Stable pre-fusion HMPV fusion proteins

Abstract

The present invention relates to stable pre-fusion human pneumovirus (HMPV) F proteins, nucleic acid molecules encoding said HMPV F proteins and uses thereof.

Description

[0001] The present invention relates to the field of medicine, and in particular to recombinant pre-fusion HMPV F protein and fragments thereof, and nucleic acid molecules encoding HMPV F protein and fragments thereof, and uses thereof, for example, in vaccines. Background of the Invention

[0003] Human metapneumovirus (HMPV) belongs to the Pneumovirinae, which also includes RSV. Genetic analysis of HMPV isolates has revealed two major groups A and B and four minor groups (A1, A2, B1, and B2) based mainly on the diversity of attachment proteins (G) and fusion proteins (F) (van Hoogen et al., Emerg. Infect Dis. 10(4): 658–666, 2004). Recently, Noa et al. (Microorganisms. August 21, 2020; 8(9): 1280.) described the subdivision of A2 into A2a and A2b, the latter of which is currently circulating.

[0004] In order to infect host cells, HMPV, like other enveloped viruses such as influenza virus, RSV and HIV, requires fusion of the viral membrane with the host cell membrane. For HMPV, the conserved fusion protein (HMPV F protein) fuses the cell membranes of the virus and the host cell. The HMPV F protein initially folds into a "pre-fusion" conformation. This metastable structure has recently been solved (Battles et al., Nat Commun. Nov 16; 8 (1): 1528, 2017.) During cell entry, the pre-fusion conformation undergoes refolding and conformational changes to its "post-fusion" conformation (McLellan, J. Virol. 85 (15): 7788-7796, 2010; Swanson, PNAS 108 (23): 9619-9624, 2011). Thus, the HMPV F protein is a metastable protein that drives membrane fusion by coupling irreversible protein refolding to membrane apposition by initially folding into a metastable form (prefusion conformation) that subsequently undergoes discrete / stepwise conformational changes to a lower energy conformation (postfusion conformation).

[0005] HMPV was first identified in 2001 in clinical samples from pediatric patients with a disease similar to human respiratory syncytial virus (RSV), but not in samples from which RSV could not be identified (van den Hoogen et al., Nat. Med. 7(6):719-724, 2001). Subsequent studies have shown that HMPV is a major cause of both upper and lower respiratory tract infections in infants, young children, the elderly, and those who are immunocompromised or have underlying chronic medical conditions. The clinical manifestations of HMPV infection are similar to those caused by RSV, ranging from mild respiratory illness to bronchiolitis and pneumonia. HPMV infection appears to be ubiquitous, as almost all children are seropositive by the age of 5 years. Previous epidemiological studies have suggested that HMPV infection causes lower respiratory tract infections in 5-15% of otherwise healthy infants (Falsey et al., J. Infect. Dis. 187:785-790, 2003). Among children under 5 years of age hospitalized for acute respiratory tract infection or fever, HMPV was detected in 4.9% of children, a population similar to that of influenza and higher than that of parainfluenza virus type 3 (PIV-3) (Williams et al., J Infect Dis. 201(12):1890-1898, 2010). The incidence of disease is highest for children under 0-6 months of age. Recurrent infection with HMPV has also been described.

[0006] To date, most infection data are derived from studies in children, however, HMPV can also cause evidence accumulation of severe disease in adults. Like influenza and RSV, infections in adults are most severe in the elderly and patients with chronic underlying medical conditions. The incidence of symptomatic infection in the adult population is usually less than 5% in most studies (Falsey, Pediatr. Infect. Dis. J. 27: S80–83, 2008). It has been quantified by Gaunt et al.: influenza A, influenza B and RSV are the main viral causes of respiratory diseases in the elderly (> 65 years old), followed by HMPV, which contributes to 2.1 disability-adjusted life years (DALY) / 1000 hospitalized populations (Gaunt et al., J. Clin. Virol. 52 (3): 215-221, 2011).

[0007] There are currently no approved therapeutic or prophylactic treatments for the management of HMPV infection. As HPMV represents the leading cause of acute viral respiratory infections after RSV, an effective therapy or vaccine against HMPV is needed. SUMMARY OF THE INVENTION

[0009] The present invention provides a recombinant stable trimeric prefusion human pneumovirus (HMPV) fusion (F) protein, i.e., a recombinant HMPV F protein that is stabilized in a prefusion conformation. The HMPV F protein of the present invention comprises at least one epitope specific to the prefusion conformation of the F protein. The present invention provides a full-length HMPV F protein and a soluble HMPV F protein. In certain embodiments, the prefusion hMPV F protein is a soluble protein (i.e., it is not membrane-bound and lacks a transmembrane region and a cytoplasmic region). The present invention also provides a nucleic acid molecule encoding a prefusion HMPV F protein according to the present invention and a vector comprising such a nucleic acid molecule. The present invention also relates to a pharmaceutical composition, preferably a vaccine composition, comprising one or more HMPV F proteins, nucleic acid molecules and / or vectors according to the present invention, and its use in inducing an immune response to the HMPV F protein, in particular its use as a vaccine. The present invention also relates to a method for inducing an anti-human pneumovirus (HMPV) immune response in a subject, comprising administering to the subject an effective amount of a prefusion HMPV F protein, a nucleic acid molecule encoding the HMPV F protein, and / or a vector comprising the nucleic acid molecule. Preferably, the induced immune response is characterized by neutralizing antibodies, T cells and / or protective immunity against HMPV F. In a specific aspect, the present invention relates to a method for inducing neutralizing anti-human pneumovirus (HMPV) F protein antibodies in a subject, comprising administering to the subject an effective amount of an immunogenic composition comprising a prefusion HMPV F protein, a nucleic acid molecule encoding the HMPV F protein and / or a vector comprising the nucleic acid molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings.It is to be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0012] Figure 1 (a) Simplified schematic diagram of full-length HMPV F with indicated residue numbers and (b) soluble HMPV F based on the F ectodomain fused to the Foldon domain. Immature F0 is processed in F2 and F1 fragments by TMPRSS2 or other / related proteases starting at position 103, between residue 102 in the F1 domain and the N-terminus of the fusion peptide (FP). The signal peptide (SP), HR1 domain (HR1), HR2 domain (HR2), transmembrane (TM) and cytoplasmic portion (CM) are indicated.

[0013] Figure 2(A) Alignment of segments of HMPV F at the borders of F2 (italics) and F1 (bold) for non-stabilized HMPV F variants (MPV201285) and stable variants (MPV210531, MPV210502, MPV210500, MPV210507, and MPV210509). Stable variants contain T69Y, A116H, A140C / A147C, D185P, H468N, E453Q substitutions). Stable variants have C-terminal truncations of F2 and / or additional furin sites. (B) Reduced SDS-PAGE followed by Western blotting with anti-hMPV polyclonal sera from supernatants of HEK293 cells transfected with plasmids corresponding to stable soluble HMPV F ectodomains with F2-F1 borders according to (A). Incomplete processing was observed and marked with F0. (C) Analytical SEC analysis of cell culture supernatants showed a trimer peak with a retention time between 4.2 and 4.4 minutes (left) and trimer stability after 30 minutes of heat treatment (right). (D) Biolayer interferometry of supernatants using Octet showed binding to pre-fusion specific antibody ADI-14448 and post-fusion specific antibody DS7 (binding to non-preF conformation) (transfections were performed with n=2. Pools were analyzed on analytical SEC. Individual transfections were analyzed on Octet and mean values / design are reported, with error bars indicating standard deviations). The dashed line in the right figure indicates the lower limit of quantification.

[0014] Figure 3 .(A) Alignment of HMPV F sequences at the border of F2 (italics) and F1 (bold), with the RSV p27 domain (double underlined) and additional furin sites for the F2 C-terminus (underlined) inserted. (B) Reduced SDS-PAGE followed by Western blotting of anti-hMPV polyclonal sera from supernatants of HEK293 cells transfected with plasmids corresponding to stable soluble HMPV F ectodomains with F2-F1 borders according to (A). Complete processing is indicated by F1, and incomplete processing is indicated by residual F0. (C) Analytical SEC analysis of cell culture supernatants shows trimer levels (left) and trimer stability (right) after 30 minutes of heat treatment. (D) Biolayer interferometry of supernatants using Octet shows binding to pre-fusion specific antibody ADI-14448 and post-fusion specific antibody DS7 (binding to non-preF conformation). The dashed line in the right panel indicates the lower limit of quantitation.

[0015] Figure 4.HMPV F (MPV210751) with wild-type HR2 region and foldon was compared with a variant without foldon or HR2 modification (MPV211241), and a variant with modification of the HR2 position truncated at residue 489 (MPV21142-MPV211292). HMPV F variants with F2 truncated at residue 89, furin protease cleavage site, and RSV p27 with indicated HR2 regions were expressed in Expi-HEK cells. Trimer content relative to MPV210751 after harvest and storage at 4°C for 6-8 weeks (left). Relative trimer content after heat stress compared to 4°C control samples (right). Transfection was performed with n=2. Mean values / designs are reported, with error bars indicating standard deviations.

[0016] Figure 5 .(A) HMPV F (MPV210751) with wild-type HR2 region and foldon was compared with a variant without foldon or HR2 modification (MPV211241), and a variant with modification of the HR2 position truncated at residue 489 (MPV211247, MPV211287, MPV211917 and MPV211918). HMPV F variants with F2 truncated at residue 89, furin cleavage site and RSV p27 with indicated HR2 region were expressed in Expi-HEK cells. Supernatants were tested for the amount (middle figure) and thermal stability (right figure) of F trimers. (B) Supernatants were further evaluated for binding to pre-fusion specific antibody ADI-14448, vertex interface binder MPV458 and antibody DS7 (binding to non-preF conformation) using biolayer interferometry on Octet. The dotted line in the right figure indicates the lower limit of quantification.

[0017] Figure 6 .(A) Comparison of HMPV F variants with truncated F2, furin cleavage site and RSV p27 domain, stabilized HR2 (I473W, S477I and A484I) and additional stabilizing substitutions (E453Q or E453P, V112R, D209E), and natural variant V231I. The left histogram shows the amount of trimer in the supernatant of HEK293 cells after transfection with the indicated F variants as measured by analytical SEC. The right figure shows the thermal stability of the trimer relative to 4°C after incubation at 58 or 63°C for 30 minutes. (B) The supernatant was further evaluated by binding to pre-fusion specific antibody ADI-14448, vertex interface antibody MPV458 and antibody DS7 (binding to non-preF conformation) using biolayer interferometry on Octet. The dotted line in the right figure indicates the lower limit of quantification.

[0018] Figure 7 (A) Comparison of HMPV F variants with truncated F2, furin cleavage site and RSV p27 domain, stable HR2 (L473W, D475R, Q476K, S477F, N478D, R479E, A484I) and additional stable substitutions (E453Q or E453P, V112R, D209E) and stable substitutions (V231I) based on natural HMPV F variants. The left histogram shows the amount of trimers in the supernatant of HEK293 cells after transfection with the indicated F variants as measured by analytical SEC. The right figure shows the thermal stability of trimers relative to 4°C after incubation at 58 or 63°C for 30 minutes. (B) Supernatants were further evaluated for binding to prefusion specific antibody ADI-14448, apex interface antibody MPV458 and antibody DS7 (binding to non-preF conformation) using biolayer interferometry on Octet. The dashed line in the right panel indicates the lower limit of quantitation.

[0019] Figure 8 (A) Comparison of HMPV F variants with truncated F2, furin cleavage site and RSV p27 domain, stabilized HR2 (L473W, D475R, Q476K, S477F, N478D, R479E, A484I) with additional stabilizing substitutions (E453Q, V112R, T69Y, S149Y, N313W and S445Y) and stabilizing substitutions (N404P) based on natural HMPV F variants. The histogram shows the amount of trimer in the supernatant of HEK293 cells after transfection with the indicated F variants as measured by analytical SEC. (B) Supernatants were further evaluated for binding to prefusion specific antibody ADI-14448, apex interface antibody MPV458 and antibody DS7 (binding to non-preF conformation) using biolayer interferometry on Octet. The dashed line in the right panel indicates the lower limit of quantitation. (C) T by DSF m50 As an indication of the thermal stability of the trimer in the supernatant of HEK293 cells.

[0020] Fig. 9.(A) Comparison of HMPV F variants with truncated F2, furin cleavage site and RSV p27 domain, stabilizing substitutions (E453P, V112R, D209E) and substitutions based on the natural variant V231I, and HR2 stabilizing mutations (positions 473, 474, 477, 484 and 485). The histogram shows the amount of trimer in the supernatant of HEK293 cells after transfection with the indicated F variants as measured by analytical SEC. (B) Supernatants were further evaluated for binding to prefusion-specific antibody ADI-14448, apex interface antibody MPV458 and antibody DS7 (binding to non-preF conformation) using biolayer interferometry on Octet. The dashed line in the right figure indicates the lower limit of quantification. (C) T of DSF by m50 As an indication of the thermal stability of the trimer in the supernatant of HEK293 cells. The vertical line indicates the detection of the Tm50. The derivative is plotted for each sample and the assigned melting temperature is indicated in °C. For MPV220123, no Tm50 could be assigned.

[0021] Fig.10 .(A) Comparison of HMPV F variants with truncated F2, furin cleavage site and RSV p27 domain, stabilizing substitutions (E453P, V112R, D209E), natural variant V231I, and HR2 stabilizing mutations S477I and A484I and further stabilizing mutations T69W, S149Y, N313W, S445Y and N404P. The histogram shows the amount of trimer in the supernatant of HEK293 cells after transfection with the indicated F variants as measured by analytical SEC. (B) Supernatants were further evaluated for binding to prefusion-specific antibody ADI-14448, apex interface antibody MPV458 and antibody DS7 (binding to non-preF conformation) using biolayer interferometry on Octet. The dashed line in the right figure indicates the lower limit of quantification. (C) T of DSF by m50 As an indication of the thermal stability of the trimer in the supernatant of HEK293 cells. The vertical line indicates the detection of Tm50. The derivative is plotted for each sample and the assigned melting temperature is indicated in °C.

[0022] Fig.11.(A). Reduced SDS-PAGE Coomassie staining of purified HMPV F protein. (B) Analytical SEC-MALS of purified HMPV F protein as trimer after purification and storage at 4°C (species smaller than trimer are indicated by D). (C) Heat stress at 37°C for 2 and / or 8 weeks (aggregates are indicated by A). (D) Analytical SEC after one, five or ten snap freezing cycles. (E) Melting temperatures of HMPV-F protein measured by DSF. Derivatives are plotted for each sample and the assigned melting temperatures are indicated in °C. (F) Binding to prefusion-specific antibody ADI-14448, apex interface antibodies MPV458 and MPV465, preF and postF binding antibody ADI-18992 and non-preF-specific antibody DS7 were measured using biolayer interferometry on Octet. (G) 2D class averages of negatively stained EM images of MPV212047.

[0023] Fig.12 Cryo EM structure of prefusion HMPV F MPV212047 without the foldon trimerization domain. One protomer is depicted as a cartoon representation, while two protomers are depicted as surface representations.

[0024] Fig.13 Naive cotton rats were immunized with the indicated doses of AS01B-adjuvanted prefusion (PreF: MPV212047) or postfusion (PostF: MPV190470) HMPV F protein on days 0 and 21, followed by 10 5 PFU HMPV A2 challenge and sacrifice on day 46. Negative controls were immunized twice with PBS, and positive controls were immunized with 10 4 PFU HMPV A2 challenge once. (A) HMPV prefusion F binding antibody titer at day 42 in ELISA. (B) HMPV A2 neutralizing antibody titer at day 42 in HMPV A2-GFP VNA. (C) Rhinitis virus load at day 46 as measured by plaque reduction neutralization test (PRNT). Each point depicts the value of an individual animal, while the horizontal line indicates the median response of the group. The dotted line indicates the lower limit of detection (LLoD).

[0025] Fig.14.(A) Analytical size exclusion chromatography (SEC) profile of purified HMPV F trimer.(B) Purified HMPV F trimer bound to pre-fusion (ADI-14448) and vertex interface (MPV458 and MPV465) HMPV F antibodies using quantitative Octet. Initial binding rates are plotted.(C) Balb / c mice were immunized with indicated doses of AS01B adjuvanted pre-fusion open (MPV220215) or closed (MPV212047) HMPV F proteins on days 0 and 28. Negative controls were immunized twice with PBS.(C) HMPV pre-fusion F binding antibody titers at day 42 in ELISA.(D) HMPV A2 neutralizing antibody titers at day 42 in HMPV A2-GFP VNA. Each point depicts the value of each animal, while the horizontal line indicates the median response of the group. The dashed horizontal line indicates the lower limit of detection (LLoD). Significant differences across doses are indicated above the groups as determined by the Tobit model with a Bonferroni correction for multiple comparisons.

[0026] Fig.15 Balb / c mice were treated with at least 1x10 3 Up to 3x10 5 PFU HMPV A2 was pre-exposed, followed by immunization with 15 μg non-adjuvanted prefusion HMPV F protein (MPV212047) at week 12. HMPV prefusion F binding antibody titers at weeks 14 and 16 in ELISA. Each point depicts the value of an individual animal, while the horizontal line indicates the median response of the group. The dotted line indicates the lower limit of detection (LLoD).

[0027] Fig.16 (A) Trimer peak height (black bars) and melting temperature (Tm50) (grey bars) of cell culture supernatants expressing HMPV A2 F protein as determined by analytical SEC and differential scanning fluorimetry (DSF), respectively. The bars depict the mean + SD of n = 2 replicates. (B) Table depicts the subtype, strain identifier and Genbank ID of HMPV F variants. (C) Analytical SEC of cell culture supernatants expressing HMPV A2, B1 and B2 F proteins from (B). Trimers are indicated by 'T' and monomers are indicated by 'M'. (D) Tm50 of cell culture supernatants from (C) as determined by DSF. The bars depict the mean + SD of n = 3 replicates. (E) Pre-fusion (ADI-61026), apex interface (MPV458) and non-fusion (DS7) HMPV F antibody binding in cell culture supernatants from (C) using quantitative Octet. The initial binding rates are plotted.

[0028] Fig.17 .(A) Expression levels of HMPV F MPV220759 and MPV221190 trimers in cell culture supernatants as determined by analytical SEC. (B) Analytical SEC profile of purified HMPV F trimers. (C) Characterization of HMPV F trimers based on MALS signals. (D) Melting temperature (Tm50) of purified HMPV F trimers as determined by DSF. N=3 replicates were measured, and individual values ​​and averages are reported as gray solid lines and black solid lines, respectively. (E) Binding of purified HMPV F trimers to pre-fusion (ADI-14448 and ADI-61026), apex interface (MPV458) and non-fusion pre-(DS7) HMPV F antibodies using quantitative Octet. Initial binding rates are plotted as the average + SD of n=3-6 measurements. (F) Standardized trimer peaks of HMPV F trimers subjected to 1-10 quick freezing (SF) cycles in liquid nitrogen and supercooling stress to -20°C as determined by analytical SEC. Untreated trimer was set to 100%. The bars depict the mean ± SD of n = 5 supercooled samples or depict n = 1 SF sample. The open circles depict individual measurements. (G) Balb / c mice were immunized intramuscularly with 1.5, 5 or 15 μg AS01B adjuvanted prefusion HMPV F protein or PBS at days 0 and 28. HMPV prefusion F binding antibody titer ELISA at day 43. Each point depicts the value of each animal, and the horizontal line indicates the median response of the group.

[0029] Fig.18 .(A) Expression levels of HMPV F MPV221190 and MPV220552 trimers in cell culture supernatants as determined by analytical SEC. The trimer peak is indicated by a black arrow for MPV221190 and a gray arrow for MPV220552. (B) Characterization of MPV220552 trimers based on MALS signals. (C) Melting temperature (Tm50) of MPV220552 trimers as determined by DSF. N=3 replicates were measured, and individual values ​​and averages were reported as gray solid lines and black solid lines, respectively. (D) Binding of HMPV F trimers to pre-fusion (ADI-14448 and ADI-61026), apex interface (MPV458), and non-fusion pre-(DS7) HMPV F antibodies using quantitative Octet. Initial binding rates are plotted. (E) Analytical SEC profiles of purified MPV221190 and MPV220552 trimers after purification (t=0), after 2 weeks storage at 4°C, or after 2 weeks storage at 37°C.

[0030] Fig.19(A) Analytical SEC of cell culture supernatants expressing backbone MPV220847 and variants carrying substitutions at position 477. (B) Melting temperatures (Tm50) of cell culture supernatants from (A) as determined by DSF. N = 3 replicates, and individual and average values ​​are reported as solid grey and black lines, respectively. (C) Pre-fusion (ADI-14448) and non-fusion (DS7) HMPV F antibody binding in cell culture supernatants from (A) using quantitative Octet. Initial binding rates are plotted.

[0031] Fig. 20 Analytical SEC of cell culture supernatants expressing backbone MPV211241 and variants carrying substitutions at position 477. The black line depicts the supernatant at harvest (4°C) and the grey line depicts the supernatant after 30 minutes incubation at 58°C. The retention time of the HMPV F trimer is indicated with 'T' and the monomer with 'M'.

[0032] Fig.21 Median fluorescence intensity of prefusion (ADI-61026) and non-prefusion (DS7) antibodies binding to full-length HMPV F protein expressed on the cell surface as determined by flow cytometry.

[0033] Fig. 22 (A) Alignment of HMPV F p27 variants. Glycosylation sites are underlined, N to Q mutations are in bold. Deletions are indicated by dashed lines. (B) Analytical SEC of cell culture supernatants from (A) expressing HMPV F protein co-transfected with (grey) or without (black) 20% furin. (C) Reduced SDS-PAGE followed by Western blotting with anti-hMPV polyclonal sera from selected supernatants from (B). DETAILED DESCRIPTION OF THE INVENTION

[0035] The fusion protein (F) of human pneumovirus (HMPV or hMPV) is a trimeric class I fusion protein involved in the fusion of the viral membrane with the host cell membrane, which is required for infection. HPMV F mRNA is translated into a 539 amino acid precursor protein designated as F0, which contains a signal peptide sequence at the N-terminus (i.e., amino acid residues 1-18 of SEQ ID NO: 1) (Ulbrandt et al., Journal of General Virology (2008), 89, 3113-3118), which is removed by signal peptidases in the endoplasmic reticulum.

[0036] To become active, the precursor form F0 is proteolytically cleaved (processed) by cellular proteases to generate two domains (F1 and F2) in a metastable disulfide-linked heterodimer (F1+F2). The newly formed N-terminus of F1 is considered to be a fusion peptide. Three F2-F1 dimers bind to form a mature F protein that adopts a metastable prefusogenic ("pre-fusion") conformation that is triggered to undergo a conformational change upon contact with the target cell membrane. This conformational change exposes the fusion peptide, which binds to the host cell membrane and promotes fusion of the membrane of the virus or infected cell with the target cell membrane. The fusion peptide and the transmembrane domain (see Figure 1 ) are two heptad repeat (HR) regions, HR1 and HR2. Once cleaved, HMPV F can be triggered to undergo an essentially irreversible and energetically favorable conformational change from a prefusion form to a postfusion state, where the released potential energy drives membrane fusion.

[0037] The F1 domain (corresponding to amino acid residues 103-539 of SEQ ID NO: 1) contains a 23 hydrophobic fusion peptide at its N-terminus (corresponding to amino acid residues 103-126 of SEQ ID NO: 1), a refolding region 2 (RR2) (corresponding to amino acids 426-491 of SEQ ID NO: 1), wherein HR2 comprises amino acids 453 to 484, and the C-terminus contains a transmembrane region (TM) (corresponding to amino acid residues 492-513 of SEQ ID NO: 1) and a cytoplasmic region (corresponding to amino acid residues 514-539) (Ulbrandt et al., Journal of General Virology (2008), 89, 3113-3118).

[0038] After cleavage, the F2 domain (corresponding to amino acid residues 19-102 of SEQ ID NO: 1) is covalently linked to F1 via two disulfide bridges (Ulbrandt et al., Journal of General Virology (2008), 89, 3113-3118). The F1-F2 heterodimers assemble as homotrimers in the virion.

[0039] Vaccines for HMPV infection are currently unavailable but desirable. A potential method for producing vaccines is a subunit vaccine based on the HMPV F protein of purification. However, for this method, it is desirable that the conformation of the purified HMPV F protein is similar to the conformation of the pre-fusion state of the HMPV F protein, and is stable over a period of time, and can be produced in sufficient quantities. In addition, for vaccines based on subunits, the HMPV F protein needs to be truncated by the disappearance of the transmembrane (TM) region and the cytoplasmic region to produce the F protein (F or sF) of soluble secretion. Because the TM region is responsible for membrane anchoring and trimerization, the soluble F protein without anchoring is obviously more unstable than the full-length protein, and will be easily refolded into the final state after fusion. In order to obtain the soluble F protein in the stable pre-fusion conformation showing high expression level and high stability, the pre-fusion conformation therefore needs to be stabilized. Since the full-length (membrane-bound) HMPV F protein is also metastable, stabilization of the prefusion conformation is also desirable for the full-length HMPV F protein (ie, including the TM and cytoplasmic regions), e.g., for any vector-based live attenuated vaccine approach or RNA vaccine.

[0040] The present invention provides a trimeric recombinant pre-fusion HMPV F protein, i.e., a HMPV F protein that is stable in a pre-fusion conformation. In the research leading to the present invention, several modifications compared to the amino acid sequence of the wild-type HMPV F protein (particularly the amino acid sequence of SEQ ID NO: 1) were introduced, such as mutations, deletions, insertions and / or fusions of amino acids, so as to obtain the stable pre-fusion HMPV F protein. The stable pre-fusion HMPV F protein of the present invention is in a pre-fusion conformation, i.e., they contain (display) at least one epitope specific for the pre-fusion conformation F protein. The epitope specific for the pre-fusion conformation F protein is an epitope that does not exist in the post-fusion conformation. Without wishing to be bound by any particular theory, it is believed that the pre-fusion conformation of the HMPV F protein may contain the same epitopes as those present on the HMPV F protein expressed on the natural HMPV virion, and therefore may provide advantages in terms of eliciting protective neutralizing antibodies. In certain embodiments, the protein of the present invention comprises at least one epitope recognized by a pre-fusion specific anti-HMPV monoclonal antibody. Examples of such prefusion HMPV antibodies are MPE8 (Corti et al., Nature 50(7467):439-443, 2013) and ADI-14448 (Gilman et al., SciImmunol. 2016 Dec 16; 1(6):eaaj1879.doi:10.1126 / SciImmunol.aaj1879.Epub 2016 Dec 9). In certain embodiments, the recombinant prefusion HMPV F protein comprises at least one epitope recognized by at least one prefusion-specific monoclonal antibody as described above, and is a trimer. In certain embodiments, the stable prefusion HMPV F protein according to the invention is soluble and thus comprises a truncated F1 domain (i.e., the transmembrane region and the cytoplasmic region have been (partially) deleted).

[0041] The present invention particularly provides a pre-fusion human pneumovirus (HMPV) F precursor F0 protein comprising F1 and F2 domains and comprising at least one modification in the amino acid sequence of the F1 and / or F2 domains.

[0042] In certain embodiments, at least one modification stabilizes the prefusion conformation and / or increases trimer formation and / or (thermo)stability of the HMPV F protein.

[0043] With respect to the primary amino acid sequence of the HMPV F0 protein, the following terms are used to describe the structural features of the F protein. The term F0 refers to the full-length translated HMPV F protein precursor. During maturation, the F0 polypeptide undergoes proteolytic cleavage at a cleavage site located between F2 and F1 (i.e., between amino acids at positions 102 and 103).

[0044] As used herein, the F2 domain includes at least a portion of amino acids 19-102, and the F1 domain includes at least a portion of amino acids 103-539. The soluble F protein includes the F2 domain and the F1 domain of the HMPV F protein, but does not include the transmembrane domain of the HMPV F protein. The soluble portion of the F1 domain includes at least a portion of and up to all of the amino acids 103-491 of the F0 protein. As indicated above, these amino acid positions (and all subsequent amino acid positions specified herein) are given with reference to the exemplary HMPV F protein precursor polypeptide (F0) of SEQ ID NO: 1.

[0045] According to the present invention, the HMPV F protein comprises at least one modification that stabilizes the prefusion conformation of the F protein, such that the HMPV F protein retains at least one immunodominant epitope of the prefusion conformation of the F protein and / or increases trimer formation and / or the (thermo)stability of the HMPV F protein.

[0046] In certain embodiments, at least one modification is the introduction of at least one non-native cleavage site. Thus, in certain embodiments, the invention provides a stable pre-fusion human pneumovirus (HMPV) F precursor (F0) protein comprising F1 and F2 domains, and comprising a first non-native cleavage site between the F1 and F2 domains, e.g., at the C-terminal end of the F2 domain.

[0047] Additionally or alternatively, the F2 domain can be C-terminally truncated. Therefore, in certain embodiments, the first non-natural cleavage site is placed at the C-terminal end of the truncated F2 domain, for example, the F2 domain can be C-terminally truncated after position 89, and the first non-natural cleavage site is placed after the C-terminal amino acid residue at position 89. In the research leading to the present invention, it has been shown that the cleavage site is between residues 92-93. Therefore, if, for example, a furin protease site containing 4 residues is added after residue 89, the position of the terminal is ~92, but the last residue at the C-terminus is not natural.

[0048] According to the present invention, it has been shown that by introducing a non-native cleavage site between the F1 and F2 domains, the processing (cleavage) of the HMPV F0 protein is improved compared to the HMPV F protein with the native cleavage site. According to the present invention, it has been shown that the improved processing increases the stability and antigenicity of the HMPV F protein.

[0049] According to the present invention, the term non-native cleavage site refers to a cleavage site that is not present in the F protein of naturally occurring HMPV.

[0050] The cleavage site is a proteolytic site utilized by a cell protease, which activates a wide range of precursor proteins, including type I viral fusion proteins, such as HMPV F. Therefore, various cell proteases (such as furin, TMPRSS2, cathepsin and other transmembrane serine proteases) known to catalyze the proteolytic activation process cut various viral cell surface proteins, which are necessary for the virus to enter the host cell. The cleavage site generally comprises or consists of a specific amino acid sequence recognized by a cell protease. Therefore, the protease recognizes the site where it cuts via the specific amino acid sequence along the polypeptide chain. For example, furin-like proteases preferably cut proteins just after the basic amino acid target sequence (standardly, Arg-X-(Arg / Lys)-Arg). According to the present invention, a non-natural cleavage site can be introduced from the N-terminal of the natural cleavage site, i.e., in addition to the natural cleavage site. In a preferred embodiment, the first non-natural cleavage site replaces the natural cleavage site.

[0051] In certain embodiments, the protein of the present invention further comprises a second non-natural cleavage site in the F2 domain. According to the present invention, the second non-natural cleavage site is introduced into the F2 domain positioned at the N-terminus of the first non-natural cleavage site, wherein there is a spacer sequence between the first non-natural cleavage site and the second non-natural cleavage site. Thus, the second non-natural cleavage site is introduced into the F2 domain positioned at the N-terminus of the first natural or non-natural cleavage site, and the spacer sequence is present between the first non-natural cleavage site and the second non-natural cleavage site, i.e., the first cleavage site and the second cleavage site are separated by the spacer sequence, but not directly adjacent. The spacer sequence may be part of the native sequence of the F2 domain, or may be a heterologous sequence that separates the first non-natural cleavage site from the second non-natural cleavage site.

[0052] In certain embodiments, the second non-natural cleavage site is placed after amino acid residue 88 of the F2 domain, or after amino acid residue 89, or after amino acid residue 90 of the F2 domain, and the first non-natural cleavage site is placed at the C-terminal position of the F2 domain. In a preferred embodiment, the second non-natural cleavage site is placed after amino acid 88 or 89 of the F2 domain. In a preferred embodiment, the second non-natural cleavage site is placed after amino acid 88 or 89 of the F2 domain, such that it is cleaved after residue 92 or 93.

[0053] In certain embodiments, the first non-natural cleavage site comprises the amino acid sequence RX1X2R, wherein X1 and X2 can be any amino acids.

[0054] In a preferred embodiment, the first non-native cleavage is a furin cleavage site comprising the sequence RX1[K / R]R.

[0055] In certain preferred embodiments, the F0 protein is included in the p27 peptide of the RSV F protein between the F1 and (truncated) F2 domains, wherein the p27 peptide is included in the first cleavage site at its C-terminal end. In this case, the spacer sequence is therefore (part of) the p27 peptide sequence. The p27 peptide can be any p27 peptide of any RSV F protein, or a variant thereof. In a specific embodiment, the p27 peptide is from RSV A and comprises the amino acid sequence ELPRFMNYTLNNAKKTNVTLSKK R (SEQ ID NO:2, cleavage site underlined), or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2. In certain embodiments, the p27 peptide has an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, and most preferably at least 99% sequence identity to SEQ ID NO:2. In these embodiments, the p27 peptide comprises a first non-natural cleavage site at its C-terminus, and a second non-natural cleavage site is placed at the N-terminus of the p27 peptide. Again, the F2 domain may be truncated at its C-terminal side. The F domain may be truncated after the amino acid residue at position 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 101. In a preferred embodiment, the F2 domain is truncated after the amino acid residue at position 89, followed by a cleavage site, which results in the inclusion of an Arg at position 91 at the C-terminus of F2.

[0056] In other embodiments, the p27 peptide is from RSVB and comprises the amino acid sequence EAPQYMNYTINTTKNLNVSISKK R (SEQ ID NO: 150), or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 150. In certain embodiments, the p27 peptide has an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, and most preferably at least 99% sequence identity to SEQ ID NO: 150.

[0057] In certain embodiments, the F0 protein comprises an optimized variant of the p27 peptide of the RSV F protein positioned between the F1 and (truncated) F2 domains, wherein the p27 peptide comprises a first cleavage site at its C-terminal end.

[0058] In certain embodiments, the p27 peptide has been modified by deleting one or more glycosylation sites in the p27 peptide sequence. The p27 peptide typically has 2 or 3 N-linked glycosylation sites. The glycosylation site is typically an 'NXS / T' motif that allows N-linked glycosylation in mammalian cells. The side of this motif may or may not be a short linker (Gly / Ser). Preferably, all glycosylation sites in the p27 sequence have been deleted. The deletion of the glycosylation site can be accomplished by mutation of certain amino acids in the NXS / T motif so that the glycosylation site no longer exists, or by deleting one or more amino acids so that the glycosylation site no longer exists.

[0059] According to the present invention, it has been shown that using a deglycosylated variant of the p27 peptide, cleavage is improved without the addition of furin.

[0060] In other embodiments, the p27 peptide has been modified by deleting 1-11 amino acids, preferably 9 or 11 amino acids, from the p27 sequence.

[0061] In certain preferred embodiments, the p27 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 185 and 186.

[0062] In certain embodiments, the second non-natural cleavage site comprises the sequence RX1X2R, wherein X1 and X2 can be any amino acids (https: / / doi.org / 10.1371 / journal.pone.0054290).

[0063] In a preferred embodiment, the second non-natural cleavage is a furin cleavage site comprising the sequence RX1[K / R]R, preferably RRRR.

[0064] In addition or alternatively, the protein may include a C-terminal truncated F1 domain. Therefore, in order to obtain a soluble F protein, the TM and cytoplasmic region can be removed to provide an HMPV F extracellular domain. Therefore, in certain embodiments, the protein includes a truncated F1 domain. In a specific embodiment, the truncated F1 domain does not include a transmembrane region and a cytoplasmic region. The F1 domain can be truncated after the amino acid at position 481, 482, 483, 484, 485, 486, 487, 488 or 489. In certain embodiments, the F1 domain is truncated after the amino acid residue at position 481 or 489. Preferably, the F1 domain is truncated after the amino acid residue at position 489. In a preferred embodiment, the truncated F1 domain therefore includes amino acids 103-481 or 103-489 of the HMPV F protein, preferably amino acids 103-489 of the HMPV F protein or is composed thereof.

[0065] As used throughout this application, the positions of amino acid residues (or amino acid positions) are given with reference to the sequence of the HMPV F protein of SEQ ID NO: 1. As used in the present invention, the phrase "amino acid residue at a position such as 88 of the HMPV F protein" therefore means the amino acid corresponding to the amino acid at position 88 in the HMPV F protein of SEQ ID NO: 1. It should be noted that in the numbering system used throughout this application, 1 refers to the N-terminal amino acid of the immature F0 protein (SEQ ID NO: 1), i.e. including the signal peptide. When using HMPV strains other than strain TN / 00 / 3-14 of SEQ ID NO: 1, the amino acid positions of the F protein are numbered with reference to the numbering of the F protein of the strain of SEQ ID NO: 1 by aligning the sequences of the other HMPV strains with the F protein of SEQ ID NO: 1, with the insertion of spaces when necessary. Sequence alignment can be accomplished using methods well known in the art, such as by CLUSTALW, Bioedit or CLC Workbench.

[0066] The amino acid according to the present invention may be any of the twenty naturally occurring (or 'standard' amino acids) or a variant thereof, such as a D-amino acid (the D-enantiomer of an amino acid having a chiral center), or any variant not found naturally in proteins, such as, for example, norleucine. Standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties, such as cysteine, which can form covalent disulfide bonds (or disulfide bridges) with other cysteine ​​residues, proline, which can induce rotation of the protein backbone, and glycine, which is more flexible than other amino acids. Table 2 shows the abbreviations and properties of standard amino acids. The skilled person will appreciate that proteins can be mutated by conventional molecular biology procedures.

[0067] As described above, in certain embodiments (e.g., in the case of soluble F proteins), the proteins of the present invention comprise truncated F1 domains. As used herein, a "truncated" F1 domain refers to an F1 domain that is not a full-length F1 domain, i.e., one or more amino acid residues at the C-terminus have been deleted. In certain embodiments, at least the transmembrane domain and the cytoplasmic domain have been deleted to allow expression as a soluble extracellular domain. Because the TM region is responsible for membrane anchoring and trimerization, "anchorless" (i.e., without TM and cytoplasmic domains) soluble F proteins are monomeric and show low expression. In order to obtain a soluble trimeric F protein in a stable prefusion conformation, the prefusion conformation therefore needs to be stabilized. In order to promote trimerization, it is known to replace the TM / CT region with a heterologous trimerization domain, such as a folding subdomain.

[0068] According to the present invention, a soluble trimeric HMPV pre-fusion F protein is provided that does not contain a heterologous trimerization domain. Therefore, in certain embodiments, the protein according to the present invention comprises one or more stabilizing amino acid residues in the HR2 domain, the HR2 domain comprising amino acids 453 to 484 of the HMPV F precursor (F0) protein. According to the present invention, it has been surprisingly found that, compared to an HMPV F protein that does not contain one or more stabilizing mutations in the HR2 domain, the trimer content is increased by the presence of one or more stabilizing mutations in the HR2 domain, even in the absence of a heterologous trimerization domain.

[0069] As indicated above, the HMPV F protein is typically a homotrimer, i.e., a macromolecular complex formed by three protein monomers (or protomers) that are typically non-covalently associated. In certain embodiments, the one or more stabilizing amino acids in the HR2 domain optimize interprotomer interactions between one or more amino acid residues in the HR2 domains of different HMPV F protomers in the trimer.

[0070] In a preferred embodiment, the amino acid at position 477 is I, V, L or F or M.

[0071] In certain embodiments, in addition, the amino acid residue at position 473 is I, F or W, and / or the amino acid residue at position 474 is or I, and / or the amino acid residue at position 475 is R, and / or the amino acid residue at position 476 is K, and / or the amino acid residue at position 478 is D, and / or the amino acid residue at position 479 is E, and / or the amino acid residue at position 480 is L, and / or the amino acid residue at position 484 is I, and / or the amino acid residue at position 488 is I.

[0072] In a preferred embodiment, the amino acid residue at position 473 is W, the amino acid residue at position 477 is I, and the amino acid residue at position 484 is I.

[0073] In another preferred embodiment, the amino acid residue at position 473 is W, the amino acid residue at position 476 is K, the amino acid residue at position 477 is F, and the amino acid residue at position 484 is I.

[0074] In yet another specific embodiment, the amino acid residue at position 477 is I, V, L or F or M, the amino acid residue at position 473 is I, F or W, the amino acid residue at position 475 is R, the amino acid residue at position 476 is K, the amino acid residue at position 478 is D, the amino acid residue at position 479 is E, and the amino acid residue at position 484 is I.

[0075] To further stabilize the trimeric prefusion HMPV F protein, in certain embodiments, the amino acid residue at position 112 is R, and / or the amino acid residue at position 209 is E, and / or the amino acid residue at position 453 is P or Q.

[0076] In a further embodiment, the amino acid residue at position 149 is Y, and / or the amino acid residue at position 313 is W, and / or the amino acid residue at position 445 is Y.

[0077] In a preferred embodiment, the amino acid residue at position 112 is R, the amino acid residue at position 209 is E, and the amino acid residue at position 453 is P or Q.

[0078] According to the present invention, the HMPV F protein is stabilized (in the pre-trimeric fusion conformation) by introducing one or more modifications, such as addition, deletion or substitution of one or more amino acids. One such stabilizing modification is the addition of at least one non-native cleavage site in the amino acid sequence of the HMPV F0 protein.

[0079] Another stabilizing modification is the introduction of one or more stabilizing amino acids into the HR2 domain.

[0080] According to the present invention, a stable trimeric HMPV protein in a prefusion conformation is therefore provided. Compared to HMPV F proteins that do not contain these modifications, the modifications according to the present invention preferably result in an increase in the stability of the prefusion conformation of the HMPV F trimer. Compared to HMPV F proteins that do not contain modifications, the modifications according to the present invention preferably result in a stable "closed" prefusion F trimer with reduced binding to antibodies (e.g., DS7) for postfusion conformations and / or reduced binding to apex interface binding antibodies MPV458 and MPV465. Additionally or alternatively, compared to HMPV F proteins that do not contain these modifications, the modifications according to the present invention result in increased trimer content, melting temperature and / or trimer stability after storage for two weeks at 4°C and / or 37°C or after a quick-freeze cycle. In particular, compared to HMPV F proteins that do not contain these modifications, the modifications according to the present invention result in increased trimer content and trimer stability after storage for at least 6 months at 4°C.

[0081] Additionally or alternatively, the modifications of the invention result in increased expression levels of the prefusion HMPV F trimer compared to an HMPV F protein not comprising these modifications.

[0082] According to the invention, it has thus been demonstrated that the presence of a specific amino acid at the indicated position increases the stability of the trimeric protein in the prefusion conformation. According to the invention, the specific amino acid may already be present at the indicated position (e.g., in a naturally occurring variant of the HMPV F protein) or may be introduced according to the invention by substitution (mutation) of the amino acid at that position into a specific amino acid residue. According to the invention, the protein comprises one or more mutations in its amino acid sequence, i.e., one or more naturally occurring amino acids at the indicated position have been substituted by another amino acid.

[0083] Certain stabilizing amino acid residues occurring in naturally occurring HMPV F proteins include 231I, 404P, and 368N. Thus, in certain embodiments, the amino acid residue at position 231 is I, the amino acid residue at position 404 is P, and / or the amino acid residue at position 368 is N.

[0084] The protein according to the invention may further comprise one or more additional stabilizing mutations, for example one or more stabilizing mutations that have been described in co-pending patent application EP 21215259. Thus, in certain embodiments, the amino acid residue at position 69 is Y or W, and / or the amino acid residue at position 73 is W, and / or the amino acid residue at position 185 is P, and / or the amino acid residue at position 191 is I, and / or the amino acid residue at position 116 is H, and / or the amino acid residue at position 342 is P.

[0085] Additionally or alternatively, the protein may further comprise one or more non-natural intra-protomer or inter-protomer disulfide bonds, such as described in EP 21215259. In certain embodiments, the one or more disulfide bonds are selected from an intra-protomer disulfide bond between amino acid residues 140 and 147, and / or an intra-protomer disulfide bond between amino acid residues 141 or 161, and / or an intra-protomer disulfide bond between amino acid residues 360 and 459.

[0086] Any and / or all stabilizing modifications can be used individually and / or in combination with any other stabilizing modifications disclosed herein to produce an HMPV F protein according to the present invention. In an exemplary embodiment, the HMPV F protein comprises at least one non-native cleavage site. Additionally or alternatively, the HMPV F protein comprises one or more stabilizing mutations in the HR2 domain. Additionally or alternatively, the HMPV F protein comprises an amino acid sequence in which the amino acid residue at position 112 is R, and / or the amino acid residue at position 209 is E, and / or the amino acid residue at position 453 is P or Q, and / or the amino acid residue at position 149 is Y, and / or the amino acid residue at position 313 is W, and / or the amino acid residue at position 445 is Y.

[0087] Note again that reference is made to SEQ ID NO: 1 for the positions of the amino acid residues. The skilled person will be able to determine the corresponding amino acid residues in the F protein of other HMPV strains.

[0088] As described above, in certain embodiments (e.g., in the case of soluble F proteins), the proteins of the invention comprise a truncated F1 domain. As used herein, a "truncated" F1 domain refers to an F1 domain that is not a full-length F1 domain, i.e., one or more amino acid residues at the C-terminus have been deleted. In certain embodiments, at least the transmembrane domain and the cytoplasmic domain have been deleted to allow expression as a soluble extracellular domain. In certain embodiments, the F1 domain is truncated after amino acid residues 481, 482, 483, 484, 485, 486, 487, 488, or 489.

[0089] In a preferred embodiment, the truncated F1 domain comprises or consists of amino acids 103-489 of the HMPV F protein.

[0090] In a preferred embodiment, the soluble protein according to the invention does not contain a heterologous trimerization domain. However, according to the invention, a heterologous trimerization domain can be linked to a truncated F1 domain, if desired, optionally via a linker sequence. In certain other embodiments, the heterologous trimerization domain is a foldon domain comprising the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 136).

[0091] In a preferred embodiment, the HMPV F0 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-135, SEQ ID NOs: 151-159, SEQ ID NOs: 161-182 and SEQ ID NO: 184, or a fragment thereof. In a particularly preferred embodiment, the HMPV F0 protein comprises an amino acid sequence of SEQ ID NOs: 30, 70, 81, 82, 83, 96, 111, 119, 131, 132, 133, 134, 135, 155, 156, 159, 170, 180 and 184, or a fragment thereof. In a particularly preferred embodiment, the HMPV F0 sequence comprises an amino acid sequence of SEQ ID NOs: 111, 159, 180 or 184, or a fragment thereof. In a preferred embodiment, the HMPV F0 sequence comprises an amino acid sequence of SEQ ID NO: 111, or a fragment thereof. In a preferred embodiment, the HMPV F0 sequence comprises an amino acid sequence of SEQ ID NO: 159, or a fragment thereof. In another preferred embodiment, the HMPV F0 sequence comprises the amino acid sequence of SEQ ID NO: 180 or a fragment thereof. In another preferred embodiment, the HMPV F0 sequence comprises the amino acid sequence of SEQ ID NO: 184 or a fragment thereof. In a preferred embodiment, the amino acid sequence does not comprise any heterologous C-terminal tag sequence.

[0092] The present invention further provides an HMPV F protein comprising at least one modification in the amino acid sequence of the F1 and / or F2 domains, wherein the protein has been cleaved at one or more cleavage sites, resulting in F2 and F1 domains covalently linked by one or more natural disulfide bridges, and wherein the protein is a trimer. As described above, the present invention provides an immature (or inactive) HMPV F0 protein. After expression, in order to be activated, the protein is usually cleaved (or processed) by a protease. The present invention also encompasses processed HMPV F proteins, i.e., HMPV F proteins after cleavage (or processing). Therefore, the present invention also provides HMPV F proteins based on the processing of the HMPV F0 protein as described above, wherein the processed protein has been cleaved at one or more cleavage sites, resulting in an HMPV F protein comprising (truncated) F2 domains and (truncated) F1 domains, the domains being covalently linked by one or more disulfide bridges, thereby forming an F1-F2 dimer. Then, three F1-F2 dimers (each F1-F2 dimer is a protomer) form a homotrimer. It should be understood that in embodiments where the inactive F0 protein comprises a second non-natural cleavage site, the spacer sequence between the first non-natural site and the second non-natural site is removed by cleavage.

[0093] It will also be appreciated that the mature HMPV F protein will not contain a signal sequence.

[0094] In a preferred embodiment, the processed HMPV F protein according to the present invention comprises an F1 domain comprising amino acids 103-481, preferably amino acids 103-489, of the HMPV F0 protein, and an F2 domain comprising amino acids 19-88 of the HMPV F0 protein. In a particularly preferred embodiment, the processed HMPV F protein according to the present invention comprises an F1 domain comprising amino acids 103-489 of the HMPV F0 protein, and an F2 domain comprising amino acids 19-89 of the HMPV F0 protein.

[0095] In a specific embodiment, the F1 domain consists of amino acids 103-489 of the HMPV F0 protein, and the F2 domain consists of amino acids 19-89 of the HMPV F0 protein. According to the present invention, the amino acid sequence of the F1 and / or F2 domain contains one or more modifications compared to the amino acid sequence of the F1 and / or F2 domain of the wild-type HMPV F protein.

[0096] According to the present invention, it has been found that when the F2 domain contains amino acids 19-89, it is preferred that (after cleavage) the amino acid at position 91 is R (as it is in the native sequence). Thus, in certain embodiments, the amino acid at position 89+2 is R: the F2 domain has been truncated after the amino acid at position 89, but the addition of a non-native cleavage site (such as RRRR) preferably results in the retention of R at position 91.

[0097] In certain embodiments, the processed HMPV F protein is derived from an HMPV F0 sequence selected from the group consisting of SEQ ID NOs: 4-135, SEQ ID NOs: 151-159, SEQ ID NOs: 161-182, and SEQ ID NO: 184, i.e., comprises the F1 and F2 domains of an F0 sequence selected from the group consisting of SEQ ID NOs: 4-135, SEQ ID NOs: 151-159, SEQ ID NOs: 161-182, and SEQ ID NO: 184.

[0098] In a particularly preferred embodiment, the processed HMPV F protein comprises an F1 domain comprising amino acids 121-507 of an amino acid sequence selected from SEQ ID NO: 30, 70, 81, 82, 83, 96, 111, 119, 131, 132, 133, 134, 135, 155, 156, 159, 170, 180 and 184, and an F2 domain comprising amino acids 1-89, preferably amino acids 19-89. In a particularly preferred embodiment, the HMPV F protein comprises an F1 domain comprising amino acids 121-507 of an amino acid sequence of SEQ ID NO: 111, 159, 180 or 184, and an F2 domain comprising amino acids 1-89, preferably 19-89.

[0099] As used throughout this application, nucleotide sequences are provided from 5' to 3' direction, and amino acid sequences are provided from N-terminus to C-terminus as is customary in the art. The skilled artisan will appreciate that proteins can be mutated by conventional molecular biology procedures.

[0100] As described above, according to the present invention, the HMPV F protein is stabilized in the prefusion conformation, as measured by, for example, reduced binding to post-fusion specific HMPV F antibodies, such as DS7, and / or reduced binding to open apex interface binders, such as MPV458 and MPV465, compared to an HMPV F protein without the modifications of the present invention.

[0101] Additionally or alternatively, the HMPV F protein according to the invention has an increased trimer content compared to an HMPV F protein without the modification of the invention, as measured by an increase in trimer content in the supernatant or the trimer yield of the purified protein as detected, for example, via analytical SEC.

[0102] Additionally or alternatively, the thermostability of the HMPV F protein is increased compared to an HMPV F protein without the modification of the invention, as measured by, for example, trimer content or melting temperature in the supernatant or melting temperature of the purified protein after heat stress.

[0103] The present invention further provides a nucleic acid molecule encoding an HMPV F protein according to the present invention. The nucleic acid molecule can be a DNA or RNA polynucleotide. In a preferred embodiment, the nucleic acid molecule encoding the protein according to the present invention is codon-optimized for expression in mammalian cells, preferably human cells. The method of codon optimization is known and has been described previously (e.g., WO 96 / 09378). If at least one non-preferred codon is replaced by a more preferred codon compared to the wild-type sequence, the sequence is considered to be codon-optimized. In this article, a non-preferred codon is a codon that is less frequently used in an organism than another codon encoding the same amino acid, and a more preferred codon is a codon that is more frequently used in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in a codon frequency table, such as http: / / www.kazusa.or.jp / codon. Preferably more than one non-preferred codon, preferably most or all non-preferred codons are replaced by more preferred codons. Preferably, the most frequently used codons in an organism are used in a codon-optimized sequence. Replacement by a preferred codon generally results in higher expression.

[0104] It will be appreciated by the skilled person that, due to the degeneracy of the genetic code, numerous different polynucleotides and nucleic acid molecules can encode the same protein. It will also be appreciated that the skilled person can use conventional techniques to perform nucleotide substitutions that do not affect the protein sequence encoded by the nucleic acid molecule to reflect the codon usage of any particular host organism in which the protein is to be expressed. Therefore, unless otherwise indicated, "nucleotide sequences or nucleic acid molecules encoding amino acid sequences" include all nucleotide sequences or nucleic acid molecules that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may or may not include introns.

[0105] In certain embodiments, the nucleic acid molecule according to the invention encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-135, SEQ ID NO: 151-159, SEQ ID NO: 161-182 and SEQ ID NO: 184, or a processed HMPV F protein or fragment thereof derived therefrom. In a particularly preferred embodiment, the nucleic acid molecule encodes a protein comprising an amino acid sequence of SEQ ID NO: 30, 70, 81, 82, 83, 96, 111, 119, 131, 132, 133, 134, 135, 155, 156, 159, 170, 180 and 184, or a processed HMPV F protein or fragment thereof derived therefrom. In a particularly preferred embodiment, the nucleic acid encodes a protein comprising an amino acid sequence of SEQ ID NO: 111, 159, 180 or 184, or a processed HMPV F protein derived therefrom. In a preferred embodiment, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 111 or a processed HMPV F protein derived therefrom. In a preferred embodiment, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 159 or a processed HMPV F protein derived therefrom. In another preferred embodiment, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 180 or a processed HMPV F protein derived therefrom. In another preferred embodiment, the nucleic acid encodes a protein comprising the amino acid sequence of SEQ ID NO: 184 or a processed HMPV F protein derived therefrom.

[0106] Nucleic acid sequences can be cloned using conventional molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using conventional procedures by service companies operating in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScripts, Invitrogen, Eurofins).

[0107] As mentioned above, the nucleic acid molecule as described herein can be an RNA polynucleotide (or RNA). The RNA can be mRNA, modified mRNA, self-replicating RNA or circular mRNA.

[0108] Preferred RNA is self-replicating.Even when being delivered to the vertebrate cell that does not contain any protein, the RNA molecule (replicon) of self-replication can also cause by the multiple sub-RNA generation of transcription (antisense copy generated by itself via it) from itself.The RNA molecule of self-replication is usually + chain molecule, it can be directly translated after being delivered to the cell, and this translation provides RNA dependent RNA polymerase, and it produces antisense transcript and sense transcript by the RNA delivered subsequently.Therefore, the RNA delivered causes the generation of multiple sub-RNA.These sub-RNAs and colinear sub-genomic transcripts can be self-translated to provide the in situ expression of encoding immunogen, or can be transcribed to provide further transcripts with the same meaning as the RNA delivered, and it is translated to provide the in situ expression of immunogen.The overall result of this transcription sequence is the substantial amplification of the replicon RNA number introduced, and therefore the coded immunogen becomes the main polypeptide product of cell.

[0109] A suitable system for realizing self-replication is to use an RNA replicon based on alphavirus. These + chain replicons are translated after being delivered to the cell to provide replicase (or replicase-transcriptase). Replicase is translated into a polyprotein that self-cuts to provide replication complex, and the replication complex produces + genome-chain copies of chain delivery RNA. These-chain transcripts themselves can be transcribed to provide more copies of + chain parent RNA, and also provide subgenomic transcripts of encoding immunogens. Therefore, the translation of subgenomic transcripts results in the in situ expression of the immunogen by infected cells. Suitable alphavirus replicons can use replicase from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild-type virus sequences can be used, for example, the attenuated TC83 mutant of VEEV has been used in replicons.

[0110] Thus, a preferred self-replicating RNA molecule encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule, and (ii) an HPMV F protein according to the invention. The polymerase may be an alphavirus replicase, e.g. comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3 and nsP4.

[0111] Although the natural alphavirus genome encodes structural virion proteins in addition to the nonstructural replicase polyprotein, it is preferred that the self-replicating RNA molecules of the present invention do not encode alphavirus structural proteins. Therefore, the preferred self-replicating RNA can lead to the production of its own genomic RNA copies in the cell, but does not produce RNA-containing virions. The inability to produce these virions means that, unlike the wild-type alphavirus, the self-replicating RNA molecules cannot continue themselves in an infectious form. The alphavirus structural proteins required for continuation in the wild-type virus are not present in the self-replicating RNA of the present invention, and their positions are replaced by genes encoding the immunogen of interest, so that the subgenomic transcripts encode the immunogen instead of the structural alphavirus virion proteins.

[0112] Thus, a self-replicating RNA molecule useful for the present invention may have two open reading frames. The first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes an immunogen. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, e.g., encoding further immunogens (see below) or encoding additional polypeptides.

[0113] The self-replicating RNA molecule may have a 5' sequence that is compatible with the encoded replicase. The self-replicating RNA molecules may have a variety of lengths, but they are typically 5000-25000 nucleotides long, such as 8000-15000 nucleotides, or 9000-12000 nucleotides.

[0114] RNA molecules useful for the invention may have a 5' cap (e.g., 7-methylguanosine). The cap may enhance the in vivo translation of the RNA. The 5' nucleotides of RNA molecules useful for the invention may have a 5' triphosphate group. In the capped RNA, this may be linked to the 7-methylguanosine via a 5'-5' bridge.

[0115] The RNA molecule can have a 3' polyadenylic acid tail. It can also include a polyadenylic acid polymerase recognition sequence (e.g., AAUAAA) near its 3' end. The RNA molecule useful for the present invention is usually single-stranded. Single-stranded RNA can generally initiate adjuvant effect by binding to TLR7, TLR8, RNA helicase and / or PKR. The RNA (dsRNA) delivered in double-stranded form can bind to TLR3, and the receptor can also be triggered by the dsRNA formed during the replication of the single-stranded RNA or in the secondary structure of the single-stranded RNA.

[0116] RNA molecules useful for the present invention can be conveniently prepared by in vitro transcription (IVT). IVT can use a (cDNA) template that is produced and propagated in bacteria as a plasmid, or a (cDNA) template that is synthetically produced (e.g., by gene synthesis and / or polymerase chain reaction modification methods). For example, a DNA-dependent RNA polymerase (e.g., bacteriophage T7, T3, or SP6 RNA polymerase) can be used to transcribe RNA from a DNA template. Appropriate end-capping and polyadenylation addition reactions can be used as required (although the polyadenylation of the replicon is usually encoded within the DNA template). These RNA polymerases can have strict requirements for the 5' nucleotides of the transcription, and in some embodiments, these requirements must be matched with those of the encoded replicase to ensure that the RNA transcribed by the IVT can effectively serve as a substrate for its self-encoded replicase.

[0117] The self-replicating RNA may include (in addition to any 5' cap structure) one or more nucleotides having a modified nucleobase. Thus, the RNA may contain m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), mlA (1-methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycylamino)adenosine); =Methionyl adenosine); t6A (N6-threonylaminoformyl adenosine); ms2t6A (2-methylthio-N6-threonylaminoformyl adenosine); m6t6A (N6-methyl-N6-threonylaminoformyl adenosine); hn6A (N6.-hydroxynorvalylaminoformyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyl adenosine); Ar(p) (2'-O-ribosyl adenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4- Acetylcytidine); f5C (5-formylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4 acetyl 2TO methylcytidine); k2C (lysine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (whitingoside); o2yW (peroxywhitingoside); OHyW (hydroxywhitingoside); OHyW * (unmodified hydroxy wyoside); imG (wyoside); mimG (methylguanosine); Q (quercetin); oQ (epoxyquercetin); galQ (galactosylquercetin); manQ (mannosylquercetin); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaurine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl) uridine); ho5U (5-hydroxyuridine);mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine) glycoside); mnm5U (5-methylaminomethyl uridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-seleno-uridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine); cmnm5U (5-carboxymethylaminomethyl uridine); cnmm5Um (5-carboxymethylaminomethyl-2-LO-methyl uridine); cmnm5s2U (5-carboxymethylaminomethyl methyl-2-thiouridine); m62A (N6, N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4, 2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6, TO-dimethyladenosine); rn62Am (N6, N6, 0-2-trimethyladenosine); m2'7G (N2, 7-dimethylguanosine); m2'2 '7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); m1Gm (1,2'-O-dimethylguanosine); m'Am (1,2-O-dimethyladenosine)irinomethyluridine); tm5s2U (S-taurine methyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine);or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxoadenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2 In some embodiments, the self-replicating RNA may include 2-deaza purines, ... However, in some embodiments, the RNA does not include modified nucleobases, and may not include modified nucleotides, i.e., all nucleotides in the RNA are standard A, C, G, and U ribonucleotides (except for any 5' cap structure that may include 7'-methylguanosine). In other embodiments, the RNA may include a 5' cap comprising 7'-methylguanosine, and the first 1, 2, or 3 5' ribonucleotides may be methylated at the 2' position of the ribose. The RNA used with the present invention ideally includes only phosphodiester linkages between nucleosides, but in some embodiments, it may contain phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. Ideally, the liposome includes less than 10 different RNA species, such as 5, 4, 3, or 2 different species; most preferably, the liposome includes a single RNA species, i.e., all RNA molecules in the liposome have the same sequence and the same length. ;

[0118] In certain embodiments, the nucleic acid molecule is an RNA polynucleotide having an open reading frame encoding the HPMV F protein or a fragment thereof according to the present invention, and can be formulated into cationic lipid nanoparticles, cationic nanoemulsions, or polymer-based formulations, or any combination or alternative formulations suitable for bringing saRNA into human or animal cells in vitro or in vivo in different species, including humans.

[0119] In some embodiments, the RNA replicons of the present disclosure can be formulated using one or more liposomes, lipid complexes and / or lipid nanoparticles. In some embodiments, the liposome or lipid nanoparticle formulations described herein can include a polycationic composition. In some embodiments, formulations including polycationic compositions can be used for in vivo and / or ex vivo delivery of the RNA replicons described herein.

[0120] The present invention therefore further provides an RNA replicon encoding a recombinant pre-fusion HMPV F protein or a fragment thereof, wherein the HMPV F protein comprises an amino acid sequence selected from SEQ ID NO: 180 or 184, or a fragment or variant thereof.

[0121] In certain aspects, the RNA replicon comprises, from 5' end to 3' end:

[0122] (1) The 5' untranslated region (5'-UTR) required for nonstructural protein-mediated RNA virus amplification;

[0123] (2) a polynucleotide sequence encoding at least one, preferably all, non-structural proteins of an RNA virus;

[0124] (3) Subgenomic promoters of RNA viruses;

[0125] (4) a polynucleotide sequence encoding a recombinant pre-fusion HMPV F protein or a fragment or variant thereof; and

[0126] (5) 3' untranslated region (3'-UTR) required for nonstructural protein-mediated RNA virus amplification.

[0127] In certain aspects, the RNA replicon comprises, from 5' end to 3' end:

[0128] (1) 5' untranslated region (5'-UTR) of alphavirus,

[0129] (2) 5' replication sequence of the nonstructural gene nsp1 of alphavirus,

[0130] (3) Downstream loop (DLP) motif of viral species,

[0131] (4) a polynucleotide sequence encoding an autoprotease peptide,

[0132] (5) a polynucleotide sequence encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4,

[0133] (6) Alphavirus subgenomic promoter,

[0134] (7) a polynucleotide sequence encoding the recombinant pre-fusion HMPV F protein or a fragment thereof,

[0135] (8) alphavirus 3' untranslated region (3'UTR), and

[0136] (9) Optional polyadenylation sequence.

[0137] In certain aspects, provided herein is an RNA replicon comprising, from the 5' end to the 3' end:

[0138] (1) a 5′-UTR having a polynucleotide sequence of SEQ ID NO: 136,

[0139] (2) a 5' replicating sequence having the polynucleotide sequence of SEQ ID NO: 137,

[0140] (3) a DLP motif comprising the polynucleotide sequence of SEQ ID NO: 138,

[0141] (4) a polynucleotide sequence encoding the P2A sequence of SEQ ID NO: 139,

[0142] (5) polynucleotide sequences encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4 having the nucleic acid sequences of SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142 and SEQ ID NO: 143, respectively,

[0143] (6) a subgenomic promoter having a polynucleotide sequence of SEQ ID NO: 144,

[0144] (7) a polynucleotide sequence encoding a pre-fusion HMPV F protein having an amino acid sequence selected from SEQ ID NO: 180 and SEQ ID NO: 184, or a fragment or variant thereof, and

[0145] (8) A 3′UTR having the polynucleotide sequence of SEQ ID NO: 145.

[0146] In certain aspects, (a) the polynucleotide sequence encoding the P2A sequence comprises SEQ ID NO: 146, and the RNA replicon further comprises a poly(A) sequence, preferably, the poly(A) sequence has SEQ ID NO: 147 at the 3' end of the replicon.

[0147] In certain aspects, the RNA replicon comprises a polynucleotide sequence of SEQ ID NO: 187 or 188. In a preferred embodiment, the polynucleotide sequence 7972-9649 of SEQ ID NO: 187 is a coding sequence of an HMPV F protein (including a stop codon), in particular an HMPV protein of SEQ ID NO: 184. In another preferred embodiment, the polynucleotide sequence 7972-9500 of SEQ ID NO: 188 is a coding sequence of an HMPV F protein (including a stop codon), in particular an HMPV protein of SEQ ID NO: 184.

[0148] Also provided is a nucleic acid comprising a DNA sequence encoding the RNA replicon described herein, preferably, the nucleic acid further comprises a T7 promoter operably linked to the 5' end of the DNA sequence, more preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO:148.

[0149] As used herein, the term "fragment" refers to a protein or (poly)peptide having an amino-terminal and / or carboxyl-terminal and / or internal deletion, but wherein the remaining amino acid sequence is identical to the sequence of the HMPV F protein, e.g., the corresponding position in the full-length sequence of the HMPV F protein. It will be appreciated that in order to induce an immune response and in general for vaccination purposes, the protein need not be full-length, nor need it have all its wild-type functions, and that fragments of the protein are equally useful.

[0150] The fragment according to the invention is an immunologically active fragment and generally comprises at least 15 amino acids or at least 30 amino acids of the HMPV F protein. In certain embodiments, it comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or 550 amino acids of the HMPV F protein.

[0151] It will also be appreciated by those skilled in the art that proteins may be altered, for example, by amino acid substitutions, deletions, additions, etc., for example, using conventional molecular biology procedures. Generally, conservative amino acid substitutions may be applied without loss of function or immunogenicity of the polypeptide. This can be readily checked according to conventional procedures well known to the skilled artisan.

[0152] It will be appreciated by the skilled person that, due to the degeneracy of the genetic code, numerous different nucleic acids can encode the same polypeptide or protein. It will also be appreciated that the skilled person can use conventional techniques to perform nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acid to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed. Therefore, unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

[0153] Nucleic acid sequences can be cloned using conventional molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using conventional procedures by service companies operating in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Invitrogen, Eurofins).

[0154] The present invention also provides the vector comprising nucleic acid molecule as above.In certain embodiments, therefore, nucleic acid molecule according to the present invention is the part of vector.Such vector can be easily operated by the method well known to those skilled in the art, and can be for example designed to be able to replicate in prokaryotic cells and / or eukaryotic cells.In addition, many vectors can be used to transform eukaryotic cells, and will be integrated into the genome of such cells in whole or in part, resulting in a stable host cell comprising the desired nucleic acid in its genome.The vector used can be any vector suitable for cloning DNA and can be used to transcribe the nucleic acid of interest.

[0155] Preferably, the vector is a self-replicating RNA replicon.

[0156] As used herein, "self-replicating RNA molecules", which can be used interchangeably with "self-amplifying RNA molecules" or "RNA replicons" or "replicon RNAs" or "saRNAs", refer to RNA molecules modified from the genome of positive-strand RNA viruses, which contain all the genetic information required to direct their own amplification or self-replication in permissive cells. Self-replicating RNA molecules are similar to mRNA. It is single-stranded, 5' capped and 3' polyadenylated and has positive orientation. In order to direct its own replication, the RNA molecule 1) encodes a polymerase, a replicase or other protein that can interact with proteins, nucleic acids or ribonucleoproteins derived from viruses or host cells to catalyze the RNA amplification process; and 2) contains cis-acting RNA sequences required for the replication and transcription of subgenomic replicon-encoding RNAs. Therefore, the delivered RNA results in the production of multiple sub-RNAs. These sub-RNAs, as well as colinear subgenomic transcripts, can be translated by themselves to provide in situ expression of the gene of interest, or can be transcribed to provide further transcripts with the same meaning as the delivered RNA, which are translated to provide in situ expression of the gene of interest. The overall result of this transcription sequence is a substantial amplification of the number of introduced replicon RNAs, and thus the encoded gene of interest becomes the major polypeptide product of the cell.

[0157] In one embodiment, the RNA replicon of the present application comprises, from the 5' end to the 3' end, the following: (1) a 5' untranslated region (5'-UTR) required for non-structural protein-mediated amplification of RNA viruses; (2) a polynucleotide sequence encoding at least one, preferably all, non-structural proteins of an RNA virus; (3) a subgenomic promoter of an RNA virus; (4) a polynucleotide sequence encoding a recombinant pre-fusion HMPV F protein or a fragment or variant thereof; and (5) a 3' untranslated region (3'-UTR) required for non-structural protein-mediated amplification of RNA viruses.

[0158] In certain embodiments, the self-replicating RNA molecule encodes an enzyme complex (replicase polyprotein) for self-amplification, which includes RNA-dependent RNA polymerase function, helicase, capping and polyadenylation activity. The viral structural gene downstream of the replicase under the control of the subgenomic promoter can be replaced by the HMPV F protein or its fragment or variant before fusion described herein. After transfection, the replicase is immediately translated, interacts with the 5' and 3' ends of the genomic RNA, and synthesizes complementary genomic RNA copies. They serve as templates for synthesizing novel positive strands, capping and polyadenylated genomic copies and subgenomic transcripts. Amplification ultimately results in an extremely high RNA copy number of up to 2 × 105 copies / cell. Therefore, compared with conventional mRNA, much less saRNA is sufficient to achieve effective gene transfer and protective vaccination (Beissert et al., Hum Gene Ther. 2017, 28 (12): 1138–1146).

[0159] Subgenomic RNA is an RNA molecule that is smaller in length or size than the genomic RNA from which it is derived. Viral subgenomic RNA can be transcribed by an internal promoter, whose sequence is located within the genomic RNA or its complement. Transcription of subgenomic RNA can be mediated by a virally encoded polymerase associated with a host cell-encoded protein, ribonucleoprotein, or a combination thereof. Numerous RNA viruses generate subgenomic mRNA (sgRNA) for expression of their 3'-proximal genes.

[0160] In some embodiments of the present disclosure, the HMPV F protein or fragment thereof before fusion described herein is expressed under the control of a subgenomic promoter. In certain embodiments, instead of a natural subgenomic promoter, the subgenomic RNA may be placed under the control of an internal ribosome entry site (IRES) derived from encephalomyocarditis virus (EMCV), bovine viral diarrhea virus (BVDV), poliovirus, foot-and-mouth disease virus (FMD), enterovirus 71, or hepatitis C virus. Subgenomic promoters range from 24 nucleotides (Sindbis virus) to more than 100 nucleotides (beet necrotic yellow vein virus), and are typically found upstream of transcription initiation.

[0161] In some embodiments, the RNA replicon includes a coding sequence for at least one, at least two, at least three or at least four nonstructural viral proteins (e.g., nsP1, nsP2, nsP3, nsP4). The alphavirus genome encodes nonstructural proteins nsP1, nsP2, nsP3 and nsP4, which are produced as a single polyprotein precursor, sometimes referred to as P1234 (or nsP1-4 or nsP1234), and are processed and cleaved into mature proteins by proteolytic processing. The size of nsP1 can be about 60 kDa, and may have methyltransferase activity and be involved in viral capping reactions. nsP2 has a size of about 90 kDa, and may have helicase and protease activity, while nsP3 is about 60 kDa and contains three domains: a macrodomain, a central (or alphavirus-specific) domain and a hypervariable domain (HVD). The size of nsP4 is about 70 kDa, and contains a core RNA-dependent RNA polymerase (RdRp) catalytic domain. After infection, the alphavirus genomic RNA is translated to produce the P1234 polyprotein, which is cleaved into individual proteins.When disclosing nucleic acid or polypeptide sequences herein, such as sequences of nsP1, nsP2, nsP3, nsP4, sequences considered to be based on or derived from the original sequences are also disclosed.

[0162] In some embodiments, the RNA replicon includes a coding sequence for a portion of at least one nonstructural viral protein. For example, the RNA replicon may include about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or any two of these values ​​of the coding sequence for at least one nonstructural viral protein. In some embodiments, the RNA replicon may include a coding sequence for a substantial portion of at least one nonstructural viral protein. As used herein, the "substantial portion" of a nucleic acid sequence encoding a nonstructural viral protein includes sufficient nucleic acid sequences encoding nonstructural viral proteins to provide the protein or by manually evaluating the sequence through a person skilled in the art, or by using an algorithm such as BLAST's computer automated sequence comparison and identification (see, e.g., in "Basic Local Alignment Search Tool"; Altschul SF et al., J. Mol. Biol. 215: 403-410, 1993) of the putative identification. In some embodiments, the RNA replicon may include the entire coding sequence for at least one nonstructural protein. In some embodiments, the RNA replicon comprises substantially all coding sequences for natural viral nonstructural proteins. In certain embodiments, one or more nonstructural viral proteins are derived from the same virus. In other embodiments, one or more nonstructural proteins are derived from different viruses.

[0163] RNA replicon can be derived from any suitable positive-strand RNA virus, such as alphavirus or flavivirus. Preferably, RNA replicon is derived from alphavirus. The term "alphavirus" describes an enveloped single-stranded positive RNA virus of the Togaviridae family. The genus Alphavirus contains about 30 members, which can infect humans and other animals. Alphavirus particles usually have a diameter of 70nm, tend to be spherical or slightly polymorphic, and have a 40nm equidistant nucleocapsid. The total genome length of alphavirus ranges from 11,000 to 12,000 nucleotides, and has a 5' cap and a 3' polyadenylic acid tail. There are two open reading frames (ORFs) in the genome, non-structural (ns) and structural. The ns ORF encodes proteins (nsP1-nsP4) required for viral RNA transcription and replication. The structural ORF encodes three structural proteins: core nucleocapsid protein C, and envelope proteins P62 and E1 combined as heterodimers. The surface glycoprotein anchored by the viral membrane is responsible for receptor recognition and enters the target cell through membrane fusion. The four ns protein genes are encoded by genes in the 5′ two-thirds of the genome, while the three structural proteins are translated from subgenomic mRNAs that are colinear with the 3′ one-third of the genome.

[0164] In some embodiments, the self-replicating RNA that can be used for the present invention is an RNA replicon derived from an alphavirus species. In some embodiments, the alphavirus RNA replicon is an alphavirus belonging to the VEEV / EEEV group, or the SF group or the SIN group. Non-limiting examples of SF group alphaviruses include Semliki Forest Virus, O'Nyong-Nyongvirus, Ross River Virus, Middelburg Virus, Chikungunya Virus, Bama Forest Virus, Geta Virus, Mayaro Virus, Lushan Virus, Bebaru Virus and Una Virus. Non-limiting examples of SIN group alphaviruses include Sindbis virus, Girdwood SA virus, South African arbovirus No.86, Ockelbo virus, Aola virus, Babanken virus, Whataroa virus and Kyz virus. Non-limiting examples of VEEV / EEEV group alphaviruses include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Evergladesvirus (EVEV), Mukambu virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), Oneonion virus (ONNV), Ross River virus (RRV), Bama Forest virus (BF), Geta virus (GET), Sao Mountain virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), and Una virus (UNAV).

[0165] Non-limiting examples of Alphavirus species include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mukumbu virus (MUCV), Semliki Forest virus (SFV), Pixona virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), Oneonion virus (ONNV), Ross River virus (RRV), Bama Forest virus (BF), Geta virus (GET), Sao Mountain virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Ora virus (AURAV), Wodaro River virus (WHAV), Babancon virus (BABV), Kyz virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus. Both virulent and avirulent alphavirus strains are suitable. In some embodiments, the alphavirus RNA replicon is a replicon of Sindbis virus (SIN), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), or Eastern equine encephalitis virus (EEEV). In some embodiments, the alphavirus RNA replicon is Venezuelan equine encephalitis virus (VEEV).

[0166] In certain embodiments, the self-replicating RNA molecule comprises a polynucleotide encoding one or more nonstructural proteins nsp1-4, a subgenomic promoter, such as a 26S subgenomic promoter, and a gene of interest encoding a prefusion HMPV F protein or a fragment thereof described herein.

[0167] The self-replicating RNA molecule may have a 5' cap (eg, 7-methylguanosine). The cap may enhance the in vivo translation of the RNA.

[0168] The 5' nucleotide of the self-replicating RNA molecule useful for the invention may have a 5' triphosphate group. In capped RNA, this may be linked to 7-methylguanosine via a 5'-to-5' bridge. The 5' triphosphate may enhance RIG-I binding.

[0169] The self-replicating RNA molecule may have a 3' poly(A) tail. It may also include a poly(A) polymerase recognition sequence (eg, AAUAAA) near its 3' end.

[0170] In any embodiment of the present disclosure, the RNA replicon may lack (or contain) the coding sequence of at least one (or all) structural viral proteins (e.g., nucleocapsid protein C and envelope proteins P62, 6K and E1). In these embodiments, the sequence encoding one or more structural genes may be replaced by one or more heterologous sequences, such as the coding sequence of the pre-fusion HMPV F protein or fragment thereof as described herein. In a preferred embodiment, the RNA replicon lacks (or contains) all coding sequences for structural viral proteins.

[0171] In certain embodiments, the self-replicating RNA vectors of the present application comprise one or more features to confer resistance to translational inhibition by the innate immune system or otherwise increase expression of a GOI (e.g., a prefusion HMPV F protein or fragment or variant thereof described herein).

[0172] In certain embodiments, RNA sequences can be codon optimized to improve translation efficiency. In view of the present disclosure, RNA molecules can be modified by any method known in the art to enhance stability and / or translation, such as by adding a polyadenylic acid tail of, for example, at least 30 adenosine residues; and / or capping the 5-end with a modified nucleotide, such as a 7-methylguanosine cap, which can be incorporated during RNA synthesis or enzymatically modified after RNA transcription.

[0173] In certain embodiments, the RNA replicon of the present application comprises, in order from the 5' end to the 3' end: (1) an alphavirus 5' untranslated region (5'-UTR), (2) a 5' replication sequence of the alphavirus nonstructural gene nsp1, (3) a downstream loop (DLP) motif of the virus species, (4) a polynucleotide sequence encoding a self-protease peptide, (5) a polynucleotide sequence encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4, (6) an alphavirus subgenomic promoter, (7) a polynucleotide sequence encoding a recombinant pre-fusion HMPV F protein or a fragment or variant thereof, (8) an alphavirus 3' untranslated region (3'UTR), and (9) an optional polyadenylation sequence.

[0174] In certain embodiments, the self-replicating RNA vector of the present application comprises a downstream loop (DLP) motif of a viral species. As used herein, "downstream loop" or "DLP motif" refers to a polynucleotide sequence comprising at least one RNA stem loop, when placed downstream of the start codon of an open reading frame (ORF), compared to an otherwise identical construct without the DLP motif, the polynucleotide sequence provides an increase translation of the ORF. For example, members of the genus Alphavirus can resist the activation of antiviral RNA activated protein kinase (PKR) by means of the dominant RNA structure present in the viral 26S transcript, which allows the eIF2-independent translation initiation of these mRNAs. The structure is called a downstream loop (DLP), which is located downstream of AUG in SINV 26S mRNA. DLP is also detected in Semliki Forest Virus (SFV). Similar DLP structures have been reported in at least 14 other members of the genus Alphavirus, including New World (e.g., MAYV, UNAV, EEEV (NA), EEEV (SA), AURAV) and Old World (SV, SFV, BEBV, RRV, SAG, GETV, MIDV, CHIKV, and ONNV) members. The predicted structures of these alphavirus 26S mRNAs were constructed based on SHAPE (selective 2'-hydroxyl acylation and primer extension) data (Toribio et al., Nucleic Acids Res. May 19; 44(9): 4368-80, 2016), the contents of which are hereby incorporated by reference). Stable stem-loop structures were detected in all cases except CHIKV and ONNV, while MAYV and EEEV showed less stable DLPs (Toribio et al., 2016 supra). In the case of Sindbis virus, the DLP motif is found in the first 150 nt of the Sindbis genomic RNA. The hairpin is located downstream of the AUG start codon of the Sindbis capsid (AUG is located at nt50 of the Sindbis genomic RNA). Previous studies of sequence comparison and structural RNA analysis have revealed the evolutionary conservation of DLP in SINV, and predicted that there are equivalent DLP structures in many members of the genus Alphavirus (see, e.g., Ventoso, J. Virol. 9484-9494, Vol. 86, September 2012). Examples of self-replicating RNA vectors comprising DLP motifs are described in U.S. Patent Application Publication US2018 / 0171340 and International Patent Application Publication WO2018106615, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the replicon RNA of the present application comprises a DLP motif showing at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 138.

[0175] In one embodiment, the self-replicating RNA molecule also contains a coding sequence for a self-protease peptide operably connected to the DLP motif downstream and the non-structural protein (e.g., one or more of nsp1-4) or the coding sequence upstream of the gene of interest (e.g., HMPV F protein or its fragment before fusion described herein). Examples of self-protease peptides include, but are not limited to, peptide sequences selected from: porcine Teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis virus A (ERAV) 2A (E2A), thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie virus (Flacherie Virus) 2A (BmIFV2A) and combinations thereof. In some embodiments, the replicon RNA of the present application comprises a coding sequence for P2A having an amino acid sequence of SEQ ID NO: 139. Preferably, the coding sequence exhibits at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence shown in SEQ ID NO:146.

[0176] Any replicon of the present invention may also include 5' and 3' untranslated regions (UTRs). UTRs may be wild-type New World or Old World alphavirus UTR sequences, or sequences derived therefrom. In various embodiments, 5'UTRs may have any suitable length, such as about 60nt or 50-70nt or 40-80nt. In some embodiments, 5'UTRs may also have a conservative primary structure or secondary structure (e.g., one or more stem loops), and may participate in the replication of alphavirus or replicon RNA. In some embodiments, 3'UTRs may be as many as several hundred nucleotides, such as 50-900 or 100-900 or 50-800 or 100-700 or 200nt-700nt. 3'UTRs may also have a secondary structure, such as a stem loop (step loop), and may be followed by a polyadenylic acid bundle or a polyadenylic acid tail. In any embodiment of the present invention, 5' and 3' untranslated regions may be operably linked to any other sequence encoded by the replicon. The UTR can be operably linked to a promoter and / or a sequence encoding a heterologous protein or peptide by providing sequences and intervals required for recognition and transcription of other coding sequences. In view of the present disclosure, any polyadenylation signal known to those skilled in the art can be used. For example, the polyadenylation signal can be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal.

[0177] In another embodiment, the self-replicating RNA replicon of the present application comprises a modified 5' untranslated region (5'-UTR), preferably, the RNA replicon lacks at least a portion of the nucleic acid sequence encoding the viral structural protein. For example, the modified 5'-UTR may be included in one or more nucleotide substitutions at positions 1, 2, 4 or a combination thereof. Preferably, the modified 5'-UTR includes a nucleotide substitution at position 2, and more preferably, the modified 5'-UTR has a U->G or U->A substitution at position 2. Examples of such self-replicating RNA molecules are described in U.S. Patent Application Publication US2018 / 0104359 and International Patent Application Publication WO2018075235, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the replicon RNA of the present application comprises a 5'-UTR showing at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the sequence shown in SEQ ID NO:136.

[0178] In some embodiments, the RNA replicon of the present application comprises a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream or at the 5' end of the polynucleotide sequence encoding the HMPV F protein or its fragment before fusion. The signal peptide generally guides the localization of the protein, promotes the secretion of the protein from the cell in which it is produced, and / or improves antigen expression and cross-presentation to antigen-presenting cells. When expressed from a replicon, the signal peptide may be present at the N-terminus of the HMPV F protein or its fragment before fusion, but, for example, when secreted from a cell, it is cut off by a signal peptidase. The expressed protein in which the signal peptide has been cut is often referred to as a "mature protein". In view of the present disclosure, any signal peptide known in the art can be used. For example, the signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as an Ig heavy chain γ signal peptide SPIgG, an Ig heavy chain ε signal peptide SPIgE or a short leader peptide sequence of HMPV. An exemplary nucleic acid sequence encoding a signal peptide is shown in SEQ ID NO: 149.

[0179] In various embodiments, the RNA replicons disclosed herein can be modified, synthetic or recombinant RNA replicons. As non-limiting examples, the RNA replicons can be one or more of the following: 1) synthesized or modified in vitro, for example, using chemical or enzymatic techniques, such as by using chemical nucleic acid synthesis, or by using enzymes for replication, polymerization, exonuclease digestion, endonuclease digestion, ligation, reverse transcription, transcription, base modification (including, for example, methylation) or recombination (including homologous and site-specific recombination) of nucleic acid molecules; 2) contiguous nucleotide sequences that are not contiguous in nature; 3) modified using molecular cloning techniques so that it lacks one or more nucleotides relative to a naturally occurring nucleotide sequence; and 4) manipulated using molecular cloning techniques so that it has one or more sequence changes or rearrangements relative to a naturally occurring nucleotide sequence.

[0180] Any component or sequence of RNA replicon can be operably connected to any other component or sequence.The component or sequence of RNA replicon can be operably connected, for expressing the ability of gene of interest and / or replicon self-replication in the organism of host cell or treatment.As used herein, the term "operably connected" should be understood in its most extensive reasonable context, and refers to the connection of polynucleotide elements with functional relationship.When a polynucleotide is placed in a functional relationship with another polynucleotide, it is "operably connected".For example, when there is a suitable enzyme, a promoter or UTR operably connected to a coding sequence can affect the transcription and expression of the coding sequence.Promoter need not be adjacent to the coding sequence, as long as it functions to guide its expression.Therefore, the operably connected between the RNA sequence encoding heterologous protein or peptide and a regulatory sequence (for example, promoter or UTR) is a functional connection that allows the expression of polynucleotides of interest. Operably linked can also refer to sequences such as encoding RdRp (e.g., nsP4), nsP1-4, UTR, promoter and other coding sequences in the RNA replicon being linked so that they allow transcription and translation of the HMPV F protein before fusion and / or replication of the replicon. The UTR can be operably linked by providing sequences and spacing required for ribosomal recognition and translation by other coding sequences.

[0181] The immunogenicity of a prefusion HMPV F protein, or a fragment or variant thereof, expressed by an RNA replicon can be determined by a variety of assays known to those of ordinary skill in the art in view of the present disclosure.

[0182] Another general aspect of the present application relates to a nucleic acid comprising a DNA sequence encoding the RNA replicon of the present application. The nucleic acid can be, for example, a fragment of a DNA plasmid or a linearized DNA plasmid. Preferably, the nucleic acid further comprises a promoter, such as a T7 promoter, operably linked to the 5' end of the DNA sequence. More preferably, the T7 promoter comprises the nucleotide sequence of SEQ ID NO:148. In view of the present disclosure, the nucleic acid can be used to produce the RNA replicon of the present application using methods known in the art. For example, the RNA replicon can be obtained by in vivo or in vitro transcription of the nucleic acid.

[0183] Host cells containing RNA replicons or nucleic acids encoding RNA replicons of the present application also form part of the present invention. HMPV F protein or its fragments or variants can be produced by recombinant DNA technology, which involves expressing molecules in host cells, or transgenic animals or plants, such as Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells or yeast, fungi, insect cells, etc. In certain embodiments, cells are from multicellular organisms, and in certain embodiments, they have vertebrate or invertebrate origins. In certain embodiments, cells are mammalian cells such as human cells or insect cells. In general, producing recombinant proteins such as HMPV F protein of the present invention or its fragments or variants in host cells includes introducing heterologous nucleic acid molecules encoding proteins in an expressible form into host cells, culturing cells under conditions conducive to the expression of nucleic acid molecules, and allowing the expression of proteins or their fragments or variants in the cells. Nucleic acid molecules encoding proteins in an expressible form can be in the form of expression cassettes, and sequences capable of reaching nucleic acid expression, such as enhancers, promoters, polyadenylation signals, etc. are generally required. Those skilled in the art know that various promoters can be used to obtain expression of genes in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viral, prokaryotic or eukaryotic sources, or artificially designed.

[0184] In certain embodiments of the present invention, the vector is an adenovirus vector. The adenovirus according to the present invention belongs to the Adenoviridae family, and is preferably an adenovirus belonging to the genus Mastadenovirus. It can be a human adenovirus, or it can be an adenovirus that infects other species, including but not limited to bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), canine adenovirus (e.g., CAdV2), porcine adenovirus (e.g., PAdV3 or 5), or simian adenovirus (which includes monkey adenovirus and simian adenovirus, such as chimpanzee adenovirus or gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV or AdHu), or a simian adenovirus such as a chimpanzee or gorilla adenovirus (ChAd, AdCh or SAdV), or a rhesus adenovirus (RhAd). In the present invention, if it is referred to as Ad without indicating a species, it means a human adenovirus, for example, the abbreviated notation "Ad26" means the same as HAdV26, which is a human adenovirus serotype 26. In addition, as used herein, the notation "rAd" means recombinant adenovirus, for example, "rAd26" means recombinant human adenovirus 26.

[0185] The most advanced studies have been performed using human adenoviruses, and human adenoviruses are preferred according to certain aspects of the present invention. In certain preferred embodiments, the recombinant adenovirus according to the present invention is based on a human adenovirus. In preferred embodiments, the recombinant adenovirus is based on human adenovirus serotypes 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to a particularly preferred embodiment of the present invention, the adenovirus is a human adenovirus with serotype 26. The advantages of these serotypes include low seroprevalence in the population and / or pre-existing low neutralizing antibody titers, as well as experience in clinical trials for human subjects. In some embodiments, the adenovirus is replication-deficient, for example, because it contains a deletion in the E1 region of the genome. For adenoviruses derived from non-group C adenoviruses, such as Ad26 or Ad35, the E4-orf6 coding sequence of the adenovirus is usually exchanged with the E4-orf6 of adenovirus of the human subgroup C, such as Ad5. This allows such adenoviruses to propagate in well-known complementing (or packaging) cell lines that express the E1 gene of Ad5, such as 293 cells, PER.C6 cells, etc. (see, e.g., Haveng, et al., 2006, J Gen Virol 87:2135-43; WO 03 / 104467). However, such adenoviruses cannot replicate in non-complementing cells that do not express the E1 gene of Ad5.

[0186] Host cells containing nucleic acid molecules encoding HMPV F protein before fusion also form part of the present invention. HMPV F protein before fusion can be produced by recombinant DNA technology, which involves expressing molecules in host cells, or transgenic animals or plants, such as Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells or yeast, fungi, insect cells, etc. In certain embodiments, cells are from multicellular organisms, and in certain embodiments, they have vertebrate or invertebrate origins. In certain embodiments, cells are mammalian cells. In certain embodiments, cells are human cells. In general, producing recombinant proteins such as HMPV F protein before fusion of the present invention in host cells includes introducing heterologous nucleic acid molecules encoding proteins in an expressible form into host cells, culturing cells under conditions conducive to the expression of nucleic acid molecules, and allowing the expression of proteins in the cells. Nucleic acid molecules encoding proteins in an expressible form can be in the form of expression cassettes, and sequences that can reach nucleic acid expression, such as enhancers, promoters, polyadenylation signals, etc. are generally required. Those skilled in the art know that various promoters can be used to obtain expression of genes in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viral, prokaryotic or eukaryotic sources, or artificially designed.

[0187] Cell culture media are available from a variety of suppliers, and an appropriate medium can be routinely selected for host cells expressing the protein of interest, here the pre-fusion HMPV F protein. An appropriate culture medium may or may not contain serum.

[0188] "Heterologous nucleic acid molecule" (also referred to herein as "transgene") is a nucleic acid molecule that does not naturally occur in a host cell. It is introduced into, for example, a vector by standard molecular biology techniques. The transgene is generally operably linked to an expression control sequence. This can be accomplished, for example, by placing the nucleic acid encoding the transgene under the control of a promoter. Further regulatory sequences can be added. Many promoters can be used for the expression of transgenes and are known to the skilled person, for example, these promoters can include viral, mammalian, synthetic promoters, and the like. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is the CMV promoter (US 5,385,839), such as the CMV immediate early promoter, for example, comprising nt.-735 to +95 from the CMV immediate early gene enhancer / promoter. A polyadenylation signal, such as a bovine growth hormone polyadenylic acid signal (US 5,122,458), can be present after the transgene. Alternatively, several widely used expression vectors are available in the art and can be obtained from commercial sources, such as the pcDNA and pEF vector series from Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc., which can be used to recombinantly express the protein of interest, or to obtain appropriate promoter and / or transcription terminator sequences, polyadenylation sequences, etc.

[0189] Cell culture can be any type of cell culture, including adherent cell culture, such as cells attached to the surface of a culture vessel or microcarriers, and suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes, because they are the easiest to operate and scale up. Today, the continuous process based on the perfusion principle becomes more common, and is also suitable. Suitable culture medium is also well-known to technicians, and generally can be obtained from commercial sources in large quantities, or customized according to standard protocols. Cultivation can be completed, for example, in a culture dish, a roller bottle or a bioreactor, using batch, fed-batch, continuous system, etc. The suitable conditions for culturing cells are known (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, ed. (1973), and RI Freshney, Culture of animal cells: A manual of basic technique, the fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

[0190] The present invention further provides a pharmaceutical composition comprising a pre-fusion HMPV F protein, and / or a fragment thereof, and / or a nucleic acid molecule and / or a vector as described herein. Therefore, the present invention provides a composition comprising a pre-fusion HMPV F protein or a fragment thereof, wherein the pre-fusion HMPV F protein or a fragment thereof exhibits an epitope present in the pre-fusion conformation of the HMPV F protein, but not present in the post-fusion conformation. The present invention also provides a composition comprising a nucleic acid molecule and / or a vector encoding such a pre-fusion HMPV F protein or a fragment. The present invention particularly provides a pharmaceutical composition, such as a vaccine composition, comprising a pre-fusion HMPV F protein, a HMPV F protein fragment, and / or a nucleic acid molecule and / or a vector as described above and one or more pharmaceutically acceptable excipients.

[0191] The present invention also provides the use of a stabilized prefusion HMPV F protein (fragment), nucleic acid molecule and / or vector according to the present invention for vaccinating a subject against HMPV.

[0192] The present invention also provides the use of a stable pre-fusion HMPV F protein (fragment), a nucleic acid molecule and / or a vector according to the present invention to induce an immune response to an HMPV F protein in a subject. A method for inducing an immune response to an HMPV F protein in a subject is further provided, comprising administering a pre-fusion HMPV F protein (fragment), and / or a nucleic acid molecule and / or a vector according to the present invention to the subject. Further provided is the use of a pre-fusion HMPV F protein (fragment), and / or a nucleic acid molecule and / or a vector according to the present invention for the manufacture of a medicament, the medicament being used to induce an immune response to an HMPV F protein in a subject. The present invention particularly provides a pre-fusion HMPV F protein (fragment), and / or a nucleic acid molecule and / or a vector according to the present invention for use as a vaccine.

[0193] The pre-fusion HMPV F protein (fragment), nucleic acid molecule or vector of the present invention can be used for the prevention (prevention) and / or treatment of HMPV infection. In certain embodiments, the prevention and / or treatment can target a patient population susceptible to HMPV infection. Such patient populations include, but are not limited to, for example, the elderly (e.g., ≥50 years old, ≥60 years old, and preferably ≥65 years old), young people (e.g., ≤5 years old, ≤1 year old), pregnant women (for maternal immunity), and hospitalized patients and patients who have been treated with antiviral compounds but have shown insufficient antiviral responses.

[0194] The prefusion HMPV F proteins, fragments, nucleic acid molecules and / or vectors according to the invention can be used in the treatment and / or prevention of diseases or conditions caused by HMPV alone, or in combination with other prophylactic and / or therapeutic treatments such as (existing or future) vaccines, antiviral agents and / or monoclonal antibodies.

[0195] The present invention further provides a method for preventing and / or treating HMPV infection in a subject using a pre-fusion HMPV F protein or fragment thereof, a nucleic acid molecule and / or a vector according to the present invention. In a specific embodiment, the method for preventing and / or treating HMPV infection in a subject comprises administering an effective amount of a pre-fusion HMPV F protein (fragment), a nucleic acid molecule and / or a vector as described above to a subject in need thereof. A therapeutically effective amount refers to the amount of a protein, nucleic acid molecule or vector that effectively prevents, improves and / or treats a disease or condition caused by infection by HMPV. Prevention encompasses the inhibition or reduction of the spread of HMPV or the inhibition or reduction of the occurrence, development or progression of one or more symptoms associated with infection by HMPV. As used herein, improvement may refer to a reduction in visible or perceptible disease symptoms, viremia, or any other measurable manifestation of HMPV infection.

[0196] For administration to a subject, such as a human, the present invention may employ a pharmaceutical composition comprising a pre-fusion HMPV F protein (fragment), a nucleic acid molecule and / or a vector as described herein, and a pharmaceutically acceptable carrier or excipient. In this context, the term "pharmaceutically acceptable" means that the carrier or excipient does not cause any unwanted or harmful effects in the subject to which it is applied at the dosage and concentration employed. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, ed., Mack Publishing Company

[1990] ; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, ed., Taylor & Francis

[2000] ; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, ed., Pharmaceutical Press

[2000] ). The HMPV F protein or nucleic acid molecule is preferably formulated and administered as a sterile solution, although a lyophilized formulation may also be utilized. The sterile solution is prepared by sterile filtration or other methods known in the art per se. The solution is then lyophilized or filled into a pharmaceutical dosage container. The pH of the solution is generally in the range of pH 3.0 to 9.5, for example pH 5.0 to 7.5. The HMPV F protein is generally in a solution with a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt. Optionally, a stabilizer, such as albumin, may be present. In certain embodiments, a detergent is added. In certain embodiments, the HMPV F protein may be formulated into an injectable formulation.

[0197] In certain embodiments, the composition according to the present invention further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to the applied antigenic determinants. The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to the HMPV F protein of the present invention. Examples of suitable adjuvants include aluminum salts, such as aluminum hydroxide and / or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, e.g., WO 90 / 14837); saponin preparations, such as QS21 and immunostimulatory complexes (ISCOMS) (see, e.g., US 5,057,540; WO 90 / 03184, WO 96 / 11711, WO 2004 / 004762, WO 2005 / 002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacetyl MPL (3dMPL), oligonucleotides containing CpG motifs, ADP-ribosylating bacterial toxins or mutants thereof, such as Escherichia coli heat-labile enterotoxin LT, cholera toxin CT, etc.; eukaryotic proteins (such as antibodies or fragments thereof (for example, against the antigen itself or CD1a, CD3, CD7, CD80) and ligands for receptors (such as CD40L, GMCSF, GCSF, etc.), which stimulate immune responses when interacting with receptor cells. In certain embodiments, the composition of the present invention comprises aluminum as an adjuvant, for example in the form of aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate or a combination thereof, with a concentration of 0.05-5 mg, for example, 0.075-1.0 mg of aluminum content / dose.

[0198] In other embodiments, the composition does not include an adjuvant.

[0199] In certain embodiments, the present invention provides a method for preparing a vaccine against respiratory syncytial virus (HMPV), comprising providing an HMPV F protein (fragment), a nucleic acid or a vector according to the present invention, and formulating it into a pharmaceutically acceptable composition. The term "vaccine" refers to a medicament or composition containing an active component that effectively induces a certain degree of immunity against a certain pathogen or disease in a subject, which will result in at least a reduction (until complete elimination) in the severity, duration or other manifestations of symptoms associated with infection or disease by a pathogen. In the present invention, the vaccine comprises an effective amount of a pre-fusion HMPV F protein (fragment) and / or a nucleic acid molecule encoding a pre-fusion HMPV F protein, and / or a vector comprising the nucleic acid molecule, which results in an effective immune response against HMPV. This provides a method for preventing severe lower respiratory tract diseases that lead to hospitalization, and a reduction in the frequency of complications such as pneumonia and bronchiolitis due to HMPV infection and replication in a subject. The term "vaccine" according to the present invention implies that it is a pharmaceutical composition, and therefore generally includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not contain further active ingredients. In certain embodiments, it may be a combination vaccine further comprising other components which induce an immune response, for example, against other proteins of HMPV and / or other infectious agents, for example against RSV, HMPV and / or influenza. The administration of further active components can be accomplished, for example, by separate administration or by administering a combination product of the vaccine of the invention and further active components.

[0200] Administration of the compositions according to the present invention can be carried out using standard routes of administration. Non-limiting embodiments include intramuscular injection.

[0201] As used herein, a subject is preferably a mammal, such as a rodent, such as a mouse, a cotton rat, or a non-human primate, or a human. Preferably, the subject is a human subject.

[0202] In addition, the proteins of the invention can be used as diagnostic tools, for example to test the immune status of an individual by determining the presence of antibodies capable of binding to the proteins of the invention in the serum of such individuals. Therefore, the present invention also relates to an in vitro diagnostic method for detecting the presence of HMPV infection in a patient, the method comprising the steps of: a) contacting a biological sample obtained from the patient with a protein according to the invention; and b) detecting the presence of an antibody-protein complex.

[0203] The present invention is further described in the following examples. The examples do not limit the present invention in any way. They only serve to illustrate the present invention. Example

[0204] Example 1: Introduction of non-natural F2-F1 cleavage site

[0205] The schematic structures of full-length and soluble HMPV F (HMPV F ectodomain) are shown in Figure 1 a and b (see SEQ ID NO: 1 for numbering). The F1 domain in the soluble variant is C-terminally truncated and (optionally) has a Foldon trimerization domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL; SEQ ID NO: 2) fused to the F ectodomain, similar to many other soluble trimeric viral fusion proteins.

[0206] Based on the F protein of wild-type HMPV strain TN / 00 / 3-14 (SEQ ID NO: 1), a DNA fragment encoding the hMPV protein was synthesized (Genscript, Piscataway, NJ) and cloned into the pcDNA2004 expression vector (a modified pcDNA3 plasmid with an enhanced CMV promoter).

[0207] HEK293 cells were transfected with a plasmid corresponding to the HMPV F ectodomain of strain TN / 00 / 3-14 with a C-terminal foldon domain and four stabilizing mutations and a non-native disulfide bridge (T69Y, A116H, A140C, A147C, D185P and E453Q) as described in co-pending application EP21215259 and with Figure 2 A natural variant H368N of the F2 C-terminus defined in (as found in TN / 85 / 6-3; Genbank ID ACJ53577.1). The native TMPRSS2 cleavage site (RQSR) was changed to a non-natural polybasic furin site (RRRR), as well as truncation of the F2 C-terminus (after position 90) to increase the cleavage efficiency of the HMPV F0 protein. In addition, in some proteins, a second non-natural polybasic furin site (RRRR) cleavage site was introduced in the F2 domain, placed N-terminally to the first cleavage site and separated from the F2 domain by a spacer sequence of one or more amino acid residues.

[0208] The expression platform used was the Expi293F expression system in a 96-well format (Thermo Fisher Scientific, Waltham, USA). The HMPV F encoding plasmid was co-transfected with a plasmid encoding furin at a HMPV F-furin DNA ratio of 5:1. The constructs were transfected in duplicate or quadruplicate.

[0209] After 3 days, supernatants were harvested and analyzed by SDS-PAGE 4–12% (w / v) Bis–Tris NuPAGE gels, 1× MOPS (Life Technologies) under reducing conditions, followed by Western blotting to examine the efficacy of cleavage ( Figure 2 B). Western blot analysis was performed as follows: Semi-dry blotting was performed using iBlot2: program P0 according to the manufacturer's recommendations. After blocking for 1 hour in Odyssey blocking buffer, the primary antibody (anti-hMPV-F A2 / B2 polyclonal serum, as generated after peptide immunization of rabbits at Genscript, Piscataway, NJ) was incubated for 1 hour at 1:10.000 in Odyssey blocking buffer, and the secondary antibody (α-Rabbit IRDYE CW800 (Rockland Immunochemicals, Inc., Limerick, PA, US) was incubated for 1 hour at 1:5000 in Odyssey blocking buffer. All incubations were performed at room temperature on a roller platform. After the primary and secondary antibodies, the blots were washed 3x with 50 ml TBS / 0.05% Tween20 for 5 minutes each, followed by a final wash with 50 ml 1xPBS. The blots were visualized by scanning on an Odyssey scanner using both 700CW and 800CW channels.

[0210] The harvested crude cell supernatants were also analyzed for trimer content and trimer stability after heat stress on an analytical size exclusion chromatography (SEC) in an ultra-high performance liquid chromatography (UHPLC) system using a Vanquish system (ThermoFisher Scientific, Waltham, USA) with a Sepax Unix-C SEC-300 4.6X150mm 1.8μm column (Sepax (231300-4615), injection volume 20μL, flow rate 0.35mL / min). Thermal stability is reported as the percentage of trimers after heat stress relative to a 4°C control. Constructs transfected in duplex or quadruplex were combined for SEC analysis. Elution was monitored by a UV detector. SEC profiles were analyzed by Chromeleon software version (version 7.2.7, Thermo Fisher Scientific). Graphs were plotted in GraphPad Prism (version 9.0.0, GraphPad Software). ( Figure 2 C).

[0211] The antigenicity of HMPV F protein in crude cell supernatants was also evaluated using biolayer interferometry on the Octet (ForteBio, Portsmouth, UK) using the monoclonal antibodies ADI-14448 (Gilman et al. Sci Immunol. 2016 Dec 16;1(6):eaaj1879. doi:10.1126 / sciimmunol.aaj1879. Epub 2016 Dec 9.) and DS7 (Wen et al. Nat Struct Mol Biol. 2012 Mar 4;19(4):461-3. doi:10.1038 / nsmb.2250.)). Figure 2 D). ADI-14448 has been described as a cross-neutralizing antibody against RSV and HMPV that is pre-fusion specific and binds to antigenic site III of RSV preF. Therefore, ADI-14448 binding indicates a pre-fusion conformation. The X-ray structure of Mab DS7 in complex with the HMPV fusion protein has been described (Wen et al., Nat Struct Mol Biol. 2012 Mar 4; 19(4):461-3. doi:10.1038 / nsmb.2250.). The complex of HMPV F+DS7 shows that the heptad repeat 2 (HR2), which is part of the refolding region 2 (RR2), is in a non-preF conformation because the beta sheet 22 is moved away, which is more like a post-fusion conformation than a pre-fusion conformation. Therefore, DS7 binding indicates a non-pre-fusion or post-fusion conformation.

[0212] For the preferred pre-fusion HPMV F protein according to the present invention, DS7 binding is therefore undesirable. For Octet determination, antibodies were fixed to anti-human Fc biosensors (ForteBio, Portsmouth, UK). After balancing the sensor in kinetic buffer (ForteBio, Portsmouth, UK) for 600 seconds, the sensor was transferred to a kinetic buffer with 5ug / ml of the desired antibody. Subsequently, another balancing step was included in the simulated cell culture medium. Finally, the sensor was transferred to a solution of the cell culture supernatant containing the pre-fusion HMPV F protein. The initial slope (also referred to as the binding phase; curve fitting was performed at the initial 300 seconds) and the binding in nm at 300 seconds were reported. Data analysis was completed using ForteBioData Analysis 8.2 software (ForteBio, Portsmouth, UK). Bar graphs were drawn in GraphPad Prism (version 9.0.0, GraphPad Software).

[0213] According to analytical SEC, all proteins showed expression ( Figure 2 B) and is a trimer ( Figure 2 C). Processing of all proteins was incomplete as detected by Western blotting ( Figure 2 B).

[0214] With the introduction of stabilizing mutations and the conversion of the native TMPRSS2-like cleavage site to a non-native furin cleavage site by replacing QS with RR, the expression level of MPV210531 was significantly increased compared with MPV201285 without the stabilizing mutations. Figure 2 C, left histogram panel) and thermal stability ( Figure 2 C, right histogram panel) is increased. The expression level of proteins with shorter (truncated) F2-C termini (MPV210502, MPV210500, MPV210507 and MPV210509) is reduced compared to MPV210531. The thermal stability of MPV210500, MPV210507 and MPV210509 with short F2 C termini at 60°C is increased compared to MPV210531 with long F2 C termini.

[0215] In the octet, the protein showed both anti-pre-F (bound to ADI-14448) and anti-non-pre-F binding (bound to DS7), indicating that the protein is not completely in the prefusion trimer conformation ( Figure 2 D) Anti-non pre-F binding is reduced for designs with a truncated F2 C-terminus and two non-native cleavage sites (MPV210500, MPV210507 and MPV210509).

[0216] A stable HMPV design with a truncated F2 C-terminus and an additional furin cleavage site showed improved F0 cleavage ( Figure 2 B), improved thermal stability ( Figure 2 C) and lower binding to postF-specific Mab DS7 ( Figure 2 D).

[0217] Example 2 : Optimized processing of F0

[0218] Since improved F2 C-terminal processing and truncation increase the stability and quality of the HMPV prefusion F protein (e.g., improved F0 cleavage, improved thermal stability, lower binding to the post-F specific antibody DS7), the F2 C-terminus was systematically truncated from position 102 to 88. In addition, a p27 peptide (comprising a first non-native cleavage site) and a second non-native cleavage site were introduced between the (truncated) F2 domain and the F1 domain.

[0219] The p27 region using RSV F is shown, namely the amino acid sequence ELPRFMNYTLNNAKKTNVTLSKK R (SEQ ID NO: 2; cleavage site underlined) Improved processing of F0. The p27 peptide of RSV F is known to be very efficiently cleaved by furin-like proteases (Krarup et al. Nat Commun. 2015 Sep 3; 6:8143. doi: 10.1038 / ncomms9143.; Gonzales-Reyes et al. 2001, Proc Natl Acad Sci USA 98:9859-9864).

[0220] According to the present invention, according to Figure 3 A design was presented, with the furin site RRRR and the RSV F p27 domain introduced between the (truncated) F2 domain and the F1 domain. HEK293 cells were cultured with a protein corresponding to the HMPV F ectodomain as described in Example 1 and with Figure 3 Addition of the RSV p27 domain C-terminus to the introduced furin site showed very efficient increase in HMPV F cleavage ( Figure 3 B). According to analytical SEC, the thermal stability of MPV210498 and MPV210751 at 60 °C was the highest, as measured by the trimer content remaining after heat stress ( Figure 3 C right), and the antigenic mass (i.e. prefusion conformation) of both variants was also the highest, as they showed the lowest binding to the postF-specific Mab DS7 in the Octet ( Figure 3 D).

[0221] Conclusion: Introduction of a second furin cleavage site and RSV-p27 domain (containing the first non-native cleavage site) between F1 and F2 resulted in successful complete processing. Additionally, truncation of the F2 C-terminus after residues 89 or 90 (in addition to the second furin cleavage site and RSV-p27 domain of 4 arginines (R)) resulted in the most stable HMPV preF protein with the highest prefusion quality, as indicated by low DS7 binding.

[0222] Embodiment 3: Stabilize the HR2 region

[0223] It is known that soluble HMPV is fused before F extracellular domain and heterologous trimerization domain such as foldon trimerization. However, adding foldon to introduce other heterologous protein domain, except F trimerization, does not have other benefits for vaccine immunogen. Because immunogens in other vaccines or other vaccine components may also use foldon for trimerization, preferred vaccine components are only based on viral proteins, and do not contain any other heterologous non-viral protein domains. By optimizing the interaction in the HR2 region of the stem before fusion of F protein, the invention provides a trimeric conformation of soluble HMPV protein without foldon. In variants of RSV F p27 domains with optimized F2 truncation (after amino acid residue 89), the furin cleavage site introduced at the F2 end of truncation and the best cutting, the stable preF trimer by HR2 stem region is evaluated. Further, F1 C-terminal is truncated after position 481 or 489. Protein also has a joint and C-tag for purification purposes. When the foldon domain was deleted (MPV211241), almost no trimer was detected in analytical SEC ( Figure 4 A (left histogram sub-figure, 5A (left histogram sub-figure). However, several different stable strategies in HR2 (according to Figure 4 and 5 A) restored and even improved the expression of trimers without the foldon. Trimers without the foldon were stored at 4°C for 6 to 8 weeks ( Figure 4 Left histogram inset) and heating at 58°C for 30 min ( Figure 4 The histogram on the right and Figure 5 A (the histogram on the right) remains relatively stable.

[0224] To measure antigen quality, supernatants were tested using Octet with MAbs directed against pre-F (ADI-14448), post-F (DS7), and an interface at the pre-fusion F apex that is recognized only when the trimer is open or able to 'breathe' (open transiently or partially) (MPV458; Huang et al., PLoS Pathog. 2020 Oct 9;16(10):e1008942. doi:10.1371 / journal.ppat.1008942.eCollection 2020 Oct). HMPV F variants with a stabilized HR2 showed anti-pre-F, anti-apex interface (MPV458), and a small amount of anti-non-pre-F binding (bound to DS7) ( Figure 5 B).

[0225] Conclusions: HR2 stabilizing mutations increase trimerization without the foldon and even increase trimer expression.

[0226] Example 4 : Substitutions that stabilize the prefusion conformation of HMPV

[0227] In this embodiment, alternative stabilizing mutations were evaluated in a framework corresponding to the design of MPV211287 and MPV211918, but without stabilizing substitutions T69Y, A116H, disulfide bonds A140C+A147C and D185P (mutations as described in co-pending application EP21215259). The framework has an F2 truncation after amino acid position 89, an introduced furin cleavage site and p27 of RSV also comprising a furin cleavage site. The protein further comprises H368N, stabilizing mutation E453Q and HR2 stabilizing substitutions (L473W, D475R, Q476K, S477F, N478D, R479E, A484I) or HR2 stabilizing substitutions (L473W, S477I, A484I), a linker and a C-tag. One stabilizing substitution (V231I) was obtained from strain B2 / 3817 / 04 (Genbank ID AGL74059.1). A novel stabilizing substitution (E453P) replaced the stabilizing substitution E453Q (described earlier in co-pending application EP21215259). Two other novel stabilizing substitutions are V112R and D209E. When the previous stabilizing substitutions at positions 69, 116, 140, 147 and 185 (described in co-pending application EP21215259) were mutated back to wild type ( Figure 6 MPV211940 and MPV211942 in A ( Figure 7 A), expression of the preF trimer was lost. All single stabilizing substitutions except Q453P increased trimer expression, especially V112R. Combinations of substitutions further increased expression and improved thermal stability ( Figure 6 A, 7A), especially the addition of the D209E substitution. Octet analysis showed that the variants with the novel stabilizing substitutions had improved antigenic quality compared to the reference MPV211940 and MPV211942, accompanied by relatively lower binding to DS7 ( Figure 6 B, 7B). According to the present invention, improved antigen quality refers to pre-fusion quality, as measured by increased binding of pre-F specific antibodies (e.g. ADI-14448) and decreased binding of post-F binding antibodies (e.g. DS7) and anti-interface antibodies (e.g. MPV458). These last two antibodies have very low neutralizing activity.

[0228] Since some binding to DS7 or MPV458 might also be caused by impurities in the cell culture supernatant, several HMPV F proteins were produced on a larger scale and purified for further thorough analysis (see Example 8).

[0229] Embodiment 5: Additional substitutions that stabilize the HMPV prefusion conformation

[0230] Additional stabilizing mutations T69W, S149Y, N313W and S445Y as well as the natural variant N404P were evaluated by introducing F2 truncation after amino acid position 89, furin site + p27 of RSV, wild-type variant H368N, stabilizing mutation E453Q, HR2 stabilization (L473W, D475R, Q476K, S477F, N478D, R479E, A484I), and linker and C-tag into the MPV212033 backbone containing only the stabilizing mutation V112R. The combination of substitutions further increased expression and improved thermal stability ( Figure 8 A and C) (see Example 1 for methods). Differential scanning fluorimetry (DSF) of cell culture supernatants showed that all additional substitutions except N404P increased the melting temperature (Tm50) of HMPV F as measured by DSF (see Example 4 for method details). Octet analysis showed that variants with the additional substitutions S149Y, T69W, S445Y and N313W had improved antigenic quality compared to reference MPV212033, accompanied by relatively lower binding to DS7 ( Figure 8 B).

[0231] Since some binding to DS7 might also be caused by impurities in the cell culture supernatant, several HMPV F proteins were produced on a larger scale and purified for further analysis (see Example 8).

[0232] Example 6 : HR2 variants in the backbone with V112R, D209E, V231I and E453P

[0233] Further HR2 variants were evaluated in a backbone with an F2 truncation after amino acid position 89, an introduced furin cleavage site, p27 of RSV, an F1 truncation after amino acid position 489 and no foldon. The protein further contained H368N, V112R, D209E, V231I and E453P mutations.

[0234] All tested variants showed increased trimer expression compared to MPV210571 ( Fig. 9A) (see Example 1 for methods). Compared to MPV212047 with HR2 stabilizing mutations L473W, D475R, Q476K, S477F, N478D, R479E, A484I (see Example 3), the trimer content of MPV212032 (L473W, S477I, A484I), MPV220115 (S477I), MPV220120 (L473Y, S477I, A484I) and MPV220121 (L473I, S477I, A484I) was in a similar range. The binding profiles in octet were comparable to those of MPV210751 and MPV212047 ( Fig. 9 B). Differential scanning fluorimetry (DSF) of cell culture supernatants showed that all HR2 designs (except MPV220123 where the Tm50 could not be defined due to low signal) had improved thermal stability compared to MPV210571, as described by the melting temperature in DSF (see Example 4 for method details) in the supernatant.

[0235] in conclusion:

[0236] Compared with MPV210751, HR2 stabilizing mutations in MPV212032 (L473W, S477I, A484I), MPV220115 (S477I), MPV220120 (L473Y, S477I, A484I) and MPV220121 (L473I, S477I, A484I) improved trimer content and thermal stability. Since some binding to DS7 may also be caused by impurities in the cell culture supernatant, several HMPV F proteins were produced on a larger scale and purified for further analysis (see Example 8).

[0237] Embodiment 7: Further stabilization of HR2 variants with S477I and A484I

[0238] MPV220116 with the HR2 stabilizing mutations S477I, A484I and F2 truncation after amino acid position 89, the introduced furin cleavage site, p27 of RSV, no foldon and the mutations H368N, V112R, D209E, V231I and E453P (+ linker and C-tag) was selected to test the additional stabilizing mutations T69W, S149Y, N313W and S445Y and the natural variant N404P.

[0239] Combinations of substitutions further increased expression ( Fig.10A) (for methods see Example 1). Octet analysis showed that variants with the additional substitutions S149Y, T69W, S445Y and N313W had improved antigenic quality compared to the reference MPV220116, accompanied by relatively lower binding to DS7 ( Fig.10 B). Differential scanning fluorimetry (DSF) of cell culture supernatants showed that all additional substitutions improved the melting temperature (Tm50) thermostability of HMPV F, as described by the melting temperature (Tm50) in DSF (see Example 4 for method details) in the supernatant. Fig.10 C).

[0240] in conclusion:

[0241] Additional substitutions S149Y, T69W, S445Y and N313W improved trimer content, antigenic quality and thermal stability.Since some binding to DS7 may also be caused by impurities in the cell culture supernatant, several HMPV F proteins were produced on a larger scale and purified for further analysis (see Example 8).

[0242] Example 8 : Production and purification of selected proteins

[0243] Production and purification

[0244] Several HMPV proteins were produced and purified according to Table 1. Most of the selected HMPV F proteins are described in previous examples: MPV210530 ( Figure 3 )、MPV210751( Figure 4 )、MPV211918( Figure 4 )、MPV212017、MPV212047( Figure 6 )、MPV212043( Figure 6 )、MPV212044( Figure 6 )、MPV212045( Figure 6 )、MPV212046( Figure 6 ), MPV220087 and MPV220092.

[0245] Cells were transiently transfected using ExpiFectamine 293 (Thermo Fisher Scientific, Waltham, USA) at a 300 ml scale according to the manufacturer's instructions and cultured for 5 days in a shaking incubator at 37° C. and 10% CO 2 . The culture supernatant was harvested, centrifuged at 600 rpm for 10 minutes, and filtered on a 0.22 μm PVDF filter to remove cells and cell debris.

[0246] Protein is purified by means of two-step or three-step scheme. First, the culture supernatant of harvest and clarification is loaded onto a pre-packaged C-tagXL 5-ml column (Thermo Fisher Scientific, catalog #494307205, Waltham, USA). The column is pre-loaded with affinity resin (Capture Select), which is composed of C-tag specific single domain antibodies fixed to beads based on agarose. This resin is highly specific for binding proteins with C-tags. Elution of proteins with C-tags is performed using TRIS buffer containing 2MMgCl. Based on UV signal (A280), the eluted fractions are merged and concentrated using Amicon Ultracel 50kDa MWCO centrifugal filter device (Merck Millipore, catalog #UFC805024, Darmstadt, Germany). Protein is purified by means of two-step or three-step scheme. First, the harvested and clarified culture supernatant was loaded onto a pre-packaged CaptureSelect C-tagXL 5-ml column (ThermoFisher Scientific, catalog # 494307205, Waltham, USA). The column was pre-loaded with an affinity resin (CaptureSelect) consisting of a C-tag specific single domain antibody fixed to agarose-based bead. This resin is highly specific for binding to proteins with C-tags. Elution of C-tagged proteins was performed using TRIS buffer containing 2M MgCl. Based on the UV signal (A280), the eluted fractions were combined and concentrated using an Amicon Ultracel 50kDa MWCO centrifugal filter device (Merck Millipore, catalog # UFC805024, Darmstadt, Germany). Subsequently, the concentrated collected elution peak is applied to a Superdex200 Increase 10 / 300 column (Cytiva, catalog # 28-9909-44, Marlborough, Massachusetts, the U.S.), which is balanced in a running buffer (20 mM Tris, 150 mM NaCl, pH 7.4) for refining purposes, i.e., removing minimal multimeric and monomeric proteins. The three-step protocol includes an additional run before Superdex200, on a Superose 6 Increase 10 / 300 column (Cytiva, catalog # 29-0915-96, Marlborough, Massachusetts, the U.S.), on a C- tag pool (applicable only to MPV210751, MPV211918, MPV212017, and MPV212047).The quality after the design purification indicated that this step was not necessary due to the lack of larger aggregates and was therefore not applicable to other purifications.

[0247] In addition, the postFhMPV protein (SEQ ID NO: 3) as described by Mas et al., PLoS Pathog. 2016 Sep 9; 12(9): e1005859.doi: 10.1371 / journal.ppat.1005859.eCollection 2016 Sep 9) was also produced and purified. The expression plasmid encoding the recombinant post-fusion hMPV F protein was prepared as described in Example 2. On a 300 ml scale, cells were transiently transfected and subsequently purified by a two-step protocol (see above for details). Subsequently, TEV cleavage was performed to remove the foldon and c-tag. For 15 μg of protein, 1 μL TEV (10 000 units / mL) was used. The protein-TEV mixture was incubated overnight at 4°C. TEV-His protease was removed from the protein sample by pulling down with Ni Sepharose excel beads (GE Healthcare, 17-3712-03). Ni Sepharose excel beads are added to the protein-TEV mixture and incubated at room temperature for 2 hours. Flowthroughs are collected via micro bio-spin columns (Bio Rad, 7326204). The cut protein samples are heat stressed at 45 ° C for 30 minutes (Hsieh et al., Nat Commun March 14, 2022; 13 (1): 1299.doi: 10.1038 / s41467-022-28931-3.). Subsequently, the protein sample is applied to Superose 6Increase 10 / 300 columns (GE Healthcare, Chicago, USA), which are balanced in running buffer (20mM Tris, 150mM NaCl, pH 7.4) for refining purposes, i.e., removing minimal multi-protein and monomeric proteins.

[0248] The proteins were then analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions ( Fig.11 A) Proteins were visualized on the gel after staining with Instant Blue.

[0249] Results and Conclusions

[0250] The yields of the different variants are shown in Table 1. Truncation of the F2 C-terminus reduced the yield, deletion of the HR2 stabilizer and foldon increased the yield. Improved expression was obtained by introducing V112R, D209E, V231I and E453P (MPV212047). Introduction of further S149Y, N404P (MPV220087) and subsequently S445P, N313W (MPV220092) resulted in increased expression for MPV220092. The major band on the reduced SDS-PAGE corresponded to the F1 domain, with no residual F0 ( Fig.11 A), fully processed protein is indicated.

[0251] Table 1: Purified proteins of Example 8

[0252]

[0253] *Residues at the C-terminus of F2 are located N-terminal to the furin site; NA = not available; **After 3 weeks, # also contained smaller species

[0254] Trimer content

[0255] The purified protein was evaluated by analytical size exclusion chromatography (SEC) after purification and storage at 4°C to investigate the trimer content ( Fig.11 B). SEC was performed using an ultra-high performance liquid chromatography (UHPLC) system (ThermoFisher Scientific) with a Sepax Unix-C SEC-300 4.6X150mm 1.8μm column (Sepax (231300-4615), injection volume 20μL, flow rate 0.3mL / min). Elution was monitored by a UV detector (Thermo Fisher Scientific), a μDawn light scattering (LS) detector (Wyatt Technologies), a μT-rEx refractive index (RI) detector (Wyatt Technologies), and a Nanostar dynamic light scattering (DLS) detector (Wyatt Technologies). SEC profiles were analyzed by the Astra 7.3.2.19 software package (Wyatt Technology). Chromatograms were plotted in Graph Pad Prism (version 9.0.0, GraphPad Software).

[0256] Results and Conclusions

[0257] On analytical SEC, all purified proteins were highly trimerized after purification ( Fig.11 B), and had the expected molecular weight of a trimeric protein (Table 1). After 7 weeks of storage, the high trimer content was retained for all proteins. MPV212044 showed a peak next to the trimer after 7 weeks at 37°C, with a peak for species with a molecular weight smaller than the trimer.

[0258] In general, all purified proteins were trimers ( Fig.11 B and Table 1), and MPV212047 showed high trimer content after 7 weeks at 4°C.

[0259] Stability at 37°C

[0260] The purified protein was aliquoted and the aliquots were incubated at 37°C (all proteins and control samples at time points were kept at 4°C) and subsequently analyzed using SEC-MALS (see above section for methods). The relative trimer content of samples stored at 37°C compared to control samples is listed in Table 1. Chromatograms were plotted in Graph PadPrism (Version 9.0.0, GraphPad Software).

[0261] Results and Conclusions

[0262] Chromatograms of control samples and samples stored at 37°C for 2 weeks were overlaid for each purified protein ( Fig.11 C). MPV210530 showed reduced trimer stability after 2 weeks at 37°C compared to designs with F2 truncated after position 89, as indicated by a reduced trimer peak and an additional peak reflecting aggregates. All proteins with F2 C-terminus truncated after position 89 showed the same trimer content after storage at 37°C compared to the control. All proteins were stable at 37°C for at least 2 weeks.

[0263] Freeze stability

[0264] The proteins were snap frozen once, five times or ten times. After thawing, the trimer content was compared to non-frozen samples by SEC-MALS (see above section for methods). The relative trimer content for stressed samples and control samples is listed in Table 1. The chromatograms were drawn in Graph Pad Prism (Version 9.0.0, GraphPad Software).

[0265] Results and Conclusions

[0266] The proteins were tested in a cryoprotectant-free buffer (buffer composition: 20 mM Tris, 150 mM NaCl, pH 7.4), and after one quick freeze, all proteins remained highly trimerized. Fig.11 D), differences in freezing stability can be observed. Mutations that increase freeze / thaw stability can be arranged in the following order: D209E>V112R>V321I>Q453P. For MPV212044, some aggregates were detected. In general, the HMPV F protein of the present invention shows high freezing stability.

[0267] Thermal stability of proteins

[0268] The thermal stability of the purified prefusion HMPV F protein was determined by differential scanning fluorimetry (DSF) by monitoring the fluorescence emission of Sypro Orange Dye (ThermoFisher Scientific) in a 96-well optical qPCR plate. 15 μl of a 66.67 μg / ml protein solution (buffer as described in Example 2) was used per well ( Fig.11 E). 5 μl of 20x Sypro orange solution was added to each well. After the temperature was gradually increased from 25°C to 95°C (0.015°C / s), the protein unfolded and the fluorescent dye bound to the exposed hydrophobic residues, resulting in a characteristic change in emission. Melting curves were measured using a ViiA7 real-time PCR instrument (Applied BioSystems). The first derivative of the fluorescence signal (au) of three individual samples (technical triplicates) with respect to temperature (°C) and the average melting curve were plotted using Graphpad Prism software (Dan Diego, CA, US). Tm50 (the lowest point on the curve) was deducted from the average melting curve. The Tm50 value represents the temperature at which 50% of the protein unfolds, and is therefore a measure of the temperature stability of the protein.

[0269] Results and Conclusions

[0270] The design with a truncated F2 C terminus at 89 (MPV210751), which further contained a furin site and RSV p27, as well as T69Y, A116H, A140C, A147C, D185P, H368N, E453Q, showed an increase in melting temperature of ∼5°C compared to the design with a long F2 C terminus, MPV210530 ( Fig.11E). Removal of the foldon and introduction of stabilizing mutations in HR2 in MPV211918 reduced the melting temperature by about 3°C ​​compared to MPV210751. Introduction of stabilizing substitutions V112R, D209E, V231I and 453P (MPV212017) increased the melting temperature by about 10°C compared to MPV211918. The disulfide bridge 140-147 and the stabilizing substitutions T69Y, A116H and D185P had no additional stabilizing effect and removal did not reduce the melting temperature (compare MPV212047 to MPV212017).

[0271] Conclusions: HMPV F with a truncated F2 C-terminus combined with stabilizing and addition of novel stabilizing mutations to HR2 resulted in high yields of highly stable trimers in the absence of the Foldon domain.

[0272] In vitro antigenicity

[0273] The selected purified proteins were tested for their in vitro antigenic quality using biolayer interferometry by Octet ( Fig.11 F). In addition to the antibodies described in Example 2, the anti-apex interface binder MPV465 (Huang et al., PLoS Pathog. 2020 Oct 9; 16(10):e1008942.doi:10.1371 / journal.ppat.1008942.eCollection 2020 Oct), and the antibody ADI-18992 that recognizes the preF and postF conformations and is described as binding to site IV (Gilman et al. Sci Immunol. 2016 Dec 16; 1(6):eaaj1879.doi:10.1126 / sciimmunol.aaj1879.Epub 2016 Dec 9) were used. The antibodies were immobilized as described in Example 2. After equilibration of the sensor in kinetic buffer (ForteBio) for 600 seconds, the sensor was transferred to kinetic buffer with 5ug / ml of the desired antibody. Subsequently, another equilibration step was included in the kinetic buffer. Finally, the sensor was transferred into the protein solution (20 μg / mL in 1×KB). The analysis was performed as described in Example 2.

[0274] Results and Conclusions

[0275] For the HMPV F variant in which the F2 C-terminus was not truncated (MPV210530), binding to non-preF antibodies and anti-interface antibodies was higher compared to the purified protein with F2 truncation. Stabilized prefusion F proteins with the novel stabilizing mutations MPV212017 and MPV212047 showed the most favorable binding and bound only to preF-specific antibodies, confirming the finding about Figure 5 The lower antigen mass observed in the supernatant of, for example, MPV212047 in B was caused by impurities.

[0276] Structural characterization by nsTEM

[0277] The purified proteins were analyzed by negative stain transmission electron microscopy (nsTEM) ( Fig.11 G).

[0278] Continuous carbon grid (copper, EMS) was glow-discharged for 30 seconds in an easiglow plasma cleaner. 4 microliters of sample diluted (20mMTris, 150mM NaCl, pH7.4) to a concentration range of 5 to 25μg / ml were applied to the glow-discharged grid and incubated for 1 minute. The sample solution was partially absorbed by gentle side blotting, and then the grid was immediately dyed by depositing it on 40μl drops of 2% (w / v) uranyl acetate solution for a total of 1 minute. After dyeing, the grid was blotted and stored at room temperature before imaging. The prepared grid was imaged in a Talos L120CTEM (Thermo Fisher Scientific) equipped with a Ceta camera. Depending on the imaging conditions, the resulting pixel range was 2.4 to 2.8ang / pixel. CTFFIND4 was used to estimate the parameters of the contrast transfer function (CTF) on each micrograph, and the remainder of the processing (selection and 2D classification) was completed in RELION (version 3 or 4).

[0279] Results and Conclusions

[0280] Two-dimensional (2D) class averages obtained by electron microscopy (EM) analysis of negatively stained samples using an acidic stain of purified MPV212047 revealed a regular, homogeneous, closed trimer ( Fig.11 G)

[0281] Structural characterization of MPV212047 by cryo-electron microscopy

[0282] The cryo-electron microscopy (Cryo-EM) structure of HMPV F engineered MPV212047 was solved to confirm the correct trimeric prefusion conformation of the protein ( Fig.12). For this purpose, 3.5 μL of 0.8–1.0 mg / ml purified preF protein (MPV212047) complexes were applied to plasma-cleaned (Gatan Solarus) Quantifoil 1.2 / 1.3 porous gold grids and subsequently vitrified using a Vitrobot Mark IV (FEI Company). The cryogrids were loaded into a Glacios transmission electron microscope (ThermoFisher Scientific) operated at 200 keV in a Nanoprobe with a Falcon IV direct electron detector. Images were recorded in counting mode using an EPU with The image was recorded in EER format with a 5.7 s exposure, corresponding to ~40 electrons. Total dose. Movies were subjected to beam-induced motion correction, contrast transfer function (CTF) parameter estimation, automated reference particle picking, particle extraction with a box size of 280 pixels, and two-dimensional (2D) classification in CryoSPARClive during data acquisition. Particle images with clear HA features were merged and subjected to de novo 3D reconstruction, followed by 3D heterogeneous refinement with C3 symmetry in CryoSPARC. The particles were then refined using non-uniform (UN) refinement within CryoSPARC with C3 symmetry. Before visualization, all density maps were sharpened using an automated program to apply different negative temperature factors, together with half maps for model building. Local resolution was determined using ResMap. The initial template for the HMPV F trimer was derived from SWISS-MODEL. The model was docked into the EM density map using Chimera, followed by manual adjustments using COOT. The model geometry was further improved using Rosetta. The geometric parameters of the final model were verified in COOT and using MolProbity and EMRinger. These refinements were performed iteratively until no further improvement was observed. Model overfitting was assessed by its refinement for one cryo-EM half-image. FSC curves were calculated between the resulting model and the working half-image and between the resulting model and the free half-image and the full image for cross-validation. Figures were generated using PyMOL ( Fig.12 ). One protomer is depicted as a cartoon representation, while two protomers are depicted as surface representations.

[0283] Based on earlier SEC-MALS, BLI and nsTEM data ( Fig.11 ) confirmed that MPV212047 is a pre-fusion trimer with its apex (distal to HR2) in a closed conformation.

[0284] Embodiment 9: Immunogenicity of the closed prefusion HMPV F protein MPV212047.

[0285] Adjuvanted HMPV prefusion F protein is more immunogenic than postfusion HMPV F in naive cotton rats Sexual and effective.

[0286] The immunogenicity of prefusion HMPV F (preF) MPV212047 was compared with postfusion HMPV F (postF) MPV190470 by intramuscular immunization of naive cotton rats with 16, 3 or 0.6 μg preF or 10 μg postF protein adjuvanted with 50 μL AS01B / animal (n=8 / dose). Proteins were purified as described in Example 8. As controls, cotton rats were treated with phosphate buffered saline (PBS) (negative control, n=8) or 10 4 Plaque forming units (PFU) of HMPV A2 (positive control, n=6) were injected intramuscularly. Animals were immunized twice with the indicated doses on days 0 and 21. The positive control group was immunized once on day 0. Animals were immunized on day 42 with 10 5 PFU HMPV A2 were challenged and sacrificed on day 46. ELISA binding antibody titers were determined against HMPV preF (site-specific biotinylated MPV212047:MPV220554) in sera isolated on day 42 ( Fig.13 A). Titers are shown as log10 of relative potency. The assay was exploratory and the LLoD was based on the use of the 99% quantile limit of all expected negative samples. Each point depicts the value of an individual animal, while the horizontal line indicates the median response of the group. Neutralizing antibody titers were determined against HMPV A2 (TN / 94-49) in sera isolated on day 42 by plaque reduction neutralization test (PRNT) ( Fig.13 B). Titers are shown as log2 of the 50% inhibitory concentration (IC50). Each point depicts the value of an individual animal, while the horizontal line indicates the median response of the group. HMPV A2 viral loads on day 4 post infection (day 46) were measured in nasal homogenates by plaque assay and expressed as log10 pfu / gram tissue ( Fig.13 C). Each point depicts an individual animal, and the median response for each group is indicated by a horizontal line. Open symbols indicate animals with no detectable levels of preF binding antibodies in day 42 serum.

[0287] Compared with the 10 μg postF protein group, the levels of HMPV preF binding antibodies were significantly higher in animals immunized with 3 μg preF protein, corresponding to significantly higher levels of neutralizing antibodies ( Fig.13A, B). In addition, protection against HMPV A2 virus in the nose was significantly lower if animals were immunized with postF protein compared to PreF protein ( Fig.13 C).

[0288] In conclusion, adjuvanted HMPV preF protein was more immunogenic and effective than HMPV postF protein in naive cotton rats.

[0289] Adjuvanted closed prefusion HMPV F protein is more immunogenic than open prefusion HMPV F protein.

[0290] The in vivo immunogenicity of the closed prefusion HMPV FMPV212047 (closed F) was compared with that of the HMPV F protein with an open conformation (MPV220215; open F). To this end, the MPV220215 HMPV F trimer was purified by C-tag affinity purification and SEC, and the HMPV F trimer was characterized as described in Example 8. MPV220215 was designed as a single-chain construct carrying a foldon trimerization domain and stabilizing substitutions V112R, S149Y, V231I, and E453P. The trimer conformation was confirmed by SEC-MALS analysis, eluting at a retention time of approximately 4 minutes with a size of 161 kDa ( Fig.14 A). The open prefusion conformation of MPV220215 was confirmed in BLI by detecting ADI-14448 prefusion-specific antibody binding and enhanced MPV458 and MPV465 vertex interface binding compared to the closed MPV212047HMPV F ( Fig.14 B).

[0291] The immunogenicity of purified open MPV220215 and closed MPV212047 HMPV prefusion F trimers was evaluated by intramuscular immunization of female Balb / C mice (n=5) with 1.5, 5 or 15 μg of protein adjuvanted with 10 μL AS01B / animal, or a negative control group immunized with PBS (n=3) on days 0 and 28. In sera isolated 2 weeks after the second immunization (day 42), ELISA binding antibody titers against HMPV preF were determined as described above ( Fig.14 C). A dose-dependent increase in the levels of HMPV preF binding antibodies was observed after immunization with both proteins, and reached significantly higher levels for the closed MPV212047 protein compared to the open MPV220215 variant. In addition, significantly higher HMPV A2 neutralization titers were detected after immunization with the closed prefusion HMPV F compared to the open MPV220215 ( Fig.14 D).

[0292] These results demonstrate that the closed prefusion HMPV F conformation induces more potent neutralizing antibodies in mice compared to the HMPV F protein in the open conformation.

[0293] Immunogenicity of HMPV prefusion F in pre-exposed mice.

[0294] The immunogenicity of HMPV prefusion F (MPV212047) was evaluated in the context of HMPV A2 pre-exposure by intramuscular immunization of female Balb / C mice (n=7) with 15 μg of unadjuvanted MPV212047 protein (purified as described in Example 8). 3 Until 3x10 5 PFU HMPV HMPV A2 were pre-exposed and immunized with MPV212047 at week 12. In sera isolated 2 and 4 weeks after immunization (weeks 14 and 16), ELISA binding antibody titers against HMPV preF were determined as described in Example 9 above ( Fig.15 ). Levels of HMPV preF binding antibodies were dramatically enhanced by immunization with MPV212047, with a 186-fold geometric mean increase in titer, confirming the in vivo immunogenicity of unadjuvanted HMPV prefusion F in the context of preexposure.

[0295] Embodiment 10: The stabilizing substitutions were transferred to HMPV F A2, B1 and B2 strain variants.

[0296] Prefusion HMPV subtype A2 (2000) F protein with minimal amino acid substitutions.

[0297] To generate an HMPV prefusion F protein with minimal HR2 stabilizing substitutions, several HR2 amino acids were returned to wild type in the stabilizing backbone MPV212047. The resulting construct MPV220558 does carry the HR2 substitutions L473W, Q476K, S477F, and A484I, but has wild type amino acids D475, N478, and R479. In addition, starting with variant MPV220558, the surface exposed H368N substitution was returned to wild type, resulting in MPV220647. In summary, MPV220647 is based on HMPV A2 strain TN / 00 / 3-14 (A22000), carries the p27 peptide, the F2 domain is truncated at amino acid 89, and contains the following set of stabilizing mutations combined with four stabilizing HR2 mutations L473W, Q476K, S477F and A484I: V112R, D209E, V231I and E453P.

[0298] As described in Example 1, the expression of HMPV F trimer was evaluated after transient expression of MPV212047, MPV220558 and MPV220647. The melting temperature (Tm50) of the HMPV F trimer in the supernatant was determined by differential scanning fluorimetry (DSF). To this end, the fluorescence emission of Sypro Orange Dye (ThermoFisherScientific) in the HMPV F protein added to the solution was monitored. The measurement was performed with a starting temperature of 25°C and a final temperature of 95°C (54°C increase / hour). The melting curve was measured using a ViiA7 real-time PCR instrument (Applied Biosystems), and the Tm50 value was derived from the negative first-order derivative, as previously described (Rutten et al. (2020) Cell Rep 30:4540-4550).

[0299] By subsequent restoration of the three HR2 stabilizing substitutions and H368N back to wild type, an effect on trimer peak height was observed ( Fig.16 A, black bars). However, the melting temperatures of the three HMPV F proteins were not negatively affected, as demonstrated by the average Tm50 of 72.3°C, 73.2°C, and 73.5°C for MPV212047, MPV220558, and MPV220647, respectively ( Fig.16 A, gray bars).

[0300] Transfer of stabilizing substitutions to HMPV F strain variants.

[0301] Next, the transfer of stable substitutions in MPV220647 to the most recent A2 strains and to the most recent subtype B1 and B2 strains was assessed as described above. Fig.16 B). The stabilizing effect of these mutations was confirmed by comparison of wild-type B2 (MPV23362) with the stable B2 variant MPV220641. Wild-type HMPV F B2 eluted at the expected monomer retention time in analytical SEC ( Fig.16 C, gray line), while the stable HMPV FB2 MPV220641 was expressed at significantly higher levels and eluted as a trimer at a retention time of approximately 4.5 minutes ( Fig.16 C, black line). Consistent with this, the average melting temperature of wild-type HMPV FB2 increased from 57.9°C to 72.7°C for stable MPV220641 as determined by DSF ( Fig.16 D). Finally, stabilization of prefusion HMPV F was confirmed in biolayer interferometry (BLI) measurements using quantitative Octet using HMPV F prefusion (ADI-61026), apex interface (MPV458) and non-prefusion (DS7) antibodies as described in Example 1.

[0302] Compared to wild-type HMPV FB2, stabilized HMPV FB2 MPV220641 showed improved binding to the prefusion-specific ADI-61026, and reduced binding to the apex interface MPV458 and non-prefusion DS7 antibodies ( Fig.16 E).

[0303] A trimer expression pattern was also observed for stable HMPV F A2 2019 (MPV220639) and HMPV F B1 2020 (MPV220640), albeit at slightly reduced expression levels compared to A2 2000 (MPV220647). The melting temperatures of recent HMPV F A2 and B1 strains were comparable to or higher than those of MPV220639 (73.6°C and 75.8°C for MPV220640 and MPV22647, respectively) ( Fig.16 D). All stable HMPV F proteins were expressed in the prefusion conformation as confirmed by binding of the ADI-61026 prefusion antibody in combination with low MPV458 and DS7 binding in the Octet ( Fig.16 E).

[0304] In summary, the stabilizing HMPV F substitutions discovered in the HMPV A2 (2000) backbone were successfully transferred to other recent HMPV subtypes, resulting in a stable trimeric prefusion HMPV F protein.

[0305] Example 11. Stabilization, purification and immunogenicity of the prefusion F protein of HMPV subtype A2 (2019).

[0306] Further stable trimer expression of the tag-free HMPV F variant MPV221190 was improved.

[0307] The stable HMPV subtype A2 (2019) prefusion F variant MPV220639 from Example 10 was produced without the C-tag purification tag to obtain variant MPV220759. In this backbone, additional substitutions S149Y and N404P were introduced to obtain variant MPV221190, and their effect on trimer expression was evaluated after transient transfection in Expi293F cells as described in Example 1. Analytical SEC of cell culture supernatants confirmed the increased trimer expression ( ) for MPV221190 carrying both S149Y and N404P over the backbone MPV220759. Fig.17 A).

[0308] Purification and characterization of untagged MPV220759 and MPV221190H MPVF trimers.

[0309] Next, MPV220759 and MPV221190 HMPV F variants were transiently transfected into Expi293F cells using ExpiFectamine (Life Technologies) according to the manufacturer's instructions and cultured for 5 days at 37°C and 10% CO2. The culture supernatant was harvested and spun at 600 g for 10 minutes to remove cells and cell debris, and then sterile filtered using a 0.22 μm vacuum filter. The HMPV F protein was purified using a two-step purification scheme that included ion exchange (Cation) purification at pH 5.0 and polishing via size exclusion chromatography using a Superdex 200 16 / 600 pg column. The trimer fractions were combined and further characterized by using an online Nanostar DLS reader (Wyatt) in combination with an ultra-high performance liquid chromatography system (Vanquish, Thermo Scientific) and an analytical SEC-MALS of a μDAWN TREOS instrument (Wyatt) coupled to an Optilab μT-rEX refractive index detector (Wyatt). The protein was loaded onto a Unix-C SEC-300 15 cm column (Sepax Technologies) with a corresponding guard column (Sepax Technologies) equilibrated in a running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 mL / min. The analytical SEC data were analyzed using the Chromeleon 7.2.8.0 software package, and the molecular weight of the HMPV F trimer was calculated by Astra software and compared to the calculated weight, confirming the trimer conformation ( Fig.17 B, C).

[0310] The melting temperature (Tm50) of the purified HMPV F trimer was determined by DSF as described in Example 10. MPV221190 carrying the substitutions S149Y and N404P had a higher melting temperature (Tm50) of 75.5°C compared to 73.5°C for MPV220759 without these substitutions. Fig.17 D).

[0311] The prefusion conformation of the purified HMPV F protein was confirmed in biolayer interferometry (BLI) measurements using quantitative Octet using HMPV F prefusion (ADI-14448 and ADI-61026), apex interface (MPV458), and non-prefusion (DS7) antibodies. Antibodies were immobilized to anti-human IgG sensors at a concentration of 5 μg / ml, and the initial binding rate of HMPV F bound at 20 μg / ml for 300 seconds was plotted as the mean + SD of 3-6 individual measurements ( Fig.17 E) Binding of the two HMPV F proteins to the prefusion-specific ADI-14448 and ADI-61026 antibodies, as well as the absence of binding to the apex interface binding antibody MPV458 and the non-prefusion DS7 were confirmed.

[0312] The stress resilience of two purified HMPV F proteins, MPV220759 and MPV221190, to repeated snap freezing (SF) cycles in liquid nitrogen and supercooling stress towards -20°C was evaluated by measuring the relative trimer content compared to untreated protein in analytical SEC ( Fig.17 F). SF stress spikes reduced the trimer content of both proteins, but less significantly for MPV221190 ( Fig.17 F, solid bars). Consistent with this, the average reduction in trimer content from five cold stress measurements was 23% for MPV220759 and 11% for MPV221190 ( Fig.17 F, open bars).

[0313] In summary, two stable trimeric HMPV prefusion F proteins based on the A2(2019) sequence without trimerization domains or purification tags were generated. Variant MPV221190 with additional stabilizing substitutions S149Y and N404P showed higher trimer expression and improved temperature stress resilience compared to MPV220759.

[0314] Immunogenicity of HMPV prefusion F MPV220759 and MPV221190.

[0315] The in vivo immunogenicity of purified MPV220759 and MPV221190 HMPV prefusion F trimers was evaluated by intramuscular immunization of female Balb / C mice (n=8) with 1.5, 5 or 15 μg of protein adjuvanted with 10 μL AS01B / animal on days 0 and 28 and compared with controls immunized with 15 μg AS01B-adjuvanted MPV212047 (n=8) or PBS (n=3). In sera isolated 2 weeks after the second immunization (day 43), ELISA binding antibody titers against HMPV preF were determined as described in Example 9 ( Fig.17 G). Dose-dependent increases in HMPV preF binding antibody levels were observed after immunization with MPV220759 and MPV221190 and reached levels comparable to MPV212047 at the 15-μg protein dose, demonstrating the immunogenicity of these HMPV prefusion F proteins in mice.

[0316] Example 12. In vitro comparison of differentially stabilized HMPV prefusion F proteins.

[0317] A pre-fusion stabilized HMPV F protein is described by Hsieh et al. (Nat Commun. 2022 Mar 14; 13(1): 1299). Expression of this so-called DS-CavEs2 design (MPV220552) was compared to MPV221190 after transient transfection in Expi293F cells as described in Example 1. DS-CavEs2 (MPV220552) has a foldon trimerization domain and elutes after a retention time of approximately 4 minutes, while MPV221190 without a foldon elutes after a retention time of approximately 4.5 minutes and strongly increased trimer expression was confirmed ( Fig.18 A).

[0318] Next, MPV220552 trimers were purified after transient expression in Expi293F cells as described in Example 11 by using StrepTag affinity chromatography (GE Healthcare, 28-9075), followed by polishing via size exclusion chromatography using a Superose 6 (GE Healthcare) column. Trimeric fractions were pooled and further characterized by SEC-MALS, DSF, and Octet as described in Example 11. The trimeric conformation of the DS-CavEs2 design was confirmed with a melting temperature of 71.1°C, comparable to an earlier report (Hsieh et al., 2022), but lower than the alternatively stable HMPVF MPV221190 ( Fig.17 D, 18B, C). Both proteins bound to ADI-14448 and ADI-61026 prefusion antibodies, but not to non-prefusion DS7. However, MPV220552 demonstrated high binding to the apex interface antibody MPV458, indicating a more open apex structure compared to MPV221190 ( Fig.18 D).

[0319] The stability of both purified proteins was evaluated by prolonged incubation at 4°C or 37°C followed by analytical SEC. Examination of the elution pattern of HMPV F confirmed a stable trimer peak for MPV221190, while a reduced trimer peak was observed for MPV220552 with the appearance of smaller species eluting between retention times of 4.5-5 minutes after 2 weeks of storage at 37°C ( Fig.18 E).

[0320] In summary, compared to the previously published DS-CavEs2 (MPV220552) HMPV prefusion F, the MPV221190 HMPV prefusion F stable trimer had higher expression levels, a more closed trimer structure as determined by BLI, and enhanced stability as determined by DSF and stress resilience.

[0321] Example 13. Stabilization of the stable HMPV prefusion F trimer without the heterologous trimerization domain and without the HR2 region.

[0322] The trimer expression of HMPV pre-fusion F protein was evaluated in the framework without heterologous trimerization domain and without stable substitution in HR2 region to investigate the requirement of HR2 stability under the presence of multiple alternative stable substitutions in the head domain. Except for the introduction of seven HR2 substitutions, the framework is comparable to MPV212047. In short, the framework MPV220847 has the F2 truncation after amino acid position 89, the introduced furin cleavage site and the p27 of RSV also comprising the furin cleavage site and the C-terminal truncation at position 489. The variant further comprises substitutions V112R, D209E, V231I, H368N, E453P, joints and C-tags.

[0323] Trimeric expression of MPV220847 was compared with two variants carrying S477I (MPV220115) or S477L (MPV220851) substitutions in the HR2 region, which were previously shown to improve the trimeric content of HMPV F ( Figure 4 As described in Example 1, after transient expression in Expi293F cells, all three HMPV F variants showed a trimerization peak eluting at a retention time of approximately 4.5 minutes ( Fig.19 A), indicating that HR2 stabilization is not required when sufficient alternative substitutions are introduced. However, although a single melting event was measured for all three HMPV F variants at around 70°C, the stabilizing effect of the S447I and S477L substitutions was still evident by the increased melting temperatures of MPV220115 and MPV220851 compared to the backbone MPV220847 in DSF ( Fig.19 B). The prefusion conformation of all three HMPV F variants was confirmed in BLI by binding of the prefusion-specific antibody ADI-14448 and the absence of binding to the non-prefusion antibody DS7 ( Fig.19 C) (DSF and BLI were performed as described in Example 11).

[0324] In summary, the trimeric HMPV prefusion F protein can be stabilized without the need for a heterologous trimerization domain and without the need for stabilization of the HR2 region, although the latter provides a clear benefit in terms of the temperature stability of the HMPV F protein.

[0325] Example 14. The HMPV prefusion F trimer without a heterologous trimerization domain is stabilized by stabilization of the HR2 region at position 477.

[0326] Optimized stabilization of the prefusion HMPV F by the HR2 stem region identified position S477 as a suitable target for stabilization ( Figure 4 The effects of hydrophobic amino acid substitutions were systematically evaluated in the backbone MPV211241, which was previously shown to express little trimeric HMPV F ( Figure 4 A left histogram panel, 5A left histogram panel). To this end, HMPV F variants carrying wild-type S477, or S477I, S477L, S477F, S477V, S477M, S477Y or S477W were expressed in Expi293F cells and analyzed as described in Example 1. To evaluate the stability of HMPV F trimer expression, culture supernatants were subjected to 30 minutes of incubation at 58°C and evaluated in analytical SEC. As previously demonstrated, backbone MPV211241 carrying wild-type S477 did not express HMPV F trimers, but eluted with a retention time between 4.5-5 minutes, corresponding to HMPV F monomers ( Fig. 20 , black line). Under heat stress, aggregates with a retention time of approximately 3 minutes appeared ( Fig. 20 , gray line). Substitutions S477I, S477L, S477F, S477V, and S477M (MPV211247, MPV211249, MPV23278, MPV211248, and MPV23279, respectively) successfully restored the expression of the HMPV F trimer, whereas substitutions S477Y and S477W (MPV23280 and MPV23281) partially restored the expression of the HMPV F trimer, expressed as a mixture of trimer and monomer ( Fig. 20 , black line). Under 58°C heat stress, HMPV F trimers expressed from variants carrying S477I or S477L substitutions remained trimerized (MPV211247 and MPV211249, Fig. 20 , gray line; Figure 4 A), whereas all other S477 variants were affected, had reduced trimer content and showed the presence of aggregates ( Fig. 20 , gray line).

[0327] In conclusion, introduction of a hydrophobic amino acid residue at position 477 in the HR2 region of HMPV F was shown to enhance expression of the trimer, with S477I and S477L substitutions resulting in the most stable trimer.

[0328] Example 15. Stabilization of the full-length HMPV prefusion F trimer.

[0329] The effects of stabilizing substitutions in the head domain (wherein the 'head domain' is defined here as the portion of the mature, processed protein that is N-terminal to the HR2 stem region) and the HR2 region of full-length HMPV F were evaluated by flow cytometry. To this end, a full-length wild-type HMPV A2 (2019) F was designed, which has an F2 truncation after amino acid position 89, an introduced furin cleavage site, and p27 of RSV (MPV221364) that also contains a furin cleavage site. This wild-type backbone was compared with MPV221376, which additionally carries four HR2 substitutions: L473W, Q476K, S477F, and A484I. Similarly, the wild-type backbone was compared with MPV221371, which carries the head domain substitutions V112R, D209E, V231I, and E453P, but is not HR2 stable. Finally, variant MPV221377 carries both the head and HR2 stabilization as described above.

[0330] HMPV F encoding plasmids were co-transfected into Expi293F cells with HMPV F: furin protease: GFP plasmid DNA at a ratio of 3: 2: 5. Two days after transfection, cells were collected, washed, stained and fixed, and then subjected to flow cytometry (FACSCanto II, Becton Dickinson). The staining step included live / dead purple dye (Thermo Fisher) and HMPV F antibodies AD-61026 and DS7, followed by staining with anti-human IgG detection antibodies labeled with Alexa Fluor 647. The median fluorescence intensity (MFI) of the HMPV F antibody signal was determined by applying a single cell, live, GFP-positive cell gate. HMPV pre-fusion F-specific antibody ADI-61026 was detected at comparable levels for all four HMPV F variants, confirming that the processed HMPV F expressed by the full-length membrane was present on the surface of the transfected Expi293F cells in a pre-fusion conformation ( Fig.21). However, the presence of HMPV non-fusion pre-F was also confirmed in these cells, with the highest DS7 binding detected for wild-type backbone MPV221364. The introduction of HR2 substitutions reduced DS7 binding, as did the introduction of head domain substitutions. The combination of both head domain and HR2 region substitutions gave the strongest reduction in DS7 binding, confirming the stabilizing effect of substitutions in both HMPV F protein regions in full-length HMPV F ( Fig.21 ).

[0331] Example 16. Alternative p27 sequences improve HMPV F processing without the need for co-expression of exogenous furin.

[0332] Introduction of a second furin cleavage site between F1 and F2 of HMPV and the RSV p27 domain resulted in complete processing of F0 upon co-transfection with furin (Example 2). To achieve complete processing while eliminating the requirement for co-transfection of exogenous furin, the RSV p27 sequence was optimized.

[0333] Variants of RSV p27 include sequences based on representative RSV A (MPV23259) or B (MPV23260) sequences introduced into the stabilizing backbone MPV221190 ( Fig. 22 A). HMPV F encoding plasmids were co-transfected as described in Example 1 with either a furin encoding plasmid at a 5:1 HMPV F: furin DNA ratio (20% furin) or with a vector encoding plasmid at a 5:1 HMPV F: vector DNA ratio (0% furin), and trimer expression was assessed by analytical SEC as described in Example 1. When co-transfected with 20% furin, backbone MPV221190 and p27 RSV A and RSV B variants (MPV23259 and MPV232660, respectively) eluted as trimers at a retention time of approximately 4.5 minutes ( Fig. 22 B, gray line). However, without furin co-transfection, the elution pattern shifted to a shorter retention time ( Fig. 22 B, black line), indicating incompletely processed HMPV F. The latter was confirmed by detection of HMPV F1 and F2 by Western blot analysis of cell lysates under reducing conditions, as described in Example 1. Without furin co-transfection, an additional band was detected between the F1 and F2 fragments, which probably corresponds to F2 with attached p27 fragments. This additional band was absent in HMPV F cell lysates co-transfected with 20% furin ( Fig. 22 C).

[0334] Additional variations on the RSV p27 segment are based on the backbone MPV23259 with the p27 RSV A sequence and include systematic deletions of glycosylation sites in the p27 sequence (MPV23261-MPV23267) and p27 sequence deletions (MPV23268, MPV23269), such as Fig. 22 Indicated in A. These variants were evaluated as described above and showed comparable trimer elution patterns when co-transfected with 20% furin ( Fig. 22 B, gray line), but showed a different elution profile when no additional furin was transfected ( Fig. 22 C, black line). Expression of p27 variants without co-transfection of furin resulted in incomplete processing of HMPV F for variants with one or two glycan deletions (MPV23261-MPV23266) ( Fig. 22 B, C). However, removal of all three glycosylation sites in MPV23267 resulted in the same trimer retention time, regardless of whether the variant was co-transfected with furin ( Fig. 22 B), and complete HMPV F processing was shown on Western blot ( Fig. 22 C), without the need for exogenous furin expression. A comparable effect was observed by removing 'NNTKNTNVTLS' from the p27 sequence in variant MPV23269, whereas removal of 'LPRFMNYTL' in MPV23268 was unsuccessful.

[0335] In summary, by optimizing the RSV p27 sequence inserted between F1 and F2 of HMPV F, complete HMPV F processing can be achieved without the need for exogenous furin expression.

[0336] Table 2. Standard amino acids, abbreviations and properties

[0337]

[0338]

[0339] sequence

[0340] SEQ ID NO: 1 hMPV A2 fusion protein (TN / 00 / 3-14) full length (signal peptide bold; wild-type cleavage site region italicized, F2 domain underlined)

[0341]

[0342] SEQ ID NO:2:p27 RSVF

[0343]

[0344] SEQ ID NO:3MPV190470 (postF); optimized furin cleavage site in bold; Joint italic; TEVCS bold italic; fold sub bold underlined; Strep tag underlined italic; C-tag underlined

[0345]

[0346] SEQ ID NO 4: MPV201285 (F1 is truncated after 481, linker is italicized; foldon is underlined; C-tag is bold)

[0347]

[0348] SEQ ID NO 5:MPV210498

[0349]

[0350] SEQ ID NO 6:MPV210500

[0351]

[0352] SEQ ID NO7:MPV210502

[0353]

[0354] SEQ ID NO8: MPV210505

[0355]

[0356] SEQ ID NO9: MPV210507

[0357]

[0358] SEQ ID NO 10:MPV210509

[0359]

[0360] SEQ ID NO11:MPV210530

[0361]

[0362] SEQ ID NO12:MPV210531

[0363]

[0364]

[0365] SEQDNO13:MP210742

[0366]

[0367] SEQ ID NO14:MPV210743

[0368]

[0369] SEQ ID NO15:MPV210744

[0370]

[0371]

[0372] SEQ ID NO16:MPV210745

[0373]

[0374] SEQ ID NO17:MPV210746

[0375]

[0376] SEQ ID NO 18:MPV210 / 47

[0377]

[0378] SEQ ID NO19:MPV210748

[0379]

[0380] SEO ID NO 20:MPV210749

[0381]

[0382]

[0383] SEQ ID NO21:MPV210750

[0384]

[0385] SEQ ID NO22:MPV210751

[0386]

[0387] SEQ ID NO23:MPV210752

[0388]

[0389]

[0390] SEQ ID NO 24: MPV211241 (F1 cut after 489, italicized linker, C-tag bold)

[0391]

[0392] SEQ ID NO25:MPV211242

[0393]

[0394] SEQ ID NO26:MPV211243

[0395]

[0396]

[0397] SEQ ID NO27:MPV211244

[0398]

[0399] SEQ ID NO28: MPV211245

[0400]

[0401] SEQ ID NO29:MPV211246

[0402]

[0403]

[0404] SEQ ID NO30:MPV211247

[0405]

[0406] SEQ ID NO31:MPV211248

[0407]

[0408] SEQ ID NO32:MPV211249

[0409]

[0410]

[0411] SEQ ID NO33:MPV211250

[0412]

[0413] SEQ ID NO34:MPV211251

[0414]

[0415] SEQ ID NO35:MPV211252

[0416]

[0417]

[0418] SEQ ID NO36:MPV211253

[0419]

[0420] SEQ ID NO37:MPV211254

[0421]

[0422] SEQ ID NO38:MPV211255

[0423]

[0424]

[0425] SEQ ID NO39:MPV211256

[0426]

[0427] SEQ ID NO40:MPV211257

[0428]

[0429] SEQ ID NO41:MPV211258

[0430]

[0431]

[0432] SEQ ID NO42:MPV211259

[0433]

[0434] SEQ ID NO43:MPV211260

[0435]

[0436] SEQ ID NO44: MPV211261

[0437]

[0438]

[0439] SEQ ID NO45:MPV211262

[0440]

[0441] SEQ ID NO46:MPV211263

[0442]

[0443] SEQ ID NO47:MPV211264

[0444]

[0445]

[0446] SEQ ID NO48:MPV211265

[0447]

[0448] SEQ ID NO49:MPV211266

[0449]

[0450] SEQ ID NO50:MPV211267

[0451]

[0452]

[0453] SEQ ID NO51:MPV211268

[0454]

[0455] SEQ ID NO52:MPV211269

[0456]

[0457] SEQ ID NO53:MPV211270

[0458]

[0459]

[0460] SEQ ID NO54:MPV211271

[0461]

[0462] SEQ ID NO55:MPV211272

[0463]

[0464] SEQ ID NO56:MPV211273

[0465]

[0466]

[0467] SEQ ID NO57:MPV211274

[0468]

[0469] SEQ ID NO58:MPV211275

[0470]

[0471] SEQ ID NO59:MPV211276

[0472]

[0473]

[0474] SEQ ID NO60:MPV211277

[0475]

[0476] SEQ ID NO61:MPV211278

[0477]

[0478] SEQ ID NO62:MPV211279

[0479]

[0480]

[0481] SEQ ID NO63:MPV211280

[0482]

[0483] SEQ ID NO64:MPV211281

[0484]

[0485] SEQ ID NO65:MPV211282

[0486]

[0487]

[0488] SEQ ID NO66:MPV211283

[0489]

[0490] SEQ ID NO67:MPV211284

[0491]

[0492] SEQ ID NO68:MPV211285

[0493]

[0494]

[0495] SEQ ID NO69:MPV211286

[0496]

[0497] SEQ ID NO70:MPV211287

[0498]

[0499] SEQ ID NO71:MPV211288

[0500]

[0501]

[0502] SEQ ID NO72:MPV211289

[0503]

[0504] SEQ ID NO73:MPV211290

[0505]

[0506] SEQ ID NO 74:MPV211291

[0507]

[0508]

[0509] SEQ ID NO75:MPV211292

[0510]

[0511] SEQ ID NO76:MPV211917

[0512]

[0513] SEQ ID NO77:MPV211918

[0514]

[0515]

[0516] SEQ ID NO78:MPV211919

[0517]

[0518] SEQ ID NO79:MPV211940

[0519]

[0520] SEQ ID NO80:MPV211942

[0521]

[0522]

[0523] SEQ ID NO81:MPV212017

[0524]

[0525] SEQ ID NO82:MPV212018

[0526]

[0527] SEQDNO83:MPV212019

[0528]

[0529]

[0530] SEQ ID NO84:MPV212020

[0531]

[0532] SEQ ID NO85:MPV212021

[0533]

[0534] SEQ ID NO86:MPV212022

[0535]

[0536]

[0537] SEQ ID NO87:MPV212023

[0538]

[0539] SEQ ID NO88:MPV212024

[0540]

[0541] SEQ ID NO89:MPV212025

[0542]

[0543]

[0544] SEQ ID NO90:MPV212026

[0545]

[0546] SEQ ID NO91:MPV212027

[0547]

[0548] 5EQ ID NO92:MPV212028

[0549]

[0550]

[0551] SEQ ID NO93:MPV212029

[0552]

[0553] SEQ ID NO94:MPV212030

[0554]

[0555] SEQ ID NO95:MPV212031

[0556]

[0557]

[0558] SEQ ID NO96:MPV212032

[0559]

[0560] SEQ ID NO97:MPV212033

[0561]

[0562] SEQ ID NO98:MPV212034

[0563]

[0564]

[0565] SEQ ID NO99:MPV212035

[0566]

[0567] SEQ ID NO100:MPV212036

[0568]

[0569] SEQ ID NO101:MPV212037

[0570]

[0571]

[0572] SEQ ID NO102:MPV212038

[0573]

[0574] SEQ ID NO103:MPV212039

[0575]

[0576] SEQ ID NO104:MPV212040

[0577]

[0578]

[0579] SEQ ID NO105:MPV212041

[0580]

[0581] SEQ ID NO106:MPV212042

[0582]

[0583] SEQ ID NO107:MPV212043

[0584]

[0585]

[0586] SEQ ID NO108:MPV212044

[0587]

[0588] SEQ ID NO109:MPV212045

[0589]

[0590] SEQ ID NO110:MPV212046

[0591]

[0592]

[0593] SEQ ID NO111:MPV212047

[0594]

[0595] SEQ ID NO112:MPV220078

[0596]

[0597] SEQ ID NO113:MPV220079

[0598]

[0599]

[0600] SEQ ID NO114:MPV220081

[0601]

[0602] SEQ ID NO115:MPV220083

[0603]

[0604] SEQ ID NO116:MPV220084

[0605]

[0606]

[0607] SEQ ID NO117:MPV220087

[0608]

[0609] SEQ ID NO118:MPV220092

[0610]

[0611] SEQ ID NO119:MPV220115

[0612]

[0613]

[0614] SEQ ID NO120:MPV220116

[0615]

[0616] SEQ ID NO121:MPV220117

[0617]

[0618] SEQ ID NO122:MPV220118

[0619]

[0620]

[0621] SEQ ID NO123:MPV220119

[0622]

[0623] SEQ ID NO124:MPV220120

[0624]

[0625] SEQ ID NO125:MPV220121

[0626]

[0627]

[0628] SEQ ID NO126:MPV220122

[0629]

[0630] SEQ ID NO127:MPV220123

[0631]

[0632] SEQ ID NO128:MPV220124

[0633]

[0634]

[0635] SEQ ID NO129:MPV220125

[0636]

[0637] SEQ ID NO130:MPV220126

[0638]

[0639] SEQ ID NO131:MPV220127

[0640]

[0641]

[0642] SEQ ID NO132:MPV220128

[0643]

[0644] SEQ ID NO133:MPV220129

[0645]

[0646] SEQ ID NO134:MPV220130

[0647]

[0648]

[0649] SEQ ID NO135:MPV220131

[0650]

[0651] SEQ ID NO:136:5'UTR

[0652]

[0653] SEQ ID NO: 137: α5′ replication sequence from nsP1

[0654] SEQ ID NO:138:Gdlp

[0655]

[0656] SEQ ID NO: 139: P2A

[0657]

[0658] SEQ ID NO: 140: nsp1 coding sequence

[0659]

[0660] SEQ ID NO: 141: nsp2 coding sequence

[0661]

[0662] SEQ ID NO: 142: nsp3 coding sequence

[0663]

[0664] SEQ ID NO: 143: nsp4 coding sequence

[0665]

[0666] SEQ ID NO:144:26S minimal promoter

[0667] SEQ ID NO: 145: 3'-UTR

[0668]

[0669] SEQ ID NO: 146: P2A

[0670]

[0671] SEQ ID NO: 147: Polyadenylation site

[0672] SEQ ID NO:148: T7 promoter

[0673] SEQ ID NO: 149: Signal peptide HMPV

[0674]

[0675] SEQ ID NO: 150: Sequence of p27 peptide from RSVB

[0676] SEQ ID NO:151:MPV220554

[0677]

[0678] SEQ ID NO:152:MPV220558

[0679]

[0680] SEQ ID NO: 153: MPV220647

[0681]

[0682] SEQ ID NO: 154: MPV220639

[0683]

[0684] SEQ ID NO: 155: MPV220640

[0685]

[0686] SEQ ID NO: 156: MPV220641

[0687]

[0688] SEQ ID NO:157: MPV23362 (wild type B)

[0689]

[0690] SEQ ID NO:158: MPV220759

[0691]

[0692] SEQ ID NO:159: MPV221190

[0693]

[0694] SEQ ID NO:160: MPV220552

[0695]

[0696] SEQ ID NO:161: MPV220847

[0697]

[0698] SEQ ID NO:162: MPV220115

[0699]

[0700] SEQ ID NO:163: MPV220851

[0701]

[0702] SEID NO:164: IPV23278

[0703]

[0704] SEQ ID NO:165: MPV23279

[0705]

[0706] SEQ ID NO:166: MPV23280

[0707]

[0708] SEQ ID NO:167: MPV23281

[0709]

[0710] SEQ ID NO:168:MPV221364

[0711]

[0712] SEQ ID NO:169:MPV221376

[0713]

[0714]

[0715] SEQ ID NO:170:MPV221371

[0716]

[0717] SEQ ID NO:171:MPV221377

[0718]

[0719] SEQ ID NO:172:MPV23259

[0720]

[0721]

[0722] SEQ ID NO:173:MPV23260

[0723]

[0724] SEQ ID NO:174:MPV23261

[0725]

[0726] SEQ ID NO:175:MPV23262

[0727]

[0728]

[0729] SEQ ID NO:176:MPV23263

[0730]

[0731] SEQ ID NO:177:MPV23264

[0732]

[0733] SEQ ID NO:178:MPV23265

[0734]

[0735]

[0736] SEQ ID NO:179:MPV23266

[0737]

[0738] SEQ ID NO:180:MPV23267

[0739]

[0740] SEQ ID NO: 181: MPV23268

[0741]

[0742]

[0743] SEQ ID NO:182:MPV23269

[0744]

[0745] SEQ ID NO: 183: MPV220215

[0746]

[0747] SEQ ID NO:184:MPV23324

[0748]

[0749]

[0750] SEQ ID NO: 185: Optimized RSVAP27 sequence

[0751]

[0752] SEQ ID NO: 186: Optimized RSVB P27 sequence

[0753]

[0754] SEQ ID NO: 187: pSMARRT_MPV23324_RNA restriction cloning ligation product

[0755]

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763] SEQ ID NO: 188: pSMARRT_MPV23267 (soluble) - RNA restriction cloning ligation product.

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

Claims

1. A pre-fusion human pneumovirus (HMPV) F precursor (F0) protein comprising F1 and F2 domains and comprising at least one modification in the amino acid sequence of the F1 and / or F2 domains.

2. The protein of claim 1, wherein the at least one modification stabilizes the pre-fusion conformation and / or increases trimer formation.

3. The protein according to claim 1 or 2, wherein the at least one modification is the introduction of at least one non-native cleavage site.

4. The protein of claim 3, wherein the protein further comprises a second non-native cleavage site in the F2 domain positioned N-terminal to the first cleavage site, wherein there is a spacer sequence between the first non-native cleavage site and the second non-native cleavage site.

5. The protein according to claim 3 or 4, wherein the first non-native cleavage site and / or the second non-native cleavage site comprises the amino acid sequence RXXR.

6. The protein of claim 3, 4 or 5, wherein the first non-native cleavage and / or the second non-native cleavage comprises the sequence RX[K / R]R.

7. The protein of any one of claims 1-6, comprising the p27 peptide of the RSV F protein between the F1 and F2 domains.

8. The protein of claim 7, wherein the p27 peptide comprises the amino acid sequence ELPRFMNYTLNNAKKTNVTLSKKRKRR (SEQ ID NO: 2), or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

2.

9. The protein of claim 7, wherein the p27 peptide comprises the amino acid sequence of a RSV B p27 peptide comprising the amino acid sequence EAPQYMNYTINTTKNLNVSISKKRKRR (SEQ ID NO: 150), or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

150.

10. The protein of any one of claims 7-9, wherein one or more glycosylation sites in the p27 peptide sequence have been deleted.

11. The protein of claim 10, wherein all glycosylation sites in the p27 sequence have been deleted.

12. The protein according to any one of claims 7 to 11, wherein the p27 peptide sequence comprises a deletion of amino acid sequences 1 to 11 from the p27 peptide sequence.

13. The protein of any one of claims 7-12, wherein the p27 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 185 and SEQ ID NO:

186.

14. The protein according to any one of the preceding claims 1 to 13, comprising a C-terminally truncated F2 domain.

15. The protein of claim 14, wherein the F2 domain is truncated after the amino acid at position 89.

16. The protein according to any one of the preceding claims, comprising a C-terminally truncated F1 domain.

17. The protein of claim 16, wherein the F1 domain is truncated after the amino acid residue at position 489 of the HMPV F precursor (F0) protein.

18. The protein of any one of the preceding claims, comprising one or more stabilizing amino acid residues in the HR2 domain comprising amino acids 453 to 484 of the HMPV F precursor (F0) protein.

19. The protein of claim 18, wherein the one or more stabilizing amino acids optimize inter-protomer interactions between one or more amino acid residues in the HR2 domains of different HMPVF protomers.

20. The protein of claim 18 or 19, wherein the amino acid at position 477 is I, V, L or F or M.

21. The protein according to claim 18, 19 or 20, wherein in addition, the amino acid residue at position 473 is I, F or W, and / or the amino acid residue at position 474 is or I, and / or the amino acid residue at position 475 is R, and / or the amino acid residue at position 476 is K, and / or the amino acid residue at position 478 is D, and / or the amino acid residue at position 479 is E, and / or the amino acid residue at position 480 is L, and / or the amino acid residue at position 484 is I, and / or the amino acid residue at position 488 is I.

22. The protein of any one of claims 18-21, wherein the amino acid residue at position 473 is W, the amino acid residue at position 477 is I, and the amino acid residue at position 484 is I.

23. The protein of any one of claims 18-21, wherein the amino acid residue at position 473 is W, the amino acid residue at position 476 is K, the amino acid residue at position 477 is F, and the amino acid residue at position 484 is I.

24. The protein according to any one of claims 18 to 21, wherein the amino acid residue at position 473 is W, the amino acid residue at position 475 is R, the amino acid residue at position 476 is K, the amino acid at position 477 is I, the amino acid residue at position 478 is D, the amino acid residue at position 479 is E, and the amino acid residue at position 484 is I.

25. The protein according to any one of the preceding claims, wherein the amino acid residue at position 112 is R, and / or the amino acid residue at position 209 is E, and / or the amino acid residue at position 453 is P or Q.

26. The protein according to any one of the preceding claims, wherein the amino acid residue at position 149 is Y, and / or the amino acid residue at position 313 is W, and / or the amino acid residue at position 445 is Y.

27. The protein of claim 26, wherein the amino acid residue at position 112 is R, the amino acid residue at position 209 is E, and the amino acid residue at position 453 is P or Q.

28. The protein of any one of the preceding claims, wherein the amino acid residue at position 231 is 1.

29. The protein of any one of the preceding claims, wherein the amino acid residue at position 404 is P.

30. The protein of any one of the preceding claims, wherein the amino acid residue at position 368 is N.

31. The protein according to any of the preceding claims, wherein the amino acid residue at position 69 is Y or W, and / or the amino acid residue at position 73 is W, and / or the amino acid residue at position 185 is P, and / or the amino acid residue at position 191 is I, and / or the amino acid residue at position 116 is H, and / or the amino acid residue at position 342 is P.

32. The protein of any one of the preceding claims, further comprising one or more non-native intra-protomer or inter-protomer disulfide bonds.

33. The protein of claim 32, wherein the one or more disulfide bonds are selected from an intra-protomer disulfide bond between amino acid residues 140 and 147, and / or an intra-protomer disulfide bond between amino acid residues 141 or 161, and / or an intra-protomer disulfide bond between amino acid residues 360 and 459.

34. The protein according to any one of the preceding claims, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-135, SEQ ID NOs: 151-159, SEQ ID NOs: 161-182 and SEQ ID NO: 184, or a fragment thereof.

35. The protein of claim 34, comprising the amino acid sequence of SEQ ID NO: 111, SEQ ID NO: 159, SEQ ID NO: 180 or SEQ ID NO: 184 or a fragment thereof.

36. The protein of any one of claims 1-35, wherein the protein has been cleaved at one or more cleavage sites resulting in F2 and F1 domains being covalently linked by one or more native disulfide bridges, and wherein the protein is trimeric.

37. The protein of claim 36, wherein the F1 domain comprises amino acids 103-489 of the HMPV F0 protein and the F2 domain comprises amino acids 19-88 of the HMPV F0 protein.

38. A nucleic acid molecule encoding a protein according to any one of the preceding claims 1-37.

39. The nucleic acid of claim 38, wherein the nucleic acid molecule is DNA or RNA.

40. The nucleic acid according to claim 38 or 39, which encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-135, SEQ ID NOs: 151-159, SEQ ID NOs: 161-182 and SEQ ID NO: 184, or a fragment thereof.

41. The nucleic acid of claim 40, encoding a protein comprising the amino acid sequence of SEQ ID NO: 111, the amino acid sequence of SEQ ID NO: 159, the amino acid sequence of SEQ ID NO: 180, or the amino acid sequence of SEQ ID NO:

184.

42. A vector comprising a nucleic acid according to any one of claims 38 to 41.

43. A pharmaceutical composition comprising a protein according to any one of claims 1 to 37, a nucleic acid according to any one of claims 38 to 41 and / or a vector according to claim 42.

44. A method for vaccinating a subject against HMPV, the method comprising administering to the subject the pharmaceutical composition of claim 43.

45. A method for preventing HMPV infection and / or replication in a subject, comprising administering to the subject a pharmaceutical composition according to claim 43.

46. ​​An isolated host cell comprising the nucleic acid of any one of claims 38-41.

Images