WO2022234283A1 - 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
WO2022234283A1
WO2022234283A1 PCT/GB2022/051147 GB2022051147W WO2022234283A1 WO 2022234283 A1 WO2022234283 A1 WO 2022234283A1 GB 2022051147 W GB2022051147 W GB 2022051147W WO 2022234283 A1 WO2022234283 A1 WO 2022234283A1
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composition
dose
suitably
seq
sequence
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PCT/GB2022/051147
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French (fr)
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David TABOR
Helen BRIGHT
Michael ABRAM
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Astrazeneca Uk Limited
Oxford University Innovation Limited
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Publication of WO2022234283A1 publication Critical patent/WO2022234283A1/en

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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
    • A61K39/12—Viral 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to induction of immune responses, suitably protective immune responses, against SARS-CoV2 (nCoV-19).
  • Coronavirus 19 (SARS-CoV2; sometimes referred to as nCoV-19 or as COVID-19) is the virus responsible for an outbreak of coronavirus disease that was first reported from Wuhan, China, on 31 December 2019.
  • Symptoms of the disease include fever, dry cough, muscle pain, and respiratory problems such as breathing difficulties / shortness of breath. In more severe cases, infection can cause pneumonia, severe acute respiratory syndrome, kidney failure and even death. Mortality rates have been estimated by the World Health Organisation (WHO) at up to 3.4% of infected individuals, with many commentators agreeing on a mortality rate of approx. 1-2% of infected individuals once figures are adjusted taking into account the mildest cases which are not always reported (e.g. if individuals did not seek treatment or diagnosis).
  • WHO World Health Organization
  • a combination which comprises a simian adenoviral vector (such as ChAdOxi) delivering a SARS-CoV2 antigen (the spike protein).
  • a simian adenoviral vector such as ChAdOxi
  • SARS-CoV2 antigen the spike protein
  • the invention relates to a composition
  • a composition comprising a viral vector, wherein the viral vector is an adenovirus based vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide, said polypeptide having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, characterised in that said polypeptide comprises the following substitutions relative to SEQ ID NO: 1: a) L18F b) D80A c) G215D d) L242 A e) A243 D f) L244 D g) K417N h) E484K i) N501Y j) D614G; and k) A701V.
  • the invention relates to a composition as described above wherein said polypeptide further comprises the following substitutions relative to SEQ ID NO: 1: 1) F814P m) A889P n) A896P 0) A939P p) K983P; and q) V984P.
  • said adenovirus based vector is a simian adenovirus based vector.
  • said adenovirus based vector is ChAdOx 1.
  • said polypeptide is a spike protein polypeptide.
  • said polypeptide comprises the spike protein receptor binding domain (RBD).
  • RBD spike protein receptor binding domain
  • said polypeptide comprises the spike protein receptor binding domain (RBD), the spike protein N-terminal Domain (NTD) and the spike protein STEM.
  • polypeptide is full length spike protein.
  • said polypeptide is present as a fusion with the tissue plasminogen activator (tPA) sequence in the order N-terminus - tPA - polypeptide - C-terminus.
  • tissue plasminogen activator tPA
  • tPA has the amino acid sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
  • polypeptide has the amino acid sequence SEQ ID NO: 3 or SEQ ID NO:
  • said polynucleotide sequence comprises the sequence of SEQ ID NO: 23 (encoding 2816) or SEQ ID NO: 24 (encoding 3990), preferably SEQ ID NO: 23 (encoding 2816).
  • said viral vector sequence is as in ECACC accession number 12052403.
  • the invention relates to use of a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2.
  • said immune response is an immune response in a mammalian subject.
  • the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein a single dose of said composition is administered to said subject.
  • the invention in another aspect relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein two doses of said composition are administered to said subject.
  • the invention in another aspect relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject.
  • the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein said composition is administered once.
  • the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein said composition is administered twice.
  • the invention in another aspect relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject.
  • composition is administered once per 12 months.
  • composition is administered once per 60 months.
  • the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein a first dose of said composition is administered to said subject after one or more doses of a different composition suitable for preventing,
  • Non limiting examples of such different compostions are; mRNA vaccines (e.g. BNTi62b2 or tozinameran; mRNA- 1273 or CX-024414), Adenovirus vector vaccines (e.g. AZD1222; Gam-COVID-Vac; Ad26.C0V2.S or JNJ-78436735), inactivated and subunit vaccines.
  • mRNA vaccines e.g. BNTi62b2 or tozinameran
  • Adenovirus vector vaccines e.g. AZD1222; Gam-COVID-Vac; Ad26.C0V2.S or JNJ-78436735
  • inactivated and subunit vaccines e.g. AZD1222; Gam-COVID-Vac; Ad26.C0V2.S or JNJ-78436735
  • the invention in another aspect relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection.
  • the invention in another aspect relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein a single dose of said composition is administered.
  • the invention in another aspect relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein two doses of said composition are administered to said subject.
  • the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject.
  • the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein said composition is administered once.
  • the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein said composition is administered twice.
  • the invention in another aspect relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject.
  • a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject.
  • said composition is administered once per 12 months.
  • composition is administered once per 60 months.
  • the invention relates to use of a composition as described above in medicine.
  • the invention relates to a composition as described above for use in medicine. In another aspect the invention relates to a composition as described above for use as a medicament. In another aspect the invention relates to use of a composition as described above in the preparation of a medicament for prevention of, or for use in prevention of, SARS- CoV2 infection.
  • Suitably prevention of SARS-CoV2 infection is prevention of SARS-CoV2 infection in a mammalian subject.
  • the invention in another aspect relates to a method of inducing an immune response against SARS-CoV2 in a mammalian subject, the method comprising administering a composition as described above to said subject. In another aspect the invention relates to a method of inducing an immune response against SARS-CoV2 in a mammalian subject, the method comprising administering a dose of a composition as described above to said subject.
  • composition is administered once.
  • invention relates to a method as described above wherein two doses of said composition are administered to said subject.
  • the invention in another aspect relates to a method as described above wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject.
  • composition is administered twice.
  • composition is administered once per 12 months.
  • composition is administered once per 60 months.
  • composition is administered by a route of administration selected from a group consisting of intranasal, aerosol, intradermal and intramuscular.
  • said administration is intranasal or intramuscular.
  • said administration is intramuscular.
  • said spike protein is full length spike protein.
  • nucleic acid encoding the spike protein antigen, and/or encoding the tPA- spike protein antigen fusion is codon optimised for humans.
  • nucleic acid encoding the spike protein antigen, and/or encoding the tPA- spike protein antigen fusion is substituted to eliminate runs of repeat nucleotides such as 5 or more consecutive occurrences of the same nucleotide.
  • nucleic acid encoding the spike protein antigen, and/or encoding the tPA- spike protein antigen fusion is codon optimised for humans and is substituted to eliminate runs of repeat nucleotides such as 5 or more consecutive occurrences of the same nucleotide.
  • said polynucleotide sequence comprises the sequence of SEQ ID NO: 23 (encoding tPA-2816) or SEQ ID NO: 24 (encoding tPA-3990 ). These present the preferred nucleotide sequences as revised (i.e. after codon optimisation for humans introduced runs of same bases and after those runs of same bases were revised to retain the same coding sequence but remove the repeats) with tPA encoded. These are highly preferred aspects of the invention.
  • the nucleotide sequence encoding tPA is 1-96 in SEQ ID NO: 23 and in SEQ ID NO: 24.
  • the primary vaccination regimen is one dose. In some aspects it may be desired to re-administer at a later date. Intervals between first and second doses are disclosed in the examples. In some aspects it may be desired to re-administer at a later date, not less than 6 months after the first immunisation. Suitably it may be desired to re-administer at a later date, such as about 12 months after the first immunisation. Suitably it maybe desired to re-administer at a later date, such as about 12 to 60 months after the first immunisation. In one aspect suitably a second or further administration is given at about 12 months after the first immunisation. In one aspect suitably a second or further administration is given at about 60 months after the first immunisation.
  • a second or further administration is given more than 60 months after the first immunisation.
  • the invention relates to use of a composition as described above in inducing an immune response against SARS-CoV2. In another aspect, the invention relates to use of a composition as described above in immunising a subject against SARS-CoV2. In another aspect, the invention relates to use of a composition as described above in prevention of SARS-CoV2 infection.
  • the invention in another aspect, relates to a method of inducing an immune response against SARS-CoV2 in a mammalian subject, the method comprising administering a composition as described above to said subject.
  • administration is intramuscular or intranasal. Most suitably said administration is intramuscular.
  • the invention relates to a method of raising an immune response by administering the adeno-based viral vector as described above.
  • the invention relates to the adeno-based viral vector as described above for use in raising an anti- SARS-CoV2 immune response.
  • the invention relates to a method as described above, (e.g. a method of inducing an immune response against SARS-CoV2 in a mammalian subject, the method comprising administering a dose of a composition as described above to said subject) wherein the dose is administered after one or more doses of a second composition suitable for inducing an immune response against SARS-CoV2 in a mammalian subject.
  • said second composition is selected from mRNA vaccines (e.g. BNTi62b2 or tozinameran; mRNA-1273 or CX-024414), Adenovirus vector vaccines (e.g. AZD1222; Gam-COVID-Vac; Ad26.C0V2.S or JNJ-78436735), inactivated and subunit vaccines.
  • the dose of the composition as described above is administered less than 4 months, less than 6 months, less than 12 months, or less than 24 months after the one or more doses of the second composition.
  • antibodies induced as described herein are neutralising antibodies i.e. antibodies capable of neutralising SARS-CoV2 viral particles. Unless otherwise apparent from the context, ‘about’ means ⁇ 1% of the stated value.
  • the vaccine design comprises the complete SARS-CoV2 Spike protein expressed under the control of a strong mammalian promoter, which includes Tet repressor sequences to allow for repression of antigen expression during vaccine manufacture, improving vaccine yields.
  • composition of the invention comprises ChAdOxi :: SARS-CoV2 spike protein i.e. ChAdOxi comprising a nucleic acid insert having a nucleotide sequence encoding the SARS-CoV2 spike protein.
  • ChAdOxi comprising a nucleic acid insert having a nucleotide sequence encoding the SARS-CoV2 spike protein.
  • the full length spike protein is used.
  • One innovation provided is the intellectual choice of the particular amino acid sequence/variant of the viral spike protein which has been selected.
  • the spike protein has the sequence 2816 (e.g. the spike protein sequence from the fusion protein of SEQ ID NO: 3) or has the sequence 3990 (e.g. the spike protein sequence from the fusion protein of SEQ ID NO: 12), most suitably has the sequence 2816 (the spike protein sequence from the fusion protein of SEQ ID NO: 3).
  • the spike protein is present as a tPA-spike fusion and has the sequence of SEQ ID NO: 3 or SEQ ID NO: 12, most suitably SEQ ID NO: 3.
  • first codon optimisation of the coding sequence of the antigen for human codon usage is carried out. More specifically, codon optimisation of the nucleotide sequence encoding the tPA-SARS-CoV2 spike protein antigen fusion for human codon usage is carried out. Then, the sequence is analysed e.g. for patches in which the human codon optimisation process has resulted in runs of identical nucleotides. For example, runs of 5 consecutive “C” bases (cytosine bases) maybe identified. These repetitive sequences might cause problems in expression, leading to problems of vaccine performance, and/or polymerase “slippage” events, leading to problems in viral vector vaccine production due to nucleic acid instability (e.g.
  • the already mutated codon optimised sequence is further mutated.
  • the process designs and makes further substitutions in the nucleotide sequence, carefully preserving the encoded amino acids using the universal genetic code, whilst changing the nucleotide bases and selecting alternate codons to remove the slippage prone repeat sequences whilst ensuring the coding sequence still accurately encodes the desired antigen.
  • This approach delivers the technical benefit of facilitating viral vector vaccine production, obtaining good yields of virus.
  • WO2018/215766 describes a vaccine for MERS (Middle Eastern Respiratory
  • Aim is to increase spike antigen expression which may improve immunogenicity.
  • Figure 1 shows key mutations (substitutions/ deletions of AZD2816 (SEQ ID NO: 3) and AZD3990 (SEQ ID NO: 12) relative to AZDi222(SEQ ID NO: 1).
  • SARS-CoV2 spike protein is a clear exception to this rule and is further evidence towards inventive step.
  • a leader sequence/secretory sequence such as the tissue plasminogen activator (tPA) amino acid sequence fused to the N-terminus of the SARS- CoV2 spike protein antigen.
  • tPA tissue plasminogen activator
  • This combination delivers enhanced immunogenicity. This is especially true for the triple combination (ChAdOxi + tPA + SARS-CoV2 spike protein).
  • 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.
  • 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 ChAdOxi nCoV-19 (AZD2816).
  • 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.
  • the boosting (second or further) immunisation may comprise MVA, RNA or protein.
  • the boost (second or further immunisation) may comprise RNA or protein.
  • boosting regimes 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), ChAdOxi/MVA or ChAdOxi/RNA or ChAdOxi/protein as prime/boost regimes maybe used.
  • a homologous prime-boost regime such as ChAdOxi/ ChAdOxi, or such as ChAdOxi nCoV-19 (AZD3990)/ ChAdOxi nCoV-19 (AZD3990), most suitably ChAdOxi nCoV-19 (AZD2816)/ ChAdOxi nCoV-19
  • ChAdOxi nCoV-19 (AZD2816) finds particular application as boosting composition.
  • ChAdOxi nCoV-19 (AZD3990) finds particular application as boosting composition.
  • the invention relates to ChAdOxi nCoV-19 (AZD2816) for use as a boosting composition.
  • the invention relates to ChAdOxi nCoV-19 (AZD3990) for use as a boosting composition.
  • a heterologous prime-boost regime may be used such as when the priming composition (i.e. first 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, and the boosting composition (i.e. second or further immunisation) comprises ChAdOxi nCoV-19 (AZD2816).
  • the priming composition i.e. first immunisation
  • the boosting composition i.e. second or further immunisation
  • the boosting composition comprises ChAdOxi nCoV-19 (AZD2816).
  • a heterologous prime-boost regime may be used such as ChAdOxi nCoV- 19 (AZD1222)/ ChAdOxi nCoV-19 (AZD2816).
  • a heterologous prime-boost regime may be used such as ChAdOxi nCoV- 19 (AZD1222)/ ChAdOxi nCoV-19 (AZD3990).
  • the first composition i.e. priming composition
  • the second composition i.e. first boosting composition
  • the second composition maybe one or more compositions selected from MVA
  • the first composition i.e. priming composition
  • the second composition i.e. first boosting composition
  • the third composition i.e. second boosting composition
  • the first composition i.e. priming composition
  • the second composition i.e. first boosting composition
  • the third composition i.e. second boosting composition
  • the first composition maybe ChAdOxi nCoV-19 (AZD1222)
  • the second composition i.e. first boosting composition
  • the third composition is ChAdOxi nCoV-19 (AZD2816).
  • the first composition i.e. priming composition
  • the second composition i.e. first boosting composition
  • the third composition i.e. second boosting composition
  • composition for use as described above wherein said use comprises:
  • said first dose and said second dose and said third dose each comprise about the same number of viral particles.
  • the first administration comprises, or consists of, a composition according to the present invention comprising a viral vector capable of expressing the SARS-CoV2 Spike protein.
  • the second or further (‘boost’) administration comprises exactly the same antigen as for viral vector.
  • 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 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.
  • first administration Participants included in the analysis were divided into groups which received two different dose levels as first dose (i.e. as first administration (prime)). The doses of the first administration (prime) were given to first dose.
  • the invention relates to a dual administration regime where a first administration and a second administration are given to a single subject, wherein the ratio of the dose of the first administration to the dose of the second administration is
  • the invention relates to a dual administration regime where a first administration and a second administration are given to a single subject, wherein the ratio of the dose of the first administration to the dose of the second administration is 1:1.
  • the vaccine can be stored, transported and handled at normal refrigerated conditions (2-8 degrees Celsius/ 36-46 degrees Fahrenheit) for at least six months and administered within existing healthcare settings.
  • the invention also provides a method of inducing an immune response against SARS- CoV2 in a mammalian subject, or a method of preventing SARS-CoV2 infection in a mammalian subject, the method comprising
  • composition for use as described above wherein said use comprises:
  • the invention also provides a method of inducing an immune response against SARS- CoV2 in a mammalian subject, or a method of preventing SARS-CoV2 infection in a mammalian subject, or a compoistion for use in such a method, the method comprising (i) administering a first dose of a composition as described above to said subject; and
  • said second dose is administered at an interval of a) less than 6 weeks, b) 6 to 8 weeks, c) 9 to 11 weeks, or d) 12 weeks or more, after administration of said first dose.
  • said first dose comprises about 2.5 x to 10 viral particles.
  • said first dose comprises about 5 x to 10 viral particles and said second dose comprises about 5 x to 10 viral particles.
  • 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 neutralising antibody response.
  • the immune response comprises a cell mediated response.
  • the immune response comprises cell mediated immunity (CMI).
  • CMI cell mediated immunity
  • the immune response comprises induction of CD8+ T cells.
  • the immune response comprises induction of a CD8+ cytotoxic T cell (CTL) response.
  • CTL cytotoxic T cell
  • composition is an immunogenic composition.
  • COVID19 is the disease caused by the SARS-CoV2 virus in humans.
  • the invention further relates to a method for preventing COVID19 in a subject, the method comprising administering a composition as described above to said subject.
  • Sequences deposited in databases can change over time.
  • the current version of sequence database(s) are relied upon.
  • the release in force at the date of filing is relied upon.
  • the accession numbers may be version/dated accession numbers.
  • the citeable accession numbers for the current database entry are the same as above, but omitting the decimal point and any subsequent digits.
  • 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.
  • the current release is relied upon. More suitably the release available at the effective filing date is relied upon.
  • GenBank database release referred to is NCBI-GenBank Release 241: 15 December 2020.
  • the invention possesses the advantage of inducing protective immunity after single dose (single administration).
  • 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.
  • a protective immune response protects against subsequent infection or disease caused by SARS-CoV2.
  • the spike protein is a large type I transmembrane protein. This protein is highly glycosylated, containing numerous N-glycosylation sites. Spike proteins assemble into trimers on the virion surface to form the distinctive "corona", or crown- like appearance.
  • the ectodomains of all CoV spike proteins share the same organization in two domains: a N-terminal domain named SI that is responsible for receptor binding and a C-terminal S2 domain responsible for fusion. CoV diversity is reflected in the variable spike proteins (S proteins).
  • a polypeptide which ‘comprises the spike protein receptor binding domain (RBD)’ means a polypeptide comprising, or consisting of, amino acid sequence corresponding to amino acids 319-541 of SEQ ID NO: 1. The same applies to the other domains/sub- domains mentioned above.
  • ‘Corresponding to’ has its natural meaning in the art i.e. for identification of the domains/sub-do mains within different spike protein sequences. For example ‘Corresponding to’ may not mean 100% identical to. Sequence identity levels/substitutions relative to SEQ ID NO: 1 are as explained herein.
  • the antigen is the SARS-CoV2 spike protein.
  • the full length spike protein is used.
  • a reference spike protein is as disclosed in SEQ ID NO: 1.
  • Exemplary spike proteins according to the present invention are as disclosed in SEQ ID NO: 3 and/or SEQ ID NO: 12.
  • a further advantage of using the full length spike protein is that it allows for better T- cell responses. Without wishing to be bound by theoiy, 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 spike protein is included in the antigen of the invention.
  • tPA tPA tissue plasminogen activator
  • tPA leader sequence is suitably fused to the SARS-CoV2 spike protein antigen of the invention.
  • tPA is fused to the N-terminus of the spike protein sequence.
  • tPA leader sequence means the tPA amino acid sequence of SEQ ID NO: 5 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.
  • 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):
  • the antigen of the invention is provided as a fusion with tPA.
  • the tPA is fused to the N-terminus of the spike protein antigen.
  • the antigen does not comprise any further sequence tags.
  • antigen does not comprise any further linker sequences.
  • Adeno-based viral vectors are particularly preferred.
  • 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 AdHus such as human embryonic kidney cells 293 (HEK 293 cells).
  • Any suitable adeno-based viral vector may be used.
  • any replication-deficient viral vector, for human use preferably derived from a non-human adenovirus may be used.
  • Ads may be used for veterinary use.
  • ChAdOxi and ChAd0x2 are examples of a suitable non-human adenovirus vector for human use.
  • the adeno-based viral vector is ChAdOxi.
  • the nucleotide sequence of the ‘empty' ChAdOxi vector is shown in SEQ ID NO: 14. This is a viral vector based on Chimpanzee adenovirus C68. ChAdOxi 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): 640385, and in WO2012/172277. Both these documents are hereby incorporated herein by reference, in particular for the specific teachings of the ChAdOxi vector, including its construction and manufacture.
  • 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. It is also possible to insert antigens at the E3 site, or close to the inverted terminal repeat sequences, if desired.
  • a clone of ChAdOxi 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 AdChOxi (E4 modified) TIPeGFP, cell line name "AdChOxi (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
  • 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,
  • a related vaccine vector ChAd63
  • ChAd63 may be used if desired.
  • ChAdOxi nCoV-19 variants may be produced by any method known in the art.
  • ChAdOxi nCoV-19 variants we mean “ChAdOxi nCoV-19 AZD1222” or “ChAdOxi nCoV-19 AZD2816” or “ChAdOxi nCoV-19 AZD3990” or other ChAdOxi vector comprising a nCoV-19 spike protein sequence variant.
  • ChAdOxl nCoV-19 variants may be produced as described in the examples, for example as described for ChAdOxl nCoV-19 AZD1222.
  • the spike protein (S) of SARS-Cov-2 (Genbank accession number YP 009724390.1) was codon optimised for expression in human cell lines and synthesised by GeneArt Gene Synthesis (Thermo Fisher Scientific). The sequence encoding amino acids 2-1273 were cloned into a shuttle plasmid following InFusion cloning (Clontech).
  • the shuttle plasmid encodes a modified human cytomegalovirus major immediate early promoter (IE CMV) with tetracycline operator (TetO) sites, poly adenylation signal from bovine growth hormone (BGH) and a tPA signal sequence upstream of the inserted gene.
  • IE CMV human cytomegalovirus major immediate early promoter
  • TetO tetracycline operator
  • BGH bovine growth hormone
  • ChAdOxl nCoV-19 means AZD1222 i.e. the ChAdOxl adenoviral vector as described in Dicks et al. (2012) PLoS ONE 7(7): 640385, and/or in WO2012/172277, comprising the nucleotide sequence of SEQ ID NO: 4 (encoding 32aa tPA leader fused to SARS-Cov-2 spike protein) inserted at the El locus of the ChAdOxl adenoviral vector under the control of the CMV (cytomegalovirus) ‘long’ promoter. This is as described in PCT/GB2021/050602.
  • this “ChAdOxl nCoV-19” (ChAdOxl nCoV-19 AZD1222) has the nucleotide sequence as shown in SEQ ID NO: 25 in PCT/GB2021/050602 (44104nt).
  • This “ChAdOxl nCoV-19” / AZD1222 viral vector is not itself part of the current invention, but does form part of the invention where described for use in immunisation methods/prime-boost regimes, or as a component of multi-part kits / compositions and the like which are disclosed herein.
  • ChAdOxl nCoV-19 AZD2816 (sometimes referred to as “AZD2816”) means the ChAdOxl adenoviral vector as described in Dicks et al. (2012) PLoS ONE 7(7): 640385, and/or in WO2012/172277, comprising the nucleotide sequence of SEQ ID NO: 3 (encoding 32aa tPA leader (SEQ ID NO: 5) fused to SARS-Cov-2 ‘2816’ spike protein) inserted at the El locus of the ChAdOxl adenoviral vector under the control of the CMV (cytomegalovirus) ‘long’ promoter. Most suitably this “ChAdOxl nCoV-19 AZD2816” (AZD2816) has the nucleotide sequence as shown in SEQ ID NO: 13.
  • the AZD2816 spike protein amino acid sequence may be taken from SEQ ID NO: 3 by removing the tPA sequence (underlined) and replacing it with a single methionine.
  • the invention relates to an isolated spike protein polypeptide having the AZD2816 amino acid sequence.
  • ChAdOxl nCoV-19 AZD3990 (sometimes referred to as “AZD3990”) means the ChAdOxl adenoviral vector as described in Dicks et al. (2012) PLoS ONE 7(7): 640385, and/or in WO2012/172277, comprising the nucleotide sequence of SEQ ID NO: 12 (encoding 32aa tPA leader (SEQ ID NO: 5) fused to SARS-Cov-2 ‘3990’ spike protein) inserted at the El locus of the ChAdOxl adenoviral vector under the control of the CMV (cytomegalovirus) ‘long’ promoter. Most suitably this “ChAdOxl nCoV-19 AZD3990” (AZD3990) has the nucleotide sequence as shown in SEQ ID NO: 25.
  • AZD3990 spike protein amino acid sequence this may be taken from SEQ ID NO: 12 by removing the tPA sequence (underlined) and replacing it with a single methionine.
  • the invention relates to an isolated spike protein polypeptide having the AZD3990 amino acid sequence.
  • any suitable route of administration may be used.
  • the invention may be administered by aerosol delivery to the respiratory tract using a widely available device commonly used for drug delivery.
  • This may be a suitable route of vaccine delivery for respiratory pathogens such as coronaviruses.
  • the composition may comprise a MVA-vectored vaccine, wherein aerosol delivery may result in strong immune responses in the respiratory tract at low doses.
  • a further advantage of aerosol deliver is avoidance of needles.
  • the route of administration is selected from group consisting of subcutaneous, intranasal, aerosol, nebuliser, intradermal and intramuscular.
  • the route of administration is selected from a group consisting of intranasal, aerosol, intradermal and intramuscular.
  • the route of administration is selected from a group consisting of intranasal, aerosol and intramuscular.
  • the route of administration is selected from a group consisting of intranasal and intramuscular.
  • the route of administration is intramuscular.
  • the route of administration maybe 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 10 9 to 10 11 viral particles.
  • the range is from 2.5X 10 10 vp to 5x 10 10 vp.
  • the dose(s)/range of dose(s) maybe derived from the examples below.
  • 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 container containing a composition as described above is provided.
  • said container may be a vial.
  • said container may be a syringe.
  • 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.
  • 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.
  • Formulations for other administration routes such as aerosol will be adjusted accordingly by the skilled operator.
  • sequence is, or is derived from, amino acid sequence provided herein, such as SEQ ID NO. 3 or SEQ ID NO: 12.
  • sequence used in the vector of the invention comprises or encodes amino acid sequence having at least 95% sequence identity, suitably having at least 96% sequence identity , suitably having at least 97% sequence identity, suitably having at least 98% sequence identity, suitably having at least 98.7% sequence identity, suitably having at least 99% sequence identity, suitably having at least 99.1% sequence identity to the reference amino acid sequence, for example the reference amino acid sequence provided as SEQ ID NO. 1.
  • a sequence identity level of 99% compared to SEQ ID NO. 1 corresponds to approximately 12 to 13 substitutions across the full length of the spike protein sequence provided as SEQ ID NO. 1.
  • the spike protein sequence used has 17 or fewer substitutions relative to SEQ ID NO: 1, suitably 16 or fewer substitutions relative to SEQ ID NO: 1, suitably 15 or fewer substitutions relative to SEQ ID NO: 1, suitably 14 or fewer substitutions relative to SEQ ID NO: 1, suitably 13 or fewer substitutions relative to SEQ ID NO: 1, suitably 12 or fewer substitutions relative to SEQ ID NO: 1, suitably 11 or fewer substitutions relative to SEQ ID NO: 1, suitably 10 or fewer substitutions relative to SEQ ID NO: 1, suitably 9 substitutions relative to SEQ ID NO: 1.
  • SEQ ID NO: 12 has the same mutations as SEQ ID NO: 3, PLUS a further 6 substitutions to proline (‘hexapro’).
  • deletion of an amino acid is regarded as a substitution.
  • the spike protein sequence used e.g. SEQ ID NO: 12 -
  • AZD3990 has 17 substitutions relative to SEQ ID NO: 1 (98.7% sequence identity to SEQ ID NO: 1).
  • the spike protein sequence used e.g. SEQ ID NO: 3 - AZD2816
  • the spike protein sequence used has 11 substitutions relative to SEQ ID NO: 1 (99.1% sequence identity to SEQ ID NO: 1).
  • spike protein amino acid sequence used is as encoded by the relevant section of the nucleotide sequence of SEQ ID NO: 13 (AZD2816 viral genome sequence).
  • the spike protein amino acid sequence used is as encoded by the relevant section of the nucleotide sequence of SEQ ID NO: 25 (AZD3990 viral genome sequence).
  • any amino acid substitutions are not in the receptor binding domain.
  • any amino acid substitutions are outside the receptor binding domain.
  • sequence identity of the nucleotide sequence encoding the spike protein is considered by comparison to reference sequence provided as SEQ ID NO. 11 or SEQ ID NO: 4.
  • SEQUENCE IDENTITY For example suitably the sequence identity of the nucleotide sequence encoding the spike protein is considered by comparison to reference sequence provided as SEQ ID NO. 11 or SEQ ID NO: 4.
  • 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.
  • Percent identity maybe calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
  • the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied. It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • sequence identity is considered for a segment of spike protein comprising at least the receptor binding domain (RBD), or at least the N-terminal domain (NTD), or at least the STEM; more suitably sequence identity is considered for a segment of spike protein comprising at least the receptor binding domain (RBD) and the N-terminal domain (NTD); more suitably sequence identity is considered for a segment of spike protein comprising at least the receptor binding domain (RBD) and the N-terminal domain (NTD), and the STEM. Most suitably sequence identity is considered for full length spike protein, e.g. the full length spike protein of SEQ ID NO: 1. ADVANTAGES AND APPLICATIONS
  • Figure 1 shows a table
  • Figure 2 shows a flow chart
  • Figure 5 shows bar charts of Immune response induced by vaccination with either wildtype (Jenner E ; AZD2816) or stabilised (Jenner E6; AZD3990) Spike sequence.
  • Figure 6 shows bar charts of Cross-reactive antibodies are increased following a booster dose with AZD2816 vaccine
  • the actual ELISPOT protocol is a standard technique which is typically always carried out in the same manner.
  • the specificity for the validated ELISPOT protocol comes from the peptides used.
  • the peptides used are derived from the SARS-CoV2 spike protein.
  • a series of overlapping peptides are synthesised beginning with the first amino acid of the spike protein.
  • 2omer peptides are synthesised.
  • adenovirus El gene must be supplied in trans by the cell line used for vaccine manufacture. In HEK293 cells, this gene is flanked by other sequences from adenovirus 5 which are present in the Ads vaccine vector, such that in rare cases a double crossover event result in the generation of replication-competent adenovirus. This is undesirable and has been solved by either the use of a different adenoviral vector such as ChAdOxi, in which the homology between the vector and the cell line is too low to allow for recombination, or the use of a cell line which expresses Ads El with no flanking sequences such as PerC6, or others developed by different companies.
  • Quality control tests include concentration (which is the potency assay), sterility, DNA sequence of vaccine antigen and absence of adventitious agents.
  • concentration which is the potency assay
  • sterility DNA sequence of vaccine antigen
  • adventitious agents DNA sequence of vaccine antigen and absence of adventitious agents.
  • the use of deep sequencing greatly accelerates characterisation of vaccine seed stocks, to confirm clonality without lengthy rounds of virus cloning, and also in detection of adventitious agents. Thus the time taken for release testing may be greatly shortened.
  • Example 3 Growth and Quantification of Viral Vectors
  • Repressed MOI 3 8m1 Blasticidin + virus at a multiplicity of infection (MOI) of 3
  • De-repressed MOI 3 8qm1 of lmg/ml tetracycline + virus MOI 3 Flasks were returned to incubate (37°C, 5% CO 2 )
  • IU was quantified using a titre immunoassay. Briefly, a black walled / clear flat bottomed 96 well plate (Corning) was seeded with adherent HEK293 TREx cells in standard growth media (below) to obtain a 95% confluent monolayer on the day required.
  • ChAdOx1 plasmid DNA of a known concentration was diluted to generate sample of a given copy number per well.
  • qPCR master mix was prepared using 2x Luna probe mix (NEB), ChAd0x2 specific primers (Thermo Fisher), ChAdOxi specific universal probe (TAMRA / FAM) (Applied Biosystems) and nuclease free water to a final volume of 15 m ⁇ per sample. Mastermix was mixed and 15 m ⁇ added to the relevant wells of a 96 well MicroAmp FAST Optical PCR plate. Template / plasmid standard / samples were added (5m1 per well) to relevant test wells. Optical film was used to cover the plate before the relevant qPCR programme was run on a StepOne qPCR machine.
  • PCR programme 95°C for 10 mins, 45 cycle of 95°C for 15 sec, 6o°C for 1 min. Recovered data was analysed using the standard curve results to generate viral genome copy number per well, which was further calculated to give genome copy per ml culture. To compare the IU titre between de-repressed and repressed, the genome copy number values of the de-repressed culture were set at 100% and the difference of the repressed culture compared to this. Repressed and de-repressed cultures gave a similar IU of virus at all time points tested.
  • ChAdOxi nCoV-19 (AZD1222)
  • ChAdOx1 nCoV-19 spike protein vaccines e.g. AZD1222 (reference) AZD2816
  • inventions AZD3990 (invention) described herein consist of the replication-deficient simian adenovirus vector ChAdOxi, containing the structural surface glycoprotein (Spike protein) antigen of the SARS CoV-2 (nCoV-19) expressed under the control of the CMV promoter, with a leading tissue plasminogen activator (tPA) signal sequence.
  • the tPA leader sequence has been shown to be beneficial in enhancing immunogenicity.
  • the different vaccines comprise different spike protein sequences/ mutants as described.
  • the code name for Drug Substance AZD1222 is ChAdOxi nCoV-19. There is no recommended International Non-proprietary Name (INN).
  • the ChAdOxi nCoV-19 (AZD1222) drug substance has a genome size of 35,542bp and is a slightly opaque frozen liquid, essentially free from visible particulates. The appearance is dependent upon the concentration of the virus and the buffer that the virus is formulated in.
  • the ChAdOx1 vector is replication-deficient as the El gene region, essential for viral replication, has been deleted. This means the virus will not replicate in cells within the human body.
  • the E3 locus is additionally deleted in the ChAdOx1 vector.
  • ChAdOx1 propagates only in cells expressing El, such as HEK293 cells and their derivatives or similar cell lines such as Per.C6 (Crucell).
  • the vaccine consists of the attenuated chimpanzee adenovirus vector ChAdOx1, expressing the SARS CoV-2 spike protein under the control of the CMV promoter.
  • Pre- adenoviral plasmid pBAC ChAdOx1 nCoV19 was generated.
  • the SARS CoV-2 Spike cDNA including a 32 amino acid N-terminal tPA leader sequence, obtained from GeneArt, was inserted into the El locus of ChAdOx1 by Gateway recombination.
  • the “long CMV promoter” is used. This is known in the art, and is described in PCT/GB2008/001262 (WO/2008/122811).
  • CMVLP Long CMV promoter
  • TO Tet operator
  • #p57io pENTR plasmid vector containing the CoV Spike antigen AZD1222 between the ‘long’ CMVLP TO promoter and BGH poly A sequences.
  • #p2503 pBAC ChAdOxi vector with El and E3 deleted, and E4 modified to improve yield and hexon expression for markerless titration. It was generated at the Jenner Institute, and its complete genome sequence is known The SARS CoV-2 Spike antigen was excised from #p5727 using Notl and Kpnl and ligated into #1990 cut with the same enzymes to obtain #p57io. The insert was verified by restriction mapping and sequencing. Gateway recombination was then performed between #5710 and #2563.
  • the sequence of the transgene region in ChAdOxi nCoV-19 (AZD1222) has been verified by sequencing directly from phenol purified viral genomic DNA.
  • the P5713 pDEST-ChAdOxi-nCOV-19 plasmid is used in the manufacture of the composition according to the present invention.
  • the plasmid encodes a viral vector according to the invention.
  • the viral sequence is excised from P5713 pDEST-ChAdOxi-nCOV-19 and the linear viral DNA is subsequently used to transfect El expressing cells, such as HEK293-TRex cells, for viral vaccine production.
  • ChAdOxi nCoV-19 (AZD2816) is constructed as above EXCEPT it is prepared so as to contain the tPA-spike fusion protein antigen of SEQ ID NO: 3.
  • ChAdOxi nCoV-19 (AZD3990) is constructed as above EXCEPT it is prepared so as to contain the tPA-spike fusion protein antigen of SEQ ID NO: 12.
  • AZD3990 sequence (Sequence: SEQ ID NO: 25) is used in the manufacture of the composition (instead of the P5713 pDEST-ChAdOxi-nCOV-19 used for AZD1222).
  • Example 5 AZD2816 D7220 C00001 Ph 2/3 Safety and Immunogenicity Vaccines (viral vectors as described above) are evaluated as follows:
  • Study Design AZ-sponsored, multi-center, multi- country, partially double blind, randomized controlled Phase 2/3 study in adults.
  • Example 8 Evaluation of Wildtype and Stabilised SARS-CoV-2 Spike sequences as vaccine imm unogens
  • T cell responses measured 21 days later. It was surprising to note that, despite the stabilised spike protein giving superior cell expression levels in vitro, levels of anti - spike antibodies induced by vaccination with AZD3990 (stabilized spike) were similar to those induced by AZD2816 (wildtype spike sequence) as shown in Figure 5A and B. Also surprising was the observation that T cell responses where significantly higher in mice vaccinated with AZD2816 expressing the wildtype Spike sequence compared to mice vaccinated with AZD3990 expressing the hexapro stabilised spike sequence (Figure 5C).
  • BALB/c mice were vaccinated intramuscularly with a dose response (10 8 iu to 10 5 iu) of either AZD2816 (wildtype sequence Jenner E) or AZD3990 (hexapro stabilised Jenner E6). 21 days later, total IgG levels were measured by ELISA against original spike protein (NC_045512) or B.1.351 spike protein. IFNg secreting cells measured by ELISpot with splenocytes stimulated with whole spike protein.
  • Example 9 Evaluation of AZD2816 as a novel ChAdOxl vectored B.1.351 variant vaccine BALB/c mice were immunised with 10 8 iu AZD1222 (ChAdOx1 nCoV-19), AZD2816 (ChAdOx1 nCoV-19 B.1.351) or with 10 8 iu of each vaccine mixed together prior to injection in prime only or prime and boost regimens. Functional ability of antibodies to neutralise pseudotyped virus expressing original spike, B.1.351 or B.1.617 spike protein was measured in the serum of vaccinated mice. AZD2816 was shown to induce cross- reactive neutralising antibodies in mice when given as a single dose, in a mixture with AZD1222 or as a boost dose following AZD1222 vaccination regimen (Table below).
  • AZD2816 When delivered as a single prime vaccination, AZD2816 induced neutralising antibodies against original Wuhan and B.1.351 virus. When compared to the original AZD1222 prototype vaccine, AZD2816 induced slightly lower neutralising titres to the original Wuhan spike protein but higher neutralisation against the B.1.351 spike protein. Mixing both vaccines together did not compromise the antibody response to either protein.
  • AZD2816 When given as a vaccine boost to mice previously vaccinated with AZD1222 , AZD2816 was observed to significantly increase the neutralising antibody titres measured against original Wuhan, B.1.351 and B.1.617 variants. In addition, boosting AZD1222 primed mice with AZD2816 increased the binding antibody titre against variant proteins P.i and B.1.429 when compared to a single dose of AZD1222 ( Figure 6). Graph A.
  • Graph B shows the Microneutralisation titre of serum (ND80) collected day 16 post-vaccination (animals Figure 5) and 21 days after prime-boost vaccination against pseudotyped virus expressing original (NC_045512), B.1.351 or B.1.617.1 spike protein. Limit of detection in the assay is defined as a titre of 40 (dotted line).
  • D614G is found in nearly all strains now. It is derived from the original defined A lineage. For example, all of B.1.1.7, P-i, B.1.429 contain D614G.
  • Example 10 Evaluation of AZD2816 as a novel ChAdOxl vectored vaccine against different variants of concerns
  • a multi-center, multi-country, randomized controlled Phase 2/3 study was conducted to determine the safety and immunogenicity of AZD2816 in previously unvaccinated adults, as well as the safety and immunogenicity of a booster dose of AZD1222 or AZD2816 in previously vaccinated adults.
  • the third dose boosters of AZD1222 or AZD2816 were administered >3 months after receipt of the second dose of either AZD1222 or an mRNA vaccine.
  • the immunogenicity was measured 28 days after the third dose booster.
  • the reactogenicity (solicited local and systemic events) were recorded for first 7 days after third dose booster. All unsolicited AEs were recorded for 28 days after third dose booster.
  • a schematic representation of the study design can be found in figure 2.
  • Neutralising antibodies were measured using a Focus Reduction Neutralization Test (FRNT). Briefly, serially diluted Ab or plasma was mixed with SARS-CoV2 strain. Mixtures were then transferred to 96-well, cell culture-treated, flat-bottom microplates containing confluent Vero cell monolayers in duplicate and incubated for a further 2 hours followed by the addition of carboxymethyl cellulose overlay medium. A focus forming assay was then performed by staining Vero cells with human anti-NP mAh (mAb2o6) followed by peroxidase-conjugated goat anti-human IgG (A0170; Sigma). Finally, the foci (infected cells), approximately too per well in the absence of antibodies, were visualized by adding TrueBlue Peroxidase Substrate.
  • FRNT Focus Reduction Neutralization Test
  • Virus-infected cell foci were counted on an AID EliSpot reader using AID ELISpot software. The percentage of focus reduction was calculated, and IC 50 was determined using the probit program from the SPSS package. SARS-CoV-2 live neutralizing antibody titers against the different variants were assessed in the participants. Day 29 values were plotted. Boxplots were created based on the lognormal distribution. Geometric mean titers at Baseline and Day 29 were tabulated. Titers assessed as less than the lower limit of quantitation were set to half of the starting dilution (20 or 80, depending on the sample).
  • AZD1222 and AZD2816 third dose boosters increased neutralising antibody titres to Wuhan and Beta variants in participants previously vaccinated with AZD1222
  • AZD1222 and AZD2816 third dose boosters increased breadth of humoral response to variants of concern in participants previously vaccinated with AZD1222
  • Figure 7 B AZD1222 and AZD2816 third dose boosters increased neutralizing antibody titres to Wuhan and Beta variants in participants previously vaccinated with mRNA vaccines
  • Figure 7C AZD1222 and AZD2816 third dose boosters increased breadth of humoral response to variants of concern in participants previously vaccinated with mRNA vaccines
  • AZD1222 and AZD2816 show neutralization of Omicron variant after 3rd dose booster with a trend toward increased geometric mean titer (GMT) after AZD2816 boost (Figure 7 E).
  • GTT geometric mean titer
  • AZD1222 and AZD2816 were generally well-tolerated following boosting of subjects that had previously received 2 doses of AZD1222 or an mRNA vaccine.
  • Third dose boosting with AZD1222 increased neutralizing titres against the original Wuhan strain as well as variants of concern (Beta, Gamma, Delta, Omicron) as compared to the two-dose primary series.
  • Third dose of AZD2816 increases neutralizing titres against Beta to a higher magnitude than a third dose of AZD1222.
  • SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22 please note that these are spike protein sequences without N-terminal tPA fusions.
  • the N-terminal Methionine of the spike protein sequence is omitted and replaced with the tPA amino acid sequence (which includes an initating Methionine - see for example SEQ ID NO: 5).
  • compositions comprising a viral vector, wherein the viral vector is an adenovirus based vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide, said polypeptide having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, characterised in that said polypeptide comprises the amino acid sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21 or 22.
  • said polypeptide is present as a fusion with the tissue plasminogen activator (tPA) sequence in the order N-terminus - tPA - polypeptide - C- terminus.
  • tissue plasminogen activator tPA
  • said tPA sequence comprises, or consists of, the amino acid sequence of SEQ ID NO: 5.
  • the lead methionine of the amino acid sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21 or 22 is omitted as noted above.
  • the descriptions of viral vector construction, insertion cargo sequence, promoters, etc relate equally to these aspects.

Abstract

The invention relates to A composition comprising a viral vector, wherein the viral vector is an adenovirus based vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide, said polypeptide having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, characterised in that said polypeptide comprises the following substitutions relative to SEQ ID NO: 1: L18F, D80A, G215D, L242 Δ, A243 Δ, L244 Δ, K417N, E484K, N501Y, D614G; and A701V. The invention also related to uses and methods.

Description

Compositions and Methods for Inducing an Immune Response
FIELD OF THE INVENTION
The invention relates to induction of immune responses, suitably protective immune responses, against SARS-CoV2 (nCoV-19).
BACKGROUND TO THE INVENTION
Coronavirus 19 (SARS-CoV2; sometimes referred to as nCoV-19 or as COVID-19) is the virus responsible for an outbreak of coronavirus disease that was first reported from Wuhan, China, on 31 December 2019.
Symptoms of the disease include fever, dry cough, muscle pain, and respiratory problems such as breathing difficulties / shortness of breath. In more severe cases, infection can cause pneumonia, severe acute respiratory syndrome, kidney failure and even death. Mortality rates have been estimated by the World Health Organisation (WHO) at up to 3.4% of infected individuals, with many commentators agreeing on a mortality rate of approx. 1-2% of infected individuals once figures are adjusted taking into account the mildest cases which are not always reported (e.g. if individuals did not seek treatment or diagnosis).
This is the first ever pandemic caused by a coronavirus. According to the World Health Organisation report as of 21 April 2021, the global number of new cases of COVID-19 in the previous week was 5.2 million, and the number of new deaths in that week was over 83,000.
The emergence of viral isolates/strains bearing mutations associated with increased rates of transmission, and in some cases associated with suspected increased mortality rates, is a serious problem. For example, B.1.1.7 ‘UK’ strains have evolved for fitness, and B.1.351 ‘S. Africa’ strains have evolved for immune escape. There are concerns about the efficacy of existing vaccines against these viral strains. For example there maybe a 2.9-fold drop in neutralising titres against B.1.1.7 in vitro for existing vaccines; there maybe decreased efficacy against mild-to-moderate COVID-19 and/or 9-fold drop in neutralising titres against against B.1.351 in vitro; there maybe decreased neutralization of the South Africa variant by AZD1222 and Pfizer anti-sera. Thus there is a need for further vaccines against SARS-CoV2, in particular to induce immune responses against these new viral strains. The present seeks to overcome problem(s) associated with the prior art. SUMMARY OF THE INVENTION
We describe a combination which comprises a simian adenoviral vector (such as ChAdOxi) delivering a SARS-CoV2 antigen (the spike protein). In more detail, the particular selection of the antigenic sequences of the invention was an important intellectual choice which had to be made.
The first sequence for SARS-CoV-2 was released on Friday 10th January 2020. An enormous number of further viral sequences have been released over the following weeks and months. For example, searching the NCBI GenBank sequence database for “SARS-CoV2 Complete Genome” reveals 160783 entries, and even narrowing this to “SARS-CoV2 spike protein” reveals 2182 entries. This is clearly an overwhelming number of possibilities.
The inventors have analysed these and have made many challenging intellectual choices in selecting the particular SARS-CoV2 antigen, and in choosing the specific sequences of that antigen which have actually been incorporated into the compositions of the invention, as well as the fusion of that selected antigen as explained in detail below. The technical problem was excacerbated by the emergence of numerous viral variants in different populations around the world, together with the need to promote cross-protection between specific viral strains thought to be of greatest clinical priority, and yet at the same time generate an immune response that is as ‘universal’ as possible. These opposing priorities added a further layer of complexity to the task facing the inventors. Thus, these factors, together with the huge array of possibilities available to a skilled worker without knowledge of the invention, clearly illustrate the inventive step associated with the invention described herein.
Thus, in one aspect the invention relates to a composition comprising a viral vector, wherein the viral vector is an adenovirus based vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide, said polypeptide having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, characterised in that said polypeptide comprises the following substitutions relative to SEQ ID NO: 1: a) L18F b) D80A c) G215D d) L242 A e) A243 D f) L244 D g) K417N h) E484K i) N501Y j) D614G; and k) A701V.
In another aspect the invention relates to a composition as described above wherein said polypeptide further comprises the following substitutions relative to SEQ ID NO: 1: 1) F814P m) A889P n) A896P 0) A939P p) K983P; and q) V984P.
Suitably said adenovirus based vector is a simian adenovirus based vector.
Suitably said adenovirus based vector is ChAdOx 1. Suitably said polypeptide is a spike protein polypeptide.
Suitably said polypeptide comprises the spike protein receptor binding domain (RBD).
Suitably said polypeptide comprises the spike protein receptor binding domain (RBD), the spike protein N-terminal Domain (NTD) and the spike protein STEM.
Suitably said polypeptide is full length spike protein.
Suitably said polypeptide is present as a fusion with the tissue plasminogen activator (tPA) sequence in the order N-terminus - tPA - polypeptide - C-terminus.
Suitably said tPA has the amino acid sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Suitably said polypeptide has the amino acid sequence SEQ ID NO: 3 or SEQ ID NO:
12. Suitably said polynucleotide sequence comprises the sequence of SEQ ID NO: 23 (encoding 2816) or SEQ ID NO: 24 (encoding 3990), preferably SEQ ID NO: 23 (encoding 2816).
Suitably said viral vector sequence is as in ECACC accession number 12052403.
Suitably administration of a single dose of a composition as described above to a mammalian subject induces protective immunity in said subject. Suitably administration of two doses of a composition as described above to a mammalian subject induces protective immunity in said subject.
Suitably administration of a first dose of a composition as described above to a mammalian subject, followed by subsequent administration of a second dose of said composition to said subject, induces protective immunity in said subject.
In another aspect the invention relates to use of a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2. Suitably said immune response is an immune response in a mammalian subject. In another aspect the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein a single dose of said composition is administered to said subject.
In another aspect the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein two doses of said composition are administered to said subject.
In another aspect the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject.
In another aspect the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein said composition is administered once. In another aspect the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein said composition is administered twice.
In another aspect the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject.
Suitably said composition is administered once per 12 months. Suitably said composition is administered once per 60 months.
In another aspect the invention relates to a composition as described above for induction of, or for use in induction of, an immune response against SARS-CoV2 in a mammalian subject, wherein a first dose of said composition is administered to said subject after one or more doses of a different composition suitable for preventing,
SARS-CoV2 infection had been administered previously. Non limiting examples of such different compostions are; mRNA vaccines (e.g. BNTi62b2 or tozinameran; mRNA- 1273 or CX-024414), Adenovirus vector vaccines (e.g. AZD1222; Gam-COVID-Vac; Ad26.C0V2.S or JNJ-78436735), inactivated and subunit vaccines.
In another aspect the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection.
Suitably preventing SARS-CoV2 infection is preventing SARS-CoV2 infection in a mammalian subject.
In another aspect the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein a single dose of said composition is administered.
In another aspect the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein two doses of said composition are administered to said subject.
In another aspect the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject. In another aspect the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein said composition is administered once.
In another aspect the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein said composition is administered twice.
In another aspect the invention relates to a composition as described above for preventing, or for use in preventing, SARS-CoV2 infection in a mammalian subject, wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject. Suitably said composition is administered once per 12 months.
Suitably said composition is administered once per 60 months.
In another aspect the invention relates to use of a composition as described above in medicine.
In another aspect the invention relates to a composition as described above for use in medicine. In another aspect the invention relates to a composition as described above for use as a medicament. In another aspect the invention relates to use of a composition as described above in the preparation of a medicament for prevention of, or for use in prevention of, SARS- CoV2 infection.
Suitably prevention of SARS-CoV2 infection is prevention of SARS-CoV2 infection in a mammalian subject.
In another aspect the invention relates to a method of inducing an immune response against SARS-CoV2 in a mammalian subject, the method comprising administering a composition as described above to said subject. In another aspect the invention relates to a method of inducing an immune response against SARS-CoV2 in a mammalian subject, the method comprising administering a dose of a composition as described above to said subject.
In another aspect the invention relates to a method as described above wherein a single dose of said composition is administered to said subject.
Suitably said composition is administered once. In another aspect the invention relates to a method as described above wherein two doses of said composition are administered to said subject.
In another aspect the invention relates to a method as described above wherein a first dose of said composition is administered to said subject, and subsequently a second dose of said composition is administered to said subject.
Suitably said composition is administered twice.
Suitably said composition is administered once per 12 months.
Suitably said composition is administered once per 60 months.
Suitably said composition is administered by a route of administration selected from a group consisting of intranasal, aerosol, intradermal and intramuscular.
Suitably said administration is intranasal or intramuscular.
Suitably said administration is intramuscular.
Suitably said spike protein is full length spike protein.
Suitably the nucleic acid encoding the spike protein antigen, and/or encoding the tPA- spike protein antigen fusion, is codon optimised for humans.
Suitably the nucleic acid encoding the spike protein antigen, and/or encoding the tPA- spike protein antigen fusion, is substituted to eliminate runs of repeat nucleotides such as 5 or more consecutive occurrences of the same nucleotide.
Suitably the nucleic acid encoding the spike protein antigen, and/or encoding the tPA- spike protein antigen fusion, is codon optimised for humans and is substituted to eliminate runs of repeat nucleotides such as 5 or more consecutive occurrences of the same nucleotide.
Most suitably said polynucleotide sequence comprises the sequence of SEQ ID NO: 23 (encoding tPA-2816) or SEQ ID NO: 24 (encoding tPA-3990 ). These present the preferred nucleotide sequences as revised (i.e. after codon optimisation for humans introduced runs of same bases and after those runs of same bases were revised to retain the same coding sequence but remove the repeats) with tPA encoded. These are highly preferred aspects of the invention. The nucleotide sequence encoding tPA is 1-96 in SEQ ID NO: 23 and in SEQ ID NO: 24.
Suitably the primary vaccination regimen is one dose. In some aspects it may be desired to re-administer at a later date. Intervals between first and second doses are disclosed in the examples. In some aspects it may be desired to re-administer at a later date, not less than 6 months after the first immunisation. Suitably it may be desired to re-administer at a later date, such as about 12 months after the first immunisation. Suitably it maybe desired to re-administer at a later date, such as about 12 to 60 months after the first immunisation. In one aspect suitably a second or further administration is given at about 12 months after the first immunisation. In one aspect suitably a second or further administration is given at about 60 months after the first immunisation.
In one aspect suitably a second or further administration is given more than 60 months after the first immunisation.
In one aspect suitably an even later second or further administration is even better.
In one aspect, the invention relates to use of a composition as described above in medicine. In one aspect, the invention relates to use of a composition as described above in the preparation of a medicament for prevention of SARS-CoV2 infection.
In another aspect, the invention relates to use of a composition as described above in inducing an immune response against SARS-CoV2. In another aspect, the invention relates to use of a composition as described above in immunising a subject against SARS-CoV2. In another aspect, the invention relates to use of a composition as described above in prevention of SARS-CoV2 infection.
In another aspect, the invention relates to a method of inducing an immune response against SARS-CoV2 in a mammalian subject, the method comprising administering a composition as described above to said subject.
Suitably a single dose of said composition is administered to said subject.
Suitably said composition is administered once.
Suitably said composition maybe administered once per 6 months.
More suitably said composition is administered once per 12 months. More suitably said composition is administered once per 60 months.
In another aspect the invention relates to a method as described above, or a composition for use as described above, wherein said first dose comprises AZD1222 and wherein said second dose comprises AZD2816. Suitably said composition is administered by a route of administration selected from a group consisting of subcutaneous, intranasal, aerosol, nebuliser, intradermal and intramuscular.
Most suitably said administration is intramuscular or intranasal. Most suitably said administration is intramuscular.
In one aspect, the invention relates to a method of raising an immune response by administering the adeno-based viral vector as described above.
In one aspect, the invention relates to the adeno-based viral vector as described above for use in preventing SARS-CoV2 infection.
In one aspect, the invention relates to the adeno-based viral vector as described above for use in raising an anti- SARS-CoV2 immune response.
In one aspect, the invention relates to a method as described above, (e.g. a method of inducing an immune response against SARS-CoV2 in a mammalian subject, the method comprising administering a dose of a composition as described above to said subject) wherein the dose is administered after one or more doses of a second composition suitable for inducing an immune response against SARS-CoV2 in a mammalian subject. In one embodiment suitably said second composition is selected from mRNA vaccines (e.g. BNTi62b2 or tozinameran; mRNA-1273 or CX-024414), Adenovirus vector vaccines (e.g. AZD1222; Gam-COVID-Vac; Ad26.C0V2.S or JNJ-78436735), inactivated and subunit vaccines.
In one embodiment suitably the dose of the composition as described above is administered less than 4 months, less than 6 months, less than 12 months, or less than 24 months after the one or more doses of the second composition.
DETAILED DESCRIPTION Coronavirus 19 (SARS-CoV2; nCoV-19; sometimes referred to as COVID-19) means the virus responsible for an outbreak of coronavirus disease in humans that was first reported from Wuhan, China, on 31 December 2019. The virus is now properly known as SARS-CoV2. The disease it causes is COVID-19. More specifically SARS-CoV2 means the virus having a genome comprising the nucleotide sequence of accession number MN908947 or MG772933.1, most suitably MG772933.1. Virus variants (i.e. virus strains/isolates bearing mutations relative to the above exemplary viral genome(s)) arise naturally in the population. If appropriate, specific virus variants are mentioned by name/designation in the text. Unless otherwise apparent from the context, discussion of SARS-CoV2/COVID-19 is understood to embrace the spectrum of viral variants arising in the population.
Suitably antibodies induced as described herein are neutralising antibodies i.e. antibodies capable of neutralising SARS-CoV2 viral particles. Unless otherwise apparent from the context, ‘about’ means ±1% of the stated value.
The vaccine design comprises the complete SARS-CoV2 Spike protein expressed under the control of a strong mammalian promoter, which includes Tet repressor sequences to allow for repression of antigen expression during vaccine manufacture, improving vaccine yields.
Suitably the composition of the invention comprises ChAdOxi :: SARS-CoV2 spike protein i.e. ChAdOxi comprising a nucleic acid insert having a nucleotide sequence encoding the SARS-CoV2 spike protein. Suitably the full length spike protein is used.
Suitably a human tPA leader sequence is added at the N-terminal end (i.e. 5’ end of the nucleotide sequence encoding same).
Suitably the nucleotide sequence is codon optimised for human codon use.
One innovation provided is the intellectual choice of the particular amino acid sequence/variant of the viral spike protein which has been selected.
In one aspect suitably the spike protein has the sequence 2816 (e.g. the spike protein sequence from the fusion protein of SEQ ID NO: 3) or has the sequence 3990 (e.g. the spike protein sequence from the fusion protein of SEQ ID NO: 12), most suitably has the sequence 2816 (the spike protein sequence from the fusion protein of SEQ ID NO: 3).
In one aspect suitably the spike protein is present as a tPA-spike fusion and has the sequence of SEQ ID NO: 3 or SEQ ID NO: 12, most suitably SEQ ID NO: 3.
In addition, runs of repeated bases were specifically identified and removed from the sequence. Special attention has been paid to the nucleotide sequences encoding the antigen and in particular addressing technical problems of genetic stability and sequence rearrangements/mutations. This approach has delivered surprising technical benefits including efficient high yield production without the need for Tet repression, as well as intact virus being successfully rescued with correct cargo sequences preserved.
In more detail, first codon optimisation of the coding sequence of the antigen for human codon usage is carried out. More specifically, codon optimisation of the nucleotide sequence encoding the tPA-SARS-CoV2 spike protein antigen fusion for human codon usage is carried out. Then, the sequence is analysed e.g. for patches in which the human codon optimisation process has resulted in runs of identical nucleotides. For example, runs of 5 consecutive “C” bases (cytosine bases) maybe identified. These repetitive sequences might cause problems in expression, leading to problems of vaccine performance, and/or polymerase “slippage” events, leading to problems in viral vector vaccine production due to nucleic acid instability (e.g. mutations, rearrangements such as truncations etc). In order to address these technical problems, the already mutated codon optimised sequence is further mutated. Thus, the process designs and makes further substitutions in the nucleotide sequence, carefully preserving the encoded amino acids using the universal genetic code, whilst changing the nucleotide bases and selecting alternate codons to remove the slippage prone repeat sequences whilst ensuring the coding sequence still accurately encodes the desired antigen. This approach delivers the technical benefit of facilitating viral vector vaccine production, obtaining good yields of virus.
The inventors were surprised that:
- Vaccine yields of the SARS-CoV2 viral vector composition appear to be the same with and without tet repression. The inventors found this to be astonishing. WO2018/215766 describes a vaccine for MERS (Middle Eastern Respiratory
Syndrome) coronavirus (MERS-CoV). One vector mentioned in this document is ChAdOxi. The vaccine comprises the full length MERS CoV spike protein with a human tPA leader added at the 5’ end. In one aspect the relevant part of the nucleotide sequence is codon optimised for human use. In view of the problems and drawbacks encountered in preparing GMP manufacture of the MERS-CoV vaccine described in WO2018/215766, the view before this invention was that for all viral glycoproteins tet repression would be needed. The view was that these viral glycoproteins are toxic, hence the requirement for Tet repression during manufacture. The invention demonstrates the surprising benefit that Tet repression is NOT required for manufacture of the SARS-CoV2 viral vector composition.
Especially suitable aspects include AZD2816 and AZD3990. AZD2816 is an authentic viral sequence with a total of 11 changes (amino acid substitutions and 3 amino acid deletions) compared to the AZD1222 prototype vaccine sequence (SEQ ID NO: 1), including:
• 3 amino acid changes in the RBD that are found in the B.1.1.7 (UK) and Pi (Brazil) variants
• 3 amino acids substitutions in the N Terminal Domain (NTD), including a deletion of 3 amino acids in an exposed loop
• 2 changes in the stem; D614G mutation that now predominates and A701V AZD3QQO is as above and further includes an additional 6 proline substitutions in the stem
• Potential increase in protein stability, antigen expression and immunogenicity
• Aim is to increase spike antigen expression which may improve immunogenicity.
• Appears as a well-folded trimer and has 10-fold higher expression levels on cell surface
We refer to Figure 1 which shows key mutations (substitutions/ deletions of AZD2816 (SEQ ID NO: 3) and AZD3990 (SEQ ID NO: 12) relative to AZDi222(SEQ ID NO: 1).
In more detail, a further problem experienced by the inventors in different areas of their research had lead them to the conclusion that viral glycoproteins were consistently toxic in the viral particle production systems used for manufacture. The inventors had therefore concluded that Tet repression would always be necessary in order to avoid toxicity issues. This hypothesis was reinforced by their observations working with internal viral protein antigens, which did not appear to suffer from the same toxicity problems as viral glycoproteins. The present invention employing the
SARS-CoV2 spike protein is a clear exception to this rule and is further evidence towards inventive step.
Optional incorporation of a leader sequence/secretory sequence such as the tissue plasminogen activator (tPA) amino acid sequence fused to the N-terminus of the SARS- CoV2 spike protein antigen is disclosed. This combination (tPA + SARS-CoV2 spike protein) delivers enhanced immunogenicity. This is especially true for the triple combination (ChAdOxi + tPA + SARS-CoV2 spike protein). 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 another aspect, 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.
In one aspect suitably the heterologous boost (i.e. the second or further immunisation) comprises ChAdOxi nCoV-19 (AZD2816).
In one aspect 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.
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), ChAdOxi/MVA or ChAdOxi/RNA or ChAdOxi/protein as prime/boost regimes maybe used.
More suitably if a two dose regimen is required, a homologous prime-boost regime such as ChAdOxi/ ChAdOxi, or such as ChAdOxi nCoV-19 (AZD3990)/ ChAdOxi nCoV-19 (AZD3990), most suitably ChAdOxi nCoV-19 (AZD2816)/ ChAdOxi nCoV-19
(AZD2816), may be used.
ChAdOxi nCoV-19 (AZD2816) finds particular application as boosting composition. ChAdOxi nCoV-19 (AZD3990) finds particular application as boosting composition.
Thus in one aspect the invention relates to ChAdOxi nCoV-19 (AZD2816) for use as a boosting composition. Thus in one aspect the invention relates to ChAdOxi nCoV-19 (AZD3990) for use as a boosting composition.
In one aspect a heterologous prime-boost regime may be used such as when the priming composition (i.e. first 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, and the boosting composition (i.e. second or further immunisation) comprises ChAdOxi nCoV-19 (AZD2816).
In one aspect a heterologous prime-boost regime may be used such as ChAdOxi nCoV- 19 (AZD1222)/ ChAdOxi nCoV-19 (AZD2816).
In one aspect a heterologous prime-boost regime may be used such as ChAdOxi nCoV- 19 (AZD1222)/ ChAdOxi nCoV-19 (AZD3990).
In one aspect a triple-administration immunisation regime may be used.
In this aspect suitably the first composition (i.e. priming composition) maybe 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; suitably the second composition (i.e. first boosting composition) maybe 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; suitably the third composition (i.e. second boosting composition) is ChAdOxi nCoV-19 (AZD2816).
In this aspect more suitably the first composition (i.e. priming composition) maybe ChAdOxi nCoV-19 (AZD1222) or ChAdOxi nCoV-19 (AZD2816); suitably the second composition (i.e. first boosting composition) maybe ChAdOxi nCoV-19 (AZD1222) or ChAdOxi nCoV-19 (AZD2816); suitably the third composition (i.e. second boosting composition) is ChAdOxi nCoV-19 (AZD2816).
In one aspect suitably the first composition (i.e. priming composition) maybe ChAdOxi nCoV-19 (AZD1222); suitably the second composition (i.e. first boosting composition) maybe ChAdOxi nCoV-19 (AZD1222) or ChAdOxi nCoV-19 (AZD2816); suitably the third composition (i.e. second boosting composition) is ChAdOxi nCoV-19 (AZD2816). In one aspect suitably the first composition (i.e. priming composition) maybe ChAdOxi nCoV-19 (AZD1222); suitably the second composition (i.e. first boosting composition) maybe ChAdOxi nCoV-19 (AZD1222); suitably the third composition (i.e. second boosting composition) is ChAdOxi nCoV-19 (AZD2816).
Thus in one aspect the invention relates to a composition for use as described above wherein said use comprises:
(i) administering a first dose of said composition to said subject;
(ii) administering a second dose of said composition to said subject, and (iii) administering a third dose of said composition to said subject.
Suitably said first dose and said second dose and said third dose each comprise about the same number of viral particles.
In boost aspects suitably the first administration comprises, or consists of, a composition according to the present invention comprising a viral vector capable of expressing the SARS-CoV2 Spike protein.
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 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.
PRIME-BOOST DOSES
Participants included in the analysis were divided into groups which received two different dose levels as first dose (i.e. as first administration (prime)). The doses of the first administration (prime) were
2.5 x 1010 vp (‘low dose’ / ‘half dose’ group) and - 5.0 x 1010 vp (‘standard dose’ / ‘full dose’ group). Thus in one aspect the invention relates to a dual administration regime where a first administration and a second administration are given to a single subject, wherein the ratio of the dose of the first administration to the dose of the second administration is
0.5:1.
Thus in another aspect the invention relates to a dual administration regime where a first administration and a second administration are given to a single subject, wherein the ratio of the dose of the first administration to the dose of the second administration is 1:1.
The vaccine can be stored, transported and handled at normal refrigerated conditions (2-8 degrees Celsius/ 36-46 degrees Fahrenheit) for at least six months and administered within existing healthcare settings. The invention also provides a method of inducing an immune response against SARS- CoV2 in a mammalian subject, or a method of preventing SARS-CoV2 infection in a mammalian subject, the method comprising
(i) administering a first dose of a composition as described above to said subject; and (ii) administering a second dose of a composition as described above to said subject, wherein said second dose comprises about twice the number of viral particles of said first dose. The invention also provides a composition for use as described above wherein said use comprises:
(i) administering a first dose of said composition to said subject; and
(ii) administering a second dose of said composition to said subject, wherein said first dose and said second dose each comprise about the same number of viral particles.
The invention also provides a composition for use as described above wherein said use comprises:
(i) administering a first dose of said composition to said subject; and (ii) administering a second dose of said composition to said subject, wherein said second dose comprises about twice the number of viral particles of said first dose. The invention also provides a method of inducing an immune response against SARS- CoV2 in a mammalian subject, or a method of preventing SARS-CoV2 infection in a mammalian subject, or a compoistion for use in such a method, the method comprising (i) administering a first dose of a composition as described above to said subject; and
(ii) administering a second dose of a composition as described above to said subject, wherein said first dose comprises about half the number of viral particles of said second dose.
The invention also provides a method of inducing an immune response against SARS- CoV2 in a mammalian subject, or a method of preventing SARS-CoV2 infection in a mammalian subject, or a compoistion for use in such a method, the method comprising (i) administering a first dose of a composition as described above to said subject; and
(ii) administering a second dose of a composition as described above to said subject, wherein the ratio of the number of viral particles in said first dose to the number of viral particles in said second dose is 0.5:1.
The invention also provides a method of inducing an immune response against SARS- CoV2 in a mammalian subject, or a method of preventing SARS-CoV2 infection in a mammalian subject, the method comprising
(i) administering a first dose of a composition as described above to said subject; and
(ii) administering a second dose of a composition as described above to said subject, wherein the ratio of the number of viral particles in said first dose to the number of viral particles in said second dose is 1:2.
Suitably said second dose is administered at an interval of a) less than 6 weeks, b) 6 to 8 weeks, c) 9 to 11 weeks, or d) 12 weeks or more, after administration of said first dose. In one aspect suitably said first dose comprises about 2.5 x to10 viral particles. (LD)
In one aspect suitably said first dose comprises about 5 x 1010 viral particles. (SD) Suitably said second dose comprises about 5 x 1010 viral particles. (SD)
In one aspect suitably said first dose comprises about 2.5 x to10 viral particles and said second dose comprises about 5 x to10 viral particles. (LD-SD)
In one aspect suitably said first dose comprises about 5 x to10 viral particles and said second dose comprises about 5 x to10 viral particles. (SD-SD)
Suitably said composition is administered by a route of administration selected from a group consisting of intranasal, aerosol, intradermal and intramuscular. More suitably said administration is intramuscular.
APPLICATIONS It is a technical benefit that the invention delivers immunity with only a single dose. Immunity may be enhanced (boosted) with a second or further dose.
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 neutralising 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. 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.
COVID19 is the disease caused by the SARS-CoV2 virus in humans. Suitably the invention further relates to a method for preventing COVID19 in a subject, the method comprising administering a composition as described above to said subject. DATABASE RET .EASE
Sequences deposited in databases can change over time. Suitably the current version of sequence database(s) are relied upon. Alternatively, the release in force at the date of filing is relied upon. As the skilled person knows, the accession numbers may be version/dated accession numbers. The citeable accession numbers for the current database entry are the same as above, but omitting the decimal point and any subsequent digits.
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 current release is relied upon. More suitably the release available at the effective filing date is relied upon. Most suitably the GenBank database release referred to is NCBI-GenBank Release 241: 15 December 2020.
UniProt (Universal Protein Resource) is a comprehensive catalogue of information on proteins (‘UniProt: a hub for protein information’ Nucleic Acids Res. 43: D204-D212 (2015).). Suitably the current release is relied upon. More suitably the release available at the effective filing date is relied upon. Most suitably, the UniProt consortium
European Bioinformatics Institute (EBI), SIB Swiss Institute of Bioinformatics and Protein Information Resource (PIR)’s UniProt Knowledgebase (UniProtKB) Release 2021 _ 02 of April 2021 is relied upon. ADVANTAGES
In some aspects the invention possesses the advantage of inducing protective immunity after single dose (single administration). 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 SARS-CoV2.
SPIKE PROTEIN
The spike protein (S protein) is a large type I transmembrane protein. This protein is highly glycosylated, containing numerous N-glycosylation sites. Spike proteins assemble into trimers on the virion surface to form the distinctive "corona", or crown- like appearance. The ectodomains of all CoV spike proteins share the same organization in two domains: a N-terminal domain named SI that is responsible for receptor binding and a C-terminal S2 domain responsible for fusion. CoV diversity is reflected in the variable spike proteins (S proteins).
Spike Protein Domains
The total length of SARS-CoV-2 Spike protein is 1273 aa, including the lead Methionine - it will be noted that the reference sequence for spike protein of SEQ ID NO: 1 shows the lead methionine and so SEQ ID NO: 1 is 1273 amino acids in length. Occasionally some sequences are shown without a lead methionine. It is a routine matter for the skilled reader to identify the amino acid numbers (addresses) below and identify the corresponding amino acids in SEQ ID NO: 1 or other spike protein sequences by adjusting for sequence differences (such as presence or absence of lead methionine) using routine knowledge in the art. The spike protein consists of a signal peptide (amino acids 1-13) located at the N- terminus, the Si subunit (14-685 residues), and the S2 subunit (686-1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. In the Si subunit, there is an N-terminal domain (14-305 residues) and a receptor-binding domain (RBD, 319-541 residues). In the S2 subunit, there is a fusion peptide (FP) (788-806 residues), heptapeptide repeat sequence 1 (HRi) (912-984 residues), HR2 (1163-1213 residues), TM domain (1213-1237 residues), and cytoplasm domain (1237-1273 residues). The STEM domain suitably comprises residues 614-984, more suitably residues 701-984.
Suitably Si subunit means a polypeptide having, or consisting of, amino acid sequence corresponding to amino acids 14-685 of SEQ ID NO: 1.
Suitably the N-Terminal domain (NTD) means a polypeptide having, or consisting of, amino acid sequence corresponding to amino acids 14-305 of SEQ ID NO: 1. Suitably the receptor-binding domain (RBD) means a polypeptide having, or consisting of, amino acid sequence corresponding to amino acids 319-541 of SEQ ID NO: 1. Suitably the STEM domain (STEM) means a polypeptide having, or consisting of, amino acid sequence corresponding to amino acids 614-984 of SEQ ID NO: 1, more suitably a polypeptide having, or consisting of, amino acid sequence corresponding to amino acids 701-984 of SEQ ID NO: 1.
Thus a polypeptide which ‘comprises the spike protein receptor binding domain (RBD)’ means a polypeptide comprising, or consisting of, amino acid sequence corresponding to amino acids 319-541 of SEQ ID NO: 1. The same applies to the other domains/sub- domains mentioned above.
‘Corresponding to’ has its natural meaning in the art i.e. for identification of the domains/sub-do mains within different spike protein sequences. For example ‘Corresponding to’ may not mean 100% identical to. Sequence identity levels/substitutions relative to SEQ ID NO: 1 are as explained herein.
Suitably the antigen is the SARS-CoV2 spike protein.
Suitably the full length spike protein is used.
Suitably full length means each amino acid in the spike protein is included.
A reference spike protein is as disclosed in SEQ ID NO: 1. Exemplary spike proteins according to the present invention are as disclosed in SEQ ID NO: 3 and/or SEQ ID NO: 12.
By choosing the full length spike 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 spike protein is that it allows for better T- cell responses. Without wishing to be bound by theoiy, 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 spike protein is included in the antigen of the invention. tPA tPA (tissue plasminogen activator) - more specifically the tPA leader sequence - is suitably fused to the SARS-CoV2 spike protein antigen of the invention. Suitably tPA is fused to the N-terminus of the spike protein 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.
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 tPAto the N-terminus of the spike 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 spike protein antigen. Suitably the antigen does not comprise any further sequence tags.
Suitably the antigen does not comprise any further linker sequences. Adeno-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 AdHus such as human embryonic kidney cells 293 (HEK 293 cells).
Any suitable adeno-based viral vector may be used.
In more detail, any replication-deficient viral vector, for human use preferably derived from a non-human adenovirus may be used. For veterinary use Ads may be used. ChAdOxi and ChAd0x2 are examples of a suitable non-human adenovirus vector for human use.
Most suitably the adeno-based viral vector is ChAdOxi. ChAdOxi
The nucleotide sequence of the ‘empty' ChAdOxi vector (NGS-verified viral genome sequence with a Gateway™ cassette in the El locus) is shown in SEQ ID NO: 14. This is a viral vector based on Chimpanzee adenovirus C68. ChAdOxi 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): 640385, and in WO2012/172277. Both these documents are hereby incorporated herein by reference, in particular for the specific teachings of the ChAdOxi vector, including its construction and manufacture.
For insertion of the nucleotide sequence encoding spike protein, 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 ChAdOxi 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 AdChOxi (E4 modified) TIPeGFP, cell line name "AdChOxi (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 ‘empty1 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.
ChAd6.2
In one aspect a related vaccine vector, ChAd63, may be used if desired.
Production of ChAdOxi nCoV-19 variants
ChAdOxi nCoV-19 variants may be produced by any method known in the art. By ‘ChAdOxi nCoV-19 variants’ we mean “ChAdOxi nCoV-19 AZD1222” or “ChAdOxi nCoV-19 AZD2816” or “ChAdOxi nCoV-19 AZD3990” or other ChAdOxi vector comprising a nCoV-19 spike protein sequence variant. For example ChAdOxl nCoV-19 variants may be produced as described in the examples, for example as described for ChAdOxl nCoV-19 AZD1222.
In overview, for ChAdOxl nCoV-19 AZD1222 the spike protein (S) of SARS-Cov-2 (Genbank accession number YP 009724390.1) was codon optimised for expression in human cell lines and synthesised by GeneArt Gene Synthesis (Thermo Fisher Scientific). The sequence encoding amino acids 2-1273 were cloned into a shuttle plasmid following InFusion cloning (Clontech). The shuttle plasmid encodes a modified human cytomegalovirus major immediate early promoter (IE CMV) with tetracycline operator (TetO) sites, poly adenylation signal from bovine growth hormone (BGH) and a tPA signal sequence upstream of the inserted gene.
For the avoidance of doubt, “ChAdOxl nCoV-19” means AZD1222 i.e. the ChAdOxl adenoviral vector as described in Dicks et al. (2012) PLoS ONE 7(7): 640385, and/or in WO2012/172277, comprising the nucleotide sequence of SEQ ID NO: 4 (encoding 32aa tPA leader fused to SARS-Cov-2 spike protein) inserted at the El locus of the ChAdOxl adenoviral vector under the control of the CMV (cytomegalovirus) ‘long’ promoter. This is as described in PCT/GB2021/050602. Most suitably this “ChAdOxl nCoV-19” (ChAdOxl nCoV-19 AZD1222) has the nucleotide sequence as shown in SEQ ID NO: 25 in PCT/GB2021/050602 (44104nt). This “ChAdOxl nCoV-19” / AZD1222 viral vector is not itself part of the current invention, but does form part of the invention where described for use in immunisation methods/prime-boost regimes, or as a component of multi-part kits / compositions and the like which are disclosed herein.
“ChAdOxl nCoV-19 AZD2816” (sometimes referred to as “AZD2816”) means the ChAdOxl adenoviral vector as described in Dicks et al. (2012) PLoS ONE 7(7): 640385, and/or in WO2012/172277, comprising the nucleotide sequence of SEQ ID NO: 3 (encoding 32aa tPA leader (SEQ ID NO: 5) fused to SARS-Cov-2 ‘2816’ spike protein) inserted at the El locus of the ChAdOxl adenoviral vector under the control of the CMV (cytomegalovirus) ‘long’ promoter. Most suitably this “ChAdOxl nCoV-19 AZD2816” (AZD2816) has the nucleotide sequence as shown in SEQ ID NO: 13.
SEQ ID NO: 3 - amino acid sequence of tPA - 2816 Spike Protein fusion ftPA underlined! (AZD2816) MDAMKRGLCCVLLLCGAVFVSASOEIHARFRRFVFLVLLPLVSSOCVNFrTRTOLPPA YTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFANPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVY YHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYF KIYSKHTPINLVRGLPQGFSALEPLVDLPIGINITRFQTLHRSYLTPGDSSSGWTAGAAA YYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPT ESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNL DSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPT Y GVGY QPYRVWLSFELLHAPATV CGPKKSTNLVKNKCVNFNFN GLTGTGVLTESNKK
FLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNC TEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQT QTNSPRRARSVASQSIIAYTMSLGVENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVD CTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKD FGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFN GLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQN VLYENQKLIAN QFN SAIGKIQDSLSSTASALGKLQDWN QNAQALNTLVKQLSSNFGAI SSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECV LGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFP REGVFVSNGTHWFVTQRNFYEPQinTDNTFVSGNCDWIGIVNNTVYDPLQPELDSF KEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYE QYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLK GVKLHYT
Also disclosed is the AZD2816 spike protein amino acid sequence - this may be taken from SEQ ID NO: 3 by removing the tPA sequence (underlined) and replacing it with a single methionine. Thus in one aspect the invention relates to an isolated spike protein polypeptide having the AZD2816 amino acid sequence.
“ChAdOxl nCoV-19 AZD3990” (sometimes referred to as “AZD3990”) means the ChAdOxl adenoviral vector as described in Dicks et al. (2012) PLoS ONE 7(7): 640385, and/or in WO2012/172277, comprising the nucleotide sequence of SEQ ID NO: 12 (encoding 32aa tPA leader (SEQ ID NO: 5) fused to SARS-Cov-2 ‘3990’ spike protein) inserted at the El locus of the ChAdOxl adenoviral vector under the control of the CMV (cytomegalovirus) ‘long’ promoter. Most suitably this “ChAdOxl nCoV-19 AZD3990” (AZD3990) has the nucleotide sequence as shown in SEQ ID NO: 25.
SEQ ID NO: 12 - amino acid sequence of tPA - 3990 Spike Protein fusion ftPA underlined) (Proline substitutions relative to SEQ ID NO: 1/SEQ ID NO: 3 in bold) (AZD3990) MDAMKRGLCCVLLLCGAVFVSASOEIHARFRRFVFLVLLPLVSSOCVNFTTRTOLPPA YTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFANPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVY YHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYF KIYSKHTPINLVRGLPQGFSALEPLVDLPIGINITRFQTLHRSYLTPGDSSSGWTAGAAA YYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPT ESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS
PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNL
DSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPT
Y GVGY QPYRVWLSFELLHAPATV CGPKKSTNLVKNKCVNFNFN GLTGTGVLTESNKK
FLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNC
TEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQT
QTNSPRRARSVASQSIIAYTMSLGVENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVD
CTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKD
FGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKF
NGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGPALQIPFPMQMAYRFNGIGVTQ
NVLYENQKLIAN QFN SAIGKIQDSLSSTP SALGKLQDWN QNAQALNTLVKQLSSNFG
AISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE
CVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAH
FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELD
SFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGK
YEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPV
LKGVKLHYT
Also disclosed is the AZD3990 spike protein amino acid sequence - this may be taken from SEQ ID NO: 12 by removing the tPA sequence (underlined) and replacing it with a single methionine. Thus in one aspect the invention relates to an isolated spike protein polypeptide having the AZD3990 amino acid sequence.
ADMINISTRATION ROUTE
In principle any suitable route of administration may be used.
The invention may be administered by aerosol delivery to the respiratory tract using a widely available device commonly used for drug delivery. This may be a suitable route of vaccine delivery for respiratory pathogens such as coronaviruses. In one aspect the composition may comprise a MVA-vectored vaccine, wherein aerosol delivery may result in strong immune responses in the respiratory tract at low doses. A further advantage of aerosol deliver is avoidance of needles.
Suitably the route of administration is selected from group consisting of subcutaneous, intranasal, aerosol, nebuliser, intradermal and intramuscular.
Suitably the route of administration is selected from a group consisting of intranasal, aerosol, intradermal and intramuscular.
Suitably the route of administration is selected from a group consisting of intranasal, aerosol and intramuscular.
More suitably the route of administration is selected from a group consisting of intranasal and intramuscular.
Most suitably the route of administration is intramuscular. The route of administration maybe 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 humans, in one aspect the range is from 109 to 1011 viral particles.
For humans, in one aspect the range is from 2.5X 1010 vp to 5x 1010 vp.
For humans, in one aspect the dose(s)/range of dose(s) maybe derived from the examples below.
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
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 SARS-CoV2 spike protein, optionally fused to the tPA protein, and incorporating said nucleic acid into an adeno-based viral vector, suitably a ChAdOxi vector. Suitably the nucleic acid is operably linked to a promoter suitable for inducing expression of said SARS-CoV2 spike protein (or SARS- CoV2 spike protein-tPA fusion protein) when in a mammalian cell such as a human cell.
FORMULATION
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 ChAdOxi formulation buffer, as used for the clinical product is:
FORMULATION BUFFER COMPONENTS
1. 10 mM Histidine
2. 7.5 % Sucrose
3. 35 mM Sodium chloride
4. 1 mM Magnesium chloride
5. 0.1 % Polysorbate 80
6. o.i mM EDTA
7. 0.5% Ethanol
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
It may be possible to use only the Si domain of the spike protein, or only the soluble part of the spike protein, or only the receptor binding domain of the spike protein. Thus, in one aspect only the receptor binding domain of the spike protein is used. Suitably this has the tPA fusion. REFERENCE SEQUENCE
One example of a CoV spike protein reference sequence (not part of the invention) is vCoV-19 spike protein from Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-i i.e. the spike protein encoded by the viral genome with GenBank accession number MN908947.
More suitably said spike protein reference sequence (not part of the invention) has the amino acid sequence as in (or as encoded in) the SARS-CoV2 genome of GenBank accession number MG772933.1 (Bat SARS-like coronavirus isolate bat-SL-CoVZC45). Suitably the SARS-CoV2 may be isolate bat-SL-CoVZC45.
Most suitably said spike protein reference sequence (not part of the invention) has the amino acid sequence of SEQ ID NO: 1.
SEQ ID NO: 1 - Amino acid sequence of SARS-CoV2 Spike protein only (no tPA fusion) (also referred to as ‘prototype7wild-type/AZ1222/P10697GBWO i.e. reference sequence - not part of the invention)
FVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSN VTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNWIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLM DLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDP LSETKCTLKSFTVEKGIYQTSNFRVQPTESrVRFPNITNLCPFGEVFNATRFASVYAWN RKRISN CVADY SVLYN SASFSTFKCY GVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTE IY QAGSTPCN GVEGFN CYFPLQ SY GFQPTN GVGY QPYRVWLSFELLHAPATV CGPKKS TNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDIT PCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTR AGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVA YSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRAL TGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVT LADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGW TFGAGAALQIPFAMQMAYRFN GIGVTQNVLYEN QKLIAN QFN SAIGKIQDSLSSTASAL GKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSL QTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWF LHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQinTDNT FVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNI QKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCM
TSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
SEQ ID NO: 11: Nucleotide sequence for spike protein from nCoV 19 genome (also referred to as ‘prototype7wild-type/AZi222/Pio697GBWO i.e. reference sequence - not part of the invention)
(From GenBank Accession number MG772933.1)
ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAAC
CAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGGTGTTTATTACCCTG
ACAAAGTTTTCAGATCCTCAGTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTT
TCCAATGTTACTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGT
TTGATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAGAAGTCT
AACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCAGTCCCTAC
TTATTGTTAATAACGCTACTAATGTTGTTATTAAAGTCTGTGAATTTCAATTTTGTAAT
GATCCATTTTTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGATGGAAAGTGAGT
Figure imgf000032_0001
ATGGACCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGA
ATATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGTGCGTGAT
CTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGCCAATAGGTATTAACA
TCACTAGGTTTCAAACTTTACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCT
TCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAACCTAGGA
CTTTTCTATTAAAATATAATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACT
TGACCCTCTCTCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATC
TATCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATAT
Figure imgf000032_0002
GCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAAT
CGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTT
ACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATT
ATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATAT
TTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAAT
TGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACC
Figure imgf000032_0003
CCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTT
AACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCAACAATTT
GGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCACAGACACTTGAGATTC TTCTAACCAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAAGTCCCTGTTGCT
ATTCATGCAGATCAACTTACTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTT
TCAAACACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGT
GACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTC
GGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGC
AGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTA
GTGTTACCACAGAAATTCTACCAGTGTCTATGACCAAGACATCAGTAGATTGTACAAT
GTACATTTGTGGTGATTCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTT
TGTACACAATTAAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCC
AAGAAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGATTTTGG
TGGTTTTAATmTCACAAATATTACCAGATCCATCAAAACCAAGCAAGAGGTCATTT
ATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTTCATCAAACAAT
ATGGTGATTGCCTTGGTGATATTGCTGCTAGAGACCTCATTTGTGCACAAAAGTTTAA
CGGCCTTACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGCTCAATACACTTCTG
CACTGTTAGCGGGTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACA
AATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAAT
GTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAA
TTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAAAACTTCAAGATGTGGTCAA
CCAAAATGCACAAGCTTTAAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTGCA
ATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGC
AAATTGATAGGTTGATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACA
ATTAATTAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGTCA
GAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGCTATCATCTTA
TGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTTGCATGTGACTTATGTCCCT
GCACAAGAAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGATGGAAAAGCACACT
TTCCTCGTGAAGGTGTCTTTGTTTCAAATGGCACACACTGGTTTGTAACACAAAGGAA
TTTTTATGAACCACAAATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGAT
GTTGTAATAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGACT
CATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTT
AGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGACCGC
CTCAATGAGGTTGCCAAGAATTTAAATGAATCTCTCATCGATCTCCAAGAACTTGGAA
AGTATGAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTT
GATTGCCATAGTAATGGTGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGT
CTCAAGGGCTGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGC
CAGTGCTCAAAGGAGTCAAATTACATTACACATAA SEQUENCE VARIATION
Suitably the sequence is, or is derived from, amino acid sequence provided herein, such as SEQ ID NO. 3 or SEQ ID NO: 12. A degree of sequence variation may be tolerated. Suitably the sequence used in the vector of the invention comprises or encodes amino acid sequence having at least 95% sequence identity, suitably having at least 96% sequence identity , suitably having at least 97% sequence identity, suitably having at least 98% sequence identity, suitably having at least 98.7% sequence identity, suitably having at least 99% sequence identity, suitably having at least 99.1% sequence identity to the reference amino acid sequence, for example the reference amino acid sequence provided as SEQ ID NO. 1.
A sequence identity level of 99% compared to SEQ ID NO. 1 (having 1273 amino acids) corresponds to approximately 12 to 13 substitutions across the full length of the spike protein sequence provided as SEQ ID NO. 1.
Suitably the spike protein sequence used has 17 or fewer substitutions relative to SEQ ID NO: 1, suitably 16 or fewer substitutions relative to SEQ ID NO: 1, suitably 15 or fewer substitutions relative to SEQ ID NO: 1, suitably 14 or fewer substitutions relative to SEQ ID NO: 1, suitably 13 or fewer substitutions relative to SEQ ID NO: 1, suitably 12 or fewer substitutions relative to SEQ ID NO: 1, suitably 11 or fewer substitutions relative to SEQ ID NO: 1, suitably 10 or fewer substitutions relative to SEQ ID NO: 1, suitably 9 substitutions relative to SEQ ID NO: 1. It is possible to regard SEQ ID NO: 3 as having 9 changes relative to SEQ ID NO: 1 - however, it should be noted that one such change is a deletion of three amino acids relative to SEQ ID NO: 1 (deletion of amino acids L242, A243 and L244 relative to SEQ ID NO: 1). Therefore as discussed herein this should correctly be regarded as three changes (three ‘substitutions’) - therefore SEQ ID NO: 3 has a total of 11 mutations relative to SEQ ID NO: 1 (i.e. 8 amino acid substitutions plus three amino acid deletions = 11 mutations (‘substitutions’) in total.)
Similarly SEQ ID NO: 12 has the same mutations as SEQ ID NO: 3, PLUS a further 6 substitutions to proline (‘hexapro’). This SEQ ID NO: 12 should correctly be regarded as having a total of 17 mutations relative to SEQ ID NO: 1 (i.e. (8+6=) 14 amino acid substitutions plus three amino acid deletions = 17 mutations (‘substitutions’) in total.) Thus for the purposes of assessing sequence identity/ counting mutations (‘substitutions’) relative to the reference sequence (SEQ ID NO: 1), deletion of an amino acid is regarded as a substitution. In one aspect suitably the spike protein sequence used (e.g. SEQ ID NO: 12 -
AZD3990) has 17 substitutions relative to SEQ ID NO: 1 (98.7% sequence identity to SEQ ID NO: 1).
In one aspect suitably the spike protein sequence used (e.g. SEQ ID NO: 3 - AZD2816) has 11 substitutions relative to SEQ ID NO: 1 (99.1% sequence identity to SEQ ID NO: 1).
In one aspect suitably the spike protein amino acid sequence used is as encoded by the relevant section of the nucleotide sequence of SEQ ID NO: 13 (AZD2816 viral genome sequence).
In one aspect suitably the spike protein amino acid sequence used is as encoded by the relevant section of the nucleotide sequence of SEQ ID NO: 25 (AZD3990 viral genome sequence). Suitably any amino acid substitutions are not in the receptor binding domain. Suitably any amino acid substitutions are outside the receptor binding domain.
Suitably counting of substitutions does not include addition of the tPA sequence. The above applies equally to nucleotide sequences. For example suitably the sequence used in the vector of the invention comprises nucleotide sequence having at least 95% sequence identity, suitably having at least 96% sequence identity , suitably having at least 97% sequence identity, suitably having at least 98% sequence identity, suitably having at least 98.7% sequence identity, suitably having at least 99% sequence identity, suitably having at least 99.1% sequence identity to the reference nucleotide sequence.
For example suitably the sequence identity of the nucleotide sequence encoding the spike protein is considered by comparison to reference sequence provided as SEQ ID NO. 11 or SEQ ID NO: 4. SEQUENCE IDENTITY
It may be desired to consider sequence relationships in terms of sequence identity. 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.
Percent identity maybe calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
Although this is a very simple and consistent method, it fails to take into consideration that, for example in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in percent homology (percent identity) when a global alignment (an alignment across the whole sequence) is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology (identity) score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology/identity.
These more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum percent homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et ah, 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 ah, 1990, J. Mol. Biol. 215:403- 410) and the GENEWORKS suite of comparison tools.
Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied. It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
Suitably sequence identity is considered for a segment of spike protein comprising at least the receptor binding domain (RBD), or at least the N-terminal domain (NTD), or at least the STEM; more suitably sequence identity is considered for a segment of spike protein comprising at least the receptor binding domain (RBD) and the N-terminal domain (NTD); more suitably sequence identity is considered for a segment of spike protein comprising at least the receptor binding domain (RBD) and the N-terminal domain (NTD), and the STEM. Most suitably sequence identity is considered for full length spike protein, e.g. the full length spike protein of SEQ ID NO: 1. ADVANTAGES AND APPLICATIONS
The inventors realised that Pi and P2 ‘Brazil’ strains share many similar mutations with B.1.351 variants. The inventors observed that strains with a common set of mutations are arising independently, and that variants of concern are arising due to evolution of the Spike protein. The inventors combined their insights in making the intellectual decisions in arriving at the invention.
Figure imgf000037_0001
Figure imgf000038_0001
~ This is the correct PANGO lineage corresponding to the Alpha variant of concern, initiated detected in Kent, United Kingdom in Sept 2020.
* As of 3i-May-202i, this variant was only known as B.1.617 and Delta. The sub- lineages B.1.617.1 and B.1.617.2 came later. As of the filing date, B.1.617.2 is known as Delta (variant of concern) while B.1.617.1 is known as Kappa (variant of interest).
** As of 3i-May-202i, B.1.427/B.1.429 was known as a variant of interest (not a variant of concern); later it became known as Epsilon. Throughout the text, references to the ‘original Wuhan spike protein’ or ‘original spike’ mean the spike protein of SEQ ID NO: 1 (AZD1222). See for instance Examples 8, 9 and 10 and elsewhere.
Please note that the following Table below was generated on i7-May-202i based on available information and nomenclature at the. This represents how SARS-CoV-2 variants were called in May 2021.
Table: SARS-CoV-2 Variants of Concern and Variants of Interest, as of 11 May 2021:
Figure imgf000039_0001
(*) = detected in some sequences but not all
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. 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 a table Figure 2 shows a flow chart
Figure 3 shows a DNA map of ChAdOxi nCoV-19 (AZD1222)
Figure 4 shows graphs of Plasma Membrane expression of wildtype and proline stabilised SARS-CoV-2 Spike sequences
Figure 5 shows bar charts of Immune response induced by vaccination with either wildtype (Jenner E ; AZD2816) or stabilised (Jenner E6; AZD3990) Spike sequence.
Figure 6 shows bar charts of Cross-reactive antibodies are increased following a booster dose with AZD2816 vaccine
Figure 7 shows box charts of live neutralisation studies against different SARS-CoV2 virus variants of concern following a booster dose with AZD1222 and AZD2816 vaccine
EXAMPLES
Example 1: Seed stock & Manufacture
For ChAdOxi SARS-CoV2 AZD2816, vaccine seed stock preparation is carried out. Manufacture is then transferred to GMP manufacturers. Suitably one manufacturer (Advent) produces material (initially 1000 doses). Suitably one manufacturer (CanSino) manufactures in China, at 200L scale, 20,000 doses per batch. High capacity filling lines may be used, with or without lyophilisation. Example 2: ELISpot and CMC
In clinical studies, blood samples are taken to test for IgG antibodies using a validated ELISA and T cell responses using a validated ELISpot protocol at baseline and following vaccination.
Regarding the validated ELISPOT protocol, it should be noted that the actual ELISPOT protocol is a standard technique which is typically always carried out in the same manner. The specificity for the validated ELISPOT protocol comes from the peptides used. In this invention, the peptides used are derived from the SARS-CoV2 spike protein. In one aspect, a series of overlapping peptides are synthesised beginning with the first amino acid of the spike protein. In this aspect, 2omer peptides are synthesised. Therefore, the first peptide comprises the amino acid sequence of amino acids 1 to 20 of the SARS-CoV2 spike protein; the second peptide synthesised comprises amino acids 11 to 30 of the SARS-CoV2 spike protein; the third peptide synthesised comprises the amino acid sequence of amino acids 21 to 40 of the SARS- CoV2 spike protein and so on. This collection of peptides maybe grouped together in pools to facilitate carrying out of the ELISPOT protocol. Any suitable approach to the pooling of the peptides may be adopted by the skilled operator.
Chemistry. Manufacturing & Control (CMC) Development
Replication-deficient adenoviral vectored vaccines are known. The adenovirus El gene must be supplied in trans by the cell line used for vaccine manufacture. In HEK293 cells, this gene is flanked by other sequences from adenovirus 5 which are present in the Ads vaccine vector, such that in rare cases a double crossover event result in the generation of replication-competent adenovirus. This is undesirable and has been solved by either the use of a different adenoviral vector such as ChAdOxi, in which the homology between the vector and the cell line is too low to allow for recombination, or the use of a cell line which expresses Ads El with no flanking sequences such as PerC6, or others developed by different companies.
A further refinement of the cell line is to include the ability to repress expression of the vaccine antigen during manufacture. The vaccine antigen is under the control of a strong mammalian promoter in order to provide high level antigen expression after vaccination. Expression of the antigen during manufacture may have a deleterious effect on vaccine yield. By preventing vaccine expression during manufacture, the yield is no longer affected by the choice of antigen and the process maybe standardised. A cGMP cell bank may be used.
The upstream process consists of expanding the cell bank, infecting with the seed virus and allowing the adenovirus to replicate within the cells. After harvest, detergent lysis, clarification and further downstream purification is achieved by standard methods. The purified Drug Substance is then diluted into formulation buffer, filter sterilised and filled into vials which may be stored as liquid or lyophilised.
Quality control tests include concentration (which is the potency assay), sterility, DNA sequence of vaccine antigen and absence of adventitious agents. The use of deep sequencing greatly accelerates characterisation of vaccine seed stocks, to confirm clonality without lengthy rounds of virus cloning, and also in detection of adventitious agents. Thus the time taken for release testing may be greatly shortened. Example 3 : Growth and Quantification of Viral Vectors
HEK293 TREx suspension cells were cultured in the following media:
Figure imgf000042_0001
HEK 293 TREx cells express the tetracycline repressor protein which binds to sites in the CMV promoter of the recombinant adenovirus and prevent expression of the spike protein (e.g. tPA-spike protein 2816 fusion) during production of the viral vector (e.g. ChAOxi nCoV-19 (AZD2816)) in these cells. Expresssion of the tet repressor protein is switched off when tetracycline is added to the culture medium, allowing the spike protein to be expressed.
The day prior to infection, HEK293 TREx cells were pelleted and re-suspended in minimal media (CD293, 1% FBS, smM L-Glutamine and pen / strep), counted by trypan blue exclusion and seeded at ixioe6/ml. The culture flask was left to grow overnight (37°C, 5% CO2, within an orbital incubator).
On the day of infection the cells were counted by trypan blue exclusion and adjusted to ixioe6/ml with minimal media. Cells were aliquoted into 80ml volumes in fresh culture flasks and various additions made to each flask:
Repressed MOI 3: 8m1 Blasticidin + virus at a multiplicity of infection (MOI) of 3 De-repressed MOI 3: 8qm1 of lmg/ml tetracycline + virus MOI 3 Flasks were returned to incubate (37°C, 5% CO2)
From uninfected cells, a 5qqm1 volume was taken and pelleted. The pellet and supernatant were stored at -8o°C separately to be used as a negative control in qPCR.
Quantification of infectious units (IU):
IU was quantified using a titre immunoassay. Briefly, a black walled / clear flat bottomed 96 well plate (Corning) was seeded with adherent HEK293 TREx cells in standard growth media (below) to obtain a 95% confluent monolayer on the day required.
Figure imgf000042_0002
Figure imgf000043_0001
Samples to titrate were thawed, vortexed and a 10μl aliquot taken to test. This was mixed with 9qm1 growth media to produce a 10-1 dilution. Further dilutions in standard growth media (to-2 to 1.1x10-7) were made in duplicate per sample across an empty V- bottomed 96 well plate. Media from the assay plate was removed and 5qm1 of each test sample / dilution was plated. Plates were incubated for 24h (37°C, 8% CO2) before a further 5qm1 standard growth media was added. Plate was returned to the incubator for a further 24h. After a total of 48h, all well contents were aspirated and the cells fixed with ioomΐ per well pre-chilled Methanol. Plates were placed at -20°C overnight.
To immunostain all incubation steps were performed at room temperature: plates were washed (x5) with PBS before blocking first with ioomΐ per well Bloxall (Vector Labs) for 30 mins and then 200m1 per well 1% casein solution (Thermo Fisher) for 15 mins after washing (x5) with PBS. Anti-adenoviral hexon antibody (AbCam) diluted in 1% casein solution was added to wells (iqqmΐ / well). After 1 h the primary antibody was removed and plates washed (x8) with lx TBS (Tris Buffered Saline - Sigma). Secondary antibody (goat anti-mouse IgG whole molecule, Sigma) was diluted in TBS containing 3% skim milk powder. This was added ioomΐ / well before a further lh incubation. Plates were again washed (x8) in lxTBS before Iqqmΐ per well BCIP / NBT was added per well to visualise infected cells. Once ‘spots’ had stained well, BCIP / NBT was removed, plate washed (x5) in tap water and left to dry overnight. Images were obtained of each well using the AID Elispot reader and distinct spots counted in wells where 20-200 could be seen. The IU titre was assessed by calculating the dilution factor of each given sample and the number of spots counted at that dilution.
Quantification of genome copy number within cultures:
Samples were taken from storage at -8o°C and thawed at room temperature. Pellet samples were re-suspended in 5qqm1 molecular grade water to return them to their previous concentration volume in culture.
All samples were diluted Iqmΐ in I5m1 DNArealeasy (Anachem) and the following PCR programme used to generate viral DNA template: 05°C for 15 mins, 96°C for 2 mins, 65°C for 4 mins, 96°C for 1 min, 65°C for imin, 96°C for 30 secs.
For a standard curve ChAdOx1 plasmid DNA of a known concentration was diluted to generate sample of a given copy number per well. qPCR master mix was prepared using 2x Luna probe mix (NEB), ChAd0x2 specific primers (Thermo Fisher), ChAdOxi specific universal probe (TAMRA / FAM) (Applied Biosystems) and nuclease free water to a final volume of 15 mΐ per sample. Mastermix was mixed and 15 mΐ added to the relevant wells of a 96 well MicroAmp FAST Optical PCR plate. Template / plasmid standard / samples were added (5m1 per well) to relevant test wells. Optical film was used to cover the plate before the relevant qPCR programme was run on a StepOne qPCR machine.
PCR programme: 95°C for 10 mins, 45 cycle of 95°C for 15 sec, 6o°C for 1 min. Recovered data was analysed using the standard curve results to generate viral genome copy number per well, which was further calculated to give genome copy per ml culture. To compare the IU titre between de-repressed and repressed, the genome copy number values of the de-repressed culture were set at 100% and the difference of the repressed culture compared to this. Repressed and de-repressed cultures gave a similar IU of virus at all time points tested.
Example 4 : Assembly Of Vaccine
Physical. Chemical and Pharmaceutical Properties and Formulation Description of ChAdOxi nCoV-19 (AZD1222) ChAdOx1 nCoV-19 spike protein vaccines (e.g. AZD1222 (reference) AZD2816
(invention) AZD3990 (invention)) described herein consist of the replication-deficient simian adenovirus vector ChAdOxi, containing the structural surface glycoprotein (Spike protein) antigen of the SARS CoV-2 (nCoV-19) expressed under the control of the CMV promoter, with a leading tissue plasminogen activator (tPA) signal sequence. The tPA leader sequence has been shown to be beneficial in enhancing immunogenicity. The different vaccines comprise different spike protein sequences/ mutants as described.
The code name for Drug Substance AZD1222 is ChAdOxi nCoV-19. There is no recommended International Non-proprietary Name (INN). The ChAdOxi nCoV-19 (AZD1222) drug substance has a genome size of 35,542bp and is a slightly opaque frozen liquid, essentially free from visible particulates. The appearance is dependent upon the concentration of the virus and the buffer that the virus is formulated in.
ChAdOxl Vector
The ChAdOx1 vector is replication-deficient as the El gene region, essential for viral replication, has been deleted. This means the virus will not replicate in cells within the human body. The E3 locus is additionally deleted in the ChAdOx1 vector. ChAdOx1 propagates only in cells expressing El, such as HEK293 cells and their derivatives or similar cell lines such as Per.C6 (Crucell).
ChAdOxl nCoV-19 (AZD1222) Vaccine Strain Assembly
The vaccine consists of the attenuated chimpanzee adenovirus vector ChAdOx1, expressing the SARS CoV-2 spike protein under the control of the CMV promoter. Pre- adenoviral plasmid pBAC ChAdOx1 nCoV19 was generated. The SARS CoV-2 Spike cDNA including a 32 amino acid N-terminal tPA leader sequence, obtained from GeneArt, was inserted into the El locus of ChAdOx1 by Gateway recombination. Suitably the “long CMV promoter” is used. This is known in the art, and is described in PCT/GB2008/001262 (WO/2008/122811).
Notable features
• “Long” CMV promoter (CMVLP) containing intron A, and Tet operator (TO) sites for repression of transgene expression in cells expressing the Tet repressor
• Synthetic codon-optimised SARS CoV-2 spike protein open reading frame
• BGH polyA signal
• Flanking site-specific recombination sequences utilised for transgene insertion.
• Chloramphenicol resistance gene in BAC vector backbone
• Pmel sites for release of viral genome
The following DNA constructs were used:
• #p5727: SARS CoV-2 Spike cDNA in DNA vector pMK
• #pi990: pENTR plasmid vector containing the CMV ‘long’ promoter (with intron A and Tet operator sites; CMVLP TO) and the BGH poly A sequence.
• #p57io: pENTR plasmid vector containing the CoV Spike antigen AZD1222 between the ‘long’ CMVLP TO promoter and BGH poly A sequences. • #p2503: pBAC ChAdOxi vector with El and E3 deleted, and E4 modified to improve yield and hexon expression for markerless titration. It was generated at the Jenner Institute, and its complete genome sequence is known The SARS CoV-2 Spike antigen was excised from #p5727 using Notl and Kpnl and ligated into #1990 cut with the same enzymes to obtain #p57io. The insert was verified by restriction mapping and sequencing. Gateway recombination was then performed between #5710 and #2563.
The sequence of the transgene region in ChAdOxi nCoV-19 (AZD1222) has been verified by sequencing directly from phenol purified viral genomic DNA.
The DNA map of #P5713 pBAC ChAdOxi nCoV-19 (AZD1222) used to generate the recombinant viral vector vaccine is shown in Figure 3.
In more detail, the P5713 pDEST-ChAdOxi-nCOV-19 plasmid is used in the manufacture of the composition according to the present invention. Specifically, the plasmid encodes a viral vector according to the invention. The viral sequence is excised from P5713 pDEST-ChAdOxi-nCOV-19 and the linear viral DNA is subsequently used to transfect El expressing cells, such as HEK293-TRex cells, for viral vaccine production. SEQ ID NO: 15 - P5713 pDEST-ChAdOxi nCoV-19 (AZD1222) DNA Sequence. Format: DNA (top strand), 44104 nucleotides.
ChAdOxi nCoV-19 (AZD2816) Vaccine Strain Assembly
ChAdOxi nCoV-19 (AZD2816) is constructed as above EXCEPT it is prepared so as to contain the tPA-spike fusion protein antigen of SEQ ID NO: 3.
The procedure is followed exactly as described above for AZD1222 Vaccine Strain Assembly, except: the AZD2816 plasmid P5841 pDEST ChAdOxi nCoV-i9E DNA (Sequence of plasmid: SEQ ID NO: 13) is used in the manufacture of the composition (instead of the P5713 pDEST-ChAdOxi-nCOV-19 used for AZD1222).
ChAdOxi nCoV-19 (AZD3990) Vaccine Strain Assembly
ChAdOxi nCoV-19 (AZD3990) is constructed as above EXCEPT it is prepared so as to contain the tPA-spike fusion protein antigen of SEQ ID NO: 12.
The procedure is followed exactly as described above for AZD1222 Vaccine Strain Assembly, except: the AZD3990 sequence (Sequence: SEQ ID NO: 25) is used in the manufacture of the composition (instead of the P5713 pDEST-ChAdOxi-nCOV-19 used for AZD1222).
Example 5: AZD2816 D7220 C00001 Ph 2/3 Safety and Immunogenicity Vaccines (viral vectors as described above) are evaluated as follows:
• Spike protein expression in infected cells
• Transcriptomic and proteomic studies of infected cells
• Mouse immunogenicity studies assessing B and T cell responses following various vaccination regimens · Viral challenge studies in animals following vaccination
Study Objectives: Show immunogenicity of the AZD2816 against SA B.135.1. Show AZD2816 retains immunogenicity against Wuhan strain
Study Design: AZ-sponsored, multi-center, multi- country, partially double blind, randomized controlled Phase 2/3 study in adults. Two study populations: Na'ive and immunized individuals (AZD1222 or mRNA External datasets will support contextualization Study powered for precision in consideration of published guidance We do not define formal non-inferiority criteria Descriptive analyses will support decision making Study Design shown in Figure 2.
Example 6 : Mouse Immunogenicity Studies
Study 1
Figure imgf000047_0001
Groups of 5-6 Balb/c mice
Minimum of 3 weeks between vaccinations lxio8 IU dose - IM route
Study lb = repeat with AZD3990
Study 2
Figure imgf000048_0002
(N.B. Throughout this document, standard notation may be used e.g. 108 may be written 10
Figure imgf000048_0001
8 meaning io-to-the-power-of-8.)
Immunological endpoints measured 3 weeks after Dose 1 Immunological Endpoints T cell ELISpot
• Spike specific IgG binding (ELISA)
• Breadth of IgG binding (octet)
• Neutralisation titre (live virus and pseudotype)
Example 7: Syrian hamster challenge study
Study Protocol Groups of 4/5 hamsters 21-28 days between doses Challenge 28 days after second dose
Intranasal inoculation with 104 TCID50 of challenge virus
Figure imgf000048_0003
Challenge viruses B.1.1.7 (UK) B.1.351 (S. Africa)
Pi (Brazil)
Example 8 : Evaluation of Wildtype and Stabilised SARS-CoV-2 Spike sequences as vaccine imm unogens
Wildtype and proline stabilised Spike sequences of Wuhan and B.1.351 (Jenner E) were evaluated in HeLa cells for cell surface expression. As shown by others, proline stabilised SARs-CoV-2 Spike protein is expressed to higher levels on the surface of cells compared to wildtype Spike protein_(Figure 4).
Here S2P; spike sequence were stabilised with 2 prolines. HexaPro; spike sequence stabilised with 6 prolines. HeLa cells were transfected with mRNAs encoding SARS- CoV-2 spike protein variants (Wuhan and South African (Jenner E) variants with the mutations). 24)1 post-transfection cells were stained with human anti-SARS-CoV-2 antibody and signal detected by europium-labelled anti-human IgG. The amount of signal, expressed in relative fluorescent units (RFU), is directly proportionate to the amount of spike protein present at the cell surface. Pairwise comparisons between plasma membrane expression levels yielded by different variants were conducted by the t-test ensuring normal distribution (Shaprio-Wilk test) and equality of variances (F test). Significance levels: ns. - not statistically significant; * - p<0.05; *** - p<0.01. (Astrazeneca ELN: MS01368-08).
Stabilised (Jenner E6; AZD3990) and wildtype (Jenner E; AZD2816) B.1.351 (South Africa) Spike sequence were cloned into the ChAdOxi vector and evaluated as immunogens. BALB/c mice were immunised with either AZD2816 (wildtype sequence Jenner E) or AZD3990 (hexapro stabilised Jenner E6) and Spike-specific antibody and
T cell responses measured 21 days later. It was surprising to note that, despite the stabilised spike protein giving superior cell expression levels in vitro, levels of anti - spike antibodies induced by vaccination with AZD3990 (stabilized spike) were similar to those induced by AZD2816 (wildtype spike sequence) as shown in Figure 5A and B. Also surprising was the observation that T cell responses where significantly higher in mice vaccinated with AZD2816 expressing the wildtype Spike sequence compared to mice vaccinated with AZD3990 expressing the hexapro stabilised spike sequence (Figure 5C). Here, BALB/c mice were vaccinated intramuscularly with a dose response (10 8 iu to 10 5 iu) of either AZD2816 (wildtype sequence Jenner E) or AZD3990 (hexapro stabilised Jenner E6). 21 days later, total IgG levels were measured by ELISA against original spike protein (NC_045512) or B.1.351 spike protein. IFNg secreting cells measured by ELISpot with splenocytes stimulated with whole spike protein. Example 9 : Evaluation of AZD2816 as a novel ChAdOxl vectored B.1.351 variant vaccine BALB/c mice were immunised with 10 8 iu AZD1222 (ChAdOx1 nCoV-19), AZD2816 (ChAdOx1 nCoV-19 B.1.351) or with 10 8 iu of each vaccine mixed together prior to injection in prime only or prime and boost regimens. Functional ability of antibodies to neutralise pseudotyped virus expressing original spike, B.1.351 or B.1.617 spike protein was measured in the serum of vaccinated mice. AZD2816 was shown to induce cross- reactive neutralising antibodies in mice when given as a single dose, in a mixture with AZD1222 or as a boost dose following AZD1222 vaccination regimen (Table below).
Table : Evaluation of the neutralising antibody response induced by AZD2816 in mice
Figure imgf000050_0001
Figure imgf000051_0001
When delivered as a single prime vaccination, AZD2816 induced neutralising antibodies against original Wuhan and B.1.351 virus. When compared to the original AZD1222 prototype vaccine, AZD2816 induced slightly lower neutralising titres to the original Wuhan spike protein but higher neutralisation against the B.1.351 spike protein. Mixing both vaccines together did not compromise the antibody response to either protein.
Functional ability of antibodies to neutralise pseudotyped virus expressing original spike, B.1.351 or B.1.617 spike protein was measured in the serum of vaccinated mice. Pseudotyped virus neutralization titres are expressed as the reciprocal of the serum dilution that inhibited luciferase expression by 50% (ID50) or 80% (ID80). Table shows the median (min to max) per group.
When given as a vaccine boost to mice previously vaccinated with AZD1222 , AZD2816 was observed to significantly increase the neutralising antibody titres measured against original Wuhan, B.1.351 and B.1.617 variants. In addition, boosting AZD1222 primed mice with AZD2816 increased the binding antibody titre against variant proteins P.i and B.1.429 when compared to a single dose of AZD1222 (Figure 6). Graph A. shows the total IgG response against original spike protein (NC_045512) or B.1.351 measured in the serum of mice collected 16 days after vaccination with AZD1222 (n=5) (animals from Figure 5) or a prime-boost regimen of AZD1222 followed 4 weeks later by AZD2816 (n=6). Graph B shows the Microneutralisation titre of serum (ND80) collected day 16 post-vaccination (animals Figure 5) and 21 days after prime-boost vaccination against pseudotyped virus expressing original (NC_045512), B.1.351 or B.1.617.1 spike protein. Limit of detection in the assay is defined as a titre of 40 (dotted line). Data was log-transformed and analysed with a 2-way anova (repeated measure) and post-hoc positive test, statistically significant differences (p<0.05) between groups are indicated. Graph C shows total IgG responses measured against B.1.17, P.i, B.1.429 or D614G spike proteins in serum collected 16 days and 3 weeks after the final vaccination. All ELISAs were performed simultaneously, data log transformed and analysed with a 2-way anova (repeated measure) with a post-hoc positive test, statistically significant differences between groups (p<0.05) are indicated. Taken together this data shows that a booster dose with AZD2816 can further enhance antibody responses and provide broad cross-reactivity against variant proteins.
It should be noted that D614G is found in nearly all strains now. It is derived from the original defined A lineage. For example, all of B.1.1.7, P-i, B.1.429 contain D614G.
Example 10 : Evaluation of AZD2816 as a novel ChAdOxl vectored vaccine against different variants of concerns
A multi-center, multi-country, randomized controlled Phase 2/3 study was conducted to determine the safety and immunogenicity of AZD2816 in previously unvaccinated adults, as well as the safety and immunogenicity of a booster dose of AZD1222 or AZD2816 in previously vaccinated adults. Whereby the third dose boosters of AZD1222 or AZD2816 were administered >3 months after receipt of the second dose of either AZD1222 or an mRNA vaccine. The immunogenicity was measured 28 days after the third dose booster. The reactogenicity (solicited local and systemic events) were recorded for first 7 days after third dose booster. All unsolicited AEs were recorded for 28 days after third dose booster. A schematic representation of the study design can be found in figure 2.
Neutralising antibodies were measured using a Focus Reduction Neutralization Test (FRNT). Briefly, serially diluted Ab or plasma was mixed with SARS-CoV2 strain. Mixtures were then transferred to 96-well, cell culture-treated, flat-bottom microplates containing confluent Vero cell monolayers in duplicate and incubated for a further 2 hours followed by the addition of carboxymethyl cellulose overlay medium. A focus forming assay was then performed by staining Vero cells with human anti-NP mAh (mAb2o6) followed by peroxidase-conjugated goat anti-human IgG (A0170; Sigma). Finally, the foci (infected cells), approximately too per well in the absence of antibodies, were visualized by adding TrueBlue Peroxidase Substrate. Virus-infected cell foci were counted on an AID EliSpot reader using AID ELISpot software. The percentage of focus reduction was calculated, and IC50 was determined using the probit program from the SPSS package. SARS-CoV-2 live neutralizing antibody titers against the different variants were assessed in the participants. Day 29 values were plotted. Boxplots were created based on the lognormal distribution. Geometric mean titers at Baseline and Day 29 were tabulated. Titers assessed as less than the lower limit of quantitation were set to half of the starting dilution (20 or 80, depending on the sample).
AZD1222 and AZD2816 third dose boosters increased neutralising antibody titres to Wuhan and Beta variants in participants previously vaccinated with AZD1222
(Figure 7 A). AZD1222 and AZD2816 third dose boosters increased breadth of humoral response to variants of concern in participants previously vaccinated with AZD1222 (Figure 7 B) AZD1222 and AZD2816 third dose boosters increased neutralizing antibody titres to Wuhan and Beta variants in participants previously vaccinated with mRNA vaccines (Figure 7C). AZD1222 and AZD2816 third dose boosters increased breadth of humoral response to variants of concern in participants previously vaccinated with mRNA vaccines (Figure 7 D). AZD1222 and AZD2816 show neutralization of Omicron variant after 3rd dose booster with a trend toward increased geometric mean titer (GMT) after AZD2816 boost (Figure 7 E).
The results of the study show that AZD1222 and AZD2816 were generally well-tolerated following boosting of subjects that had previously received 2 doses of AZD1222 or an mRNA vaccine. Third dose boosting with AZD1222 increased neutralizing titres against the original Wuhan strain as well as variants of concern (Beta, Gamma, Delta, Omicron) as compared to the two-dose primary series. Third dose of AZD2816 increases neutralizing titres against Beta to a higher magnitude than a third dose of AZD1222.
Although illustrative aspects of the invention have been disclosed in detail herein, the invention is not limited to those precise aspects. Various changes and modifications can be effected by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.
The specification/description of this application comprises a sequence listing in WIPO ST.25/ST.26 format. To assist the reader, we refer to the table of sequences below.
Figure imgf000054_0001
Figure imgf000055_0001
For SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22 please note that these are spike protein sequences without N-terminal tPA fusions. To create N-terminal tPA fusions from any of these sequences, the N-terminal Methionine of the spike protein sequence is omitted and replaced with the tPA amino acid sequence (which includes an initating Methionine - see for example SEQ ID NO: 5).
Thus we expressly describe compositions comprising a viral vector, wherein the viral vector is an adenovirus based vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide, said polypeptide having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, characterised in that said polypeptide comprises the amino acid sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21 or 22.
In another aspect suitably said polypeptide is present as a fusion with the tissue plasminogen activator (tPA) sequence in the order N-terminus - tPA - polypeptide - C- terminus. Most suitably said tPA sequence comprises, or consists of, the amino acid sequence of SEQ ID NO: 5. When present as a fusion protein, the lead methionine of the amino acid sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21 or 22 is omitted as noted above. The descriptions of viral vector construction, insertion cargo sequence, promoters, etc relate equally to these aspects.

Claims

1. A composition comprising a viral vector, wherein the viral vector is an adenovirus based vector, the viral vector comprising nucleic acid having a polynucleotide sequence encoding a polypeptide, said polypeptide having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, characterised in that said polypeptide comprises the following substitutions relative to SEQ ID NO: 1: a) L18F b) D80A c) G215D d) L242 A e) A243 D f) L244 D g) K417N h) E484K i) N501Y j) D614G; and k) A701V.
2. A composition according to claim 1 wherein said polypeptide further comprises the following substitutions relative to SEQ ID NO: 1: l) F814P m) A889P n) A896P
0) A939P p) K983P; and q) V984P.
3. A composition according to claim 1 or claim 2 wherein said adenovirus based vector is ChAdOx 1.
4. A composition according to any of claims 1 to 3 wherein said polypeptide comprises the spike protein receptor binding domain (RBD).
5. A composition according to any of claims 1 to 4 wherein said polypeptide comprises the spike protein receptor binding domain (RBD), the spike protein N- terminal Domain (NTD) and the spike protein STEM.
6. A composition according to any of claims 1 to 5 wherein said polypeptide is full length spike protein.
7. A composition according to any preceding claim wherein said polypeptide is present as a fusion with the tissue plasminogen activator (tPA) sequence in the order N-terminus - tPA - polypeptide - C-terminus.
8. A composition according to claim 7 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.
9. A composition according to any preceding claim wherein said polypeptide has the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 12.
10. A composition according to any preceding claim wherein said polynucleotide sequence comprises the sequence of SEQ ID NO: 23 or SEQ ID NO: 24, preferably SEQ ID NO: 23.
11. A composition according to any of claims 2 to 10 wherein said viral vector sequence is as in ECACC accession number 12052403.
12. A composition according to any of claims 1 to 11 wherein administration of a single dose of said composition to a mammalian subject induces protective immunity in said subject.
13. A composition according to any of claims 1 to 11 wherein administration of a first dose of said composition to a mammalian subject followed by administration of a second dose of said composition to said mammalian subject induces protective immunity in said subject.
14. A composition according to any preceding claim for use in induction of an immune response against SARS-CoV2 in a mammalian subject.
15. A composition according to any preceding claim for use in preventing SARS- CoV2 infection in a mammalian subject.
16. Use of a composition according to any of claims 1 to 15 in medicine.
17. Use of a composition according to any of claims 1 to 15 in the preparation of a medicament for prevention of SARS-CoV2 infection in a mammalian subject.
18. A method of inducing an immune response against SARS-CoV2 in a mammalian subject, the method comprising administering a dose of a composition according to any of claims 1 to 15 to said subject.
19. A composition for use according to claim 14 or a composition for use according to claim 15 wherein said use comprises: (i) administering a first dose of said composition to said subject; and
(ii) administering a second dose of said composition to said subject.
20. A method according to claim 18, or a composition for use according to claim 19, wherein said first dose and said second dose each comprise about the same number of viral particles.
21. A method according to claim 18, or a composition for use according to claim 19, wherein each said dose comprises about 5 x to10 viral particles.
22. A method according to claim 18, or a composition for use according to claim 19, wherein said second dose comprises about twice the number of viral particles of the first dose.
23. A method according to claim 18, or a composition for use according to claim 19, wherein said first dose comprises about 2.5 x to10 viral particles, and said second dose comprises about 5 x to10 viral particles.
24. A method according to claim 18 or claim 20, or a composition for use according to claim 19 or claim 20, wherein said second dose is administered at an interval of a) less than 6 weeks, b) 6 to 8 weeks, c) 9 to 11 weeks, or d) 12 weeks or more, after administration of said first dose.
25. A method according to claim 18 or any of claims 20 to 24, or a composition for use according to any of claims 19 to 24, wherein said first dose comprises AZD1222 and wherein said second dose comprises AZD2816.
26. A method according to claim 18 or any of claims 20 to 25 wherein said composition is administered by a route of administration selected from a group consisting of intranasal, aerosol, intradermal and intramuscular.
27. A method according to claim 26 wherein said administration is intramuscular.
28. A method according to claim 18, wherein the dose is administered after one or more doses of a second composition suitable for inducing an immune response against SARS-CoV2 in a mammalian subject.
29. A method according to claim 28, wherein second composition is selected from mRNA vaccines (e.g. BNTi62b2 or tozinameran; mRNA-1273 or CX-024414), Adenovirus vector vaccines (e.g. AZD1222; Gam-COVID-Vac; Ad26.C0V2.S or JNJ- 78436735), inactivated and subunit vaccines.
30. A method according to claim 28 or 29, wherein the dose of the composition according to any of claims 1 to 15 is administered less than 4 months, less than 6 months, less than 12 months, or less than 24 months after the one or more doses of the second composition.
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