 amino acid sequence: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITI ELSNIKEIKCNGTDAKVKLIKOELDKYKNAVTELOLLMOSTPATNNRARRELPRFMNYTLNNAK KTNVTLSKK RKRRFLGFLLGVGSAISGVAVSKVLHLEGEVNKIKSALLSTNKAWSLSNGVSV LTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYm nTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYWQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTR TDRGWYCflHKGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEV NLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVS NKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIR KSDELLHNVNAVKSTTNIMITTIIIVIIV ILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIA FSN

- 16 043031

Table 2 Nucleotide sequence of preferred nucleic acid molecules of the present invention

SEQ ID NO: 3: Codon-optimized nucleic acid encoding RSV F pre-F2.1 protein before fusion

PreF2.1

ATGGAGCTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTT CTGCTTCGCCAGCGGCCAGAACATCACCGAGGAGTTCTACCAGAGCACCTGCAGCGCCGTGAGC AAGGGCTACCTGGGCGCCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCA GAAGCTGATCAAGCAGGAGCTGGA CAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCACCAACAAC CGCCAT CGCCAGCGGCGTGGCCGTGAGCAAGGTGCTGCACCTGGAGGCGAGGTGAACAAGATCAAGAGC GCCCTGCTGAGCACCAACAAGGCCGTGGTGAGCCTGAGCAACGGCGTGAGCGTGCTGACCAGCA AGGTGCTGGACCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTG CAG CAT C C C CAACAT C GAGAC C G T GAT C GAG T T C CAG CAGAAGAAC AACAGAC T G C T G GAGAT C ACCAGAGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGAGCACCTACATGCTGACCAACA GCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAGAAGCTGATGAGCAA CAACGTGCAGATCGTGAGACAGCAGAGCTACAGCATCATGAGCATCAAGGAGGAGTGCTG GCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGA AC G T G GA CAT C T T CAAC C C CAAG TAC GAC T G CAAGAT CAT GAC CAG CAAGAC C GAC G T GAG CAG CAGCGTGATCACCAGCCTGGGCGCCATCGTGAGCTGCTACGGCAAGACCAAGTGCACCGCCAGC AACAAGAACAGAGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGAGCAACAAGGGCG TGGACACCGTGAGCGTGGGCACAC CCTGTACTACGTGAACAAGCAGGAGGCAAGAGCCTGTA CGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGAC GCCAGCATCAGCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACG AGTGCTGCACAACGTGAACGCCGTGAAGAGCACCACCAACATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCATCAT ATCATCGTGATCCTGCTGAGCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCAGA AGCACCCCCGTGACCCTGAGCAAGGACCAGCTGAGCGGCATCAACAACATCGCCTTTCAGCAACT GA

SEQ ID NO: 4: Codon-optimized nucleic acid encoding RSV F pre-F2.2 protein before fusion

PreF2.2

ATGGAGCTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTT CTGCTTCGCCAGCGGCCAGAACATCACCGAGGAGTTCTACCAGAGCACCTGCAGCGCCGTGAGC AAGGGCTACCTGAGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCA AGCTGATCAAGCAGGAGCTGGA CAAGTACAAGAACGCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAAC GCCAT CGCCAGCGGCGTGGCCGTGCAAGGTGCTGCACCTGGAGGCGAGGTGAACAAGATCAAGAGC GCCCTGCTGAGCACCAACAAGGCCGTGGTGAGCCTGAGCAACGGCGTGAGCGTGCTGACCAGCA AGGTGCTGGACCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTG CAG CAT C C C CAACAT C GAGAC C G T GAT C GAG T T C CAG CAGAAGAACAACAGAC T G C T G GAGAT C ACCAGAAGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGAGCACCTACATGCTGACCAACA GCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAGAAGCTGATGAGCAA CAACGTGCAGATCGTGAGACAGCAGAGCTACAGCATCATGAGCATCATCAAGGAGGAGTGTGCTG GCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACA AC G T G GACAT C T T CAAC C C CAAG TAC GAC T G CAAGAT CAT GAC CAG CAAGAC C GAC G T GAG CAG CAGCGTGATCACCAGCCTGGGCGCCATCGTGAGCTGCTACGGCAAGACCAAGTGCACCGCCAGC AACAAGAACAGAGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGAGCAACAAGGGCG TGGACACCGTGAGCGTGGGCAACACCC TGTACTACGTGAACAAGCAGGAGGGCAAGAGCCTGTA CGTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCAACGAGTTCGAC GCCAGCATCAGCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGAAAGAGCGACG AGTGCTGCACAACGTGAACGCCGTGAAGAGCACCACCAACATCATCATCATCATCATCATCATCATCATCAT CGTGAT CATCGTGATCCTGCTGAGCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCAGA AGCACCCCCGTGACCCTGAGCAAGGACCAGCTGAGCGGCATCAACAACATCGCCTTTCAGCAACT GA

- 17 043031

SEQUENCE LIST <110> Crucell Holland B.V.

<120> RSV vaccine <130> 0270 EP P00 PRI <140> EP16163807 <141> 2015-04-05 <160>4 < 170> PatentIn version 3.5 < 210>1 < 211>574 < 212> PROTEIN < 213> Artificial sequence <220>

<223> RSV preF2.1 amino acid sequence <400>1

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1.5 1015

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 2530

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Gly Ala Leu

4045

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 5560

Lys Glu Ile Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 7580

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

9095

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135140

- 18 043031

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val

180 185190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200205

Lys Gln Ser Cys Ser Ile Pro Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360365

Thr Met Asn Ser Leu

Thr Leu Pro Ser Glu

370

375

Val Asn Leu Cys Asn Val 380

- 19 043031

Asp 385

ile

Phe

Asn

Pro Lys

390

Tyr

asp

Cys

Lys

Ile 395

Met

Thr

Ser

Lys Thr

400

asp

Val

Ser

Ser

Ser Val

405

ile

Thr

Ser

Leu

Gly 410

Ala

ile

Val

Ser Cys

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

450

Ser

Val

gly

Asn

Thr

455

Leu

Tyr

Tyr

Val

Asn

460

Lys

Gln

Glu

gly

Lys

465

Ser

Leu

Tyr

Val

Lys 470

gly

Glu

Pro

ile

ile

475

Asn

Phe

Tyr

asp

Pro

480

Leu

Val

Phe

Pro

Ser

Asp 485

Glu

Phe

asp

Ala

Ser 490

ile

Ser

Gln

Val

Asn 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

530

ile

ile

Val

ile

ile

535

Val

ile

Leu

Leu

Ser

540

Leu

ile

Ala

Val

Gly 545

Leu Leu

Leu

Tyr

Cys 550

Lys Ala

Arg

Ser

Thr

555

ProVal

Thr

Leu

Ser

560

Lys

AspGln

Leu

Ser

Gly 565

Ile Asn

Asn

ile

Ala 570

Phe Ser

Asn<210>

<211>

<212>

<213>

574

PROTEIN

Artificial sequence <220>

<223>

amino acid sequence of RSV preF2.2 <400>

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile

Leu

Thr

- 20 043031

Asn Ile Thr Glu Glu Phe

1.5 10

Ala Val Thr Phe Cys Phe Ala Ser Gly

2

Tyr Gln Ser Thr Cys Ser Ala Val Ser

40

Arg Thr Gly Trp Tyr Thr Ser Val Ile

55

Lys Glu Ile Lys Cys Asn Gly Thr Asp 65 70

Gln Glu Leu Asp Lys Tyr Lys Asn Ala 85

Met Gln Ser Thr Pro Ala Thr Asn Asn

100 1

Arg Phe Met Asn Tyr Thr Leu Asn Asn

115 120

Leu Ser Lys Lys Arg Lys Arg Arg Phe

130 135

Gly Ser Ala Ile Ala Ser Gly Val Ala 145 150

Glu Gly Glu Val Asn Lys Ile Lys Ser

165

Ala Val Val Ser Leu Ser Asn Gly Val

180 1

Leu Asp Leu Lys Asn Tyr Ile Asp Lys

195 200

Lys Gln Ser Cys Ser Ile Pro Asn Ile

210 215

Gln Lys Asn Asn Arg Leu Leu Glu Ile 225 230

Ala Gly Val Thr Thr Pro Val Ser Thr

Gly Tyr Leu Ser Ala Leu 45

Ile Glu Leu Ser Asn Ile 60

Lys Val Lys Leu Ile Lys 75 80

Thr Glu Leu Gln Leu Leu

95

Ala Arg Arg Glu Leu Pro

110

Lys Lys Thr Asn Val Thr

125

Gly Phe Leu Leu Gly Val 140

Ser Lys Val Leu His Leu

155 160

Leu Leu Ser Thr Asn Lys

170 175

Val Leu Thr Ser Lys Val

190

Leu Leu Pro Ile Val Asn

205

Thr Val Ile Glu Phe Gln

220

Arg Glu Phe Ser Val Asn

235 240

Met Leu Thr Asn Ser Glu

- 21 043031

Met Pro Ile

265

Asn

245

250

255

Leu

Leu

Met

Leu 305

Leu

Thr

Pro

Thr

Asp 385

asp

Tyr

Lys

Thr

Lys 465

Leu

Leu

Met

Ser Leu Ile

260

Ser Asn

275

Ser

290

Tyr

Cys

asp

Gln

ile

ile

gly

Thr

Arg

Ala

Met

370

ile

Val

gly

Thr

Asn

asp

Thr

Asp Gln Lys

270

Lys

Asn

Val

Gln

Ile Val

280

Arg

Gln

Gln

Ser Tyr 285

Ser

ile

Lys

Glu

Glu

295

Val

Leu

Ala

Tyr

Val

300

Val

Gln

Leu

Pro

Val

Thr

gly

ile

Asp 310

Thr

Pro

Cys

trp

Lys

315

Leu

His

Thr

Ser

Pro

320

Asn

Thr 325

Lys

Glu

gly

Ser

Asn

330

ile

Cys

Leu

Thr

Arg 335

trp

Tyr

Cys

asp

Asn

Ala

gly

Ser

Val

Ser

Phe

Phe

340

345

350

Glu

Thr

Cys

Lys

Val

Gln

Ser

Asn

Arg

Val

Phe

Cys

asp

355

360

365

Asn

Phe

Ser

Lys

Phe

Val

450

Ser

Leu

Thr

Leu

375

Pro

Ser

Glu

Val

Asn

380

Leu

Cys

Asn

Val

Asn

Pro

Lys 390

Tyr

asp

Cys

Lys

Ile 395

Met

Thr

Ser

Lys

Thr

400

Ser

Ser

Val 405

ile

Thr

Ser

Leu

Gly 410

Ala

ile

Val

Ser

Cys 415

Thr

Lys

Cys

Thr

Ala

Ser

Asn

Lys

Asn

Arg

gly

ile

ile

420

425

430

Ser

435

Ser

Val

Asn

gly

Cys

asp

Tyr

440

Val

Ser

Asn

Lys

gly

445

Val

asp

gly

Asn

Thr

455

Leu

Tyr

Tyr

Val

Asn

460

Lys

Gln

Glu

gly

Ser Leu

Val Phe

Tyr

Pro

Val

Lys 470

Gly Glu

Pro

ile

ile

475

Asn Phe

Tyr

asp

Pro

480

Ser

Asn

Glu Phe

asp

Ala

Ser

Ile Ser

Gln

Val

Asn

- 22 043031

Glu Lys

485 490 495

Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys

500 505

Ser Asp Glu Leu 510

Leu His Asn Val Asn Ala Val Lys Ser

515 520

Thr Thr Asn Ile Met Ile Thr

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

545550

Lys Asp Gln Leu Ser Gly Ile Asn Asn

565

Ser Thr Pro Val Thr Leu Ser

555 560

Ile Ala Phe Ser Asn

570 <210>3 <211>1725 <212> DNA <213> Artificial sequence <220>

<223> codon-optimized nucleic acid encoding RSV F pre-F2.1 protein before fusion <400> 3 atggagctgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60 tgcttcgcca gcggccagaa catcaccgag gagttctacc agagcacctg cagcgcc gtg 120 agcaagggct acctgggcgc cctgagaacc ggctggtaca ccagcgtgat caccatcgag 180 ctgagcaaca tcaaggagat caagtgcaac ggcaccgacg ccaaggtgaa gctgatcaag 240 caggagctgg acaagtacaa gaacgccgtg accgagctgc agctgctgat gcagagcacc 300 cccgccacca acaacagagc cagaagag ctgcccagat tcatgaacta caccctgaac 360 aacgccaaga agaccaacgt gaccctgagc aagaagagaa agagaagatt cct gggcttc 420 ctgctgggcg tgggcagcgc catcgccagc ggcgtggccg tgagcaaggt gctgcacctg 480 gagggcgagg tgaacaagat caagagcgcc ctgctgagca ccaacaaggc cgtggtgagc 540

- 23 043031 ctgagcaacg gcgtgagcgt gctgaccagc aaggtgctgg acctgaagaa 600 aagcagctgc tgcccatcgt gaacaagcag agctgcagca tccccaacat 660 atcgagttcc agcagaagaa caacagactg ctggagatca ccagagagtt 720 gccggcgtga ccacccccgt gagcacctac atgctgacca acagcgagct 780 atcaacgaca tgcccatcac caacgaccag aagaagctga tgagcaacaa 840 gtgagacagc agagctacag catcatgagc atcatcaagg aggaggtgct 900 gtgcagctgc ccctgtacgg cgt gatcgac accccctgct ggaagctgca 960 ctgtgcacca ccaacaccaa ggagggcagc aacatctgcc tgaccagaac 1200 g acgtgagca gcagcgtgat caccagcctg ggcgccatcg tgagctgcta 1260 aagtgcaccg ccagcaacaa gaacagaggc atcatcaaga ccttcagcaa 1320 tacgtgagca acaagggcgt ggacaccgtg agcgtgggca acaccctgta 1380 aagca ggagg gcaagagcct gtacgtgaag ggcgagccca tcatcaactt 1440 ctggtgttcc ccagcgacga gttcgacgcc agcatcagcc aggtgaacga 1500 cagagcctgg ccttcatcag aaagagcgac gagctgctgc acaacgtgaa 1560 agcaccacca acatcatgat caccaccatc atcatcgtga tcatcgtgat 1620 caacaacatc gccttcagca actga 1725 ctacatcgac cgagaccgtg cagcgtgaac gctgagcctg cgtgcagatc ggcctacgtg caccagcccc cgacagaggc ctgcaaggtg cgaggtgaac cagcaagacc cggcaagacc cggctgcgac ctacgtgaac ctacgacccc gaagatca ac cgccgtgaag cctgctgagc gaccctgagc

- 24 043031 <210> 4 <211> 1725 <212> DNA <213> Artificial sequence <220>

< 223> codon-optimized nucleic acid encoding RSV F pre-F2.2 protein before fusion < 400> 4 gtg 120 agcaagggct acctgagcgc cctgagaacc ggctggtaca ccagcgtgat caccatcgag 180 ctgagcaaca tcaaggagat caagtgcaac ggcaccgacg ccaaggtgaa gctgatcaag 240 caggagctgg acaagtacaa gaacgccgtg accgagctgc agctgctgat gcagagcacc 300 cccgccacca acaacagagc cagaagag ctgcccagat tcatgaacta caccctgaac 360 aacgccaaga agaccaacgt gaccctgagc aagaagagaa agagaagatt cct gggcttc 420 ctgctgggcg tgggcagcgc catcgccagc ggcgtggccg tgagcaaggt gctgcacctg 480 gagggcgagg tgaacaagat caagagcgcc ctgctgagca ccaacaaggc cgtggtgagc 540 ctgagcaacg gcgtgagcgt gct gaccagc aaggtgctgg acctgaagaa ctacatcgac 600 aagcagctgc tgcccatcgt gaacaagcag agctgcagca tccccaacat cgagaccgtg 660 atcgagttcc agcagaagaa caacagactg ctggagatca ccagagagtt cagcgtgaac 720 gccggcgtga ccacccccgt gagcacctac atgctgacca acagcgagct gctgagcctg gcagatc 840 gtgagacagc agagctacag catcatgagc atcatcaagg aggaggtgct ggcctacgtg 900 gtgcagctgc ccctgtacgg cgtgatcgac accccctgct ggaagctgca caccagcccc 960 ctgtgcacca ccaacaccaa ggagggcagc aacatctgcc t gaccagaac cgacagaggc

- 25 043031

1020 tggtactgcg acaacgccgg cagcgtgagc ttcttccccc aggccgagac ctgcaaggtg

1080 cagagcaaca gagtgttctg cgacaccatg aacagcctga ccctgcccag cgaggtgaac 1140 ctgtgcaacg tggacatctt caaccccaag tacgactgca agatcatgac cagcaagacc tgagctgcta cggcaagacc 1260 aagtgcaccg ccagcaacaa gaacagaggc atcatcaaga ccttcagcaa cggctgcgac 1320 tacgtgaag ggcgagccca tcatcaactt ctacgacccc 1440 ctggtgttcc ccagcaacga gttcgacgcc agcatcagcc aggtgaacga gaagatcaac 1500 cagagcctgg ccttcatcag aaagagcgac gagctgctgc acaacgtgaa cgccgtgaag 1560 agcaccacca acatcatgat caccaccatc atcatcgtga tcatcgtgat cctgctgagc 1620 ctgatcgccg tgggcctgct gctgtactgc aaggccagaa gcacccccg t gaccctgagc 1680 aaggaccagc tgagcggcat caacaacatc gccttcagca actga 1725

Claims (1)

A method for vaccinating a subject against RSV, the method comprising administering to the subject a vector based on recombinant human adenovirus serotype 26 containing a nucleic acid molecule encoding an RSV F protein that is stabilized in a pre-fusion conformation (RSV F protein before fusion), where F is the RSV protein contains the amino acid sequence of SEQ ID NO: 1 or 2, wherein the recombinant human adenovirus has a deletion in the E1 region, a deletion in the E3 region, or a deletion in both the E1 and E3 regions of the adenovirus genome.