WO2018210871A1

WO2018210871A1 - Methods and compositions for inducing protective immunity against rsv infection

Info

Publication number: WO2018210871A1
Application number: PCT/EP2018/062604
Authority: WO
Inventors: Olivier GODEAUX; Jerald C. Sadoff; Macaya Julie DOUOGUIH
Original assignee: Janssen Vaccines & Prevention B.V.
Priority date: 2017-05-17
Filing date: 2018-05-15
Publication date: 2018-11-22

Abstract

Compositions, vaccines and methods using adenovirus vectors for inducing protective immunity against a respiratory syncytial virus (RSV) infection are described.

Description

Methods and compositions for inducing protective immunity against RSV infection

Field of the invention

This invention relates to compositions, vaccines and methods for inducing protective immunity against RSV infection.

Background of the invention

Respiratory syncytial virus (RSV) is considered to be the most important cause of serious acute respiratory illness in infants and children under 5 years of age. Globally, in 2005, RSV was responsible for an estimated 3.4 million hospitalizations worldwide in children under 5 years of age. Furthermore, 66,000 to 199,000 children younger than 5 years died from RSV-associated acute lower respiratory tract infection (LRTI) in 2005, and 99% of these deaths occurred in developing countries. Nevertheless, the disease burden due to RSV in developed countries is substantial, with RSV infection during childhood linked to the development of wheezing, airway hyperreactivity and asthma. In the United States (US), the infection rate was 68.8% in children younger than 12 months of age and 82.6% during the second year of life. Virtually all children had been infected at least once by 24 months of age, and about one half had experienced 2 infections. In the US, RSV infection in children under 5 years of age is the cause of 57,000 to 175,000 hospitalizations, 500,000 emergency room visits, and approximately 500 deaths each year. In children under 1 year of age, RSV is the most important cause of bronchiolitis, and RSV hospitalization is highest among children under 6 months of age. Major risk factors for severe RSV disease are premature birth and concurrent heart or lung disease; other risk factors include siblings in the household, male sex, and lack of breastfeeding. Although it is clear that most severe disease occurs during primary infection in infancy, symptomatic upper respiratory tract infections (URTI) occur throughout life. RSV repeatedly re-infects individuals throughout life, and it has been hypothesized that reinfection could be due to the fact that infection with RSV does not induce durable protective immunity or that variations in the virus enable evasion of the immune system.

In addition to children, RSV is an important cause of respiratory infections in the elderly, immunocompromised, and those with underlying chronic cardio-pulmonary conditions. In long-term care facilities, RSV is estimated to infect 5-10% of the residents per year with significant rates of pneumonia (10 to 20%) and death (2 to 5%). In one

epidemiology study of RSV burden, it was estimated that 11 ,000 elderly persons die annually ofRSV in the US.

Despite the high disease burden and a strong interest in RSV vaccine development, no licensed vaccine is available for RSV. In the 1960s, a formalin- inactivated RSV (FI-RSV) vaccine was associated with enhanced respiratory disease (ERD) in young children, characterized by an increased rate of RSV-mediated, severe LRTI in the vaccinated individuals compared with the control group. In addition to the FI-RSV vaccine, several live- attenuated and subunit RSV vaccines have been examined in animal models and human studies. Live-attenuated vaccines have been specifically challenged by difficulties related to over- and under-attenuation in infants. Several vaccine candidates are in development based on a variety of platform technologies including replication incompetent vectors. However, still no licensed vaccines for protection against RSV exists. There thus is an unmet need for safe and effective vaccines that elicit immune responses against RSV viruses. Brief summary of the invention

It is discovered in the present invention that various prime-boost combinations of replication incompetent vectors encoding antigenic RSV protein generate effective immune protection against RSV virus infections in different target populations, in particular in young children.

Accordingly, one general aspect of the present invention relates to a combination vaccine comprising:

(i) a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic respiratory syncytial virus (RSV) protein; and

(ii) a second composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein, wherein one of the compositions is a priming composition and the other composition is a boosting composition. In another aspect, the present invention relates to a combination vaccine comprising:

(i) a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein;

(ii) a second composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein; and (iii) a third composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein, wherein one of the compositions is a priming composition and the other compositions are boosting compositions.

Another general aspect of the present invention relates to the use of a first

composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein; and a second composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein, for generating a protective immune response against a RSV virus.

Another aspect of the present invention relates to the use of a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein; and a second composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein; and a third composition comprising a nucleic acid encoding an antigenic RSV protein, for generating a protective immune response against an RSV virus.

In a preferred embodiment of the invention, the adenovirus vector in the first composition (i) comprises a nucleic acid encoding an antigenic RSV protein having the amino acid sequence of SEQ ID NO: 1 or 2, and the adenovirus vector in the second composition (ii) comprises a nucleic acid encoding an antigenic RSV protein having the amino acid sequence of SEQ ID NO: 1 or 2.

In another preferred embodiment, the adenovirus vector in the third composition comprises a nucleic acid encoding an RSV protein having the amino acid sequence of SEQ ID NO: 1 or 2. In certain embodiments, the nucleic acid encoding the antigenic RSV protein comprises the nucleic acid sequence of SEQ ID NO: 3 or 4.

It is further contemplated that compositions described herein can be embodied in a kit. For example, in one embodiment, the present invention can include a kit comprising: (i) a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein; and

(ii) a second composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein; and, optionally,

(iii) a third composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein.

In a preferred embodiment, the present invention relates to a combination vaccine, a kit or a use as described herein, wherein the adenovirus vector in composition (i) comprises a nucleic acid encoding an RSV F protein having the amino acid sequence of SEQ ID NO: 1 or 2; and wherein the adenovirus vector in composition (ii) comprises a nucleic acid encoding an RSV F protein having the amino acid sequence of SEQ ID NO: 1 or 2.

In another embodiment, the adenovirus vector in composition (iii) comprises a nucleic acid encoding an RSV F protein having the amino acid sequence of SEQ ID NO: 1 or 2.

In a preferred embodiment, the adenovirus vector in composition (i), (ii) and (iii) comprises a nucleic acid encoding an RSV F protein having the amino acid sequence of SEQ ID NO: 1

In certain embodiments, the nucleic acid encoding the antigenic RSV protein comprises the nucleic acid sequence of SEQ ID NO: 3 or 4. In a preferred embodiment the adenovirus vectors are recombinant Ad26 (rAd26) vectors.

One additional general aspect of the present invention relates to a method of inducing an immune response against an RSV virus in a subject, the method comprising: (i) administering to the subject a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein; and

(ii) administering to the subject a second composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein; and, optionally,

(iii) administering to the subject a third composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein of an RSV virus.

In a preferred embodiment, the adenovirus vector in the first, second and the optional third composition comprises a nucleic acid encoding an antigenic RSV protein having the amino acid sequence of SEQ ID NO: 1 or 2.

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

In a preferred embodiment, the adenovirus vector in composition (i), (ii) and (iii) comprises a nucleic acid encoding an RSV F protein having the amino acid sequence of SEQ ID NO: 1.

In a preferred embodiment, the adenovirus vectors used in the method of the present invention are recombinant Ad26 vectors.

In certain embodiments, the second composition is administered 1-12 weeks after the first composition, preferably 2-10 weeks, more preferably 4-8 weeks.

In a preferred embodiment, the second composition is administered 4 weeks (i.e. 1 month) or 8 weeks (i.e. 2 months) after the first composition.

In certain embodiment, the third composition is administered 1-12 weeks after the second composition, preferably 2-10 weeks, more preferably 4-8 weeks.

In a preferred embodiment, the third composition, if any, is administered 4 weeks (i.e. 1 month) or 8 weeks (i.e. 2 months) after the first composition.

In a preferred embodiment of the present invention, the method comprises a first (e.g. priming) vaccination with an immunologically effective amount of an Ad26 vector expressing an antigenic RSV protein, followed by a second (e.g. boosting) vaccination with an immunologically effective amount of an Ad26 vector expressing said antigenic RSV protein.

In another embodiment of the present invention, the method comprises a first

(e.g.priming) vaccination with an immunologically effective amount of an Ad26 vector expressing an antigenic RSV protein, followed by a second (e.g. boosting) vaccination with an immunologically effective amount of an Ad26 vector expressing an antigenic RSV protein, followed by a third (e.g. boosting) vaccination with an immunologically effective amount of an Ad26 vector, expressing an antigenic RSV protein.

In a preferred embodiment, said RVS antigenic protein is an RSV F protein in a precision conformation, preferably the RSV F protein comprising the amino acid sequence of SEQ ID NO: 1 or 2, preferably SEQ ID NO: 1. In certain preferred embodiments, the subject is a child, e.g. an infant or baby, between the age of 0 months and about 24 months. In certain embodiments, the subject is a child between the age of 0 and about 12 months, preferably the subject is a child between the age of about 2 and 6 months. In a preferred embodiment, the first composition is administered to an infant at birth.

In another preferred embodiment, the first composition is administered to an infant of about 0-8 weeks of age, in particular an infant that is about 1, 2, 3, 4, 5, 6, 7 or 8 weeks old.

In a preferred embodiment, the first composition is administered to an infant of about 2 months of age. In a preferred embodiment, the first composition is administered to an infant at about

2 months of age and the second composition is administered to said infant at about 3 or 4 months of age.

In another preferred embodiment, the first composition is administered to an infant at about 2 months of age, and the second composition is administered to said infant at about 3 or 4 months of age, and the third composition is administered to said infant at about 4 or 6 months of age.

Detailed description of the invention

As used herein, "subject" means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the invention. The term "mammal" as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human. As used herein, the term "protective immunity" or "protective immune response" means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a "protective immune response" develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a "protective immune response" or "protective immunity" against a certain agent will not die as a result of the infection with said agent.

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, an adjuvant is used to enhance an immune response to the adenovirus vectors of the invention.

It is discovered in the present invention that homologous prime-boost combinations using recombinant adenovectors, in particular, Ad26 priming followed by Ad26 boosting, are surprisingly effective in generating protective immune responses against RSV viruses in young children. In addition, the vaccines do not result in enhanced respiratory disease and thus are safe.

An adenovirus according to the invention belongs to the family of the Adenoviridae and preferably is one that belongs to the genus Mastadeno virus. It can be a human adenovirus, but also an adenovirus that infects other species, including but not limited to a bovine adenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g. CAdV2), a porcine adenovirus (e.g. PAdV3 or 5), or a simian adenovirus (which includes a monkey adenovirus and an ape adenovirus, such as a chimpanzee adenovirus or a gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV, or AdHu; in the present invention a human adenovirus is meant if referred to Ad without indication of species, e.g. the brief notation "Ad5" means the same as HAdV5, which is human adenovirus serotype 5), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV).

Most advanced studies have been performed using human adenoviruses, and human adenoviruses are preferred according to certain aspects of the invention. In certain preferred embodiments, the recombinant adenovirus according to the invention is based upon a human adenovirus. In preferred embodiments, the recombinant adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49 or 50. According to a particularly preferred embodiment of the invention, an adenovirus is a human adenovirus of one of the serotypes 26 or 35, more preferably the adenovirus is a human adenovirus of one of the serotypes 26.

An advantage of these serotypes is a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population. Preparation of recombinant Ad26 vectors is described, for example, in WO 2007/104792 and in Abbink et al, (2007) Virol 81(9): 4654-63, both of which are incorporated by reference herein in their entirety.

Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792. Preparation of recombinant Ad35 vectors is described, for example, in US Patent No. 7,270,811, in WO 00/70071, and in Vogels et al, (2003) J Virol 77(15): 8263-71, all of which are incorporated by reference herein in their entirety. Exemplary genome sequences of Ad35 are found in GenBank Accession AC_ 000019 and in Fig. 6 of WO 00/70071.

Simian adenoviruses generally also have a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and a significant amount of work has been reported using chimpanzee adenovirus vectors (e.g. US6083716; WO 2005/071093; WO 2010/086189; WO 2001/085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al, 2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346: 394-40 I; Tats is et al, 2007, Molecular Therapy 15: 608-17; see also review by Bangari and Mittal, 2006, Vaccine 24: 849- 62; and review by Lasaro and Ertl, 2009, Mol Ther 17: 1333-39). Hence, in other preferred embodiments, the recombinant adenovirus according to the invention is based upon a simian adenovirus, e.g. a chimpanzee adenovirus. In certain embodiments, the recombinant adenovirus is based upon simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or SA7P.

In certain embodiments according to the present invention the adenoviral vectors comprise capsid proteins from two rare serotypes: Ad26 and Ad35. In a typical embodiment, the vector is a recombinant Ad26 or Ad35 virus. An "adenovirus capsid protein" refers to a protein on the capsid of an adenovirus (e.g., Ad 26 or Ad 35) that is involved in determining the serotype and/or tropism of a particular adenovirus. Adenoviral capsid proteins typically include the fiber, penton and/or hexon proteins. As used herein a "Ad26 capsid protein" or a "Ad35 capsid protein" can be, for example, a chimeric capsid protein that includes at least a part of an Ad26 or Ad35 capsid protein. In certain embodiments, the capsid protein is an entire capsid protein of Ad26 or of Ad35. In certain embodiments, the hexon, penton and fiber are of Ad26 or of Ad35. Thus, the vectors that can be used in the invention comprise an Ad26 or Ad35 capsid protein (e.g., a fiber, penton or hexon protein). One of skill will recognize that it is not necessary that an entire Ad26 or Ad35 capsid protein be used in the vectors of the invention. Thus, chimeric capsid proteins that include at least a part of an Ad26 or Ad35 capsid protein can be used in the vectors of the invention. The vectors of the invention can also comprise capsid proteins in which the fiber, penton, and hexon proteins are each derived from a different serotype, so long as at least one capsid protein is derived from Ad26 or Ad35. In certain embodiments, the fiber, penton and hexon proteins are each derived from Ad26 or each from Ad35. One of skill will recognize that elements derived from multiple serotypes can be combined in a single recombinant adenovirus vector. Thus, a chimeric adenovirus that combines desirable properties from different serotypes can be produced. Thus, in some embodiments, a chimeric adenovirus of the invention could combine the absence of preexisting immunity of the Ad26 and Ad35 serotypes with characteristics such as temperature stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like.

In certain embodiments the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad26 or from Ad35 (i.e., the vector is Ad26 or Ad35). In some embodiments, the adenovirus is replication deficient, e.g. because it contains a deletion in the El region of the genome. For the adenoviruses of the invention, being derived from Ad26 or Ad35, it is typical to exchange the E4-orf6 coding sequence of the adenovirus with the E4-orf6 of an adenovirus of human subgroup C such as Ad5. This allows propagation of such adenoviruses in well-known complementing cell lines that express the El genes of Ad5, such as for example 293 cells, PER.C6 cells, and the like (see, e.g. Havenga et al, 2006, J Gen Virol 87: 2135-43; WO 03/104467). In certain embodiments, the adenovirus is a human adenovirus of serotype 35, with a deletion in the El region into which the nucleic acid encoding the antigen has been cloned, and with an E4 orf6 region of Ad5. In certain embodiments, the adenovirus is a human adenovirus of serotype 26, with a deletion in the El region into which the nucleic acid encoding the antigen has been cloned, and with an E4 orf6 region of Ad5. For the Ad35 adenovirus, it is typical to retain the 3' end of the E 1 B 55K open reading frame in the adenovirus, for instance the 166 bp directly upstream of the pIX open reading frame or a fragment comprising this such as a 243 bp fragment directly upstream of the pIX start codon, marked at the 5' end by a Bsu36I restriction site, since this increases the stability of the adenovirus because the promoter of the pIX gene is partly residing in this area (see, e.g. Havenga et al, 2006, supra; WO 2004/001032). Typically, a vector useful in the invention is produced using a nucleic acid comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector). Thus, the invention also provides isolated nucleic acid molecules that encode the adenoviral vectors of the invention. The nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded.

The adenovirus vectors useful for the invention are typically replication defective. In these embodiments, the virus is rendered replication-defective by deletion or inactivation of regions critical to replication of the virus, such as the El region. The regions can be substantially deleted or inactivated by, for example, inserting the gene of interest (usually linked to a promoter). In some embodiments, the vectors of the invention can contain deletions in other regions, such as the E2, E3 or E4 regions or insertions of heterologous genes linked to a promoter. For E2- and/or E4-mutated adenoviruses, generally E2- and/or E4-complementing cell lines are used to generate recombinant adenoviruses. Mutations in the E3 region of the adenovirus need not be complemented by the cell line, since E3 is not required for replication.

A packaging cell line is typically used to produce sufficient amount of adenovirus vectors of the invention. A packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication-defective vector, thus allowing the virus to replicate in the cell. Suitable cell lines include, for example, PER.C6, 911, 293, and El A549.

As indicated above, in a preferred embodiment, the adenovectors according to the invention comprise a nucleic acid encoding an antigenic RSV protein. The adenovirus in the first composition and the adenovirus in the second composition and the adenovirus in the third composition can be identical or different. In a preferred embodiment, the adenovirus in the first and second composition, and the adenovirus in the optional third composition are identical.

In certain embodiments, the antigenic RSV protein is an RSV fusion (F) protein. The RSV F protein is conserved between RSV strains making it an attractive vaccine candidate able to elicit broadly neutralizing antibodies. The RSV F protein facilitates infection by fusing the viral and host-cell membranes. In the process of fusion, the F protein refolds irreversibly from a labile pre-fusion conformation to a stable post-fusion conformation. Because of the instability of the RSV F protein, the RSV F protein has the propensity to prematurely refold into its more stable post- fusion conformation. This phenomenon is an intrinsic feature of the protein both in solution and on the surface of the virions. In human sera most RSV neutralizing antibodies are, however, directed against the RSV F in the the pre-fusion conformation. In a preferred embodiment of the invention, the antigenic RSV protein therefore is an RSV F protein in the pre-fusion conformation.

In a preferred embodiment of the invention, the adenovirus vector in the first, second and the optional third composition comprises a nucleic acid encoding a RSV fusion (F) protein, preferably a RSV F protein in the pre-fusion conformation, preferably an RSV F protein having the amino acid sequence of SEQ ID NO: l or 2, preferably SEQ ID NO: 1.

If required, the heterologous gene encoding the RSV proteins can be codon-optimized to ensure proper expression in the treated host (e.g., human). Codon-optimization is a technology widely applied in the art. Typically, the heterologous gene is cloned into the El and/or the E3 region of the adenoviral genome.

In certain embodiments, the nucleic acid molecule encoding the RSV pre-fusion F protein comprises the nucleic acid sequence of SEQ ID NO: 3 or 4. The heterologous RSV gene can be under the control of (i.e., operably linked to) an adenovirus-derived promoter (e.g., the Major Late Promoter) or can be under the control of a heterologous promoter. Examples of suitable heterologous promoters include the CMV promoter and the RSV promoter. Preferably, the promoter is located upstream of the heterologous gene of interest within an expression cassette.

The compositions of the invention can be formulated as a vaccine (also referred to as an "immunogenic composition") according to methods well known in the art. Such compositions can include adjuvants to enhance immune responses. The optimal ratios of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present disclosure. The preparation and use of immunogenic

compositions are well known to those of skill in the art. Adjuvants suitable for coadministration in accordance with the present invention should be ones that are potentially safe, well tolerated and effective in people including QS-21, MPL-SE, CpG ODN, Alum, and MF59. Other adjuvants that can be administered include lectins, growth factors, cytokines and lymphokines such as alpha-interferon, gamma interferon, platelet derived growth factor (PDGF), granulocyte-colony stimulating factor (gCSF), granulocyte macrophage colony stimulating factor (gMCSF), tumor necrosis factor (TNF), epidermal growth factor (EGF), IL1, IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12 or encoding nucleic acids therefore. It is also possible to use vector-encoded adjuvant, e.g. by using heterologous nucleic acid that encodes a fusion of the oligomerization domain of C4-binding protein (C4bp) to the antigen of interest (e.g. Solabomi et al, 2008, Infect Immun 76: 3817-23). In certain embodiments the compositions of the invention comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or

combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose. The compositions of the invention can comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes.

The present invention further provides a method of priming and boosting an immune response to a RSV virus in a subject using one or more adenoviral vectors for priming and boosting administrations.

According to one general aspect of the present invention, a method of inducing an immune response against a RSV virus in a subject comprises:

(i) administering to the subject a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein;

(ii) administering to the subject a second composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein, and, optionally

(iii) administering to the subject a third composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein.

In a preferred embodiment the later step is conducted 1-12 weeks after the previous step, preferably 2-10 weeks, more preferably 4-8 weeks. In a more preferred embodiment the later step is conducted 4 or 8 weeks after the previous step. In a preferred embodiment, step (ii) is conducted 4 weeks (i.e 1 month) or 8 weeks (i.e. 2 months) after step (i).

In a preferred embodiment, step (iii) is conducted 4 weeks (i.e 1 month) or 8 weeks (i.e. 2 months) after step (ii). A subject as used herein preferably is a mammal, for instance a rodent, e.g. a mouse, a cotton rat, or a non-human-primate, or a human. Preferably, the subject is a human subject.

In certain preferred embodiments, the subject is a child, for example an infant or baby, between the age of 0 months and about 24 months. In certain embodiments, the subject is a child between the age of 0 and about 12 months, preferably the subject is a child between the age of 0 and 6 months, preferably between about 2 and 6 months.

In a preferred embodiment, the first composition is administered to an infant at birth.

In a preferred embodiment, the first composition is administered to an infant of about 2 months of age.

In a preferred embodiment, the first composition is administered to an infant at about 2 months of age and the second composition is administered to said infant at about 3 or 4 months of age.

In another preferred embodiment, the first composition is administered to an infant at about 2 months of age, and the second composition is administered to said infant at about 3 or 4 months of age, and the third composition is administered to said infant at about 4 or 6 months of age. The subject may be seronegative or seropositive. Seropositive subjects typically show a significant level of serum antibodies, or other immunologic marker in the serum, indicating previous exposure to RSV. In certain embodiments, the subject is a seronegative subject, i.e. showing no significant level of serum antibodies, or other immunologic marker in the serum, that would indicate previous exposure to RSV.

In a preferred embodiment according to this method, an Ad26 vector is used for the priming followed by one or more boosting steps with an Ad26 vector. Preferably, the boosting composition is administered 1-12 weeks after priming, more preferably 4 or 8 weeks after priming. In a preferred embodiment, the boosting composition is administered 8 weeks after priming. In another preferred embodiment, the boosting composition is administered 4 weeks after priming.

For administering to humans, the invention may employ compositions comprising the adenovirus vectors together with a pharmaceutically acceptable carrier or excipient. In the present context, the term "Pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful 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 adenovector preferably is formulated and administered as a sterile solution although it is also possible to utilize lyophilized

preparations. 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 generally is in the range of pH 3.0 to 9.5, e.g pH 5.0 to 7.5. The adenovector typically is in a solution having a suitable pharmaceutically acceptable buffer, and the solution may also contain a salt. Optionally stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, the compositions may be formulated into an injectable preparation. For instance, adenovirus may be stored in the buffer that is 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% glycerol. Another useful formulation buffer suitable for administration to humans is 20 mM Tris, 2 mM MgC12, 25 mM NaCl, sucrose 10%) w/v, polysorbate-80 0.02%> w/v. Obviously, many other buffers can be used, and several examples of suitable formulations for the storage and for pharmaceutical administration of purified (adeno)virus preparations can for instance be found in European patent no. 0853660, US patent 6,225,289 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.

In another preferred embodiment, use is made of an adenovirus formulation as described in WO2015/040002. Thus, in a preferred embodiment of the inventions, the first, second and optional third composition comprising the adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein comprise: a recombinant adenovirus; a citrate buffer, wherein the citrate concentration is ranging between about 5mM and 30 mM;

hydroxypropyl-beta-cyclodextrin (HBCD), wherein the concentration of HBCD is ranging between about 1% (w/w) and 10%> (w/w); a salt, e.g. sodium chloride in a concentration between about 20 mM and about 200 mM; and non-ionic detergent, e.g. Polysorbate-80 in a concentration ranging from about 0.005%) (w/w) to about 0.5%> (w/w); wherein said formulation has a pH ranging between 5.5 and 6.5.

In certain embodiments, the compositions have a pH ranging between about 5.7 and 6.3, and comprise citrate at a concentration ranging between about 5 and 30 mM; HBCD at a concentration ranging between 1% (w/w) and 10% (w/w); NaCl at a concentration ranging between 20 mM and 200 mM; Polysorbate-80 at a concentration ranging between about 0.01% (w/w) and 0.05% (w/w).

In certain embodiments, the compositions comprise citrate at a concentration of about 15 mM; HBCD at a concentration of about 5% (w/w); NaCl at a concentration of about 75 mM, and Polysorbate-80 at a concentration of about 0.03% (w/w).

In certain embodiments, the compositions further comprise ethanol, wherein the ethanol concentration is ranging between about 0.1 % (w/w) to 1% (w/w).

In a preferred embodiment, the compositions comprise citrate at a concentration of about 15 mM; HBCD at a concentration of about 5% (w/w); NaCl at a concentration of about 75 mM, Polysorbate-80 at a concentration of about 0.03% (w/w) and ethanol at a concentration of about 0.4% (w/w).

Administration of the immunogenic compositions comprising the vectors is typically intramuscular or subcutaneous. However other modes of administration such as intravenous, cutaneous, intradermal or nasal can be envisaged as well. Intramuscular administration of the immunogenic compositions can be achieved by using a needle to inject a suspension of the adenovirus vector. An alternative is the use of a needleless injection device to administer the composition (using, e.g., Biojector(TM)) or a freeze-dried powder containing the vaccine.

The immunogenic compositions containing the adenovirus vectors are administered to a subject, giving rise to an anti-RSV virus immune response in the subject. An amount of a composition sufficient to induce a detectable immune response is defined to be an

"immunologically effective dose." As shown below, the immunogenic compositions of the invention induce a humoral as well as a cell-mediated immune response. In a typical embodiment the immune response is a protective immune response.

Following production of adenovirus vectors and optional formulation of such particles into immunogenic compositions, the vectors can be administered to an individual, particularly human or other primate. Administration can be to humans, or another mammal, e.g., mouse, rat, hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, monkey, dog or cat. Delivery to a non-human mammal need not be for a therapeutic purpose, but can be for use in an experimental context, for instance in investigation of mechanisms of immune responses to the adenovirus vectors. In one exemplary regimen, the adenovirus vector is administered (e.g., intramuscularly) in a volume ranging between about 100 μΐ to about 10 ml containing concentrations of about 10⁴ to 10¹² virus particles/ml. Preferably, the adenovirus vector is administered in a volume ranging between 0.25 and 1.0 ml. More preferably the adenovirus vector is administered in a volume of 0.5 ml. Typically, the adenovirus is administered in an amount of about 10⁹ to about 10¹² viral particles (vp) to a human subject during one administration, more typically in an amount of about 10¹⁰ to about 10¹² vp. The human adenovirus vector can be administered in a concentration of about 10⁷ vp/ml, 10⁸ vp/ml, 10⁹ vp/ml, 10¹⁰ vp/ml, 5xl0¹⁰ vp/ml, 10¹¹ vp/ml, or 10¹² vp/ml. In a preferred embodiment, the adenovirus vector is administered in an amount of about lxlO¹¹ vp. In another preferred embodiment, the adenovirus vector is administered in an amount of about 5xl0¹⁰ vp.

The composition can, if desired, be presented in a kit, pack or dispenser, which can contain one or more unit dosage forms containing the active ingredient. The kit, for example, can comprise metal or plastic foil, such as a blister pack. The kit, pack, or dispenser can be accompanied by instructions for administration.

Examples

The following examples are provided to illustrate, but not to limit the claimed invention.

EXAMPLE 1

A clinical study will be performed in humans for evaluating the safety, tolerability and immunogenicity of Ad26.RSV.preF in Adults 18 to 50 Years of Age, RSV-seropositive and RSV-seronegative Toddlers 12 to 24 Months of Age.

The adeno virus-vectored vaccine candidate assessed in this study is:

Ad26.RSV.preF, a replication-incompetent adenovirus serotype 26 (Ad26) containing a deoxyribonucleic acid transgene that encodes for the pre-fusion conformation-stabilized F protein (pre-F) derived from the respiratory syncytial virus (RSV) A2 strain having an amino acid sequence of SEQ ID NO : 1.

Safety will be assessed by collection of solicited local and systemic adverse events, unsolicited adverse events and serious adverse events, and by physical examination. In addition, standard chemistry, hematologic (including coagulation parameters) and urinalysis parameters may be assessed at multiple time points. Immunogenicity will be assessed using the immunologic assays summarized in Tables 1 and 2. Table 1: Summary of Immunogenicity Assays (Humoral)

Assay Purpose

Secondary endpoints

RSV neutralization A Analysis of neutralizing antibodies to the A strain

F-protein antibody

Analysis of antibodies binding to RSV F protein in

(ELISA; pre- and/or post- fusion and/or pre-fusion form

fusion)

Exploratory endpoints

RSV strain cross- Analysis of cross-neutralizing antibodies to B and/or a neutralization different A strain(s)

Pre- and post-F specificity by binding or functional assays as

F-protein antibody ELISA, and/or competition ELISA. Adsorption of serum or specificity characterization plasma with pre-F and post-F protein before any antibody assay, epitope mapping, functional VNA

Adenovirus neutralization

Analysis of neutralizing antibodies to adenovirus

assay

Analysis of antibody characteristics including ADCC, ADCP,

Functional and molecular

avidity, Fc characteristics, Ig isotype, functional VNA and antibody characterization

protective antibody assessments

ADCC = antibody-dependent cell-mediated cytotoxicity; ADCP = antibody-dependent cellular phagocytosis; ELISA = enzyme-linked immunosorbent assay; F = fusion; Ig = immunoglobulin; RSV = respiratory syncytial virus; VNA = virus neutralizing antibody

Table 2: Summary of Immunogenicity Assays (Cellular)

Assay Purpose

Secondary endpoints

Flow cytometry Analysis of T-cell responses to RSV F-protein peptides for Thl/Th2

(ICS)* subtyping

Exploratory endpoints

IFNy ELISpot T-cell IFNy responses to RSV F-protein peptides

Analysis of T-cell responses to RSV F-protein peptide-stimulated

ICS PBMCs (including but not limited to, CD4/CD8, IL2, INFy, TNFa, activation markers and memory)

Analysis of secreted cytokines in RSV F peptide-stimulated PBMC

Cytokine analysis supernatant, including, but not limited to, measurement of Thl/Th2 cytokine balance

ELISpot = enzyme-linked immunospot; F = fusion; ICS = intracellular cytokine staining; IFNy = interferon gamma; IL = interleukin; PBMC = peripheral blood mononuclear cell; Th = T-helper (cell); RSV = respiratory syncytial virus; TNFa = tumor necrosis factor alpha * Cytokine analysis for Thl/Th2 profiling will be done to replace samples in cases where no ICS data can be generated or no data are available SEQUENCES

Amino acid sequences of the RSV pre-fusion F proteins encoded by the nucleic acid molecules of the invention

SEQ ID NO: 1 : RSV preF2.2 amino acid sequence:

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIE

LSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNY

TLNNAK TNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLST

NKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEI

TREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSII

KEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGS

VSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV

ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQE

GKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKST

TNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

SEQ ID NO: 2: RSV preF2.1 amino acid sequence:

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITI

ELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMN

YTLNNAK TNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLS

TNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLE

ITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSI

IKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGS

VSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSV

ITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQE

GKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKST

TNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

Nucleotide sequence of preferred nucleic acid molecules of the invention

SEQ ID NO: 3: codon optimized nucleic acid encoding the RSV F pre-F2.2 pre-fusion protein

ATGGAGCTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTG ACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAGTTCTACCAGAGCACCT GCAGCGCCGTGAGCAAGGGCTACCTGAGCGCCCTGAGAACCGGCTGGTACACCA GCGTGATCACCATCGAGCTGAGCAACATCAAGGAGATCAAGTGCAACGGCACCG ACGCCAAGGTGAAGCTGATCAAGCAGGAGCTGGACAAGTACAAGAACGCCGTG ACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACAGAGCCAGA AGAGAGCTGCCCAGATTCATGAACTACACCCTGAACAACGCCAAGAAGACCAAC GTGACCCTGAGCAAGAAGAGAAAGAGAAGATTCCTGGGCTTCCTGCTGGGCGTG GGCAGCGCCATCGCCAGCGGCGTGGCCGTGAGCAAGGTGCTGCACCTGGAGGGC GAGGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGAGC CTGAGCAACGGCGTGAGCGTGCTGACCAGCAAGGTGCTGGACCTGAAGAACTAC ATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAAC ATCGAGACCGTGATCGAGTTCCAGCAGAAGAACAACAGACTGCTGGAGATCACC AGAGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGAGCACCTACATGCTG ACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAG AAGAAGCTGATGAGCAACAACGTGCAGATCGTGAGACAGCAGAGCTACAGCATC ATGAGCATCATCAAGGAGGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTAC GGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACC AACACCAAGGAGGGCAGCAACATCTGCCTGACCAGAACCGACAGAGGCTGGTAC TGCGACAACGCCGGCAGCGTGAGCTTCTTCCCCCAGGCCGAGACCTGCAAGGTG CAGAGCAACAGAGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCAGCGAG GTGAACCTGTGCAACGTGGACATCTTCAACCCCAAGTACGACTGCAAGATCATG ACCAGCAAGACCGACGTGAGCAGCAGCGTGATCACCAGCCTGGGCGCCATCGTG AGCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACAGAGGCATCATC AAGACCTTCAGCAACGGCTGCGACTACGTGAGCAACAAGGGCGTGGACACCGTG AGCGTGGGCAACACCCTGTACTACGTGAACAAGCAGGAGGGCAAGAGCCTGTAC GTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCAACG AGTTCGACGCCAGCATCAGCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCCT TCATCAGAAAGAGCGACGAGCTGCTGCACAACGTGAACGCCGTGAAGAGCACCA CCAACATCATGATCACCACCATCATCATCGTGATCATCGTGATCCTGCTGAGCCT GATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCAGAAGCACCCCCGTGACCCT GAGCAAGGACCAGCTGAGCGGCATCAACAACATCGCCTTCAGCAACTGA SEQ ID NO: 4: codon optimized nucleic acid encoding the RSV F pre-F2.1 pre-fusion protein

ATGGAGCTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTG ACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAGTTCTACCAGAGCACCT GCAGCGCCGTGAGCAAGGGCTACCTGGGCGCCCTGAGAACCGGCTGGTACACCA GCGTGATCACCATCGAGCTGAGCAACATCAAGGAGATCAAGTGCAACGGCACCG ACGCCAAGGTGAAGCTGATCAAGCAGGAGCTGGACAAGTACAAGAACGCCGTG ACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACAGAGCCAGA AGAGAGCTGCCCAGATTCATGAACTACACCCTGAACAACGCCAAGAAGACCAAC GTGACCCTGAGCAAGAAGAGAAAGAGAAGATTCCTGGGCTTCCTGCTGGGCGTG GGCAGCGCCATCGCCAGCGGCGTGGCCGTGAGCAAGGTGCTGCACCTGGAGGGC GAGGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGAGC CTGAGCAACGGCGTGAGCGTGCTGACCAGCAAGGTGCTGGACCTGAAGAACTAC ATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCCCCAAC ATCGAGACCGTGATCGAGTTCCAGCAGAAGAACAACAGACTGCTGGAGATCACC AGAGAGTTCAGCGTGAACGCCGGCGTGACCACCCCCGTGAGCACCTACATGCTG ACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAG AAGAAGCTGATGAGCAACAACGTGCAGATCGTGAGACAGCAGAGCTACAGCATC ATGAGCATCATCAAGGAGGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTAC GGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACC AACACCAAGGAGGGCAGCAACATCTGCCTGACCAGAACCGACAGAGGCTGGTAC TGCGACAACGCCGGCAGCGTGAGCTTCTTCCCCCAGGCCGAGACCTGCAAGGTG CAGAGCAACAGAGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCAGCGAG GTGAACCTGTGCAACGTGGACATCTTCAACCCCAAGTACGACTGCAAGATCATG ACCAGCAAGACCGACGTGAGCAGCAGCGTGATCACCAGCCTGGGCGCCATCGTG AGCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACAGAGGCATCATC AAGACCTTCAGCAACGGCTGCGACTACGTGAGCAACAAGGGCGTGGACACCGTG AGCGTGGGCAACACCCTGTACTACGTGAACAAGCAGGAGGGCAAGAGCCTGTAC GTGAAGGGCGAGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACG AGTTCGACGCCAGCATCAGCCAGGTGAACGAGAAGATCAACCAGAGCCTGGCCT TCATCAGAAAGAGCGACGAGCTGCTGCACAACGTGAACGCCGTGAAGAGCACCA CCAACATCATGATCACCACCATCATCATCGTGATCATCGTGATCCTGCTGAGCCT GATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCAGAAGCACCCCCGTGACCCT GAGCAAGGACCAGCTGAGCGGCATCAACAACATCGCCTTCAGCAACTGA

Claims

1. A vaccine combination comprising:

(i) a first composition comprising an immunologically effective amount of a first adenovirus vector comprising a nucleic acid encoding an antigenic respiratory syncytial virus (RSV) protein; and

(ii) a second composition comprising an immunologically effective amount of a second adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein, wherein one of the compositions is a priming composition and the other composition is a boosting composition.

2. The vaccine combination according to claim 1, further comprising:

(iii) a third composition comprising an immunologically effective amount of a third adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein, wherein one of the compositions is a priming composition and the other compositions are boosting compositions.

3. The vaccine combination according to claim 1 or 2, wherein the first and second adenovirus vector comprise a nucleic acid encoding an RSV F protein having an amino acid sequence of SEQ ID NO: 1 or 2.

4. The vaccine combination according to claim 2 or 3, wherein the third adenovirus vector comprise a nucleic acid encoding an RSV F protein having an amino acid sequence of SEQ ID NO: 1 or 2.

5. The vaccine combination according to any one of the claims 1 to 4, wherein the adenovirus vectors are recombinant Ad26 vectors.

6. A method of inducing an immune response against RSV in a subject, the method comprising:

(i) administering to the subject a first composition comprising an immunologically effective amount of a first adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein; and

(ii) administering to the subject a second composition comprising an immunologically effective amount of a second adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein.

7. The method according to claim 6, further comprising: (iii) administering to the subject a third composition comprising an immunologically effective amount of a second adenovirus vector comprising a nucleic acid encoding an antigenic RSV protein.

8. The method according to claim 6 or 7, wherein the adenovirus vector in the first composition (i) comprises a nucleic acid encoding an RSV protein having the amino acid sequence of SEQ ID NO: 1 or 2, and the adenovirus vector in the second composition (ii) comprises a nucleic acid encoding an RSV protein having the amino acid sequence of SEQ ID NO: 1 or 2, and, optionally, wherein the adenovirus vector in composition (iii) comprises a nucleic acid encoding an RSV F protein having the amino acid sequence of SEQ ID NO: 1 or 2.

9. The method according to claim 6, 7 or 8, wherein the adenovirus vectors are Ad26 vectors.

10. The method according to any one of the claims 6-9, wherein step (i) and step (ii) of the method are conducted 1-12 weeks apart, preferably 2-10 weeks, more preferably 4-8 weeks, more preferably 4 or 8 weeks apart.

11. The method according to any one of the claims 6-10, wherein step (ii) and step (iii) of the method are conducted 1-12 weeks apart, preferably 2-10 weeks, more preferably 4-8 weeks, more preferably 4 or 8 weeks apart.

12. The method according to any one of the claims 6-11, wherein the subject is a child of about 0 to 24 months of age, preferably of about 0-12 months of age, more preferably of about 0-6 months of age, more preferably of about 2 to 6 months of age.

13. The method according to any one of the claims 6-12, wherein step (i) is conducted in an infant of about 0-2 months of age.

14. The method according to any one of the claims 6-13, wherein the first composition is administered to an infant at about 2 months of age and the second composition is

administered to said infant at about 3 or 4 months of age.

15. The method according to any one of the claims 6-14, wherein the first composition is administered to an infant at about 2 months of age and the second composition is

administered to said infant at about 3 or 4 months of age. and the third composition is administered to said infant at about 4 or 6 months of age.