‘Yin is not specifically directed to RSV nor is it concerned with vaccine development. It would not provide an obvious RSV vaccination strategy to the Skilled Team. It is a report of F protein structures of PIV5 from the perspective of its structural biology. It discusses the conformation of pre and postfusion F proteins of PIV5 and PIV3, respectively. The Skilled Team would know such conformation changes and their mechanisms were not well understood at the Relevant Dates and that Yin is trying to develop that understanding. Yin discloses nothing which would be read as being relevant to vaccination against RSV and gives the Skilled Team interested in developing vaccines no reason to produce a RSV F protein stabilised in the pre-fusion conformation. Furthermore Yin does not expressly teach how to stabilise the RSV F protein in the prefusion conformation and I do not believe the Skilled Team would assume that, because PIV5 had been stabilised, a similar approach would necessarily work with the RSV F protein. That would require further research. For these reasons I do not consider that the Yin paper makes the contents of the Relevant Claims obvious.’
‘The common general knowledge did not include the approach of developing as a vaccine an F protein stabilised in the prefusion conformation. The idea of vaccinating with the F protein in its prefusion conformation had not occurred to me at the Relevant Dates. I was aware that the F protein had two conformations and I was familiar with the Calder paper. I had by the Relevant Dates been focused on the problem of RSV vaccination for over 25 years but at no point did I make a connection between the two conformations and the design of HRSV (or BRSV) vaccines. Furthermore I have no recollection of having discussed this idea with others working in the field or having heard this being discussed. I have addressed my mind to whether my thinking was typical of people working in the field at the Relevant Dates and believe it was. There are numerous review articles in the field around the Relevant Dates and, as far as I am aware, none of them draw attention to the potential relevance of different forms of the F protein in developing a vaccination strategy and none suggest the approach of vaccinating with a subunit vaccine comprising the F protein stabilised in the prefusion conformation.’
‘The relevance of prefusion and postfusion conformations of the RSV F protein to a vaccination strategy was not part of the Skilled Team's thinking at the Relevant Dates, as reflected in the review articles which I referred to at Taylor 1 paragraph 5.55.’
‘None of the review articles above nor any of the other review articles before the Relevant Dates that I reviewed in preparing Taylor 1 listed in Exhibit GT-11 refer to the prefusion or postfusion RSV F protein as being something to consider in vaccine development.’
‘Despite half a century of dedicated research, there remains no licensed vaccine product. Herein are described past and current efforts to harness innate and adaptive immune potentials to combat RSV. A plethora of candidate vaccine products and strategies are reviewed.’
‘In her summary on purified F protein she explains that despite modest neutralizing antibody activity: ‘Purified F proteins remain a topic of interest, with recent attention paid to the protein’s postfusion structure [169]’
‘I also disagree further in paragraph 201 that "it would have been clear to the RSV Vaccinologist that immunizing with an RSV F protein that adopts a postfusion conformation might not lead to induction of antibodies that recognize the prefusion conformation". As I have already stated RSV vaccinologists at the priority date were not addressing themselves to the question of antigenic differences between the pre- and post-fusion forms of the F protein. Yin does not address this subject. It is only with hindsight that we know that there are relevant antigenic differences between the two forms of the F protein. Furthermore I am not aware of any theory in the field at the Relevant Dates that binding of neutralising antibodies to the prefusion conformation of RSV F "would prevent transition to the postfusion state and block fusion of the membranes, thereby blocking virus infection". I do not recall any such theory being proposed or discussed. … ‘The difference between Dr Johnson and me is that she contends that a prefusion form of the virus was an obvious target and would have been seen to be advantageous. I disagree for the reasons I have given.’
‘112. In support of its case that the claims are not obvious in the light of Yin GSK rely upon the secondary evidence of what was being reported in the literature and Dr Taylor's evidence. The contemporaneous literature around the priority date does not address the option of vaccinating with the prefusion form of the F protein. If Dr Johnson is right and at the priority date, even before reading Yin it was known that there was a prefusion and postfusion form of the F protein and it was known or obvious that it was advantageous to vaccinate with the prefusion form, one would expect that to be stated somewhere in the literature. The first time this is mentioned (other than in the Patents) is in 2011 (see Taylor III paragraph 2.22).’
‘The meeting brought together leading experts from all over the world, working on the development and use of vaccines against the most important virus infections of humans and animals.’
‘José Melero, using state of the art technology, has identified the fusion properties and fusion sites of RSV fusion protein (F-protein) [31]. Future research will be aimed at the development of compounds or antibodies capable of binding to the fusogenic intermediate of the F-protein.’
‘Dr Melero was indicating that this was a promising area of his research.’
‘Inhibitors of HRSV entry have been the topic of intense research in recent years. Besides the afore-mentioned humanized anti-F monoclonal antibody (Groothuis and Nishida, 2002), small inhibitors of F protein activity have been actively searched. By analogy with synthetic peptides that inhibit human immunodeficiency virus (HIV) replication (Wild et al., 1994), synthetic peptides that reproduce sequences of the heptad repeats (HR) regions of HRSV were synthesized and tested for inhibition of virus infectivity. Peptides containing partial sequences of HRB were found to be highly active inhibitors of HRSV infectivity (Lambert et al., 1996). These peptides presumably bind to the HRA core of an F protein intermediate during the process of virus—cell membrane fusion, blocking latter stages of the fusion process.’
‘At any rate, inhibition of the F protein activity by synthetic drugs seems a feasible approach for the development of anti-HRSV compounds that may find some future application in the clinic.’
"Cleavage Activation. As discussed 15 previously, the precursor F0 molecule", this is on the 16 right-hand side of 1472, "is biologically inactive and 17 cleavage of the F0 to the disulfide linked chains F1 and F2 18 activates the protein, rendering the molecule fusion-active 19 and permitting viral infectivity."
‘It is possible.’ ii) Line 12: ‘Not necessarily, because the Calder paper suggests that conformational epitopes in antigenic areas 2 and 4 are not disrupted.’ iii) As for the answer at Line 21, she said ‘I said ‘Yes’ but I was not clear what the approach to what was at that stage.’
‘It would also be CGK that as a matter of first principles an antigen which better mimics the native, prefusion form of the F protein (present on infectious virions) would be more likely to generate antibodies that interfere with cell entry and so could serve as better immunogens. This concept was well-known in the HIV field. Indeed, by the Priority Date, a number of stabilization strategies had been used to try to preserve the native trimeric conformation of the HIV-1 Env protein. These approaches were summarised in a number of review articles available at the time, including for example articles by Burton 2002 (citing “Neutralizing antibody (*) binds to native envelope spikes..”), Burton 2006 (review of different Env stabilization approaches to generate native Env antigens), Haynes and Montefiori 2006 (citing “the lack of current immunogens that mirror the native envelope structures needed to induce neutralizing antibodies ..”), Nabel 2002 (citing “..expression in a conformation that more closely resembles that of the native protein..”).’
“The F protein is believed to drive membrane fusion by coupling irreversible protein refolding to membrane juxtaposition and by initially folding into a metastable form that subsequently undergoes discrete/stepwise conformational changes to a lower energy state (176,203). The F protein found on virions is considered to be in a prefusion form, and after membrane fusion has occurred, the F protein is considered to be in a postfusion form.” ii) Second, in Cane at p50, under the heading ‘Antibody recognition of RSV proteins’ the paragraph concludes with this: ‘Analysis of the repertoire of F-specific antibodies induced by RSV infection demonstrated that highly neutralising antibodies recognised the mature F protein on the cell surface and on virions, whereas poorly neutralising antibodies appeared to recognise immature F protein (Sakurai et al., 1999).’ iii) Third, at p488 in Fields, Chapter 15 (Immunization against viral diseases): “First, antiviral Abs against the extracellular domain of surface proteins predominantly recognize conformational epitopes. Such conformational epitopes are difficult to mimic with peptides or other forms of an immunogen in which the surface protein antigenic sites are denatured. An immunogen that possesses the structures of the native protein most effectively induces Abs that recognize the conformational epitopes that mediate immunity.”
“The present invention relates to a recombinant respiratory syncytial virus (RSV) antigen comprising a soluble F protein polypeptide comprising an F2 domain and an F1 domain of an RSV F protein polypeptide, wherein there is no furin cleavage site between the F2 domain and the F1 domain, and wherein the polypeptide further comprises a heterologous trimerization domain positioned C-terminal to the F1 domain.”
"The present disclosure concerns recombinant respiratory syncytial virus (RSV) antigens that solve problems encountered with RSV antigens previously used in vaccines, and improve the immunological as well as manufacturing properties of the antigen. The recombinant RSV antigens disclosed herein involve a Fusion (F) protein analog that include a soluble F protein polypeptide, which has been modified to stabilize the prefusion conformation of the F protein, that is, the conformation of the mature assembled F protein prior to fusion with the host cell membrane."
‘[0015] The PreF antigens are stabilized (in the trimeric prefusion conformation) by introducing one or more modifications, such as the addition, deletion or substitution, of one or more amino acids. One such stabilizing modification is the addition of an amino acid sequence comprising a heterologous stabilizing domain. In exemplary embodiments, the heterologous stabilizing domain is a protein multimerization domain. One particularly favorable example of such a protein multimerization domain is a coiled-coil domain, such as an isoleucine zipper domain that promotes trimerization of multiple polypeptides having such a domain. An exemplary isoleucine zipper domain is depicted in SEQ ID NO:11. Typically, the heterologous stabilizing domain is positioned C-terminal to the F1 domain.’
‘[0017] Another stabilizing modification is the elimination of a furin recognition and cleavage site that is located between the F2 and F1 domains in the native F0 protein. One or both furin recognition sites, located at positions 105-109 and at positions 133-136 can be eliminated by deleting or substituting one or more amino acid of the furin recognition sites, such that the protease is incapable of cleaving the PreF polypeptide into its constituent domains. Optionally, the intervening pep27 peptide can also be removed or substituted, e.g., by a linker peptide. Additionally, or optionally, a non-furin cleavage site (e.g., a metalloproteinase site at positions 112-113) in proximity to the fusion peptide can be removed or substituted.’
‘[0018] Another example of a stabilizing mutation is the addition or substitution of a hydrophilic amino acid into a hydrophobic domain of the F protein. Typically, a charged amino acid, such as lysine, will be added or substituted for a neutral residue, such as leucine, in the hydrophobic region. For example, a hydrophilic amino acid can be added to, or substituted for, a hydrophobic or neutral amino acid within the HRB coiled-coil domain of the F protein extracellular domain. By way of example, a charged amino acid residue, such as lysine, can be substituted for the leucine present at position 512 of the F protein. Alternatively, or in addition, a hydrophilic amino acid can be added to, or substituted for, a hydrophobic or neutral amino acid within the HRA domain of the F protein. For example, one or more charged amino acids, such as lysine, can be inserted at or near position 105-106 (e.g., following the amino acid corresponding to residue 105 of reference SEQ ID NO:2, such as between amino acids 105 and 106) of the PreF antigen). Optionally, hydrophilic amino acids can be added or substituted in both the HRA and HRB domains. Alternatively, one or more hydrophobic residues can be deleted, so long as the overall conformation of the PreF antigen is not adversely impacted.’
"Fig. 10 depicts cartoons of the mature RSV protein" "and the three RSV soluble fusion (sF) protein constructs ... used in our studies."
‘[0024] To enhance the immune response produced following administration, the immunogenic composition typically also includes an adjuvant. In the case of immunogenic compositions for eliciting a protective immune response against RSV (e.g., vaccines), the compositions favorably include an adjuvant that predominantly elicits a Th1 immune response (a Th1 biasing adjuvant). [0025] The immunogenic compositions described herein are favorably employed as vaccines for the reduction or prevention of infection with RSV, without inducing a pathological response (such as vaccine enhanced viral disease) following administration or exposure to RSV.’
“The F1 fragment contains at least two heptad repeat domains, designated HRA and HRB, and situated in proximity to the fusion peptide and transmembrane anchor domains, respectively. In the prefusion conformation, the F2-F1 dimer forms a globular head and stalk structure, in which the HRA domains are in a segmented (extended) conformation in the globular head. In contrast, the HRB domains form a three-stranded coiled coil stalk extending from the head region. During transition from the prefusion to the postfusion conformations, the HRA domains collapse and are brought into proximity to the HRB domains to form an anti-parallel six helix bundle. In the postfusion state the fusion peptide and transmembrane domains are juxtaposed to facilitate membrane fusion.”
“The PreF antigens disclosed herein are designed to stabilize and maintain the prefusion conformation of the RSV F protein, such that in a population of expressed protein, a substantial portion of the population of expressed protein is in the prefusogenic (prefusion) conformation (e.g., as predicted by structural and/or thermodynamic modeling or as assessed by one or more of the methods disclosed above). Stabilizing modifications are introduced into a native (or synthetic) F protein, such as the exemplary F protein of SEQ ID NO:2, such that the major immunogenic epitopes of the prefusion conformation of the F protein are maintained following introduction of the PreF antigen into a cellular or extracellular environment (for example, in vivo, e.g., following administration to a subject).”
‘(i) that the PreF sequence in SEQ ID NO:6 includes an engineered lysine residue which connects the F2 and F1 regions. It is at position 106 of the SEQ ID NO: 6 sequence. Given the pep27 region is removed and furin cleavage sites have been deleted, the lysine residue was engineered into the sequence to link the F2 and F1 sequences together. That same modification is described by the patent as “another example of a stabilizing mutation” at paragraph [0018]. Because the lysine residue might be exposed, it could contribute to solubility of the protein, being a positively charged amino acid, and it may also serve as a "short" linker that contributes to stability because its presence may prevent distorting the region linking F2 and F1. (ii) The amino acid substitution at position 482, referred to in paragraph [0065] as another stabilizing mutation, is also present in the SEQ ID NO:6 sequence. A modification to improve periodicity in the HRB region could also potentially have an impact on the stability of the protein.’
“a greater proportion of the F protein is in the prefusion conformation than would be the case in a preparation of an antigen in which the F protein does not have a heterologous trimerization domain positioned C-terminal to the F1 domain.”
‘The term "polypeptide" refers to a polymer in which the monomers are amino acid residues which are joined together through amide bonds.’
‘In relation to that construct, the Patents use the term “soluble F protein polypeptide” which is the same feature as used in the claims. Therefore, the patentee is itself using the term “polypeptide” to encompass constructs such as Pfizer’s RSVpreF product.’
“1. A recombinant respiratory syncytial virus (RSV) antigen comprising a soluble F protein polypeptide comprising at least one modification that stabilizes the prefusion conformation of the F protein. 2. A recombinant respiratory syncytial virus (RSV) antigen comprising a soluble F protein polypeptide, which F protein polypeptide comprises at least one modification selected from: (i) an addition of an amino acid sequence comprising a heterologous trimerization domain; (ii) a deletion of at least one furin cleavage site; (iii) a deletion of at least one non-furin cleavage site; (iv) a deletion of one or more amino acids of the pep27 domain; and (v) at least one substitution or addition of a hydrophilic amino acid in a hydrophobic domain of the F protein extracellular domain.”
“66. The whole approach to interpretation and scope of protection therefore involves the following steps, considered through the eyes of the notional addressee: (i) Does the variant infringe any of the claims as a matter of normal interpretation? (ii) If not, does the variant nevertheless infringe because it varies from the invention in a way or ways which is or are immaterial? This is to be determined by asking these three questions: (a) Notwithstanding that it is not within the literal (that is to say, I interpolate, normal) meaning of the relevant claim(s) of the patent, does the variant achieve substantially the same result in substantially the same way as the invention, i.e. the inventive concept revealed by the patent? (b) Would it be obvious to the person skilled in the art, reading the patent at the priority date, but knowing that the variant achieves substantially the same result as the invention, that it does so in substantially the same way as the invention? (c) Would such a reader of the patent have concluded that the patentee nonetheless intended that strict compliance with the literal meaning of the relevant claim(s) of the patent was an essential requirement of the invention?” (a) Notwithstanding that it is not within the literal (that is to say, I interpolate, normal) meaning of the relevant claim(s) of the patent, does the variant achieve substantially the same result in substantially the same way as the invention, i.e. the inventive concept revealed by the patent? (b) Would it be obvious to the person skilled in the art, reading the patent at the priority date, but knowing that the variant achieves substantially the same result as the invention, that it does so in substantially the same way as the invention? (c) Would such a reader of the patent have concluded that the patentee nonetheless intended that strict compliance with the literal meaning of the relevant claim(s) of the patent was an essential requirement of the invention?”
“257. Apple relies on the “result” being tracking the same sets of resources, namely sequence number usage and buffer memory usage. Its submissions essentially treated this as the “way” as well. Apple’s approach was based on, and arose from, evidence Mr Kubota had given on equivalence for the purpose of infringement/essentiality, where he referred to tracking those resources. 258. Optis disagreed. It pointed out that in addition to tracking those resources, Mr Kubota had referred to simplicity of implementation and (claim 9) avoidance of superfluous polling. It pointed out that InterDigital is more complex (as windows based mechanisms will always be) and does not avoid superfluous polling. I agree with those points, and I have already analysed why above.”
‘110. Paragraphs [0009] and [0010] of the Patent discuss counter-based and windows-based mechanisms in such a way as to contrast them.’
‘116. Another way of looking at this is that counter-based mechanisms operate in units of PDUs and bytes, whereas windows-based mechanisms operate in terms of percentages, because they have regard to how many PDUs or bytes there are in the buffer compared with the maximum PDUs or bytes allowed. 117. In my view, “counting” in claim 1 is clearly intended to take its meaning from this rather particular context. It means maintaining a count of transmitted PDUs and bytes, in such units.’
‘The critical passage in KCI is as follows. Arnold J had held that on its true construction the relevant agreement there did convey the legal title to the applicant but he went on to hold that even if that was wrong, the agreement was effective to transfer the entire beneficial interest. The applicant had an enforceable legal right to call for a conveyance of the bare legal title and that made the applicant the “successor in title” for the purposes of a claim to priority under Article 87(1) of the EPC and Article 4(A)(1) of the Paris Convention even if KC Inc had not acquired the bare legal title at the relevant date. After referring to a decision of the EPO Case J19/87 Burr-Brown /Assignment [1988] EPOR 350, Arnold J held: ‘71. To my mind, this makes sense. Article 4(A) of the Paris Convention and Article 87(1) of the EPC are provisions in international treaties whose operation cannot depend upon the distinction drawn by English law, but not most other laws, between legal and equitable title. When determining whether a person is a "successor in title" for the purposes of the provisions, it must be the substantive rights of that person, and not his compliance with legal formalities, that matter.’’
‘I find that the legal principles applicable to priority entitlement are settled at this first instance level. They are: i) Usually the right to claim priority goes with the right to the invention. That is uncontroversial. ii) The right to claim priority must be with the person making the patent application in which that right is claimed when they make that claim, i.e. when the application is filed. A later acquisition of that right cannot make good a lack of it on the relevant date. If the right was not in place at the time then the right is lost for all time. That is Edwards v Cook. iii) But if the local law applicable to rights of the applicant and the patent application at the place and time when it was made allows for a splitting of property rights into legal and equitable interests, then it will be sufficient to establish an entitlement to priority if the applicant holds the entire equitable interest at the relevant date. That is KCI, HTC and FujiFilm and was held in the Court of Appeal in Idenix provisionally to be correct. iv) A person with a legally enforceable right to call for the assignment of the legal title to a piece of property such as an invention (or a right to claim priority) has the equitable title to that property. When the cases refer to the applicant holding the substantive right and title to the invention, they are referring to this legal/equitable distinction.’
“6. On or before2 December 2022 , the Claimant shall file and serve a Statement of Case on Entitlement to Claim Priority and Belgian Law setting out all facts and matters relied on in support of its case that WO456 is not entitled to priority. 7. On or before20 January 2023 , the Defendants shall file and serve a Statement of Case on Entitlement to Claim Priority and Belgian Law in reply.”
‘A claim lacks novelty if it covers something that formed part of the state of the art at the priority date’. ii) Second, at [112]: ‘The teaching of the specification, once construed, is a pure question of fact, as is what the skilled man would do with that teaching without the exercise of inventive ingenuity.’ iii) Third, at [128]: ‘As ever, the question is what is explicitly disclosed and what also is necessarily implicit in the teaching. The skilled man must be taken to read documents in an intelligent way, seeking to find what is disclosed as a matter of substance.’
“As described above, the sF protein may also be physically stabilized by adding a GCNt segment to clamp the C terminus, or by adding cysteines that will cross-link the trimer C termini.”
“The three versions of the RSV sF protein (cartoon in Fig. 10; sequences in Fig.11) were constructed from MP340 by replacing the transmembrane and cytoplasmic domain of the F protein gene: 1) with a FLAG tag followed by a 6-histidine (6HIS) tag (SC-2); 2) and the last two amino acids of the HR2 helix (523 and 524) with two cysteine residues to allow the C terminus of the F sequence in the trimer to covalently link the monomers, followed by a FLAG tag followed by a 6HIS tag (HC-1); and 3) with a TEV protease cleavage site followed by a GCNt trimerization domain followed by a FLAG tag followed by a Factor Xa cleavage site followed by a 6HIS tag (sMP340-A). These novel sequences replacing the C terminus of the RSV F protein were designed to purify the sF protein released into the medium of transfected cells (6HIS tag or FLAG tag), enable easy detection of the sF proteins (6HIS tag or FLAG tag), or to clamp this end of the molecule to stabilize it (covalently with cysteines or non-covalently with the GCNt trimerization domain).”
“In our initial experiments, both SC-2 and sMP340-A migrated further into the sucrose gradients than expected (Fig. 14), indicating that they were aggregated. We had expected SC-2 to migrate in this manner, indicative of aggregation, but not sMP340-A. We hypothesized that freezing the protein between the time of production and purification and the sucrose gradient might be responsible for the sMP340-A migration indicating aggregation.”
“According to our hypothesis, any mouse monoclonal antibody (MAb) against the F protein that neutralizes RSV infectivity in cell culture would bind to the virion form of the F protein and probably to the pre-triggered form of the F protein. If the SC-2 or sMP340- A protein represents the pre-triggered form of the sF protein, neutralizing MAbs should recognize it.”
“All 11 of these MAbs immunoprecipitated the SC-2 sF protein efficiently (Fig. 16, "-" lanes) suggesting that this sF protein is in the native F protein conformation. The same 11 MAbs did not immunoprecipitate the sMP340-A sF protein efficiently, suggesting that sMP340-A may not be in the native conformation.”
“The heated SC-2 sF protein lost its ability to be recognized efficiently by all 11 of the Mabs (Fig. 16, "+" lanes), indicating that heating had caused major conformational changes in the SC-2 sF protein, consistent with it being triggered by the heat treatment. Heating the sMP340-A sF protein had no effect on MAb binding (Fig. 16 "+" lanes), indicating that sMP340-A is not triggered by mild heat.”
‘The positions and structural transitions of key parts of the fusion machinery, including the hydrophobic fusion peptide and two helical heptad repeat regions, clarify the mechanism of membrane fusion mediated by the F protein.’
"[77] It generally only comes into play when one is considering the question 'if it was obvious, why was it not done before?' That question itself can have many answers showing it was nothing to do with the invention, for instance that the prior art said to make the invention obvious was only published shortly before the date of the patent, or that the practical implementation of the patent required other technical developments. But once all other reasons have been discounted and the problem is shown to have been long-standing and solved by the invention, secondary evidence can and often does, play an important role. If a useful development was, in hindsight, seemingly obvious for years and the apparently straightforward technical step from the prior art simply was not taken, then there is likely to have been an invention."
"[85] It would be wrong to read this decision as saying that secondary evidence is always of minor importance. That would be to throw away a vast mass of jurisprudence, including many House of Lords cases, (e.g. Vickers, Sons & Co v Siddell and Technograph). It would indeed involve disregarding some of the approach actually used in Mölnlycke."
‘442. Where the issue is one of obviousness the courts in this jurisdiction are very used to separating primary evidence (that of the experts) from secondary evidence, such as the experience of real world workers who did or did not make the invention, and applying appropriate caution to the latter, based for example on whether they represented the ordinary skilled person, whether they had the cited art, and so on.’ ‘446. I also identify at this stage that the usefulness of secondary evidence must depend in significant part on how complete and how testable it is. In the present case I did not hear oral evidence from any of the real world workers relied on, and the documentary record is patchy, including because the documentation created at the time was poor (in the case of Mr Clark). This makes it especially hard to assess why the workers in question succeeded or failed, as the case may be. Secondary evidence on obviousness is often discounted by a trial judge on the basis that it is simply unknown why (for example) the invention was not made before and in my view the same should apply to undue burden, as part of the overall exercise of assessing the secondary evidence.’
“Increasing knowledge of the structure and antigenic properties of the HRSV F protein and improved methods of expression and purification may allow for the production of more stable and more immunogenic HRSV F vaccine preparations. As of this writing, the only HRSV subunit vaccine currently being evaluated in clinical trials is an F protein particle vaccine developed by Novavax (clinicaltrials.gov NCT01290419), which is currently undergoing phase 1 evaluation in healthy adults. In other recent work, the HRSV F protein was engineered to remove the fusion peptide, transmembrane region, and cytoplasmic tail, yielding an expressed protein that formed a postfusion trimeric structure that was homogenous, stable, and highly immunogenic.378, 536”
‘We undertook structural and functional studies of the inter- action between 101F and its epitope on the RSV F glycopro- tein to investigate the mechanism of antibody-mediated RSV neutralization. Here we present the crystal structure of the antigen-binding fragment (Fab) of 101F in complex with its F glycoprotein-derived epitope peptide. The structure defined the length of the linear epitope and allowed for modeling of 101F binding to pre- and postfusion F trimers. Hypotheses based on these models were tested to investigate the mechanism of 101F neutralization and the extent of the epitope. These results are analyzed and discussed in the context of known antibody escape mutations, mechanisms of antibody-mediated virus neutralization, and applicability to epitope-specific vaccine design.’
‘During the entry process, there are several steps which neutralizing antibodies can block, including attachment, triggering, and transition of the fusion glycoprotein to the postfusion state. We have demonstrated that 101F does not block virus attachment over a wide range of concentrations (Fig. 5B), which agrees with recently published data showing a similar result at a single antibody concentration (29). We have also shown that 101F is capable of preventing infection once the virus has attached to the cell (Fig. 5C). This narrows the window of 101F neutralization to some point between triggering of the fusion glycoprotein and transition to the postfusion state. In addition, since our modeling studies show that 101F binding is compatible with both the pre- and postfusion states (Fig. 3), 101F is predicted to bind all forms of the fusion glycoprotein, including intermediates. Thus, we propose that 101F also does not prevent triggering but rather prevents adoption of the postfusion conformation due to its bulk in the context of the cell and viral membranes.’
‘This epitope [the context is antigenic site IV] is C-terminal to the cysteine-rich region and is part of domain II, which in homologous paramyxovirus F glycoproteins remains structurally unchanged between pre- and postfusion conformations (46).’
‘The prefusion and postfusion forms of RSV F each have potential shortcomings as vaccine antigens. Large structural differences between the lollipop-shaped prefusion F trimer and the crutch-shaped postfusion F trimer are apparent even at the resolution of electron microscopy of negatively stained specimens, suggesting that prefusion and postfusion F may be antigenically distinct (11). To prevent viral entry, F-specific neutralizing antibodies presumably must bind the prefusion conformation of F on the virion, before the viral envelope fuses with a cellular membrane. Therefore, it might be expected that RSV F must be presented in the prefusion conformation to elicit neutralizing antibodies efficiently. However, prefusion F is a “metastable” structure that readily rearranges into the lower energy postfusion state, which aggregates due to exposure of a hydrophobic fusion peptide (12), and efforts to generate a soluble, stabilized prefusion F subunit antigen have not yet yielded candidates suitable for testing in humans.’
‘We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-Å X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigen’s efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.’
‘The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs.’
“Thus, 101F and motavizumab can likely bind the fusion glycoprotein in the prefusion, postfusion, and intermediate states, which results from their epitopes residing in domains that are not expected to undergo large structural rearrangements during the fusion process (44).”
‘It is all of a piece with the abovementioned papers, showing that an appreciation of the problem lying in the postfusion conformation of the existing F subunit vaccines formed no part of the prevailing view. Additionally, we remark that the fact that the report of postfusion was picked up so quickly in the review papers further tells against the suggestion that an approach to vaccination based on conformation had been taken earlier.’
“No one can tell whether live attenuated, subunit, VLP, replicating, or nonreplicating vectored vaccines will turn out to be successful in phase 3, but hopefully this new competition will help to bring much needed respiratory vaccines to the market sooner. Every year without them will be a lost year, especially for those who don’t receive the care they need.”
‘The prefusion state of respiratory syncytial virus (RSV) fusion (F) glycoprotein is the target of most RSV-neutralizing activity in human sera, but its metastability has hindered characterization. To overcome this obstacle, we identified prefusion-specific antibodies that were substantially more potent than the prophylactic antibody palivizumab. The cocrystal structure for one of these antibodies, D25, in complex with the F glycoprotein revealed D25 to lock F in its prefusion state by binding to a quaternary epitope at the trimer apex. Electron microscopy showed that two other antibodies, AM22 and 5C4, also bound to the newly identified site of vulnerability, which we named antigenic site Ø. These studies should enable design of improved vaccine antigens and define new targets for passive prevention of RSV-induced disease.’
‘The proven success of palivizumab (3) has spurred vaccine efforts aimed at eliciting protective RSV F–directed antibodies. These efforts have been complicated by the conformational diversity of RSV F (4–8 [including McLellan 2011, Swanson 2011 and McLellan 2013A]), a type I fusion glycoprotein that merges virus and host-cell membranes by using the difference in folding energy between two substantially different states: a metastable state adopted before virus-cell interaction (prefusion) and a stable state that occurs after merging of virus and cell membranes (postfusion). Both states exhibit epitopes targeted by neutralizing antibodies, and postfusion RSVF is being developed as a vaccine candidate (6 [Swanson 2011], 9). Recently, however, the major target of RSV-neutralizing antibodies elicited by natural infection was found to reside primarily on the prefusion conformation of RSV F (10 [Magro 2012]). Antibodies such as 5C4 (7 [McLellan 2013A]), AM22, and D25 (11, 12) are substantially more potent than palivizumab and target antigenic site 0/ (zero), a metastable site located at the membrane-distal apex of the prefusion RSV F trimer (7 [McLellan 20132A]). To enhance elicitation of similarly potent antibodies, we engineered soluble variants of RSV F with stably exposed antigenic site 0/. These variants were characterized antigenically and crystallographically and tested for immunogenicity in mice and nonhuman primates (rhesus macaques). Structure-Based Vaccine Strategy We and others have engineered antigenicity (13–17) through structure-based design of the epitopes recognized by template neutralizing antibodies. For example, the crystal structure of motavizumab (a variant of palivizumab) bound to its F glycoprotein epitope (18 [McLellan 2010]) allowed us to create epitope scaffolds, which stably presented the motavizumab epitope on heterologous proteins (19 [McLellan 2011]). Although motavizumab-epitope scaffolds could elicit immune responses that recognized F, substantial neutralizing activity was not induced (19). We hypothesized that instead of a single epitope recognized by a single template antibody, it would be advantageous to present a “supersite” (20), comprising a collection of overlapping epitopes recognized by multiple antibodies. Even more preferable would be for such a site to be ultrasensitive to neutralization. These considerations led to a “neutralization-sensitive site” strategy: (i) to identify a viral site targeted by multiple antibodies with extremely potent neutralizing activity, (ii) to determine the structure of the site in complex with a representative antibody, (iii) to engineer the stable presentation of the site in the absence of recognizing antibody, and (iv) to elicit high-titer protective responses through immunization with engineered antigens that stably present the neutralization-sensitive site (fig. S1). Engineering of RSV F Antigens Antigenic site Ø was chosen as the target site because of its recognition by RSV-neutralizing antibodies that are 10- to 100-fold more potent than palivizumab (7, 11, 12). We previously determined the structure of antigenic site Ø in complex with the D25 antibody (7). Structure determination involved appending the T4-phage fibritin trimerization domain (“foldon”) (21, 22) to the C terminus of the RSV F ectodomain (5) and binding of the prefusion-specific D25 antibody. Although these approaches stabilized antigenic site Ø, D25 binding sterically occluded the target site. To stably present antigenic site Ø in the absence of D25, we retained the C-terminal trimerization domain and combined it with other means of stabilization, including the introduction of cysteine pairs or cavity-filling hydrophobic substitutions.’
‘In 2012, however, a group in Spain showed that the majority of neutralizing activity in rabbits inoculated with a recombinant vaccinia expressing the F protein targeted the flighty pre-fusion form of F, not the stable post-fusion form. Not much was known about pre-fusion F because it was so transient, but soon afterwards, Graham and McLellan and their teams at NIAID’s Vaccine Research Center figured out the structure of the pre-fusion F protein as it was bound to a powerful antibody called D25. In particular, Graham and colleagues saw that D25 and other strong neutralizing antibodies attach to the apex of the pre-fusion F protein, each at a different angle, interfering with the protein rearrangement required for the virus to fuse with and enter cells. They named this apical region antigenic site 0, and obtained high-resolution X-ray diffraction data on the complex’s crystals, solving the structure with molecular replacement. Because of its location on the pre-fusion F’s apex, antigenic site 0 is accessible to antibodies even on the crowded surface of a virus, helping to explain why the strongest natural antibodies to RSV target pre-fusion F. “That’s when we really got serious about trying to do the protein engineering steps to stabilize the molecule in the pre-fusion form,” in order to use it as a vaccine antigen, Graham says. Within a year, they’d cracked it. In a 2013 Science paper, the researchers reported that if they added cysteine residues to certain sites and filled some cavities in the protein structure, the protein remained in the pre-fusion state. Injecting this stabilized pre-fusion F protein, which they called DS-Cav1, into mice and macaques generated an RSV-specific neutralizing antibody response many times higher than what is needed to thwart RSV infection. “It was much more immunogenic in terms of inducing neutralizing activity than anything we had [ever seen] before,” says Graham, who is named with McLellan, Kwong and others as a coinventor on patents for pre-fusion F protein antigen design, and consults for RSV vaccine developers.’
"The specification must disclose the invention clearly and completely enough for it to be performed by a person skilled in the art. The key elements of this requirement which bear on the present case are these: i) the first step is to identify the invention and that is to be done by reading and construing the claims; ii) in the case of a product claim that means making or otherwise obtaining the product; iii) in the case of a process claim, it means working the process; iv) sufficiency of the disclosure must be assessed on the basis of the specification as a whole including the description and the claims; v) the disclosure is aimed at the skilled person who may use his common general knowledge to supplement the information contained in the specification; vi) the specification must be sufficient to allow the invention to be performed over the whole scope of the claim; vii) the specification must be sufficient to allow the invention to be so performed without undue burden."
“[The skilled person] must seek success. He may need to carry out the ordinary methods of trial and error, which involve no inventive step and generally are necessary in applying the particular discovery to produce a practical result. In each case, it is a question of fact, depending on the nature of the invention, as to whether the steps needed to perform the invention are ordinary steps of trial and error which a skilled man would realise would be necessary and normal to produce a practical result.”
“Even though a reasonable amount of trial and error is permissible when it comes to the sufficiency of disclosure in an unexplored field or – as it is in this case – where there are many technical difficulties, there must then be available adequate instructions in the specification or on the basis of common general knowledge which would lead the skilled person necessarily and directly towards success through the evaluation of initial failures or through an acceptable statistical expectation rate in case of random experiments.”
“The House of Lords did not throw any doubt on the principle that a claim is not rendered insufficient because there is some room for doubt, or fuzziness, at the edge of the claim. The claim in Kirin-Amgen was insufficient because it was conceptually uncertain.”
“If the court cannot ascertain the boundary, having used all the interpretative tools at its disposal, it must conclude that the specification does not disclose the invention clearly enough and completely enough for it to be performed by a person skilled in the art.”
“… we do not consider that there is any issue of principle which prevents the granting of Arrow declarations in appropriate cases. Drawing the threads together: (i) A declaration that a product, process or use was old or obvious at a particular date does not necessarily offend against s.74 of the Act. (ii) Such a declaration may offend against the Act where it is a disguised attack on the validity of a granted patent. (iii) Such declarations do not offend against the scheme of the EPC or the Act simply because the declaration is sought against the background of pending divisional applications by the counter-party. (iv) On the other hand the existence of pending applications cannot itself be a sufficient justification for granting a declaration. (v) Whether such a declaration is justified depends on whether a sufficient case can be made for the exercise of the court's discretion in accordance with established principles.”