Improved chimeric polypeptides and uses thereof

Abstract

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 structure-stabilizing portion, as well as complexes comprising these chimeric polypeptides. The present disclosure also relates to the use of these chimeric polypeptides and their complexes in compositions, as well as methods for eliciting an immune response against a microbial polypeptide (preferably, an enveloped virus fusion protein or a bacterial outer membrane polypeptide) or its respective complex, and / or for treating or preventing an associated microbial infection (preferably, an enveloped virus infection or a bacterial infection). Furthermore, the present disclosure relates to compositions and methods for producing antigen-binding molecules that specifically bind to such microbial polypeptides or complexes thereof (preferably, an enveloped virus fusion protein or complex thereof, or a bacterial outer membrane polypeptide or complex thereof).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from earlier European Patent Application No. EP 22 166 085.5, filed together with a sequence listing on March 31, 2022. The contents of this earlier application and the accompanying sequence listing are incorporated herein by reference in their entirety.

[0002] 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 structure-stabilizing portion, as well as complexes comprising these chimeric polypeptides. The present disclosure also relates to the use of these chimeric polypeptides and complexes thereof in compositions, and to methods for eliciting an immune response against the microbial polypeptide (preferably, an enveloped virus fusion protein or a bacterial outer membrane polypeptide) or its respective complex, and / or for treating or preventing an associated microbial infection (preferably, an enveloped virus infection or a bacterial infection). Furthermore, the present disclosure relates to compositions and methods for producing antigen-binding molecules that specifically bind to such microbial polypeptides or complexes thereof (preferably, an enveloped virus fusion protein or complex thereof, or a bacterial outer membrane polypeptide or complex thereof). [Background technology]

[0003] Enveloped viruses, such as respiratory syncytial virus (RSV), influenza virus, and human immunodeficiency virus (HIV), require fusion of the viral membrane with the host cell membrane to enter and infect host cells. Viral fusion proteins facilitate this process by undergoing energetically favorable structural rearrangements from a metastable "pre-fusion" conformation to a highly stable "post-fusion" conformation. This structural change triggers fusion of the viral and host cell membranes, resulting in the release of the viral genome into the host cell.

[0004] Viral fusion proteins are currently classified into three major classes based on their individual structural architectures and the molecular characteristics that drive the fusion process. Class I and Class III fusion proteins are trimers in both their pre-fusion and post-fusion conformations, while Class II fusion proteins are dimers in their pre-fusion conformations, which then rearrange into trimeric post-fusion forms. However, in the future, new classes of viral fusion proteins may be identified that share several important characteristics in common with these currently defined classes. Class I and Class III fusion proteins share significant structural features, including an N-terminal signal sequence and C-terminal transmembrane and cytoplasmic domains. They also share a similar fusion mechanism, in which the initial pre-fusion trimer undergoes partial dissociation to allow for the extensive structural rearrangements necessary to form the post-fusion trimer.

[0005] Viral fusion proteins are excellent subunit vaccine candidates because they are the primary targets of protective neutralizing antibody responses against many medically important enveloped viruses. However, the inherent metastable nature of these fusion proteins, especially when isolated and recombinantly expressed as soluble proteins, presents a major obstacle to effective subunit vaccine design. Evidence indicates that the majority of broadly cross-reactive and potently neutralizing antibodies elicited during natural infection primarily target the prefusion form rather than the postfusion form. In addition, the prefusion form of viral envelope fusion proteins has been shown to contain epitopes that are either absent or structurally inaccessible in the postfusion form (e.g., Magro et al., 2012. Proc. Natl. Acad. Sci. USA 109(8):3089-3094). Given these known observations, stabilized prefusion forms are generally considered antigenically more desirable for vaccine development. However, conventional recombinant expression of these proteins typically results in premature triggering and a conformational shift to the structurally more stable post-fusion form. Since then, strategies have been explored that would overcome these previous obstacles posed by the intrinsic structural tendencies of this particular class of proteins, mostly through stabilization of the antigenically more potent pre-fusion state.

[0006] The accumulation of structural information on a large number of related viral targets has paved the way for the structure-based design of viral fusion protein antigens stabilized in the prefusion state. One reported advance toward this goal is the development of an engineered form of the respiratory syncytial virus (RSV) fusion (F) protein (RSV F), also known as DS-Cav1 Foldon, in which stabilization of the prefusion state is achieved through the introduction of stabilizing mutations, including structure-induced artificial disulfide bonds (DS) and hydrophobic cavity-filling (Cav1) mutations, as well as C-terminal fusion to the T4 bacteriophage fibritin trimerization domain (commonly known 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).

[0007] However, a major drawback of such "structural vaccinology"-based approaches is that they rely on high-resolution structural data from each individual viral target, thus limiting the process for vaccine development against targets whose structures remain unknown. Given the continuing emergence of new viral variants, as is strikingly 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 without the prior availability of respective 3D structural information, i.e., universally applicable platform technologies.

[0008] To address this need, some of the present inventors have recently developed approaches to stabilize viral fusion protein antigens in their pre-fusion conformation (International Publication Nos. WO 2018 / 176103 and WO 2022 / 043908). Their technology is based on the discovery that viral fusion proteins can be maintained in their pre-fusion form by operably attaching a heterologous moiety containing complementary heptad repeat region (HRR) pairs downstream of the virion surface-exposed domain of the fusion protein. These HRRs promote trimerization with two additional chimeric polypeptide subunits, thereby associating the HRR pairs of each of the three subunits into a six-helix bundle structure. The resulting trimeric structure acts as a kind of "molecular clamp" that "locks" the fusion ectodomain polypeptide in the pre-fusion conformation, thereby preventing it from reconstituting into a post-fusion conformation. The first generation of this technology focused on utilizing the clamp derived from the six-helix bundle of human immunodeficiency virus (HIV) glycoprotein 41 (gp41).

[0009] This technology was successfully applied to the development of a SARS-CoV-2 vaccine candidate. Subsequent Phase 1 clinical trials showed that the vaccine candidate elicited potent neutralizing antibody responses against the enveloped viral fusion protein ectodomain; however, all recipients were also found to have generated additional antibodies against the clamp domain derived from the small HIV gp41. Although the titers of these antibodies were low, they were sufficient to generate false-positive results in several rapid HIV diagnostic tests. Due to this observed interference with HIV diagnostics, further development and clinical investigation of this vaccine candidate was discontinued.

[0010] Therefore, there remains an urgent need for new viral fusion protein subunit vaccines that avoid the risk of HIV diagnostic interference caused by the clamp domain while at the same time having equal or even improved ability to elicit neutralizing antibody responses against the targeted antigen, particularly new vaccine designs that can be easily applied to viral targets as a universal platform technology in the absence of structural information.

[0011] Furthermore, given the increasing threat of antibiotic resistance and the resurgence of numerous related infectious diseases, there is an urgent need for novel vaccines against bacterial pathogens. In recent years, bacterial outer membrane proteins have become of great interest for vaccine development because they are proteins that interact with the extracellular environment. A specific class of outer membrane proteins found in many pathologically relevant Gram-negative bacteria are so-called trimeric autotransporter adhesins (TAAs), which mediate initial adhesion to host cells during the infection process. TAAs therefore appear to constitute important virulence factors and have attracted increasing interest as potential vaccine targets. Most relevant bacterial infections, however, are not covered by any of the current vaccines, and new developments have slowed in recent decades (see, e.g., the discussion by 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). Thus, there is a need in the art for vaccines against relevant bacterial targets.

[0012] The present invention addresses these needs and provides related advantages as well. Summary of the Invention [Means for solving the problem]

[0013] Accordingly, in a first aspect, the present invention provides a chimeric polypeptide comprising a microbial polypeptide (preferably (a) an enveloped virus fusion ectodomain polypeptide or (b) a bacterial outer membrane polypeptide) operably connected downstream to 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) FHRR has an amino acid sequence having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO: 80 or 81. and / or (ii) FHRR comprises or consists of an amino acid sequence having at least 90% sequence similarity to the amino acid sequence set forth 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 set forth in SEQ ID NO: 82 or 83.

[0014] In a second aspect, the present invention provides a chimeric polypeptide comprising a first (poly)peptide operably connected downstream to a structure-stabilising moiety, wherein said structure-stabilising moiety is as defined in relation to the first aspect of the invention, and preferably wherein the first polypeptide is a therapeutic polypeptide.

[0015] In a third aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding a chimeric polypeptide as defined in any embodiment disclosed herein in relation to the first or second aspect of the invention.

[0016] In a fourth aspect, the present invention provides a host cell comprising nucleic acid as defined according to the third aspect of the invention. In a fifth aspect, the present invention provides a method for producing a chimeric polypeptide complex, the method comprising combining chimeric polypeptides as defined according to the first or second aspect of the invention under conditions suitable for the formation of a chimeric polypeptide complex, whereby a chimeric polypeptide complex is produced 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.

[0017] In a sixth aspect, the present invention provides a chimeric polypeptide complex comprising three chimeric polypeptide subunits, each subunit being a chimeric polypeptide as defined according to the first or second aspect of the invention, wherein the complex is characterized by a six-helix bundle formed by homotrimerization of the structure-stabilizing portions of the three chimeric polypeptides.

[0018] In a seventh aspect, the present invention provides a composition comprising a chimeric polypeptide as defined according to the first or second aspect of the invention or a chimeric polypeptide complex as defined according to the sixth aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0019] In an eighth aspect, the present invention provides 1. A method for identifying an agent that binds to a microbial polypeptide or complex thereof, comprising: The method is (i) contacting a candidate agent with a chimeric polypeptide comprising a microbial polypeptide as defined according to the first aspect of the invention or a chimeric polypeptide complex comprising a microbial polypeptide as defined according to the sixth aspect of the invention; (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex; Including, Preferably, the candidate agent is part of a compound library (e.g., a small molecule or macromolecule library). A method is provided.

[0020] In a preferred embodiment of the latter aspect, (a) the microbial polypeptide or complex thereof is a fusion protein or a complex of fusion proteins of an enveloped virus, respectively, and the method comprises the steps of: (i) contacting a candidate agent with a chimeric polypeptide comprising an enveloped virus fusion ecto-domain polypeptide as defined according to the first aspect of the present invention or a chimeric polypeptide complex comprising an enveloped virus fusion ecto-domain polypeptide as defined according to the sixth aspect of the present invention, wherein the enveloped virus fusion ecto-domain polypeptide corresponds to the fusion protein of the enveloped virus; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or the chimeric polypeptide complex. or (b) the microbial polypeptide or its complex is a bacterial outer membrane polypeptide or a complex of an outer membrane polypeptide, respectively, and the method comprises the steps of (i) contacting a candidate agent with a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined according to the first aspect of the invention or a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined according to the sixth aspect of the invention, wherein the bacterial outer membrane polypeptide corresponds to the bacterial outer membrane polypeptide; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or the chimeric polypeptide complex.

[0021] In a preferred embodiment of the latter embodiment, (a) the bacterial outer membrane polypeptide or complex thereof is a bacterial trimeric autotransporter adhesin (TAA) polypeptide or a complex of TAA polypeptides, respectively, and the method comprises the steps of: (i) contacting a candidate agent with a chimeric polypeptide comprising a TAA polypeptide as defined according to the first aspect of the invention or a chimeric polypeptide complex comprising a TAA polypeptide as defined according to the sixth aspect of the invention, wherein the TAA polypeptide corresponds to a bacterial TAA polypeptide; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex. Including, or (b) the bacterial outer membrane polypeptide or complex thereof is a major outer membrane protein (MOMP) polypeptide or complex thereof, respectively, of a chlamydial bacterium, and the method comprises the steps of: (i) contacting a candidate agent with a chimeric polypeptide comprising a chlamydial MOMP polypeptide as defined according to the first aspect of the invention or a chimeric polypeptide complex comprising a chlamydial MOMP polypeptide as defined according to the sixth aspect of the invention, wherein the chlamydial MOMP polypeptide corresponds to a MOMP polypeptide of a chlamydial bacterium; and (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex. Includes:

[0022] In a ninth aspect, the present invention provides A method for producing an antigen-binding molecule that specifically binds to a microbial polypeptide or a complex thereof, comprising: (1) immunizing a subject with a chimeric polypeptide comprising a microbial polypeptide as defined according to the first aspect of the invention, or a chimeric polypeptide complex comprising a microbial polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention; (2) identifying and / or isolating B cells from the immunized subject that specifically bind to the microbial polypeptide or complex thereof; (3) producing an antigen-binding molecule expressed by the B cells; The present invention provides a method comprising:

[0023] In a preferred embodiment of the latter aspect, (a) the microbial polypeptide or complex thereof is a fusion protein or an ectodomain of a fusion protein complex of an enveloped virus, respectively, and the method comprises the steps of: (1) immunizing a subject with a chimeric polypeptide comprising an ectodomain polypeptide as defined according to the first aspect of the present invention, or a chimeric polypeptide complex comprising an ectodomain polypeptide as defined according to the sixth aspect of the present invention, or a composition thereof as defined according to the seventh aspect of the present invention, wherein the ectodomain polypeptide corresponds to the fusion protein of the enveloped virus; (2) identifying and / or isolating B cells from the immunized subject that specifically bind to the ectodomain of the fusion protein or complex thereof; and (3) producing an antigen-binding molecule expressed by the B cells. or (b) the microbial polypeptide or complex thereof is a bacterial outer membrane polypeptide or a complex of outer membrane polypeptides, and the method comprises the steps of: (1) immunizing a subject with a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined according to the first aspect of the invention, or a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, wherein the bacterial outer membrane polypeptide corresponds to a bacterial outer membrane polypeptide; (2) identifying and / or isolating B cells from the immunized subject that specifically bind to the bacterial outer membrane polypeptide or complex thereof; and (3) producing an antigen-binding molecule expressed by the B cells.

[0024] In a preferred embodiment of the latter embodiment, (i) the outer membrane polypeptide or complex thereof is a bacterial TAA polypeptide or complex thereof, respectively, and the method comprises the steps of: (1) immunizing a subject with a chimeric polypeptide comprising a TAA polypeptide as defined according to the first aspect of the present invention, or a chimeric polypeptide complex comprising a TAA polypeptide as defined according to the sixth aspect of the present invention, or a composition thereof as defined according to the seventh aspect of the present invention, wherein the TAA polypeptide corresponds to a bacterial TAA polypeptide; (2) identifying and / or isolating B cells from the immunized subject that specifically bind to the TAA polypeptide or complex thereof; and (3) producing an antigen-binding molecule expressed by the B cells. or (ii) the outer membrane polypeptide or complex thereof is a major outer membrane protein (MOMP) polypeptide or complex thereof of a chlamydial bacterium, respectively, and the method comprises the steps of: (1) immunizing a subject with a chimeric polypeptide comprising a chlamydial MOMP polypeptide as defined according to the first aspect of the present invention, or a chimeric polypeptide complex comprising a chlamydial MOMP polypeptide as defined according to the sixth aspect of the present invention, or a composition thereof as defined according to the seventh aspect of the present invention, wherein the chlamydial MOMP polypeptide corresponds to a MOMP polypeptide of a chlamydial bacterium; (2) identifying and / or isolating B cells from the immunized subject that specifically bind to the chlamydial MOMP polypeptide or complex thereof; and (3) producing an antigen-binding molecule produced by the B cells.

[0025] In a tenth aspect, the present invention provides a method for producing a pharmaceutical composition comprising: A chimeric polypeptide comprising a microbial polypeptide as defined according to the first aspect of the present invention and / or a microbial polypeptide as one or more subunits of a chimeric polypeptide complex comprising a microbial polypeptide as defined according to the sixth aspect of the present invention. The present invention provides an antigen-binding molecule that specifically binds to

[0026] In a preferred embodiment of the latter aspect, the antigen-binding molecule comprises: (i) the ectodomain of a chimeric polypeptide comprising an enveloped virus fusion ectodomain polypeptide as defined according to the first aspect of the present invention, and / or the ectodomain of one or more subunits of a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain polypeptide as defined according to the sixth aspect of the present invention; or (ii) a bacterial outer membrane polypeptide of a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined according to the first aspect of the invention, and / or a bacterial outer membrane polypeptide of a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined according to the sixth aspect of the invention. It specifically binds to

[0027] In a preferred embodiment of the latter embodiment, the antigen-binding molecule comprises: (i) a TAA polypeptide of a chimeric polypeptide comprising a TAA polypeptide as defined according to the first aspect of the invention, and / or a TAA polypeptide of a chimeric polypeptide complex comprising a TAA polypeptide as defined according to the sixth aspect of the invention, or (ii) a chlamydial major outer membrane protein (MOMP) polypeptide of a chimeric polypeptide comprising a chlamydial MOMP polypeptide as defined according to the first aspect of the invention, and / or a chlamydial MOMP polypeptide of a chimeric polypeptide complex comprising a chlamydial MOMP polypeptide as defined according to the sixth aspect of the invention. It specifically binds to

[0028] 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. In a twelfth aspect, the present invention provides a composition comprising an antigen-binding molecule defined according to the tenth or eleventh aspect of the invention and a pharmaceutically acceptable carrier, diluent or adjuvant.

[0029] In a thirteenth aspect, the present invention provides a composition comprising a nucleic acid as defined according to the third aspect of the invention. In a fourteenth aspect, the present invention provides a chimeric polypeptide as defined according to the first or second aspect of the invention, a nucleic acid as defined according to the third aspect of the invention, a host cell as defined according to the fourth aspect of the invention, a chimeric polypeptide complex as defined according to the sixth aspect of the invention, a composition as defined according to the seventh aspect of the invention, an antigen-binding molecule as defined according to the tenth or eleventh aspect of the invention, or a composition as defined according to the twelfth or thirteenth aspect of the invention, for use as a medicament.

[0030] In a fifteenth aspect, the present invention provides a method for producing a pharmaceutical composition comprising: 1. A method of eliciting an immune response in a subject against a microbial polypeptide or complex thereof, comprising administering to the subject: (i) a chimeric polypeptide comprising a microbial polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or (ii) a composition defined according to the thirteenth aspect of the present invention The present invention provides a method comprising administering

[0031] In a preferred embodiment of the latter aspect, (a) the microbial polypeptide or complex thereof is a fusion protein or complex of fusion proteins, respectively, of an enveloped virus, and the method comprises administering to a subject (i) a chimeric polypeptide comprising an enveloped virus fusion ectodomain as defined according to the first aspect of the present invention, a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain as defined according to the sixth aspect of the present invention, or a composition thereof as defined according to the seventh aspect of the present invention, or (ii) a composition as defined according to the thirteenth aspect of the present invention, wherein the ectodomain polypeptide subunit of the chimeric polypeptide complex corresponds to the fusion protein of the enveloped virus; or (b) the microbial polypeptide or complex thereof is a bacterial outer membrane polypeptide or a complex of outer membrane polypeptides, respectively, and the method comprises the step of administering to a subject (i) a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or (ii) a composition as defined according to the thirteenth aspect of the invention, wherein the bacterial outer membrane polypeptide subunit of the chimeric polypeptide complex corresponds or substantially corresponds to a bacterial outer membrane polypeptide expressed by the bacterium.

[0032] In a preferred embodiment of the latter embodiment, (a) the bacterial outer membrane polypeptide or complex thereof is a bacterial TAA polypeptide or complex of TAA polypeptides, respectively, and the method comprises administering to a subject (i) a chimeric polypeptide comprising a TAA polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a TAA polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or (ii) a composition as defined according to the thirteenth aspect of the invention, wherein the TAA polypeptide subunits of the chimeric polypeptide complex correspond or substantially correspond to a TAA polypeptide expressed by the bacterium, or (b) the bacterial outer membrane polypeptide or complex thereof is a chlamydial major outer membrane protein (MOMP) polypeptide or a complex of chlamydial MOMP polypeptides, respectively, and the method comprises the step of administering to a subject (i) a chimeric polypeptide comprising a chlamydial MOMP polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a chlamydial MOMP polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or (ii) a composition as defined according to the thirteenth aspect of the invention, wherein the chlamydial MOMP polypeptide subunit of the chimeric polypeptide complex corresponds or substantially corresponds to a chlamydial MOMP polypeptide expressed by the bacterium.

[0033] In a sixteenth aspect, the present invention provides a method for treating or preventing a microbial infection in a subject, comprising administering to the subject an effective amount of (i) a chimeric polypeptide comprising a microbial polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention; (ii) an antigen-binding molecule as defined according to the tenth or eleventh aspect of the invention, or a composition thereof as defined according to the twelfth aspect of the invention; or (iii) a composition defined according to the thirteenth aspect of the present invention The present invention provides a method comprising administering

[0034] In a preferred embodiment of the latter aspect, (a) the microbial infection is an enveloped virus infection in the subject, and the method comprises administering to the subject an effective amount of (i) a chimeric polypeptide comprising an enveloped virus fusion ectodomain as defined according to the first aspect of the present invention, a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain as defined according to the sixth aspect of the present invention, or a composition thereof as defined according to the seventh aspect of the present invention, (ii) an antigen-binding molecule as defined according to the tenth or eleventh aspect of the present invention, or a composition thereof as defined according to the twelfth aspect of the present invention, or (iii) a composition as defined according to the thirteenth aspect of the present invention, or (b) the microbial infection is a bacterial infection in the subject, and the method comprises the step of administering to the subject an effective amount of (i) a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, (ii) an antigen-binding molecule as defined according to the tenth or eleventh aspect of the invention, or a composition thereof as defined according to the twelfth aspect of the invention, or (iii) a composition as defined according to the thirteenth aspect of the invention.

[0035] In a preferred embodiment of the latter embodiment, (a) the bacterial infection is an infection caused by a bacterium expressing a TAA, and the method comprises administering to the subject an effective amount of (i) a chimeric polypeptide comprising a TAA polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a TAA polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, (ii) an antigen-binding molecule as defined according to the tenth or eleventh aspect of the invention, or a composition thereof as defined according to the twelfth aspect of the invention, or (iii) a composition as defined according to the thirteenth aspect of the invention; or (b) the bacterial infection is an infection caused by a Chlamydia bacterium, and the method comprises administering to the subject an effective amount of (i) a chimeric polypeptide comprising a Chlamydia MOMP polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a Chlamydia MOMP polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, (ii) an antigen-binding molecule as defined according to the tenth or eleventh aspect of the invention, or a composition thereof as defined according to the twelfth aspect of the invention, or (iii) a composition as defined according to the thirteenth aspect of the invention.

[0036] In a seventeenth aspect, the present invention provides a method for inducing an immune response to a microbial polypeptide or a complex of microbial polypeptides in a subject, comprising: (i) a chimeric polypeptide comprising a microbial polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or (ii) a composition defined according to the thirteenth aspect of the present invention The present invention provides a vaccine comprising:

[0037] In a preferred embodiment of the latter aspect, (a) the microbial polypeptide is an enveloped virus fusion ectodomain polypeptide, and the vaccine comprises (i) a chimeric polypeptide comprising an enveloped virus fusion ectodomain polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or (ii) a composition as defined according to the thirteenth aspect of the invention, wherein the ectodomain polypeptide subunit of the chimeric polypeptide complex corresponds to the fusion protein of the enveloped virus, for use in a method of eliciting an immune response in a subject against a fusion protein or complex of fusion proteins of the enveloped virus; or (b) the microbial polypeptide is a bacterial outer membrane polypeptide, and the vaccine comprises (i) a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or (ii) a composition as defined according to the thirteenth aspect of the invention, for use in a method of eliciting an immune response in a subject against a bacterial outer membrane polypeptide or a complex of outer membrane polypeptides, wherein the bacterial outer membrane polypeptide subunit of the chimeric polypeptide complex corresponds to the bacterial outer membrane polypeptide.

[0038] In a preferred embodiment of the latter embodiment, (a) the bacterial outer membrane polypeptide is a trimeric autotransporter adhesin (TAA) polypeptide, and the vaccine comprises (i) a chimeric polypeptide comprising a TAA polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a TAA polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or (ii) a composition as defined according to the thirteenth aspect of the invention, wherein the TAA polypeptide subunit of the chimeric polypeptide complex corresponds to the bacterial TAA polypeptide, for use in a method of eliciting an immune response in a subject against a bacterial TAA polypeptide or a complex of TAA polypeptides. or (b) the bacterial outer membrane polypeptide is a chlamydial MOMP polypeptide, and the vaccine comprises (i) a chimeric polypeptide comprising a chlamydial MOMP polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a chlamydial MOMP polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or (ii) a composition as defined according to the thirteenth aspect of the invention, for use in a method of eliciting an immune response in a subject against a MOMP polypeptide or a complex of MOMP polypeptides of a chlamydial bacterium, wherein the MOMP polypeptide subunit of the chimeric polypeptide complex corresponds to a MOMP polypeptide of a chlamydial bacterium.

[0039] In an eighteenth aspect, the present invention provides a chimeric polypeptide comprising a microbial polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a microbial polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or an antigen-binding molecule as defined according to the tenth or eleventh aspect of the invention, or a composition thereof as defined according to the twelfth aspect of the invention, or a composition as defined according to the thirteenth aspect of the invention, for use in a method for treating or preventing a microbial infection in a subject.

[0040] In a preferred embodiment of the latter aspect, (a) the microbial polypeptide is an enveloped virus fusion ectodomain polypeptide; and there is provided a chimeric polypeptide comprising an enveloped virus fusion ectodomain polypeptide as defined according to the first aspect of the present invention, a chimeric polypeptide complex comprising an enveloped virus fusion ectodomain as defined according to the sixth aspect of the present invention, or a composition thereof as defined according to the seventh aspect of the present invention, or an antigen-binding molecule as defined according to the tenth or eleventh aspect of the present invention, or a composition thereof as defined according to the twelfth aspect of the present invention, or a composition as defined according to the thirteenth aspect of the present invention, for use in a method for treating or preventing an enveloped virus infection in a subject. or (b) the microbial polypeptide is a bacterial outer membrane polypeptide; and there is provided a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or an antigen-binding molecule as defined according to the tenth or eleventh aspect of the invention, or a composition thereof as defined according to the twelfth aspect of the invention, or a composition as defined according to the thirteenth aspect of the invention, for use in a method for treating or preventing a bacterial infection in a subject.

[0041] In a preferred embodiment of the latter embodiment, (a) the bacterial outer membrane polypeptide is a bacterial trimeric autotransporter adhesin (TAA) polypeptide; and there is provided a chimeric polypeptide comprising a bacterial trimeric autotransporter adhesin (TAA) polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a TAA polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or an antigen-binding molecule as defined according to the tenth or eleventh aspect of the invention, or a composition thereof as defined according to the twelfth aspect of the invention, or a composition as defined according to the thirteenth aspect of the invention, for use in a method for treating or preventing a bacterial infection by a bacterium expressing the TAA in a subject. or (b) the bacterial outer membrane polypeptide is a chlamydial MOMP polypeptide; and there is provided a chimeric polypeptide comprising a chlamydial MOMP polypeptide as defined according to the first aspect of the invention, a chimeric polypeptide complex comprising a chlamydial MOMP polypeptide as defined according to the sixth aspect of the invention, or a composition thereof as defined according to the seventh aspect of the invention, or an antigen-binding molecule as defined according to the tenth or eleventh aspect of the invention, or a composition thereof as defined according to the twelfth aspect of the invention, or a composition as defined according to the thirteenth aspect of the invention, for use in a method for treating or preventing chlamydial infection in a subject. [Brief explanation of the drawings]

[0042] [Figure 1] Nineteen putative clamp sequences were designed from the trimerization domains of proteins derived from animal viruses known not to commonly infect humans. Potential second-generation clamp sequences are aligned with the original HIV-based clamp, designated HIV clamp. CD and CK codes were assigned to the various second-generation clamps. The virus from which the sequence was derived is indicated. [Figure 2]Size-exclusion HPLC (SE-HPLC) analysis of selected VL22_66K clamp antigens. AUC = area under the curve. Antigens labeled CDX or CK8 are VL22_66K CDX or VL22_K66 CK8, respectively. Fsol is a non-stabilized soluble form of the RSV ectodomain and is the post-fusion F control (SEQ ID NO: 27). F HIV clamp is a control antigen stabilized by the HIV clamp (SEQ ID NO: 26). [Figure 3] Transmission electron microscopy (TEM) of selected VL22_66K clamp antigens. Samples were coated onto glow-discharged carbon-coated grids at 10 μg / ml. The antigen labeled CDX is VL22_K66 CDX. [Figure 4] Thermostability analysis of selected VL22_66K clamps by SE-HPLC. Antigens were incubated at 4°C or 25°C for 72 hours. Antigens labeled CDX are VL22_66K CDX. [Figure 5] Comparison of recovered antigen yields from transient ExpiCHO (Thermo Fisher) protein expression of VL22_66K clamp antigen (A) and VL22_66E clamp antigen (B). ND = not determined. In each graph, the dashed line indicates the recovered yield of the F HIV clamp. For brevity, VL22 is omitted from the VL22 clamp antigen name in the graph labels. [Figure 6] Thermal stability of VL22_CD10 and VL22_CD11 and F HIV clamps assessed by binding of RSV F-specific mAbs (101F and MPE8) via ELISA. Prior to ELISA, antigens were incubated at 4°C, 25°C, 40°C, and 60°C for 72 hours. [Figure 7] Thermostability of VL22_CD10 and VL22_CD11 and the F HIV clamp assessed by duplicate SE-HPLC analysis. Prior to SE-HPLC, antigens were incubated at 4°C, 25°C, 40°C, and 60°C for 72 hours. [Figure 8]Cryo-electron microscopy of the VL22_CD11 trimer and a low-resolution model generated from the image. [Figure 9] Cryo-electron microscopy of the VL22_CD11 trimer in complex with motavizumab Fab and the resulting high-resolution model. [Figure 10] Binding of sera from eight mice immunized with SARS-CoV-2 S HIV clamp to VL22_CD10, VL22_CD11, and F HIV clamp by ELISA (Panels A-C). Endpoint titers (EPTs) are plotted as geometric means with geometric standard deviations (Panel D). [Figure 11] Analysis of binding of Bio-Rad Geenius HIV 1 / 2 control human plasma (panels A and B) and NIBSC International HIV Reference Standard (panels C and D) to SARS-CoV-2 S HIV clamp and SARS-CoV-2 S CD11 by ELISA. [Figure 12] Analysis by ELISA of binding of sera from eight mice immunized with VL22_CD10 (A), VL22_CD11 (B), and F HIV clamp (C) to HIV gp41 (#ab49070, Abcam, Cambridge, United Kingdom), the same antigen used for vaccination (self-antigen), the clamp domains within the antigens when paired with alternative ectodomains (SARS-CoV-2 S HIV clamp, SARS-CoV-2 S CD10, and SARS-CoV-2 S CD11), and the ectodomains within the antigens when paired with alternative clamps (F HIV clamp SARS-CoV-2 S CD11). [Figure 13]Serum neutralization of RSV A2 was assessed by Plaque Reduction Neutralization Test (PRNT) after two vaccinations of BALB / c mice (n=5 or n=3 for PBS) with 25 μl of AddaVax (Invivogen) and 5 μg of VL22_CD10, VL22_CD11, or F HIV clamp, or PBS control, three weeks apart. The international standard refers to the WHO 1st International Standard for Antiserum to Respiratory Syncytial Virus (NIBSC, #16-284). [Figure 14] SE HPLC of a partial CD panel with SARS-CoV-2 spike antigen. Dashed lines indicate the elution times of molecular weight standards: A = thyroglobulin (670 kDa), B = gamma globulin (158 kDa), C = ovalbumin (44 kDa). [Figure 15] SE-FPLC of viral antigens stabilized with HIV clamp and CD11. Comparison between SARS-CoV-2 S HIV clamp and SARS-CoV-2 S CD11 on Superose 6 Increase 10 / 300GL (Cytiva) (Panel A). Comparison between Nipah virus F HIV clamp and Nipah F CD11 on Superdex 200 Increase 10 / 300GL (Cytiva) (Panel B). Comparison between influenza HA HIV clamp and influenza HA CD11 on Superdex 200 Increase 10 / 300GL (Cytiva) (Panel c). [Figure 16]Serum neutralization assessed by PRNT or pseudovirus neutralization after two vaccinations of BALB / c mice (n=8) 3 weeks apart with doses consisting of 25μl AddaVax (Invivogen) and 5μg of each antigen or PBS control. Neutralization was assessed by PRNT against live virus, SARS-CoV-2 614G mutant, and influenza A H1N1pdm California09, and with lenti-pseudovirus for Nipah virus. [Figure 17] SE-FPLC of SARS-CoV-2 S CD11(GSG-linker) (SEQ ID NO: 29) and SARS-CoV-2 S CD11-SG(G-linker) (SEQ ID NO: 34) on Superose 6 Increase 10 / 300GL (Cytiva). Arrows indicate the predicted sizes of the trimeric and monomeric proteins. [Figure 18] Model of the CD11 trimer (gray) with selected regions for incorporation of potential N-linked glycosylation sites (black). [Figure 19] Alignment of silenced CD11 sequences. Shading indicates modified residues, arrows indicate glycosylation sites, and numbering is used to define each site. The designation of the variants reflects the site numbering (e.g., 159 has glycosylation sites 1, 5, and 9). Gray bars indicate samples incubated at 4°C and 40°C for 1 week and subsequently analyzed by SE-HPLC. [Figure 20] Binding affinity (KD) of 101F and MPE8 binding to VL22 antigen containing CD11, CD11-silenced variants, or non-stabilized Fsol. [Figure 21] Protein yields of VL22 antigens containing CD11 or CD11-silenced variants. Average yields of duplicate antigen expression runs are graphed with standard deviation, except for CD11_12456, CD11_1245T68, CD11_15T, CD11_1, and CD11_5T, which were obtained from only a single expression run. [Figure 22]SE-HPLC of VL22 antigen containing CD11 or CD11 silenced variants. Duplicate traces are overlaid for each sample. [Figure 23] SE-HPLC traces of VL22 antigen containing CD11 or CD11 silenced variants after 1 week of incubation at 4°C or 40°C. [Figure 24] SE-HPLC traces of VL22 antigen containing CD11 or CD11 silenced variants after 38 days of incubation at 4° C. or 40° C. Duplicate runs are plotted for each temperature incubation. [Figure 25] Glycan composition of recombinant antigens. (A) Alignment of N-linked sites in each vaccine candidate, and (B) N-linked site-specific occupancy. Note that CD11 refers to VL22_CD11, and CD11 145T8 and CD11 1245T8 refer to silenced clamp variants of VL22_CD11. N518 = site 1, N523 = site 2, N530 = site 4, N534 = site 5, and N544 = site 8 (e.g., 145T8 has N518, N530, N534, and N544). [Figure 26] SE-HPLC traces of CD11-based lead antigens VL22_CD11_1245T8 and VL22_CD11_145T8, as well as non-silenced VL22_CD11, F HIV clamp, and control DS-Cav1 foldon and SC9-10 DS-Cav AY foldon. [Figure 27] Yields of VL22_CD11_145T8, VL22_CD11, VL22_HIV clamp, DS-Cav1 foldon, and SC9-10 DS-Cav AY foldon. Average yields and standard deviations obtained from 3–5 separate transient expression runs are shown. [Figure 28]RSV A2 PRNT analysis of final serum from mice receiving 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 mixed with Addavax adjuvant. Error bars represent geometric mean titers and standard deviations; geometric mean titers are also labeled above each data set. Significance compared to PBS-vaccinated controls was determined by one-way ANOVA with Dunn's multiple comparison test using GraphPad Prism Windows version 9.2.0. Significant differences indicated: ****P<0.0001, ***P<0.001, **P<0.01, *P≦0.05, and ns=not significant. The international standard refers to the WHO 1st International Standard for Antiserum to Respiratory Syncytial Virus (NIBSC, #16-284). LoD indicates the limit of detection of the assay. [Figure 29] Binding of final serum obtained from mice receiving 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 mixed with Addavax adjuvant to either the same antigen (self-antigen) used for vaccination, the RSV F ectodomain with an alternative stabilization domain (CD11 or HIV clamp), or the Nipah F antigen (Foldon, HIV clamp, CD11, CD11_145T8, or CD11_1245T8) containing the same stabilization domain was measured by ELISA. Geometric mean endpoint titers (EPTs) are plotted along with the geometric standard deviation. [Figure 30] Alignment of the amino acid sequences of CD11 and site-directed modifications (SEQ ID NOs: 55-72), with the HIV clamp sequence provided for reference. [Figure 31]Expression levels of CD11 site-specific modifications compared to unmodified CD11 controls. Three ELISA measurements are shown for each of the SARS-CoV-2 S CD11 panel, SARS-CoV-2(Delta)S CD11 panel, and SARS-CoV-2(Delta)S CD11-QS panel, and two post-purification Nanodrop measurements are shown for each of the SARS-CoV-2(Delta)S CD11 panel and SARS-CoV-2(Delta)S CD11-QS panel. Geometric means are plotted along with geometric standard deviations. [Figure 32] Percentage of antigens found in trimeric conformation, HMW aggregates, and LMW products for the SARS-CoV-2(Delta)S CD11 site-directed modification panel (A) and the SARS-CoV-2(Delta)S CD11-QS site-directed modification panel (B) by SEC. [Figure 33] Percentage of antigen found in trimeric conformation, HMW aggregates, and LMW products of the SARS-CoV-2(Delta)S CD11 site-directed modified panel (A) and the SARS-CoV-2(Delta)S CD11-QS site-directed modified panel (B) by SEC after 48 hours of incubation at 40°C. [Figure 34] Antigenic integrity of VL22 clamp 2 (D25 mAb) or SARS2-S clamp 2 (CR3022 mAb) assessed by conformation-specific ELISA, either as each antigen alone or as a mixture, at time 0 and after 6 weeks of incubation at 4°C. nr = not available. [Figure 35] Serum neutralization of RSV A2 (A) or SARS-CoV-2 (B) assessed by PRNT after two vaccinations, 3 weeks apart, of BalB / C mice (n=8) with 25 μl of AddaVax (Invivogen) and respective doses consisting of 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. [Figure 36]Expression of CD11-stabilizing bacterial autotransporters analyzed by SDS-PAGE (A) of E. coli lysates or Western blot (WB) (B) with anti-CD11 monoclonal antibody. Lane 1 = lysate from negative control E. coli transformed with only the pSU vector backbone; lane 2 = molecular weight markers (molecular weights listed on the left); lanes 3–9 = E. coli lysates transformed with pSU expression vectors containing the CD11-stabilizing antigens EhaG, HiA, NadA, NhhA, YadA, EibD, and UpaG. [Figure 37] CD11-stabilized bacterial autotransporters, EhaG (A), UpaG (B), and HiA (C), purified by affinity chromatography and analyzed by SDS-PAGE. [Figure 38] Purified CD11-stabilized bacterial autotransporters, EhaG (A), UpaG (B), and HiA (C), analyzed by size-exclusion chromatography on Superose 6 Increase 10 / 300GL (Cytiva). Also shown are linear regressions of logarithmic marker molecular weights and elution volumes, as well as a table comparing the predicted and observed molecular weights of each autotransporter. [Figure 39] Purified CD11-stabilized autotransporter, UpaG, analyzed by negative staining transmission electron microscopy (TEM). [Figure 40] Serum neutralization of RSV A2 assessed by PRNT after two vaccinations of BalB / C mice (n = 8) 3 weeks apart with 25 μl of AddaVax (Invivogen) and respective doses consisting of 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. [Figure 41]Examples of membrane-tethered clamp-stabilized antigens to be delivered by LNP-encapsulated mRNA, DNA, or alternative vaccine vectors include: A) a standard soluble RSV F clamp 2 design, in which the encoded antigen is secreted from cells into the extracellular medium after administration; B) a membrane-tethered RSV F clamp 2 design in which a hydrophobic sequence is inserted into the linker region between the FHRR and SHRR regions of clamp 2, in which the encoded antigen is secreted but remains tethered to the cell surface after administration; C) a membrane-embedded RSV F clamp 2 design in which the clamp 2 sequence is inserted after the native transmembrane domain so that the clamp is present on the cytoplasmic side of the membrane, in which the encoded antigen is secreted but remains tethered to the cell surface after administration. [Figure 42] Overview of candidate membrane-tethering (poly)peptides, including origin, sequence, and structural information. [Figure 43] Quantitative assessment of soluble and membrane-bound clamp 2-stabilizing proteins. A) Soluble antigen measured in cell supernatants by capture ELISA. B) Cell surface-bound antigen measured by flow cytometry. [Figure 44] Western blots of CHO cell extracts after transfection of membrane-tethered clamp constructs and controls. Each cell extract was run on SDS-PAGE with or without pretreatment with PNGase to remove N-linked glycans. Gel 1 (left to right)—molecular weight marker, purified VL22-Clamp2 (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 (left to right) - molecular weight marker, purified VL22-clamp2 (sequence number 75), cell extract VL22_CT5s_alpha (sequence number 225), cell extract VL22_CT5s_gamma (sequence number 227), cell extract VL22_CT5s_eta (sequence number 232), cell extract VL22_CT5s_epsilon (sequence number 229), cell extract untransfected. [Figure 45]Quantification of expression levels of membrane-tethered clamp constructs. Detergent extracts from cell membranes quantified by capture ELISA: A) Motavizumab, B) D25. ELISA curves and 50% effective concentrations (EC50) evaluated by nonlinear regression are shown. DETAILED DESCRIPTION OF THE INVENTION

[0043] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Although any method and material similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described.For the purposes of this disclosure, the following terms are defined below.

[0044] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more than one element.

[0045] 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 combinations when interpreted in the alternative ("or").

[0046] Furthermore, as used herein, the terms "about" and "approximately," when referring to a measurable value, e.g., amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, location, length, etc., are meant to encompass a variation of, for example, ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, location, length, etc. In instances where the terms "about" and "approximately" are used in reference to the location or position of a region within a reference polypeptide, these terms encompass a variation of, for example, up to ±20 amino acid residues, up to ±15 amino acid residues, up to ±10 amino acid residues, up to ±5 amino acid residues, up to ±4 amino acid residues, up to ±3 amino acid residues, up to ±2 amino acid residues, or even ±1 amino acid residue. Furthermore, whenever the term "about" or "approximately" is used, the invention also specifically relates to the corresponding exact value (without variation).

[0047] As used herein, the term "adjuvant" refers to a compound that, when used in combination with a particular immunogen (e.g., a (poly)peptide, chimeric polypeptide, chimeric polypeptide complex, polynucleotide, or nucleic acid construct of the present disclosure) in a composition, will increase the resulting immune response, including strengthening and / or broadening the specificity of either or both antibody and cellular immune responses. In the context of the present disclosure, an adjuvant will preferably enhance the specific immunogenic effect of an active agent of the present disclosure. The term "adjuvant" is understood to typically not include an agent that confers immunity by itself. An adjuvant nonspecifically assists the immune system to enhance antigen-specific immune responses, for example, by promoting antigen presentation to the immune system or induction of an innate immune response. Furthermore, an adjuvant can preferably modulate antigen-specific immune responses, for example, by shifting a predominantly Th2-based antigen-specific response to a more Th1-based antigen-specific response, or vice versa. Thus, an adjuvant can preferably modulate cytokine expression / secretion, antigen presentation, the type of immune response, and the like.

[0048] The term "agent," used interchangeably with "compound" herein, refers to any compound or substance, including, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, and the like. An "agent" can be any chemical entity, including, without limitation, 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, nucleic acid oligomers, amino acids, carbohydrates, oligonucleotides, ribozymes, DNAzymes, glycoproteins, glycolipids, siRNAs, lipoproteins, and modified forms and combinations thereof. In some embodiments, the nucleic acid is DNA or RNA, and the nucleic acid analog can be selected from, for example, PNA, pcPNA, and LNA. The nucleic acid can be single-stranded or double-stranded and can be selected from nucleic acids, oligonucleotides, and the like, encoding proteins of interest. Such nucleic acids include, for example, nucleic acids encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small molecule inhibitory nucleic acid sequences, including, but not limited to, RNAi, shRNA, siRNA, microRNA, and antisense oligonucleotides. The protein can be any protein of interest, for example, a mutant protein, a therapeutic protein, or a truncated protein, where the protein is normally absent or expressed at low levels in cells. Proteins and peptides 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, disaccharide, oligosaccharide, or polysaccharide.

[0049] 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 specific product of humoral or cellular immunity, such as an antibody molecule or a T-cell receptor. Antigens can be any type of molecule, including, for example, haptens, simple intermediate metabolites, sugars (e.g., oligosaccharides), lipids, and hormones, as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins. General categories 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 various other antigens.

[0050] "Antigen-binding molecule" refers to a molecule having binding affinity for a target antigen. This term is understood to extend to immunoglobulins, immunoglobulin fragments, and non-immunoglobulin-derived proteins or non-protein frameworks that exhibit antigen-binding activity. Exemplary antigen-binding molecules useful in the practice of the present disclosure include polyclonal and monoclonal antibodies and their fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) and domain antibodies (including, e.g., shark and camel antibodies), as well as fusion proteins containing antibodies and any other modified configuration of an immunoglobulin molecule that contains an antigen binding / recognition site. Antibodies include antibodies of any class, e.g., IgG, IgA, or IgM (or subclass thereof), and antibodies need not be of any particular class. Depending on the amino acid sequence of the constant region of their heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Antigen-binding molecules, or dimeric antibodies, and multivalent forms of antibodies are also encompassed.In some embodiments, antigen-binding molecules are chimeric antibodies, in which a portion of the heavy and / or light chain is identical to 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 to or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855). Humanized antibodies are also contemplated, and are generally produced by transferring complementarity-determining regions (CDRs) from the heavy and light chain variable regions of a non-human (e.g., rodent, preferably mouse) immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted for the framework regions of the non-human counterpart. The use of antibody components derived from humanized antibodies obviates potential problems associated with the immunogenicity of non-human constant regions. General techniques for cloning non-human, 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, in 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.), pages 399-434 (John Wiley & Sons, Inc. 1996), and Queen et al., U.S. Patent No. 5,693,762. (1997). Humanized antibodies include "primatized" antibodies, in which the antigen-binding region of the antibody is derived from an antibody produced by immunizing a rhesus monkey with an antigen of interest. Humanized antibodies are also contemplated as antigen-binding molecules. Further examples of "antigen-binding molecules" include any of the agents described above, which can be obtained, for example, by using the method according to the eighth aspect of the present invention.

[0051] The term "antiparallel," as used herein, refers to a proteinaceous polymer in which regions or segments of the polymer are in a parallel orientation but have opposite polarities. As used herein, the term "specifically binds" refers to a binding reaction that determines the presence of a chimeric polypeptide or complex of the present disclosure in the presence of a heterogeneous population of molecules, including macromolecules, e.g., proteins and other biologics. In certain embodiments, the term "specifically binds" when referring to an antigen-binding molecule is used interchangeably with terms such as "specifically immunointeractive," referring to a binding reaction that determines the presence of a chimeric polypeptide or complex of the present disclosure in the presence of a heterogeneous population of proteins and other biologics. Under specified assay conditions, the molecule specifically binds to a chimeric polypeptide or complex of the present disclosure and does not bind in significant amounts to other molecules (e.g., proteins or antigens) present in the sample. In embodiments of antigen-binding molecules, various immunoassay formats can be used to select antigen-binding molecules that are specifically immunointeractive with a chimeric polypeptide or complex of the present disclosure. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunointeractive with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immune reactivity, see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0052] The term "chimeric," when used in reference to a molecule, means that the molecule contains portions derived from, obtained or isolated from, or based on, two or more different origins or sources. Thus, a polypeptide is chimeric if it contains two or more amino acid sequences of different origins and (1) polypeptide sequences that are not found together in nature (i.e., at least one of the amino acid sequences is heterologous to at least one of the other amino acid sequences), or (2) amino acid sequences that are not naturally adjacent.

[0053] By "coding sequence" is meant 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). In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to encoding the polypeptide product of a gene or the final mRNA product of a gene.

[0054] The terms "coiled coil" or "coiled-coil structure" are used interchangeably 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 proteins are involved in important biological functions, such as regulating gene expression, e.g., transcription factors. Coiled coils often, but not always, contain a repeating pattern of hydrophobic (h) and polar (p) amino acid residues called a heptad repeat, hpphppp or hppphpp (see below in this specification). This repeating pattern in (poly)peptide sequences results in a naturally folded α-helical secondary structure, resulting in the presentation of hydrophobic residues along one face of the helix and hydrophilic residues along the opposite face, forming an amphipathic structure. The most favorable way for two or three such helices to arrange themselves in a water-filled environment is for the hydrophobic faces of the helices to wrap around or occlude one another, leaving the hydrophilic amino acids exposed to the solvent. This, therefore, is the burial of the hydrophobic surfaces, thereby providing the thermodynamic driving force for α-helix oligomerization and structural stability. The packing at the coiled-coil interface is extremely tight. The α-helices can be parallel or antiparallel, with left-handed supercoils usually employed. Although not preferred, some right-handed coiled coils have also been observed in natural and designed proteins. The terms "coiled coil" or "coiled-coil structure" will be clear to those skilled in the art based on common general knowledge.In this regard, review articles on coiled-coil structures, such as 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), among others.

[0055] 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) that can hybridize to each other, oligomerize (e.g., dimerize), interact, or otherwise form a complex with each other. For example, "complementary regions of polypeptides" can come together to form a complex, which in certain embodiments is characterized by an antiparallel two-helix bundle.

[0056] As used herein, the term "complex" refers to an assembly or aggregation of molecules (e.g., peptides, polypeptides, etc.) in direct and / or indirect contact with each other. In certain embodiments, "contact" or more specifically "direct contact" means that two or more molecules are in sufficient proximity that non-covalent attractive interactions, such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions, govern the interaction of the molecules. In such embodiments, a complex of molecules (e.g., peptides and polypeptides) forms under conditions in which the complex is thermodynamically favorable (e.g., compared to the unaggregated or uncomplexed states of its constituent molecules). As used herein, the term "complex," unless otherwise specified, refers to an assembly of two or more molecules (e.g., peptides, polypeptides, or a combination thereof). In certain embodiments, the term "complex" refers to an assembly of three polypeptides.

[0057] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising," as well as "contain," "contains," and "containing," will be understood to refer to the inclusion of the indicated step or element or group of steps or elements, but not to the exclusion of any other step or element or group of steps or elements. Thus, use of terms such as "comprise" indicates that the recited elements are required or mandatory, while other elements are optional and may or may not be present. "Consisting of" means including and limited to everything that the phrase "consisting of" follows. Thus, the phrase "consisting of" indicates that the recited elements are required or mandatory, and that there are no other elements that may be present. "Consisting essentially of" means including all elements listed before this phrase, and limited to other elements that do not interfere with or interfere with the activity or function of the recited elements as described in this disclosure. Thus, the term "consisting essentially of" indicates that the recited elements are required or essential, while other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements. Throughout this specification, the term "comprising" (or "containing", etc.) also includes the narrower meanings of "consisting essentially of" and "consisting of." Thus, whenever the term "comprising" (or "containing", etc.) is used, the invention specifically relates to the corresponding subject matter defined by the term "consisting essentially of" (instead of "comprising" or "containing") as well as the corresponding subject matter defined by the term "consisting of."

[0058] As used herein, the terms "conjugated," "linked," "fused," or "fusion," and their grammatical equivalents, are used interchangeably in the context of joining two or more elements or components or domains together by some means, including chemical conjugation or recombinant means (e.g., genetic fusion). Methods of chemical conjugation (e.g., using heterobifunctional crosslinkers) 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, preferably in a metastable pre-fusion conformation.

[0059] In certain embodiments, the structurally stabilizing moiety is indirectly fused to the polypeptide, e.g., via a hinge, particularly a flexible linker comprising, e.g., one or more glycine (Gly) and / or one or more serine (Ser) residues. In other embodiments, the structurally stabilizing moiety is directly fused to the polypeptide disclosed herein.

[0060] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally subclassified as shown in Table 1.

[0061] [Table 1]

[0062] Conservative amino acid substitution also includes grouping based on side chain.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-hydroxyl 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 the replacement of leucine with isoleucine or valine, the replacement of aspartic acid with glutamic acid, the replacement of threonine with serine, or similar replacement of an amino acid with a structurally related amino acid will not have a significant effect on the properties of the resulting variant polypeptide.Whether an amino acid change results in a functional polypeptide can be easily determined by assaying its activity. Conservative substitutions are shown in Table 2 under the headings of exemplary and preferred substitutions. Amino acid substitutions that fall within the scope of the present disclosure are generally achieved by selecting substitutions that do not significantly alter (a) the structure of the peptide backbone in the range of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintain the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

[0063] [Table 2]

[0064] The term "construct" refers to a recombinant genetic molecule containing one or more isolated nucleic acid sequences derived from different sources. Thus, a construct is a chimeric molecule in which two or more nucleic acid sequences of different origins are assembled into a single nucleic acid molecule, including any construct containing (1) a nucleic acid sequence containing regulatory and coding sequences not found together in nature (i.e., at least one of the nucleotide sequences is heterologous to at least one of the other nucleotide sequences), or (2) a sequence encoding a portion of a functional RNA molecule or protein that is not naturally contiguous, or (3) a portion of a promoter that is not naturally contiguous. Representative constructs include any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single- or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, and containing one or more operably linked nucleic acid molecules. Constructs of the present disclosure will generally include elements necessary to induce expression of a desired nucleic acid sequence, such as a target nucleic acid sequence or a modulator nucleic acid sequence, also contained within the construct. Such elements may include control elements, such as a promoter operably linked to the nucleic acid sequence of interest (to induce its transcription), and often also include a polyadenylation sequence. Within certain embodiments of the present disclosure, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, e.g., prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs may be contained within a single nucleic acid molecule, e.g., a single vector, or may be contained within two or more separate nucleic acid molecules, e.g., two or more separate vectors.An "expression construct" generally comprises at least one control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, a promoter operably linked to the nucleotide sequence to be expressed is provided in the expression construct for expression in an organism or a part thereof, including a host cell. Conventional compositions and methods for preparing and using constructs and host cells for the implementation of the present disclosure are well known to those skilled in the art, and are described, for example, in Molecular Cloning: A Laboratory Manual, 3. rd See, e.g., J.F. Sambrook, D.W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0065] "Corresponds to" or "corresponding to" refers to a nucleic acid or amino acid sequence that exhibits substantial sequence similarity or identity, respectively, to a reference nucleic acid or amino acid sequence. Generally, a nucleic acid 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, in order of increasing preference, to at least a portion of a reference nucleic acid or amino acid sequence or the entire reference nucleic acid or amino acid sequence.

[0066] The term "domain," as used herein, refers to a portion of a molecule or structure that shares common physicochemical characteristics, such as, but not limited to, hydrophobic, polar, globular, and helical domains, or properties such as ligand binding, membrane fusion, signal transduction, and cell permeability. A domain has a folded protein structure that has the ability to retain its tertiary structure independent of the rest of the protein. Generally, domains are responsible for distinct functional properties of a protein and can often be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or domain. A domain may be coextensive with a region or portion thereof, or it may comprise distinct, non-contiguous regions of a molecule. Examples of protein domains include cellular or extracellular localization domains (e.g., signal peptides, SPs), immunoglobulin (Ig) domains, membrane fusion (e.g., fusion peptides, FPs), ectodomains, membrane-proximal external regions (MPERs), transmembrane (TM) domains, and cytoplasmic (C) domains.

[0067] "Effective amount" in the context of treating, inhibiting the onset of, or preventing a condition means administering to an individual in need of such treatment, inhibition, or prevention, an amount of a drug or composition, either as a single dose or as part of a series, that is effective in preventing the onset of symptoms of the condition, blocking such symptoms, and / or treating existing symptoms.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, evaluation of the medical condition, and other relevant factors.It is expected that the amount will fall within a relatively broad range that can be determined by routine testing.

[0068] The term "endogenous" refers to a polypeptide or portion thereof that is present and / or naturally expressed within a host organism or its cells. For example, an "endogenous" ectodomain polypeptide or portion thereof refers to the ectodomain polypeptide of an envelope fusion protein, or a portion of that ectodomain that is naturally expressed in an enveloped virus.

[0069] The term "endogenous production" refers to expression from 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 certain embodiments, the organism is multicellular (e.g., a vertebrate, preferably a mammal, more preferably a primate, e.g., a human), and the nucleic acid is expressed within a cell or tissue of the multicellular organism.

[0070] The terms "epitope" and "antigenic determinant" are used interchangeably herein to refer to an antigen, typically a protein determinant, that can specifically bind to an antibody (such an epitope is often referred to as a "B cell epitope") or can be presented to a T cell receptor by a major histocompatibility complex (MHC) protein (e.g., class I or class II) (such an epitope is often referred to as a "T cell epitope"). When a B cell epitope is a peptide or polypeptide, it typically contains three or more amino acids, generally at least five, and more usually at least 8-10 amino acids. The amino acids may be adjacent amino acid residues in the primary structure of the polypeptide (often referred to as a contiguous peptide sequence) or may be spatially juxtaposed in the folded protein (often referred to as a non-contiguous peptide sequence). T cell epitopes may bind to MHC class I or MHC class II molecules. Typically, T cell epitopes that bind to MHC class I are 8-11 amino acids in length. Class II molecules bind peptides that are 10-30 residues long or can be longer, with an optimal length of 12-16 residues. The ability of putative T cell epitopes to bind to MHC molecules can be predicted and confirmed experimentally (Dimitrov et al., 2010. Bioinformatics 26(16):2066-8).

[0071] The term "flexible linker," as used herein, refers to a proteinaceous molecule comprising at least one amino acid residue, usually at least two amino acid residues, linked by a peptide bond, which allows two polypeptides linked thereby to move more freely relative to each other than they would move without the flexible linker. In certain embodiments, the flexible linker provides the two polypeptides linked thereby with increased rotational freedom than the two polypeptides would have in the absence of the flexible linker. Such relative freedom of movement or rotational freedom allows the polypeptides linked by the flexible linker to perform their individual functions or elicit activities with less structural hindrance. Flexible linkers are characterized by the absence of secondary structure, e.g., the absence of helices or beta sheets, or a secondary structure content of up to 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). Additional flexible linker sequences are well known in the art. In various embodiments, the flexible linker comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 may 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%, and even more preferably 100%) of the amino acid residues are selected from glycine, serine, and alanine, and 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%, and even more preferably 100%) of the amino acid residues are selected from glycine and serine. In some embodiments, all of the amino acid residues are glycine.

[0072] The term "helical bundle" refers to multiple peptide helices that fold substantially parallel or antiparallel to one another. A two-helix bundle has two helices that fold substantially parallel or antiparallel to one another. Similarly, a six-helix bundle has six helices that fold substantially parallel or antiparallel to one another. "Substantially parallel or antiparallel" means that the helices are folded so that their side chains can interact with one another. For example, the hydrophobic side chains of the helices can interact with one another to form a hydrophobic core.

[0073] The term "heterologous," when used with reference to portions of a nucleic acid or protein, indicates 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 typically produced recombinantly, having two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or multiple coding regions from different sources. Similarly, a heterologous protein indicates 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").

[0074] The term "host" refers to any organism or cell thereof, whether eukaryotic or prokaryotic, into which a construct of the present disclosure can be introduced. In specific embodiments, the term "host" refers to eukaryotic organisms, e.g., unicellular eukaryotes, e.g., yeast and fungi, and multicellular eukaryotes, e.g., animals, including, but not limited to, invertebrates (e.g., insects, cnidarians, echinoderms, nematodes, etc.), eukaryotic parasites (e.g., malaria parasites, e.g., Plasmodium falciparum, helminths, etc.), vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and mammals (e.g., rodents, primates, e.g., humans and non-human primates). Thus, the term "host cell" preferably encompasses cells of such eukaryotic organisms, as well as cell lines derived from such eukaryotic organisms.

[0075] Reference herein to "immune interactive" includes reference to any interaction, reaction, or other form of association between molecules, and particularly where one of the molecules is or mimics a component of the immune system.

[0076] As used herein, the term "immunogenic composition" or "immunogenic formulation" refers to a preparation that will induce an immune response when administered to a vertebrate, particularly an animal, e.g., a mammal.

[0077] The term "linker" or "flexible linker" refers to a molecule or group of molecules (e.g., a monomer or polymer) that connects two molecules and often functions to arrange the two molecules in a desired configuration.

[0078] As used herein, the term "metastable," when used in the context of a protein (e.g., an enveloped virus ectodomain polypeptide), refers to an unstable but constrained conformational state that is rapidly converted to a more stable conformational state upon changing conditions. For example, an enveloped virus fusion protein in its pre-fusion form is in an unstable metastable conformation that is converted to a more stable post-fusion conformation upon fusion, e.g., to a host cell.

[0079] As used herein, the term "moiety" refers to a portion of a molecule, which may be a functional group, a set of functional groups, and / or a particular grouping of atoms within a molecule that is responsible for the molecule's characteristic chemical, biological, and / or pharmaceutical properties.

[0080] The term "neutralizing antigen-binding molecule" refers to an antigen-binding molecule that binds to or interacts with a target molecule or ligand, preventing the target antigen from binding or associating with a binding partner, e.g., a receptor or substrate, thereby preventing a biological response that would normally result from the molecular interaction. In the context of the present disclosure, the neutralizing antigen-binding molecule suitably associates with the metastable or prefusion form of an enveloped virus fusion protein, and preferably prevents or reduces the binding and / or fusion of the spike protein to a cell membrane.

[0081] The term "oligomer" refers to a molecule consisting of more than one but a limited number of monomer units, as opposed to a polymer, which is composed of, at least in principle, an unlimited number of monomers. Oligomers include, but are not limited to, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, and the like. An oligomer may be a macromolecular complex formed by the noncovalent association of macromolecules, such as proteins. In this sense, homo-oligomers are formed by identical molecules; in contrast, hetero-oligomers will be made up of at least two different molecules. In certain embodiments, the oligomers of the present disclosure are trimeric polypeptide complexes consisting of three polypeptide subunits. In these embodiments, the trimeric polypeptide may be a "homotrimeric polypeptide complex" consisting of three identical polypeptide subunits, or a "heterotrimeric polypeptide complex" consisting of three polypeptide subunits, in which at least one subunit polypeptide is non-identical. A "polypeptide subunit" is a single amino acid chain or monomer that combines with two other polypeptide subunits to form a trimeric polypeptide complex.

[0082] The terms "operably connected" or "operably linked," as used herein, refer to a juxtaposition of the components so described in a relationship permitting them to function in their intended manner. For example, a "regulatory sequence (e.g., a promoter) operably linked" to a nucleotide sequence of interest (e.g., a coding and / or non-coding sequence) refers to the positioning and / or orientation of the regulatory sequence relative to the nucleotide sequence of interest that permits expression of that sequence under conditions compatible with the regulatory sequence. Regulatory sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct expression of that nucleotide sequence. Thus, for example, intervening non-coding sequences (e.g., non-translated but transcribed sequences) can be present between the promoter and coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Similarly, "operably linking" an enveloped virus fusion ectodomain polypeptide to a heterologous structural stabilizing moiety (SSM) includes positioning and / or orienting the structural stabilizing moiety (SSM) such that, under suitable conditions (e.g., in aqueous solution and / or physiological conditions), it can associate with the structural stabilizing moieties (SSMs) of two additional chimeric polypeptides to form a trimer, preferably in which the FHRRs and SHRRs of the three SSMs associate in the form of a six-helix bundle.

[0083] The terms "patient," "subject," "host," or "individual," as used interchangeably herein, refer to any subject for whom treatment or prevention is desired, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrates within the scope of the present disclosure include primates (e.g., humans, monkeys, and apes, including monkey species such as the genus Macaque (e.g., cynomolgus monkeys, e.g., Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta)), and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the tribe Miridae), and tamarins (species from the genus Saguinus), and ape species such as chimpanzees (species from the genus Pan troglodytes)), rodents (e.g., Examples of suitable animals include, but are not limited to, any member of the subphylum Chordata, including, for example, mice, rats, and guinea pigs, lagomorphs (e.g., rabbits and hares), cattle (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (e.g., chickens, turkeys, ducks, geese, or other birds of prey, pet birds such as canaries and budgerigars), marine mammals (e.g., dolphins and whales), reptiles (snakes, frogs, lizards, and the like), and fish. A preferred subject is a human, particularly a human in need of eliciting an immune response against a fusion protein or complex thereof of an enveloped virus. However, it is understood that the foregoing terms do not imply the presence of the condition.

[0084] "Pharmaceutically acceptable carrier" refers to a solid or liquid filler, diluent, or encapsulating substance that can be safely used in local or systemic administration to animals, preferably mammals, including humans. Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and similar materials, and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Any conventional carrier is contemplated for use in pharmaceutical compositions, except insofar as it is incompatible with the active ingredient.

[0085] The term "polynucleotide" or "nucleic acid," as used herein, encompasses any molecule containing two or more nucleotides, particularly a polymer of nucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The nucleotides can be, for example, deoxyribonucleotides, ribonucleotides, or nucleotide analogs, which may optionally be substituted or modified. The nucleotides can be linked by phosphodiester bonds / linkages, or by, for example, phosphorothioate, methylphosphonate, or boranophosphate linkages. The term "polynucleotide" or "nucleic acid" specifically relates to DNA or RNA, such as mRNA, cRNA, or cDNA. The term typically refers to a polymeric form of nucleotides at least 10 bases in length, e.g., either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. A "polynucleotide" or "nucleic acid" can be single-stranded or double-stranded.

[0086] The terms "peptide," "polypeptide," "(poly)peptide," and "protein" are used interchangeably herein and refer to a polymer of two or more amino acids linked by an amide bond (i.e., peptide bond) formed between the amino group of one amino acid and the carboxyl group of another amino acid. The amino acids contained in a peptide, polypeptide, (poly)peptide, or protein, also referred to as amino acid residues, 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), although non-proteinogenic and / or non-standard α-amino acids (e.g., ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, α-methylalanine ( Preferably, the amino acid residues contained in the peptide, polypeptide, or protein are selected from α-amino acids, more preferably from the 20 standard proteinogenic α-amino acids (which may exist as L- or D-isomers, preferably all L-isomers). A peptide, polypeptide, or protein may be modified or unmodified, for example, at its N-terminus, at its C-terminus, and / or in the functional side chains of any of its amino acid residues (in particular in the functional side chains of one or more Lys, His, Ser, Thr, Tyr, Cys, Asp, Glu, and / or Arg residues).Such modifications may include, for example, the attachment of any of the protecting groups described for the corresponding functional groups in Wuts PG & Greene TW, Greene's protective groups in organic synthesis, John Wiley & Sons, 2006. Such modifications may also include the covalent attachment of one or more polyethylene glycol (PEG) chains (to form a PEGylated peptide, polypeptide, or protein), the covalent attachment of albumin, glycosylation, and / or the attachment of one or more fatty acids (e.g., one or more C. 8-30 The modified peptides, polypeptides, or proteins may also include acylation with alkanoic or alkenoic acids (forming fatty acid-acylated peptides, polypeptides, or proteins). Furthermore, such modified peptides, polypeptides, or proteins may also include peptidomimetics, 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 a 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 consisting of two or more identical or different molecules. Thus, peptides, polypeptides, and proteins may form dimers, trimers, and higher-order oligomers, where the peptide, polypeptide, or protein molecules forming such dimers, trimers, etc., may be identical or non-identical. The corresponding higher order structures are consequently referred to as homo- or hetero-dimers, homo- or hetero-trimers, and homo- or hetero-oligomers, etc. Such dimers, trimers, and oligomers are similarly encompassed by the terms "peptide," "polypeptide," "(poly)peptide," and "protein."

[0087] The term "amino acid" specifically refers 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 includes 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, telluricine (te), and the like. "amino acid" also refers to amino acids such as α-leucine, ...

[0088] As used herein, the term "post-fusion conformation" of an enveloped virus fusion protein refers to the structure of the enveloped virus fusion protein in its final conformation (i.e., formed at the end of the fusion process) and in its most energetically favorable state. In the post-fusion conformation, the fusion peptide or loop of the fusion protein is in close proximity to the transmembrane domain of the fusion protein. The specific structural elements that promote the formation of a hairpin structure vary depending on the class of envelope fusion protein. For example, the post-fusion conformation of a class I fusion protein is characterized by the interaction between the endogenous FHRR and SHRR regions of each class I fusion protein, resulting in the formation of a hairpin structure characterized by a six-helix bundle containing three endogenous SHRR and three endogenous FHRR regions. Alternatively, the post-fusion conformation of a class III fusion protein is characterized by the interaction between the internal fusion loop and the C-terminal transmembrane region, which promotes the formation of a hairpin structure. The post-fusion conformations of individual viral fusion proteins have been determined by electron microscopy and / or X-ray crystallography, and such structures are readily identifiable by confirmation in negative-stained electron micrographs and / or the absence of pre-fusion epitopes.

[0089] As used herein, the term "pre-fusion conformation" of an enveloped virus fusion protein refers to a structure of the enveloped virus fusion protein that is in a metastable conformation (i.e., a semistable conformation that is not the most energetically favorable final conformation) and can undergo conformational rearrangement to the final post-fusion conformation upon appropriate triggering. Typically, the pre-fusion conformation of a viral fusion protein contains a hydrophobic sequence called a fusion peptide or fusion loop, which is located inside the pre-fusion conformation and cannot interact with either the viral membrane or the host cell membrane. Upon triggering, this hydrophobic sequence is inserted into the host cell membrane, disrupting the fusion protein and forming a post-fusion hairpin-like conformation. The pre-fusion conformation of a viral fusion protein varies depending on the class of envelope fusion protein. Each class is characterized by non-interacting structural elements that subsequently assemble into an energetically favorable post-fusion conformation. For example, the prefusion conformation of class I fusion proteins relies on an endogenous FHRR region that does not interact with the endogenous SHRR region of each fusion protein in 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 a-helical coiled-coil that does not interact with the fusion loop in the C-terminal region of each fusion protein in the trimer, thereby preventing the formation of a hairpin structure. The prefusion conformations of individual viral fusion proteins have been determined by electron microscopy and / or X-ray crystallography, and such structures are readily identifiable by negative-stain electron micrographs and / or by the presence of prefusion epitopes that are not present in the postfusion conformation.

[0090] The terms "regulatory element," "regulatory sequence," "control element," "control sequence," and the like are used interchangeably herein to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that either directly or indirectly influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory elements include enhancers, promoters, translation leader sequences, introns, Rep recognition elements, intergenic regions, and polyadenylation signal sequences. These include sequences that may be natural or synthetic, as well as combinations of synthetic and natural sequences.

[0091] The term "replicon" refers to any genetic element, e.g., plasmid, chromosome, virus, cosmid, etc., that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control.

[0092] "Self-assembly" refers to the process of spontaneous assembly of higher-order structures (e.g., molecules) that relies on the natural attraction of the components of the higher-order structures to one another. This typically occurs through the random movement of molecules and the formation of bonds based on size, shape, composition, or chemical properties.

[0093] The term "sequence identity" as used herein refers to the sequence identity between two (poly)peptides or nucleic acids.The (poly)peptide or nucleic acid sequences to be compared are aligned to obtain 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 July;47(W1):W636-W641. DOI: 10.1093 / nar / gkz268).If the same position in the sequences to be compared is occupied by the same nucleic acid base or amino acid residue, then the respective molecules are identical at that position.Therefore, "percent identity" is a function of the number of matching positions divided by the number of positions to be compared, multiplied by 100%. For example, if 6 out of 10 sequence positions are identical, the identity is 60%. "Identity" or "percent identity (%)" between two amino acid sequences can be determined, for example, using the Needleman-Wunsch algorithm incorporated into EMBOSS Needle (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.) using a BLOSUM62 matrix with a "gap opening penalty" of 10, a "gap extension penalty" of 0.5, an "end gap penalty" of mismatch, an "end gap opening penalty" of 10, and an "end gap extension penalty" of 0.5.The percent identity (%) is typically determined over the entire length of the query sequence being analyzed. Two molecules with the same primary amino acid or nucleic acid sequence are identical, regardless of any chemical and / or biological modifications. For example, two antibodies with the same primary amino acid sequence but different glycosylation patterns are identical by this definition. In the case of nucleic acids, two molecules with the same sequence but different linking moieties, for example, thiophosphates instead of phosphates, are identical by this definition.

[0094] "Similar" (poly)peptide sequences are those that, when aligned, share similar, and most frequently, but not necessarily, identical, amino acid residues at the same positions in the sequences being compared. Similar amino acid residues are grouped into families based on the chemical characteristics of their side chains. Such families are described below with respect to "conservative amino acid substitutions." The "similarity" or "percent similarity" between sequences is calculated by dividing the number of positions containing identical or similar residues at the same sequence position in the sequences being compared by the total number of positions compared, multiplied by 100%. For example, if 6 of 10 sequence positions have identical amino acid residues and 2 of 10 positions contain similar residues, the sequences have 80% similarity. The percent similarity between two sequences can be determined, for example, using EMBOSS Needle (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ) using a BLOSUM62 matrix with a "gap opening penalty" of 10, a "gap extension penalty" of 0.5, an "end gap penalty" of mismatch, an "end gap opening penalty" of 10, and an "end gap extension penalty" of 0.5. The percent similarity (%) is typically determined over the entire length of the query sequence being analyzed.

[0095] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linked in a linear chain by peptide bonds. 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 that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it partially or completely cures the disease and / or the adverse effects attributable to the disease. "Treatment," as used herein, encompasses any treatment of disease in mammals, particularly humans, including (a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease, (b) inhibiting the disease, i.e., halting its development, and (c) alleviating the disease, i.e., causing regression of the disease.

[0096] 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., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form of the gene or gene product.

[0097] Each embodiment described herein applies mutatis mutandis to each and every embodiment unless otherwise indicated. The term "enveloped virus fusion ectodomain polypeptide," as used herein, refers to a polypeptide that comprises 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. 2. Chimeric Polypeptides The present invention is based on further advances in strategies for artificially stabilizing or "clamping" enveloped virus fusion protein ecto-domain polypeptides in a pre-fusion conformation. Generally, this "molecular clumping" strategy utilizes the fusion or linkage of structurally stabilizing moieties (SSMs) to the ecto-domain polypeptide to form a chimeric polypeptide.

[0098] The structure-stabilizing portion (SSM) is typically a single-chain polypeptide containing complementary heptad repeats that lack complementarity to the ecto-domain polypeptide and therefore preferentially associate with each other rather than with the structural elements of the ecto-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 antiparallel two-helix bundles that inhibit the reconstitution of the ecto-domain polypeptide into its post-fusion conformation. The two-helix bundles of the structure-stabilizing portion can trimerize to form highly stable six-helix bundles, thereby enabling the self-assembly of chimeric polypeptides to form artificial enveloped virus fusion protein complexes. The complex assembled in this manner can mimic the pre-fusion conformation of natural enveloped virus fusion protein complexes and includes three chimeric polypeptides characterized by six-helix bundles formed by the coiled-coil structures of the structure-stabilizing portions of each chimeric polypeptide.

[0099] While the initially established clamp technology was found to be unfavorably associated with the induction of antibodies leading to HIV diagnostic interference due to the use of structural stabilizing moieties (SSMs) derived from human immunodeficiency virus (HIV) glycoprotein 41 (gp41), the present inventors aimed to overcome these obstacles while simultaneously stabilizing targeted viral fusion protein antigens in their "pre-fusion" conformation, thus providing at least the same or even improved ability to induce protective neutralizing antibody responses during vaccination. To this end, the present inventors designed a panel of 19 putative clamp sequences derived from the trimerization domains of fusion proteins derived from selected viruses known not to commonly infect humans. The 19 candidate clamps were fused to the RSV fusion protein ectodomain. Subsequent evaluation with thorough biophysical characterization led to the identification of lead molecules derived from the fusion proteins of two genetically related caprine lentiviruses: two constructs (CD9, CD11, see Example 1) derived from Visna virus (also known as Visna-Maedi virus, Maedi-visna virus (MVV), and ovine lentivirus) and one additional construct (CD10, see Example 1) derived from Caprine Arthritis-Encephalitis Virus (CAEV).

[0100] The results presented herein demonstrate that the novel vaccine leads generated are viable alternatives to previous HIV clamp-based molecules. Furthermore, the data demonstrate that these leads are superior in stability and at least comparable in ability to elicit neutralizing antibody responses compared to previously developed HIV clamp-based vaccine candidates.

[0101] According to the newly identified lead structures, the chimeric polypeptides defined by the invention disclosed herein are characterized in that they comprise a microbial polypeptide, preferably an enveloped virus fusion ectodomain polypeptide, operably connected downstream to a heterologous structural stabilizing moiety (SSM) having specific sequence characteristics detailed below.

[0102] Similarly, the "molecular clamping" strategy disclosed herein can also be suitably utilized 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 structurally stabilizing moiety (SSM). The two-helix bundle of the structurally stabilizing moiety can then trimerize to form a highly stable six-helix bundle, thereby enabling the self-assembly of a trimer of the bacterial outer membrane polypeptide (e.g., a trimer of a TAA polypeptide).

[0103] 2.1 Structural stabilization part A "structure-stabilizing moiety (SSM)," as utilized in the context of the present invention, 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 may optionally be interconnected by a linker region (preferably, in the following order from N-terminal to C-terminal: FHRR-linker-SHRR), as further defined herein below (e.g., in Section 2.1.2).

[0104] 2.1.1 Heptad repeat Alpha-helical coiled coils are characterized at the amino acid sequence level; each helix is ​​composed of a series of heptad repeats. A heptad repeat (heptad unit, heptad) is a seven-residue sequence motif that can be coded as hpphppp, where each "h" represents a hydrophobic residue and each "p" represents a polar (i.e., hydrophilic) residue. In some cases, p residues are observed at the h position, and vice versa. Heptad repeats are also often coded by the pattern abcdefg (abcdefg) or defgabc (defgabc), where the identifiers "a" through "g" refer to the conventional heptad positions where typical amino acid types are observed. By convention, the identifiers "a" and "d" indicate the positions of core residues (central, embedded residues) in the coiled coil. The typical amino acid types observed at the core a and d positions are hydrophobic amino acid residue types, while at all other positions (non-core positions) predominantly polar (hydrophilic) residue types are observed. Thus, the conventional heptad pattern "hpphppp" corresponds to the pattern notation "abcdefg" (the "hppphpp" pattern corresponds to the pattern notation "defgabc", which is used for coiled-coils starting with a hydrophobic residue at the d position).

[0105] A heptad repeat region (HRR) as referred to in accordance with the present invention comprises at least two, and preferably three or more (preferably consecutive, i.e., uninterrupted) heptad repeats in an individual α-helix of a coiled-coil structure. Each series of consecutive heptad repeats in a helix is ​​designated a "heptad repeat sequence" (HRS). The start and end of a heptad repeat sequence are preferably determined based on an experimentally determined three-dimensional (3-D) structure, if available. If a 3-D structure is not available, the start and end of a heptad repeat sequence are preferably determined based on an experimentally determined three-dimensional (3-D) structure, if available. If a 3-D structure is not available, the start and end of a heptad repeat sequence are preferably determined based on an experimentally determined three-dimensional (3-D) structure, if available. n or (hppphpp) nThe pattern is determined based on optimal overlay with the actual amino acid sequence, where "h" and "p" refer to hydrophobic and polar (hydrophilic) residues, respectively, and "n" is a number equal to or greater than 2. The beginning and end of each heptad repeat sequence are considered to be the first and last hydrophobic residues at the a and d positions, respectively. Conventional H residues are preferably selected from the group consisting of valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, threonine, serine, and alanine, more preferably selected from the group consisting of valine, isoleucine, leucine, and methionine, and most preferably isoleucine. Conventional p residues are preferably selected from the group consisting of glycine, alanine, cysteine, serine, threonine, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, and arginine. If this method does not allow a clear 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).

[0106] In the chimeric polypeptides according to the invention, the "structure stabilizing moiety (SSM)" comprises, in order from N-terminus to C-terminus, a first heptad repeat region (FHRR) and a second heptad repeat region (SHRR), wherein: (i) FHRR has a sequence identity of at least 60% (or, in ascending order of 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% or more) with respect to the amino acid sequence set forth in SEQ ID NO: 80 or 81; , 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 set forth in SEQ ID NO: 82 or 83, and SHRR comprises or consists of an amino acid sequence having at least 40% (or in ascending order of 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%, or 99%, most preferably 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 82 or 83. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 100%), and / or (ii) the FHRR comprises or consists of an amino acid sequence having a sequence identity of at least 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%, or 99%, most preferably 100%) to the amino acid sequence set forth in SEQ ID NO: 80 or 81. and SHRR comprises or consists of an amino acid sequence having at least 70% (or in ascending order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 100%) sequence similarity to the amino acid sequence set forth in SEQ ID NO: 82 or 83.89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and most preferably 100%.

[0107] According to the present invention, (i) FHRR has at least 60% (or in increasing order of 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 set forth in SEQ ID NO: 80. and SHRR comprises or consists of an amino acid sequence having at least 40% (or in ascending order of 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%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 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 set forth in SEQ ID NO: 80; and / or (ii) the FHRR comprises or consists of an amino acid sequence having at least 90% (or in increasing order of preference at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 80. and SHRR comprises or consists of an amino acid sequence having at least 70% sequence similarity (or, in order of 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%) to the amino acid sequence set forth in SEQ ID NO: 82.

[0108] In some embodiments, (i) FHRR is at least 60% (or, in order of 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%) identical to the amino acid sequence set forth in SEQ ID NO: 80. and SHRR comprises or consists of an amino acid sequence having sequence identity of at least 40% (or in ascending order of 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%) to the amino acid sequence set forth in SEQ ID NO: 82. , 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 set forth in SEQ ID NO: 80; and / or (ii) FHRR comprises or consists of an amino acid sequence having at least 90% (or in increasing order of preference at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 80. and SHRR comprises or consists of an amino acid sequence having sequence similarity of at least 70% (or in ascending order of 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%) to the amino acid sequence set forth in SEQ ID NO: 82.

[0109] In some embodiments, the structural stabilizing portion is capable of homotrimerizing with the structural stabilizing portions of two additional chimeric polypeptides, wherein the homotrimerization preferably results in the formation of a six-helix bundle consisting of an inner trimer of three parallel-oriented substantially α-helical FHRRs packed with three substantially α-helical SHRRs in an antiparallel orientation relative to the FHRRs.

[0110] In some embodiments, FHRR and SHRR are each (abcdefg-) n or (defgabc-) n wherein elements "a" through "g" of the pattern refer to the positions at which the amino acids are placed, n is a number equal to or greater than 2, and wherein 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.

[0111] With regard to which amino acids are classified as hydrophilic (polar) or hydrophobic (non-polar) amino acids, respectively, in the context of the present disclosure, reference is made to Table 1 above. In a preferred embodiment of the latter embodiment, the FHRR comprises four independently selected seven-residue motifs repeated, and the SHRR comprises five independently selected seven-residue motifs repeated, wherein the seven-residue motif is characterized by a pattern of amino acids represented as (abcdefg-) or (defgabc-), where pattern elements "a" through "g" indicate positions at which amino acids are placed, and where 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.

[0112] In some embodiments, the structure-stabilizing moiety has a glutamine at a position corresponding to position 17 of SEQ ID NO:80. As evidenced by the experimental data disclosed herein, the presence of the corresponding mutation (Gln17) results in a slight additional increase in soluble protein yield (see Example 4, Table 4, CD11 vs. CD9), which indicates a stabilizing effect on protein folding and trimer assembly. Additional data consistently demonstrated that this mutation also beneficially enhances thermostability (see Example 5, Figure 4). Given these beneficial effects provided by glutamine at position 17, substitution with asparagine, or similarly glutamic acid or aspartic acid, is expected to have an equivalent positive effect, and these are also contemplated herein as alternative preferred embodiments. However, constructs with serine or threonine at position 17 also provide favorable trimer stabilization, and having a serine or threonine in the structure-stabilizing portion at the position corresponding to position 17 of SEQ ID NO: 80 is also contemplated as a further alternative embodiment.

[0113] In an alternative preferred embodiment, the structure-stabilizing moiety has a leucine at a position corresponding to position 17 of SEQ ID NO: 80. As evidenced by the data presented in Example 16, the corresponding mutation (Leu17, see construct designated "CT9") resulted in a substantial improvement in the presence of trimers in solution as evidenced by size exclusion chromatography (SEC) analysis (Figure 32) and in the thermal stability of the trimers (Figure 33).

[0114] In specific embodiments, the structure-stabilizing portion comprises at least one immune-silencing portion that reduces or inhibits elicitation of an immune response against the structure-stabilizing portion. These embodiments are advantageous because they can enable the generation of a selective and / or enhanced immune response against a microbial polypeptide, such as (i) an enveloped virus fusion ectodomain polypeptide or complex thereof, or (ii) a bacterial outer membrane polypeptide (e.g., a TAA polypeptide) or complex thereof, if the chimeric polypeptide comprises a bacterial outer membrane polypeptide (e.g., a TAA polypeptide).

[0115] The immune silencing moiety can be a glycosylation site that is specifically recognized and glycosylated by one or more glycosylation enzymes, particularly glycosyltransferases. Glycosylation can be N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences NXS and NXT (represented in the three-letter code as Asn-Xaa-Ser and Asn-Xaa-Thr, respectively), where X (Xaa) is any amino acid other than P (Pro), are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine (Asn) side chain, and these sequences are generally referred to as "glycosylation site" or "sequon." 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), but 5-hydroxyproline or 5-hydroxylysine can also be used, which can be further extended with additional monosaccharide units, such as Gal, GalNAc, or N-acetylglucosamine (GlcNAc). The immune silencing moiety can be inserted into the structure-stabilizing portion, including one or both of the heptad repeat regions.

[0116] In a particularly preferred embodiment of the latter embodiment, at least one immunosilencing moiety is an N-linked glycosylation site. In an even more preferred embodiment, the N-linked glycosylation site is post-translationally modified to attach a glycan (e.g., a polysaccharide, oligosaccharide, or monosaccharide). The attached glycan may correspond to one known to be commonly utilized in the particular mammal (preferably Homo sapiens) to which the chimeric polypeptide or complex thereof or any of the compositions provided herein is intended to be administered, in order to reduce or avoid an immune response to the corresponding protein sequence. Those skilled in the art can select suitable host cells for recombinant expression of the chimeric polypeptide and complexes thereof, where the host cells possess glycosylation machinery that produces such N-glycans that are endogenous to the targeted mammalian subject species and are therefore likely to provide an immune silencing effect.

[0117] In some embodiments, the structurally stabilizing moiety comprises at least one glycosylation site, wherein preferably the at least one glycosylation site is an N-linked glycosylation site selected from the group consisting of (1)-Asn-Xaa-Ser- and (2)-Asn-Xaa-Thr-, where Xaa is an amino acid other than Pro, and preferably the glycosylation site is glycosylated at an occupancy level of at least 50%.

[0118] Generally, the "occupancy level" of a glycosylation site in a given protein molecule refers to the percentage (%) of this glycosylation site that is actually glycosylated within a given population of that protein molecule. Those skilled in the art will recognize that this level depends, inter alia, on the essential sequence characteristics and, consequently, structural characteristics of the protein, but also on the organism (expression host) and expression pathway (cytoplasmic or secretory) used for recombinant expression of 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 easily applied by those skilled in the art. One exemplary method for determining the "occupancy level%" of an N-glycosylation site is also described herein, see attached Example 14.

[0119] In some embodiments, the structurally stabilizing portion 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-, and Xaa is an amino acid other than Pro.

[0120] In some embodiments, the structure-stabilizing moiety is located at (i) (ia) positions 5 to 7 of SEQ ID NO: 80, (ib) positions 1 to 3 of SEQ ID NO: 82, and (ic) positions 17 to 19 of SEQ ID NO: 82, or (ii) (ii-a) positions 5 to 7 of SEQ ID NO: 80, (ii-b) positions 1 to 3 of SEQ ID NO: 82, (ii-c) positions 17 to 19 of SEQ ID NO: 82, (ii-d) positions 27 to 29 of SEQ ID NO: 82, or (iii) (iii-a) positions 5 to 7 of SEQ ID NO: 80, (iii-b) positions 1 to 3 of SEQ ID NO: 82, (iii-c) positions 13 to 15 of SEQ ID NO: 82, (iii-d) positions 17 to 29 of SEQ ID NO: 82. (iv-c) positions 6 to 8 of SEQ ID NO: 82, (iv-d) positions 13 to 15 of SEQ ID NO: 82, (iv-e) positions 17 to 19 of SEQ ID NO: 82, and (iv-f) positions 27 to 29 of SEQ ID NO: 82, wherein preferably, each N-linked glycosylation site is independently -Asn-Xaa-Thr-, and Xaa is an amino acid other than Pro.

[0121] In some embodiments, the structurally stabilizing portion 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.

[0122] In preferred embodiments of the latter embodiment, the structure-stabilizing portion 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. As evidenced by the size exclusion chromatography (SEC) data presented in Example 16, particularly Figures 32 and 33, each of the above-described site-specific mutations (i) to (v) (referred to as CT1, CT5, CT6, CT9, and CT10, respectively, in Example 16) provides further enhancement of trimer stabilization compared to unmodified CD11.

[0123] In a preferred embodiment, the structure-stabilizing portion has a deletion of (i) an arginine at the amino acid position corresponding to glutamine 22 of SEQ ID NO:80, and (ii) amino acid residues at positions corresponding to glutamine 1 and serine 2 of SEQ ID NO:80.

[0124] In a preferred embodiment, the structure-stabilizing moiety has (i) a leucine at amino acid position corresponding to position 20 of SEQ ID NO: 82, and / or (ii) a glutamine at amino acid position corresponding to position 11 of SEQ ID NO: 80. As evidenced by the mutagenesis studies in Example 16, maintaining these amino acids as in the wild-type sequence of CD11 is beneficial in terms of trimer stabilization.

[0125] In some embodiments, the structurally stabilizing moiety comprises one or more unnatural amino acids.As used herein, "unnatural" or "non-natural amino acid" refers to an amino acid that is neither one of the 20 common naturally occurring amino acids nor a rare naturally occurring amino acid, such as selenocysteine ​​(Sec) or pyrrolysine (Pyl). Other terms that can be used synonymously with the term "unnatural amino acid" are non-naturally encoded amino acids, unnatural amino acids, non-naturally occurring amino acids, and various linked and unlinked versions thereof. The term "non-natural amino acid" includes, but is not limited to, amino acids that occur naturally by modification of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into growing (poly)peptide chains 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 in nature and can be obtained synthetically or by modifying unnatural amino acids. Unnatural amino acids can be incorporated into the chimeric polypeptides contemplated herein (e.g., one or both of the heptad repeat regions contained therein) or any other (poly)peptide by using the extended genetic code. Unnatural amino acids are biosynthetically incorporated into the desired position using a tyrosyl-tRNA / aminoacyl-tRNA synthetase orthogonal pair and a nonsense codon at the desired site. Unnatural amino acids are supplied from an external source to cells expressing a construct that can express a chimeric polypeptide. This strategy allows for the incorporation of side chains with a wide range of physical attributes, including, but not limited to, chemical crosslinking groups (e.g., azide or haloalkane), traceable markers (e.g., fluorescent or radioactive), and photosensitive groups, to enable temporally controlled modification.To these unnatural amino acids, various moieties can be covalently linked by chemical attachment to structure-stabilizing moieties to provide beneficial properties.

[0126] In preferred embodiments of the latter embodiment, the one or more unnatural amino acids allow for the coupling of (i) polyethylene glycol (PEG), (ii) an immunostimulatory moiety, or (iii) a lipid.

[0127] Further embodiments may include any possible combination of the above examples, or additional non-natural chemical additions covalently linked to the structure-stabilizing moieties. Optionally, one or more additional cysteine ​​residues can 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.

[0128] 2.1.2 Linker The structure stabilising moiety (SSM) of the present invention may suitably comprise a linker that separates the first and second heptad repeat regions (also referred to herein as FHRR and SHRR, respectively).

[0129] Linkers generally include any amino acid residues that cannot be clearly assigned to a heptad repeat sequence. Linkers are frequently used in the field of protein engineering to interconnect different functional units, for example, in the generation of single-chain variable fragment (scFv) constructs derived from antibody variable light chains (VL) and variable heavy chains (VH). They are generally conformationally flexible in solution and are preferably composed primarily of polar amino acid residue types. Typical (frequently used) amino acids in flexible linkers are serine and glycine. Although less preferred, flexible linkers may also contain alanine, threonine, and proline. Therefore, the intervening linker of the structure-stabilizing moiety is preferably conformationally flexible to ensure loose (unhindered) association of the FHRR and SHRR as a two-helix bundle, preferably adopting an α-helical coiled-coil structure. Linkers suitable 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 link amino acid sequences, so long as they are conformationally flexible, in the sense that they allow, and preferably do not interfere with, the assembly of the characteristic two-helix bundle of the structure-stabilizing moieties.

[0130] Those skilled in the art will be able to determine the optimal linker, optionally after a limited number of routine experiments. The intervening linker is preferably an amino acid sequence, generally consisting of at least one amino acid residue, usually at least two amino acid residues, with a non-critical upper limit of about 100 amino acid residues selected for convenience. 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, usually about 1 to about 40 amino acid residues, and typically about 1 to about 30 amino acid residues. In a non-limiting example, the linker has approximately the same number of amino acids connecting the complementary FHRR and SHHR regions of a class I enveloped viral fusion protein. In a specific, non-limiting embodiment, at least 50% of the amino acid residues in the linker sequence are selected from the group consisting of proline, glycine, and serine. In a further non-limiting embodiment, at least 60%, for example at least 70%, for example 80%, more specifically 90% of the amino acid residues of the linker sequence are selected from the group consisting of proline, glycine, and serine. In another specific embodiment, the linker sequence is essentially composed of polar amino acid residues, and in such specific embodiments, at least 50%, for example at least 60%, for example 70% or more 80%, more specifically 90%, or up to 100% of the amino acid residues of the linker sequence are selected from the group consisting of glycine, serine, threonine, alanine, proline, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, and arginine. In a specific embodiment, the linker sequence is [GGSG] n GG (SEQ ID NOs: 161-170), [GGGGS] n (SEQ ID NOs: 152-155, 171-176), [GGGGGG] n (SEQ ID NOs: 160, 177-185), [GGGKGGGG] n (SEQ ID NOs: 186-195), [GGGNGGGG] n (SEQ ID NOs: 196-205), [GGGCGGGG] n(SEQ ID NOs: 206 to 215), where n is an integer of 1 to 10, preferably 1 to 5, and more preferably 1 to 3.

[0131] In a preferred embodiment, the FHRR and SHRR contained in the structure-stabilizing moiety (SSM) are connected by a linker. Preferably, the linker comprises or consists of a peptide having an amino acid sequence identical to SEQ ID NO: 84 or 85, more preferably the amino acid sequence defined by SEQ ID NO: 84.

[0132] In addition to introducing structural flexibility to facilitate spacing of the heptad repeat regions (i.e., FHRR and SHRR) of the structural stabilizing moiety (SSM) and preferably antiparallel association of these regions, the linker may contain one or more auxiliary functionalities.

[0133] For example, the linker can include 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.

[0134] The purification moiety typically comprises a stretch of amino acids that allows recovery of the chimeric polypeptide through affinity binding. Numerous purification moieties or "tags" are known in the art, illustrative examples of which include biotin carboxyl carrier protein-tag (BCCP-tag), Myc-tag (c-myc-tag), calmodulin-tag, FLAG-tag, HA-tag, His-tag (hexahistidine-tag, His6, 6H, 6xHis), maltose binding protein-tag (MBP-tag), Nus-tag, chitin binding protein-tag (CBP-tag), glutathione-S-transferase-tag (GST-tag), green fluorescent protein-tag (GFP-tag), polyglutamate-tag, amyloid beta-tag, thioredoxin-tag, S-tag, Softag 1, Softag 3, Strep-tag, streptavidin-binding peptide-tag (SBP-tag), biotin-tag, streptavidin-tag, and V5-tag.

[0135] An immunomodulatory moiety can be introduced into the linker to modulate the immune response elicited by the chimeric polypeptide or its complex. Non-limiting examples of such moieties include the immune silencing or suppressing moieties 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, as well as amoebas. In certain embodiments, the antigenic moiety is derived from an antigen of a pathogenic virus. Exemplary viruses implicated in disease include measles, mumps, rubella, poliomyelitis, 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, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Epstein-Barr virus, and other herpes viruses, e.g., Antigens include, but are not limited to, papillomavirus, Ebola virus, influenza virus, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue fever (e.g., GenBank Accession No. M24444), hantavirus, Sendai virus, respiratory syncytial virus, orthomyxovirus, vesicular stomatitis virus, visna virus, cytomegalovirus, and human immunodeficiency virus (HIV) (e.g., GenBank Accession No. U18552). Any suitable antigens derived from such viruses are useful in the practice of the present invention. For example, exemplary retroviral antigens derived from HIV include, but are not limited to, antigens from the gag, pol, and env genes, Nef protein, reverse transcriptase, and other HIV components. Illustrative examples of hepatitis virus antigens include, but are not limited to, the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and antigens from other hepatitis viruses, such as hepatitis A, hepatitis B, and hepatitis C.Illustrative examples of influenza virus antigens include, but are not limited to, antigens such as hemagglutinin and neuraminidase, and other influenza virus components. Illustrative examples of measles virus antigens include, but are not limited to, antigens such as measles virus fusion protein and other measles virus components. Illustrative examples of rubella virus antigens include, but are not limited to, antigens such as proteins E1 and E2 and other rubella virus components, and rotavirus antigens, such as VP7sc and other rotavirus components. Illustrative 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 virus antigens include antigens such as 9PI, gpII, and other varicella-zoster virus antigen components. Non-limiting examples of Japanese encephalitis virus antigens include antigens such as protein E, ME, ME-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitis virus antigen components.

[0136] Representative examples of rabies virus antigens include, but are not limited to, antigens such as rabies glycoprotein, rabies nucleoprotein, and other rabies virus 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, Second Edition, eds. Fields, BN and Knipe, DM, 1991, Raven Press, New York. In a specific embodiment, the viral antigen is an antigen of the enveloped virus to which the ectodomain polypeptide corresponds. In other embodiments, the viral antigen is an antigen of a different enveloped virus to which the ectodomain polypeptide corresponds.

[0137] In some embodiments, one or more cancer or tumor-associated antigens are inserted into the linker. Such antigens include 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 MOV 18, TUAN, alpha-fetoprotein (AFP), OFP, CA-125, CA-50, CA-19-9, kidney tumor-associated antigen G250, EGP-40 (also known as EpCAM), S100 (malignant melanoma-associated antigen), p53, prostate tumor-associated antigen (e.g., PSA and PSMA), p21ras, Her2 / neu, EGFR, EpCAM, VEGFR, FGFR, MUC-I, CA Antigens specific for 125, CEA, MAGE, CD20, CD19, CD40, CD33, A3, A33 antibodies, BrE3 antigen, CD1, CD1a, CD5, CD8, CD14, CD15, CD16, CD21, CD22, CD23, CD30, CD33, CD37, CD38, CD40, CD45, CD46, CD52, CD54, CD74, CD79a, CD126, CD138, 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, Ba 733, 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, and the like.

[0138] The antigenic moiety or moieties included in the linker may correspond to a full-length or partial antigen. When a partial antigen is utilized, the partial antigen may include one or more epitopes of the antigen of interest, including B cell epitopes and / or T cell epitopes (e.g., cytotoxic T lymphocyte (CTL) epitopes and / or helper T (Th) epitopes). 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 may include another cell targeting moiety that can provide delivery to specific cell types within the immunized individual. Targeted cell populations include, but are not limited to, B cells, fold cells, and antigen-presenting cells (APCs). In the latter example, the targeting moiety facilitates enhanced recognition of the chimeric polypeptide or its complex by APCs, such as dendritic cells or macrophages. Such targeting sequences can enhance APC presentation of epitopes of the associated ecto-domain polypeptide, which can increase the resulting immune response, including enhancing or broadening the specificity of either or both of the antibody and cellular immune responses to the ecto-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, which comprises, consists of, or consists 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).

[0139] 2.1.3 Particularly Preferred Structure-Stabilizing Moieties (SSMs) Two structure-stabilizing moieties, designated "clamp 2" and "clamp 2s", were selected as lead constructs by the inventors: Clamp 2 (alternatively referred to herein as "CD11-QS") is defined by SEQ ID NO: 265: LANATAAQQEVLEAQYAMVQHIAKGIRILEARVARGGSGGNHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 265] Clamp2s (alternatively referred to herein as "CD11_145T8-QS_CT5" and corresponding to the silencing variant of clamp2) is defined by SEQ ID NO: 266: LANATAAQQEVLEAQYAMVQHIAKGIRILEARVARGGSGGNHTTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI [SEQ ID NO: 266]

[0140] 2.1.4 Membrane tethering In certain embodiments, it may be beneficial to provide the chimeric polypeptide or a complex thereof tethered to the cell surface, for example, the cell surface of a cell that expresses the chimeric polypeptide.

[0141] For example, in a chimeric polypeptide according to the first aspect of the invention intended to be utilized as a vaccine, membrane tethering to the cell surface (e.g., of a host cell expressing the chimeric polypeptide by vaccination with mRNA, a 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 presented on the cell surface.

[0142] Without wishing to be bound by any theory, a corresponding configuration may be beneficial in terms of providing enhanced stimulation of immune responses and thus eliciting stronger, more sustained, and broadly neutralizing antibody responses. This is because cells and / or cell-derived organelles expressing and presenting high concentrations and / or densities of chimeric polypeptides may help recruit and activate naive and memory B cells, thereby enhancing the stimulation of potent B cell-mediated neutralizing antibody responses. Furthermore, interaction with B cells may be further amplified through avidity effects resulting from the presentation of multiple chimeric polypeptides or multiple complexes thereof on the cell surface. Efficient formation of neutralizing antibodies by B cells also requires helper function from T cells (especially CD4 T cells). While induction of B cell responses requires direct interaction with an antigen, CD4 T cells are stimulated by peptides derived from the same antigen in complex with MHC II molecules. Thus, tethering the chimeric polypeptide or its complex to the surface of cells and / or cell-derived organelles can indirectly result in higher epitope presentation through MHCII, which may also serve to recruit CD4 T cells, thereby further enhancing the immune response, and to form potent neutralizing antibodies.

[0143] Two general types of embodiments of membrane-tethered constructs specifically contemplated herein include: (i) the embodiment in which the SSM further comprises a membrane tethering polypeptide; and (ii) The embodiment in which the chimeric polypeptide further comprises a transmembrane (TM) domain upstream of (i.e., N-terminal to) the SSM. Examples include:

[0144] Representative embodiments relating to (i) and (ii) are described herein below in sections 2.1.4.1 and 2.1.3.2, respectively.

[0145] 2.1.4.1 Membrane-tethering (poly)peptides In a particularly preferred embodiment, the chimeric polypeptide of the present invention further comprises a "membrane tethering (poly)peptide." As used herein, the terms "membrane tethering (poly)peptide," "membrane anchoring (poly)peptide," "membrane tether," or "membrane anchor," "membrane-localizing (poly)peptide," "membrane-targeting (poly)peptide," or further equivalents thereof refer 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 broad sense, the term "membrane tethering (poly)peptide" or its equivalents referred to herein encompasses (poly)peptides whose ability to insert into a membrane (e.g., the lipid bilayer of a cell or (nano)lipid particle) is at least partially due to the intrinsic properties and physicochemical properties of specific amino acid residues contained therein (e.g., amino acids with side chains characterized by a high degree of intrinsic hydrophobicity, such as, specifically, tryptophan, phenylalanine, tyrosine, isoleucine, leucine, and valine). However, the term also encompasses (poly)peptides that derive this ability through the presence of certain chemical or post-translational modifications of one or more of the amino acid residues they contain. For example, one or more amino acids may have a covalently bound lipid / fatty acid. Such amino acids may be, for example, but not limited to, myristoylated, palmitoylated, or prenylated.

[0146] Generally, a membrane tethering (poly)peptide can be suitably included at any position within a chimeric polypeptide, as long as its presence does not negatively interfere, or at least substantially, with the folding of the remainder of the chimeric polypeptide and / or does not sterically impair its ability to trimerize. Furthermore, those skilled in the art will understand that in instances where a chimeric polypeptide includes a microbial polypeptide (preferably, an enveloped virus fusion ectodomain polypeptide or a bacterial outer membrane polypeptide (e.g., a TAA polypeptide)), the membrane tethering (poly)peptide should only be included in the chimeric polypeptide at a position where its presence does not sterically interfere, or at least substantially sterically interfere, with the surface accessibility of the microbial polypeptide (preferably, an enveloped virus fusion ectodomain polypeptide or a bacterial outer membrane polypeptide (e.g., a TAA polypeptide)), so as not to negatively interfere with the immunogenic potential of these antigenic portions.

[0147] As demonstrated by the experimental evidence disclosed herein (see, e.g., Example 21), the inventors found that constructs having a membrane-tethering (poly)peptide inserted as a linker between the FHRR and SHRR of an SSM were particularly effective at tethering a chimeric polypeptide or complex thereof to the surface of cells expressing the chimeric polypeptide. The corresponding construct appears to result in the structural arrangement illustrated in Figure 41B, in which the SSM is oriented proximal to the cell membrane and the portion of the chimeric polypeptide (e.g., an enveloped viral fusion ectodomain polypeptide or a bacterial outer membrane polypeptide (e.g., a TAA polypeptide)) upstream of the SSM is shown projecting distally relative to the cell membrane.

[0148] Thus, in a preferred embodiment of the embodiment in which the FHRR and SHRR comprised in the SSM are connected by a linker, the linker comprises or consists of a membrane tethering (poly)peptide.

[0149] To be particularly suitable for constructs according to the latter embodiment, the membrane-tethering (poly)peptide should be of a length and structural conformation compatible with the structural arrangement of the FHRR and SSHR in the SSM. More specifically, the membrane-tethering (poly)peptide should be of sufficient length to allow its stable insertion into the cell membrane, but its N- and C-termini should be spatially oriented / separated in a manner consistent with the C- and N-termini of the FHRR and SSHR, respectively, of the SSM. In addition, this may be beneficial in providing further stabilization and / or rigidity if the membrane-tethering (poly)peptide, or at least a substantial portion thereof, is itself capable of trimerization.

[0150] In a preferred embodiment, the membrane tethering polypeptide is (i) comprising or consisting of, in increasing order of preference, 10 to 50, 15 to 40, 16 to 30, 18 to 28, 20 to 26, 22 to 24, and most preferably 23 amino acid residues; and / or (ii) 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, in ascending order of preference.

[0151] 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 chimeric polypeptides to the outer surface of the cell membrane (see Example 21 and Figures 41B and 42). Thus, in particularly preferred embodiments, the membrane tethering (poly)peptide comprises or consists of an amino acid sequence having at least 60% (or, in ascending order of 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 set forth in SEQ ID NOs: 236-243.

[0152] In the specific constructs defined by SEQ ID NOS: 225-232 and utilized in Example 21, a membrane-tethering (poly)peptide was included in the linker region interconnecting the FHRR and SHRR of the SSM. Thus, in these constructs, the included membrane-tethering (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, which originate from insertion into the original linker sequence GGSGG (defined by SEQ ID NOS: 84). Without wishing to be bound by any theory, the inclusion of one or more amino acids, e.g., glycine or serine (or other small amino acid residues), as flanking moieties may help separate the membrane-tethering (poly)peptide from the rest of the chimeric polypeptide, allowing the former to insert into the cell membrane while the latter to be displayed on the surface, thereby enabling optimal antigen presentation.

[0153] Thus, in a preferred embodiment, the membrane tethering (poly)peptide comprises or consists of an amino acid sequence having at least 60% sequence identity (or, in ascending order of 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%) to any one of the amino acid sequences set forth in SEQ ID NOs: 244-251.

[0154] Although each of the eight membrane tethering (poly)peptides tested proved effective in tethering the respective chimeric polypeptides to the cell membrane, three constructs bearing membrane tethering (poly)peptides designated herein as "alpha," "gamma," or "epsilon" (defined by SEQ ID NOs: 236, 238, and 240, respectively) resulted in the highest levels of surface-bound chimeric polypeptides (as assessed by flow cytometry using antigen-specific monoclonal antibodies; see Figure 43B).

[0155] Thus, in a preferred embodiment, the membrane tethering (poly)peptide is (a) any one of the amino acid sequences set forth in SEQ ID NOs: 236, 238, and 240; or (b) any one of the amino acid sequences set forth in SEQ ID NOs: 244, 246, and 248; and / or consisting of an amino acid sequence having at least 60% (or in ascending order of 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%, and most preferably 100%) sequence identity to

[0156] Further analysis of the panel of exemplified constructs also revealed that the inclusion of a membrane tethering (poly)peptide designated herein as "gamma" (defined by SEQ ID NO: 238) was most effective in stabilizing the viral fusion protein (RSV F) in a pre-fusion conformation. Thus, in an even more preferred embodiment, the membrane tethering (poly)peptide comprises or consists of an amino acid sequence having at least 60% (or, in order of 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. In an alternative, but less preferred, embodiment, however, the membrane-tethering (poly)peptide may be included in the C-terminal portion of the chimeric polypeptide, i.e., C-terminal to the SHRR of the SSM. For example, in an exemplary embodiment of the latter embodiment, the chimeric polypeptide may include a (poly)peptide containing a glycosylphosphatidylinositol (GPI)-anchor C-terminal to the SHRR of the SSM.

[0157] 2.1.4.2 Membrane tethering by inclusion of a transmembrane domain upstream of the SSM In an alternative embodiment, membrane tethering of the chimeric polypeptide of the present invention can be achieved through the inclusion of a transmembrane (poly)peptide upstream (i.e., N-terminal) of the SSM. The corresponding construct is envisioned to result in the structural arrangement illustrated in Figure 41C, in which the SSM is oriented internally (i.e., cytoplasmically) within the cell membrane, and the portion of the chimeric polypeptide upstream (i.e., N-terminal) of the SSM, e.g., an enveloped viral fusion protein ectodomain polypeptide or a TAA polypeptide, is displayed extracellularly, i.e., protruding from it on the cell surface.

[0158] The term "transmembrane (poly)peptide" or "transmembrane domain," as used herein, refers in its broadest sense to any (poly)peptide that can span (i.e., cross) the lipid bilayer of a cell membrane and thus function to link the extracellular and intracellular portions of a polypeptide chain. It can be a single alpha helix, a transmembrane beta barrel, a beta helix, or any other structure. Typically, a transmembrane domain refers to the single transmembrane alpha helix of a transmembrane protein, also known as an integral membrane protein.

[0159] For example, in related embodiments, 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-terminal to the microbial polypeptide (e.g., the enveloped virus fusion ectodomain polypeptide or the bacterial surface polypeptide) and N-terminal to the SSM. In particularly preferred embodiments of the latter embodiment, the transmembrane (poly)peptide is included in the chimeric polypeptide immediately C-terminal to the microbial polypeptide (e.g., the C-terminal to the enveloped virus fusion ectodomain polypeptide or the bacterial surface polypeptide) and N-terminal to the SSM. In even more preferred embodiments, if applicable, the transmembrane (poly)peptide corresponds to or substantially corresponds to a transmembrane (poly)peptide naturally included in the microbial polypeptide (e.g., the enveloped virus fusion polypeptide).

[0160] In preferred embodiments of the chimeric polypeptide of the first or second aspect of the present invention, the chimeric polypeptide further comprises a hinge region operably connecting the microbial polypeptide (preferably an enveloped virus fusion ectodomain polypeptide or a bacterial outer membrane polypeptide) to the structural stabilizing moiety (SSM). Thus, in particularly preferred embodiments of the latter embodiment, if a transmembrane (poly)peptide is envisaged for inclusion in the chimeric polypeptide, the hinge region may comprise, further comprise, or consist of a transmembrane (poly)peptide.

[0161] Furthermore, in those instances where the chimeric polypeptide comprises an enveloped virus fusion ectodomain polypeptide, the transmembrane (poly)peptide preferably corresponds to the transmembrane (poly)peptide naturally contained in the enveloped virus fusion polypeptide. In any of the latter embodiments, the transmembrane (poly)peptide may be operably connected to the SSM through a peptide consisting of 3 to 5 amino acid residues independently selected from serine and glycine.

[0162] As shown in the examples disclosed herein, such a construct (referred to as "mVL22-TM-CD11" in Example 21 and defined by SEQ ID NO:233) has been generated as a "proof of concept" and shown to be expressed with high yield and to be capable of constraining an enveloped viral fusion protein ectodomain in a pre-fusion conformation. Thus, in a particularly preferred embodiment of the chimeric polypeptide according to the first aspect of the invention, where the ectodomain polypeptide corresponds to or is a variant of the ectodomain of a fusion protein derived from respiratory syncytial virus (RSV), the chimeric polypeptide further comprises a hinge region operably connecting the ectodomain polypeptide to the SSM, the hinge region comprising 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.

[0163] 2.2 Microbial polypeptides 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).

[0164] The term "microbial polypeptide," as used herein, broadly refers to a polypeptide that corresponds to or substantially corresponds to a polypeptide of (or derived from) a microorganism, where the microorganism is preferably selected from the group consisting of bacteria, archaea, protists, fungi, and viruses.

[0165] In a particularly preferred embodiment, the microorganism is a pathogenic microorganism (i.e., the microorganism is a mammalian pathogen, preferably a human pathogen), preferably one selected from the group consisting of bacteria and viruses.

[0166] Given the ability of the structurally stabilizing moieties (SSMs) disclosed herein to trimerize, and thus to present other polypeptides in a trimeric conformation when utilized as fusion partners for these, the technology disclosed herein will be particularly suitable for achieving stabilization of such polypeptides that also naturally exist as trimers. Thus, in a preferred embodiment, the microbial polypeptide (or any of the more specific forms defined hereinafter) is a polypeptide that is capable of trimerizing.

[0167] Preferably, the chimeric polypeptides of the present invention are single polypeptide chains in which the structural stabilizing moiety (SSM) is C-terminal to (downstream of) the microbial polypeptide, although the present disclosure also contemplates alternative arrangements in which the structural stabilizing moiety (SSM) is N-terminal to (upstream of) the microbial polypeptide. In either instance, the structural stabilizing moiety (SSM) and the microbial polypeptide may be connected by a hinge as defined herein.

[0168] In other or even more preferred embodiments, the microbial polypeptide is a viral surface polypeptide or a bacterial surface polypeptide. 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.

[0169] When reference is made to a "variant" of a microbial polypeptide (including any of the specific microbial polypeptides described herein), this preferably refers to a polypeptide having at least 70%, or, in ascending order of preference, at least 80%, 85%, 90%, 95%, or 98% sequence identity to the corresponding microbial polypeptide.

[0170] 2.2.1 Enveloped Virus Fusion Proteins and Ectodomain Polypeptides In some embodiments, the enveloped virus fusion ectodomain polypeptide corresponds to or is a variant of (i) a class I enveloped virus fusion protein ectodomain (preferably, the ectodomain is derived from a virus selected from an orthomyxovirus, a paramyxovirus, an orthopneumovirus, a metapneumovirus, a retrovirus, a coronavirus, a filovirus, and an arenavirus), or (ii) a class III enveloped virus fusion protein ectodomain (preferably, the ectodomain is derived from a virus selected from a rhabdovirus and a herpesvirus).

[0171] In preferred embodiments of the latter embodiment, the enveloped virus fusion ectodomain polypeptide corresponds to or is a variant of a fusion protein derived from (i) respiratory syncytial virus (RSV), (ii) metapneumovirus, (iii) a coronavirus, preferably a betacoronavirus, more preferably severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or Middle East respiratory syndrome-related coronavirus (MERS-CoV), (iv) a henipavirus, preferably Hendra virus (HeV) or Nipah virus (NiV), (v) an influenza virus, preferably influenza A or influenza B, (vi) a parainfluenza virus (PIV), preferably a human parainfluenza virus (HPIV), (vii) an arenavirus, preferably Lassa fever virus, or (viii) a retrovirus, preferably human T-lymphotropic virus-1 (HTLV-1).

[0172] In some embodiments, the chimeric polypeptide further comprises a hinge region operably connecting the microbial polypeptide (preferably, a bacterial or viral surface polypeptide, more preferably, an enveloped viral fusion ectodomain polypeptide or a bacterial outer membrane polypeptide (e.g., a TAA polypeptide)) to the structure-stabilizing moiety, 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.

[0173] Most preferably, the chimeric polypeptides of the present invention are single polypeptide chains in which the structural stabilizing moiety (SSM) is C-terminal to (downstream from) the enveloped virus fusion ectodomain polypeptide, although the present disclosure also contemplates alternative arrangements in which the structural stabilizing moiety is N-terminal to (upstream from) the enveloped virus fusion protein ectodomain. In either instance, the structural stabilizing moiety and the enveloped virus fusion protein ectodomain may be connected by a hinge as defined herein.

[0174] Non-limiting examples of enveloped virus fusion ectodomain polypeptides are provided below:

[0175] 2.2.1.1 Inf A HA Non-limiting examples of Inf A HA ectodomain polypeptides include: Ectodomains 1-529: [SEQ ID NO: 86] (GenPept gbAEC23340.1) This sequence contains the following domains / portions: SP=1~16 Ectodomain = 17~529 Furin cleavage site = 345-346 FP=346~355 MPER=470~529 Head area = 51 to 328, 403 to 444 Stem region = 17–58, 327–401, 442–509. SP-free ectodomain, 18–529: KLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRICNSPHQILDGKNCTLIDALLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWTGVAQD GSSYACRRGSVNSFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIVSTKRSQQTVIPNIGSRPWVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKS SIMRSDAHIDECNSECITPNGSIPNDKPFQNVNKITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMSKLFERTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDIYRNEALNNRFQIKGVQLKSGYKD [SEQ ID NO: 87] (GenPept gbAEC23340.1) SP-free and MPER-free ectodomain, 18–469: KLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRICNSPHQILDGKNCTLIDALLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNL EFTQESFNWTGVAQDGSSYACRRGSVNSFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIVSTKRSQQTVIPNIGSRPWVRGVSSIISIYWT IVKPGDILLINSTGNLIAPRGYFKIQSGKSSIMRSDAHIDECNSECITPNGSIPNDKPFQNVNKITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMSKLFERTRR [SEQ ID NO: 88] (GenPept gbAEC23340.1) Ectodomains 18–341, 346–529, containing an altered furin cleavage site: KLPGSDNSMATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGRICNSPHQILDGKNCTLIDALLGDPHCDDFQNKEWDLFVERSTAYSNCYPYYVPDYATLRSLVASSGNLEFTQESFNWTGVAQD GSSYACRRGSVNSFFSRLNWLYNLNYKYPEQNVTMPNNDKFDKLYIWGVHHPGTDKDQTNLYVQASGRVIVSTKRSQQTVIPNIGSRPWVRGVSSIISIYWTIVKPGDILLINSTGNLIAPRGYFKIQSGKSS IMRSDAHIDECNSECITPNGSIPNDKPFQNVNKITYGACPRYVKQNTLKLATGMRNVPERRRKKRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQITGKLNRVIKKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMSKLFERTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDIYRNEALNNRFQIKGVQLKSGYKD [SEQ ID NO: 89] (GenPept gbAEC23340.1) Linker region included: stem domains 1-58, 327-401, 442-509: MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELGFGQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMLDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGMLNRLIGSGGSGELLVALLNQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKD [SEQ ID NO: 90] (GenPept gbAEC23340.1) Head domains 1-18, 51-328, and 403-444, including the linker region: MKTIIALSYILCLVFAQKEVTNATELVQNSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRGSNNSFFSRLNWLTHSKFKYPALNVTMPNNEEFDKLYIWGVHHPGTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNGSGGSGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELL [SEQ ID NO: 91] (GenPept gbAEC23340.1)

[0176] 2.2.1.2 Inf B HA Non-limiting examples of Inf B HA ectodomain polypeptides include: Ectodomains 1-547: [SEQ ID NO: 92] (GenPept gbAFH57854.1) This sequence contains the following domains / portions: SP=1~16 Ectodomain = 17~547 Furin cleavage site = 361-362 FP=362~382 MPER=488~547 Head area = 48~344, 418~456 Stem region = 17-47, 345-417, 457-547 SP-free ectodomain, 17–547: [SEQ ID NO: 93] (GenPept gbAFH57854.1) SP-free and MPER-free ectodomain, 17–487: RICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTS NVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGR IVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKML [SEQ ID NO: 94] (GenPept gbAFH57854.1) Ectodomains 17–355 and 362–547, which do not contain SP but contain an altered furin cleavage site: [SEQ ID NO: 95] (GenPept gbAFH57854.1) Stem domains 1-47, 345-417, and 457-547, including the linker region: MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLGSGLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNYLSGSGGSGIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFNAGDFSLPTFDSLNITAASLNDDGLDNHT [SEQ ID NO: 96] (GenPept gbAFH57854.1) Head domains 1-17, 48-344, and 418-456, including the linker region: MKAIIVLLMVVTSNADRTTTPTKSHFANLKGTQTRGKLCPNCFNCTDLDVALGRPKCMGNTPSAKVSILHEVKPATSGCFPIMHDRTKIRQLPNLLRGYENIRLSTSNVINTETAPGGPYKVGTSGSCPNVANGNGFFNTMAWVIPKDNNKTAINPVTVEVPYICSEGEDQITVWGFHSDDKTQMERLYGDSNPQKFTSSANGVTTHYVSQIGGFPNQTEDEGLKQSGRIVVDYMVQKPGKTGTIVYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKGSGGSGELEVKNLQRLSGAMNELHDEILELDEKVDDLRADTISSQ [SEQ ID NO: 97] (GenPept gbAFH57854.1)

[0177] 2.2.1.3 RSV F Non-limiting examples of RSV F ectodomain polypeptides include: Ectodomains 1-524: [SEQ ID NO: 98] (GenPept gbAHL84194.1) This sequence contains the following domains / portions: SP=1~23 Ectodomain = 24~524 Furin cleavage sites: 109–110, 136–137 FP=137~163 D25 interaction domain = 61-97, 193-240 RSV F ectodomain (1-520): MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKK RKRRFLGFLLGVGSAIASGGAVVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDM PITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGK [SEQ ID NO: 99] SP-free ectodomain, 24–524: SGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGV AVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVR QQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN [SEQ ID NO: 100] (GenPept gbAHL84194.1) Ectodomain, 24–524, without SP and containing an altered furin cleavage site: SGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNNANNELPRFMNYTLNNAKKTNVTLSNNNNNNFLGFLLGVGSAIASGV AVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVR QQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCEIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN [SEQ ID NO: 101] (GenPept gbAHL84194.1) D25 interaction domain including linker region, 61-97, 193-240: LSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMGGLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVN [SEQ ID NO: 102] (GenPept gbAHL84194.1)

[0178] 2.2.1.4 hMPV F An exemplary hMPV F precursor has the following amino acid sequence: [SEQ ID NO: 103] (GenPept gbAAN52913.1). This sequence contains the following domains / portions: SP=1~19 Ectodomain = 1 to 490 Furin cleavage site = 102-103 FP=103~125 FHRR=126~169 SHRR=456~490 TM=491~514 C=515~539 Non-limiting examples of hMPV F ectodomain polypeptides include: Ectodomain 1-490: MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAK TIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRR KGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTG [SEQ ID NO: 104] (GenPept gbAAN52913.1) SP-free ectodomain, 20–490: KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNAL KKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTG [SEQ ID NO: 105] (GenPept gbAAN52913.1) Ectodomain, 20-490, without SP and containing an altered furin cleavage site: KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPNQSNFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNAL KKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTG [SEQ ID NO: 106] (GenPept gbAAN52913.1)

[0179] 2.2.1.5 PIV F Non-limiting examples of PIV F ectodomain polypeptides include: Ectodomains 1-493: MPTSILLIITTMIMASFCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVSNQESNENTDPRTKRFFGGVIGTIALGVATSAQ ITAAVALVEAKQARSDIEKLKEAAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYD LLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTT [SEQ ID NO: 107] (GenPept gbAAB21447.1) This sequence contains the following domains / portions: SP=1~19 Ectodomain = 1 to 493 Furin cleavage site = 109-110 FP=110~135 SP-free ectodomain, 20–493: IDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVSNQESNENTDPRTKRFFGGVIGTIALGVATSAQITAAVALVEAKQAR SDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKV RVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTT [SEQ ID NO: 108] (GenPept gbAAB21447.1) ectodomain, 20–493, without SP and containing an altered furin cleavage site: IDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVSNQESNENTDPNTKNFFGGVIGTIALGVATSAQITAAVALVEAKQAR SDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKV RVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTVVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPNDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTT [SEQ ID NO: 109] (GenPept gbAAB21447.1) 2.2.1.6 MeV F Non-limiting examples of MeV F ectodomain polypeptides include: Ectodomains 1-493: MGLKVNVSAIFMAVLLTLQTPTGQIHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVAT AAQITAGIALHQSMLNSQAIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGD LLGILESRGIKARITHVDTESYLIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSST [SEQ ID NO: 110] (GenPept dbjBAB60865.1) This sequence contains the following domains / portions: SP=1~24 Ectodomain = 1 to 493 Furin cleavage site = 112-113 FP=113~137 SP-free ectodomain, 25–493: IHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRFAGVVLAGAALGVATAAQITAGIALHQSMLNSQ AIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKA RITHVDTESYLIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSST [SEQ ID NO: 111] (GenPept dbjBAB60865.1) Ectodomain without SP and containing an altered furin cleavage site: IHWGNLSKIGVVGIGSASYKVMTRSSHQSLVIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSNNHKNFAGVVLAGAALGVATAAQITAGIALHQSMLNSQ AIDNLRASLETTNQAIEAIRQAGQEMILAVQGVQDYINNELIPSMNQLSCDLIGQKLGLKLLRYYTEILSLFGPSLRDPISAEISIQALSYALGGDINKVLEKLGYSGGDLLGILESRGIKA RITHVDTESYLIVLSIAYPTLSEIKGVIVHRLEGVSYNIGSQEWYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQDPDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPILLERLDVGTNLGNAIAKLEDAKELLESSDQILRSMKGLSST [SEQ ID NO: 112] (GenPept dbjBAB60865.1)

[0180] 2.2.1.7 HeV F Non-limiting examples of HeV F ectodomain polypeptides include: Ectodomains 1-487: MATQEVRLKCLLCGIIVLVLSLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNVSKCTGTVMENYKSRLTGILSPIKGAIELYNNNTHDLVGDVKLAGVVMAGIAAIGIATAA QITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATED FDDLLESDSIAGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQELLPVSFNNDNSEWISIVPNFVLIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSHVPRFALSGGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPPVYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPS [SEQ ID NO: 113] (GenPept NP_047111.2) This sequence contains the following domains / portions: SP=1~20 Ectodomain = 1 to 487 Furin cleavage site = 109-110 FP=110~135 FHRR=136~169 SHRR=456~587 TM=488~518 C=519~546 SP-free ectodomain, 21–487: SLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNVSKCTGTVMENYKSRLTGILSPIKGAIELYNNNTHDLVGDVKLAGVVMAGIAIGIATAAQITAGVALYEAMKNA DNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSI AGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQELLPVSFNNDNSEWISIVPNFVLIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSHVPRFALSGGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPPVYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPS [SEQ ID NO: 114] (GenPept NP_047111.2) Ectodomain, 21–487, without SP and containing an altered furin cleavage site: SLEGLGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNVSKCTGTVMENYKSRLTGILSPIKGAIELYNNNTHDLVGDVNLAGVVMAGIAIGIATAAQITAGVALYEAMKNA DNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDQISCKQTELALDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSI AGQIVYVDLSSYYIIVRVYFPILTEIQQAYVQELLPVSFNNDNSEWISIVPNFVLIRNTLISNIEVKYCLITKKSVICNQDYATPMTASVRECLTGSTDKCPRELVVSSHVPRFALSGGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCTTVVLGNIIISLGKYLGSINYNSESIAVGPPVYTDKVDISSQISSMNQSLQQSKDYIKEAQKILDTVNPS [SEQ ID NO: 115] (GenPept NP_047111.2)

[0181] 2.2.1.8 NiV F Non-limiting examples of NiV F ectodomain polypeptides include: Ectodomains 1-487: MVVILDKRCYCNLLILMISECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAA QITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATED FDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPS [SEQ ID NO: 116] (GenPept NP 112026) This sequence contains the following domains / portions: SP=1~20 Ectodomain = 1 to 487 Furin cleavage site = 109-110 FP=110~135 SP-free ectodomain: SECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNA DNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSI TGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPS [SEQ ID NO: 117] (GenPept NP 112026) Ectodomain without SP and containing an altered furin cleavage site: SECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVNLAGVIMAGVAIGIATAAQITAGVALYEAMKNA DNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSI TGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPS [SEQ ID NO: 118] (GenPept NP 112026)

[0182] 2.2.1.9 HIV GP160 Non-limiting examples of HIV GP160 ectodomain polypeptides include: Ectodomains 1-688: [SEQ ID NO: 119] (GenPept dbjBAF31430.1) This sequence contains the following domains / portions: SP=1~28 Ectodomain = 1 to 688 Furin cleavage site = 508-509 FP=509~538 MPER=668~688 GP120=1~508 SP-free ectodomain: [SEQ ID NO: 120] (GenPept dbjBAF31430.1) SP-free, MPER-free ectodomain: [SEQ ID NO: 121] (GenPept dbjBAF31430.1) Ectodomain without SP and containing an altered furin cleavage site: [SEQ ID NO: 122] (GenPept dbjBAF31430.1) GP41 ectodomain 509-688: VVQREKRAVGTIGAMFLGFLGAAGSTMGAASLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGRLICTTAVPWNASWSNKSLDDIWNNMTWMQWEKEIDNYTGLIYRLIEESQTQQEKNEQDLLQLDTWASLWNWFSISNWLWYIK [SEQ ID NO: 123] (GenPept dbjBAF31430.1)

[0183] 2.2.1.10 EBOV GP Non-limiting examples of EBOV GP ectodomain polypeptides include: Ectodomain 1-650: [SEQ ID NO: 124] (GenPept NP_066246.1) This sequence contains the following domains / portions: SP=1~27 Ectodomain = 1 to 650 Furin cleavage site = 501-502 Cathepsin cleavage sites: 191-192, 201-202 FP=511~556 MPER=636~650 Mucin-like domain = 312-461 SP-free ectodomain: [SEQ ID NO: 125] (GenPept NP_066246.1) SP-free, MPER-free ectodomain: [SEQ ID NO: 126] (GenPept NP_066246.1) Ectodomain without SP and containing an altered furin cleavage site: [SEQ ID NO: 127] (GenPept NP_066246.1) Ectodomain without SP and without mucin-like domain: QRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGD FAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKL IWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVGGNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQ [SEQ ID NO: 128] (GenPept NP_066246.1) Ectodomain without mucin-like domain: MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGS ECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDN LTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTL [SEQ ID NO: 129]

[0184] 2.2.1.11 MARV GP Non-limiting examples of MARV GP ectodomain polypeptides include: Ectodomain 1-650: [SEQ ID NO: 130] (GenPept YP_001531156.1) This sequence contains the following domains / portions: SP=1~19 Ectodomain = 1 to 650 Furin cleavage site = 434-435 FP=526~549 MPER=628~650 Mucin-like domain = 244-425 SP-free ectodomain, 20–650: [SEQ ID NO: 131] (GenPept YP_001531156.1) SP-free and MPER-free ectodomain, 20–627: [SEQ ID NO: 132] (GenPept YP_001531156.1) Ectodomain without SP and containing an altered furin cleavage site: [SEQ ID NO: 133] (GenPept YP_001531156.1) Ectodomain without SP and without mucin-like domain: PILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIA LHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTARPEIKLGGAQHLVYFR RKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAVLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWG [SEQ ID NO: 134] (GenPept YP_001531156.1)

[0185] 2.2.1.12 SARS-CoV S Non-limiting examples of SARS-CoV S ectodomain polypeptides include: Ectodomains 1-1199: This sequence contains the following domains / portions: SP=1~13 Ectodomain = 1 to 1199 Human airway trypsin-like protease cleavage site = 667-668 FP=770~788 MPER=1188~1199 SP-free ectodomain: SP-free, MPER-free ectodomain:

[0186] 2.2.1.13 MERS-CoV S Non-limiting examples of MERS-CoV S ectodomain polypeptides include: Ectodomains 1-1301: This sequence contains the following domains / portions: SP=1~21 Ectodomain = 1~1301 Furin cleavage sites: 751-752, 887-888 FP=888~891, 951~980 MPER=1292~1301 SP-free ectodomain, 22–1301: SP-free and MPER-free ectodomain, 22–1291: Ectodomain without SP and containing an altered furin cleavage site:

[0187] 2.2.1.14 VSV G Non-limiting examples of VSV G ectodomain polypeptides include: Ectodomains 1-462: MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGT WLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQY CKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWK [SEQ ID NO: 142] (GenPept gbADX53329.1) This sequence contains the following domains / portions: SP=1~17 Ectodomain = 1 to 462 MPER=421~462 SP-free ectodomain: FTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYAT VTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGV WFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWK [SEQ ID NO: 143] (gbADX53329.1) SP-free, MPER-free ectodomain: FTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVF [SEQ ID NO: 144] (GenPept gbADX53329.1)

[0188] 2.2.1.15 RABV GP Non-limiting examples of RABV GP ectodomain polypeptides include: Ectodomains 1-458: MIPQTLLFVPLLVFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYN WKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVSDLDPYDKSLHSRVFPSGKCSGITVSSTYCPTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKS LKGACKLKLCGVLGLRLMDGTWAAIQTSDEAKWCPPDQLVNIHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEAEDFVEVHLPDVHKQVSGVDLGLPSWGK [SEQ ID NO: 145] (GenPept gbAFM52658.1) This sequence contains the following domains / portions: SP=1~20 Ectodomain = 1 to 458 SP-free ectodomain: FPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHN PYPDYHWLRTVKTTKESLVIISPSVSDLDPYDKSLHSRVFPSGKCSGITVSSTYCPTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKGACKLKLC GVLGLRLMDGTWAAIQTSDEAKWCPPDQLVNIHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEAEDFVEVHLPDVHKQVSGVDLGLPSWGK [SEQ ID NO: 146] (GenPept gbAFM52658.1)

[0189] 2.2.1.16 HSV1 Gb Non-limiting examples of HSV1 Gb ectodomain polypeptides include: Ectodomains 1-774: [SEQ ID NO: 147] (GenPept gbAAF04615.1) This sequence contains the following domains / portions: SP=1~24 Ectodomain = 1 to 774 SP-free ectodomain, 25–774: [SEQ ID NO: 148] (GenPept gbAAF04615.1)

[0190] 2.2.2 Bacterial outer membrane polypeptides In a preferred embodiment, the bacterial outer membrane polypeptide is (i) a chlamydial major outer membrane protein (MOMP) polypeptide, or (ii) a trimeric autotransporter adhesin (TAA) polypeptide is.

[0191] 2.2.2.1 Chlamydial major outer membrane protein (MOMP) polypeptide Chlamydiae are obligate intracellular pathogens in eukaryotic cells and are known to be the most common cause of bacterial sexually transmitted diseases worldwide. Although infections can be resolved by antibiotic treatment, this is often left untreated due to the high frequency of asymptomatic infections, leading to disease progression and severe sequelae. Therefore, the development of a vaccine against chlamydia is considered important. The "chlamydial major outer membrane protein" (commonly referred to as "MOMP" or "chlamydial MOMP") has become one of the important target molecules for vaccine development (see, for example, the review by Madico G et al., Structural and Immunological Characterization of Novel Recombinant MOMP-Based Chlamydial Antigens. Vaccines (Basel). 2017;6(1):2. doi: 10.3390 / vaccines6010002).

[0192] Chlamydia MOMP is a surface-exposed trimeric porin with a putative 16-stranded barrel-spanning core region, eight surface-exposed loops, and eight short periplasmic loops per monomer. Given that MOMP naturally exists in a trimeric conformation, the techniques disclosed herein likely offer advantages in stabilizing the trimeric state of polypeptides derived from or corresponding to MOMP. The corresponding trimers formed by trimerization of the molecular clamps disclosed herein may enable antigens derived from MOMP to be presented in a conformation similar to the native trimeric state of MOMP (or fragments / portions derived therefrom). Therefore, when the respective chimeric polypeptides are used as vaccines, antigen presentation resembling the native trimeric conformation may result in the generation of a stronger, broadly neutralizing antibody response compared to antigens not presented in a trimeric state.

[0193] Molecular characterization and topology modeling of MOMP identified four serotype-specific domains of sequence variability (variable domains, VDs) along with constant domains (CDs) in loops 2, 3, 5, and 6. VDs have been shown to contain B- and T-cell epitopes capable of eliciting humoral responses (monoclonal and polyclonal), while CDs can induce T-cell responses (see, e.g., Madico G et al Vaccines (Basel). 2017;6(1):2).

[0194] The terms "Chlamydia major outer membrane protein (MOMP) polypeptide" or "Chlamydia major outer membrane porin (MOMP) polypeptide," as used interchangeably herein, are intended to refer to both the full-length polypeptide corresponding to the monomer subunit of MOMP, as well as to fragments or portions of the latter polypeptide.

[0195] Within the family Chlamydiaceae, there are three known species that infect humans: C. trachomatis, C. pneumoniae, and C. psittaci, the genetic sequences of each of which are publicly available.

[0196] Thus, in a preferred embodiment, the chlamydial major outer membrane protein (MOMP) polypeptide corresponds to or is a variant of a chlamydial MOMP polypeptide from a species selected from C. trachomatis, C. pneumoniae, and C. sittaki.

[0197] In particularly preferred embodiments, the chlamydia MOMP polypeptide comprises or consists of an amino acid sequence having at least 70% (or, in ascending order of 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. SEQ ID NO: 216 Chlamydia trachomatis major outer membrane protein (MOMP) (included in SEQ ID NO: 263) MKKLLKSVLVFAALSSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPCTTWCDAISMRVGYYGDFVFDRVLKTDVNKEFQMGAAPTTSDVAGLQNDPTINVARPNPAYGKHMQDAEMFTNAAYMALNIWDRFDVFCTLGATTGYLKGNSASFNLVGLFGTKTQSSSFNTAKLIPNTALNEAVVELYINTTFAWSV GARAALWECGCATLGASFQYAQSKPKVEELNVLCNASEFTINKPKGYVGAEFPLNITAGTEAATGTKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRVSFDADTIRIAQPKLAEAILDVTTLNRTTAGKGSVVSAGTDNELADTMQIVSLQLNKMKSRKSCGIAVGTTIVDADKYAVTVEARLIDERAAHVNAQFRF The letters in italics correspond to the leader sequence. SEQ ID NO: 217 Chlamydia pneumoniae major outer membrane protein (MOMP), Uniprot entry: P27455 MKKLLKSALLSAAFAGSVGSLQALPVGNPSDPSLLIDGTIWEGAAGDPCDPCATWCDAISLRAGFYGDYVFDRILKVDAPKTFSMGAKPTGSAAANYTTAVDRPNPAYNKHLHDAEWFTNAGFIALNIWDRFDVFCTLGASNGYIRGNSTAFNLVGLFGVKGTTVNANELPNVSLSNGVVELYTDTSFSWSVGA RGALWECGCATLGAEFQYAQSKPKVEELNVICNVSQFSVNKPKGYKGVAFPLPTDAGVATATGTKSATINYHEWQVGASLSYRLNSLVPYIGVQWSRATFDADNIRIAQPKLPTAVLNLTAWNPSLLGNATALSTTDSFSDFMQIVSCQINKFKSRKACGVTVGATLVDADKWSLTAEARLINERAAHVSGQFRF The letters in italics correspond to the leader sequence. In an even more preferred embodiment, the chimeric polypeptide comprises or consists of an amino acid sequence having at least 70% (or, in order of 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.

[0198] Preferably, the chimeric polypeptides of the present invention are single polypeptide chains in which the structural stabilizing moiety (SSM) is C-terminal to (i.e., downstream of) the bacterial surface polypeptide, although the present disclosure also contemplates alternative arrangements in which the structural stabilizing moiety (SSM) is N-terminal to (i.e., upstream of) the bacterial surface polypeptide. In either instance, the structural stabilizing moiety (SSM) and bacterial surface polypeptide may be connected by a hinge as defined herein.

[0199] Chlamydial PGP3 Polypeptide Another surface polypeptide and prominent antigen of chlamydia expressly contemplated herein to be included in the chimeric polypeptide as a bacterial surface polypeptide is the so-called "plasmid gene protein 3 (PGP3)."

[0200] Thus, in a preferred embodiment, the bacterial surface polypeptide is a chlamydial PGP3 polypeptide, more preferably a chlamydial PGP3 polypeptide derived from Chlamydia trachomatis.

[0201] 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, in ascending order of 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. SEQ ID NO: 259 Chlamydia trachomatis PGP3 (sequence corresponds to amino acid residues 3 to 264 of the full-length protein) NSGFYLYNTENCVFADNIKVGQMTEPLKDQQIILGTTSTPVAAKMTASDGISLTVSNNSSTNASITIGLDAEKAYQLILEKLGNQILDGIADTIVDSTVQDILDKITTDPSLGLLKAFNNFPITNKIQCNG LFTPSNIETLLGGTEIGKFTVTPKSSGSMFLVSADIIASRMEGSVVLALVREGDSKPCAISYGYSSGVPNLCSLRTSITNTGLTPTTYSLRVGGLESGVVWVNALSNGNDILGITNTSNVSFLEVIPQTNA Based on an evaluation of the structural topology of the chlamydial PGP3 polypeptide, the inventors believe that a configuration in which the SSM is N-terminal to (i.e., upstream of) the chlamydial PGP3 polypeptide would be particularly advantageous in terms of resulting in a stably folded fusion protein and providing effective presentation of the chlamydial PGP3 polypeptide as an antigen.

[0202] Thus, in an alternative, more particularly preferred embodiment of the present invention, there is provided a chimeric polypeptide comprising a heterologous structure-stabilizing moiety (SSM) operably connected downstream to a chlamydial 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 set forth in 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 set forth in 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 set forth in 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 set forth in SEQ ID NO: 82 or 83.

[0203] In particularly preferred embodiments of the latter aspect, the chimeric polypeptide comprises or consists of an amino acid sequence having at least 70% (or, in order of 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.

[0204] Those skilled in the art will readily appreciate that the embodiments disclosed herein relating to chimeric polypeptides according to the first aspect of the invention, and embodiments of further aspects relating to chimeric polypeptides according to the first aspect of the invention, apply mutatis mutandis to chimeric polypeptides defined according to the alternative aspects described above, to the extent applicable.

[0205] 2.2.2.2 Trimeric Autotransporter Adhesin (TAA) Polypeptides As shown by the evidence disclosed herein (see, e.g., Example 19 and corresponding Figures 36-39), the applicability of the structure-stabilizing moieties of the present invention is in no way limited to stabilizing antigens derived from enveloped virus fusion protein ectodomains, but can also be suitably utilized to stabilize other (poly)peptides, particularly those that naturally occur in a trimeric state and whose native conformation can therefore also be stabilized by the SSMs disclosed herein.

[0206] Additional (poly)peptides that also constitute vaccine target antigens are of particular interest, including those of bacterial origin, e.g., bacterial outer membrane proteins (OMPs). Among the latter are the so-called "trimeric autotransporter adhesins (TAA)," which have recently been advocated as promising new vaccine targets (see, e.g., the review by Thibau A. et al. 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).

[0207] The term "trimeric autotransporter adhesins (TAAs)," also commonly known as "bacterial autotransporters," "trimeric autotransporters," "non-fimbrial adhesins (NFAs)," or "oligomeric coiled-coil adhesins (Oca)," refers to a group of Gram-negative bacterial outer membrane proteins that play important roles in bacterial infection and host colonization. TAAs are generally constructed from three identical polypeptide chains (fibers) assembled into long, filamentous trimeric proteins. Each monomer subunit typically consists of an N-terminal extracellular portion commonly referred to as the "passenger domain" and a C-terminal membrane anchor commonly 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 host cells. Passenger domains typically contain one or more head and neck domains and one or more coiled-coil stalk domains. Depending on the arrangement of the head domain, TAA can be classified as either a "lollipop" or a "beads-on-a-string"-like structure. The C-terminal "translocator domain" typically consists of three subunits, each consisting of a long amphipathic helix followed by a four-stranded β-meander, which assembles into a 12-stranded β-barrel (each monomer subunit contributes four strands) embedded in the bacterial outer membrane. The translocator domain is thought to be responsible for the insertion and translocation of the passenger domain to the outside of the cell. In some TAAs, such as YadA from Yersinia enterocolitica, the passenger domain consists of only one head and one stalk region, but more complex TAAs exist that contain multiple head and stalk regions in various arrangements. Furthermore, although all TAAs contain a translocator domain, not all of them contain both a stalk and a head region.TAAs are synthesized as precursors containing three functional domains: an N-terminal signal sequence, a passenger domain, and a C-terminal translocator domain. The signal sequence, typically approximately 20–50 amino acid residues long (hence often referred to as an "extended signal peptide"), targets these proteins to the Sec transport machinery at the cytoplasmic membrane. This translocation across the membrane then proceeds via the Sec pathway, utilizing ATP as an energy source, leading to loss of the signal peptide. From within the periplasm, the translocator domain inserts into the outer membrane as a β-barrel structure, forming a pore through which the passenger domain translocates onto the bacterial cell surface. At this point, the passenger domain can remain associated with the outer membrane either through covalent attachment to the translocator domain or noncovalently after cleavage from the translocator domain. Alternatively, the passenger domain may be released 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 autotransporter adhesins. Med Microbiol Immunol. 2020;209(3):233-242).

[0208] As shown in Example 19 and corresponding Figures 36-39, this technique could also be successfully utilized to generate chimeric polypeptides and complexes thereof comprising TAA polypeptides fused to SSMs of the present invention in place of enveloped virus fusion ectodomain polypeptides. Collectively, a panel of seven constructs employing TAAs derived from major bacterial pathogens could be solublely and stably expressed and purified from E. coli, and subsequent analysis (by TEM) demonstrated overall shapes consistent with their native, long, filamentous conformations. These results therefore raise the possibility that this molecular clamping technique can also favorably stabilize TAAs in a near-native conformation, and thus that each construct will likely prove effective as a vaccine, and in eliciting potent immune responses specifically against these important classes of virulence factors.

[0209] Although TAAs naturally fold into trimers, those skilled in the art will understand that whenever a "TAA polypeptide" is referred to herein, this term refers to a single polypeptide chain that corresponds to a monomer subunit of a TAA polypeptide or is a variant or fragment thereof.

[0210] Thus, in a preferred embodiment, the TAA polypeptide is (i) a TAA polypeptide derived from a bacterium of a genus selected from Neisseria, Escherichia coli, Haemophilus, Yersinia, Salmonella, Bartonella, Vibrio, Acinetobacter, and Moraxella; and / or (ii) a TAA polypeptide selected from Neisseria meningitidis adhesin (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); corresponds to or is a variant of

[0211] In a preferred embodiment, the bacterium of the genus Neisseria is one of the species N. meningitidis. In another preferred embodiment, the bacterium of the genus Escherichia coli is selected from the group consisting of enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), adherent invasive E. coli (AIEC), and uropathogenic E. coli (UPEC).

[0212] In other preferred embodiments, the Haemophilus bacterium is selected from the group consisting of H. influenzae, H. ducreyi, H. aegyptius, H. parainfluenzae, H. parasuis, and H. paragallinarum. In other preferred embodiments, the bacteria of the genus Yersinia is selected from the group consisting of Y. enterocolitica and Y. pseudotuberculosis.

[0213] In other preferred embodiments, the Salmonella bacterium is selected from the group consisting of S. typhi, S. enterica, and S. enteritidis. In other preferred embodiments, the Bartonella bacterium is selected from the group consisting of B. bacilliformis, B. quintana, B. clarigiae, B. elizabeta, B. grahami, B. henselae, B. coerella, B. naantariensis, B. vinsonii, B. bachoensis, and B. localime.

[0214] In other preferred embodiments, the Vibrio bacterium is selected from the group consisting of V. vulnificus, V. parahaemolyticus, V. cholerae, and V. camperi. In other preferred embodiments, the bacterium of the genus Acinetobacter is selected from the group consisting of A. calcoaceticus, A. baumannii, A. haemolyticus, A. junii, A. johnsonii, A. rwofi, A. radioresistens, A. schnindleri, A. ursingii, A. bailli, A. buvechii, A. gerneri, A. grimonti, A. tandoi, A. chernbergia, A. tounelli, and A. parvus.

[0215] In other preferred embodiments, the bacteria of the genus Moraxella is selected from the group consisting of M. catarrhalis, M. lactata, and M. bovis. In Example 19 disclosed herein, chimeric polypeptides were designed containing either the full-length TAA polypeptide (without the signal peptide) or a C-terminally truncated version thereof (lacking the translocator domain partially or even entirely).

[0216] In other or even more preferred embodiments, the TAA polypeptide is (i) Passenger Domains, and / or (ii) Translocator domain Comprises or consists of

[0217] However, it is also contemplated that constructs containing only one (i.e., a single) head, neck, or stalk domain, or constructs containing only the translocator domain, may also be produced in similar yields. Given that the head domain often serves an adhesive function, constructs containing only the head domain as the TAA polypeptide fused to the SSM may be particularly effective at eliciting potent neutralizing antibody responses against the effector protein and related bacteria expressing the protein or similar variants thereof.

[0218] In an even more preferred embodiment, the TAA polypeptide comprises or consists of at least one of a head domain, a neck domain, and / or a stalk domain, or an antigenic fragment of any of these.

[0219] In preferred embodiments, the TAA polypeptide comprises or consists of an amino acid sequence having at least 70% (or, in ascending order of 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: 252-258.

[0220] In an even more preferred embodiment, the chimeric polypeptide comprises or consists of an amino acid sequence having at least 70% (or, in ascending order of 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.

[0221] In addition, the present technology can also be advantageously utilized to achieve trimerization of other (poly)peptides, e.g., therapeutic (poly)peptides, which do not naturally adopt a trimeric state but whose presentation as a trimer may offer advantages in terms of their activity and / or interactions with other molecules / binding partners, e.g., when applied as pharmaceuticals. Exemplary embodiments are described in Section 2.4 below.

[0222] 2.3 Representative Chimeric Polypeptide Constructs Non-limiting examples of chimeric polypeptides of the present invention are described below. RSV fusion protein 66K-T 103 -GS-G 145 -E 511 -GSG-VISNA based SSM MELLILKANAITTILTAVTFFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVSKV LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 22] or a corresponding amino acid sequence thereto (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); where: The letters in italics correspond to the signal peptide of the RSV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to the VISNA-based SSM (CD11).

[0223] RSV fusion protein 66E-T 103 -GS-G 145 -E 511 -GSG-VISNA based SSM MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVSKV LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 24] or a corresponding amino acid sequence thereto (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). where: The letters in italics correspond to the signal peptide of the RSV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0224] SARS-CoV-2 spike protein-N 679 -GSG-S 691 -G1204 -VISNA-based SSM CD11 QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 29] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the SARS-CoV-2 spike protein, Letters in bold are flexible linkers, The underlined letters correspond to the VISNA-based SSM CD11.

[0225] Nipah virus fusion-VISNA-based SSM MVVILDKRCYCNLLILMISECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIG IATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLL RTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCP RELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI[SEQ ID NO: 31] or a corresponding amino acid sequence thereof (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). where: · The letters in italics correspond to the signal peptide of the Nipah virus fusion protein, Letters in bold are flexible linkers, The underlined letters correspond to the VISNA-based SSM CD11.

[0226] Influenza hemagglutinin protein R 526 -VISNA-based SSM MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFE IFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGN LVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKV NSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI[SEQ ID NO: 33] or a corresponding amino acid sequence thereto (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). where: The letters in italics correspond to the signal peptide of the influenza hemagglutinin protein, Letters in bold are flexible linkers, The underlined letters correspond to the VISNA-based SSM CD11.

[0227] SARS-CoV-2 spike protein-N 679 -GSG-S 691 -G 1204 -VISNA-based SSM CD11(-SG) QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 34] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the SARS-CoV-2 spike protein, Letters in bold are flexible linkers, The underlined letters correspond to the VISNA-based SSM CD11.

[0228] RSV fusion protein 66E-T 103 -GS-G 145 -E 511 -GSG - VISNA-based SSM (CD11_1245T8) with an additional N-linked glycosylation sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVSKV LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVARGGSGG NHTWQNWTEEIENHTGNLTLLLREAANQTHIAQRDARRI [SEQ ID NO: 51] or a corresponding amino acid sequence thereto (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). where: The letters in italics correspond to the signal peptide of the RSV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0229] RSV fusion protein 66E-T 103 -GS-G 145 -E 511 -GSG - VISNA-based SSM (CD11_145T8) with an additional N-linked glycosylation sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVSKV LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI[SEQ ID NO: 52] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the RSV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0230] SARS-CoV-2 (Delta) Spike Protein-N 679 -GSG-S 691 -G 1204 -VISNA-based SSM CD11 QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 73] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the SARS-CoV-2 spike protein, Letters in bold are flexible linkers, The underlined letters correspond to the VISNA-based SSM CD11.

[0231] SARS-CoV-2 (Delta) Spike Protein-N 679 -GSG-S 691 -G 1204 -VISNA-based SSM in which QS is removed from the CD11 FHRR (CD11) LANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 74] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the influenza hemagglutinin protein, The underlined letters correspond to the VISNA-based SSM CD11.

[0232] RSV fusion protein 66E-T 103 -GS-G 145 -E 511 - VISNA-based SSM in which GSG-QS was removed from the CD11 FHRR (CD11) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVSKV LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEGSG LANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI[SEQ ID NO: 75] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the RSV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0233] RSV fusion protein 66E-T 103 -GS-G 145 -E 511 -GSG - A VISNA-based SSM with an additional N-linked glycosylation sequence and QS removed from the CD11 FHRR (CD11_145T8-QS) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVSKV LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI[SEQ ID NO: 76] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the RSV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0234] RSV fusion protein 66E-T 103 -GS-G 145 -E 511 -GSG- A VISNA-based SSM with an additional N-linked glycosylation sequence, QS removed from the CD11 FHRR, and CT1 mutation (CD11_145T8-QS) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVSKV LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEGSG QSLANATAAQQEVLEAQYAMVRHIAKGIRILEARVAR GGSGG NHTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI[SEQ ID NO: 77] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the RSV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0235] RSV fusion protein 66E-T 103 -GS-G 145 -E 511 -GSG- A VISNA-based SSM with an additional N-linked glycosylation sequence, QS removed from the CD11 FHRR, and CT5 mutation (CD11_145T8-QS) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVSKV LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEGSG LANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI[SEQ ID NO: 78] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the RSV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0236] RSV fusion protein 66E-T 103 -GS-G 145 -E 511 -GSG- A VISNA-based SSM with an additional N-linked glycosylation sequence, QS removed from the CD11 FHRR, and CT9 mutation (CD11_145T8-QS) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVSKV LHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSN NVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKI MTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI[SEQ ID NO: 79] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the RSV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0237] E. coli EhaG-VISNA-based SSM CD11 QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 218] or a corresponding amino acid sequence thereof (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). where: Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0238] E. coli EibD-VISNA-based SSM CD11 MQNGTYSVLQDDSQKSGPVKYGSTYEVVKTVDNGNFRYEVKEKKNDKRTLFKFDSEGNVTVKGKGITHTLHDPALKDFARTAEGKKNEQNGNTPPHKLTDSAVRGVYNK VYGLEKTEITGFSVEDGENGKVSLGSDAKASGEFSVAVGNGARATEKASTAVGSWAAADGKQSTALGVGTYAYANASTALGSVAFVDNTATYGTAAGNRAKVDKDATEG TALGAKATVTNKNSVALGANSVTTRDNEVYIGYKTGTESDKTYGTRVLGGLSDGTRNSDAATVGQLNRKVGGVYDDVKARITVESEKQKKYTDQKTSEVNEKVEARTTV GVDSDGKLTRAEGATKTIAVNDGLVALSGRTDRIDYAVGAIDGRVTRNTQSIEKNSKAIAANTRTLQQHSARLDSQQRQINENHKEMKRAAAQSAALTGLFQPYSVGGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 219] or a corresponding amino acid sequence thereof (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). where: Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0239] E. coli UpaG-VISNA-based SSM CD11 QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 220] or a corresponding amino acid sequence thereof (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). where: Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0240] H. influenzae HiA-VISNA-based SSM CD11 MNNNTPVTNKLKAYGDANFNFTNNSIADAEKQVQEAYKGLLNLNEKNASDKLLVEDNTAATVGNLRKLGWVLSSKNGTRNEKSQQVKHADEVLFEGKGGVQVTSTSENGKHTITFALAKDLGVKTATVSDTLTIGGGAAAGATTTPKVNVTSTTDGLKFAKDAAGANGDTTVHLNGIGSTLTDTLVGSPATHIDGGDQSTHYTRAASIKDVLNAGWNIKGVKAGSTTGQSENVDFVHTYDTVEFLSADTETTTVTVDSKENGKRTEVKIGAKTSVIKEKDGKLFTGKANKETNKVDGANATEDADEGKGLVTAKDVDIVADVNKTGWRIKTTDANGQNGDFATVASGTNVTFASGNGTTATVTNGTDGITVKYDAKVGDGLKLDGDKIAADTTALTVNDGKNANNPKGKVADVASTDEKKLVTAKGLVTALNSLSWTTTAAEADGGTLDNASEQEVKAGDKVTFKAGKNLKVKQEGANFTYSLQDALTGLTSITLGTGNN GAKTEINKDGLTITPANGAGANNANTISVTKDGISAGGQSVKNVVSGLKKFGDANFDPLTSSADNLTKQNDDAYKGLTNLDEKGTDKQTPVVADNTAATVGDLRGLGWVISADKTGGSTEYHDQVRNANEVKFKSGNGINVSGKTVNGRREITFELAKGEVVKSNEFTVKETNGKETSLVKVGDKYYSKEDIDLTTGQPKLKDGNTVAAKYQDKGGKVVSVTDNTEATITNKGSGYVTGNQVADAIAKSGFELGLADEADAKAAFDDKTKALSAGTTEIVNAHDKVRFANGLNTKVSAATVESTDANGDKVTTTFVKTDVELPLTQIYNTDANGKKITKVVKDGQTKWYELNADGTADMTKEVTLGNVDSDGKKVVKDNDGKWYHAKADGTADKTKGEVSNDKVSTDEKHVVSLDPNDQSKGKGVVIDNVANGDISATSTDAINGSQLYAVAKGVTNLAGQVNNLEGKVNKVGKRADAGTASALAASQLPQATMPGGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 221] or a corresponding amino acid sequence thereof (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). where: Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0241] N. meningitidis NadA-VISNA-based SSM CD11 MATNDDDVKKAATVAIAAAYNNGQEINGFKAGETIYDIDEDGTITKKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDATTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDIAD SLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKDNIAKKANSADVYTREESDSKFVRIDGLNATTEKLDTRLASAEKSIADHDTRLNGLDKTVSDLRKETRQGLAEQAALSGLFQPYNVGGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 222] or a corresponding amino acid sequence thereof (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). where: Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0242] N. meningitidis NhhA-VISNA-based SSM CD11 MNNEEQEEDLYLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQNGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTN GDTTVHLNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKG ENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMD ETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 223] or a corresponding amino acid sequence thereof (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). where: Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0243] Y. enterocolitica YadA-VISNA-based SSM CD11 MDDYDGIPNLTAVQISPNADPALGLEYPVRPPVPGAGGLNASAKGIHSIAIGATAEAAKGAAVAVGAGSIATGVNSVAIGPLSKALGDSAVTYGAASTAQKDGVAIGARASTSDTGVAVGFNSKADAKNSVAIGHSSHVAANHGYSIAIGDRSKTDRENSVSIGHESLNRQLTHLAAGTKDTDAVNVAQL KKEIEKTQENTNKRSAELLANANAYADNKSSSVLGIANNYTDSKSAETLENARKEAFAQSKDVLNMAKAHSNSVARTTLETAEEHANSVARTTLETAEEHANKKSAEALASANVYADSKSSHTLKTANSYTDVTVSNSTKKAIRESNQYTDHKFRQLDNRLDKLDTRVDKGLASSAALNSLFQPYGVGGSG QSLANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 224] or a corresponding amino acid sequence thereof (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). where: Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0244] hMPV fusion protein clamp 2s MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTA GVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQI KLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCK VSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEQFNVALDQVFESIENSQALVDQSNRIGSG LANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI [SEQ ID NO: 260] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the hMPV fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0245] HTLV-1 glycoprotein clamp 2s MGWSCIILFLVATATGVHSESRCTLTIGVSSYHSKPCNPAQPVCSWTLDLLALSADQALQPPCPNLVSYSNYHATYSLYLFPHWIKCPNRNGGGYYSASYSDPCSLKCPYLGCQSWTCPYTGAVSSPYWKFQQDVNFTQEVSRLNINLHFSKCGFPFSLLVDAPGYDPIWLLNTEPSQLPPTAPPLLPHSNLDHILEPSIPWKSKLL TLVQLTLQSTNYTCIVCIDRASLSTWHVLYSPNISIPSSSSTPLLYPSLALPAPHLTLPFNWTHCFDPQIQAIVSSPCHNSLILPPFSLSPVPTLRSRSRRGGGGVSALAMGTGIAGGITGSMSLASGKNLLHEVDKDISQLTQAIVKNHKNLLKIAQYAAQNRRGLDLLFWEQGGLCKALQEQCCFLNITNSHVSILQERPPLEGSG LANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI [SEQ ID NO: 261] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the HTLV-1 glycoprotein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0246] hPIV3 fusion protein clamp 2s MPTSILLIITTMIMASFCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVTNQESNENTDPRTERFFGGVIGTIAL GVATSAQITAAVALVEAKQAKSDIEKLKEAIRDTNKAVQSVQSSVGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITE IFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQ CPRTTVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKGSG LANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTTWQQWEEEIENHTGNLTLLLREAANQTHIAQRDARRI [SEQ ID NO: 262] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the signal peptide of the hPIV3 fusion protein. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0247] Chlamydia trachomatis major outer membrane protein clamp 2 MKKLLKSVLVFAALSSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPCTTWCDAISMRVGYYGDFVFDRVLKTDVNKEFQMGAAPTTSDVAGLQNDPTINVARPNPAYGKHMQDAEMFTNAAYMALNIWDRFDVFCTLGATTGYLKGNSASFNLVGLFGTKTQSSSFNTAKLIPNTALNEAVVELYINTTFAWSVGA RAALWECGCATLGASFQYAQSKPKVEELNVLCNASEFTINKPKGYVGAEFPLNITAGTEAATGTKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRVSFDADTIRIAQPKLAEAILDVTTLNRTTAGKGSVVSAGTDNELADTMQIVSLQLNKMKSRKSCGIAVGTTIVDADKYAVTVEARLIDERAAHVNAQFRFGSG LANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRI [SEQ ID NO: 263] or a corresponding amino acid sequence thereof (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). where: The letters in italics correspond to the leader sequence. Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0248] Chlamydia trachomatis PGP3 clamp 2 MANATAAQQEVLEAQYAMVQHIAKGIRILEARVAR GGSGG NHTWQQWEEEIEQHEGNLSLLLREAALQVHIAQRDARRIGGSGGNSGFYLYNTENCVFADNIKVGQMTEPLKDQQIILGTTSTPVAAKMTASDGISLTVSNNSSTNASITIGLDAEKAYQLILEKLGNQILDGIADTIVDSTVQDILDKITTDPSLGLLKAFNNFPITNKIQCNGLFTPSNIETLLGGTEIGKFTVTPKSSGSMFLVSADIIASRMEGSVVLALVREGDSKPCAISYGYSSGVPNLCSLRTS ITNTGLTPTTYSLRVGGLESGVVWVNALSNGNDILGITNTSNVSFLEVIPQTNA [SEQ ID NO: 264] or a corresponding amino acid sequence thereof (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). where: Letters in bold are flexible linkers, · The underlined letters correspond to VISNA-based SSMs.

[0249] 2.4 Use of structure-stabilizing moieties as universal oligomerization domains In addition to their utility in stabilizing the ecto-domain polypeptides of the present invention against rearrangement into a post-fusion conformation, the structurally stabilizing moiety is useful as a universal oligomerization domain (UOD) for oligomerizing any heterologous molecule of interest into oligomers, particularly trimers. In certain embodiments, the UOD is fused upstream or downstream of a heterologous proteinaceous molecule (referred to herein as a "first (poly)peptide") to form a chimeric polypeptide. Typically, the UOD is fused downstream of the heterologous proteinaceous molecule. As in the ecto-domain embodiments described herein, the complementary heptad repeats of the UOD associate with each other under conditions favorable for association (e.g., in aqueous solution), resulting in the formation of an antiparallel two-helix bundle, which trimerizes to form a highly stable six-helix bundle, thereby enabling trimerization of the chimeric polypeptide to form a trimeric polypeptide complex.

[0250] Accordingly, in a second aspect, the present invention provides a chimeric polypeptide comprising a first (poly)peptide operably connected downstream to a structure-stabilising moiety, said structure-stabilising moiety being as defined in relation to the first aspect of the invention, and preferably wherein the first polypeptide is a therapeutic polypeptide.

[0251] The heterologous proteinaceous molecule (i.e., the "first (poly)peptide") may be a naturally occurring or non-naturally occurring polypeptide. In certain embodiments, the heterologous (poly)peptide is or comprises a therapeutic (poly)peptide. A wide variety of therapeutic (poly)peptides, including both ligands and receptors, are known in the art to be useful for treating or preventing various diseases.

[0252] 2.5 Methods for preparing chimeric polypeptides and their conjugates The chimeric polypeptides of the present disclosure can be prepared by chemical synthesis or recombinant means. Typically, the polypeptides are prepared by expression of a recombinant construct encoding the 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). ni), mammalian cells (e.g., human, non-human primate, horse, cow, sheep, dog, cat, and rodent (e.g., hamster), avian cells (e.g., chicken, duck, and goose), bacteria (e.g., Escherichia coli, Bacillus subtilis, and Streptococcus species), yeast cells (e.g., Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorphs, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii), Suitable insect and mammalian cell culture media include, for example, S. guillerimondii, Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica, Tetrahymena cells (e.g., Tetrahymena thermophila), or combinations thereof. Many suitable insect and mammalian cells are 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 nii 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 Accession No. 96022940), Hep G2 cells, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), rhesus 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, DSM ACC 2219), baby hamster kidney (BHK) cells, e.g., BHK21-F, HKCC cells, and the like. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx® cells), chicken fetal fibroblasts, chicken embryonic germ cells, duck cells (e.g., AGE1.CR and AGE1.CR.pIX cell lines (ProBioGen), which are described, for example, in Vaccine 27:4975-4982 (2009) and International Publication No. WO2005 / 042728), EB66 cells, and the like.

[0253] Suitable insect cell expression systems, such as baculovirus systems, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kits from, inter alia, Invitrogen, San Diego, Calif. Avian cell expression systems are also known to those skilled in the art and are described, for example, in 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. EP 03291813.8, International Publication No. WO 03 / 043415, and International Publication No. WO 03 / 076601. Similarly, bacterial 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.

[0254] Recombinant constructs encoding the modified or chimeric polypeptides of the present disclosure can be prepared in a suitable vector using conventional methods. Several vectors suitable for expressing recombinant proteins in insect or mammalian cells are well known and conventional in the art. Suitable vectors may contain several 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, as well as a signal or leader sequence (e.g., of mammalian origin or from a heterologous mammalian or non-mammalian species) for targeting to the secretory pathway in a selected host cell. For example, for expression in insect cells, a suitable baculovirus expression vector, 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 the recombinant protein. For expression in mammalian cells, a vector is used that will drive expression of the construct in the desired mammalian host cell (eg, Chinese hamster ovary cell).

[0255] The modified or chimeric polypeptide can be purified using any applicable method. Suitable methods for purifying desired proteins are well known in the art, including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating, and size exclusion. An appropriate purification scheme can be created using two or more of these or other suitable methods. If desired, the modified or chimeric polypeptide can include a purification moiety or "tag" that facilitates purification, for example, as described above. Such tagged polypeptides can be conventionally purified from conditioned medium, for example, by chelating or affinity chromatography.

[0256] A modified or chimeric polypeptide may contain additional sequences, for example, for expression purposes, the native leader peptide of a heterologous polypeptide of interest (e.g., the native leader peptide of a bacterial or viral surface polypeptide, such as the leader peptide of an enveloped viral fusion protein or bacterial outer membrane polypeptide) may be replaced with a different one.

[0257] In a fifth aspect, the present invention provides a method for producing a chimeric polypeptide complex, the method comprising the step of combining chimeric polypeptides of the present invention under conditions suitable for the formation of 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.

[0258] 3. Polynucleotides and Nucleic Acid Constructs for Endogenous or Heterologous Production of Chimeric Polypeptides The present disclosure also contemplates polynucleotides and nucleic acid constructs for endogenous or heterologous (i.e., recombinant) production of the chimeric polypeptides in a host organism, suitably a vertebrate, preferably a mammal, such as a human.

[0259] 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.

[0260] In a preferred embodiment, the nucleic acid further comprises a promoter operably linked to the polynucleotide sequence encoding the chimeric polypeptide, the promoter preferably being a mammalian promoter. Those 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.

[0261] Generally, polynucleotides contemplated herein comprise coding sequences for the chimeric polypeptides of the present disclosure. These polynucleotides are useful for generating nucleic acid constructs from which the chimeric polypeptide coding sequences can be expressed to immunize a subject. In some embodiments, these polynucleotides are themselves useful for directly immunizing a subject. In representative embodiments of this type, the polynucleotide comprises 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 messenger RNA (mRNA) having an open reading frame encoding a polypeptide disclosed herein.

[0262] 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 the polypeptides disclosed herein. In some embodiments, codon optimization can be used to match the codon usage in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base counts that can damage gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in encoded proteins; add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translation rates to allow various domains of proteins to fold properly; 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, the open reading frame (ORF) sequence is optimized using an optimization algorithm. In some embodiments, the codon-optimized RNA can have, for example, an enhanced level of G / C. The G / C content of a nucleic acid molecule can affect the stability of the RNA. RNA with increased amounts of guanine (G) and / or cytosine (C) residues can be more functionally stable than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. International Publication No. WO 02 / 098443 discloses pharmaceutical compositions comprising mRNA stabilized by sequence modifications in the translated region.Due to the degeneracy of the genetic code, the modification works by replacing existing codons with ones that promote greater RNA stability without changing the resulting amino acid. This approach is limited to the coding regions of RNA.

[0263] In some embodiments, the RNA polynucleotides of the present disclosure may further comprise additional sequences, such as sequences containing or encoding 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 that may or may not incorporate unnatural bases (or unnatural nucleotides) to reduce triggering of the innate immune response, and a 3' UTR element, a poly(A) sequence, and / or a polyadenylation signal, wherein the RNA is modified or unmodified.

[0264] RNA polynucleotides can be transcribed in vitro from a template DNA referred to as an "in vitro transcription template." In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, encodes a 3' UTR, and encodes 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.

[0265] "5' untranslated region" (UTR) refers to the region of an mRNA immediately upstream (i.e., 5') of the start codon (i.e., the first codon of an mRNA transcript that is translated by ribosomes) that does not encode a polypeptide.

[0266] "3' untranslated region" (UTR) refers to the region of an mRNA immediately downstream (i.e., 3') of the stop codon (i.e., the codon in the mRNA transcript that signals the end of translation) that does not encode a polypeptide.

[0267] An "open reading frame" is a stretch of contiguous codons that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA) that encodes a polypeptide.

[0268] A "poly-A tail" is a region of an mRNA that contains multiple consecutive adenosine monophosphates and is downstream, e.g., immediately downstream (i.e., 3'), from the 3' UTR. A poly-A tail can contain 10 to 300 adenosine monophosphates. For example, a poly-A 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, a poly-A tail contains 50 to 250 adenosine monophosphates. In relevant biological contexts (e.g., intracellular, in vivo), poly(A) tails function, for example, to protect mRNA from enzymatic degradation in the cytoplasm, and assist in transcription termination, nuclear export of mRNA, and translation.

[0269] In some embodiments, RNA polynucleotides are formulated in lipid nanoparticles (LNPs). 5'-capping of polynucleotides can be completed simultaneously during in vitro transcription reactions by generating a 5'-guanosine cap structure using the following chemical RNA cap analogs according to the manufacturer's protocol: 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.). 5'-capping of modified RNAs can be completed post-transcriptionally by generating a "Cap 0" structure using vaccinia virus capping enzyme: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). The Cap 1 structure can be generated by using both vaccinia virus capping enzyme and a 2'-O-methyltransferase to generate m7G(5')ppp(5')G-2'-O-methyl. The Cap 2 structure can be generated by Cap 1 construction followed by 2'-O-methylation of the third-to-last 5' nucleotide using a 2'-O-methyltransferase. The Cap 3 structure can be generated by Cap 2 construction followed by 2'-O-methylation of the fourth-to-last 5' nucleotide using a 2'-O-methyltransferase. The enzymes can be derived from recombinant sources.

[0270] The present disclosure also contemplates nucleic acid constructs for endogenous production of the polypeptides disclosed herein. The nucleic acid constructs can be self-replicating extrachromosomal vectors / replicons (e.g., plasmids) or vectors that integrate into the host genome. In certain embodiments, the nucleic acid constructs are viral vectors. Exemplary viral vectors include retroviral vectors, lentiviral vectors, poxvirus vectors, vaccinia virus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, flavivirus vectors, and alphavirus vectors. Viral vectors can be live, attenuated, replication-conditional, or replication-defective, and are typically non-pathogenic (defective), replication-competent viral vectors.

[0271] 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 in 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), and Weir et al. (1983, J. Virol. 46:530). Suitable promoters for use with 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 are more tolerated for use in humans, including Lister, NYVAC, which contains specific genomic deletions (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), who describe the use of Yaba-like disease virus as a vector for cancer therapy, and 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.

[0272] In certain embodiments, adenoviral vectors can be used to express the chimeric polypeptide of interest. Adenoviruses that can serve as the basis for viral transfer vectors can be from any origin, any subgroup, any subtype, a mixture of subtypes, or any serotype. For example, the adenovirus may 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), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenovirus serotype. Adenovirus serotypes 1 through 51 are available from the American Type Culture Collection (ATCC, Manassas, Va.). Non-group C adenovirus, and even non-human adenovirus, can be used to prepare replication-defective adenovirus vector.Non-group C adenovirus vector, the method of producing non-group C adenovirus vector, and the method of using non-group C adenovirus vector are disclosed in, for example, United States Patent No. 5,801,030, United States Patent No. 5,837,511 and United States Patent No. 5,849,561, and International Patent Application No. WO 97 / 12986 and International Patent Application No. WO 98 / 53087.Any adenovirus, and even chimeric adenovirus, can be used as the source of the viral genome of adenovirus vector.For example, human adenovirus can be used as the source of the viral genome of replication-defective adenovirus vector.Further examples of adenoviral vectors are described 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, 20090088398, and U.S. Patent Nos. 6,143,290, 6,596,535, 6,855,317, 6,936,257, 7,125,717, 7,378,087, and 7,550,296.

[0273] Viral vectors can also be based on adeno-associated viruses (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. The AAV vector may also be a self-complementary (sc) AAV vector, which is described, for example, in U.S. Patent Publication Nos. 2007 / 01110724 and 2004 / 0029106, and U.S. Patent Nos. 7,465,583 and 7,186,699.

[0274] Herpes simplex virus (HSV)-based viral vectors are also suitable for the endogenous production of the chimeric polypeptides of the present disclosure. Many replication-deficient HSV vectors contain deletions that remove one or more intermediate-early genes to prevent replication. The advantage of herpes vectors is their ability to enter latency, which can result in long-term DNA expression, and their large viral DNA genome, which can accommodate up to 25 kb of exogenous DNA. For descriptions of HSV-based vectors, see, for example, U.S. Patent Nos. 5,837,532, 5,846,782, 5,849,572, and 5,804,413, as well as International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583.

[0275] 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 (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).

[0276] In certain embodiments, the retroviral vector is a lentiviral vector. As will be understood by those skilled in the art, a viral vector, e.g., a lentiviral vector, generally refers to a viral vector particle containing a viral vector genome. For example, a lentiviral vector particle may contain a lentiviral vector genome. With respect to lentiviral vectors, the vector genome may be derived from any of a number of suitable available lentiviral genome-based vectors, including those specific for human gene therapy applications (see, e.g., 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 Maedi-Visna virus. A desirable feature of lentiviruses is their ability to infect both dividing and non-dividing cells, although target cells do not necessarily need to be dividing or stimulated to divide. Generally, the genome and envelope glycoproteins will be based on different viruses so that the resulting viral vector particles are pseudotyped. It is desirable to incorporate safety features of the viral vector. Safety features include self-inactivating LTRs and integration defects, as described in more detail herein. In certain embodiments, the integration defect may be conferred by an element of the vector genome, but may also be derived from an element of the packaging system (e.g., a non-functional integrase protein that may not be part of the vector genome but may be supplied in trans). An exemplary vector includes a packaging signal (psi), a Rev-responsive element (RRE), a splice donor, a splice acceptor, optionally a central polypurine tract (cPPT), and a WPRE element.In certain exemplary embodiments, the viral vector genome comprises sequences derived from a lentivirus genome, such as an HIV-1 genome or an SIV genome. The viral genome construct may comprise sequences derived from the 5' and 3' LTRs of a lentivirus, particularly the R and U5 sequences derived from the 5' LTR of a lentivirus, and an inactivated or self-inactivating 3' LTR derived from a lentivirus. The LTR sequences may be LTR sequences 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.

[0277] The vector genome may contain an inactivated or self-inactivating 3' LTR (see, for example, Zufferey et al., 1998. J. Virol. 72:9873; Miyoshi et al., 1998. J. Virol. 72:8150). Self-inactivating vectors generally have a deletion of enhancer and promoter sequences from the 3' long terminal repeat (LTR), which are copied to the 5' LTR upon vector integration. In one example, the U3 element of the 3' LTR contains a deletion of its enhancer sequence, TATA box, Spl, and NF-kappa B site. As a result of the self-inactivating 3' LTR, the provirus generated after entry and reverse transcription contains an inactivated 5' LTR. The rationale is to improve safety by reducing the risk of vector genome migration and the effect of the LTR on nearby cellular promoters. Self-inactivating 3' LTRs can be constructed by any method known in the art.

[0278] Optionally, the U3 sequence from lentivirus 5' LTR can be replaced with a promoter sequence, for example, a heterologous promoter sequence, in the viral construct. This can increase the titer of the virus recovered from the packaging cell line. An enhancer sequence can also be included. Enhancer / promoter combinations that increase the expression of viral RNA genome in the packaging cell line can be used. In one example, a CMV enhancer / promoter sequence is used (see, for example, U.S. Patent Nos. 5,385,839 and 5,168,062).

[0279] In certain embodiments, the risk of insertional mutagenesis is minimized by constructing lentiviral vectors to be integration-deficient. Various approaches can be pursued to produce non-integrating vector genomes. These approaches involve engineering mutations 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 to prevent integration, for example, by mutating or deleting one or both binding sites or by rendering the 3' LTR-proximal polypurine tract (PPT) nonfunctional through deletion or modification. In addition, non-genetic approaches are available, including pharmacological agents that inhibit one or more functions of integrase. These approaches are not mutually exclusive; more than one of them can be used at a time. For example, both the integrase and binding sites can be nonfunctional, or the integrase and PPT sites can be nonfunctional, or the binding site and PPT site can be nonfunctional, or all of them can be nonfunctional. Exemplary lentiviral vectors are described, for example, in U.S. Publication Nos. 20150224209, 20150203870, 20140335607, 20140248306, 20090148936, and 20080254008.

[0280] Viral vectors may also be based on alphaviruses. Alphaviruses include Sindbis virus (and Venezuelan equine encephalitis virus (VEEV)), Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Kabaso virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Nudum virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus (SFV), Southern elephant seal virus, and Tonate virus. Examples of alphavirus vectors include Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Wataroa virus. Generally, the genomes of such viruses encode nonstructural proteins (e.g., replicons) that can be translated in the cytoplasm of host cells, as well as 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 transgene delivery. Pseudotyped viruses can be formed by combining alphavirus envelope glycoproteins and retroviral capsids. Examples of alphavirus vectors can be found in U.S. Publication Nos. 20150050243, 20090305344, and 20060177819.

[0281] Alternatively, the viral vector can be based on a flavivirus, including Japanese encephalitis virus, dengue virus (e.g., dengue-1, dengue-2, dengue-3, dengue-4), yellow fever virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, West Nile virus, Kunjin virus, Rocio encephalitis virus, ileus 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, louping ill virus, Powassan virus, Negishi virus, Absetalob virus, Hansalova virus, Apoivirus, and Hypr virus. Examples of flavivirus vectors are described in U.S. Publication Nos. 20150231226, 20150024003, 20140271708, 20140044684, 20130243812, 20120294889, 20120128713, and 2011013568. 6, 20110014229, 20110003884, 20100297167, 20100184832, 20060159704, 20060088937, 20030194801, and 20030044773.

[0282] 4.Host cells The present invention also contemplates, in a fourth aspect, a host cell comprising nucleic acid as defined according to the third aspect of the invention.

[0283] In some embodiments, the host cell is (i) a prokaryotic host cell, or (ii) a eukaryotic host cell. Exemplary eukaryotic host cells include, 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 cells (e.g., cell line HEK-293), cell lines derived from Chinese hamster ovary (CHO) cells, such as the ExpiCHO (Thermo Fisher) cell line used in the examples disclosed herein. Preferably, the host cell has glycosylation machinery (endogenous or engineered) that allows glycosylation (preferably N-glycosylation) of the expressed polypeptide with glycans commonly expressed in the particular mammalian subject species (e.g., Homo sapiens) to which the thus-expressed polypeptide (i.e., a chimeric polypeptide or conjugate thereof contemplated herein) is intended to be administered.

[0284] In a preferred embodiment, the host cell is a cell (preferably a mammalian cell) stably transfected with a nucleic acid contemplated herein. In the case of stably transfected cells, the expression system is integrated into the genome of the target cell and remains there in a stable manner. In contrast to transient transfection, the transferred gene is not only not degraded but also doubles with each cell division and is passed on to daughter cells. The latter therefore retain the ability to prepare the desired protein for a long period of time. Processes for transfecting, particularly for preparing stably transfected cells, are known in the art. Host cells can be transformed, for example, using electroporation, which allows the uptake of nucleic acids into cells due to the short-term application of an electric field by permeabilization of the cell membrane, or by transfection or infection with a viral vector, as also described herein. In addition to the transient expression of recombinant proteins, the expression system used can also enable clonal selection of transfected host cells, thereby allowing the selection of clonal cell lines with suitable expression efficiency.

[0285] Exemplary prokaryotic host cells include, but are not limited to, Escherichia coli, Shigella, Klebsiella, Xanthomonas, Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus, and Clostridium. A particularly preferred prokaryotic host cell is Escherichia coli.

[0286] 5. Chimeric Polypeptide Conjugates and Methods for Their Production In a fifth aspect, the present invention provides a method for producing a chimeric polypeptide complex, the method comprising combining chimeric polypeptides as defined according to the first or second aspect of the invention under conditions suitable for the formation of a chimeric polypeptide complex, whereby a chimeric polypeptide complex is produced 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.

[0287] Preferably, the six-helix bundle consists of an inner trimer of three parallel-oriented substantially α-helical FHRRs packed with three substantially α-helical SHRRs in an antiparallel orientation relative to the FHRRs.

[0288] In a sixth aspect, the present invention provides a chimeric polypeptide complex comprising three chimeric polypeptide subunits, each subunit being a chimeric polypeptide as defined according to the first or second aspect of the invention, wherein the complex is characterized by a six-helix bundle formed by homotrimerization of the structure-stabilizing portions of the three chimeric polypeptides.

[0289] In some embodiments, the six-helix bundle consists of an inner trimer of three parallel-oriented substantially α-helical FHRRs packed with three substantially α-helical SHRRs in an antiparallel orientation relative to the FHRRs.

[0290] Although it is generally preferred that the chimeric polypeptide complex so produced comprises three identical chimeric polypeptides, complexes in which the individual chimeric polypeptides differ from one another with respect to any one or more of the components included in the chimeric polypeptides defined herein (e.g., structural stabilizing moieties (SSMs) and / or microbial polypeptides (e.g., enveloped viral fusion ectodomain polypeptides or bacterial outer membrane polypeptides)) are also envisioned and are expressly contemplated herein.

[0291] In some embodiments, the chimeric polypeptide subunits each comprise an enveloped virus fusion ectodomain polypeptide, and the complex comprises at least one pre-fusion epitope of an enveloped virus fusion protein.

[0292] 6. Screening Method The present invention also encompasses methods of screening for agents that bind, preferably specifically, to microbial polypeptides (preferably, fusion proteins of enveloped viruses or bacterial outer membrane polypeptides (e.g., bacterial TAA polypeptides) and / or their respective complexes (e.g., complexes of fusion proteins or TAA polypeptides). In certain embodiments, compound libraries are screened for binding to chimeric polypeptides comprising microbial polypeptides (preferably, chimeric polypeptides comprising enveloped virus fusion ectodomain polypeptides or chimeric polypeptides comprising bacterial outer membrane polypeptides), or complexes thereof.

[0293] Thus, in an eighth aspect, the present invention provides a method for identifying an agent that binds to a microbial polypeptide or a complex thereof, comprising the steps of: (i) contacting a candidate agent with a chimeric polypeptide comprising a microbial polypeptide as defined according to the first aspect of the invention or a chimeric polypeptide complex comprising a microbial polypeptide as defined according to the sixth aspect of the invention; (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex; Including, Preferably, methods are provided wherein the candidate agent is part of a compound library (eg, a small molecule or macromolecule library).

[0294] In a preferred embodiment, the method further comprises: (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; and / or (ii) isolating the candidate drug Includes:

[0295] In a preferred embodiment of the latter aspect, (a) the microbial polypeptide or complex thereof is a fusion protein or complex of fusion proteins, respectively, of an enveloped virus, and the method comprises: (i) contacting a candidate agent with a chimeric polypeptide comprising an enveloped virus fusion ecto-domain polypeptide as defined according to the first aspect of the present invention, or a chimeric polypeptide complex comprising an enveloped virus fusion ecto-domain polypeptide as defined according to the sixth aspect of the present invention, wherein the enveloped virus fusion ecto-domain polypeptide corresponds to a fusion protein of an enveloped virus; (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex; Including, or (b) the microbial polypeptide or complex thereof is a bacterial outer membrane polypeptide or complex of outer membrane polypeptides, respectively, and the method comprises: (i) contacting a candidate agent with a chimeric polypeptide comprising a bacterial outer membrane polypeptide as defined according to the first aspect of the invention, or a chimeric polypeptide complex comprising a bacterial outer membrane polypeptide as defined according to the sixth aspect of the invention, wherein the bacterial outer membrane polypeptide corresponds to a bacterial outer membrane polypeptide; (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex; Includes:

[0296] In a preferred embodiment of the latter embodiment, the method comprises: (i)(ia) contacting a candidate agent according to item (a) with the fusion protein or fusion protein complex and detecting binding of the candidate agent to the fusion protein or fusion protein complex; or (ib) contacting a candidate agent according to item (b) with an outer membrane polypeptide or a complex of the outer membrane polypeptide and detecting binding of the candidate agent to the outer membrane polypeptide or the complex of the outer membrane polypeptide; and / or (ii) isolating the candidate drug Further includes:

[0297] In a preferred embodiment, (a) the bacterial outer membrane polypeptide or complex thereof is a bacterial trimeric autotransporter adhesin (TAA) polypeptide or complex of TAA polypeptides, and the method comprises: (i) contacting a candidate agent with a chimeric polypeptide comprising a TAA polypeptide as defined according to the first aspect of the invention, or a chimeric polypeptide complex comprising a TAA polypeptide as defined according to the sixth aspect of the invention, wherein the TAA polypeptide corresponds to a bacterial TAA polypeptide; (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex; Including, or (b) the bacterial outer membrane polypeptide or complex thereof is a major outer membrane protein (MOMP) polypeptide or complex thereof of Chlamydia bacteria, respectively, and the method comprises: (i) contacting a candidate agent with a chimeric polypeptide comprising a chlamydial MOMP polypeptide as defined according to the first aspect of the invention, or a chimeric polypeptide complex comprising a chlamydial MOMP polypeptide as defined according to the sixth aspect of the invention, wherein the chlamydial MOMP polypeptide corresponds to a MOMP polypeptide of a chlamydial bacterium; (ii) detecting binding of the candidate agent to the chimeric polypeptide or chimeric polypeptide complex; Includes:

[0298] In a preferred embodiment of item (a) of the latter embodiment, the method comprises: (i)(ia) contacting a candidate agent with a TAA polypeptide or a complex of TAA polypeptides and detecting binding of the candidate agent to the TAA polypeptide or a complex of TAA polypeptides; or (ib) contacting the candidate agent with a MOMP polypeptide or a complex of a MOMP polypeptide; and / or (ii) isolating the candidate drug Further includes:

[0299] In a preferred embodiment of the latter embodiment, the candidate agent specifically binds to the chimeric polypeptide or chimeric polypeptide complex. In other or even more preferred embodiments, the candidate agents specifically bind to a microbial polypeptide or complex thereof.

[0300] In an even more preferred embodiment, the candidate agent is: (i) an enveloped virus fusion protein or a complex of fusion proteins; or (ii) an outer membrane polypeptide or a complex of outer membrane polypeptides It specifically binds to

[0301] In an even more preferred embodiment of item (ii) of the latter embodiment, the candidate agent is (i) a TAA polypeptide or a complex of TAA polypeptides, or (ii) MOMP polypeptide or MOMP polypeptide complex It specifically binds to

[0302] Candidate agents encompass numerous chemical classes, including small molecules, e.g., small organic compounds, and macromolecules, e.g., peptides, polypeptides, and polysaccharides. Candidate agents contain functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, typically including at least an amine, carbonyl, hydroxyl, or carboxyl group, and desirably, at least two of the functional chemical groups. Candidate compounds may contain cyclic carbon or heterocyclic structures, or aromatic or polyaromatic structures, substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including, but not limited to, peptides, polypeptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Compound libraries may include naturally occurring compounds in the form of bacterial, fungal, plant, and animal extracts. Alternatively, or in addition, compound libraries may include naturally occurring or synthetically produced compounds.

[0303] In particularly preferred embodiments also contemplated herein, the candidate agent is a peptide, antibody, antibody fragment (e.g., single-chain variable fragment (scFv)), or any alternative binding protein scaffold, expressed as part of a combinatorial library used in high-throughput (HT) combinatorial library-based display and selection methods, 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, e.g., a bacterial outer membrane polypeptide (e.g., a TAA polypeptide)) or complex thereof can be routinely selected by using such known display and selection approaches with a chimeric polypeptide or complex thereof comprising the microbial polypeptide as the respective antigen against which selection is performed (e.g., a chimeric polypeptide or complex thereof comprising an enveloped virus fusion ectodomain polypeptide, or a chimeric polypeptide or complex thereof comprising a bacterial surface polypeptide, e.g., a chimeric polypeptide or complex thereof comprising a bacterial outer membrane polypeptide (e.g., a chimeric polypeptide or complex thereof comprising a TAA polypeptide)).

[0304] Methods for determining whether a drug binds to a target protein and / or for determining the affinity of a drug for a target protein are known in the art. For example, the binding of a drug to a target protein can be detected and / or quantified using various techniques, such as, but not limited to, biolayer interferometry (BLI), Western blot, dot blot, surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), AlphaScreen® or AlphaLISA® assay, or mass spectrometry-based methods.

[0305] In some embodiments, an agent can be assayed using any surface plasmon resonance (SPR)-based assay known in the art to characterize the kinetic parameters of the interaction 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 ectodomain polypeptide or a complex thereof, or a chimeric polypeptide comprising a bacterial surface polypeptide, e.g., a chimeric polypeptide comprising a bacterial outer membrane polypeptide (e.g., a chimeric polypeptide comprising a TAA polypeptide)). Any SPR instrument can be used in the methods described herein, including, but not limited to, a BIAcore instrument (Biacore AB, Uppsala, Sweden), an IAsys instrument (Affinity Sensors, Franklin, Mass.), an IBIS system (Windsor Scientific Limited, Berks, UK), an SPR-CELLIA system (Nippon Laser and Electronics Lab, Hokkaido, Japan), and an SPR Detector Spreeta (Texas Instruments, Dallas, Tex.). See, for example, 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.

[0306] In some embodiments, biomolecular interactions between a drug and a chimeric polypeptide comprising a microbial polypeptide or a complex thereof (e.g., a chimeric polypeptide comprising an enveloped virus fusion ecto-domain polypeptide or a complex thereof, or a chimeric polypeptide comprising a bacterial surface polypeptide, e.g., 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 assayed using BLI on an Octet (ForteBio Inc.). BLI is a label-free optical analysis technique that senses binding between a ligand (e.g., a chimeric polypeptide comprising an ecto-domain polypeptide of the present invention or a complex thereof) immobilized on a biosensor chip in solution and an analyte (e.g., a test compound) by measuring changes in the thickness of the protein layer on the biosensor chip in real time.

[0307] In some embodiments, the AlphaScreen (PerkinElmer) assay can be used to characterize the binding of a test agent to a chimeric polypeptide or complex thereof comprising a microbial polypeptide (e.g., a chimeric polypeptide or complex thereof comprising an enveloped virus fusion ectodomain polypeptide, or a chimeric polypeptide or complex thereof comprising a bacterial surface polypeptide, e.g., a chimeric polypeptide or complex thereof comprising a bacterial outer membrane polypeptide (e.g., a chimeric polypeptide or complex thereof comprising a TAA polypeptide)). The acronym ALPHA stands for Amplified Luminescent Proximity Homogeneous Assay. AlphaScreen is a bead-based proximity assay that detects binding between molecules bound to donor and acceptor beads (e.g., between a subject chimeric polypeptide or complex and a test compound) by measuring a signal generated by energy transfer between the donor and acceptor beads. (See, e.g., Eglen et al. (2008) Curr Chem Genomics 1:2-10).

[0308] In some embodiments, the AlphaLISA® (PerkinElmer) assay can be used to characterize the binding of test agents to the chimeric polypeptides or complexes of the present invention. AlphaLISA is modified from the AlphaScreen assay described above to include 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.)

[0309] Various immunoassay techniques can be used, including competitive and non-competitive immunoassays. The term "immunoassay" encompasses techniques including, but not limited to, flow cytometry, FACS, enzyme immunoassays (EIAs), such as enzyme-linked multiplex immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA), as well as capillary electrophoresis immunoassays (CEIA), radioimmunoassays (RIA), immunoradiometric assays (IRMA), fluorescence polarization immunoassays (FPIA), and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used with laser-induced fluorescence. Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. In addition, nephelometric assays, for example, in which the formation of a protein / antibody complex results in an increase in light scattering that is converted into a peak rate signal as a function of marker concentration, are suitable for use in the methods of the present invention.

[0310] In some embodiments, binding of a test agent to a subject chimeric polypeptide or complex can be assayed using thermal denaturation methods, including differential scanning fluorimetry (DSF) and differential static light scattering (DSLS).

[0311] In some embodiments, binding of a test agent to a chimeric polypeptide or complex of the invention can be assayed using mass spectrometry-based methods, such as, but not limited to, affinity selection coupled with mass spectrometry (AS-MS) platforms, which are label-free methods in which the protein and test compound are incubated, unbound molecules are washed away, and the protein-ligand complex is analyzed by MS for ligand identity after a decomplexation step.

[0312] In some embodiments, binding of a test agent to a subject chimeric polypeptide or complex can be quantified by immunoassay or by chromatographic detection, e.g., using a detectably labeled protein, e.g., a radiolabeled (e.g., 32P, 35S, 14C, or 3H), fluorescently labeled (e.g., FITC), or enzyme-labeled chimeric polypeptide or complex or test compound.

[0313] In some embodiments, the present invention contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in measuring the extent of interaction, either direct or indirect, between a chimeric polypeptide or complex and a test compound.

[0314] Any of the above embodiments are suitable for development onto a high throughput platform. Compounds may further be tested in animal models to identify those with the most potent in vivo effects, e.g., those that specifically bind to a microbial polypeptide or complex thereof (e.g., an enveloped viral fusion protein or complex of fusion proteins, or a bacterial surface polypeptide or complex thereof, e.g., a bacterial outer membrane polypeptide or complex thereof (e.g., a chimeric polypeptide or complex thereof comprising a TAA polypeptide), and preferably stimulate or enhance a therapeutically useful effect, e.g., reduction of microbial (e.g., viral or bacterial) load, reduction of infection or symptoms associated therewith. These molecules may serve as "lead compounds" for further pharmaceutical development, e.g., by subjecting the compounds to sequential modification, molecular modeling, and other routine procedures utilized in rational drug design.

[0315] 7. Antigen-...

Claims

1. A chimeric polypeptide comprising a microbial polypeptide operably connected downstream or upstream to a heterogeneous structural stabilization portion (SSM), wherein the structural stabilization portion comprises a polypeptide comprising a first heptad repeat region (FHRR) and a second heptad repeat region (SHRR) in the order from the N-terminus to the C-terminus, (i) The FHRR contains or consists of an amino acid sequence having at least 60% sequence identity with the amino acid sequence described in SEQ ID NO: 80 or 81, and the SHRR contains or consists of an amino acid sequence having at least 40% sequence identity with the amino acid sequence described in SEQ ID NO: 82 or 83, and / or (ii) The FHRR contains or comprises an amino acid sequence having at least 90% sequence similarity to the amino acid sequence described in SEQ ID NO: 80 or 81, and the SHRR contains or comprises an amino acid sequence having at least 70% sequence similarity to the amino acid sequence described in SEQ ID NO: 82 or 83. Chimeric polypeptide.

2. (i) The FHRR contains or consists of an amino acid sequence having at least 60% sequence identity with the amino acid sequence described in SEQ ID NO: 80, and the SHRR contains or consists of an amino acid sequence having at least 40% sequence identity with the amino acid sequence described in SEQ ID NO: 82, and / or (ii) The FHRR comprises or consists of an amino acid sequence having at least 90% sequence similarity to the amino acid sequence described in SEQ ID NO: 80, and the SHRR comprises or consists of an amino acid sequence having at least 70% sequence similarity to the amino acid sequence described in SEQ ID NO:

82. The chimeric polypeptide according to claim 1.

3. (i) The structural stabilizing portion can homotrimerize with the structural stabilizing portions of two further chimeric polypeptides, Preferably, the homotrimerization forms a 6-helix bundle, the 6-helix bundle consisting of three parallel-oriented, substantially α-helix FHRR internal trimers, with three substantially α-helix SHRRs packed in an antiparallel orientation relative to the FHRRs, and / or (ii) The FHRR and SHRR are, respectively, (a-b-c-d-e-f-g-) ｎ or (d-e-f-g-a-b-c-) ｎ It comprises an independently selected seven-residue motif that is repeated n times and characterized by an amino acid pattern represented as, where elements "a" to "g" of the pattern refer to positions where amino acids are located, n is 2 or a number greater than that, 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. The chimeric polypeptide according to claim 1.

4. The chimeric polypeptide according to claim 1, wherein the structural stabilizing portion is located at the C-terminus of the microbial polypeptide.

5. The chimeric polypeptide according to claim 1, wherein the structural stabilizing portion has glutamine at a position corresponding to position 17 of sequence number 80.

6. (a) The structural stabilizing portion includes at least one immune silencing portion that reduces or inhibits the induction of an immune response to the structural stabilizing portion, Preferably, the immunosilencing portion is a glycosylation site. (b) The structural stabilizing portion includes at least one glycosylated site, Preferably, the at least one glycosylated site is (1) -Asn-Xaa-Ser-, and (2) -Asn-Xaa-Thr- An N-linked glycosylation site selected from the group consisting of the following: Xaa is an amino acid other than Pro, Preferably, the glycosylated site is glycosylated at a level of at least 50% occupancy. (c) The structural stabilization portion is (i) Positions 5-7 of Sequence ID No. 80, (ii) The first to third positions of sequence number 82, (iii) Positions 6-8 of sequence number 82, (iv) Positions 13-15 of sequence number 82, (v) Positions 17-19 of Sequence ID No. 82, and / or (vi) Positions 27-29 of Sequence ID No. 82 The amino acid position corresponding to the amino acid position contains one or more N-linked glycosylation sites, Preferably, each N-linked glycosylation site is independently -Asn-Xaa-Thr-, where Xaa is an amino acid other than Pro, or (d) The structural stabilization portion, (i) (i-a) Positions 5-7 of Sequence ID No. 80, (i-b) Positions 1-3 of sequence number 82, (i-c) Positions 17-19 of sequence number 82, or (ii)(ii-a) Positions 5-7 of Sequence ID No. 80, (ii-b) Positions 1-3 of sequence number 82, (ii-c) Positions 17-19 of sequence number 82, and (ii-d) Positions 27-29 of sequence number 82, or (iii)(iii-a) Positions 5-7 of Sequence ID No. 80, (iii-b) Positions 1-3 of sequence number 82, (iii-c) Positions 13-15 of sequence number 82, (iii-d) Positions 17-19 of sequence number 82, and (iii-e) Positions 27-29 of sequence number 82, or (iv)(iv-a) Positions 5-7 of sequence number 80, (iv-b) Positions 1-3 of sequence number 82, (iv-c) Positions 6-8 of sequence number 82, (iv-d) Positions 13-15 of sequence number 82, (iv-e) Positions 17-19 of sequence number 82, (iv-f) Positions 27-29 of sequence number 82, The amino acid position corresponding to this contains an N-linked glycosylation site, Preferably, each N-linked glycosylation site is independently -Asn-Xaa-Thr-, where Xaa is an amino acid other than Pro. The chimeric polypeptide according to claim 1.

7. (a) The structural stabilization portion is (i) Arginine at the amino acid position corresponding to glutamine 22 in Sequence ID No. 80, (ii) Histidine at the amino acid position corresponding to asparagine 1 in SEQ ID NO: 82, (iii) Threonine at the amino acid position corresponding to histidine 2 in Sequence ID No. 82, (iv) Serine at the amino acid position corresponding to alanine 25 in Sequence ID No. 82, (v) Glutamine at the amino acid position corresponding to alanine 26 in SEQ ID NO: 82, (vi) Asparagine at the amino acid position corresponding to leucine 27 in SEQ ID NO: 82, (vii) Leucine at the amino acid position corresponding to glutamine 17 in Sequence ID No. 80, (viiii) Having a deletion of an amino acid residue at the position corresponding to arginine 37 in SEQ ID NO: 80, (b) The structural stabilizing portion has a deletion of an amino acid residue at the position corresponding to glutamine 1 and serine 2 in SEQ ID NO: 80, and / or (c) The structural stabilizing portion comprises one or more non-natural amino acids, preferably the one or more non-natural amino acids (i) Polyethylene glycol (PEG), (ii) Immune stimulating portion, or (iii) Lipids Enables coupling The chimeric polypeptide according to claim 1.

8. The chimeric polypeptide according to claim 1, wherein the microbial polypeptide is a virus or bacterial surface polypeptide.

9. (i) The virus surface polypeptide is an enveloped virus fusion ectodomain polypeptide, or (ii) The bacterial surface polypeptide is a bacterial outer membrane polypeptide, according to claim 8.

10. The envelope virus fusion ectodomain polypeptide is (i) A class I enveloped virus fusion protein ectodomain, preferably derived from a virus selected from orthomyxovirus, paramyxovirus, orthopneumovirus, metapneumovirus, retrovirus, coronavirus, filovirus, and arenavirus, or (ii) Class III enveloped virus fusion protein ectodomain, preferably derived from a virus selected from rhabdoviruses and herpesviruses. A chimeric polypeptide according to claim 9, which corresponds to or is a variant thereof.

11. The envelope virus fusion ectodomain polypeptide is (i) Respiratory syncytial virus, (ii) Metapneumovirus, (iii) Coronavirus, preferably beta-coronavirus, more preferably severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or Middle East respiratory syndrome-related coronavirus (MERS-CoV), (iv) Henipah virus, 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 virus, (viiii) Retrovirus, preferably human T-lymphophobic virus-1 (HTLV-1) The chimeric polypeptide according to claim 10, which corresponds to or is a variant thereof of a fusion protein derived from.

12. The bacterial outer membrane polypeptide, (i) Chlamydia major outer membrane protein (MOMP) polypeptide, or (ii) Trimeric autotransporter adhesin (TAA) polypeptide And here preferably, the TAA polypeptide is (i) TAA polypeptides derived from bacteria of a genus selected from Neisseria, Escherichia coli, Haemophilus, Yersinia, Salmonella, Bartonella, Vibrio, Acinetobacter, and Moraxella, and / or (ii) TAA polypeptides selected from Neisseria meningitidis adhesin (NadA), Neisseria meningitidis hia / hsf homolog (NhhA), Escherichia coli autotransporter G (EhaG), Escherichia coli IgG-binding protein D (EibD), urinary tract pathogenic Escherichia coli autotransporter G (UpaG), Haemophilus influenzae adhesin (HiA), and Yersinia enterocolitica adhesin (YadA). Corresponding to, or a variant thereof, The chimeric polypeptide according to claim 9.

13. The FHRR and SHRR included in the SSM are connected by a linker. Preferably, the linker contains or comprises a (poly)peptide having the same amino acid sequence as SEQ ID NO: 84 or 85. Here, optionally, the linker contains or consists of a membrane tethering (poly)peptide. The chimeric polypeptide according to claim 1.

14. The aforementioned structural stabilization portion is the amino acid sequence of Sequence ID No.

266. The chimeric polypeptide according to claim 1.

15. The aforementioned structural stabilization portion is the amino acid sequence of Sequence ID No.

265. The chimeric polypeptide according to claim 1.

16. The invention further includes a hinge region that operably connects the microbial polypeptide to the structural stabilizing portion, Preferably, the hinge region is (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 Includes or consists of Here, optionally, the hinge region contains or consists of a transmembrane (poly)peptide. The chimeric polypeptide according to claim 1.

17. A chimeric polypeptide comprising a first (poly)peptide operably connected downstream or upstream to a structural stabilizing portion, wherein the structural stabilizing portion is as defined in claim 1, Preferably, the first (poly)peptide is a therapeutic (poly)peptide. Preferably, the first (poly)peptide is operably connected downstream of the structural stabilization portion. Chimeric polypeptide.

18. A nucleic acid comprising a polynucleotide sequence encoding a chimeric polypeptide as defined in claim 1, Optionally, 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. Nucleic acid.

19. A host cell containing the nucleic acid described in claim 18.

20. A method for producing a chimeric polypeptide complex, comprising the step of combining the chimeric polypeptide described in claim 1 under conditions suitable for the formation of 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 structural stabilizing portions of the three chimeric polypeptides.

21. A chimeric polypeptide complex comprising three chimeric polypeptide subunits, wherein each subunit is the chimeric polypeptide described in claim 1, and the complex is characterized by a six-helix bundle formed by homotrimerization of the structural stabilizing portions of the three chimeric polypeptides.

22. The method according to claim 20 or the chimeric polypeptide complex according to claim 21, wherein the six-helix bundle comprises three internal trimers of substantially α-helix FHRRs oriented parallel to each other, with three substantially α-helix SHRRs packed in an antiparallel orientation relative to the FHRRs.

23. The chimeric polypeptide complex according to claim 21, wherein each of the chimeric polypeptide subunits comprises an enveloped virus fusion ectodomain polypeptide, and the complex comprises at least one pre-fusion epitope of an enveloped virus fusion protein.

24. A composition comprising a chimeric polypeptide according to claim 1 or a chimeric polypeptide complex according to claim 21, and a pharmaceutically acceptable carrier, diluent, or adjuvant.

25. A method for identifying a drug that binds to a microbial polypeptide or its complex, The method described above is (i) The step of contacting a candidate drug with a chimeric polypeptide comprising the microbial polypeptide described in claim 1 or a chimeric polypeptide complex comprising the microbial polypeptide described in claim 21, (ii) A step of detecting the binding of the candidate drug to the chimeric polypeptide or chimeric polypeptide complex; Includes, Preferably, the candidate drug is part of a compound library (e.g., a small molecule or macromolecule library). method.

26. A method for producing antigen-binding molecules that specifically bind to microbial polypeptides or their complexes, (1) The step of immunizing a target with a chimeric polypeptide containing the microbial polypeptide described in claim 1, a chimeric polypeptide complex containing the microbial polypeptide, or a composition thereof, (2) The step of identifying and / or isolating B cells that specifically bind to the microbial polypeptide or its complex from the immunized subject, (3) The step of producing the antigen-binding molecule expressed by the B cell A method that includes this.

27. (a) The microbial polypeptide or its complex is the ectodomain of an enveloped virus fusion protein or the complex of the fusion protein, and the method is (1) A step of immunizing a target with a chimeric polypeptide comprising the ectodomain polypeptide described in claim 9, or a chimeric polypeptide complex comprising the ectodomain polypeptide described in claim 21, or a composition thereof, wherein the ectodomain polypeptide corresponds to the fusion protein of the enveloped virus, (2) The step of identifying and / or isolating B cells from the immunized subject that specifically bind to the ectodomain or complex of the fusion protein, (3) The step of producing the antigen-binding molecule expressed by the B cell including, or (b) The microbial polypeptide or complex thereof is a bacterial outer membrane polypeptide or a complex of outer membrane polypeptides, and the method is (1) A step of immunizing a target with a chimeric polypeptide comprising the bacterial outer membrane polypeptide described in claim 9, or a chimeric polypeptide complex comprising the bacterial outer membrane polypeptide described in claim 21, or a composition thereof, wherein the bacterial outer membrane polypeptide corresponds to the outer membrane polypeptide of the bacterium, (2) The step of identifying and / or isolating B cells that specifically bind to the bacterial outer membrane polypeptide or its complex from the immunized subject, (3) The step of producing antigen-binding molecules expressed by the B cells including, The method according to claim 26.

28. A microbial polypeptide of a chimeric polypeptide comprising the microbial polypeptide described in claim 1, and / or a microbial polypeptide of one or more subunits of a chimeric polypeptide complex comprising the microbial polypeptide described in claim 21. An antigen-binding molecule that specifically binds to a target.

29. A composition comprising the antigen-binding molecule described in claim 28 and a pharmaceutically acceptable carrier, diluent, or adjuvant.

30. A composition comprising the nucleic acid described in claim 18.

31. The chimeric polypeptide encoded by the polynucleotide sequence contained in the nucleic acid, The composition according to claim 30, which is a chimeric polypeptide comprising the microbial polypeptide described in claim 1.

32. The chimeric polypeptide containing the microbial polypeptide is (i) A chimeric polypeptide comprising the envelope virus fusion ectodomain polypeptide described in claim 9, or (ii) Chimeric polypeptide comprising the bacterial outer membrane polypeptide described in claim 9 The composition according to claim 31.

33. The composition according to claim 30, wherein the nucleic acid includes or consists of RNA.

34. The RNA comprises at least one modified nucleotide, Preferably, the at least one modified nucleotide is a modified uridine, more preferably a methylated derivative of uridine, and most preferably N1-methyl-pseudouridine. The composition according to claim 33.

35. The RNA is formulated in a delivery vehicle which is a liposome, lipoplex, or lipid nanoparticle (LNP). Preferably, the lipid nanoparticles (LNPs) include cationic lipids, neutral lipids, steroids, and / or PEGylated lipids. The composition according to claim 33.

36. (i) a chimeric polypeptide comprising the microbial polypeptide described in claim 1, a chimeric polypeptide complex comprising the microbial polypeptide described in claim 21, or a composition thereof, (ii) The composition according to claim 31, Use in the manufacture of pharmaceuticals for inducing an immune response against microbial polypeptides or their complexes in a target.

37. Use of a chimeric polypeptide comprising a microbial polypeptide, a chimeric polypeptide complex comprising the microbial polypeptide, or a composition thereof in the manufacture of a pharmaceutical product for inducing an immune response to a microbial polypeptide or a complex thereof in a subject, The microbial polypeptide or its complex is (a) The microbial polypeptide or complex thereof is, each, a fusion protein of an enveloped virus or a complex of the fusion protein, and the pharmaceutical is (i) a chimeric polypeptide comprising the enveloped virus fusion ectodomain described in claim 9, a chimeric polypeptide complex comprising the enveloped virus fusion ectodomain described in claim 21, or a composition thereof, (ii) A composition comprising a nucleic acid containing a polynucleotide sequence encoding the chimeric polypeptide described in (i) above. Includes, The ectodomain polypeptide subunit of the chimeric polypeptide complex corresponds to the fusion protein of the enveloped virus. or (b) The microbial polypeptide or complex thereof is, respectively, a bacterial outer membrane polypeptide or a complex of outer membrane polypeptides, and the pharmaceutical is (i) a chimeric polypeptide comprising the bacterial outer membrane polypeptide described in claim 9, a chimeric polypeptide complex comprising the bacterial outer membrane polypeptide described in claim 21, or a composition thereof, (ii) A composition comprising a nucleic acid comprising a polynucleotide sequence encoding the chimeric polypeptide described in (i) above, Includes, The bacterial outer membrane polypeptide subunit of the chimeric polypeptide complex corresponds to or substantially corresponds to the bacterial outer membrane polypeptide expressed by the bacterium. The aforementioned use.

38. (i) A chimeric polypeptide comprising the microbial polypeptide described in claim 1, a chimeric polypeptide complex comprising the microbial polypeptide described in claim 21, or a composition thereof. (ii) The composition according to claim 29, or (iii) The composition according to claim 31, Use in the manufacture of pharmaceuticals for treating or preventing microbial infections in a target population.

39. Use of a chimeric polypeptide comprising a microbial polypeptide in the manufacture of a pharmaceutical for treating or preventing a microbial infection in a subject, The aforementioned microbial infections are (a) The microbial infection is an enveloped virus infection in the subject, and the pharmaceutical is (i) a chimeric polypeptide comprising the enveloped virus fusion ectodomain described in claim 9, a chimeric polypeptide complex comprising the enveloped virus fusion ectodomain described in claim 21, or a composition thereof, (ii) A composition comprising a nucleic acid containing a polynucleotide sequence encoding the chimeric polypeptide described in (i) above. including, or (b) The microbial infection is a bacterial infection in the subject, and the pharmaceutical is (i) a chimeric polypeptide comprising the bacterial outer membrane polypeptide described in claim 9, a chimeric polypeptide complex comprising the bacterial outer membrane polypeptide described in claim 21, or a composition thereof, (iii) A composition comprising a nucleic acid containing a polynucleotide sequence encoding the chimeric polypeptide described in (i) above. including, The aforementioned use.

40. A pharmaceutical composition for treating or preventing a microbial infection in a subject, wherein the pharmaceutical composition comprises a chimeric polypeptide containing the microbial polypeptide described in claim 1, or a chimeric polypeptide complex containing the microbial polypeptide described in claim 21.

41. A pharmaceutical composition for treating or preventing a microbial infection in a subject, wherein the pharmaceutical composition comprises a chimeric polypeptide containing a microbial polypeptide, or a chimeric polypeptide complex containing the microbial polypeptide, The microbial polypeptide, (a) an enveloped virus fusion ectodomain polypeptide, a chimeric polypeptide comprising the enveloped virus fusion ectodomain polypeptide described in claim 9, and a chimeric polypeptide complex comprising the enveloped virus fusion ectodomain described in claim 21 are provided, wherein the pharmaceutical composition is for treating or preventing an enveloped virus infection in a subject. ,or (b) A bacterial outer membrane polypeptide is provided, which is a chimeric polypeptide comprising the bacterial outer membrane polypeptide described in claim 9, and a chimeric polypeptide complex comprising the bacterial outer membrane polypeptide described in claim 21. The aforementioned pharmaceutical composition is for treating or preventing bacterial infections in the subject. The aforementioned pharmaceutical composition.

42. A pharmaceutical composition for inducing an immune response against a microbial polypeptide or its complex in a target, A chimeric polypeptide comprising the microbial polypeptide described in claim 1, or a chimeric polypeptide complex comprising the microbial polypeptide described in claim 21, The aforementioned pharmaceutical composition.

43. A pharmaceutical composition for inducing an immune response to a microbial polypeptide or a complex thereof in a subject, wherein the pharmaceutical composition comprises a chimeric polypeptide containing a microbial polypeptide, or a chimeric polypeptide complex containing the microbial polypeptide, The microbial polypeptide or its complex, (a) A chimeric polypeptide comprising the enveloped virus fusion ectodomain described in claim 9, and a chimeric polypeptide complex comprising the enveloped virus fusion ectodomain described in claim 21, Here, the ectodomain polypeptide subunit of the chimeric polypeptide complex corresponds to the fusion protein of the enveloped virus, or (b) A chimeric polypeptide comprising the bacterial outer membrane polypeptide described in claim 9, or a chimeric polypeptide complex comprising the bacterial outer membrane polypeptide described in claim 21, Here, the bacterial outer membrane polypeptide subunit of the chimeric polypeptide complex corresponds to or substantially corresponds to the bacterial outer membrane polypeptide expressed by the bacterium. The aforementioned pharmaceutical composition.

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