Improved chimeric polypeptides and uses thereof

Abstract

The present disclosure relates generally to chimeric polypeptides comprising a microbial polypeptide (preferably (a) a virion surface exposed portion of an enveloped viral fusion protein or (b) a bacterial outer membrane polypeptide) and a heterologous structure stabilizing portion, and to complexes comprising those chimeric polypeptides. The disclosure also relates to the use of these chimeric polypeptides and complexes thereof in compositions and methods for eliciting an immune response against a microbial polypeptide, preferably a fusion protein of an enveloped virus or a bacterial outer membrane polypeptide, or a corresponding complex thereof, and / or for treating or preventing a related microbial infection, preferably an enveloped virus infection or a bacterial infection. Furthermore, the disclosure relates to compositions and methods for producing antigen-binding molecules that specifically bind such microbial polypeptides or complexes thereof (preferably binding to enveloped viral fusion proteins or complexes thereof, or binding to bacterial outer membrane polypeptides or complexes thereof).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority from the prior European patent application EP 22 166 085.5 filed on March 31, 2022, and the accompanying sequence listing. The contents of that prior application and the accompanying sequence listing are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure generally relates to chimeric polypeptides comprising a microbial polypeptide (preferably (a) a virion surface-exposed portion of an enveloped virus fusion protein or (b) a bacterial outer membrane polypeptide) and a heterologous structural stabilizing portion, and to complexes comprising those chimeric polypeptides. The present disclosure also relates to the use of these chimeric polypeptides and their complexes in compositions and methods for eliciting an immune response against a microbial polypeptide (preferably an enveloped virus fusion protein or a bacterial outer membrane polypeptide) or its corresponding complex and / or for treating or preventing a related microbial infection (preferably an enveloped virus infection or a bacterial infection). In addition, the present disclosure also relates to compositions and methods for producing antigen-binding molecules that specifically bind to such a microbial polypeptide or its complex (preferably an enveloped virus fusion protein or its complex, or a bacterial outer membrane polypeptide or its complex). Background Art

[0004] Enveloped viruses, such as respiratory syncytial virus (RSV), influenza virus, and human immunodeficiency virus (HIV), require the fusion of the viral membrane with the host cell membrane to enter and infect the host cell. The viral fusion protein facilitates this process by undergoing an energetically favorable structural rearrangement from a metastable "prefusion" conformation to a highly stable "postfusion" conformation. This structural change drives the fusion of the viral and host cell membranes, leading to the release of the viral genome into the host cell.

[0005] Viral fusion proteins are currently divided into three main categories based on their respective structural architecture and the molecular characteristics that drive the fusion process. Class I and Class III fusion proteins are trimers in their pre-fusion and post-fusion conformations, while Class II fusion proteins are dimers in their pre-fusion conformations and are then rearranged into trimeric post-fusion forms. However, in the future, it may be possible to identify viral fusion proteins of new categories that have some key features in common with these currently defined categories. Class I and Class III fusion proteins have basic structural features, including N-terminal signal sequences and C-terminal transmembrane and cytoplasmic domains. They also have similar fusion mechanisms, in which the initial pre-fusion trimer undergoes partial dissociation to allow for the formation of the obvious structural rearrangement required for the trimer after fusion.

[0006] Viral fusion proteins are excellent subunit vaccine candidates because they are the main targets of protective neutralizing antibody responses for many medically important enveloped viruses. However, the inherent metastable nature of these fusion proteins, especially when recombinantly expressed as soluble proteins in an isolated state, is a major obstacle to the design of effective subunit vaccines. Evidence shows that most of the broad cross-reactivity and potent neutralizing antibodies elicited during natural infection primarily target the pre-fusion form, rather than the post-fusion form. In addition, it has been shown that the pre-fusion form of viral envelope fusion proteins contains epitopes that are not present or structurally inaccessible in the post-fusion form (e.g., Magro et al., 2012. Proc. Natl. Acad. Sci. USA 109 (8): 3089-3094). Due to these known observations, for the development of vaccines, it is generally believed that stabilized pre-fusion forms are more desirable in terms of antigenicity. However, conventional recombinant expression of these proteins often leads to premature triggering and conformational transition to the more structurally stable post-fusion form. The search has since continued for strategies that would overcome these obstacles previously posed by the inherent structural tendencies of this particular type of protein, primarily by stabilizing them in a more antigenic prefusion state.

[0007] The accumulation of structural information of many relevant viral targets has paved the way for the design of viral fusion protein antigens with stable pre-fusion state based on structure. According to reports, one progress is the development of an engineered form of respiratory syncytial virus (RSV) fusion (F) protein (RSV F), also known as DS-Cav1 Foldon, in which the pre-fusion state is stabilized by introducing stabilizing mutations guided by structure, including artificial disulfide bonds (DS) and hydrophobic cavity filling (Cav1) mutations, and C-terminal fusion to the T4 phage fiber protein trimerization domain (commonly referred to as "Foldon") (McLellan et al., Science.2013; 342(6158): 592-598; Zhang et al., Vaccine.2018; 36(52): 8119-8130.DOI: 10.1016 / j.vaccine.2018.10.032).

[0008] However, a major drawback of such approaches based on "structural vaccinology" is their reliance on high-resolution structural data from each individual viral target, which limits the development process of vaccines against targets whose structures are not yet known. Given the continuous emergence of new viral variants, as is evident for example for SARS-CoV-2 in the ongoing COVID-19 pandemic, there is a particular need for vaccine design strategies that can be easily applied, i.e. as a generally applicable platform technology, without the need for prior access to the corresponding 3D structural information.

[0009] To address this need, some of the present inventors have recently developed a method for stabilizing viral fusion protein antigens in their pre-fusion conformations (WO 2018 / 176103; WO 2022 / 043908). Their technology is based on the discovery that viral fusion proteins can be maintained in their pre-fusion form by operably linking heterologous moieties comprising a pair of complementary heptad repeat regions (HRRs) downstream of the virion surface-exposed domain of the fusion protein. These HRRs promote trimerization with two other chimeric polypeptide subunits, whereby the HRR pairs of each of the three subunits associate into a six-helix bundle structure. The resulting trimeric structure acts as a "molecular clamp" that "locks" the fusion ectodomain polypeptide in the pre-fusion conformation, thereby inhibiting its rearrangement into the post-fusion conformation. The first generation of this technology focused on utilizing a clamp derived from the 6-helix bundle of human immunodeficiency virus (HIV) glycoprotein 41 (gp41).

[0010] Although this technology was successfully applied to the development of a SARS-CoV-2 vaccine candidate, which was shown in a subsequent Phase 1 clinical trial to elicit potent neutralizing antibody responses against the extracellular domain of the enveloped virus fusion protein, additional antibodies against the small HIV gp41-derived clamp domain were also found in all recipients. Although the titers of these antibodies were low, they were sufficient to produce false positive results in some HIV point-of-care diagnostic tests. Due to this observed interference with HIV diagnosis, further development and clinical studies of this vaccine candidate were suspended.

[0011] Therefore, there remains an urgent need for new viral fusion protein subunit vaccines that circumvent the risk of HIV diagnostic interference caused by the clamp domain while having comparable or even improved ability to elicit neutralizing antibody responses against the target antigen, and in particular, new vaccine designs that can be easily applied as general platform technologies to viral targets in the absence of structural information.

[0012] In addition, due to the increasing threat of antibiotic resistance and the resurgence of many related infections, new vaccines for bacterial pathogens are urgently needed. In recent years, bacterial outer membrane proteins have become a major focus of vaccine development because they are proteins that interact with the extracellular environment. A specific type of outer membrane protein found in many pathologically relevant Gram-negative bacteria is the so-called trimeric autotransporter adhesins (TAA), which mediate the first adhesion to the host cell during infection. Therefore, TAA is considered to be an important virulence factor and has received increasing attention as a potential vaccine target. However, most related bacterial infections are not covered by any existing vaccines, and new developments have even slowed down in the past few decades (see, for example, review: Thibau A. et al., Immunogenicity of trimeric autotransporter adhesins and their potential as vaccine targets. Med Microbiol Immunol. 2020; 209(3): 243-263. doi: 10.1007 / s00430-019-00649-y). Therefore, there is a need in the art for vaccines against relevant bacterial targets.

[0013] The present invention satisfies these needs and provides related advantages. Summary of the Invention

[0014] Thus, in a first aspect, the present invention provides a chimeric polypeptide comprising a microbial polypeptide (preferably (a) an enveloped viral fusion ectodomain polypeptide or (b) a bacterial outer membrane polypeptide) operably linked downstream of a heterologous structure stabilizing moiety (SSM), wherein the structure stabilizing moiety is a polypeptide comprising, in N-terminal to C-terminal order, a first heptad repeat region (FHRR) and a second heptad repeat region (SHRR), wherein (i) the FHRR comprises or consists of an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 80 or 81, and the SHRR comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence of SEQ ID NO: 82 or 83; and / or (ii) the FHRR comprises or consists of an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 80 or 81, and the SHRR comprises or consists of an amino acid sequence having at least 70% sequence similarity to the amino acid sequence of SEQ ID NO: 82 or 83.

[0015] In a second aspect, the present invention provides a chimeric polypeptide comprising a first (poly)peptide operably linked downstream of a structure stabilizing portion, wherein the structure stabilizing portion is as defined in the first aspect of the present invention; wherein preferably, the first polypeptide is a therapeutic polypeptide.

[0016] In a third aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a chimeric polypeptide as defined in the embodiments disclosed herein in relation to the first or second aspect of the invention.

[0017] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid defined according to the third aspect of the present invention.

[0018] In a fifth aspect, the present invention provides a method for producing a chimeric polypeptide complex, wherein the method comprises: combining the chimeric polypeptides defined according to the first or second aspect of the present invention under conditions suitable for forming the chimeric polypeptide complex, thereby producing a chimeric polypeptide complex, the chimeric polypeptide complex comprising three chimeric polypeptide subunits and being characterized by a six-helix bundle formed by homotrimerization of the structure-stabilizing portions of the three chimeric polypeptides.

[0019] In a sixth aspect, the present invention provides a chimeric polypeptide complex comprising three chimeric polypeptide subunits, wherein each subunit is a chimeric polypeptide as defined in the first or second aspect of the invention, and wherein the complex is characterized by a six-helix bundle formed by homotrimerization of the structure-stabilizing portions of the three chimeric polypeptides.

[0020] In a seventh aspect, the present invention provides a composition comprising a chimeric polypeptide defined according to the first or second aspect of the present invention, or a chimeric polypeptide complex defined according to the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or adjuvant.

[0021] In an eighth aspect, the present invention provides a method for identifying an agent that binds to a microbial polypeptide or a complex thereof, wherein the method comprises:

[0022] (i) contacting the candidate agent with the chimeric polypeptide comprising a microbial polypeptide as defined in the first aspect of the present invention or the chimeric polypeptide complex comprising a microbial polypeptide as defined in the sixth aspect of the present invention; and

[0023] (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex;

[0024] Preferably, the candidate agent is part of a compound library (eg, a small molecule or large molecule library).

[0025] In a preferred embodiment of the latter aspect, the microbial polypeptide or complex thereof is:

[0026] (a) respectively, a fusion protein of an enveloped virus or a complex of such a fusion protein, wherein the method comprises: (i) contacting a candidate agent with a chimeric polypeptide comprising an enveloped virus fusion ectodomain polypeptide as defined in the first aspect of the present invention or a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain polypeptide as defined in the sixth aspect of the present invention, wherein the enveloped virus fusion ectodomain polypeptide corresponds to a fusion protein of an enveloped virus; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or the chimeric polypeptide complex;

[0027] or

[0028] (b) respectively, an outer membrane polypeptide of a bacterium or a complex of said outer membrane polypeptide, wherein the method comprises: (i) contacting a candidate agent with a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined in the first aspect of the present invention or a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined in the sixth aspect of the present invention, wherein the bacterial outer membrane polypeptide corresponds to an outer membrane polypeptide of the bacterium; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or the chimeric polypeptide complex.

[0029] In a preferred embodiment of the latter embodiment, the bacterial outer membrane polypeptide or complex thereof is:

[0030] (a) a trimeric autotransporter adhesin (TAA) polypeptide of a bacterium or a complex of said TAA polypeptide, respectively, wherein the method comprises: (i) contacting a candidate agent with a chimeric polypeptide comprising a TAA polypeptide as defined in the first aspect of the present invention or a chimeric polypeptide complex comprising a TAA polypeptide as defined in the sixth aspect of the present invention, wherein the TAA polypeptide corresponds to a TAA polypeptide of a bacterium; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or the chimeric polypeptide complex;

[0031] or

[0032] (b) respectively, a major outer membrane protein (MOMP) polypeptide of a Chlamydia bacterium or a complex thereof, wherein the method comprises: (i) contacting a candidate agent with a chimeric polypeptide comprising a Chlamydia MOMP polypeptide as defined in the first aspect of the present invention or a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide as defined in the sixth aspect of the present invention, wherein the Chlamydia MOMP polypeptide corresponds to the MOMP polypeptide of a Chlamydia bacterium; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or the chimeric polypeptide complex.

[0033] In a ninth aspect, the present invention provides a method for producing an antigen-binding molecule that specifically binds to:

[0034] A microbial polypeptide or a complex thereof, wherein the method comprises:

[0035] (1) immunizing a subject with a chimeric polypeptide comprising a microbial polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined in the sixth aspect of the present invention, or a combination thereof as defined in the seventh aspect of the present invention;

[0036] (2) identifying and / or isolating B cells from the immunized subject that specifically bind to the microbial polypeptide or complex thereof; and

[0037] (3) Production of antigen-binding molecules expressed by the B cells.

[0038] In a preferred embodiment of the latter aspect, the microbial polypeptide or complex thereof is:

[0039] (a) respectively, an extracellular domain of a fusion protein of an enveloped virus or a complex of said fusion protein, wherein the method comprises: (1) immunizing a subject with a chimeric polypeptide comprising an extracellular domain polypeptide as defined in the first aspect of the invention, or a chimeric polypeptide complex comprising an extracellular domain polypeptide as defined in the sixth aspect of the invention, or a combination thereof as defined in the seventh aspect of the invention, wherein the extracellular domain polypeptide corresponds to a fusion protein of an enveloped virus; (2) identifying and / or isolating B cells from the immunized subject, said B cells specifically binding to the extracellular domain of the fusion protein or the complex thereof; and (3) producing an antigen binding molecule expressed by the B cells;

[0040] or

[0041] (b) an outer membrane polypeptide of a bacterium or a complex of said outer membrane polypeptide, wherein the method comprises: (1) immunizing a subject with a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined in the first aspect of the present invention, or a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined in the sixth aspect of the present invention, or a combination thereof as defined in the seventh aspect of the present invention, wherein the bacterial outer membrane polypeptide corresponds to the outer membrane polypeptide of the bacterium; (2) identifying and / or isolating B cells from the immunized subject, said B cells specifically binding to the bacterial outer membrane polypeptide or its complex; and (3) producing antigen binding molecules expressed by the B cells.

[0042] In a preferred embodiment of the latter embodiment, the outer membrane polypeptide or complex thereof is:

[0043] (i) respectively, a TAA polypeptide or a complex thereof of a bacterium, wherein the method comprises: (1) immunizing a subject with a chimeric polypeptide comprising a TAA polypeptide as defined in the first aspect of the invention, or a chimeric polypeptide complex comprising a TAA polypeptide as defined in the sixth aspect of the invention, or a combination thereof as defined in the seventh aspect of the invention, wherein the TAA polypeptide corresponds to a TAA polypeptide of a bacterium; (2) identifying and / or isolating B cells from the immunized subject, said B cells specifically binding to the TAA polypeptide or the complex thereof; and (3) producing an antigen binding molecule expressed by the B cells.

[0044] or

[0045] (ii) respectively, a major outer membrane protein (MOMP) polypeptide of a Chlamydia bacterium or a complex thereof, wherein the method comprises: (1) immunizing a subject with a chimeric polypeptide comprising a Chlamydia MOMP polypeptide as defined in the first aspect of the invention, or a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide as defined in the sixth aspect of the invention, or a combination thereof as defined in the seventh aspect of the invention, wherein the Chlamydia MOMP polypeptide corresponds to the MOMP polypeptide of a Chlamydia bacterium; (2) identifying and / or isolating B cells from the immunized subject, said B cells specifically binding to the Chlamydia MOMP polypeptide or a complex thereof; and (3) producing an antigen binding molecule expressed by the B cells.

[0046] In a tenth aspect, the present invention provides an antigen binding molecule that specifically binds to:

[0047] The chimeric polypeptide comprising a microbial polypeptide as defined in the first aspect of the present invention and / or the microbial polypeptide comprising one or more subunits of the chimeric polypeptide complex comprising a microbial polypeptide as defined in the sixth aspect of the present invention.

[0048] In preferred embodiments of the latter aspect, the antigen binding molecule specifically binds:

[0049] (i) an extracellular domain of a chimeric polypeptide comprising an enveloped virus fusion extracellular domain polypeptide as defined in the first aspect of the present invention; and / or an extracellular domain of one or more subunits of a chimeric polypeptide complex comprising an enveloped virus fusion extracellular domain polypeptide as defined in the sixth aspect of the present invention;

[0050] or

[0051] (ii) a bacterial outer membrane polypeptide of a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined in the first aspect of the present invention; and / or a bacterial outer membrane polypeptide of a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined in the sixth aspect of the present invention.

[0052] In preferred embodiments of the latter embodiment, the antigen binding molecule specifically binds:

[0053] (i) a TAA polypeptide of a chimeric polypeptide comprising a TAA polypeptide as defined in the first aspect of the present invention; and / or a TAA polypeptide of a chimeric polypeptide complex comprising a TAA polypeptide as defined in the sixth aspect of the present invention; or

[0054] (ii) a Chlamydia major outer membrane protein (MOMP) polypeptide comprising a chimeric polypeptide of a Chlamydia MOMP polypeptide as defined in the first aspect of the present invention; and / or a Chlamydia MOMP polypeptide comprising a chimeric polypeptide complex of a Chlamydia MOMP polypeptide as defined in the sixth aspect of the present invention.

[0055] In an eleventh aspect, the present invention provides an antigen-binding molecule obtainable by the method according to the ninth aspect of the present invention.

[0056] In a twelfth aspect, the present invention provides a composition comprising an antigen binding molecule as defined in the tenth or eleventh aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or adjuvant.

[0057] In a thirteenth aspect, the present invention provides a composition comprising the nucleic acid defined according to the third aspect of the present invention.

[0058] In the fourteenth aspect, the present invention provides a chimeric polypeptide as defined in the first or second aspect of the present invention, a nucleic acid as defined in the third aspect of the present invention, a host cell as defined in the fourth aspect of the present invention, a chimeric polypeptide complex as defined in the sixth aspect of the present invention, a composition as defined in the seventh aspect of the present invention, an antigen-binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition as defined in the twelfth or thirteenth aspect of the present invention, for use as a drug.

[0059] In a fifteenth aspect, the present invention provides a method of eliciting an immune response against a microbial polypeptide or a complex thereof in a subject, wherein the method comprises administering to the subject:

[0060] (i) a chimeric polypeptide comprising a microbial polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention; or

[0061] (ii) The composition defined in the thirteenth aspect of the present invention.

[0062] In a preferred embodiment of the latter aspect, the microbial polypeptide or complex thereof is:

[0063] (a) respectively, a fusion protein of an enveloped virus or a complex of said fusion protein, wherein the method comprises administering to the subject (i) a chimeric polypeptide comprising a fusion extracellular domain of an enveloped virus as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a fusion extracellular domain of an enveloped virus as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention; or (ii) a composition as defined in the thirteenth aspect of the present invention; wherein the extracellular domain polypeptide subunit of the chimeric polypeptide complex corresponds to a fusion protein of an enveloped virus;

[0064] or

[0065] (b) respectively, an outer membrane polypeptide or a complex of outer membrane polypeptides of a bacterium, wherein the method comprises administering to the subject: (i) a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined in the first aspect of the invention, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined in the sixth aspect of the invention, or a composition thereof as defined in the seventh aspect of the invention; or (ii) a composition as defined in the thirteenth aspect of the invention; wherein the bacterial outer membrane polypeptide subunit of the chimeric polypeptide complex corresponds to or substantially corresponds to a bacterial outer membrane polypeptide expressed by the bacterium.

[0066] In a preferred embodiment of the latter embodiment, the bacterial outer membrane polypeptide or complex thereof is:

[0067] (a) a TAA polypeptide or a complex of TAA polypeptides of a bacterium, respectively, wherein the method comprises administering to a subject: (i) a chimeric polypeptide comprising a TAA polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a TAA polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention; or (ii) a composition as defined in the thirteenth aspect of the present invention; wherein the TAA polypeptide subunit of the chimeric polypeptide complex corresponds to or substantially corresponds to a TAA polypeptide expressed by the bacterium;

[0068] or

[0069] (b) a Chlamydia major outer membrane protein (MOMP) polypeptide or a complex of Chlamydia MOMP polypeptides, respectively, wherein the method comprises administering to the subject: (i) a chimeric polypeptide comprising a Chlamydia MOMP polypeptide as defined in the first aspect of the invention, a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide as defined in the sixth aspect of the invention, or a composition thereof as defined in the seventh aspect of the invention; or (ii) a composition as defined in the thirteenth aspect of the invention; wherein the Chlamydia MOMP polypeptide subunit of the chimeric polypeptide complex corresponds to or substantially corresponds to a Chlamydia MOMP polypeptide expressed by the bacterium.

[0070] In a sixteenth aspect, the present invention provides a method for treating or preventing a microbial infection in a subject, wherein the method comprises administering to the subject an effective amount of:

[0071] (i) a chimeric polypeptide comprising a microbial polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined in the sixth aspect of the present invention, or a combination thereof as defined in the seventh aspect of the present invention;

[0072] (ii) an antigen-binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention; or

[0073] (iii) The composition defined in the thirteenth aspect of the present invention.

[0074] In a preferred embodiment of the latter aspect, the microbial infection is: (a) an enveloped virus infection in a subject, wherein the method comprises administering to the subject an effective amount of: (i) a chimeric polypeptide comprising a fusion extracellular domain of an enveloped virus as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a fusion extracellular domain of an enveloped virus as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention; (ii) an antigen binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention; or (iii) a composition as defined in the thirteenth aspect of the present invention;

[0075] or

[0076] (b) a bacterial infection in a subject, wherein the method comprises administering to the subject an effective amount of: (i) a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention; (ii) an antigen binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention; or (iii) a composition as defined in the thirteenth aspect of the present invention.

[0077] In a preferred embodiment of the latter embodiment, the bacterial infection is:

[0078] (a) infection by bacteria expressing TAA, wherein the method comprises administering to the subject an effective amount of: (i) a chimeric polypeptide comprising a TAA polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a TAA polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention; (ii) an antigen-binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention; or (iii) a composition as defined in the thirteenth aspect of the present invention;

[0079] or

[0080] (b) Chlamydia bacterial infection, wherein the method comprises administering to the subject an effective amount of: (i) a chimeric polypeptide comprising a Chlamydia MOMP polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention; (ii) an antigen binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention; or (iii) a composition as defined in the thirteenth aspect of the present invention.

[0081] In a seventeenth aspect, the present invention provides a vaccine comprising:

[0082] (i) a chimeric polypeptide comprising a microbial polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention; or

[0083] (ii) The composition defined in the thirteenth aspect of the present invention; the vaccine is used in a method for eliciting an immune response against a microbial polypeptide or a microbial polypeptide complex in a subject.

[0084] In a preferred embodiment of the latter aspect, the microbial polypeptide is:

[0085] (a) an enveloped virus fusion extracellular domain polypeptide, the vaccine comprising: (i) a chimeric polypeptide comprising an enveloped virus fusion extracellular domain polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising an enveloped virus fusion extracellular domain polypeptide as defined in the sixth aspect of the present invention, or a combination thereof as defined in the seventh aspect of the present invention; or (ii) a composition as defined in the thirteenth aspect of the present invention; the vaccine is used in a method for eliciting an immune response against an enveloped virus fusion protein or a complex of the fusion protein in a subject, and wherein the extracellular domain polypeptide subunit of the chimeric polypeptide complex corresponds to a fusion protein of the enveloped virus;

[0086] or

[0087] (b) a bacterial outer membrane polypeptide, and the vaccine comprises: (i) a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined in the sixth aspect of the present invention, or a combination thereof as defined in the seventh aspect of the present invention; or (ii) a composition as defined in the thirteenth aspect of the present invention; the vaccine is used in a method for eliciting an immune response against a bacterial outer membrane polypeptide or a complex of the outer membrane polypeptide in a subject, and wherein the bacterial outer membrane polypeptide subunit of the chimeric polypeptide complex corresponds to the outer membrane polypeptide of the bacterium.

[0088] In a preferred embodiment of the latter embodiment, the bacterial outer membrane polypeptide is:

[0089] (a) a trimeric autotransporter adhesin (TAA) polypeptide, and the vaccine comprises: (i) a chimeric polypeptide comprising a TAA polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a TAA polypeptide as defined in the sixth aspect of the present invention, or a combination thereof as defined in the seventh aspect of the present invention; or (ii) a composition as defined in the thirteenth aspect of the present invention; the vaccine is used in a method for eliciting an immune response against a bacterial TAA polypeptide or a complex of the TAA polypeptide in a subject, and wherein the bacterial TAA polypeptide subunit of the chimeric polypeptide complex corresponds to the bacterial TAA polypeptide.

[0090] or

[0091] (b) a Chlamydia MOMP polypeptide, the vaccine comprising: (i) a chimeric polypeptide comprising a Chlamydia MOMP polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide as defined in the sixth aspect of the present invention, or a combination thereof as defined in the seventh aspect of the present invention; or (ii) a composition as defined in the thirteenth aspect of the present invention; the vaccine is used in a method for eliciting an immune response against a MOMP polypeptide of a Chlamydia bacterium or a complex of the MOMP polypeptide in a subject, and wherein the MOMP polypeptide subunit of the chimeric polypeptide complex corresponds to a MOMP polypeptide of the Chlamydia bacterium.

[0092] In the eighteenth aspect, the present invention provides a chimeric polypeptide comprising a microbial polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention, or an antigen binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention, or a composition as defined in the thirteenth aspect of the present invention, for use in a method for treating or preventing a microbial infection in a subject.

[0093] In a preferred embodiment of the latter aspect, the microbial polypeptide is:

[0094] (a) an enveloped virus fusion ectodomain polypeptide, and providing a chimeric polypeptide comprising an enveloped virus fusion ectodomain polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention, or an antigen-binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention, or a composition as defined in the thirteenth aspect of the present invention, for use in a method for treating or preventing an enveloped virus infection in a subject;

[0095] or

[0096] (b) a bacterial outer membrane polypeptide, and provides a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention, or an antigen binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention, or a composition as defined in the thirteenth aspect of the present invention, for use in a method for treating or preventing a bacterial infection in a subject.

[0097] In a preferred embodiment of the latter embodiment, the bacterial outer membrane polypeptide is:

[0098] (a) a bacterial trimeric autotransporter adhesin (TAA) polypeptide, and providing a chimeric polypeptide comprising a bacterial trimeric autotransporter adhesin (TAA) polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a TAA polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention, or an antigen binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention, or a composition as defined in the thirteenth aspect of the present invention, for use in a method for treating or preventing a bacterial infection by a bacterium expressing TAA in a subject;

[0099] or

[0100] (b) Chlamydia MOMP polypeptide, and provides a chimeric polypeptide comprising a Chlamydia MOMP polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide as defined in the sixth aspect of the present invention, or a composition thereof as defined in the seventh aspect of the present invention, or an antigen binding molecule as defined in the tenth or eleventh aspect of the present invention, or a composition thereof as defined in the twelfth aspect of the present invention, or a composition as defined in the thirteenth aspect of the present invention, for use in a method for treating or preventing a Chlamydia bacterial infection in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 Nineteen putative clamp sequences were designed, derived from protein trimerization domains of animal viruses known not to normally infect humans. Potential second-generation clamp sequences were identical to the original HIV-based clamp sequence and were designated HIV-clamp. CD and CK codes were assigned to different second-generation clamps. The virus from which the sequence originated is indicated.

[0102] Figure 2Size exclusion HPLC (SE-HPLC) analysis of selected VL22_66K clamp antigens. AUC = area under the curve. Antigens labeled with CDX or CK8 are VL22_66K CDX or VL22_K66 CK8, respectively. Fsol is a non-stabilized soluble form of the RSV ectodomain and a post-fusion F control (SEQ ID NO: 27). F HIV-Clamp is a control antigen stabilized by HIV-Clamp (SEQ ID NO: 26).

[0103] Figure 3 : Transmission electron microscopy (TEM) of selected VL22_66K clamp antigens. Samples were plated at 10 μg / ml on glow-discharged carbon-coated grids. The antigen-labeled CDX was VL22_K66 CDX.

[0104] Figure 4 : Thermal stability analysis of selected VL22_66K clamps by SE-HPLC. Antigens were incubated at 4°C or 25°C for 72 hours. The antigen-labeled CDX was VL22_66K CDX.

[0105] Figure 5 Comparison of antigen yields recovered from transient ExpiCHO (ThermoFisher) protein expression of the VL22_66K clamp antigen (A) and the VL22_66E clamp antigen (B). ND = Not Determined. In each figure, the dashed line represents the yield recovered from the F HIV-clamp. For simplicity, VL22 was omitted from the VL22 clamp antigen designation in the figure labels.

[0106] Figure 6 : The thermal stability of VL22_CD10, VL22_CD11 and F HIV-clamp was evaluated by binding to RSV F specific mAbs (101F and MPE8) in ELISA. Prior to ELISA, antigens were incubated at 4, 25, 40 and 60°C for 72 hours.

[0107] Figure 7 : The thermal stability of VL22_CD10 and VL22_CD11 as well as FHIV-clamp was evaluated by repeated SE-HPLC analysis. Prior to SE-HPLC, the antigens were incubated at 4, 25, 40 and 60°C for 72 hours.

[0108] Figure 8 : Cryo-electron microscopy of the VL22_CD11 trimer and a low-resolution model generated from the images.

[0109] Figure 9 Cryo-electron microscopy of the VL22_CD11 trimer in complex with Motavizumab Fab and the resulting high-resolution model

[0110] Figure 10 : Sera from 8 mice immunized with SARS-CoV-2 S HIV-clamp were tested by ELISA for binding to VL22_CD10, VL22_CD11, and F HIV-clamp (Figures AC). Endpoint titers (EPT) were plotted as geometric mean versus geometric standard deviation (Figure D).

[0111] Figure 11 : Bio-Rad Geenius HIV 1 / 2 Control Human Plasma (Panels A and B) and the NIBSC International HIV Reference Standard (Panels C and D) were analyzed by ELISA for binding to SARS-CoV-2S HIV-clamp and SARS-CoV-2S CD11.

[0112] Figure 12 : Sera from 8 mice immunized with VL22_CD10 (A), VL22_CD11 (B) and F HIV-clamp (C) were analyzed by ELISA for binding to the same antigen used for vaccination (autoantigen), HIV gp41 (#ab49070, Abcam, Cambridge, UK), to the clamp domain within the antigen when paired with alternative ectodomains (SARS-CoV-2S HIV-clamp, SARS-CoV-2S CD10 and SARS-CoV-2S CD11), and to the ectodomain within the antigen when paired with alternative clamps (FHIV-clamp or SARS-CoV-2S CD11).

[0113] Figure 13 BALB / c mice (n=5 or PBS group n=3) were vaccinated twice, three weeks apart, with each dose consisting of 25 μl AddaVax (Invivogen) and 5 μg VL22_CD10, VL22_CD11, or FHIV-clamp, or a PBS control. Serum neutralization of RSV A2 was assessed by plaque reduction neutralization test (PRNT). The international standard was the WHO First International Standard for Respiratory Syncytial Virus Antiserum (NIBSC; #16-284).

[0114] Figure 14 : SE HPLC of a portion of the CD panel using SARS-CoV-2 spike antigen. Dashed lines indicate the elution times of molecular weight markers. A = thyroglobin (670 kDa), B = gammaglobulin (158 kDa), C = ovalbumin (44 kDa).

[0115] Figure 15SE-FPLC of viral antigens stabilized with HIV-clamp and CD11. Comparison of SARS-CoV-2 S HIV-clamp and SARS-CoV-2 S CD11 on Superose 6 Increase 10 / 300 GL (Cytiva) (Panel A). Comparison of Nipah virus F HIV-clamp and Nipah F CD11 on Superdex 200 Increase 10 / 300 GL (Cytiva) (Panel B). Comparison of influenza HA HIV-clamp and influenza HA CD11 on Superdex 200 Increase 10 / 300 GL (Cytiva) (Panel C).

[0116] Figure 16 BALB / c mice (n=8) were vaccinated twice, three weeks apart, with each dose consisting of 25 μl AddaVax (Invivogen) and 5 μg of each antigen or PBS control. Serum neutralization was assessed by PRNT or pseudovirus neutralization. Neutralization of live virus, SARS-CoV-2 614G mutant, and influenza A (H1N1) pdm California 09, as well as neutralization of Nipah virus with a lentiviral pseudovirus, was assessed by PRNT.

[0117] Figure 17 : SE-FPLC of SARS-CoV-2S CD11 (GSG-linker) (SEQ ID NO: 29) and SARS-CoV-2S CD11-SG (G-linker) (SEQ ID NO: 34) on Superose 6 Increase 10 / 300 GL (Cytiva). Arrows indicate the expected sizes of trimeric and monomeric proteins.

[0118] Figure 18 : Model of the CD11 trimer (grey) with selected regions potentially incorporating N-linked glycosylation sites (black).

[0119] Figure 19 : Alignment of silent CD11 sequences. Shading indicates modified residues, arrows indicate glycosylation sites, and numbering is used to define each site. The nomenclature of the variants reflects the site number (e.g., 159 has glycosylation sites 1, 5, and 9). Gray bars represent samples incubated at 4 and 40°C for 1 week and subsequently analyzed by SE-HPLC.

[0120] Figure 20 : Binding affinity (KD) of 101F and MPE8 to VL22 antigen containing CD11, CD11 silent variants or to non-stabilized Fsol.

[0121] Figure 21 : Protein yield of VL22 antigen containing CD11 or CD11 silent variants. Except for CD11_12456, CD11_1245T68, CD11_15T, CD11_1 and CD11_5T (where the results are from only one expression run), the average yield and standard deviation of two expression runs of the antigen are plotted.

[0122] Figure 22 : SE-HPLC of VL22 antigen containing CD11 or CD11 silent variants. Duplicate traces are overlaid for each sample.

[0123] Figure 23 : SE-HPLC traces of VL22 antigen containing CD11 or CD11 silent variants after incubation at 4 or 40°C for 1 week.

[0124] Figure 24 : SE-HPLC traces of VL22 antigen containing CD11 or CD11 silent variants after 38 days of incubation at 4 or 40° C. Duplicate runs were plotted for each temperature incubation.

[0125] Figure 25 Figure 2: Glycan composition of recombinant antigens. (A) Alignment of N-linked sites in each candidate vaccine and (B) N-linked site-specific occupancy. Note that CD11 refers to VL22_CD11, and CD11 145T8 and CD11 1245T8 refer to silent clamp variants of VL22_CD11. N518 = site 1; N523 = site 2; N530 = site 4; N534 = site 5; N544 = site 8 (e.g., 145T8 has N518, N530, N534, and N544).

[0126] Figure 26 : SE-HPLC traces of lead-silenced CD11-based antigens VL22_CD11_1245T8 and VL22_CD11_145T8, as well as non-silenced VL22_CD11, F HIV-clamp, and controls DS-Cav1 Foldon and SC9-10 DS-Cav AY Foldon.

[0127] Figure 27 : Yields of VL22_CD11_145T8, VL22_CD11, VL22_HIV-Clamp, DS-Cav1 Foldon and SC9-10 DS-Cav AY Foldon. Shown are the average yields and standard deviations of 3-5 individual transient expression runs.

[0128] Figure 28RSV A2PRNT analysis was performed on terminal sera from mice that received two intramuscular injections of 1 μg VL22_CD11_1245T8, VL22_CD11_145T8, VL22_CD11, F HIV-clamp, DS-Cav1 Foldon, or SC9-10DS-Cav AY Foldon mixed with Addavax adjuvant. Error bars represent geometric mean titers and standard deviations, where geometric mean titers are also marked on each data set. Significance relative to the PBS-vaccinated control was determined by one-way analysis of variance and Dunn's multiple comparison test using GraphPad Prism version 9.2.0 for Windows. Significance: ****P<0.0001; ***P<0.001; **P<0.01; *P≤0.05, ns=not significant. International standard refers to the first WHO international standard for respiratory syncytial virus antiserum (NIBSC; #16-284). LoD represents the limit of detection of the assay.

[0129] Figure 29 Binding of terminal sera from mice that received two intramuscular injections of 1 μg of VL22_CD11_1245T8, VL22_CD11_145T8, VL22_CD11, F HIV-clamp, DS-Cav1 Foldon, or SC9-10 DS-Cav AY Foldon adjuvanted with Addavax to the same antigen used for vaccination (autoantigen), RSV F ectodomain with an alternative (stabilizing domain (CD11 or HIV-clamp)), or Nipah F antigen containing the same stabilizing domain (Foldon, HIV-clamp, CD11, CD11_145T8, or CD11_1245T8). Geometric mean endpoint titers (EPTs) and geometric standard deviations are plotted.

[0130] Figure 30 : Alignment of the amino acid sequence of CD11 and site-directed modifications (SEQ ID NOs: 55-72) with the HIV-clamp sequence provided for reference.

[0131] Figure 31 : Expression levels of site-specific modifications of CD11 relative to unmodified CD11 controls. Three measurements were performed by ELISA for each of the SARS-CoV-2S CD11 group, the SARS-CoV-2(Δ)S CD11 group, and the SARS-CoV-2(Δ)S CD11-QS group, and two measurements were performed by Nanodrop (after purification) for each of the SARS-CoV-2(Δ)S CD11 group and the SARS-CoV-2(Δ)S CD11-QS group. Geometric means and geometric standard deviations are plotted.

[0132] Figure 32 : The percentages of antigens in trimeric conformations, HMW aggregates, and LMW products of the SARS-CoV-2(δ)S CD11 site-directed modification group (A) and the SARS-CoV-2(δ)SCD11-QS site-directed modification group (B) were found by SEC.

[0133] Figure 33 : After incubation at 40°C for 48 h, the percentages of antigens in trimeric conformations, HMW aggregates, and LMW products were found in the SARS-CoV-2(δ)S CD11 site-directed modification group (A) and the SARS-CoV-2(δ)S CD11-QS site-directed modification group (B) by SEC.

[0134] Figure 34 : Antigenic integrity of VL22 clamp 2 (D25 mAb) or SARS2-S clamp 2 (CR3022 mAb) was assessed by conformation-specific ELISA at 4°C for 0 or 6 weeks, either individually or as a mixture. nr = not found.

[0135] Figure 35 BalB / C mice (n=8) were vaccinated twice, three weeks apart, with each vaccine dose consisting of 25 μl AddaVax (Invivogen) and 1 μg VL22 clamp 2, 1 μg SARS2-S clamp 2, or 1 μg VL22 clamp 2 + 1 μg SARS2-S clamp 2 or PBS control. Neutralization of RSV A2 (A) or SARS-CoV-2 (B) in serum was assessed by PRNT.

[0136] Figure 36 : Expression of CD11-stabilized bacterial autotransporters was analyzed by SDS-PAGE of E. coli lysates (A) or Western blotting (WB) with an anti-CD11 monoclonal antibody (B). Lane 1 = lysate of negative control E. coli transformed with the pSU vector backbone alone, lane 2 = molecular weight marker (MW listed on the left), lanes 3-9 = lysates of E. coli transformed with the pSU expression vector containing the CD11-stabilizing antigens EahG, HiA, NadA, NhhA, YadA, EibD, and UpaG.

[0137] Figure 37 : CD11-stabilized bacterial autotransporters, Eag (A), UpaG (B), and HiA (C), were purified by affinity chromatography and analyzed by SDS-PAGE.

[0138] Figure 38: Purified CD11-stabilized bacterial autotransporters, EhaG (A), UpaG (B), and HiA (C), were analyzed by size exclusion chromatography on Superose 6 Increase 10 / 300GL (Cytiva). Also shown are the linear regressions of the logarithmic MW and elution volume, and a table comparing the expected and observed MW for each autotransporter.

[0139] Figure 39 : Purified CD11-stabilized bacterial autotransporter UpaG was analyzed by negative staining transmission electron microscopy (TEM).

[0140] Figure 40 BalB / C mice (n=8) were vaccinated twice, three weeks apart, with each vaccine dose consisting of 25 μl AddaVax (Invivogen) and 1 μg VL22 clamp 2, 1 μg SARS2-S clamp 2, 1 μg VL22 clamp 2 + 1 μg SARS2-S clamp 2 1 (CD11), or PBS control. RSV A2 neutralization in serum was assessed by PRNT.

[0141] Figure 41 : Schematic representation of the generation of membrane-tethered clamp-stabilized antigens delivered by LNP-encapsulated mRNA, DNA, or alternative vaccine vectors. A) Standard soluble RSV F clamp 2 design—upon administration, the encoded antigen will be secreted from cells into the extracellular medium. B) Design of a membrane-tethered RSV F clamp 2 in which a hydrophobic sequence is inserted within the linker region between the FHRR and SHRR domains of clamp 2—upon administration, the encoded antigen will be secreted but remain tethered to the cell surface. C) Design of a membrane-embedded RSV F clamp 2 in which the clamp 2 sequence is inserted after the native transmembrane domain, such that the clamp is present on the cytoplasmic side of the membrane—upon administration, the encoded antigen will be secreted but remain tethered to the cell surface.

[0142] Figure 42 : An overview of candidate membrane-bound (poly)peptides, including sources, sequences and structural information.

[0143] Figure 43 : Qualitative assessment of soluble and membrane-bound CLAMP2-stabilized proteins. A) Soluble antigen in cell supernatants was measured by capture ELISA. B) Cell surface-bound antigen was measured by flow cytometry.

[0144] Figure 44Western blot analysis of CHO cell extracts following transfection with membrane-tethering clamp constructs and controls. Each cell extract was run on SDS-PAGE with or without PNGase pretreatment to remove N-linked glycans. Gel 1 (left to right) - molecular weight marker, purified VL22-Clamp 2 (SEQ ID NO: 75), cell extract mFwt (SEQ ID NO: 234), cell extract VL22-CT5s (SEQ ID NO: 78), cell extract mVL22-TM-CD11 (SEQ ID NO: 233), mDSCavAY (SEQ ID NO: 235), second cell extract mFwt (SEQ ID NO: 234). Gel 2 (from left to right) - molecular weight marker, purified VL22-clamp 2 (SEQ ID NO: 75), cell extract VL22_CT5s_α (SEQ ID NO: 225), cell extract VL22_CT5s_γ (SEQ ID NO: 227), cell extract VL22_CT5s_η (SEQ ID NO: 232), cell extract VL22_CT5s_ε (SEQ ID NO: 229), untransfected cell extract.

[0145] Figure 45 : Quantification of expression levels of membrane tethering clamp constructs. Detergent extracts from cell membranes quantified by capture ELISA, A) Motavizumab, B) D25. ELISA curves were evaluated by nonlinear regression, showing the 50% effective concentration (EC 50 ). DETAILED DESCRIPTION

[0146] 1. Definition

[0147] Unless otherwise specified, all terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used to implement or test the present disclosure, preferred methods and materials are described. For the purposes of this disclosure, the following terms are defined below.

[0148] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0149] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when read in the alternative (or).

[0150] In addition, when referring to a measurable value such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length, etc., the terms "about" and "approximately" as used herein are intended to encompass variations of, for example, ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specifically specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length, etc. In cases where the terms "about" and "approximately" are used in connection with the location or position of regions within a reference polypeptide, these terms encompass variations of, for example, ±20 amino acid residues, ±15 amino acid residues, ±10 amino acid residues, ±5 amino acid residues, ±4 amino acid residues, ±3 amino acid residues, ±2 amino acid residues, or even ±1 amino acid residue. In addition, whenever the terms "about" or "approximately" are used, the present invention also specifically relates to the corresponding exact value (without variation).

[0151] As used herein, the term "adjuvant" refers to a compound that, when used in combination with a specific immunogen in a composition (e.g., a (poly)peptide, chimeric polypeptide, chimeric polypeptide complex, polynucleotide, and nucleic acid construct of the present disclosure), will enhance the resulting immune response, including strengthening and / or broadening the specificity of one or both of the antibody and cellular immune response. In the context of the present disclosure, an adjuvant will preferably enhance the specific immunogenic effect of the active agent of the present disclosure. The term "adjuvant" is generally understood to include agents that do not confer immunity themselves. Adjuvants non-specifically assist the immune system to enhance antigen-specific immune responses by, for example, promoting antigen presentation to the immune system or inducing an innate immune response. In addition, adjuvants may preferably, for example, modulate antigen-specific immune responses, for example, by converting a predominantly Th2-based antigen-specific response to a more Th1-based antigen-specific response, or vice versa. Thus, adjuvants can advantageously modulate cytokine expression / secretion, antigen presentation, immune response type, and the like.

[0152] As used herein, the terms "agent" and "compound" are used interchangeably to refer to any compound or substance, such as, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, and the like. An "agent" can be any chemical entity, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, the agent is selected from nucleic acids, nucleic acid analogs, proteins, antibodies, peptides, aptamers, oligomers of nucleic acids, amino acids, carbohydrates, oligonucleotides, ribozymes, deoxyribozymes, glycoproteins, glycolipids, siRNA, lipoproteins, and modifications and combinations thereof. In some embodiments, the nucleic acid is DNA or RNA; the nucleic acid analog, for example, can be selected from PNA, pcPNA, and LNA. The nucleic acid can be single-stranded or double-stranded and can be selected from nucleic acids encoding a protein of interest, oligonucleotides, and the like. These nucleic acids include, for example, nucleic acids encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, such as, but not limited to, RNAi, shRNA, siRNA, microRNA, antisense oligonucleotides, and the like. The protein can be any protein of interest, such as a mutant protein; a therapeutic protein; or a truncated protein, wherein the protein is not normally present in the cell or is expressed at a low level in the cell. The protein and peptide can be selected from mutant proteins, genetically engineered proteins, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins, and fragments thereof. The carbohydrate can be, for example, a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide.

[0153] As used herein, the term "antigen" and its grammatical equivalents (e.g., "antigenic") refer to a compound, composition, or substance that can be specifically bound by a product of specific humoral or cellular immunity (e.g., an antibody molecule or a T-cell receptor). Antigens can be any type of molecule, including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones, as well as macromolecules such as complex sugars (e.g., polysaccharides), phospholipids, and proteins. Common classes of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoan and other parasitic antigens, tumor antigens, antigens involved in autoimmune diseases, allergies, and transplant rejection, toxins, and other miscellaneous antigens.

[0154] "Antigen binding molecules" means molecules with binding affinity to target antigens. It should be understood that this term extends to immunoglobulins, immunoglobulin fragments, and proteins or other non-protein frameworks of non-immunoglobulin sources that exhibit antigen binding activity. Representative antigen binding molecules that can be used to implement the present disclosure include polyclonal antibodies and monoclonal antibodies and fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) and domain antibodies (for example, including shark antibodies and camelid antibodies), and fusion proteins comprising antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen binding / recognition sites. Antibodies include antibodies of any class, such as IgG, IgA or IgM (or their subclasses), and antibodies do not have to be of any specific class. Depending on the antibody amino acid sequence of its heavy chain constant region, immunoglobulins can be classified into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional configuration of the immunoglobulins of different classes are well known. Antigen binding molecules also encompass dimeric antibodies, and antibodies in multivalent form. In some embodiments, the antigen binding molecule is a chimeric antibody in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as a fragment of such an antibody, as long as they exhibit the desired biological activity (see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855). Humanized antibodies are also contemplated, generally produced by transferring complementary determining regions (CDRs) from variable heavy and variable light chains of non-human (e.g., rodent, preferably mouse) immunoglobulins to human variable domains. Common human antibody residues are subsequently replaced in the framework regions of non-human counterparts. The use of antibody components derived from humanized antibodies avoids potential problems associated with the immunogenicity of non-human constant regions. General techniques for cloning non-human immunoglobulin variable domains, particularly murine immunoglobulin variable domains, are described, for example, by Orlandi et al. (1989, Proc. Natl. Acad. Sci. USA 86:3833).Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al. (1986, Nature 321:522), Carter et al. (1992, Proc. Natl. Acad. Sci. USA 89:4285), Sandhu (1992, Crit. Rev. Biotech. 12:437), Singer et al. (1993, J. Immun. 150:2844), Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc. 1995), Kelley ("Engineering Therapeutic Antibodies," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pp. 399-434 (Johnson et al.). Wiley & Sons, Inc. 1996) and Queen et al. U.S. Patent No. 5,693,762 (1997) describe humanized antibodies. Humanized antibodies include "primatized" antibodies, in which the antigen-binding region of the antibody is derived from antibodies produced by immunizing macaques with the antigen of interest. Humanized antibodies are also contemplated as antigen-binding molecules. Other examples of "antigen-binding molecules" include any of the above-mentioned agents, which can be obtained, for example, by using the method according to the eighth aspect of the present invention.

[0155] As used herein, the term "antiparallel" refers to proteinaceous polymers in which regions or segments of the polymer are in a parallel orientation but with opposite polarity.

[0156] As used herein, the term "specific binding" refers to a binding reaction that determines the presence of a chimeric polypeptide of the present invention or a complex in the presence of a heterogeneous population of molecules (including macromolecules such as proteins and other biological products). In a specific embodiment, when referring to antigen binding molecules, the term "specific binding" is used interchangeably with the term "specific immune interaction" to refer to a binding reaction that determines the presence of a chimeric polypeptide of the present invention or a complex in the presence of a heterogeneous population of proteins and other biological products. Under specified assay conditions, the molecule specifically binds to the chimeric polypeptide of the present invention or a complex and does not bind to other molecules (e.g., proteins or antigens) present in the sample in an appreciable amount. In antigen binding molecule embodiments, a variety of immunoassay patterns can be used to select antigen binding molecules that interact specifically with the chimeric polypeptide of the present invention or a complex. For example, solid phase ELISA immunoassays are conventionally used to select monoclonal antibodies that interact specifically with proteins. For descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0157] The term "chimeric," when used in reference to a molecule, means a molecule that contains portions derived from, obtained from, isolated from, or based on two or more different origins or sources. Thus, a polypeptide is chimeric when it comprises two or more amino acid sequences from different sources and comprises (1) polypeptide sequences that do not occur together in nature (i.e., at least one of the amino acid sequences is heterologous with respect to at least one of the other amino acid sequences), or (2) amino acid sequences that are not naturally contiguous.

[0158] "Coding sequence" means any nucleic acid sequence that contributes to encoding the polypeptide product of a gene or the final mRNA product of a gene (e.g., the mRNA product of a gene after splicing). Conversely, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to encoding the polypeptide product of a gene or does not contribute to the final mRNA product of a gene.

[0159] The terms "coiled-coil" or "coiled-coil structure" are used interchangeably herein to refer to a structural motif in proteins in which two or more α-helices (most often 2-7 α-helices) are coiled together like the strands of a rope (dimers and trimers are the most common types). Many coiled-coil-type proteins are involved in important biological functions, such as regulating gene expression, for example, transcription factors. Coiled-coils often, but not always, contain a repeating pattern of hydrophobic (h) and polar (p) amino acid residues, hpphppp or hppphpp, known as a heptad repeat (see below). This repeating pattern in (poly)peptide sequences naturally folds into an α-helical secondary structure, resulting in hydrophobic residues appearing along one side of the helix and hydrophilic residues along the opposite side, forming an amphipathic structure. For two or three such helices, the most favorable way to arrange themselves in an aqueous environment is to wrap or isolate the hydrophobic faces of the helices from each other, while exposing the hydrophilic amino acids to the solvent. Thus, it is the burial of the hydrophobic surfaces that provides the thermodynamic driving force for α-helical oligomerization and the stability of the structure. The coiling at the coiled-coil interface is unusually tight. α-Helices can be parallel or antiparallel and typically adopt a left-handed supercoiled form. Although unfavorable, some right-handed coiled coils have also been observed in nature and in designed proteins. The terms "coiled-coil" or "coiled-coil structure" are readily apparent to those skilled in the art based on common knowledge. Reference is made to the specific literature in this regard for review of papers involving coiled-coil structures, such as, for example, Cohen and Parry (1990. Proteins 7:1-15); Kohn and Hodges (1998. Trends Biotechnol 16:379-389); Schneider et al. (1998. Fold Des 3:R29-R40); Harbury et al. (1998. Science 282:1462-1467); Mason and Arndt (2004. Chem-BioChem 5:170-176); Lupas and Gruber (2005. Adv Protein Chem 70:37-78); Woolfson (2005. Adv Protein Chem 70:79-112); Parry et al. (2008. J Struct Biol 163:258-269); and Mcfarlane et al. (2009. Eur J Pharmacol 625:101-107).

[0160] As used herein, the term "complementary" and its grammatical equivalents refer to the characteristic of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, or portions thereof, etc.) that are capable of hybridizing, oligomerizing (e.g., dimerizing), interacting, or otherwise forming a complex with each other. For example, "complementary regions of polypeptides" are capable of forming together a complex characterized by an antiparallel double helix bundle in specific embodiments.

[0161] As used herein, the term "complex" refers to an aggregate or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with each other. In specific embodiments, "contact" or more specifically, "direct contact" means that two or more molecules are close enough so that attractive non-covalent interactions, such as van der Waals forces, hydrogen bond formation, ionic interactions, and hydrophobic interactions, dominate the interaction of the molecules. In such embodiments, complexes of molecules (e.g., peptides and polypeptides) are formed under such conditions that the complex is thermodynamically favorable (e.g., compared to the unaggregated or uncomplexed state of its component molecules). As used herein, unless otherwise described, the term "complex" refers to an aggregate of two or more molecules (e.g., peptides, polypeptides, or a combination thereof). In specific embodiments, the term "complex" refers to an aggregate of three polypeptides.

[0162] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," "comprising," and "contain," "contains," and "containing" will be understood to indicate the inclusion of a recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. Thus, use of the terms "comprising" and the like indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present. "Consisting of" is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and other elements may not be present. By "consisting essentially of" is meant to include any elements listed after the phrase, and be limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements. In this specification, the term "comprising" (or "containing", etc.) also includes the narrow meanings of "consisting essentially of" and "consisting of". Therefore, whenever the term "comprising" (or "containing", etc.) is used, the present invention also specifically relates to the corresponding subject matter defined by the term "consisting essentially of" and the corresponding subject matter defined by the term "consisting of" (instead of "comprising" or "containing").

[0163] As used herein, the terms "conjugated", "connected", "fused" or "fusion" and their grammatical equivalents are used interchangeably where two or more elements or components or domains are joined together by any means, including chemical conjugation or recombinant means (e.g., by genetic fusion). Methods of chemical conjugation (e.g., using heterobifunctional cross-linkers) are known in the art. More specifically, as used herein, a "(poly)peptide"-"structure stabilizing moiety" fusion or conjugate refers to the genetic or chemical conjugation of a (poly)peptide to a structure stabilizing moiety, the (poly)peptide being suitably in a metastable, pre-fusion conformation.

[0164] In specific embodiments, the structure stabilizing portion is indirectly fused to the polypeptide, for example, via a hinge, particularly a flexible linker, comprising, for example, one or more glycine (Gly) and / or one or more serine (Ser) residues. In other embodiments, the structure stabilizing portion is directly fused to the polypeptide disclosed herein.

[0165] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, and these families can generally be subdivided as shown in Table 1:

[0166] Table 1

[0167] Amino acid subclassification

[0168]

[0169] Conservative amino acid substitutions also include groupings based on side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxy side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similarly replacing an amino acid with a structurally related amino acid will not significantly affect the performance of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can be easily determined by analyzing the activity of the polypeptide. Conservative substitutions are shown in Table 2 under the heading "Exemplary Substitutions and Preferred Substitutions." Generally, amino acid substitutions within the scope of the present disclosure are accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

[0170] Table 2

[0171] Exemplary and preferred amino acid substitutions

[0172]

[0173]

[0174] The term "construct" refers to a recombinant genetic molecule comprising one or more isolated nucleic acid sequences from different sources. Thus, a construct is a chimeric molecule in which two or more nucleic acid sequences from different sources are assembled into a single nucleic acid molecule and includes any construct containing (1) nucleic acid sequences that do not occur together in nature (i.e., at least one of the nucleotide sequences is heterologous to at least one of the other nucleotide sequences), including regulatory sequences and coding sequences that do not occur together in nature, or (2) sequences encoding portions of functional RNA molecules or proteins that are not naturally contiguous, or (3) portions of promoters that are not naturally contiguous. Representative constructs include any recombinant nucleic acid molecule derived from any source that is capable of genomic integration or autonomous replication, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, including nucleic acid molecules in which one or more nucleic acid molecules have been operatively linked. The constructs of the present disclosure will generally contain the necessary elements to direct the expression of a nucleic acid sequence of interest, which is also contained in the construct, such as, for example, a target nucleic acid sequence or a regulator nucleic acid sequence. In some embodiments, the present invention provides a kind of construct that can be used for the expression of the gene of the present invention.This type of element can include control elements, such as the promoter that is effectively connected to the target nucleic acid sequence (thereby instructing its transcription), and often also includes a polyadenylation sequence.Within the scope of some embodiments of the present invention, construct can be contained in the inside of the vector.Except the component of construct, the vector can also for example include one or more selection markers, one or more replication origins (such as prokaryotic replication origin and eukaryotic replication origin), at least one multiple cloning site and / or promote the stable integration of the construct into the element of the host cell genome.Two or more constructs can be contained in a single nucleic acid molecule (such as a single vector) inside, or can be contained in two or more nucleic acid molecules (such as two or more vectors that separate) inside." expression construct " usually includes at least one control sequence that is effectively connected to the target nucleotide sequence.In this way, for example, a promoter that is effectively connected to the nucleotide sequence to be expressed is provided in the expression construct for expression in biology or its part (comprising host cell). Conventional compositions and methods for making and using constructs and host cells for practicing the present invention are well known to those skilled in the art, see, for example, Molecular Cloning: A Laboratory Manual, 3rd Edition, Vols. 1, 2, and 3, JF Sambrook, DW Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0175] "Corresponds to" or "corresponding to" refers to a nucleic acid sequence or amino acid sequence that exhibits substantial sequence similarity or identity with a reference nucleic acid sequence or amino acid sequence, respectively. Typically, a nucleic acid sequence or amino acid sequence will exhibit at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or even up to 100% sequence similarity or identity, with increasing preference, to at least a portion of a reference nucleic acid sequence or amino acid sequence, or to the entire reference nucleic acid sequence or amino acid sequence.

[0176] As used herein, the term "domain" refers to a portion of a molecule or structure that shares common physicochemical characteristics, such as, but not limited to, hydrophobicity, polarity, globular and helical domains or properties, such as ligand binding, membrane fusion, signal transduction, cell penetration, and the like. Typically, a domain has a folded protein structure that has the ability to retain its tertiary structure, independent of the rest of the protein. Typically, a domain is responsible for independent functional properties of a protein, and in many cases, it can be added, removed, or transferred to other proteins without losing the function of the rest of the protein and / or domain. A domain can coexist with multiple regions or portions thereof; a domain can also include different discrete regions of a molecule. Examples of protein domains include, but are not limited to, cellular localization domains or extracellular localization domains (e.g., signal peptides; SPs), immunoglobulin (Ig) domains, membrane fusion (e.g., fusion peptides; FP) domains, extracellular domains, membrane proximal external regions (MPER) domains, transmembrane (TM) domains, and cytoplasmic (C) domains.

[0177] In the context of treating a condition, inhibiting the development of a condition, or preventing a condition, an "effective amount" refers to an amount of a pharmaceutical agent or composition administered to an individual in need of such treatment, inhibition, or prevention, in a single dose or as part of a series of doses, that is effective in preventing the onset of symptoms, controlling such symptoms, and / or treating existing symptoms of the condition. The effective amount will vary depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, the assessment of the medical condition, and other relevant factors. It is expected that this amount will fall within a relatively wide range that can be determined by routine experimentation.

[0178] The term "endogenous" refers to a polypeptide or portion thereof that is present and / or naturally expressed inside a host organism or cell thereof. For example, an "endogenous" extracellular domain polypeptide or portion thereof refers to an extracellular domain polypeptide or portion of an envelope fusion protein that is naturally expressed in an enveloped virus.

[0179] The term "endogenously produced" refers to expression of a nucleic acid in an organism and the associated production and / or secretion of an expression product of the nucleic acid in the organism. In specific embodiments, the organism is multicellular (e.g., a vertebrate, preferably a mammal, more preferably a primate such as a human) and the nucleic acid is expressed in a cell or tissue of the multicellular organism.

[0180] The terms "epitope" and "antigenic determinant" are used interchangeably herein to refer to an antigen that is capable of specifically binding to an antibody (such epitopes are often referred to as "B cell epitopes") or presenting a major histocompatibility complex (MHC) protein (e.g., class I or class II) to a T cell receptor (such epitopes are often referred to as "T cell epitopes"), generally referring to a protein determinant. Where a B cell epitope is a peptide or polypeptide, it generally comprises three or more amino acids, typically at least 5 and more generally at least 8 to 10 amino acids. Amino acids can be adjacent amino acid residues in the primary structure of a polypeptide (often referred to as contiguous peptide sequences), or can become spatially close in a folded protein (often referred to as non-contiguous peptide sequences). T cell epitopes can be bound to MHC class I or MHC class II molecules. Typically, T cell epitopes that bind to MHC class I are 8 to 11 amino acids long. Class II molecules bind to peptides that can be 10 to 30 residues or longer, with an optimal length of 12 to 16 residues. The ability of putative T cell epitopes to bind to MHC molecules can be predicted and experimentally confirmed (Dimitrov et al., 2010. Bioinformatics 26(16):2066-8).

[0181] As used herein, the term "flexible linker" refers to a protein molecule containing at least one amino acid residue, typically at least two amino acid residues connected by a peptide bond, which allows the two polypeptides connected thereby to move more freely relative to each other than in the absence of the flexible linker. In certain embodiments, the flexible linker provides an increased degree of rotational freedom for the two polypeptides connected thereby compared to the two polypeptides connected in the absence of the flexible linker. This freedom of relative movement or rotational freedom allows the polypeptides connected by the flexible linker to perform their respective functions or induce their activity with less structural hindrance. Flexible linkers can be characterized by the absence of secondary structure, such as helices or beta-sheets, or a maximum secondary structure content of 10%, 20%, 30% or 40%. Non-limiting examples of flexible linkers include the amino acid sequences GS, GSG, GGSGG (SEQ ID NO: 84), GGSG (SEQ ID NO: 149), GSGS (SEQ ID NO: 150), AS, GGGS (SEQ ID NO: 151), G4S (SEQ ID NO: 152), (G4S)2 (SEQ ID NO: 153), (G4S)3 (SEQ ID NO: 154), (G4S)4 (SEQ ID NO: 155), G4SG (SEQ ID NO: 156), GSGG (SEQ ID NO: 157), and GSGGS (SEQ ID NO: 158). In alternative preferred embodiments, the linker comprises or consists of the amino acid sequence G, GG, GGG, GGGG (SEQ ID NO: 159), or GGGGG (SEQ ID NO: 160). Other flexible linker sequences are well known in the art. In various embodiments, the flexible linker contains or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues. In some of the same and other embodiments, the flexible linker contains or consists of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues.In some of the same and other embodiments, the flexible linker contains or consists of about 1 to about 30 amino acid residues, about 1 to about 25 amino acid residues, about 1 to about 20 amino acid residues, about 1 to about 15 amino acid residues, about 1 to about 12 amino acid residues, about 1 to about 10 amino acid residues, about 1 to about 8 amino acid residues, about 1 to about 6 amino acid residues, about 1 to about 5 amino acid residues, about 1 to about 4 amino acid residues, or about 1 to about 3 amino acid residues. In some of the same and other embodiments, the flexible linker contains or consists of about 2 to about 30 amino acid residues, about 2 to about 25 amino acid residues, about 2 to about 20 amino acid residues, about 2 to about 15 amino acid residues, about 2 to about 12 amino acid residues, about 2 to about 10 amino acid residues, about 2 to about 8 amino acid residues, about 2 to about 6 amino acid residues, about 2 to about 5 amino acid residues, or about 2 to about 4 amino acid residues. In some of the same and other embodiments, the flexible linker contains or consists of about 3 to about 30 amino acid residues, about 3 to about 25 amino acid residues, about 3 to about 20 amino acid residues, about 3 to about 15 amino acid residues, about 3 to about 12 amino acid residues, about 3 to about 10 amino acid residues, about 3 to about 8 amino acid residues, about 3 to about 6 amino acid residues, or about 3 to about 5 amino acid residues. In certain embodiments, the flexible linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. Specifically, the flexible linker can be composed of amino acid residues (e.g., having any of the exemplary numbers of amino acid residues described above), wherein preferably at least about 70% (more preferably at least about 80%, even more preferably at least about 90%, even more preferably at least about 95%, still more preferably 100%) of the amino acid residues are selected from glycine, serine, and alanine; more preferably, at least about 70% (more preferably at least about 80%, even more preferably at least about 90%, even more preferably at least about 95%, still more preferably 100%) of the amino acid residues are selected from glycine and serine. In some embodiments, the amino acid residues are all glycine.

[0182] The term "helix bundle" refers to a plurality of peptide helices that are folded such that the helices are substantially parallel or antiparallel to one another. A two-helix bundle has two helices that are folded such that they are substantially parallel or antiparallel to one another. Similarly, a six-helix bundle has six helices that are folded such that they are substantially parallel or antiparallel to one another. "Substantially parallel or antiparallel" means that the helices are folded such that the side chains of the helices can interact with one another. For example, the hydrophobic side chains of the helices can interact with one another to form a hydrophobic core.

[0183] The term "heterologous," when used to refer to portions of a nucleic acid or protein, means that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are often recombinantly produced, having two or more sequences from unrelated genes arranged to form a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein means that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0184] The term "host" refers to any organism or cell thereof, whether eukaryotic or prokaryotic, into which the constructs of the present disclosure can be introduced. In a specific embodiment, the term "host" refers to a eukaryotic organism, including unicellular eukaryotic organisms such as yeast and fungi and multicellular eukaryotic organisms such as animals, non-limiting examples of which include invertebrates (e.g., insects, coelenterates, echinoderms, nematodes, etc.); eukaryotic parasites (e.g., malarial parasites such as Plasmodium falciparum, worms, etc.); vertebrates (e.g., fish, amphibians, reptiles, birds, mammals); and mammals (e.g., rodents, primates such as humans and non-human primates). Therefore, the term "host cell" appropriately encompasses cells of such eukaryotic organisms and cell lines derived from such eukaryotic organisms.

[0185] Reference herein to "immunological interaction" includes reference to any interaction, reaction or other form of association between molecules and in particular where one of the molecules is or mimics a component of the immune system.

[0186] As used herein, the term "immunogenic composition" or "immunogenic preparation" refers to a preparation that will induce an immune response when administered to a vertebrate, particularly an animal such as a mammal.

[0187] The term "linker" or "flexible linker" refers to a molecule or group of molecules (such as a monomer or polymer) that connects two molecules and often serves to place the two molecules in a desired configuration.

[0188] As used herein, the term "metastable," as used in the context of proteins (e.g., enveloped viral ectodomain polypeptides), refers to an unstable but constrained conformational state that rapidly transitions to a more stable conformational state when conditions change. For example, an enveloped viral fusion protein in its prefusion form is in an unstable metastable conformation and, when fused to a host cell, transitions to a more stable postfusion conformation.

[0189] As used herein, the term "moiety" refers to a portion of a molecule, which may be a functional group, a group of functional groups, and / or a specific group of atoms within a molecule that is responsible for the characteristic chemical, biological, and / or medical properties of the molecule.

[0190] The term "neutralizing antigen binding molecule" refers to an antigen binding molecule that binds to or interacts with a target molecule or ligand and prevents the target antigen from binding to or associating with a binding partner such as a receptor or substrate, thereby suspending a biological response that would otherwise occur due to the interaction of the molecules. In the context of the present disclosure, the neutralizing antigen binding molecule suitably associates with a metastable or prefusion form of an enveloped viral fusion protein and preferably interferes with or reduces the binding and / or fusion of the spike protein to the cell membrane.

[0191] In contrast to polymers, which are composed of an unlimited number of monomers, at least in principle, the term "oligomer" refers to a molecule composed of more than one but limited number of monomeric units. Oligomers include, but are not limited to, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, and the like. An oligomer can be a macromolecular complex formed by non-covalent bonding of macromolecules (such as proteins). In this sense, homo-oligomers will be formed by identical molecules, and in contrast, hetero-oligomers will be composed of at least two different molecules. In a specific embodiment, the oligomer of the present disclosure is a trimeric polypeptide complex composed of three polypeptide subunits. In these embodiments, the trimeric polypeptide can be a "homotrimeric polypeptide complex" composed of three identical polypeptide subunits or a "heterotrimeric polypeptide complex" composed of three polypeptide subunits in which at least one subunit polypeptide is different. A "polypeptide subunit" is a single amino acid chain or monomer that is combined with two other polypeptide subunits to form a trimeric polypeptide complex.

[0192] As used herein, the term "operably connected" or "operably linked" refers to a juxtaposition wherein the components so described are in a relationship that permits them to function in their intended manner. For example, a regulatory sequence (e.g., a promoter) that is "operably linked" to a nucleotide sequence of interest (e.g., a coding sequence and / or non-coding sequence) means that the regulatory sequence is positioned and / or oriented relative to the nucleotide sequence of interest to permit expression of the sequence of interest under conditions compatible with the regulatory sequences. Regulatory sequences do not have to be contiguous with the nucleotide sequence of interest, as long as they function to direct its expression. Thus, for example, an intervening non-coding sequence (e.g., a sequence that is not translated but transcribed) can be present between a promoter and a coding sequence, and the promoter sequence can still be considered "operably linked to the coding sequence." Likewise, "operably linking" an enveloped viral fusion ectodomain polypeptide to a heterologous structure stabilizing moiety (SSM) includes positioning and / or orienting the structure stabilizing moiety (SSM) such that it can associate with the structure stabilizing moieties (SSMs) of two additional chimeric polypeptides under appropriate conditions (e.g., in aqueous solution and / or physiological conditions) to form a trimer, wherein preferably, in the trimer, the FHRRs and SHRRs of the three SSMs are associated in the form of a six-helix bundle.

[0193] The terms "patient," "subject," "host," or "individual," as used interchangeably herein, refer to any subject in need of treatment or prevention, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrates within the scope of the present disclosure include, but are not limited to, any member of the subphylum Chordata, including primates (e.g., humans, monkeys, and apes, and including monkey species such as from the genus Macaca (e.g., cynomolgus monkeys, such as Macaca fascicularis and / or rhesus monkeys (Macaca mulatta)) and baboons (Papioursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri), and leucocephalians (species from the genus Saguinus), and ape species such as chimpanzees (Panthera fascicularis). Troglodytes))), rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), cattle (e.g., cattle), sheep (e.g., sheep), goats (e.g., goats), pigs (e.g., pigs), horses (e.g., horses), dogs (e.g., dogs), cats (e.g., cats), birds (e.g., chickens, turkeys, ducks, geese, or other poultry, pet birds such as canaries, parrots, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.) and fish. Preferred subjects are humans, particularly those in need of stimulating an immune response against an enveloped virus fusion protein or its complex. However, it should be understood that the aforementioned terms do not indicate the presence of symptoms.

[0194] "Pharmaceutically acceptable carrier" means a solid or liquid filler, diluent or encapsulated material that can be used safely when applied topically or systemically to an animal, preferably a mammal (including a human). Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterials, antifungals), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavorings (flavoring agents), dyes, similar materials of this type and combinations thereof, as known to those of ordinary skill in the art (e.g., referring to Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except where any conventional carrier is incompatible with the active ingredient, the use of the carrier in a pharmaceutical composition is contemplated.

[0195] As used herein, the terms "polynucleotide" or "nucleic acid" encompass any molecule containing two or more nucleotides, particularly polymers of nucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleotides can be, for example, deoxyribonucleotides, ribonucleotides or nucleotide analogs, and they can optionally be substituted or modified. The nucleotides can be linked by phosphodiester bonds / bondings or, for example, by phosphorothioate bonds, methylphosphonate bonds or borophosphate bonds. The terms "polynucleotide" or "nucleic acid" particularly relate to DNA or RNA, such as mRNA, cRNA or cDNA. The term generally refers to a polymeric form of nucleotides (either ribonucleotides or deoxynucleotides or modified forms of any type of nucleotides), for example, of at least 10 bases in length. "Polynucleotide" or "nucleic acid" can be single-stranded or double-stranded.

[0196] The terms "peptide," "polypeptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymer of two or more amino acids linked by amide bonds (i.e., peptide bonds) formed between the amino group of one amino acid and the carboxyl group of another amino acid. The amino acids (also referred to as amino acid residues) comprised in the peptide, polypeptide, (poly)peptide or protein may be selected from the 20 standard proteinogenic α-amino acids (i.e. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val), but may also be selected from non-proteinogenic and / or non-standard α-amino acids (e.g. ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, α-methylalanine (i.e. 2-aminoisobutyric acid), norvaline, norleucine, tert-leucine, labonion or alanine or glycine substituted with a cyclic group on the side chain, such as cyclopentylalanine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, thienylalanine, cyclohexylglycine or phenylalanine). Preferably, the amino acid residues contained in the peptide, polypeptide or protein are selected from α-amino acids, more preferably selected from 20 standard proteinogenic α-amino acids (which can exist in L-isomers or D-isomers, preferably all in L-isomers). The peptide, polypeptide or protein may be unmodified or modified, for example at its N-terminus, its C-terminus and / or at a functional group in the side chain of any amino acid residue thereof (particularly at a side chain functional group of one or more Lys, His, Ser, Thr, Tyr, Cys, Asp, Glu and / or Arg residues). Such modifications may include, for example, attachment of any protecting group to the corresponding functional group described in the following literature: Wuts et al. PG & Greene TW, Greene's protective groups in organic synthesis, John Wiley & Sons, 2006. These modifications may also include covalent attachment of one or more polyethylene glycol (PEG) chains (forming a PEGylated peptide, polypeptide or protein), covalent attachment of albumin, glycosylation and / or acylation with one or more fatty acids (e.g., one or more C 8-30Alkanonic or alkenoic acids; forming fatty acid acylated peptides, polypeptides, or proteins). In addition, such modified peptides, polypeptides, or proteins may also include peptide mimetics, provided that they contain at least two amino acids linked by an amide bond (formed between the amino group of one amino acid and the carboxyl group of another amino acid). The amino acid residues contained in the peptide, polypeptide, or protein may, for example, exist as a linear molecular chain (forming a linear peptide, polypeptide, or protein), or may form one or more rings (corresponding to a cyclic peptide, polypeptide, or protein). Peptides, polypeptides, or proteins may also form oligomers composed of two or more identical or different molecules. Thus, peptides, polypeptides, and proteins may form dimers, trimers, and higher oligomers, wherein the peptide, polypeptide, or protein molecules forming such dimers, trimers, etc. may be identical or different. Therefore, the corresponding higher-order structures are referred to as homodimers or heterodimers, homotrimers or heterotrimers, and homo-oligomers or hetero-oligomers (etc.). Such dimers, trimers, and oligomers are also encompassed by the terms "peptide," "polypeptide," "(poly)peptide," and "protein."

[0197] The term "amino acid" refers in particular to any of the 20 standard proteinogenic α-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val), but also to non-proteinogenic and / or non-standard α-amino acids (e.g., ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, α-methylalanine (i.e., 2-aminoisobutyric acid), norvaline, norleucine, tert-leucine, labonion, or any amino acid replaced by a cyclic group on the side chain). The term "amino acid" refers to a substituted alanine or glycine, for example cyclopentylalanine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, thienylalanine, cyclohexylglycine or phenylalanine, as well as β-amino acids (e.g. β-alanine), γ-amino acids (e.g. γ-aminobutyric acid, isoglutamine or statins) and / or δ-amino acids, and any other compound comprising at least one carboxylic acid group and at least one amino group. Unless otherwise defined, "amino acid" preferably refers to an α-amino acid, more preferably any one of the 20 standard proteinogenic α-amino acids (which can exist as an L-isomer or a D-isomer, preferably as an L-isomer).

[0198] As used herein, the "post-fusion conformation" of the term enveloped virus fusion protein refers to the structure of the enveloped virus fusion protein, which is in the terminal conformation (i.e., formed at the end of the fusion process) and is the most energetically favorable state. In the post-fusion conformation, the fusion peptide or loop of the fusion protein is in close proximity to the fusion protein transmembrane domain. The specific structural elements that promote the formation of a hairpin structure vary according to the category of the envelope fusion protein. For example, the post-fusion conformation of Class I fusion proteins is characterized by the interaction between the endogenous FHRR region and the endogenous SHRR region of each Class I fusion protein to form a hairpin structure, and the hairpin structure is characterized by a six-helix bundle comprising three endogenous SHRR regions and three endogenous FHRR regions. Alternatively, the post-fusion conformation of Class III fusion proteins is characterized by the interaction between the internal fusion loop and the C-terminal transmembrane region that promotes the formation of a hairpin structure. The postfusion conformation of each viral fusion protein has been determined by electron microscopy and / or X-ray crystallography, and such structures are readily recognizable when viewed in negative-stained electron micrographs and / or by the absence of a prefusion epitope.

[0199] As used herein, the term "pre-fusion conformation" of an enveloped viral fusion protein refers to the structure of an enveloped viral fusion protein that is in a metastable conformation (i.e., in a semistable conformation that is not the most energetically favorable terminal conformation) and that can undergo conformational rearrangement to a terminal post-fusion conformation when appropriately triggered. Typically, the pre-fusion conformation of a viral fusion protein contains a hydrophobic sequence, referred to as a fusogenic peptide or fusion loop, that is located within the pre-fusion conformation and that cannot interact with the viral membrane or host cell membrane. Once triggered, this hydrophobic sequence inserts into the host cell membrane and the fusion protein collapses into a post-fusion hairpin conformation. The pre-fusion conformation of a viral fusion protein varies according to the class of the envelope fusion protein. Each class is characterized by non-interacting structural elements that subsequently associate in an energetically favorable post-fusion conformation. For example, the pre-fusion conformation of class I fusion proteins relies on an endogenous FHRR region that does not interact with the endogenous SHRR region of the respective fusion protein of the trimer, thereby preventing the formation of a hairpin structure characterized by a six-helix bundle. Alternatively, the prefusion conformation of class III fusion proteins relies on a central α-helical coiled coil that does not interact with the fusion loop at the C-terminal region of the respective fusion proteins of the trimer, thereby not allowing hairpin structure formation. The prefusion conformation of various viral fusion proteins has been determined by electron microscopy and / or X-ray crystallography, and such structures are readily recognizable when viewed in negative-stained electron micrographs and / or based on the absence of prefusion epitopes in the postfusion conformation.

[0200] "Regulatory element," "regulatory sequence," "control element," "control sequence," and the like are used interchangeably herein to refer to nucleotide sequences that are located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that directly or indirectly affect the transcription, RNA processing or stability, or translation of the bound coding sequence. Regulatory elements include enhancers, promoters, translation leader sequences, introns, Rep recognition elements, intergenic regions, and polyadenylation signal sequences. They include both natural and synthetic sequences, as well as sequences that can be a combination of synthetic and natural sequences.

[0201] The term "replicon" refers to any genetic element that behaves as an autonomous unit of polynucleotide replication inside a cell, ie, capable of replication under its own control, such as a plasmid, chromosome, virus, cosmid, etc.

[0202] "Self-assembly" refers to the process by which higher-level structures spontaneously assemble, relying on the natural attraction of the components (e.g., molecules) of the higher-level structure to each other. It generally occurs through the random motion of molecules and forms bonds based on size, shape, composition, or chemical properties.

[0203] As used herein, the term "sequence identity" refers to the sequence matching between two (poly)peptides or nucleic acids. The (poly)peptide or nucleic acid sequences to be compared are aligned to provide maximum identity, for example, using a bioinformatics tool for pairwise alignment, such as EMBOSS Needle (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ; see also Madeira F, et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Research. 2019 Jul; 47 (W1): W636-W641. DOI: 10.1093 / nar / gkz268). When the same position in the sequences to be compared is occupied by the same nucleobase or amino acid residue, the corresponding molecules are identical at that position. Therefore, "percent identity" is a function of the number of matching positions divided by the number of compared positions and multiplied by 100%. For example, if 6 of 10 sequence positions are identical, the identity is 60%. "Identity" or "percent (%) identity" between two amino acid sequences can be determined, for example, by using the Needleman-Wunsch algorithm (Needleman, SB and Wunsch, CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970; 48(3): 443-53. DOI: 10.1016 / 0022-2836(70)90057-4.) which has been incorporated into EMBOSS Needle, using the BLOSUM62 matrix, a "gap opening penalty" of 10, a "gap extension penalty" of 0.5, a pseudo "end gap penalty," a "terminal gap opening penalty" of 10, and a "terminal gap extension penalty" of 0.5. The percent (%) identity is typically determined over the entire length of the query sequence being analyzed. Two molecules having the same primary amino acid or nucleic acid sequence are identical regardless of any chemical and / or biological modifications that have occurred therein. For example, two antibodies with the same primary amino acid sequence but different glycosylation patterns are identical according to this definition. For example, in the case of nucleic acids, two molecules with the same sequence but different linking components, such as phosphorothioate instead of phosphate, are identical according to this definition.

[0204] "Similar" (poly)peptide sequences are sequences that, when aligned, have similar amino acid residues, and typically (but not necessarily) have identical amino acid residues at the same position in the sequences being compared. Similar amino acid residues are divided into families based on the chemical properties of their side chains. Such families are described below under "conservative amino acid substitutions". The "similarity" or "percent (%) similarity" between sequences is the number of positions containing identical or similar residues at the same sequence position in the sequences being compared, divided by the total number of positions compared and multiplied by 100%. For example, if 6 of 10 sequence positions have identical amino acid residues and 2 of 10 sequence positions contain similar residues, then the sequences have 80% similarity. For example, the percent similarity between two sequences can be determined using EMBOSS Needle (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ) using the BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extension penalty" of 0.5, a pseudo "end gap penalty," a "terminal gap open penalty" of 10, and a "terminal gap extension penalty" of 0.5. The percent similarity (%) is typically determined over the entire length of the query sequence being analyzed.

[0205] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds.

[0206] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and / or therapeutic in terms of partially or completely curing a disease and / or the side effects attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be predisposed to developing the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its progression; and (c) alleviating the disease, i.e., causing regression of the disease.

[0207] The terms "wild-type," "native," and "naturally occurring" are used interchangeably herein to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type, native, or naturally occurring gene or gene product (e.g., polypeptide) is that which is most frequently observed in a population and is therefore arbitrarily designated as the "normal" or "wild-type" form of the gene or gene product.

[0208] Each embodiment described herein should apply mutatis mutandis to each and every embodiment unless specifically stated otherwise.

[0209] As used herein, the term "enveloped virus fusion ectodomain polypeptide" refers to a polypeptide that contains the virion surface-exposed portion of a mature enveloped virus fusion protein, with or without a signal peptide, but lacks the transmembrane domain and cytoplasmic tail of a naturally occurring enveloped virus fusion protein.

[0210] 2. Chimeric polypeptides

[0211] The present invention is based on a further development of a strategy for artificially stabilizing or "clamping" enveloped virus fusion protein extracellular domain polypeptides in a pre-fusion conformation. Typically, this "molecular clamping" strategy utilizes the fusion or bonding of a structure stabilizing moiety (SSM) to an extracellular domain polypeptide to form a chimeric polypeptide.

[0212] The structural stabilizing moiety (SSM) is typically a single-chain polypeptide comprising complementary heptad repeats that lack complementarity with the extracellular domain polypeptide and therefore preferentially associate with each other rather than with the structural elements of the extracellular domain polypeptide. The complementary heptad repeats associate with each other under conditions suitable for their association (e.g., in aqueous solution) resulting in the formation of an antiparallel double helix bundle that inhibits the rearrangement of the extracellular domain polypeptide into a post-fusion conformation. The double helix bundle of the structural stabilizing moiety can trimerize to form a highly stable six-helix bundle, thereby allowing the chimeric polypeptide to self-assemble to form an artificial enveloped virus fusion protein complex. The complex so assembled can mimic the pre-fusion conformation of the natural fusion protein complex of the enveloped virus and comprises three chimeric polypeptides characterized by a six-helix bundle, formed by the coiled-coil structure of each structural stabilizing moiety of the chimeric polypeptide.

[0213] Although the clamp technology initially established was found to be adversely associated with antibody induction that interfered with HIV diagnosis due to the use of a structural stabilizing portion (SSM) derived from human immunodeficiency virus (HIV) glycoprotein 41 (gp41), the inventors set out to develop alternative clamp constructs that overcome these obstacles while providing at least comparable or even improved stability in the ability of the targeted viral fusion protein antigen in its "pre-fusion" conformation, and therefore inducing protective neutralizing antibody responses during vaccination. To this end, the inventors designed a group of nineteen putative clamp sequences derived from the trimerization domains of the fusion proteins of selected viruses known to not normally infect humans. Nineteen candidate clamps were fused to the RSV fusion protein ectodomain. Subsequently, evaluation through comprehensive biophysical characterization identified lead molecules that were fusion proteins derived from two genetically related goat lentiviruses, two constructs derived from Vesna virus (also known as Visna-Maedi virus, Maedi-Visna virus (MVV) and Ovine lentivirus) (CD9, CD11; see Example 1) and another construct derived from caprine arthritis-encephalitis virus (CAEV) (CD10; see Example 1).

[0214] The results presented herein demonstrate that the generated novel vaccine leads are viable alternatives to previous HIV-clamp-based molecules. Furthermore, the data suggest that these leads are superior in stability and at least equivalent in their ability to elicit neutralizing antibody responses compared to earlier developed HIV-clamp-based vaccine candidates.

[0215] Based on the newly identified lead structure, the chimeric polypeptides defined in the present invention are characterized as comprising a microbial polypeptide, preferably an enveloped viral fusion ectodomain polypeptide, operably downstream linked to a heterologous structure stabilizing moiety (SSM) having specific sequence properties as detailed below.

[0216] Similarly, the "molecular clamp" strategy disclosed herein can also be appropriately used to maintain other (poly)peptides in a trimeric state. For example, according to a preferred embodiment of the first aspect of the present invention, a bacterial outer membrane polypeptide (preferably a bacterial trimeric autotransporter adhesin (TAA) polypeptide) is fused to a structure stabilizing portion (SSM). The double helix bundle of the structure stabilizing portion can then also be trimerized to form a highly stable six-helix bundle, thereby also allowing the self-assembly of bacterial outer membrane polypeptide trimers (e.g., TAA polypeptide trimers).

[0217] 2.1 Structural stabilization

[0218] As used herein, a "structure stabilizing moiety (SSM)" is a polypeptide comprising, in N-terminal to C-terminal order, a first heptad repeat region (FHRR) and a second heptad repeat region (SHRR), wherein the FHRR and SHRR are optionally linked to each other via a linker region (preferably in the following N-terminal to C-terminal order: FHRR-Linker-SHRR), as further defined below (e.g., in Section 2.1.2).

[0219] 2.1.1 Heptad repeat sequence

[0220] The α-helical coiled-coil has been characterized at the amino acid sequence level, characterized by each helix consisting of a series of heptad repeats. A heptad repeat (septet unit, septet) is a seven-residue sequence motif that can be coded as hpphppp, where each 'h' represents a hydrophobic residue and each 'p' is a polar (i.e., hydrophilic) residue. Occasionally, a p residue is observed at the h position, and vice versa. Heptad repeats are also often coded according to the patterns abcdefg (abcdefg) or defgabc (defgabc), in which case the designations 'a' through 'g' refer to the conventional seven-fold positions at which common amino acid types are observed. By convention, the designations 'a' and 'd' refer to the positions of core residues (central, buried residues) in the coiled-coil. The common amino acid types observed at the core a- and d-positions are hydrophobic; at all other positions (non-core positions), polar (hydrophilic) residue types are predominantly observed. Thus, the conventional septet pattern 'hpphppp' matches the pattern notation 'abcdefg' (the 'hppphpp' pattern matches the pattern notation 'defgabc', which is used for coiled coils starting with a hydrophobic residue at the d-position).

[0221] The heptad repeat region (HRR) of the present invention comprises at least 2 and appropriately 3 or more (preferably continuous, i.e., uninterrupted) heptad repeat sequences in each α-helical coiled-coil structure. Each series of continuous heptad repeat sequences in a helix is ​​referred to as a 'heptad repeat sequence' (HRS). If available, the start and end points of the heptad repeat sequence are preferably determined based on an experimentally determined three-dimensional (3-D) structure. If a 3-D structure is not available, the start and end points of the heptad repeat sequence are determined based on the best coverage of the actual amino acid sequence to the (hpphppp)n or (hppphpp)n pattern, where 'h' and 'p' refer to hydrophobic residues and polar (i.e., hydrophilic) residues, respectively, and where 'n' is a number equal to or greater than 2. The start and end points of each heptad repeat sequence are taken as the first hydrophobic residue at the a-position or the last hydrophobic residue at the d-position, respectively. Conventional H residues are preferably selected from valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, threonine, serine and alanine, more preferably from valine, isoleucine, leucine and methionine, and most preferably isoleucine. Conventional p-residues are preferably selected from glycine, alanine, cysteine, serine, threonine, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine and arginine. If this method does not allow unambiguous assignment of amino acid residues to heptad repeat sequences, more specialized analytical methods can be applied, such as the COILS method of Lupas et al. (1991. Science 252: 1162-1164; http: / / www.russell.embl-heidelberg.de / cgi-bin / coils-svr.pl).

[0222] In the chimeric polypeptide according to the present invention, the "structure stabilizing moiety (SSM)" comprises a first heptad repeat region (FHRR) and a second heptad repeat region (SHRR) in N-terminal to C-terminal order, wherein (i) FHRR comprises the residue corresponding to SEQ ID 80 or 81 having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity, or consisting of an amino acid sequence having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity, and the SHRR comprises The amino acid sequence shown in NO:82 or 83 has at least 40% (or, with increasing preference, at least 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 200%, 201 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity; and / or (ii) FHRR comprises or consists of an amino acid sequence having a sequence identity to SEQ 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence similarity to the amino acid sequence shown in SEQ ID NO:80 or 81, and the SHRR comprises or consists of an amino acid sequence having at least 70% (or, with increasing preference, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence similarity to the amino acid sequence shown in SEQ ID NO:82 or 83.

[0223] According to the present invention, (i) FHRR comprises or consists of an amino acid sequence having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity to the amino acid sequence shown in SEQ ID NO: 80, and (ii) SHRR comprises or consists of an amino acid sequence having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity to the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in NO:82 has at least 40% (or, with increasing preference, at least 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity; and / or (ii) FHRR comprises or consists of an amino acid sequence identical to SEQ 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence similarity to the amino acid sequence shown in SEQ ID NO:80, and the SHRR comprises or consists of an amino acid sequence having at least 70% (or, with increasing preference, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence similarity to the amino acid sequence shown in SEQ ID NO:82.

[0224] In some embodiments, (i) FHRR comprises, or consists of, an amino acid sequence having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 100%) sequence identity to the amino acid sequence shown in SEQ ID NO:80, and (ii) SHRR comprises, or consists of, an amino acid sequence having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 100%) sequence identity to the amino acid sequence shown in SEQ ID NO:80 The amino acid sequence shown in NO:82 has at least 40% (or, with increasing preference, at least 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity; and / or (ii) FHRR comprises or consists of an amino acid sequence identical to SEQ 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence similarity to the amino acid sequence shown in SEQ ID NO:80, and the SHRR comprises or consists of an amino acid sequence having at least 70% (or, with increasing preference, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence similarity to the amino acid sequence shown in SEQ ID NO:82.

[0225] In some embodiments, the structure stabilizing moiety is capable of homotrimerizing with the structure stabilizing moieties of two additional chimeric polypeptides; wherein preferably, a six-helix bundle is formed by homotrimerization, wherein the six-helix bundle is composed of an inner trimer of three substantially alpha-helical FHRRs oriented in parallel, and the three substantially alpha-helical SHRRs are packed in an antiparallel orientation relative to the FHRRs.

[0226] In some embodiments, the FHRR and SHRR each comprise an independently selected 7-residue motif repeated n times, characterized by the sequence (abcdefg-) n or (defgabc-) n An amino acid pattern represented by , wherein pattern elements 'a' to 'g' represent the positions at which amino acids are located, n is a number equal to or greater than 2, and at least 50% of positions 'a' and 'd' are occupied by hydrophobic amino acids, and at least 50% of positions 'b', 'c', 'e', ​​'f' and 'g' are occupied by polar (hydrophilic) amino acids.

[0227] As to which amino acids are classified as belonging to the group of hydrophilic (polar) or hydrophobic (non-polar) amino acids, respectively, in the context of the present disclosure, reference is made to Table 1 above.

[0228] In a preferred embodiment of the latter embodiment, the FHRR comprises four independently selected repeats of a 7-residue motif and the SHRR comprises five independently selected repeats of a 7-residue motif, wherein the 7-residue motif is characterized by an amino acid pattern represented by (abcdefg-) or (defgabc-), wherein the pattern elements 'a' to 'g' represent the positions at which amino acids are located, and at least 50% of positions 'a' and 'd' are occupied by hydrophobic amino acids, and at least 50% of positions 'b', 'c', 'e', ​​'f' and 'g' are occupied by hydrophilic amino acids.

[0229] In some embodiments, the structure stabilizing moiety has a glutamine at a position corresponding to position 17 of SEQ ID NO:80.

[0230] It is evident from the experimental data disclosed herein that the presence of the corresponding mutation (Gln17) results in a slight increase in soluble protein production (see Example 4, Table 4; CD11 vs. CD9), indicating a stabilizing effect on protein folding and trimer binding. Additional data consistently indicate that this mutation also advantageously enhances thermal stability (see Example 5, Figure 4). In view of these favorable effects provided by the glutamine at position 17, it is expected that similar positive effects will be provided by substitution of asparagine or substitutions similar to glutamic acid or aspartic acid, which are also considered as alternative preferred embodiments herein. However, since constructs with serine or threonine at position 17 also provide suitable trimer stabilization, it is also contemplated as an additional alternative embodiment that the structure stabilizing moiety has a serine or threonine at the position corresponding to position 17 of SEQ ID NO: 80.

[0231] In an alternative preferred embodiment, the structure stabilizing moiety has a leucine at the position corresponding to position 17 of SEQ ID NO: 80. As is apparent from the data provided in Example 16, the presence of the corresponding mutation (Leu17; see construct denoted "CT9") results in a significant improvement in the presence of trimers in solution, as revealed by size exclusion chromatography (SEC) analysis ( Figure 32 ), and the thermal stability of the trimer was also significantly improved ( Figure 33 ).

[0232] In certain embodiments, the structural stabilizing portion comprises at least one immunologically silencing portion that can reduce or inhibit the induction of an immune response to the structural stabilizing portion. These embodiments are advantageous because they can allow for the generation of a selective and / or enhanced immune response to a microbial polypeptide, such as, for example, (i) an enveloped virus fusion ectodomain polypeptide or a complex thereof; or (ii) in the case where the chimeric polypeptide comprises a bacterial outer membrane polypeptide (e.g., a TAA polypeptide) or a complex thereof.

[0233] The immunologically silent portion can be a glycosylation site that is specifically recognized and glycosylated by one or more glycosylases (particularly glycosyltransferases). Glycosylation can be N-linked or O-linked. N-linked refers to the connection of the sugar moiety to the side chain of an asparagine residue. The tripeptide sequences NXS and NXT (represented in three-letter codes as Asn-Xaa-Ser and Asn-Xaa-Thr, respectively), where X (Xaa) is any amino acid except P (Pro), are recognition sequences for the enzymatic attachment of the sugar moiety to the asparagine (Asn) side chain, and these sequences are often referred to as "glycosylation sites" or "glycosylation sequences." O-linked glycosylation refers to the attachment of one of the monosaccharide units N-acetylgalactosamine (GalNAc) or galactose (Gal) to a hydroxyamino acid, most commonly serine (Ser) or threonine (Thr), although 5-hydroxyproline or 5-hydroxylysine may also be used, which may be further extended by additional monosaccharide units such as Gal, GalNAc or N-acetylglucosamine (GlcNAc). The immunologically silencing moiety may be inserted into the structurally stabilizing moiety, comprising one or two heptad repeat regions.

[0234] In particularly preferred embodiments of the latter embodiment, the at least one immunologically silent moiety is an N-linked glycosylation site.

[0235] In an even more preferred embodiment, the N-linked glycosylation sites are post-translationally modified and glycans (e.g., polysaccharides, oligosaccharides, or monosaccharides) are attached. The attached glycans may correspond to those known to be commonly used in a particular mammal (preferably Homo sapiens) that are envisioned for administration with the chimeric polypeptide or its complex or with any composition provided herein to reduce or circumvent an immune response to the corresponding protein sequence. One skilled in the art will be able to select a suitable host cell for recombinant expression of the chimeric polypeptide and its complex, wherein the host cell has a glycosylation machinery that produces N-glycans that are endogenous to the targeted mammalian subject species and therefore may provide an immune silencing effect.

[0236] In some embodiments, the structure stabilizing portion comprises at least one glycosylation site; wherein preferably, the at least one glycosylation site is an N-linked glycosylation site selected from: (1) -Asn-Xaa-Ser-; and (2) -Asn-Xaa-Thr-; wherein Xaa is an amino acid other than Pro; wherein preferably, the glycosylation site is glycosylated at an occupancy level of at least 50%.

[0237] Generally, the "occupancy level" of a glycosylation site in a given protein molecule refers to the percentage (%) level of the glycosylation site that is actually glycosylated in a given population of the protein molecule. Those skilled in the art will appreciate that this level depends on the intrinsic sequence and structural properties of the protein, as well as on the organism (expression host) and expression pathway (cytoplasmic vs. secretory) used to recombinantly express the protein. Means and methods for determining and quantifying the "occupancy level %" of a given glycosylation site are known in the art and can be readily applied by a skilled person. An exemplary method for determining the "occupancy level %" of an N-glycosylation site is also described herein; see the accompanying Example 14.

[0238] In some embodiments, the structure stabilizing moiety comprises one or more N-linked glycosylation sites at amino acid positions corresponding to: (i) positions 5-7 of SEQ ID NO:80; (ii) positions 1-3 of SEQ ID NO:82; (iii) positions 6-8 of SEQ ID NO:82; (iv) positions 13-15 of SEQ ID NO:82; (v) positions 17-19 of SEQ ID NO:82; and / or (vi) positions 27-29 of SEQ ID NO:82; wherein preferably, each N-linked glycosylation site is independently -Asn-Xaa-Thr-, wherein Xaa is an amino acid other than Pro.

[0239] In some embodiments, the structure stabilizing portion comprises an N-linked glycosylation site at an amino acid position corresponding to: (i) (ia) positions 5-7 of SEQ ID NO: 80; (ib) positions 1-3 of SEQ ID NO: 82; and (ic) positions 17-19 of SEQ ID NO: 82; or (ii) (ii-a) positions 5-7 of SEQ ID NO: 80; (ii-b) positions 1-3 of SEQ ID NO: 82; (ii-c) positions 17-19 of SEQ ID NO: 82; and (ii-d) positions 27-29 of SEQ ID NO: 82; or (iii) (iii-a) positions 5-7 of SEQ ID NO: 80; (iii-b) positions 1-3 of SEQ ID NO: 82; (iii-c) positions 13-15 of SEQ ID NO: 82; (iii-d) positions 17-19 of SEQ ID NO: 82; and (iii-e) positions 27-29 of SEQ ID NO: 82; or (iv) (iv-a) positions 5-7 of SEQ ID NO: 80; (iii-b) positions 1-3 of SEQ ID NO: 82; (iii-c) positions 13-15 of SEQ ID NO: 82; NO:80 positions 5-7; (iv-b) positions 1-3 of SEQ ID NO:82; (iv-c) positions 6-8 of SEQ ID NO:82; (iv-d) positions 13-15 of SEQ ID NO:82; (iv-e) positions 17-19 of SEQ ID NO:82; and (iv-f) positions 27-29 of SEQ ID NO:82; wherein preferably, each N-linked glycosylation site is independently -Asn-Xaa-Thr-, wherein Xaa is an amino acid other than Pro.

[0240] In some embodiments, the structure stabilizing moiety has (i) an arginine at the amino acid position corresponding to glutamine 22 of SEQ ID NO:80; (ii) a histidine at the amino acid position corresponding to asparagine 1 of SEQ ID NO:82; (iii) a threonine at the amino acid position corresponding to histidine 2 of SEQ ID NO:82; (iv) a serine at the amino acid position corresponding to alanine 25 of SEQ ID NO:82; (v) a glutamine at the amino acid position corresponding to alanine 26 of SEQ ID NO:82; (vi) a glutamine at the amino acid position corresponding to leucine 27 of SEQ ID NO:82; (vii) a leucine at the amino acid position corresponding to glutamine 17 of SEQ ID NO:80; (viii) a deletion of the amino acid residue at the position corresponding to arginine 37 of SEQ ID NO:80; and / or (ix) a deletion of the amino acid residues at the positions corresponding to glutamine 1 and serine 2 of SEQ ID NO:80.

[0241] In preferred embodiments of the latter embodiment, the structure stabilizing moiety has (i) an arginine at the amino acid position corresponding to glutamine 22 of SEQ ID NO: 80; (ii) a histidine at the amino acid position corresponding to asparagine 1 of SEQ ID NO: 82 and a threonine at the amino acid position corresponding to histidine 2 of SEQ ID NO: 82; (iii) a serine at the amino acid position corresponding to alanine 25 of SEQ ID NO: 82, a glutamine at the amino acid position corresponding to alanine 26 of SEQ ID NO: 82, and a glutamine at the amino acid position corresponding to leucine 27 of SEQ ID NO: 82; (iv) a leucine at the amino acid position corresponding to glutamine 17 of SEQ ID NO: 80; (v) a deletion of the amino acid residue at the position corresponding to arginine 37 of SEQ ID NO: 80; and / or (vi) a deletion of the amino acid residues at the positions corresponding to glutamine 1 and serine 2 of SEQ ID NO: 80. From Example 16, in particular Figure 32 and 33 It is apparent from the size exclusion chromatography (SEC) data shown in that each of the above site-specific mutations (i) to (v) (referred to as CT1, CT5, CT6, CT9 and CT10 in Example 16, respectively) further enhances the stability of the trimer relative to unmodified CD11.

[0242] In preferred embodiments, the structure stabilizing moiety has (i) an arginine at the amino acid position corresponding to glutamine 22 of SEQ ID NO:80, and (ii) deletions of the amino acid residues at the positions corresponding to glutamine 1 and serine 2 of SEQ ID NO:80.

[0243] In preferred embodiments, the structure stabilizing moiety has (i) a leucine at the amino acid position corresponding to position 20 of SEQ ID NO: 82; and / or (ii) a glutamine at the amino acid position corresponding to position 11 of SEQ ID NO: 80. From the mutagenesis studies of Example 16, it is apparent that maintaining these amino acids in the wild-type sequence of CD11 is beneficial in stabilizing the trimer.

[0244] In some embodiments, the structure stabilizing moiety comprises one or more unnatural amino acids.

[0245] As used herein, "unnatural" or "non-natural amino acid" refers to an amino acid that is not one of the 20 common naturally occurring amino acids or rare naturally occurring amino acids such as selenocysteine ​​(Sec) or pyrrolysine (Pyl). Other terms that may be used synonymously with the term unnatural amino acid are non-naturally encoded amino acids, non-natural amino acids, non-naturally occurring amino acids, and their various hyphenated and non-hyphenated forms. The term "unnatural amino acid" includes, but is not limited to, amino acids that are naturally produced by modifying naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but which themselves are not incorporated into a growing (poly)peptide chain by the translation complex. Examples of non-naturally encoded naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. In addition, the term unnatural amino acid includes, but is not limited to, amino acids that do not occur naturally and can be obtained synthetically or can be obtained by modification of unnatural amino acids. Non-natural amino acids can be incorporated into chimeric polypeptides (e.g., into one or two of the heptad repeat regions contained therein) or any other (poly)peptide contemplated herein by using an expanded genetic code. Non-natural amino acids are biosynthetically incorporated into the target site using tyrosyl-tRNA / aminoacyl-tRNA synthetase orthogonal pairs and nonsense codons at the target site. Non-natural amino acids are supplied to the cells of the expression construct from an external source, and chimeric polypeptides can be expressed from the construct, and this strategy can incorporate side chains with a wide range of physical properties, including but not limited to chemical cross-linking groups (e.g., azide or haloalkyl), traceable markers (e.g., fluorescent or radioactive), and photosensitive groups to achieve temporally controlled modification. For these non-natural amino acids, each moiety can be covalently linked to the structure stabilizing moiety by chemical addition to provide favorable properties.

[0246] In preferred embodiments of the latter embodiment, the one or more unnatural amino acids allow for conjugation to (i) polyethylene glycol (PEG); (ii) an immunostimulatory moiety; or (iii) a lipid.

[0247] Further embodiments may include any possible combination of the above examples of covalent attachment of structure stabilizing moieties, or additional non-natural chemical additions.

[0248] Optionally, one or more additional cysteine ​​residues may be inserted into the FHRR and / or SHRR to form disulfide bonds and further stabilize the antiparallel α-helical coiled-coil structure of the structure stabilizing moiety.

[0249] 2.1.2 Connectors

[0250] The structure stabilising moiety (SSM) of the present invention may suitably comprise a linker separating the first and second heptad repeat regions (also referred to herein as FHRR and SHRR, respectively).

[0251] The linker typically comprises any amino acid residue that cannot be unambiguously attributed to a heptad repeat sequence. Linkers are frequently used in the field of protein engineering to interconnect different functional units, for example, for the production of single-chain variable fragment (scFv) constructs derived from antibody variable light chains (VL) and variable heavy chains (VH). In solution, they are generally conformationally flexible and appropriately and advantageously comprise polar amino acid residue types. Common (frequently used) amino acids in flexible linkers are serine and glycine. Less preferably, flexible linkers may also comprise alanine, threonine, and proline. Therefore, the intervening linker of the structure stabilizing portion is preferably conformationally flexible to ensure that the relaxed (unhindered) association of the FHRR and SHRR is a double helical bundle that appropriately adopts an α-helical coiled coil structure. Suitable linkers for use in the polypeptides contemplated herein will be apparent to those skilled in the art and may generally be any linker used in the art to connect amino acid sequences, as long as the linker is structurally flexible, meaning that it allows and appropriately does not impair the assembly of the characteristic double helical bundle structure of the structure stabilizing portion.

[0252] One of ordinary skill in the art will be able to determine the optimal linker, optionally after performing a limited number of routine experiments. The intervening linker is suitably an amino acid sequence generally consisting of at least 1 amino acid residue and generally consisting of at least 2 amino acid residues, with a non-critical upper limit selected for convenience being about 100 amino acid residues. In a specific embodiment, the linker consists of about 1 to about 50 amino acid residues, or about 50 to about 100 amino acid residues, generally about 1 to about 40 amino acid residues, and generally about 1 to about 30 amino acid residues. In a non-limiting example, the linker has approximately the same number of amino acids as the number of amino acids connecting the FHRR region and the SHHR region of the Class I enveloped virus fusion protein. In a specific non-limiting embodiment, at least 50% of the amino acid residues of the linker sequence are selected from proline, glycine, and serine. In other non-limiting embodiments, at least 60%, such as at least 70%, such as, for example, 80%, and more specifically 90% of the amino acid residues of the linker sequence are selected from proline, glycine, and serine. In other embodiments, the linker sequence consists essentially of polar amino acid residues; in such embodiments, at least 50%, such as at least 60%, such as for example 70% or 80% and more particularly 90% or up to 100% of the amino acid residues of the linker sequence are selected from glycine, serine, threonine, alanine, proline, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine and arginine. In a particular embodiment, the linker sequence may comprise [GGSG] n GG(SEQ ID NO:161-170),[GGGGS] n (SEQ ID NO:152-155; 171-176), [GGGGG] n (SEQ ID NO:160;177-185), [GGGKGGGG] n (SEQ ID NO:186-195), [GGGNGGGG] n (SEQ ID NO:196-205), [GGGCGGGG] n (SEQ ID NO: 206-215), wherein n is an integer from 1 to 10, suitably from 1 to 5, more suitably from 1 to 3.

[0253] In a preferred embodiment, the FHRR and SHRR comprised in the structure stabilizing moiety (SSM) are connected via a linker. Preferably, the linker comprises or consists of a peptide having the same amino acid sequence as SEQ ID NO: 84 or 85, more preferably comprises or consists of the amino acid sequence defined by SEQ ID NO: 84.

[0254] In addition to spacing the heptad repeat regions (ie, FHRR and SHRR) of the structure stabilizing moiety (SSM), and preferably introducing structural flexibility to promote antiparallel association of these regions, the linker may also comprise one or more auxiliary functional groups.

[0255] For example, the linker can comprise a purification moiety that facilitates purification of the chimeric polypeptide and / or at least one immunomodulatory moiety that modulates the immune response to the chimeric polypeptide.

[0256] The purification moiety generally comprises a stretch of amino acids that enables recovery of the chimeric polypeptide by affinity binding. Numerous purification moieties or 'tags' are known in the art, illustrative examples of which include a biotin carboxyl carrier protein tag (BCCP tag), a Myc tag (c-myc tag), a calmodulin tag, a FLAG-tag, an HA tag, a His tag (hexahistidine tag, His6, 6H, 6×His), a maltose binding protein tag (MBP tag), a Nus tag, a chitin binding protein tag (CBP tag), a glutathione-S-transferase tag (GST tag), a green fluorescent protein tag (GFP tag), a polyglutamic acid tag, an amyloid beta tag, a thioredoxin tag, an S tag, a Softag1, a Softag3, a Strep tag, a streptavidin binding peptide tag (SBP tag), a biotin tag, a streptavidin tag, and a V5 tag.

[0257] Immunomodulatory moieties can be introduced into linkers to modulate the immune response stimulated by the chimeric polypeptide or its complex. Non-limiting examples of such moieties include immunosilencing or suppressive moieties as described above, antigenic moieties, including antigenic moieties derived from pathogenic organisms, or other disease-related antigenic moieties, such as cancer or tumor-associated antigens. Exemplary pathogenic organisms include, but are not limited to, viruses, bacteria, fungi, parasites, algae, and protozoa and amoebas. In specific embodiments, the antigenic moiety is derived from an antigen of a pathogenic virus. Illustrative viruses that cause disease include, but are not limited to, measles, mumps, rubella, polio, hepatitis A, hepatitis B (e.g., GenBank Accession No. E02707), and hepatitis C (e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenoviruses (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Epstein-Barr, and other herpes viruses such as papillomavirus, Ebola, influenza, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBank Accession No. M24444), hantavirus, Sendai, respiratory syncytial virus, orthomyxovirus, vesicular stomatitis, vesina, cytomegalovirus, and human immunodeficiency virus (HIV) (e.g., GenBank Accession No. U18552). Any suitable antigen derived from such viruses can be used to implement the present invention. For example, illustrative retroviral antigens derived from HIV include, but are not limited to, antigens such as gene products of gag, pol, and env genes, Nef protein, reverse transcriptase, and other HIV components. Schematic examples of hepatitis virus antigens include, but are not limited to, antigens such as hepatitis B virus S, M, and L proteins, hepatitis B virus, and other hepatitis viruses (e.g., hepatitis A, B, and C virus) pre-S antigens. Schematic examples of influenza virus antigens include, but are not limited to, antigens such as hemagglutinin and neuraminidase and other influenza virus components. Schematic examples of measles virus antigens include, but are not limited to, antigens such as measles virus fusion protein and other measles virus components. Schematic examples of rubella virus antigens include, but are not limited to, antigens such as E1 and E2 proteins and other rubella virus components; rotavirus antigens such as VP7sc and other rotavirus components. Schematic examples of cytomegalovirus antigens include, but are not limited to, antigens such as envelope glycoprotein B and other cytomegalovirus antigen components. Non-limiting examples of respiratory syncytial virus antigens include antigens such as RSV fusion protein, M2 protein and other respiratory syncytial virus antigen components. Illustrative examples of herpes simplex virus antigens include, but are not limited to, antigens such as immediate early protein, glycoprotein D and other herpes simplex virus antigen components.Non-limiting examples of varicella-zoster viral antigens include antigens such as 9PI, gpII and other varicella-zoster viral antigen components. Non-limiting examples of Japanese encephalitis viral antigens include antigens such as protein E, ME, ME-NS1, NS1, NS1-NS2A, 80% E and other Japanese encephalitis viral antigen components.

[0258] Representative examples of rabies viral antigens include, but are not limited to, antigens such as rabies glycoprotein, rabies nucleoprotein, and other rabies viral antigen components. Illustrative examples of papillomavirus antigens include, but are not limited to, LI and L2 capsid proteins and E6 / E7 antigens associated with cervical cancer. For additional examples of viral antigens, see Fundamental Virology, 2nd Edition, Fields, BN and Knipe, DM, 1991, Raven Press, New York. In specific embodiments, the viral antigen is an antigen corresponding to an extracellular domain polypeptide of an enveloped virus. In other embodiments, the viral antigen is an antigen corresponding to an extracellular domain polypeptide of a different enveloped virus.

[0259] In some embodiments, one or more cancer-associated or tumor-associated antigens are inserted into the linker. Such antigens include, but are not limited to, MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, high molecular weight melanoma-associated antigen MAA, GD2, carcinoembryonic antigen (CEA), TAG-72, ovarian-associated antigens OV-TL3 and MOV18, TUAN, alpha-fetoprotein (AFP), OFP, CA-125, CA-50, CA-19-9, renal tumor-associated antigen G250, EGP-40 (also known as EpCAM), S100 (malignant melanoma-associated antigen), p53, prostate tumor-associated antigens (e.g., PSA and PSMA), p21ras, Her2 / neu, EGFR, EpCAM, VEGFR, FGFR, MUC-1, CA 125, CEA, MAGE, CD20, CD19, CD40, CD33, A3, antigen specific for A33 antibody, BrE3 antigen, CD1, CD1a, CD5, CD8, CD14, CD15, CD16, CD21, CD22, CD23, CD30, CD33, CD37, CD38, CD40, CD45, CD46, CD52, CD54, CD74, CD79a, CD126, CD 138, CD154, B7, Ia, Ii, HMI.24, HLA-DR (e.g., HLA-DR10), NCA95, NCA90, HCG and subunits, CEA (CEACAM5), CEACAM-6, CSAp, EGP-I, EGP-2, Ba733, KC4 antigen, KS-I antigen, KS1-4, Le-Y, MUC2, MUC3, MUC4, PIGF, ED-B fibronectin, NCA 66a-d, PAM-4 antigen, PSA, PSMA, RS5, SIOO, TAG-72, TO, TAG TRAIL-RI, TRAIL-R2, p53, tenascin, insulin growth factor-1 (IGF-I), Tn antigen, etc.

[0260] The antigenic portion or portions contained in the linker can correspond to a full-length antigen or a partial antigen. When a partial antigen is used, the partial antigen can comprise one or more epitopes of the target antigen, including B cell epitopes and / or T cell epitopes (e.g., cytotoxic T lymphocyte (CTL) epitopes and / or helper T cell (Th) epitopes). The epitopes of numerous antigens are known in the literature or can be determined using routine techniques known to those skilled in the art. In other embodiments, the linker can include another cell targeting moiety that can provide delivery to specific cell types within the vaccinated individual. The target cell population includes, but is not limited to, B cells, microfold cells, and antigen presenting cells (APCs). In subsequent examples, the targeting moiety promotes enhanced recognition of the chimeric polypeptide or its complex by APCs, such as dendritic cells or macrophages. Such targeting sequences can enhance the presentation of epitopes of the associated extracellular domain polypeptide by APCs, which can in turn enhance the immune response generated, including strengthening or broadening the specificity of either or both of the antibody immune response and the cellular immune response to the extracellular domain polypeptide. Non-limiting examples of APC targeting moieties include ligands that bind to APC surface receptors, such as, but not limited to, mannose-specific lectin (mannose receptor), IgG Fc receptor, DC-SIGN, BDCA3 (CD141), 33D1, SIGLEC-H, DCIR, CD11c, heat shock protein receptors, and scavenger receptors. In specific embodiments, the APC targeting moiety is a dendritic cell targeting moiety comprising, consisting of, or consisting essentially of the sequence FYPSYHSTPQRP (Uriel et al., J. Immunol. 2004 172:7425–7431) or NWYLPWLGTNDW (Sioud et al., FASEB J 2013 27(8):3272-83).

[0261] 2.1.3 Particularly Preferred Structure Stabilizing Moieties (SSMs)

[0262] The inventors selected two structure-stabilizing moieties as lead constructs and named them "Clamp 2" and "Clamp 2s":

[0263] Clamp 2 (also referred to herein as "CD11-QS") is represented by SEQ ID NO:

[0264] 265 Definition:

[0265] LANATAAQQEVLEAQYAMVQHIAKGIRILEARVARGGSGGNHTWQQWEEEIEQHEGNSLLLREAALQVHIAQRDARRI[SEQ ID NO:265]

[0266] ● Clamp 2s (also referred to herein as "CD11_145T8-QS_CT5",

[0267] which corresponds to a silent variant of clamp 2) is defined by SEQ ID NO: 266:

[0268] LANATAAQQEVLEAQYAMVQHIAKGIRILEARVARGGSGGNHTTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI[SEQ ID NO:266]

[0269] 2.1.4 Membrane binding

[0270] In certain embodiments, it may be advantageous to provide a chimeric polypeptide, or complex thereof, that is tethered to the surface of a cell, such as the surface of a cell expressing the chimeric polypeptide.

[0271] For example, in a chimeric polypeptide according to the first aspect of the invention, which is intended for use as a vaccine, membrane tethering to a cell surface (e.g., the cell surface of a host cell that expresses the chimeric polypeptide following vaccination with an mRNA, viral vector or other nucleic acid encoding the chimeric polypeptide) will allow the chimeric polypeptide (or at least an antigenic portion thereof, i.e., a microbial polypeptide, preferably an enveloped viral fusion ectodomain polypeptide or a bacterial outer membrane polypeptide (e.g., a TAA polypeptide)) to be displayed on the cell surface.

[0272] Without wishing to be bound by any theory, it is believed that a corresponding arrangement may be advantageous in providing enhanced stimulation of the immune response and thus for eliciting a more effective, long-lasting broadly neutralizing antibody response. This is because cells and / or cell-derived organelles that express and thus display high concentrations and / or high densities of the chimeric polypeptide may help to recruit and activate the initial And memory B cells, thereby enhancing the stimulation of potent B cell-mediated neutralizing antibody response. In addition, the interaction with B cells can also be amplified by the affinity effect produced by displaying a variety of chimeric polypeptides or their multiple complexes on the cell surface. B cells also need the auxiliary function of T cells (particularly CD4 T cells) to effectively form neutralizing antibodies. Although the induction of B cell responses requires direct interaction with antigens, CD4 T cells are stimulated by peptides derived from the same antigen complexed with MHCII molecules. Therefore, it is speculated that tethering chimeric polypeptides or their complexes to the surface of cells and / or cell-derived organelles can indirectly lead to the presentation of higher epitopes by MHCII, which can also help to recruit CD4 T cells, thereby further enhancing the immune response and forming effective neutralizing antibodies.

[0273] Two general types of embodiments of membrane-tethered constructs are particularly contemplated herein include:

[0274] (i) Embodiment, wherein the SSM further comprises a membrane-tethering polypeptide; and

[0275] (ii) Embodiment, wherein the chimeric polypeptide further comprises a transmembrane (TM) domain upstream (ie, N-terminal) of the SSM.

[0276] Representative embodiments involving (i) and (ii) are listed below in Sections 2.1.4.1 and 2.1.3.2, respectively.

[0277] 2.1.4.1 Membrane-bound (poly)peptides

[0278] In a particularly preferred embodiment, the chimeric polypeptide of the present invention further comprises a "membrane-tethered (poly)peptide". As used herein, the term "membrane-tethered (poly)peptide", "membrane-anchored (poly)peptide", "membrane-tethered" or "membrane anchor", "membrane-localized (poly)peptide", "membrane-targeted (poly)peptide" or another equivalent thereof refers to a (poly)peptide sequence that can act as an anchor to tether the chimeric polypeptide of the present invention to the extracellular surface of a cell membrane (e.g., the lipid bilayer of a cell membrane). In its broadest sense, the term "membrane-tethered (poly)peptide" or its equivalents as referred to herein encompasses (poly)peptides that are able to at least partially insert into a membrane (e.g., the lipid bilayer of a cell or (nano)lipid particle) due to the inherent and physicochemical properties of the specific amino acid residues contained therein (e.g., amino acids having side chains characterized by high intrinsic hydrophobicity, such as, in particular, tryptophan, phenylalanine, tyrosine, isoleucine, leucine, and valine). However, the term also includes (poly)peptides in which this ability is conferred by the presence of certain chemical or post-translational modifications of one or more of the contained amino acid residues. For example, one or more amino acids may carry a covalently linked lipid / fatty acid. Such amino acids may be, for example, but not intended to be limiting, myristoylated, palmitoylated or isoprenate.

[0279] In general, the membrane-bound (poly)peptide may be suitably included at any position within the chimeric polypeptide, so long as its presence does not negatively interfere, or at least does not substantially negatively interfere, with the folding of the remainder of the chimeric polypeptide and / or sterically conferring its ability to trimerize. Furthermore, those skilled in the art will appreciate that where the chimeric polypeptide comprises a microbial polypeptide, preferably an enveloped viral fusion ectodomain polypeptide or a bacterial outer membrane polypeptide (e.g., a TAA polypeptide), the membrane-bound (poly)peptide should only be included at positions in the chimeric polypeptide where its presence does not sterically interfere, or at least does not substantially sterically interfere, with the surface accessibility of the microbial polypeptide, preferably an enveloped viral fusion ectodomain polypeptide or a bacterial outer membrane polypeptide (e.g., a TAA polypeptide), so as not to negatively interfere with the immunogenic capacity of these antigenic portions.

[0280] As shown by experimental evidence disclosed herein (see, for example, Example 21), the present inventors have found that a construct in which a membrane-tethered (poly)peptide is inserted as a linker between the FHRR and SHRR of SSM is particularly effective in tethering a chimeric polypeptide or a complex thereof to the surface of a cell expressing the chimeric polypeptide. Figure 41 The structural arrangement shown in B, wherein the SSM is oriented proximal to the cell membrane, while the portion of the chimeric polypeptide located upstream of the SSM (e.g., an enveloped virus fusion ectodomain polypeptide or a bacterial outer membrane polypeptide (e.g., a TAA polypeptide)) is displayed distal to the cell surface and protrudes from the cell surface.

[0281] Thus, in preferred embodiments of those embodiments in which the FHRR and SHRR comprised in the SSM are connected via a linker, said linker comprises or consists of a membrane tethered (poly)peptide.

[0282] In order to be particularly suitable for constructs according to the latter embodiment, the membrane-tethered (poly)peptide should have a length and structural conformation that is compatible with the structural arrangement of the FHRR and SSHR in SSM. More specifically, the membrane-tethered (poly)peptide should be of sufficient length to allow its stable insertion into the cell membrane, while its N-terminus and C-terminus should be spatially oriented / separated in a manner that matches the C-terminus and N-terminus of the FHRR and SHRR of SSM, respectively. In addition, if the membrane-tethered (poly)peptide or at least a substantial portion thereof is itself capable of trimerization, it may be beneficial in providing further stabilization and / or rigidification.

[0283] In preferred embodiments, the membrane-tethered polypeptide:

[0284] (i) comprises or consists, with increasing preference, of 10 to 50, 15 to 40, 16 to 30, 18 to 28, 20 to 26, 22 to 24, most preferably 23 amino acid residues; and / or

[0285] (ii) with increasing preference, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid residues selected from tryptophan, phenylalanine, tyrosine, isoleucine, leucine and / or valine.

[0286] In the examples disclosed herein, a panel of eight different membrane-tethering (poly)peptides (defined by SEQ ID NOs: 236-243, respectively) was developed, each of which was subsequently found to be effective in tethering the chimeric polypeptide to the outer surface of the cell membrane (see, Examples 21 and 22). Figure 41 B and 42). Thus, in particularly preferred embodiments, the membrane tethered (poly)peptide comprises or consists of an amino acid sequence having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity to one of the amino acid sequences shown in SEQ ID NOs: 236-243.

[0287] In the specific constructs defined by SEQ ID NOs: 225-232 employed in Example 21, the membrane-tethered (poly)peptide is contained within the linker region connecting the FHRR and SHRR of the SSM. Thus, in these constructs, the membrane-tethered (poly)peptides (defined by SEQ ID NOs: 236-243, respectively) are flanked at the N- and C-termini by two (GG) and three (SGG) amino acids, respectively, derived from the insertion of the original linker sequence GGSGG (defined by SEQ ID NO: 84). Without wishing to be bound by theory, it is speculated that the addition of one or more amino acids, such as glycine or serine (or other small amino acid residues), as flanking moieties will help separate the membrane-tethered (poly)peptide from the rest of the chimeric polypeptide, allowing the former to be inserted into the cell membrane while the latter will be displayed on the surface, thereby allowing for optimal antigen presentation.

[0288] Thus, in preferred embodiments, the membrane tethered (poly)peptide comprises or consists of an amino acid sequence having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity to any one of the amino acid sequences shown in SEQ ID NOs: 244-251.

[0289] While each of the eight membrane-tethered (poly)peptides tested proved effective in tethering the corresponding chimeric polypeptide to the cell membrane, three constructs bearing membrane-tethered (poly)peptides designated herein as "α," "γ," or "ε" (defined by SEQ ID NOs: 236, 238, and 240, respectively) resulted in the highest levels of surface-bound chimeric polypeptide (assessed by flow cytometry using antigen-specific monoclonal antibodies; see Figure 43 B).

[0290] Thus, in preferred embodiments, the membrane tethered (poly)peptide comprises or consists of an amino acid sequence having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity to:

[0291] (a) any one of the amino acid sequences shown in SEQ ID NOs: 236, 238 and 240; or

[0292] (b) any one of the amino acid sequences shown in SEQ ID NOs: 244, 246 and 248.

[0293] Further analysis of the exemplary set of constructs indicated that the addition of the membrane-tethered (poly)peptide designated herein as "γ" (defined by SEQ ID NO: 238) was also most effective in stabilizing the prefusion conformation of the viral fusion protein (RSV F). Thus, in an even more preferred embodiment, the membrane tethered (poly)peptide comprises or consists of an amino acid sequence having at least 60% (or, with increasing preference, at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 100%) sequence identity to SEQ ID NO: 238 or SEQ ID NO: 246. However, in alternative, but less preferred, embodiments, the membrane-bound (poly)peptide may be included at the C-terminal portion of the chimeric polypeptide, i.e., at the C-terminus of the SHRR of the SSM. For example, in an exemplary embodiment of the latter embodiment, the chimeric polypeptide may include a (poly)peptide comprising a glycosylphosphatidylinositol (GPI)-anchor located at the C-terminus of the SHRR of the SSM.

[0294] 2.1.4.2 Membrane tethering by adding a transmembrane region upstream of SSM

[0295] In an alternative embodiment, membrane tethering of the chimeric polypeptide of the present invention can be established by adding a transmembrane (poly)peptide upstream (i.e., N-terminal) of the SSM. Figure 41 The structural arrangement shown in C, wherein the SSM is oriented toward the interior (i.e., cytoplasmic side) of the cell membrane, and the portion of the chimeric polypeptide located upstream (i.e., N-terminus) of the SSM, such as an enveloped viral fusion protein extracellular domain polypeptide or a TAA polypeptide, is displayed extracellularly, i.e., located on the cell surface and protruding therefrom.

[0296] As used herein, the term "transmembrane (poly)peptide" or "transmembrane domain" refers in its broadest sense to any (poly)peptide that can span (i.e., pass through) the lipid bilayer of the cell membrane and thus function to connect the extracellular and intracellular parts of the polypeptide chain. This can be a single α-helix, a transmembrane β-barrel, a β-helix, or any other structure. Typically, a transmembrane domain represents a single transmembrane α-helix of a transmembrane protein, also known as an integral protein.

[0297] For example, in a related embodiment, when the chimeric polypeptide comprises a microbial polypeptide (e.g., an enveloped virus fusion ectodomain polypeptide or a bacterial surface polypeptide (e.g., a bacterial outer membrane polypeptide)), the chimeric polypeptide may further comprise a transmembrane (poly)peptide C-terminus of the microbial polypeptide (e.g., an enveloped virus fusion ectodomain polypeptide or a bacterial surface polypeptide) and an N-terminus of the SSM. In particularly preferred embodiments of the latter embodiment, the transmembrane (poly)peptide is contained in the chimeric polypeptide immediately following the C-terminus of the microbial polypeptide (e.g., the C-terminus of the enveloped virus fusion ectodomain polypeptide or the bacterial surface polypeptide) and the N-terminus of the SSM. In even more preferred embodiments, the transmembrane (poly)peptide corresponds to or substantially corresponds to a transmembrane (poly)peptide naturally contained in a microbial polypeptide (e.g., an enveloped virus fusion polypeptide).

[0298] In a preferred embodiment of the chimeric polypeptide of the first or second aspect of the invention, the chimeric polypeptide further comprises a hinge region operably linking the microbial polypeptide (preferably an enveloped viral fusion ectodomain polypeptide or a bacterial outer membrane polypeptide) and the structure stabilizing moiety (SSM). Thus, in particularly preferred embodiments of the latter embodiment, wherein a transmembrane (poly)peptide is envisioned to be incorporated into the chimeric polypeptide, the hinge region may comprise, further comprise, or consist of a transmembrane (poly)peptide.

[0299] Furthermore, in the case where the chimeric polypeptide comprises an enveloped viral fusion extracellular domain polypeptide, the transmembrane (poly)peptide preferably corresponds to the transmembrane (poly)peptide naturally contained in the enveloped viral fusion polypeptide. In any of the latter embodiments, the transmembrane (poly)peptide may be operably linked to the SSM via a peptide consisting of 3-5 amino acid residues independently selected from serine and glycine.

[0300] As demonstrated in the Examples disclosed herein, such a construct (referred to as "mVL22-TM-CD11" in Example 21 and as defined by SEQ ID NO: 233) has been produced as a "proof-of-concept" and has been shown to be expressed in high yields and to be able to stabilize the extracellular domain of the enveloped viral fusion protein in a pre-fusion conformation. Therefore, in particularly preferred embodiments of the chimeric polypeptide according to the first aspect of the present invention, wherein the extracellular domain polypeptide corresponds to the extracellular domain of a fusion protein from respiratory syncytial virus (RSV) or is a variant thereof, the chimeric polypeptide further comprises a hinge region operably connecting the extracellular domain polypeptide to the SSM, and the hinge region comprises a transmembrane (poly)peptide corresponding to amino acid residues 486-512 of SEQ ID NO: 233. In an even more preferred embodiment, the hinge region comprises or consists of a transmembrane (poly)peptide corresponding to amino acid residues 486-514 of SEQ ID NO: 233.

[0301] 2.2 Microbial peptides

[0302] According to the most general embodiment of the first aspect of the invention, the chimeric polypeptide comprises a microbial polypeptide operably linked downstream to a heterologous structure stabilizing moiety (SSM).

[0303] As used herein, the term "microbial polypeptide" broadly refers to a polypeptide that corresponds or substantially corresponds to a polypeptide of (or derived from) a microorganism, wherein the microorganism is preferably one selected from bacteria, archaea, protists, fungi and viruses.

[0304] In a particularly preferred embodiment, the microorganism is a pathogenic microorganism (ie, the microorganism is a mammalian pathogen, preferably a human pathogen); preferably one selected from bacteria and viruses.

[0305] In view of the trimerization ability of the structure stabilizing moieties (SSMs) disclosed herein, it is believed that the technology disclosed herein is particularly suitable for achieving stabilization of polypeptides that also naturally exist as trimers when used as fusion partners for other polypeptides that exist in a trimeric conformation. Therefore, in a preferred embodiment, the microbial polypeptide (or any more specific form thereof, as defined below) is a polypeptide capable of trimerization.

[0306] Although it is preferred that the chimeric polypeptide of the present invention is a single polypeptide chain in which the structure stabilizing moiety (SSM) is at the C-terminus (downstream) of the microbial polypeptide, the present disclosure also contemplates an alternative arrangement in which the structure stabilizing moiety (SSM) is at the N-terminus (upstream) of the microbial polypeptide. In both cases, the structure stabilizing moiety (SSM) and the microbial polypeptide can be connected by a hinge as defined herein.

[0307] In other or even more preferred embodiments, the microbial polypeptide is a viral surface polypeptide or a bacterial surface polypeptide.

[0308] In even more preferred embodiments, (i) the viral surface polypeptide is an enveloped viral fusion ectodomain polypeptide; or (ii) the bacterial surface polypeptide is a bacterial outer membrane polypeptide.

[0309] When referring to a "variant" of a microbial polypeptide (including any specific microbial polypeptide described herein), this preferably refers to a polypeptide having at least 70% or, with increasing preference, at least 80%, 85%, 90%, 95% or 98% sequence identity to the corresponding microbial polypeptide.

[0310] 2.2.1 Enveloped Virus Fusion Proteins and Extracellular Domain Peptides

[0311] In some embodiments, the enveloped virus fusion ectodomain polypeptide corresponds to the following sequence or is a variant of the following sequence: (i) a class I enveloped virus fusion protein ectodomain; wherein preferably, the ectodomain is from a virus selected from the group consisting of orthomyxoviruses, paramyxoviruses, orthopneumoviruses, metapneumoviruses, retroviruses, coronaviruses, filoviruses and arenaviruses; or (ii) a class III enveloped virus fusion protein ectodomain; or wherein preferably, the ectodomain is from a virus selected from the group consisting of rhabdoviruses and herpesviruses.

[0312] In preferred embodiments of the latter embodiment, the enveloped viral fusion ectodomain polypeptide corresponds to or is a variant of a fusion protein ectodomain, wherein the fusion protein is from: (i) respiratory syncytial virus (RSV); (ii) metapneumovirus; (iii) coronavirus, preferably betacoronavirus, more preferably severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or Middle East respiratory syndrome-related coronavirus (MERS-CoV); (iv) henipavirus, preferably Hendra virus (HeV) or Nipah virus (NiV); (v) influenza virus, preferably influenza A or influenza B; (vi) parainfluenza virus (PIV), preferably human parainfluenza virus (HPIV); (vii) arenavirus, preferably Lassa Fever virus; or (viii) retrovirus, preferably human T-lymphotropic virus-1 (HTLV-1).

[0313] In some embodiments, the chimeric polypeptide further comprises a hinge region that operably links a microbial polypeptide (preferably a bacterial or viral surface polypeptide, more preferably an enveloped virus fusion extracellular domain polypeptide or a bacterial outer membrane polypeptide (e.g., a TAA polypeptide)) to a structure-stabilizing portion; wherein preferably, the hinge region comprises or consists of: (i) a (poly)peptide consisting of 3 to 5 amino acid residues independently selected from serine and glycine; (ii) serine and glycine residues; (iii) GGSG (SEQ ID NO: 149); (iv) GSG; or (v) G.

[0314] Although the most preferred chimeric polypeptide of the present invention is a single polypeptide chain in which the structure stabilizing moiety (SSM) is C-terminal (downstream) of the enveloped virus fusion protein extracellular domain polypeptide, the present disclosure also contemplates another arrangement in which the structure stabilizing moiety is N-terminal (upstream) of the enveloped virus fusion protein extracellular domain. In both cases, the structure stabilizing moiety and the enveloped virus fusion protein extracellular domain can be connected by a hinge as defined herein.

[0315] Non-limiting examples of enveloped virus fusion ectodomain polypeptides are provided below:

[0316] 2.2.1.1 Inf A HA

[0317] Non-limiting examples of Inf A HA extracellular domain polypeptides include:

[0318] Extracellular domain 1-529:

[0319] [SEQ ID NO:86](GenPept gbAEC23340.1).

[0320] The sequence contains the following domains / parts:

[0321] SP=1-16

[0322] Extracellular domain = 17 - 529

[0323] Furin cleavage site = 345 - 346

[0324] FP = 346 - 355

[0325] MPER = 470 - 529

[0326] Head region = 51 - 328, 403 - 444

[0327] Stem region = 17 - 58, 327 - 401, 442 - 509

[0328] Ectodomain minus SP18-529:

[0329] KLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRICNSPHQILDGKNCTLIDALLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWTGVAQDGSSYACRRGSVNSFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIVSTKRSQQTVIPNIGSRPWVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKSSIMRSDAHIDECNSECITPNGSIPNDKPFQNVNKITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMSKLFERTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDIYRNEALNNRFQIKGVQLKSGYKD[SEQ ID NO:87](GenPept gbAEC23340.1).

[0330] Ectodomain minus SP, minus MPER18-469:

[0331] KLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRICNSPHQILDGKNCTLIDALLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWTGVAQDGSSYACRRGSVNSFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIVSTKRSQQTVIPNIGSRPWVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKSSIMRSDAHIDECNSECITPNGSIPNDKPFQNVNKITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMSKLFERTRR[SEQ ID NO:88](GenPept gbAEC23340.1).

[0332] Extracellular domain 18-341, 346-529 plus altered furin cleavage site:

[0333] KLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRICNSPHQILDGKNCTLIDALLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWTGVAQDGSSYACRRGSVNSFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIVSTKRSQQTVIPNIGSRPWVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKSSIMRSDAHIDECNSECITPNGSIPNDKPFQNVNKITYGACPRYVKQNTLKLATGMRNVPERRRKKRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMSKLFERTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDIYRNEALNNRFQIKGVQLKSGYKD[SEQ ID NO:89](GenPept gbAEC23340.1).

[0334] Stem domains 1-58, 327-401, 442-509 plus linker region:

[0335] MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELGFGQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMLDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGMLNRLIGSGGSGELLVALLNQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD[SEQ ID NO:90](GenPept gbAEC23340.1).

[0336] Head domains 1-18, 51-328, 403-444 plus linker region:

[0337] MKTIIALSYILCLVFAQKEVTNATELVQNSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRGSNNSFFSRLNWLTHSKFKYPALNVTMPNNEEFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNGSGGSGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELL[SEQ ID NO:91](GenPeptgbAEC23340.1).

[0338] 2.2.1.2 Inf B HA

[0339] Non-limiting examples of the extracellular domain polypeptide of Inf B HA include:

[0340] Extracellular domain 1-547:

[0341] [SEQ ID NO:92](GenPept gbAFH57854.1).

[0342] The sequence contains the following domains / parts:

[0343] SP=1-16

[0344] Extracellular domain = 17-547

[0345] Furin cleavage site = 361-362

[0346] FP=362-382

[0347] MPER=488-547

[0348] Head area = 48-344, 418-456

[0349] Stem region = 17-47, 345-417, 457-547

[0350] Ectodomain minus SP17-547:

[0351] RICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLNDDGLDNHT[SEQ ID NO:93](GenPept gbAFH57854.1).

[0352] Extracellular domain minus SP, minus MPER 17-487:

[0353] RICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKML[SEQ ID NO:94](GenPept gbAFH57854.1).

[0354] Ectodomain minus SP plus altered furin cleavage sites 17-355, 362-547:

[0355] RICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPARRRKKRAGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLNDDGLDNHT[SEQ ID NO:95](GenPept gbAFH57854.1).

[0356] Stem domains 1-47, 345-417, 457-547 plus linker region:

[0357] MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLGSGLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSGSGGSGIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLNDDGLDNHT[SEQ ID NO:96](GenPept gbAFH57854.1).

[0358] Head domains 1-17, 48-344, 418-456 plus linker region:

[0359] MKAIIVLLMVVTSNADRTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKGSGGSGELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQ[SEQ ID NO:97](GenPept gbAFH57854.1).

[0360] 2.2.1.3 RSV F

[0361] Non-limiting examples of RSV F extracellular domain polypeptides include:

[0362] Extracellular domain 1 - 524:

[0363] [SEQ ID NO:98](GenPept gbAHL84194.1).

[0364] The sequence contains the following domains / parts:

[0365] SP=1-23

[0366] Extracellular domain = 24-524

[0367] Furin cleavage sites = 109-110, 136-137

[0368] FP=137-163

[0369] D25 interaction domain = 61-97, 193-240

[0370] RSV F extracellular domain (1-520):

[0371] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRRLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGK[SEQID NO:99].

[0372] Extracellular domain minus SP 24-524:

[0373] SGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRRLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN[SEQ ID NO:100](GenPeptgbAHL84194.1).

[0374] Ectodomain minus SP plus altered furin cleavage site 24-524:

[0375] SGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNNANNELPRFMNYTLNNAKKTNVTLSNNNNNNFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN[SEQ ID NO:101](GenPept gbAHL84194.1).

[0376] D25 interaction domain 61-97, 193-240 plus linker region:

[0377] LSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMGGLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVN[SEQ ID NO:102](GenPept gbAHL84194.1).

[0378] 2.2.1.4 hMPV F

[0379] The exemplary hMPV F precursor has the following amino acid sequence:

[0380] [SEQ ID NO:103](GenPept gbAAN52913.1).

[0381] The sequence contains the following domains / parts:

[0382] SP=1-19

[0383] Extracellular domain = 1-490

[0384] Furin cleavage site = 102-103

[0385] FP=103-125

[0386] FHRR=126-169

[0387] SHRR=456-490

[0388] TM=491-514

[0389] C=515-539

[0390] Non-limiting examples of hMPV F ectodomain polypeptides include:

[0391] Extracellular domain 1-490:

[0392] MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTG[SEQ ID NO:104](GenPeptgbAAN52913.1).

[0393] Ectodomain minus SP20-490:

[0394] KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTG[SEQ ID NO:105](GenPept gbAAN52913.1).

[0395] Ectodomain minus SP plus altered furin cleavage site 20-490:

[0396] KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPNQSNFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTG[SEQ ID NO:106](GenPept gbAAN52913.1).

[0397] 2.2.1.5 PIV F

[0398] Non-limiting examples of the extracellular domain polypeptide of PIV F include:

[0399] Extracellular domain 1-493:

[0400] MPTSILLIITTMIMASFCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVSNQESNENTDPRTKRFFGGVIGTIALGVAT SAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTV DKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDI VPRYAFVNGGVVANCITTTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTT[SEQ ID NO:107](GenPeptgbAAB21447.1).

[0401] The sequence contains the following domains / parts:

[0402] SP=1-19

[0403] Extracellular domain = 1-493

[0404] Furin cleavage site = 109-110

[0405] FP=110-135

[0406] Extracellular domain minus SP 20-493:

[0407] IDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVSNQESNENTDPRTKRFFGGVIGTIALGVATSAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTT[SEQ ID NO:108](GenPept gbAAB21447.1).

[0408] Ectodomain minus SP plus altered furin cleavage site 20-493:

[0409] IDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVSNQESNENTDPNTKNFFGGVIGTIALGVATSAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTT[SEQ ID NO:109](GenPept gbAAB21447.1).

[0410] 2.2.1.6 MeV F

[0411] Non-limiting examples of the extracellular domain polypeptides of MeV F include:

[0412] Extracellular domain 1–493:

[0413] MGLKVNVSAIFMAVLLTLQTPTGQIHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALG VATAAQITAGIALHQSMLNSQAIDNLRASLETNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEK LGYSGGDLLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVS GSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSST[SEQ ID NO:110](GenPeptdbjBAB60865.1).

[0414] The sequence contains the following domains / parts:

[0415] SP=1-24

[0416] Extracellular domain = 1-493

[0417] Furin cleavage site = 112-113

[0418] FP=113-137

[0419] Ectodomain minus SP25-493:

[0420] IHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSST[SEQ ID NO:111](GenPept dbjBAB60865.1)

[0421] Ectodomain minus SP plus altered furin cleavage site:

[0422] IHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSNNHKNFAGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSST[SEQ ID NO:112](GenPept dbjBAB60865.1).

[0423] 2.2.1.7 HeV F

[0424] Non-limiting examples of the extracellular domain polypeptide of HeV F include:

[0425] Extracellular domain 1-487:

[0426] MATQEVRLKCLLCGIIVLVLSLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNVSKCTGTVMENYKSRLTGILSPIKGAIELYNNNTHDLVGDVKLAGVVMAGIAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSIAGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQELLPVSFNNDNSEWISIVPNFVLIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSHVPRFALSGGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPPVYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPS[SEQ ID NO:113](GenPept NP_047111.2).

[0427] This sequence contains the following domains / parts:

[0428] SP = 1 - 20

[0429] Extracellular domain = 1 - 487

[0430] Furin cleavage site = 109 - 110

[0431] FP = 110 - 135

[0432] FHRR = 136 - 169

[0433] SHRR = 456 - 587[[ID=二十二]]

[0434] TM = 488 - 518

[0435] C = 519 - 546

[0436] Extracellular domain minus SP 21-487:

[0437] SLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNVSKCTGTVMENYKSRLTGILSPIKGAIELYNNNTHDLVGDVKLAGVVMAGIAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSIAGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQELLPVSFNNDNSEWISIVPNFVLIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSHVPRFALSGGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPPVYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPS[SEQ ID NO:114](GenPept NP_047111.2).

[0438] Ectodomain minus SP plus altered furin cleavage site 21-487:

[0439] SLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNVSKCTGTVMENYKSRLTGILSPIKGAIELYNNNTHDLVGDVNLAGVVMAGIAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSIAGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQELLPVSFNNDNSEWISIVPNFVLIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSHVPRFALSGGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPPVYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPS[SEQ ID NO:115](GenPept NP_047111.2).

[0440] 2.2.1.8 NiV F

[0441] Non-limiting examples of NiV F extracellular domain polypeptides include:

[0442] Extracellular domain 1-487:

[0443] MVVILDKRCYCNLLILILMISECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPS[SEQ ID NO:116](GenPept NP 112026). <0ooo1105>This sequence contains the following domains / parts:

[0445] SP = 1 - 20

[0446] Extracellular domain = 1 - 487

[0447] Furin cleavage site = 109 - 110 [[ID=I4]]

[0448] FP = 110 - 135

[0449] Extracellular domain minus SP:

[0450] It should be noted that there may be a typo in the original text where "0ooo1105" should probably be "

[0444] ". This has been corrected in the translation as best as possible while maintaining the integrity of the original text structure.SECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPS[SEQ ID NO:117](GenPept NP 112026).

[0451] Ectodomain minus SP plus altered furin cleavage site:

[0452] SECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVNLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPS[SEQ ID NO:118](GenPept NP 112026).

[0453] 2.2.1.9 HIV GP160

[0454] Non-limiting examples of extracellular domain polypeptides of HIV GP160 include:

[0455] Extracellular domain 1-688:

[0456] MRVKGTRKNYWWRWGTMLLGMLMICSAAEQLWVTVYYGVPVWKEATTTLFCASDAKAVNTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCTNWDGRNGTMNTTSTRNTTTANISRWEMEGEIKNCSFNVTTSIRNKMHKEYALFYKLDVMPIDNGSSYTLINCNTSVITQACPKVSFEPIPIHYCTPAGFALLKCNDKKFNGTGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVQLNETVVINCTRPGNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNLSEASWNKTLKQIATKLREQFVNKTIIFNQSSGGDPEIVMHSFNCGGEFFYCDTTQLFNSAWFSNNTGLNYNNGSNTGGNITLPCRIKQIVNRWQEVGKAMYAPPIRGNITCSSNITGLLLTRDGGNNVTNESEIFRPGGGNMKDNWRSELYKYKVVKIEPLGVAPTRAKRRVVQREKRAVGTIGAMFLGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGRLICTTAVPWNASWSNKSLDDIWNNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQLDTWASLWNWFSISNWLWYIK[SEQ ID NO:119](GenPept dbjBAF31430.1).

[0457] This sequence contains the following domains / parts:

[0458] SP = 1 - 28

[0459] Extracellular domain = 1 - 688

[0460] Furin cleavage site = 508 - 509

[0461] FP = 509 - 538

[0462] MPER = 668 - 688

[0463] GP120 = 1 - 508

[0464] Extracellular domain minus SP:

[0465] EQLWVTVYYGVPVWKEATTTLFCASDAKAVNTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCTNWDGRNGTMNTTSTRNTTTANISRWEMEGEIKNCSFNVTTSIRNKMHKEYALFYKLDVMPIDNGSSYTLINCNTSVITQACPKVSFEPIPIHYCTPAGFALLKCNDKKFNGTGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVQLNETVVINCTRPGNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNLSEASWNKTLKQIATKLREQFVNKTIIFNQSSGGDPEIVMHSFNCGGEFFYCDTTQLFNSAWFSNNTGLNYNNGSNTGGNITLPCRIKQIVNRWQEVGKAMYAPPIRGNITCSSNITGLLLTRDGGNNVTNESEIFRPGGGNMKDNWRSELYKYKVVKIEPLGVAPTRAKRRVVQREKRAVGTIGAMFLGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGRLICTTAVPWNASWSNKSLDDIWNNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQLDTWASLWNWFSISNWLWYIK[SEQ ID NO:120](GenPept dbjBAF31430.1).

[0466] Extracellular domain minus SP, minus MPER:

[0467] [SEQ ID NO:121](GenPeptdbjBAF31430.1).

[0468] Ectodomain minus SP plus altered furin cleavage site:

[0469] EQLWVTVYYGVPVWKEATTTLFCASDAKAVNTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNDMVEQMQEDIISLWDQSLKPCVKLTPLCVTLNCTNWDGRNGTMNTTSTRNTTTANISRWEMEGEIKNCSFNVTTSIRNKMHKEYALFYKLDVMPIDNGSSYTLINCNTSVITQACPKVSFEPIPIHYCTPAGFALLKCNDKKFNGTGPCKNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENLTDNAKTIIVQLNETVVINCTRPGNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNLSEASWNKTLKQIATKLREQFVNKTIIFNQSSGGDPEIVMHSFNCGGEFFYCDTTQLFNSAWFSNNTGLNYNNGSNTGGNITLPCRIKQIVNRWQEVGKAMYAPPIRGNITCSSNITGLLLTRDGGNNVTNESEIFRPGGGNMKDNWRSELYKYKVVKIEPLGVAPTNANNNVVQREKRAVGTIGAMFLGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGRLICTTAVPWNASWSNKSLDDIWNNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQLDTWASLWNWFSISNWLWYIK[SEQ ID NO:122](GenPept dbjBAF31430.1).

[0470] GP41 extracellular domain 509-688:

[0471] VVQREKRAVGTIGAMFLGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGRLICTTAVPWNASWSNKSLDDIWNNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQLDTWASLWNWFSISNWLWYIK[SEQ ID NO:123](GenPept dbjBAF31430.1).

[0472] 2.2.1.10 EBOV GP

[0473] Non-limiting examples of EBOV GP ectodomain polypeptides include:

[0474] Extracellular domain 1-650:

[0475] [SEQ ID NO:124](GenPept NP_066246.1)

[0476] The sequence contains the following domains / parts:

[0477] SP=1-27

[0478] Extracellular domain = 1-650

[0479] Furin cleavage site = 501-502

[0480] Cathepsin cleavage sites = 191 - 192, 201 - 202

[0481] FP = 511 - 556

[0482] MPER = 636 - 650

[0483] Mucin-like domain = 312 - 461

[0484] Extracellular domain minus SP:

[0485] QRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQ[SEQ ID NO:125](GenPept NP_066246.1). Extracellular domain minus SP, minus MPER:

[0486] QRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTL[SEQ ID NO:126](GenPept NP_066246.1).

[0487] Ectodomain minus SP plus altered furin cleavage site:

[0488] [SEQ ID NO:127](GenPept NP_066246.1).

[0489] Extracellular domain minus SP, minus mucin-like domain:

[0490] QRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVGGNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQ[SEQ ID NO:128](GenPept NP_066246.1).

[0491] Extracellular domain minus mucin-like domain:

[0492] MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTL[SEQ ID NO:129]

[0493] 2.2.1.11 MARV GP

[0494] Non-limiting examples of MARV GP extracellular domain polypeptides include:

[0495] Extracellular domain 1-650:

[0496] MKTTCFLISLILIQGTKNLPILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTARPEIKLTSTPTDATKLNTTDPSSDDEDLATSGSGSGEREPHTTSDAVTKQGLSSTMPPTPSPQPSTPQQGGNNTNHSQDAVTELDKNNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNDNTQSTITENEQTSAPSITTLPPTGNPTTAKSTSSKKGPATTAPNTTNEHFTSPPPTPSSTAQHLVYFRRKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAVLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWG[SEQ ID NO:130](GenPept YP_001531156.1).

[0497] This sequence contains the following domains / parts:

[0498] SP = 1-19

[0499] Extracellular domain = 1-650

[0500] Furin cleavage site = 434-435

[0501] FP = 526-549

[0502] MPER = 628-650

[0503] Mucin-like domain = 244-425

[0504] Ectodomain minus SP 20-650:

[0505] [SEQ ID NO:131](GenPept YP_001531156.1).

[0506] Extracellular domain minus SP, minus MPER 20-627:

[0507] [SEQ ID NO:132](GenPept YP_001531156.1).

[0508] Ectodomain minus SP plus altered furin cleavage site:

[0509] [SEQ ID NO:133](GenPept YP_001531156.1).

[0510] Extracellular domain minus SP, minus mucin-like domain:

[0511] PILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTARPEIKLGGAQHLVYFRRKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAVLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWG[SEQ ID NO:134](GenPept YP_001531156.1).

[0512] 2.2.1.12 SARS-CoV S

[0513] Non-limiting examples of the extracellular domain polypeptide of SARS-CoV S include:

[0514] Extracellular domain 1-1199:

[0515]

[0516] The sequence contains the following domains / parts:

[0517] SP=1-13

[0518] Extracellular domain = 1-1199

[0519] Human airway trypsin-like protease cleavage site = 667-668

[0520] FP = 770-788

[0521] MPER=1188-1199

[0522] Extracellular domain minus SP:

[0523]

[0524] Extracellular domain minus SP, minus MPER:

[0525]

[0526] 2.2.1.13 MERS-CoV S

[0527] Non-limiting examples of MERS-CoV S ectodomain polypeptides include:

[0528] Extracellular domain 1-1301:

[0529]

[0530] The sequence contains the following domains / parts:

[0531] SP=1-21

[0532] Extracellular domain = 1-1301

[0533] Furin cleavage site = 751-752, 887-888

[0534] FP=888-891,951-980

[0535] MPER=1292-1301

[0536] Extracellular domain minus SP 22-1301:

[0537]

[0538] Ectodomain minus SP, minus MPER22-1291:

[0539]

[0540] Ectodomain minus SP plus altered furin cleavage site:

[0541]

[0542] 2.2.1.14 VSV G

[0543] Non-limiting examples of VSV G ectodomain polypeptides include:

[0544] Extracellular domain 1-462:

[0545] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTK QGTWLNPGFPPQSCGYATVTDAEAAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGK ACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRV DIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWK[SEQ ID NO:142](GenPept gbADX53329.1).

[0546] The sequence contains the following domains / parts:

[0547] SP=1-17

[0548] Extracellular domain = 1-462

[0549] MPER=421-462

[0550] Extracellular domain minus SP:

[0551] FTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWK[SEQID NO:143](gbADX53329.1).

[0552] Extracellular domain minus SP, minus MPER:

[0553] FTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVF[SEQ ID NO:144](GenPept gbADX53329.1).

[0554] 2.2.1.15 RABV GP

[0555] Non-limiting examples of RABV GP ectodomain polypeptides include:

[0556] Extracellular domain 1-458:

[0557] MIPQTLLFVPLLVFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRA AYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVSDLDPYDKSLHSRVFPSGKCSGITVSSTYCPTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDE RGLYKSLKGACKLKLCGVLGLRLMDGTWAAIQTSDEAKWCPPDQLVNIHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADA HYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEAEDFVEVHLPDVHKQVSGVDLGLPSWGK[SEQ ID NO:145] (GenPept gbAFM52658.1).

[0558] The sequence contains the following domains / parts:

[0559] SP=1-20

[0560] Extracellular domain = 1-458

[0561] Extracellular domain minus SP:

[0562] FPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVSDLDPYDKSLHSRVFPSGKCSGITVSSTYCPTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWAAIQTSDEAKWCPPDQLVNIHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEAEDFVEVHLPDVHKQVSGVDLGLPSWGK [SEQ ID NO:146](GenPept gbAFM52658.1).

[0563] 2.2.1.16 HSV1 Gb

[0564] Non-limiting examples of the extracellular domain polypeptides of HSV1 Gb include:

[0565] Extracellular domain 1-774:

[0566] MRQGAPARGCRWFVVWALLGLTLGVLVASAAPSSPGTPGVAAATQAANGGPATPAPPALGAAPTGDPKPKKNKKPKNPTPPRPAGDNATVAAGHATLREHLRDIKAESTDANFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLETTAFHRDDHETDMELKPANAATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFYGYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTVAWDWVPKRPSVCTMTKWQEVDEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGDCIGKDARDAMDRIFARRYNATHIKVGQPQYYLANGGFLIAYQPLLSNTLAELYVREHLREQSRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRVAIAWCELQNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSMRISSRPGACYSRPLVSFRYEDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIHADANAAMFAGLGAFFEGMGDLGRAVGKVVMGIVGGVVSAVSGVSSFMSNP[SEQ ID NO:147](GenPept gbAAF04615.1).

[0567] This sequence contains the following domains / parts:

[0568] SP = 1 - 24

[0569] Extracellular domain = 1 - 774

[0570] Extracellular domain minus SP25-774:

[0571] [SEQ ID NO:148](GenPept gbAAF04615.1).

[0572] 2.2.2 Bacterial outer membrane polypeptides

[0573] In a preferred embodiment, the bacterial outer membrane polypeptide is:

[0574] (i) Chlamydia major outer membrane protein (MOMP) polypeptide; or

[0575] (ii) Trimeric autotransporter adhesin (TAA) polypeptides.

[0576] 2.2.2.1 Chlamydia major outer membrane protein (MOMP) polypeptide

[0577] Chlamydia bacteria are obligate intracellular pathogens of eukaryotic cells and are known to be the most common cause of bacterial sexually transmitted infections worldwide. Although infection can be cured with antibiotic treatment, due to frequent asymptomatic infection, this is usually overlooked, leading to disease progression and serious sequelae. Therefore, it is considered to be crucial to develop an anti-Chlamydia vaccine. "Chlamydia major outer membrane protein" (also commonly referred to as "MOMP" or "Chlamydia MOMP") has become one of the main target molecules for vaccine development (see, for example, Madico G et al., Review, Structural and Immunological Characterization of Novel Recombinant MOMP-Based Chlamydial Antigens. Vaccines (Basel). 2017; 6 (1): 2. doi: 10.3390 / vaccines6010002).

[0578] Chlamydia MOMP is a surface-exposed trimeric porin, and each monomer has a barrel-shaped transmembrane core region of presumed 16-chains, 8 surface-exposed rings and 8 short periplasmic loops. Therefore, in view of the fact that MOMP is naturally in a trimeric conformation, technology disclosed herein may also provide benefits in terms of the trimeric state of the polypeptide stabilizing the origin or corresponding to MOMP. The corresponding trimer formed after the trimerization of molecular clamps disclosed herein can make the antigen in MOMP source exist with the conformation of the natural trimeric state similar to MOMP (or the fragment / part in its source). Therefore, when using respective chimeric polypeptide as a vaccine, compared with the antigen not existing with trimeric state, antigen presentation similar to the natural trimeric conformation can cause the generation of more effective broad neutralizing antibody responses.

[0579] Molecular characterization and topological modeling of MOMP have identified four serovar-specific domains (variable domains, VDs) and a constant domain (CD) with sequence variations in loops 2, 3, 5, and 6. While the VDs have been shown to contain B-cell and T-cell epitopes (monoclonal and polyclonal) that can elicit humoral responses, the CDs can induce T-cell responses (see, e.g., Madico G et al., Vaccines (Basel). 2017; 6(1):2).

[0580] The terms "Chlamydia major outer membrane protein (MOMP) polypeptide" or "Chlamydia major outer membrane porin (MOMP) polypeptide" as used interchangeably herein refer to full-length polypeptides corresponding to monomeric subunits of MOMP, as well as fragments or portions of the latter polypeptides.

[0581] There are three known species of the family Chlamydia that infect humans: C. trachomatis, C. pneumoniae, and C. psittaci. The genome sequences of each of these are publicly available.

[0582] Thus, in preferred embodiments, the Chlamydia major outer membrane protein (MOMP) polypeptide corresponds to a Chlamydia MOMP polypeptide from a species selected from the group consisting of Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci, or is a variant thereof.

[0583] In particularly preferred embodiments, the Chlamydia MOMP polypeptide comprises or consists of an amino acid sequence having at least 70% (or, with increasing preference, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably 100%) sequence identity to one of the amino acid sequences defined by SEQ ID NO: 216 or 217.

[0584] SEQ ID NO:216

[0585] Chlamydia trachomatis major outer membrane protein (MOMP) (as contained in SEQ ID NO: 263)

[0586] MKKLLKSVLVFAALSSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPCTTWCDAISMRVGYYGDFVFDRVLKTDVNKEFQMGAAPTTSDVAGLQNDPTINVARPNPAYGKHMQDAEMFTNAAYMALNIWDRFDVFCTLGATTGYLKGNSASFNLVGLFGTKTQSSSFNTAKLIPNTALNEAVVELYINTTFAWSVGARAALWECGCATLGASFQYAQSKPKVEELNVLCNASEFTINKPKGYVGAEFPLNITAGTEAATGTKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRVSFDADTIRIAQPKLAEAILDVTTLNRTTAGKGSVVSAGTDNELADTMQIVSLQLNKMKSRKSCGIAVGTTIVDADKYAVTVEARLIDERAAHVNAQFRF

[0587] · The italicized text corresponds to the leader sequence

[0588] SEQ ID NO:217

[0589] Chlamydia pneumoniae major outer membrane protein (MOMP); Uniprot accession number: P27455

[0590] MKKLLKSALLSAAFAGSVGSLQALPVGNPSDPSLLIDGTIWEGAAGDPCDPCATWCDAISLRAGFYGDYVFDRILKVDAPKTFSMGAKPTGSAAANYTTAVDRPNPAYNKHLHDAEWFTNAGFIALNIWDRFDVFCTLGASNGYIRGNSTAFNLVGLFGVKGTTVNANELPNVSLSNGVVELYTDTSFSWSVGARGALWECGCATLGAEFQYAQSKPKVEELNVICNVSQFSVNKPKGYKGVAFPLPTDAGVATATGTKSATINYHEWQVGASLSYRLNSLVPYIGVQWSRATFDADNIRIAQPKLPTAVLNLTAWNPSLLGNATALSTTDSFSDFMQIVSCQINKFKSRKACGVTVGATLVDADKWSLTAEARLINERAAHVSGQFRF

[0591] · The italicized text corresponds to the leader sequence

[0592] In an even more preferred embodiment, the chimeric polypeptide comprises or consists of an amino acid sequence having at least 70% (or, with increasing preference, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably 100%) sequence identity to the amino acid sequence defined by SEQ ID NO: 263.

[0593] Although it is preferred that the chimeric polypeptide of the present invention is a single polypeptide chain in which the structure stabilizing moiety (SSM) is C-terminal to the bacterial surface polypeptide (i.e., downstream), the present disclosure also contemplates an alternative arrangement in which the structure stabilizing moiety (SSM) is N-terminal to the bacterial surface polypeptide (i.e., upstream). In both cases, the structure stabilizing moiety (SSM) and the bacterial surface polypeptide can be connected by a hinge as defined herein.

[0594] Chlamydia PGP3 polypeptide

[0595] Another surface polypeptide and major antigen of Chlamydia that is specifically contemplated for inclusion in the chimeric polypeptide as a bacterial surface polypeptide is the so-called "plasmid gene protein 3 (PGP3)".

[0596] Thus, in a preferred embodiment, the bacterial surface polypeptide is a Chlamydia PGP3 polypeptide, more preferably a Chlamydia PGP3 polypeptide from Chlamydia trachomatis.

[0597] In particularly preferred embodiments of the latter embodiment, the Chlamydia trachomatis PGP3 polypeptide comprises or consists of an amino acid sequence having at least 70% (or, with increasing preference, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably 100%) sequence identity to one of the amino acid sequences defined by SEQ ID NO: 259.

[0598] SEQ ID NO:259

[0599] Chlamydia trachomatis PGP3 (sequence corresponding to amino acid residues 3-264 of the full-length protein)

[0600] NSGFYLYNTENCVFADNIKVGQMTEPLKDQQIILGTTSTPVAAKMTASDGISLTVSNNSSTNASITIGLDAEKAYQLILEKLGNQILDGIADTIVDSTVQDILDKITTDPSLGLLKAFNNFPITNKIQCNG LFTPSNIETLLGGTEIGKFTVTPKSSGSMFLVSADIIASRMEGSVVLALVREGDSKPCAISYGYSSGVPNLCSLRTSITNTGLTPTTYSLRVGGLESGVVWVNALSNGNDILGITNTSNVSFLEVIPQTNA

[0601] Based on an assessment of the structural topology of the Chlamydia PGP3 polypeptide, the present inventors believe that the arrangement of the SSM at the N-terminus (i.e., upstream) of the Chlamydia PGP3 polypeptide will be particularly suitable for producing a stably folded fusion protein and for providing efficient display of the Chlamydia PGP3 polypeptide as an antigen.

[0602] Thus, in an alternative but particularly preferred aspect of the invention, there is provided a chimeric polypeptide comprising a heterologous structure stabilizing moiety (SSM) operably linked downstream of a Chlamydia PGP3 polypeptide, wherein the structure stabilizing moiety is a polypeptide comprising, in N-terminal to C-terminal order, a first heptad repeat region (FHRR) and a second heptad repeat region (SHRR), wherein (i) the FHRR comprises or consists of an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 80 or 81, and the SHRR comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence of SEQ ID NO: 82 or 83; and / or (ii) the FHRR comprises or consists of an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 80 or 81, and the SHRR comprises or consists of an amino acid sequence having at least 70% sequence similarity to the amino acid sequence of SEQ ID NO: 82 or 83.

[0603] In particularly preferred embodiments of the latter aspect, the chimeric polypeptide comprises or consists of an amino acid sequence that has at least 70% (or, with increasing preference, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably 100%) sequence identity to the amino acid sequence defined by SEQ ID NO: 264.

[0604] Those skilled in the art will readily appreciate that the embodiments disclosed herein relating to the chimeric polypeptide according to the first aspect of the invention, as well as the embodiments of other aspects relating to the chimeric polypeptide according to the first aspect of the invention, are applicable, where appropriate, to the chimeric polypeptide defined according to the above alternative aspects.

[0605] 2.2.2.2 Trimeric autotransporter adhesin (TAA) peptides

[0606] As demonstrated by the evidence disclosed herein (see, e.g., Example 19 and corresponding Figures 36 to 39 ), the applicability of the structural stabilizing portion of the present invention is by no means limited to the stabilization of antigens derived from the extracellular domain of enveloped viral fusion proteins, but can also be appropriately used to stabilize other (poly)peptides, in particular those that also naturally exist in a trimer state and whose natural conformation can therefore also be stabilized by the SSM disclosed herein.

[0607] Of particular interest are other (poly)peptides that also constitute target antigens for vaccines, including those of bacterial origin, such as bacterial outer membrane proteins (OMPs). Among the latter are the so-called "trimeric autotransporter adhesins (TAAs)", which have recently been considered as promising new vaccine targets (e.g., see Thibau A. et al., review, Immunogenicity of trimeric autotransporter adhesins and their potential as vaccine targets. Med Microbiol Immunol. 2020 Jun; 209(3): 243-263. doi: 10.1007 / s00430-019-00649-y).

[0608] The term "trimeric autotransporter adhesin (TAA)", also commonly referred to as "bacterial autotransporter", "trimeric autotransporter", "non-ciliary adhesin (NFAs)", or "oligomeric coiled-coil adhesins (Oca)", refers to a group of outer membrane proteins of Gram-negative bacteria that play an important role in bacterial infection and host colonization. TAAs are typically composed of three identical polypeptide chains (fibers) assembled into long filamentous trimeric proteins. Each monomeric subunit is typically composed of an N-terminal extracellular portion, often referred to as the "passenger domain", and a C-terminal membrane anchor, often referred to as the "translocator domain", "translocation domain" or "β-domain". The N-terminal "passenger domain" is responsible for specific effector functions, such as adhesion to specific molecular components on the host cell. The passenger domain typically includes one or more head and neck domains, and one or more coiled-coil stalk domains. Depending on the arrangement of the head domain, TAAs can be classified as either "lollipop" or "bead-on-a-string" structures. The C-terminal "translocator domain" typically consists of three subunits, each consisting of a long amphipathic helix followed by a four-stranded β-zigzag structure, which assemble into a 12-stranded β-barrel (each monomeric subunit contributes four strands) that is embedded in the bacterial outer membrane. The translocator domain is believed to be responsible for the insertion and translocation of the passenger domain to the outside of the cell. While in some TAAs, such as YadA of Yersinia enterocolitica, the passenger domain consists of only a head region and a stalk region, there are more complex TAAs that include multiple head and stalk regions in various arrangements. Furthermore, although all TAAs have a translocator domain, not all TAAs contain both a stalk and a head region.

[0609] TAA is synthesized as a precursor containing three functional domains: an N-terminal signal sequence, a passenger domain, and a C-terminal translocator domain. The signal sequence is typically about 20-50 amino acid residues long (hence often referred to as an "extended signal peptide") and targets these proteins to the Sec transport mechanism of the cytoplasmic membrane. Transmembrane translocation then proceeds through the Sec pathway, utilizing ATP as an energy source, ultimately leading to the loss of the signal peptide. The translocator domain then inserts into the outer membrane from the periplasm in a β-barrel structure and forms a hole through which the passenger domain is translocated to the bacterial cell surface. At this point, the passenger domain can remain connected to the outer membrane by covalently linking to the translocator domain or by non-covalently cleaving from the translocator domain. Alternatively, the passenger domain may be released freely into the extracellular environment after cleavage from the β domain (see, e.g., Linke D et al., Trimeric autotransporter adhesins: variable structure, common function. Trends Microbiol. 2006; 14(6): 264-70; Bassler J et al., A domain dictionary of trimeric autotransporter adhesins. Int J Med Microbiol. 2015; 305(2): 265-75; Kiessling AR et al., Recent advances in the understanding of trimeric autotransporteradhesins. Med Microbiol Immunol. 2020; 209(3): 233-242).

[0610] As in Example 19 and the corresponding Figures 36 to 39 As shown, the present technology can also be successfully used to generate chimeric polypeptides and complexes thereof, which contain TAA polypeptides fused to the SSM of the present invention, rather than enveloped virus fused ectodomain polypeptides. In summary, a set of seven constructs with TAAs derived from major bacterial pathogens can be soluble and stably expressed and purified from E. coli, and subsequent analysis (by TEM) showed an overall shape consistent with their native filamentous conformation. These results indicate that the current molecular clamp technology can also appropriately stabilize TAAs in a near-native conformation, such that the corresponding constructs can also prove effective as vaccines, particularly in eliciting strong immune responses against these important classes of virulence factors.

[0611] Although TAA naturally folds into a trimer, those skilled in the art will understand that whenever a "TAA polypeptide" is referred to herein, the term refers to a single polypeptide chain that corresponds to a monomeric subunit of a TAA polypeptide or is a variant or fragment of a monomeric subunit of a TAA polypeptide.

[0612] Thus, in a preferred embodiment, the TAA polypeptide corresponds to the following sequence or is a variant of the following sequence:

[0613] (i) a TAA polypeptide from a bacterium of the genus Neisseria, Escherichia, Haemophilus, Yersinia, Salmonella, Bartonella, Vibrio, Acinetobacter and Moraxella; and / or

[0614] (ii) a TAA polypeptide selected from the group consisting of Neisseria meningitidis adhesin A (NadA), Neisseria meningitidis hia / hsf homolog (NhhA), Escherichia coli autotransporter G (EhaG), Escherichia coli IgG binding protein D (EibD), uropathogenic Escherichia coli autotransporter G (UpaG), Haemophilus influenzae adhesin (HiA), and Yersinia enterocolitica adhesin (YadA).

[0615] In a preferred embodiment, the bacterium of the genus Neisseria is one of the species Neisseria meningitidis.

[0616] In other preferred embodiments, the bacteria of the genus Escherichia are selected from the group consisting of enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enteroaggregative Escherichia coli (EAEC), enteroinvasive Escherichia coli (EIEC), enterohemorrhagic Escherichia coli (EHEC), adherent-invasive Escherichia coli (AIEC) and uropathogenic Escherichia coli (UPEC).

[0617] In other preferred embodiments, the Haemophilus bacterium is selected from the group consisting of: Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus parasuis, and Haemophilus paragallinarum.

[0618] In other preferred embodiments, the bacterium of the genus Yersinia is selected from the group consisting of: Y. enterocolitica and Y. pseudotuberculosis.

[0619] In other preferred embodiments, the bacteria of the genus Salmonella are selected from the group consisting of: Salmonella typhi (S. typhi), Salmonella enterica (S. enterica) and Salmonella enteritidis (S. enteritidis).

[0620] In other preferred embodiments, the bacterium of the genus Bartonella is selected from the group consisting of: B. bacilliformis, B. quintana, B. clarridgeiae, B. elizabethae, B. grahamii, B. henselae, B. koehlerae, B. naantaliensis, B. vinsonii, B. washoensis and B. rochalimae.

[0621] In other preferred embodiments, the bacterium of the genus Vibrio is selected from the group consisting of: V. vulnificus, V. parahaemolyticus, V. cholerae and V. campbellii.

[0622] In other preferred embodiments, the Acinetobacter bacterium is selected from the group consisting of: A. calcoaceticus, A. baumannii, A. haemolyticus, A. junii, A. johnsonii, A. lwoffii, A. radioresistance tens, A. schindleri, A. ursingii, A. baylyi, A. bouvetii, A. gerneri, A. grimontii, A. tandoii, A. tjernbergiae, A. towneri, and A. parvus.

[0623] In other preferred embodiments, the bacterium of the genus Moraxella is selected from the group consisting of: M. catarrhalis, M. lacunata, M. bovis.

[0624] In Example 19 disclosed herein, chimeric polypeptides were designed to comprise the full-length TAA polypeptide (without the signal peptide) or a C-terminal truncated form thereof (which partially or even completely lacks the translocator domain).

[0625] In other or even more preferred embodiments, the TAA polypeptide comprises or consists of:

[0626] (i) a passenger domain; and / or

[0627] (ii) Translocator domain.

[0628] However, it is also believed that constructs containing only one (i.e., a single) head, neck, or stem domain, or only the translocator domain, can also be produced with similar yields. Given that the head domain is generally responsible for adhesion function, individual constructs containing only the head domain as a TAA polypeptide fused to an SSM may be particularly effective in eliciting potent neutralizing antibody responses against the effector protein and related bacteria expressing the protein or similar variants thereof.

[0629] In an even more preferred embodiment, the TAA polypeptide comprises or consists of at least one head domain, neck domain and / or stalk domain; or any antigenic fragment thereof.

[0630] In preferred embodiments, the TAA polypeptide comprises or consists of an amino acid sequence having at least 70% (or, with increasing preference, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably 100%) sequence identity to one of the amino acid sequences set forth in SEQ ID NOs: 252 to 258.

[0631] In an even more preferred embodiment, the chimeric polypeptide comprises or consists of an amino acid sequence having at least 70% (or, with increasing preference, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably 100%) sequence identity to one of the amino acid sequences set forth in SEQ ID NOs: 218-224.

[0632] In addition, the present technology can also be appropriately used to achieve trimerization of other (poly)peptides, such as therapeutic (poly)peptides, which are naturally not adapted to a trimeric state, but whose presentation as a trimer can provide benefits in terms of their activity and / or interaction with other molecules / binding partners, for example, when used as drugs. Section 2.4 below describes exemplary embodiments.

[0633] 2.3 Representative chimeric polypeptide constructs

[0634] Non-limiting examples of chimeric polypeptides of the present invention are shown below:

[0635] Based on RSV fusion protein 66K-T 103 -GS-G 145 —E 511 -GSG-VISNA's SSM

[0636] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity);

[0637] in:

[0638] Italic text corresponds to the signal peptide of the RSV fusion protein;

[0639] Bold text is a flexible joint; and

[0640] • The underlined text corresponds to the VISNA-based SSM (CD11).

[0641] Based on RSV fusion protein 66E-T 103 -GS-G 145 —E 511 -GSG-VISNA's SSM

[0642] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0643] in:

[0644] Italic text corresponds to the signal peptide of the RSV fusion protein;

[0645] Bold text is a flexible joint; and

[0646] • The underlined text corresponds to the VISNA-based SSM.

[0647] Based on the SARS-CoV-2 spike protein–N 679 -GSG-S 691 —G 1204 -VISNA's SSM CD11

[0648] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0649] in:

[0650] Italicized text corresponds to the signal peptide of the SARS-CoV-2 spike protein;

[0651] Bold text is a flexible joint; and

[0652] • The underlined text corresponds to the VISNA-based SSM CD11.

[0653] Nipah virus-based viral fusion-VISNA SSM

[0654] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0655] in:

[0656] Italicized text corresponds to the signal peptide of the Nipah virus fusion protein; and

[0657] Bold text is a flexible joint; and

[0658] • The underlined text corresponds to the VISNA-based SSM CD11.

[0659] Based on influenza hemagglutinin protein R 526 -VISNA's SSM

[0660] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0661] in:

[0662] Italicized text corresponds to the signal peptide of the influenza hemagglutinin protein; and

[0663] Bold text is a flexible joint; and

[0664] • The underlined text corresponds to the VISNA-based SSM CD11.

[0665] Based on the SARS-CoV-2 spike protein–N 679 -GSG-S 691 —G 1204 -VISNA's SSM CD11(-SG)

[0666] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0667] in:

[0668] Italicized text corresponds to the signal peptide of the SARS-CoV-2 spike protein;

[0669] Bold text is a flexible joint; and

[0670] • The underlined text corresponds to the VISNA-based SSM CD11.

[0671] RSV fusion protein 66E-T with additional N-linked glycosylation sequence 103 -GS-G 145 —E 511 -GSG-VISNA's SSM (CD11_1245T8)

[0672] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0673] in:

[0674] Italic text corresponds to the signal peptide of the RSV fusion protein;

[0675] Bold text is a flexible joint; and

[0676] • The underlined text corresponds to the VISNA-based SSM.

[0677] RSV fusion protein 66E-T with additional N-linked glycosylation sequence 103 -GS-G 145 —E 511 -GSG-VISNA's SSM (CD11_145T8)

[0678] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0679] in:

[0680] Italic text corresponds to the signal peptide of the RSV fusion protein;

[0681] Bold text is a flexible joint; and

[0682] • The underlined text corresponds to the VISNA-based SSM.

[0683] Based on SARS-CoV-2 (δ) spike protein – N 679 -GSG-S 691 —G 1204 -VISNA's SSM CD11

[0684] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0685] in:

[0686] Italicized text corresponds to the signal peptide of the SARS-CoV-2 spike protein;

[0687] Bold text is a flexible joint; and

[0688] • The underlined text corresponds to the VISNA-based SSM CD11.

[0689] Based on SARS-CoV-2 (δ) spike protein – N 679 -GSG-S 691 —G 1204 - SSM of VISNA (CD11), and QS removed from CD11 FHRR

[0690] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0691] in:

[0692] Italicized text corresponds to the signal peptide of the influenza hemagglutinin protein; and

[0693] • The underlined text corresponds to the VISNA-based SSM CD11.

[0694] Based on RSV fusion protein 66E-T 103 -GS-G 145 —E 511 -GSG-VISNA SSM (CD11), and QS removed from CD11FHRR

[0695] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0696] in:

[0697] Italic text corresponds to the signal peptide of the RSV fusion protein;

[0698] Bold text is a flexible joint; and

[0699] • The underlined text corresponds to the VISNA-based SSM.

[0700] RSV fusion protein 66E-T with additional N-linked glycosylation sequence 103 -GS-G 145 —

[0701] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0702] in:

[0703] Italic text corresponds to the signal peptide of the RSV fusion protein;

[0704] Bold text is a flexible joint; and

[0705] • The underlined text corresponds to the VISNA-based SSM.

[0706] RSV fusion protein 66E-T with additional N-linked glycosylation sequence 103 -GS-G 145 —E 511 - SSM of GSG-VISNA with QS removed from CD11 FHRR and including the CT1 mutation (CD11_145T8-QS)

[0707] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0708] in:

[0709] Italic text corresponds to the signal peptide of the RSV fusion protein;

[0710] Bold text is a flexible joint; and

[0711] • The underlined text corresponds to the VISNA-based SSM.

[0712] RSV fusion protein 66E-T with additional N-linked glycosylation sequence 103 -GS-G 145 —E 511 - SSM of GSG-VISNA with QS removed from CD11 FHRR and including the CT5 mutation (CD11_145T8-QS)

[0713] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0714] in:

[0715] Italic text corresponds to the signal peptide of the RSV fusion protein;

[0716] Bold text is a flexible joint; and

[0717] • The underlined text corresponds to the VISNA-based SSM.

[0718] RSV fusion protein 66E-T with additional N-linked glycosylation sequence 103 -GS-G 145 —E 511 - SSM of GSG-VISNA with QS removed from CD11 FHRR and including the CT9 mutation (CD11_145T8-QS)

[0719] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0720] in:

[0721] Italic text corresponds to the signal peptide of the RSV fusion protein;

[0722] Bold text is a flexible joint; and

[0723] • The underlined text corresponds to the VISNA-based SSM.

[0724] SSM CD11 based on E. coli EhaG-VISNA

[0725] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0726] in:

[0727] Bold text is a flexible joint; and

[0728] • The underlined text corresponds to the VISNA-based SSM.

[0729] SSM CD11 based on E. coli EibD-VISNA

[0730] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0731] in:

[0732] Bold text is a flexible joint; and

[0733] • The underlined text corresponds to the VISNA-based SSM.

[0734] SSM CD11 based on Escherichia coli UpaG-VISNA

[0735] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0736] in:

[0737] Bold text is a flexible joint; and

[0738] • The underlined text corresponds to the VISNA-based SSM.

[0739] SSM CD11 based on Haemophilus influenzae HiA-VISNA

[0740] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0741] in:

[0742] Bold text is a flexible joint; and

[0743] • The underlined text corresponds to the VISNA-based SSM.

[0744] SSM CD11 based on Neisseria meningitidis NadA-VISNA

[0745] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0746] in:

[0747] Bold text is a flexible joint; and

[0748] • The underlined text corresponds to the VISNA-based SSM.

[0749] SSM CD11 based on NhhA-VISNA of Neisseria meningitidis

[0750] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0751] in:

[0752] Bold text is a flexible joint; and

[0753] • The underlined text corresponds to the VISNA-based SSM.

[0754] SSM CD11 based on Yersinia enterocolitica YadA-VISNA

[0755] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0756] in:

[0757] Bold text is a flexible joint; and

[0758] • The underlined text corresponds to the VISNA-based SSM.

[0759] hMPV fusion protein clamp 2s

[0760] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0761] in:

[0762] Italic text corresponds to the signal peptide of the hMPV fusion protein;

[0763] Bold text is a flexible joint; and

[0764] • The underlined text corresponds to the VISNA-based SSM.

[0765] HTLV-1 glycoprotein clamp 2s

[0766] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0767] in:

[0768] Italic text corresponds to the signal peptide of the HTLV-1 glycoprotein;

[0769] Bold text is a flexible joint; and

[0770] • The underlined text corresponds to the VISNA-based SSM.

[0771] hPIV3 fusion protein clamp 2s

[0772] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0773] in:

[0774] Italic text corresponds to the signal peptide of the hPIV3 fusion protein;

[0775] Bold text is a flexible joint; and

[0776] • The underlined text corresponds to the VISNA-based SSM.

[0777] Chlamydia trachomatis major outer membrane protein clamp 2

[0778] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0779] in:

[0780] Italic text corresponds to the leader sequence;

[0781] Bold text is a flexible joint; and

[0782] • The underlined text corresponds to the VISNA-based SSM.

[0783] Chlamydia trachomatis PGP3 clamp 2

[0784] or its corresponding amino acid sequence (e.g., an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity)

[0785] in:

[0786] Bold text is a flexible joint; and

[0787] • The underlined text corresponds to the VISNA-based SSM.

[0788] 2.4 Use of structure-stabilizing moieties as universal oligomerization domains

[0789] In addition to its effectiveness in stabilizing the extracellular domain polypeptides of the present invention to resist rearrangement into the post-fusion conformation, the structural stabilizing portion can be used as a universal oligomerization domain (UOD) for oligomerizing any heterologous molecule of interest into oligomers (particularly trimers). In a specific embodiment, the UOD is fused upstream or downstream of a heterologous protein molecule (referred to herein as "first (poly) peptide") to form a chimeric polypeptide. Typically, the UOD is fused downstream of the heterologous protein molecule. As described in the extracellular domain embodiments described herein, the complementary heptapeptide repeats of the UOD bind to each other under conditions suitable for their binding (e.g., in aqueous solution) to form an antiparallel double helical bundle, which trimerizes to form a highly stable six-helix bundle, thereby allowing the chimeric polypeptide trimerization to form a trimeric polypeptide complex.

[0790] Therefore, in a second aspect, the present invention provides a chimeric polypeptide comprising a first (poly)peptide operably linked downstream of a structure stabilizing portion, wherein the structure stabilizing portion is as defined in the first aspect of the present invention; wherein preferably, the first (poly)peptide is a therapeutic (poly)peptide.

[0791] The heterologous protein molecule (i.e., the "first (poly)peptide") can be a natural or non-natural polypeptide. In certain embodiments, the heterologous (poly)peptide is or comprises a therapeutic (poly)peptide. It is known in the art that a variety of therapeutic (poly)peptides, including ligands and receptors, can be used to treat or prevent a variety of diseases.

[0792] 2.5 Methods for preparing chimeric polypeptides and their complexes

[0793] The chimeric polypeptides of the present disclosure can be prepared by chemical synthesis or recombinant means. Typically, chimeric polypeptides are prepared by expressing a recombinant construct encoding a modified or chimeric polypeptide in a suitable host cell, although any suitable method can be used. Suitable host cells include, for example, insect cells (e.g., Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni), mammalian cells (e.g., humans, non-human primates, horses, cows, sheep, dogs, cats, and rodents (e.g., hamsters), avian cells (e.g., chickens, ducks, and geese), bacteria (e.g., Escherichia coli, Bacillus subtilis, and Streptococcus spp.), yeast cells (e.g., Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha), and yeast cells. Polymorphs), Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica), Tetrahymena cells (e.g., Tetrahymena thermophile), or a combination thereof. Many suitable insect cells and mammalian cells are well known in the art. Suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a clonal isolate derived from the parent Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)).Suitable mammalian cells include, for example, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 cells), NIH-3T3 cells, 293-T cells, Vero cells, HeLa cells, PERC.6 cells (ECACC deposit number 96022940), Hep G2 cells, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), rhesus monkey fetal lung cells (ATCC CL-160), Madin-Darby bovine kidney ("MDBK") cells, Madin-Darby canine kidney ("MDCK") cells (e.g., MDCK (NBL2), ATCC CCL34; or MDCK 33016, DSMACC 2219), baby hamster kidney (BHK) cells, such as BHK21-F, HKCC cells, etc. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., . cells), chicken embryo fibroblasts, chicken embryonic germ cells, duck cells (e.g., AGE1.CR and AGE1.CR.pIX cell lines (ProBioGen), described in, e.g., Vaccine 27:4975-4982 (2009) and WO2005 / 042728), EB66 cells, etc.

[0794] Suitable insect cell expression systems, such as baculovirus systems, are known to those skilled in the art and are described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insert cell expression systems are commercially available in kit form from companies such as Invitrogen, San Diego, Calif. Avian cell expression systems are also known to those skilled in the art and are described in, for example, U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; European Patent No. EP 0787180B; European Patent Application No. EP03291813.8; WO 03 / 043415; and WO 03 / 076601. Similarly, bacterial cell expression systems and mammalian cell expression systems are also known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al. eds., 1989) Butterworths, London.

[0795] Conventional methods can be used to prepare recombinant constructs encoding modified polypeptides or chimeric polypeptides of the present disclosure in suitable vectors. A variety of suitable vectors for expressing recombinant proteins in insect cells or mammalian cells are well known and conventional in the art. Suitable vectors can contain numerous components, including but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as transcription control elements (e.g., promoters, enhancers, terminators); and / or one or more translation signals; and (e.g., mammalian-derived or from heterologous mammals or non-mammalian species) a signal sequence or leader sequence for targeting the secretory pathway in a selected host cell. For example, for expression in insect cells, suitable baculovirus expression vectors such as pFastBac (Invitrogen) can be used to produce recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express recombinant proteins. For expression in mammalian cells, a vector that drives the expression of the construct in the target mammalian host cell (e.g., Chinese hamster ovary cells) is used.

[0796] The modified polypeptide or chimeric polypeptide can be purified using any suitable method. Suitable methods for purifying proteins of interest are well known in the art and include precipitation and various types of chromatography, such as hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, chelate chromatography, and size exclusion chromatography. Two or more of these or other suitable methods can be used to create a suitable purification scheme. If desired, the modified polypeptide or chimeric polypeptide can include a purification moiety or "tag" to facilitate purification, such as those described above. Such tagged polypeptides can be conveniently purified, for example, from conditioned medium, by chelate chromatography or affinity chromatography.

[0797] Modified polypeptides or chimeric polypeptides can include additional sequences. For example, for expression purposes, the natural leader peptide of a heterologous polypeptide of interest (e.g., a natural leader peptide of a bacterial or viral surface polypeptide, e.g., a leader peptide of an enveloped virus fusion protein or a bacterial outer membrane polypeptide) can be replaced by a different leader peptide.

[0798] In a fifth aspect, the present invention provides a method for producing a chimeric polypeptide complex, wherein the method comprises: combining the chimeric polypeptides of the present invention under conditions suitable for forming the chimeric polypeptide complex, thereby producing a chimeric polypeptide complex comprising three chimeric polypeptide subunits and characterized by a six-helix bundle formed by homotrimerization of the structure-stabilizing portions of the three chimeric polypeptides.

[0799] 3. Polynucleotides and nucleic acid constructs for endogenous or heterologous production of chimeric polypeptides

[0800] The present disclosure also contemplates polynucleotides and nucleic acid constructs for the endogenous or heterologous (ie, recombinant) production of chimeric polypeptides in a host organism, suitably a vertebrate, preferably a mammal, such as a human.

[0801] More specifically, in a third aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a chimeric polypeptide as defined in the embodiments disclosed herein in relation to the first or second aspect of the invention.

[0802] In a preferred embodiment, the nucleic acid further comprises a promoter operably linked to the polynucleotide sequence encoding the chimeric polypeptide; wherein the promoter is preferably a mammalian promoter. One skilled in the art will be able to select a promoter that functions appropriately to control expression of the chimeric polypeptide in the particular host organism envisioned.

[0803] Typically, the polynucleotides contemplated herein comprise the coding sequence of a chimeric polypeptide of the present disclosure. These polynucleotides can be used to prepare nucleic acid constructs from which the chimeric polypeptide coding sequence can be expressed for use in immunizing a subject. In some embodiments, these polynucleotides themselves can be used to directly immunize a subject. In representative embodiments of this type, the polynucleotides comprise at least one ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) having an open reading frame encoding a polypeptide of the present disclosure. In some embodiments, the RNA is a messenger RNA (mRNA) having an open reading frame encoding a polypeptide disclosed herein.

[0804] In some embodiments, the polynucleotides of the present disclosure are codon optimized. Codon optimization methods are known in the art and can be used to optimize the expression of polypeptides disclosed herein. In some embodiments, codon optimization can be used to match the codon frequency in the target and host organism to ensure correct folding; bias GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codons or base runs that may damage gene construction or expression; customize transcription and translation control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (such as glycosylation sites) in the encoded protein; add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; regulate translation rate to allow the various domains of the protein to fold appropriately; or reduce or eliminate problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art. Non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.), and / or proprietary methods. In some embodiments, open reading frame (ORF) sequences are optimized using optimization algorithms. In some embodiments, codon optimized RNA can be for example RNA in which the G / C level is improved. The G / C content of nucleic acid molecules can affect the stability of RNA. RNA with an increased amount of guanine (G) and / or cytosine (C) residues is more stable functionally than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 discloses a pharmaceutical composition comprising an mRNA that is stable by sequence modification in a translation region. Due to the degeneracy of the genetic code, modification is achieved by replacing existing codons with those codons that can improve RNA stability but do not change the amino acid produced. This method is limited to the coding region of RNA.

[0805] In some embodiments, the RNA polynucleotides of the present disclosure may further comprise sequences containing or encoding additional sequences, such as one or more functional domains, one or more additional regulatory sequences, and / or an engineered 5' cap. Thus, in some embodiments, the RNA vaccine comprises a 5'UTR element; an optionally codon-optimized open reading frame with or without the incorporation of non-natural bases (or non-natural nucleotides) to reduce innate immune response triggering; and a 3'UTR element, a poly(A) sequence, and / or a polyadenylation signal, wherein the RNA is modified or unmodified.

[0806] RNA polynucleotides can be transcribed in vitro from template DNA, referred to as "in vitro transcription templates." In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a polyA tail. The specific nucleotide sequence composition and length of the in vitro transcription template will depend on the mRNA encoded by the template.

[0807] "5' untranslated region" (UTR) refers to the region of an mRNA that is immediately upstream (ie, 5') of the start codon (ie, the first codon of the mRNA transcript translated by the ribosome) and does not encode a polypeptide.

[0808] "3' untranslated region" (UTR) refers to the region of an mRNA immediately downstream (ie, 3') of the stop codon (ie, the codon of the mRNA transcript that signals the termination of translation) that does not encode a polypeptide.

[0809] An "open reading frame" is a contiguous sequence of codons that encodes a polypeptide, beginning with a start codon (eg, methionine (ATG)) and ending with a stop codon (eg, TAA, TAG, or TGA).

[0810] A "polyA tail" is a region of an mRNA that is located downstream of, for example, directly downstream (i.e., 3') the 3' UTR and contains multiple consecutive adenosine monophosphates. The polyA tail can contain 10 to 300 adenosine monophosphates. For example, the polyA tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, the polyA tail contains 50-250 adenosine monophosphates. In relevant biological contexts (e.g., in cells, in vivo), the function of the poly(A) tail is to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and to aid in transcription termination, mRNA export from the nucleus, and translation.

[0811] In some embodiments, the RNA polynucleotide is formulated within a lipid nanoparticle (LNP). 5'-capping of the polynucleotide can be accomplished simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.) according to the manufacturer's protocol. 5'-capping of the modified RNA can be accomplished post-transcriptionally using a vaccinia virus capping enzyme to generate a "CapO" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). The Cap 1 construct can be generated using vaccinia virus capping enzyme and 2'-O methyltransferase: m7G(5')ppp(5')G-2'-O-methyl. The Cap 2 construct can be generated from the Cap 1 construct, followed by 2'-O-methylation of the 5'-penultimate nucleotide using 2'-O methyltransferase. The Cap 3 construct can be generated from the Cap 2 construct, followed by 2'-O-methylation of the 5'-penultimate nucleotide using 2'-O methyltransferase. The enzymes can be derived from recombinant sources.

[0812] The present disclosure also contemplates nucleic acid constructs for endogenous production of the polypeptides disclosed herein. The nucleic acid construct can be a self-replicating extrachromosomal vector / replicon (e.g., a plasmid) or a vector integrated into the host genome. In a specific embodiment, the nucleic acid construct is a viral vector. Exemplary viral vectors include retroviral vectors, lentiviral vectors, poxvirus vectors, vaccinia virus vectors, adenoviral vectors, adeno-associated virus vectors, herpes virus vectors, flavivirus vectors, and alphavirus vectors. The viral vector can be live, attenuated, replication-conditional, or replication-defective, and is typically a non-pathogenic (defective), replication-competent viral vector.

[0813] For example, when the viral vector is a vaccinia virus vector, the polynucleotide encoding the chimeric polypeptide of the present disclosure can be inserted into a non-essential site of the vaccinia virus vector genome. Such non-essential sites are described, for example, in Perkus et al. (1986. Virology 152:285); Hruby et al. (1983. Proc. Natl. Acad. Sci. USA 80:3411); Weir et al. (1983. J. Virol. 46:530). Suitable promoters for vaccinia virus include, but are not limited to, P7.5 (see, e.g., Cochran et al. 1985. J. Virol. 54:30); P11 (see, e.g., Bertholet, et al., 1985. Proc. Natl. Acad. Sci. USA 82:2096); and CAE-1 (see, e.g., Patel et al., 1988. Proc. Natl. Acad. Sci. USA 85:9431). Highly attenuated strains of vaccinia virus are more suitable for use in humans, including Lister, NYVAC (see, e.g., Guerra et al., 2006. J. Virol. 80:985-998); Tartaglia et al., 1992. AIDS Research and Human Retroviruses 8:1445-1447), or MVA (see, e.g., Gheradi et al., 2005. J. Gen. Virol. 86:2925-2936); Mayr et al., 1975. Infection 3:6-14). See also Hu et al. (2001. J. Virol. 75:10300-10308), which describes the use of Yaba-like disease viruses as cancer therapy vectors; U.S. Patent Nos. 5,698,530 and 6,998,252. See also, e.g., U.S. Patent No. 5,443,964. See also U.S. Patent Nos. 7,247,615 and 7,368,116.

[0814] In certain embodiments, an adenoviral vector can be used to express a chimeric polypeptide of interest. The adenovirus upon which the viral transfer vector is based can be from any source, any subgroup, any subtype, a mixture of subtypes, or any serotype. For example, the adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), unclassified serogroups (e.g., serotypes 49 and 51), or any other adenoviral serotype. Adenoviral serotypes 1 to 51 are available from the American Type Culture Collection (ATCC, Manassas, Va.). Non-group C adenoviruses, even non-human adenoviruses, can be used to prepare replication-defective adenoviral vectors. Non-group C adenoviral vectors, methods for producing non-group C adenoviral vectors, and methods for using non-group C adenoviral vectors are disclosed, for example, in U.S. Patent Nos. 5,801,030, 5,837,511, and 5,849,561, and International Patent Applications WO 97 / 12986 and WO 98 / 53087. Any adenovirus, even a chimeric adenovirus, can be used as a source of viral genome for an adenoviral vector. For example, human adenovirus can be used as a source of viral genome for a replication-defective adenoviral vector. Other examples of adenoviral vectors can be found in Molin et al. (1998. J. Virol. 72: 8358-8361), Narumi et al. (1998. Am J. Respir. Cell Mol. Biol. 19: 936-941), Mercier et al. (2004. Proc. Natl. Acad. Sci. USA 101:6188-6193), U.S. Publication Nos. 20150093831, 20140248305, 20120283318, 20100008889, 20090175897, and 20090088398, and U.S. Patent Nos. 6,143,290, 6,596,535; 6,855,317; 6,936,257; 7,125,717; 7,378,087; 7,550,296.

[0815] Viral vectors can also be based on adeno-associated virus (AAV). For descriptions of AAV-based vectors, see, for example, U.S. Patent Nos. 8,679,837, 8,637,255, 8,409,842, 7,803,622, and 7,790,449, and U.S. Publication Nos. 20150065562, 20140155469, 20140037585, 20130096182, 20120100606, and 20070036757. AAV vectors can also be self-complementary (sc) AAV vectors, which are described in, for example, U.S. Patent Publications 2007 / 01110724 and 2004 / 0029106 and U.S. Patent Nos. 7,465,583 and 7,186,699.

[0816] Viral vectors based on herpes simplex virus (HSV) are also suitable for endogenous production of chimeric polypeptides of the present disclosure. Many replication-defective HSV vectors contain deletions to remove one or more immediate early genes (early-mid gene) to prevent replication. The advantage of herpes simplex virus vectors is that they can enter the latent period, thereby causing long-term expression of DNA, and their viral DNA genome is large and can accommodate up to 25kb of exogenous DNA. For descriptions of vectors based on HSV, see, for example, U.S. Patent Nos. 5,837,532, 5,846,782, 5,849,572 and 5,804,413, and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637 and WO 99 / 06583.

[0817] Retroviral vectors can include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SW), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., 1992. J. Virol. 66:2731-2739; Johann et al., 1992. J. Virol. 66:1635-1640; Sommerfelt et al., 1990. Virology 176:58-59; Wilson et al., 1989. J. Virol. 63:2374-2378; Miller et al., 1991. J. Virol. 65:2220-2224; Miller et al., 1990. Mol. Cell Biol. 10:4239; Kolberg, 1992. NIH Res. 4:43; Cornetta et al., 1991. Hum. Gene Ther. 2:215).

[0818] In a specific embodiment, the retroviral vector is a lentiviral vector. As understood by the skilled artisan, a viral vector, such as a lentiviral vector, generally refers to a viral vector particle comprising a viral vector genome. For example, a lentiviral vector particle may comprise a lentiviral vector genome. For lentiviral vectors, the vector genome may be derived from any of a large number of suitable, available lentiviral genome-based vectors, including those identified for human gene therapy applications (see, for example, Pfeifer et al., 2001.Annu.Rev.Genomics Hum.Genet.2:177-211). Suitable lentiviral vector genomes include those based on human immunodeficiency virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, simian immunodeficiency virus (SIV), and Madi-Visna virus. The desirable feature of lentiviruses is that they are able to infect dividing and non-dividing cells, but the target cells do not necessarily have to be dividing cells or are not necessarily stimulated to divide. Typically, the genome and envelope glycoproteins will be based on different viruses, so that the resulting viral vector particles are pseudotyped. The safety features of the viral vector are desirably incorporated. Safety features include self-inactivation LTR and integration defects as described in more detail herein. In certain embodiments, the integration defect can be conferred by elements of the vector genome, but can also be derived from elements of the packaging system (e.g., a non-functional integrase protein that may not be part of the vector genome but is supplied in a trans manner). Exemplary vectors contain packaging signals (psi), Rev-responsive elements (RRE), splice donors, splice acceptors, optionally central polypurine regions (cPPT) and WPRE elements. In certain exemplary embodiments, the viral vector genome comprises sequences from a lentiviral genome (such as an HIV-1 genome or an SIV genome). The viral genome construct can include sequences from lentiviral 5' and 3' LTRs, and can particularly include R sequences and U5 sequences from lentiviral 5' LTRs and inactivated or self-inactivated 3' LTRs from lentiviral 5' LTRs. The LTR sequence can be an LTR sequence from any lentivirus derived from any species. For example, they may be LTR sequences from HIV, SIV, FIV or BIV. Typically, the LTR sequences are HIV LTR sequences.

[0819] The vector genome may comprise an inactivated or self-inactivated 3'LTR (e.g., see Zufferey et al., 1998. J. Virol. 72: 9873; Miyoshi et al., 1998. J. Virol. 72: 8150). Self-inactivated vectors typically have a deletion of the enhancer sequence and promoter sequence from the 3' long terminal repeat (LTR), which is copied into the 5'LTR during vector integration. In one embodiment, the U3 element of the 3'LTR contains a deletion of its enhancer sequence, TATA box, Sp1 site, and NF-κB site. As a result of the self-inactivated 3'LTR, the provirus generated after entry and reverse transcription will contain an inactivated 5'LTR. Safety is improved by reducing the risk of vector genome movement and reducing the effect of the LTR on nearby cellular promoters. The self-inactivated 3'LTR can be constructed by any method known in the art.

[0820] Optionally, the U3 sequence derived from the 5'LTR of the lentivirus can be replaced with a promoter sequence in the viral construct, such as a heterologous promoter sequence. This can increase the titer of the virus recovered from the packaging cell line. An enhancer sequence can also be included. Any enhancer / promoter combination that increases viral RNA genome expression in the packaging cell line can be used. In one example, a CMV enhancer / promoter sequence is used (e.g., see U.S. Patent Nos. 5,385,839 and 5,168,062).

[0821] In certain embodiments, the risk of insertional mutagenesis is minimized by constructing a lentiviral vector with an integration defect. A variety of approaches can be sought to produce non-integrating vector genomes. These approaches require that (all) mutations be engineered into the integrase enzyme component of the pol gene so that it encodes a protein with an inactive integrase. The vector genome itself can be modified, for example, to prevent integration by mutating or deleting one or two attachment sites or by deleting or modifying the 3'LTR-proximal polypurine region (PPT). In addition, non-genetic approaches are available; these approaches include pharmacological substances that inhibit one or more integrase functions. These approaches are not mutually exclusive, that is, more than one of these approaches can be used at a time. For example, both the integrase and the attachment site can be non-functional, or the integrase and PPT site can be non-functional, or the attachment site and the PPT site can be non-functional, or all of them can be non-functional. Exemplary lentiviral vectors are described, for example, in U.S. Pub. Nos. 20150224209, 20150203870, 20140335607, 20140248306, 20090148936, and 20080254008.

[0822] Viral vectors can also be based on alphaviruses. Viral vectors can also be based on alphaviruses. Alphaviruses include Sindbis virus (and Venezuelan equine encephalitis virus (VEEV)), Aura virus, Babanki virus, Bama Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mossodas Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, salmon pancreatic disease virus, Semliki Forest virus (SFV), Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus and Whataroa virus. Typically, the genome encoding of this type of virus can be translated in the cytoplasm of the host cell. Non-structural proteins (e.g., replicon) and structural proteins (e.g., capsid and envelope). Ross River virus, Sindbis virus, SFV and VEEV have all been used to develop viral transfer vectors for delivering transgenes. Pseudotyped viruses can be formed by combining envelope glycoproteins with retroviral capsids. Examples of alphavirus vectors can be found in U.S. Publication Nos. 20150050243, 20090305344 and 20060177819.

[0823] Alternatively, the viral vector can be based on a flavivirus. Flaviviruses include Japanese encephalitis virus, dengue virus (e.g., dengue-1, dengue-2, dengue-3, dengue-4), yellow fever virus, Australian Murray Valley encephalitis virus, St. Louis encephalitis virus, West Nile virus, Kunjin virus, Rocio encephalitis virus, Ilheus virus, tick-borne encephalitis virus, Central European encephalitis virus, Siberian encephalitis virus, Russian spring-summer encephalitis virus, Kyasanur Forest disease virus, Omsk hemorrhagic fever virus, jumping disease virus, Powass virus, Negishi virus, Absettarov virus, Hansalova virus, Apoi virus, and Hypr virus. Examples of flavivirus vectors can be found in U.S. Pub. Nos. 20150231226, 20150024003, 20140271708, 20140044684, 20130243812, 20120294889, 20120128713, 20110135686, 20110014229, 20110003884, 20100297167, 20100184832, 20060159704, 20060088937, 20030194801, and 20030044773.

[0824] 4. Host cells

[0825] In a fourth aspect, the present invention also contemplates a host cell comprising a nucleic acid as defined in the third aspect of the invention.

[0826] In some embodiments, the host cell is (i) a prokaryotic host cell; or (ii) a eukaryotic host cell.

[0827] Exemplary eukaryotic host cells are, but are not limited to, yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris), insect cells (e.g., Spodoptera frugiperda), or mammalian cells. Examples of preferred mammalian host cells are human embryonic kidney cell lines (e.g., cell line HEK-293), and cell lines derived from Chinese hamster ovary (CHO) cells, e.g., the ExpiCHO (Thermo Fisher) cell line used in the Examples disclosed herein. Preferably, the host cell has a glycosylation machinery (endogenous or genetically engineered) that allows glycosylation (preferably N-glycosylation) of the expressed polypeptide with glycans that are typically expressed in a specific mammalian subject species (e.g., humans) and is intended to be administered with the polypeptide so expressed (i.e., a chimeric polypeptide or complex thereof as contemplated herein).

[0828] In a preferred embodiment, the host cell is those cells (preferably mammalian cells) that have been stably transfected with the nucleic acid considered herein. In the case of stably transfected cells, the expression system is integrated into the genome of the target cell and is retained in the genome in a stable manner. In contrast to transient transfection, the transferred gene is not only not degraded here, but also doubles and is passed on to daughter cells as each cell division occurs. Therefore, the latter maintains the ability to prepare the desired protein for a long time. The method for preparing transfected cells, particularly stably transfected cells, is known in the art. The host cell can be transformed, for example, by electroporation, wherein the cell membrane is permeable due to the short application of an electric field, thereby allowing nucleic acid to be absorbed into the cell, or by transfection or infection of a viral vector as described herein. In addition to the transient expression of the recombinant protein, the expression system used can also allow clones to select the transfected host cell so that a clonal cell line with a suitable expression efficiency can be selected.

[0829] Exemplary prokaryotic host cells include, but are not limited to, Escherichia species, Shigella species, Klebsiella species, Xhantomonas species, Salmonella species, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species. Particularly preferred prokaryotic host cells are Escherichia coli.

[0830] 5. Chimeric polypeptide complex and preparation method thereof

[0831] In a fifth aspect, the present invention provides a method for producing a chimeric polypeptide complex, wherein the method comprises: combining the chimeric polypeptides defined according to the first or second aspect of the present invention under conditions suitable for forming the chimeric polypeptide complex, thereby producing a chimeric polypeptide complex, the chimeric polypeptide complex comprising three chimeric polypeptide subunits and being characterized by a six-helix bundle formed by homotrimerization of the structure-stabilizing portions of the three chimeric polypeptides.

[0832] Preferably, the six-helix bundle consists of an inner trimer of three substantially α-helical FHRRs oriented in parallel, while the three substantially α-helical SHRRs are packed in an antiparallel orientation relative to the FHRRs.

[0833] In a sixth aspect, the present invention provides a chimeric polypeptide complex comprising three chimeric polypeptide subunits, wherein each subunit is a chimeric polypeptide as defined in the first or second aspect of the invention, and wherein the complex is characterized by a six-helix bundle formed by homotrimerization of the structure-stabilizing portions of the three chimeric polypeptides.

[0834] In some embodiments, the six-helix bundle is composed of an inner trimer of three substantially α-helical FHRRs oriented in parallel, while three substantially α-helical SHRRs are packed in an antiparallel orientation relative to the FHRRs.

[0835] While it is generally preferred that the chimeric polypeptide complexes so generated comprise three identical chimeric polypeptides, complexes are also contemplated and expressly contemplated in which the individual chimeric polypeptides differ from one another, for example, with respect to any one or more components comprised in the chimeric polypeptides defined herein (e.g., a structure stabilizing moiety (SSM) and / or a microbial polypeptide (e.g., an enveloped viral fusion ectodomain polypeptide or a bacterial outer membrane polypeptide)).

[0836] In some embodiments, the chimeric polypeptide subunits each comprise an enveloped viral fusion ectodomain polypeptide, and the complex comprises at least one prefusion epitope of the enveloped viral fusion protein.

[0837] 6. Screening Methods

[0838] The present invention also includes methods for screening for agents that bind, preferably specifically bind, to a microbial polypeptide (preferably an enveloped viral fusion protein or a bacterial outer membrane polypeptide (e.g., a bacterial TAA polypeptide) and / or a corresponding complex (e.g., a fusion protein or a complex of a TAA polypeptide). In specific embodiments, a compound library is screened for binding to a chimeric polypeptide comprising a microbial polypeptide (preferably a chimeric polypeptide comprising an enveloped viral fusion extracellular domain polypeptide or a chimeric polypeptide comprising a bacterial outer membrane polypeptide) or a complex thereof.

[0839] Therefore, in an eighth aspect, the present invention provides a method for identifying an agent that binds to a microbial polypeptide or a complex thereof, wherein the method comprises:

[0840] (i) contacting the candidate agent with the chimeric polypeptide comprising a microbial polypeptide as defined in the first aspect of the present invention or the chimeric polypeptide complex comprising a microbial polypeptide as defined in the sixth aspect of the present invention; and

[0841] (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex;

[0842] Preferably, the candidate agent is part of a compound library (eg, a small molecule or large molecule library).

[0843] In a preferred embodiment, the method further comprises:

[0844] (i) contacting a candidate agent with a microbial polypeptide or a complex thereof, and detecting binding of the candidate agent to the microbial polypeptide or a complex thereof;

[0845] and / or

[0846] (ii) Isolating candidate agents.

[0847] In a preferred embodiment of the latter aspect, the microbial polypeptide or complex thereof is:

[0848] (a) respectively, a fusion protein of an enveloped virus or a complex of such fusion proteins, wherein the method comprises:

[0849] (i) contacting the candidate agent with a chimeric polypeptide comprising an enveloped virus fusion ectodomain polypeptide as defined in the first aspect of the present invention or a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain polypeptide as defined in the sixth aspect of the present invention, wherein the enveloped virus fusion ectodomain polypeptide corresponds to a fusion protein of the enveloped virus; and

[0850] (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex;

[0851] or

[0852] (b) respectively, an outer membrane polypeptide of a bacterium or a complex of said outer membrane polypeptides, wherein said method comprises:

[0853] (i) contacting the candidate agent with a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined in the first aspect of the present invention or a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined in the sixth aspect of the present invention, wherein the bacterial outer membrane polypeptide corresponds to an outer membrane polypeptide of a bacterium; and

[0854] (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex.

[0855] In a preferred embodiment of the latter embodiment, the method further comprises:

[0856] (i)(ia) contacting the candidate agent of item (a) with the fusion protein or the complex of the fusion protein, and detecting the binding of the candidate agent to the fusion protein or the complex of the fusion protein; or

[0857] (ib) contacting the candidate agent of item (b) with the outer membrane polypeptide or the complex of the outer membrane polypeptide, and detecting the binding of the candidate agent to the outer membrane polypeptide or the complex of the outer membrane polypeptide;

[0858] and / or

[0859] (ii) Isolating candidate agents.

[0860] In a preferred embodiment, the bacterial outer membrane polypeptide or complex thereof is:

[0861] (a) a trimeric autotransporter adhesin (TAA) polypeptide of a bacterium or a complex of said TAA polypeptide, wherein said method comprises:

[0862] (i) contacting the candidate agent with a chimeric polypeptide comprising a TAA polypeptide as defined in the first aspect of the present invention or a chimeric polypeptide complex comprising a TAA polypeptide as defined in the sixth aspect of the present invention, wherein the TAA polypeptide corresponds to a TAA polypeptide of a bacterium; and

[0863] (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex;

[0864] or

[0865] (b) a major outer membrane protein (MOMP) polypeptide of a Chlamydia bacterium or a complex thereof, wherein the method comprises:

[0866] (i) contacting the candidate agent with a chimeric polypeptide comprising a Chlamydia MOMP polypeptide as defined in the first aspect of the present invention or a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide as defined in the sixth aspect of the present invention, wherein the Chlamydia MOMP polypeptide corresponds to a MOMP polypeptide of a Chlamydia bacterium; and

[0867] (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex.

[0868] In a preferred embodiment of item (a) of the latter embodiment, the method further comprises:

[0869] (i) (ia) contacting a candidate agent with a TAA polypeptide or a complex of a TAA polypeptide, and detecting binding of the candidate agent to the TAA polypeptide or the complex of the TAA polypeptide; or

[0870] (ib) contacting the candidate agent with a MOMP polypeptide or a complex of a MOMP polypeptide;

[0871] and / or

[0872] (ii) Isolating candidate agents.

[0873] In preferred embodiments of the latter embodiment, the candidate agent specifically binds to the chimeric polypeptide or chimeric polypeptide complex.

[0874] In other or even more preferred embodiments, the candidate agent specifically binds to a microbial polypeptide or a complex thereof.

[0875] In an even more preferred embodiment, the candidate agent specifically binds to:

[0876] (i) an enveloped viral fusion protein or a complex of such fusion proteins; or

[0877] (ii) an outer membrane polypeptide or a complex of said outer membrane polypeptide.

[0878] In an even more preferred embodiment of item (ii) of the latter embodiment, the candidate agent specifically binds to:

[0879] (i) a TAA polypeptide or a complex of said TAA polypeptide; or

[0880] (ii) a MOMP polypeptide or a complex of said MOMP polypeptide.

[0881] Candidate agents include many chemical classes, including small molecules, such as small organic compounds and macromolecules, such as peptides, polypeptides and polysaccharides. Candidate agents contain functional groups necessary for interaction with protein structures, particularly hydrogen bonds, and typically include at least amine, carbonyl, hydroxyl or carboxyl groups, preferably at least two chemical functional groups. Candidate compounds may include cyclic carbon or heterocyclic structures or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate drugs also exist in biomolecules, including but not limited to: peptides, polypeptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Compound libraries may include natural compounds in the form of bacterial extracts, fungal extracts, plant extracts and animal extracts. Alternatively, or in addition, compound libraries may include natural compounds or synthetically produced compounds.

[0882] In particularly preferred embodiments also contemplated herein, the candidate drug is a peptide, antibody, antibody fragment (e.g., a single chain variable fragment (scFv)), or any alternative binding protein scaffold, and is expressed as part of a combinatorial library, such as for display and selection methods for high throughput (HT) combinatorial libraries, such as, but not limited to, phage display, ribosome display, mRNA display, and cell surface display (e.g., yeast display). Candidate agents that specifically bind to a microbial polypeptide (e.g., an enveloped virus fusion protein or complex thereof, or a bacterial surface polypeptide, such as a bacterial outer membrane polypeptide, or complex thereof (e.g., a TAA polypeptide)) or a complex thereof can be routinely selected by using known display and selection methods in conjunction with a chimeric polypeptide comprising a microbial polypeptide or complex thereof (e.g., a chimeric polypeptide comprising an enveloped virus fusion extracellular domain polypeptide, or a chimeric polypeptide comprising a bacterial surface polypeptide, such as a bacterial outer membrane polypeptide, or complex thereof (e.g., a chimeric polypeptide comprising a TAA polypeptide)) as the corresponding antigen for selection.

[0883] Methods for determining whether an agent binds to a target protein and / or for determining the affinity of an agent for a target protein are known in the art. For example, the binding of an agent to a target protein can be detected and / or quantified using a variety of techniques, such as, but not limited to, biofilm interferometry (BLI), Western blotting, dot blotting, surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), or Assay or mass spectrometry-based methods.

[0884] In some embodiments, any surface plasmon resonance (SPR)-based assay known in the art can be used to assay an agent to characterize the kinetic parameters of the interaction between the agent and a chimeric polypeptide comprising a microbial polypeptide, or a complex thereof (e.g., a chimeric polypeptide comprising an enveloped viral fusion extracellular domain polypeptide, or a complex thereof, or a chimeric polypeptide comprising a bacterial surface polypeptide, or a complex thereof, such as a chimeric polypeptide comprising a bacterial outer membrane polypeptide, or a complex thereof (e.g., a chimeric polypeptide comprising a TAA polypeptide, or a complex thereof). Any commercially available SPR instrument, including but not limited to a BIAcore instrument (Biacore AB; Uppsala, Sweden); an IAsys instrument (affinity sensor; Franklin, Massachusetts); an IBIS system (Windsor Scientific Limited; Berkshire, United Kingdom), an SPR-CELLIA system (Nippon Laser and Electronics Lab; Hokkaido, Japan), and an SPR Detector Spreata (Texas Instruments; Dallas, Texas), can be used in the methods described herein. See, e.g., Mullett et al. (2000) Methods 22:77-91; Dong et al. (2002) Reviews in Mol Biotech 82:303-323; Fivash et al. (1998) Curr Opin Biotechnol 9:97-101; and Rich et al. (2000) Curr Opin Biotechnol 11:54-61.

[0885] In some embodiments, biomolecular interactions between an agent and a chimeric polypeptide comprising a microbial polypeptide, or a complex thereof (e.g., a chimeric polypeptide comprising an enveloped virus fusion extracellular domain polypeptide, or a complex thereof, or a chimeric polypeptide comprising a bacterial surface polypeptide, or a complex thereof, such as a chimeric polypeptide comprising a bacterial outer membrane polypeptide, or a complex thereof (e.g., a chimeric polypeptide comprising a TAA polypeptide, or a complex thereof)) can be measured on an Octet using BLI (ForteBio). BLI is a label-free optical analytical technique that detects binding between a ligand (e.g., a chimeric polypeptide comprising an extracellular domain polypeptide of the present invention, or a complex thereof) immobilized on a biosensor tip and an analyte (e.g., a test compound) in solution by measuring changes in the thickness of a protein layer on the biosensor tip in real time.

[0886] In some embodiments, the AlphaScreen (PerkinElmer) assay can be used to characterize the binding of a test agent to a chimeric polypeptide comprising a microbial polypeptide, or a complex thereof (e.g., a chimeric polypeptide comprising an enveloped viral fusion ectodomain polypeptide, or a complex thereof, or a chimeric polypeptide comprising a bacterial surface polypeptide, or a complex thereof, such as a chimeric polypeptide comprising a bacterial outer membrane polypeptide, or a complex thereof (e.g., a chimeric polypeptide comprising a TAA polypeptide, or a complex thereof). The acronym ALPHA stands for Amplified Luminescent Proximity Homogeneous Assay. AlphaScreen is a bead-based proximity assay that senses binding between molecules attached to donor and acceptor beads (e.g., a subject chimeric polypeptide or complex and a test compound) by measuring the signal generated by energy transfer between the donor and acceptor beads (see, e.g., Eglen et al. (2008) Curr Chem Genomics 1:2-10).

[0887] In some embodiments, The ELISA (PerkinElmer) assay can be used to characterize the binding of a test agent to a chimeric polypeptide or complex of the invention. The AlphaLISA is a modification of the AlphaScreen assay described above that includes europium-containing acceptor beads and serves as an alternative to traditional ELISA assays (see, e.g., Eglen et al. (2008) Curr Chem Genomics 1:2-10.).

[0888] A variety of immunoassay techniques can be used, including competitive and non-competitive immunoassays. The term "immunoassay" encompasses numerous techniques, including but not limited to flow cytometry, FACS, enzyme immunoassays (EIA), such as enzyme amplified immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA), in addition to capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), fluorescence polarization immunoassay (FPIA), and chemiluminescence assay (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser-induced fluorescence. Liposomal immunoassays, such as flow injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. In addition, nephelometry assays are suitable for use in the methods of the present invention, where, for example, the formation of a protein / antibody complex results in an increase in light scattering, which is converted into a peak rate signal that is a function of marker concentration.

[0889] In some embodiments, binding of a test agent to a subject chimeric polypeptide or complex can be determined using thermal denaturation methods, including differential scanning fluorimetry (DSF) and differential static light scattering (DSLS).

[0890] In some embodiments, binding of a test agent to a chimeric polypeptide or complex of the invention can be determined using a mass spectrometry-based method, such as, but not limited to, an affinity selection coupled to mass spectrometry (AS-MS) platform. This is a label-free method in which a protein is incubated with a test compound, unbound molecules are washed away, and after a decomplexation step, the protein-ligand complex is analyzed by MS to identify the ligand.

[0891] In some embodiments, binding of a test substance to a subject chimeric polypeptide or complex can be quantified using, for example, a detectably labeled protein, such as a radiolabeled (e.g., 32P, 35S, 14C, or 3H), fluorescently labeled (e.g., FITC), or enzyme-labeled chimeric polypeptide or complex or test compound, by immunoassay or by chromatography.

[0892] In some embodiments, the present invention contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in directly or indirectly measuring the extent of interaction between a chimeric polypeptide or complex and a test compound.

[0893] All of the above embodiments are suitable for development into high-throughput platforms.

[0894] Compounds can be further tested in animal models to identify those with the strongest in vivo effects, e.g., compounds that specifically bind to a microbial polypeptide or complex thereof (e.g., a fusion protein of an enveloped virus, or a complex of fusion proteins, or a bacterial surface polypeptide or complex thereof, such as a bacterial outer membrane polypeptide or complex thereof (e.g., a chimeric polypeptide containing a TAA polypeptide or complex thereof) and preferably stimulate or enhance a therapeutically useful effect, e.g., reducing microbial (e.g., viral or bacterial) load, reducing infection or symptoms associated therewith. These molecules can serve as "lead compounds" for further drug development, e.g., through serial modification of compounds, molecular modeling, and other conventional procedures employed in rational drug design.

[0895] 7. Antigen-binding molecules and methods for preparing them

[0896] In a ninth aspect, the present invention provides a method for producing an antigen-binding molecule that specifically binds to:

[0897] A microbial polypeptide or a complex thereof, wherein the method comprises:

[0898] (1) immunizing a subject with a chimeric polypeptide comprising a microbial polypeptide as defined in the first aspect of the present invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined in the sixth aspect of the present invention, or a combination thereof as defined in the seventh aspect of the present invention;

[0899] (2) identifying and / or isolating B cells from the immunized su...

Claims

1. A chimeric polypeptide comprising a microbial polypeptide operably linked downstream of a heterologous structure stabilizing moiety (SSM), wherein the structure stabilizing moiety is a polypeptide comprising a first heptad repeat region (FHRR) and a second heptad repeat region (SHRR) in N-terminal to C-terminal order, wherein (i) FHRR comprises or consists of an amino acid sequence having at least 60% sequence identity to the amino acid sequence shown in SEQ ID NO:80 or 81, and SHRR comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence shown in SEQ ID NO:82 or 83; and / or (ii) FHRR comprises or consists of an amino acid sequence having at least 90% sequence similarity to the amino acid sequence shown in SEQ ID NO: 80 or 81, and SHRR comprises or consists of an amino acid sequence having at least 70% sequence similarity to the amino acid sequence shown in SEQ ID NO:82 or 83.

2. The chimeric polypeptide according to claim 1, wherein: (i) FHRR comprises or consists of an amino acid sequence having at least 60% sequence identity to the amino acid sequence shown in SEQ ID NO:80, and SHRR comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence shown in SEQ ID NO:82; and / or (ii) FHRR comprises or consists of an amino acid sequence having at least 90% sequence similarity to the amino acid sequence shown in SEQ ID NO:80, and SHRR comprises or consists of an amino acid sequence having at least 70% sequence similarity to the amino acid sequence shown in SEQ ID NO:

82.

3. The chimeric polypeptide according to claim 1 or 2, wherein the structure stabilizing moiety is capable of homotrimerizing with the structure stabilizing moieties of two further chimeric polypeptides; Preferably, the six-helix bundle is formed by homotrimerization, wherein the six-helix bundle consists of an inner trimer of three substantially α-helical FHRRs oriented in parallel, while three substantially α-helical SHRRs are packed in an antiparallel orientation relative to the FHRRs.

4. The chimeric polypeptide according to any one of claims 1 to 3, wherein FHRR and SHRR each comprise an independently selected 7-residue motif repeated n times, characterized in that (abcdefg-) n or (defgabc-) n An amino acid pattern represented by wherein pattern elements 'a' to 'g' represent the positions of amino acids, n is a number equal to or greater than 2, and at least 50% of positions 'a' and 'd' are occupied by hydrophobic amino acids, and at least 50% of positions 'b', 'c', 'e', ​​'f' and 'g' are occupied by hydrophilic amino acids.

5. The chimeric polypeptide according to any one of claims 1 to 4, wherein the structure stabilizing moiety has a glutamine at the position corresponding to position 17 of SEQ ID NO:

80.

6. The chimeric polypeptide of any one of claims 1 to 5, wherein the structural stabilizing portion comprises at least one immunologically silencing portion that reduces or inhibits the induction of an immune response to the structural stabilizing portion; Preferably, the immunologically silent portion is a glycosylation site.

7. The chimeric polypeptide according to any one of claims 1 to 6, wherein the structure stabilizing moiety comprises at least one glycosylation site; Preferably, the at least one glycosylation site is an N-linked glycosylation site selected from: (1)-Asn-Xaa-Ser-; and (2) -Asn-Xaa-Thr-; Where Xaa is an amino acid other than Pro; Preferably, the glycosylation sites are glycosylated at an occupancy level of at least 50%.

8. The chimeric polypeptide according to any one of claims 1 to 7, wherein the structure stabilizing moiety comprises one or more N-linked glycosylation sites at the amino acid positions corresponding to: (i) positions 5-7 of SEQ ID NO:80; (ii) positions 1-3 of SEQ ID NO:82; (iii) positions 6-8 of SEQ ID NO:82; (iv) positions 13-15 of SEQ ID NO:82; (v) positions 17-19 of SEQ ID NO:82; and / or (vi) positions 27-29 of SEQ ID NO:82; Preferably, each N-linked glycosylation site is independently -Asn-Xaa-Thr-, wherein Xaa is an amino acid other than Pro.

9. The chimeric polypeptide according to claim 8, wherein the structure stabilizing moiety comprises an N-linked glycosylation site at the amino acid position corresponding to: (i)(ia) positions 5-7 of SEQ ID NO:80; (ib) positions 1-3 of SEQ ID NO:82; and (ic) positions 17-19 of SEQ ID NO:82; or (ii) (ii-a) positions 5-7 of SEQ ID NO:80; (ii-b) positions 1-3 of SEQ ID NO:82; (ii-c) positions 17-19 of SEQ ID NO:82; and (ii-d) positions 27-29 of SEQ ID NO:82; or (iii)(iii-a) positions 5-7 of SEQ ID NO:80; (iii-b) positions 1-3 of SEQ ID NO:82; (iii-c) positions 13-15 of SEQ ID NO:82; (iii-d) positions 17-19 of SEQ ID NO:82; and (iii-e) positions 27-29 of SEQ ID NO:82; or (iv) (iv-a) positions 5-7 of SEQ ID NO:80; (iv-b) positions 1-3 of SEQ ID NO:82; (iv-c) positions 6-8 of SEQ ID NO:82; (iv-d) positions 13-15 of SEQ ID NO:82; (iv-e) positions 17-19 of SEQ ID NO:82; and (iv-f) positions 27-29 of SEQ ID NO:82; Preferably, each N-linked glycosylation site is independently -Asn-Xaa-Thr-, wherein Xaa is an amino acid other than Pro.

10. The chimeric polypeptide according to any one of claims 1 to 9, wherein the structure stabilizing portion has (i) arginine at the amino acid position corresponding to glutamine 22 of SEQ ID NO:80; (ii) a histidine at the amino acid position corresponding to asparagine 1 of SEQ ID NO:82; (iii) threonine at the amino acid position corresponding to histidine 2 of SEQ ID NO:82; (iv) serine at the amino acid position corresponding to alanine 25 of SEQ ID NO:82; (v) glutamine at the amino acid position corresponding to alanine 26 of SEQ ID NO:82; (vi) asparagine at the amino acid position corresponding to leucine 27 of SEQ ID NO:82; (vii) leucine at the amino acid position corresponding to glutamine 17 of SEQ ID NO:80; (viii) a deletion of the amino acid residue at the position corresponding to arginine 37 of SEQ ID NO: 80; and / or (ix) Deletion of the amino acid residues at the positions corresponding to Glutamine 1 and Serine 2 of SEQ ID NO:

80.

11. The chimeric polypeptide according to any one of claims 1 to 10, wherein the structure stabilizing moiety comprises one or more unnatural amino acids.

12. The chimeric polypeptide according to claim 11, wherein the one or more unnatural amino acids allow for coupling to (i) polyethylene glycol (PEG); (ii) an immunostimulatory moiety; or (iii) Lipids.

13. The chimeric polypeptide according to any one of claims 1 to 12, wherein the microbial polypeptide is a viral or bacterial surface polypeptide.

14. The chimeric polypeptide according to claim 13, wherein (i) the viral surface polypeptide is an enveloped virus fusion ectodomain polypeptide; or (ii) the bacterial surface polypeptide is a bacterial outer membrane polypeptide.

15. The chimeric polypeptide according to claim 14, wherein the enveloped viral fusion ectodomain polypeptide corresponds to the following sequence or is a variant of the following sequence: (i) Class I enveloped virus fusion protein ectodomain; Preferably, the extracellular domain is from a virus selected from the group consisting of an orthomyxovirus, a paramyxovirus, an orthopneumovirus, a metapneumovirus, a retrovirus, a coronavirus, a filovirus and an arenavirus; or (ii) Class III enveloped virus fusion protein ectodomain; Preferably, the extracellular domain is from a virus selected from rhabdovirus and herpesvirus.

16. The chimeric polypeptide according to claim 15, wherein the enveloped virus fusion ectodomain polypeptide corresponds to the ectodomain of a fusion protein or is a variant of the ectodomain of a fusion protein derived from: (i) Respiratory syncytial virus; (ii) Metapneumovirus; (iii) a coronavirus, preferably a beta coronavirus, more preferably severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or Middle East respiratory syndrome-related coronavirus (MERS-CoV); (iv) henipavirus, preferably Hendra virus (HeV) or Nipah virus (NiV); (v) influenza virus, preferably influenza A or influenza B; (vi) parainfluenza virus (PIV), preferably human parainfluenza virus (HPIV); (vii) an arenavirus, preferably a Lassa Fever virus; or (viii) a retrovirus, preferably human T-lymphotropic virus-1 (HTLV-1).

17. The chimeric polypeptide according to claim 14, wherein the bacterial outer membrane polypeptide is: (i) Chlamydia major outer membrane protein (MOMP) polypeptide; or (ii) Trimeric autotransporter adhesin (TAA) polypeptides.

18. The chimeric polypeptide according to claim 17, wherein the TAA polypeptide corresponds to the following sequence or is a variant of the following sequence: (i) a TAA polypeptide from a bacterium of the genus selected from the group consisting of Neisseria, Escherichia, Haemophilus, Yersinia, Salmonella, Bartonella, Vibrio, Acinetobacter and Moraxella; and / or (ii) a TAA polypeptide selected from the group consisting of Neisseria meningitidis adhesin A (NadA), Neisseria meningitidis hia / hsf homolog (NhhA), Escherichia coli autotransporter G (EhaG), Escherichia coli IgG binding protein D (EibD), uropathogenic Escherichia coli autotransporter G (UpaG), Haemophilus influenzae adhesin (HiA) and Yersinia enterocolitica adhesin (YadA).

19. The chimeric polypeptide according to claim 17 or 18, wherein the TAA polypeptide comprises or consists of: (i) a passenger domain; and / or (ii) Translocator domain.

20. The chimeric polypeptide according to any one of claims 1 to 19, wherein the FHRR and SHRR comprised in the SSM are connected via a linker; Preferably, the linker comprises or consists of a (poly)peptide having the same amino acid sequence as SEQ ID NO: 84 or 85.

21. The chimeric polypeptide according to claim 20, wherein the linker comprises or consists of a membrane tethered (poly)peptide.

22. The chimeric polypeptide according to any one of claims 1 to 21, further comprising a hinge region, which operably links a microbial polypeptide, preferably an enveloped viral fusion ectodomain polypeptide according to any one of claims 14 to 16, 20 and 21 or a bacterial outer membrane polypeptide according to any one of claims 14 and 17 to 21, to the structure stabilizing moiety; Preferably, the hinge region comprises or consists of: (i) a (poly)peptide consisting of 3 to 5 amino acid residues independently selected from serine and glycine; (ii) serine and glycine residues; (iii) GGSG; (iv) GSG; or (v) G.

23. The chimeric polypeptide according to claim 22, wherein the hinge region comprises or consists of a transmembrane (poly)peptide.

24. A chimeric polypeptide comprising a first (poly)peptide operably linked downstream to a structure stabilizing moiety, wherein the structure stabilizing moiety is as defined in any one of claims 1 to 12, 20 and 21; Preferably, the first (poly)peptide is a therapeutic (poly)peptide.

25. A nucleic acid comprising a polynucleotide sequence encoding a chimeric polypeptide as defined in any one of claims 1 to 24.

26. The nucleic acid according to claim 25, further comprising a promoter operably linked to the polynucleotide sequence encoding the chimeric polypeptide; wherein the promoter is preferably a mammalian promoter.

27. A host cell comprising the nucleic acid of claim 25 or 26.

28. according to the host cell of claim 27, wherein said host cell is (i) a prokaryotic host cell; or (ii) Eukaryotic host cells.

29. A method of producing a chimeric polypeptide complex, wherein the method comprises: The chimeric polypeptides according to any one of claims 1 to 24 are combined under conditions suitable for forming a chimeric polypeptide complex, thereby producing a chimeric polypeptide complex comprising three chimeric polypeptide subunits and characterized by a six-helix bundle formed by homotrimerization of the structure stabilizing portions of the three chimeric polypeptides.

30. The method of claim 29, wherein the six-helix bundle consists of an inner trimer of three substantially α-helical FHRRs oriented in parallel, and three substantially α-helical SHRRs packed in an antiparallel orientation relative to the FHRRs.

31. A chimeric polypeptide complex comprising three chimeric polypeptide subunits, wherein each subunit is a chimeric polypeptide according to any one of claims 1 to 24, and wherein the complex is characterized by a six-helix bundle formed by homotrimerization of the structure-stabilizing portions of the three chimeric polypeptides.

32. The chimeric polypeptide complex of claim 31, wherein the six-helix bundle is composed of an inner trimer of three substantially α-helical FHRRs oriented in parallel, and three substantially α-helical SHRRs are packed in an antiparallel orientation relative to the FHRRs.

33. The chimeric polypeptide complex according to claim 31 or 32, wherein the chimeric polypeptide subunits each comprise an enveloped virus fusion ectodomain polypeptide, and wherein the complex comprises at least one prefusion epitope of an enveloped virus fusion protein.

34. A composition comprising the chimeric polypeptide according to any one of claims 1 to 24, or the chimeric polypeptide complex according to any one of claims 31 to 33, and a pharmaceutically acceptable carrier, diluent or adjuvant.

35. A method for identifying an agent that binds to a microbial polypeptide or a complex thereof, wherein the method comprises: (i) contacting the candidate agent with a chimeric polypeptide comprising a microbial polypeptide according to any one of claims 1 to 23 or a chimeric polypeptide complex comprising a microbial polypeptide according to any one of claims 31 to 33; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex; Preferably, the candidate agent is part of a compound library (eg, a small molecule or macromolecule library).

36. The method according to claim 35, wherein the microbial polypeptide or complex thereof is: (a) respectively, a fusion protein of an enveloped virus or a complex of said fusion protein, wherein said method comprises: (i) contacting the candidate agent with a chimeric polypeptide comprising an enveloped virus fusion ectodomain polypeptide according to any one of claims 14 to 16 or 20 to 23 or a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain polypeptide according to any one of claims 31 to 33, wherein the enveloped virus fusion ectodomain polypeptide corresponds to a fusion protein of the enveloped virus; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex; or (b) respectively, an outer membrane polypeptide of a bacterium or a complex of said outer membrane polypeptides, wherein said method comprises: (i) contacting the candidate agent with a chimeric polypeptide comprising a bacterial outer membrane polypeptide according to any one of claims 14 and 17 to 23 or a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide according to claim 31 or 32, wherein the bacterial outer membrane polypeptide corresponds to an outer membrane polypeptide of a bacterium; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex.

37. The method according to claim 36(b), wherein the bacterial outer membrane polypeptide or a complex thereof is: (a) a trimeric autotransporter adhesin (TAA) polypeptide of a bacterium or a complex of said TAA polypeptide, wherein said method comprises: (i) contacting the candidate agent with a chimeric polypeptide comprising a TAA polypeptide according to any one of claims 17 to 23 or a chimeric polypeptide complex comprising a TAA polypeptide according to claim 31 or 32, wherein the TAA polypeptide corresponds to a TAA polypeptide of bacteria; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex; or (b) a major outer membrane protein (MOMP) polypeptide of a Chlamydia bacterium or a complex thereof, wherein the method comprises: (i) contacting the candidate agent with a chimeric polypeptide comprising a Chlamydia MOMP polypeptide according to any one of claims 17 to 23 or a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide according to claim 31 or 32, wherein the Chlamydia MOMP polypeptide corresponds to a MOMP polypeptide of a Chlamydia bacterium; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex.

38. The method according to claim 35, further comprising: (i) contacting the candidate agent of claim 35 with a microbial polypeptide or a complex thereof, and detecting the binding of the candidate agent to the microbial polypeptide or a complex thereof; and / or (ii) isolating the candidate agent.

39. The method according to claim 36, further comprising: (i)(ia) contacting the candidate agent of claim 36(a) with the fusion protein or the complex of the fusion protein, and detecting binding of the candidate agent to the fusion protein or the complex of the fusion protein; or (ib) contacting the candidate agent of claim 36(b) with the outer membrane polypeptide or the complex of the outer membrane polypeptide, and detecting the binding of the candidate agent to the outer membrane polypeptide or the complex of the outer membrane polypeptide; and / or (ii) isolating the candidate agent.

40. The method according to claim 37, further comprising: (i)(ia) contacting the candidate agent of claim 37(a) with a TAA polypeptide or a complex of a TAA polypeptide, and detecting binding of the candidate agent to the TAA polypeptide or the complex of the TAA polypeptide; or (ib) contacting the candidate agent of 37(b) with a MOMP polypeptide or a complex of a MOMP polypeptide; and / or (ii) isolating the candidate agent.

41. The method according to any one of claims 35 to 40, wherein the candidate agent specifically binds to the chimeric polypeptide or chimeric polypeptide complex.

42. The method according to any one of claims 35 to 41, wherein the candidate agent specifically binds to the microbial polypeptide or a complex thereof.

43. The method of claim 36 or 39, wherein the candidate agent specifically binds to: (i) an enveloped virus fusion protein or a complex of such fusion proteins; or (ii) an outer membrane polypeptide or a complex of said outer membrane polypeptide.

44. The method of claim 37 or 40, wherein the candidate agent specifically binds to: (i) a TAA polypeptide or a complex of said TAA polypeptide; or (ii) a MOMP polypeptide or a complex of said MOMP polypeptide.

45. A method for producing an antigen binding molecule that specifically binds to a microbial polypeptide or a complex thereof, wherein the method comprises: (1) immunizing a subject with a chimeric polypeptide comprising a microbial polypeptide according to any one of claims 1 to 23, or a chimeric polypeptide complex comprising a microbial polypeptide according to any one of claims 31 to 33, or a composition thereof according to claim 34; (2) identifying and / or isolating B cells from the immunized subject that specifically bind to the microbial polypeptide or its complex; as well as (3) producing antigen-binding molecules expressed by the B cells.

46. ​​The method according to claim 45, wherein the microbial polypeptide or complex thereof is: (a) the extracellular domain of a fusion protein of an enveloped virus or a complex of said fusion protein, wherein said method comprises: (1) immunizing a subject with a chimeric polypeptide comprising an extracellular domain polypeptide according to any one of claims 14 to 16 or 20 to 23, or a chimeric polypeptide complex comprising an extracellular domain polypeptide according to any one of claims 31 to 33, or a composition thereof according to claim 34, wherein the extracellular domain polypeptide corresponds to a fusion protein of an enveloped virus; (2) identifying and / or isolating B cells from the immunized subject, said B cells specifically binding to the extracellular domain of the fusion protein or its complex; and (3) producing antigen-binding molecules expressed by the B cells. or (b) an outer membrane polypeptide of a bacterium or a complex of said outer membrane polypeptide, wherein said method comprises: (1) immunizing a subject with a chimeric polypeptide comprising a bacterial outer membrane polypeptide according to any one of claims 14 and 17 to 23, or a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide according to claim 31 or 32, or a composition thereof according to claim 34, wherein the bacterial outer membrane polypeptide corresponds to an outer membrane polypeptide of a bacterium; (2) identifying and / or isolating B cells from the immunized subject, said B cells specifically binding to a bacterial outer membrane polypeptide or a complex thereof; and (3) producing antigen-binding molecules expressed by the B cells.

47. The method according to claim 46(b), wherein the outer membrane polypeptide or complex thereof is: (i) respectively, a TAA polypeptide or a complex thereof of a bacterium, wherein the method comprises: (1) immunizing a subject with a chimeric polypeptide comprising a TAA polypeptide according to any one of claims 17 to 23, or a chimeric polypeptide complex comprising a TAA polypeptide according to claim 31 or 32, or a composition thereof according to claim 34, wherein the TAA polypeptide corresponds to a TAA polypeptide of a bacterium; (2) identifying and / or isolating B cells from the immunized subject, wherein the B cells specifically bind to the TAA polypeptide or a complex thereof; as well as (3) producing antigen-binding molecules expressed by the B cells. or (ii) respectively, a major outer membrane protein (MOMP) polypeptide of a Chlamydia bacterium or a complex thereof, wherein the method comprises: (1) immunizing a subject with a chimeric polypeptide comprising a Chlamydia MOMP polypeptide according to any one of claims 17 to 23, or a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide according to claim 31 or 32, or a composition thereof according to claim 34, wherein the Chlamydia MOMP polypeptide corresponds to a MOMP polypeptide of a Chlamydia bacterium; (2) identifying and / or isolating B cells from the immunized subject that specifically bind to the Chlamydia MOMP polypeptide or a complex thereof; as well as (3) producing antigen-binding molecules expressed by the B cells.

48. An antigen-binding molecule that specifically binds to: A microbial polypeptide of a chimeric polypeptide comprising a microbial polypeptide according to any one of claims 1 to 23 and / or a microbial polypeptide of one or more subunits of a chimeric polypeptide complex comprising a microbial polypeptide according to any one of claims 31 to 33.

49. An antigen binding molecule according to claim 48, wherein the antigen binding molecule specifically binds: (i) an extracellular domain of a chimeric polypeptide comprising an enveloped virus fusion extracellular domain polypeptide according to any one of claims 14 to 16 or 20 to 23; and / or an extracellular domain of one or more subunits of a chimeric polypeptide complex comprising an enveloped virus fusion extracellular domain polypeptide according to any one of claims 31 to 33; or (ii) a bacterial outer membrane polypeptide of a chimeric polypeptide comprising a bacterial outer membrane polypeptide according to any one of claims 14 and 17 to 23; and / or a bacterial outer membrane polypeptide of a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide according to claim 31 or 32.

50. An antigen binding molecule according to claim 49(ii), wherein the antigen binding molecule specifically binds: (i) a TAA polypeptide of a chimeric polypeptide comprising a TAA polypeptide according to any one of claims 17 to 23; and / or a TAA polypeptide of a chimeric polypeptide complex comprising a TAA polypeptide according to claim 31 or 32; or (ii) a Chlamydia major outer membrane protein (MOMP) polypeptide comprising a chimeric polypeptide of a Chlamydia MOMP polypeptide according to any one of claims 17 to 23; and / or a Chlamydia MOMP polypeptide comprising a chimeric polypeptide complex of a Chlamydia MOMP polypeptide according to claim 31 or 32.

51. An antigen-binding molecule, which can be obtained by the following steps: (i) the method according to any one of claims 45 to 47; (ii) the method of claim 46(a); (iii) the method of claim 46(b); (iv) the method of claim 47(i); or (v) The method of claim 47(ii).

52. A composition comprising an antigen binding molecule according to any one of claims 48 to 51 and a pharmaceutically acceptable carrier, diluent or adjuvant.

53. A composition comprising a nucleic acid according to claim 25 or 26.

54. The composition according to claim 53, wherein the chimeric polypeptide encoded by the polynucleotide sequence contained in the nucleic acid is: (i) a chimeric polypeptide comprising a microbial polypeptide according to any one of claims 1 to 23; or (ii) A chimeric polypeptide comprising a first (poly)peptide according to claim 24.

55. The composition of claim 54(i), wherein the chimeric polypeptide comprising a microbial polypeptide is: (i) a chimeric polypeptide comprising an enveloped virus fusion ectodomain polypeptide according to any one of claims 14 to 16 and 20 to 23; or (ii) a chimeric polypeptide comprising a bacterial outer membrane polypeptide according to any one of claims 14 and 17 to 23.

56. The composition of claim 55(ii), wherein the chimeric polypeptide comprising a bacterial outer membrane polypeptide is: (i) a chimeric polypeptide comprising a TAA polypeptide according to any one of claims 17 to 23; or (ii) A chimeric polypeptide comprising a Chlamydia major outer membrane protein (MOMP) polypeptide according to any one of claims 17 to 23.

57. A composition according to any one of claims 53 to 56, wherein the nucleic acid comprises or consists of RNA.

58. A composition according to claim 57, wherein the RNA comprises at least one modified nucleotide; wherein preferably, the at least modified nucleotide is a modified uridine, more preferably a methylated derivative of uridine, most preferably N1-methyl-pseudouridine.

59. The composition according to claim 57 or 58, wherein the RNA is formulated in a delivery vehicle, the delivery vehicle being a liposome, a lipoplex or a lipid nanoparticle (LNP); Preferably, the lipid nanoparticle (LNP) comprises cationic lipids, neutral lipids, steroids and / or pegylated lipids.

60. A composition according to any one of claims 53 to 56, wherein the nucleic acid comprises or consists of DNA; Preferably, the DNA is contained in a plasmid.

61. A chimeric polypeptide according to any one of claims 1 to 24, a nucleic acid according to claim 25 or 26, a host cell according to claim 27 or 28, a chimeric polypeptide complex according to any one of claims 31 to 33, a composition according to claim 34, an antigen binding molecule according to any one of claims 48 to 51 or a composition according to any one of claims 52 to 60 for use as a medicament.

62. A method of eliciting an immune response in a subject to a microbial polypeptide or a complex thereof, wherein the method comprises administering to the subject: (i) a chimeric polypeptide comprising a microbial polypeptide according to any one of claims 1 to 23, a chimeric polypeptide complex comprising a microbial polypeptide according to any one of claims 31 to 33, or a combination thereof according to claim 34; or (ii) A composition according to claim 54(i) or any one of claims 55 to 60 as dependent thereon.

63. The method according to claim 62, wherein the microbial polypeptide or complex thereof is: (a) respectively, a fusion protein of an enveloped virus or a complex of said fusion protein, wherein said method comprises administering to a subject: (i) a chimeric polypeptide comprising a fusion extracellular domain of an enveloped virus according to any one of claims 14 to 16 or 20 to 23, a chimeric polypeptide complex comprising a fusion extracellular domain of an enveloped virus according to any one of claims 31 to 33, or a combination thereof according to claim 34; or (ii) a composition according to claim 55(i) or any one of claims 57 to 60 as dependent thereon; wherein the extracellular domain polypeptide subunit of the chimeric polypeptide complex corresponds to a fusion protein of the enveloped virus; or (b) respectively, an outer membrane polypeptide or a complex of outer membrane polypeptides of a bacterium, wherein the method comprises administering to a subject: (i) a chimeric polypeptide comprising a bacterial outer membrane polypeptide according to any one of claims 14 and 17 to 23, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide according to claim 31 or 32, or a combination thereof according to claim 34; or (ii) a composition according to claim 55(ii) or any one of claims 56 to 60 as dependent thereon; wherein the bacterial outer membrane polypeptide subunit of the chimeric polypeptide complex corresponds to or substantially corresponds to a bacterial outer membrane polypeptide expressed by the bacterium.

64. The method according to claim 63(b), wherein the bacterial outer membrane polypeptide or complex thereof is: (a) a TAA polypeptide or a complex of TAA polypeptides of a bacterium, respectively, wherein the method comprises administering to a subject: (i) a chimeric polypeptide comprising a TAA polypeptide according to any one of claims 17 to 23, a chimeric polypeptide complex comprising a TAA polypeptide according to claim 31 or 32, or a combination thereof according to claim 34; or (ii) a composition according to claim 56(i) or any one of claims 57 to 60 as dependent thereon; wherein the TAA polypeptide subunit of the chimeric polypeptide complex corresponds to or substantially corresponds to a TAA polypeptide expressed by the bacterium; or (b) a Chlamydia major outer membrane protein (MOMP) polypeptide or a complex of Chlamydia MOMP polypeptides, respectively, wherein the method comprises administering to a subject: (i) a chimeric polypeptide comprising a Chlamydia MOMP polypeptide according to any one of claims 17 to 23, a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide according to claim 31 or 32, or a combination thereof according to claim 34; or (ii) a composition according to claim 56(ii) or any one of claims 57 to 60 as dependent thereon; wherein the Chlamydia MOMP polypeptide subunit of the chimeric polypeptide complex corresponds to or substantially corresponds to the Chlamydia MOMP polypeptide expressed by the bacterium.

65. A method for treating or preventing a microbial infection in a subject, wherein the method comprises administering to the subject an effective amount of: (i) a chimeric polypeptide comprising a microbial polypeptide according to any one of claims 1 to 23, a chimeric polypeptide complex comprising a microbial polypeptide according to any one of claims 31 to 33, or a combination thereof according to claim 34; (ii) an antigen binding molecule according to claims 48 to 51, or a composition thereof according to claim 52; or (iii) A composition according to claim 54(i) or any one of claims 55 to 60 as dependent thereon.

66. The method according to claim 65, wherein the microbial infection is: (a) an enveloped virus infection in a subject, wherein the method comprises administering to the subject an effective amount of: (i) a chimeric polypeptide comprising a fusion ectodomain of an enveloped virus according to any one of claims 14 to 16 or 20 to 23, a chimeric polypeptide complex comprising a fusion ectodomain of an enveloped virus according to any one of claims 31 to 33, or a combination thereof according to claim 34; (ii) an antigen-binding molecule according to claim 49(i) or 51(ii), or a composition thereof according to claim 52; or (iii) a composition according to claim 55(i) or any one of claims 57 to 60 as dependent thereon; or (b) a bacterial infection in a subject, wherein the method comprises administering to the subject an effective amount of: (i) a chimeric polypeptide comprising a bacterial outer membrane polypeptide according to any one of claims 14 and 17 to 23, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide according to claim 31 or 32, or a combination thereof according to claim 34; (ii) an antigen binding molecule according to claims 49(ii), 50 and 51(iii-v), or a composition thereof according to claim 52; or (iii) A composition according to claim 55(ii) or any one of claims 56 to 60 as dependent thereon.

67. The method according to claim 66(b), wherein the bacterial infection is: (a) infection with a bacterium expressing a TAA, wherein the method comprises administering to the subject an effective amount of: (i) a chimeric polypeptide comprising a TAA polypeptide according to any one of claims 17 to 23, a chimeric polypeptide complex comprising a TAA polypeptide according to claim 31 or 32, or a combination thereof according to claim 34; (ii) an antigen-binding molecule according to claim 50(i) or 51(iv), or a composition thereof according to claim 52; or (iii) a composition according to claim 56(i) or any one of claims 57 to 60 as dependent thereon; or (b) Chlamydia bacterial infection, wherein the method comprises administering to the subject an effective amount of: (i) a chimeric polypeptide comprising a Chlamydia MOMP polypeptide according to any one of claims 17 to 23, a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide according to claim 31 or 32, or a combination thereof according to claim 34; or (ii) an antigen-binding molecule according to claim 50(ii) or 51(v), or a composition thereof according to claim 52; or (iii) A composition according to claim 56(ii) or any one of claims 57 to 60 as dependent thereon.

68. A vaccine comprising: (i) a chimeric polypeptide comprising a microbial polypeptide according to any one of claims 1 to 23, a chimeric polypeptide complex comprising a microbial polypeptide according to any one of claims 31 to 33, or a combination thereof according to claim 34; or (ii) a composition according to claim 54(i) or any one of claims 55 to 60 as dependent thereon; The vaccine is used in a method of eliciting an immune response in a subject against a microbial polypeptide or a microbial polypeptide complex.

69. A vaccine for use according to claim 68, wherein the microbial polypeptide is: (a) an enveloped virus fusion extracellular domain polypeptide, and the vaccine comprises: (i) a chimeric polypeptide comprising an enveloped virus fusion ectodomain according to any one of claims 14 to 16 or 20 to 23, a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain polypeptide according to any one of claims 31 to 33, or a combination thereof according to claim 34; or (ii) a composition according to claim 55(i) or any one of claims 57 to 60 as dependent thereon; The vaccine is for use in a method of eliciting an immune response in a subject against a fusion protein of an enveloped virus or a complex of said fusion protein, and wherein the ectodomain polypeptide subunit of the chimeric polypeptide complex corresponds to a fusion protein of said enveloped virus; or (b) a bacterial outer membrane polypeptide, and the vaccine comprises: (i) a chimeric polypeptide comprising a bacterial outer membrane polypeptide according to any one of claims 14 and 17 to 23, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide according to claim 31 or 32, or a combination thereof according to claim 34; or (ii) a composition according to claim 55(ii) or any one of claims 56 to 60 as dependent thereon; The vaccine is used in a method of eliciting an immune response in a subject against an outer membrane polypeptide of a bacterium or a complex of said outer membrane polypeptide, and wherein the outer membrane polypeptide subunit of the chimeric polypeptide complex corresponds to an outer membrane polypeptide of said bacterium.

70. A vaccine for use according to claim 69, wherein the bacterial outer membrane polypeptide is: (a) a bacterial trimeric autotransporter adhesin (TAA) polypeptide, and the vaccine comprises: (i) a chimeric polypeptide comprising a TAA polypeptide according to any one of claims 17 to 23, a chimeric polypeptide complex comprising a TAA polypeptide according to claim 31 or 32, or a combination thereof according to claim 34; or (ii) a composition according to claim 56(i) or any one of claims 57 to 60 as dependent thereon; The vaccine is used in a method of eliciting an immune response in a subject against a TAA polypeptide or a complex of TAA polypeptides of a bacterium, and wherein the chimeric polypeptide complex TAA polypeptide subunit corresponds to a TAA polypeptide of the bacterium; or (b) a Chlamydia MOMP polypeptide, the vaccine comprising: (i) a chimeric polypeptide comprising a Chlamydia MOMP polypeptide according to any one of claims 17 to 23, a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide according to claim 31 or 32, or a combination thereof according to claim 34; or (ii) a composition according to claim 56(ii) or any one of claims 57 to 60 as dependent thereon; The vaccine is used in a method of eliciting an immune response in a subject against a MOMP polypeptide or a complex of MOMP polypeptides of a Chlamydia bacterium, and wherein the chimeric polypeptide complex MOMP polypeptide subunit corresponds to a MOMP polypeptide of the Chlamydia bacterium.

71. A chimeric polypeptide comprising a microbial polypeptide according to any one of claims 1 to 23, a chimeric polypeptide complex comprising a microbial polypeptide according to any one of claims 31 to 33, or a composition thereof according to claim 34, or an antigen binding molecule according to any one of claims 48 to 51, or a composition thereof according to claim 52, or a composition according to claim 54(i) or any one of claims 55 to 60 dependent therefrom, for use in a method of treating or preventing a microbial infection in a subject.

72. A chimeric polypeptide comprising a microbial polypeptide for use according to claim 71, wherein the microbial polypeptide is: (a) an enveloped virus fusion ectodomain polypeptide, and providing a chimeric polypeptide comprising an enveloped virus fusion ectodomain polypeptide according to any one of claims 14 to 16 and 20 to 23, a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain according to any one of claims 31 to 33, or a composition thereof according to claim 34, or an antigen binding molecule according to claim 49(i) or 51(ii) or a composition thereof according to claim 52, or a composition according to claim 55(i) or any one of claims 57 to 60 dependent thereon, for use in a method of treating or preventing an enveloped virus infection in a subject; or (b) a bacterial outer membrane polypeptide, and provides a chimeric polypeptide comprising a bacterial outer membrane polypeptide according to any one of claims 14 and 17 to 23, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide according to claim 31 or 32, or a composition thereof according to claim 34, or an antigen binding molecule according to any one of claims 49(ii), 50 and 51(iii-v) or a composition thereof according to claim 52, or a composition according to claim 55(ii) or any one of claims 56 to 60 dependent therefrom, for use in a method for treating or preventing a bacterial infection in a subject.

73. The chimeric polypeptide comprising a microbial polypeptide according to claim 72(b), wherein the bacterial outer membrane polypeptide is: (a) a bacterial trimeric autotransporter adhesin (TAA) polypeptide, and providing a chimeric polypeptide comprising a bacterial TAA polypeptide according to any one of claims 17 to 23, a chimeric polypeptide complex comprising a TAA polypeptide according to claim 31 or 32, or a composition thereof according to claim 34, or an antigen binding molecule according to claim 50(i) or 51(iv) or a composition thereof according to claim 52, or a composition according to claim 56(i) or any one of claims 57 to 60 dependent therefrom, for use in a method of treating or preventing a bacterial infection by a bacterium expressing TAA in a subject; or (b) a Chlamydia major outer membrane protein (MOMP) polypeptide, and a chimeric polypeptide comprising a Chlamydia MOMP polypeptide according to any one of claims 17 to 23, a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide according to claim 31 or 32, or a composition thereof according to claim 34, or an antigen binding molecule according to claim 50(ii) or 51(v) or a composition thereof according to claim 52, or a composition according to claim 56(ii) or any one of claims 57 to 60 dependent therefrom, for use in a method for treating or preventing a Chlamydia infection in a subject.

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