WO2022084663A1 - Compositions and methods for inducing an immune response - Google Patents

Compositions and methods for inducing an immune response Download PDF

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Publication number
WO2022084663A1
WO2022084663A1 PCT/GB2021/052692 GB2021052692W WO2022084663A1 WO 2022084663 A1 WO2022084663 A1 WO 2022084663A1 GB 2021052692 W GB2021052692 W GB 2021052692W WO 2022084663 A1 WO2022084663 A1 WO 2022084663A1
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fhbp
antigen
vaccine
suitably
sequence
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PCT/GB2021/052692
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French (fr)
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Adrian V.S. Hill
Christine S. ROLLIER
Christina DOLD
David Wyllie
Andrew J. POLLARD
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The Chancellor, Masters And Scholars Of The University Of Oxford
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/099—Bordetella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • N. m eningitidis is a leading cause of bacterial meningitis and septicaemia worldwide.
  • the clinical manifestations of the disease are variable, with severe cases being associated with fulminant invasive disease and mortality rates that can reach 8-10%. Those who survive are often left with devastating sequelae, including marked neurological deficits and limb amputation.
  • the severity of the disease is compounded by the difficulty in recognition in the early stages, which can lead to delays in effective and life saving therapy. Prevention of disease by effective vaccination is therefore a crucial protective strategy.
  • - 4CMenB (Bexsero®). licensed in the UK, contains a combination of four subcapsular antigens: Three components are recombinant proteins consisting of one variant of factor H binding protein (FHbp), neisserial heparin binding antigen (NHBA), and neisserial adhesin A (NadA). The fourth component is the outer membrane vesicle (0MV) from a group B strain responsible for an outbreak in New Zealand.
  • FHbp factor H binding protein
  • NHBA neisserial heparin binding antigen
  • NadA neisserial adhesin A
  • the fourth component is the outer membrane vesicle (0MV) from a group B strain responsible for an outbreak in New Zealand.
  • ChAdOx1.85A novel chimpanzee adenovirus
  • MVA85A modified vaccinia Ankara virus
  • the present seeks to overcome problem(s) associated with the prior art.
  • the invention relates to induction of immune responses, suitably protective immune responses, against Neisseria m eningitidis, more suitably Neisseria m eningitidis group B ("MenB").
  • MenB Neisseria m eningitidis group B
  • the inventors have designed genetic constructs for use in immunogenic compositions.
  • the approach taken is distinct from known work due to the inventors deliberately choosing to present a bacterial antigen in a viral vector.
  • the exemplary antigen (fHbp) is itself a bacterial outer membrane protein.
  • presenting a bacterial protein in a viral vector (which entails expression by the mammalian cells in the subject to which the viral vector is administered) is itself an innovative approach. This is especially true as an approach for generation of functional antibodies, which requires the bacterial antigen to adopt a conformation corresponding to that adopted when expressed naturally in bacteria.
  • the invention provides a composition
  • a composition comprising a viral vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide antigen, wherein said antigen comprises Factor H Binding Protein (fHbp) from Neisseria m eningitidis, characterised in that said viral vector is an adenovirus based vector.
  • said antigen comprises Factor H Binding Protein (fHbp) from Neisseria m eningitidis group B.
  • said adenovirus based vector is a non-human adenovirus based vector.
  • said adenovirus based vector is selected from the group consisting of ChAdOx1 and ChAd0x2.
  • said adenovirus based vector is ChAdOx1.
  • said Factor H Binding Protein comprises an arginine substitution at the amino acid position corresponding to serine 223 in the wild type Factor H Binding Protein (fHbp).
  • Factor H Binding Protein comprises the amino acid sequence of SEQ ID NO: 2.
  • said signal sequence is a mammalian signal sequence (e.g. a human signal sequence) or a bacterial signal sequence.
  • the signal sequence when it is a bacterial signal sequence, it may be homologous (e.g. the naturally occurring signal sequence integral to fHbp) or heterologous (e.g. a bacterial signal sequence derived from another protein (i.e. from a non-fHbp protein) from the same bacterium such as Neisseria m eningitidis, and/or derived from another bacterium (i.e. derived from a non-Neisseria m eningitidis bacterium).
  • homologous e.g. the naturally occurring signal sequence integral to fHbp
  • heterologous e.g. a bacterial signal sequence derived from another protein (i.e. from a non-fHbp protein) from the same bacterium such as Neisseria m eningitidis, and/or derived from another bacterium (i.e. derived from a non-Neisseria m eningitidis bacter
  • said antigen comprises at least two signal sequences.
  • said at least two signal sequences comprise at least one bacterial signal sequence and at least one mammalian (e.g. human) signal sequence.
  • said antigen is present as a fusion with the tissue plasminogen activator (tPA) sequence in the order N-terminus - tPA - Factor H Binding Protein - C-terminus.
  • tPA tissue plasminogen activator
  • Suitably 'signal sequence' means an amino acid sequence which directs secretion of the polypeptide from the cell in which is it expressed such as a mammalian cell e.g. a human cell in the subject to which the composition of the invention is administered.
  • the invention relates to a composition as described above wherein said tPA has the amino acid sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
  • the invention relates to a composition as described above wherein said antigen has the amino acid sequence SEQ ID NO: 3.
  • the invention relates to a composition as described above wherein said viral vector sequence is as in ECACC accession number 12052403.
  • the invention relates to a composition as described above for use in induction of an immune response against Neisseria m eningitidis.
  • the invention relates to a composition as described above for use in boosting of an immune response against Neisseria m eningitidis.
  • the invention in another embodiment relates to a composition as described above for use in preventing Neisseria m eningitidis infection.
  • a single dose of said composition is administered.
  • suitably two doses of said composition are administered.
  • a single priming dose of said composition is administered.
  • a single boosting dose of said composition is administered.
  • a single priming dose of said composition is administered, followed by one or more further dose(s) of said composition.
  • a single boosting dose of said composition is administered, followed by one or more further dose(s) of said composition.
  • suitably said composition is administered once.
  • suitably said composition is administered twice.
  • the invention relates to use of a composition as described above in medicine.
  • the invention relates to use of a composition as described above in the preparation of a medicament for prevention of Neisseria meningitidis infection.
  • the invention in another embodiment relates to a method of inducing an immune response against Neisseria meningitidis in a mammalian subject, the method comprising administering a as described above to said subject.
  • a single dose of said composition is administered to said subject.
  • composition is administered once.
  • the invention relates to a method as described above further comprising administration of a second or further dose of said composition subsequent to administration of the first dose.
  • composition is administered by a route of administration selected from a group consisting of intranasal, aerosol, sublingual, intradermal and intramuscular. More suitably said administration is intramuscular.
  • a second dose of a composition as described above and instructions for administration to a mammalian subject.
  • administration of a single dose of the composition of the invention to a mammalian subject induces protective immunity in said subject.
  • a second or further dose of the composition of the invention is administered to the mammalian subject.
  • said second or further dose is administered 6 months after the first or preceding dose.
  • said second or further dose induces or maintains protective immunity in said subject.
  • protective immune response means that the composition is capable of generating a protective response in a host organism, such as a human or a non-human mammal, to whom it is administered according to the invention.
  • a protective immune response protects against subsequent infection or disease caused by Neisseria m eningitidis.
  • fHbp which is an outer membrane protein.
  • fHbp an outer membrane protein.
  • use of a viral vector to deliver a bacterial outer membrane protein as an antigen can induce a functional antibody response.
  • a viral vector to deliver such a protein delivers it "out of context" i.e. it is produced in the mammalian subject in their own mammalian cells transduced by the viral vector which is administered.
  • Those eukaryotic cells (rather than prokaryotic bacterial cells) produce the protein, and it is subsequently presented to the immune system of the subject in a manner dramatically different to its natural presentation on the surface/outer membrane of the bacterial pathogen from which the antigen is derived.
  • this approach is effective in induction of strong antibody responses, such as functional antibody responses.
  • the inventors teach use of a mutant fHbp (the S223R mutant) in a viral vector. This goes against conventional thinking in the art. Clearly it would normally be assumed that keeping as close as possible to the wild type protein would be desirable, since the wild type protein is what will be encountered if the subject comes into contact with the wild type pathogen (i.e. a MenB bacterium). However, choosing to produce compositions of the invention using a mutant antigen with the S223R substitution is surprisingly effective, which is an advantage of the invention.
  • the inventors teach the use of non-human adenovirus based vectors. This offers the advantage of avoiding pre-existing immune responses which might be present against human adenovirus vectors. This also brings advantages in terms of ease of manufacture.
  • the inventors thoughtfully selected the fHbp (including mutant S223R fHbp) as antigen. This represents creative thinking and contributes to supporting inventive step. This is because conventional candidate antigens from MenB such as PorA and FetA might naturally be selected first by a skilled worker. However, the inventors disclose that although use of these popular PorA/FetA antigens appears to induce an antibody response when delivered to a mammalian subject using a viral vector, it appears to induce no functional immune response. In this regard we refer to the Examples section where data is presented in support of this.
  • a viral vector to deliver a bacterial protein is very different to the natural presentation of the protein on bacteria and/ or very different to the natural presentation of the protein by antigen presenting cells (APCs) in vivo.
  • APCs antigen presenting cells
  • Essentially delivering the bacterial antigen using a viral vector in this manner makes the bacterial protein "look like" it is expressed by a virus.
  • This cryptic approach is an intellectual contribution made by the inventors, and demonstrates inventive step over the known art.
  • a further advantage of this ingenious approach is that it retains the conformation of fHbp. This is especially surprising since this retention of conformation did not occur when attempting to deliver MenB antigens such as PorA/FetA using a viral vector approach (see Examples). Therefore, this advantageous technical benefit arises from the inventors' choice of combination of a viral vector with the fHbp antigen.
  • Adenoviruses are attractive vectors for human vaccination. They possess a stable genome so that inserts of foreign genes are not deleted and they can infect large numbers of cells without any evidence of insertional mutagenesis.
  • Replication defective adenovirus can be engineered by deletion of genes from the El locus, which is required for viral replication, and these viruses can be propagated easily with good yields in cell lines expressing El from AdHu 5 such as human embryonic kidney cells 293 (HEK 293 cells).
  • Human adenoviruses are under development as vectors for malaria, HIV and hepatitis C vaccines, amongst others. They have been used extensively in human trials with excellent safety profile mainly as vectors for HIV vaccines.
  • a limiting factor to widespread use of human adenovirus as vaccine vectors has been the level of anti-vector immunity present in humans where adenovirus is a ubiquitous infection.
  • the prevalence of immunity to human adenoviruses prompted the consideration of simian adenoviruses as vectors, as they exhibit hexon structures homologous to human adenoviruses.
  • Simian adenoviruses are not known to cause pathological illness in humans and the prevalence of antibodies to chimpanzee origin adenoviruses is less than 5% in humans residing in the US. Any suitable adeno-based viral vector may be used.
  • the adenovirus may comprise a simian or human adenovirus.
  • the adenovirus may comprise a Group E adenovirus.
  • the adenovirus may comprise ChAd63 or ChAd3 or ChAdOx1 or ChAd0x2 or a gorilla-derived adenovirus.
  • the adenovirus may comprise ChAdOx1.
  • the adenovirus may comprise a group A, B, C , D or E adenovirus.
  • the adenovirus may comprise Ad35, Ad 5 , Ad6, Ad26, or Ad28.
  • the adenovirus may be of simian (e.g. chimpanzee, gorilla or bonobo) origin.
  • the adenovirus may comprise any of ChAd63, ChAdOx1, ChAd0x2, 15 C6, C7, C9, PanAds, or ChAd3.
  • the adenovirus may be a human adenovirus, for example Ad 5 - (see “Adenoviruses as vaccine vectors” Nia Tatsis & Hildegund C.J. Ertl (2004) Molecular Therapy https://www.sciencedirect.com/science/article/pii/S1525001604013425 ), Ad35 - (see Vogels R., et al. 2003. "Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity.” J. Virol.
  • Ad6 - see “Characterization of Species C Human Adenovirus Serotype 6 (Ad6)", Eric A. Weaver et al (2011), Virology https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056908/ ), Ad26 - (see “Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D", Abbink et al (2007), J.
  • any replication-deficient viral vector for human use preferably derived from a non-human adenovirus may be used.
  • Ad 5 may be used for veterinary use.
  • the adenovirus may comprise a non-human adenovirus such as a chimpanzee adenovirus.
  • the adenovirus may comprise a chimpanzee adenovirus such as disclosed in US8216834B2 'Chimpanzee adenovirus vaccine carriers' which provides details of numerous chimp adenovirus vectors, including ChAd3 & ChAd63, and is incorporated herein by reference specifically for the teachings of chimpanzee adenoviruses.
  • ChAd0x2 is an example of a suitable non-human adenovirus vector for human use.
  • the adeno-based viral vector is ChAdOx1.
  • ChAdOx1 is a replication-deficient simian adenoviral vector. Vaccine manufacturing maybe achieved at small or large scale. Pre-existing antibodies to the vector in humans are very low, and the vaccines induce strong antibody and T cell responses after a single dose, whilst the lack of replication after immunisation results in an excellent safety profile in subjects of all ages.
  • the El site may be used, suitably with the hCMV IE promoter.
  • the short or the long version maybe used; most suitably the long version as described in WO2008/122811, which is specifically incorporated herein by reference for the teaching of the promoters, particularly the long promoter.
  • a clone of ChAdOx1 containing GFP is deposited with the ECACC: a sample of E. coli strain SW1029 (a derivative of DH10B) containing bacterial artificial chromosomes (BACs) containing the cloned genome of AdChOXi (pBACe3.6 AdChOx1 (E4 modified) TIPeGFP, cell line name "AdChOx1 (E4 modified) TIPeGFP" was deposited by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 oJG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052403. Isis Innovation Limited is the former name of the proprietor/applicant of this patent/ application.
  • BACs bacterial artificial chromosomes
  • ChAd0x2 The nucleotide sequence of the ChAd0x2 vector (with a GatewayTM cassette in the El locus) is shown in SEQ ID NO. 2 This is a viral vector based on Chimpanzee adenovirus C68. (This is the sequence of SEQ ID NO: 10 in gb patent application number 1610967.0).
  • ChAd0x2 containing GFP is deposited with the ECACC: deposit accession number 16061301 was deposited by Isis Innovation Limited on 13 June 2016 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 oJG, United Kingdom under the Budapest Treaty. Isis Innovation Limited is the former name of the proprietor/applicant of this patent/application.
  • a related vaccine vector ChAd63, maybe used if desired.
  • the polypeptide antigen of the invention comprises a signal sequence.
  • said signal sequence is a secretion sequence.
  • the inventors have discovered that inclusion of signal sequences into fusion proteins with the antigen of interest can enhance performance.
  • use of mammalian signal sequences such as the tPA (tissue plasminogen activator) signal sequence is known.
  • tPA tissue plasminogen activator
  • the inventors believe that it has never been disclosed to use bacterial signal sequences in antigens intended for inducing an immune response in a mammal, in particular the inventors believe that it has never been disclosed to use bacterial signal sequences in addition to mammalian signal sequences such as the tPA signal sequence in antigens intended for inducing an immune response in a mammal.
  • the invention relates to the use of a bacterial signal sequence fused to an antigen, in particular use of a bacterial signal sequence in addition to a mammalian signal sequence, fused to an antigen for use in inducing an immune response in a mammal.
  • fusion protein comprising two signal sequences is particularly advantageous.
  • the inventors have discovered that creating a fusion protein comprising two heterologous signal sequences fused to the antigen of interest is especially advantageous. Moreover, the inventors have realised that fusing a bacterial signal sequence and a mammalian signal sequence and the antigen of interest is especially advantageous.
  • heterologous signal sequences decreases degradation of the fusion protein.
  • this new direction is especially cryptic since it teaches directly against the view in the art which is that bacterial signal sequences should be removed from bacterial antigens before they are introduced to mammals.
  • the inventors teach directly against this prior art view by specifically including the bacterial signal sequence (despite the destination being a mammalian environment) and find that this surprisingly generates better results.
  • the inventors teach a generally applicable principle of use of bacterial signal sequence to promote an effective immune response in a mammalian environment.
  • the inventors teach that the inclusion of dual signal sequences (e.g. one mammalian signal sequence and one bacterial signal sequence) on a single protein with the antigen of interest is especially effective.
  • the bacterial signal sequence may provide an adjuvant effect, and/or may provide a "danger" or enhancing signal to the immune system, thereby provoking a more effective response.
  • the antigen with dual signal sequences i.e. with two signal sequences.
  • a bacterial signal sequence can be added to the antigen and the second signal sequence can be provided as a heterologous signal sequence in the same fusion protein.
  • this naturally occurring bacterial signal sequence can be deleted and replaced with a superior bacterial signal sequence as taught by the inventors.
  • An example of a superior bacterial signal sequence to be substituted into the antigen in this manner includes signal sequence such as:
  • the construct comprises a second signal sequence in the form of a mammalian signal sequence such a tPA signal sequence.
  • the immature form of the signal sequence is retained, or inserted, or substituted into (or fused to) the antigen.
  • one of those signal sequences is tPA.
  • tPA tPA tissue plasminogen activator
  • tPA leader sequence means the tPA amino acid sequence of SEQ ID NO: 5
  • the C terminal 'RR' is not actually part of the tPA leader sequence. It comes from the fusion of two restriction sites.
  • the tPA leader sequence may be used with or without the C terminal 'RR' e.g. SEQ ID NO: 7 or SEQ ID NO: 8. Most suitably the sequence is used as shown in SEQ ID NO: 5.
  • the underlined A is P in the naturally occurring tPA leader sequence.
  • the P->A mutation has the advantage of improved antigen secretion.
  • the tPA leader sequence may be used with or without the P->A mutation, i.e. suitably the tPA leader sequence maybe used as SEQ ID NO: 5 or SEQ ID NO: 6.
  • sequence is used with the P->A mutation (with or without the C terminal 'RR'). Most suitably the sequence is used as shown in SEQ ID NO: 5.
  • amino acid sequence is introduced internally in making a fusion protein, it would be usual to remove the lead methionine (so that no internal methionine is introduced into the fusion protein).
  • the tPA signal sequence of SEQ ID NO: 5 without the lead methionine is presented as SEQ ID NO: 24.
  • nucleotide sequence encoding tPA which has been codon optimised for human codon usage, is as shown in SEQ ID NO: 9 (this is the sequence encoding SEQ ID NO: 5):
  • tPA promotes secretion of proteins to which it is fused. It is believed that tPA increases expression of proteins to which it is fused. Notwithstanding the underlying mechanism, the advantage in the invention of fusing tPA to the N-terminus of the protein antigen is that improved immunogenicity is achieved.
  • the antigen of the invention is provided as a fusion with tPA.
  • the tPA is fused to the N-terminus of the protein antigen.
  • the antigen does not comprise any further sequence tags.
  • the antigen does not comprise any further linker sequences.
  • two signal sequences are included in the polypeptide comprising the antigen such as fHbp antigen.
  • those two signal sequences comprise the tPA signal sequence and the fHbp signal sequence.
  • the skilled operator may introduce some 'spacer' or 'linker' amino acids in between the first and second signal sequences.
  • a four amino acid spacer having the sequence maybe used having the sequence:
  • the resulting dual signal sequence fusion comprises (spacer of SEQ ID NO: 16 underlined):
  • the signal sequences maybe arranged in order from the N-terminus of the protein to the C-terminus of the protein as follows:
  • the elements may be N-terminus - tPA signal sequence - fHbp signal sequence - antigen - C-terminus.
  • nucleic acids of the invention when the nucleotide sequences encoding the polypeptides as noted above are arranged in order from 5' to 3' on a single contiguous nucleic acid.
  • the antigen is capable of inducing an immune response in a human.
  • the antigen may be from any source.
  • the antigen is from a pathogen of humans, or may be a tumour antigen. More suitably the antigen is a bacterial antigen or a viral antigen. Most suitably the antigen is a bacterial antigen.
  • the Factor H Binding Protein is from from from Neisseria m eningitidis group B.
  • the antigen is a bacterial antigen.
  • the bacterial antigen is a gram- negative bacterial antigen.
  • the antigen is a surface exposed antigen.
  • the antigen protein can be varied.
  • the signal sequence such as bacterial signal sequence can be varied.
  • Sequence comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate percent homology (such as percent identity) between two or more sequences.
  • a suitable computer program for carrying out such analysis is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Altschul et al., 1990, J. Mol. Biol. 215:403-410) and the GENEWORKS suite of comparison tools.
  • any heterologous signal sequence /signal peptide insertions or fusions are excluded from percent identity calculations.
  • sequence identity is judged against the full length of the reference sequence.
  • sequence identity is judged against the full length of SEQ ID NO: 1 or SEQ ID NO: 2.
  • amino acid sequence discussed comprises amino acid sequence derived from the reference sequence or organism/ genome as specified.
  • sequence identity it will be noted that the reference sequences may not be 100 amino acids in length. Therefore each single substitution is equivalent to more than 1% or less than 1% change in identity depending on the length of the reference sequence when all amino acids of the reference sequence are considered. The values are given to nearest whole percentage point and should be understood accordingly given that it is not possible to substitute partial amino acids within a polypeptide sequence. Unless otherwise apparent from the context, the same sequence identity levels as noted above for amino acid sequences also apply to nucleotide sequences herein.
  • nucleic acid When particular amino acid residues are referred to herein using numeric addresses, the numbering is taken with reference to the wild type fHbp amino acid sequence (or to the polynucleotide sequence encoding same if referring to nucleic acid).
  • UniProt release 2020_04 is relied upon.
  • EBI European Bioinformatics Institute
  • SIB SIB Swiss Institute of Bioinformatics and Protein Information Resource
  • UniProtKB UniProt Knowledgebase Release 2020_04 published 12 August 2020 is relied upon.
  • UniProt Universal Protein Resource
  • UniProt is a comprehensive catalogue of information on proteins (“UniProt: the universal protein knowledgebase” Nucleic Acids Res. 45: D158-D169 (2017)).
  • GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences (National Center for Biotechnology Information, U.S.).
  • GenBank database release referred to is 15 June 2020, NCBI-GenBank Release 238.
  • Mutating has it normal meaning in the art and may refer to the substitution or truncation or deletion of one or more residues, motifs or domains. Mutation may be effected at the polypeptide level, for example, by synthesis of a polypeptide having the mutated sequence, or maybe effected at the nucleotide level, for example, by making a polynucleotide encoding the mutated sequence, which polynucleotide may be subsequently translated to produce the mutated polypeptide.
  • the mutations to be used are as set out herein. Unless otherwise apparent from the context, mutations mentioned herein are substitutions. For example 'S223R' means that the residue corresponding to 'S223' in the wild type fHbp (SEQ ID NO: 1) is substituted with R.
  • the antigen sequence is, or is derived from, amino acid sequence provided herein, such as SEQ ID NO. 1.
  • a degree of sequence variation maybe tolerated.
  • the antigen sequence used in the vector of the invention comprises amino acid sequence having at least 80%, suitably at least 85%, suitably at least 90%, suitably at least 92%, suitably at least 94%, suitably at least 96%, suitably at least 98%, most suitably 99% sequence identity to the reference amino acid sequence, for example the reference amino acid sequence provided as SEQ ID NO. 1.
  • the fHbp sequence may have a S223R mutation relative to the wild type fHbp of SEQ ID NO: 1. This is advantageous in reducing or eliminating binding of fHbp to Factor H.
  • the amino acid sequence of fHbp with the S223R mutation is provided in SEQ ID NO: 2.
  • the antigen sequence is, or is derived from, amino acid sequence provided herein, such as SEQ ID NO. 2. A degree of sequence variation may be tolerated.
  • the antigen sequence used in the vector of the invention comprises amino acid sequence having at least 80%, suitably at least 85%, suitably at least 90%, suitably at least 92%, suitably at least 94%, suitably at least 96%, suitably at least 98%, most suitably 99% sequence identity to the reference amino acid sequence, for example the reference amino acid sequence provided as SEQ ID NO. 2.
  • SEQ ID NO: 1 has S at amino acid 223 of SEQ ID NO: 1. This is the wild type amino acid at position 223 of fHbp.
  • SEQ ID NO: 2 has R at the position corresponding to amino acid 223 of SEQ ID NO: 1.
  • the antigen sequence used has R at the position corresponding to amino acid 223 of SEQ ID NO: 1.
  • Variants of factor H binding protein may be used as antigen.
  • One composition of the invention may comprise more than one fHbp variant. This helps to increase coverage i.e. helps to raise an immune response against different MenB variants which the subject might encounter.
  • fHBps There are three groups of fHBps (group 1, 2 & 3) and variants exist within each of these (e.g. 1.1 as in the exemplary adenovirus based vector construct of the invention).
  • stabilising mutations may advantageously be made in the expressed antigen (and/or in the nucleotide sequence encoding it) to improve transcript stability when expressed from the adenovirus based vector.
  • fHbp variants are described in W02016/014719A1 (Children's Hospital & Research Center Oakland) which is incorporated herein by reference for the teachings of fHbp variants.
  • a variant of fHbp wherein the variant comprises an amino acid substitution selected from at least one of: a) an amino acid substitution of the glutamine at amino acid 38 (Q38); b) an amino acid substitution of the glutamic acid at amino acid 92 (E92); c) a substitution of glycine for arginine at amino acid 130 (R130G); d) an amino acid substitution of the serine at amino acid 223 (S223); and e) a substitution of histidine for leucine at amino acid 248 (H248L), wherein the amino acid substitutions are relative to wild type fHbp (SEQ ID NO: 1), wherein the variant comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:1.
  • the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of the affinity of wild type fHbp (SEQ ID NO: 1) for human fH.
  • the variant induces a functional antibody response (such as a bactericidal antibody response) to at least one strain of Neisseria m eningitidis in a mammalian host.
  • a functional antibody response such as a bactericidal antibody response
  • the amino acid substitution at Q38 is Q38R, Q38K, Q38H, Q38F, Q38Y, or Q38W.
  • the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W.
  • the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W.
  • the variant fHbp may further include a R41S or a R41A substitution relative to SEQ ID NO: 1.
  • the variant fHbp may include a R41S or a R41A substitution and a substitution at S223, e.g., R41S/S223R, relative to SEQ ID NO: 1.
  • the variant fHbp may further include a R41S or a R41A substitution and a H248L substitution relative to SEQ ID NO: 1.
  • the variant fHbp may include two, three, or more of the substitutions disclosed herein.
  • the variant fHbp may include the following substitutions: S223R and H248L relative to SEQ ID NO: 1.
  • the full length fHbp protein is used.
  • 'full length means each amino acid in the fHbp protein is included.
  • full length means an fHbp protein having a length (i.e. total number of amino acids) corresponding to that of SEQ ID NO: 1 or SEQ ID NO: 2.
  • a further advantage of using the full length fHbp protein is that it allows for better T- cell responses. Without wishing to be bound by theory, it is believed that the more amino acid sequences present, then the more potential targets there are for the T-cell responses. Thus, suitably every amino acid of the fHbp protein is included in the antigen of the invention.
  • sequence identity level of 99% compared to SEQ ID NO. 1 or SEQ ID NO. 2 corresponds to approximately 2 to 3 substitutions across the full length of the fHbp antigen amino acid sequence provided as SEQ ID NO. 1 or SEQ ID NO. 2.
  • the antigen construct used has 3 or fewer substitutions, suitably 2 or fewer substitutions, suitably one substitution relative to SEQ ID NO: 1 or SEQ ID NO. 2.
  • the invention provides polypeptides, nucleic acids encoding said polypeptides, and/or vectors carrying said nucleic acids, for use in immunising a subject against a disease.
  • the invention provides polypeptides, nucleic acids encoding said polypeptides, and/or vectors carrying said nucleic acids, for use in inducing an immune response against Neisseria m eningitidis, more suitably against Neisseria m eningitidis group B.
  • the invention relates to use of a composition as described above in inducing an immune response against Neisseria m eningitidis, suitably Neisseria m eningitidis group B.
  • the invention relates to use of a composition as described above in immunising a subject against Neisseria m eningitidis, suitably Neisseria m eningitidis group B.
  • the disease is Meningococcus infection.
  • the disease is infection by meningococcal group B bacteria.
  • the disease is infection ⁇ jy N eisseria m eningitidis, more suitably infection by Ne isseria m eningitidis group B.
  • the disease is Meningitis.
  • the disease is Meningitis B.
  • the subject is a human.
  • the method is a method of immunising.
  • the immune response comprises a humoral response.
  • the immune response comprises an antibody response.
  • the immune response comprises a functional antibody response.
  • the immune response comprises a bactericidal antibody response.
  • the immune response comprises both a humoral response and a cell mediated response.
  • the immune response comprises protective immunity.
  • composition is an antigenic composition.
  • composition is an immunogenic composition.
  • composition is a vaccine composition.
  • composition is a pharmaceutical composition.
  • composition is formulated for administration to mammals, suitably to primates, most suitably to humans.
  • Suitable promoters include the hCMV IE promoter.
  • the short or the long version maybe used; most suitably the long version as described in WO2008/122811, which is specifically incorporated herein by reference for the teaching of the promoters, particularly the long promoter.
  • nucleic acid encoding the antigen, and/or encoding the signal sequenceantigen fusion protein is codon optimised for mammals, most suitably codon optimised for humans.
  • compositions and methods provided are extremely advantageous in that they achieve induction of immune responses in a subject with only a single dose of composition ('vaccine'). This is simpler and easier. This saves costs. This saves labour. This improves compliance (i.e. subjects do not have to attend for a second or further administration).
  • the invention relates to a single dose of composition as described above.
  • the invention relates to a single administration of composition as described above.
  • the invention also finds application in prime-boost immunisation regimes. For example, if after a period of time the immune response declines, as naturally tends to happen for many immune responses, then it maybe desired to boost the response in a patient back to useful levels such as protective levels.
  • the boosting immunisation may be carried out using a different composition to the composition used for the original priming immunisation.
  • This is referred to as heterologous prime boost.
  • the heterologous boost i.e. the second or further immunisation
  • the heterologous boost comprises one or more compositions selected from MVA, RNA, DNA, protein, adenovirus based viral vector, simian adenovirus based viral vector, gorilla-based adenovirus based viral vector, or human adenovirus based viral vector.
  • the boosting (second or further) immunisation may comprise MVA, RNA or protein.
  • the boost (second or further immunisation) may comprise RNA or protein.
  • the invention relates to a heterologous prime boost regime comprising a (protein +/- outer membrane vesicle) prime using either one of the licence vaccine(s) and a boost with the Adenovirus based vector described herein, such as ChAdOx1 MenB.1 (sometimes called MenBOx1).
  • a heterologous prime boost regime comprising a (protein +/- outer membrane vesicle) prime using either one of the licence vaccine(s) and a boost with the Adenovirus based vector described herein, such as ChAdOx1 MenB.1 (sometimes called MenBOx1).
  • the invention relates to a heterologous prime boost regime comprising (protein +/- outer membrane vesicle) prime using either one of the licenced vaccines i.e. Bexsero® from GlaxoSmithKline (GSK) or Trumenba® from Pfizer, and a boost with the Adenovirus based vector described herein, such as ChAdOx1 MenB.1 (sometimes called MenBOx1).
  • the invention relates to a heterologous prime boost regime comprising a prime using either one of the licenced vaccines i.e.
  • the invention relates to a method of inducing an immune response against Neisseria m eningitidis in a mammalian subject, the method comprising
  • composition comprising (protein +/- outer membrane vesicle), suitably Bexsero® from GlaxoSmithKline (GSK) or Trumenba® from Pfizer, to said subject; and
  • composition comprising adenovirus based vector as described herein, suitably ChAdOx1 MenB.1, to said subject.
  • the invention in another embodiment relates to a kit comprising: a) a dose of a composition comprising (protein +/- outer membrane vesicle), suitably Bexsero® from GlaxoSmithKline (GSK) or Trumenba® from Pfizer; and b) a dose of a composition comprising adenovirus based vector as described herein, suitably ChAdOx1 MenB.1; and c) instructions for administration to a mammalian subject.
  • a composition comprising (protein +/- outer membrane vesicle), suitably Bexsero® from GlaxoSmithKline (GSK) or Trumenba® from Pfizer
  • instructions for administration to a mammalian subject comprising: a) a dose of a composition comprising (protein +/- outer membrane vesicle), suitably Bexs
  • boosting regimes include raising the level of immune response in the subject, and/or increasing the duration of the immune response.
  • ChAdOx1/MVA or ChAdOx1/RNA or ChAdOx1/ protein as prime/boost regimes maybe used.
  • a homologous ChAdOx1 / ChAdOx1 prime/boost regime may be used.
  • Typical modified RNA or Self-amplifying mRNA vaccination regimen Two doses of vaccine administered, typically 4-8 weeks between each dose
  • Advantageous viral vector vaccination regimen according to the invention One dose of vaccine administered.
  • the first administration comprises, or consists of, a composition according to the present invention comprising a viral vector capable of expressing Men B fHbp.
  • the second or further ('boost') administration comprises exactly the same antigen as for viral vector.
  • the second or further ('boost') administration comprises an RNA vaccine.
  • the second or further ('boost') administration comprises IM administration.
  • the second or further ('boost') administration comprises adjuvant
  • said adjuvant is selected by the operator depending on platform.
  • the second or further ('boost') administration comprises saRNA no adjuvant is needed.
  • the dose is suitably in the range of 0.001 to 1 microgrammes.
  • the dose is suitably in the range of 1 to 15 microgrammes.
  • a boost composition may be administered about 6 months after administration of the original priming immunisation.
  • the invention provides a method of inducing an immune response against Neisseria m eningitidis in a mammalian subject, the method comprising
  • composition comprising a viral vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding the fHbp protein from Neisseria m eningitidis, characterised in that said viral vector is an adenovirus based vector to said subject, and
  • step (i) is a priming composition.
  • step (ii) is a boosting composition.
  • step (ii) is carried out 6 months weeks after the step (i).
  • any suitable route of administration may be used.
  • the route of administration is selected from group consisting of subcutaneous, intranasal, aerosol, sublingual, nebuliser, intradermal and intramuscular.
  • the route of administration is selected from a group consisting of intradermal and intramuscular.
  • the route of administration is intramuscular.
  • the route of administration may be applied to humans and/or other mammals.
  • Viral particles - vp/mL This refers to the count of total viral particles administered.
  • Infectious units - i.u./mL. This refers to the number of infectious units administered, and can be correlated more accurately with immunogenicity.
  • Preferred doses according to the present invention are:
  • the range is from io 9 to io 11 viral particles.
  • the range is from 2.5X io 10 vp to 5x io 10 vp.
  • doses apply to an average adult human of 70 Kg weight.
  • adults of different weights receive the same dose, i.e. there is no adjustment per kg. Doses may be determined by a physician using the guidance provided herein.
  • the dose(s)/range of dose(s) maybe derived from the examples below.
  • a dose of 5x10 10 vp may be used.
  • a dose of io n vp may be used.
  • the viral vector of the invention is formulated with simple buffer.
  • An exemplary buffer may be as shown below under the heading 'Formulation'.
  • nucleic acid sequence is codon optimised for mammalian codon usage, most suitably for human codon usage.
  • a nebuliser containing a composition as described above is provided.
  • a nasal applicator containing a composition as described above is provided.
  • an inhaler containing a composition as described above is provided.
  • a pressurised canister containing a composition as described above is provided.
  • a method of making a composition as described above comprising preparing a nucleic acid encoding the antigen (suitably the MenB fHbp protein), optionally fused to the tPA protein, and incorporating said nucleic acid into an adeno-based viral vector, suitably a ChAdOx1 vector.
  • the nucleic acid is operably linked to a promoter suitable for inducing expression of said antigen (or antigen-tPA fusion protein) when in a mammalian cell such as a human cell.
  • compositions disclosed such as ChAdOx vector directing expression of MenB fHbp
  • they have the ability to induce high levels of functional antibody following a single shot (single administration/single immunisation).
  • composition is formulated taking into account its route of administration.
  • composition is formulated to be suitable for the route of administration specified.
  • composition is formulated to be suitable for the route of administration selected by the operator or physician.
  • ChAdOx1 formulation buffer as used for the clinical product is:
  • Formulations for other administration routes such as aerosol will be adjusted accordingly by the skilled operator.
  • composition and/or formulation does not comprise adjuvant.
  • adjuvant is omitted from the composition and/or formulation of the invention.
  • the invention provides an adenoviral vector comprising a Men B antigen, preferably Men B fHbp.
  • a viral vector comprising an antigen for inducing an immune response in a mammal against a disease antigen, wherein said antigen is fused to a bacterial signal sequence.
  • said antigen is further fused to a second signal sequence.
  • said second signal sequence is a mammalian signal sequence.
  • the mammalian signal sequence is tPA.
  • said viral vector is an MVA viral vector. More suitably the viral vector is an adenoviral vector.
  • the antigen is selected from the group comprising (Fi+V) and (fHbp). More suitably the antigen is fHbp.
  • FIG. 1 shows schematic diagrams representing the different types of constructs generated for PorA or FetA antigens:
  • a For full-length constructs full-length genes were inserted in frame after the tPA leader, and a V5 marker peptide was added at the C-terminus.
  • B A single PorA VR loop was fused to a FliC scaffold (with or without flanking cysteines, Cys), with a V5 peptide sequence at the C-terminus.
  • C Two PorA VR loops were inserted into the FliC scaffold with a flexible polylinker sequence, SGMPGSGPAY, between the VR regions.
  • FIG. 2 shows graphs - evaluation of antibody responses against PorA (P1.7,16) induced by adenoviral vectors.
  • Mean values with SDs are displayed. Dashed lines indicate negative cut-off values (resulting with sera from naive mice). Groups of 4 or 5 Balb/C or NIH mice were used for the experiments.
  • Figure 3 shows graphs - comparison of antibody responses induced by Ad-F 3-3 vaccines versus an 0MV vaccine.
  • Balb/c mice received 10 9 IU of full length Ad-F 3-3 , Ad-FliC- VR3-3 or 5 ⁇ g of 0MV (44/76-FetA on PorA off ).
  • Antibody responses against recombinant rF 3-3 (A) or against 44/76-FetA on PorA off whole cells (B, pooled sera from each group) 6 weeks post vaccination are shown. Mean values with SDs for each group are displayed (A). Groups of 8 mice were used for the experiments. Kruskal Wallis test with Dunn's multiple comparison was used to perform comparisons between the groups * P ⁇ 0.05 and *** P ⁇ 0.001
  • Figure 4 shows a bar chart - protective efficacy of Ad and MVA vectored vaccines expressing PT Si-220 in immunised mice.
  • Mice were immunised with one or two doses of vaccines at eight-week interval. Challenge was performed two weeks after the booster immunisation (week 10).
  • Five mice in the control group, at day zero, were sacrificed three hours after aerosol challenge with the virulent bacteria to establish the successful infection by analysing bacterial counts in their lungs. The remaining five mice were sacrificed on day seven along other groups to determine viable counts in the mouse lungs. Results for each group are shown as individual counts and geometric mean ⁇ 95% CI (n 6 mice).
  • One-way ANOVA followed by Tukey's multiple comparison test was used to compare data and determine different differences between each group. Only significant differences between the groups are indicated in the graph.
  • Figure 5 shows a diagram - the DNA map of pBAC ChAdOx1 MenB.1 (sometimes called MenBOx1) used to generate the recombinant viral vector vaccine.
  • Figure 7 shows charts. Immunogenicity of ChAdOx1 MenB.1 in CD1 mice at week 2 and 6 post a single injection as compared with a HuAd 5 counterpart. Each mouse is represented by a dot and the geometric mean with 95% confidence interval of the group are displayed (horizontal lines). The red horizontal dashed line represent the threshold for protection (titre of 1:4).
  • Figure 8 shows plots. Dose responses of ChAdOx1 MenB.1 in BALB/c and CD1 mice at week 6 post a single injection. Each mouse is represented by a dot, the geometric means with 95% confidence interval are displayed (horizontal lines). The red horizontal dashed line represents the threshold for protection (titre of 1:4).
  • Figure 9 shows plots. Dose responses of ChAdOx1 MenB.1 in 3 mouse strains at week 6 post a single injection. Each mouse is represented by a dot, the geometric means with 95% confidence interval are displayed (horizontal lines). The red horizontal dashed line represents the threshold for protection (titre of 1:4).
  • Figure 11 shows plots. Immunogenicity of the clinical lot ChAdOx1 MenB.1 in CD1 outbred mice. Each mouse is represented by a dot and the geometric means with 95% confidence interval of the groups are displayed (horizontal lines). The red horizontal dashed line represent the threshold for protection (titre of 1:4).
  • Figure 12 shows photographs and bar charts. Antigen expression and imm unogenicity of Ad5 vectors expressing different versions of NadA and fHbp in m ice.
  • A Antigen expression in cells infected with Ad 5 containing either a NadA transgene flanked with a C-terminal V5 tag or, fHbp, or no transgene, detected by immunofluorescence against V5 or against fHbp, in green.
  • Antibodies were detected in serum samples by ELISA against heat inactivated 2996 (B and C) or H44/76 (D and E) bacteria.
  • B Serum antibody endpoint titers elicited against whole cells from strain 2996, 2 and 6 weeks post a single injection of mice with the vaccines indicated in the X-axis.
  • C Endpoint IgG subclass titers induced by Ad 5 -NadA immunization as compared with 29960MV immunization, against strain 2996 whole cells, at week 6.
  • Statistical differences observed using Kruskal- Wallis (B) and Mann-Whitney tests (C) are noted, with *** p ⁇ o.ooi, ** p ⁇ o.oi and * p ⁇ 0.05.
  • FIG. 13 shows a table and graphs.
  • Ad-fHbp induces high, cross-protective and long-lasting SBA responses.
  • A Bactericidal antibodies were detected in serum samples at weeks 6 and 42 by hSBA against strains H44/76, BZ83 or BZ198.
  • B Groups of mice were immunized once with decreasing doses of Ad-fHbp or H44/76 OMVs.
  • mice Bactericidal antibodies were detected in serum samples at week 6 post immunization by hSBA against strains H44/76 or BZ83 (heterologous PorA, low fHbp 1.1 expression).
  • C Groups of mice were immunized once with 10 9 iu Ad-fHbp or with the licensed vaccine 4CMenB (Bexsero®, 1/5* or 1/10* of a human dose). Bactericidal antibodies were detected in serum samples three weeks post injection, by hSBA against strain H44/76 (fHbp vi.i).
  • D Groups of Balb/c and CDi mice were immunized as described above.
  • Bactericidal antibodies were detected in serum samples, 6 weeks post-immunization, by hSBA against strains H44/76 or BZ83.
  • serum samples from each group were pooled, the bactericidal titres are expressed as the dilution giving a survival below 50% as compared to bacteria incubated with buffer, as examplified with the raw data of two assays detailed in the graphs in panel A (pi.10 is a monoclonal antibody with known bactericidal activity, used here as a control in the assay).
  • Figure 14 shows plots. Effect of heterologous prim e boost compliance en on the SBA responses, (A) using protein-based com ponents, and (B and C) using Ad and MVA com binations .
  • SBA was measured 2 weeks post last immunization against a strain expressing the homologous fHbp as used in the vaccines (variant 1.1, strain H44/76).
  • Control mice were immunized with 4CMenB at day o, 21 and 35.
  • SBA was measured 2 weeks post last immunization (panel B) or at several time points up to week 28 (panel C).
  • panel B and C the SBA titers for each individual mouse, and the geometric mean and 95% confidence interval of the group, are presented.
  • the horizontal doted line denotes the putative threshold associated with protection (titer of 1:4).
  • Figure 15 shows plots. Im pact of clinically relevant m odifications on the SBA response in m ice.
  • Figure 16 shows plots.
  • a point m utation in the transgene abrogates binding to hum an factor H (fH) and increases SBA responses in the presence of hum an fH.
  • Point- mutations were introduced in the transgene (Ml, H248L) and (M2, S223R).
  • a and B In vitro expression of the resulting protein. Hela cells were infected with the adenoviruses serotype 5 as mentioned (Ad empty as negative control, or Ad fHbp wild type sequence, or either one of the mutant), and expression of the antigen was measured by flow cytometry using an anti-fHbp monoclonal antibody, and expressed as % of positive cells.
  • Transgenic mice expressing human fH were immunized once with the adenoviruses mentioned in the X axis, or one, two (G) or three times with 4CmenB (H), and SBA titers were measured at several time points post injection (at weeks 2 and 6 for experiment in panel F, at weeks 3 and 8 for panel G, and 6 times over 21 weeks for the experiment in panel H).
  • Individual serum SBA titers against strain H44/76 are shown, with geometric means and confidence intervals. * denotes p ⁇ 0.05, *8 p ⁇ o.oi and *” p ⁇ o.ooi.
  • Figure 17 shows bar charts. Kinetics of SBA responses against different strains.
  • Serum SBA titers were measured at the time points indicated in the X axis, serum samples from each group were pooled, the bactericidal titres are expressed as the dilution giving a survival below 50% as compared to bacteria incubated with buffer.
  • the horizontal doted red line denotes the putative threshold associated with protection (titer of 1:4).
  • Figure 19 shows plots Figure 20 shows photgraphs Figure 21 shows plots Figure 22 shows plots Figure 23 shows plots Figure 24 shows plots Figure 25 shows plots Figure 26 shows plots Figure 27 shows plots Figure 28 shows a graph
  • Figure 29 shows graphs. In more detail, shown is time trend of hSBA titre .
  • ChAdOx1 MenB.1 black
  • Bexsero® at day o (dotted light and dark grey)
  • ChAdOx1 MenB.1 black for the homologous ChAdOx1 ChAdOx1 prime boost, and medium grey for the heterologous Bexsero® followed by ChAdOx1 prime boost
  • Bexsero® light grey, for the Bexsero® - Bexsero® prime boost
  • Data shown are median and IQR for each group.
  • Figure 30 shows graphs. In more detail, shown is time trend of T cell response. Eight to 10 participants were immunized once with ChAdOx1 MenB.1 at day o at either 2.5x10 10 viral particles (vp) (black), or 5x10 10 vp (dotted grey) or twice with 4CMenB (Bexsero®) at day o and day 28 (light grey) as indicated in the legends (A), and fHbp-specific IFN-gamma producing T cells enumerated at days o, 14, 180 and 208.
  • vp 2.5x10 10 viral particles
  • 5x10 10 vp dotted grey
  • 4CMenB Bosero®
  • ChAdOx1 MenB.1 black
  • Bexsero® at day o (medium and light grey)
  • ChAdOx1 MenB.1 black for the homologous ChAdOx1 ChAdOx1 prime boost, medium grey for the heterologous Bexsero® followed by ChAdOx1 prime boost
  • Bexsero® light grey, for the Bexsero® - Bexsero® prime boost
  • Data shown are median and IQR for each group.
  • Figure 31 shows graphs. In m ore detail, shown is tim e trend of h SB A titre (A) and T-cell response (B) in m erged group. 16 to 20 participants were immunized once with ChAdOx1 MenB.1 at day o with 5x10 lo vp (dotted black line), or with 4CMenB (Bexsero®) at day o (light grey), or twice with Bexsero® at day o and 28 (medium grey) as indicated in the legends. Data shown are median and IQR.
  • Figure 32 shows MAIT cell-deficient mice and humans with weak MAIT cell activation have impaired vaccine-induced T cell responses following ChAdOx1 immunization.
  • B Spearman rank correlation analysis in the change in CD69 expression on MAIT cells from pre-boost to day 1 post-boost versus the change in frequency of IFN-g-producing PBMCs measured by peptide ELISPOT from pre-boost to day 14 post-boost.
  • Figure 33 shows (A) Gating scheme for the identification of MAIT cells (MR1/5-OP- RU++ T cells) in PBMCs of healthy human volunteers immunized with ChAdOx1.
  • C Frequencies of MAIT cells in peripheral blood one day pre- and one day postimmunization.
  • E Pearson correlation of change in CCL2 chemokine level following vaccination and the change in expression of CD69 on MAIT cells. *, P ⁇ 0.05; ***, P ⁇ o.ooi; Wilcoxon rank-sum test. Symbols indicate individual donors, and group mean is shown.
  • Figure 34 shows plots of SBA titers against strain H44/76-SL. Individual data and geometric mean for each group are indicated, prior to vaccination (A), 1 month after a single vaccine injection (B) and 6 months after 1 or 2 immunizations, as indicated in the X-axis. The horizontal dotted line indicates the putative protective threshold titer of 1:4.
  • Figure 35 shows plots of SBA titers against strain H44/76-SL. Individual data and geometric mean for each group are indicated, at day 208 (A) and 365 (B), after 1 or 2 immunizations, as indicated in the X-axis, with the interval between first and second injection indicated in parenthesis. The horizontal dotted line indicates the putative protective threshold titer of 1:4.
  • Figure 36 shows plots of SBA titers against strain Moi 240355. Individual data and geometric mean for each group are indicated, prior to vaccination (A), 1 month after a single vaccine injection (B) and 6 months after 1 or 2 immunizations, as indicated in the X-axis. The horizontal dotted line indicates the putative protective threshold titer of 1:4.
  • Figure 37 shows plots of SBA titers against strain Moi 240355. Individual data and geometric mean for each group are indicated, at day 208 (A) and 365 (B), after 1 or 2 immunizations, as indicated in the X-axis with the interval between first and second injection indicated in parenthesis. The horizontal dotted line indicates the putative protective threshold titer of 1:4.
  • Prior to Day 180 participants are grouped according to the priming regimen (A).
  • At day 180 groups were further divided according to the different boosting regimen (B).
  • Figure 39 shows graphs of IgG memory B cell (A) and IFN-gamma secreting T-cell (B) responses against fHbp 1.1. geometric means and CI are indicated, after 1 or 2 immunizations, as indicated in the legends.
  • participants Prior to Day 180, participants are grouped according to the priming regimen. At day 180, groups were further divided according to the different boosting regimen as indicated.
  • Figure 40 shows plots of plasma cytokine quantification by MSD. Individual data, geometric means and CI are indicated, after 1 or 2 immunizations, as indicated in the legends. Prior to Day 180, participants are grouped according to the priming regimen. At day 180, groups were further divided according to the different boosting regimen as indicated.
  • Example 1 Com parative Data (PorA/FetA)
  • Adenoviral vectored vaccines are able to induce both strong cellular and antibody responses against viruses, parasites and the intracellular pathogen Mycobacterium tuberculosis.
  • the capacity of adenovirus vectors to induce antibody responses to transmembrane outer membrane proteins from bacteria has not been previously established.
  • PorA and FetA adenoviral vaccines were designed using sequences with the Genbank accession numbers X52995.1 (strain 44/76), AF226337.1 (NZ98/254) and X89755.i (44/76). Where appropriate, N to Q amino acid substitutions were performed to remove potential sites of N-linked glycosylation. Native sequences were codon optimised for expression in humans (GeneArt, Regensburg, Germany). Transgenes were cloned into plasmids containing attRi and attR2 recombination sites (Gateway® Life technologies, CA, USA) under control of a CMV promotor.
  • tPA human tissue plasminogen activator
  • variable regions from PorA and FetA were also cloned into a flagellin scaffold: the epitope sequences were fused to replace the central portion (D3) of flagellin protein from E, coli as described previously (Lu 1995 DOI: io.iO38/nbtO495-366). Cysteine residues flanking the variable loops were engineered where indicated to further constrain the conformation of the epitopes.
  • Transgenes were recombined with pAd-PL DEST using LR clonase (Invitrogen) to generate recombinant El and E3 deficient human adenovirus serotype 5 (AdHu 5 ).
  • viruses were produced as previously described (Dicks 2012 DOI: 10.1371/journal.pone.oo4O385). Viral vectored vaccines were formulated in endotoxin-free PBS. Outer membrane vesicle (0MV) vaccines were produced from the MenB strains NZ98/254, 44/76-FetA, supplementPorA, ruin and 44/76- FetA on PorA off (Marsay 2015) by detergent extraction as previously described (Frasch 2001); the latter two strains are mutants of 44/76 that were created to assess bactericidal activity against PorA and FetA individually. All 0MV vaccines were formulated to contain either 2.5 ⁇ g or 5 ⁇ g of total protein in 20 mM TRIS buffer pH 7.0- 7.5 (Sigma Aldrich, MO, USA) with 8 5 ⁇ g of AlhydrogelTM per dose.
  • HeLa cells were seeded into 6 well culture plates containing rat collagen coated coverslips (BD Bioscience, NJ, USA). Cells were transfected with plasmid DNA containing the transgenes (stated in the figure legends) using Lipofectamine 2000 according to the manufacturer's instructions (Life technologies, CA, USA). Alternatively, HeLa cells were infected with adenoviruses expressing transgenes at a MOI of 100. Transfected and infected HeLa cells were left overnight at 37°C with 5% C0 2 for protein expression. Cells were fixed with 4% paraformaldehyde and permeabilised with 0.2% triton X-100 in PBS.
  • Transgene proteins were detected using an appropriate mouse primary monoclonal (anti-P.1.7, anti-Pi.16, anti-Pi.4 or anti-F3- 3) or polyclonal mouse FetA F 3-3 immunised sera (all provided by NIBSC, UK) followed by goat anti-mouse IgG conjugated to Alexafluor 488 (Life technologies, CA, USA). Cell nuclei were counterstained with DAPI and visualised using a Leica DMI3000 B microscope.
  • mice were housed in specific pathogen-free conditions. Mice were immunized with a single injection of to 8 or 10 9 infectious units of each vaccine. All vaccines were given intramuscularly to the hind thigh muscle of both legs, with an 8-week interval between priming and boosting immunizations when boosting was performed. Blood was collected from tail bleeds or terminal cardiac bleeds at various time points and allowed to clot then centrifuged at 15,000 x g for 10 minutes. Sera were aliquoted and stored at -20°C until use.
  • Immulon 2HB Plates (Thermo Fisher Scientific, MA, USA) were coated with either heat killed whole cell preparations of N. m eningitidis in PBS (OD 6oonm 0.1), or recombinant PorA or FetA proteins in carbonate bicarbonate buffer (Sigma Aldrich, MO, USA) at 2 ⁇ g/ml at 4°C overnight. Plates were washed with PBS Tween 20 at 0.05% before being blocked with 1% BSA in PBS (all Sigma Aldrich, MO, USA) for 2 hours at 37°C. Sera were diluted 1:2000 in blocking buffer before being serially diluted in duplicate and left at 4°C overnight.
  • Plates were washed before addition of anti P1.7, anti Pi.16 or anti F 3-3 monoclonal antibodies (NIBSC, UK) and incubated for 1 hour at room temperature (RT). Plates were washed and HRP-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch inc. PA, USA) was added at 1:10,000 dilution and incubated for 1 hour at RT. Plates were developed with TMB solution (Sigma Aldrich, MO, USA) and stopped with 2M H 2 SO 4 .
  • the serum bactericidal assay measures complement-dependent bacterial lysis mediated by antibodies and in humans is the correlate of protection used for licensure of meningococcal vaccines.
  • SBA serum bactericidal assay
  • Full-length PorA and FetA proteins can be expressed in HeLa cells from DNA and adenoviral vectors
  • Several designs of the bacterial OMPs PorA (P1.7,16 and P1.7-2,4) and FetA (F 3-3 ) including full length proteins, VR loops within FliC scaffolds, tPA leader fused and non- tPA leader fused (cytosolic) were generated to assess immunogenicity when delivered by adenoviral vectors. Schematics for the transgene designs are shown in Fig. 1.
  • PorA PI.7,16 expressing vectors induce antibody responses in m ice
  • the PorA P1.7, 16 vaccines Ad-P1.7,16, Ad-cytosolic-P1.7,16, Ad-FliC- VR1.7 and Ad-FliC- VR1.7-C were assessed for immunogenicity in mice, with Ad expressing unmodified FliC as a negative control.
  • specific antibody responses were elicited by the full-length Ad-P1.7,16 and Ad-cytosolic-P1.7,16 vectors, as early as two weeks after vaccination, and were maintained for up to 32 weeks (Figure 2A).
  • immunisation with Ad-P1.7,16 with the tPA leader induced consistently higher antibody titres than the Ad-cytosolic-P1.7,16 without the tPA leader.
  • FetA F3-3 expressing vectors induce antibody responses in m ice
  • the functional capacity of the antibody responses induced by the vectored PorA/FetA vaccines was measured by SBA. None of the viral vectors induced a detectable bactericidal response despite the particularly high antibody concentrations detected in sera after immunisation with the full length vaccines Ad-P1.7,16, Ad-cytosolic-P1.7,16 and Ad-FliC-F 3-3 - see table below showing SBA titres at 16 weeks post vaccination for Ad-P1.7,16 and Ad-F 3-3 expressing adenoviral vaccines measured against target strain 44/76-SL, using baby rabbit complement and pooled mouse sera - serum bactericidal titres after a single dose of P1.7,16 and F 3-3 Adenoviral vectors:
  • Antibody subclass ELISAs were performed on sera taken from mice immunised with a single dose of 10 9 IU of Ad-P1.7,16 or Ad-FliC-VR1.7 or two doses of 5 ⁇ g of OMV comparator (44/76-FetA, administratPorA, neglect) against rP1.7,16. Addition of 1M sodium thiocyanate to the diluent was performed to assess relative antibody avidity induced by the adenovirus and 0MV vaccines. The levels of IgG3 antibodies were low or non- detectable in all vaccine groups (data not shown).
  • mice immunized with Ad-FliC-VR1.7 only had IgG2a antibody titres that were detectable above the negative cut-off.
  • the antibody endpoint- loads were reduced in the adenovirus and 0MV vaccine groups across all three subclasses when 1M sodium thiocyanate was included in the diluent. However, this was only significant in the 0MV immunized mouse sera for IgG2b antibodies (P ⁇ 0.01, data not shown).
  • PorA subtype Pl.7-2,4 expressing adenoviral vaccines also induce strong antibody responses but no bactericidal activity
  • the antibody levels induced by adenoviral vaccines coding for P1.7-2,4 antigens were compared with homologous (PorA subtype) OMVs (NZ98/254).
  • Ad-Pi.7-2,4 induced high levels of antibodies detected against recombinant PorA rP1.7- 2,4 by ELISA 6 weeks post immunisation, which were comparable to the levels of P1.7- 2,4-specific antibodies elicited in 0MV immunised mice.
  • Immunisation with Ad-FliC- VR1.4-C induced detectable but weak antibody responses 6 weeks post immunisation.
  • Boosting with OMVs at week 8 resulted in increased end-point titres in mice primed with Ad-FliC-VRi.4 or OMVs after the booster (week 6 vs. week 14, P ⁇ 0.001 and P ⁇ 0.01 respectively; week 6 vs. week 20, P ⁇ 0.05 for both groups).
  • Mice that received Ad- P1.7-2,4 as the priming vaccine did not elicit significantly higher P1.7-2,4-specific antibody levels following an 0MV boost. No hSBA activity was detected 6 weeks after a single immunisation with Ad-P1.7-2,4 or Ad-FliC- VR1.4 (data not shown).
  • Bordetella pertussis is the causative agent of the highly contagious respiratory infection whooping cough (pertussis) .
  • Pertussis is a vaccine-preventable disease and although there is high vaccination coverage in many countries, there are reports of increased numbers of pertussis cases globally.
  • Most developed countries replaced the whole-cell pertussis vaccines with less reactogenic acellular pertussis vaccines that are effective at preventing severe pertussis disease, but are unable to prevent bacterial colonisation and subsequent transmission.
  • the acellular pertussis vaccines also do not induce longterm protection in vaccinated individuals and their main component is pertussis toxin, which is a complex toxin made of the Si subunit and the B oligomer.
  • the viral vectored Bordetella pertussis toxin SI subunit vaccines are not protective in vivo
  • mice immunised with the aP vaccine had significantly lower bacterial counts in their lungs than the control group and mice vaccinated with the viral vectored vaccines on day 7 post challenge (p ⁇ 0.0001, comparison with control group on day 7, mice primed with Ad Si-220, prime-boosted with MVA + Ad (Si-220) and those prime-boosted with Ad + MVA (Si-220)), bacterial counts in the lungs of mice vaccinated with the Ad and or MVA were similar to those found in PBS-immunised mice at day seven ( Figure 4).
  • OMP Outer membrane protein OMV Outer Membrane Vesicles PBS Phosphate-buffered saline PCR Polymerase chain reaction PI Principal Investigator SBA Serum bactericidal antibody SAE Serious Adverse Event SOP Standard operating procedure ST Sequence type SUSAR Suspected unexpected serious adverse reaction
  • TPA Tissue plasminogen activator
  • TSA Tryptic Soy Agar vp Virus particles
  • VSS Virus seed stock WHO World Health Organisation Wt Wild type 4CMenB
  • Bexsero® Four component meningococcus B vaccine
  • ChAdOx1 MenB.l is a replication-deficient (El and E3 deleted) simian adenovirus, which contains the Neisseria m eningitidis (MenB) gene encoding for antigen factor H binding protein (fHbp), with a point mutation of a serine to an arginine at the amino acid position
  • the antigen, fHbp is an outer membrane protein and is a component of the two vaccines licensed for MenB (Bexsero® from GSK and Trumenba® from Pfizer).
  • wild type (wt) fHbp binds human factor H (fH), a complement inhibitor, with very high affinity, leading to a potential dampening of the immune response to the vaccine and a theoretical risk of raising an autoimmune response against fH.
  • fH human factor H
  • fH human factor H
  • ChAdOx1 Chimpanzee adenoviruses (ChAd) have been developed as viral vectors following concerns that preexisting immunity to human serotypes (such as serotype 5) could limit future widespread use of these viruses.
  • Phylogenetic studies show that simian and human adenoviruses fall into the same eight species. ChAdOx1, like many simian adenoviruses isolated to date, is a member of species E, which also contains one human virus (Human adenovirus serotype 4). Because of the El deletion, the virus can only propagate in cells expressing El functions, and thus virus is unable to replicate within vaccinated animals or humans.
  • meningococcal antigen fHbp from ChAdOx1 MenB.l was detected in mammalian cells, as assessed by immunofluorescence with a fHbp-specific monoclonal antibody. In addition, binding of fH to fHbp mutant was decreased as compared to the wt fHbp.
  • ChAdOx1 MenB.l induced high SBA titres in different murine strains.
  • - ChAdOx1 MenB.1 induced SBA responses in different strains of mice and in a dose-dependent manner.
  • ChAdOx1 vaccines containing different inserts from H. influenza, from M. tuberculosis, and from prostate cancer have been used in clinical trials at the University of Oxford. Safety data from these trials are favourable with no safety concerns raised.
  • ChAdOx1 MenB.1 The clinical development of ChAdOx1 MenB.1 is aimed towards the production of an effective
  • ChAdOx1 MenB.1 Phase I trial will evaluate the safety and immunogenicity of various intramuscular doses of ChAdOx1 MenB.1 in healthy adults, and provide a proof of concept that a bacterial outer membrane protein expressed from an adenovirus vector can induce bactericidal response in human.
  • the clinical development will eventually progress to a formulation that will include another variant of fHbp, in order to increase the coverage of the vaccine, and to the target populations (infants and adolescents), that may have been primed with the licensed vaccine 4CMenB.
  • SBA serum bactericidal activity
  • Factor H binding protein is an important virulence factor expressed on the surface of /V. m eningitidis.
  • the function of fHbp is to bind human complement factor H (fH), an important down-regulator of the host alternative complement pathway. Binding of human fH to the bacterial surface via fHbp interferes with complement- mediated lysis of the bacteria and is an important immune evasion strategy of N. m eningitidis.
  • Strains in which fHbp has been deleted have reduced binding to fH and reduced survival in human complement mediated bacterial killing assay compared to wild type strains.
  • fHbp incorporated in the vaccine is expected to form a complex with human fH, and this interaction between fH and fHbp adversely affected the immunogenicity of the 4CMenB vaccine in transgenic mice expressing human fH (lower serum IgG anti-FHbp antibody responses and 15 fold lower SBA response).
  • human fH lower serum IgG anti-FHbp antibody responses and 15 fold lower SBA response.
  • the ability of anti-fHbp antibodies to inhibit binding of fHbp to fH appears to be crucial to the breadth of bactericidal activity(6)(7).
  • Binding of human fH to the fHbp within the vaccine may mask epitopes in the fH binding site that are required for generation of bactericidal antibodies. Theoretically, the resulting antibodies in vaccinees could fail to block binding of fH to the bacteria. Moreover, the formation of a complex between fH and fHbp upon vaccination may lead to the aberrant generation of antibodies against fH.
  • Anti-fH IgM were detected in 2 of 15 fH transgenic mice (whose fH binds fHbp) after three doses of 4CMenB, whereas no such antibodies were found in wild type mice (whose fH does not bind fHbp) following the same vaccination schedule(8). Anti-fH antibodies have not been reported in any 4CMenB vaccine studies involving humans, although they were not looked for, and are not known to be produced after natural N. meningitidis infection.
  • the challenge for meningococcal vaccines is to induce serum bactericidal antibodies (SBA).
  • SBA serum bactericidal antibodies
  • the majority of subcapsular antigens are variable and induce strain-specific protection, limiting their use as strain-specific formulations designed to counter clinical outbreaks.
  • Giuliani et al. highlighted the intriguing possibility that adjuvants inducing T-helper type-1 responses contribute to broader protection against MenB in animal models(n).
  • Adenovirus vectored vaccines have extensively been shown to induce robust IFN- gamma responses with a variety of antigens, in pre-clinical models and in numerous clinical trials(i2). Therefore an attractive hypothesis is that concomitant induction of IFN-gamma due to the intrinsic properties of the Adenovirus may induce higher levels of protective antibodies and broader coverage against MenB.
  • Adenoviruses can infect several cell types but no evidence of insertional mutagenesis has been observed.
  • the adenoviral genome is well characterized and easy to manipulate. Adenoviruses cause mild disease but deletion of key genes (El which is required for viral replication) renders them replication-defective.
  • Replication-deficient adenoviruses can be propagated in cell lines approved by regulatory agencies for human product development (human embryonic kidney cells 293) and following Good Manufacturing Practice.
  • Recombinant Adenoviral vectors expressing antigens from HIV-i, TB, malaria, influenza, RSV and hepatitis C virus are in phase I/II clinical trials, and elicit strong antibody responses in humans with an excellent safety record. Recently, excellent safety and immunogenicity was observed in a phase I trial for malaria in 10 week-old Gambian infants(i4).
  • Chimpanzee adenovirus Ox1 was developed in Oxford University from the group E chimpanzee adenovirus ⁇ 258(16).
  • the cellular immunogenicity of recombinant El- E3- deleted vector ChAdOx1 is comparable to that of other species E derived chimpanzee adenovirus vectors including ChAd63, and the prevalence of virus neutralizing antibodies in human was lower than for ChAd63 in British and Gambian infants.
  • no individual possessed a neutralisation titre above 200 (the threshold for a positive titre during routine pre-vaccination screening).
  • the low seroprevalence of ChAdOx1 in humans suggests that this new vector could be particularly efficacious in a clinical setting.
  • the vaccine construct was generated at the Oxford Vaccine Group, University of Oxford. Manufacture of the vaccine was carried out in accordance with the requirements of cGMP by:
  • ChAdOx1 vector is replication-deficient as the El gene region, essential for viral replication, has been deleted. The virus will not replicate in cells within the human body. In addition the E3 locus, which promotes viral particle release and inhibits the host's antiviral response, is also deleted. ChAdOx1 propagates only in cells expressing Ei, such as HEK293 cells and their derivatives or similar cell lines such as Per.C6 (Crucell). ChAdOxl fHbp S223R (ChAdOxl MenB.l) assem bly
  • OVG98 pENTR plasmid vector containing the fHbpi.i-S223R antigen between the 'long' CMVLP TO promoter and BGH poly A sequences.
  • FHbpi.i S223R gene was cloned from a GeneArt DNA string, using Hindlll and Notl restriction enzymes and ligated to backbone (from plasmid OVG33). The size of restricted fHbpi.i S223R gene was 826 bp.
  • the linearised DNA vector and the DNA insert were gel- purified and the ratio 1:3 for ligation reaction was used (T4 Ligase). A DNA mini prep from recombinant clones (Kanamycin resistant) was performed. The correct size of the insert was verified by restriction mapping and the antigen was sequenced.
  • the structural fHbpi.i S223R cassette contained in pmono-fHbpi.i S223R was cloned into ChAdOxl destination vector (P2563) using Gateway® LR Clonase®, to generate ChAdOxl MenB.l.
  • ChAdOxl destination vector P2563
  • Gateway® LR Clonase® Gateway® LR Clonase®
  • ChAdOxl vector used to derive ChAdOxl MenB.l was generated at the Jenner Institute, and its complete genome sequence is known.
  • the sequence of the transgene region in ChAdOxl MenB.l has been verified by (i) sequencing directly from phenol purified viral genomic DNA; and (ii) sequencing of DNA amplified by PCR. The primers for both sequencing methods were the same.
  • the DNA map of pBAC ChAdOxl MenB.l (also called MenBOx1) used to generate the recombinant viral vector vaccine is shown in Figure 5.
  • Pre-GMP Pre-GMP Starting Material
  • adenoviral destination vector plasmid number OVG 98-11 (ChAdOx1-fHbpi.i S223R), was obtained from OVG.
  • the plasmid was transformed into competent E. coli DH5C1 and transformants plated onto
  • LAgar Vegitone plates containing chloramphenicol A single colony from the transformation plate was re-streaked onto a chloramphenicol plate and a single colony picked for plasmid preparation.
  • the plasmid was grown up in LB Vegitone and DNA was isolated using a Qiagen
  • the purified destination plasmid was digested with Pmei to release the Non-Viral Plasmid Sequence.
  • HEK 293 cells were transfected with 10 ⁇ g of unpurified digest and approximately 300 ng of purified adenoviral genome fragment. Both transfections were harvested, the digest transfection material frozen down and the fragment transfection expanded in HEK 293 cells. Three Hyperflasks were each infected with 1 mL of lysate. The cells were harvested when cytopathic effect (CPE) was evident.
  • CPE cytopathic effect
  • the cells were harvested by centrifugation.
  • the pellets were resuspended in cell lysis buffer, frozen, thawed and treated with Benzonase® endonuclease to reduce host cell DNA.
  • After two further rounds of freezing and thawing the lysate was clarified by centrifugation to remove cell debris.
  • the resulting clarified lysate was purified by two rounds of caesium chloride ultracentrifugation.
  • the virus band was harvested. This was dialysed against three changes of formulation buffer (10 mM Histidine, 35 mM NaCl, 1 mM MgC12, 0.1 mM EDTA,
  • ChAdOx1 MenB.1 pre-GMP starting material was designated as ChAdOx1 MenB.1 pre-GMP starting material.
  • the pre-GMP Starting Material was used to infect a GMP-manufactured, suspension HEK293 cell line, at a Multiplicity of Infection (MOI) of 2.5. After 47.5 hours the virus- infected cells were harvested by centrifugation to remove their culture supernatant, resuspended in lysis buffer (io mM Tris, 135 mM NaCl, 1 mM MgC12) and frozen at -8o°C (nominal). A proportion of the cells were lysed by thawing and freezing three times in total to release the virus particles.
  • MOI Multiplicity of Infection
  • the lysed cells were centrifuged to remove cell debris and the clarified lysate stored in small aliquots at -8o°C as the ChAdOx1 MenB.1 Master Virus Seed Stock (MVSS).
  • MVSS ChAdOx1 MenB.1 Master Virus Seed Stock
  • the remaining frozen cells were thawed and treated with Benzonase® endonuclease to reduce host cell DNA levels.
  • the lysis procedure continued by freezing and thawing for a total of three times. After centrifugation the clarified lysate was stored at -8o°C as the ChAdOx1 MenB.1 Bulk Harvest Lot (BHL) until further processing.
  • Formulation Buffer The purified 10 mM Histidine, 35 mM NaCl, 1 mM MgC12, 0.1 mM EDTA,
  • the BPL was sterile-filtered (0.22 micron) in a EU GMP Grade A pharmaceutical isolator to generate the Bulk Product which was then manually dispensed into 0.45 mL aliquots in sterile glass vials. These were stoppered and sealed and removed from the isolator for inspection. All accepted and rejected vials were labelled accordingly and stored at - 8o°C as Final Drug Product.
  • the stability testing was carried out on the Toxicology and Stability batch reference D525- P24 produced by the Process and Development team at the CBF. Fifty vials were produced and an aliquot of this material was used for infectivity and viral particles assays which were determined to be 3.52 X 109 ifu/mL and 5.03 X 1011 vp/mL, respectively. This gave this batch reference D525-P24 a P to I ratio of 142.9 : 1.
  • ChAdOx1 MenB.1 vaccine is stored according to GMP at -80 °C (nominal) at the Clinical BioManufacturing Facility, University of Oxford. Considering prior experience with other adenoviruses and the internal stability study, the clinical lot (ChAdOx1 batch 03D16-01, date of manufacture 24 November 2016) was assigned an expiry date of 24 November 2017. CBF continues to carry out a shelflife extension program on clinical vials stored at the same temperature. Prior to the expiry date of the clinical lot, virus was retested using a potency assay based on in vitro infectivity in HEK 293 cells using an anti-hexon immunostaining assay.
  • the vaccine vials are single use.
  • fHbp antigen in mammalian cells infected with ChAdOx1 MenB.1, and its capacity to bind human fH was investigated using a flow cytometry-based assay. HeLa cells were infected overnight with the ChAdOx1 MenB.1 adenovirus at a MOI of 500. Any adenovirus that had not infected the cells was subsequently removed by washing each sample. Expression of fHbp on the cell surface and intracellular compartment was detected using a monoclonal antibody specific to fHbp variant 1.1. Results show that ChAdOx1 MenB.1 is able to express fHbp ( Figure 6 A).
  • fHbp expressed in infected cells to bind human fH was also verified: cells infected with recombinant adenoviruses were incubated with human fH, and bound fH was detected with a monoclonal antibody against human fH. Results show that fH binding was detected in up to 73% of live cells infected with an adenovirus expressing wt fHbp, as expected, but not with adenoviruses (HuAd 5 or ChAdOx1) expressing the mutated fHbp S223R ( Figure 6B).
  • SBA was performed on serum samples from groups of mice immunized with ChAdOx1 MenB.1 to measure the immunogenicity and functionality of the vaccine-induced antibody response.
  • the SBA is a measure of efficacy as a bactericidal titre superior to 1:4 is expected to correlate with protection.
  • the two licensed MenB vaccines (4CMenB and rLP2o86) were progressed to clinical development and obtained licensure based on the results of this assay.
  • SBA assay was performed according to the current version of SOP OVGLO56 (MenB serum bactericidal assay vi.o), which follows the standardized method developed by PHE (17). Blood samples were centrifuged and serum separated and stored at -200C until use in SBA.
  • mice expressing human fH were used to assess the SBA activity elicited by ChAdOx1 MenB.1 in the presence of human fH (better reflecting the human situation).
  • the comparator group was given a 3rd dose of 4CMenB to reflect the schedule recommended for infants by the manufacturer.
  • mice immunized with ChAdOx1 MenB.1 elicit a SBA response by week 5 post injection, while this is only achieved after 2 injections of 4CMenB, which contains the wt fHbp that binds human fH in these transgenic mice.
  • the SBA titers in mice immunized with ChAdOx1 MenB.1 persisted at high levels until week 21 when the experiment was terminated ( Figure 10 ).
  • AdCh63 MSP-i another malaria antigen
  • AdCh63 MSP-i another malaria antigen
  • Ad6NSmut was barely detected only in the regional lymph nodes. There was no evidence of replication of the virus or presence of a disseminated infection. The results were consistent with the injection of a nonreplicating virus.
  • the strain and species used in the nonclinical repeat dose toxicology study was Balb/c mouse.
  • WBC white blood cell counts
  • Group mean triglyceride concentration for treated males males and females was slightly lower than control (males 0.79X control; females 0.68X control), however examination of the individual data revealed considerable overlap in the concentrations.
  • Group mean pottasium concentrations were higher for treated males and females (males 1.2X; females 1.3X control), with the difference from controls attaining a level of statistical significance.
  • the group mean phosphorus concentration was higher for treated females (1.2X control),
  • control and treated group means including those attaining a level of statistical significance were slight in degree or there was considerable overlap between groups in the range of the individual data. Further comment awaits the results of the histopathological examination.
  • ChAdOx1 MenB.1 in human
  • ChAdOx1 the vector ChAdOx1 has been used in previous clinical trials at the university of Oxford (ChAdOx1)
  • three variants of the antigen fHbp have been used in thousands of individuals including 2 months old infants during clinical development of 4CMenB and rLP2o86, and since post-licensure use in the UK (including 2 months old infants) and in the US (in adolescents and adults).
  • Both the vectors and the antigen have been shown to be safe, moderately reactogenic and able to induce strong immune responses in different age groups.
  • the described phase I study (ChAdOx1 MenB.1 Phase I trial, 0VG2017/04) will evaluate the safety and immunogenicity of various intramuscular doses of ChAdOx1 MenB.1 in healthy adults, and provide a proof of concept that a bacterial outer membrane protein expressed from an adenovirus vector can induce bactericidal response in human.
  • the study will assess the safety and immunogenicity of ChAdOx1 MenB.1 in man. Specifically, the immunogenicity following a single or two doses of ChAdOx1 MenB.1 will be evaluated for:
  • ChAdOx1 MenB.1 vaccine There have been no human studies conducted with the ChAdOx1 MenB.1 vaccine. The following adverse events may occur in some volunteers following vaccination with ChAdOx1 MenB.1, based on previous experience with other simian adenovirus viral vectored vaccines:
  • phase I single centre, open-label dose-escalation study will assess the safety and immunogenicity of two doses of 2.5XIO 10 vp or 5x10 lo vp of meningococcal capsular group B vaccine ChAdOx1 MenB.1 administered intramuscularly to healthy subjects. It will be the first planned administration of the vaccine in man.
  • the data from this phase I study will assist in the design of future clinical studies in decreasing age groups until safety and efficacy can be assessed in infants, the ultimate target population for the ChAdOx1 MenB.1 vaccine.
  • ChAdOx1 MenB.1 vaccine is similar to vaccines based on chimpanzee serotype replication deficient adenoviruses that have been used safely in humans.
  • the production process, formulation and manufacturing site is similar to that as for ChAdOx1 NP+Mi, ChAdOx1 Ag8 5 A and ChAdOx1. 5 T 4 .
  • Reports from clinical studies of ChAdOx1 NP+Mi and ChAdOx1 Ag8 5 A vaccines have demonstrated that they are moderately reactogenic, and safe.
  • ChAdOx1 MenB.1 expresses an antigen that has been used in thousands of individuals in clinical trials and post licensure.
  • ChAdOx1 MenB.1 should not be administered to individuals with known or presumed hypersensitivity to any component of the vaccines. Prior to administration of any dose of any of the vaccines, the volunteer should be questioned about the occurrence of any possible adverse events following any previous dose(s). Do not administer ChAdOx1 MenB.1 intravenously. This vaccine is intended for IM administration.
  • Safety data will be collected as part of the main objective of the phase I study.Participants will be seen and examined frequently during the first 28 days after vaccination to evaluate the physical manifestations of the immunisation, and selected subjects will have blood drawn to assess the biochemical parameters associated with organ toxicity. They will be followed for 6 months or longer depending on the protocol and will enrol in a product development registry to provide long-term safety follow-up.
  • Example 5 Vambox clinical trial: additional data
  • the examples above already presented the following data from the clinical trial, for the groups immunized with the novel vaccine ChAdOx1 MenB.1 (sometimes referred to as 'Vambox') or with a known licensed vaccine 4CMenB (Bexsero®) :
  • Vaccination with ChAdOx1 MenB.1 elicited protective responses similar to one or two doses of 4CMenB and higher responses than one dose of rLP2o86 against strain H44/76-SL (Fig. 38A).
  • SBA responses > 1:4 were maintained in more than 75% of participants at day 365 against this strain (Fig. 38B).
  • Lower responses were elicited against strain M01240355 (Fig. 38 A and B).
  • Adenoviral vectors are at the forefront of vaccine development for cancer, viruses and parasitic diseases.
  • the expression of a bacterial protein in a eukaryote cell may impact on the antigen's localization and, more importantly, conformation. Nevertheless, their potential to induce T Helper type i and high antibody responses after a single dose in humans is attractive to combat the disease and disability caused by capsular group B meningococcus (MenB). Therefore, the potential of adenovirus (Ad) viral vectors as a delivery platform for MenB antigens factor H binding protein (fHbp) and Neisserial Adhesin A (NadA) was investigated. The Ad vectored vaccines generated high antigen-specific antibody responses in mice after a single dose.
  • Ad-fHbp candidate was optimized for human use by the use of judicious variants and progressed to clinical development.
  • AdHu 5 vaccine based on another MenB protein (NadA) but, as with PorA and FetA (see Example 1), the anti- NadA antibodies were not functional by SBA.
  • Meningococcal bacteria are the leading cause of childhood meningitis and septicaemia in the UK . Given the dramatically rapid progression of this life-threatening infection, vaccination is considered the only strategy to conquer one of the most devastating infectious diseases of childhood.
  • Effective vaccines against the capsular groups A, C, W and Y leaves group B meningococcus (MenB) as the major cause of meningococcal disease and deaths in several developed countries (Ladhani 2016 DOI: 10.1136/archdischild-20i5-3o8928).
  • the serogroup B polysaccharide cannot be used as a vaccine target mainly because it is poorly immunogenic in non-human primates (Devi 1997 PMID: 9038314).
  • MenB vaccines are based on subcapsular protein antigens (Bjune 1991 doi: 10.1016/0140- 6736(91)91961-s, Oster 2005 doi: 10.1016/j.vaccine.2005.01.063, Lewis 2009 doi: 10.1586/erv.O9.3O, rollier 2015 doi: 10.1517/14712598.2015.983897, Findlow 2019 doi: 10.1080/14760584.2019.1578217).
  • Two MenB vaccines are licensed, 4-component MenB vaccine (4CMenB, Bexsero®), and rLP2o86 (Trumemba®), based on one or several of those subcapsular antigens.
  • the former also contains an outer membrane vesicle, used to tackle an outbreak in New Zealand (Andrews 2014 doi: 10.1016/S1473- 3099(13)70341-4). Both vaccines are licensed for adolescents, but none are included in an adolescent program. Meningococcal carriage is highest in teenagers and young adults (Christensen 2010 DOI: 10.1016/81473-3099(10)70251-6), and preventing carriage in this population could drive herd protection.
  • viral based vaccine platforms such as adenoviral and poxviral vectors could provide a potent solution: they induce both innate and adaptive immune responses in mammalian hosts (Tatsis 2004 DOI: i0.i0i6/j.ymthe.2004.07.0i3). They were originally developed for their well-recognised ability to induce potent cellular immunity.
  • adenovirus-based vaccine has been shown to induce potent and rapid neutralizing antibodies, as was demonstrated initially with rabies (Xiang 1996 doi: 10.1006/viro.1996.0239), and confirmed since with several pathogens including in clinical trials, such as SARS-C0V-2 (van doremaleen 2020), malaria (Draper 2008) and Ebola (Miligan 2016).
  • Adenoviral vectored vaccines have been extensively shown to induce strong IFN-gamma producing T cell responses which should provide the ideal conditions for switching to high levels of complement-fixing bactericidal antibody unlike conventional aluminium-based adjuvanted vaccines (Giuliani 2006 https://d0i.0rg/10.1073/pnas.0603940103).
  • nucleotide sequences for the antigens NadA 3 and fHbp 1.1 were obtained from the GenBank sequence database (https://www.ncbi.nlm.nih.gov/genbank/). The sequences were codon optimized for mammalian tissue.
  • Recombinant adenoviruses (Ad 5 , ChAdOx1 and ChAd0x2) were generated as described previously using a Gatewaycompatible entry vector, (Rollier 2019 DOI: 10.1038/S41598-020-61730-8, Folegatti doi: 10.1016/80140-6736(20)31604-4, Wang doi: io.i37i/journal.pntd.ooo687o), using a CMV pro motor and a tissue plasminogen activator signal sequence.
  • NadA inserts were constructed with a C-terminal deletion A 351 - 4o5 to remove the membrane anchoring domain, and a C-terminal V5 tag to allow antigen detection.
  • the antigens were inserted as 'full length' using the immature sequence, including the signal sequence that is cleaved in the mature protein, or truncated versions (labelled t) where the bacterial signal sequence was omitted (mature protein). Empty or irrelevant adenoviral vectors were used as controls.
  • the modified vaccinia Ankara (MVA) vectors encoding the same antigens were generated as described previously (https://doi.org/10.1038/nm881).
  • OMVs Native and deoxycholate Outer membrane vesicles
  • HeLa cells were seeded overnight on high binding protein coverslips (BD Bioscience, NJ, USA) placed in polystyrene six-well culture plates (5x10 5 cells per well) in complete Dio medium (Dulbecco's modified eagles medium with added penicillin, streptomycin, L-glutamine and foetal calf serum) at 37°C.
  • Dio medium Dulbecco's modified eagles medium with added penicillin, streptomycin, L-glutamine and foetal calf serum
  • Adenoviral vectors were added at a molarity of infection of 100, at 37°C overnight.
  • Cells were fixed with 4% paraformaldehyde, permeabilised with 0.2% Triton X-100 for five minutes.
  • Antigen expression (meningococcal antigen fHbp 1.1) was detected with the anti-fHbp monoclonal antibody, JAR4 (supplied by NIBSC) diluted at 1:500 in 1% BSA in PBS, followed by Alexa Fluor 488 conjugated goat-anti-mouse IgG (Life technologies, CA, USA). Cell nuclei were counterstained with DAPI and slides visualised using a Leica DMI3000 B microscope.
  • Immulon 2HB plates (Thermo Fisher Scientific, MA, USA) were coated with heat killed whole cell preparations of N. meningitidis in PBS (OD 6oonm 0.1), or recombinant fHbp or NadA proteins at 2.5 ⁇ g/ml in carbonate bicarbonate buffer (Sigma Aldrich, MO, USA). Samples were serially diluted in 1% BSA PBST-0.05%.
  • Antibody binding was detected with Horseradish peroxidase-conjugated goat anti-mouse (Jackson ImmunoResearch inc. PA, USA) and visualised with 3, 3', 5,5'- Tetramethylbensidine substrate (TMB, Sigma Aldrich, MO, USA). The reaction was stopped with 50pl H 2 SO 4 and optical densities (OD) were measured at 450nm with reduction at 6oonm. End-point titres for total IgG antibodies were defined as the serum dilution corresponding to the final OD reading above two times the average of naive negative control readings.
  • the serum bactericidal assay was performed as described previously using 25% (vol/vol) human serum as complement source (Marsay 2015), on complement heat- inactivated murine sera, serially diluted in bactericidal buffer (Hanks Balanced Salt Solution supplemented with 0.5% BSA ).
  • a titre was defined as the reciprocal of the highest dilution of serum that yielded >50% decrease in colony forming units relative to that of control wells within 6omins at 37°C without CO 2 .
  • ELISPOT enzyme- linked im mune-spot assay
  • 96 well filtration ELISPOT plate (MilliporeTM) were coated with recombinant fHbp at 2.5ug/ml or 1:1000 dilution of goat-anti-mouse IgG (BiolegendTM, positive controls), or PBS (blank wells). Plates were blocked with complete Dio media, and splenocytes or bone marrow cells added in duplicates at a concentration of 4x10 5 , 2x10 5 and 1x10 5 cells per well. Alkaline phosphatase conjugated goat-anti-mouse (InvitrogenTM) was added followed by alkaline phosphatase substrate (Bio-RAD).
  • NadA and fHbp encoding adenoviral vaccine vectors are imm unogenic in mice
  • Recombinant replication deficient adenovirus encoding known vaccine antigen targets (PorA, FetA, NadA and fHbp) were created, but the PorA and FetA encoding vectors failed to elicit bactericidal antibody responses (Marsay hopefully 2020).
  • two versions of the NadA and of the fHbp genes were constructed, full length or truncated as described above, and inserted into Ei-deleted Ad 5 .
  • Ad 5 vectors encoding the full length or the truncated versions of the NadA gene were able to produce the antigen in mammal cells, as evidenced by the detection of the V5 sequence, tagged to the C-term of NadA, in infected HeLa cells, above the background observed in cells infected with an empty Ad 5 (Fig.i2A).
  • Both vectors encoding the full length and truncated NadA were able to induce antibody responses in mice after a single dose against whole cells from strain 2996, containing a homologous NadA (Fig.i2B).
  • the antibody responses induced by the Ad 5 vectors to the single NadA antigen were of comparable magnitude to the response detected in mice immunized with 2996 outer membrane vesicles (0MV), containing many more homologous antigens including the immunodominant PorA (Fig. 12B).
  • IgG subclasses were measured against the homologous 2996 whole cells, and both the Ad- NadA-f and 0MV immunization elicited IgGi, 2a, 2b and 3. Higher levels of IgGi, 2b and 3 were detected in the OMV-immunized mice (Fig. 12C, p ⁇ 0.01 for IgG1, IgG2b and IgG3).
  • Ad 5 vectors encoding a full length and a truncated version of fHbp were able to produce fHbp in target cells, as determined by binding to anti-fHbp monoclonal antibody (Fig. 12A).
  • both vectors were able to induce antibody responses in mice as soon as two weeks post a single injection, as evidenced by antibody responses against whole cells containing a homologous fHbp variant (Fig. 13A).
  • the antibody titers were of similar magnitude to those induced by homologous native 0MV, containing many more antigens including PorA: endpoint ELISA titres reached 32, GOO- 256, 000 at week 6 after Ad-fHbp immunisation, and 16,000-128,000 with native 0MV (nOMV).
  • the IgG2a titers were superior in the Ad-immunized mice (Mann-Whitney, p ⁇ 0.05), while the IgG2b and the IgG3 responses were superior in the nOMV-immunized mice (p ⁇ o.ooi and p ⁇ o.oi, respectively).
  • the IgG2b and IgG3 responses elicited by the nOMV vaccines could be directed against any of the immunogenic antigens comprised within OMVs.
  • Bactericidal antibody responses are generated by the fHbp-expressing Ad5 vectors While no SBA response could be detected in the Ad-NadA immunized mice (against strain 2996, data not shown) or 5/99, both full length and truncated fHbp-encoding Ad 5 elicited functional antibody responses, as evidenced by the bactericidal activity detected in mice (Fig.isA).
  • strain BZ83 contains low levels of fHbp variant 1.1, but does not express the PorA contained in the nOMVs used for immunizing mice (H44/76), and thus allows a fair comparison of the fHbp-specific bactericidal antibodies.
  • the strain BZ198 expresses heterologous fHbp 1.5, and the mutant strain mBZi98 expresses fHbp 1.4.
  • the SBA titers were still detected 42 weeks post a single dose, when the response induced by the nOMV to the homologous strain had decreased to undetectable levels (Fig. 13A).
  • the Ad-induced SBA titers were dose dependent (Fig. 13B), and were also elicited in outbred mice (Fig. 13C).
  • SBA responses induced by a single dose ChAdOx1-fHbp were higher than those induced by a single dose of the licensed vaccine 4CMenB (Fig. 13D).
  • a Thi biased T-cell response maybe associated with better functional responses to protein based meningococcal vaccines, as evidenced by higher SBA titers when using a Thi-inducing adjuvant (Giuliani 2006 https://d0i.org/10.1073/pnas.0603940103). Therefore the heterologous vectored prime boost approach was explored, using an MVA vector encoding the same fHbp 1.1 sequence used in the adenovirus prime. Results showed that two weeks post the last immunization, the prime boost regimen, whether Ad-MVA or MVA- Ad, did not induce significantly higher titers as compared with Ad alone, and that MVA alone induced higher variability and lower SBA responses (Fig. 14B).
  • adenovirus serotypes such as the serotype 5 used here can neutralize the vaccine and thus dampen its immunogenicity, and one solution is to use adenoviruses that do not circulate in humans, such as chimpanzee serotypes (Dicks 2012 doi: io.i37i/journal.pone.oo4O385). Two such vectors were developed previously, ChAdOx1 and Chad0x2 (Dicks 2012 doi: io.i37i/journal.pone.oo4O385; Folegatti 2019 doi: io.339O/vaccines7O2004O) and their immunogenicity compared in mice.
  • a needle free delivery such as liquid mucosal immunization is relevant to immunization of babies and adolescents, and may provide higher levels of protection due to inducing the response at the entry site of the pathogen
  • intranasal and sublingual delivery of the adenovirus in liquid form was investigated in mice, as a single dose (Fig. 15C).
  • the SBA titers in serum were lower when the mucosal routes were used, but nothing is known from the contribution of potential mucosal responses in protection, or in carriage. Nevertheless, as licensure of meningococcal vaccines is based on the SBA titers in serum, the intramuscular route was selected for clinical proof of concept with ChAdOx1 fHbp.
  • Mutations of the fHbp transgene can increase the bactericidal response In humans, fHbp binds to the human complement inhibitor factor H, thus decreasing the innate response to the invading bacteria (Schneider 2009 doi: io.iO38/natureO7769). This may affect the anti-fHbp antibody repertoire when fHbp is used as vaccine antigen, and decrease serum bactericidal activity by covering important fHbp epitopes, and led to the generation of mutants fHbp proteins with expected lower binding to human fH (Beernink 2011, Rossi 2013 DOI: 10.1128/IAI.01491-15, Costa 2014 DOI: 10.1128/mBio.oi625-i4).
  • SBA were measured at different time points after a single dose of adenovirus vaccine, as well as after 1, 2 or 3 doses of the protein-based licenced vaccines. None of the vaccines induced SBA against a strain expressing a low amount of fHbp 1.1, homologous to the fHbp contained in the adenovirus and the qCMenB vaccines (Fig. 17A). However, while qCMenB contains other antigens susceptible to induce SBA responses, a single dose of Ad fHbp induced earlier SBA, and titers at least equivalent to those induced by the protein-based vaccine against strains expressing middle and high amount of homologous fHbp 1.1 (Fig. 17B, C).
  • a novel adenovirus-based vaccine encoding the MenB factor H binding protein (fHbp) with an N-terminal signal sequence (Ad- fHbp) induces high titres of protective antibody after a single dose in mice.
  • Ad- fHbp MenB factor H binding protein
  • a panel of N- terminal signal sequence insertion/deletion variants of the antigen were comparatively assessed for in vitro expression from mammalian cells and for immunogenicity in mice.
  • a novel MenB vaccine has been developed that consists of a recombinant, replication deficient chimpanzee adenovirus viral vector, ChAdOx1, encoding the immature form of the fHbp antigen that includes the complete original sequence, which comprises the bacterial lipobox domain (LTAC) at the C-terminus of the pre-lipoprotein signal peptide.
  • the construct also contains a second signal sequence: a mammalian tissue plasminogen activator (tPA) signal peptide at the N-terminus of the fHbp signal sequence, to ensure addressing the resulting protein to the secretory pathway within the mammalian cells (12).
  • tPA tissue plasminogen activator
  • the fHbp sequence also contains and a point mutation to decrease the affinity of the antigen to human factor H, as this was demonstrated to elicit higher titres of protective antibody in macaques (13).
  • the ChAdOx1 fHbp vectored vaccine candidate has entered a phase I trial to assess its safety and immunogenicity in humans.
  • the FL SS fHbp construct contains the mammalian signal peptide NRTAFCCLSLTTALI and the bacterial lipobox motif LTAC, so variants lacking one or both of these sequences as well as single amino acid variants of these sequences were assessed side- by-side to determine their relative contributions to the enhanced functional immune response.
  • the present study focuses on the comparative assessment of SS variants using a combination of in vitro expression assays and in vivo immunogenicity data in mice to determine the link between transgene expression and the enhanced functional antibody response associated with the candidate vaccine antigen.
  • this SS to other bacterial antigens is explored with a view to developing a generalisable sequence to boost the immunogenicity of viral vectored vaccines currently undergoing pre-clinical development.
  • Fig. 18 Overnight expression of human adenovirus serotype 5-encoded factor H binding protein N-terminal sequence variants from HeLa cells.
  • HeLa cells (1 x 10 6 per sample) were infected overnight with 5 x 10 8 infectious units of one of a series of human adenovirus serotype 5 (AdHu 5 ) constructs encoding an N-terminal sequence variant of the factor H binding protein (fHbp) and expression was quantified by flow cytometry after surface and intracellular staining of harvested cells with an anti-fHbp antibody (JAR5) and a fluorescently-tagged detection antibody.
  • the y-axis corresponds to the percentage of total fluorescent (fHbp-expressing) HeLa cells after overnight infection.
  • the three-hour timepoint appears to coincide with a window of increased antigen expression while expression appears to plateau at the five-hour timepoint for this construct.
  • These timepoints were chosen for further comparisons of early antigen expression between the mutated constructs.
  • HeLa cells were stimulated with brefeldin A, infected with each SS variant construct, and analysed by flow cytometry for intracellular expression of fHbp between three and five hours post-infection (fig. 19A).
  • the FL SS fHbp construct was the only construct that expressed at high levels at both timepoints, while most constructs which expressed highly at three hours post-infection failed to maintain these expression levels at the five-hour timepoint (fig. 19B).
  • Fig. 26 Side-by-side comparison of factor H binding protein N-terminal sequence variants, with and without enhanced green fluorescent protein tags, expressed from human adenovirus serotype 5 vectors after overnight infection HeLa cells.
  • HeLa cells (1 x 10 6 per sample) were infected overnight with 5 x 10 8 infectious units of one of a series of human adenovirus serotype 5 ( AdHu 5 ) constructs encoding an N-terminal sequence variant of the factor H binding protein (fHbp), fHbp fused with enhanced green fluorescent protein (eGFP), or GFP only, and expression was quantified by flow cytometry after surface and intracellular staining of non-GFP-expressing cells with an anti-fHbp antibody (JAR5) and a fluorescently-tagged detection antibody.
  • AdHu 5 human adenovirus serotype 5 constructs encoding an N-terminal sequence variant of the factor H binding protein (fHbp), fHbp fused with enhanced green fluorescent protein (eGFP), or
  • the y-axis corresponds to the percentage of total fluorescent (fHbp- and/or GFP-expressing) HeLa cells after overnight infection.
  • the differences in expression levels between the antigen variants tested is conserved for the eGFP fusion antigens and lower for the eGFP fusion antigens than the eGFP only positive control, confirming that the differences in antigen expression levels are attributable to the fHbp N-terminal signal sequence.
  • the eGFP-expressing constructs were detected in a greater proportion of infected cells, possibly due to the greater natural fluorescence intensity of these antigens compared with the detection of antibody-labelled antigens from the original constructs which may be less efficient. Overall, the trend in expression level differences was replicated for the eGFP constructs, confirming that the impact of the SS remains apparent even in the fusion antigens. The lower expression levels of the fHbp-eGFP fusion antigens compared with that of the eGFP only positive control confirms that the observed differences in expression between eGFP-containing constructs are due to the N- terminal fHbp SS variant antigens.
  • DAPI 4',6-diamidino-2-phenylindole
  • the FL and SP KO SS variants displayed similar antigen expression kinetics.
  • the SP + LTA KO SS variant was expressed at a marginally later time and at a lower level, while the LTA KO resulted in the lowest levels of antigen expression.
  • mice were immunized once with The adenoviral constructs encoding the different fHbp with N-terminal SS variants at a dose of 1 x 10 7 infectious units,. Sera collected at several timepoints were assessed for fHbp-specific IgG titres by indirect ELISA. Statistically significant differences in anti-fHbp antibody titres were observed between signal sequence variant groups at weeks two and four post-immunisation. The highest titres were induced by the FL SS and SP KO SS constructs; the lowest titres were induced by the truncated SS, lipobox KO and qCMenB groups (fig. 27 A-B). By weeks six and 14, the only statistically significant differences observed were between 4CMenB and several signal sequence variant groups, as the response had matured to reached plateau (fig. 27 C-D).
  • Enzyme-linked immunosorbent assays were performed on serum samples taken at weeks (A) two, (B) four, (C) six, and (D) 14 post-immunisation to determine the titres of anti-fHbp IgG in sera.
  • Statistical comparisons were made using a Mann-Whitney U-test. * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
  • anti-fHbp antibodies The functionality of anti-fHbp antibodies was assessed by performing SBA assays against the H44/76-SL reference strain that naturally expresses fHbp 1.1 using the sera taken at weeks two, four, and 6 post-immunisation. Significant differences in SBA titre were measured at each timepoint, with greatest differences observed at weeks two and four post-immunisations (fig. 21 A-B).
  • a single dose of AdHu 5 expressing the FL SS fHbp antigen induced the highest titres of bactericidal antibody at week two, significantly higher (p ⁇ 0.01) than the truncated SS fHbp, while 4CMenB failed to induce protective titres after a single dose at this timepoint (fig. 21A).
  • heterologous FL SS appeared to significantly increase anti-Fi IgG titres compared with the AdHu 5 F1 construct at week four post-immunisation, though the titres were still significantly lower than those associated with the F1 antigen with native SS (fig. 22). These data suggest that, the native SS may perform better than the meningococcal fHbp SS.
  • Fig. 22 Anti-Fi antigen IgG titres in sera from mice immunised with human adenovirus serotype 5 vectors encoding the full-length F1 antigen, with or without the N-terminal amino acid signal sequence, or the truncated form of the antigen.
  • Groups of 12 BALB/c mice were immunised with a sub-optimal dose of 1 x 10 7 infectious units of one of the human adenovirus serotype 5 (AdHu 5 ) vectors encoding the Yersinia pestis F1 antigen with native signal sequence (SS), a heterologous N-terminal full-length (FL) SS, or a truncated form of the F1 antigen lacking any SS.
  • AdHu 5 human adenovirus serotype 5 vectors encoding the Yersinia pestis F1 antigen with native signal sequence (SS), a heterologous N-terminal full-length (FL) SS, or a truncated form
  • Enzyme-linked immunosorbent assays were performed on serum samples taken at weeks A. two and B. four post-immunisation to determine the titres of anti-F1 antigen IgG in sera.
  • the humoral response induced by the F1 antigen with native SS was superior to that induced by the incorporation of the heterologous FL SS to this antigen or its truncated form at both timepoints.
  • Statistical comparisons were made using a Mann-Whitney U- test. * p ⁇ 0.05; ” p ⁇ 0.01; ”* p ⁇ 0.001; **** p ⁇ 0.0001.
  • antigen-specific T cell responses were compared between these two antigens and several Salmonella antigens - CdtB, SipD, and SseB. Plasmids were designed to include the SS ahead of these Salmonella Paratyphi antigens, cloned, and incorporated intoAdHu 5 vectors. Groups of six BALB/c mice were immunised with 1 x 10 7 IU of eachAdHu 5 vaccine encoding each of the bacterial antigens with or without the N-terminal SS.
  • Fig. 23 Antigen-specific T cell responses induced in mice two weeks after immunisation with human adenovirus serotype 5 vectors encoding bacterial antigens with or without an N-terminal signal sequence.
  • Groups of six BALB/c mice were immunised with human adenovirus serotype 5 (Ad H 115) vaccines expressing one of a series of bacterial antigens, with or without an N-terminal signal sequence (SS).
  • Spleens were harvested two weeks post-immunisation, processed, and stimulated at a concentration of 1:100 with the relevant peptide pool.
  • IFN- y/interleukin (IL)-17A dual colour fluorospot was performed to assess antigen-specific T cell responses associated with these cytokines.
  • This study provides a functional exploration of the distinct elements of a novel signal sequence included ahead of a MenB lipoprotein expressed from a viral vector-encoded transgene.
  • the relative contribution of each of these elements was assessed through in vitro expression assays in a mammalian cell line and validated with the use of high- resolution microscopy.
  • the elucidation of the transgene expression kinetics provides a potential link with differences observed between these constructs through comparative immunogenicity analyses.
  • the inclusion of the SS ahead of several other transgenes encoding heterologous bacterial antigens demonstrates the utility of this design in promoting early antigen-specific cellular immune responses.
  • a mammalian SP was elected to promote expression of the lipoprotein within mammalian tissues.
  • the results of the expression assays presented here confirm that the FL SS is superior to the truncated SS for the promotion of fHbp 1.1 antigen expression in mammalian cells. It has also been demonstrated that these SS elements were judiciously chosen fortheir combined influence on transgene expression and immunogenicity; the expression assay data provides rationale for the inclusion of the FL SS within the vaccine antigen sequence as the FL sequence promotes the most consistent levels of early transgene expression across each of the chosen timepoints.
  • fHbp-eGFP transgene expression was predominantly located within the cytoplasm in apparent association with membrane structures and no fluorescence detected in the extracellular media. This is in accordance with the typical role of SSs in targeting antigens to the membranes of the ER and cell surface.
  • the FL SS also demonstrates superiority to the truncated SS variant, demonstrating its ability to enhance the antigenicity of the fHbp antigen.
  • the level of antigen expression in APCs has been highlighted as an important contributing factor to the formation of antibodies against the transgene product from adenovirus vectors (29).
  • the induction of antigen-specific humoral immunity is primarily mediated by the interaction of CD 4 + TH cells with MHC class Il-presented peptides on the surface of these cells.
  • the ability of the FL SS to enhance these distinct CD 4 + T cell responses against the neisserial fHbp, the plague F1 antigen, and the Salm onella antigens CdtB, SseB, and SipD was investigated and found to significantly increase some of these responses.
  • the increase in T H 1-mediated antigen- specific responses against SipD and SseB were significant, as was the T H 17-mediated response against SseB.
  • Both of these proteins are constituents of the Salm onella type III secretion system (T3SS) and their potential as candidate vaccine antigens has been highlighted by studies in mice and humans (33, 34 ).
  • CD4 + T cells of the IFN-y and IL- 17-producing variety also contributed to the response induced by the live-attenuated Salm onella Typhi vaccine, Ty2ia (35), in humans.
  • the findings of the antigen-specific T cell responses provide compelling evidence that the FL SS can be incorporated ahead of adenovirus-encoded heterologous bacterial antigen transgene sequences to boost antigen-specific immune responses.
  • Plasm id construct design The nucleotide sequence for the desired antigen was obtained from the GenBank sequence database (httpsfy/wvyy ⁇ The sequence was run through a glycosylation site finder
  • kit buffer was added to the 2 mL tube at a ratio of 3:1 (100 mg gel ⁇ 100 ⁇ L) and incubated at 50 °C on a heating block for 10 mins or until dissolved, one volume of isopropanol was added and the gel digest was filtered
  • the reaction was run overnight on a thermocycler set to 16 °C.
  • thermocompetent DH5C1 cells After overnight ligation, 5 ⁇ L of sample was added to 50 ⁇ L of thermocompetent DH5C1 cells in a 2 mL Eppendorf tube on ice. A negative control was set up by adding 5 ⁇ L of dH 2 0 to the same volume of cells. Samples were incubated at 4 °C for 30 mins, heat- shocked at 42 °C for 30 s, and returned to 4 °C for 2 mins. 250 ⁇ L of pre-warmed (37 °C) SOC recovery medium was added to each tube and samples were placed on a 37 °C shaking incubator for 1 hour before plating 200 ⁇ L on LB agar containing 30 ⁇ g/mL Kanomycin using an L-shaped spreader.
  • proteinase K 1 ⁇ L was added to each sample the following day and incubated at 37 °C for 10 mins.
  • the proteinase K-digested samples were transformed using DH5C1 cells as outlined in section 2.5.5, but this time plating 200 ⁇ L on LB agar containing 100 ⁇ g/ mL ampicillin and adding 10 ⁇ L streaks of up to four samples per plate of LB agar containing 15 ⁇ g/mL chloramphenicol.
  • Cells were harvested at approximately 70-80% confluency. Where brefeldin stimulation was required, cells were resuspended in media containing 3 ⁇ g/mL of brefeldin A solution. 1 x to 6 cells per sample were infected with the appropriate volume of adenovirus construct to obtain a concentration of 5 x to 8 . Infected cells were incubated for the required time (overnight or specific time points), harvested and surface stained and/ or stained intracellularly with anti-fHbp monoclonal antibody JAR5 (National Institute of Biological Standards and Controls) and GFP-tagged IgG detection antibody using a Fixation/Permeabilization Solution Kit.
  • JAR5 National Institute of Biological Standards and Controls
  • CFU colony-forming units
  • a Mouse IFN-Y/IL-17A FluoroSpot kit was used to measure antigen-specific T cell responses in mouse spleen tissue.
  • Anti-IFN-y and anti-IL-17A monoclonal capture antibodies were diluted in PBS to a concentration of 15 ⁇ g/ml and 10 ⁇ g/ml, respectively.
  • the IPFL plate membrane was washed with 15 ⁇ L of 35% ethanol per well for no more than 60 s and then washed three times with 200 ⁇ L of sterile H 2 0 per well, too ⁇ L of capture antibody was added to each well and the plate was sealed for overnight incubation at 4 °C.
  • the plate was washed three times with 200 ⁇ L of sterile PBS per well the following day and the wells were then blocked with DMEM containing 10% FBS for 30 mins. Stimuli, either DMS0 (1:100) as a negative control, ConA (1:200) as a positive control, or reconstituted peptide pools (1:100) corresponding to the vaccine antigen, were added to the appropriate wells, followed by 2 x 10 5 splenocytes from the appropriate sample to each corresponding well. The plate was sealed and incubated overnight at 37 °C in a 5% CO 2 incubator. Cells were removed from the wells the following day and the plate was washed five times with 200 ⁇ L of PBS per well.
  • Anti-IFN-y-R4-6A2-BAM and anti-IL-i7A-MT227O (biotinylated) detection antibodies were diluted in PBS containing 0.1% BSA in the same tube to a concentration of 1:200 and 1:250, respectively. 100 ⁇ L of detection antibody mixture was added to each well and incubated for two hours at room temperature.
  • Anti-BAM-490 and SA-550 fluorophore conjugates were both diluted to a concentration of 1:200 in the same tube with PBS containing 0.1% BSA and, after washing the plate five times with PBS, 100 ⁇ L of this mixture was added to each well. The plate was wrapped in aluminium foil and incubated for one hour in the dark at room temperature.
  • the plate was washed five times with PBS before adding 50 ⁇ L of fluorescence enhancer to each well and incubating in the same manner for 15 mins. The plate was emptied of all liquid and the underdrain was removed. Plates were completely dried in the dark at room temperature prior to spot counting with an IRISTM FluoroSpot reader (Mabtech). Excitation 490 nm/emission 510 nm (FITC) and excitation 550 nm/emission 570 nm (Cy3) wavelengths were used to measure IFN-y and IL-17A, respectively.
  • FITC nm/emission 510 nm
  • Cy3 excitation 550 nm/emission 570 nm
  • 300 ⁇ L of 300 nM DAPI (5 mg/mL stock solution diluted to 300 nM in PBS) was added to the fixed/permeabilised cells for 5 mins at 4 °C, washed again and slowly transferred, using forceps and dabbing off excess liquid, to microscope slides with a drop of ProLong Gold Antifade Mountant in the centre. Slides were cured on a flat surface overnight in the dark and imaged the following day using a Zeiss 780 inverted confocal microscope (Zeiss) with ZEN Lite image acquisition software (Zeiss). Cells were located in brightfield and then the interface was switched to 'acquisition mode' where 'smart settings' were applied. The fluorophore-specific settings were manually refined, and images were taken across multiple planes of focus for each sample triplicate.
  • Any other fHbp variant including stabilising mutations and/ or mutations affecting the binding to human factor H, may be used in the composition of the invention.
  • Figure 28 shows that fHbp variants 2.19 and 1.4 are immunogenic.
  • Example 8 Im munogenicity in Humans
  • ChAdOx1 MenB.1 is immunogenic in humans.
  • the subjects are adults aged 18 to 50 years old who are in good health.
  • Participants are allocated to one of eight sub-groups. Those in Group 1 receive a single low dose of 2.5 x10 ⁇ 10 VP of ChAdOx1 MenB.1. Those in Group 2 receive a single high dose 5X10 ⁇ 10 VP of ChAdOx1 MenB.1. Group 3 participants receive a high dose of ChAdOx1 MenB.1 plus a repeat booster dose at six months. Group 4 participants receive a dose of Bexsero® at baseline with a high dose booster of ChAdOx1 MenB.1 at six months. Those in Group 5 receive two doses of Bexsero® at baseline and 28 days later. Those in Groups 6 and 7 receive Bexsero® at baseline and six months. Participants in Group 8 receive two doses of Trumenba® at baseline and six months. Participants in Groups 6, 7 and 8 will be asked to consent to a blood donation for making serum standards. Symptoms of the shots are recorded in participant diaries.
  • Serum bactericidal antibody titers were measured against meningococcal strain H44/76-SL, and enumeration of the fHbp-specific interferon-gamma (IFN-y) secreting T cells was performed on peripheral blood mononuclear cells by FLUOROSPOT. Results show that a single dose of ChAdOx1 MenB.1 vaccine elicits protective hSBA titers ( Figure 29A).
  • ChAdOx1 menB.1 induced a higher IFN-gamma secreting antigen-specific T cell response than two injections of the comparator ( Figure 30), and this T-cell response is still detectable six months post single injection.
  • ChAdOx1 MenB.1 was able to induce a hSBA antibody response, as well as a T cell response in participants previously primed with the licenced comparator 4CMenB. This is especially beneficial as the composition of the invention may be used in adolescents vaccinated in infancy with the licensed vaccine 4CmenB - which is currently in the UK infant vaccination schedule.
  • the T cell response to Ad MenB (ChAdOx1 MenB.1) is shown in Fig 32.
  • figures 33D and 33E show stimulation of an innate response by Ad MenB (ChAdOx1 MenB.1).

Abstract

The invention relates to a composition comprising a viral vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide antigen, wherein said antigen comprises Factor H Binding Protein (fHbp) from Neisseria meningitidis, characterised in that said viral vector is an adenovirus based vector. The invention also relates to uses, methods and kits.

Description

Compositions and Methods for Inducing an Immune Response
BACKGROUND TO THE INVENTION
N. m eningitidis is a leading cause of bacterial meningitis and septicaemia worldwide. The annual incidence in European countries stands at 0.94 per 100,000. The clinical manifestations of the disease are variable, with severe cases being associated with fulminant invasive disease and mortality rates that can reach 8-10%. Those who survive are often left with devastating sequelae, including marked neurological deficits and limb amputation. The severity of the disease is compounded by the difficulty in recognition in the early stages, which can lead to delays in effective and life saving therapy. Prevention of disease by effective vaccination is therefore a crucial protective strategy. The introduction of conjugate vaccines derived from meningococcal A, C, Y and W capsular polysaccharide into the routine infant or adolescent immunization schedule, in countries such as the United Kingdom, has led to dramatic reductions in the burden of disease. Therefore the majority of cases of invasive meningococcal disease are now caused by capsular group B strains (87% of cases in the United Kingdom, 76% in Europe). In 2012/2013, there were 800 reported cases of invasive meningococcal disease in the UK, of which 621 were due to capsular group B strains (Public Health England, 2014). The development of effective vaccines against capsular group B N. m eningitidis however has long been an unmet need and has necessitated the development of novel approaches to vaccine design, because the group B capsule is poorly immunogenic in humans due to its similarity with human antigens.
The search for comprehensive protection against meningococcal group B disease therefore necessarily focused on subcapsular antigens. The current vaccines licensed are:
- rLP2o86 (Trumenba®). licensed in the US, contains combinations of two variants of the protein fHbp. It is relatively reactogenic, requires 2 to 3 injections and is only developed for adolescents; therefore this vaccine will not target disease in the most at risk cohort.
- 4CMenB (Bexsero®). licensed in the UK, contains a combination of four subcapsular antigens: Three components are recombinant proteins consisting of one variant of factor H binding protein (FHbp), neisserial heparin binding antigen (NHBA), and neisserial adhesin A (NadA). The fourth component is the outer membrane vesicle (0MV) from a group B strain responsible for an outbreak in New Zealand. The efficacy of qCMenB vaccine was extrapolated from effectiveness data obtained from use of the 0MV component in New Zealand, immunogenicity data from clinical trials and estimation of coverage from in vitro and epidemiology studies (Rollier CS, Dold C, Marsay L, Sadarangani M, Pollard AJ. The capsular group B meningococcal vaccine, 4CMenB : clinical experience and potential efficacy. ExpertOpin Biol Ther [Internet]. Informa Healthcare; 2015 Jan 8;15(1):131-42). While these data suggest that the vaccine will prevent a proportion of invasive meningococcal disease cases, the potential coverage of these vaccines is debated and will not be 100%. This multicomponent vaccine is expensive to produce. It is reactogenic in infants (high fever rates) and requires up to 3 injections to elicit a relatively short-lived coverage. The vaccine has borderline cost-effectiveness.
Stylianou et al (Vaccine 33 (2015) pages 6800-6808) disclose Improvement of BCG protective efficacy with a novel chimpanzee adenovirus (ChAdOx1.85A) and a modified vaccinia Ankara virus (MVA85A), both expressing Ag85A. Although intranasally administered ChAdOx1.85A induced strong immune responses in the lungs, it failed to consistently protect against aerosol M.tb challenge. In contrast, ChAdOx1.85A followed by MVA85A administered either mucosally or systemically, induced strong immune responses and was able to improve the protective efficacy of BCG.
Mercado, N. B. et al. (Single-shot Ad26 vaccine protects against SARS-C0V-2 in rhesus macaques. Nature https://d0i.0rg/10.1038/s41586-020-2607-z (2020) discloses that a single-shot Ad26 vaccine protects against SARS-C0V-2 in rhesus macaques. Use of wildtype leader sequence as well as tissue plasminogen activator (tPA) leader sequence is disclosed.
The present seeks to overcome problem(s) associated with the prior art.
SUMMARY OF THE INVENTION
The invention relates to induction of immune responses, suitably protective immune responses, against Neisseria m eningitidis, more suitably Neisseria m eningitidis group B ("MenB").
The inventors have designed genetic constructs for use in immunogenic compositions.
The approach taken is distinct from known work due to the inventors deliberately choosing to present a bacterial antigen in a viral vector. In particular, the exemplary antigen (fHbp) is itself a bacterial outer membrane protein. Firstly presenting a bacterial protein in a viral vector (which entails expression by the mammalian cells in the subject to which the viral vector is administered) is itself an innovative approach. This is especially true as an approach for generation of functional antibodies, which requires the bacterial antigen to adopt a conformation corresponding to that adopted when expressed naturally in bacteria. In addition, the choice of a bacterial outer membrane protein - which will be expressed by a virus, inside a mammalian cell, and surface exposed/secreted in the absence of the usual background of the bacterial outer membrane components - makes the inventors' approach a very surprising and unexpected route to immunisation.
Notwithstanding these complications, the data presented herein clearly convey to the skilled reader that the approach is successful. The invention is based on these surprising findings.
Thus in one aspect the invention provides a composition comprising a viral vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide antigen, wherein said antigen comprises Factor H Binding Protein (fHbp) from Neisseria m eningitidis, characterised in that said viral vector is an adenovirus based vector. Suitably said antigen comprises Factor H Binding Protein (fHbp) from Neisseria m eningitidis group B.
Suitably said adenovirus based vector is a non-human adenovirus based vector.
Suitably said adenovirus based vector is a simian adenovirus based vector, preferably a chimp adenovirus based vector.
Suitably said adenovirus based vector is selected from the group consisting of ChAdOx1 and ChAd0x2.
Suitably said adenovirus based vector is ChAdOx1.
Suitably said Factor H Binding Protein (fHbp) comprises an arginine substitution at the amino acid position corresponding to serine 223 in the wild type Factor H Binding Protein (fHbp).
Suitably said Factor H Binding Protein (fHbp) comprises the amino acid sequence of SEQ ID NO: 2.
Suitably said antigen further comprises a signal sequence.
Suitably said signal sequence is a mammalian signal sequence (e.g. a human signal sequence) or a bacterial signal sequence.
When the signal sequence is a bacterial signal sequence, it may be homologous (e.g. the naturally occurring signal sequence integral to fHbp) or heterologous (e.g. a bacterial signal sequence derived from another protein (i.e. from a non-fHbp protein) from the same bacterium such as Neisseria m eningitidis, and/or derived from another bacterium (i.e. derived from a non-Neisseria m eningitidis bacterium).
In another embodiment suitably said antigen comprises at least two signal sequences. Suitably said at least two signal sequences comprise at least one bacterial signal sequence and at least one mammalian (e.g. human) signal sequence.
Suitably said antigen is present as a fusion with the tissue plasminogen activator (tPA) sequence in the order N-terminus - tPA - Factor H Binding Protein - C-terminus.
Suitably 'signal sequence' means an amino acid sequence which directs secretion of the polypeptide from the cell in which is it expressed such as a mammalian cell e.g. a human cell in the subject to which the composition of the invention is administered.
In another embodiment the invention relates to a composition as described above wherein said tPA has the amino acid sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
In another embodiment the invention relates to a composition as described above wherein said antigen has the amino acid sequence SEQ ID NO: 3.
In another embodiment the invention relates to a composition as described above wherein said viral vector sequence is as in ECACC accession number 12052403.
In another embodiment the invention relates to a composition as described above for use in induction of an immune response against Neisseria m eningitidis.
In another embodiment the invention relates to a composition as described above for use in boosting of an immune response against Neisseria m eningitidis.
In another embodiment the invention relates to a composition as described above for use in preventing Neisseria m eningitidis infection.
In one embodiment suitably a single dose of said composition is administered.
In one embodiment suitably two doses of said composition are administered.
In one embodiment suitably a single dose of said composition is administered, followed by one or more further dose(s) of said composition.
In one embodiment suitably a single priming dose of said composition is administered.
In one embodiment suitably a single boosting dose of said composition is administered. In one embodiment suitably a single priming dose of said composition is administered, followed by one or more further dose(s) of said composition.
In one embodiment suitably a single boosting dose of said composition is administered, followed by one or more further dose(s) of said composition.
In one embodiment suitably said composition is administered once.
In one embodiment suitably said composition is administered twice.
In another embodiment the invention relates to use of a composition as described above in medicine.
In another embodiment the invention relates to use of a composition as described above in the preparation of a medicament for prevention of Neisseria meningitidis infection.
In another embodiment the invention relates to a method of inducing an immune response against Neisseria meningitidis in a mammalian subject, the method comprising administering a as described above to said subject.
Suitably a single dose of said composition is administered to said subject.
Suitably said composition is administered once.
In another embodiment the invention relates to a method as described above further comprising administration of a second or further dose of said composition subsequent to administration of the first dose.
Suitably administration of said second or further dose of said composition is carried out approximately 6 months after administration of the first dose
Suitably said composition is administered by a route of administration selected from a group consisting of intranasal, aerosol, sublingual, intradermal and intramuscular. More suitably said administration is intramuscular.
In another embodiment the invention relates to a kit comprising:
-a first dose of a composition as described above; and optionally
-a second dose of a composition as described above; and instructions for administration to a mammalian subject. In one embodiment suitably administration of a single dose of the composition of the invention to a mammalian subject induces protective immunity in said subject. In one embodiment suitably a second or further dose of the composition of the invention is administered to the mammalian subject. Suitably said second or further dose is administered 6 months after the first or preceding dose. Suitably said second or further dose induces or maintains protective immunity in said subject.
The skilled reader will appreciate from the data presented when protective immunity in a mammalian subject such as a mammal is attained or expected to be attained. For example antibody responses are demonstrated herein. For example T-cell responses are demonstrated herein. For example serum bactericidal assay (SBA) experiments are demonstrated herein.
DETAILED DESCRIPTION OF THE INVENTION
The phrase "protective immune response" or "protective immunity" as used herein means that the composition is capable of generating a protective response in a host organism, such as a human or a non-human mammal, to whom it is administered according to the invention. Suitably a protective immune response protects against subsequent infection or disease caused by Neisseria m eningitidis. The data presented herein convey to the skilled person that the claimed compositions and methods/uses can be understood to be effective in this regard.
It is an advantage of the invention that immune responses are generated against fHbp, which is an outer membrane protein. Thus, it is a surprise in itself that use of a viral vector to deliver a bacterial outer membrane protein as an antigen can induce a functional antibody response. This is because using a viral vector to deliver such a protein delivers it "out of context" i.e. it is produced in the mammalian subject in their own mammalian cells transduced by the viral vector which is administered. Those eukaryotic cells (rather than prokaryotic bacterial cells) produce the protein, and it is subsequently presented to the immune system of the subject in a manner dramatically different to its natural presentation on the surface/outer membrane of the bacterial pathogen from which the antigen is derived. It is a further surprise that this approach is effective in induction of strong antibody responses, such as functional antibody responses.
The inventors teach use of a mutant fHbp (the S223R mutant) in a viral vector. This goes against conventional thinking in the art. Clearly it would normally be assumed that keeping as close as possible to the wild type protein would be desirable, since the wild type protein is what will be encountered if the subject comes into contact with the wild type pathogen (i.e. a MenB bacterium). However, choosing to produce compositions of the invention using a mutant antigen with the S223R substitution is surprisingly effective, which is an advantage of the invention.
The inventors teach the use of non-human adenovirus based vectors. This offers the advantage of avoiding pre-existing immune responses which might be present against human adenovirus vectors. This also brings advantages in terms of ease of manufacture.
The inventors thoughtfully selected the fHbp (including mutant S223R fHbp) as antigen. This represents creative thinking and contributes to supporting inventive step. This is because conventional candidate antigens from MenB such as PorA and FetA might naturally be selected first by a skilled worker. However, the inventors disclose that although use of these popular PorA/FetA antigens appears to induce an antibody response when delivered to a mammalian subject using a viral vector, it appears to induce no functional immune response. In this regard we refer to the Examples section where data is presented in support of this.
It is an advantage of the invention that both T-cell responses and antibody responses are induced. This leads to a more robust/more effective immune response.
The fHbp S223R mutation reduces/eliminates binding of fHbp to human factor H (fH). fH is a complement inhibitor, and binding of fHbp to fH is a very high affinity interaction which can lead to dampening of the immune response and/or to a possible risk of raising an immune response against fH itself. Therefore, using the S223R mutant fHbp provides several advantages.
The inventors assert that choosing an adenovirus based vector to deliver the antigen is advantageous for the reason of already triggering an innate immune response against the viral vector, thereby facilitating the directing of that immune response against the fHbp antigen introduced by the inventors. Without wishing to be bound by theory, it is thought that this combination provides a "perfect mix" to trigger a strong and useful immune response. In addition, it is believed that the antigen may be retained in the cell longer/ degraded more slowly when delivered in this manner, which assists in generating and/ or maintaining the immune response.
It should be noted that using a viral vector to deliver a bacterial protein is very different to the natural presentation of the protein on bacteria and/ or very different to the natural presentation of the protein by antigen presenting cells (APCs) in vivo. Essentially delivering the bacterial antigen using a viral vector in this manner makes the bacterial protein "look like" it is expressed by a virus. This is a completely different approach to prior art attempts to induce immune response against MenB antigens. This cryptic approach is an intellectual contribution made by the inventors, and demonstrates inventive step over the known art.
A further advantage of this ingenious approach is that it retains the conformation of fHbp. This is especially surprising since this retention of conformation did not occur when attempting to deliver MenB antigens such as PorA/FetA using a viral vector approach (see Examples). Therefore, this advantageous technical benefit arises from the inventors' choice of combination of a viral vector with the fHbp antigen.
It is an advantage of the invention that the inventors demonstrate robust T-cell responses directed against the fHbp antigen when delivered using a viral vector as taught herein.
ADENOVIRUS-BASED VIRAL VECTORS
Adenoviruses are attractive vectors for human vaccination. They possess a stable genome so that inserts of foreign genes are not deleted and they can infect large numbers of cells without any evidence of insertional mutagenesis.
Replication defective adenovirus can be engineered by deletion of genes from the El locus, which is required for viral replication, and these viruses can be propagated easily with good yields in cell lines expressing El from AdHu5 such as human embryonic kidney cells 293 (HEK 293 cells).
Previous mass vaccination campaigns in over 2 million adult US military personnel using orally administered live human adenovirus serotype 4 and 7 have shown good safety and efficacy data. Human adenoviruses are under development as vectors for malaria, HIV and hepatitis C vaccines, amongst others. They have been used extensively in human trials with excellent safety profile mainly as vectors for HIV vaccines.
A limiting factor to widespread use of human adenovirus as vaccine vectors has been the level of anti-vector immunity present in humans where adenovirus is a ubiquitous infection. The prevalence of immunity to human adenoviruses prompted the consideration of simian adenoviruses as vectors, as they exhibit hexon structures homologous to human adenoviruses. Simian adenoviruses are not known to cause pathological illness in humans and the prevalence of antibodies to chimpanzee origin adenoviruses is less than 5% in humans residing in the US. Any suitable adeno-based viral vector may be used.
The adenovirus may comprise a simian or human adenovirus. The adenovirus may comprise a Group E adenovirus. The adenovirus may comprise ChAd63 or ChAd3 or ChAdOx1 or ChAd0x2 or a gorilla-derived adenovirus. The adenovirus may comprise ChAdOx1. The adenovirus may comprise a group A, B, C , D or E adenovirus. The adenovirus may comprise Ad35, Ad5, Ad6, Ad26, or Ad28. The adenovirus may be of simian (e.g. chimpanzee, gorilla or bonobo) origin. The adenovirus may comprise any of ChAd63, ChAdOx1, ChAd0x2, 15 C6, C7, C9, PanAds, or ChAd3.
In one embodiment the adenovirus may be a human adenovirus, for example Ad 5 - (see "Adenoviruses as vaccine vectors" Nia Tatsis & Hildegund C.J. Ertl (2004) Molecular Therapy https://www.sciencedirect.com/science/article/pii/S1525001604013425 ), Ad35 - (see Vogels R., et al. 2003. "Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity." J. Virol. 77:8263-8271 https://jvi.asm.org/content/77/15/8263), Ad6 - (see "Characterization of Species C Human Adenovirus Serotype 6 (Ad6)", Eric A. Weaver et al (2011), Virology https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056908/ ), Ad26 - (see "Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D", Abbink et al (2007), J. Virology https://pubmed.ncbi.nlm.nih.gov/1732934o/ ), or Ad28 - (see "Potent immune responses and in vitro pro-inflammatory cytokine suppression by a novel adenovirus vaccine vector based on rare human serotype 28", Kahl et al (2010) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2927224/).
In more detail, any replication-deficient viral vector, for human use preferably derived from a non-human adenovirus may be used. For veterinary use Ad5 may be used.
More suitably the adenovirus may comprise a non-human adenovirus such as a chimpanzee adenovirus. In one embodiment the adenovirus may comprise a chimpanzee adenovirus such as disclosed in US8216834B2 'Chimpanzee adenovirus vaccine carriers' which provides details of numerous chimp adenovirus vectors, including ChAd3 & ChAd63, and is incorporated herein by reference specifically for the teachings of chimpanzee adenoviruses. ChAd0x2 is an example of a suitable non-human adenovirus vector for human use.
Most suitably the adeno-based viral vector is ChAdOx1.
ChAdOx1
ChAdOx1 is a replication-deficient simian adenoviral vector. Vaccine manufacturing maybe achieved at small or large scale. Pre-existing antibodies to the vector in humans are very low, and the vaccines induce strong antibody and T cell responses after a single dose, whilst the lack of replication after immunisation results in an excellent safety profile in subjects of all ages.
ChAdOx1 is described in Dicks MDJ, Spencer AJ, Edwards NJ, Wadell G, Bojang K, et al. (2012) A Novel Chimpanzee Adenovirus Vector with Low Human Seroprevalence: Improved Systems for Vector Derivation and Comparative Immunogenicity. PLoS ONE 7(7): 040385, and in WO2012/172277. Both these documents are hereby incorporated herein by reference, in particular for the specific teachings of the ChAdOx1 vector, including its construction and manufacture.
For insertion of the nucleotide sequence encoding the antigen, suitably the El site may be used, suitably with the hCMV IE promoter. Suitably the short or the long version maybe used; most suitably the long version as described in WO2008/122811, which is specifically incorporated herein by reference for the teaching of the promoters, particularly the long promoter.
It is also possible to insert antigens at the E3 site, or close to the inverted terminal repeat sequences, if desired.
In addition, a clone of ChAdOx1 containing GFP is deposited with the ECACC: a sample of E. coli strain SW1029 (a derivative of DH10B) containing bacterial artificial chromosomes (BACs) containing the cloned genome of AdChOXi (pBACe3.6 AdChOx1 (E4 modified) TIPeGFP, cell line name "AdChOx1 (E4 modified) TIPeGFP") was deposited by Isis Innovation Limited on 24 May 2012 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 oJG, United Kingdom under the Budapest Treaty and designated by provisional accession no. 12052403. Isis Innovation Limited is the former name of the proprietor/applicant of this patent/ application.
ChAd0x2 The nucleotide sequence of the ChAd0x2 vector (with a Gateway™ cassette in the El locus) is shown in SEQ ID NO. 2 This is a viral vector based on Chimpanzee adenovirus C68. (This is the sequence of SEQ ID NO: 10 in gb patent application number 1610967.0).
In addition, a clone of ChAd0x2 containing GFP is deposited with the ECACC: deposit accession number 16061301 was deposited by Isis Innovation Limited on 13 June 2016 with the European Collection of Cell Cultures (ECACC) at the Health Protection Agency Culture Collections, Health Protection Agency, Porton Down, Salisbury SP4 oJG, United Kingdom under the Budapest Treaty. Isis Innovation Limited is the former name of the proprietor/applicant of this patent/application.
ChAd63
In one embodiment a related vaccine vector, ChAd63, maybe used if desired.
SIGNAL SEQUENCES
Suitably the polypeptide antigen of the invention comprises a signal sequence. Suitably said signal sequence is a secretion sequence.
The inventors have discovered that inclusion of signal sequences into fusion proteins with the antigen of interest can enhance performance. In the prior art, use of mammalian signal sequences such as the tPA (tissue plasminogen activator) signal sequence is known. However, in the prior art the inventors believe that it has never been disclosed to use bacterial signal sequences in antigens intended for inducing an immune response in a mammal, in particular the inventors believe that it has never been disclosed to use bacterial signal sequences in addition to mammalian signal sequences such as the tPA signal sequence in antigens intended for inducing an immune response in a mammal. Thus, in a broad aspect, the invention relates to the use of a bacterial signal sequence fused to an antigen, in particular use of a bacterial signal sequence in addition to a mammalian signal sequence, fused to an antigen for use in inducing an immune response in a mammal.
Moreover, the inventors have discovered that creating a fusion protein comprising two signal sequences is particularly advantageous.
Moreover, the inventors have discovered that creating a fusion protein comprising two heterologous signal sequences fused to the antigen of interest is especially advantageous. Moreover, the inventors have realised that fusing a bacterial signal sequence and a mammalian signal sequence and the antigen of interest is especially advantageous.
Without wishing to be bound by theory, it is believed that the use of heterologous signal sequences in this manner decreases degradation of the fusion protein. Moreover, this new direction is especially cryptic since it teaches directly against the view in the art which is that bacterial signal sequences should be removed from bacterial antigens before they are introduced to mammals. In contrast, the inventors teach directly against this prior art view by specifically including the bacterial signal sequence (despite the destination being a mammalian environment) and find that this surprisingly generates better results.
Thus, the inventors teach a generally applicable principle of use of bacterial signal sequence to promote an effective immune response in a mammalian environment. In particular, the inventors teach that the inclusion of dual signal sequences (e.g. one mammalian signal sequence and one bacterial signal sequence) on a single protein with the antigen of interest is especially effective.
Data provided in the Examples section demonstrate that constructs according to the invention perform better than licensed vaccines in the same disease area. This is true for both the magnitude of the response, and the duration of the response for diseases such as MenB.
There is no incentive in the prior art for anybody to produce a viral vector comprising a bacterial antigen and a signal sequence. The inventors' approach is contrary to the view in the art. In particular, a skilled person incorporating antigen into any commercially available viral vector is likely already to have the mammalian signal sequence such as the tPA signal sequence present in the vector and so any further signal sequence is simply not required, or is potentially counter-productive when the resulting polypeptide progresses into the eukaryotic translation machinery. The view in the art is that simply using the mammalian signal sequence is sufficient.
Without wishing to be bound by theory, it is believed that the bacterial signal sequence may provide an adjuvant effect, and/or may provide a "danger" or enhancing signal to the immune system, thereby provoking a more effective response. In one aspect of the invention it is taught to provide the antigen with dual signal sequences i.e. with two signal sequences.
In one embodiment, if the antigen does not have a naturally occurring signal sequence, then a bacterial signal sequence can be added to the antigen and the second signal sequence can be provided as a heterologous signal sequence in the same fusion protein.
In another embodiment, when the antigen has a naturally occurring bacterial signal sequence, this naturally occurring bacterial signal sequence can be deleted and replaced with a superior bacterial signal sequence as taught by the inventors. An example of a superior bacterial signal sequence to be substituted into the antigen in this manner includes signal sequence such as:
SEQ ID NO. 11:
MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGSKLNRTAFCCLSLTTALILTA
Whether the bacterial signal sequence is by addition or by substitution as explained above, suitably the construct comprises a second signal sequence in the form of a mammalian signal sequence such a tPA signal sequence.
There are mature and immature forms of signal sequences. Suitably the immature form of the signal sequence is retained, or inserted, or substituted into (or fused to) the antigen.
Most suitably when two signal sequences are present in the same fusion protein as the antigen of interest, one of those signal sequences is tPA.
Exemplary Signal Sequences fHbp signal sequence
Suitably SEQ ID NO: 4 (fHbp signal sequence): NRTAFCCLSLTTALILTA is fused to the protein antigen of the invention
Suitably this is located after the tPA and before the C that gets lipidated when in the mature form of the fHbp in bacteria. tPA tPA (tissue plasminogen activator) - more specifically the tPA leader sequence - is suitably fused to the protein antigen of the invention. Suitably tPA is fused to the N- terminus of the protein antigen sequence. Suitably tPA leader sequence means the tPA amino acid sequence of SEQ ID NO: 5
SEQ ID NO: 5
MDAMKRGLCCVLLLCGAVFVSASQEIHARFRR
In the above SEQ ID NO: 5 the C terminal 'RR' is not actually part of the tPA leader sequence. It comes from the fusion of two restriction sites. Suitably the tPA leader sequence may be used with or without the C terminal 'RR' e.g. SEQ ID NO: 7 or SEQ ID NO: 8. Most suitably the sequence is used as shown in SEQ ID NO: 5.
The underlined A is P in the naturally occurring tPA leader sequence. The P->A mutation has the advantage of improved antigen secretion.
Suitably the tPA leader sequence may be used with or without the P->A mutation, i.e. suitably the tPA leader sequence maybe used as SEQ ID NO: 5 or SEQ ID NO: 6.
SEQ ID NO: 6
MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRR
SEQ ID NO: 7 (=SEQ ID NO: 5 without C-terminal 'RR') MDAMKRGLCCVLLLCGAVFVSASQEIHARF
SEQ ID NO: 8 (=SEQ ID NO: 6 without C-terminal 'RR') MDAMKRGLCCVLLLCGAVFVSPSQEIHARF
More suitably the sequence is used with the P->A mutation (with or without the C terminal 'RR'). Most suitably the sequence is used as shown in SEQ ID NO: 5.
These signal sequences maybe used with or without the lead methionine. For example if the amino acid sequence is introduced internally in making a fusion protein, it would be usual to remove the lead methionine (so that no internal methionine is introduced into the fusion protein). For example the tPA signal sequence of SEQ ID NO: 5 without the lead methionine is presented as SEQ ID NO: 24.
Alternate tPA signal sequences are presented below in SEQ ID NO:s 18 to 21.
An exemplary nucleotide sequence encoding tPA, which has been codon optimised for human codon usage, is as shown in SEQ ID NO: 9 (this is the sequence encoding SEQ ID NO: 5):
ATGGACGCCATGAAGAGGGGCCTGTGCTGCGTGCTGCTGCTGTGTGGCGCCGTGTTT GTGTCCGCCAGCCAGGAAATCCACGCCCGGTTCAGACGG It is believed that tPA promotes secretion of proteins to which it is fused. It is believed that tPA increases expression of proteins to which it is fused. Notwithstanding the underlying mechanism, the advantage in the invention of fusing tPA to the N-terminus of the protein antigen is that improved immunogenicity is achieved. Thus, most suitably the antigen of the invention is provided as a fusion with tPA. Most suitably the tPA is fused to the N-terminus of the protein antigen.
Suitably the antigen does not comprise any further sequence tags. Suitably the antigen does not comprise any further linker sequences.
Alternate Signal Sequences
In addition to, or instead of, the exemplary signal sequences disclosed above, it is possible to use one of more of the following:
Figure imgf000017_0001
Figure imgf000017_0002
Dual Signal Sequence
In a preferred embodiment, two signal sequences are included in the polypeptide comprising the antigen such as fHbp antigen. Most suitably those two signal sequences comprise the tPA signal sequence and the fHbp signal sequence. When two signal sequences are fused in this manner, the skilled operator may introduce some 'spacer' or 'linker' amino acids in between the first and second signal sequences. For example, a four amino acid spacer having the sequence maybe used having the sequence:
SEQ ID NO: 16: GSKL
In this embodiment suitably the resulting dual signal sequence fusion comprises (spacer of SEQ ID NO: 16 underlined):
SEQ ID NO: 17:
MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGSKLNRTAFCCLSLTTALILTA
Order of Elements
The signal sequences maybe arranged in order from the N-terminus of the protein to the C-terminus of the protein as follows:
• N-terminus - mammalian signal sequence - bacterial signal sequence - antigen
- C-terminus; or
• N-terminus - bacterial signal sequence - mammalian signal sequence - antigen
- C-terminus.
Most suitably, the order of elements is:
• N-terminus - mammalian signal sequence - bacterial signal sequence - antigen
- C-terminus.
In one example, the elements may be N-terminus - tPA signal sequence - fHbp signal sequence - antigen - C-terminus.
The same comments on order of elements apply to nucleic acids of the invention when the nucleotide sequences encoding the polypeptides as noted above are arranged in order from 5' to 3' on a single contiguous nucleic acid.
When the antigen is a bacterial antigen and possesses an existing bacterial signal sequence, it may be that the existing naturally occurring bacterial signal sequence of that antigen performs well in the construct of the invention, particularly when the mammalian signal sequence is added. However in one embodiment enhanced performance maybe achieved by replacing the naturally occurring bacterial signal sequence with the bacterial signal sequence of fHbp, and then optionally adding the second signal sequence in the form of the mammalian signal sequence to prepare the overall construct.
It should be pointed out that the idea of using a second signal sequence in these constructs is in itself contrary to what is taught in the art. In the art provision of a single signal sequence has been shown to be effective. Thus there is no motivation for a skilled worker to go to the additional labour and complication of adding a further signal sequence when the art teaches that possession of a single signal sequence is already sufficient and effective. It is therefore a surprising approach contrary to what is taught in the art to expend the extra effort and cost in provision of second signal sequences as taught herein.
Suitably the antigen is capable of inducing an immune response in a human.
ANTIGEN
The antigen may be from any source. Suitably the antigen is from a pathogen of humans, or may be a tumour antigen. More suitably the antigen is a bacterial antigen or a viral antigen. Most suitably the antigen is a bacterial antigen.
Suitably the antigen maybe from Meningococcus such as from Meningococcus B ("Men B").
Suitably the antigen comprises Factor H Binding Protein (fHbp). Suitably the antigen consists essentially of Factor H Binding Protein (fHbp). Suitably the antigen consists of Factor H Binding Protein (fHbp).
Suitably the Factor H Binding Protein (fHbp) is from Neisseria m eningitidis.
More suitably the Factor H Binding Protein (fHbp) is from from Neisseria m eningitidis group B.
It should be noted that in prior art discussions of fHbp, the fHbp signal sequence is already known. However, the view in the art is that the fHbp signal sequence should be removed. In contrast, the inventors teach to use the fHbp signal sequence.
Suitably the antigen is a bacterial antigen. Suitably the bacterial antigen is a gram- negative bacterial antigen. Suitably the antigen is a surface exposed antigen.
It is an advantage of the invention that the antigen protein can be varied.
It is an advantage of the invention that the signal sequence such as bacterial signal sequence can be varied.
SEQUENCE IDENTITY / SEQUENCE VARIATION
Sequence comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate percent homology (such as percent identity) between two or more sequences. A suitable computer program for carrying out such analysis is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Altschul et al., 1990, J. Mol. Biol. 215:403-410) and the GENEWORKS suite of comparison tools.
Suitably any heterologous signal sequence /signal peptide insertions or fusions are excluded from percent identity calculations.
Suitably sequence identity is judged against the full length of the reference sequence. In one embodiment suitably sequence identity is judged against the full length of SEQ ID NO: 1 or SEQ ID NO: 2.
Suitably 'from' means that the amino acid sequence discussed comprises amino acid sequence derived from the reference sequence or organism/ genome as specified.
In all discussions of sequence identity, it will be noted that the reference sequences may not be 100 amino acids in length. Therefore each single substitution is equivalent to more than 1% or less than 1% change in identity depending on the length of the reference sequence when all amino acids of the reference sequence are considered. The values are given to nearest whole percentage point and should be understood accordingly given that it is not possible to substitute partial amino acids within a polypeptide sequence. Unless otherwise apparent from the context, the same sequence identity levels as noted above for amino acid sequences also apply to nucleotide sequences herein.
Reference Sequence
Suitably sequences herein are discussed with reference to specified sequence(s) provided. For example fHbp is discussed with reference to wild-type fHbp having the sequence of SEQ ID NO: 1.
When particular amino acid residues are referred to herein using numeric addresses, the numbering is taken with reference to the wild type fHbp amino acid sequence (or to the polynucleotide sequence encoding same if referring to nucleic acid).
This is to be used as is well understood in the art to locate the residue of interest. This is not always a strict counting exercise - attention must be paid to the context. For example, if the protein of interest is of a slightly different length, then location of the correct residue in that sequence may require the sequences to be aligned and the equivalent or corresponding residue picked. This is well within the ambit of the skilled reader.
Suitably the current version of sequence database(s) are relied upon. Alternatively, the release in force at the date of filing is relied upon. For the avoidance of doubt, UniProt release 2020_04 is relied upon. In more detail, the UniProt consortium European Bioinformatics Institute (EBI), SIB Swiss Institute of Bioinformatics and Protein Information Resource (PIR)'s UniProt Knowledgebase (UniProtKB) Release 2020_04 published 12 August 2020 is relied upon. UniProt (Universal Protein Resource) is a comprehensive catalogue of information on proteins ("UniProt: the universal protein knowledgebase" Nucleic Acids Res. 45: D158-D169 (2017)).
GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences (National Center for Biotechnology Information, U.S.
National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA; Nucleic Acids Research, 2013 Jan;4i(Di):D36-42) and accession numbers provided relate to this unless otherwise apparent. Suitably the GenBank database release referred to is 15 June 2020, NCBI-GenBank Release 238.
Mutating has it normal meaning in the art and may refer to the substitution or truncation or deletion of one or more residues, motifs or domains. Mutation may be effected at the polypeptide level, for example, by synthesis of a polypeptide having the mutated sequence, or maybe effected at the nucleotide level, for example, by making a polynucleotide encoding the mutated sequence, which polynucleotide may be subsequently translated to produce the mutated polypeptide. Suitably, the mutations to be used are as set out herein. Unless otherwise apparent from the context, mutations mentioned herein are substitutions. For example 'S223R' means that the residue corresponding to 'S223' in the wild type fHbp (SEQ ID NO: 1) is substituted with R.
Suitably the antigen sequence is, or is derived from, amino acid sequence provided herein, such as SEQ ID NO. 1. A degree of sequence variation maybe tolerated. Suitably the antigen sequence used in the vector of the invention comprises amino acid sequence having at least 80%, suitably at least 85%, suitably at least 90%, suitably at least 92%, suitably at least 94%, suitably at least 96%, suitably at least 98%, most suitably 99% sequence identity to the reference amino acid sequence, for example the reference amino acid sequence provided as SEQ ID NO. 1.
The fHbp sequence may have a S223R mutation relative to the wild type fHbp of SEQ ID NO: 1. This is advantageous in reducing or eliminating binding of fHbp to Factor H. The amino acid sequence of fHbp with the S223R mutation is provided in SEQ ID NO: 2. Suitably the antigen sequence is, or is derived from, amino acid sequence provided herein, such as SEQ ID NO. 2. A degree of sequence variation may be tolerated. Suitably the antigen sequence used in the vector of the invention comprises amino acid sequence having at least 80%, suitably at least 85%, suitably at least 90%, suitably at least 92%, suitably at least 94%, suitably at least 96%, suitably at least 98%, most suitably 99% sequence identity to the reference amino acid sequence, for example the reference amino acid sequence provided as SEQ ID NO. 2.
SEQ ID NO: 1 has S at amino acid 223 of SEQ ID NO: 1. This is the wild type amino acid at position 223 of fHbp.
SEQ ID NO: 2 has R at the position corresponding to amino acid 223 of SEQ ID NO: 1. Suitably the antigen sequence used has R at the position corresponding to amino acid 223 of SEQ ID NO: 1.
Further fHbp Variants
Variants of factor H binding protein (fHbp) may be used as antigen. One composition of the invention may comprise more than one fHbp variant. This helps to increase coverage i.e. helps to raise an immune response against different MenB variants which the subject might encounter.
There are three groups of fHBps (group 1, 2 & 3) and variants exist within each of these (e.g. 1.1 as in the exemplary adenovirus based vector construct of the invention). In some applications, stabilising mutations may advantageously be made in the expressed antigen (and/or in the nucleotide sequence encoding it) to improve transcript stability when expressed from the adenovirus based vector. fHbp variants are described in W02016/014719A1 (Children's Hospital & Research Center Oakland) which is incorporated herein by reference for the teachings of fHbp variants. For example a variant of fHbp wherein the variant comprises an amino acid substitution selected from at least one of: a) an amino acid substitution of the glutamine at amino acid 38 (Q38); b) an amino acid substitution of the glutamic acid at amino acid 92 (E92); c) a substitution of glycine for arginine at amino acid 130 (R130G); d) an amino acid substitution of the serine at amino acid 223 (S223); and e) a substitution of histidine for leucine at amino acid 248 (H248L), wherein the amino acid substitutions are relative to wild type fHbp (SEQ ID NO: 1), wherein the variant comprises an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO:1.
Suitably the variant fHbp binds human factor H (fH) with an affinity that is 50% or less of the affinity of wild type fHbp (SEQ ID NO: 1) for human fH.
Suitably the variant induces a functional antibody response (such as a bactericidal antibody response) to at least one strain of Neisseria m eningitidis in a mammalian host.
In one embodiment, the amino acid substitution at Q38 is Q38R, Q38K, Q38H, Q38F, Q38Y, or Q38W. In one embodiment, the amino acid substitution at E92 is E92K, E92R, E92H, E92F, E92Y, or E92W. In one embodiment, the amino acid substitution at S223 is S223R, S223K, S223H, S223F, S223Y, or S223W. In one embodiment, the variant fHbp may further include a R41S or a R41A substitution relative to SEQ ID NO: 1. For example the variant fHbp may include a R41S or a R41A substitution and a substitution at S223, e.g., R41S/S223R, relative to SEQ ID NO: 1. In other cases, the variant fHbp may further include a R41S or a R41A substitution and a H248L substitution relative to SEQ ID NO: 1. In certain cases, the variant fHbp may include two, three, or more of the substitutions disclosed herein. In a specific example, the variant fHbp may include the following substitutions: S223R and H248L relative to SEQ ID NO: 1.
Suitably the full length fHbp protein is used.
Suitably 'full length' means each amino acid in the fHbp protein is included.
Suitably full length means an fHbp protein having a length (i.e. total number of amino acids) corresponding to that of SEQ ID NO: 1 or SEQ ID NO: 2. By choosing the full length fHbp protein, advantageously the correct confirmation of the protein in assured. Truncated proteins can assume unnatural conformations. This drawback is avoided by using the full length protein.
A further advantage of using the full length fHbp protein is that it allows for better T- cell responses. Without wishing to be bound by theory, it is believed that the more amino acid sequences present, then the more potential targets there are for the T-cell responses. Thus, suitably every amino acid of the fHbp protein is included in the antigen of the invention.
The sequence identity level of 99% compared to SEQ ID NO. 1 or SEQ ID NO. 2 (having 255 amino acids) corresponds to approximately 2 to 3 substitutions across the full length of the fHbp antigen amino acid sequence provided as SEQ ID NO. 1 or SEQ ID NO. 2. Suitably the antigen construct used has 3 or fewer substitutions, suitably 2 or fewer substitutions, suitably one substitution relative to SEQ ID NO: 1 or SEQ ID NO. 2.
Suitably counting of substitutions does not include addition of signal sequence such as addition of tPA signal sequence sequence.
IMMUNISATION/ADMINISTRATION
The invention provides polypeptides, nucleic acids encoding said polypeptides, and/or vectors carrying said nucleic acids, for use in immunising a subject against a disease. The invention provides polypeptides, nucleic acids encoding said polypeptides, and/or vectors carrying said nucleic acids, for use in inducing an immune response against Neisseria m eningitidis, more suitably against Neisseria m eningitidis group B.
In another aspect, the invention relates to use of a composition as described above in inducing an immune response against Neisseria m eningitidis, suitably Neisseria m eningitidis group B. In another aspect, the invention relates to use of a composition as described above in immunising a subject against Neisseria m eningitidis, suitably Neisseria m eningitidis group B.
In one embodiment suitably the disease is Meningococcus infection.
In one embodiment suitably the disease is infection by meningococcal group B bacteria. In one embodiment suitably the disease is infection \jy N eisseria m eningitidis, more suitably infection by Ne isseria m eningitidis group B.
In one embodiment suitably the disease is Meningitis.
In one embodiment suitably the disease is Meningitis B. Suitably the subject is a human.
Suitably the method is a method of immunising.
Suitably the immune response comprises a humoral response. Suitably the immune response comprises an antibody response. Suitably the immune response comprises a functional antibody response. Most suitably the immune response comprises a bactericidal antibody response.
Suitably the immune response comprises a cell mediated response. Suitably the immune response comprises cell mediated immunity (CMI). Suitably the immune response comprises induction of CD8+ T cells. Suitably the immune response comprises induction of a CD8+ cytotoxic T cell (CTL) response.
Suitably the immune response comprises both a humoral response and a cell mediated response.
Suitably the immune response comprises protective immunity.
Suitably the composition is an antigenic composition.
Suitably the composition is an immunogenic composition.
Suitably the composition is a vaccine composition.
Suitably the composition is a pharmaceutical composition.
Suitably the composition is formulated for administration to mammals, suitably to primates, most suitably to humans.
PROMOTERS
In principle the nucleotide sequence encoding the antigen such as fHbp may be placed under the control of any suitable promoter such as a promoter capable of directing expression of the antigen in a mammalian cell following transduction by the viral vector.
Suitable promoters include the hCMV IE promoter. Suitably the short or the long version maybe used; most suitably the long version as described in WO2008/122811, which is specifically incorporated herein by reference for the teaching of the promoters, particularly the long promoter.
CODON OPTIMISATION
Suitably the nucleic acid encoding the antigen, and/or encoding the signal sequenceantigen fusion protein, is codon optimised for mammals, most suitably codon optimised for humans. SINGLE DOSE
The compositions and methods provided are extremely advantageous in that they achieve induction of immune responses in a subject with only a single dose of composition ('vaccine'). This is simpler and easier. This saves costs. This saves labour. This improves compliance (i.e. subjects do not have to attend for a second or further administration).
Thus suitably the invention relates to a single dose of composition as described above. Thus suitably the invention relates to a single administration of composition as described above.
PRIME-BOOST
The invention also finds application in prime-boost immunisation regimes. For example, if after a period of time the immune response declines, as naturally tends to happen for many immune responses, then it maybe desired to boost the response in a patient back to useful levels such as protective levels.
Boosting maybe homologous boosting i.e. maybe attained using a second administration of the same composition as used for the original priming immunisation. In a preferred embodiment a homologous ChAdOx1 / ChAdOx1 prime/boost regime may be used.
In another embodiment, the boosting immunisation may be carried out using a different composition to the composition used for the original priming immunisation. This is referred to as heterologous prime boost. Suitably the heterologous boost (i.e. the second or further immunisation) comprises one or more compositions selected from MVA, RNA, DNA, protein, adenovirus based viral vector, simian adenovirus based viral vector, gorilla-based adenovirus based viral vector, or human adenovirus based viral vector. More suitably the boosting (second or further) immunisation may comprise MVA, RNA or protein. Most suitably, the boost (second or further immunisation) may comprise RNA or protein.
In a preferred embodiment the invention relates to a heterologous prime boost regime comprising a (protein +/- outer membrane vesicle) prime using either one of the licence vaccine(s) and a boost with the Adenovirus based vector described herein, such as ChAdOx1 MenB.1 (sometimes called MenBOx1).
In a highly preferred embodiment the invention relates to a heterologous prime boost regime comprising (protein +/- outer membrane vesicle) prime using either one of the licenced vaccines i.e. Bexsero® from GlaxoSmithKline (GSK) or Trumenba® from Pfizer, and a boost with the Adenovirus based vector described herein, such as ChAdOx1 MenB.1 (sometimes called MenBOx1). In other words in a most highly preferred embodiment the invention relates to a heterologous prime boost regime comprising a prime using either one of the licenced vaccines i.e. Bexsero® from GlaxoSmithKline (GSK) or Trumenba® from Pfizer, and a boost with the Adenovirus based vector described herein, such as ChAdOx1 MenB.1 (sometimes called MenBOx1).
In another embodiment the invention relates to a method of inducing an immune response against Neisseria m eningitidis in a mammalian subject, the method comprising
(i) administering a composition comprising (protein +/- outer membrane vesicle), suitably Bexsero® from GlaxoSmithKline (GSK) or Trumenba® from Pfizer, to said subject; and
(ii) administering a composition comprising adenovirus based vector as described herein, suitably ChAdOx1 MenB.1, to said subject.
Suitably administration of (ii) is subsequent to administration of (i).
More suitably administration of (ii) is carried out approximately 6 months after administration of (i).
In another embodiment the invention relates to a kit comprising: a) a dose of a composition comprising (protein +/- outer membrane vesicle), suitably Bexsero® from GlaxoSmithKline (GSK) or Trumenba® from Pfizer; and b) a dose of a composition comprising adenovirus based vector as described herein, suitably ChAdOx1 MenB.1; and c) instructions for administration to a mammalian subject.
Advantages of boosting regimes (i.e. involving a second or further administration/immunisation) include raising the level of immune response in the subject, and/or increasing the duration of the immune response.
If a two dose regimen is required, e.g. for particular applications such as sustained immunity (e.g. in healthcare workers), ChAdOx1/MVA or ChAdOx1/RNA or ChAdOx1/ protein as prime/boost regimes maybe used.
In a preferred embodiment a homologous ChAdOx1 / ChAdOx1 prime/boost regime may be used.
Typical modified RNA or Self-amplifying mRNA vaccination regimen Two doses of vaccine administered, typically 4-8 weeks between each dose
Typical protein vaccination regimen
Two or three doses of vaccine administered, typically 4-8 weeks between each dose and adjuvant must also be administered at immunisation
Advantageous viral vector vaccination regimen according to the invention: One dose of vaccine administered.
In boost embodiments suitably the first administration comprises, or consists of, a composition according to the present invention comprising a viral vector capable of expressing Men B fHbp.
Suitably the second or further ('boost') administration comprises exactly the same antigen as for viral vector.
Suitably the second or further ('boost') administration comprises an RNA vaccine.
Suitably the second or further ('boost') administration comprises a self amplifying RNA vaccine.
Suitably the second or further ('boost') administration comprises IM administration.
Suitably when the second or further ('boost') administration comprises adjuvant, said adjuvant is selected by the operator depending on platform. When the second or further ('boost') administration comprises saRNA no adjuvant is needed.
Suitably when the second or further ('boost') administration comprises RNA, the dose is suitably in the range of 0.001 to 1 microgrammes.
Suitably when the second or further ('boost') administration comprises protein, the dose is suitably in the range of 1 to 15 microgrammes.
Suitably a boost composition may be administered about 6 months after administration of the original priming immunisation.
Suitably boosting is homologous i.e. using the same composition as used in the first or previous administration (e.g. priming administration). Thus in one aspect the invention provides a method of inducing an immune response against Neisseria m eningitidis in a mammalian subject, the method comprising
(i) administering a composition comprising a viral vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding the fHbp protein from Neisseria m eningitidis, characterised in that said viral vector is an adenovirus based vector to said subject, and
(ii) administering a composition comprising a viral vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding the fHbp protein from Neisseria meningitidis, characterised in that said viral vector is an adenovirus based vector to said subject.
Suitably step (i) is a priming composition.
Suitably step (ii) is a boosting composition.
Suitably step (ii) is carried out 6 months weeks after the step (i).
ADMINISTRATION ROUTE
In principle any suitable route of administration may be used.
Disclosures of administration/formulation/dose etc apply equally to prime-only ('single shot' or single administration embodiments) as to prime-boost ('boosting' or dual administration embodiments).
Suitably the route of administration is selected from group consisting of subcutaneous, intranasal, aerosol, sublingual, nebuliser, intradermal and intramuscular.
Suitably the route of administration is selected from a group consisting of intradermal and intramuscular.
Most suitably the route of administration is intramuscular.
The route of administration may be applied to humans and/ or other mammals.
DOSE
It should be noted that there are alternate ways of describing the dose for adenoviral vectors.
• Viral particles - vp/mL. This refers to the count of total viral particles administered.
• Infectious units - i.u./mL. This refers to the number of infectious units administered, and can be correlated more accurately with immunogenicity.
By convention, clinical trials in the UK tend to provide the dose in terms of viral particles.
Preferred doses according to the present invention are:
For adult humans, in one embodiment the range is from io9 to io11 viral particles.
For adult humans, in one embodiment the range is from 2.5X io10 vp to 5x io10 vp. This assumes an adult human of average weight 70 Kg (adult human average weights vary by region e.g. average for Africa = 60 Kg, average for Europe = 70 kg, average for North America = 80 Kg.) Unless otherwise apparent, doses apply to an average adult human of 70 Kg weight. Thus in one embodiment adults of different weights receive the same dose, i.e. there is no adjustment per kg. Doses may be determined by a physician using the guidance provided herein.
For adult humans, in one embodiment the dose(s)/range of dose(s) maybe derived from the examples below.
In infant humans a dose of 5x1010 vp may be used. In human children a dose of ionvp may be used.
Suitably no adjuvant is administered with the viral vector of the invention.
Suitably the viral vector of the invention is formulated with simple buffer. An exemplary buffer may be as shown below under the heading 'Formulation'.
FURTHER FEATURES AND ADVANTAGES
Suitably the nucleic acid sequence is codon optimised for mammalian codon usage, most suitably for human codon usage.
Suitably a container containing a composition as described above is provided. Suitably said container may be a vial. Suitably said container may be a syringe.
Suitably a nebuliser containing a composition as described above is provided. Suitably a nasal applicator containing a composition as described above is provided. Suitably an inhaler containing a composition as described above is provided.
Suitably a pressurised canister containing a composition as described above is provided.
A method of making a composition as described above is provided, said method comprising preparing a nucleic acid encoding the antigen (suitably the MenB fHbp protein), optionally fused to the tPA protein, and incorporating said nucleic acid into an adeno-based viral vector, suitably a ChAdOx1 vector. Suitably the nucleic acid is operably linked to a promoter suitable for inducing expression of said antigen (or antigen-tPA fusion protein) when in a mammalian cell such as a human cell. It is a surprising property of the exemplary compositions disclosed (such as ChAdOx vector directing expression of MenB fHbp) that they have the ability to induce high levels of functional antibody following a single shot (single administration/single immunisation).
FORMULATION
Suitably the composition is formulated taking into account its route of administration. Suitably the composition is formulated to be suitable for the route of administration specified. Suitably the composition is formulated to be suitable for the route of administration selected by the operator or physician.
Vaccine formulation may be liquid, suitably stable for at least 1 year at 2-8°C, or may be lyophilised, suitably stable at ambient temperatures e.g. room temperature 18-22 °C.
The ChAdOx1 formulation buffer, as used for the clinical product is:
FORMULATION BUFFER COMPONENTS
1. 10 mM Histidine
2. 7.5 % Sucrose (w/v)
3. 35 mM Sodium chloride
4. 1 mM Magnesium chloride
5. 0.1 % Polysorbate 80 (w/v)
6. o.i mM EDTA
7. 0.5% Ethanol (v/v)
8. Hydrochloric acid (for pH adjustment to ~pH 6.6)
Formulated in Water for Injection Ph Eur.
Formulations for other administration routes such as aerosol will be adjusted accordingly by the skilled operator.
Suitably the composition and/or formulation does not comprise adjuvant. Suitably adjuvant is omitted from the composition and/or formulation of the invention.
FURTHER ASPECTS
In a broad aspect the invention provides an adenoviral vector comprising a Men B antigen, preferably Men B fHbp. In a broad aspect is described a viral vector comprising an antigen for inducing an immune response in a mammal against a disease antigen, wherein said antigen is fused to a bacterial signal sequence. Suitably said antigen is further fused to a second signal sequence. Suitably said second signal sequence is a mammalian signal sequence. Suitably the mammalian signal sequence is tPA.
In one embodiment suitably said viral vector is an MVA viral vector. More suitably the viral vector is an adenoviral vector.
In one embodiment the antigen is selected from the group comprising (Fi+V) and (fHbp). More suitably the antigen is fHbp.
Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.
Where an apparatus feature is described as being operable to provide a function, it will be appreciated that this includes an apparatus feature which provides that function or which is adapted or configured to provide that function.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will now be described by way of example, with reference to the accompanying drawings, in which:
Figure 1 shows schematic diagrams representing the different types of constructs generated for PorA or FetA antigens: A For full-length constructs, full-length genes were inserted in frame after the tPA leader, and a V5 marker peptide was added at the C-terminus. B A single PorA VR loop was fused to a FliC scaffold (with or without flanking cysteines, Cys), with a V5 peptide sequence at the C-terminus. C Two PorA VR loops were inserted into the FliC scaffold with a flexible polylinker sequence, SGMPGSGPAY, between the VR regions. D A molecular adjuvant (IMX313) was added at the C-terminus.
Figure 2 shows graphs - evaluation of antibody responses against PorA (P1.7,16) induced by adenoviral vectors. Antibody responses to a single dose of 109 infectious units (IU) of Ad-vectors in Balb/C mice against rP1.7,16 overtime (A) and against 44/76-SL whole cells in Balb/C and NIH mice 2 weeks post vaccination with to8 IU (Low) or 109 IU (high) of Ad-P1.7,16 (B). Mean values with SDs are displayed. Dashed lines indicate negative cut-off values (titres obtained with sera from naive mice). Groups of 4 or 5 Balb/C or NIH mice were used for the experiments. * P < 0.05, ” P < 0.01 and **** P < 0.0001 respectively (comparison between Ad-P1.7,16 and Ad-P1.7,16- cytosolic).
Figure 3 shows graphs - comparison of antibody responses induced by Ad-F3-3 vaccines versus an 0MV vaccine. Balb/c mice received 109 IU of full length Ad-F3-3, Ad-FliC- VR3-3 or 5μg of 0MV (44/76-FetAonPorAoff). Antibody responses against recombinant rF3-3 (A) or against 44/76-FetAonPorAoff whole cells (B, pooled sera from each group) 6 weeks post vaccination are shown. Mean values with SDs for each group are displayed (A). Groups of 8 mice were used for the experiments. Kruskal Wallis test with Dunn's multiple comparison was used to perform comparisons between the groups * P < 0.05 and *** P < 0.001
Figure 4 shows a bar chart - protective efficacy of Ad and MVA vectored vaccines expressing PT Si-220 in immunised mice. Mice were immunised with one or two doses of vaccines at eight-week interval. Challenge was performed two weeks after the booster immunisation (week 10). Five mice in the control group, at day zero, were sacrificed three hours after aerosol challenge with the virulent bacteria to establish the successful infection by analysing bacterial counts in their lungs. The remaining five mice were sacrificed on day seven along other groups to determine viable counts in the mouse lungs. Results for each group are shown as individual counts and geometric mean ± 95% CI (n = 6 mice). One-way ANOVA followed by Tukey's multiple comparison test was used to compare data and determine different differences between each group. Only significant differences between the groups are indicated in the graph.
Figure 5 shows a diagram - the DNA map of pBAC ChAdOx1 MenB.1 (sometimes called MenBOx1) used to generate the recombinant viral vector vaccine.
Figure 6 shows plots and charts. Fig 6A - Expression of fHbp antigen Fig 6B - Binding of human fH binding. Expression of fHbp (6A) and binding of human fH (6B) in ix106 cells infected with recombinant adenoviruses as indicated below, by flow cytometry. Flow panels: the X axis represents fHbp expression (6A) or human fH binding (6B) on the cells. The Y axis represents the cell viability. Graphs represent the percentages of cells positive for fHbp expression (6A) or human fH binding (6B) in two negative control and five test samples replicates.
Figure 7 shows charts. Immunogenicity of ChAdOx1 MenB.1 in CD1 mice at week 2 and 6 post a single injection as compared with a HuAd5 counterpart. Each mouse is represented by a dot and the geometric mean with 95% confidence interval of the group are displayed (horizontal lines). The red horizontal dashed line represent the threshold for protection (titre of 1:4).
Figure 8 shows plots. Dose responses of ChAdOx1 MenB.1 in BALB/c and CD1 mice at week 6 post a single injection. Each mouse is represented by a dot, the geometric means with 95% confidence interval are displayed (horizontal lines). The red horizontal dashed line represents the threshold for protection (titre of 1:4).
Figure 9 shows plots. Dose responses of ChAdOx1 MenB.1 in 3 mouse strains at week 6 post a single injection. Each mouse is represented by a dot, the geometric means with 95% confidence interval are displayed (horizontal lines). The red horizontal dashed line represents the threshold for protection (titre of 1:4).
Figure 10 shows plots. Immunogenicity of ChAdOx1 MenB.1 (shortened MenBOx1, dark blue in the figure legend) in human fH transgenic mice at several time points (indicated below the X axis) post a single adenovirus injection, compared to mice immunized once, two or three times with 4CMenB (in dark red). Each mouse is represented by a dot and the geometric means with 95% confidence interval of the groups are displayed (horizontal lines). The red horizontal dashed line represent the threshold for protection (titre of 1:4). Statistical comparison of the SBA titers was performed by AN0VA at each time point, * p<0.05, ** p<o.oi, ”* p<o.ooi and **** p<o,oooi.
Figure 11 shows plots. Immunogenicity of the clinical lot ChAdOx1 MenB.1 in CD1 outbred mice. Each mouse is represented by a dot and the geometric means with 95% confidence interval of the groups are displayed (horizontal lines). The red horizontal dashed line represent the threshold for protection (titre of 1:4).
Figure 12 shows photographs and bar charts. Antigen expression and imm unogenicity of Ad5 vectors expressing different versions of NadA and fHbp in m ice. (A) Antigen expression in cells infected with Ad5 containing either a NadA transgene flanked with a C-terminal V5 tag or, fHbp, or no transgene, detected by immunofluorescence against V5 or against fHbp, in green. (B, C, D and E) Groups of mice (n=8 to 16) were immunized once with 109 infectious units (iu) of Ad5 or 2.5 microg of OMVs. Antibodies were detected in serum samples by ELISA against heat inactivated 2996 (B and C) or H44/76 (D and E) bacteria. (B) Serum antibody endpoint titers elicited against whole cells from strain 2996, 2 and 6 weeks post a single injection of mice with the vaccines indicated in the X-axis. (C) Endpoint IgG subclass titers induced by Ad5 -NadA immunization as compared with 29960MV immunization, against strain 2996 whole cells, at week 6. (D) Serum antibody end-point titers and (E) IgG subclasses elicited against whole cells from strain H44/76, 2 and 6 weeks post a single injection of mice with the vaccines indicated in the X-axis. For (B), (C), (D) and (E) the titers for each individual mouse, and the geometric mean and 95% confidence interval of the group, are presented. Statistical differences observed using Kruskal- Wallis (B) and Mann-Whitney tests (C) are noted, with *** p<o.ooi, ** p<o.oi and * p<0.05.
Figure 13 shows a table and graphs. Ad-fHbp induces high, cross-protective and long-lasting SBA responses. Groups of mice (n=6 to 12) were immunized once on day o with Ad fHbp or fhbp t, or H44/76 nOMV or 4CmenB. (A) Bactericidal antibodies were detected in serum samples at weeks 6 and 42 by hSBA against strains H44/76, BZ83 or BZ198. (B) Groups of mice were immunized once with decreasing doses of Ad-fHbp or H44/76 OMVs. Bactericidal antibodies were detected in serum samples at week 6 post immunization by hSBA against strains H44/76 or BZ83 (heterologous PorA, low fHbp 1.1 expression). (C) Groups of mice were immunized once with 109 iu Ad-fHbp or with the licensed vaccine 4CMenB (Bexsero®, 1/5* or 1/10* of a human dose). Bactericidal antibodies were detected in serum samples three weeks post injection, by hSBA against strain H44/76 (fHbp vi.i). (D) Groups of Balb/c and CDi mice were immunized as described above. Bactericidal antibodies were detected in serum samples, 6 weeks post-immunization, by hSBA against strains H44/76 or BZ83. For all panels, serum samples from each group were pooled, the bactericidal titres are expressed as the dilution giving a survival below 50% as compared to bacteria incubated with buffer, as examplified with the raw data of two assays detailed in the graphs in panel A (pi.10 is a monoclonal antibody with known bactericidal activity, used here as a control in the assay).
Figure 14 shows plots. Effect of heterologous prim e boost regim en on the SBA responses, (A) using protein-based com ponents, and (B and C) using Ad and MVA com binations . (A) Mice (n=5 to 6 per group) were immunized at day o, or o and 21 or o, 21 and 35 with the regimen indicated in the X axis, combining Ad MenB and 4CMenB, or Ad MenB and OMVs. SBA was measured 2 weeks post last immunization against a strain expressing the homologous fHbp as used in the vaccines (variant 1.1, strain H44/76). (B and C) Mice (n=5 or 6 per group) were immunized at day o with wither the Ad and MVA single dose, or at day o and boosted at week 8 for the prime boost combinations indicated in the X axis. Control mice were immunized with 4CMenB at day o, 21 and 35. SBA was measured 2 weeks post last immunization (panel B) or at several time points up to week 28 (panel C). For panels A, B and C, the SBA titers for each individual mouse, and the geometric mean and 95% confidence interval of the group, are presented. The horizontal doted line denotes the putative threshold associated with protection (titer of 1:4). Statistically significant differences are noted with * p<0.05, ** p<o.oi and ***p<o.ooi. (D) Enum eration of fHbp- specific antibody secreting B cells in spleen and bone m arrow , at week 28 post immunization with the vaccines described and in the X axis and legend. Data for each individual mouse is presented, with the geometric mean and 95% confidence interval of the group.
Figure 15 shows plots. Im pact of clinically relevant m odifications on the SBA response in m ice. Groups of mice (n= 4 or 5) were immunized with a single dose Ad, (A) with different serotypes and either a short or a longer version of the CMV prom oter, or (B) 5xl0 e8 IU ChAdOxl vaccine in different m ouse strains, as com pared with 2 doses of l/5th of a hum an dose of 4 CMenB, or (C) via intram uscular or m ucosal, needle free, routes as indicated in the X axis. The SBA titers were measured in serum 2, io or 20 weeks post injection (panel B, C and A respectively). (D) Kinetics of SBA response induced by ChAdOxl vaccine. Mice were injected with 109 IU ChAdOxl fHbp, or three injections of i/5th of the human dose of 4CMenB as indicated in the legend. The SBA titers for each individual mouse, and the geometric mean and 95% confidence interval of the group, are presented at the time points indicated in the X axis. Individual titers, and the geometric mean and 95% confidence interval of the group are presented. The horizontal doted line denotes the putative threshold associated with protection (titer of 1:4). * p<o.O5. ** p<o.oi (Kruskall-Wallis and multiple comparison correction).
Figure 16 shows plots. A point m utation in the transgene abrogates binding to hum an factor H (fH) and increases SBA responses in the presence of hum an fH. Point- mutations were introduced in the transgene (Ml, H248L) and (M2, S223R). (A and B) In vitro expression of the resulting protein. Hela cells were infected with the adenoviruses serotype 5 as mentioned (Ad empty as negative control, or Ad fHbp wild type sequence, or either one of the mutant), and expression of the antigen was measured by flow cytometry using an anti-fHbp monoclonal antibody, and expressed as % of positive cells. Flow panels from an individual experiment are shown in panel A, and the individual results in replicated experiments, with geometric means and confidence interval are shown in panel B. (C and D) In v itro binding to hum an fH. Hela cells were infected with the adenoviruses as mentioned, followed by incubation with human fH. Detection of bound human fH was performed using a commercial anti-human fH antibody by flow cytometry. Individual flow panels are shown in panel A, while replicates are shown in panel B with geometric mean and confidence intervals. (E) Im m unogenicity of the m utant expressing vectors in the absence of hum an fH . Groups of Balb/c mice (n=5) were immunized once with the adenovirus as mentioned in the X axis, and the individual serum SBA titers against strain H44/76 were measured at week 6 and 14. Individual titers are shown at each time point, with geometric mean and confidence intervals. (F, G, H) SBA titers in the presence of hum an fH. Transgenic mice expressing human fH (n=i2) were immunized once with the adenoviruses mentioned in the X axis, or one, two (G) or three times with 4CmenB (H), and SBA titers were measured at several time points post injection (at weeks 2 and 6 for experiment in panel F, at weeks 3 and 8 for panel G, and 6 times over 21 weeks for the experiment in panel H). Individual serum SBA titers against strain H44/76 are shown, with geometric means and confidence intervals. * denotes p<0.05, *8 p<o.oi and *” p<o.ooi. Figure 17 shows bar charts. Kinetics of SBA responses against different strains. SBA using human complement was performed against nine strains relevant to the UK and Europe, including strains expressing the homologous fHbp to the vaccine antigen in the adenovirus vaccine and licensed vaccine 4CMenB (A, B and C), and strains expressing heterologous fHbp (D to I) , at low, medium or high quantities, as indicated below the X axis of each graph. Mice were immunized with a single dose of Ad fHbp i.i M2 (black bars), or up to three doses of the licensed vaccines qCMenB (blue bars) or rLP2o86 (grey bars). Serum SBA titers were measured at the time points indicated in the X axis, serum samples from each group were pooled, the bactericidal titres are expressed as the dilution giving a survival below 50% as compared to bacteria incubated with buffer. The horizontal doted red line denotes the putative threshold associated with protection (titer of 1:4).
Figure 18 shows plots
Figure 19 shows plots Figure 20 shows photgraphs Figure 21 shows plots Figure 22 shows plots Figure 23 shows plots Figure 24 shows plots Figure 25 shows plots Figure 26 shows plots Figure 27 shows plots Figure 28 shows a graph
Figure 29 shows graphs. In more detail, shown is time trend of hSBA titre .
Eight to 10 participants were immunized once ChAdOx1 MenB.1 at day o (black) or twice with 4CMenB (Bexsero®) at day o and day 28 (dotted grey) as indicated in the legends (A), and hSBA titers measured against strain H44/76-SL at days o, 14, 28, 180 and 208.
In panel B, eight to 10 participants were immunized with ChAdOx1 MenB.1 (black), or Bexsero® at day o (dotted light and dark grey), and then boosted at day 180 with either ChAdOx1 MenB.1 (black for the homologous ChAdOx1 ChAdOx1 prime boost, and medium grey for the heterologous Bexsero® followed by ChAdOx1 prime boost) or Bexsero® (light grey, for the Bexsero® - Bexsero® prime boost) as indicated in the legend, and hSBA antibody titers measured at day o, 14, 28, 180. 187 and 208). Data shown are median and IQR for each group.
Figure 30 shows graphs. In more detail, shown is time trend of T cell response. Eight to 10 participants were immunized once with ChAdOx1 MenB.1 at day o at either 2.5x1010 viral particles (vp) (black), or 5x1010vp (dotted grey) or twice with 4CMenB (Bexsero®) at day o and day 28 (light grey) as indicated in the legends (A), and fHbp-specific IFN-gamma producing T cells enumerated at days o, 14, 180 and 208.
In panel B, eight to 10 participants were immunized with ChAdOx1 MenB.1 (black), or Bexsero® at day o (medium and light grey), and then boosted at day 180 with either ChAdOx1 MenB.1 (black for the homologous ChAdOx1 ChAdOx1 prime boost, medium grey for the heterologous Bexsero® followed by ChAdOx1 prime boost), or Bexsero® (light grey, for the Bexsero® - Bexsero® prime boost) as indicated in the legend, and fHbp-specific IFN-gamma producing T cells enumerated at day o, 14, 180 and 208). Data shown are median and IQR for each group.
Figure 31 shows graphs. In m ore detail, shown is tim e trend of h SB A titre (A) and T-cell response (B) in m erged group. 16 to 20 participants were immunized once with ChAdOx1 MenB.1 at day o with 5x10 lovp (dotted black line), or with 4CMenB (Bexsero®) at day o (light grey), or twice with Bexsero® at day o and 28 (medium grey) as indicated in the legends. Data shown are median and IQR.
Figure 32 shows MAIT cell-deficient mice and humans with weak MAIT cell activation have impaired vaccine-induced T cell responses following ChAdOx1 immunization. (A) Frequency of IFN-g-producing PBMCs measured by peptide ELISPOT in ChAdOx1 vaccinated volunteers either pre-boost (N=14), or on day 14 post-boost (N=13). (B) Spearman rank correlation analysis in the change in CD69 expression on MAIT cells from pre-boost to day 1 post-boost versus the change in frequency of IFN-g-producing PBMCs measured by peptide ELISPOT from pre-boost to day 14 post-boost. (C) C57BL/6J (N=12) or MRi KO (N=9) mice were immunized intramuscularly with 108 IU of ChAdOx1 expressing an HCV-GTi-6_D_TM-Ii+L transgene, and on day 16 post- immunization splenocytes were collected. Group averages of CDioya expression, IFN-g production, TNF production, or dual production of IFN-g and TNF by CD8+ T cells following 5 h restimulation with an overlapping peptide pool of the HCV genotype lb proteome. (D) C57BL/6J (N=12) or MRi KO (N=i) mice were immunized intramuscularly with 108 IU of ChAdOx1 expressing an nCoV-19 transgene, and on day 13 post-immunization splenocytes were collected. Group averages of CDioya expression, IFN-g production, TNF production, or dual production of IFN-g and TNF by CD8+ T cells following 5 h restimulation with an overlapping peptide pool of the SARS-C0V-2 SI and S2 proteins. (E,F) C57BL/6J (N=12) or MRi KO (N=12, N=n postboost) were primed intramuscularly with 107 IU of ChAd63 expressing OVA and boosted intravenously on day 28 with 108 IU (squares) or 109 IU (circles) of ChAd63- 0VA. Group averages of CDioya expression, IFN-g production, TNF production, or dual production of IFN-g and TNF by CD8+ T cells following 6 h restimulation with the SIINFEKL peptide either 3 weeks post-prime (E) or 3 weeks post-boost (F). *, P<0.05; P<o.oi; ***, P<o.ooi. Wilcoxon rank-sum test (A), or two-way ANOVA (C-F). Symbols indicate individual volunteers/mice, dotted line indicates level of expression in absence of peptide stimulation, and mean ± SEM are shown.
Figure 33 shows (A) Gating scheme for the identification of MAIT cells (MR1/5-OP- RU++ T cells) in PBMCs of healthy human volunteers immunized with ChAdOx1. (B) Healthy human volunteers (N=14) were immunized with a 5XIO10 vp dose of ChAdOx1. (C) Frequencies of MAIT cells in peripheral blood one day pre- and one day postimmunization. (D) Concentration of plasma cytokine levels in healthy human volunteers (N=14) one day pre- and one day post-immunization with 5XIO10 vp of ChAdOx1. (E) Pearson correlation of change in CCL2 chemokine level following vaccination and the change in expression of CD69 on MAIT cells. *, P<0.05; ***, P<o.ooi; Wilcoxon rank-sum test. Symbols indicate individual donors, and group mean is shown.
Figure 34 shows plots of SBA titers against strain H44/76-SL. Individual data and geometric mean for each group are indicated, prior to vaccination (A), 1 month after a single vaccine injection (B) and 6 months after 1 or 2 immunizations, as indicated in the X-axis. The horizontal dotted line indicates the putative protective threshold titer of 1:4.
Figure 35 shows plots of SBA titers against strain H44/76-SL. Individual data and geometric mean for each group are indicated, at day 208 (A) and 365 (B), after 1 or 2 immunizations, as indicated in the X-axis, with the interval between first and second injection indicated in parenthesis. The horizontal dotted line indicates the putative protective threshold titer of 1:4.
Figure 36 shows plots of SBA titers against strain Moi 240355. Individual data and geometric mean for each group are indicated, prior to vaccination (A), 1 month after a single vaccine injection (B) and 6 months after 1 or 2 immunizations, as indicated in the X-axis. The horizontal dotted line indicates the putative protective threshold titer of 1:4.
Figure 37 shows plots of SBA titers against strain Moi 240355. Individual data and geometric mean for each group are indicated, at day 208 (A) and 365 (B), after 1 or 2 immunizations, as indicated in the X-axis with the interval between first and second injection indicated in parenthesis. The horizontal dotted line indicates the putative protective threshold titer of 1:4.
Figure 38 shows bar charts of SBA titers against strains H44/76-SL and Moi 240355. Proportion of participants with a titer < or = to 1:4 are indicated, after 1 or 2 immunizations, as indicated in the legends. Prior to Day 180, participants are grouped according to the priming regimen (A). At day 180, groups were further divided according to the different boosting regimen (B). Figure 39 shows graphs of IgG memory B cell (A) and IFN-gamma secreting T-cell (B) responses against fHbp 1.1. geometric means and CI are indicated, after 1 or 2 immunizations, as indicated in the legends. Prior to Day 180, participants are grouped according to the priming regimen. At day 180, groups were further divided according to the different boosting regimen as indicated.
Figure 40 shows plots of plasma cytokine quantification by MSD. Individual data, geometric means and CI are indicated, after 1 or 2 immunizations, as indicated in the legends. Prior to Day 180, participants are grouped according to the priming regimen. At day 180, groups were further divided according to the different boosting regimen as indicated.
EXAMPLES
Example 1 - Com parative Data (PorA/FetA)
Here we disclose development of another chimp Adeno vectored vaccine for MenB based on different proteins from Meningococcus - the PorA & FetA proteins. This is not part of the invention. This is provided as comparative data.
We show expression of the proteins in mammalian cells when infected with the viral vector and induction of high titres of antigen-specific antibody. However, the antibodies generated were found not to be functional by serum bactericidal assay, possibly due to conformational changes in the epitope and/or post-translational modifications caused by expression in the mammalian cells. This demonstrates that a chimpanzee adeno-based vector approach for viral vectored vaccines does not work for all bacterial antigens.
Adenoviral vectored vaccines are able to induce both strong cellular and antibody responses against viruses, parasites and the intracellular pathogen Mycobacterium tuberculosis. The capacity of adenovirus vectors to induce antibody responses to transmembrane outer membrane proteins from bacteria has not been previously established. Here we assess the immune responses to viral vectors encoding Porin A (PorA) and Ferric enterobactin receptor A (FetA) of capsular group B Neisseria m eningitidis. The vaccine vectors expressed the bacterial proteins in vitro, induced higher titres (>io5 end-point titre) and longer lasting (>32 weeks) transgene-specific antibody responses in vivo in mice as compared with the antigens expressed in outer membrane vesicles (OMVs). These findings suggest that the short-lived immune responses to OMVs in current use might be overcome by use of viral vectors. However, bactericidal activity was undetectable. Taken together, these results demonstrate that while it is possible to express transmembrane bacterial proteins through a viral vector and induce strong and persistent antigen-specific antibodies, the conformation or post- translational modifications of bacterial outer membrane antigens produced in eukaryote cells may not result in presentation of the necessary epitopes for induction of functional antibody.
Generation of vaccines
PorA and FetA adenoviral vaccines were designed using sequences with the Genbank accession numbers X52995.1 (strain 44/76), AF226337.1 (NZ98/254) and X89755.i (44/76). Where appropriate, N to Q amino acid substitutions were performed to remove potential sites of N-linked glycosylation. Native sequences were codon optimised for expression in humans (GeneArt, Regensburg, Germany). Transgenes were cloned into plasmids containing attRi and attR2 recombination sites (Gateway® Life technologies, CA, USA) under control of a CMV promotor. The human tissue plasminogen activator (tPA) leader was fused to the transgenes in frame to promote secretion of the antigens unless otherwise stated, to address the antigens to the mammalian secretory pathway. For several eukaryotic antigens this has been shown to increase the production and presentation of the antigen to the immune system (Biswas 2011 DOI: 10.1371/journal.pone.oo20977). For expression of only the bactericidal epitopes, variable regions from PorA and FetA were also cloned into a flagellin scaffold: the epitope sequences were fused to replace the central portion (D3) of flagellin protein from E, coli as described previously (Lu 1995 DOI: io.iO38/nbtO495-366). Cysteine residues flanking the variable loops were engineered where indicated to further constrain the conformation of the epitopes. Transgenes were recombined with pAd-PL DEST using LR clonase (Invitrogen) to generate recombinant El and E3 deficient human adenovirus serotype 5 (AdHu5). The viruses were produced as previously described (Dicks 2012 DOI: 10.1371/journal.pone.oo4O385). Viral vectored vaccines were formulated in endotoxin-free PBS. Outer membrane vesicle (0MV) vaccines were produced from the MenB strains NZ98/254, 44/76-FetA,„PorA,„ and 44/76- FetAonPorAoff (Marsay 2015) by detergent extraction as previously described (Frasch 2001); the latter two strains are mutants of 44/76 that were created to assess bactericidal activity against PorA and FetA individually. All 0MV vaccines were formulated to contain either 2.5μg or 5μg of total protein in 20 mM TRIS buffer pH 7.0- 7.5 (Sigma Aldrich, MO, USA) with 85μg of Alhydrogel™ per dose.
Imm unofluorescence Assay (IF A)
HeLa cells were seeded into 6 well culture plates containing rat collagen coated coverslips (BD Bioscience, NJ, USA). Cells were transfected with plasmid DNA containing the transgenes (stated in the figure legends) using Lipofectamine 2000 according to the manufacturer's instructions (Life technologies, CA, USA). Alternatively, HeLa cells were infected with adenoviruses expressing transgenes at a MOI of 100. Transfected and infected HeLa cells were left overnight at 37°C with 5% C02 for protein expression. Cells were fixed with 4% paraformaldehyde and permeabilised with 0.2% triton X-100 in PBS. Transgene proteins were detected using an appropriate mouse primary monoclonal (anti-P.1.7, anti-Pi.16, anti-Pi.4 or anti-F3- 3) or polyclonal mouse FetA F3-3 immunised sera (all provided by NIBSC, UK) followed by goat anti-mouse IgG conjugated to Alexafluor 488 (Life technologies, CA, USA). Cell nuclei were counterstained with DAPI and visualised using a Leica DMI3000 B microscope.
Imm unisation experim ents in mice
Procedures were performed according to the U.K. Animals (Scientific Procedures) Act 1986 and were approved by the University of Oxford Animal Care and Ethical Review Committee. Six to 8-week-old female BALB/c-OlaHsd and NIH-OlaHsd mice (Harlan, UK) were housed in specific pathogen-free conditions. Mice were immunized with a single injection of to8 or 109 infectious units of each vaccine. All vaccines were given intramuscularly to the hind thigh muscle of both legs, with an 8-week interval between priming and boosting immunizations when boosting was performed. Blood was collected from tail bleeds or terminal cardiac bleeds at various time points and allowed to clot then centrifuged at 15,000 x g for 10 minutes. Sera were aliquoted and stored at -20°C until use.
Detection of anti-PorA or FetA antibodies by ELISA
Immulon 2HB Plates (Thermo Fisher Scientific, MA, USA) were coated with either heat killed whole cell preparations of N. m eningitidis in PBS (OD 6oonm 0.1), or recombinant PorA or FetA proteins in carbonate bicarbonate buffer (Sigma Aldrich, MO, USA) at 2μg/ml at 4°C overnight. Plates were washed with PBS Tween 20 at 0.05% before being blocked with 1% BSA in PBS (all Sigma Aldrich, MO, USA) for 2 hours at 37°C. Sera were diluted 1:2000 in blocking buffer before being serially diluted in duplicate and left at 4°C overnight. For assessment of relative avidity of antibodies, 1M sodium thiocyanate (Sigma Aldrich, MO, USA) with 0.05% tween 20 was added to the dilution buffer of samples where stated as avidity. Plates were washed and HRP- conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch inc. PA, USA) was added at 1:10,000 (total IgG) or 1:20,000 (IgGi, IgG2a, IgG2b or IgG3 subclass) and incubated for 2 hours at 37°C. Plates were developed with TMB solution (Sigma Aldrich, MO, USA) and stopped with 2M H2SO4. Endpoint-titres were determined as the reciprocal of the dilution giving an OD45O nm reading above that obtained for naive control wells plus 2 x SD of 6 replicates for each plate. Indirect ELISA against V5 tag
MaxiSorp ELISA plates (Thermo Fisher Scientific, MA, USA) were coated with anti-V5 tag antibody (Abeam, UK) in PBS (Sigma Aldrich, MO, USA) at 2μg/ml at 4°C overnight. Plates were washed with PBS Tween 20 at 0.05% before being blocked with 1% BSA in PBS (all Sigma Aldrich, MO, USA) for 1 hour at room temperature. Supernatants from individual transfection supernatants were serially diluted 1:7 in blocking buffer down 96 well plates in duplicate and left at 4°C overnight. Plates were washed before addition of anti P1.7, anti Pi.16 or anti F3-3 monoclonal antibodies (NIBSC, UK) and incubated for 1 hour at room temperature (RT). Plates were washed and HRP-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch inc. PA, USA) was added at 1:10,000 dilution and incubated for 1 hour at RT. Plates were developed with TMB solution (Sigma Aldrich, MO, USA) and stopped with 2M H2SO4.
Serum bactericidal assay (SBA)
The serum bactericidal assay (SBA) measures complement-dependent bacterial lysis mediated by antibodies and in humans is the correlate of protection used for licensure of meningococcal vaccines. The SBA was performed as described previously (Marsay 2015). Exogenous rabbit or human complement with no intrinsic bactericidal activity was used at 25% (vol/vol) against the target bacterial strains. Human complement was sourced from consenting healthy adults. Test sera were heat inactivated at 56°C for 30 minutes to remove intrinsic complement activity. Bacterial strains were grown overnight for single colonies on blood agar plates at 37°C and 5% C02. Approximately 50 colonies were then sub-cultured for 4 hours before being reconstituted in Hanks buffered salt solution (Gibco) with 0.5% bovine serum albumin (Sigma Aldrich, MO, USA). The bacteria were further diluted to give approximately too colony forming units per topi used for the assay. A titre was defined as the reciprocal of the highest dilution of serum that yielded >50% decrease in colony forming units relative to that of control wells within 6omins at 37°C without C02.
Statistics
Statistical analysis of differences between antibody endpoint titres were performed using two-way ANOVA with Bonferroni post-tests, 1 way ANOVA with Dunns multiple comparisons test or by Mann Whitney U test as stated in figure legends, using Prism 5 (Graphpad, software Inc. CA, USA).
Full-length PorA and FetA proteins can be expressed in HeLa cells from DNA and adenoviral vectors Several designs of the bacterial OMPs PorA (P1.7,16 and P1.7-2,4) and FetA (F3-3) including full length proteins, VR loops within FliC scaffolds, tPA leader fused and non- tPA leader fused (cytosolic) were generated to assess immunogenicity when delivered by adenoviral vectors. Schematics for the transgene designs are shown in Fig. 1. Full length PorA P1.7, 16 and FetA F3-3 proteins were detected in HeLa cells by anti-PorA and anti-FetA specific antibodies; however, the VR1.16 epitope of PorA P1.7, 16 contained an N-linked glycosylation motif (NLT). To prevent the glycosylation of this protective epitope during transit through the Golgi apparatus, full length P1.7,16 without a tPA leader (Cytosolic-P1.7,16) was created. Expression of this transgene was also detected by IFA in HeLa cells using anti-P1.7 mAb targeting the 1.7 VR region and bactericidal epitope (data not shown).
PorA PI.7,16 expressing vectors induce antibody responses in m ice
The PorA P1.7, 16 vaccines Ad-P1.7,16, Ad-cytosolic-P1.7,16, Ad-FliC- VR1.7 and Ad-FliC- VR1.7-C were assessed for immunogenicity in mice, with Ad expressing unmodified FliC as a negative control. After a single injection, specific antibody responses were elicited by the full-length Ad-P1.7,16 and Ad-cytosolic-P1.7,16 vectors, as early as two weeks after vaccination, and were maintained for up to 32 weeks (Figure 2A). In accordance with previous findings, immunisation with Ad-P1.7,16 with the tPA leader induced consistently higher antibody titres than the Ad-cytosolic-P1.7,16 without the tPA leader. The difference was significant at 6, 8 and 32 weeks after vaccination (P < 0.05, P < 0.01 and P < 0.001 respectively). In comparison, the Ad-FliC-VR1.7 and Ad- FHC-VR1.7-C vectors, which only expressed the VR1.7 epitope, elicited low antibody titres that were only significantly higher in the Ad-VR1.7 group as compared with the Ad-FliC-control group, two weeks post-vaccination (P < 0.001, Figure 2A). The addition of flanking cysteines at the base of the P1.7 epitope in the Ad-FliC- VR1.7-C vaccine did not enhance antibody titres. To confirm that antibodies generated by immunisation with Ad-P1.7,16 could bind PorA P1.7, 16 naturally expressed by meningococci, ELISA against whole cell preparations of strain 44/76-SL were performed. Balb/c and NIH mice immunised with a low (108 IU) or high (109 IU) dose of Ad-P1.7,16 elicited detectable antibody responses against the whole bacterial cells two weeks post vaccination (Figure 2B).
FetA F3-3 expressing vectors induce antibody responses in m ice
FetA F3-3 expressing vectors Ad-F3-3 (full length) and Ad-FliC-VR3-3 (VR3-3 only) elicited FetA-specific antibody responses in mice as measured against rF3-3 protein (Figure 3A). An 0MV comparator that did not contain the immunodominant PorA but had a defined quantity of FetA (7.8% of total OMPs), was used to assess the FetA response to an OMV vaccine (OMV-44/76FetAonPorAoff). Immunisation with Ad -F3-3 resulted in significantly higher antibody titres compared with the OMV comparator and Ad-FliC- VR3-3 immunised mice 6 weeks post immunisation (P < 0.05 and P < 0.001 respectively). Mice in the OMV comparator group elicited F3-3 specific antibody titres that were not significantly greater as compared with those elicited by Ad-FliC- VR3-3- immunization. Ad-F3-3 induced anti-FetA antibodies bound naturally expressed FetAF3-3 present in whole cell preparations (Figure 3B). As with the Pl.7,16 adenoviral vaccines, greater levels of antibodies were detected in response to the OMVs when whole bacterial cells were used for antibody capture, likely due to induction of antibodies against multiple bacterial antigens in the OMV vaccine.
Anti-Pl.7,16 and anti-F3-3 antibodies generated by imm unisation w ith adenoviral vectors are not bactericidal
The functional capacity of the antibody responses induced by the vectored PorA/FetA vaccines was measured by SBA. None of the viral vectors induced a detectable bactericidal response despite the particularly high antibody concentrations detected in sera after immunisation with the full length vaccines Ad-P1.7,16, Ad-cytosolic-P1.7,16 and Ad-FliC-F3-3 - see table below showing SBA titres at 16 weeks post vaccination for Ad-P1.7,16 and Ad-F3-3 expressing adenoviral vaccines measured against target strain 44/76-SL, using baby rabbit complement and pooled mouse sera - serum bactericidal titres after a single dose of P1.7,16 and F3-3 Adenoviral vectors:
Figure imgf000045_0001
Antibody subclass ELISAs were performed on sera taken from mice immunised with a single dose of 109 IU of Ad-P1.7,16 or Ad-FliC-VR1.7 or two doses of 5μg of OMV comparator (44/76-FetA,„PorA,„) against rP1.7,16. Addition of 1M sodium thiocyanate to the diluent was performed to assess relative antibody avidity induced by the adenovirus and 0MV vaccines. The levels of IgG3 antibodies were low or non- detectable in all vaccine groups (data not shown). Sera from Ad-P1.7,16 and 0MV immunized mice had detectable levels of IgGi, IgG2a and IgG2b, whereas mice immunized with Ad-FliC-VR1.7 only had IgG2a antibody titres that were detectable above the negative cut-off. The antibody endpoint-titres were reduced in the adenovirus and 0MV vaccine groups across all three subclasses when 1M sodium thiocyanate was included in the diluent. However, this was only significant in the 0MV immunized mouse sera for IgG2b antibodies (P < 0.01, data not shown).
PorA subtype Pl.7-2,4 expressing adenoviral vaccines also induce strong antibody responses but no bactericidal activity
The absence of bactericidal activity induced by Ad-P1.7,16 could have been due to N- glycosylation of the VR2 epitope (P1.16), as there is evidence that the P1.7 epitope is less effective at inducing bactericidal antibodies than P1, 16 (van der Ley 1993 PMID: 7691745). Therefore, vectors encoding a different PorA (P1.7-2,4) that does not contain an N-linked glycosylation motif in the VR and protective regions were created. Bactericidal activity against this PorA is primarily to the P1.4 VR2 epitope (Martin 2006). The antibody levels induced by adenoviral vaccines coding for P1.7-2,4 antigens were compared with homologous (PorA subtype) OMVs (NZ98/254). In Balb/C mice, Ad-Pi.7-2,4 induced high levels of antibodies detected against recombinant PorA rP1.7- 2,4 by ELISA 6 weeks post immunisation, which were comparable to the levels of P1.7- 2,4-specific antibodies elicited in 0MV immunised mice. Immunisation with Ad-FliC- VR1.4-C induced detectable but weak antibody responses 6 weeks post immunisation. Boosting with OMVs at week 8 resulted in increased end-point titres in mice primed with Ad-FliC-VRi.4 or OMVs after the booster (week 6 vs. week 14, P < 0.001 and P < 0.01 respectively; week 6 vs. week 20, P < 0.05 for both groups). Mice that received Ad- P1.7-2,4 as the priming vaccine did not elicit significantly higher P1.7-2,4-specific antibody levels following an 0MV boost. No hSBA activity was detected 6 weeks after a single immunisation with Ad-P1.7-2,4 or Ad-FliC- VR1.4 (data not shown).
Summary of Comparative Data
We demonstrate here that adenoviral vectors coding for PorA or FetA meningococcal antigens induce strong and long-lived antibody responses in mice after a single dose. However, no serum bactericidal activity was detected in mice immunised with any of the PorA/FetA adenoviral vaccines alone and there was no appreciable increase in bactericidal antibodies in response to combining Ad-prime with 0MV boost as compared with using 0MV only. Example 2 - Com parative Data (Bordetella pertussis)
Here we show development of a chimp adeno/MVA prime-boost approach for the prevention of infection/disease caused by Bordetella pertussis (whooping cough). As in Example 1, experiments using the chimp adeno-vectored vaccine expressing the target antigen induced antigen-specific antibodies in immunised mice but these were not protective.
Bordetella pertussis is the causative agent of the highly contagious respiratory infection whooping cough (pertussis) . Pertussis is a vaccine-preventable disease and although there is high vaccination coverage in many countries, there are reports of increased numbers of pertussis cases globally. Most developed countries replaced the whole-cell pertussis vaccines with less reactogenic acellular pertussis vaccines that are effective at preventing severe pertussis disease, but are unable to prevent bacterial colonisation and subsequent transmission. The acellular pertussis vaccines also do not induce longterm protection in vaccinated individuals and their main component is pertussis toxin, which is a complex toxin made of the Si subunit and the B oligomer.
In this study, we used recombinant adenovirus and modified vaccinia virus Ankara vaccine vectors to develop a novel vaccine that would have a great potential to induce both humoral and cellular immune responses. A single injection of adenoviral vectored vaccine expressing the toxin Si subunit, containing the enzymatic activity, induced antigen- specific IgG antibodies in mice, whereas immunisation with viral vectors based on modified vaccinia virus Ankara did not. However, heterologous prime - boost strategies significantly improved immunogenicity in mice primed with modified Vaccinia virus Ankara. However, the various immunisation strategies failed to induce protective responses in mice, highlighting the limitations of the viral vectored vaccine to raise functional antibody responses against bacterial targets.
The viral vectored Bordetella pertussis toxin SI subunit vaccines are not protective in vivo
Protective efficacy of Ad and MVA vectors expressing Si-220 was assessed using an aerosol challenge model with a virulent B. pertussis 18-323 strain (1 x 108 CFU/ml) via aerosolised droplets. Mice immunised with phosphate buffered saline (PBS) served as a negative control group, and mice immunised with a commercial aP vaccine (Pediacel®) as positive control.
Mice immunised with the aP vaccine had significantly lower bacterial counts in their lungs than the control group and mice vaccinated with the viral vectored vaccines on day 7 post challenge (p < 0.0001, comparison with control group on day 7, mice primed with Ad Si-220, prime-boosted with MVA + Ad (Si-220) and those prime-boosted with Ad + MVA (Si-220)), bacterial counts in the lungs of mice vaccinated with the Ad and or MVA were similar to those found in PBS-immunised mice at day seven (Figure 4).
Example 3 - Manufacture and Characteristics of Exem plary Vector ChAdOxl MenB.l
LIST OF ABBREVIATIONS
AE Adverse Event
BAC Bacterial artificial chromosome
BHL Bulk harvest lots cGMP Current good manufacturing practice
ChAd Chimpanzee adenovirus
ChAdOxl Chimpanzee adenovirus vaccine vector Ox1
DNA Deoxyribonucleic acid
DMC Data Monitoring Committee
ELISA Enzyme-linked Immunosorbent Assay fH Factor H fHbp Factor H Binding Protein
GLP Good Laboratory Practices
GMP Good Manufacturing Practice
HEK Human embryonic kidney cells
IM Intramuscular in Infection units
IgG Immunoglobulin G
IMPD Investigational Medicinal Product Dossier kDa Kilodalton
MenB Capsular group B meningococcus
MenBOx1 Shorter acronym for Meningococcus group B Ox1-based vaccine
(ChAdOxl MenB.l)
Nanometre
OMP Outer membrane protein OMV Outer Membrane Vesicles PBS Phosphate-buffered saline PCR Polymerase chain reaction PI Principal Investigator SBA Serum bactericidal antibody SAE Serious Adverse Event SOP Standard operating procedure ST Sequence type SUSAR Suspected unexpected serious adverse reaction
TPA Tissue plasminogen activator TSA Tryptic Soy Agar vp Virus particles VSS Virus seed stock WHO World Health Organisation Wt Wild type 4CMenB Four component meningococcus B vaccine (trade-name Bexsero®) General characteristics of ChAdOxi MenB.l vector
ChAdOx1 MenB.l is a replication-deficient (El and E3 deleted) simian adenovirus, which contains the Neisseria m eningitidis (MenB) gene encoding for antigen factor H binding protein (fHbp), with a point mutation of a serine to an arginine at the amino acid position
223. The antigen, fHbp is an outer membrane protein and is a component of the two vaccines licensed for MenB (Bexsero® from GSK and Trumenba® from Pfizer). However wild type (wt) fHbp binds human factor H (fH), a complement inhibitor, with very high affinity, leading to a potential dampening of the immune response to the vaccine and a theoretical risk of raising an autoimmune response against fH. We have introduced a point mutation that decreases the binding of fHbp to fH, while retaining immunogenicity. Chimpanzee adenoviruses (ChAd) have been developed as viral vectors following concerns that preexisting immunity to human serotypes (such as serotype 5) could limit future widespread use of these viruses. Phylogenetic studies show that simian and human adenoviruses fall into the same eight species. ChAdOx1, like many simian adenoviruses isolated to date, is a member of species E, which also contains one human virus (Human adenovirus serotype 4). Because of the El deletion, the virus can only propagate in cells expressing El functions, and thus virus is unable to replicate within vaccinated animals or humans.
Pre-clinical Experience with ChAdOxi MenB.l
Expression of the meningococcal antigen fHbp from ChAdOx1 MenB.l was detected in mammalian cells, as assessed by immunofluorescence with a fHbp-specific monoclonal antibody. In addition, binding of fH to fHbp mutant was decreased as compared to the wt fHbp.
ChAdOx1 MenB.l has been demonstrated in pre-clinical studies to be safe, non-toxic and immunogenic. The vaccine efficacy was measured using a serum bactericidal assay (SBA): antibodies against MenB are functional when they mediated killing of the bacteria using this assay. Bactericidal activity was evaluated using the gold-standard serum bactericidal assay (SBA), using a human complement source.
- ChAdOx1 MenB.l elicited robust bactericidal activity in mice after a single immunization.
- ChAdOx1 MenB.l induced high SBA titres in different murine strains. - ChAdOx1 MenB.1 induced SBA responses in different strains of mice and in a dose- dependent manner.
- Comparison was performed with the four component licensed vaccine (4CMenB). The bactericidal response induced after a single injection of ChAdOx1 MenB.1 exceeds the response induced with the licensed comparator 4CMenB, despite the fact that this vaccine contains more antigens.
- The bactericidal response was observed in transgenic mice expressing human fH.
Clinical Experience with ChAdOxi MenB.i
There is not yet clinical experience with this vaccine. However, three ChAdOx1 vaccines containing different inserts (from H. influenza, from M. tuberculosis, and from prostate cancer) have been used in clinical trials at the University of Oxford. Safety data from these trials are favourable with no safety concerns raised.
General investigational plan
The clinical development of ChAdOx1 MenB.1 is aimed towards the production of an effective
MenB vaccine for infants, adolescents and adults to protect against capsular group B invasive disease. ChAdOx1 MenB.1 Phase I trial will evaluate the safety and immunogenicity of various intramuscular doses of ChAdOx1 MenB.1 in healthy adults, and provide a proof of concept that a bacterial outer membrane protein expressed from an adenovirus vector can induce bactericidal response in human. The clinical development will eventually progress to a formulation that will include another variant of fHbp, in order to increase the coverage of the vaccine, and to the target populations (infants and adolescents), that may have been primed with the licensed vaccine 4CMenB. Owing to the very low incidence of capsular group B invasive disease, efficacy trials are not possible, but the rise of serum bactericidal antibodies above the protective level of 1:4 is accepted as an indication of efficacy and was used for licensure of the two licensed vaccines.
Serum bactericidal assay (SBA)
A laboratory method termed "serum bactericidal activity (SBA) analysis" has been designed to assess the ability of serum antibodies to kill meningococci in presence of complement. Studies have demonstrated that individuals whose serum is found to be positive in the SBA analysis are more likely to be protected against meningococcal disease^). Phase III clinical trials with capsular group B and C vaccines have also found that there is a similar correlation between lack of SBA and susceptibility to meningococcal disease. Therefore, when assessing whether a vaccine candidate is likely to offer protection against meningococcal disease, the SBA analysis is the gold standard, and the two licensed vaccines have reached licensure using this method.
The o uter m em brane protein fHbp as va ccine antigen
Factor H binding protein (fHbp) is an important virulence factor expressed on the surface of /V. m eningitidis. The function of fHbp is to bind human complement factor H (fH), an important down-regulator of the host alternative complement pathway. Binding of human fH to the bacterial surface via fHbp interferes with complement- mediated lysis of the bacteria and is an important immune evasion strategy of N. m eningitidis. Strains in which fHbp has been deleted have reduced binding to fH and reduced survival in human complement mediated bacterial killing assay compared to wild type strains. The discovery of meningococcal fHbp has led to the development of the two recombinant meningococcal group B vaccines that incorporate fHbp (4CMenB or Bexsero® by Novartis/ GSK and a vaccine composed of two fHbp variants manufactured by Pfizer inc.).
Following vaccination, fHbp incorporated in the vaccine is expected to form a complex with human fH, and this interaction between fH and fHbp adversely affected the immunogenicity of the 4CMenB vaccine in transgenic mice expressing human fH (lower serum IgG anti-FHbp antibody responses and 15 fold lower SBA response). In mice immunised with 4CMenB, the ability of anti-fHbp antibodies to inhibit binding of fHbp to fH appears to be crucial to the breadth of bactericidal activity(6)(7). While serum anti-fHbp antibodies elicited by wt fHbp in wt mice inhibited binding of fH to fHbp, the corresponding antibodies elicited in human fH transgenic mice enhanced fH binding(8). These results suggested that a wt fHbp is not the ideal antigen for a vaccine composition to be used in humans. Several groups therefore engineered fHbp mutants to eliminate fH binding, and showed that some mutants have enhanced protective antibody responses in the presence of human fH (9X10). It is unknown whether the anti-fHbp antibody repertoire of immunised humans block fH binding. Binding of human fH to the fHbp within the vaccine may mask epitopes in the fH binding site that are required for generation of bactericidal antibodies. Theoretically, the resulting antibodies in vaccinees could fail to block binding of fH to the bacteria. Moreover, the formation of a complex between fH and fHbp upon vaccination may lead to the aberrant generation of antibodies against fH. Anti-fH IgM were detected in 2 of 15 fH transgenic mice (whose fH binds fHbp) after three doses of 4CMenB, whereas no such antibodies were found in wild type mice (whose fH does not bind fHbp) following the same vaccination schedule(8). Anti-fH antibodies have not been reported in any 4CMenB vaccine studies involving humans, although they were not looked for, and are not known to be produced after natural N. meningitidis infection.
Adeno v irus based im m unization strategies
The challenge for meningococcal vaccines is to induce serum bactericidal antibodies (SBA). The majority of subcapsular antigens are variable and induce strain-specific protection, limiting their use as strain-specific formulations designed to counter clinical outbreaks. Giuliani et al. highlighted the intriguing possibility that adjuvants inducing T-helper type-1 responses contribute to broader protection against MenB in animal models(n). Adenovirus vectored vaccines have extensively been shown to induce robust IFN- gamma responses with a variety of antigens, in pre-clinical models and in numerous clinical trials(i2). Therefore an attractive hypothesis is that concomitant induction of IFN-gamma due to the intrinsic properties of the Adenovirus may induce higher levels of protective antibodies and broader coverage against MenB.
Adenoviruses have been investigated as vaccine deliveiy platform since the 1970s. Vaccination of 2 million US military personnel using orally administered live human adenovirus serotype 4 and 7 have shown good safety and efficacy data(i3).
Adenoviruses can infect several cell types but no evidence of insertional mutagenesis has been observed. The adenoviral genome is well characterized and easy to manipulate. Adenoviruses cause mild disease but deletion of key genes (El which is required for viral replication) renders them replication-defective. Replication-deficient adenoviruses can be propagated in cell lines approved by regulatory agencies for human product development (human embryonic kidney cells 293) and following Good Manufacturing Practice. Recombinant Adenoviral vectors expressing antigens from HIV-i, TB, malaria, influenza, RSV and hepatitis C virus, are in phase I/II clinical trials, and elicit strong antibody responses in humans with an excellent safety record. Recently, excellent safety and immunogenicity was observed in a phase I trial for malaria in 10 week-old Gambian infants(i4).
Pre-existing immunity against highly prevalent human serotypes, present in up to 80% of individuals, may render certain serotype such as human serotype 5 vectors partially ineffective. Therefore other adenoviruses based on simian serotypes have now been developed, including Ox1, developed by Oxford University. Such vectors have hexon structures homologous to human serotype 4, but do not circulate at detectable levels in human populations and neutralizing antibody prevalence is very low in humans on all continents(i5). These vectors are highly immunogenic: the most developed, based on ChAd63 serotype, has showed very good safety and exceptional immunogenicity in 14 human clinical trials involving 682 volunteers. Chimpanzee adenovirus Ox1 was developed in Oxford University from the group E chimpanzee adenovirus ¥258(16). The cellular immunogenicity of recombinant El- E3- deleted vector ChAdOx1 is comparable to that of other species E derived chimpanzee adenovirus vectors including ChAd63, and the prevalence of virus neutralizing antibodies in human was lower than for ChAd63 in British and Gambian infants. In a UK cohort of 100 people, no individual possessed a neutralisation titre above 200 (the threshold for a positive titre during routine pre-vaccination screening). The low seroprevalence of ChAdOx1 in humans suggests that this new vector could be particularly efficacious in a clinical setting. Therefore ChAdOx1 now offers an attractive option for vaccine development against MenB and we elected to employ this novel replication-deficient viral vector to develop an adenovirus-vectored vaccine expressing a mutated fHbp with reduced binding to human fH in order to induce protective antibody responses to MenB.
Description ofExemplary Vector ChAdOxi MenB.l
An exemplary composition of the invention, such as a vaccine composition, comprises a viral vector such as the replication-deficient (El and E3 deleted) simian adenovirus vector ChAdOx1, containing a genetic cassette encoding for the meningococcal capsular group B antigen fHbp variant 1.1 with a point mutation to prevent binding to the natural human ligand, fH (S223R), codon optimized for mammalian expression, under the control of the strong cytomegalovirus (CMV) immediate early promoter. This vector- fHbp combination is sometimes referred to as 'the vaccine construct' (or even occasionally 'the vaccine').
Generation of ChAdOx1 MenB.l adeno v irus v ector
The vaccine construct was generated at the Oxford Vaccine Group, University of Oxford. Manufacture of the vaccine was carried out in accordance with the requirements of cGMP by:
Clinical BioManufacturing Facility (CBF) Old Road, Headington Oxford OX3 7JT, UK MIA (IMP) 21584
ChAdOxi v ector
The ChAdOx1 vector is replication-deficient as the El gene region, essential for viral replication, has been deleted. The virus will not replicate in cells within the human body. In addition the E3 locus, which promotes viral particle release and inhibits the host's antiviral response, is also deleted. ChAdOx1 propagates only in cells expressing Ei, such as HEK293 cells and their derivatives or similar cell lines such as Per.C6 (Crucell). ChAdOxl fHbp S223R (ChAdOxl MenB.l) assem bly
Pre-adenoviral plasmid pBAC ChAdOxl MenB.l (OVG98) was generated and prepared at the Oxford Vaccine Group, University of Oxford. The fHbpi.i-S223R cDNA, obtained from GeneArt, was PCR amplified and inserted into the El locus of ChAdOXi by BAC GalK recombineering:
The following DNA constructs were used:
OVG65: fHbpi.i S223R cDNA ligated to backbone restricted from OVG33 P2563: pENTR plasmid vector containing the CMV 'long' promoter (with intron A and Tet operator sites) and the BGH poly A sequence.
OVG98: pENTR plasmid vector containing the fHbpi.i-S223R antigen between the 'long' CMVLP TO promoter and BGH poly A sequences.
FHbpi.i S223R gene was cloned from a GeneArt DNA string, using Hindlll and Notl restriction enzymes and ligated to backbone (from plasmid OVG33). The size of restricted fHbpi.i S223R gene was 826 bp. The linearised DNA vector and the DNA insert were gel- purified and the ratio 1:3 for ligation reaction was used (T4 Ligase). A DNA mini prep from recombinant clones (Kanamycin resistant) was performed. The correct size of the insert was verified by restriction mapping and the antigen was sequenced.
The structural fHbpi.i S223R cassette contained in pmono-fHbpi.i S223R (OVG65) was cloned into ChAdOxl destination vector (P2563) using Gateway® LR Clonase®, to generate ChAdOxl MenB.l. After transforming competent bacteria (NEB 5-alpha), recombinant colonies were selected using antibiotic selection (Chloramphenicol). The selected colony was expanded and a midi DNA purification was performed. DNA sequencing was used to verify the construct. The characterized ChAdOxl MenB.l (OVG98) was digested with Pmel enzyme for linearization. Linearised plasmid was submitted to CBF.
The ChAdOxl vector used to derive ChAdOxl MenB.l was generated at the Jenner Institute, and its complete genome sequence is known. The sequence of the transgene region in ChAdOxl MenB.l has been verified by (i) sequencing directly from phenol purified viral genomic DNA; and (ii) sequencing of DNA amplified by PCR. The primers for both sequencing methods were the same. The DNA map of pBAC ChAdOxl MenB.l (also called MenBOx1) used to generate the recombinant viral vector vaccine is shown in Figure 5.
Manufacturing of clinical lot
Pre-GMP Starting Material The adenoviral destination vector, plasmid number OVG 98-11 (ChAdOx1-fHbpi.i S223R), was obtained from OVG.
The plasmid was transformed into competent E. coli DH5C1 and transformants plated onto
LAgar Vegitone plates containing chloramphenicol. A single colony from the transformation plate was re-streaked onto a chloramphenicol plate and a single colony picked for plasmid preparation. The plasmid was grown up in LB Vegitone and DNA was isolated using a Qiagen
Endofree Plasmid Maxi kit. The purified destination plasmid was digested with Pmei to release the Non-Viral Plasmid Sequence.
HEK 293 cells were transfected with 10 μg of unpurified digest and approximately 300 ng of purified adenoviral genome fragment. Both transfections were harvested, the digest transfection material frozen down and the fragment transfection expanded in HEK 293 cells. Three Hyperflasks were each infected with 1 mL of lysate. The cells were harvested when cytopathic effect (CPE) was evident.
The cells were harvested by centrifugation. The pellets were resuspended in cell lysis buffer, frozen, thawed and treated with Benzonase® endonuclease to reduce host cell DNA. After two further rounds of freezing and thawing the lysate was clarified by centrifugation to remove cell debris. The resulting clarified lysate was purified by two rounds of caesium chloride ultracentrifugation. The virus band was harvested. This was dialysed against three changes of formulation buffer (10 mM Histidine, 35 mM NaCl, 1 mM MgC12, 0.1 mM EDTA,
0.5 % (v/v) ethanol, 7.5 % (w/v) sucrose, 0.1 % (w/v) PS80, in Water for Injection, at pH
6.6) to remove caesium chloride. The virus particle concentration was established and the virus filtered through a 0.22 micron sterilising filter and frozen at -8o°C (nominal). This material was designated as ChAdOx1 MenB.1 pre-GMP starting material.
All tests undertaken on the ChAdOx1 MenB.1 pre-GMP starting material meet the specifications defined in the ChAdOx1 MenB.1 Product Specification
Clinical Lot
The pre-GMP Starting Material was used to infect a GMP-manufactured, suspension HEK293 cell line, at a Multiplicity of Infection (MOI) of 2.5. After 47.5 hours the virus- infected cells were harvested by centrifugation to remove their culture supernatant, resuspended in lysis buffer (io mM Tris, 135 mM NaCl, 1 mM MgC12) and frozen at -8o°C (nominal). A proportion of the cells were lysed by thawing and freezing three times in total to release the virus particles. The lysed cells were centrifuged to remove cell debris and the clarified lysate stored in small aliquots at -8o°C as the ChAdOx1 MenB.1 Master Virus Seed Stock (MVSS). The remaining frozen cells were thawed and treated with Benzonase® endonuclease to reduce host cell DNA levels. The lysis procedure continued by freezing and thawing for a total of three times. After centrifugation the clarified lysate was stored at -8o°C as the ChAdOx1 MenB.1 Bulk Harvest Lot (BHL) until further processing.
Aliquots of BHL were thawed and purified by two rounds of caesium chloride density ultracentrifugation. The resulting pure virus band was removed from each ultracentrifuge tube, pooled and dialysed three times to remove caesium chloride and buffer-exchange into
Formulation Buffer. The purified 10 mM Histidine, 35 mM NaCl, 1 mM MgC12, 0.1 mM EDTA,
0.5 % (v/v) ethanol, 7.5 % (w/v) sucrose, 0.1 % (w/v) PS80, pH 6.6 virus was then adjusted for concentration by the addition of Formulation Buffer and filtered (0.45 micron) to remove any larger aggregates. The resulting Purification Lot (PL) was stored at -8o°C.
Once the PLs were tested and shown to meet the specifications defined in the Product Specification they were thawed, pooled and filtered (0.45 micron) to become the Bulk Purified Lot (BPL).
The BPL was sterile-filtered (0.22 micron) in a EU GMP Grade A pharmaceutical isolator to generate the Bulk Product which was then manually dispensed into 0.45 mL aliquots in sterile glass vials. These were stoppered and sealed and removed from the isolator for inspection. All accepted and rejected vials were labelled accordingly and stored at - 8o°C as Final Drug Product.
Validation of analytical Procedures: The methods used are the same as for the drug substance and have been qualified to be fit for purpose (viral particle and infectious titres, pH, and the ELISA based assays to detect residual host cell protein and Benzonase). All tests carried out by external contractors have been validated and carried out to meet the requirements of cGMP or GLP for the in vivo assays. Stability testing of ChAdOxl MenB.l (ATA)
The stability testing was carried out on the Toxicology and Stability batch reference D525- P24 produced by the Process and Development team at the CBF. Fifty vials were produced and an aliquot of this material was used for infectivity and viral particles assays which were determined to be 3.52 X 109 ifu/mL and 5.03 X 1011 vp/mL, respectively. This gave this batch reference D525-P24 a P to I ratio of 142.9 : 1. Five vials were pooled and diluted to 2.0 X 1011 vp/mL, and the rationale behind this was that the concentration of viral particles in the Toxicity & Stability batch should be the closest possible to the anticipated vp/ml of the clinical batch, which was expected to be between 1.0 X 1011 and 2.02 X 1011 vp/mL. The resulting 12 vials were labelled ChAdOxl MenB.l, Tox & Stability batch, ATA, 15 Nov. 2016,
0.8 mL. Diluted, RPT-16-037 Vial 51-62 were stored at the accelerated degradation temperature of 2 to 8 °C. The stability study commenced on 15 Nov. 2016, which was set as time point o. The time line for stability study and the results obtained so far are depicted in the table below:
Figure imgf000057_0001
Figure imgf000058_0001
The ChAdOx1 MenB.1 vaccine is stored according to GMP at -80 °C (nominal) at the Clinical BioManufacturing Facility, University of Oxford. Considering prior experience with other adenoviruses and the internal stability study, the clinical lot (ChAdOx1 batch 03D16-01, date of manufacture 24 November 2016) was assigned an expiry date of 24 November 2017. CBF continues to carry out a shelflife extension program on clinical vials stored at the same temperature. Prior to the expiry date of the clinical lot, virus was retested using a potency assay based on in vitro infectivity in HEK 293 cells using an anti-hexon immunostaining assay. The result were between plus or minus half a log from the release assay and so the clinical lot was deemed suitable for continued clinical use as there is no evidence of deterioration of the product or of the stability test material. The clinical material was assigned a new expiry date of a further 12 months (Current date of expiry is 24 November 2018). This process will be followed up to 36 months without submitting a substantial amendment.
Va ccine form ulation a nd p ackaging
The ChAdOx1 MenB.1 vaccine is formulated in 10 mM Histidine, 35 mM NaCl, 1 mM MgC12,
0.1 mM EDTA, 0.5 % (v/v) ethanol, 7.5 % (w/v) sucrose, 0.1 % (w/v) PS80, in Water for Injection, at pH 6.6.
The vaccine is stored frozen (-8o°C nominal) in Type 1 glass, particle free (as per USP or Ph. Eur. Method), sterile and depyrogenated vials each containing 0.45 mL. They are stoppered with bromobutyl rubber stoppers and sealed with aluminium crimps.
The vaccine vials are single use.
No nclinical Studies
Four types of non clinical studies have been performed:
1. Expression of the antigen in mammalian cells upon infection with ChAdOx1 MenB.1, and capacity to bind human fH. 2. Immunology and biological activity studies of ChAdOx1 MenB.1 have been performed in mouse models (outbred, inbred and transgenic for human fH).
3. Study of biodistribution has not been performed with ChAdOx1 MenB.1, given the results of other biodistribution studies of replication-deficient simian adenoviruses, which are presented in this brochure.
4. A separate study was performed to evaluate the toxicity of ChAdOx1 MenB.1 administered in a more intensive manner than anticipated in the clinical protocols.
Biological activ ity of ChAdOxi MenB.i in m am m alian cells
Expression of fHbp antigen in mammalian cells infected with ChAdOx1 MenB.1, and its capacity to bind human fH was investigated using a flow cytometry-based assay. HeLa cells were infected overnight with the ChAdOx1 MenB.1 adenovirus at a MOI of 500. Any adenovirus that had not infected the cells was subsequently removed by washing each sample. Expression of fHbp on the cell surface and intracellular compartment was detected using a monoclonal antibody specific to fHbp variant 1.1. Results show that ChAdOx1 MenB.1 is able to express fHbp (Figure 6 A). For negative control, cells were infected with an adenovirus expressing an irrelevant antigen (NadA). In these cells, no expression of fHbp was detected. The positive controls were cells infected with human serotype 5 recombinant adenovirus expressing wt fHbp or the mutated fHbp S223R, and in both cases expression of fHbp was detected (Figure 6 A).
The capacity of fHbp expressed in infected cells to bind human fH was also verified: cells infected with recombinant adenoviruses were incubated with human fH, and bound fH was detected with a monoclonal antibody against human fH. Results show that fH binding was detected in up to 73% of live cells infected with an adenovirus expressing wt fHbp, as expected, but not with adenoviruses (HuAd5 or ChAdOx1) expressing the mutated fHbp S223R (Figure 6B).
Im m unogenicity and b iologica l activ ity of ChAdOxi MenB.i in m o use m odels
SBA was performed on serum samples from groups of mice immunized with ChAdOx1 MenB.1 to measure the immunogenicity and functionality of the vaccine-induced antibody response. The SBA is a measure of efficacy as a bactericidal titre superior to 1:4 is expected to correlate with protection. The two licensed MenB vaccines (4CMenB and rLP2o86) were progressed to clinical development and obtained licensure based on the results of this assay. SBA assay was performed according to the current version of SOP OVGLO56 (MenB serum bactericidal assay vi.o), which follows the standardized method developed by PHE (17). Blood samples were centrifuged and serum separated and stored at -200C until use in SBA. Human complement-mediated SBA titres were measured in individual sera, using wild-type 44/76-SL as the target strain (gift from Prof R. Borrow, PHE North West Laboratory, Manchester). This strain expresses fHbp variant 1.1. The complement source was obtained from healthy donors who had provided written informed consent (Ethics number 10/H0102/23). The donation trial was conducted in accordance with the clinical trial protocol and the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization (ICH) Good Clinical Practices standards. The analyses were performed in microtiter plates, and the colony forming units (CFU) were counted using an automated counter (Synbiosis Protocol 3 colony counter). The bactericidal titres were defined as the reciprocal of the serum dilution that killed at least 50% of the organisms.
Im m unogenicity and functional activ ity elicited in o utbred m ice (CD1) im m unized w ith a single dose of Ch Ad 0x1 MenB.l as comp ared to Hu Ad 5 v ector
Groups of mice (n=5) were immunized with 1x109 iu ChAdOx1 MenB.l or the human serotype 5 counterpart containing the same insert (HuAd5-fHbpi.iS223R).
Immunization was given once by IM route. Serum samples were collected at 2 and 6 weeks, and SBA measured as described above. Results show that ChAdOx1 MenB.l elicited a robust SBA response as soon as two weeks post injection, and the response was higher at week 6 (Figure 7).
Dose-resp onse studies in o utbred m ice (BALB/c) and inbred m ice (CD1) im m unized w ith a single injection of ChAdOxi MenB. l
Groups of mice (n=6) were immunized with different doses of ChAdOx1 MenB.l once by IM. Naive mice were not immunized and a group of mice immunized with the licensed vaccine 4CMenB (1/ 10th of the human dose) was added as a comparator and positive control. This group received a second injection at week 3 to reflect the minimum 2-dose schedule recommended by the manufacturer, as one dose of this vaccine is not able to elicit SBA in humans. Blood samples were obtained 6 weeks post adenovirus injection. Results showed that in inbred BALB/c mice, the SBA response is dependent of the dose administered and all mice respond to a minimum dose of ix108iu. In outbred mice, the variability between mice is higher than in inbred, as expected, and all mice respond to a minimum dose of 5x108iu. (Figure 8).
Comp arison of im m unogenicity in three m o use strains : o utbred m ice (CD1) and inbred m ice (BALB/c and C57BL6)
Mice were immunized with 5x10s iu of ChAdOx1 MenB.l, and bled 6 weeks after. The comparator licensed vaccine 4CMenB was also used as described above (2 doses at weeks o and 3). This study showed that ChAdOx1 MenB.1 is immunogenic in all strains
(Figure 9)
Im m unogenicity and functional activ ity elicited in transgenic m ice expressing hum a n fH
Transgenic BALB/c mice expressing human fH were used to assess the SBA activity elicited by ChAdOx1 MenB.1 in the presence of human fH (better reflecting the human situation). Groups of mice (n=io) were immunized with ChAdOx1 MenB.1 or the human serotype 5 counterpart encoding the same antigen, once by IM, with 109 iu/mouse. Naive mice were not immunized. This experiment lasted 21 weeks in order to observe the persistence of the antibody responses. The comparator group was given a 3rd dose of 4CMenB to reflect the schedule recommended for infants by the manufacturer. All mice immunized with ChAdOx1 MenB.1 elicit a SBA response by week 5 post injection, while this is only achieved after 2 injections of 4CMenB, which contains the wt fHbp that binds human fH in these transgenic mice. The SBA titers in mice immunized with ChAdOx1 MenB.1 persisted at high levels until week 21 when the experiment was terminated (Figure 10 ).
Imm unopotency of ChAdOxi MenB.i (clinical lot) he immunopotency of ChAdOx1 MenB.1 clinical vials was confirmed: four CD1 outbred mice were immunized IM with 108 iu, sacrificed after 6 weeks and blood collected for use in SBA as described above and following SOP OVGL 056. The experiment comprised naive animals and a comparator group immnunized twice with 4CMenB (Figure 11). The pivotal, non-clinical studies were conducted in laboratories in the UK working to the principles of GLP. The laboratories are not part of a formal GLP accreditation program, however a fully functional quality management system and the work was conducted according to approved SOPs by qualified personnel who are experts in these laboratory assays.
Biodistrib ution of rep lication deficient sim ian adeno v iruses
A biodistribution study for ChAdOx1 MenB.1 was not included in this Example. Biodistribution studies have been performed with three recombinant viral vectored vaccines based on El, E3-deleted simian adenovirus C63, as well as one human adenovirus 6 vectored vaccine.
In a study conducted in 2006, tissue samples were obtained to assess the potential tissue distribution of the test substance AdCh63 ME-TRAP (group E chimpanzee adenovirus expressing a malaria antigen) in BALB/c mice up to 8 days following a single intradermal injection into each pinna (3.3 x 109 viral particles (vp). The preclinical GLP study was performed at Huntingdon Life Sciences Ltd with analyses for the biodistribution study being performed at the University of Oxford. No infectious AdCh63 ME-TRAP virus particles were detected in any internal organ (reproductive organs, spleen, liver, cervical lymph nodes). The results demonstrated that one week after intradermal injection, AdCh63 ME-TRAP was only detected at the injection site. The amount of AdCh63 ME-TRAP virus detected after one week was considerably reduced from that detected on day 1 samples. There was no evidence of replication of the virus or presence of disseminated infection. The results were therefore consistent with the injection of a non-replicating virus.
In a separate study, AdCh63 MSP-i (another malaria antigen) was administered intramuscularly (the chosen route now used in clinical trials of adenoviruses), and virus was detected at the injection site immediately after injection, but not in organs or injection site at one week. As above, these results were consistent with the injection of a replication deficient virus as virus was no longer detectable at any site after 24 hours. A study was also conducted with the vaccine AdChsNSmut (encoding a hepatitis C virus antigen). Animals were injected IM with 6.08 x 109 vp into each quadriceps. The results showed that one hour post-injection, AdChsNSmut particles were found in the quadriceps (injection site) and in regional lymph nodes, and not in any other organs (liver, spleen, reproductive organs). One week after intramuscular injection, AdChsNSmut was barely detected only in the regional lymph nodes. There was no evidence of replication of the virus or presence of a disseminated infection. The results were consistent with the injection of a non-replicating virus.
Another study was conducted with the replication deficient human adenovirus vectored hepatitis C vaccine Ad6NSmut. One hour post-injection, Ad6NSmut particles could be found in the quadriceps (injection site) and in regional lymph nodes, and not in any other organs.
One week after intramuscular injection, Ad6NSmut was barely detected only in the regional lymph nodes. There was no evidence of replication of the virus or presence of a disseminated infection. The results were consistent with the injection of a nonreplicating virus.
Four other replication-deficient simian adenovirus vectored vaccines, ChAdV63.HIVconsv
AdCh63 AMAi, ChAdOx1 NP+M1, and ChAdOx1 Ag85A have been used in approved clinical studies without performing separate biodistribution studies as it was considered that sufficient information concerning the biodistribution of vaccines based on replication-
6o deficient El and E3 deleted simian adenoviruses was available and that further studies would involve use of animals in experiments without providing any useful information.
Toxicology study of ChAdOxl MenB.l (En v igo rep ort KH75CL)
Study Design and Structure
Identity of treatm ent groups (M - Male ; F - Fem ale)
Figure imgf000063_0001
The strain and species used in the nonclinical repeat dose toxicology study was Balb/c mouse.
The study was conducted in compliance with the following Good Laboratory Practice standards and the data generated considered to be valid.
The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994).
OECD Principles of Good Laboratory Practice (as revisedin 1997), ENV/MC/CHEM(98)17.
EC Commission Directive 2004/10/EC (Official Journal No L 1504).
These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), Japan (MHLW, MAFF and METI), and other countries that are signatories to the OECD Mutual Acceptance of Data Agreement.
Laboratory investigations
Figure imgf000063_0002
Res ults Mortality
There were no unscheduled deaths on the study.
Clinical signs
There were no clinical signs considered clearly related to treatment and there was no apprent reaction to treatment at the dose site.
Body we ight
There was no effect of treatment on the overall group mean weight gain during the study.
Food consum ption
There was no effect of treatment on food consumption.
Haematology
Haematological investigations performed on Day 28 revealed slightly higher group mean circulating total white blood cell counts (WBC) for treated males (1.8X control) with all differential cell types affected (between 1.4X and 2.0X control) and with the difference from controls for neutrophil, lymphocyte, eosinophil and large unstained cell counts attaining statistical significance.
A slightly higher than control group mean red cell distribution width was observed for treated males and females (males 1.05X control; females 1.04X control).
Values for other parameters, some of which attained a level of statistical significance were considered to be within the expected ranges for mice of this age and strain. Further comment awaits the results of the histopathological examination.
Blood chem istry
A slightly lower group mean alkaline phosphatase activity was observed for treated females (0.90X control). Group mean alanine aminotransferase and aspartate aminotransferase activity was higher (2.5X and 1.8X control respectively) for treated females.
Group mean creatinine concentration for treated males males and females was slightly higher than control (males 1.2X control; females 1.6X control).
Group mean triglyceride concentration for treated males males and females was slightly lower than control (males 0.79X control; females 0.68X control), however examination of the individual data revealed considerable overlap in the concentrations. Group mean pottasium concentrations were higher for treated males and females (males 1.2X; females 1.3X control), with the difference from controls attaining a level of statistical significance. The group mean phosphorus concentration was higher for treated females (1.2X control),
Other differences between the control and treated group means, including those attaining a level of statistical significance were slight in degree or there was considerable overlap between groups in the range of the individual data. Further comment awaits the results of the histopathological examination.
Organ weights
Analysis of organ weights obtained from animals killed on Day 28 did not reveal any changes considered to be related to treatment.
All intergroup differences in organ weights were considered to fall within the expected range for this age and strain of animal.
Macropathology
- Anim als Killed After 2 Weeks of Treatment
The macroscopic examination performed after 2 weeks of treatment revealed the following changes in the right lumbar lymph node:
Right lum bar lymph node
Enlargement was noted in one male and one female treated animal.
Sum mary of findings in the right lum bar lymph node for anim als killed after 2 weeks of treatment
Figure imgf000065_0001
The incidence and distribution of all other findings were considered to be unrelated to treatment.
Example 4 - Effects in Humans
Previous and current clinical studies
There is no previous study of ChAdOx1 MenB.1 in human, however the vector ChAdOx1 has been used in previous clinical trials at the university of Oxford (ChAdOx1), and three variants of the antigen fHbp have been used in thousands of individuals including 2 months old infants during clinical development of 4CMenB and rLP2o86, and since post-licensure use in the UK (including 2 months old infants) and in the US (in adolescents and adults). Both the vectors and the antigen have been shown to be safe, moderately reactogenic and able to induce strong immune responses in different age groups.
The described phase I study (ChAdOx1 MenB.1 Phase I trial, 0VG2017/04) will evaluate the safety and immunogenicity of various intramuscular doses of ChAdOx1 MenB.1 in healthy adults, and provide a proof of concept that a bacterial outer membrane protein expressed from an adenovirus vector can induce bactericidal response in human. The study will assess the safety and immunogenicity of ChAdOx1 MenB.1 in man. Specifically, the immunogenicity following a single or two doses of ChAdOx1 MenB.1 will be evaluated for:
Serum bactericidal activity of antibodies
Antibody concentrations against wt and mutated fHbp Antibody concentration against human fH
Circulating B-cells and T-cell responses specific for fHbp Serum opsonophagocytic activity of antibodies
Gene expression profile and genetic host factors in relation to vaccine response
Marketing Exp erience
ChAdOx1 MenB.1 vaccine is not licensed in any country.
Reference Safety Inform ation
There have been no human studies conducted with the ChAdOx1 MenB.1 vaccine. The following adverse events may occur in some volunteers following vaccination with ChAdOx1 MenB.1, based on previous experience with other simian adenovirus viral vectored vaccines:
• Injection site pain
• Injection site erythema
• Injection site warmth
• Injection site swelling
• Injection site pruritus
• Myalgia
• Arthralgia
• Headache
• Fatigue
• Fever
• Feverishness
• Malaise
• Nausea
These adverse events are expected to be primarily mild in severity, however occasional moderate or severe adverse events have been reported. These adverse events are expected to last for approximately 24-48 hours following vaccination, though adverse events of longer duration have also been reported. As this is a first in human vaccine, there is no specific RSI for ChAdOx1 MenB.1 as yet. All SAEs at least possibly related to ChAdOx1 MenB.1 will be considered unexpected and be reported to the MHRA and REC as SUSARs within the regulatory timelines (15 days for all SUSARs unless life threatening in which case 7 days, with a final report within a further 8 days).
SUMMARY DATA AND GUIDANCE FOR THE INVESTIGATOR
The described phase I, single centre, open-label dose-escalation study will assess the safety and immunogenicity of two doses of 2.5XIO10 vp or 5x10 lovp of meningococcal capsular group B vaccine ChAdOx1 MenB.1 administered intramuscularly to healthy subjects. It will be the first planned administration of the vaccine in man. The data from this phase I study will assist in the design of future clinical studies in decreasing age groups until safety and efficacy can be assessed in infants, the ultimate target population for the ChAdOx1 MenB.1 vaccine.
Characteristics of the ChAdOxi MenB.i IMP
The vaccine consists of the replication-deficient (El and E3 deleted) simian adenovirus vector ChAdOx1, containing a genetic cassette encoding for the meningococcal capsular group B antigen fHbp variant 1.1 with a point mutation to prevent binding to the natural human ligand, fH (S223R). The drug product is composed of a drug substance (purified adenovirus particles) suspended in a 10 mM Histidine, 35 mM NaCl, 1 mM MgC12, 0.1 mM EDTA, 0.5 % (v/v) ethanol, 7.5 % (w/v) sucrose, 0.1 % (w/v) PS80, in Water for Injection, at pH 6.6 buffer. Release testing of the vaccine product confirmed it passed all pre-defined specification criteria, e.g. identity, potency, endotoxin content and sterility. The product was found to be stable and passed the specification after 3 months stability studies both under normal and accelerated storage conditions.
In the non-clinical studies there were no toxicological or other findings indicating any safety issues beyond those expected for an adenovirus-based vaccine product. Animal studies showed that no systemic toxicity was observed, and the vaccine was immunogenic and effective in generating bactericidal responses that correlate with protection.
Non-clinical studies performed with the ChAdOx1 MenB.1 vaccine do not indicate any potential risks that require further evaluation.
There have been no human studies conducted with the ChAdOx1 MenB.1 vaccine. However ChAdOx1 MenB.1 vaccine is similar to vaccines based on chimpanzee serotype replication deficient adenoviruses that have been used safely in humans. The production process, formulation and manufacturing site is similar to that as for ChAdOx1 NP+Mi, ChAdOx1 Ag85A and ChAdOx1.5T4. Reports from clinical studies of ChAdOx1 NP+Mi and ChAdOx1 Ag85A vaccines have demonstrated that they are moderately reactogenic, and safe. In addition, ChAdOx1 MenB.1 expresses an antigen that has been used in thousands of individuals in clinical trials and post licensure.
Guidance for the Investigator: contraindications and precautions for use The study vaccine (ChAdOx1 MenB.1) should not be administered to individuals with known or presumed hypersensitivity to any component of the vaccines. Prior to administration of any dose of any of the vaccines, the volunteer should be questioned about the occurrence of any possible adverse events following any previous dose(s). Do not administer ChAdOx1 MenB.1 intravenously. This vaccine is intended for IM administration.
Inclusion and exclusion criteria for study participants, procedures for supervision following vaccine administration and emergency procedures are described in the phase I clinical protocol.
Safety data will be collected as part of the main objective of the phase I study.Participants will be seen and examined frequently during the first 28 days after vaccination to evaluate the physical manifestations of the immunisation, and selected subjects will have blood drawn to assess the biochemical parameters associated with organ toxicity. They will be followed for 6 months or longer depending on the protocol and will enrol in a product development registry to provide long-term safety follow-up.
Allergic and hypersensitivity reactions to the vaccine are a reasonable expectation. Severe hypersensitivity reactions, including shock and respiratory insufficiency, are always a theoretical possibility with any immunisation. Subjects will be monitored for 60 minutes after administration of study vaccine to ensure no acute allergic events are in process and to see if a febrile response is developing. All clinical staff are trained and can provide evidence of competency in the acute management of anaphylaxis reactions including the use of intra- muscular adrenaline.
Nam es and address of com panies involved in manufacture and regulation
Manufacturing of Product Name: Clinical BioManufacturing Facility (CBF) Address: Old Road, Headington, Oxford OX3 7JT, UK
Product toxicity testing Name: Envigo CRS imited Address: Woodley Road, Alconbury, PE284HS UK
Release of Product for trial Name: Clinical BioManufacturing Facility (CBF) Address:
Old Road, Headington, Oxford OX3 7JT, UK
Example 5 - Vambox clinical trial: additional data The examples above already presented the following data from the clinical trial, for the groups immunized with the novel vaccine ChAdOx1 MenB.1 (sometimes referred to as 'Vambox') or with a known licensed vaccine 4CMenB (Bexsero®) :
Serum bactericidal antibody titers (SBA) against strain H44/76, conducted in house, up to day 208 (Figure 29 and 31)
T cell responses to the antigen fHbp up to day 208, Figures 30 and 31
Samples are now available for participants vaccinated with another licensed vaccine rLP2o86 (Trumenba®). Serum antibody assays were performed by an independent laboratory (PHE, Vaccine evaluation Unit, Manchester, UK), against the homologous strain H44/76-SL, as well as against strain M01240355, expressing a mismatched fHbp variant (3.4). We refer to Findlow et al 2010 (Clin Infect Dis. 2010 Nov 15551 (io):ii27-37) - specifically Table 1 of Findlow et al 2020, for details of these known strains, especially strain M01240355. As previously, the study size is not powered to detect statistical differences.
SBA titers against strain H44/76-SL indicate that some of the participants had SBA responses at baseline, as expected due to asymptomatic carriage (Fig. 34A). A single injection of either ChadOx1 MenB.1 or 4CMenB increased SBA titers one and six months later (Fig. 34B and 34C). At that time, groups were further divided to receive either no boost, or a second dose of homologous vaccine, or a heterologous boost with ChadOx1 MenB.1 as indicated in the X-axis (Figure 35). Results at day 208 and 365 indicate that a second dose of ChadOx1 MenB.1 induced higher SBA titers than a single dose.
Vaccination with ChAdOx1 MenB.1 elicited lower SBA responses to strain M01240355: a single injection of ChAdOx1 MenB.1 mildly increased the SBA response at day 28 (Fig. 36B). and a further injection at six months did not modify the SBA responses (Fig.37).
Results were also expressed as proportion of participants with a SBA titer > or = 1:4 (Fig. 38). Vaccination with ChAdOx1 MenB.1 elicited protective responses similar to one or two doses of 4CMenB and higher responses than one dose of rLP2o86 against strain H44/76-SL (Fig. 38A). SBA responses > 1:4 were maintained in more than 75% of participants at day 365 against this strain (Fig. 38B). Lower responses were elicited against strain M01240355 (Fig. 38 A and B).
IgG Memory B cell responses to fHbp were elicited after the first vaccine dose, and were increased after a second dose. ChAdOx1 MenB.1 was able to increase the B cell response induced by a previous 4CMenB dose (Fig. 39A). High IFN-gamma responses were elicited by ChAdOx1 MenB.1 immunization, either alone or after 4CMenB prime (Fig.
39B).
Cytokine responses were quantified 1 day after the first or second dose of vaccine (Fig.40). ChAdOx MenB.1 vaccination increased the plasma levels of interferon (IFN) gamma, interleukin (IL) 6, IL-10 and tumour necrosis factor (TNF) alpha.
References
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Prevention and control of meningococcal disease: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep [Internet]. 2013;62(RR-2):I-28.
3. Finne J, Leinonen M, Makelii PH. ANTIGENIC SIMILARITIES BETWEEN BRAIN COMPONENTS AND BACTERIA CAUSING MENINGITIS. Implications for Vaccine Development and Pathogenesis. Lancet. i983;322(8346):355-7.
4. Rollier CS, Dold C, Marsay L, Sadarangani M, Pollard AJ. The capsular group B meningococcal vaccine, 4CMenB : clinical experience and potential efficacy. Expert Opin Biol Ther [Internet]. Informa Healthcare; 2015 Jan 8 [cited 2016 May 5];15(1): 131-42.
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6. Giuntini S, Reason DC, Granoff DM. Complement-mediated bactericidal activity of anti- factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding. Infect Immun. 2011;79(9):3751-9.
7. Konar M, Granoff DM, Beernink PT. Importance of inhibition of binding of complement factor H for serum bactericidal antibody responses to meningococcal factor H-binding protein vaccines. J Infect Dis. 2013;208(4):627-36. 8. Costa I, Pajon R, Granoff DM. Human factor H (FH) impairs protective meningococcal anti-FHbp antibody responses and the antibodies enhance FH binding. MBio.
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9. Beernink PT, Shaughnessy J, Braga EM, Liu Q, Rice PA, Ram S, et al. A meningococcal factor H binding protein mutant that eliminates factor H binding enhances protective antibody responses to vaccination. J Immunol [Internet]. 2on;i86(6):36o6-i4.
10. Granoff DM, Ram S, Beernink PT. Does binding of complement factor H to the meningococcal vaccine antigen, factor H binding protein, decrease protective serum antibody responses? Vol. 20, Clinical and Vaccine Immunology. 2013. p. 1099-107.
11. Giuliani MM, Adu-Bobie J, Comanducci M, Arico B, Savino S, Santini L, et al. A universal vaccine for serogroup B meningococcus. Proc Natl Acad Sci U S A [Internet]. 20o6;iO3(29):io834-9.
12. Ewer KJ, Lambe T, Rollier CS, Spencer AJ, Hill AVS, Dorrell L. Viral vectors as vaccine platforms: From immunogenicity to impact. Vol. 41, Current Opinion in Immunology.
2016. p. 47-54-
13. Top FH. Control of adenovirus acute respiratory disease in U.S. Army trainees. Vol. 48, Yale Journal of Biology and Medicine. 1975. p. 185-95.
14. Afolabi MO, Tiono AB, Adetifa UJ, Yaro JB, Drammeh A, Nebie I, et al. Safety and
Immunogenicity of ChAd63 and MVA ME-TRAP in West African Children and Infants. Mol Ther. 2016; 1-8.
15. Tatsis N, Blejer A, Lasaro MO, Hensley SE, Cun A, Tesema L, et al. A CD46- binding chimpanzee adenovirus vector as a vaccine carrier. Mol Ther [Internet]. 2007;15(3):6O8-17.
16. Dicks MDJ, Spencer AJ, Edwards NJ, Wadell G, Bojang K, Gilbert SC, et al. A novel chimpanzee adenovirus vector with low human seroprevalence: Improved systems for vector derivation and comparative immunogenicity. PLoS One. 2012;7(7).
17. Borrow R, Balmer P, Miller E. Meningococcal surrogates of protection - Serum bactericidal antibody activity. In: Vaccine. 2005. p. 2222-7 Example 5 - An Exem plary Viral Vector Construct ('Vaccine ') Against
Capsular Group B Meningococcal Disease
Here we describe a novel adenovirus vaccine against Meningococcus based on an adenoviral vector. We show the preclinical development, evaluation and optimization for clinical development.
Adenoviral vectors are at the forefront of vaccine development for cancer, viruses and parasitic diseases. However, the expression of a bacterial protein in a eukaryote cell may impact on the antigen's localization and, more importantly, conformation. Nevertheless, their potential to induce T Helper type i and high antibody responses after a single dose in humans is attractive to combat the disease and disability caused by capsular group B meningococcus (MenB). Therefore, the potential of adenovirus (Ad) viral vectors as a delivery platform for MenB antigens factor H binding protein (fHbp) and Neisserial Adhesin A (NadA) was investigated. The Ad vectored vaccines generated high antigen-specific antibody responses in mice after a single dose. A subset of the vaccines expressing fHbp variants induced functional serum bactericidal responses, with protective titres superior or equal to the titres induced by two doses of protein-based licensed comparators, that also persisted longer. The Ad-fHbp candidate was optimized for human use by the use of judicious variants and progressed to clinical development.
Here we show preclinical development and testing of the exemplary ChAdOx1 MenB vector construct based on the fHbp antigen. We show data with several variants of the vectors of the invention, including using AdHu5 as the vector before moving to the preferred ChAdOx1. A single dose of the candidate vaccine was shown to induce surprisingly high levels of antibody that were functional by SBA. Comparative experiments showed that a single dose of the adeno vectored vaccine induced antibody levels that were higher than a licensed vaccine (qCMenB "Bexsero"), that these levels were not significantly improved on by using heterologous prime-boost approaches and that the immunity induced by a single dose of the Ad-fHbp vaccine appears to be more durable than that induced by qCMenB. We describe the development of the preferred exemplary construct based on the ChAdOx1 vector. We also tested a construct based on ChAd0x2 and found comparable results.
For comparative purposed we also describe attempts to make an AdHu5 vaccine based on another MenB protein (NadA) but, as with PorA and FetA (see Example 1), the anti- NadA antibodies were not functional by SBA. Introduction
Meningococcal bacteria are the leading cause of childhood meningitis and septicaemia in the UK . Given the dramatically rapid progression of this life-threatening infection, vaccination is considered the only strategy to conquer one of the most devastating infectious diseases of childhood. Effective vaccines against the capsular groups A, C, W and Y leaves group B meningococcus (MenB) as the major cause of meningococcal disease and deaths in several developed countries (Ladhani 2016 DOI: 10.1136/archdischild-20i5-3o8928). Unlike other serogroups, the serogroup B polysaccharide cannot be used as a vaccine target mainly because it is poorly immunogenic in non-human primates (Devi 1997 PMID: 9038314). Therefore, MenB vaccines are based on subcapsular protein antigens (Bjune 1991 doi: 10.1016/0140- 6736(91)91961-s, Oster 2005 doi: 10.1016/j.vaccine.2005.01.063, Lewis 2009 doi: 10.1586/erv.O9.3O, rollier 2015 doi: 10.1517/14712598.2015.983897, Findlow 2019 doi: 10.1080/14760584.2019.1578217). Two MenB vaccines are licensed, 4-component MenB vaccine (4CMenB, Bexsero®), and rLP2o86 (Trumemba®), based on one or several of those subcapsular antigens. The former also contains an outer membrane vesicle, used to tackle an outbreak in New Zealand (Andrews 2014 doi: 10.1016/S1473- 3099(13)70341-4). Both vaccines are licensed for adolescents, but none are included in an adolescent program. Meningococcal carriage is highest in teenagers and young adults (Christensen 2010 DOI: 10.1016/81473-3099(10)70251-6), and preventing carriage in this population could drive herd protection. However both vaccines require 2 doses in adolescents and adults (rollier 2015); the persistence of the protective response appears limited (Vesikari 2019 DOI: io.ioi6/j.vaccine.2018.11.073) while carriage and disease is spread over several years in this age groups (Christensen 2013 doi: io.ioi6/j.vaccine.2013.03.034, 2017 doi: 10. ioi6/j.vaccine.2016.11.076); there is no evidence of impact of qCmenB on carriage (Marshall 2020 doi: 10.1056/NEJMoa1900236), thus limiting their cost effectiveness beyond infancy, highlighting the need for an improved solution for adolescents, such as a cheap, single dose vaccine capable of inducing longer persistence of protective antibodies.
In this context, viral based vaccine platforms such as adenoviral and poxviral vectors could provide a potent solution: they induce both innate and adaptive immune responses in mammalian hosts (Tatsis 2004 DOI: i0.i0i6/j.ymthe.2004.07.0i3). They were originally developed for their well-recognised ability to induce potent cellular immunity. However a single dose of adenovirus-based vaccine has been shown to induce potent and rapid neutralizing antibodies, as was demonstrated initially with rabies (Xiang 1996 doi: 10.1006/viro.1996.0239), and confirmed since with several pathogens including in clinical trials, such as SARS-C0V-2 (van doremaleen 2020), malaria (Draper 2008) and Ebola (Miligan 2016). Adenoviral vectored vaccines have been extensively shown to induce strong IFN-gamma producing T cell responses which should provide the ideal conditions for switching to high levels of complement-fixing bactericidal antibody unlike conventional aluminium-based adjuvanted vaccines (Giuliani 2006 https://d0i.0rg/10.1073/pnas.0603940103).
However using viral vectors to induce antibody responses to bacterial outer membrane proteins is hampered by the differences between prokaryotic and eukaryotic expression systems, that may result in a lack of expression of bacterial antigens in mammalian cells, or a loss of protective epitopes due to misfolding or aberrant post-transcriptional modifications (Marsay, hopefully 2020). However, because of successes obtained for bacterial antigens from S. pneum oniae (Arevalo 2009) and Y. pestis (Sha 2016), we designed a series of vectors expressing the protective antigens factor H binding protein (fHbp) (Fletcher 2004, Schneider 2006, Madico 2006, Giuliani 2010), included in both of the licensed vaccines, and Neisserial Adhesin A (NadA), included in 4CMenB (Pizza, Science 2000, Comanducci 2002). The vectors were assessed for antigen expression in mammal cells, and for immunogenicity and induction of protective bactericidal activity. One exemplary candidate was selected and optimized for human use.
Vaccines, recom binant proteins and meningococcal strains
The nucleotide sequences for the antigens NadA 3 and fHbp 1.1 were obtained from the GenBank sequence database (https://www.ncbi.nlm.nih.gov/genbank/). The sequences were codon optimized for mammalian tissue. Recombinant adenoviruses (Ad5, ChAdOx1 and ChAd0x2) were generated as described previously using a Gatewaycompatible entry vector, (Rollier 2019 DOI: 10.1038/S41598-020-61730-8, Folegatti doi: 10.1016/80140-6736(20)31604-4, Wang doi: io.i37i/journal.pntd.ooo687o), using a CMV pro motor and a tissue plasminogen activator signal sequence. NadA inserts were constructed with a C-terminal deletion A 351-4o5 to remove the membrane anchoring domain, and a C-terminal V5 tag to allow antigen detection. The antigens were inserted as 'full length' using the immature sequence, including the signal sequence that is cleaved in the mature protein, or truncated versions (labelled t) where the bacterial signal sequence was omitted (mature protein). Empty or irrelevant adenoviral vectors were used as controls.
The modified vaccinia Ankara (MVA) vectors encoding the same antigens were generated as described previously (https://doi.org/10.1038/nm881).
Native and deoxycholate Outer membrane vesicles (OMVs) were generated and purified as described previously (https://doi.org/10.1371/journal.pone.0148840 and dOMV).
Recombinant NadA and fHbp proteins were produced as previously described. Imm unofluorescence Assay (IF A)
HeLa cells were seeded overnight on high binding protein coverslips (BD Bioscience, NJ, USA) placed in polystyrene six-well culture plates (5x105 cells per well) in complete Dio medium (Dulbecco's modified eagles medium with added penicillin, streptomycin, L-glutamine and foetal calf serum) at 37°C. Adenoviral vectors were added at a molarity of infection of 100, at 37°C overnight. Cells were fixed with 4% paraformaldehyde, permeabilised with 0.2% Triton X-100 for five minutes. Antigen expression (meningococcal antigen fHbp 1.1) was detected with the anti-fHbp monoclonal antibody, JAR4 (supplied by NIBSC) diluted at 1:500 in 1% BSA in PBS, followed by Alexa Fluor 488 conjugated goat-anti-mouse IgG (Life technologies, CA, USA). Cell nuclei were counterstained with DAPI and slides visualised using a Leica DMI3000 B microscope.
Imm unisation experim ents in mice
Procedures were performed according to the U.K. Animals (Scientific Procedures) Act 1986 and were approved by the University of Oxford Animal Care and Ethical Review Committee. Six to 8-week-old female BALB/c-OlaHsd and NIH-OlaHsd, CD1 outbred mice (Harlan, UK) were housed in specific pathogen-free conditions. Blood was collected from tail bleeds or terminal cardiac bleeds at various time points and allowed to clot then centrifuged at 15000 x g for 10 minutes. Sera were aliquoted and stored at - 20°C until use. Spleen, lymph-nodes and bone marrow where applicable were harvested following cervical dislocation under sedation.
Detection of antibodies by ELISA against whole cells or recombinant proteins
Immulon 2HB plates (Thermo Fisher Scientific, MA, USA) were coated with heat killed whole cell preparations of N. meningitidis in PBS (OD 6oonm 0.1), or recombinant fHbp or NadA proteins at 2.5 μg/ml in carbonate bicarbonate buffer (Sigma Aldrich, MO, USA). Samples were serially diluted in 1% BSA PBST-0.05%. High, medium, and low positive quality controls were used in each plate, to ensure assay reproducibility: for the whole cell ELISA, PorA monoclonal antibody P1.7 at 1:20,000; 1:40,000; 1:80,000 dilution respectively; for the rfHbp ELISA, Jar4 monoclonal antibody at 1:100,000, 1:200,000 and 1:300,000. Serum from naive BALB/C mice was used as negative control along with buffer only, diluted 1:4,000 for whole cell ELISA, at dilution 1:400 for subclasses IgG 1 and 2a to rfHbp, and 1:1,000 for IgG, IgG2b, IgG3 to rfHbp. Antibody binding was detected with Horseradish peroxidase-conjugated goat anti-mouse (Jackson ImmunoResearch inc. PA, USA) and visualised with 3, 3', 5,5'- Tetramethylbensidine substrate (TMB, Sigma Aldrich, MO, USA). The reaction was stopped with 50pl H2SO4 and optical densities (OD) were measured at 450nm with reduction at 6oonm. End-point titres for total IgG antibodies were defined as the serum dilution corresponding to the final OD reading above two times the average of naive negative control readings.
Serum bactericidal assay (SBA)
The serum bactericidal assay (SBA) was performed as described previously using 25% (vol/vol) human serum as complement source (Marsay 2015), on complement heat- inactivated murine sera, serially diluted in bactericidal buffer (Hanks Balanced Salt Solution supplemented with 0.5% BSA ). A titre was defined as the reciprocal of the highest dilution of serum that yielded >50% decrease in colony forming units relative to that of control wells within 6omins at 37°C without CO2.
Enum eration of antigen-specific antibody secreting B cells by enzyme- linked im mune-spot assay (ELISPOT)
96 well filtration ELISPOT plate (Millipore™) were coated with recombinant fHbp at 2.5ug/ml or 1:1000 dilution of goat-anti-mouse IgG (Biolegend™, positive controls), or PBS (blank wells). Plates were blocked with complete Dio media, and splenocytes or bone marrow cells added in duplicates at a concentration of 4x105, 2x105 and 1x105 cells per well. Alkaline phosphatase conjugated goat-anti-mouse (Invitrogen™) was added followed by alkaline phosphatase substrate (Bio-RAD). Spot counts performed using an AID ELISpot Reader ELR03 and ELISpot software as described previously (Khatami 2014). Results were expressed as number of antigen-specific spots detected per million cells, minus the number of spots counted in the absence of antigen (medium only). A negative result was recorded as 1 for calculation purposes.
Detection of fHbp expression and human factor H binding was assessed by flow cytometry.
Statistics
Statistical analysis of differences between antibody titres were performed using either Kruskall -Wallis, Mann-Whitney T test, two-way AN0VA with Bonferroni post-tests, or 1 way AN0VA with Dunns multiple comparisons test when appropriate and as stated, using Prism 5 (Graphpad, software Inc. CA, USA),
Both NadA and fHbp encoding adenoviral vaccine vectors are imm unogenic in mice Recombinant replication deficient adenovirus encoding known vaccine antigen targets (PorA, FetA, NadA and fHbp) were created, but the PorA and FetA encoding vectors failed to elicit bactericidal antibody responses (Marsay hopefully 2020). In this study, two versions of the NadA and of the fHbp genes were constructed, full length or truncated as described above, and inserted into Ei-deleted Ad5. Both the resulting Ad5 vectors encoding the full length or the truncated versions of the NadA gene (Ad5-NadA- f and Ad5-Nad-t) were able to produce the antigen in mammal cells, as evidenced by the detection of the V5 sequence, tagged to the C-term of NadA, in infected HeLa cells, above the background observed in cells infected with an empty Ad5 (Fig.i2A). Both vectors encoding the full length and truncated NadA were able to induce antibody responses in mice after a single dose against whole cells from strain 2996, containing a homologous NadA (Fig.i2B). Remarkably, the antibody responses induced by the Ad5 vectors to the single NadA antigen were of comparable magnitude to the response detected in mice immunized with 2996 outer membrane vesicles (0MV), containing many more homologous antigens including the immunodominant PorA (Fig. 12B). IgG subclasses were measured against the homologous 2996 whole cells, and both the Ad- NadA-f and 0MV immunization elicited IgGi, 2a, 2b and 3. Higher levels of IgGi, 2b and 3 were detected in the OMV-immunized mice (Fig. 12C, p<0.01 for IgG1, IgG2b and IgG3).
Similarly, Ad5 vectors encoding a full length and a truncated version of fHbp were able to produce fHbp in target cells, as determined by binding to anti-fHbp monoclonal antibody (Fig. 12A). In addition, both vectors were able to induce antibody responses in mice as soon as two weeks post a single injection, as evidenced by antibody responses against whole cells containing a homologous fHbp variant (Fig. 13A). The antibody titers were of similar magnitude to those induced by homologous native 0MV, containing many more antigens including PorA: endpoint ELISA titres reached 32, GOO- 256, 000 at week 6 after Ad-fHbp immunisation, and 16,000-128,000 with native 0MV (nOMV). An analysis of the IgG subclasses at week 6 indicated that the Ad5-fHbp induced IgGi and IgG2a mainly, while mice immunized with the native H44/76 OMVs induced responses that also included high IgG2b and IgG3 (Fig. 12B). The IgG2a titers were superior in the Ad-immunized mice (Mann-Whitney, p<0.05), while the IgG2b and the IgG3 responses were superior in the nOMV-immunized mice (p<o.ooi and p<o.oi, respectively). The IgG2b and IgG3 responses elicited by the nOMV vaccines could be directed against any of the immunogenic antigens comprised within OMVs.
Bactericidal antibody responses are generated by the fHbp-expressing Ad5 vectors While no SBA response could be detected in the Ad-NadA immunized mice (against strain 2996, data not shown) or 5/99, both full length and truncated fHbp-encoding Ad5 elicited functional antibody responses, as evidenced by the bactericidal activity detected in mice (Fig.isA). Ad-fHbp elicited average SBA titres of 1:512 in mice after a single immunization (ranging from 1:256 to >1:1024 in different independent experiments, data not shown), consistently higher than nOMV-induced responses (1:32 to 1:64, Fig. 13A). Remarkably, the SBA assay against strain H44/76 detects bactericidal antibodies directed to all antigens present in the OMVs, and thus greatly advantages the OMV vaccine. Therefore, other strains were used as targets in the SBA assay: strain BZ83 contains low levels of fHbp variant 1.1, but does not express the PorA contained in the nOMVs used for immunizing mice (H44/76), and thus allows a fair comparison of the fHbp-specific bactericidal antibodies. The strain BZ198 expresses heterologous fHbp 1.5, and the mutant strain mBZi98 expresses fHbp 1.4. The SBA results demonstrate that bactericidal antibody titres elicited by Ad5-fHbp to a single antigen are higher than titres elicited by nOMVs against both homologous and heterologous strains. In addition, OMVs were unable to elicit bactericidal antibody responses against strains with low levels of fHbp, even if homologous (BZ83), or against strains containing heterologous fHbp variants (BZ198, variants 1.5 and 1.4). By contrast, the Ad5-fHbp elicited SBA responses against the low fHbp expressing strain and against strains expressing heterologous fHbp. The SBA titers were still detected 42 weeks post a single dose, when the response induced by the nOMV to the homologous strain had decreased to undetectable levels (Fig. 13A). The Ad-induced SBA titers were dose dependent (Fig. 13B), and were also elicited in outbred mice (Fig. 13C). Interestingly, SBA responses induced by a single dose ChAdOx1-fHbp were higher than those induced by a single dose of the licensed vaccine 4CMenB (Fig. 13D).
Impact of prim e boost regimen using different vaccine platform s Heterologous prime boost regimen using a vectored vaccine construct were shown to induce higher antibody responses as compared with single dose or homologous prime boost modalities (De cassan 2015 doi: 10.3389/fimmu.20i5.00348). Combinations of Ad fHbp prime followed by protein boost, either as recombinant fHbp contained in the licensed vaccine 4CMenB, or using native outer membrane vesicles vaccines (OMVs), thus using fHbp natural presentation, was assessed in mice, and compared with homologous prime-boost or prime-boost-boost of 4CMenB or OMV. Remarkably, the results show that SBA responses induced by multi-dose approaches did not reach higher titers as compared with a single Ad fHbp injection, two weeks post last injection (Fig. 14A), suggesting that the response induced in this mouse model are reaching a plateau at that time point. One of the most immunogenic vaccine regimen with regard to induction of T-cell responses is based on heterologous adenovirus prime, poxvirus boost regimen (Reyes Sandoval DOI: 10.1128/IAI.00740-09). A Thi biased T-cell response maybe associated with better functional responses to protein based meningococcal vaccines, as evidenced by higher SBA titers when using a Thi-inducing adjuvant (Giuliani 2006 https://d0i.org/10.1073/pnas.0603940103). Therefore the heterologous vectored prime boost approach was explored, using an MVA vector encoding the same fHbp 1.1 sequence used in the adenovirus prime. Results showed that two weeks post the last immunization, the prime boost regimen, whether Ad-MVA or MVA- Ad, did not induce significantly higher titers as compared with Ad alone, and that MVA alone induced higher variability and lower SBA responses (Fig. 14B). The effect of vectored combinations regimen on persistence of the SBA response was explored up to week 28 (Fig. 14C), and showed that the responses persisted in all groups, and the mice immunized with MVA alone or three doses of the licensed comparator 4CMenB elicited lower functional antibody responses. There was no statistically significant differences between mice immunized with Ad alone, as compared with Ad-MVA or MVA- Ad at any of the time points tested, although a trend towards higher titers in the latter groups was observed. We further explored whether the prime boost regimen were associated with higher numbers of antigen-specific antibody producing B-cell in the bone marrow and spleen, as these are associated with longer persistence of circulating antibodies (Fig. 14D). While the number of antigen-specific antibody-producing B cells were variable in both organs, higher numbers were detected in the mice that received at least one Ad injection (Ad alone, Ad-MVA or MVA- Ad, Fig. 14D).
Modifications of the vaccine candidate to create a clinically relevant vaccine
Pre-existing immunity to human adenovirus serotypes such as the serotype 5 used here can neutralize the vaccine and thus dampen its immunogenicity, and one solution is to use adenoviruses that do not circulate in humans, such as chimpanzee serotypes (Dicks 2012 doi: io.i37i/journal.pone.oo4O385). Two such vectors were developed previously, ChAdOx1 and Chad0x2 (Dicks 2012 doi: io.i37i/journal.pone.oo4O385; Folegatti 2019 doi: io.339O/vaccines7O2004O) and their immunogenicity compared in mice. In addition, two different CMV promotors were considered, a long and a shorter version described previously (Sridhar DOI: 10.1128/JVL02568-07). The results showed that SBA titers tended to be higher when mice were vaccinated with Ad5, and there was no statistically significant difference between the two clinically relevant backbones (Fig. 15A), and the ChAdOx1 backbone was selected for clinical development. Induction of SBA responses by a single dose of ChAdOx1 fHbp was confirmed in three strains of mice, including an outbred strain (Fig. 15B).
Because a needle free delivery such as liquid mucosal immunization is relevant to immunization of babies and adolescents, and may provide higher levels of protection due to inducing the response at the entry site of the pathogen, intranasal and sublingual delivery of the adenovirus in liquid form was investigated in mice, as a single dose (Fig. 15C). The SBA titers in serum were lower when the mucosal routes were used, but nothing is known from the contribution of potential mucosal responses in protection, or in carriage. Nevertheless, as licensure of meningococcal vaccines is based on the SBA titers in serum, the intramuscular route was selected for clinical proof of concept with ChAdOx1 fHbp. Remarkably, SBA responses induced by a single dose ChAdOx1 fHbp were similar to those induced by qCMenB administered 3 times, 3 weeks apart (Fig. 15D). However, the SBA responses induced by qCMenB decreased faster than the responses induced by a single dose of chAdOx1 fHbp, as evidenced at weeks 21 and 32 (15 and 26 weeks post third qCMenB dose, Fig. 15D).
Mutations of the fHbp transgene can increase the bactericidal response In humans, fHbp binds to the human complement inhibitor factor H, thus decreasing the innate response to the invading bacteria (Schneider 2009 doi: io.iO38/natureO7769). This may affect the anti-fHbp antibody repertoire when fHbp is used as vaccine antigen, and decrease serum bactericidal activity by covering important fHbp epitopes, and led to the generation of mutants fHbp proteins with expected lower binding to human fH (Beernink 2011, Rossi 2013 DOI: 10.1128/IAI.01491-15, Costa 2014 DOI: 10.1128/mBio.oi625-i4). The binding of fHbp to the human complement inhibitor factor H has been shown to decrease the potential SBA response to vaccines containing fHbp (Granoff 2013 doi: 10.1128/CVI.00260-13), and mutations preventing such binding are associated with higher SBA titers in the presence of human factor H (Beernink 2011 doi: 10.4049/jimmunol.1003470; Granoff 2016 doi: 10.1172/jci.insight.889O7). We thus explored if the same would occur with the adenoviral vectored platform. Two vectors were constructed, containing mutations in the fHbp sequences previously described (Rossi 2016 DOI: 10.1128/IAI.01491-15). These mutations were demonstrated to decrease factor H binding when tested as recombinant proteins. The expression of the fHbp mutants was confirmed in vitro to be at least equivalent to the expression of the wild type antigen in infected Hela cells (21 to 32% of infected cells, Fig. 16A and B). The resulting fHbp mutants, expressed by the adenovirus vectors in infected Hela cells had reduced binding to recombinant human factor H, and the reduction was independent of the adenoviral backbone used (Fig. 16C and D). Both vectors induced SBA titers comparable with those elicited by the wild-type counterpart in wild-type mice (Fig. 16E). As fHbp binding is specific to human factor H, SBA titers were assessed in transgenic mice expressing human fH at levels similar to those found in healthy humans (Beernink J Immunol 2011). In this model, the Ad vector expressing the mutant M2 (S223R) induced superior functional SBA titers in the presence of human fH as compared with vectors containing the wild type sequence or the other mutation (Fig. 16F). Moreover, ChAdOx1 fHbp M2 induced early and higher responses than a single or two doses of qCMenB in the presence of human fH (Fig. 16G). In an independent longitudinal study in the human fH expressing transgenic mouse model, a single ChAdOx1 fHbp M2 adenovirus vaccine dose elicited comparable titers to three injection of qCMenB, that persisted up to 21 weeks post injection (Fig. 16G).
Capacity of the novel vaccine candidate to protect against different strains Many variants of fHbp circulate in invasive meningococcal strains (Hoiseth et al. (2013) The Pediatric Infectious Disease Journal. 32 (10): 1096), therefore the capacity of the vaccine candidate to protect against strains expressing different variants, and in different quantities was measured by SBA. Nine target strains were selected, varying either by the variant expressed or by the putative quantity of fHbp expressed on their surface. Groups of mice were immunized with a single dose of the adenovirus vaccine, or up to three doses of the licensed vaccines qCMenB or rLP2o86. SBA were measured at different time points after a single dose of adenovirus vaccine, as well as after 1, 2 or 3 doses of the protein-based licenced vaccines. None of the vaccines induced SBA against a strain expressing a low amount of fHbp 1.1, homologous to the fHbp contained in the adenovirus and the qCMenB vaccines (Fig. 17A). However, while qCMenB contains other antigens susceptible to induce SBA responses, a single dose of Ad fHbp induced earlier SBA, and titers at least equivalent to those induced by the protein-based vaccine against strains expressing middle and high amount of homologous fHbp 1.1 (Fig. 17B, C). For responses against strains carrying heterologous fHbp, both Ad fHbp and qCMenB were able to induce SBA responses against strains expressing variant 1.13 (Fig. 17D), and 1.15 (fig. 17F), but none against 1.14 (Fig. 17E). Responses were induced against strains expressing low and medium amounts of variant 1.4 (Fig. 17G and H). Unsurprisingly, none of the fHbp 1.1-based vaccines were able to induce SBA against a strain expressing a variant from the family 2 (2.19, Fig. 17I). The absence of cross-reactivity across families were previously observed in fHbp- protein based vaccines (Feavers 2017 DOI: 10.1128/CVI.00566-16). Altogether, these results show that the strain coverage induced by a single dose of fHbp inserted in the adenovirus delivery platform was similar to the one induced by three doses of recombinant fHbp protein formulated with other antigens in Alum. References
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Example 6 - Signal Sequences
A novel adenovirus-based vaccine encoding the MenB factor H binding protein (fHbp) with an N-terminal signal sequence (Ad- fHbp) induces high titres of protective antibody after a single dose in mice. To explore the immune mechanism behind the early, sustained protective immune response induced by Ad-fHbp, a panel of N- terminal signal sequence insertion/deletion variants of the antigen were comparatively assessed for in vitro expression from mammalian cells and for immunogenicity in mice. The full-length sequence demonstrated superior early expression of the antigen in infected mammal cells, associated with induction of higher bactericidal antibody, and was also found to significantly boost antigen-specific T cell responses when incorporated ahead of other adenovirus-encoded bacterial antigens, highlighting its potential as an immune-enhancing sequence element for other viral vectored vaccine transgenes. Taken together, these findings provide an in-depth assessment of the mechanisms underlying the robust immune response to a novel meningococcal vaccine ahead of its characterisation in a phase I clinical trial in humans.
Here we investigate the mechanism by which inclusion of both the native (bacterial) signal sequence and the mammalian tPA in the clinical vaccine candidate appears to have resulted in better immunogenicity. This is counterintuitive to standard practice in the field; usually the native signal sequence would be removed.
A novel MenB vaccine has been developed that consists of a recombinant, replication deficient chimpanzee adenovirus viral vector, ChAdOx1, encoding the immature form of the fHbp antigen that includes the complete original sequence, which comprises the bacterial lipobox domain (LTAC) at the C-terminus of the pre-lipoprotein signal peptide. The construct also contains a second signal sequence: a mammalian tissue plasminogen activator (tPA) signal peptide at the N-terminus of the fHbp signal sequence, to ensure addressing the resulting protein to the secretory pathway within the mammalian cells (12). The fHbp sequence also contains and a point mutation to decrease the affinity of the antigen to human factor H, as this was demonstrated to elicit higher titres of protective antibody in macaques (13). The ChAdOx1 fHbp vectored vaccine candidate has entered a phase I trial to assess its safety and immunogenicity in humans. To determine the specific element of the bacterial signal sequence, from here on referred to as the "full-length (FL) signal sequence (SS)", that contributes most to the stronger and persistent functional antibody response induced by the vaccine in mice, a panel of fHbp i.i N-terminal amino acid sequence variant plasmids with distinct deletions or mutations were constructed, and incorporated into AdHu5 vectors for in vitro and in vivo analyses (Table 1):
Figure imgf000086_0001
Table i - Panel of factor H binding protein signal sequence variants for comparative assessments of immunogenicity. N-terminal amino acid "signal sequences" for factor H binding protein (fHbp) i.i transgenes encoded by human adenovirus serotype 5 viral vector vaccines. The signal peptide portion of the sequence is highlighted in underline, the LTA portion of the lipobox in bold, the lipobox cysteine in |boxing|, the N-terminal methionine in italic, and the SSG amino acids that remains following the natural cleavage of the signal peptide in plain text. KO = knockout.
The FL SS fHbp construct contains the mammalian signal peptide NRTAFCCLSLTTALI and the bacterial lipobox motif LTAC, so variants lacking one or both of these sequences as well as single amino acid variants of these sequences were assessed side- by-side to determine their relative contributions to the enhanced functional immune response. The present study focuses on the comparative assessment of SS variants using a combination of in vitro expression assays and in vivo immunogenicity data in mice to determine the link between transgene expression and the enhanced functional antibody response associated with the candidate vaccine antigen. In addition, the potential application of this SS to other bacterial antigens is explored with a view to developing a generalisable sequence to boost the immunogenicity of viral vectored vaccines currently undergoing pre-clinical development.
Modifying the SS ahead of the Factor H binding protein sequence impacts on the antigen expression levels at early timepoints in vitro
Expression of the variants at early timepoints was assessed by infecting HeLa cells overnight with AdHu5 vectors encoding one of each of the fHbp SS variants outlined in Table 1 and measuring fHbp expression by flow cytometry. The construct with signal peptide (SP) knockout (KO) SS was expressed by a higher percentage of cells (25-30% of cells) after overnight infection, while the truncated SS fHbp was expressed by <10% of cells (fig. 18). This implies that the lipobox (LTAC) portion of the SS is sufficient to induce the difference in early antigen expression levels observed between the FL and truncated SS fHbp constructs. The levels of expression of the lipobox KO SS construct suggests that the mammalian signal peptide does boost expression levels relative to the truncated SS fHbp, but not to the same degree as the lipobox itself. The results obtained with the LTA amino acids KO indicates that the C residue is important for this increase in expression, further evidenced by the low expression levels observed for the C to A mutation SS construct. However, knocking out both the signal peptide and the LTA portion of the lipobox results in a similarly low level of expression, highlighting the importance of an intact lipobox for boosting expression. The inclusion of a methionine (M) amino acid at the beginning of the bacterial native signal sequence, indicating a start codon at this position, also appears to negatively impact upon expression despite an otherwise unaltered SS.
Fig. 18 . Overnight expression of human adenovirus serotype 5-encoded factor H binding protein N-terminal sequence variants from HeLa cells. HeLa cells (1 x 106 per sample) were infected overnight with 5 x 108 infectious units of one of a series of human adenovirus serotype 5 (AdHu5) constructs encoding an N-terminal sequence variant of the factor H binding protein (fHbp) and expression was quantified by flow cytometry after surface and intracellular staining of harvested cells with an anti-fHbp antibody (JAR5) and a fluorescently-tagged detection antibody. The y-axis corresponds to the percentage of total fluorescent (fHbp-expressing) HeLa cells after overnight infection. The amino acid composition of the N-terminal sequence impacts upon the expression of the transgene-encoded antigen within the first 24 hours of infection. Statistical comparisons were made using a Mann-Whitney U-test. * p < 0.05; ” p < 0.01; p < 0.001.
By comparing the percentage of cells expressing fHbp after surface-staining only or intracellular-only staining, the intracellular expression of the antigen was shown to contribute to the majority of total antigen expression after overnight infection (fig. 24). Fig. 24. Intracellular and surface expression of human adenovirus serotype 5-encoded factor H binding protein with a full-length N terminal signal sequence on HeLa cells after overnight infection. HeLa cells (1 x 106 per sample) were infected overnight with 5 x 108 infectious units of a human adenovirus serotype 5 (AdHu5) construct encoding the factor H binding protein (fHbp) with a full-length signal sequence and expression was quantified by flow cytometry after surface only, intracellular only, or both surface and intracellular staining of harvested cells with an anti-fHbp antibody (JAR5) and a fluorescently-tagged detection antibody. The y-axis corresponds to the percentage of total fluorescent (fHbp-expressing) HeLa cells after overnight infection. The vast majority of antigen is expressed within the cell at this timepoint.
To further examine the intracellular antigen expression levels at early timepoints, cells were treated with brefeldin A, an inhibitor that blocks protein transport to the Golgi apparatus causing protein to accumulate instead in the endoplasmic reticulum (ER), and flow cytometry was performed hourly for six hours post-infection with a single AdHu5 construct (FL SS fHbp), thus determining key timepoints for peak antigen expression (fig. 25).
Fig. 25. Time-course expression assay of human-adenovirus serotype 5-encoded factor H binding protein with a full-length N-terminal signal sequence on HeLa cells stimulated with brefeldin. A. HeLa cells (1 x 106 per sample) were stimulated with brefeldin A to stop protein transport within the cells and subsequently infected with 5 x to8 infectious units of a human adenovirus serotype 5 (AdHu5) construct encoding the factor H binding protein (fHbp) with a full-length signal sequence and expression was quantified by flow cytometiy after intracellular only staining of harvested cells with an anti-fHbp antibody (JAR5) and a fluorescently-tagged detection antibody. The y-axis corresponds to the percentage of total fluorescent (fHbp-expressing) HeLa cells after each hourly timepoint post-infection (x-axis) up to six hours. The antigen begins to be expressed at high levels at three hours post-infection and expression levels plateau around five hours.
Based on the expression levels measured, the three-hour timepoint appears to coincide with a window of increased antigen expression while expression appears to plateau at the five-hour timepoint for this construct. These timepoints were chosen for further comparisons of early antigen expression between the mutated constructs. HeLa cells were stimulated with brefeldin A, infected with each SS variant construct, and analysed by flow cytometry for intracellular expression of fHbp between three and five hours post-infection (fig. 19A). The FL SS fHbp construct was the only construct that expressed at high levels at both timepoints, while most constructs which expressed highly at three hours post-infection failed to maintain these expression levels at the five-hour timepoint (fig. 19B). Several constructs which did not express at three hours post-infection showed modest levels of expression at the later timepoint. Surprisingly, the truncated SS fHbp construct expresses highly after three hours, but by five hours post-infection the antigen level has declined. This suggests that the SS may play a role in promoting sustained expression of antigen within cells, or slows its degradation. Similarly to the truncated SS fHbp, antigen levels from the other variants that express highly at three hours post-infection decline over time. The LTA KO vector induced expression at a consistently low level at each timepoint, indicating that the mammalian signal peptide portion of the SS does not appear to enhance the early expression of the antigen by itself in this mammalian cell line. The fact that the FL SS exhibits the most consistent levels of expression during these early timepoints, and the expression levels of each variant fluctuate between timepoints highlights variable contributions to the timing and persistency of antigen expression associated with each element of the entire SS, but strengthens the hypothesis that the inclusion of the entire SS may boost the immunogenicity of the fHbp antigen.
Fig. 19. Intracellular expression levels of human adenovirus serotype 5-encoded factor H binding protein N-terminal sequence variants from HeLa cells at early timepoints post-infection. HeLa cells (1 x 106 per sample) were stimulated with brefeldin A to stop protein transport within the cells and subsequently infected with 5 x 108 infectious units of one of a series of human adenovirus serotype 5 (AdHu5) constructs encoding an N-terminal sequence variant of the factor H binding protein (fHbp). Cells were harvested at A. three-hour and B. five-hour timepoints post-infection to measure early expression levels by flow cytometry after intracellular only staining with an anti-fHbp antibody (JAR5) and a fluorescently-tagged detection antibody. The y-axis corresponds to the percentage of total fluorescent (fHbp-expressing) HeLa cells after infection. Each portion of the N-terminal signal sequence exhibits a variable contribution to the early expression of the fHbp antigen, but the full-length signal sequence induces early and sustained levels of expression. Statistical comparisons were made using a Mann- Whitney U-test. * p < 0.05; " p < 0.01; *” p < 0.001.
The am ino acid composition of the N -term inal signal sequence influences the dynam ics of antigen expression from HeLa cells by promoting early expression As the in vitro expression assays alluded to differential contributions of distinct SS elements to the transgene expression kinetics, this phenomenon was further explored by selecting four SS variants - FL SS, SP KO SS, LTA KO SS, and SP + LTA KO SS - and employing microscopy techniques to visualise expression of these antigens in HeLa cells. To this end, plasmids encoding each of these antigen variants fused with eGFP via a flexible linker peptide were designed, cloned, and incorporated into AdHu5 vectors. To ensure accurate recapitulation of antigen expression, overnight expression from HeLa cells was compared between these eGFP fusion constructs and their corresponding original constructs (fig. 26).
Fig. 26. Side-by-side comparison of factor H binding protein N-terminal sequence variants, with and without enhanced green fluorescent protein tags, expressed from human adenovirus serotype 5 vectors after overnight infection HeLa cells. HeLa cells (1 x 106 per sample) were infected overnight with 5 x 108 infectious units of one of a series of human adenovirus serotype 5 ( AdHu5) constructs encoding an N-terminal sequence variant of the factor H binding protein (fHbp), fHbp fused with enhanced green fluorescent protein (eGFP), or GFP only, and expression was quantified by flow cytometry after surface and intracellular staining of non-GFP-expressing cells with an anti-fHbp antibody (JAR5) and a fluorescently-tagged detection antibody. The y-axis corresponds to the percentage of total fluorescent (fHbp- and/or GFP-expressing) HeLa cells after overnight infection. The differences in expression levels between the antigen variants tested is conserved for the eGFP fusion antigens and lower for the eGFP fusion antigens than the eGFP only positive control, confirming that the differences in antigen expression levels are attributable to the fHbp N-terminal signal sequence.
The eGFP-expressing constructs were detected in a greater proportion of infected cells, possibly due to the greater natural fluorescence intensity of these antigens compared with the detection of antibody-labelled antigens from the original constructs which may be less efficient. Overall, the trend in expression level differences was replicated for the eGFP constructs, confirming that the impact of the SS remains apparent even in the fusion antigens. The lower expression levels of the fHbp-eGFP fusion antigens compared with that of the eGFP only positive control confirms that the observed differences in expression between eGFP-containing constructs are due to the N- terminal fHbp SS variant antigens. Confocal microscopy was employed to visualise the expression of these fHbp-eGFP fusion antigens from AdHu5-infected HeLa cells. To relate the microscopy results to the flow cytometry data, 3 x 105 cells seeded on glass coverslips placed at the bottom of six-well plates were first infected overnight with 1.5 x 108 IU (to obtain an multiplicity of infection (MOI) of 500) of each eGFP-expressing AdHu5 vaccine, and then fixed, DAPI-stained, and transferred to microscope slides for imaging using a Zeiss 780 inverted confocal microscope. In accordance with the relative differences in expression levels quantified for the four constructs as measured by flow cytometry, a similar pattern was observed in the confocal microscopy images (fig. 20). All constructs displayed a greater intensity of eGFP expression relative to a negative control of uninfected HeLas (fig. 20A), with the eGFP only construct expressing at the greatest intensity (fig. 20B). The FL SS and SP KO SS constructs displayed similar levels of eGFP intensity, while both the LTA KO SS and SP + LTA KO SS constructs displayed similarly low levels of eGFP intensity (fig. 20C-20F). These results serve only as a visual confirmation that overnight expression of the fHbp-eGFP antigen could be detected by fluorescence microscopy.
Fig. 20 . Confocal microscopy of HeLa cells infected overnight with human adenovirus serotype 5 expressing factor H binding protein N-terminal amino acid sequence variants fused with enhanced green fluorescent protein. HeLa cells were grown overnight on glass coverslips placed in the wells of a six-well plate at a concentration of 3 x 105 cells per well. Cells were then infected overnight the following day with a proportional dose (1.5 x 108 infectious units) of human adenovirus serotype 5 (AdHu5) encoding one of the four factor H binding protein (fHbp) N-terminal signal sequence (SS) variants fused with enhanced green fluorescent protein (eGFP). Cells on the coverslips were fixed, 4',6-diamidino-2-phenylindole (DAPI)-stained and the coverslips were transferred to microscope slides for imaging on a Zeiss 780 inverted confocal microscope. Images were taken at multiple fields of vision and representative images for each construct are displayed here. A. Uninfected cells (negative control). B. eGFP only (positive control). C. Full-length SS fHbp 1.1-eGFP D. LTA knockout (KO) SS fHbp 1.1-eGFP. E. Signal peptide (SP) KO SS fHbp 1.1-eGFP. F. SP + LTA KO SS fHbp 1.1-eGFP. Blue fluorescence indicates DAPI-stained nuclei, green fluorescence indicates antigen-eGFP expression. 16 pm scale bars are shown in the bottom right corner of each image.
To compare the expression dynamics of these fluorescent antigen variants in a longitudinal manner, 1 x 105 HeLa cells were seeded overnight in each well of an eightwell chambered coverslip. The following day the cells were stained using far-red fluorogenic SiR-DNA, infected with 5 x 107 IU (to obtain an MOI of 500) of each eGFP- expressing AdHu5 vaccine, and imaged every ten minutes over the course of 14 hours using a Zeiss Spinning Disc microscope. Time-lapse images for each infection were obtained across three separate fields for each well of AdHu5-infected cells (Supplementary Materials). Differences were observed between the timings of antigen expression between the constructs, particularly at early timepoints post-infection. The FL and SP KO SS variants displayed similar antigen expression kinetics. The SP + LTA KO SS variant was expressed at a marginally later time and at a lower level, while the LTA KO resulted in the lowest levels of antigen expression. These images confirm the in vitro antigen expression data and provide further evidence for the differential contribution of these specific SS elements to early antigen expression from mammalian cells.
Factor H binding protein N -term inal am ino acid sequence variants induce differential functional antibody titres at early timepoints in m ice
Groups of mice were immunized once with The adenoviral constructs encoding the different fHbp with N-terminal SS variants at a dose of 1 x 107 infectious units,. Sera collected at several timepoints were assessed for fHbp-specific IgG titres by indirect ELISA. Statistically significant differences in anti-fHbp antibody titres were observed between signal sequence variant groups at weeks two and four post-immunisation. The highest titres were induced by the FL SS and SP KO SS constructs; the lowest titres were induced by the truncated SS, lipobox KO and qCMenB groups (fig. 27 A-B). By weeks six and 14, the only statistically significant differences observed were between 4CMenB and several signal sequence variant groups, as the response had matured to reached plateau (fig. 27 C-D).
Fig. 27. Anti-factor H binding protein IgG titres in sera from mice immunised with human adenovirus serotype 5 vectors encoding N-terminal amino acid sequence variants of the antigen. Groups of six BALB/c mice were immunised with a sub-optimal dose of 1 x 107 infectious units of one of the human adenovirus serotype 5 (AdHu5) vectors encoding factor H binding protein (fHbp) with N-terminal signal sequence variants or 1/10 of the human dose of qCmenB as a comparator. Enzyme-linked immunosorbent assays were performed on serum samples taken at weeks (A) two, (B) four, (C) six, and (D) 14 post-immunisation to determine the titres of anti-fHbp IgG in sera. Statistical comparisons were made using a Mann-Whitney U-test. * p < 0.05; ** p < 0.01; *** p < 0.001.
The functionality of anti-fHbp antibodies was assessed by performing SBA assays against the H44/76-SL reference strain that naturally expresses fHbp 1.1 using the sera taken at weeks two, four, and 6 post-immunisation. Significant differences in SBA titre were measured at each timepoint, with greatest differences observed at weeks two and four post-immunisations (fig. 21 A-B). A single dose of AdHu5 expressing the FL SS fHbp antigen induced the highest titres of bactericidal antibody at week two, significantly higher (p < 0.01) than the truncated SS fHbp, while 4CMenB failed to induce protective titres after a single dose at this timepoint (fig. 21A). The differences between the SBA titres associated with each construct closely resembled the differences in antigen expression levels observed after overnight infection of HeLa cells, with the exception of the lipobox KO SS which was highly expressed in this assay but induced lower SBA titres at this timepoint. This trend was still apparent by week 4 postimmunisation, with most constructs inducing SBA titres above the threshold for protection (fig. 21B). Low titres were induced by 4CMenB, even one week after a second dose of this vaccine. By week six post-immunisation, all constructs had induced SBA titres of > 1:4, the putative threshold of protection for meningococcal vaccines (fig. 21C). At this timepoint, the truncated SS fHbp was associated with the lowest titres, significantly lower than the FL SS and lipobox KO SS fHbp variants, while similar titres were obtained from the FL SS fHbp and 4CMenB groups.
Fig. 21. Serum bactericidal antibody titres in sera of mice immunised with human adenovirus serotype 5 encoding N-terminal amino acid sequence variants of the factor H binding protein compared with 4CMenB. Groups of six BALB/c mice were immunised with a sub-optimal dose of 1 x 107 infectious units of one of the signal sequence variant constructs or 1/10 of the human dose of qCMenB and serum bactericidal antibody (SBA) assays were performed against the H44/76 strain of Neisseria meningitidis using sera derived from blood samples taken at different timepoints post-immunisation. A. Week two SBA titres. B. Week four SBA titres. C. Week six SBA titres. The dotted red line represents the cut-off titre of 1:4 deemed sufficient for protection. Differences in the SBA titre between constructs are attributable to the N-terminal signal sequence variants of the factor H binding protein, particularly at early timepoints post-immunisation. Statistical comparisons were made using a Mann-Whitney U-test. * p < 0.05; " p < 0.01.
Taken together, these results demonstrate that the N-terminal SS impacts on the functional antibody titres induced by the adenoviral-driven expression of the fHbp transgene. The pattern of bactericidal antibody response suggests that the early expression associated with these SS variants influence their immunogenicity. Additionally, the FL fHbp SS is capable of inducing protective titres of SBA just two weeks after a single shot of a sub-optimal dose, while the licensed qCMenB vaccine requires two doses and greater than four weeks to achieve such titres in mice. Inclusion of the full-length signal sequence at the N -term inus of other adenovirus- encoded bacterial antigens boosts antigen-specific T cell responses to the transgene product
To determine whether the SS used for fHbp could be applied to other bacterial antigens to increase immunogenicity, vectors incorporating antigens from Y. pestis, the causative organisms of the disease plague, were constructed. The plasmids encoded the F1 antigen with its native SS, or lacking its native SS, or replacing the native SS with the FL fHbp SS. Groups of 12 BALB/c mice were immunised with 1 x 107 IU of one of each of the AdHu5-plague vaccines. The anti-Fi IgG titres were measured from serum samples taken at weeks two and four post-immunisation by an indirect ELISA. The inclusion of the heterologous FL SS appeared to significantly increase anti-Fi IgG titres compared with the AdHu5 F1 construct at week four post-immunisation, though the titres were still significantly lower than those associated with the F1 antigen with native SS (fig. 22). These data suggest that, the native SS may perform better than the meningococcal fHbp SS.
Fig. 22. Anti-Fi antigen IgG titres in sera from mice immunised with human adenovirus serotype 5 vectors encoding the full-length F1 antigen, with or without the N-terminal amino acid signal sequence, or the truncated form of the antigen. Groups of 12 BALB/c mice were immunised with a sub-optimal dose of 1 x 107 infectious units of one of the human adenovirus serotype 5 (AdHu5) vectors encoding the Yersinia pestis F1 antigen with native signal sequence (SS), a heterologous N-terminal full-length (FL) SS, or a truncated form of the F1 antigen lacking any SS. Enzyme-linked immunosorbent assays were performed on serum samples taken at weeks A. two and B. four post-immunisation to determine the titres of anti-F1 antigen IgG in sera. The humoral response induced by the F1 antigen with native SS was superior to that induced by the incorporation of the heterologous FL SS to this antigen or its truncated form at both timepoints. Statistical comparisons were made using a Mann-Whitney U- test. * p < 0.05; ” p < 0.01; ”* p < 0.001; **** p < 0.0001.
To determine whether a more robust cellular response could be induced against bacterial antigens that utilise a signal peptide for expression in their native state, antigen-specific T cell responses were compared between these two antigens and several Salmonella antigens - CdtB, SipD, and SseB. Plasmids were designed to include the SS ahead of these Salmonella Paratyphi antigens, cloned, and incorporated intoAdHu5 vectors. Groups of six BALB/c mice were immunised with 1 x 107 IU of eachAdHu5 vaccine encoding each of the bacterial antigens with or without the N-terminal SS. Spleens were harvested two weeks post-immunisation to assess antigen-specific IFN-y- and IL-17A-producing T cell responses by fluorospot. Inclusion of the N- terminal SS boosted the antigen-specific IFN-y-producing T cell responses to most of the antigens tested, particularly for SipD and SseB (fig. 23A), and even led to a significant increase in SseB-specific IL-17A-producing T cells which were otherwise mostly undetectable against the native counterparts of each antigen (fig. 23B). These data highlight the additional attribute of the N-terminal SS in boosting antigen-specific T cell responses against heterologous bacterial antigens and demonstrate the utility of the SS as a broadly-applicable immune-enhancing peptide.
Fig. 23 . Antigen-specific T cell responses induced in mice two weeks after immunisation with human adenovirus serotype 5 vectors encoding bacterial antigens with or without an N-terminal signal sequence. Groups of six BALB/c mice were immunised with human adenovirus serotype 5 (Ad H 115) vaccines expressing one of a series of bacterial antigens, with or without an N-terminal signal sequence (SS). Spleens were harvested two weeks post-immunisation, processed, and stimulated at a concentration of 1:100 with the relevant peptide pool. An interferon (IFN)- y/interleukin (IL)-17A dual colour fluorospot was performed to assess antigen-specific T cell responses associated with these cytokines. A. IFN-y and B. IL-17A spot-forming units (SFU) per million cells were quantified for each antigen. Inclusion of an N- terminal SS was found to boost both types of antigen-specific T cell responses. Statistical comparisons were made using a Mann-Whitney U-test. * p < 0.05.
Discussion of Exam ple 6
This study provides a functional exploration of the distinct elements of a novel signal sequence included ahead of a MenB lipoprotein expressed from a viral vector-encoded transgene. The relative contribution of each of these elements was assessed through in vitro expression assays in a mammalian cell line and validated with the use of high- resolution microscopy. The elucidation of the transgene expression kinetics provides a potential link with differences observed between these constructs through comparative immunogenicity analyses. Furthermore, the inclusion of the SS ahead of several other transgenes encoding heterologous bacterial antigens demonstrates the utility of this design in promoting early antigen-specific cellular immune responses.
A mammalian SP was elected to promote expression of the lipoprotein within mammalian tissues. The results of the expression assays presented here confirm that the FL SS is superior to the truncated SS for the promotion of fHbp 1.1 antigen expression in mammalian cells. It has also been demonstrated that these SS elements were judiciously chosen fortheir combined influence on transgene expression and immunogenicity; the expression assay data provides rationale for the inclusion of the FL SS within the vaccine antigen sequence as the FL sequence promotes the most consistent levels of early transgene expression across each of the chosen timepoints. The application of confocal and time-lapse microscopy was more revealing as to the timing and intracellular localisation of antigen expression in a mammalian cell line and confirmed the findings of the in vitro expression assays. fHbp-eGFP transgene expression was predominantly located within the cytoplasm in apparent association with membrane structures and no fluorescence detected in the extracellular media. This is in accordance with the typical role of SSs in targeting antigens to the membranes of the ER and cell surface.
Delivery of exogeneous antigen via viral vectored vaccines has been explored for the prevention of a wide range of diseases, particularly those requiring strong CD8+ T cell responses. This vaccine platform also has strong potential to induce robust humoral responses, making them an attractive target for diseases required antibody-mediated protection. IMD is one such disease, and vaccines have been licensed on the basis of their ability to induce a human SBA titre of > 4, the putative correlate of protection against the disease (28). All fHbp 1.1 SS variants elicit protective titres of antibody in mice within four weeks of a sub-optimal dose, while the FL SS fHbp 1.1 induces protective titres by as early as week two post-immunisation. This in contrast to qCMenB which, at 1/10 of the human dose, requires two doses and more than four weeks to induce similar titres in mice. The FL SS also demonstrates superiority to the truncated SS variant, demonstrating its ability to enhance the antigenicity of the fHbp antigen. The level of antigen expression in APCs has been highlighted as an important contributing factor to the formation of antibodies against the transgene product from adenovirus vectors (29). The induction of antigen-specific humoral immunity is primarily mediated by the interaction of CD4 + TH cells with MHC class Il-presented peptides on the surface of these cells. The subset of CD4 + TH cells can influence the nature of the humoral response; IFN-y production is a hallmark of TH1 cells that promote IgG2 and IgG3 production from B cells (30). Both of these antibodies are important in the context of early childhood as this age group is known to be poor at inducing these antibody subclasses, with the former playing an important role in immunity to N. m eningitidis (31). IL-17A production indicates TH17 cell activity, the role of which is of growing interest to vaccinologists due to its association with vaccine- induced immune responses against a variety of bacterial pathogens, such as Bacillus pertussis and Streptococcus pneum oniae (32). The ability of the FL SS to enhance these distinct CD4 + T cell responses against the neisserial fHbp, the plague F1 antigen, and the Salm onella antigens CdtB, SseB, and SipD was investigated and found to significantly increase some of these responses. The increase in TH1-mediated antigen- specific responses against SipD and SseB were significant, as was the TH17-mediated response against SseB. Both of these proteins are constituents of the Salm onella type III secretion system (T3SS) and their potential as candidate vaccine antigens has been highlighted by studies in mice and humans (33, 34 ). CD4+ T cells of the IFN-y and IL- 17-producing variety also contributed to the response induced by the live-attenuated Salm onella Typhi vaccine, Ty2ia (35), in humans. The findings of the antigen-specific T cell responses provide compelling evidence that the FL SS can be incorporated ahead of adenovirus-encoded heterologous bacterial antigen transgene sequences to boost antigen-specific immune responses.
Materials and Methods
Anim al procedures
All procedures were performed in accordance with the terms of the UK Home Office Animals Act Project License. Procedures were approved by the University of Oxford Animal Care and Ethical Review Committee. General anaesthesia was induced using 3.5% isofluorane mixed with 2 L/min of O2 released into the mouse anaesthetic chamber and then via direct inhalation for each mouse through a tube while procedures were being performed. Cardiac bleeds (followed by cervical dislocation), immunisations and terminal bleeds were performed under general anaesthesia. All mice were female and aged between six- and eight-weeks of age at the beginning of each experiment. Once anaesthetised, vaccines were administered to the mouse by injecting no more than 50 μL into each of the musculus tibialis at the back of the leg, using a 29-gauge insulin syringe. Tail bleeds were performed to obtain blood samples prior to terminal bleeds. Using a 37 °C heating box, mice were pre-warmed and restrained to facilitate nicking of the tail vein to collect 8-10 drops of blood in a 2 mL tube. For terminal bleeds, mice were put under general anaesthetic and cardiac bleeds were performed followed by cervical dislocation to confirm death. Blood for RNA-seq experiments was transferred to pre-filled RNAlater™ blood collection microcentrifuge tubes.
Splenocyte isolation
Spleens were removed by incising the left-hand side of the abdomen, after cardiac bleed and cervical dislocation of the animal, and transferred to gentleMACs™ C tubes containing autoMACS® running buffer. Spleens were macerated in a gentleMACS™ Dissociator (Miltenyi Biotec) using the mouse spleen pre-set program and then tubes were centrifuged at 250 x g for 10 mins. The supernatants were discarded, and pellets resuspended in RBC lysis solution for 5 mins. Sterile PBS was added to quench the lysis solution after 5 mins and the cell suspensions were passed through 70 um filter into 50 mL Falcon tubes using Pasteur pipettes. The filtered solutions were centrifuged again at 250 x g for 10 mins, supernatants discarded, and pellets resuspend in 10 mL of complete DMEM. A Muse® Count & Viability Assay Kit was used to measure the final cell count and viability of each sample on a Muse® Cell Analyzer instrument (Millipore). Samples were diluted to 4 x to6 in complete DMEM.
Plasm id construct design The nucleotide sequence for the desired antigen was obtained from the GenBank sequence database (httpsfy/wvyy^ The sequence was run through a glycosylation site finder
(http; / / www- cbs, dtp, dk / seryices/Net NGlyc / ) to identify putative glycosylation sites
5 and remove them if the probability of glycosylation exceeds 50%. This is done to remove sequences that may be post-translationally modified if expressed from mammalian cells. The polyA tail was removed from the sequence and the GeneAit® Gene Synthesis tool (Invitrogen) (https://ww w.thermofisher.com/en/home/life- science/cloning/gene-synthesis/geneart-gene-synthesis.html) was used to upload the
10 sequence, optimise codons for mammalian tissue, and add restriction sites. The Hindlll (AAGCTT at the start of the sequence) and Notl (GCGGCCGC at the end of the sequence) restriction sites were added to the construct.
Restriction digest
To cut out the transgene from the plasmid backbone, a restriction digest
15 reaction was set up as a 50 μL reaction in a PGR tube as follows:
Figure imgf000098_0001
Agarose gel electrophoresis
DNA samples were run on a 1% agarose gel after restriction digest. The gel was
20 stained using peqGreen DNA dye and placed in an electrophoresis cassette filled with 1X TAE buffer. 10 μL of purple loading dye was added to the 50 μL samples to achieve a 1X concentration. Samples were loaded into the wells of the gel along with a 1 kb DNA ladder. The electrophoresis apparatus was connected to a voltage box set to 100V and the gel was run for 40 mins. The gel was analysed using a UV light box and bands
25 corresponding to the size of the insert were excised from the gel using a gel extractor tool and placed in a 2 mL Eppendorf tube. DNA was extracted from the gel section using a QIAquick Gel Extraction Kit. Briefly, kit buffer was added to the 2 mL tube at a ratio of 3:1 (100 mg gel ~ 100 μL) and incubated at 50 °C on a heating block for 10 mins or until dissolved, one volume of isopropanol was added and the gel digest was filtered
30 and washed as per kit instructions, eluting the DNA into a final volume of too μL dH20. The DNA concentration was measured using a NanoDrop™ 2000 Spectrophotometer (Thermo Scientific). DNA ligation
A 20 μL DNA ligation reaction was set up, to place the insert sequence into a pMONO expression plasmid with Kanomycin resistance, in a PCRtube as follows:
Figure imgf000099_0001
The reaction was run overnight on a thermocycler set to 16 °C.
Transformation
After overnight ligation, 5 μL of sample was added to 50 μL of thermocompetent DH5C1 cells in a 2 mL Eppendorf tube on ice. A negative control was set up by adding 5 μL of dH20 to the same volume of cells. Samples were incubated at 4 °C for 30 mins, heat- shocked at 42 °C for 30 s, and returned to 4 °C for 2 mins. 250 μL of pre-warmed (37 °C) SOC recovery medium was added to each tube and samples were placed on a 37 °C shaking incubator for 1 hour before plating 200 μL on LB agar containing 30 μg/mL Kanomycin using an L-shaped spreader. Plates were incubated at 37 °C overnight. The following day, colonies were picked from the plates using a pipette tip and dropped in an Erlenmeyer shaker flask containing 50 mL of LB broth with 30 μg/ mL Kanomycin to be placed in a 37 °C shaking incubator overnight.
DNA extraction
Samples were transferred from the Erlenmeyer flasks for 50 mL Falcon tubes and centrifuged at 300 x g for 10 mins. The supernatant was poured off and DNA was extracted from the pellets using a QIAGEN Plasmid Midi Kit, as per manufacturer's instructions. DNA concentrations were measured using the NanoDrop™ and subsequently restriction digested as outlined in section 2.5.2 (using insert DNA rather than plasmid DNA). Aliquots of the digested DNA samples were run on a 1% gel and, if the band corresponded to the appropriate insert size, sent to Source Bioscience for sequencing, using forward and reverse primers for the 3' cytomegalovirus (CMV) promoter and bovine growth hormone (bGH) polyA region sequences, respectively, to ensure the presence of the correct antigen sequence in these samples.
Cloning into adenovirus
Sample sequence files, forward and reverse, were aligned to the plasmid containing the transgene insert reference file using SeqMan Pro (DNASTAR) software. For samples with 100% sequence identity to the reference, DNA was treated with a Gateway™ LR Clonase™ II kit. The DNA samples, containing the antigen sequence of interest, were diluted in TE buffer to yield a solution containing 150 ng of DNA which was then combined with 1 |iL of an AdHu5 destination vector (at a concentration of 150 ng/ μL) in a PCR tube. 2 |iL of LR Clonase™ II Enzyme Mix was added to each sample and incubated at room temperature for 10 mins. 1 μL of proteinase K was added to each sample the following day and incubated at 37 °C for 10 mins. The proteinase K-digested samples were transformed using DH5C1 cells as outlined in section 2.5.5, but this time plating 200 μL on LB agar containing 100 μg/ mL ampicillin and adding 10 μL streaks of up to four samples per plate of LB agar containing 15 μg/mL chloramphenicol. Plates were incubated overnight and the following day samples with colonies on LB ampicillin plates that did not grow on LP chloramphenicol plates were picked and cultured overnight in a 37 °C shaking incubator in Erlenmeyer flasks containing 50 mL of LB broth with too μg/ mL ampicillin. DNA was extracted the following day as per section 2.5.6, the concentration was measured, and samples were again sent for sequencing with Source BioScience using the same primers. Samples with 100% sequence identity relative to a reference sequence (AdHu5 destination vector containing the antigen insert) were selected for linearization. 1 μg of DNA was restricted by 3 μL of Pad restriction enzyme in 10 μL of CutSmart® buffer and made up to too μL total volume with dH20 in a PCR tube. The reaction was run for four hours at 37 °C on a thermocycler and the reaction was stopped by exposing to 65 °C for 25 mins. 15 μL of sample was run on an agarose gel (section 2.5.3) to ensure linearization was successful and the remaining volume (85 μL) was sent to the Viral Vector Core Facility (WCF, Jenner Institute, University of Oxford) for production.
Expression assays
Cells were harvested at approximately 70-80% confluency. Where brefeldin stimulation was required, cells were resuspended in media containing 3 μg/mL of brefeldin A solution. 1 x to6 cells per sample were infected with the appropriate volume of adenovirus construct to obtain a concentration of 5 x to8. Infected cells were incubated for the required time (overnight or specific time points), harvested and surface stained and/ or stained intracellularly with anti-fHbp monoclonal antibody JAR5 (National Institute of Biological Standards and Controls) and GFP-tagged IgG detection antibody using a Fixation/Permeabilization Solution Kit. Cells were resuspended in 400 μL of permeabilization buffer and filtered before running on a FACSCalibur flow cytometer (BD Biosciences). The percentage of cells expressing GFP was measured and analysis was performed using FloJo software (TreeStar). Indirect enzym e-linked imm unosorbent assays
To quantify antibodies against fHbp in animal sera, 2.5 μg/mL recombinant fHbp 1.1 (plasmid gifted by Dr Peter Beernink, CHORI, and protein produced at Oxford Protein Production Facility) in carb/bicarb buffer was coated onto 96-well flat-bottomed microtitre plates overnight at 4 °C. Plates were washed with PBS containing 0.05% (v/v) Tween-20. Plates were blocked with 1% BSA in PBS and serial dilutions of sera or positive control (JAR5 antibody) were added. After incubation, anti-goat anti-mouse horseradish peroxidase conjugate was added at a 1:20,000 dilution. Plates were incubated for 20 mins with 100 μL per well TMB enzyme-linked immunosorbent assay (ELISA) substrate. 1M sulphuric acid was added to stop the reaction. Absorbance were read at 450 nm and 630 nm using a Multiskan MS plate reader (Biotek). Interpolated IgG concentrations were calculated for each serum sample using a standard curve.
Serum bactericidal antibody assays
H44/76-SL (provided by Public Health England, PHE) strain N. meningitidis group B bacteria were used for SBAs. Test sera (20 μL) were heated for 30 mins at 56 °C to inactivate endogenous complement. Equal volumes (10 μL) of bacterial suspension (optical density (OD)600 = 0.1 bugs diluted to 1:5000), and human complement were added to twofold serial dilutions of test serum sample. The SBA titre was the serum dilution resulting in 50% survival of the bacteria compared to the number of colony-forming units (CFU)/mL in the variable complement control, containing inactivated complement and no sera.
FluoroSpot assays
A Mouse IFN-Y/IL-17A FluoroSpot kit was used to measure antigen-specific T cell responses in mouse spleen tissue. Anti-IFN-y and anti-IL-17A monoclonal capture antibodies were diluted in PBS to a concentration of 15 μg/ml and 10 μg/ml, respectively. The IPFL plate membrane was washed with 15 μL of 35% ethanol per well for no more than 60 s and then washed three times with 200 μL of sterile H20 per well, too μL of capture antibody was added to each well and the plate was sealed for overnight incubation at 4 °C. The plate was washed three times with 200 μL of sterile PBS per well the following day and the wells were then blocked with DMEM containing 10% FBS for 30 mins. Stimuli, either DMS0 (1:100) as a negative control, ConA (1:200) as a positive control, or reconstituted peptide pools (1:100) corresponding to the vaccine antigen, were added to the appropriate wells, followed by 2 x 105 splenocytes from the appropriate sample to each corresponding well. The plate was sealed and incubated overnight at 37 °C in a 5% CO2 incubator. Cells were removed from the wells the following day and the plate was washed five times with 200 μL of PBS per well. Anti-IFN-y-R4-6A2-BAM and anti-IL-i7A-MT227O (biotinylated) detection antibodies were diluted in PBS containing 0.1% BSA in the same tube to a concentration of 1:200 and 1:250, respectively. 100 μL of detection antibody mixture was added to each well and incubated for two hours at room temperature. Anti-BAM-490 and SA-550 fluorophore conjugates were both diluted to a concentration of 1:200 in the same tube with PBS containing 0.1% BSA and, after washing the plate five times with PBS, 100 μL of this mixture was added to each well. The plate was wrapped in aluminium foil and incubated for one hour in the dark at room temperature. The plate was washed five times with PBS before adding 50 μL of fluorescence enhancer to each well and incubating in the same manner for 15 mins. The plate was emptied of all liquid and the underdrain was removed. Plates were completely dried in the dark at room temperature prior to spot counting with an IRIS™ FluoroSpot reader (Mabtech). Excitation 490 nm/emission 510 nm (FITC) and excitation 550 nm/emission 570 nm (Cy3) wavelengths were used to measure IFN-y and IL-17A, respectively.
Confocal m icroscopy
Complete DMEM containing 1 x 106 HeLa cells were seeded in 300 μL volumes per well on glass coverslips placed at the bottom of six-well plates. Cells were infected overnight with 1.5 x to8 IU (proportional to the number of cells infected) of each AdHu5 vaccine containing an antigen-eGFP transgene or appropriate control. Cells were washed the following day using sterile PBS and fixed and permeabilised using 250 μL of Fixation/Permeabilization solution from and incubated at 4 °C for 20 mins. After washing with PBS, 300 μL of 300 nM DAPI (5 mg/mL stock solution diluted to 300 nM in PBS) was added to the fixed/permeabilised cells for 5 mins at 4 °C, washed again and slowly transferred, using forceps and dabbing off excess liquid, to microscope slides with a drop of ProLong Gold Antifade Mountant in the centre. Slides were cured on a flat surface overnight in the dark and imaged the following day using a Zeiss 780 inverted confocal microscope (Zeiss) with ZEN Lite image acquisition software (Zeiss). Cells were located in brightfield and then the interface was switched to 'acquisition mode' where 'smart settings' were applied. The fluorophore-specific settings were manually refined, and images were taken across multiple planes of focus for each sample triplicate.
Tim e-lapse im aging
Complete DMEM containing 1 x 106 HeLa cells were seeded in too μL volumes per well (1 x 105 cells per well) overnight in each well of an eight-well chambered coverslip. The following day the cells were stained using far-red Anorogenic SiR-DNA 2 hours prior to infection. Cells were washed with PBS and subsequently infected with 5 x 107 IU (proportional to the number of cells) of each eGFP-expressing AdHu5 vaccine. The chambered coverslip was then secured on the stage, within a live cell stage incubator set to 37 °C and supplemented with 5% CO2, of a ZEISS Spinning Disc microscope (Zeiss). Three coordinates were set for each sample using ZEN Blue image acquisition software (Zeiss) and imaged every io mins over the course of 14 hours. The time-lapse for each sample was then constructed from these images using ImageJ software (Fiji).
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Example 7 - fHbp Variants
Any other fHbp variant, including stabilising mutations and/ or mutations affecting the binding to human factor H, may be used in the composition of the invention.
For example we refer to Figure 28 which shows that fHbp variants 2.19 and 1.4 are immunogenic.
Example 8 - Im munogenicity in Humans We demonstrate that the most preferred embodiment of the invention (ChAdOx1 MenB.1) is immunogenic in humans.
Outline
The subjects are adults aged 18 to 50 years old who are in good health.
Participants are allocated to one of eight sub-groups. Those in Group 1 receive a single low dose of 2.5 x10^10 VP of ChAdOx1 MenB.1. Those in Group 2 receive a single high dose 5X10^10 VP of ChAdOx1 MenB.1. Group 3 participants receive a high dose of ChAdOx1 MenB.1 plus a repeat booster dose at six months. Group 4 participants receive a dose of Bexsero® at baseline with a high dose booster of ChAdOx1 MenB.1 at six months. Those in Group 5 receive two doses of Bexsero® at baseline and 28 days later. Those in Groups 6 and 7 receive Bexsero® at baseline and six months. Participants in Group 8 receive two doses of Trumenba® at baseline and six months. Participants in Groups 6, 7 and 8 will be asked to consent to a blood donation for making serum standards. Symptoms of the shots are recorded in participant diaries.
Detail
To assess the immunogenicity of the composition of the invention in humans, healthy adult participants were recruited in a phase i/2b, single centred non-randomized clinical trial (EudraCT ISRCTN46336916). Immunogenicity was assessed by measuring the functional antibody response as well as cell-mediated immune response. Functional antibody responses were measured using serum bactericidal assay with human complement (hSBA) as previously described (Marsay et al., 2015), as these have been shown to correlate with protection in humans. A titre superior or equal to 1:4 is associated with protection (Goldschneider et al., 1969).
Serum bactericidal antibody titers were measured against meningococcal strain H44/76-SL, and enumeration of the fHbp-specific interferon-gamma (IFN-y) secreting T cells was performed on peripheral blood mononuclear cells by FLUOROSPOT. Results show that a single dose of ChAdOx1 MenB.1 vaccine elicits protective hSBA titers (Figure 29A). Remarkably, a single ChadOx1 MenB.1 dose elicits similar protective antibody titers as two injections of the licensed comparator 4CMenB (trade name Bexsero®) administered at Day o and Day 28, and superior to a single dose Bexsero® (Figure 29B). In contrast, previous studies with ChAdOx1-based vaccines show that two injections maybe necessary to induce functional antibody responses in all participants, as seen with the neutralizing response induced by ChAdOx1 nCoVig in healthy adults.
Moreover the single dose ChAdOx1 MenB.1 induced similar hSBA titers 6 months after vaccination as compared with two doses 4CMenB (Figure 29A). Homologous and Heterologous Prime-Boost
Moreover, a booster dose of ChAdOx1 MenB.1 given at six months was able to boost the responses elicited by a first ChAdOx1 MenB.1 dose, or by the protein-based licensed comparator vaccine 4CMenB (Figure 29B).
In addition we show that ChAdOx1 menB.1 induced a higher IFN-gamma secreting antigen-specific T cell response than two injections of the comparator (Figure 30), and this T-cell response is still detectable six months post single injection.
Heterologous Prime-Boost
Notably, ChAdOx1 MenB.1 was able to induce a hSBA antibody response, as well as a T cell response in participants previously primed with the licenced comparator 4CMenB. This is especially beneficial as the composition of the invention may be used in adolescents vaccinated in infancy with the licensed vaccine 4CmenB - which is currently in the UK infant vaccination schedule.
We also refer to figure 31.
Example 9 - Further Im munogenicity in Hum ans
The T cell response to Ad MenB (ChAdOx1 MenB.1) is shown in Fig 32. This demonstrates that the composition of the invention is immunogenic in humans. This is especially important and surprising because for some antigen inserts the T cell response is not always very high after a single dose and may require a boost. Also some antigen inserts do not produce a useful response (see comparative data in earlier examples). So it is a special benefit of the invention that for a single dose this is a remarkably strong T cell response.
Human volunteers showed significant increases in IFN-g-producing T cells following ChAdOx1 boosting immunization (Fig. 32A), and the degree of expansion was positively correlated with the degree of post vaccination MAIT cell activation (Fig. 32B).
Moreover, the activation of the MAIT cells by Ad MenB (ChAdOx1 MenB.1) at day 1, which correlate with the T cell response (Fig 32B) is a completely new finding.
In addition, figures 33D and 33E show stimulation of an innate response by Ad MenB (ChAdOx1 MenB.1).
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to those precise embodiments and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

Claims

1. A composition comprising a viral vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide antigen, wherein said antigen comprises Factor H Binding Protein (fHbp) from Neisseria m eningitidis, characterised in that said viral vector is an adenovirus based vector.
2. A composition according to claim i wherein said adenovirus based vector is a non-human adenovirus based vector.
3. A composition according to claim 1 or claim 2 wherein said adenovirus based vector is a simian adenovirus based vector, preferably a chimp adenovirus based vector.
4. A composition according to any of claims 1 to 3 wherein said adenovirus based vector is selected from the group consisting of ChAdOx1 and ChAd0x2.
5. A composition according to any of claims 1 to 4 wherein said adenovirus based vector is ChAdOx1.
6. A composition according to any of claims 1 to 5 wherein said Factor H Binding Protein (fHbp) comprises an arginine substitution at the amino acid position corresponding to serine 223 in the wild type Factor H Binding Protein (fHbp).
7. A composition according to claim 6 wherein said Factor H Binding Protein (fHbp) comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2.
8. A composition according to any preceding claim wherein said antigen further comprises a signal sequence.
9. A composition according to claim 8 wherein said signal sequence is a human signal sequence or a bacterial signal sequence.
10. A composition according to claim 9 wherein said antigen comprises at least two signal sequences.
11. A composition according to claim 10 wherein said at least two signal sequences comprise at least one bacterial signal sequence and at least one human signal sequence.
12. A composition according to any preceding claim wherein said antigen is present as a fusion with the tissue plasminogen activator (tPA) sequence in the order N- terminus - tPA - Factor H Binding Protein - C-terminus.
13. A composition according to claim 5 wherein said tPA has the amino acid sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
14. A composition according to any preceding claim wherein said antigen has the amino acid sequence SEQ ID NO: 3.
15. A composition according to any of claims 2 to 14 wherein said viral vector sequence is as in ECACC accession number 12052403.
16. A composition according to any of claims 1 to 15 for use in induction of an immune response against Ne isseria meningitidis.
17. A composition according to any of claims 1 to 15 for use in boosting of an immune response against Neisseria meningitidis.
18. A composition according to any of claims 1 to 15 for use in preventing Neisseria meningitidis infection.
19. A composition according to claim 16 or claim 17 or claim 18 wherein a single dose of said composition is administered.
20. A composition according to claim 16 or claim 17 or claim 18 wherein said composition is administered once.
21. Use of a composition according to any of claims 1 to 15 in medicine.
22. Use of a composition according to any of claims 1 to 15 in the preparation of a medicament for prevention of Neisseria meningitidis infection.
23. A method of inducing an immune response against Neisseria meningitidis in a mammalian subject, the method comprising administering a composition according to any of claims 1 to 15 to said subject.
24. A method according to claim 23 wherein a single dose of said composition is administered to said subject.
25. A method according to claim 23 or 24 wherein said composition is administered once.
26. A method according to claim 23 further comprising administration of a second or further dose of said composition subsequent to administration of the first dose.
27. A method according to claim 26 wherein administration of said second or further dose of said composition is carried out approximately 6 months after administration of the first dose
28. A method according to any of claims 23 to 27 wherein said composition is administered by a route of administration selected from a group consisting of intranasal, aerosol, sublingual, intradermal and intramuscular.
29. A method according to claim 28 wherein said administration is intramuscular.
30. A kit comprising: a first dose of a composition according to any of claims 1 to 15; and optionally a second dose of a composition according to any of claims 1 to 15; and instructions for administration to a mammalian subject.
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