WO2016040556A1 - Virosomes containing respiratory syncytial virus strain line 19 fusion protein and uses thereof - Google Patents

Abstract

The invention provides Respiratory Syncytial Virus (RSV) vaccines and, in particular, RSV virosomes and related compositions and methods.

Description

Virosomes Containing Respiratory Syncytial Virus Strain Line 19 Fusion Protein and Uses Thereof

Field of the Invention

This invention relates to Respiratory Syncytial Virus (RSV) vaccines and, in particular, RSV virosomes and related compositions and methods.

Background of the Invention

Respiratory Syncytial Virus (RSV) is a respiratory tract pathogen that infects the lungs of people of all ages. Although most otherwise healthy people typically recover from RSV infection in 1 to 2 weeks, after suffering only minor symptoms, infection can be particularly severe in infants and older adults, such as those having chronic pulmonary or cardiovascular disease. Indeed, in such people, RSV is a significant cause of hospitalization and even death.

At present, there is no drug approved for the treatment of RSV. Therapy is, in general, supportive, and can include approaches such as hydration with intravenous fluids, humidified oxygen, mechanical ventilation, and nebulized bronchodilators. Medical approaches to preventing RSV infection at this time are limited to the use of Palivizumab (Synagis®), an IgG monoclonal antibody against the F glycoprotein of RSV. Use of Palivizumab is limited to infants that are at high risk because of prematurity or other medical problems, such as congenital heart disease.

There is no licensed vaccine against RSV. The development of RSV vaccines based on killed virus and subunit proteins has been hindered by issues relating to sufficiency of immunogenicity, as well as the induction of enhanced respiratory disease (ERD). With respect to the latter, an early developed RSV vaccine, including formalin-inactivated RSV and alum adjuvant, resulted in ERD severe enough to require hospitalization in an unusually high percentage of vaccinated infants upon subsequent natural RSV exposure. Use of this vaccine also caused two deaths. The development of live attenuated and recombinant virus vaccines against RSV has been met with challenges including, for example, over- attenuation and difficulty in propagation, and includes as a potentially confounding factor the possiblity of reversion.

In view of the potential severity of RSV infection in certain patients, as well as limitations on the use of Palivizumab, there is a need for a vaccine against RSV infection.

Summary of the Invention

The invention provides virosomes including a fusion (F) protein of Respiratory Syncytial Virus (RSV) strain Line 19 (e.g., a protein having the amino acid sequence of SEQ ID NO:1 , or a protein with greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a protein having the amino acid sequence of SEQ ID NO:1 ). The F protein can have, e.g., one or more substitutions relative to the sequence of SEQ ID NO:1 . Exemplary substitutions include a substitution at I557 of, e.g., SEQ ID NO:1 (e.g., an I557V substitution).

Additionally, or alternatively, the F protein can have a substitution in one or more (e.g., two, three, or four) of the following positions: 79, 191 , 357, and 371 (e.g., M79I, R191 K, K357T, and Y371 N), as compared to, e.g., SEQ ID NO:1 . In certain embodiments, the F protein includes one or more (e.g., two or more, or three or more) of the following amino acids: methionine at residue 79, arginine at residue 191 , lysine at residue 357, and tyrosine at residue 371 . Additionally, or alternatively, the F protein can include one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the following amino acids: proline at residue 4, alanine at residue 16, serine at residue 25, valine at residue 76, alanine at residue 103, threonine at residue 122, isoleucine at residue 152, arginine at residue 213, asparagine at residue 515, and glycine at residue 519. In all of the foregoing embodiments, the F protein does not have the complete sequence of the RSV A2 strain F protein.

The virosomes can further include a G protein, a small hydrophobic (SH) protein, and/or a matrix (M) protein of RSV. The virosomes of the invention can also include lipids and proteins extracted from the membrane of an RSV strain (e.g., a chimeric RSV strain including an RSV strain (e.g., RSV strain A2) in which the F protein is replaced with an RSV Line 19 F protein as described herein).

The F protein, as present in the virosomes of the invention, can be substantially in the pre-fusion conformation. For example, the 5C4/Palivizumab ratio of the virosome as calculated by division of the slopes of the curves can be at least 20% greater (e.g., at least 25%, 30%, 35%, 40%, 45%, 50%, or greater) than the 5C4/Palivizumab ratio of a virosome generated from an RSV A2 strain, as calculated by division of the slopes of the curves.

The virosomes of the invention can also include an adjuvant such as, e.g., a saponin, PHAD

(phosphorylated hexaacyl disaccharide), 3-D-PHAD (3-O-desacyl derivative of phosphorylated hexaacyl disaccharide), 3-O-D MPLA (3-O-desacyl derivative of monophosphoryl lipid A), or MPLA

(monophosphoryl lipid A).

Additionally, or alternatively, the virosomes can further include one or more additional lipids. For example, the virosomes can include a phospholipid such as, for example, a phosphatidylcholine (PC) species (e.g., one or more synthetic or essentially pure PC species) and/or a phosphatidylethanolamine (PE) species (e.g., one or more synthetic or essentially pure PE species). In certain embodiments, the at least one synthetic or essentially pure PC species and the at least one synthetic or essentially pure PE species are the only non-viral phospholipids in the virosomes. Furthermore, the virosomes can optionally include a sterol or sterol derivative (e.g., cholesterol at a ratio of 0-30 mol% (e.g., 5-30 mol%, 10-25 mol%, or about 20 mol%) of total added phospholipid).

The PC species and PE species can be, e.g., at a molar ratio of 3:1 to 1 :3 (e.g., between 2:1 to 1 :2, 3:1 to 1 :1 , 2:1 to 1 :1 , 1 :1 to 1 :3, or 1 :1 to 1 :2) and/or can contain acyl chains with unsaturated bonds (e.g., each of the acyl chains of PC and PE all contain one unsaturated bond or the total number of unsaturated bonds in the acyl chains can be four). Additionally, or alternatively, the acyl chains can have between 14 and 18 carbon atoms (e.g., 16 or 18 carbon atoms).

In certain embodiments, the virosomes of the invention can include one or more (e.g., two or more) of the following: synthetic 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dioleoyl-sn- glycero-3-phosphoetanolamine (DOPE), 1 ,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (PPPE), and 1 -palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (PLPE). In these embodiments, the virosomes of the invention can include, e.g., about 400-450 nmol DOPC, about 800-900 nmol DOPE, about 250-350 nmol 3-D-PHAD, and about 200-300 nmol cholesterol per mg of viral membrane protein.

In virosomes of the invention that include an adjuvant (e.g., a synthetic adjuvant), the adjuvant can be present at a ratio of 0.01 -2 mg of adjuvant per mg of viral protein (e.g., at a ratio of 0.1 -2 mg per mg of viral protein, a ratio of 0.5-2 mg per mg of viral protein, or at about 1 mg per mg of viral protein). The molar ratio of total synthetic phospholipid to adjuvant can be, e.g., between 3 and 6 or between 3.5 and 5. In certain embodiments, the molar ratio of total synthetic phospholipid to adjuvant is between 1 .5 and 10.

In any of the foregoing embodiments of the invention, the virosomes can have, e.g., a narrow size distribution with a modal diameter of between 55-90 nm with less than 15% having a particle size above 150 nm, and less than 15% below 50 nm.

In another aspect, the invention provides pharmaceutical compositions (e.g., vaccines) containing any of the virosomes of the invention (e.g., a composition also including a pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant).

The invention also provides methods of inducing an immune response to RSV in a subject by administering a pharmaceutical composition of the invention to a subject (e.g., a subject that does not have, but is at risk of developing, RSV infection). In certain embodiments, the subject can be a human subject.

In another aspect, the invention provides methods of making virosomes of the invention. These methods include (i) solubilizing the viral envelope of an RSV strain including the F protein of RSV strain Line 19 (e.g., a chimeric RSV strain including RSV strain A2 in which the A2 F protein is replaced with the RSV Line 19 F protein), and (ii) reconstituting the viral envelope in the absence of viral nucleic acid.

In yet another aspect, the invention features virosomes of the invention for use as medicaments, for inducing an immune response to RSV, for use in methods of preventing or treating RSV infection, and/or for vaccinating a subject against RSV.

By "fusion (F) protein of Respiratory Syncytial Virus (RSV) strain Line 19 (RSV Line 19 F protein,

RSV L19 F protein, or simply L19F)" is meant a protein having the sequence of SEQ ID NO:1 or substantial amino acid sequence identity (e.g., greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity) to the sequence of SEQ ID NO:1 (but not having the sequence of the RSV A2 F protein). An RSV L19 F protein can optionally include one or more substitution selected from, for example, the group consisting of: M79I, R191 K, K357T, Y371 N, and I557V (provided that it does not have the complete sequence of the RSV A2 F protein).

It is well understood that, for example, a few residues at the N-terminus might be lost during a purification process unexpectedly, and, therefore, RSV L19 F protein also includes such truncated forms, The term "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST - 2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, for a reference polypeptide of sequence A, when compared to a variant polypeptide of sequence B, the percent amino acid sequence identity is calculated as: 100 times the fraction X/Y, where X is the number of amino acid sequence residues scored as identical matches between A and B, and where Y is the total number of amino acid residues in the polypeptide sequence of B. By "conservative amino acid substitution," as used herein, is meant replacement, in an amino acid sequence, of one amino acid with another amino acid of the same family of amino acids, as based on the chemical nature of their side chains. Genetically encoded amino acids can be divided into four families: acidic (aspartate, glutamate); basic (lysine, arginine, histidine); nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes grouped as aromatic amino acids. In similar fashion, the amino acids can also be separated into the following groups: acidic (aspartate, glutamate); basic (lysine, arginine, histidine); alipathic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally grouped separately as alipathic-hydroxyl; aromatic (phenylalanine, tyrosine, tryptophan); amide (asparagine, glutamine); and sulfur-containing (cysteine, methionine).

The invention provides several advantages. For example, the virosomes of the invention, including the RSV L19 F protein, induce higher levels of neutralizing antibodies, as compared to virosomes including the RSV A2 F protein. This increase in neutralizing titers is important from a clinical perspective, as such an increase in response to vaccination is associated with a decreased likelihood of RSV-associated acute respiratory illness (see, e.g., Falsey et al., J. Infect. Dis. 198:1317-1326, 2008). In addition, virosomes of the invention exhibit increased stability, as described herein, which is useful, e.g., in the context of consideration of the shelf life of pharmaceutical compositions including the virosomes. Use of virosomes, generally, provides other advantages. For example, the production of virosomes does not involve the use of chemicals (e.g., formalin) that could possibly modify protective epitopes, resulting in decreased immunogenicity and, possibly, ERD (see above).

Other features and advantages of the invention will be apparent from the following detailed description, the examples, the drawings, and the claims. Brief Description of the Drawings

Figure 1 is a panel of three graphs showing the size distribution of virosomes produced from the three indicated viral strains as measured by the concentration of particles (particles/ml) for particle sizes ranging from 0-400 nm. The size distributions for RSV A2 (top), RSV A2 L19F (middle), and RSV A2 L19F I557V mutant (bottom) virosomes are indicated.

Figure 2 is a panel of three graphs showing the results of equilibrium density gradient centrifugation as measured by the density (g/ml; triangles), protein concentration ^g/ml; squares), and phosphate concentration (nmol/ml; circles) for virosomes derived from the three indicated virus strains for each of the indicated centrifugation fractions (Fr). The virosomes are derived from RSV A2 (top), RSV A2 L19F (middle), and RSV A2 L19F I557V mutant (bottom).

Figure 3 is graph showing RSV titer as calculated by the log₁₀ transformed TCID₅₀ per milliliter

(ml) of homogenized lung tissue for control (HNE), A2 virosomes, A2 L19F virosomes, and A2 L19F I557V virosome-infected mice.

Figure 4 is a graph showing the log₂ transformed in vitro neutralizing titers calculated from the serum of control (HNE), A2 virosome, A2 L19F virosome, and A2 L19F I557V virosome-infected mice two weeks after the first vaccination. Figure 5 is a graph showing the log₂ transformed in vitro neutralizing titers calculated from the serum of control (HNE), A2 virosome, A2 L19F virosome, and A2 L19F I557V virosome-infected mice two weeks after the second vaccination.

Figure 6 is a graph showing the log₁₀ transformed geometric mean titers (GMT) of RSV-specific IgG antibody detected in serum by ELISA for control (HNE), A2 virosome, A2 L19F virosome, and A2 L19F I557V virosome-infected mice two weeks after the second vaccination.

Figure 7 is a graph showing concentration of the lgG1 isotype given in μg/ml of serum collected from control (HNE), A2 virosome, A2 L19F virosome, and A2 L19F I557V virosome-infected mice two weeks after the second vaccination.

Figure 8 is a graph showing concentration of the lgG2A isotype given in μg/ml of serum collected from control (HNE), A2 virosome, A2 L19F virosome, and A2 L19F I557V virosome-infected mice two weeks after the second vaccination.

Figure 9 is a graph showing the weight in grams of control (HNE), A2 virosome, A2 L19F virosome, and A2 L19F I557V virosome-infected mice on study days 0 (first vaccination), 14 (+1 day post- second vaccination), 30/31 , and 35.

Figure 10 is a graph showing the results of an ELISA where the RSV strain A2 F protein (A2), RSV A2 Line19 F protein (19F), and RSV A2 mutant I557V Line19 F protein (557) are detected by the Synagis® antibody with increasing viral concentration ^g/ml), as measured by absorbance at 492 nm.

Figure 1 1 is a graph showing the results of an ELISA where the RSV strain A2 F protein (A2), RSV A2 Line19 F protein (19F), and RSV A2 mutant I557V Line19 F protein (557) are detected by the 5C4 antibody with increasing viral concentration ^g/ml), as measured by absorbance at 492 nm.

Figure 12 is a graph showing the results of an ELISA where the RSV strain A2 F protein (A2), RSV A2 Line19 F protein (19F), and RSV A2 mutant I557V Line19 F protein (557) are detected by the 5C4 antibody with increasing virosomal concentration ^g/ml), as measured by absorbance at 492 nm.

Figure 13 is a graph showing the results of an ELISA where the RSV strain A2 F protein (A2),

RSV A2 Line19 F protein (19F), and RSV A2 mutant I557V Line19 F protein (557) are detected by the Synagis® antibody with increasing virosomal concentration ^g/ml), as measured by absorbance at 492 nm.

Detailed Description of the Invention

The invention provides virosomes including the fusion (F) protein of Respiratory Syncytial Virus (RSV) strain Line 19 (L19 F protein), pharmaceutical compositions including these virosomes, and methods of using and making such virosomes and compositions. Optionally, the RSV L19 F protein of the virosomes of the invention includes one or more substitutions (e.g., I557V), as is explained further below. The virosomes, compositions, and methods of the invention are described further, as follows, after a brief description of the RSV structural proteins.

Respiratory Syncytial Virus (RSV) Structural Proteins

RSV includes four structural proteins in or associated with the membrane, the fusion (F), G, small hydrophobic (SH), and matrix (M) proteins. Embodiments of the present invention include virosomes comprising the RSV Line 19 F protein, optionally in combination with one or more of RSV G, SH, and M proteins (as well as lipid components, which are discussed below). The RSV G, SH, and/or M proteins can be those of RSV strain Line 19, or one or more of these can be from a different RSV strain, such as the RSV A2 strain. With respect to the latter case, the virosomes can be made from a chimeric RSV strain, such as an RSV A2 virus in which the A2 F sequences have been replaced with RSV Line 19 F sequences. An example of such a chimera is described by Moore et al., J. Virol. 83(9) :4185-4194, 2009.

The sequence of the RSV L19 F protein of EMBL FJ614814.1 is as follows:

1 MELPILKANA ITTILAAVTF CFASSQNITE EFYQSTCSAV

41 SKGYLSALRT GWYTSVITIE LSNIKKNKCN GTDAKVKLMK

81 QELDKYKNAV TELQLLMQST PAANNRARRE LPRFMNYTLN

121 NTKKTNVTLS KKRKRRFLGF LLGVGSAIAS GIAVSKVLHL

161 EGEVNKIKSA LLSTNKAVVS LSNGVSVLTS RVLDLKNYID

201 KQLLPIVNKQ SCRISNIETV IEFQQKNNRL LEITREFSVN

241 AGVTTPVSTY MLTNSELLSL I DMP ITNDQ KKLMSNNVQI

281 VRQQSYSIMS I IKEEVLAYV VQLPLYGVID TPCWKLHTSP

321 LCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPQAEKCKV

361 QSNRVFCDTM YSLTLPSEVN LCNVDIFNPK YDCKIMTSKT

401 DVSSSVITSL GAIVSCYGKT KCTASNKNRG I IKTFSNGCD

441 YVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPI INFYDP

481 LVFPSDEFDA SISQVNEKIN QSLAFIRKSD ELLHNVNAGK

521 STTNIMITTI IIVIIVILLS LIAVGLLLYC KARSTPITLS

561 KDQLSGINNI AFSN (SEQ ID NO : 1 )

As noted above, the RSV L19 F protein, as included in virosomes of the invention, can optionally include one or more substitution mutations. The sequence of SEQ ID NO:1 differs from that of the F protein of RSV strains A2 and Long in five amino acid positions (79, 191 , 357, 371 , and 557; noted in the sequence set forth above by underlining and boldface type; Moore et al., JVI 2009). The invention includes the use of RSV L19 F (in virosomes as described herein) including substitution of one or more of these amino acids with the corresponding amino acid present in the complete RSV A2 strain, or a conservative substitution (provided that the resulting F protein sequence is not that of the F protein of RSV strain A2). Thus, the invention includes the use of RSV L19 F with a substitution of isoleucine at position 557 with valine (I557V) or, e.g., alanine, leucine, proline, phenylalanine, methionine, or tryptophan; a substitution of methionine at position 79 with isoleucine (M79I) or, e.g., alanine, valine, leucine, proline, phenylalanine, or tryptophan; a substitution of arginine at position 191 with lysine (R191 K) or, e.g., histidine, serine, asparagine, or glutamine; a substitution of lysine at position 357 with threonine (K357T) or, e.g., arginine or histidine; and/or a substitution of tyrosine at position 371 with asparagine (Y371 N) or, e.g., glycine, glutamine, cysteine, serine, threonine, phenylalanine, or tryptophan. The RSV L19 F protein can optionally include a substitution in one, two, three, four, or all five of these positions, in any combination, provided that the resulting F protein sequence is not that of the complete F protein of RSV strain A2. The mutations can be made using methods that are known in the art including, for example, the bacterial artificial chromosome (BAC)-based method described by Hotard et al., Virology 434(1 ):129-136, 2012.

In addition to the substitution mutants noted above, the invention also includes the use of RSV L19 F proteins having sequences that have substantial amino acid sequence identity to the sequence set forth above (SEQ ID NO:1 ), with or without the indicated substitutions, provided that the sequence is not that of the complete RSV A2 F protein. Thus, for example, an RSV L19 F protein used in the virosomes of the invention can have greater than 80% amino acid sequence identity (e.g., greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or greater sequence identity) to the sequence set forth above (SEQ ID NO:1 ; with or without the indicated substitutions), provided that the sequence is not that of the complete RSV A2 F protein. The selection of loci tolerant to sequence variability can be carried out by consideration of, e.g., known structure and sequence information of different strains (e.g., conservation between strains; see, e.g., Johnson et al., J. Gen. Virol. 69:2623-2628, 1988), as can be carried out by those of skill in the art. In addition, maintenance of fusion capability can be determined by methods known in the art (see, e.g., below)

As noted above, the improved effects of the virosomes of the invention may be due, at least in part, to increased stability of the pre-fusion conformation of the RSV L19 F protein. Thus, in particular embodiments of the invention, variant RSV L19 F proteins, as described above, maintain increased or substantial pre-fusion conformation, as compared to, for example, that of the RSV A2 F protein.

Maintaining increased pre-fusion conformation means maintenance of, for example, at least 20% (e.g., at least 25%, 30%, 35%, 40%, 45%, 50%, or more) of pre-fusion conformation of F protein, when compared to RSV A2 F-containing virosomes, in an assay as described herein.

Monoclonal antibodies against an epitope unique to RSV-F in the pre-fusion form, such as 5C4,

AM22, and D25, have been found and proven instrumental in the determination of the pre-fusion conformation (McLellan, J.S. et al. Science 2013, May 31 ; 340 (6136):1 1 13). Other antibodies are capable of detecting only the post-fusion conformation or epitopes that do not change in response to the conformational change. RSV-F can be triggered to undergo the conformational change to the post-fusion conformation by exposure to low osmolarity or high temperature. Synagis® (Palivizumab; Medimmune) is a humanized mouse monoclonal antibody that recognizes a conformation-independent epitope present on the F protein of most RSV strains. 5C4 is a mouse monoclonal antibody that specifically recognizes the pre-fusion conformation of the RSV F protein. The ratio of pre-fusion vs. post-fusion, or the ratio of pre-fusion vs. constant epitope, can be used to quantify the pre-fusion conformation quantitatively. These ratios can be found through the comparison of ELISA assay results as described below. For example, the protein can exhibit a 5C4/Palivizumab ratio of greater than 1 when determined according to the method described in Example 2, below, as calculated by division of the slopes of the curves. Fusion can also be measured directly in a fusion assay; the virus is labeled with a self-quenching concentration of the fluorescent dye octadecylrhodamine, and fusion of the virus with cells is quantitated by measuring fluorescence dequenching (Srinivasakuma, N. et al. J. Virol. 65 (1991 ) 4063-69).

Other RSV structural proteins that can be included in the virosomes of the invention (together with RSV L19 F, as described herein) include RSV G, SH, and/or M proteins. These proteins can be from RSV strain Line 19 or from another RSV strain (e.g., RSV strain A2, strain A, Long strain, or a B strain). In the latter case, in which the non-F proteins are from a non-Line 19 RSV strain, the virosomes of the invention can be generated from a chimeric RSV strain (e.g., RSV A2 L19 F), as described above. In one specific example, a virosome of the invention is made from chimeric RSV A2 L19 F, with the F protein including an I557V substitution mutation (i.e., RSV A2 L19 F I557V). Furthermore, in addition to naturally occurring sequences, non-F structural proteins included in the virosomes of the invention can have sequences with substantial identity to naturally occurring sequences (see, e.g., below), as defined above.

Exemplary sequences of RSV A2 proteins that can be included in the virosomes of the invention, together with RSV Line 19 F proteins as described herein, are provided as follows.

RSV A2 G protein (UniprotKB P03423): 1 MSKNKDQRTA KTLERTWDTL NHLLFI SSCL YKLNLKSVAQ

41 ITLSILAMII STSLIIAAII FIASANHKVT PTTAI IQDAT

81 SQIKNTTPTY LTQNPQLGIS PSNPSEITSQ ITTILASTTP

121 GVKSTLQSTT VKTKNTTTTQ TQPSKPTTKQ RQNKPPSKPN

161 NDFHFEVFNF VPCSICSNNP TCWAICKRIP NKKPGKKTTT

201 KPTKKPTLKT TKKDPKPQTT KSKEVPTTKP TEEPTINTTK

241 TNIITTLLTS NTTGNPELTS QMETFHSTSS EGNPSPSQVS

281 TTSEYPSQPS SPPNTPRQ (SEQ ID NO : 2 ) RSV A2 SH protein (UniprotKB P04852)

1 MENTSITIEF SSKFWPYFTL IHMITTIISL LIIISIMIAI

41 LNKLCEYNVF HNKTFELPRA RVNT (SEQ ID NO : 3 )

RSV A2 M protein (UniprotKB P03419)

1 METYVNKLHE GSTYTAAVQY NVLEKDDDPA SLTIWVPMFQ

41 SSMPADLLIK ELANVNILVK QISTPKGPSL RVMINSRSAV

81 LAQMPSKFTI CANVSLDERS KLAYDVTTPC EIKACSLTCL

121 KSKNMLTTVK DLTMKTLNPT HDIIALCEFE NIVTSKKVII

161 PTYLRSISVR NKDLNTLENI TTTEFKNAIT NAKI IPYSGL

201 LLVITVTDNK GAFKYIKPQS QFIVDLGAYL EKESIYYVTT

241 NWKHTATRFA IKPMED (SEQ ID NO : 4 )

Virosomes

In some embodiments, virosomes are lipid bilayers containing viral surface proteins (e.g., glycoproteins and surface proteins from enveloped viruses, or truncations or fragments thereof).

Virosomes can thus, in general, be considered as reconstituted membranes of enveloped viruses.

Virosomes can be produced by extraction of membrane proteins and lipids from enveloped viruses with a detergent (e.g., C12E8, Triton X-100, or n-octyl^-D-glucopyranoside), or a short-chain phospholipid (e.g., phosphatidylcholine or a derivative thereof, e.g., 1 ,2-diheptanoyl-sn-phosphatidylcholine (DHPC) or 1 ,2- dicaproyl-sn-phosphatidylcholine (DCPC)). Typically, viral nucleocapsid is removed from extracted proteins and lipids by centrifugation. Optionally, additional components (e.g., adjuvants, additional lipids, and/or cholesterol; optionally dissolved in a solution including the detergent or short-chain phospholipid) are mixed with the extracted proteins and lipids. The detergent or short-chain phospholipid is then removed by, for example, dialysis, thus permitting reconstitution and reforming of the lipid bilayer envelope. This results in the formation of virosomes including the extracted membrane proteins and lipids, as well as any of the additional components noted above.

Details of methods for making virosomes that can be used in the present invention are described in WO 2004/071492; Stegmann et al., EMBO J. 6:2651 -2659, 1987; and Kamphuis et al., Plos One 7(5):e36812, 1 -1 1 , 2012, each of which is incorporated herein by reference in its entirety. In one specific example, in brief summary, purified virus is pelleted by ultracentrifugation and dissolved in buffer including 1 ,2-dicaproyl-sn-phosphatidylcholine (DCPC). Nucleocapsid is then removed by

ultracentrifugation. Subsequently, egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE) in, e.g., a 2:1 molar mixture, is evaporated to a dry film in a glass tube. The supernatant, including the extracted membrane lipids and proteins, is then added to the lipid mixture. Optionally, an adjuvant or other component (e.g., further lipids or cholesterol) is dissolved in DCPC and added to the protein/lipid mixture, which after incubation is filtered and dialyzed to remove the DCPC, leading to the formation of virosomes (Kamphuis et al., supra).

In addition to the specific methods described above, virosomes can be assembled, in general, from any integral membrane protein or peripheral membrane protein, or proteins conjugated to lipid anchors. Central features of virosomes include that they are particles of a size that is efficiently taken up by phagocytic cells of the immune system, and that they closely mimic the composition, surface architecture, and functional activities (particularly the membrane fusion activity) of native viral envelopes. Lipids and integral membrane proteins from a virus used to make a virosome will thus, typically, be present in a virosomal membrane.

The virosomes of the present invention, including RSV Line 19 F protein (as described herein), will typically include the lipids of the virus from which they are obtained (e.g., RSV L19, RSV A2 L19 F, or RSV A2 L19 F I557V). In addition to those lipids, the virosomes can optionally incorporate other components such as, for example, amphiphilic adjuvants (see, e.g., WO 2004/1 10486), additional lipid species (e.g., a phosphatidylcholine (PC) species and/or a phosphatidylethanolamine (PE) species), and/or sterol or a derivative thereof. To incorporate these additional components into a virosome of the invention, and consistent with the description set forth above, virus is solubilized with a detergent or short- chain phospholipid, viral nucleocapsid is removed, and then the additional component, optionally dissolved in the same detergent or short-chain phospholipid, is added to the solubilized viral membranes. The detergent or short-chain phospholipid is then removed, resulting in the formation of virosomes that include the viral membrane proteins and lipids, as well as the additional component(s).

Examples of adjuvants that can be incorporated into virosomes in this manner include MPLA (monophosphoryl lipid A) and MPLA derivatives (e.g., 3'-0-desacyl MPLA, 3D MPL™(3'-0-desacyl monophosphoryl lipid A), synthetic versions of MPLA and MPLA derivatives (e.g., phosphorylated hexa acyl disaccharide (PHAD, which is also known as glycopyranoside lipid A or GLA) and the 3'-0-desacyl derivative of PHAD (3-D-PHAD)), saponins (e.g., QS21 ), lipidated imidazoquinolines (see, e.g., WO 2010/048520 and US 201 1/0282061 ), lipidated stimulating peptides (e.g., Pam₃CSK₄, and L-18 muramyldipeptide (L18-MDP)), and vitamin D and derivatives of vitamin D. Use of MPLA in this context is described, for example, in WO 2004/1 10486; Stegmann et al., supra; and Kamphuis et al., supra. The use of Pam₃CSK₄, and L18-MDP in the context of virosomes is described by Shafique et al., PlosOne 8(4):e61287, 1 -12, 2013.

Functionally reconstituted viral membranes of the invention may comprise purified lipids from other sources, e.g., purified or synthetic lipids, in addition to the viral lipids. The other lipids can be added to the virosome membranes during preparation. Fusion activity of the virosomes is generally optimally maintained when lipids similar to those of viral origin or lipid mixtures which closely resemble the lipid composition of the viral envelope are added. Thus, in line with the present invention, a broad range of lipids can be comprised in the virosomal membrane. The group of lipids comprises neutral and charged phospholipids, steroid-derived lipids, and neutral and charged synthetic lipids. A lipid composition for the provision of the virosomes with fusion activity can thus be a composition that is obtained or obtainable from natural viral membranes. Lipid compositions for use in the present invention thus include compositions exclusively composed of natural lipids of a virus, compositions composed of natural lipids of a virus supplemented with lipids from other sources, as well as compositions composed of lipids from various sources, which mimic the lipid composition of a natural viral membrane. A virosome of the present invention may comprise one or more lipids selected from the group consisting of cationic lipids, synthetic lipids, glycolipids, phospholipids, sterols, and derivatives thereof. Preferred lipids include sterols, such as cholesterol, and phospholipids, such as phosphatidylcholine (PC), sphingomyelin (SPM), phosphatidylethanolamine (PE), and phosphatidylserine (PS). However, other phospholipids may also be added. These include, but are not limited to, phosphatidylglycerol (PG), phosphatidic acid (PA), cardiolipin (CL), and phosphatidylinositol (PI), with varying fatty acyl compositions and of natural and/or (semi) synthetic origin, and dicetyl phosphate. Ceramide and various glycolipids, such as cerebrosides or gangliosides, may also be added. Examples of sterol derivatives that can be incorporated into virosomes of the invention include cholesterol hemisuccinate, phytosterols such as lanosterol, ergosterol, and vitamin D and vitamin D related compounds.

In various embodiments, the virosomes of the invention have modal diameters that are significantly smaller than 220 nm, to facilitate filter sterilization. Virosomes of the invention thus may have diameters (particle size) in the range of 40 to 200 nm, e.g., from 50 nm to 150 nm, or 70 nm to 130 nm. In various examples, the RSV virosomes have a homogeneous size distribution with less than 10 to 15% of the virosomes having a particle size above 150 nm, and less than 10 to 15% having a particle size below 50 nm. The modal diameters thus can be, in various examples, below 90 nm or below 85 nm. For example, the modal diameters can be in the range of 55-90 nm, 58-82 nm, 59-80 nm, 65-80 nm, 65-77 nm, 68-75 nm, 68-78 nm, or 69-74 nm.

The remainder of this section describes a virosome composition that includes particular components, including adjuvants, lipids, and cholesterol, in particular amounts. This virosome composition can be used in the context of RSV virosomes including the F protein of RSV Line 19 (optionally including a substitution as described herein (e.g., I557V; also see above)) and optionally made from a chimeric RSV including RSV A2 in which F sequences are replaced with RSV Line 19 F sequences (optionally including a substitution mutation as described herein; e.g., RSV A2 L19 F I557V)). Use of this particular type of virosome composition outside of the context of the RSV L19 F protein- containing virosomes, as described herein, is not included in this invention.

A virosome according to this example comprises: (i) lipids and proteins extracted from the membrane of RSV (with the F protein being RSV Line 19 F, as described above); (ii) a synthetic adjuvant chosen from the group of PHAD and 3-D-PHAD, preferably at a ratio of 0.01 -2 mg of adjuvant per mg of viral protein; (iii) at least one synthetic or essentially pure phosphatidylcholine (PC) species and at least one synthetic or essentially pure phosphatidylethanolamine (PE) at a molar ratio of 3:1 to 1 :3, characterized in that the acyl chains have between 14 and 18 carbon atoms, the total number of unsaturated bonds in the acyl chains being four, and the molar ratio of total synthetic phospholipid to adjuvant is between 1 .5 and 10; and (iv) a sterol or sterol derivative at a ratio of 0-30 mol% of total added phospholipid.

As used in this example, the expression "synthetic or essentially pure" refers to an exogenously added, non-viral phospholipid species of defined quality, purity, and chemical structure. It does not refer to purified or semi-purified phospholipids of mixed fatty acid composition that are extracted from natural sources, such as tissue-derived, plant-derived, or egg-derived PC and PE. In one embodiment of this example, the synthetic or essentially pure PC and PE species are manufactured according to the guidelines of Good Manufacturing Practice (cGMP). Synthetic or essentially pure PC and PE species are commercially available, for example, from Avanti Polar Lipids, Alabaster, AL. In one embodiment, said at least one synthetic or essentially pure PC species and said at least one synthetic or essentially pure PE species are the only non-viral phospholipids in said virosome.

RSV virosomes with desirable properties were obtained according to this example when the acyl chains of synthetic PE and PC have between 14 and 18 carbon atoms and the total number of unsaturated bonds in the acyl chains of is four. Preferably, both PC and PE contain acyl chains with unsaturated bonds. The acyl chains can be mono- or di-unsaturated. In view of their reduced susceptibility for oxidation, mono-unsaturated acyl chains are preferred. For example, provided is a RSV virosome (having an RSV Line 19 F protein, as described herein) wherein each of the acyl chains of PC and PE all contain one unsaturated bond. In one embodiment, the PC and/or the PE species is a symmetric phospholipid, i.e., comprising identical acyl chains at the sn-1 and sn-2 position of the glycerol backbone. Whereas blends of two or more distinct synthetic PC and/or PE species are envisaged, an RSV virosome (including RSV Line 19 F protein, as described herein) according to this example of the invention typically comprises one synthetic or essentially pure PC species and one synthetic or essentially pure PE species.

In one embodiment, the acyl chains in the synthetic PC and/or PE have 16 or 18 carbon atoms, preferably 18 carbon atoms. Preferably, the total number of carbon atoms in the acyl chains of PC and PE is at least 70. Very good results are obtained if the total number of carbon atoms is 72.

Good results were obtained with RSV virosomes comprising one or more selected from the group consisting of synthetic 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dioleoyl-sn-glycero-3- phosphoetanolamine (DOPE), 1 ,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (PPPE), and 1 - palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (PLPE). Preferably, provided is a RSV virosome (including RSV Line 19 F protein, as described herein), comprising two or more selected from the group consisting of synthetic DOPC, DOPE, PLPE, and PLPC.

In a specific aspect, the RSV virosome (including RSV Line 19 F protein, as described herein) comprises synthetic PC consisting of synthetic DOPC and synthetic PE consisting of DOPE.

An improved RSV virosome (including RSV Line 19 F protein, as described herein) of this example of the invention is furthermore characterized in that the molar ratio between the synthetic or essentially pure PC species and synthetic or essentially pure PE species is between 3:1 to 1 :3.

Preferably, said ratio is between 2:1 to 1 :2.

In one embodiment, synthetic PC is used in an amount equal to or exceeding that of synthetic

PE. Accordingly, provided is an RSV virosome (including RSV Line 19 F protein, as described herein) comprising synthetic PC species and synthetic PE species at a molar ratio of between 3:1 to 1 :1 , preferably 2:1 to 1 :1 . In a specific aspect, PC is present in excess of PE, for instance at a molar ratio of between 3:1 up to 1 :1 , preferably 2:1 up to 1 :1 .

In another, preferred embodiment, synthetic PC is used in an amount equal to or less than that of synthetic PE. Accordingly, provided is an RSV virosome (including RSV Line 19 F protein, as described herein) comprising synthetic PC species and synthetic PE species at a molar ratio of between 1 :1 to 1 :3, preferably 1 :1 to 1 :2. In a specifically preferred aspect, PE is present in excess of PC, for instance at a molar ratio of between 3:1 up to 1 :1 , preferably 2:1 up to 1 :1 .

The addition of further non-viral lipids may enhance one or more desirable properties of the virosomes. For example, cholesterol can be added to increase the storage stability of the virosomes. In one embodiment, the RSV virosome (including RSV Line 19 F protein, as described herein) comprises cholesterol at a ratio of 5-30 mol% of total added phospholipid, preferably 10-25 mol%, more preferably about 20 mol%. In a preferred embodiment, the virosome comprises DOPC, DOPE, and cholesterol, preferably wherein DOPE is present in excess, e.g., at least 1 .5-fold, of DOPC.

It was found that a molar ratio of total synthetic phospholipid to synthetic adjuvant between 1 .5 and 10 is of relevance for the properties of an RSV virosome according to this example of the invention. Good results were obtained when the ratio is between 3 and 6, preferably between 3.5 and 5. In a specific aspect, the ration is between 3.7 and 4.5, like 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, or 4.5.

An RSV virosome of this example of the invention (including RSV Line 19 F protein, as described herein) is characterized by the presence of a synthetic adjuvant selected from PHAD (phosphorylated hexaacyl disaccharide) and the 3-O-desacyl derivate thereof, 3-D-PHAD. Both are known in the art as synthetic TLR-4 agonists. PHAD is also referred to in the art as Glycopyranoside Lipid A or GLA. See Lousada-Dietrich et al., Vaccine. 201 1 Apr 12;29(17):3284-92.

In one embodiment, the RSV virosome contains PHAD, which has the following structure (designations 14 indicate the total number of carbon atoms in each ac l chain):

In a preferred embodiment, the virosome contains 3-D-PHAD which has the following structure:

The synthetic adjuvant is used in an RSV virosome of this example of the invention (including RSV Line 19 F protein, as described herein) preferably at a ratio of 0.01 -2 mg per mg of viral protein, more preferably at a ratio of 0.5-2 mg per mg of viral protein, for instance at about 1 mg per mg of viral protein, optionally in combination with a molar ratio of total synthetic phospholipid to adjuvant is between 3 and 6, preferably between 3.5 and 5.

In a specific embodiment, an RSV virosome of this example of the invention (including RSV Line 19 F protein, as described herein) comprises about 400-450 nmol DOPC, about 800-900 nmol DOPE, about 250-350 nmol 3-D-PHAD, and about 200-300 nmol cholesterol per mg of viral membrane protein.

Pharmaceutical compositions and methods

The invention also provides pharmaceutical compositions (e.g., vaccines) that contain one or more virosomes of the invention (including an RSV Line 19 F protein, as described herein), and a pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant. Pharmaceutically acceptable stabilizing agents, osmotic agents, buffering agents, dispersing agents, and the like may also be included in the pharmaceutical compositions of the invention. The form used depends on the intended mode of administration and therapeutic application. The pharmaceutical compositions can include any compatible, non-toxic substance suitable to deliver the virosomes to a patient.

The term "pharmaceutically acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, etc., and methods of use thereof, can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18^th edition, Mack Publishing Company, Easton, PA, 1990; and Handbook of Pharmaceutical Excipients, 5^th edition, 2005, each of which is incorporated by reference in its entirety for all purposes.

Formulations are selected based, in part, on the route of pharmaceutical composition delivery. The pharmaceutical compositions of the present invention can be delivered by any acceptable route including, for example, mucosal (e.g., intranasal, intrapulmonary, or oral), as well as parenteral (e.g., by intramuscular, intravascular (e.g., intravenous or intraarterial), or intraperitoneal injection) routes.

Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols, as can be selected by those of skill in the art.

Formulations suitable for intranasal administration, where the carrier is a liquid, can be administered in the form of a nasal spray, nasal drops, or a nebulized aerosol. Pharmaceutically acceptable carriers for intranasal delivery are exemplified by powders containing lyophilized virosomes, water, buffered saline solutions, glycerin, polysorbate 20, cremophor EL, and aqueous mixtures of caprylic/capric glyceride, and may be buffered to provide a neutral pH environment.

Formulations suitable for pulmonary administration (e.g., by inhalation or insufflation therapy) include those presented in the form of an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.

Formulations suitable for parenteral administration (e.g., by intramuscular, intravascular, or intraperitoneal injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the virosome is suspended or otherwise provided. Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.

Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.

The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injection, immediately prior to use.

For oral administration, the virosomes can be administered in liquid dosage forms, such as elixirs, syrups, and suspensions. Liquid dosage forms for oral administration can contain excipients like coloring and flavoring to increase patient acceptance.

As noted above, adjuvants can optionally be included within the membranes of virosomes of the invention. In addition, adjuvants can optionally be included in pharmaceutical compositions that include the virosomes, but outside of the virosome membranes. Adjuvants for this purpose can be selected based on, for example, the route of administration to be used. Exemplary adjuvants include aluminum salts (e.g. potassium aluminum sulfate, alum, aluminum phosphate, aluminum

hydroxyphosphate, and aluminum hydroxide), immune stimulating matrices (e.g., saponin containing lipid and cholesterol complexes), CpG oligonucleotides, 3D-MPL, MF-59, QS21 , muramyl dipeptide, MPLA and related forms and derivatives (see above), and polyphosphazine.

Appropriate amounts of virosomes to be administered to a subject, such as a human patient, can be determined by those of skill in the art. Typically, about 1 -100 pg of antigen protein, e.g., 15-50 pg of antigen protein, is administered in a single dosage. A subject, such as a human patient, can be treated with a single dosage or, optionally, be treated with multiple dosages. In the case of multiple dosages, an appropriate regimen can be determined by those of skill in the art. In various examples, a treatment regimen can include 2-10 dosages (e.g., 3-4 dosages), with each dosage separated from another by 1 -4 days or 1 -2 weeks, depending upon the condition of the patient (e.g., whether the patient has an existing RSV infection (and the severity thereof) or if the patient is being treated prophylactically).

Subjects that can be treated according to the methods of the invention include human patients determined by those of skill in the art to be in need of such treatment. Examples of such patients include (i) elderly patients, older than about 65 years old, with or without underlying cardiac or respiratory disease; (ii) patients, older than about 50 years old, with underlying cardiac or respiratory disease; (iii) women of childbearing age, to prevent infection in infants for the first 4-6 months of age; (iv) infants less than about 6 months old; (v) infants about 6-12 months old; (vi) children from about 12 months to about 5 years of age; and (vii) immunocompromised patients.

The invention is further described in the following Examples, which are not to be interpreted to limit the scope of the invention in any way.

Examples

Throughout the following examples, the RSV L19 F sequence of SEQ ID NO:1 is used, with and without a substitution mutation (I557V), as indicated.

Example 1 : Animal Experiments

This example describes a study investigating virosome vaccines derived from three different RSV strains: RSV-A2, RSV-A2 Line 19F, and RSV-A2 Line 19F I557V. The virosome vaccines were all adjuvanted with 3-deacyl-phosphorylated hexaacyl disaccharide (3D PHAD). The virosomes were compared in mice vaccinated twice at a two-week interval. Protection of vaccinated mice after challenge with live RSV virus and immune responses (neutralizing antibody titers) after vaccination were evaluated in comparison to a negative control group (vehicle).

Methods and Materials

Preparation of Virosomes

Tangential flow filtration (TFF) columns (GE UFP-30-C-MMOA1 , 26 cm², 30 kDa cut-off), stored in 20% ethanol, were flushed with sterile phosphate buffer solution (PBS without Ca²⁺ and Mg²⁺) just prior to use in concentrating viruses.

Twenty-eight vials (approximately 39 ml) of RSV-A2 were quickly thawed, concentrated by TFF, and diafiltered versus 30 ml phosphate buffered solution (PBS) to a volume of 3.4 ml. Fifteen vials (approximately 22.5 ml) of RSV-A2 Line 19F were likewise concentrated to 3.7 ml. Ten vials

(approximately 15 ml) of RSV-A2 Line 19F I557V were concentrated to a volume of 3.2 ml. The protein concentrations according to Bradford determination were 1 .8 mg/ml (RSV-A2), 0.81 mg/ml (RSV-A2 Line 19F), and 1 .25 mg/ml (RSV-A2 Line 19F I557V). To produce virosomes, 170 μΙ of the DCPC solution was added to 1 .7 ml RSV-A2; 41 1 μΙ of the DCPC solution was added to 3.7 ml of RSV-A2 Line 19F; and 356 μΙ of the DCPC solution was added to 3.2 ml of RSV-A2 Line 19F I557V.

After 30 minutes on ice, the samples were spun for 30 minutes at 40,000 rpm in a table-top S100 AT 4 rotor (Sorvall discovery M120-SE table top ultracentrifuge). The supernatants were harvested, filtered through a 0.1 μιη Pall Acrodisc filter, and found to contain 2.3 mg/ml for RSV-A2; 0.57 mg/ml for RSV-A2 Line 19F; and 0.83 mg/ml for RSV-A2 Line 19F I557V.

Dry lipid films were prepared from mixtures of DOPC, DOPE, 3D PHAD, and cholesterol (all from Avanti Polar Lipids) to contain 425 nmol of DOPC, 850 nmol of DOPE, 300 nmol of 3D PHAD, and 255 nmol of cholesterol per mg of viral supernatant protein. The films specifically contained lipids as follows:

• Solution 1 : for RSV-A2 supernatant:

995 nmol DOPC, 1989 nmol DOPE, 702 nmol 3D PHAD, 597 nmol cholesterol

• Solution 2: for RSV-A2 Line 19F supernatant:

850 nmol DOPC, 1700 nmol DOPE, 600 nmol 3D PHAD, 510 nmol cholesterol

• Solution 3: for RSV-A2 Line 19F I557V supernatant:

1063 nmol DOPC, 2125 nmol DOPE, 750 nmol 3D PHAD, 638 nmol cholesterol

The lipid films were dried onto the walls of glass tubes by evaporation of the solvent

(chloroform/methanol) with argon, followed by 30 minutes in a vacuum desiccator.

A solution of 100 mM DCPC was prepared by diluting a 500 mM stock of DCPC with HNE buffer

(5 mM HEPES, 145 mM sodium chloride, 1 mM ethylenediamine-tetraacetic acid (EDTA), pH 7.4, containing 50 IU penicillin and 50 μg/ml streptomycin). 200 μΙ of the DCPC solution was added to each film, and the tubes were immersed in hot tap water for 30-60 seconds. Some turbidity was seen in all solutions. The viral supernatants were added, and then the solutions were vortexed, incubated on ice for 15 minutes, and filtered through a Whatman FP30 0.22 μιη filter. All solutions appeared slightly turbid before and after filtration. The solutions were then injected into gamma-sterilized slide-a-lyzers (Thermo, 10 kDa cut-off, size 0.5-3 ml) and dialyzed against 6x 2 L of PBS over 48 hours at 4°C. Afterwards, all solutions were dialyzed overnight against HNE buffer at 4°C and collected. Bradford protein

determination (Biorad) yielded 1 .2 mg (in 1 .63 ml) for solution 1 ; 1 .2 mg (in 3.66 ml) for solution 2; and 2.1 mg (in 4.6 ml) for solution 3. From each solution, 2x 75 μg was kept refrigerated until the start of animal experiments (4-10°C).

Particle size distribution was assessed by Nanosight® measurements. All analyses were done using samples stored at 4-10°C by single particle tracking on a Nanosight® LM-10 instrument using a 405 nm laser, software version 3 (beta), shutter set to 1200 and gain to 500, threshold "AUTO" at 20.0°C, controlled by Peltier cooling/heating. Dilutions of the virosomes were made in HNE buffer such that between 10 and 100 particles were visible in the observation chamber. Thus, absolute particle numbers cannot be compared between solutions. Ten sets of 90 second observations were integrated per sample, for a total of about 50,000 measured track lengths. Results were adjusted for finite track length. Sample viscosity was assumed to be 1 .00 cP.

Samples of the virosomes were analyzed by equilibrium density gradient centrifugation on 10-

60% sucrose gradients in HNE buffer. Samples were spun for 64 hours and 30 minutes in a Sorvall AH650 rotor at 50,000 rpm, and samples from the gradient were analyzed for protein (Bradford assay; Bio-Rad), phosphate, and density (by refractometry).

Test Animals, Group Assignment, Dosage Levels, and Administration

Six groups of ten female Balb/c mice (6-8 weeks old) received virosome (5 μg protein per dose) and control compositions on Day 0 and Day 13. In more detail, animals were anesthetized with 3-4.5% isoflurane/0₂ for the administrations. Protein concentrations were adjusted to 5 μg per 50 μΙ for IM injection with HNE. The virosome and control compositions were administered as IM injections into both calf muscles at 25 μΙ per leg (total of 5 μg protein). Table 1 illustrates the treatment schedule.

Table 1. Treatment schedule

^* General health was monitored on each study day (Day -1 through Day 35)

^** At Day 30, five mice from each group were terminated and blood was collected for neutralization assays. The remaining five mice per group were challenged the next day (Day 31 ).

Blood and Serum Samples

Serum from study animals was used to measure total immunoglobulin G (IgG), lgG1 , lgG2a, and virus microneutralization titers at different time points. Blood samples were collected by submandibular bleeding or retro-orbital puncture prior to vaccination on Day 0 and Day 13, on Day 30 from animals to be terminated for neutralizing antibody determination, and on Day 31 prior to challenge with RSV. At the day of terminal sacrifice (Day 35), blood was collected from the posterior vena cava. Blood was allowed to clot at room temperature for the duration of the experimental procedures and then placed in a refrigerator. Clotted blood samples were centrifuged and serum from all samples was aliquoted and stored deep frozen in polypropylene tubes at <-10°C.

Lung Samples and Virus Titration

At Day 35, the lungs from a total of five animals per study group were harvested, processed, and numbered. The right lobes of the lungs were used for measurement of RSV viral titer. In more detail, the right lobes of the lungs harvested from animals sacrificed on Day 35 were removed aseptically, and the lung tissue was then homogenized in 1 ml of 2% fetal bovine serum (FBS) containing DMEM medium using an automated Potter homogenizer Polytron-Aggregate/-/ (Thomas Scientific, Swedesboro, NJ, USA). Lung homogenates were centrifuged at 1 ,400 rpm for 10 minutes at 4°C, and supernatants, diluted to a 1 :5 starting dilution, were used to determine viral titers using the TCID₅₀ method.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 5.00 (GraphPad Software, San Diego California USA). Statistical significance was assessed using the Mann-Whitney U test. A P value of 0.05 or lower was considered to represent a statistically significant difference.

Results

Virosome Size and Size Distribution

Figure 1 shows the size distribution of virosomes between 0 and 400 nm, while Table 2 summarizes modal size, standard deviation, and percentage of particles >150 nm. The results of equilibrium density gradient centrifugation analysis of the virosomes are shown in Figure 2.

Table 2. Modal size, standard deviation, and percent of particles >150 nm

Virus Titers

Virus titers were determined from aliquots of lung homogenate supernatants from five mice per group. Figure 3 shows the lung viral titers four days after challenge (Day 35). Titers are shown as 50% endpoint titers as determined by TCID₅₀. All virosome vaccine formulations were found to protect against live virus challenge, while the mice in the control group were not protected. Viral titers observed in the group that received the control were significantly higher compared to titers in all groups that received a vaccine. There was no difference between the protections conferred by the virosomal vaccines in this assay. Statistical analysis was performed using the two-sided Mann Whitney U test.

In Vitro Neutralization

Sera from ten individual mice per group were assayed for anti-RSV antibodies by virus neutralization. Neutralizing antibody titers (log₂) two weeks after the first vaccination and two weeks after the booster vaccination are demonstrated in Figure 4 and Figure 5, respectively. Statistical analysis was performed using the two-sided Mann Whitney U test.

Significantly higher levels of virus-neutralizing antibody titers (log₂) two weeks after primary vaccination were observed for mice that received virosomes derived from RSV-A2 L19F (p<0.01 ) or RSV- A2 L19F I557V (p<0.001 ) compared to those that received RSV-A2 virosomes. Mean neutralizing antibody titers were 2.5 ± 1 .4 and 3.4 ± 1 .6 for RSV-A2 L19F virosomes and RSV-A2 L19F I557V virosomes, respectively. For virosomes derived from RSV-A2 virosomes, mean titers were 0.32 ± 0.70. Serum from control mice did not show detectable neutralizing activity.

Two weeks after the second vaccination, all mice that received a virosomal vaccine showed neutralizing antibody titers (log₂). Titers were significantly higher (p<0.001 ) in mice that received virosomes derived from RSV-A2 L19F or RSV-A2 L19F I557V compared to all other groups. Mean neutralizing antibody titers for virosomes derived from RSV-A2 L19F and RSV-A2 L19F I557V were similar (9.0 ± 0.6 and 9.19 ± 0.70, respectively). Mean titers for RSV-A2 virosomes were 6.61 ± 0.86

Over the course of this four week study, an increase of neutralizing antibody titers was observed in all vaccinated animals. While most of the animals that received either RSV-A2 L19F virosomes (8/10) or RSV-A2 L19F I557V virosomes (9/10) showed an early increase two weeks after the first vaccination, only a few animals that received RSV-A2 virosomes (1/10) had an early increase in neutralizing antibodies. The neutralizing antibodies further increased in all animals after the second vaccination. The increase (all titers are log₂) after the second vaccination for RSV-A2 L19F virosomes was from 2.5 ± 4.5 to 9.0 ± 0.6; and for RSV-A2 L19F I557V virosomes from 3.4 ± 6.4 to 9.19 ± 0.71 . The observed increase of neutralizing antibody titers for animals that received RSV-A2 virosomes was from 0.54 ± 0.93 to

6.1 ± 0.9. At both time points, 14 days following vaccination 1 and vaccination 2, the highest neutralizing antibody titers were observed for animals vaccinated with RSV-A2 L19F and RSV-A2 L19F I557V virosomes ELISA

RSV Specific Serum IgG

Sera from individual mice were assayed for anti-RSV antibodies by ELISA two weeks after the second vaccination (Day 30/31 ). The geometric mean titers (GMT) ± standard deviation (SD) for RSV- specific total IgG was obtained after the second vaccination. Statistical analysis was performed using the two-sided Mann Whitney U test.

RSV-specific IgG titers were observed in all animals that received a virosomal RSV vaccine while control animals showed no RSV-specific IgG titers two weeks after the second vaccination (Figure 6). Titers (log 10) for the groups that received a virosomal vaccine ranged between 4.3 ± 0.79 and

5.48 ± 0.24. The highest and similar GMTs were observed in animals that received RSV-A2 L19F virosomes and RSV-A2 L19F I557V virosomes (5.32 ± 0.19 and 5.4 ± 0.24, respectively). For both groups, titers were significantly higher compared to titers in groups that received RSV-A2 virosomes (p<0.001 ; 4.68 ± 0.21 ).

RSV-Specific IgG Isotype Responses

RSV-specific IgG isotype levels were measured two weeks after the second vaccination

(Day 30/31 ) in ten mice per group. Levels of RSV-specific lgG1 and lgG2a antibodies are demonstrated in Figure 7 and Figure 8, respectively, and indicated as μg/ml serum. Statistical analysis was performed using the two-sided Mann Whitney U test.

Figure 7 shows increased lgG1 antibodies two weeks after the second vaccination in all groups that were vaccinated compared to the controls group. Mean titers ^g/mL) for the animals that received RSV-A2 virosomes were 5.27 ± 3.86. The highest GMTs were found in the animals that were vaccinated with RSV-A2 L19F virosomes or RSV-A2 L19F I557V virosomes, but with large variances within the groups (1 1 .08 ± 12.87 and 15.04 ± 12.00, respectively). A significant difference for the lgG1 titer was observed (p<0.05) for the animals in the group that received the RSV-A2 L19F I557V virosomes compared to animals that received RSV-A2 virosomes.

Figure 8 shows the levels of lgG2a two weeks after the second vaccination. All groups that received virosomes showed an increased titer with large variations within the groups, while control mice showed no increase. The highest and similar GMTs ^g/mL) were found for the groups that received the RSV-A2 L19F virosomes (30.19 ± 13.85) and RSV-A2 L19F I557V virosomes (28.64 ± 13.28). However, the range of the individual mouse titers in these two groups was high (12.36 - 54.32 and 13.91 - 55.37, respectively lgG2a titers for animals that received RSV-A2 virosomes were 18.13 ± 9.996, RSV-A2 L19F virosomes were 30.19 ± 13.85, and RSV-A2 L19F l557V were; 28.64 ± 13.28).

Clinical Observations

Animals were weighed on study days 0, 14, 30/31 , and 35 (Figure 9). No peculiarities were observed during the daily general health check and weighing, even after virus challenge. All mice from the different groups gained weight during each period after the two vaccine administrations. Minimal weight loss after challenge was observed in the animals that received the HNE control. None of the study animals was found dead or in a moribund stage during the course of the study. All animals survived until the day of terminal procedures (Day 30 or Day 35). Conclusions

Virosomes derived from L19F A2 virus or L19F I557V A2 virus induced higher levels of neutralizing antibodies and ELISA serum IgG compared to levels induced by virosomes derived from A2 virus. No adverse effects with respect to general health and body weight changes were observed in any of the immunized groups throughout the study.

Example 2: RSV-F Protein Stability

As noted above, Synagis® (Palivizumab; Medimmune) is a humanized mouse monoclonal antibody that recognizes a conformation-independent epitope present on the F protein of most RSV strains. 5C4 is a mouse monoclonal antibody that specifically recognizes the prefusion conformation of the RSV F protein. Palivizumab was used to quantify the F protein on intact virus, and the ratio of 5C4 to Palivizumab was used to determine the relative concentration of the F protein that was present the prefusion conformation.

96 well ELISA plates (Greiner high-binding) were coated with a 1 :100 dilution of rabbit-anti-RSV serum in coating buffer (0.2 M sodium bicarbonate/carbonate, pH 9.6) overnight at 4°C. The plates were washed with phosphate buffered saline solution (PBS, without Ca²⁺ and Mg²⁺) and blocked with 2% bovine serum albumin (BSA) in coating buffer for 1 hour at room temperature (RT) and washed with PBS. Serial two-fold dilutions (in PBS) of purified virus stocks from RSV strain A2, 19F, and I557V, or virosomes produced from these strains (production and composition as described in Example 1 ), were applied to the plates. After a 1 .5 hour incubation at room temperature, plates were washed with PBS and incubated with 100 μΙ per well of Palivizumab at 4 μg/ml or 5C4 at 4 μg/ml in PBS with 1 % BSA; for 1 .5 hours at room temperature. The plates were washed with PBS and incubated with a 1 :2000 dilution in PBS of antibodies coupled to Horse Radish Peroxidase (HRP): goat-anti human-HRP and goat-anti- mouse-HRP for Palivizumab and 5C4 respectively (both antibodies form Bethyl Labs). After incubation for 1 hour at room temperature, the plates were washed again and developed with ortho-phenyl-diamine (OPD). The reaction was stopped with H₂S0₄ after 30 minutes, and the absorbance at 492 nm was read in an ELISA reader. Plots of absorption vs. viral protein concentration are shown for Palivizumab (Figure 10) and 5C4 (Figure 1 1 ). The lines indicate semi-logarithmic fits of the data points; for all fits, the r² was >0.95, except for the I557V (0.92). Absorption vs. virosomal protein concentration was plotted for 5C4 (Figure 12) and Palivizumab (Figure 13).

Table 3 shows the 5C4/Palivizumab ratio calculated either by division of the slopes of the curves, or the absorbance calculated from the fits for a protein concentration of 50 μg/ml. These data show increased stability of the prefusion form of the RSV F protein in virosomes made from RSV I557V and RSV 19F. Repeated measurements over the course of two months showed the stability of the 5C4 epitope on virosomes.

Table 3. 5C4/Palivizumab ratios

Other Embodiments

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features set forth herein.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated as being incorporated by reference in their entirety.

The invention does not include virosomes, compositions, and related methods described herein that do not include RSV Line 19 F protein, as described herein (whether having the sequence of SEQ ID NO:1 , having one or more substitution mutations relative to the sequence of SEQ ID NO:1 , or having substantial sequence identity to SEQ ID NO:1 (optionally with one of the substitutions), as described herein).

Use of singular forms herein, such as "a" and "the," does not exclude indication of the corresponding plural form, unless the context indicates to the contrary. Similarly, use of plural terms does not exclude indication of a corresponding singular form.

The invention is further described in the following numbered paragraphs. 1 . A virosome comprising a fusion (F) protein of Respiratory Syncytial Virus (RSV) strain Line 19.

2. The virosome of paragraph 1 , further comprising a G protein, a small hydrophobic (SH) protein, or a matrix (M) protein of RSV.

3. The virosome of paragraph 1 or 2, wherein said F protein comprises at least one substitution

mutation.

4. The virosome of paragraph 3, wherein said substitution mutation comprises an amino acid

substitution at I557.

5. The virosome of paragraph 4, wherein said substitution is an I557V amino acid substitution.

6. The virosome of any one of paragraphs 1 -5, wherein the amino acid sequence of said F protein

comprises one or more of the following amino acids: methionine at residue 79, arginine at residue

191 , lysine at residue 357, and tyrosine at residue 371 .

7. The virosome of paragraph 6, wherein the amino acid sequence of said F protein comprises two or more of the following amino acids: methionine at residue 79, arginine at residue 191 , lysine at residue 357, and tyrosine at residue 371 .

8. The virosome of paragraph 7, wherein the amino acid sequence of said F protein comprises three or more of the following amino acids: methionine at residue 79, arginine at residue 191 , lysine at residue 357, and tyrosine at residue 371 .

9. The virosome of any one of paragraphs 1 -8, wherein the amino acid sequence of said F protein

comprises one or more of the following amino acids: proline at residue 4, alanine at residue 16, serine at residue 25, valine at residue 76, alanine at residue 103, threonine at residue 122, isoleucine at residue 152, arginine at residue 213, asparagine at residue 515, and glycine at residue 519.

10. The virosome of paragraph 9, wherein the amino acid sequence of said F protein comprises the following amino acids: proline at residue 4, alanine at residue 16, serine at residue 25, valine at residue 76, alanine at residue 103, threonine at residue 122, isoleucine at residue 152, arginine at residue 213, asparagine at residue 515, and glycine at residue 519.

1 1 . The virosome of any one of paragraphs 1 -10, wherein said virosome comprises lipids and proteins extracted from the membrane of an RSV strain.

12. The virosome of paragraph 1 1 , wherein the RSV strain from which said lipids and proteins are

extracted is a chimeric RSV strain comprising an RSV strain in which the F protein is replaced with the RSV Line 19 F protein.

13. The virosome of paragraph 1 1 , wherein the RSV strain from which said lipids and proteins are

extracted is a chimeric RSV strain comprising an RSV A2 strain in which the A2 F protein is replaced with the RSV Line 19 F protein.

14. The virosome of any one of paragraphs 1 -13, wherein the F protein is substantially in the pre-fusion conformation.

15. The virosome of any one of paragraphs 1 -14, wherein the 5C4/Palivizumab ratio of said virosome as calculated by division of the slopes of the curves is at least 20% greater than the 5C4/Palivizumab ratio of a virosome generated from an RSV A2 strain, as calculated by division of the slopes of the curves.

16. The virosome of any one of paragraphs 1 -15, wherein said F protein comprises at least one

substitution mutation selected from the group consisting of: M79I, R191 K, K357T, and Y371 N.

17. The virosome of any one of paragraphs 1 -16, further comprising an adjuvant. 18. The virosome of paragraph 17, wherein the adjuvant is selected from the group consisting of a saponin, PHAD (phosphorylated hexaacyl disaccharide), 3-D-PHAD (3-O-desacyl derivative of phosphorylated hexaacyl disaccharide), 3-O-D MPLA (3-O-desacyl derivative of monophosphoryl lipid A), and MPLA (monophosphoryl lipid A).

19. The virosome of any one of paragraphs 1 -18, further comprising a phosphatidylcholine (PC) species, a phosphatidylethanolamine (PE) species, and/or sterol or sterol derivative.

20. The virosome of any one of paragraphs 1 , 2, 6-15, or 17-19, wherein said F protein comprises the sequence of SEQ ID NO:1 .

21 . The virosome of any one of paragraphs 1 -15 or 17-19, wherein said F protein comprises the

sequence of SEQ ID NO:1 with a substitution at I557.

22. The virosome of paragraph 21 , wherein said substitution at I557 is I557V.

23. A pharmaceutical composition comprising a virosome of any one of paragraphs 1 -22 and a

pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant.

24. A method of inducing an immune response to RSV in a subject, said method comprising

administering to the subject the pharmaceutical composition of paragraph 23.

25. The method of paragraph 24, wherein the subject does not have, but is at risk of developing, RSV infection.

26. A method of reducing infection and/or replication of RSV in a subject, said method comprising

administering to the subject a pharmaceutical composition of paragraph 23.

27. The method of any one of paragraphs 24-26, wherein said subject is a human subject.

28. A method of making a virosome of any one of paragraphs 1 -22, the method comprising (i) solubilizing the viral envelope of an RSV strain comprising the F protein of RSV strain Line 19, and (ii) reconstituting the viral envelope in the absence of viral nucleic acid.

29. The method of paragraph 28, wherein the viral envelope that is solubilized is that of a chimeric RSV strain comprising RSV strain A2 in which the A2 F protein is replaced with the RSV Line 19 F protein.

30. The method of paragraph 28 or 29, wherein the RSV Line 19 F protein comprises an I557V amino acid substitution.

31 . A virosome of any one of paragraphs 1 -22 for use as a medicament.

32. A virosome of any one of paragraphs 1 -22 for inducing an immune response to RSV.

33. A virosome of any one of paragraphs 1 -22 for use in a method of preventing or treating RSV infection. 34. A virosome of any one of paragraphs 1 -22 for vaccinating a subject against RSV.

Other embodiments are within the scope of the following claims.

What is claimed is:

Claims

Claims

1 . A virosome comprising a fusion (F) protein of Respiratory Syncytial Virus (RSV) strain Line 19.

2. The virosome of claim 1 , further comprising a G protein, a small hydrophobic (SH) protein, or a matrix (M) protein of RSV.

3. The virosome of claim 1 , wherein said F protein comprises at least one substitution mutation.

4. The virosome of claim 3, wherein said substitution mutation comprises an amino acid substitution at I557.

5. The virosome of claim 4, wherein said substitution is an I557V amino acid substitution.

6. The virosome of claim 1 , wherein the amino acid sequence of said F protein comprises one or more of the following amino acids: methionine at residue 79, arginine at residue 191 , lysine at residue 357, and tyrosine at residue 371 .

7. The virosome of claim 6, wherein the amino acid sequence of said F protein comprises two or more of the following amino acids: methionine at residue 79, arginine at residue 191 , lysine at residue 357, and tyrosine at residue 371 .

8. The virosome of claim 7, wherein the amino acid sequence of said F protein comprises three or more of the following amino acids: methionine at residue 79, arginine at residue 191 , lysine at residue 357, and tyrosine at residue 371 .

9. The virosome of claim 1 , wherein the amino acid sequence of said F protein comprises one or more of the following amino acids: proline at residue 4, alanine at residue 16, serine at residue 25, valine at residue 76, alanine at residue 103, threonine at residue 122, isoleucine at residue 152, arginine at residue 213, asparagine at residue 515, and glycine at residue 519.

10. The virosome of claim 9, wherein the amino acid sequence of said F protein comprises the following amino acids: proline at residue 4, alanine at residue 16, serine at residue 25, valine at residue 76, alanine at residue 103, threonine at residue 122, isoleucine at residue 152, arginine at residue 213, asparagine at residue 515, and glycine at residue 519.

1 1 . The virosome of claim 1 , wherein said virosome comprises lipids and proteins extracted from the membrane of an RSV strain.

12. The virosome of claim 1 1 , wherein the RSV strain from which said lipids and proteins are extracted is a chimeric RSV strain comprising an RSV strain in which the F protein is replaced with the RSV Line 19 F protein.

13. The virosome of claim 1 1 , wherein the RSV strain from which said lipids and proteins are extracted is a chimeric RSV strain comprising an RSV A2 strain in which the A2 F protein is replaced with the RSV Line 19 F protein.

14. The virosome of claim 1 , wherein the F protein is substantially in the pre-fusion conformation.

15. The virosome of claim 1 , wherein the 5C4/Palivizumab ratio of said virosome as calculated by division of the slopes of the curves is at least 20% greater than the 5C4/Palivizumab ratio of a virosome generated from an RSV A2 strain, as calculated by division of the slopes of the curves.

16. The virosome of claim 1 , wherein said F protein comprises at least one substitution mutation selected from the group consisting of: M79I, R191 K, K357T, and Y371 N.

17. The virosome of claim 1 , further comprising an adjuvant.

18. The virosome of claim 17, wherein the adjuvant is selected from the group consisting of a saponin, PHAD (phosphorylated hexaacyl disaccharide), 3-D-PHAD (3-O-desacyl derivative of phosphorylated hexaacyl disaccharide), 3-O-D MPLA (3-O-desacyl derivative of monophosphoryl lipid A), and MPLA (monophosphoryl lipid A).

19. The virosome of claim 1 , further comprising a phosphatidylcholine (PC) species, a

phosphatidylethanolamine (PE) species, and/or sterol or sterol derivative.

20. The virosome of claim 1 , wherein said F protein comprises the sequence of SEQ ID NO:1 .

21 . The virosome of claim 1 , wherein said F protein comprises the sequence of SEQ ID NO:1 with a substitution at I557.

22. The virosome of claim 21 , wherein said substitution at I557 is I557V.

23. A pharmaceutical composition comprising a virosome of claim 1 and a pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant.

24. A method of inducing an immune response to RSV in a subject, said method comprising

administering to the subject the pharmaceutical composition of claim 23.

25. The method of claim 24, wherein the subject does not have, but is at risk of developing, RSV infection.

26. A method of reducing infection and/or replication of RSV in a subject, said method comprising administering to the subject a pharmaceutical composition of claim 23.

27. The method of claim 24, wherein said subject is a human subject.

28. A method of making a virosome of claim 1 , the method comprising (i) solubilizing the viral envelope of an RSV strain comprising the F protein of RSV strain Line 19, and (ii) reconstituting the viral envelope in the absence of viral nucleic acid.

29. The method of claim 28, wherein the viral envelope that is solubilized is that of a chimeric RSV strain comprising RSV strain A2 in which the A2 F protein is replaced with the RSV Line 19 F protein.

30. The method of claim 28, wherein the RSV Line 19 F protein comprises an I557V amino acid substitution.

31 . A virosome of claim 1 for use as a medicament.

32. A virosome of claim 1 for inducing an immune response to RSV.

33. A virosome of claim 1 for use in a method of preventing or treating RSV infection.

34. A virosome of claim 1 for vaccinating a subject against RSV.