WO2022175479A1 - Vaccine combinations against respiratory syncytial virus strain a and b infections - Google Patents

Abstract

Methods of safely inducing a protective immune response against respiratory syncytial virus (RSV) A and B infections and methods of preventing infection and/or replication of RSV in human subjects are described.

Description

Vaccine combinations Against Respiratory Syncytial Virus Strain A and B Infections

FIELD OF THE INVENTION

The present invention is in the field of medicine. In particular, embodiments of the invention relate to vaccine combinations against Respiratory Syncytial Virus (RSV) strain A and B infections.

BACKGROUND

Human RSV (HRSV) is divided into two major antigenic groups of strains, subtypes A and B, that are largely defined by genetic variation in the G glycoprotein. These subtypes show an irregular, alternating prevalence pattern, with subtype A having a higher cumulative prevalence than subtype B. The F protein is highly conserved between RSV A and B and induces neutralizing antibodies across the two group. The RSV F protein shows a high degree of sequence identity (-95% in the mature ectodomain) between RSV A and B virus strains.

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, RSV is responsible for an estimated 3.4 million hospitalizations annually. In the United States, 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 the US, 60% of infants are infected upon initial exposure to RSV, and nearly all children will have been infected with the virus by 2-3 years of age. Immunity to RSV is transient, and repeated infection occurs throughout life (Hall et al., J Infect Dis. 1991 : 163;693-698). 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 (Centers for Disease Control and Prevention (CDC). Respiratory Syncytial Virus Infection (RSV) - Infection and Incidence. Almost all RSV-related deaths (99%) in children under 5 years of age occur in the developing world (Nair et al., Lancet. 2010:375; 1545-1555). 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 addition to children, RSV is an important cause of respiratory infections in the elderly, immunocompromised, and those with underlying chronic cardio-pulmonary conditions (Falsey et al., N Engl JMed. 2005:352;1749-1759). 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%) (Falsey et al., Clin Microbiol Rev. 2000:13;371- 384). In one epidemiology study of RSV burden, it was estimated that 11,000 elderly persons die annually of RSV in the US (Thompson et al., JAMA. 2003:289;179-186). These data support the importance of developing an effective vaccine for certain adult populations.

Prophylaxis through passive immunization with a neutralizing monoclonal antibody against the RSV fusion (F) glycoprotein (Synagis® [palivizumab]) is available, but only indicated for premature infants (less than 29 weeks gestational age), children with severe cardio-pulmonary disease or those that are profoundly immunocompromised (American Academy of Pediatrics Committee on Infectious Diseases, American Academy of Pediatrics Bronchiolitis Guidelines Committee. Updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection. Pediatrics. 2014: 134;415-420). Synagis® has been shown to reduce the risk of hospitalization by 55% (Prevention. Prevention of respiratory syncytial virus infections: indications for the use of palivizumab and update on the use of RSV-IGIV. American Academy of Pediatrics Committee on Infectious Diseases and Committee of Fetus and Newborn. Pediatrics. 1998: 102; 1211-1216). Despite the high disease burden and a strong interest in RSV vaccine development, no licensed vaccine is available for RSV. In the late 1960s, a series of studies were initiated to evaluate a formalin-inactivated RSV vaccine (FI-RSV) adjuvanted with alum, and the results of these studies had a major impact on the RSV vaccine field. Four studies were performed in parallel in children of different age groups with an FI-RSV vaccine delivered by intramuscular injection (Chin et al., Am J Epidemiol. 1969:89;449-463; Fulginiti et al., Am J Epidemiol. 1969:89;435-448; Kapikian et al., Am J Epidemiol. 1969:89;405-421; Kim et al., Am J Epidemiol. 1969:89;422-434). Eighty percent of the RSV-infected FI-RSV recipients required hospitalization and two children died during the next winter season (Chin et al., Am J Epidemiol. 1969:89;449-463). Only 5% of the children in the RSV-infected control group required hospitalization. The mechanisms of the observed enhanced respiratory disease (ERD) among the FI-RSV recipients upon reinfection have been investigated and are believed to be the result of an aberrant immune response in the context of small bronchi present in that age group. Data obtained from analysis of patient samples and animal models suggest that FI-RSV ERD is characterized by low neutralizing antibody titers, the presence of low avidity non-neutralizing antibodies promoting immune complex deposition in the airways, reduced cytotoxic CD8+ T-cell priming shown to be important for viral clearance, and enhanced CD4+ T helper type 2 (Th2)-skewed responses with evidence of eosinophilia (Beeler et al., Microb Pathog. 2013 :55;9-15; Connors et al., J Virol. 1992:66;7444-7451; De Swart et al., J Virol. 2002:76;11561-11569; Graham et al., J Immunol. 1993:151;2032-2040; Kim et al., Pediatr Res. 1976:10;75-78; Murphy et al., J Clin Microbiol . 1986:24;197-202; Murphy et al., J Clin Microbiol. 1988:26;1595-1597; Polack et al., J ExpMed. 2002:196;859- 865). It is believed that the chemical interaction of formalin and RSV protein antigens may be one of the mechanisms by which the FI-RSV vaccine promoted ERD upon subsequent RSV infection (Moghaddam et al., Nat Med. 2006:12;905-907). For these reasons, formalin is no longer used in RSV vaccine development.

In addition to the FI-RSV vaccine, several live-attenuated and subunit RSV vaccines have been examined in animal models and human studies, but many have been inhibited by the inability to achieve the right balance of safety and immunogenicity/efficacy. Live- attenuated vaccines have been specifically challenged by difficulties related to over- and under-attenuation in infants (Belshe et al., J Infect Dis. 2004:190;2096-2103; Karron et al., J Infect Dis. 2005:191;1093-1104; Luongo et al., Vaccine. 2009:27;5667-5676). With regard to subunit vaccines, the RSV fusion (F) and glycoprotein (G) proteins, which are both membrane proteins, are the only RSV proteins that induce neutralizing antibodies (Shay et al., JAMA. 1999:282; 1440-1446). Unlike the RSV G protein, the F protein is conserved between RSV strains. A variety of RSV F-subunit vaccines have been developed based on the known superior immunogenicity, and the high degree of conservation of the F protein between RSV strains (Graham, Immunol Rev. 2011 :239; 149-166). The proof-of-concept provided by the currently available anti-F protein neutralizing monoclonal antibody prophylaxis provides support for the idea that a vaccine inducing high levels of long-lasting neutralizing antibody may prevent RSV disease (Feltes et al., Pediatr Res. 2011 :70; 186-191 ; Groothuis et al., J Infect Dis. 1998:177;467-469; Groothuis et al., N Engl JMed. 1993:329;1524-1530). Several studies have suggested that decreased protection against RSV in elderly could be attributed to an age-related decline in interferon gamma (IFNy) production by peripheral blood mononuclear cells (PBMCs), a reduced ratio of CD8+ to CD4+ T cells, and reduced numbers of circulating RSV-specific CD8+ memory T cells (De Bree et al., J Infect Dis. 2005 : 191 ; 1710-1718; Lee et al , Mech Ageing Dev. 2005:126;1223-1229; Looney et al., J Infect Dis. 2002:185;682-685). High levels of serum neutralizing antibody are associated with less severe infections in elderly (Walsh and Falsey, J Infect Dis. 2004: 190;373-378). It has also been demonstrated that, following RSV infection in adults, serum antibody titers rise rapidly but then slowly return to pre-infection levels after 16 to 20 months (Falsey et al., JMed Virol. 2006:78; 1493-1497). With consideration given to the previously observed ERD in the FI-RSV vaccine studies in the 1960s, future vaccines should promote a strong antigen-specific CD8+ T-cell response and avoid a skewed Th2-type CD4+ T cell response (Graham, Immunol Rev. 2011:239; 149-166).

The RSV F protein fuses the viral and host-cell membranes by irreversible protein refolding from the labile pre-fusion conformation to the stable post-fusion conformation. Structures of both conformations have been determined for RSV F (McLellan et al., Science 2013:342, 592-598; McLellan et al., Nat Struct Mol Biol 2010:17, 248-250; McLellan et al.,

Science 340, 2013:1113-1117; Swanson et al., Proceedings of the National Academy of Sciences of the United States of America 2011 : 108, 9619-9624), as well as for the fusion proteins from related paramyxoviruses, providing insight into the mechanism of this complex fusion machine. Like many other class I fusion proteins, RSV F undergoes proteolytic processing during maturation in the secretory pathway of infected cells. RSV F is synthesized as a single-chain inactive precursor (also called F0) that contains three subunits: FI, F2, and a 27-amino acid glycopeptide called pep27. This precursor must be cleaved by a furin-like protease to release pep27 and form the mature, fusion-competent protein (FIG.1, RSV F mature processed). The C-terminal FI subunit contains the transmembrane domain, two heptad repeats, and an N-terminal fusion peptide. Residues in the F2 subunit contribute to fusogenicity of the F protein and possibly the species specificity of RSV. In the mature processed protein, the FI and F2 subunits are covalently associated via two disulfide bonds. Three F1-F2 protomers then associate via weak intermolecular interactions to form the trimeric, prefusion protein on the surface of the virion. Most neutralizing antibodies in human sera are directed against the pre-fusion conformation of the F protein, but due to its instability the pre-fusion conformation has a propensity to prematurely refold into the post-fusion conformation, both in solution and on the surface of the virions. Vaccines comprising RSV F proteins stabilized in a pre-fusion conformation, as well as vectors containing nucleic acid encoding RSV F proteins have been described. These vaccines typically are based on F proteins of RSV A strains. However, currently there is no report on safety or efficacy of such vaccines in humans.

Accordingly, there is still a high need for a safe and effective vaccine against RSV.

SUMMARY OF THE INVENTION

The present application describes compositions and methods with increased immunogenic efficacy. More specifically, the application describes efficacious combinations for concurrent administration, that elicit both potent B and T cell responses, thereby enhancing immunogenicity, and ultimately protection, against both respiratory syncytial virus (RSV) A and B infections.

In one general aspect, the present invention provides methods for inducing a protective immune response against respiratory syncytial virus (RSV) A and B infection in a human subject in need thereof, comprising administering to the subject a combination comprising:

(a) a first dose of one or more RSV FA antigen(s); and

(b) a first dose of one or more RSV FB antigen(s).

Preferably, the RSV FA and RSV FB antigens are co-administered.

In a further aspect, the invention provides immunogenic combinations for use in inducing a protective immune response against RSV A and B infection in a human subject in need thereof, comprising: (a) an effective amount of one or more RSV FA antigen(s); and

(b) an effective amount of one or more RSV FB antigen(s).

BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

FIG. 1: Schematic representation of the RSV F protein precursor F0, RSV F mature processed and RSV preF protein. The two domains (FI and F2), transmembrane domain (TM), foldon domain (FD), furin cleavage sites, N-glycan sites and interchain disulfide bonds of the proteins are shown. The 5 amino acid mutations in the RSV preF protein are also identified.

FIG 2: Immunogenicity and protective efficacy of RSV A and RSV B combination vaccines in cotton rats. Animals were intramuscularly immunized at day 0 with a mix of RSV A-based Ad26 (Ad26RSV009, encoding the RSV FA protein of SEQ ID NO: 3) (10⁶ vp) and preF_A protein RSV150042 (SEQ ID NO: 9 (not-processed), SEQ ID NO: 11 processed)) (5 pg) (Mix A), with a mix of RSV B-based Ad26 (Ad26RSV019, coding for the RSV FB protein of SEQ ID NO: 18 ) (10⁶ vp) and preF_B protein (RSV200125: SEQ ID NO: 10 (not processed), SEQ ID NO: 12 (processed)) (5 pg) (Mix B), or the combination of Mix A + Mix B. Animals were intranasally challenged at day 49 with RSV A2, or with RSV B 17-058221, a recent clinical isolate RSV B strain. Lung and nose viral load was determined by plaque assay in tissue homogenates isolated 5 days post challenge (A). Pre-challenge serum samples were analyzed for neutralizing antibodies against the RSV strains indicated by microneutralization assay (B). Symbols represent viral load or neutralizing titers of individual animals, whereas mean titers are indicated with horizontal lines. Lower limit of detection or qualification is indicated with a dotted line.

FIG 3: Immunogenicity of RSV A and RSV B combination vaccines in RSV pre exposed mice. Mice were pre-exposed with RSV A or RSV B, or remained naive. Twelve weeks after pre-exposure, animals were intramuscularly immunized with a mix of RSV A- based Ad26 (Ad26RSV009) (10⁸ vp) and preF protein RSV150042 (1.5 pg) (Mix A), with a mix of RSV B-based Ad26 (Ad26RSV019) (10⁸ vp) and preFe protein (RSV200125) (1.5 pg) (Mix B), or the combination of Mix A + Mix B. Virus neutralizing antibody titers against the RSV strains indicated were determined by firefly luciferase-based assay (A), or microneutralization assay (B) at 6 weeks after the immunization. Symbols represent neutralizing titers of individual animals, whereas mean titers are indicated with horizontal lines. Lower limit of qualification is indicated with a dotted line if available.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

It should also be understood that the terms “about,” “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension/ characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

Although respiratory syncytial virus (RSV) infects people throughout life, most individuals fail to mount a long lasting protective immune response. In addition, in the elderly, the waning immune response contributes to increased susceptibility to severe disease after RSV infection, causing significant morbidity and mortality. There are indications in the literature that both neutralizing antibodies and T-cell mediated immunity play a role in preventing RSV infection. It is therefore believed that a successful RSV vaccine, in particular a successful vaccine for the elderly, should elicit both potent neutralizing antibody levels and induce a robust T-cell response. A successful RSV vaccine further should prevent serious RSV-associated lower respiratory tract infections (LRTI) in the elderly and other adults at risk. Several strategies have recently been used in RSV vaccine development, including the generation of subunit (protein) and vector-based vaccines. No vaccine, however, has been approved as to date.

Recently, stabilized pre-fusion RSV FA proteins have been described with a unique set of amino acid mutations, as compared to the wild type RSV F protein from the RSV A2 strain (Genbank ACO83301.1) (see e.g. W02014/174018, WO2017/174564 and WO2017/174568, the content of each of which is herein incorporated by reference in its entirety). By demonstrating specific binding to pre-fusion specific antibodies in vitro, it was shown that this RSV FA protein antigen existed in the pre-fusion conformation and that the pre-fusion conformation was stable. Pre-clinical data showed that administration of the pre-fusion RSV FA proteins induced virus neutralizing antibodies in both mice and cotton rats. In addition, non-adjuvanted RSV preF_A protein induced low T cell responses in mice. In cotton rats, prime boost immunization induced protection after intranasal challenge with the RSV A2 strain 3 weeks after boost immunization. Cotton rats immunized with pre-fusion RSV FA proteins showed lower virus titer in the lung and nose 5 days after challenge compared with cotton rats immunized with post-fusion RSV F protein ((Krarup et al. Nat Comm 6, Article number: 8143, 2015). In addition, cotton rats immunized with pre-fusion RSV FA proteins were protected against subsequent challenge with an RSV B strain.

In addition, it has been shown that human recombinant adenoviral vectors comprising DNA encoding for the RSV FA protein in post-fusion confirmation induce virus neutralizing titers and T cell responses in mice after a single immunization. Prime immunization or heterologous prime boost immunization with adenoviral vector serotypes 26 and 35 encoding the post-fusion RSV F protein induced protection against intranasal challenge with RSV A2 or B15/97 in cotton rats (Widjojoatmodjo et al., Vaccine 33(41):5406-5414, 2015). Human recombinant adenoviral vectors comprising DNA encoding RSV FA proteins in the pre-fusion conformation have been described in W02014/174018 and WO2017/174564, the content of each of which is herein incorporated by reference in its entirety. In addition, it has been demonstrated that Ad26.RSV.preF had an acceptable safety profile and elicited sustained humoral and cellular immune responses after a single immunization in older adults (Williams et al., J Infect Dis 2020 Apr 22; doi: 10.1093/infdis/jiaal 93). In addition, Ad26.RSV.preF induced protection in a human challenge study (https://pubmed.ncbi.nlm.nih.gov/32851411/).

Human RSV (HRSV) is divided into two major antigenic groups of strains, subtypes A and B, that are largely defined by genetic variation in the G glycoprotein. These subtypes show an irregular, alternating prevalence pattern, with subtype A having a higher cumulative prevalence than subtype B. The F protein is highly conserved between RSV A and B and induces neutralizing antibodies across the two groups. Although the F proteins of A and B strains show a high degree of sequence identity (-95% in the mature ectodomain) and the FA protein can induce neutralizing antibodies across the two subgroups, in the research that led to the present invention, it was surprisingly found that a vaccine based on RSV FA protein had a lower efficacy against infection by RSV B strains as compared to RSV A.

The present application describes methods and immunogenic combinations with increased immunogenic efficacy. More specifically, the application describes efficacious methods and immunogenic combinations that elicit both potent antibody and T cell responses, thereby enhancing immunogenicity, and ultimately protection, against respiratory syncytial virus (RSV) A and B infections.

The present invention provides methods for inducing a protective immune response against respiratory syncytial virus (RSV) A and B infection in a human subject in need thereof, comprising administering to the subject a combination comprising:

(a) a first dose of one or more RSV FA antigen(s); and

(b) a first dose of one or more RSV F_B antigen(s).

The RSV FA and RSV F_B antigens are preferably administered concurrently (i.e. are co-administered). In certain embodiments, the RSV FA and RSV F_B antigens are formulated in different compositions, which are admixed prior to co-administration. The RSV FA and RSV F_B antigens may however also be co-formulated in one composition. In certain preferred embodiments, the RSV FA and RSV FB antigens are administered intramuscularly, i.e. by intramuscular injection.

The one or more RSV FA antigens may comprise a nucleic acid molecule encoding an RSV FA protein, a vector comprising a nucleic acid molecule encoding an RSV FA protein, and/or a soluble RSV FA protein and/or fragment thereof.

In addition, or alternatively, the one or more RSV FB antigens may comprise a nucleic acid molecule encoding an RSV FB protein, a vector comprising a nucleic molecule encoding an RSV FB protein and/or a soluble RSV FB protein and/or fragment thereof.

As used herein, the term “RSV fusion protein” or “RSV F protein,” refers to the fusion (F) protein of any group, subgroup, isolate, type, or strain of respiratory syncytial virus (RSV). As described above, RSV exists as a single serotype having two antigenic subgroups, A and B. Examples of RSV F protein thus include RSV F proteins from RSV A strains, e.g. RSV A1 and RSV A2, such proteins also referred to as RSV FA proteins, and RSV F proteins from RSV B strains, e.g. RSV B1 and RSV B2, such proteins also referred to as RSV FB proteins. As used herein, the term “RSV FA and RSV FB protein” includes RSV FA and RSV FB proteins comprising mutations, e.g., point mutations, insertions, deletions, as compared to wild-type RSV FA and RSV FB proteins, as well as fragments (e.g. ectodomains) and splice variants of full-length wild type RSV FA and RSV FB proteins.

The RSV FA and RSV FB antigens according to the invention may be any suitable RSV FA and FB antigen that is known in the art. In preferred embodiments of the invention, the RSV FA and RSV FB proteins encoded by the nucleic acid molecules and/or the soluble RSV FA and FB proteins are stabilized in the pre-fusion conformation. The RSV FA and/or RSV FB proteins that are stabilized in the pre-fusion conformation comprise at least one epitope that is recognized by a pre-fusion specific monoclonal antibody, such as the antibody CR9501. CR9501 comprises the binding regions of the antibodies referred to as 58C5 in WO201 1/020079 and W02012/006596, which binds specifically to RSV F protein in its pre fusion conformation and not to the post-fusion conformation.

Several RSV FA proteins that are stabilized in the pre-fusion conformation (pre-fusion F proteins, or pre-F proteins) are known in the art and can be used according to the invention. RSV FA proteins that have been stabilized in the pre-fusion conformation and that are particularly useful according to the invention are RSV FA proteins having at least one stabilizing mutation as compared to a wild type RSV FA protein, in particular as compared to the RSV FA protein having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the RSV FA proteins that are stabilized in the pre-fusion conformation and that are useful according to the invention comprise an amino acid sequence of an RSV FA protein, wherein the amino acid residue at position 215 is P and optionally the amino acid sequence at position 486 is N. According to particular embodiments, RSV FA proteins that are stabilized in the pre-fusion conformation and that are useful according to the invention comprise an amino acid sequence of an RSV FA protein, wherein the amino acid residue at position 66 is E, the amino acid residue at position 67 is I, the amino acid residue at position 76 is V, the amino acid residue at position 215 is P and the amino acid sequence at position 486 is N. It is again to be understood that for the numbering of the amino acid positions reference is made to SEQ ID NO: 1.

According to certain embodiments, RSV F_B proteins that have been stabilized in the pre-fusion conformation and that are useful in the application are RSV F_B proteins having at least one stabilizing mutation as compared to a wild type RSV F_B protein, in particular as compared to the RSV F_B protein having the amino acid sequence of SEQ ID NO: 2. In certain embodiments, RSV F_B proteins that are stabilized in the pre-fusion conformation that are useful according to the invention comprise an amino acid sequence of an F protein of an RSV B strain, wherein the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 215 is P, and the amino acid residue at position 486 is N.

According to particular embodiments, RSV FB proteins that are stabilized in the pre fusion conformation and that are useful according to the invention comprise an amino acid sequence of an RSV FB protein, wherein the amino acid residue at position 101 is Q, the amino acid residue at position 152 is M, the amino acid residue at position 172 is Q, the amino acid residue at position 173 is L, the amino acid at position 215 is P, the amino acid residue at position 486 is N, and wherein optionally the amino acid residue at position 191 is R, the amino acid residue at position 206 is M, the amino acid residue at position 203 is I, the amino acid residue at position 209 is R, and the amino acid residue at position 489 is Y. It is again to be understood that for the numbering of the amino acid positions for the RSV B component reference is made to SEQ ID NO: 2.

In certain preferred embodiments, the nucleic acid molecule encoding the RSV FA antigen encodes for a pre-fusion RSV FA protein having the amino acid sequence of SEQ ID NO: 3, or fragments thereof and/or the nucleic acid molecule encoding the RSV FB antigen encodes for an RSV FB protein having the amino acid sequence of SEQ ID NO: 4, 5, 18, or 20 or fragments thereof, preferably an RSV FB protein having the amino acid sequence of SEQ ID NO: 18.

In addition, or alternatively, the nucleic acid molecule encoding the RSV FA protein may comprise the nucleic acid sequence of SEQ ID NO: 6 and/or the nucleic acid molecule encoding the RSV FB protein may comprise the nucleic acid sequence of SEQ ID NO: 7, 8,

17 or 19, preferably SEQ ID NO: 17.

It is understood by a skilled person that numerous different nucleic acid molecules can encode the same protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons can, using routine techniques, make nucleotide substitutions that do not affect the protein sequence encoded by the nucleic acid molecules (or polynucleotides) described herein to reflect the codon usage of any particular host organism in which the proteins are to be expressed. Therefore, unless otherwise specified, a “nucleic acid molecule encoding an amino acid sequence” includes all nucleic acid sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleic acid sequences that encode proteins and RNA can include introns. Sequences herein are provided from 5' to 3' direction, as custom in the art.

In certain embodiments, the nucleic acid molecules encode a fragment of the pre fusion F protein of RSV A or B. The fragment can result from either or both of amino- terminal and carboxy-terminal deletions. The extent of deletion can be determined by a person skilled in the art to, for example, achieve better yield of the recombinant adenovirus. The fragment will be chosen to comprise an immunologically active fragment of the RSV FA or the FB protein, i.e. a part that will give rise to an immune response in a subject. This can be easily determined using in silico, in vitro and/or in vivo methods, all routine to the skilled person.

As described herein, the RSV FA and/or RSV FB antigens may comprise a nucleic acid molecule that encodes an RSV FA and/or RSV FB protein antigen. Both deoxy ribonucleic acids (DNA) and ribonucleic acids (RNA) are suitable. The nucleic acid can be included in a DNA or RNA vector, such as a replicable vector (e.g., a viral replicon, a self- amplifying nucleic acid), or in a virus (e.g., a live attenuated virus) or viral vector (e.g., replication proficient or replication deficient viral vector). Suitable viral vectors include but are not limited to an adenovirus, a modified vaccinia ankara virus (MV A), a paramyxovirus, a Newcastle disease virus, an alphavirus, a retrovirus, a lentivirus, an adeno-associated virus (AAV), a vesicular stomatitis virus, and a flavivirus. Optionally, the viral vector is replication defective. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.

In certain preferred embodiments, the vector is an adenoviral vector. An adenoviral vector (or adenovirus) according to the invention belongs to the family of the Adenoviridae, and preferably is one that belongs to the genus Mastadenovirus. 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), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV), or a rhesus monkey adenovirus (RhAd). In the invention, a human adenovirus is meant if referred to as Ad without indication of species, e.g. the brief notation “Ad26” means the same as HAdV26, which is human adenovirus serotype 26. Also as used herein, the notation “rAd” means recombinant adenovirus, e.g., “rAd26” refers to recombinant human adenovirus 26.

In certain embodiments, the vector is a human recombinant adenovirus, also referred to as recombinant adenoviral vectors. The preparation of recombinant adenoviral vectors is well known in the art. The term “recombinant” for an adenovirus, as used herein implicates that it has been modified by the hand of man, e.g. it has altered terminal ends actively cloned therein and/or it comprises a heterologous gene, i.e. it is not a naturally occurring wild type adenovirus.

In certain embodiments, an adenoviral vector is deficient in at least one essential gene function of the El region, e.g. the Ela region and/or the Elb region, of the adenoviral genome that is required for viral replication. In certain embodiments, an adenoviral vector is deficient in at least part of the non-essential E3 region. In certain embodiments, the vector is deficient in at least one essential gene function of the El region and at least part of the non-essential E3 region. The adenoviral vector can be “multiply deficient,” meaning that the adenoviral vector is deficient in one or more essential gene functions in each of two or more regions of the adenoviral genome. For example, the aforementioned El -deficient or E1-, E3-deficient adenoviral vectors can be further deficient in at least one essential gene of the E4 region and/or at least one essential gene of the E2 region (e.g., the E2A region and/or E2B region).

In certain embodiments, the recombinant adenovectors of the invention comprise as the 5’ terminal nucleotides the nucleotide sequence: CTATCTAT (SEQ ID NO: 15). These embodiments are advantageous because such vectors display improved replication in production processes, resulting in batches of adenovirus with improved homogeneity, as compared to vectors having the original 5’ terminal sequences (generally CATCATCA (SEQ ID NO: 16.)) (see also patent application nos. PCT/EP2013/054846 and US 13/794,318 , entitled ‘Batches of recombinant adenovirus with altered terminal ends’ filed on 12 March 2012 in the name of Crucell Holland B.V.), incorporated in its entirety by reference herein.

Most advanced studies have been performed using human adenoviruses, and human adenoviruses are preferred adenoviral vectors according to certain aspects of the invention. In certain preferred embodiments, a 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, 50, 52, etc. According to a particularly preferred embodiment of the invention, the adenoviral vector is a human adenovirus of serotype 26. Advantages of these serotypes include a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and experience with use in human subjects in clinical trials.

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 2010085984; 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: 394-401; Tatsis 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 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, the recombinant adenovirus is based upon a chimpanzee adenovirus such as ChAdOx 1 (see e.g. WO 2012/172277), or ChAdOx 2 (see e.g. WO 2018/215766). In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as BZ28 (see e.g. WO 2019/086466). In certain embodiments, the recombinant adenovirus is based upon a gorilla adenovirus such as BLY6 (see e.g. WO 2019/086456), or BZ1 (see e.g. WO 2019/086466).

In certain embodiments of the invention, the adenoviral vectors comprise capsid proteins from rare serotypes, e.g. including Ad26. An “adenovirus capsid protein” refers to a protein on the capsid of an adenovirus (e.g., Ad26, Ad35, rAd48, rAd5HVR48 vectors) 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 “capsid protein” for a particular adenovirus, such as an “Ad26 capsid protein” can be, for example, a chimeric capsid protein that includes at least a part of an Ad26 capsid protein. In certain embodiments, the capsid protein is an entire capsid protein of Ad26. In certain embodiments, the hexon, penton and fiber are of Ad26. One of ordinary skill in the art 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 pre-existing immunity of a first serotype 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. See for example WO 2006/040330 for chimeric adenovirus Ad5HVR48, that includes an Ad5 backbone having partial capsids from Ad48, and also e.g. WO 2019/086461 for chimeric adenoviruses Ad26HVRPtrl, Ad26HVRPtrl2, and Ad26HVRPtrl3, that include an Ad26 virus backbone having partial capsid proteins of Ptrl, Ptrl2, and Ptrl3, respectively)

In certain embodiments the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad26 (i.e., the vector is rAd26).

In some embodiments, the adenovirus is replication deficient, e.g., because it contains a deletion in the El region of the genome. For adenoviruses being derived from non-group C adenovirus, such as 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 ah, 2006, J Gen Virol 87: 2135-43; WO 03/104467). However, such adenoviruses will not be capable of replicating in non-complementing cells that do not express the El genes of Ad5. In certain embodiments, the adenovirus is rendered replication deficient 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 a gene of interest, such as a gene encoding an RS V F protein (usually linked to a promoter), within the region. 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 within one or more of these regions. 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.

The preparation of recombinant adenoviral vectors is well known in the art. Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et ah, (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792. Examples of vectors useful for the invention for instance include those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety.

Recombinant adenovirus can be prepared and propagated in host cells (or packaging cells) according to well-known methods, which entail cell culture of the host cells that are infected with the adenovirus. The cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture. A packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication deficient vector, thus allowing the virus to replicate in the cell. Suitable packaging cell lines for adenoviruses with a deletion in the El region include, for example, PER.C6, 911, 293, and El A549 cells.

In preferred embodiments of the invention, the vector is a human adenoviral vector, preferably a replication-incompetent recombinant adenoviral vector of serotype 26, i.e. a rAd26 vector, most preferably a rAd26 vector with at least a deletion in the El region of the adenoviral genome, e.g. such as that described in Abbink, J Virol, 2007. 81(9): p. 4654-63, which is incorporated herein by reference. Typically, the nucleic acid sequence encoding the RSV FA and/or FB protein is cloned into the El and/or the E3 region of the adenoviral genome.

As described above, the RSV fusion (F) glycoprotein typically is synthesized as a F0 precursor which contains a signal peptide, F2 and FI domains of the F protein and a peptide p27. The F0 precursor is processed by furin or related host cellular proteases into F2 and FI domains, and the signal peptide and the p27 are removed. The FI domain contains a transmembrane (TM) and cytoplasmic (CP) domain. F2 and FI domains typically are connected by disulfide bridges (Figure 1). The F2-F1 heterodimers are organized on virions as trimeric spikes.

After processing, the processed mature RSV FA protein encoded by the adenoviral vector comprises the F2 domain (i.e. the amino acids 26 to 109 of SEQ ID NO: 3) and the FI domain (i.e. the amino acids 137-574) of SEQ ID NO: 3), which are linked by one or more disulfide bridges, and the processed mature RSV FB protein encoded by the adenoviral vector comprises the F2 domain (i.e. the amino acids 26 to 109 of SEQ ID NO: 4, 5, 18 or 20 ) and the FI domains (i.e. the amino acids 137-574) of SEQ ID NO: 4, 5, 18 or 20, which are linked by one or more disulfide bridges. The processed RSV FA and FB proteins thus will not comprise the signal peptide and the p27 peptide anymore.

Soluble RSV FA and FB proteins used according to the invention typically comprise the ectodomain of the RSV FA and FB proteins. The soluble RSV FA and/or FB proteins according to the invention preferably also are stabilized in the pre-fusion conformation (also referred to as pre-fusion F proteins or preF proteins). The soluble RSV pre-fusion FA and FB proteins lack the transmembrane and cytoplasmic domains. Thus, in certain embodiments, the transmembrane and cytoplasmic domains have been removed, and, optionally, replaced by a heterologous trimerization domain linked to the C-terminus of the FI domain, either directly or through a linker. In some embodiments, the T4 bacteriophage fibritin “foldon” (Fd) trimerization domain has been added to the C-terminus of the FI domain to increase trimerization of the RSV F protein.

In certain preferred embodiments, the soluble RSV FA protein comprises the amino acid sequence of SEQ ID NO: 9 or 11.

In addition, or alternatively, and/or the soluble RSV FB protein comprises the amino acid sequence of SEQ ID NO: 10 or 12.

In certain embodiments, the soluble RSV FA protein is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 13 and/or the soluble RSV FB protein is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 14, 17 or 19, preferably SEQ ID NO: 17.

The soluble pre-fusion RSV F proteins can be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g., Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6® cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants. In certain embodiments, the cells are from a multicellular organism; in certain embodiments, they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. In general, the production of recombinant proteins in a host cell, such as the pre-fusion RSV F proteins of the disclosure, comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the protein in the cell. The nucleic acid molecule encoding a protein in expressible format can be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like. The person skilled in the art is aware that various promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed.

Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here, the pre-fusion RSV F proteins. The suitable medium may or may not contain serum.

A “heterologous nucleic acid molecule” (also referred to herein as “transgene”) is a nucleic acid molecule that is not naturally present in the host cell. It is introduced into, for instance, a vector by standard molecular biology techniques. A transgene is generally operably linked to expression control sequences. This can, for instance, be done by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Further regulatory sequences can be added. Many promoters can be used for expression of a transgene(s), and are known to the skilled person, e.g., these can comprise viral, mammalian, synthetic promoters, and the like. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is a CMV-promoter (US 5,385,839), e.g., the CMV immediate early promoter, for instance, comprising nt. -735 to +95 from the CMV immediate early gene enhancer/promoter. A polyadenylation signal, for example, the bovine growth hormone polyA signal (US 5,122,458), can be present behind the transgene(s). Alternatively, several widely used expression vectors are available in the art and from commercial sources, e.g., the pcDNA and pEF vector series of INVITROGEN®, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from STRATAGENE™, etc., which can be used to recombinantly express the protein of interest, or to obtain suitable promoters and/or transcription terminator sequences, polyA sequences, and the like.

The cell culture can be any type of cell culture, including adherent cell culture, e.g., cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done, for instance, in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems, and the like. Suitable conditions for culturing cells are known (see, e.g., 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)). In preferred embodiments of the invention, the one or more RSV FA antigens comprise a replication-incompetent recombinant human adenovirus of serotype 26 (Ad26) comprising a nucleic acid molecule that encodes a pre-fusion conformation-stabilized membrane-bound FA protein, preferably the pre-F_A protein of SEQ ID NO: 3 and a soluble pre-fusion FA protein, preferably the soluble pre-F_A protein of SEQ ID NO: 9 or 11, and the one or more RSV F_B antigens comprise a replication-incompetent recombinant adenovirus serotype 26 (Ad26) comprising a nucleic acid molecule that encodes a pre-fusion conformation-stabilized membrane-bound F_B protein, preferably the pre-Fe protein of SEQ ID NO: 4, 5, 18 or 20, preferably SEQ ID NO: 18, and a soluble pre-fusion F_B protein, preferably the soluble pre-Fe protein of SEQ ID NO: 10 or 12. According to the invention, the RSV FA and RSV F_B antigens are preferably administered concurrently. “Concurrent administration or co-administration,” in the context of the administration of the RSV FA and RSV F_B antigens to a subject, refers to the use of the RSV FA and RSV F_B antigens in combination, wherein said RSV FA and RSV F_B antigens administered to the subject within a period of 24 hours. In certain embodiments, the FA and FB antigens are co-formulated, for example, with a pharmaceutically acceptable buffer, carrier, excipient and/or adjuvant, in a single composition for administration, for example admixed, and administered to a subject together at the same time. In other embodiments, the FA and FB antigens are formulated, for example, with a pharmaceutically acceptable buffer, carrier, excipient and/or adjuvant, in separate compositions, and are administered to a subject in separate compositions within 24 hours, such as within 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or within 1 hour or less. Alternatively, the nucleic acid sequence encoding the RSV FA protein or vector comprising such nucleic acid sequence may be present in one formulation with the nucleic acid sequence encoding the RSV FB protein or vector comprising such nucleic acid, and the soluble RSV FA protein may be present with the soluble RSV FB protein in a separate formulation, which formulations may be admixed and administered to a subject together at the same time. Admixing can occur just prior to use, when the two components are manufactured and formulated, or any time between. In preferred embodiments, the FA and FB antigens are co formulated in a single composition for administration at the point of delivery shortly prior to administration, for example, bed side mixing, e.g. by using a multi -chamber syringe.

The FA and FB antigens described herein preferably are formulated as vaccines. As used herein, the term “vaccine” refers to a composition containing an active component effective to induce a certain degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease, and up to complete absence, of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease. The vaccine(s) preferably induce an immune response against RSV, preferably both a humoral and cellular immune response against the F protein of RSV A and RSV B. According to certain embodiments, the vaccine(s) can be used to prevent serious lower respiratory tract disease (LRTD) leading to hospitalization and to decrease the frequency of complications such as pneumonia, bronchitis and bronchiolitis due to RSV A or B infection and replication in a subject. In certain embodiments, the vaccine(s) can be combination vaccine(s) that further comprise other components that induce a protective immune response, e.g. against other proteins of RSV and/or against other infectious agents, such as e.g. influenza virus, or coronavirus, e.g. SARS CoV-2, HMPV, PIV-3. The administration of further active components can, for instance, be done by separate administration or by administering combination products of the vaccines of the application and the further active components.

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.

In certain embodiments, the protective immune response is characterized by the presence of neutralizing antibodies and/or a cellular response to RSV A and B. According to particular embodiments, the protective immune response is characterized by prevention or reduction of reverse transcriptase polymerase chain reaction (RT PCR)-confirmed RSV A or RSV B-mediated lower respiratory tract disease (LRTD). In certain embodiments, administration of the FA and FB antigens results in the reduction of reverse transcriptase polymerase chain reaction (RT PCR)-confirmed RSV A or RSV B- mediated lower respiratory tract disease (LRTD), as compared to subjects which have not been administered the vaccine combination.

In addition, or alternatively, the protective immune response is characterized by an absent or reduced RSV viral load in the nasal track and/or lungs of the subject upon exposure to RSV A orB. In addition, or alternatively, the protective immune response is characterized by an absent or reduced RSV clinical symptom in the subject upon exposure to RSV A or B. RSV clinical symptoms include, for example, nasal congestion, sore throat, headache; cough, shortness of breath, wheezing, coughing up phlegm(sputum), fever or feeling feverish, body aches and pains, fatigue (tiredness), neck pain and loss of appetite.

Preferably, a subject having a “protective immune response” or “protective immunity” against a certain agent will not die as a result of the infection with the agent.

As used herein, the term “induce” and variations thereof refers to any measurable increase in cellular activity. Induction of a protective immune response can include, for example, activation, proliferation, or maturation of a population of immune cells, increasing the production of a cytokine, and/or another indicator of increased immune function. In certain embodiments, induction of an immune response can include increasing the proliferation of B cells, producing antigen-specific antibodies, increasing the proliferation of antigen-specific T cells, improving dendritic cell antigen presentation and/or an increasing expression of certain cytokines, chemokines and co-stimulatory markers.

The ability to induce a protective immune response against RSV F protein can be evaluated either in vitro or in vivo using a variety of assays which are standard in the art. For a general description of techniques available to evaluate the onset and activation of an immune response, see for example Coligan et al. (1992 and 1994, Current Protocols in Immunology; ed. J Wiley & Sons Inc, National Institute of Health). Measurement of cellular immunity can be performed by methods readily known in the art, e.g., by measurement of cytokine profiles secreted by activated effector cells including those derived from CD4+ and CD8+ T-cells (e.g. quantification of IL-4 or IFN gamma-producing cells by ELISPOT), by measuring PBMC proliferation, by measuring NK cell activity, by determination of the activation status of immune effector cells (e.g. T-cell proliferation assays by a classical [3H] thymidine uptake), by assaying for antigen-specific T lymphocytes in a sensitized subject (e.g. peptide-specific lysis in a cytotoxicity assay, etc.). Additionally, IgG and IgA antibody secreting cells with homing markers for local sites which can indicate trafficking to the gut, lung and nasal tissues can be measured in the blood at various times after immunization as an indication of local immunity, and IgG and IgA antibodies in nasal secretions can be measured; Fc function of antibodies and measurement of antibody interactions with cells such as PMNs, macrophages, and NK cells or with the complement system can be characterized; and single cell RNA sequencing analysis can be used to analyze B cell and T cell repertoires.

The ability to induce a protective immune response against RSV F protein can be determined by testing a biological sample (e.g., nasal wash, blood, plasma, serum, PBMCs, urine, saliva, feces, cerebral spinal fluid, bronchoalveolar lavage or lymph fluid) from the subject for the presence of antibodies, e.g. IgG or IgM antibodies, directed to the RSV F protein(s) administered in the composition, e.g. viral neutralizing antibody against RSV A2 (VNA A2), VNA RSV A Memphis 37b, RSV B, pre-F antibodies, post-F antibodies (see for example Harlow, 1989, Antibodies, Cold Spring Harbor Press). For example, titers of antibodies produced in response to administration of a composition providing an immunogen can be measured by enzyme-linked immunosorbent assay (ELISA), other ELISA-based assays (e.g., MSD-Meso Scale Discovery), dot blots, SDS-PAGE gels, ELISPOT, measurement of Fc interactions with complement, PMNs, macrophages and NK cells, with and without complement enhancement, or Antibody-Dependent Cellular Phagocytosis (ADCP) Assay. Exemplary methods are described in Example 1. According to particular embodiments, the induced immune response is characterized by neutralizing antibodies to RSV and/or protective immunity against RSV.

According to particular embodiments, the protective immune response is characterized by the presence of neutralizing antibodies to RSV and/or a cellular immune response against RSV, preferably detected between about 8 to about 35 days after administration of the FA and FB antigens, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 days after administration the FA and FB antigens. More preferably, the neutralizing antibodies against RSV A and B are detected about 6 months to 5 years after the administration of the FA and FB antigens, such as 6 months, 1 year, 2 years, 3 years, 4 years or 5 years after administration of the FA and FB antigens.

The application also relates to methods of preventing or reducing reverse transcriptase polymerase chain reaction (RT PCR)-confirmed RSV-mediated lower respiratory tract disease (LRTD) in a human subject in need thereof, comprising prophylactically administering intramuscularly to the subject a combination comprising:

(a) a first dose of one or more RSV FA antigen(s); and

(b) a first dose of one or more RSV FB antigen(s), wherein (a) and (b) are co-administered.

The application also provides methods for vaccinating a subject against RSV infection with an acceptable safety profile, comprising administering to the subject a combination comprising:

(a) a first dose of one or more RSV FA antigen(s); and

(b) a first dose of one or more RSV FB antigen(s).

The methods described herein may comprise further comprising administering to the subject after the first dose:

(a) a second dose of one or more RSV FA antigen(s); and/or

(b) a second dose of one or more RSV FB antigen(s),

According to the invention, the first dose and second dose may comprise the same RSV FA and RSV FB antigens, e.g. one or more RSV FA and RSV FB antigens as described herein, or the first dose and the second dose may comprise different RSV FA and RSV FB antigens.

In certain embodiments, the second doses of the RSV FA and RSV FB antigens are co administered. The interval between the administrations if the first and second doses can vary. A typical regimen may comprise a first immunization with the combination as described herein followed by a second administration 1, 2, 4, 6, 8, 10 and 12 months later. Another regimen may entail one or 2 doses annually, prior to the RSV season. In certain embodiments, the first and second immunization are 2, 3, 4, 5, 6, 7, 8, 9 or 10 years apart. It is readily appreciated by those skilled in the art that regimens for first and second dose administrations can be adjusted based on the measured immune responses after the administrations. For example, second doses are generally administered weeks or months after administration of the first doses, for example, about 1 week, or 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks, or 36 weeks, or 40 weeks, or 44 weeks, or 48 weeks, or 52 weeks, or 56 weeks, or 60 weeks, or 64 weeks, or

68 weeks, or 72 weeks, or 76 weeks, or one, two, three, four, five up, or to ten years after administration of the first doses.

According to the invention, the subject can be a human subject of any age, e.g. from about 1 month to 100 or more years old, e.g. from about 2 months to about 100 years old. When the RSV FA and RSV FB antigens are administered to an infant, the composition can be administered one or more times. The first administration can be at or near the time of birth (e.g., on the day of or the day following birth), or within 1 week of birth or within about 2 weeks of birth. Alternatively, the first administration can be at about 4 weeks after birth, about 6 weeks after birth, about 2 months after birth, about 3 months after birth, about 4 months after birth, or later, such as about 6 months after birth, about 9 months after birth, or about 12 months after birth.

Preferably, the subject is a human subject that is susceptible to RSV infection. In certain embodiments, a human subject that is susceptible to RSV infection includes, but is not limited to, an elderly human subject, for example a human subject > 50 years old, > 60 years old, > 65 years old; or a young human subject, for example a human subject < 5 years old, < 1 year old; and/or a human subject that is hospitalized or a human subject that has been treated with an antiviral compound but has shown an inadequate antiviral response. In certain embodiments, a human subject that is susceptible to RSV infections includes but is not limited to a human subject between 18 and 59 suffering from chronic heart disease, chronic lung disease, asthma and/or immunodeficiency.

In certain preferred embodiments, the human subject is at least 60 years old.

In certain preferred embodiments, the human subject is at least 65 years old.

The application also provides immunogenic combinations (e.g. kits), or vaccine combinations, comprising (a) an effective amount of one or more RSV FA antigen(s) as described herein, and (b) an effective amount of one or more RSV F_B antigen(s) as described herein, for inducing a protective immune response against RSV A and B infection in a human subject in need thereof.

Preferably, the RSV FA and RSV FB antigens are for co-administration. The combination is preferably for use in methods for inducing a protective immune response against RSV A and B infection in a human subject in need thereof, as described herein. Preferably, the combination is used for the prevention of reverse transcriptase polymerase chain reaction (RT PCR)-confirmed RSV-mediated lower respiratory tract disease (LRTD). The RSV FA and RSV FB antigens can be present in co-formulated compositions (i.e. be co-formulated) or in different compositions that separately provide each component. In certain embodiments, the combinations comprise the RSV FA and RSV FB antigens in one container (i.e. are co-formulated). In other embodiments, combinations comprise the one or more RSV FA antigens and the one or more RSV FB antigens in separate containers, or at least one RSV FA antigen and RSV FB antigen in one container and the second, or further RSV FA and RSV FB antigen in separate container(s). The container(s) can be, for example, one or more pre-filled syringe. Such a syringe can be a multi-chamber (e.g., dual-chamber) syringe. Prior to administration, the components can be admixed and then administered to the subject at the same site (e.g., through a single needle).

According to particular embodiments, the RSV FA and/or RSV FB antigens are formulated as pharmaceutical compositions. According to particular embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier or excipient. As used herein, 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 Science (15th ed.), Mack Publishing Company, Easton, Pa., 1980). The preferred formulation of the pharmaceutical composition depends on the intended mode of administration and therapeutic application. The compositions can include pharmaceutically- acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer’s solutions, dextrose solution, and Hank’s solution. In addition, the pharmaceutical composition or formulation can also include other carriers, adjuvants, or non-toxic, non-therapeutic, non- immunogenic stabilizers, and the like. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.

In certain embodiments, pharmaceutical compositions according to the application further comprise one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant. 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 a protective immune response to the RSV F proteins of the pharmaceutical compositions. Examples of suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g. antibodies or fragments thereof (e.g. directed against the antigen itself or CD la, CD3, CD7, CD80) and ligands to receptors (e.g. CD40L, GMCSF, GCSF, etc.), which stimulate immune response upon interaction with recipient cells. 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 first immunogenic component is formulated with an adjuvant. In other embodiments, the second immunogenic component is formulated with an adjuvant. In certain embodiments , both immunogenic components contain an adjuvant. Typically, the adjuvant is admixed (e.g., prior to administration or stably formulated) with the antigenic component. When the immunogenic combination is to be administered to a subject of a particular age group, the adjuvant is selected to be safe and effective in the subject or population of subjects. Thus, when formulating a immunogenic combination for administration to an elderly subject (such as a subject greater than 65 years of age), the adjuvant is selected to be safe and effective in elderly subjects. Similarly, when the combination immunogenic composition is intended for administration to neonatal or infant subjects (such as subjects between birth and the age of two years), the adjuvant is selected to be safe and effective in neonates and infants. In certain embodiments the pharmaceutical compositions 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. 0.075-1.0 mg, of aluminium content per dose.

The immunogenic combinations of the invention can be used e.g. in stand-alone prophylaxis of a disease or condition caused by RSV A or B, or in combination with other prophylactic and/or therapeutic treatments, such as other vaccines (e.g. against influenza, HMPV, PIV and/or SARS-CoV2), antiviral agents and/or monoclonal antibodies. As used herein, the term “in combination,” in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. For example, a first therapy (e.g., a pharmaceutical composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks,

3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours,

6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject. Pharmaceutical compositions of the present application can be formulated according to methods known in the art in view of the present disclosure.

Preferably, the first dose and/or the second dose of the RSV FA and RSV FB antigens comprises an effective amount of said components. As used herein, the term “effective amount” refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject. Selection of a particular effective dose can be determined (e.g., via clinical trials) by those skilled in the art based upon the consideration of several factors, including the disease to be treated or prevented, the symptoms involved, the patient’s body mass, the patient’s immune status and other factors known by the skilled artisan. The precise dose to be employed in the formulation will also depend on the mode of administration, route of administration, target site, physiological state of the patient, other medications administered and the severity of disease. For example, the effective amount of the RSV FA and RSV FB antigens also depends on whether adjuvant is also administered, with higher dosages typically being required in the absence of adjuvant. According to preferred embodiments, an effective amount of the RSV FA and RSV FB antigens comprises an amount of the RSV FA and RSV FB antigens that is sufficient to induce a protective immune response against RSV FA an FB protein with an acceptable safety profile. As used herein, the term “acceptable safety profile” refers to a pattern of side effects that is within clinically acceptable limits as defined by regulatory authorities. According to preferred embodiments, an effective amount of the RSV F_A and RSV F_B antigens comprises an amount of pharmaceutical composition that is sufficient to prevent infection and/or replication of RSV with an acceptable safety profile. EXAMPLES

The following examples of the application are intended to further illustrate the nature of the application. It should be understood that the following examples do not limit the invention and that the scope of the invention is to be determined by the appended claims. Example 1: Phase 2b Study to Assess the Efficacy, Immunogenicity and Safety of an Ad26.RSV.preF-based Regimen in the Prevention of RT-PCR- confirmed RSV- mediated Lower Respiratory Tract Disease in Adults Aged 65 Years and Older

A multi-center, randomized, double-blind, placebo-controlled Phase 2b proof-of- concept study in male and female participants aged >65 years who are in stable health was performed. A target of up to 5,800 participants was to be enrolled. A schematic overview of the study design and groups is depicted below.

Group N^a Day 1

Group 1 2,900 Ad26.RSV.prcF (1 10" vp) /

RSV preF protein (150 pg)

Group 2 2,900 Placebo

Randomization: Participants are randomized in parallel in a 1 : 1 ratio to 1 of 2 groups to receive Ad26.RSV.preF/RSV preF protein vaccine or placebo. The randomization will be stratified by age categories (65-74 years, 75-84 years, >85 years) and by being at increased risk for severe RSV disease (yes/no), and done in blocks to ensure balance across arms.

Vaccination schedules/Study duration: Screening for eligible participants was performed pre-vaccination on Day 1. Participants were followed up until the end of the RSV season. The study will continue beyond the first RSV season. Primary analysis set for efficacy: The Per-protocol Efficacy (PPE) population included all randomized and vaccinated participants excluding participants with major protocol deviations expecting to impact the efficacy outcomes. Any participant with an RT-PCR-confirmed RSV-mediated ARI with onset within 14 days after vaccination was excluded, as well as participants who discontinued within 14 days after vaccination. Primary efficacy endpoint: The three primary efficacy endpoints are first occurrence of RT-PCR confirmed RSV-mediated LRTD according to each of the 3 case definitions shown in the table 1 below:

Table 1. Case Definitions

Case Definition #1 Case Definition #2 Case Definition #3

>3 symptoms of LRTI >2 symptoms of LRTI >2 symptoms of LRTI,

OR

(new onset or worsening) (new onset or worsening) > 1 symptom of LRTI combined with >1 systemic symptom

(new onset or worsening)

+ RT-PCR confirmation of RSV by GeneXpert or Local lab (in case of hospitalization) LRTI = lower respiratory tract infection

Symptoms are collected via the RiiQ, an ePRO questionnaire completed by the participant at baseline and daily during the ARI, and via a clinical assessment by the PI completed at baseline and at the day 3-5 visit during the ARI.

First occurrence of a considered endpoint is defined as the first day of symptoms of the first RSV-confirmed ARI episode where the criteria for the respective case definition are fulfilled on at least one assessment of the considered episode.

The 3 case definitions assessed in this study were designed to cover a range of RSV disease severity. The presence of a combination of 3 symptoms of lower respiratory tract infection similar to those used in this study have been associated with a 3-fold higher risk of a severe outcome (Belongia et al., Adult RSV Epidemiology and Outcomes, OFID, 2018).

Primary Objective(s):

To demonstrate the efficacy of active study vaccine in the prevention of reverse transcriptase polymerase chain reaction (RT PCR)-confirmed RSV-mediated lower respiratory tract disease (LRTD) according to one of the three case definitions, when compared to placebo.

Vaccine:

The active study vaccine was an Ad26.RSV.preF_A/RSV preF_A protein mixture, comprising: · Ad26.RSV.preF A, a replication-incompetent adenovirus serotype 26 (Ad26) containing a deoxyribonucleic acid (DNA) transgene that encodes the pre-fusion conformation-stabilized F protein (pre-F) derived from the RSV A2 strain, i.e. the pre-fusion conformation-stabilized FA protein (pre-F A) of SEQ ID NO: 3; and • Soluble RSV preF_A protein, a pre-fusion conformation-stabilized F protein derived from the RSV A2 strain, i.e. the RSV preF_A protein of SEQ ID NO: 11.

The vaccine was administered as a single injection in the deltoid muscle. All injections were 1 mL in volume.

The following doses were administered:

• Ad26.RSV.preF_A was supplied at a concentration of 2xlO^u vp (viral particles)/l mL in single-use vials. Dose levels of lxlO¹¹ vp were used.

• RSV preF_A protein was supplied at a concentration of 0.3 mg/1 mL in single-use vials. Dose levels of 150 pg were used.

• Placebo for Ad26.RSV.preF, and RSV preF protein.

Serious adverse events (SAEs) were reported from administration of study vaccine until the end of the RSV season, or 6 months after.

Solicited AEs (up to 7 days post-vaccination) and unsolicited AEs (up to 28 days post-vaccination) were captured in a subset of -700 participants (the Safety Subset). SAEs were captured in all participants. Humoral and cellular immunogenicity over time was collected for a subset of 200 participants (the Immuno Subset).

The study was considered successful as soon as vaccine efficacy (VE) is demonstrated for at least one of the primary endpoints. To control the false positive rate for multiplicity, the Spiessens and Debois method is applied. If the multiplicity corrected confidence interval (Cl) is above 0 for at least 1 of the 3 primary endpoints, the study is successful.

A total of 6673 participants were screened across 40 sites in the US. Of those, 857 were screening failures, 34 were randomized not vaccinated and 5782 participants were randomized and vaccinated (2891 in each group). 107 (3.7%) participants in the active group and 100 (3.5%) participants in the placebo group discontinued the study, the majority (129 participants) withdrew consent. All other participants were still ongoing at the time of database cut-off (May 15, 2020). In the full analysis (FA) set, 57.7% of the participants were female and 92.5% were white. The median age was 71 years, ranging from 65 to 98 years. The median BMI was 28.7kg/m², ranging from 11.7 to 41.1 kg/m². 25.4% of the participants was at increased risk for RSV disease (risk level as collected in eCRF, using CDC guidance (i.e. chronic heart and lung disease)) and 26.2% of the participants was pre-frail or frail at baseline. 92 (3.2%) participants in the Ad26/protein preF RSV vaccine group and 83 (2.9%) in the placebo group, had a major protocol deviation impacting efficacy. Those participants were excluded from the Per Protocol Efficacy (PPE) set, the primary analysis set for efficacy analyses.

Summary of Results:

Below, the topline results of the primary analysis are described. Unblinded results are presented. Data up to May 15, 2020 were included. This was the date when all participants were expected to have completed their End of Season call or had discontinued earlier. It is noted that due to the increasing incidence of COVID-19 cases in the US, the ARI surveillance period was shortened from 30 April 2020 to 20 March 2020. Three primary case definitions were tested for vaccine efficacy representing different degrees of severity of RSV- confirmed lower respiratory tract disease (case definition 1, 2 and 3 - based on number of lower respiratory tract signs and/or symptoms, CD1 being most severe). Symptoms were collected daily by a PRO questionnaire (RiiQ) and at Day 3-5 by clinical assessment.

The table below shows the observed incidence rate and vaccine efficacy in the study by case definition, overall and for RSV A and RSV B. The trial was considered successful if efficacy was shown (LL Cl >0%) for any of the 3 cases definitions.

The study was successful for all 3 case definitions, so proof of concept was demonstrated (see table 2). Table 2. Vaccine Efficacy

'RiiQ LRTI symptoms: cough, shortness of breath, coughing up phlegm and wheezing; Day 3 -5 clinical assessment LRTI symptoms: cough, dyspnea or decreased oxygen saturation, sputum production, wheezing/rhonchi/rales/other signs of consolidation and tachypnea 2RiiQ systemic symptoms: fatigue and feeling feverish; Day 3-5 clinical assessment systemic symptoms: malaise and fever 3Spiessen and Dubois correction for multiple primary endpoints 495%CI

The results showed a lower incidence and higher vaccine efficacy with increasing severity of case definitions, which is consistent with observations with other vaccines (influenza, meningococcal, varicella). In addition, all sensitivity analyses pointed into the same direction, showing the robustness of the results.

Subgroup analyses revealed robust efficacy across age groups (e.g. 65-74 years (VE CD2 73.2% and 75 to 84 years 80.2%), which is not always observed for other vaccines (e.g. Influenza or Pneumococcal vaccines). An unexpected difference in vaccine efficacy between RSV A and RSV B infections was observed. In total, 56 RSV positive cases were included in the primary analysis, 31 RSV A cases and 25 RSV B cases. The point estimate of vaccine efficacy for RSV A ranged from 85.1 to 94.4% while efficacy against RSV B was lower, from 43.5 to 58.1% (see table above). Note that the study is not powered for these endpoints: endpoints with a low number of events in the placebo group should be interpreted with caution.

The lower efficacy against RSV B is unexpected based on preclinical data showing protection with Ad26.RSV.preF_A against bovine RSV, which has less homology to RSV A than RSV B, as well as immunogenicity data in humans showing induction of a robust neutralizing antibody response against both RSV A and RSV B in in vitro assays. The ability of Ad26.RSV.preF to confer cross-protection against both RSV A and RSV B was confirmed in the naive cotton rat challenge model. In this study, where 63% of RSV cases in the placebo group were RSV A and 35% RSV B, the overall efficacy of the vaccine remains high, but the relative incidence of RSV A and RSV B varies from season to season, and this could impact the overall vaccine efficacy in case of a different distribution between RSV A and RSV B cases.

Solicited AEs (up to 7 days post-vaccination) and unsolicited AEs (up to 28 days post-vaccination) were captured in a subset of -700 participants (the Safety Subset). SAEs were captured in all participants. There were no SAEs considered related to the study vaccine. No safety concerns have been identified. The safety data are in line with previously observed safety data with the Ad26/protein vaccine, the only difference being less overall and less severe reported solicited events were observed in this study. In the Ad26/protein RSV preF vaccine group 37.9% of participants reported local solicited events (1.7% grade 3) versus 8.4% in the placebo group (0.3% grade 3). The most common reported local solicited event was pain/tenderness. Systemic solicited events were reported in 41.4% in the vaccine group (2% grade 3) and 16.4% in the placebo group (0.3% grade 3). The most common reported systemic events were fatigue, myalgia and headache. Fever was reported in 4.3% of subjects (0.3% grade 3) in the vaccine group versus no fever in the placebo group.

Immunogenicity Humoral and cellular immunogenicity over time was measured in a subset of 200 participants (the Immuno Subset). The randomization ratio in the Immunosubset was also 1:1. For the TLR, immunogenicity data was available for Day 1, 15 and Day 169 for preF_A and post F Elisa, VNA-A2, VNA-B and RSV F specific INFy ELISpot. The observed immunogenicity data are in line with the immunogenicity data previously observed with the Ad26.RSV.preFA/preFA protein vaccine with a robust antibody response against both subtypes. The decay of antibodies from Day 15 to 169 was comparable for VNA A2 and VNA_B with 2.3 fold for VNA_A2 and 2.1 fold decay for VNA_B.

Table 3 provides a summary of the immunogenicity observed in the Ad26/protein preF RSV vaccine group.

Table 3: Overview of immunogenicity; Per Protocol Immunogenicity Set

Ad26/protein preF RSV vaccine (N=97)

Assay Baseline Day 15 Day 169

VNA A2 GMT (95% Cl) 542 (457;643) 7244 (5889;8912) 3057 (2523;3703)

VNA B GMT (95% Cl) 4079 (3501;4752) 38006 (31693 ;45577) 17362 (14768;20413)

ELISpot Median (Q1,Q3) 34 (34;76) 444 (279;641) 201 (123;324)

Humoral immunogenicity samples at day 1 and 15 were also collected. A subset of these samples are still being analyzed and the relationship between the immunogenicity outcomes and efficacy outcomes will be further explored. In summary:

• The study shows high vaccine efficacy for all three primary endpoints, ranging from 80% for the most severe endpoint to 69.8% for the mildest endpoint, demonstrating the value of the Ad26/protein preF vaccine approach for the prevention of RSV disease in older adults.

• The robustness of the results is further supported by the sensitivity and subgroup analyses.

• Unexpectedly, lower vaccine efficacy against RSV B (58.3% against CD1; 37% of all RSV cases in placebo group) compared to RSV A (94.4%; 63% of all RSV cases in the placebo group) was observed, despite the induction of robust neutralizing antibody responses against both subtypes also in this study.

• Bivalent vaccine products, i.e. comprising both an RSV A and B product, may be needed. Example 2. Immunogenicity and protective efficacy of RSV A and RSV B combination vaccines in cotton rats.

Animals were intramuscularly immunized at day 0 with a mix of RSV A-based Ad26 (Ad26RSV009 coding for the RSV FA protein of SEQ ID NO: 3) (10⁶ vp) and preF_A protein RS VI 50042 (SEQ ID NO:9 (unprocessed), SEQ ID NO: 11 (processed)) (5 pg) (Mix A), with a mix of RSV B-based Ad26 (Ad26RSV019; coding for an RSV FB protein of SEQ ID NO: 18) (10⁶ vp) and preF protein (RSV200125; SEQ ID NO: SEQ ID NO: 10 (unprocessed), SEQ ID NO: 12 (processed)) (5 pg) (Mix B), or the combination of Mix A + Mix B. Animals were intranasally challenged at day 49 with RSV A2, or with RSV B 17-058221, a recent clinical isolate RSV B strain. Lung and nose viral load was determined by plaque assay in tissue homogenates isolated 5 days post challenge (see Fig. 2A). Pre-challenge serum samples were analyzed for neutralizing antibodies against the RSV strains indicated by microneutralization assay (Fig. 2B). Symbols represent viral load or neutralizing titers of individual animals, whereas mean titers are indicated with horizontal lines. Lower limit of detection or qualification is indicated with a dotted line.

Example 3. Immunogenicity of RSV A and RSV B combination vaccines in RSV pre-exposed mice.

Mice were pre-exposed with RSV A or RSV B or remained naive. Twelve weeks after pre-exposure, animals were intramuscularly immunized with a mix of RSV A-based Ad26 (26RSV009) (10⁸ vp) and preF protein RSV150042 (1.5 pg) (Mix A), with a mix of RSV B- based Ad26 (Ad26RSV019 ) (10⁸ vp) and preF protein (RSV200125) (1.5 pg) (Mix B), or the combination of Mix A + Mix B. Virus neutralizing antibody titers against the RSV strains indicated were determined by firefly luciferase-based assay (Fig. 3A), or microneutralization assay (Fig. 3B) at 6 weeks after the immunization. Symbols represent neutralizing titers of individual animals, whereas mean titers are indicated with horizontal lines. Lower limit of qualification is indicated with a dotted line if available.

Sequences

SEQ ID NO: 1 (RSV F protein A2 full length sequence)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIE L SNIKKNKCN GTD AKIKLIKQELDK YKN A VTELQLLMQ S TP ATNNRARRELPRFMN YTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLS TNKA V V SL SN GV S VLT SK VLDLKN YIDKQLLPIVNKQ S C SI SNIET VIEF QQKNNRLLE ITREF S VNAGVTTP VSTYMLTN SELLSLINDMPITNDQKKLMSNNVQIVRQQ S Y SIMSI IKEEVL AYVV QLPL Y GVIDTPCWKLHT SPLCTTNTKEGSNICLTRTDRGW Y CDNAGS V SFFPQ AETCK V Q SNRVF CDTMN SLTLPSEVNLCNVDIFNPK YDCKIMT SKTD V S S S V ITSLGAIV SC Y GKTKCT ASNKNRGIIKTF SNGCD YV SNKGVDT V S VGNTL YYVNKQE GKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKST TNIMITTIII VII VILL SLI A V GLLL Y CK ARS TP VTL SKDQL S GINNI AF SN

SEQ ID NO: 2 RSV F B full length CONSENSUS (wt) B sequence

MELLIHRS S AIFLTL AINALYLTS SQNITEEF YQSTC S AV SRGYLS ALRTGWYTS VITIE LSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMN YTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTN K A V V SL SN GV S VLT SK VLDLKN YINN QLLPI VNQQ S CRISNIET VIEF Q QKN SRLLEIT REFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIK EEVL AYVVQLPIY GVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWY CDNAGS V SF FPQ ADTCK V Q SNRVF CDTMN SLTLP SEV SLCNTDIFN SK YDCKIMTSKTDIS S S VIT SL GAI VSC Y GKTKCT ASNKNRGIIKTF SNGCD YV SNKGVDT V S VGNTL YYVNKLEGKN LYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNI

MIT AIII VII VVLL SLI AIGLLL Y CK AKNTP VTL SKDQL S GINNI AF SK SEQ ID NO: 3: amino acid sequence of FL stabilized preF-A protein

MELULKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNGTDAKVKUKQELDKYKNA VTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLST NKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLIN DMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNA GSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGII KTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLHNVNA VKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN SEQ ID NO: 4: amino acid sequence of FL stabilized preF-B protein (processed variant), with RSV-A signal peptide, as processed variant)

MELLILK ANAITTILT AVTF CF ASGQNITEEF YQ STC S AV SRGYL S ALRT GW YT S VITIE LSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQAANNRARREAPQYM NYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALQ LTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRIPNIETVIEFQQKNSRLL EITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMS IIKEEVL AYVV QLPI Y GVIDTPC WKLHT SPLCTTNIKEGSNICLTRTDRGW Y CDNAGS V SFFPQ ADTCK V Q SNRVF CDTMN SLTLP SEV SLCNTDIFN SK YDCKIMT SKTDIS S S VI T SLGAI VSC Y GKTKCT ASNKNRGIIKTF SNGCD YV SNKGVDT V S VGNTL YYVNKLEG KNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFIRRSDELLHNVNTGKSTT

NIMITAIIIVIIVVLLSLIAIGLLLY CKAKNTP VTLSKDQLSGINNIAF SN SEQ ID NO: 5: amino acid sequence of FL stabilized preF-B protein with RSV A signal peptide (SC variant)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIK

ETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQAANNQARGSGSGRSLGFLLGVGSAI

ASGMAVSKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTSKVLDLKNYINNQILPIVNQQ

SCRIPNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMS

SNV QIVRQQSY SIMSIIKEEVFAYVVQFPIY GVIDTPCWKFHTSPFCTTNIKEGSNICFTRTDRG

WYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSFTFPSEVSFCNTDIFNSKYDCKIMTSKTDIS

S S VITSFGAIV SCY GKTKCTASNKNRGIIKTFSNGCDYV SNKGVDTV SVGNTFYYVNKFEGK

NFYVKGEPIINYYDPFVFPSNEFDASISQVNEKINQSFAFIRRSDEFFHNVNTGKSTTNIMITAII

IVIIVVFFSFIAIGFFFYCKAKNTPVTFSKDQFSGINNIAFSN

SEQ ID NO: 6 (insert Ad26.preF_A)

ATGGAGCTGCTGATCCTGAAGGCCAACGCCATCACCACCATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGA

GGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAAGGGCTACCTGAGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCG

AGCTGAGCAACATCAAAGAAATCAAGTGCAACGGCACCGACGCCAAAGTGAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAACG

CCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCCAGACGCGAGCTGCCCCGGTTCATGAACTACAC

CCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCTTCCTGCTGGGCGTGGGCTCTGCC

ATTGCTAGCGGAGTGGCCGTGTCTAAAGTGCTGCACCTGGAAGGCGAAGTGAACAAGATCAAGAGCGCCCTGCTGAGCACCAACAAGG

CCGTGGTGTCCCTGAGCAACGGCGTGTCCGTGCTGACCAGCAAGGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATC

GTGAACAAGCAGAGCTGCAGCATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGCG

AGTTCAGCGTGAACGCTGGCGTGACCACCCCCGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGTCCCTGATCAATGACATGCCC

ATCACCAACGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGTCCATCATCAAAGAAG

AGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACC

AACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACCGGGGCTGGTACTGCGATAATGCCGGCTCCGTGTCATTCTTTCCACA

AGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGAACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAAC

GTGGACATCTTCAACCCTAAGTACGACTGCAAGATCATGACCrCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATC

GTGTCCTGCTACGGCAAGACCAAGTGCACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGT

CCAACAAGGGGGTGGACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCG AGCCCATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCAACGAGTTCGACGCCAGCATCAGCCAGGTCAACGAGAAGATCAACCAG

AGCCTGGCCTTCATCAGAAAGAGCGACGAGCTGCTGCACAATGTGAATGCCGTGAAGTCCACCACCAATATCATGATCACCACAATCAT

CATCGTGATCATTGTGATCCTGCTGAGCCTGATCGCCGTGGGCCTGCTGCTGTACTGCAAGGCCAGATCCACCCCTGTGACCCTGTCCAA

GGACCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTGATAA

SEQ ID NO: 7 insert Ad26.preF_B processed variant

Nucleotide sequence (from start, including the double stop codon) of Ad26.RSV-B.PreF processed (Ad26RSV021, plasmid ID 7646, Pr-QMPNY -RMR) :

ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACAATCCTGACCGCCGTGACCTTTTGCTTCGCCAGCGGCCAGAAC

ATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAGAGGATATCTGTCTGCCCTGAGAACCGGCTGGTACAC

CAGCGTGATCACCATCGAGCTGAGCAACATCAAAGAAACAAAGTGCAACGGCACCGACACCAAAGTGAAGCTGATCAAG

CAAGAGCTGGACAAGTACAAGAATGCCGTGACCGAACTGCAGCTGCTGATGCAGAATACCCAGGCCGCCAACAACCGGG

CCAGAAGAGAAGCCCCTCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAACGTGTCCATCAGCAAGAAGCGG

AAGCGGAGATTCCTGGGCTTTCTGCTCGGAGTGGGATCTGCCATTGCCTCTGGAATGGCCGTGTCTAAGGTGCTGCATCT

GGAAGGCGAAGTGAACAAGATCAAGAACGCCCTGCAGCTGACCAACAAGGCCGTGGTGTCTCTGTCTAATGGCGTGTCC

GTG CTG ACCAG CAG AGT G CTG G ACCT G AAG AACT AC AT CAACAACCAG CTG CTG CCCAT G GT CAACCG G CAG AG CT G CA

GAATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAGCAGGCTGCTGGAAATCACCCGCGAGTTTTCTGTG

AATGCCGGCGTGACAACCCCTCTGAGCACCTACATGCTGACCAATAGCGAGCTGCTGAGCCTGATCAACGACATGCCCAT

CACCAACG ACCAG AAAAAGCTGATGAGCAGCAACGTGCAGATCGTGCGGCAGCAGAGCTACAGCATCATGAGCATTATC

AAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCTATCTACGGCGTGATCGATACCCCTTGCTGGAAGCTGCACACAAG

CCC ACT GTG CACCACCAAT AT CAAAG AG G G CAG CAACAT CTG CCTG ACCAG AACCG ATAG AG G CTG GT ACT G CG AT AAT G

CCGGCAGCGTCAGCTTCTTCCCACAAGCCGATACCTGCAAGGTGCAGAGCAACAGAGTGTTCTGCGACACCATGAACAGC

CTGACACTGCCTAGCGAGGTGTCCCTGTGCAACACCGACATCTTCAACTCTAAGTACGACTGCAAGATCATGACCTCCAAG

ACCGACATCAGCTCCTCCGTGATCACATCTCTGGGCGCCATCGTGTCCTGCTACGGCAAGACAAAGTGTACCGCCAGCAAC

AAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAAGGCGTGGACACCGTGTCTGTGG

GCAACACCCTGTACTACGTGAACAAGCTGGAAGGCAAGAATCTGTACGTGAAGGGCGAGCCCATCATCAACTACTACGAC

CCTCTGGTGTTCCCCAGCAACGAGTTCTACGCCAGCATCAGCCAAGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCAT

CCGCAGATCCGATGAGCTGCTGCACAACGTGAACACCGGCAAGAGCACCACAAACATCATGATCACCGCCATCATCATCG TG AT CAT CGTCGTGCTGCTGTCCCT G ATCG CCAT CG G ACT G CTG CTGT ACT G CAAG G CCAAG AAC ACCCCT GTG ACACTG A

GCAAGGATCAGCTGAGCGGCATCAACAATATCGC CTT CTCC AACTG AT AA

SEQ ID NO: 8: insert Ad26.preF_B SC variant

Nucleotide sequence (from start, including the double stop codon) of Ad26.RSV-B.PreF single chain (Ad26RSV022, plasmid ID 7647, Sc-QMPN-RMR):

ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACAATCCTGACCGCCGTGACCTTTTGCTTCGCCAGCGGCCAGAAC

ATCACCGAGGAATTCTACCAGAGCACCTGTAGCGCCGTGTCCAGAGGATATCTGTCTGCCCTGAGAACCGGCTGGTACAC

CAGCGTGATCACCATCGAGCTGAGCAACATCAAAGAAACAAAGTGCAACGGCACCGACACCAAAGTGAAGCTGATCAAG

CAAGAGCTGGACAAGTACAAGAATGCCGTGACCGAACTGCAGCTGCTGATGCAGAATACCCAGGCCGCCAACAATCAGG

CCAGAGGCTCTGGATCTGGCAGAAGCCTGGGATTTCTGCTCGGCGTGGGATCTGCCATTGCCTCTGGAATGGCCGTGTCT

AAG GTG CTG C ATCT G G AAG G CG AAGT G AACAAG AT CAAG AACG CCCT G CAG CT G ACCAAC AAG GCCGTGGTGTCTCTGT

CTAATGGCGTGTCCGTGCTGACCAGCAGAGTGCTGGACCTGAAGAACTACATCAACAACCAGCTGCTGCCCATGGTCAAC

CGGCAGAGCTGCAGAATCCCCAACATCGAGACAGTGATCGAGTTCCAGCAGAAGAACAGCAGGCTGCTGGAAATCACCC

GCGAGTTTTCTGTGAATGCCGGCGTGACAACCCCTCTGAGCACCTACATGCTGACCAATAGCGAGCTGCTGAGCCTGATC

AACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAGCAACGTGCAGATCGTGCGGCAGCAGAGCTACAGCA

TCATGAGCATTATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCTATCTACGGCGTGATCGATACCCCTTGCTGG

AAGCTGCACACAAGCCCACTGTGCACCACCAATATCAAAGAGGGCAGCAACATCTGCCTGACCAGAACCGATAGAGGCT

GGTACTGCGATAATGCCGGCAGCGTCAGCTTCTTCCCACAAGCCGATACCTGCAAGGTGCAGAGCAACAGAGTGTTCTGC

GACACCATGAACAGCCTGACACTGCCTAGCGAGGTGTCCCTGTGCAACACCGACATCTTCAACTCTAAGTACGACTGCAA

GATCATGACCTCCAAGACCGACATCAGCTCCTCCGTGATCACATCTCTGGGCGCCATCGTGTCCTGCTACGGCAAGACAAA

GTGTACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAAGGCGTG

GACACCGTGTCTGTGGGCAACACCCTGTACTACGTGAACAAGCTGGAAGGCAAGAACCTGTACGTGAAGGGCGAGCCCA

TCATCAACTACTACGACCCTCTGGTGTTCCCCAGCAACGAGTTCGATGCCAGCATCAGCCAAGTGAACGAGAAGATCAAC

CAGAGCCTGGCCTTCATCAGACGCTCCGATGAGCTGCTGCACAACGTGAACACCGGCAAGAGCACCACAAACATCATGAT

CACCGCCATCATCATCGTGATCATCGTCGTGCTGCTGTCCCTGATCGCCATCGGACTGCTGCTGTACTGCAAGGCCAAGAA

CACCCCTGTGACACTGAGCAAGGATCAGCTGAGCGGCATCAACAATATCGCCTTCTCCAACTGATAA SEQ ID NO: 9 soluble RSV preF_A protein (precursor, i.e. not processed)

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNG TDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFL GFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIV NKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNN VQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNA GSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSC YGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVG NTLYYVNKQEGKSLYVKGEPIINFYDPLVFP SNEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL Signal peptide: double underlined

P27 peptide: underlined and bold, note that protein will be cleaved after RARR, but the C-terminal jjf? of the F2 domain will also be cleaved by endoproteases

SEQ ID NO: 10 soluble RSV preFe protein (precursor, i.e. not processed) MELLILKANAITTILTAVTFCFASGONITEEFYOSTCSAVSRGYLSALRTGWYTSVITIELSNIK ETKCNGTDTKVKLIKOELDKYKNAVTELOLLMONTOAANNRARREAPOYMNYTINTTKN LNVSISKKRKRRFLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALOLTNKAVVSLSNGV SVLTSRVLDLKNYINNQILPMVNRQSCRIPNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTY MLTNSELLSLINDMPITNDQKKLMSSNV QIVRQQSY SIMSIIKEEVLAYVV QLPIY GVIDTPCW KLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPS EV SLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCY GKTKCTASNKNRGIIKTFSNGCDYV SNKGVDTV S V GNTLYYVNKLEGKNLYVKGEPirNYYDPLVFP SNEFY A SIS Q VNEKIN Q SLAF IRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL Signal peptide: double underlined

P27 peptide: underlined and bold, note that protein will be cleaved after RARR, but the C-terminal RR of the F2 domain will also be cleaved by endoproteases

SEQ ID NO: 11 soluble RSV preF_A protein processed QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNGTDAKVKUKQELDKY KNAVTELQLLMQSTPATNNRAFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALL

STNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEIT REFSVNAGVTTPVSTYMLTNSELLSUNDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEE

VLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQ

AETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIV

SCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGE

PIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKD

GEWVLLSTFL

SEQ ID NO: 12: soluble RSV preFe protein processed

QNITEEF Y Q STC S AV SRGYL S ALRTGW YT S VITIELSNIKETKCNGTDTKVKLIKQELD KYKNAVTELQLLMQNTQAANNRAFLGFLLGVGSAIASGMAVSKVLHLEGEVNKIK NALQLTNKAVV SLSNGV S VLTSRVLDLKNYINNQILPMVNRQ SCRIPNIETVIEF QQK N SRLLEITREF S VNAGVTTPLSTYMLTN SELLSLINDMPITNDQKKLMS SNVQIVRQQ S Y SIMSIIKEEVL AYVV QLPI Y GVIDTPCWKLHT SPLCTTNIKEGSNICLTRTDRGW Y C DNAGS V SFFPQ ADTCK V Q SNRVF CDTMN SLTLPSE V SLCNTDIFN SK YDCKIMT SKT DIS SS VITSLGAIVSC Y GKTKCT ASNKNRGIIKTF SNGCD YV SNKGVDT V S VGNTLYY VNKLEGKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFIRRSDELLSAIG GYIPE APRD GQ A YVRKD GEW VLL STFL

SEQ ID NO: 13 nucleotide sequence encoding soluble RSV preF_A protein atggaactgctgatcctgaaggccaacgccatcaccaccatcctgaccgccgtgaccttctectttgccagcggccagaacatcaccgaggagttctacca gagcacctgtagcgccgtgtccaagggctacctgagcgccctgagaaccggctggtacaccagcgtgatcaccatcgagctgagcaacatcaaagaaat caagtgcaacggcaccgacgccaaagtgaagctgatcaagcaggaactggacaagtacaagaatgccgtgaccgaactgcagctgctgatgcagagca cccccgccaccaacaaccgggccagaagagaactgcccagattcatgaactacaccctgaacaacgccaaaaagaccaacgtgaccctgagcaagaa gcggaagcggcggttcctgggctttctgctgggagtgggaagcgccattgctagcggagtggccgtgtctaaggtgctgcacctggaaggcgaagtgaa caagatcaagtccgccctgctgagcaccaacaaggccgtggtgtctctgagcaacggcgtgtccgtgctgaccagcaaggtgctggatctgaagaactac atcgacaaacagctgctgcccatcgtgaacaagcagagctgcagcatccccaacatcgagacagtgatcgagttccagcagaagaacaaccggctgctg gaaatcacccgcgagttcagcgtgaacgctggcgtgaccacccccgtgtccacctacatgctgaccaacagcgagctgctgtccctgatcaacgacatgc ccatcaccaacgaccagaaaaagctgatgagcaacaacgtgcagatcgtgcggcagcagagctactccatcatgagcattatcaaagaagaggtgctgg cctacgtggtgcagctgcctctgtacggcgtgatcgacaccccctgctggaagctgcacaccagccctctgtgcaccaccaacaccaaagagggcagca acatctgcctgacccggaccgacagaggctggtactgcgataatgccggctccgtctcattctttccacaagccgagacatgcaaggtgcagagcaaccg ggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaatctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgacctc caagaccgacgtgtccagctccgtgatcacaagcctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggca tcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtctgtgggcaacaccctgtactacgtgaacaaacaggaagg caagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcaacgagttcgacgccagcatcagccaagtgaacgag aagatcaaccagagcctggccttcatcagaaagtccgatgagctgctgagcgccatcggcggctacatccctgaggcccctagagatggccaggcctatg tgcgga agga cggcga a tgggtgctgctgtcta ccttcctgtga

Signal peptide: double underlined

Antigen: no underline

SEQ ID NO: 14 nucleotide sequence encoding soluble RSV preFe protein

ATGGAGCTGCTGATCCTGAAAGCGAACGCCATCACCACCATTCTGACCGCGGTG

ACCTTTTGCTTTGCGTCTGGCCAGAACATCACCGAGGAGTTTTATCAGTCCACCT

GCTCTGCCGTGAGCAGGGGATACCTGTCCGCCCTGAGGACCGGCTGGTATACATC

CGTGATCACCATCGAGCTGTCTAATATCAAGGAGACAAAGTGTAACGGCACCGA

C AC AAAGGT GAAGCTGAT C AAGC AGGAGCTGGAT AAGT AC AAGA ATGCCGT GAC

AGAGCTGCAGCTGCTGATGCAGAACACCCAGGCCGCCAACAATAGGGCCCGGAG

AGAGGCCCCTCAGTACATGAACTATACCATCAATACCACAAAGAACCTGAATGT

GAGCATCTCCAAGAAGCGCAAGAGGCGCTTCCTGGGCTTTCTGCTGGGAGTGGG

CAGCGCCATCGCATCCGGCATGGCCGTGTCCAAGGTGCTGCACCTGGAGGGCGA

GGTGAACAAGATCAAGAATGCCCTGCAGCTGACAAATAAGGCCGTGGTGTCTCT

GAGC AACGGCGT GTCTGT GCTGACC AGCCGGGT GCTGGACCTGAAGAACT AC AT

CAACAATCAGATCCTGCCTATGGTGAATCGGCAGTCCTGCAGAATCCCCAACATC

GAGACAGTGATCGAGTTCCAGCAGAAGAATTCCCGGCTGCTGGAGATCACCAGA GAGTTTTCTGTGAACGCCGGCGTGACCACACCCCTGAGCACATACATGCTGACCA

ATTCCGAGCTGCTGTCTCTGATCAACGACATGCCTATCACCAATGATCAGAAGAA

GCTGATGTCCTCTAACGTGCAGATCGTGCGCCAGCAGTCCTATTCTATCATGAGC

ATCATCAAGGAGGAGGTGCTGGCCTACGTGGTGCAGCTGCCTATCTATGGCGTGA

TCGACACACCATGCTGGAAGCTGCACACCAGCCCCCTGTGCACCACAAACATCA

AGGAGGGCTCCAATATCTGCCTGACCAGGACAGACCGCGGCTGGTACTGTGATA

ACGCCGGCAGCGTGTCCTTCTTTCCACAGGCCGACACCTGCAAGGTGCAGTCCAA

TCGGGTGTTCTGTGATACAATGAACTCTCTGACCCTGCCCTCCGAGGTGTCTCTGT

GCAACACAGACATCTTTAATTCTAAGTACGATTGTAAGATCATGACCAGCAAGAC

AGATATCAGCTCCTCTGTGATCACCTCTCTGGGCGCCATCGTGAGCTGCTACGGC

AAGACC AAGT GT AC AGCCTCC AAC AAGAAT AGAGGC AT CAT C AAGACCTTC AGC

AATGGCTGTGACTACGTGAGCAACAAGGGCGTGGATACAGTGAGCGTGGGCAAC

ACCCTGTACTATGTGAATAAGCTGGAGGGCAAGAACCTGTACGTGAAGGGCGAG

CCCATCATCAATTACTATGACCCACTGGTGTTCCCCTCTAACGAGTTTTACGCCTC

TATCAGCCAGGTGAACGAGAAGATCAATCAGAGCCTGGCCTTCATCCGGAGAAG

CGATGAGCTGCTGTCCGCCATCGGCGGCTACATCCCAGAGGCACCTAGGGACGG

AC AGGCCT AT GTGAGAAAGGAT GGCGAGTGGGT GCTGCTGAGC ACCTTTCTGT A

G

SEQ ID NO: 15 (5’ terminal nucleotides of recombinant adenovectors)

CTATCTAT

SEQ ID NO: 16 (5’ terminal nucleotides of original adenovectors)

CATCATCA SEQ ID NO: 17: Ad26RSV019 (plasmid ID# PL07642)

ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACAATCCTGACCGCCGTG

ACCTTTTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCT

GTAGCGCCGTGTCCAGAGGATATCTGTCTGCCCTGAGAACCGGCTGGTACACCAG

CGTGATCACCATCGAGCTGAGCAACATCAAAGAAACAAAGTGCAACGGCACCGA

C AC C A A AGT G A AGCTGAT C A AGC A AGAGC T GGAC A AGT AC A AG A AT GC CGT GAC

CGAACTGCAGCTGCTGATGCAGAATACCCAGGCCGCCAACAACCGGGCCAGAAG

AGAAGCCCCTCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAACGT

GTCCATCAGCAAGAAGCGGAAGCGGAGATTCCTGGGCTTTCTGCTCGGAGTGGG

ATCTGCCATTGCCTCTGGAATGGCCGTGTCTAAGGTGCTGCATCTGGAAGGCGAA

GTGAACAAGATCAAGAACGCCCTGCAGCTGACCAACAAGGCCGTGGTGTCTCTG

TCTAATGGCGTGTCCGTGCTGACCAGCAGAGTGCTGGACCTGAAGAACTACATCA

ACAACCAGCTGCTGCCCATGGTCAACCGGCAGAGCTGCAGAATCCCCAACATCG

AGACAGTGATCGAGTTCCAGCAGAAGAACAGCAGGCTGCTGGAAATCACCCGCG

AGTTTTCTGTGAATGCCGGCGTGACAACCCCTCTGAGCACCTACATGCTGACCAA

TAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAA

GCTGAT GAGC AGC AACGT GC AGATCGT GCGGC AGC AGAGCT AC AGC AT CAT GAG

CATTATCAAAGAAGAGGTGCTGGCCTACGTGGTGCAGCTGCCTATCTACGGCGTG

ATCGATACCCCTTGCTGGAAGCTGCACACAAGCCCACTGTGCACCACCAATATCA

AAGAGGGC AGC AAC ATCTGCCTGACC AGAACCGAT AGAGGCTGGT ACTGCGAT A

ATGCCGGCAGCGTCAGCTTCTTCCCACAAGCCGATACCTGCAAGGTGCAGAGCA

ACAGAGTGTTCTGCGACACCATGAACAGCCTGACACTGCCTAGCGAGGTGTCCCT

GTGCAACACCGACATCTTCAACTCTAAGTACGACTGCAAGATCATGACCTCCAAG

ACCGACATCAGCTCCTCCGTGATCACATCTCTGGGCGCCATCGTGTCCTGCTACG

GCAAGACAAAGTGTACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCA GCAACGGCTGCGACTACGTGTCCAACAAAGGCGTGGACACCGTGTCTGTGGGCA

ACACCCTGTACTACGTGAACAAGCTGGAAGGCAAGAATCTGTACGTGAAGGGCG

AGCCCATCATCAACTACTACGACCCTCTGGTGTTCCCCAGCAACGAGTTCTACGC

CAGCATCAGCCAAGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCCGCAG

ATCCGATGAGCTGCTGCACAACGTGAACACCGGCAAGAGCACCACAAACATCAT

GATCACCGCCATCATCATCGTGATCATCGTCGTGCTGCTGTCCCTGATCGCCATC

GGACTGCTGCTGTACTGCAAGGCCAAGAACACCCCTGTGACACTGAGCAAGGAT

CAGCTGAGCGGCATCAACAATATCGCCTTCTCCAAC

SEQ ID NO: 18: protein encoded by SEQ ID NO: 17

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIE LSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQAANNRARREAPQYM NYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGMAVSKVLHLEGEVNKIKNALQ LTNKAVVSLSNGVSVLTSRVLDLKNYINNQLLPMVNRQSCRIPNIETVIEFQQKNSRL LEITREF S VNAGVTTPLST YMLTN SELL SLINDMPITNDQKKLMS SNVQIVRQQ S Y SIM SIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAG S V SFFPQ ADTCK V Q SNRVF CDTMN SLTLP SEV SLCNTDIFN SK YDCKIMT SKTDIS S S V ITSLGAIVSC Y GKTKCTASNKNRGIIKTF SNGCD YV SNKGVDTV S VGNTL YYVNKLE GKNLYVKGEPIINYYDPLVFPSNEFYASISQVNEKINQSLAFIRRSDELLHNVNTGKST TNIMIT AIII VII VVLL SLI AIGLLL Y CK AKNTP VTL SKDQL S GINNI AF SN

SEQ ID NO: 19 Ad26RSV020 (plasmid ID# PL07643)

ATGGAACTGCTGATCCTGAAGGCCAACGCCATCACCACAATCCTGACCGCCGTG

ACCTTTTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCT

GTAGCGCCGTGTCCAGAGGATATCTGTCTGCCCTGAGAACCGGCTGGTACACCAG CGTGATCACCATCGAGCTGAGCAACATCAAAGAAACAAAGTGCAACGGCACCGA

C AC C A A AGT GA AGC T GAT C A AGC A AGAGC T GGAC A AGT AC A AG A AT GC CGT G AC

CGAACTGCAGCTGCTGATGCAGAATACCCAGGCCGCCAACAATCAGGCCAGAGG

CTCTGGATCTGGCAGAAGCCTGGGATTTCTGCTCGGCGTGGGATCTGCCATTGCC

T C T GG A AT GGC C GT GT C T A AGGT GC T GC AT C T GG A AGGC G A AGT G A AC A AG AT C

AAGAACGCCCTGCAGCTGACCAACAAGGCCGTGGTGTCTCTGTCTAATGGCGTGT

CCGTGCTGACCAGCAGAGTGCTGGACCTGAAGAACTACATCAACAACCAGCTGC

TGCCCATGGTCAACCGGCAGAGCTGCAGAATCCCCAACATCGAGACAGTGATCG

AGTTCCAGCAGAAGAACAGCAGGCTGCTGGAAATCACCCGCGAGTTTTCTGTGA

ATGCCGGCGTGACAACCCCTCTGAGCACCTACATGCTGACCAATAGCGAGCTGCT

GAGCCTGATCAACGACATGCCCATCACCAACGACCAGAAAAAGCTGATGAGCAG

C AACGTGC AGATCGT GCGGC AGC AGAGCT AC AGC AT CAT GAGC ATT ATC AAAGA

AGAGGTGCTGGCCTACGTGGTGCAGCTGCCTATCTACGGCGTGATCGATACCCCT

TGCTGGAAGCTGCACACAAGCCCACTGTGCACCACCAATATCAAAGAGGGCAGC

AACATCTGCCTGACCAGAACCGATAGAGGCTGGTACTGCGATAATGCCGGCAGC

GTCAGCTTCTTCCCACAAGCCGATACCTGCAAGGTGCAGAGCAACAGAGTGTTCT

GCGACACCATGAACAGCCTGACACTGCCTAGCGAGGTGTCCCTGTGCAACACCG

ACATCTTCAACTCTAAGTACGACTGCAAGATCATGACCTCCAAGACCGACATCAG

CTCCTCCGTGATCACATCTCTGGGCGCCATCGTGTCCTGCTACGGCAAGACAAAG

TGTACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGC

GACTACGTGTCCAACAAAGGCGTGGACACCGTGTCTGTGGGCAACACCCTGTACT

ACGTGAACAAGCTGGAAGGCAAGAACCTGTACGTGAAGGGCGAGCCCATCATCA

ACTACTACGACCCTCTGGTGTTCCCCAGCAACGAGTTCGATGCCAGCATCAGCCA

AGTGAACGAGAAGATCAACCAGAGCCTGGCCTTCATCAGACGCTCCGATGAGCT

GCTGCACAACGTGAACACCGGCAAGAGCACCACAAACATCATGATCACCGCCAT CATCATCGTGATCATCGTCGTGCTGCTGTCCCTGATCGCCATCGGACTGCTGCTGT

ACTGCAAGGCCAAGAACACCCCTGTGACACTGAGCAAGGATCAGCTGAGCGGCA

TCAACAATATCGCCTTCTCCAAC SEQ ID NO: 20: protein encoded by SEQ ID NO: 19

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIE LSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTQAANNQARGSGSGRSL GFLLGV GS AI ASGMAV SKVLHLEGEVNKIKNALQLTNK AV V SL SNGV S VLT SRVLDL KNYINNQLLPMVNRQSCRIPNIETVIEF QQKN SRLLEITREF SVNAGVTTPLSTYMLTN SELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPC WKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDT MN SLTLP SEV SLCNTDIFN SK YDCKIMTSKTDIS S S VITSLGAIV SC Y GKTKCT ASNKN RGIIKTF SNGCD YV SNKGVDT V S V GNTL YYVNKLEGKNL YVKGEPIINY YDPLVFP S NEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLL L Y CKAKNTP VTL SKDQL SGINNI AF SN

Claims

Claims

1. A method for inducing a protective immune response against respiratory syncytial virus (RSV) A and B infection in a human subject in need thereof, comprising administering to the subject a combination comprising:

(a) a first dose of one or more RSV FA antigen(s); and

(b) a first dose of one or more RSV F_B antigen(s).

2. Method according to claim 1, wherein the RSV FA and RSV F_B antigens are co administered.

3. Method according to claim 1 or 2, wherein the one or more RSV FA antigens comprise a nucleic acid molecule encoding an RSV FA protein, a vector comprising a nucleic acid molecule encoding an RSV FA protein and/or a soluble RSV FA protein.

4. Method according to claim 1, 2 or 3, wherein the one or more RSV F_B antigens comprise a nucleic acid molecule encoding an RSV F_B protein, a vector comprising a nucleic acid molecule encoding an RSV F_B protein and/or a soluble RSV F_B protein.

5. Method according to any one of the preceding claims, wherein the one or more RSV FA antigens comprise a vector comprising a nucleic acid molecule encoding an RSV FA protein and a soluble RSV FA protein.

6. Method according to any one of the preceding claims, wherein the one ore more RSV FB antigens comprise a vector comprising a nucleic acid molecule encoding an RSV FB protein and a soluble RSV FB protein.

7. Method according to any one of the claims 3-6, wherein the RSV FA and FB proteins encoded by the nucleic acid molecules and/or the soluble RSV FA and FB proteins are stabilized in the pre-fusion conformation.

8. Method according to claim 7, wherein the nucleic acid molecule encoding the RSV FA protein comprises the nucleic acid sequence of SEQ ID NO: 6.

9. Method according to claim 7, wherein the nucleic acid molecule encodes an RSV FA protein having an amino acid sequence of SEQ ID NO: 3.

10. Method according to claim 7, wherein the nucleic acid molecule encoding the RSV FB protein comprises the nucleic acid sequence of SEQ ID NO: 7, 8, 17 or 19.

11. Method according to claim 10, wherein the nucleic acid molecule encoding the RSV FB protein comprises the nucleic acid sequence of 17.

12. Method according to claim 7, wherein the nucleic acid molecule encodes an RSV FB protein having an amino acid sequence of SEQ ID NO: 4, 5, 18 or 20.

13. Method according to claim 12, wherein the nucleic acid molecule encodes an RSV FB protein having an amino acid sequence of SEQ ID NO: 18.

14. Method according to any one of the preceding claims claim 3-113, wherein the vector is an adenoviral vector, preferably a replication-incompetent recombinant adenoviral vector of serotype 26.

15. Method according to any one of the preceding claims 3-14, wherein the soluble RSV

FA protein is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 13.

16. Method according to any one of the preceding claims 3-15, wherein the soluble RSV FA protein comprises the amino acid sequence of SEQ ID NO: 9 or 11.

17. Method according to any one of the preceding claims 3-16, wherein the soluble RSV FB protein is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 14.

18. Method according to any one of the preceding claims 3-17, wherein the soluble RSV FB protein comprises the amino acid sequence of SEQ ID NO: 10 or 12.

19. Method according to any one of the preceding claims, further comprising administering to the subject:

(a) a second dose of one or more RSV FA antigen(s); and/or

(b) a second dose of one or more RSV FB antigen(s).

20. Method according to claim 19, wherein the first dose and second dose comprise the same RSV FA and RSV FB antigens.

21. Method according to claim 19, wherein the first dose and the second dose comprise different RSV FA and RSV FB antigens.

22. Method according to claim 19, 20 or 21, wherein the RSV FA and RSV FB antigens are co-administered.

23. Method according to any one of the preceding claims, wherein the protective immune response is characterized by the prevention or reduction of reverse transcriptase polymerase chain reaction (RT PCR)-confirmed RSV A or B-mediated lower respiratory tract disease (LRTD).

24. Method according to any one of the preceding claims, wherein the protective immune response is characterized by an absent or reduced RSV viral load in the nasal track and/or lungs of the subject upon exposure to RSV A or B.

25. Method according to any one of the preceding claims, wherein the protective immune response is characterized by an absent or reduced RSV clinical symptom in the subject upon exposure to RSV A or B.

26. Method according to any one of the preceding claims, wherein the protective immune response is characterized by the presence of neutralizing antibodies and/or a cellular response to RSV A and B.

27. An immunogenic combination, comprising: (a) an effective amount of one or more RSV FA antigen(s), and

(b) an effective amount of one or more RSV FB antigen(s), for inducing a protective immune response against RSV A and B infection in a human subject in need thereof.

28. Immunogenic combination according to claim 25, wherein the first and second immunogen components are for co-administration.