CN117836001A

CN117836001A - Immunogenic compositions and uses thereof

Info

Publication number: CN117836001A
Application number: CN202280057049.3A
Authority: CN
Inventors: A·勒韦尔; J·布莱; F·尼古拉斯; P·威廉斯; D·居永-热兰
Original assignee: Austria Civaux Co
Current assignee: Austria Civaux Co
Priority date: 2021-08-24
Filing date: 2022-08-24
Publication date: 2024-04-05

Abstract

The present invention relates to immunogenic compositions and their use as vaccines for preventing influenza disease in human subjects. More specifically, the present invention relates to a method of preventing or treating influenza disease in a human subject in need thereof as a vaccine or immunotherapy comprising: a fusion protein comprising (i) an influenza virus nucleoprotein antigen, and (ii) a carrier protein comprising a self-assembled polypeptide derived from a C4bp oligomerization domain and a positively charged tail, wherein the fusion protein is administered to the human subject in an amount of 180 μg or more.

Description

Immunogenic compositions and uses thereof

Technical Field

The present disclosure relates to immunogenic compositions and their use as vaccines in preventing influenza disease in human subjects.

Background

Seasonal influenza is estimated to cause about 3 to 5 million severe cases of disease worldwide each year, of which about 290,000 to 650,000 die, mostly in people 65 years old or older [ Iuliano AD, et al lancet.2018;391:1285-1300].

Annual vaccination is considered the most effective method of preventing influenza. Seasonal trivalent or tetravalent influenza vaccine efficacy was limited on average to 42% in the last decade in the total population in the united states (32% in the elderly) [ CDC-past seasonal vaccine efficacy estimate. Obtainable from: https:// www.cdc.gov/flu/vaccines-work/past-search-estimates. Access date: 2021, 5, 21 ]. Similar data are available in Europe [ Kramer F, et al Influ Respir viruses 2020;14:237-243]. When the circulating virus does not match the vaccine virus, the efficacy may only drop to 10-20%, as in the quarter 2014-2015. Thus, there is a medical need to improve the efficacy of influenza vaccines.

Although the antibody threshold values for viral surface Hemagglutinin (HA) and Neuraminidase (NA) are considered as alternatives/relatives to efficacy in clinical trials of most current vaccines, cellular responses, particularly CD 4-and CD 8-mediated responses, are highly likely to contribute to protection, particularly in the elderly [ McElhaney JE, et al front immunol.2016;7:41.Trombetta CM,et al.Expert Rev Vaccines.2016;15:967-976.Pleguezuelos O,et al.Clin Vaccine Immunol.2015;22:949-956; savic M, et al immunology 2016;147:165-177].

Viral Nucleoprotein (NP) may be the target of choice in an attempt to improve currently available influenza vaccines by T-cell responses. The internal protein is highly conserved between the A-type strain and the A-type and B-type strains, and provides structural and functional support for the viral replication mechanism [ Ye Q, krug RM, tao YJ. Nature.2006;444:1078-1082]. In humans, there is increasing evidence that T cell immunity against conserved internal antigens plays a role in anti-influenza protection. Prospective cohort studies conducted during H1N1 pandemics in 2009 showed that pre-existing T cells specific for the conserved CD8 epitope were found at higher frequencies in individuals who developed less severe disease [ Sridhar S, et al nat med.2013;19:1305-1312]. Influenza monitoring cohort studies indicate that pre-existing T cell responses targeting internal viral proteins provide protective immunity against pandemic and seasonal influenza. The presence of NP-specific T cells (above the threshold of 20 spot forming units [ SFC ]/million peripheral blood mononuclear cells [ PBMC ]) was associated with fewer symptomatic Polymerase Chain Reaction (PCR) -positive cases of a-stream during pandemic and seasonal influenza prior to exposure to virus [ Hayward AC, et al am J Respir Crit Care med 2015;191:1422-1431]. These results provide rationality in developing NP-based anti-influenza vaccines.

OVX836 (OSIVAX, freon, france) is a recombinant protein developed as a broad-spectrum vaccine against all influenza strains. The antigenic portion corresponds to the NP sequence of the A/WSN/1933 (H1N 1) influenza virus. The OVX836 protein contained 7 copies of NP, each fused to OVX 313. The OVX313 sequence is derived from the C-terminal oligomerization domain of the human C4b binding protein (hC 4 BP) [ Hofmeyer T, et al J Mol biol.2013;425:1302-1317], but modified to minimize homology to human sequences (hybrid chicken sequences; less than 20% homology). OVX313 has the unique properties of heptameric antigens when fused to antigens by deoxyribonucleic acid (DNA) engineering, and after protein expression, thereby improving antigen accessibility to the immune system and increasing their humoral and cellular immune responses [ Del Campo j., et al npj vaccines.2019;4:4]. Since NP is not affected by antigenic variation, OVX836 will not need to be adjusted annually as required by current seasonal influenza vaccines. Animal studies have demonstrated that OVX836 elicits humoral and cellular immunity (including cd8+ T cells in the lung) and in mice [ Del Campo j., et al npj vaccines.2019;4:4] and ferret [ Del Campo J, et al options X Control Influenza-Singapore 2019; protection against influenza attacks in Abstract No 10936:456 ]. Importantly, OVX836 protects mice from virus attacks of three different influenza a subtypes separated by decades, and this is accompanied by a reduction in viral load. Although recent non-clinical experiments with OVX836 in mice supported CD8+ T as the most potent immune response, both CD4+ and CD8+ T-cells were likely involved in the destruction of infected cells [ Del Campo J, et al front immunol.2021.https:// www.frontiersin.org/statics/10.3389/fimmu.2021.678483/abstract. Access date: 2021, 5, 21 ].

A first human clinical study was performed to assess the safety and immunogenicity of OVX 836.

There remains a need for further improvements in dosing regimens and formulations of immunogenic compositions comprising OVX836 fusion proteins or functional variants thereof.

Summary of The Invention

One aspect of the present disclosure relates to an immunogenic composition for use as a vaccine or immunotherapy for preventing or treating influenza disease in a human subject in need thereof, the immunogenic composition comprising: a fusion protein comprising

(i) An influenza virus nucleoprotein antigen, and a nucleic acid molecule,

(ii) A carrier protein comprising a self-assembled polypeptide derived from a C4bp oligomerization domain and a positively charged tail,

wherein the human subject is administered an amount of 180 μg or more of the fusion protein, e.g. an amount of 180 μg to 1000 μg.

Another aspect of the disclosure claims an immunogenic composition comprising a fusion protein as defined above at a concentration of 300 μg/mL or more, and one or more pharmaceutically acceptable excipients, especially for use as a vaccine or immunotherapy for preventing or treating influenza disease in a human subject in need thereof.

Drawings

Fig. 1: mean, median and SD of the number of NP-specific ifnγ spot forming T cells (SFC/million PBMCs) in the four treatment groups at baseline (day 1) prior to inoculation.

Fig. 2: on day 8 post-inoculation, the average, median and SD of the numbers of NP-specific ifnγ spot-forming T cells (SFC/million PBMCs) in the four treatment groups.

Fig. 3: on day 36 post-inoculation (i.e., 8 days post-2 inoculation), the average, median and SD of NP-specific ifnγ spot-forming T cells (SFC/million PBMCs) at each time point in the four treatment groups.

Fig. 4: NP-specific IFN-gamma spot forming T cells (SFC)/10 in the pooled placebo and three OVX836 vaccinated groups (30 μg, 90 μg and 180 μg) ⁶ The number of individual cells was varied from baseline (day 1, pre-inoculation) to day 150 (day 24 months after the second administration). Results are expressed as arithmetic mean ± standard error. When Kruskal-Wallis test is significant, p compared to placebo<0.05，**p<0.01, dpass, steel, critchlow-Fligner post hoc test.

Fig. 5: graph a. NP-specific IFN-gamma Spot Forming Cells (SFC)/10 in the pooled placebo and 3 OVX836 vaccinated groups (30 μg, 90 μg and 180 μg) at baseline (day 1) and 8 days after the first (day 8) and second (day 36) administrations ⁶ Number of individual cells. * P is p<0.05，**p<0.01, dpass, steel, critchlow-Fligner test. D1 Pre-inoculation baseline, d8=8 days after the first inoculation, d36=8 days after the second inoculation. Graph B. SFC/10 of different groups on day 8 ⁶ Number of individual cells. When Kruskal-Wallis test is significant (p=0.002), p is the ratio of p<0.05，**p<0.01, dpass, steel, critchlow-Fligner post hoc test. In both figures, the results are shown in box plots, with the median (horizontal bars in the box), the quartile interval (ends of the box), and the minimum and maximum (up and down error bars) values.

Fig. 6: graph a. Evolution of NP-specific immunoglobulin G (IgG) geometric mean titers (gmt±95% confidence interval [ CI ]) in pooled placebo and 3 OVX836 vaccine groups (30 μg, 90 μg, and 180 μg) over time from baseline (day 1 before vaccination) to day 150 (4 months after 2 nd administration). When Kruskal-Wallis test is significant, p <0.05; * P <0.01, dwass, steel, critchlow-Fligner post hoc test. Graph B. In the pooled placebo and three OVX836 vaccine groups (30 μg, 90 μg and 180 μg), a four-fold increase in NP-specific IgG titers was presented as a percentage of subjects between baseline (day 1 before vaccination) and day 29 (day 28 after first administration). * p <0.05; * P <0.001, fisher's exact test.

Fig. 7: within the 3 treatment groups, on average (SFC/million PBMC) of NP-specific IFNγ spots forming T cells on days 1 and 8 in the pooled age group (intent-to-treat (ITT) cohort with two abnormal subjects eliminated in the OVX836 180 μg group (subjects 128-095 and 232-365 present high baseline values on days 1: 957 and 1630, respectively).

Fig. 8: percentage of ifnγ positive NP-specific cd4+ T cells at baseline (day 1), day 8, day 29 and day 180 in the pooled age group (day 29 according to protocol (PP-D29) cohort).

Fig. 9: the cumulative risk of nonspecific ILI during the influenza season (12 th 2019 to 9 th 2020) -ITT (intent to treat cohort) as a function of time between inoculation and ILI start date.

Fig. 10: the cumulative risk of nonspecific ILI that occurs 14 days after inoculation of ITT (intent-to-treat cohort) during the influenza season (12 th 2019, 2 th 2020, 3 th, 9 th) as a function of time between inoculation and ILI start date.

Fig. 11: influenza season (3 months 9 days ago) and ILI number exceeding 14 days-ITT (intent-to-treat cohort) post inoculation.

Fig. 12: for subjects belonging to the lowest quartile of cd8+ responses at baseline, at least the median percentage of ifnγ positive NP-specific cd8+ T cells on days 1 and 8.

Fig. 13: in placebo and three OVX836 vaccine groups (180 μg, 300 μg and 480 μg), the Geometric Mean Titer (GMT) of NP-specific immunoglobulin G (IgG) evolved over time from baseline (day 1 before vaccination) to day 29 (month 1 after immunization). P <0.001 compared to placebo; when the Anova test is significant, the postmortem Bonferroni groups are compared in pairs.

Fig. 14: graph a: average change in NP-specific total T cell responses assessed by ifnγ ELISpot-statistics on day 8 compared to day 1 for placebo and 3 OVX836 vaccine groups (180 μg, 300 μg, and 480 μg): after confirming that the ANOVA test is significant between groups (p < 0.05), fisher's LSD comparisons are made in pairs; error bars represent standard error-graph B: average change in percentage of ifnγ positive NP-specific cd4+ T cells on day 8 compared to day 1-statistics for placebo group and 3 OVX836 vaccine groups (180 μg, 300 μg, and 480 μg): after confirming that the ANOVA test is significant between groups (p < 0.05), fisher's LSD comparisons are made in pairs; error bars represent standard error—graph C: average change in percentage of NP-specific cd8+ T cells positive for at least ifnγ -compared to day 1-on day 8-placebo and 3 OVX836 vaccine groups (180 μg, 300 μg and 480 μg) -statistics: after confirming that the ANOVA test is significant between groups (p < 0.05), fisher's LSD comparisons are made in pairs; error bars represent standard error.

Fig. 15: the aggregate risk of PCR-confirmed ILI-the pooled OVX836 group (180. Mu.g, 300. Mu.g, and 480. Mu.g) of the OVX836-003 study and the ITT-OVX836 and FLU-001 study of the pooled untreated group (FLU-001 study) and placebo group (OVX 836-003 study) were used to treat the pooled cohorts.

Detailed Description

Definition of the definition

For easier understanding of the present disclosure, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids (also referred to herein as "non-naturally occurring amino acids"), e.g., amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon, a carboxyl group, an amino group, and an R group bonded to hydrogen, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid. The terms "amino acid" and "amino acid residue" are used interchangeably throughout.

Substitution refers to the replacement of a naturally occurring amino acid with another naturally occurring amino acid or with a non-naturally occurring amino acid.

As used herein, the term "protein" refers to any organic compound consisting of amino acids arranged in one or more linear chains (also referred to as "polypeptides") and folded into a spherical form. It includes proteinaceous materials or fusion proteins. The amino acids in such polypeptide chains may be linked together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The term "protein" also includes, but is not limited to, a peptide, a single chain polypeptide, or any complex protein consisting essentially of two or more amino acid chains. It also includes, but is not limited to, glycoproteins or other known post-translational modifications. It also includes known natural or artificial chemical modifications of the natural protein, such as, but not limited to, glycoengineering, pegylation, glycosylation, PAS, etc., incorporation of unnatural amino acids, amino acid modifications for chemical conjugation or other molecules, etc.

The term "recombinant protein" as used herein includes proteins prepared, expressed, produced or isolated by recombinant methods, such as fusion proteins isolated from host cells transformed to express the corresponding protein, such as fusion proteins isolated from transfectomas and the like.

As used herein, the term "fusion protein" refers to a recombinant protein comprising at least one polypeptide chain, which is obtained by genetic fusion or which may be obtained by genetic fusion, for example by genetic fusion of at least two gene segments encoding separate functional domains of different proteins. The fusion proteins of the present disclosure include, for example, at least one influenza virus nucleoprotein antigen and at least one other moiety that is a carrier protein comprising a self-assembled polypeptide derived from a C4bp oligomerization domain as described below and its positively charged tail.

As used herein, the term "antigenic" polypeptides includes immunogenic fragments and epitopes of a particular polypeptide (e.g., nucleoprotein NP of an influenza virus) that are capable of inducing an immune response (e.g., NP-specific immune response) against such antigenic polypeptide, at least when such antigenic polypeptide is fused to the carrier proteins disclosed herein.

As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% identity =number of identical positions/total number of positions x 100), and optimal alignment of the two sequences requires consideration of the number of introduced gaps and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms, as described below.

The percentage identity between two amino acid sequences can be determined using Needleman and Wunsch algorithms (NEEDLEMAN and Wunsch).

The percentage identity between two nucleotide or amino acid sequences can also be obtained using, for example, an algorithm such as EMBOSS Needle (pairing alignment; available at www.ebi.ac.uk, rice et al2000Trends Gnet 16:276-277). For example, EMBOSS Needle may be used with the BLOSUM62 matrix with a "gap opening penalty" of 10, a "gap extension penalty" of 0.5, a "false" end gap penalty ", a" end gap opening penalty "of 10, and a" end gap extension penalty "of 0.5. In general, the "percent identity" is a function of the shul of matching locations divided by the number of comparison locations and multiplied by 100. For example, if 6 of the 10 sequence positions are identical between the two comparison sequences after alignment, then the identity is 60%. The% identity is typically determined over the entire length of the query sequence being analyzed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical, irrespective of any chemical and/or biological modification.

As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" preferably includes mammals, such as non-human primates, sheep, dogs, cats, horses, etc.

As used herein, a "variant" of a polypeptide may be a naturally occurring or artificially mutated variant, e.g., typically obtained by amino acid substitution, deletion or insertion, as compared to the corresponding naturally occurring polypeptide. In certain embodiments, a variant may have a combination of amino acid deletions, insertions, or substitutions throughout its sequence as compared to the parent polypeptide.

In the context of the present disclosure, conservative substitutions may be defined by substitutions within the amino acid class reflected as follows:

fat residue I, L, V and M

Cycloalkenyl related residues F, H, W and Y

Hydrophobic residue A, C, F, G, H, I, L, M, R, T, V, W and Y

Negatively charged residues D and E

Polar residue C, D, E, H, K, N, Q, R, S and T

Positively charged residues H, K and R

Small residues A, C, D, G, N, P, S, T and V

Minimal residues A, G and S

Corner related residue A, C, D, E, G, H, K, N, Q, R, S, P and residue T related to formation

Flexible residue Q, T, K, S, G, P, D, E and R

A "functional variant" is a variant that retains the property of interest of the native polypeptide.

In preferred embodiments, the variant comprises an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90% or 95% identical to the native polypeptide sequence.

Thus, within the scope of the present disclosure are polypeptides comprising substitutions, insertions and/or additions, deletions and covalent modifications relative to a reference sequence, particularly the NP fusion proteins disclosed herein. For example, a sequence tag or amino acid, such as one or more lysines, may be added to the peptide sequence (e.g., at the N-terminus or C-terminus). Sequence tags may be used for peptide detection, purification or localization. Lysine can be used to increase peptide solubility or allow biotinylation. Alternatively, amino acid residues located in the carboxy and amino terminal regions of the amino acid sequence of the peptide or protein may optionally be deleted, providing a truncated sequence. Alternatively, certain amino acids (e.g., C-terminal residues or N-terminal residues) may be deleted depending on the use of the sequence, e.g., expression of the sequence as part of a larger sequence that is soluble or attached to a solid support.

Influenza virus nucleoprotein antigen

The term "influenza virus nucleoprotein antigen" as used herein refers to any native influenza virus nucleoprotein or antigenic variant thereof.

The natural influenza virus nucleoprotein comprises nucleoprotein of any of three types of influenza a, b and c viruses, preferably type a.

In some embodiments, the nucleoprotein antigen (NP) is derived from a strain of either a stream or a stream b, or a combination thereof. In some embodiments, the strain of a stream a or b is associated with a bird, pig, horse, dog, human, or non-human primate. In some embodiments, the strain is selected from the group consisting of H1N1, H3N2, H7N9, and H10N8.

In a specific embodiment, the influenza virus nucleoprotein antigen is an NP antigen of a influenza A virus, more specifically from strain A/wilson-Smith/1933H 1N1, comprising the polypeptide of SEQ ID NO:1.

In a specific embodiment, the antigenic variant is a fragment of an influenza virus nucleoprotein antigen having at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 490 consecutive amino acid residues of a wild-type nucleoprotein of a, b or c virus, preferably derived from SEQ ID No. 1. Fragments of influenza virus nucleoprotein antigen are defined as being at least one amino acid shorter than the full-length wild-type nucleoprotein of a, b or c virus.

In specific embodiments, the antigenic variant of an influenza virus nucleoprotein antigen is an antigenic polypeptide variant having at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity to the corresponding wild-type sequence of a nucleoprotein of a, b or c virus. Preferably, the antigenic variant of the influenza virus nucleoprotein antigen is an antigenic polypeptide variant having at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity to SEQ ID No. 1.

In a particular embodiment, the variants differ from the corresponding influenza virus nucleoprotein natural antigen only by amino acid substitutions with natural or unnatural amino acids, preferably by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions with natural amino acids only, especially compared to the natural influenza virus NP antigen of SEQ ID NO:1. In a specific embodiment, the variant is a mutant variant having 1, 2 or 3 amino acids substituted with natural amino acids as compared to the natural influenza NP antigen of SEQ ID NO. 1.

In more specific embodiments, the amino acid sequence of the mutant variant may differ from the native influenza virus NP antigen by a largely conservative amino acid substitution; for example, at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in a variant are conservative amino acid residue substitutions.

More conservative permutation packets include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. Conservation in terms of hydrophilic/hydrophilic properties and residue weight/size can also be substantially retained in the variant mutant polypeptide compared to the parent polypeptide of any influenza virus NP antigen, typically the parent polypeptide of SEQ ID No. 1.

In a specific embodiment, the mutant variant comprises the same polypeptide as SEQ ID NO. 1 except that 1, 2 or 3 amino acid residues have been replaced by another natural amino acid by conservative amino acid substitutions as defined above.

In a specific embodiment, the variant of the NP antigen does not comprise any mutation in an epitope recognized by the human immune system as compared to the parent polypeptide of SEQ ID NO. 1, as described in the IEDB database (immune epitope database) accessible under www.IEDB.org. In particular embodiments, the variant of the NP antigen does not comprise any mutation in a conserved amino acid residue between the NP of the strain A and the NP of the strain B compared to the parent polypeptide of SEQ ID NO. 1. As used herein, "conserved amino acid residues" correspond to identical amino acid residues between NPs of strains a and b when aligned using standard sequence proteins (such as those using BLAST algorithm).

Amino acid residue E ₃₃₉ And R is ₄₁₆ (numbering with N-terminal methionine, M ₁ ) Is essential for self-assembly of NPs and does not experience genetic diversity of the A-stream virus. Thus, in particular embodiments, variants of the NP antigen comprise E ₃₃₉ And R is ₄₁₆ 。

Carrier protein

As used herein, the term "carrier protein" generally refers to a protein that is conjugated or fused to an antigen to confer higher immunogenicity. The term is used herein in particular for the meaning of antigen-carrying proteins. The function of the protein is to increase the immunogenicity of the antigen to which it is conjugated or fused.

The carrier protein for the fusion protein comprises a self-assembled polypeptide derived from a C4bp oligomerization domain and a positively charged tail. Complement inhibitor C4-binding protein (C4 bp) is a rich plasma protein first found in mice. Its natural function is to inhibit the classical and lectin pathways of complement activation. The last exon of the C4bp alpha chain gene encodes the only domain of the protein that does not belong to the complement control protein family. The non-complement control protein domain contains 57 amino acid residues in humans, 54 amino acid residues in mice, and is both necessary and sufficient for oligomerization of C4 bp. It has been found that the self-assembling polypeptide is also necessary and sufficient for oligomerization of the resulting fusion protein when fused to an antigen.

PCT/IB2004/002717 and PCT/EP03/08926 describe the use of mammalian C4bp oligomerization domains to increase the immunogenicity of antigens in mammals. WO2007/062819 further describes C4bp oligomerization domains of chicken species and variants thereof.

In a preferred embodiment, the self-assembled polypeptide has less than 30%, preferably less than 20% identity to human C4bp in order to minimize autoimmune reactions.

In particular, in a specific embodiment, the self-assembled polypeptide derived from a C4bp oligomerization domain comprises or consists essentially of SEQ ID NO. 2.

In specific embodiments, the functional variant of the self-assembling polypeptide has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity to SEQ ID NO. 2.

Functional variants may include any variant having one or more amino acid additions, deletions and/or substitutions compared to SEQ ID NO. 2 that retain the self-assembly properties of the polypeptide of SEQ ID NO. 2.

In a particular embodiment, the variant differs from SEQ ID NO. 2 only in amino acid substitutions with natural or unnatural amino acids, preferably in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions with natural amino acids only. In a specific embodiment, the variant is a mutant variant having 1, 2 or 3 amino acids replaced by natural amino acids compared to SEQ ID NO. 2.

In more specific embodiments, the amino acid sequence of the mutant variant may differ from the self-assembled polypeptide of SEQ ID NO. 2 by a majority of conservative amino acid substitutions; for example, at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in a variant are conservative amino acid residue substitutions.

The carrier protein also comprises a C-terminal tail consisting of a positively charged peptide. The C-terminal tail is preferably a peptide consisting of 6-10 amino acids, which has at least 50% positively charged amino acids. Positively charged amino acids include arginine or lysine. Examples of such positively charged peptides are disclosed in WO2014/090905 and WO 2014/147087.

In a preferred embodiment, the positively charged tail comprises the sequence ZXBBBZ (SEQ ID NO: 3), wherein (i) Z is absent or any amino acid, (ii) X is any amino acid, and (iii) B is arginine or lysine, preferably the positively charged tail comprises or consists essentially of the sequence of SEQ ID NO: 4.

In a more preferred embodiment, the carrier protein consists essentially of an OVX313 polypeptide, corresponding to the polypeptide of SEQ ID NO. 5.

In a specific embodiment, the carrier protein is a functional variant of the OVX313 polypeptide of SEQ ID NO. 5 that has at least 70%, 80%, or more preferably at least 90% identity to SEQ ID NO. 5.

In other embodiments, the carrier protein is a functional variant of the OVX313 polypeptide of SEQ ID NO. 5 which differs from SEQ ID NO. 5 by only 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids by amino acid substitutions. In other embodiments, the carrier protein is a functional variant of the OVX313 polypeptide of SEQ ID NO. 5 which differs from SEQ ID NO. 5 by only 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids by conservative amino acid substitutions.

NP fusion proteins

Fusion proteins for use according to the present disclosure comprise:

(i) Influenza virus nucleoprotein antigen as defined above, and

(ii) A carrier protein as defined above comprising a self-assembled polypeptide derived from a C4bp oligomerization domain and a positively charged tail,

the resulting fusion protein with the nucleoprotein antigen is hereinafter referred to as "NP fusion protein" for ease of reading.

In specific embodiments, the carrier protein is fused at the C-terminus to a nucleoprotein antigen, optionally via a peptide linker. The peptide linker may be any short peptide linker commonly used for fusion proteins. Preferred peptide linkers include glycine-serine linkers, such as the dipeptide gly-ser, or gly-ser-ser, or (gly-ser-ser) n, where n is an integer from 1 to 4.

In particular embodiments, the NP fusion protein forms a heptameric particle upon self-assembly.

In certain embodiments, the NP fusion protein forms particles having a diameter of 15 to 100nm after self-assembly. The diameter of the particles may be measured, for example, by Dynamic Light Scattering (DLS). DLS measures the hydrodynamic diameter of particles ranging in size from about 0.3nm to 10 μm. DLS measurements are very sensitive to temperature and dispersant viscosity. Therefore, the temperature must be kept constant at 25 ℃ and the viscosity of the dispersant must be known.

In a specific embodiment, the NP fusion protein forms a particle having a molecular weight between 440 and 2200 kDa.

In a more preferred embodiment, the NP fusion protein consists essentially of an OVX836 polypeptide, corresponding to the polypeptide of SEQ ID NO. 6.

In particular embodiments, the NP fusion protein is a functional variant of an OVX836 polypeptide having at least 70%, 80%, or more preferably at least 90% identity to SEQ ID NO. 6.

In other embodiments, the NP fusion protein is a functional variant of the OVX836 polypeptide of SEQ ID NO. 6 that differs from SEQ ID NO. 6 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids by amino acid substitutions. In other embodiments, the NP fusion protein is a functional variant of an OVX836 polypeptide that differs from SEQ ID NO. 6 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids by conservative amino acid substitutions.

Method for preparing NP fusion protein

The NP fusion proteins for use according to the present disclosure may be prepared by any conventional method for preparing recombinant proteins, using nucleic acid molecules encoding the NP fusion proteins, the nucleotide sequences of which may be readily derived using the genetic code, and optionally taking into account codon preference depending on the host cell type.

Examples of nucleotide sequences that can be used to prepare NP fusion proteins are those encoding the amino acid sequences of SEQ ID NOs 1-6, as generally described in tables 2 and 3.

The nucleic acid molecules may be derived from the latter sequences and are optimized for protein expression in prokaryotic cells, such as E.coli bacterial cells.

The nucleic acid may be present in whole cells, in cell lysates, or may be in partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques including alkali/SDS treatment, csCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. The nucleic acids of the present disclosure may be, for example, DNA or RNA and may or may not contain intronic sequences. In one embodiment, the nucleic acid may be present in a vector, such as a recombinant plasmid vector.

Nucleic acids can be obtained using standard molecular biology techniques. Once the DNA fragments encoding the nucleoprotein antigen are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques. In these operations, for example, a DNA fragment encoding a nucleoprotein antigen may be operably linked to another DNA molecule, such as a fragment encoding a carrier protein and optionally a linker.

The term "operably linked" as used herein refers to the joining of two DNA fragments in a functional manner, e.g., such that the amino acid sequences encoded by the two DNA fragments remain in frame, or such that the protein is expressed under the control of a desired promoter.

NP fusion proteins (particularly OVX 836) for use according to the present disclosure may then be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art.

For example, to express an NP fusion protein (typically OVX 836), its corresponding fragment, DNA encoding a partial or full length recombinant protein may be obtained by standard molecular biology or biochemical techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning), and the DNA may be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences.

In this context, the term "operably linked" refers to the linkage of the coding polypeptide sequence into a vector such that transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the recombinant NP fusion protein. Expression vectors and expression control sequences compatible with the expression host cells used are selected. The protein coding gene is inserted into the expression vector according to the standard.

The recombinant expression vector may encode a signal peptide that facilitates secretion of the recombinant fusion protein from the host cell. The NP fusion protein encoding gene may be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the recombinant protein. The signal peptide may be a natural signal peptide of C4bp or a heterologous signal peptide (i.e., a signal peptide from a non-C4 bp protein). In a specific embodiment, the signal peptide is a methionine amino acid.

In addition to the NP fusion protein coding sequence, the recombinant expression vectors disclosed herein also carry regulatory sequences that control the expression of the recombinant fusion protein in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of a protein-encoding gene. Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. Regulatory sequences for mammalian host cell expression include viral elements that direct high protein expression levels in mammalian cells, such as promoters and/or enhancers derived from Cytomegalovirus (CMV), simian virus 40 (SV 40), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), and polyomaviruses. Alternatively, non-viral regulatory sequences such as ubiquitin promoters or beta-globin promoters may be used. Furthermore, regulatory elements consist of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of the human T cell leukemia virus type 1.

In addition to the NP fusion protein coding sequence and regulatory sequences, the recombinant expression vectors of the present disclosure may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and selectable marker genes. Selectable marker genes facilitate selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017 to Axel et al). For example, selectable marker genes typically confer resistance to drugs such as G418, hygromycin or methotrexate on host cells into which the vector is introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for DHFR-host cell selection/amplification with methotrexate) and the neo gene (for G418 selection).

For expression of the NP fusion protein, the expression vector encoding the recombinant protein is transfected into a host cell by standard techniques. The various forms of the term "transfection" include various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. It is theoretically possible to express the proteins of the present disclosure in prokaryotic or eukaryotic host cells. Expression of the NP protein may be performed in a prokaryotic cell, such as an E.coli host cell. The NP fusion protein may then be recovered by lysing the bacterial cells and further purified using standard purification methods. In a specific embodiment, NP fusion proteins are produced according to the method disclosed in DelCampo2021 (Frontiers in Immunology, doi: 10/3389/fimm.2021.678483).

Immunogenic compositions

In another aspect, the present disclosure provides compositions, e.g., immunogenic compositions, containing an NP fusion protein as described in the previous section at a concentration of 300 μg/mL or more and one or more pharmaceutically acceptable excipients.

Immunogenic compositions include any aqueous vehicle suitable for parenteral, intranasal, intramuscular, or subcutaneous administration (e.g., by intramuscular injection). These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts).

In particular embodiments, the NP fusion protein comprises at least 400 amino acid residues, such as 400 to 600 amino acid residues, such as 540 to 560 amino acid residues, optionally forming particles having a diameter of 15 to 100nm and/or a molecular weight of 440 to 2200kDa in the immunogenic compositions disclosed herein.

For example, the immunogenic composition is an aqueous composition comprising the polypeptide of SEQ ID NO. 6 (OVX 836) or a variant having at least 70%, 80%, preferably at least 90% or at least 95% identity to SEQ ID NO. 6 formulated with one or more pharmaceutically acceptable excipients at a concentration of 300 μg/mL or more.

In particular embodiments, the immunogenic composition may further comprise one or more of the following excipients, for example: buffers, salts, osmotics, antioxidants and surfactants or other agents to prevent protein loss and/or protein aggregation on the vial surface.

The form, route of administration, dosage and regimen of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.

For example, for intramuscular administration, the composition is an aqueous solution, which should be suitably buffered if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this regard, sterile aqueous media that can be used are known to those of skill in the art in light of the present disclosure. For example, a dose may be dissolved in 1ml of isotonic NaCl solution. Examples of formulations for injectable solutions are provided in remington: the Science and Practice of Pharmacy,23 ^rd Edition, 2020. Some variation in dosage may occur depending on the condition of the subject being treated.

In a specific embodiment, the pH of the composition is from 6.0 to 7.0, preferably from 6.3 to 6.6, for example about 6.5.

In a specific embodiment, the osmolality of the immunogenic composition is 300 to 600mOsm/kg, preferably 400 to 500mOsm/kg, for example about 450mOsm/kg.

In a specific embodiment, the immunogenic composition has

(i) Buffers having a pH of from 6.0 to 7.0, preferably from 6.3 to 6.6, for example about 6.5, and

(ii) An effective amount of an osmolality agent having an osmolality of 300 to 600mOsm/kg, preferably 400 to 500mOsm/kg, for example about 450mOsm/kg.

Examples of buffers having a pH of 6.0 to 7.0 include sodium citrate or sodium phosphate/potassium phosphate buffers.

In particular embodiments, the immunogenic composition comprises, in addition to the NP fusion protein (typically OVX 836), at least

Salts, for example sodium sulphate or sodium chloride, preferably sodium sulphate,

osmotics, for example sugars such as trehalose or maltose, preferably trehalose,

buffers, such as phosphate buffers and/or citrate buffers,

an optional antioxidant, such as methionine,

an optional surfactant, such as polysorbate 80,

wherein the pH of the composition is from 6.0 to 7.0, typically from 6.3 to 6.6 and the osmolality is from 300 to 600mOsm/kg.

The salt is used in the form of a salt,

trehalose is used in the form of a sugar,

Buffers having a pH of 6.0 to 7.0, such as phosphate buffers and/or citrate buffers,

an optional antioxidant, such as methionine,

an optional surfactant, such as polysorbate 80,

in a more specific embodiment, the immunogenic composition of the present disclosure comprises, in addition to at least the NP fusion protein (typically OVX 836):

sodium sulfate or sodium chloride, preferably sodium sulfate,

sugar, preferably trehalose, is used,

phosphate buffer and/or citrate buffer,

an optional antioxidant, such as methionine,

an optional surfactant, such as polysorbate 80,

wherein the osmolality is 300 to 600mOsm/kg, preferably 400 to 500mOsm/kg, typically 450mOsm/kg.

In a preferred embodiment, the immunogenic composition of the present disclosure comprises, in addition to at least the NP fusion protein (typically OVX 836):

sodium sulfate at a concentration of about 75mM,

trehalose at a concentration of about 200mM,

optionally, a surfactant, such as polysorbate 80 at a concentration of 0.02% to 0.08% (vol/vol), such as about 0.04%,

optionally, an antioxidant, such as L-methionine, at a concentration of about 5 mM.

sodium sulfate at a concentration of about 75mM,

trehalose at a concentration of about 200mM,

a concentration of 0.02% to 0.08% (vol/vol), such as about 0.04% polysorbate 80,

l-methionine at a concentration of about 5 mM.

In particular embodiments, the immunogenic composition does not comprise any adjuvants.

In particular embodiments, the immunogenic composition is formulated as a ready-to-use sterile injectable solution.

Sterile injectable solutions are prepared by incorporating the active compound (i.e., the NP fusion protein) in the required amount in the appropriate solvent with various of the other ingredients described above as required and then filter sterilizing.

Methods of using NP fusion proteins and immunogenic compositions thereof

The NP fusion protein as described in the previous section, in particular OVX836, and immunogenic compositions thereof, in particular comprising OVX836 of at least 300 μg/mL, are useful as a vaccine or immunotherapy for preventing or treating influenza disease in a human subject in need thereof.

Accordingly, the present disclosure provides compositions (e.g., immunogenic compositions as described in the previous section), methods, kits, and reagents for preventing and/or treating influenza viruses in humans and other mammals. The immunogenic compositions disclosed herein are useful as therapeutic or prophylactic agents. They are useful in medicine for the prevention and/or treatment of influenza disease. In an exemplary aspect, the immunogenic compositions of the present disclosure are used to provide prophylactic protection against influenza virus. After administration of the immunogenic compositions of the present disclosure, prophylactic protection against influenza virus is typically achieved at doses of 180 μg or more OVX836, 200 μg or more OVX836, 240 μg or more OVX836, 300 μg or more OVX836, or 480 μg or more OVX 836. The immunogenic composition may be administered once, twice, three times, four times or more, preferably as a single dose. Although less desirable, immunogenic compositions may also be administered to an infected individual to achieve a therapeutic response. The dose may need to be adjusted accordingly.

In some embodiments, the immunogenic compositions of the present disclosure are useful as methods of preventing influenza virus infection in a subject, the methods comprising administering at least one immunogenic composition provided herein to the subject, typically at a dose of 180 μg or more OVX836, 200 μg or more OVX836, 240 μg or more OVX836, 300 μg or more OVX836, or 480 μg or more OVX 836.

In some embodiments, the immunogenic compositions of the present disclosure are useful as methods of inhibiting a primary influenza virus infection in a subject, the method comprising administering at least one immunogenic composition provided herein to the subject, typically at a dose of 180 μg or more OVX836, 200 μg or more OVX836, 240 μg or more OVX836, 300 μg or more OVX836, or 480 μg or more OVX 836. In some embodiments, the immunogenic compositions of the present disclosure are useful as methods of treating influenza virus infection in a subject, the method comprising administering at least one immunogenic composition provided herein to the subject, typically at a dose of 180 μg or more OVX836, 200 μg or more OVX836, 240 μg or more OVX836, 300 μg or more OVX836, or 480 μg or more OVX 836.

In some embodiments, the immunogenic compositions of the present disclosure are useful as methods of reducing the incidence of influenza virus infection in a subject, the method comprising administering at least the immunogenic compositions provided herein to the subject, typically at a dose of 180 μg or more OVX836, 200 μg or more OVX836, 240 μg or more OVX836, 300 μg or more OVX836, or 480 μg or more OVX 836.

In some embodiments, the immunogenic compositions of the present disclosure are useful as a method of inhibiting the transmission of influenza virus from a first subject infected with influenza virus to a second subject not infected with influenza virus, the method comprising administering at least one of the immunogenic compositions provided herein to at least one of the first subject and the second subject, typically at a dose of 180 μg or more OVX836, 200 μg or more OVX836, 240 μg or more OVX836, 300 μg or more OVX836, or 480 μg or more.

Some embodiments of the present disclosure provide methods of inducing an antigen NP-specific immune response in a subject comprising administering any of the immunogenic compositions provided herein (preferably OVX 836-containing immunogenic compositions) to a subject in an amount effective to produce an NP-specific immune response. In some embodiments, the antigen NP-specific immune response comprises a total T cell response (particularly a CD4 or CD8 NP-specific T cell response) or a B cell response (a specific anti-NP IgG response).

In some embodiments, methods of generating an antigen NP-specific immune response comprise administering to a subject a single dose of an immunogenic composition of the disclosure (typically a single dose comprising OVX836, e.g., 180 μg or more, 200 μg or more, 240 μg or more, 300 μg or more, or 480 μg or more).

In some embodiments, the immunogenic composition (typically comprising OVX 836) is administered to the subject by intradermal injection, intramuscular injection, or by intranasal administration. In some embodiments, the immunogenic composition (typically comprising OVX 836) is administered to the subject by intramuscular injection.

In some embodiments, the immunogenic composition is formulated as an effective amount of an NP fusion protein (typically OVX 836) to generate an antigen NP-specific immune response in a subject.

The data presented in the examples demonstrate a significantly enhanced immune response using the immunogenic compositions disclosed herein, particularly using a single dose of OVX836 of 180 μg.

In some embodiments, an effective amount of NP fusion protein (typically OVX 836) is a single dose of 180 μg to 1000 μg,200 μg to 1000 μg,240 μg to 1000 μg, or 300 μg to 1000 μg, or 480 μg to 1000 μg. In some embodiments, an effective amount of an NP fusion protein (typically OVX 836) is a single dose of greater than 180 μg administered to a human subject. In some embodiments, an effective amount of an NP fusion protein (typically OVX 836) is 200 μg or more administered to a human subject. In some embodiments, the effective amount of NP fusion protein (typically OVX 836) is 240 μg or more administered to a human subject. In some embodiments, the effective amount of NP fusion protein (typically OVX 836) is 300 μg or more administered to a human subject. In some embodiments, an effective amount of an NP fusion protein (typically OVX 836) is 480 μg or more administered to a human subject.

In particular embodiments, the immune response can be measured by measuring NP-specific IFN-gamma Spot Forming Cells (SFC)/10 for at least 8 days (day 8 or day 29) after the first injection ⁶ PBMC with NP-grade on the day of injection (day 1)Specific IFN-gamma Spot Forming Cells (SFC)/10 ⁶ An increase in baseline number of PBMCs was determined.

In some embodiments, the subject exhibits at least 50%, 70%, 90%, 110%, 130% increased NP-specific IFN- γ Spot Forming Cells (SFC)/10% after day 8 of the first dose of the immunogenic composition compared to baseline (day 1 before injection) ⁶ The first dose of immunogenic composition, for example, comprises 180 μg or more of OVX836.

In particular embodiments, the immune response may be measured by measuring NP-specific CD4+ T Spot Forming Cells (SFC)/10 for at least 8 days (day 8 or day 29) after the first injection ⁶ An increase in the number of PBMCs compared to the baseline number on the day of injection (day 1) was determined.

In some embodiments, the subject exhibits at least 100%, 150%, 200%, 250%, 300%, or 350% increased NP-specific cd4+ Spot Forming Cells (SFC)/10 after day 8 of the first dose of the immunogenic composition compared to baseline (day 1 before injection) ⁶ The first dose of immunogenic composition, for example, comprises 180 μg or more of OVX836.

In some embodiments, the subject exhibits at least 20%, 30%, 50%, 75% or 100% increased NP-specific cd8+ Spot Forming Cells (SFC)/10 after day 8 of the first dose of the immunogenic composition compared to baseline (day 1 before injection) ⁶ The first dose of immunogenic composition, for example, comprises 180 μg or more of OVX836.

The data presented in the examples also demonstrate the significantly improved efficacy of vaccines using the immunogenic compositions as disclosed herein, particularly OVX836 at single doses of 180 μg or more, which prevented the occurrence of new cases of symptomatic influenza (ILI) compared to a single dose of 90 μg that did not provide protection against symptomatic influenza.

In some embodiments, the immunogenic compositions of the present disclosure are useful as methods of providing efficacy against influenza disease, preferably severe influenza, in a subject in need thereof, the method comprising administering an immunogenic composition as provided herein (typically comprising OVX 836) to the subject at a dose of 180 μg or more, 200 μg or more, 240 μg or more, 300 μg or more, or 480 μg or more.

In some embodiments, vaccine efficacy may be determined by a significant reduction in the number of influenza-like diseases after 14 days of injection in a patient population treated with an immunogenic composition of the present disclosure, typically at a dose of 180 μg or more OVX836, 200 μg or more OVX836, 240 μg or more OVX836, 300 μg or more OVX836, or 480 μg or more OVX836, as compared to a placebo or 90 μg dose of OVX 836.

As used herein, the term "influenza-like disease" or "ILI" refers to the clinical observation of fever or sudden onset of more than one of the following symptoms: chills, headache, malaise, myalgia, cough, pharyngitis and other respiratory diseases.

In some embodiments, the patient population exhibits at least a 20%, 40%, 60%, 80% or 95% reduction in influenza-like disease after 14 days of injection when treated with an immunogenic composition of the invention, typically with an OVX836 dose of 180 μg or more, as compared to a patient population receiving a placebo or OVX836 dose of 90 μg.

In some embodiments, the immunogenic compositions of the present disclosure (typically comprising OVX 836) for use as a vaccine protect a subject from severe influenza.

As used herein, the term "severe influenza" refers to an influenza-like disease (ILI; sudden fever and cough or sore throat) and exhibits at least one of the following clinical manifestations:

Dyspnea, shortness of breath or hypoxia

Radiology sign of lower respiratory tract diseases

Involvement of the central nervous system (e.g. encephalopathy, encephalitis)

Serious dehydration

Acute renal failure

-septic shock

Potential chronic diseases including asthma, chronic Obstructive Pulmonary Disease (COPD), chronic liver or kidney insufficiency, exacerbation of diabetes or other cardiovascular diseases

Any other influenza-related disease or clinical manifestation requiring hospitalization.

In some embodiments, the immunogenic compositions of the present disclosure (typically comprising OVX 836) for use as a vaccine protect a subject against one or more severe symptoms of severe influenza disease.

In some embodiments, the immunogenic composition used as a vaccine immunizes a subject against influenza for up to 2 years. In some embodiments, the immunogenic composition used as a vaccine immunizes a subject against influenza for more than 2 years, more than 3 years, more than 4 years, or 5-10 years.

In some embodiments, the subject is a young adult (e.g., about 20, 25, 30, 35, 40, 45, or 50 years old) from about 20 years old to about 50 years old.

In some embodiments, the subject is older than 50 years old, e.g., about 60 years old, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85, or 90 years old).

In some embodiments, the subject has been exposed to influenza; the subject is infected with influenza; or that the subject is at risk for influenza infection.

In other aspects, the disclosure relates to an immunogenic composition for use as a vaccine or a method of vaccinating a subject comprising administering to a subject an immunogenic composition as disclosed herein, typically comprising OVX836, and more preferably formulated at a concentration of at least 300 μg/mL, wherein a single dose of the NP fusion protein, typically OVX836, is administered to the subject in a range of 180 μg to 300 μg,300 μg to 480 μg, or 480 μg to 1000 μg. Preferably, in the method, the immunogenic vaccine is administered by intramuscular injection.

In other aspects, the disclosure relates to the use of a fusion protein as described above in the preparation of a vaccine for preventing influenza in a human subject, wherein the human subject is administered an amount of 180 μg or more of the fusion protein, e.g. comprising an amount of 180 μg to 1000 μg.

In some embodiments, the immunogenic composition (typically comprising OVX 836) is administered to the subject simultaneously or sequentially, preferably simultaneously, with a second immunogenic composition against influenza comprising one or more inactivated influenza strains and/or an effective amount of hemagglutinin HA antigen from one or more influenza strains. For example, the second immunogenic composition comprises a mixture of inactivated strains of a-and b-stream virus strains, such as a mixture of a-type strains H1N1, H3N2, and b-type strains. In a specific embodiment, the second immunogenic composition is Fluarix.

As used herein, the term "combination", "combined administration" or "simultaneous administration" refers to the combined administration of at least two active ingredients, e.g., two immunogenic compositions having different antigens or antigenic determinants, wherein a first immunogenic composition comprising an NP fusion protein as disclosed herein is administered simultaneously with a second vaccine or immunogenic composition separately in the same subject in need thereof or at intervals of time, wherein these intervals of time allow the combined active ingredients to exhibit a cooperative or synergistic effect of immune response or protection against influenza (typically an influenza disorder). Although these delivery methods are within the scope described herein, this does not mean that the immunogenic compositions must be administered simultaneously and/or formulated together for delivery. The term is also intended to include regimens in which the active (immunogenic) agents are not necessarily administered by the same route of administration.

In particular embodiments, a dose of 300 or 480 μg of an immunogenic composition of OVX836 is administered by intramuscular injection simultaneously with a dose of a second immunogenic composition (e.g., fluarix vaccine) comprising one or more inactivated influenza strains or influenza hemagglutinin antigens, which may also be administered by intramuscular injection.

The invention will be further illustrated by the following figures and examples. However, these examples and drawings should not be construed as limiting the scope of the present disclosure in any way.

Detailed Description

E1. An immunogenic composition for use as a vaccine or immunotherapy for the prevention or treatment of influenza disease in a human subject in need thereof,

the immunogenic composition comprises: a fusion protein comprising

(i) An influenza virus nucleoprotein antigen, and a nucleic acid molecule,

wherein the human subject is administered an amount of 180 μg or more of the fusion protein, e.g., an amount of 180 μg to 1000 μg to the human subject.

E2. The immunogenic composition for use according to embodiment E1, wherein 200 μg or more, or 240 μg or more of the fusion protein is administered to the human subject.

E3. The immunogenic composition for use according to embodiment E1, wherein an amount of 300 μg or more of the fusion protein is administered to the human subject.

E4. The immunogenic composition for use according to embodiment E1, wherein 480 μg or more of the fusion protein is administered to the human subject.

E5. The immunogenic composition for use according to any one of embodiments E1-E4, wherein the carrier protein is fused at the C-terminus to a nucleoprotein antigen, optionally via a glycine-serine linker.

E6. The immunogenic composition for use according to any one of embodiments E1-E5, wherein the fusion protein forms a heptameric particle upon self-assembly.

E7. The immunogenic composition for use according to any one of embodiments E1-E6, wherein the influenza virus nucleoprotein antigen comprises at least one nucleoprotein antigen from a influenza a, b or c strain, e.g., consisting essentially of NP antigen of influenza a virus/Wilson-Smith/1933H 1N 1.

E8. The immunogenic composition for use according to any one of embodiments E1-E7, wherein the influenza virus nucleoprotein antigen comprises

(i) The polypeptide of SEQ ID NO. 1, or

(ii) An antigenic polypeptide variant having at least 90% identity to SEQ ID No. 1.

E9. The immunogenic composition for use according to any one of embodiments E1-E8, wherein the self-assembled polypeptide derived from a C4bp oligomerization domain comprises SEQ ID No. 2, or a functional variant thereof having at least 90% identity to SEQ ID No. 2.

E10. The immunogenic composition for use according to any one of embodiments E1-E9, wherein the positively charged tail comprises the sequence ZXBBBBZ (SEQ ID NO: 3), wherein (i) Z is absent or any amino acid, (ii) X is any amino acid, and (iii) B is arginine or lysine, preferably the positively charged tail comprises the sequence SEQ ID NO:4.

E11. The immunogenic composition for use according to any one of embodiments E1-E10, wherein the carrier protein consists essentially of SEQ ID No. 5, or the carrier protein is a functional variant of SEQ ID No. 5 having at least 90% identity to SEQ ID No. 5.

E12. The immunogenic composition for use according to any one of embodiments E1-E11, wherein the fusion protein comprises or consists essentially of SEQ ID No. 6 or is a functional variant of SEQ ID No. 6 having at least 90% identity to SEQ ID No. 6.

E13. The immunogenic composition for use according to any one of embodiments E1-E12, wherein the amount of fusion protein is administered by the intramuscular route.

E14. The immunogenic composition for use according to any one of embodiments E1-E13, wherein the amount of fusion protein is administered as a single injection to a human subject, preferably by the intramuscular route.

E15. The immunogenic composition for use according to any one of embodiments E1-E14, wherein the subject is less than 50 years old.

E16. The immunogenic composition for use according to any one of embodiments E1-E15, wherein the subject is at least 50 years old or older.

E17. The immunogenic composition for use according to any one of embodiments E1-E16, wherein the use provides protection or cross-protection against an influenza symptom (influenza-like disorder), in particular against an influenza infection of an a-or b-strain, of an NP-specific total T cell response, an NP-specific CD4T cell response, an NP-specific CD 8T cell response, an anti-NP IgG (antibody response) and/or against an NP-specific antigen.

E18. The immunogenic composition for use according to any one of embodiments E1-E17, wherein the immunogenic composition is administered to a subject simultaneously or sequentially, preferably in combination with a second immunogenic composition against influenza virus comprising one or more inactivated influenza virus strains and/or an effective amount of hemagglutinin HA antigen from one or more influenza virus strains, preferably the second immunogenic composition is a Fluarix vaccine composition.

E19. An immunogenic composition comprising a fusion protein as defined in any one of embodiments E1-E12 and one or more pharmaceutically acceptable excipients at a concentration of 300 μg/mL or more.

E20. The immunogenic composition of embodiment E19, wherein the fusion protein comprises at least 400 amino acid residues, e.g., 400 to 600 amino acid residues, e.g., 540 to 560 amino acid residues, optionally the fusion protein forms a protein nanoparticle having a diameter of 20 to 100nm and/or a molecular weight of 440 to 2200 kDa.

E21. The immunogenic composition according to embodiment E19 or E20, further comprising at least

i. Salts, such as sodium sulfate or sodium chloride, preferably sodium sulfate,

osmotics, for example sugars, such as trehalose,

buffers, such as phosphate buffers and/or citrate buffers,

an optional antioxidant, such as methionine,

optional surfactants, such as polysorbate 80,

wherein the pH of the composition is from 6.0 to 7.0, typically from 6.3 to 6.6, and the osmolality is from 300 to 600mOsm/kg, preferably from 400 to 500mOsm/kg, for example about 450mOsm/kg.

E22. The immunogenic composition according to any one of embodiments E19-E21, comprising

i. Sodium sulfate at a concentration of about 75mM,

trehalose at a concentration of about 200mM,

l-methionine at a concentration of about 5 mM.

E23. The immunogenic composition according to any one of embodiments E19-E22, wherein the composition does not comprise any adjuvant.

E24. The immunogenic composition according to any one of embodiments E19-E23, formulated as a ready-to-use sterile solution.

E25. The immunogenic composition according to any one of embodiments E19-E24 for use as a vaccine or immunotherapy for the prevention or treatment of influenza disease in a human subject in need thereof, in particular as defined in any one of embodiments E1-E18.

Examples

Example 1: development of a stable formulation of OVX836 at 300. Mu.g/mL

OVX836 (SEQ ID NO: 6) is a drug substance of a candidate vaccine comprising a fusion protein of OVX313 carrier protein (SEQ ID NO: 5) and seasonal influenza virus nucleoprotein (NP alphavirus/wilson-Smith/1933) fused thereto.

OVX836 drug substance is provided as a concentrated solution in a stable formulation buffer. The first objective was to develop a stable formulation with target concentration compatible with single injection of OVX836 up to 180 μg via intramuscular route.

Among the technical problems associated with developing formulations, mention may be made of the abnormally high concentration (300 μg/mL) targeted by it and the quaternary conformation of OVX836, which is a dynamic balance of heptamers (440 kDa) and small oligo-heptamers (di-, tri-, tetra-or penta-heptamers), this feature being related to the self-association properties of NPs. In fact, NPs are highly basic internal proteins that provide structural and functional support for viral replication mechanisms. To achieve this, NPs form homooligomers and multiple copies of NPs encapsulate genomic RNA. Thus, the quaternary structure of OVX836 results in a variety of morphologies, including small oligo-heptamers, which can cause polymerization into aggregates. There are several factors such as temperature, pH, ionic strength, protein concentration that can affect this aggregation phenomenon.

The first development formulation was prepared according to the recommended pH and osmolality, which were near 7.4 and near 300mOsm, respectively.

As shown in table 1 below, formulation F1 was unstable and formed aggregates. Additional formulations with different buffers for higher acidic pH were prepared according to similar osmolality (see formulations F2, F3 and F4). However, all tested pH exhibited unsatisfactory stability and oligomerization.

Table 1: quality attributes of OVX836 drug products tested for the first 3 months under accelerated storage conditions at 25 ℃.

Stability of++ +; ++, moderately stable; ++, is not very stable

We then opted to increase the osmolality of the formulation. The results show a significant improvement in both product degradation and product oligomerization at pH 6.5.

Stability of++ +; ++, moderately stable; ++, is not very stable

After screening for different formulation buffers, excipients and pH, the following optimally stable formulations were finally developed:

more specifically, optimal solubilization is achieved at slightly acidic pH 5.5 to 7.0 (preferably pH 6.4 to 6.6). In addition, screening showed that the use of 20mM sodium citrate-based buffer (final pH 6.6) prevented the appearance of high molecular weight oligomers in the pharmaceutical product.

Furthermore, in the development studies, the presence of trehalose was mainly found to slow down the oligomer formation of OVX836 (reduce oligomerization, as measured by size exclusion chromatography analysis) and a concentration of 200mM was found to be optimal.

Salts such as sodium chloride or sodium sulfate have also been shown to stabilize OVX836, but sodium sulfate is strongly preferred as suggested by differential scanning calorimetry (DSC thermogram).

The available stability data for the selected optimal formulation showed no significant degradation of OVX836 drug product when stored at 5 ℃ for at least 36 months and at 25 ℃ for 3 months. The osmolality of the final formulation is 440 to 500mOsm/kg, typically 465 to 480Osm/kg.

Formulations with such osmolality and high protein concentration may not be safe or well tolerated in human subjects. This was evaluated by the study detailed in example 2 below.

Example 2: stage I-evaluation random, placebo-controlled, dose-escalation study of nucleoprotein-based influenza vaccine OVX 836: intramuscular results

Method

The randomized, placebo controlled, observer blind, continuous, dose escalation phase I study was performed at the university of antveepp (antveepp, belgium) according to the pharmaceutical clinical trial quality management code (Good Clinical Practice). This was approved by the university of ambroxol hospital and the ethical committee of ambroxol university and the federal pharmaceutical and health care agency (FAMHP) at belgium. The independent data and security monitoring committee periodically reviews the data. Written informed consent was obtained from all participating subjects. Eudragit is numbered 2018-000341-39 and Clinicalrils.gov is numbered NCT03594890.

Age 18 to 49 years, body mass index 18 to 25kg/m ² Is suitable for healthy adults. The main exclusion criteria are the past 6 months prior to screening for influenza vaccine, pregnancy or unwilling to carry out birth control, positive detection of human immunodeficiency virus or hepatitis b/c virus, the occurrence of acute febrile disease on the day of inoculation, treatment that may affect immune response (e.g. systemic corticosteroids, cytotoxic drugs, anti-inflammatory drugs and other immunomodulating drugs), history of significant medical illness (e.g. autoimmune disease, uncontrolled diabetes or hypertension, heart disease, kidney disease or liver disease).

12 subjects were included in three consecutive cohorts (low dose 30 μg, medium dose 90 μg, high dose 180 μg). Each group was randomized between OVX836 vaccine (n=9) and placebo (n=3) at a ratio of 3:1. The study was observer blinded. The study and subjects ignored the subject-assigned treatment group (placebo/vaccine) until the end of the study (month 5). Syringes containing study products (placebo or vaccine) were prepared and administered by a non-blind team.

Vaccine (300 μg/mL active) or placebo (consisting of 0.9% sodium chloride) was administered in low dose (30 μg in 0.1 mL), medium dose (90 μg in 0.3 mL) or high dose (180 μg in 0.6 mL) to deltoid muscle of the non-dominant arm. The study was divided into two phases, an active treatment phase from day 1 to day 57, consisting of two intramuscular inoculation, each followed by a 28-day follow-up, and a 58-day to 150-day (5-month) follow-up phase after the first administration.

The topical (site pain, redness, swelling and induration) and systemic (fever, cough, headache, joint pain, myalgia, discomfort/fatigue and vomiting) symptoms occurring within 7 days after each application were collected using a log card. Non-evoked Adverse Events (AEs) were recorded using open questions 28 days after each application. AE intensity was classified as mild, moderate, severe or potentially life threatening and monitored throughout the active period. Severe Adverse Events (SAE) were monitored throughout the study period, until month 5. A set of predetermined safety laboratory analyses (hematology and clinical chemistry, including blood coagulation parameters and evaluation of C-reactive protein (CRP)) were performed at the time of screening, followed by days 8, 29, 36 and 57.

Whole blood samples were collected on days 1, 8, 29, 36, 57 and 150 to isolate PBMCs and to determine NP-specific interferon-gamma (IFN- γ) T cell responses using enzyme-linked immunospot. Serum samples were collected on days 1, 29, 57 and 150 for determination of anti-NP, anti-OVX 313 and anti-hC 4BP IgG using enzyme-linked immunosorbent assay (ELISA). Immunoassays are described in the complementary methods.

The sample size is considered sufficient in view of the purely exploratory nature of the study. The study was not examined for any statistical hypothesis. Descriptive statistics are used to summarize all relevant parameters: the number and percentage of discrete variables, the average (arithmetic or geometric) of continuous variables, the median, the standard deviation, the 95% Confidence Interval (CI), the minimum and maximum. When each pairwise comparison was tested to be significant by the post hoc Dwass, steel, critchlow-Fligner test, a exploratory inferential analysis of the immunogenicity data was performed at each time point using the Kruskal-Wallis test to test for overall differences between treatment groups. The comparison in the group was performed using the paired Wilcoxon test. Fisher's exact test was used to evaluate differences in percentage of responders between treatment groups. Since no correction was employed to account for the diversity of endpoints and comparisons, p-values <5% were only considered an indication of potentially statistically significant differences.

A total of 36 subjects were included and 33 subjects (91.7%) completed the entire study.

Part 1: preliminary analysis

We performed a first analysis of the results of stage I. All groups had a similar baseline on day 1 prior to inoculation, meaning that we could compare them (fig. 1).

On days 8 and 36, all OVX836 groups showed higher responses than the placebo group. There was no difference between the 90 and 180 μg groups on day 8, but 30 μg appeared to be somewhat less optimal (fig. 2). On day 36 (8 days after the second inoculation), there was no statistically significant difference between 90 and 180 μg, and the 90 μg group even showed slightly better response than the 180 μg group (fig. 3).

FIG. 4 shows NP-specific IFN-gamma spot forming T cells (SFC)/10 in the pooled placebo and three OVX836 vaccinated groups (30. Mu.g, 90. Mu.g and 180. Mu.g) ⁶ The number of individual cells evolved over time from baseline (day 1, pre-inoculation) to day 150 (4 months after 2 nd administration). The results indicate that a single injection of 90 μg may be even better than 180 μg as indicated by day 29 and other sustained responses.

In summary, the preliminary analysis of the phase I study showed that:

-a dose higher than 30 μg is required;

the second injection has no significant advantage;

Both the-90 and 180 μg doses were safe and well tolerated with no dose effect;

there is no significant advantage in the 180 μg dose compared to the 90 μg dose, indicating a plateau of efficacy that may have been achieved at the 90 μg dose;

a second dose of 90 μg triggers a T cell immune response that may be higher than 180 μg, indicating that 90 μg may be better than the 180 μg dose level.

Part 2: detailed analysis of stage I results

Further analysis of the results of stage I has demonstrated the safety of the formulation and dosing regimen of 90 and 180 μg, and surprisingly extrapolates the trend of the effect of the dose response of 90 to 180 μg on the immune response, as detailed below.

Reactogenicity and safety

No evoked local symptoms were reported in placebo subjects, while most subjects vaccinated with OVX836 exhibited transient mild to moderate pain at the injection site. The dose-response relationship of OVX836 is not clear in terms of the number of evoked local symptoms and the number of affected subjects: 13 of 8 subjects with 30 μg, 22 of 7 subjects with 90 μg, and 16 of 7 subjects with 180 μg. There was no significant increase in induced local symptoms after the second inoculation compared to the first. The evoked local symptoms in both cohort 1 (30 μg) and cohort 3 (180 μg) were not severe (grade 3). 1 subject (11.1%) in cohort 2 (90 μg) had 2 evoked local symptoms (induration and oedema) that were severe. At the end of either post-vaccination observation period, the evoked local symptoms did not persist.

Neither the number of induced systemic symptoms nor the number of affected subjects had a dose-response relationship: 30 μg of 4 subjects had 12 symptoms, 90 μg of 6 subjects had 13 symptoms, and 180 μg of 6 subjects had 15 symptoms. In contrast, 5 out of 9 placebo subjects reported 12 induced systemic symptoms. Two cases of severe induced systemic symptoms were reported in two subjects vaccinated with OVX836, each after the first vaccination: 1 severe discomfort (fatigue) (30 μg) in queue 1 and 1 severe fever (± 39 ℃) in queue 3 (180 μg); the latter results in a suspension of the second administration. At the end of the observation period following vaccination, none of the induced systemic symptoms persisted.

The percentage of subjects reporting induced AEs during the 28 days post-inoculation are reported: no clear OVX836 dose-effect relationship was observed. There was no increase in induced AE after the second inoculation compared to the first.

Overall, 8 subjects in cohort 1 reported 23 non-evoked AEs (30 μg), 8 subjects in cohort 2 reported 21 AEs (90 μg), 8 subjects in cohort 3 reported 21 AEs (180 μg), and 6 subjects in the pooled placebo group reported 25 AEs. The following AEs are considered vaccine related: (i) queue 1-30 μg: bleeding at the injection site, rash at the inoculation site, musculoskeletal stiffness, and elevation of CRP; (ii) queue 2-90 μg: injection site hemorrhage, nausea oropharyngeal pain, two cases of pre-syncope, CRP elevation, drop in neutrophil count (severe), drop in White Blood Cell (WBC) count, and (iii) cohort 3-180 μg: nasopharyngitis (severe), musculoskeletal pain, neck pain, pre-syncope, oropharyngalgia, two cases of nasal congestion, elevated CRP, decreased lymphocyte count, elevated neutrophil count, elevated WBC count.

In one OVX836 90 μg recipient 1 SAE was reported, consisting of urinary tract infections occurring about 40 days after the second inoculation. SAE last 11 days and are considered vaccine independent.

In summary, OVX836 appears to be a safe and well-tolerated candidate vaccine by the intramuscular route of administration in the dose range of 30 μg to 180 μg. No clear dose-effect relationship was confirmed and the 180 μg dose did not appear to be the maximum tolerated dose.

NP-specific T cell immune response

The number of NP-specific IFN- γ producing T cells detected on day 1, day 8 (1 week after 1 vaccination) and day 36 (1 week after 2 vaccination) in the three OVX836 vaccinated groups versus placebo is shown in fig. 5. All subjects had pre-existing NP-specific IFN-gamma producing T cells at baseline ranging from 5 to 478 NP-specific IFN-gamma Spot Forming Cells (SFC)/10 ⁶ PBMCs, with no significant differences in the groups. On day 8 after the first vaccination, mean SFC/10 for each of the three OVX836 vaccine groups compared to day 1 (FIG. 5A) and compared to placebo (FIG. 5B) ⁶ Individual PBMCs increased significantly. On day 8, there was a trend of increasing response as a function of OVX836 dose level, but the effect was not significant. In addition to OVX836 90 μg group, the 2 nd vaccination did not allow further increase of response on day 36 (1 week after the 2 nd vaccination). On day 57 (28 days after the 2 nd vaccination) a significant difference was found between the three vaccine groups and the placebo group (p=0.002; kruskal-Wallis test), whereas there was no significant difference between OVX836 groups. On day 150 (4 months after 2-inoculation), the number of NP-specific IFN- γ -producing T cells in the 3 OVX836 group remained higher than in the placebo group, but the differences were statistically less significant (p=0.295 overall; kruskal-Wallis test).

NP-specific humoral immune response

Figure 6 (panel a) shows the change in Geometric Mean Titers (GMT) of anti-NP IgG over time in three OVX836 vaccine groups and placebo groups. All subjects presented with pre-existing anti-NP IgG at baseline, with individual titers ranging from 1,600 to 25,600, with no significant differences between groups. On day 29 after the first vaccination, GMT was significantly increased in the three vaccine groups compared to placebo (p=0.0008 overall; kruskal-Wallis test). The second vaccination on day 29 did not allow a further increase in anti-NP IgG GMT on day 57 (day 28 after the second vaccination), which remained higher on day 150 (4 months after the second vaccination), still significantly higher in the three vaccine groups compared to placebo (p=0.001 overall; kruskal-Wallis test). There was a trend towards an increase in anti-NP IgG GMT as a function of OVX836 dose level, but this effect was not significant.

Figure 6 (panel B) shows the percentage of subjects with 4-fold increase in anti-NP IgG titers relative to baseline at various time points post-inoculation. On day 29 (after the first inoculation) and on day 57 (28 days after the second inoculation), 44.4% to 87.5% of OVX836 vaccinated subjects exhibited a 4-fold increase in their baseline titer, whereas the placebo group was 0%. The overall difference between the groups at these two time points was significant (p=0.035 on day 29, p=0.001 on day 57; kruskal-Wallis test), and the post hoc statistical test showed significant differences between OVX836 μg and OVX836180 μg and placebo. On day 150 (4 months after 2 nd inoculation), 37.5 to 50.0% of OVX836 vaccinated subjects still exhibited a 4-fold increase in their baseline titer compared to 0% of placebo group, but the differences between the 4 groups were statistically less significant (p=0.128; kruskal-Wallis test).

In summary, detailed analysis of the results of the phase I study demonstrates:

the intramuscular route is superior to the intranasal route;

-a dose higher than 30 μg is required;

both the-90 and 180 μg doses were safe and well tolerated with no dose effect;

the second injection has no significant advantage;

-90 to 180 μg have a dose response trend.

Example 3: stage 2a study

A. Summary of the study

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B. Summary of results

In contrast to the preliminary analysis of the results of stage I, stage 2a has clearly demonstrated a significant dose response effect of 90 to 180 μg on immune response.

In particular, a strong increase in NP-specific T cell response, and more particularly, NP-specific cd4+ T cell response, was observed 8 days after injection, with a dose response of 90 to 180 μg.

In ITT cohorts (intent-to-treat cohorts), after elimination of two abnormal subjects in the OVX836180 μg group (subjects 128-095 and 232-365 presented high baseline values at day 1: 957 and 1630, respectively), the average increased from 130 SFC/million PBMC at baseline to 222 SFC/million PBMC at day 8 in terms of response kinetics (sink age) in the OVX836 90 μg group. In the OVX836180 μg group, the average increased from 149 at baseline to 288 SFC/million PBMCs at day 8. In the Influvac Tetra group, the mean remained relatively stable 131 SFC/million PBMC at baseline and 147 SFC/million PBMC at day 8. On day 8, OVX836180 μg was significantly different from OVX836, 90 μg (p=0.035), supporting the dose-response relationship in OVX836 group in all subjects. Fig. 7 shows the results on days 1 and 8.

In the protocol-D29 (PP-D29) cohort, the median (mean.+ -. SD) was increased from 90 (131.+ -. 153) SFC/million PBMC at baseline to 167 (223.+ -. 191) SFC/million PBMC and 163 (208.+ -. 183) SFC/million PBMC on days 8 and 29, respectively, in terms of the kinetics of the reaction (sink age) of the OVX836 group of 90. Mu.g. In the OVX836180 μg group, median (mean ± SD) was increased from 95 (168 ± 242) SFC/million PBMCs at baseline to 200 (294 ± 275) and 190 (278 ± 245) SFC/million PBMCs at day 8 and day 29, respectively. Then, in both OVX836 groups, the response dropped to baseline on day 180. In the Influvac Tetra group, the median (mean.+ -. SD) remained relatively stable at baseline at 96 (137.+ -. 153) SFCs per million PBMC and at 8, 29 and 180 days at 108 (147.+ -. 149), 94 (162.+ -. 206) and 81 (121.+ -. 131) SFCs per million PBMC. Subjects 18-49 years old, PP-D29 cohort: in the OVX836 90 μg group, the median (mean ± SD) increased from 102 (138 ± 148) SFC/million PBMCs at baseline to 198 (252 ± 208) and 175 (205 ± 169) SFC/million PBMCs on day 8 and day 29, respectively. In the OVX836180 μg group, median (mean ± SD) increased from 97 (152 ± 174) SFC/million PBMCs at baseline to 198 (275 ± 239) SFC/million PBMCs and 202 (260 ± 210) SFC/million PBMCs at day 8 and 29, respectively. In the Influvac Tetra group, median (mean.+ -. SD) maintained relatively stable 102 (138.+ -. 155) SFC/million PBMC at baseline and 108 (137.+ -. 134) and 106 (169.+ -. 221) SFC/million PBMC at day 8 and day 29, respectively. From a statistical perspective, the effects of time (p < 0.0001), treatment (p= 0.0471) and time-treatment interactions (p < 0.0001) are significant. There was no significant difference between the group averages on day 1 (all p > 0.05). On day 8, the difference between OVX836 90 μg and Influvac Tetra (p=0.0017) and between OVX836180 μg and Influvac Tetra (p=0.0001) was significant. On day 29, only the difference between OVX836180 μg and Influvac Tetra was significant (p= 0.0202).

Subjects 50-65 years old, PP-D29 cohort: in the OVX836 90 μg group, the median (mean ± SD) increased from 62 (114 ± 168) SFC/million PBMCs at baseline to 107 (152 ± 113) SFC/million PBMCs and 133 (216 ± 219) SFC/million PBMCs on day 8 and 29, respectively. In the OVX836 180 μg group, median (mean ± SD) was increased from 93 (209 ± 370) SFC/million PBMCs at baseline to 222 (345 ± 354) and 171 (328 ± 321) SFC/million PBMCs at day 8 and day 29, respectively. In the Influvac Tetra group, median (mean+ -SD) maintained relatively stable 84 (137+ -151) SFC/million PBMC at baseline and relatively stable 103 (172+ -186) and 79 (144+ -159) SFC/million PBMC at day 8 and day 29, respectively. From a statistical perspective, the effect of time (p=0.0004) and time-treatment interactions (p= 0.0479) is significant. The therapeutic effect was not significant (p=0.0964). There was no significant difference between the group averages on day 1 (all p > 0.05). On day 8, the difference between only two OVX836 dose levels was significant (p=0.0217). On day 29, only the difference between OVX836 180 μg and Influvac Tetra was significant (p= 0.0372).

Although not statistically significant, the OVX836 group had a trend in dose-response relationship in all subjects (also observed in both age groups).

In the protocol-D29 (PP-D29) cohort, the baseline (pre-inoculation) percentage of ifnγ -expressing NP-specific cd4+ T cells was low and very similar between treatment groups. In the Influvac Tetra group, all vaccines were not effective. In the OVX836 90 μg group, the median (mean ± SD) increased from 0.022% (0.034 ± 0.043%) at baseline to 0.075% (0.088 ± 0.063%) on day 8 and 0.075% (0.089 ± 0.057%) on day 29. In the OVX836 180 μg group, from 0.028% (0.034±0.027%) at baseline to 0.083% (0.106±0.076%) at day 8 and 0.096% (0.107±0.070%) at day 29. Then, in both OVX836 groups, on day 180, the reaction dropped to a value still slightly above baseline (0.040% [0.048% ± 0.028% ] in OVX836 90 μg group and 0.050% [0.055% ± 0.032% ] in OVX836 180 μg group). As shown in fig. 8, on days 8 and 29 OVX836 180 μg was significantly different from OVX836 90 μg (p=0.0406 and p=0.0353, respectively), supporting the dose-response relationship of total T cell responses already mentioned above (also observed in the age groups below and above 50). This NP-specific multifunctional CD 4T cell response was maintained for 6 months after inoculation. The results also show a strong and long-term increase in anti-NP IgG for all doses of OVX 836.

Security results

Furthermore, phase 2a demonstrates that there is no dose-response effect on safety, indicating good safety profile, similar to licensed seasonal influenza vaccines at 90 and 180 μg test doses.

Efficacy results

Most interestingly, a threshold effect of 180 μg OXV836 on the protective efficacy against influenza-like disease (ILI) symptoms was shown.

More specifically, kaplan-Meier survival analysis was used to evaluate the cumulative risk of nonspecific ILI as a function of time during the influenza season. Two analyses were performed. All ILIs occurring during the 2019-2020 influenza season up to 3 months 9 days 2020 were considered for the first time (fig. 9), and ILIs occurring during the same period but from 14 days post inoculation were considered for the second time (fig. 10). Indeed, it is widely accepted that vaccines begin to protect subjects approximately two weeks after vaccination. The three treatment groups were compared using a log rank test. When all ILIs occurring in the influenza season were considered, all comparisons between treatment groups were not significant (p=0.325). When the ILI occurring at the same time period but from 14 days post vaccination was considered, there was a trend of difference between the three groups, although not statistically significant (p=0.088), in particular the difference between OVX836 90 μg and OVX836180 μg (p=0.054) and between OVX836 90 μg and Influvac Tetra (p=0.130), whereas OVX836180 μg and Influvac Tetra had very similar distributions (p=0.650).

The OVX836 90 μg group reached higher values than the OVX836 180 μg and Influvac Tetra groups in terms of number of ILIs exceeding 14 days after influenza season and inoculation, which had similar distribution (8, 2 and 3 ILIs in the OVX836 90 μg, OVX836 180 μg and Influvac Tetra groups, respectively, 14 days after influenza season and inoculation, see fig. 11). This may reveal a potential efficacy signal for OVX836 at a dose of 180 μg. This of course requires exploration in further clinical trials.

Finally, in the subpopulation analysis, figure 12 shows that the median percentage of ifnγ -expressing NP-specific cd8+ T cells was significantly increased in only OVX836 180 μg group (p=0.020) in subjects belonging to the lowest quartile of cd8+ response at baseline (most likely subjects with the lowest probability of recent exposure/infection with influenza virus prior to vaccination).

In summary, the results of stage 2a underscore the powerful basis for testing OVX836 dose versions above 180 μg. The basis can be summarized as follows:

1. immunogenicity of: intense increase in NP-specific immune responses

Dose response effect on NP-specific total T cells (ELISPot), CD 4T cells (ICS) and IgG,180 μg better than 90 μg (trend towards the latter)

The dose-response effect of 180 μg was significant in the lowest quartile of cd8+ response at baseline (subpopulation analysis of fig. 12)

2. Efficacy: for ILI reduction in influenza season, OVX836 180 μg is better than 90 μg

Different trends in ILI accumulation risk at 180 to 90. Mu.g

180 and 90. Mu.g are significantly different in terms of ILI number in influenza season

3. Safety: OVX836 was well tolerated in all patients and at all doses (up to 180 μg).

No severe adverse event associated with vaccine

Comparable to commercially available vaccines

Evidence of no detected disease enhancement

Taken together, these data suggest that higher doses of OVX836 were investigated as a new development regimen as described in example 4.

Example 4: second phase I/2a study (OVX 836-003)

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Results

Safety: all doses of OVX836 (180, 300 and 480 μg) were found to be safe, well tolerated and compatible with the seasonal tetravalent influenza vaccine Influvac Tetra ^TM Equivalent. The incidence of "severe" adverse events was low (FDA toxicity rating of 3 according to vaccine clinical trials) and there was no dose limiting effect.

Immunogenicity of: in contrast to the preliminary analysis of the results of stage I, stage 2a clearly demonstrates a significant dose response effect of doses exceeding 180 μg and up to 480 μg on the immune response.

In particular, a strong increase in NP-specific T cell response, and more particularly, NP-specific cd4+ T cell response, was observed 8 days after injection, with 180 to 480 μg having a dose response. Furthermore, at dose levels of 300 μg and 480 μg, NP-specific cd8+ T cell responses (ifnγ+/il2+/tnfα -CD 8T cells) were observed 8 days post injection, unlike the observations that at dose levels of 180 μg no response was observed 8 days post injection.

The statistics of pairwise comparisons between Bonferroni groups were used according to the protocol, with OVX836480 μg significantly different from OVX836180 μg in anti-NP IgG response on day 8 (p=0.026), supporting a dose-response relationship exceeding the 180 μg dose level (fig. 13). Although not statistically significant, there was a trend of dose-response relationship between% of positive CD 4T cells on day 8 and CD 4T cells on day 1 between OVX836480 μg and 180 μg groups, particularly ifnγ+/il2+ tnfα -CD 4T cells (p=0.083) and multiple positive CD 4T cells (p=0.102).

Furthermore, when exploratory inferential analysis was applied to the immunogenicity data, a dose-effect relationship was observed between OVX836 at 180 μg and higher dose levels in terms of the change in T cell responses (total T cells, CD4 and CD 8T cells) from day 1 to day 8, which was tested for differences between treatment groups and time points using the inter-group ANOVA test, and then differences between treatment groups were assessed by comparison to Fisher's LSD when significant (p < 0.05). Since no correction was made to account for the diversity of endpoints and comparisons, p-values <5% were only considered as an indication of potential statistically significant differences:

-ELISpot ifnγ response (fig. 14A): all placebo had no effect. In the OVX836180 μg group, the mean change from day 1 to day 8 was 124 SFC/million PBMCs (p=0.002 relative to placebo), while the increase for the 300 μg and 480 μg dose levels was 201 and 223 SFC/million PBMCs, respectively (p <0.001vs for placebo at both dose levels). A significant difference was observed between 480 μg and 180 μg (p=0.014)

-CD 4T cell response (fig. 14B): all placebo had no effect. In the OVX836180 μg group, the mean change in% of ifnγ positive CD 4T cells was 0.046 (p <0.001 relative to placebo) on days 1 to 8, while the increases in dose levels of 300 μg and 480 μg were 0.048 and 0.065, respectively (p <0.001vs for placebo at both dose levels). Significant differences were observed between 480 μg and 180 μg (p=0.022) and between 480 μg and 300 μg (p=0.043)

-CD 8T cell response (fig. 14C): placebo and either 180 μg or 300 μg dose water had no effect on average. In the OVX836 480 μg group, the mean change in% of CD 8T cells positive for both ifnγ and IL2 was 0.034 (p=0.006 vs placebo) on days 1 to 8. A significant difference was observed between 480 μg and 180 μg in terms of mean change in% CD 8T cells positive for both ifnγ and IL2 from day 1 to day 8 (p=0.036).

Efficacy: the observational study (FLU-001 study) was performed in parallel with the OVX836-003 study (same location, same recruitment time, same inclusion/exclusion criteria) with the aim of combining the two cohorts with an influenza active cycle for ILI analysis on a balanced group of 200 subjects (50% OVX836 dose above 180. Mu.g; 50% placebo or untreated subjects).

2 cases of PCR-confirmed symptomatic influenza (ILI) were reported in OVX836 group (all dose levels), 9 cases were reported relative to placebo + untreated group, reflecting 79% of observed efficacy [5.4%;95.4% ] (see fig. 15).

Example 5: stage 2a study (OVX 836-004)

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Results

Safety: all groups (tetravalent inactivated influenza vaccine (QIIV), OVX836& QIIV and OVX 836) were found to be safe and well tolerated, with a low incidence of "severe" (3 grades according to the FDA vaccine clinical trial toxicity scale) adverse events (1 severe fatigue/myalgia in QIIV group and 1 severe headache in OVX836 group) whereas no "severe" (4 grades according to the FDA vaccine clinical trial toxicity scale) adverse events were reported in the study.

Efficacy: group QIIV reported 3 PCR-confirmed symptomatic influenza (ILI), group OVX836 reported 1, and group OVX836& QIIV reported 2.

Example 6: useful sequences for practicing the invention

TABLE 2 useful sequences for practicing the invention

Table 3: sequence listing

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Claims

1. An immunogenic composition for use as a vaccine or immunotherapy for the prevention or treatment of influenza disease in a human subject in need thereof,

the immunogenic composition comprises: a fusion protein comprising

(i) An influenza virus nucleoprotein antigen, and a nucleic acid molecule,

wherein 180 μg or more of the fusion protein is administered to the human subject, e.g., 200 μg, 240 μg to the human subject.

2. The immunogenic composition for use of claim 1, wherein the human subject is administered an amount of 300 μg or more, or 480 μg or more of the fusion protein.

3. The immunogenic composition for use of any one of claims 1-2, wherein the carrier protein is fused at the C-terminus to a nucleoprotein antigen, optionally via a glycine-serine linker.

4. The immunogenic composition for use of any one of claims 1-3, wherein the fusion protein forms a heptameric particle upon self-assembly.

5. The immunogenic composition for use of any one of claims 1-4, wherein the influenza virus nucleoprotein antigen comprises at least one nucleoprotein antigen from an influenza a, b or c strain, e.g., consisting essentially of NP antigen of influenza a virus/Wilson-Smith/1933H 1N 1.

6. The immunogenic composition for use of any one of claims 1-5, wherein the influenza virus nucleoprotein antigen comprises

(i) The polypeptide of SEQ ID NO. 1, or

7. The immunogenic composition for use according to any one of claims 1-6, wherein the self-assembled polypeptide derived from a C4bp oligomerization domain comprises SEQ ID No. 2, or a functional variant thereof having at least 90% identity to SEQ ID No. 2.

8. The immunogenic composition for use according to any one of claims 1-7, wherein the positively charged tail comprises the sequence ZXBBBBZ (SEQ ID NO: 3), wherein (i) Z is absent or any amino acid, (ii) X is any amino acid, and (iii) B is arginine or lysine, preferably the positively charged tail comprises the sequence SEQ ID NO:4.

9. The immunogenic composition for use of any one of claims 1-8, wherein the carrier protein consists essentially of SEQ ID No. 5 or the carrier protein is a functional variant of SEQ ID No. 5 having at least 90% identity to SEQ ID No. 5.

10. The immunogenic composition for use of any one of claims 1-9, wherein the fusion protein comprises or consists essentially of SEQ ID No. 6 or is a functional variant of SEQ ID No. 6 having at least 90% identity to SEQ ID No. 6.

11. The immunogenic composition for use of any one of claims 1-10, wherein the amount of fusion protein is administered by the intramuscular route.

12. The immunogenic composition for use according to any one of claims 1-11, wherein the amount of fusion protein is administered as a single injection to a human subject, preferably by the intramuscular route.

13. The immunogenic composition for use of any one of claims 1-12, wherein the subject is less than 50 years old.

14. The immunogenic composition for use of any one of claims 1-13, wherein the subject is at least 50 years old or older.

15. The immunogenic composition for use of any one of claims 1-14, wherein the use provides protection or cross-protection against an influenza symptom (influenza-like disorder), in particular against an influenza infection of an a-or b-strain, of an NP-specific total T cell response, an NP-specific CD4T cell response, an NP-specific CD 8T cell response, an anti-NPIgG (antibody response) and/or against an NP-specific CD4T cell response.