WO2019145739A1

WO2019145739A1 - Lassa virus antigenic composition

Info

Publication number: WO2019145739A1
Application number: PCT/GB2019/050233
Authority: WO
Inventors: Roger HEWSON; Stuart DOWALL; Emma KENNEDY
Original assignee: UK Secretary of State for Health and Social Care
Current assignee: UK Secretary of State for Health and Social Care
Priority date: 2018-01-29
Filing date: 2019-01-29
Publication date: 2019-08-01
Anticipated expiration: 2020-07-29
Also published as: GB201801437D0

Abstract

The present invention provides a viral vector or bacterial vector, said vector comprising a nucleic acid sequence encoding a Lassa Virus nucleoprotein or antigenic fragment thereof; wherein said vector is capable of inducing a protective immune response in a subject. The present invention also provides compositions and uses of the vector in methods of medical treatment.

Description

Lassa Virus Antigenic Composition

The present invention relates to viral vectors and bacterial vectors comprising Lassa virus antigens and their use in immunogenic and antigenic compositions. The present invention also relates to prophylactic uses of said compositions. The present invention also relates to immunogen for use in raising therapeutic antibodies, and methods for producing said immunogen.

Lassa virus is an emerging zoonotic virus and is characterised as a hazard group 4 pathogen. Lassa virus is the causative agent of“Lassa fever”, a zoonotic disease which is endemic throughout West Africa, where it is believed to cause 300000- 500000 infections per year. Lassa virus is typically transmitted by exposure to urine or faeces of infected Mastomys rats (the animal host of Lassa virus), and may also be transmitted via direct contact with bodily secretions of a person infected with Lassa virus.

Lassa fever can exhibit symptoms similar to those of Ebola virus. About 80% of people infected with Lassa virus are asymptomatic, but in severe cases, infection can lead to symptoms such as bleeding, deafness, shock, seizures, tremor, disorientation, and coma. According to the World Health Organisation, Lassa fever usually kills around 15 percent of those who develop severe disease, but in 2017, the case fatality rate is reported to be greater than 50 percent. Death usually occurs within 14 days of onset in fatal cases. The disease is particularly severe during the final trimester of pregnancy, where more than 80% of cases result in maternal death and/or fetal loss.

According to the WHO, the antiviral drug ribavirin seems to be an effective treatment for Lassa fever if given early on in the course of clinical illness. However, there is no evidence to support the role of ribavirin as post-exposure prophylactic treatment for Lassa fever. Various attempts have been made to produce a Lassa virus vaccine (such as the“GeoVax” vaccine candidate which is a VLP based upon the Lassa virus GP and Z proteins), but although some such studies report success at the pre-clinical stage, none have progressed beyond the pre-clinical stage of development. There is currently no vaccine that protects against Lassa fever. The urgent and unmet need for a vaccine against Lassa virus is recognised by the WHO, and a“Target Product Profile (TPP)” has been drafted identifying Lassa virus vaccines as a priority for product development. The WHO’s Lassa virus vaccine TTP follows prioritisation of Lassa fever as part of the WHO R&D blueprint for Action to Prevent Epidemics.

There is therefore an urgent need for further therapeutics for the prevention, treatment and suppression of Lassa fever.

The present invention addresses one or more of the above problems by providing viral vectors and bacterial vectors encoding Lassa virus nucleoprotein (NP) or antigenic fragments thereof, together with corresponding compositions and uses of said vectors and compositions in the prevention and treatment of Lassa virus infection.

The vectors and compositions of the invention enable an immune response against Lassa virus to be stimulated (i.e. induced) in an individual (i.e. a subject), and provide improved immunogenicity and efficacy.

In one aspect, the invention provides a viral vector or bacterial vector, said vector comprising a nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof; wherein said vector is capable of inducing an immune response in an individual. The present inventors have found that highly effective immune responses against Lassa virus can be generated in an individual by using a viral vector or bacterial vector to deliver to the subject nucleic acid sequences encoding Lassa virus NP (or antigenic fragments thereof), as described above.

In a preferred embodiment, the vector of the invention is a viral vector.

Lassa viruses are enveloped, single stranded, bisegmented, ambisense RNA viruses which belong to the genus family Arenaviridae and the genus Mammarenavirus. There are four major lineages of Lassa virus, which correlate with their geographic location, the endemic area of Lassa fever is expanding and a fifth Lassa virus lineage has been proposed. Analysis of the four LASV lineages has shown genetic heterogeneity, with up to 27% nucleotide sequence variation, and 15% amino acid sequence variation. This data shows that mutations accumulate in epitopes of the viral surface glycoprotein (GP), suggesting selection for immune escape. Lassa fever is the most commonly imported Viral Haemorrhagic Fever into Europe, the most recent case of which resulted in onward human to human transmission in Germany in 2016. The UK has received the most incursions out of all cases of Lassa fever that have been exported from West Africa, each one causing enormous burden to clinical, laboratory and public health resources.

The Lassa virus genome is contained in two RNA segments. The large (7 kb) segment encodes for a zinc binding protein (“Z”), referred to as the matrix protein, which regulates transcription and replication; and RNA polymerase (L). The small (3.5 kb) segment encodes the nucleoprotein (NP) and the surface glycoprotein precursor (GP; also known as the viral spike protein). Proteolytic cleavage of GP yields the envelope glycoproteins GP1 and GP2.

The Lassa virus NP forms the protein scaffold of the genomic ribonucleoprotein complexes and is critical for transcription and replication of the viral genome. The Lassa virus NP has been implicated in suppression of the host innate immune system and has been demonstrated to exhibit exonuclease activity, with strict specificity for double-stranded RNA substrates. This exonuclease activity is believed to be essential for the ability of NP to block activation of the innate immune system.

“GA391 Nigeria” may be used as a reference Lassa virus strain. The inventors conducted high fidelity sequencing of the GA391 Nigeria genome, and the reference nucleic acid sequence (identified by high fidelity sequencing) for Lassa virus nucleoprotein is represented by (SEQ ID NO: 1):

AT G AGTGCTT CCAAGG AAGT G AGGT CATT CTT GT GG ACT CAAT CCCT AAG AAGGGAACTATCTGGCTACTGTTCCAACATAAAGTTGCAGGTAGTTAAAG ACGCT CAAGCT CT CCTT CATGGT CTT GACTT CT CCGAGGT CAGT AAT GTT CAG AG ATT GAT G CG CAAG CAG AAAAG AG AT GAT G G CG ACCT AAAACG AC T GAG AG AT CT CAAT CAAGCAGT CAACAAT CTT GTT G AGCT CAAAT CCACA CAGCAG AAAAGT GT CTT AAGAGTTGG AACCTT AAGTT CAG ACG AT CT ACT AAT CCT G GCCGCT G ATTT AG AAAAACT G AAAT CAAAAGT CACCAG AACAG AAAGGCCTTT GAGTT CAGGAGTTTATATGGGGAATTT GAGTT CACAACAG CTT GAT CAAAGGAGAGCCCTTTT GAACAT GATTGGCAT GACTGGAGT AAG TGGAGGGGGAAAGGGTGCCAGT GATGGCATT GT GAGAGTTTGGGAT GT CAAAAATGCAGAGTT ACT CAACAAT CAGTT CGGAACAATGCCAAGCCTAA CTTTAGCAT GCCT GACCAAACAGGGGCAAGTGGACCT GAAT GATGCCGT T CAAG CTTT G ACAG ATTT AGG G CT G ATTT ACACAG CCAAAT ACCCCAACT CAT CT GAT CT CGACAG ATT GTCT CAG AGTCAT CCAATT CT GAAT AT GATT GACACT AAGAAAAGTT CACT CAACAT CT CTGGTT ACAATTT CAGTTT GGG TGCTGCTGTCAAAGCAGGGGCCTGCATGCTTGATGGTGGTAACATGTTA GAG ACT ATT AAGGTTT CACCT CAG ACCATGG AT GGTAT CTT G AAGT CAAT CTTGAAAGTTAAGAAGAGTCTGGGAATGTTTGTATCAGACACACCGGGT G AAAG G AACCCTT AT G AG AACAT CCT AT ACAAG AT CTGTCT CT CAG GAG A CGGATGGCCCTATATTGCATCAAGGACCTCGATTGTGGGAAGAGCATGG G AAAAT ACTGTG GT G G ACCTT GAG CAAG ACAACAAG CCCCAGAAAATT G G A AAT G G G G G G T CC AAC AAG TCATTACAGTCTGCAGGCTTTGCTGCAGG ATT AACTT ACT CT CAGTT GAT GACT CT CAAAG ATTT CAAGTGCTT CAACTT GATT CCCAACG CAAAAACCT G G ATGG AT ATT G AAG G AAG ACCAG AAG AC CCAGTTGAGATAGCCCTTTATCAACCGAGCTCGGGTTGCTATGTACATTT CTTTAGGGAGCCAACAGATTT GAAGCAATT CAAACAAGATGCAAAGTATT CACATGGTATT GAT GT GACT GATTT GTTTGCTGCCCAACCTGGGTTAACC AGTGCAGT GAT AGAAGCCCTT CCT CGGAACATGGT CAT CACTTGCCAAG GAT CAG AG GAT AT CAG AAAACT CCTT G AGT CACAAG G G AG G AG AG ACAT AAAACT GATT G ACAT CACT CTT AGTAAAGCAG ATT CAAGAAAGTTT GAGA ATG CTG TTTG G G AT C AATT CAAG GAT CT ATG T C AC AT GCACACTGGGGTA GTT GTGGAGAAAAAGAAGAGAGGTGGTAAAGAGGAAAT AACT CCT CATT GTGCACT GATGGATTGCATTAT GTTT GATGCAGCAGTTT CAGGAGGACTT GATGCAAAAGT CCT G AG AGCT GTG CT CCCT AG AG ACAT GGTGTT CAG AA CTT CAACACCT AAAGT CGT CCT G (SEQ ID NO: 1)

Translation of the high fidelity nucleic acid sequence (SEQ ID NO: 1) yields the reference polypeptide sequence for Lassa virus nucleoprotein, which is represented by (SEQ ID NO: 2):

MSASKEVRSFLWTQSLRRELSGYCSNIKLQVVKDAQALLHGLDFSEVSNVQ

RLMRKQKRDDGDLKRLRDLNQAVNNLVELKSTQQKSVLRVGTLSSDDLLILA

ADLEKLKSKVTRTERPLSSGVYMGNLSSQQLDQRRALLNMIGMTGVSGGG

KGASDGIVRVWDVKNAELLNNQFGTMPSLTLACLTKQGQVDLNDAVQALTD

LGLIYTAKYPNSSDLDRLSQSHPILNMIDTKKSSLNISGYNFSLGAAVKAGAC

MLDGGNMLETIKVSPQTMDGILKSILKVKKSLGMFVSDTPGERNPYEN ILYKI

CLSGDGWPYIASRTSIVGRAWENTVVDLEQDNKPQKIGNGGSNKSLQSAGF

AAGLTYSQLMTLKDFKCFNLIPNAKTWMDIEGRPEDPVEIALYQPSSGCYVH

FFREPTDLKQFKQDAKYSHGIDVTDLFAAQPGLTSAVIEALPRNMVITCQGS

EDIRKLLESQGRRDIKLIDITLSKADSRKFENAVWDQFKDLCHMHTGVVVEK

KKRGGKEEITPHCALMDCIMFDAAVSGGLDAKVLRAVLPRDMVFRTSTPKV

VL (SEQ ID NO: 2)

Reference nucleic acid and polypeptide sequence for Lassa virus nucleoprotein may be provided by GenBank Accession number X52400.1. Inventors note that the sense strand of the Lassa virus NP corresponds to nucleic acid positions 103-1812 of the reverse complement of the nucleic acid sequence provided in GenBank Accession number X52400.1. The reverse complement of the nucleic acid sequence provided in GenBank Accession number X52400.1 is provided by SEQ ID NO: 3.

CGCACAGTGGATCCTAGGCTATTGGATTGCGCTTTGCACTAATCAAACCTT

TGGAGTGACCACATTCAAGACCCACTCAAGGGTCAGACAACCATCACGAC

AATGAGTGCTTCCAAGGAAGTGAGGTCATTCTTGTGGACTCAATCCCTAA

GAAGGGAACTATCTGGCTACTGTTCCAACATAAAGTTGCAGGTAGTTAAA

GACGCTCAAGCTCTCCTTCATGGTCTTGACTTCTCCGAGGTCAGTAATGTT

CAGAGATTGATGCGCAAGCAGAAAAGAGATGATGGCGACCTAAAACGAC

TGAGAGATCTCAATCAAGCAGTCAACAATCTTGTTGAGCTCAAATCCACA

C AGC AGAA AAGTGTCTT A AGAGTT GGAACCTT AAGTTC AGACGATCT ACT

AATCCTGGCCGCTGATTTAGAAAAACTGAAATCAAAAGTCACCAGAACAG

AAAGGCCTTTGAGTTCAGGAGTTTATATGGGGAATTTGAGTTCACAACAG

CTT GAT C AAAGGAGAGCCCTTTTGAAC ATGATTGGC AT GACTGGAGT AAG

T GGAGGGGGA A AGGGT GC C AGT GAT GGC ATTGT GAGAGTTT GGGAT GT C A

AAAATGCAGAGTTACTCAACAATCAGTTCGGAACAATGCCAAGCCTAACT

TTAGCATGCCTGACCAAACAGGGGCAAGTGGACCTGAATGATGCCGTTCA

AGCTTTGACAGATTTAGGGCTGATTTACACAGCCAAATACCCCAACTCATC

TGATCTCGACAGATTGTCTCAGAGTCATCCAATTCTGAATATGATTGACAC

TAAGAAAAGTTCACTCAACATCTCTGGTTACAATTTCAGTTTGGGTGCTGC

TGT C AAAGC AGGGGCCTGC AT GCTTGAT GGTGGT AAC AT GTT AGAGACT A

TTAAGGTTTCACCTCAGACCATGGATGGTATCTTGAAGTCAATCTTGAAAG

TTAAGAAGAGTCTGGGAATGTTTGTATCAGACACACCGGGTGAAAGGAAC

CCTTATGAGAACATCCTATACAAGATCTGTCTCTCAGGAGACGGATGGCC

CTATATTGCATCAAGGACCTCGATTGTGGGAAGAGCATGGGAAAATACTG

TGGTGGACCTTGAGCAAGACAACAAGCCCCAGAAAATTGGAAATGGGGG

GTCCAACAAGTCATTACAGTCTGCAGGCTTTGCTGCAGGATTAACTTACTC

TCAGTTGATGACTCTCAAAGATTTCAAGTGCTTCAACTTGATTCCCAACGC

AAAAACCTGGATGGATATTGAAGGAAGACCAGAAGACCCAGTTGAGATA

GCCCTTTATCAACCGAGCTCGGGTTGCTATGTACATTTCTTTAGGGAGCCA

AC AGATTT GAAGC A ATT C AAAC AAGAT GC AAAGT ATTC AC AT GGT ATT GA

TGTGACTGATTTGTTTGCTGCCCAACCTGGGTTAACCAGTGCAGTGATAGA

AGCCCTTCCTCGGAACATGGTCATCACTTGCCAAGGATCAGAGGATATCA

GAAAACTCCTTGAGTCACAAGGGAGGAGAGACATAAAACTGATTGACATC

ACTCTTAGTAAAGCAGATTCAAGAAAGTTTGAGAATGCTGTTTGGGATCA

ATT C A AGGATCT AT GT C AC AT GC AC AC T GGGGT AGTT GT GG AGA A A A AGA

AG AG AGGT GGT A A AG AGG A A AT AAC TCCTCATT GT GC AC T GAT GG AT T GC

ATTATGTTTGATGCAGCAGTTTCAGGAGGACTTGATGCAAAAGTCCTGAG

AGTTGTGCTCCCTAGAGACATGGTGTTCAGAACTTCAACACCTAAAGTCGT

CCTGTAGATGGACGCCCCCGTGACCCACCGCCAATTGGCGGTGGGTCACG

GGGGCCCTGTGGGATTTCACCTCTTCCATCTGACAGGCACACCTGGCTGTT

TGTATAGACCACAGGAGCAGATGCCCATGTGGTTCAGCCTGTGGGGTTTT

GGGC AAGGTTT ACCT AC AATGT GTCT AT GGGTT GGT ATTTT GACC AGGT GG

AGAAAGAT GCTT AT C AGAT AGAAACTTGT GCT AAAAAC AAAT AGGTC AAC

TAACCCCAGTGGAGTTTTGCCCTGTCTATCTATGTATTCCTTTTGTAGCATC

TCTGTTATCATGTTGTCAGCTTGTTGTTCGATGTCATCTGAAAACTTGGTCT

CATTGAGATATGATCCATTTGAGATAAGCCAACACCTTGGTAGTGAGGTC TTTCCTGTTGATGTGTGGTTAAGGTACCAATATCTGCTGTAGTTACAATAT

GGTATGCCCATGATGTCTCTCAAGTGGTTCTTCATGATGAGCTGATCATTT

ATTAAAGCATTGACAGCCTTGTTGATCAGCTGTATGCTCATTTGGGCCTCT

GTTTTGAGCCTCCTTATGGCCTGTTTGTTGAAATCGAACAACCTTAGCATG

TCACAAAATTCTTCATCATGTTTCTCATTGCACTTGGCTACTGCAGTGTTTC

CAAAGCACTTTAATTCGGCCTCAATCAACATCCATCTAGTAAGGCAGTATC

CCCCTGGTGTTTCATTTCCCTCTGAGTCTGATAGTGTCCAGGTGAATGTCC

CCAACAACCTTCTGCTAATGTATATGTCTCTAGTTCTTTGTGAGAGAAGCC

CAAGGTAGCCAATAGGTGATGGTCTGGAAAATTGGCAGTGGTCATCCCAG

GTTGTATTCTGAATGATTAGATATTGGTAACTGGTCATTATACAGTCCCAG

TTACCGCGTCCAGAATCAAGAGCAATATAACTCCCTCCCCAAGCCATTCTC

ATAAATGTTTGTAAGACACCATTTGCAAGTGTGCCGCAGTGACCTGCTGC

ATCCACTGCGAAGCTATGACTCAGGTTGTATTGCACAGTGATTTTCCCCCC

ATTGAAATCGCAGCTCATTGCCTCATATTGATTGAAGTTGGGAATGGACA

GAT G A A AGGT AG AG AT GAT AC T CAT G AGGC T GT GAT CAT A A AG ATT C C TT

TT GTGGGC ATC AGAGAGGTT AC AGAATTTGT GATT AAGAAT GCTGGT GTT

GGTCAAGGTGAGCTCAAGTCCTGTCTCATTCCCCACCCTTATATAATGATG

ACTGTTGTTCTTTGTGCAGGATAGCGGCATGGTCATATTAAGAGTCTCCAT

ATTCAACTCAAGGGTTTGCAGCTCGTAAGTCCCTTTGTAGATCAGTGAGCA

TGACCTTCCTGAAAGTAGAAGGAATGTGACAAGCCCTATCAAGCCACACG

TGGCAACATTGTATAGTCCCTTCAGAATTGCTAGGATGGATAGTGCAATA

AGGACAATATTCATCACTTCCTCAATAACATGAGGAACTTCTTGGAAGAA

TGTCACAATCTGTCCCATCCTGATTGCGACACTTTCCAAAAAGGAGGTTTT

AAAATGCGCAATCCTAAATGCCTAGGATCCCCGGTGC (SEQ ID NO: 3)

The reverse complement of the Lassa virus nucleic acid sequence provided by GenBank Accession number X52400.1, which encodes the Lassa virus NP (corresponding to positions 103-1812 of SEQ ID NO: 3) is represented by SEQ ID NO: 4.

ATGAGTGCTTCCAAGGAAGTGAGGTCATTCTTGTGGACTCAATCCCTAAG AAGGGAACT ATCTGGCT ACTGTTCC AAC AT AAAGTT GC AGGT AGTT AAAG ACGCTCAAGCTCTCCTTCATGGTCTTGACTTCTCCGAGGTCAGTAATGTTC AG AG ATT GAT GCGC AAGC AGA AAAGAGAT GAT GGCGACCT AAAACGACT GAGAGATCTC AAT C AAGC AGTC AAC AATCTTGTT GAGCTC AA ATCC AC AC AGC AGAAAAGT GTCTT AAGAGTT GGAACCTT AAGTTC AGACGATCT ACT A ATCCTGGCCGCTGATTTAGAAAAACTGAAATCAAAAGTCACCAGAACAGA AAGGCCTTTGAGTTCAGGAGTTTATATGGGGAATTTGAGTTCACAACAGC TT GATC AAAGGAGAGCCCTTTTGAAC AT GATTGGC AT GACTGGAGT AAGT GGAGGGGGAAAGGGTGCC AGT GAT GGC ATTGTGAGAGTTT GGGAT GT C A AAAATGCAGAGTTACTCAACAATCAGTTCGGAACAATGCCAAGCCTAACT TTAGCATGCCTGACCAAACAGGGGCAAGTGGACCTGAATGATGCCGTTCA AGCTTTGACAGATTTAGGGCTGATTTACACAGCCAAATACCCCAACTCATC TGATCTCGACAGATTGTCTCAGAGTCATCCAATTCTGAATATGATTGACAC TAAGAAAAGTTCACTCAACATCTCTGGTTACAATTTCAGTTTGGGTGCTGC TGT C AAAGC AGGGGCCTGC AT GCTTGAT GGTGGT AAC AT GTT AGAGACT A TTAAGGTTTCACCTCAGACCATGGATGGTATCTTGAAGTCAATCTTGAAAG TTAAGAAGAGTCTGGGAATGTTTGTATCAGACACACCGGGTGAAAGGAAC

CCTTATGAGAACATCCTATACAAGATCTGTCTCTCAGGAGACGGATGGCC

CTATATTGCATCAAGGACCTCGATTGTGGGAAGAGCATGGGAAAATACTG

TGGTGGACCTTGAGCAAGACAACAAGCCCCAGAAAATTGGAAATGGGGG

GTCCAACAAGTCATTACAGTCTGCAGGCTTTGCTGCAGGATTAACTTACTC

TCAGTTGATGACTCTCAAAGATTTCAAGTGCTTCAACTTGATTCCCAACGC

AAAAACCTGGATGGATATTGAAGGAAGACCAGAAGACCCAGTTGAGATA

GCCCTTTATCAACCGAGCTCGGGTTGCTATGTACATTTCTTTAGGGAGCCA

AC AGATTT GAAGC A ATT C AAAC AAGAT GC AAAGT ATTC AC AT GGT ATT GA

TGTGACTGATTTGTTTGCTGCCCAACCTGGGTTAACCAGTGCAGTGATAGA

AGCCCTTCCTCGGAACATGGTCATCACTTGCCAAGGATCAGAGGATATCA

GAAAACTCCTTGAGTCACAAGGGAGGAGAGACATAAAACTGATTGACATC

ACTCTTAGTAAAGCAGATTCAAGAAAGTTTGAGAATGCTGTTTGGGATCA

ATT C A AGGATCT AT GT C AC AT GC AC AC T GGGGT AGTT GT GG AGA A A A AGA

AG AG AGGT GGT A A AG AGG A A AT A AC TCCTCATT GT GC AC T GAT GG AT T GC

ATTATGTTTGATGCAGCAGTTTCAGGAGGACTTGATGCAAAAGTCCTGAG

AGTTGTGCTCCCTAGAGACATGGTGTTCAGAACTTCAACACCTAAAGTCGT

CCTG (SEQ ID NO: 4)

The high fidelity nucleic acid sequence identified by the inventors (SEQ ID NO: 1) differs from the corresponding nucleic acid sequence provided by GenBank Accession number X52400.1 by a single nucleic acid (corresponding to the “A” residue at position 1658 in SEQ ID NO: 1, and the“T” residue at positions 1770 and 1658 of SEQ ID NOs: 3 and 4, respectively).

Translation of SEQ ID NO: 4 yields a reference polypeptide sequence for Lassa virus nucleoprotein, which is represented by SEQ ID NO: 5 :

MS ASKEVRSFLWT Q SLRREL SGY C SNIKLQ VVKD AQ ALLHGLDF SE V SNVQR LMRKQKRDD GDLKRLRDLN Q A VNNL VELK S T Q QK S VLR V GTL SSDDLLILA ADLEKLKSKVTRTERPLS SGVYMGNLS SQQLDQRRALLNMIGMTGVSGGGK GASDGIVRVWDVKNAELLNNQFGTMPSLTLACLTKQGQVDLNDAVQALTDL GLI YT AK YPN S SDLDRL S Q SHPILNMIDTKK S SLNIS GYNF SLGA A VK AGACM LDGGNMLETIKV SPQTMDGILKSILKVKKSLGMF V SDTPGERNPYENILYKIC L S GDGWP YI ASRT SI V GR AWENT VVDLEQDNKPQKIGN GGSNK SLQ S AGF A A GLT YSQLMTLKDFKCFNLIPNAKTWMDIEGRPEDP VEI AL Y QP S SGC YVHFFR EPTDLKQFKQDAKYSHGIDVTDLFAAQPGLTSAVIEALPRNMVITCQGSEDIR KLLESQGRRDIKLIDITLSKADSRKFENAVWDQFKDLCHMHTGVVVEKKKRG GKEEITPHCALMDCIMFDAAVSGGLDAKVLRVVLPRDMVFRTSTPKVVL

(SEQ ID NO: 5)

Consistent with the single nucleic acid difference at position 1658 in SEQ ID NOs: 1 and 4, the amino acid at position 553 of SEQ ID NO: 5 is Valine, as compared to Alanine in SEQ ID NO: 2. As used herein, the term“antigenic fragment” means a peptide or protein fragment of a Lassa virus NP which retains the ability to induce an immune response in an individual, as compared to the reference Lassa virus NP. An antigenic fragment may therefore include at least one epitope of the reference protein. By way of example, an antigenic fragment of the present invention may comprise (or consist of) a peptide sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550 or 570 amino acids, wherein the peptide sequence has at least 70% sequence homology over a corresponding peptide sequence of (contiguous) amino acids of the reference protein. An antigenic fragment may comprise (or consist of) at least 10 consecutive amino acid residues from the sequence of the reference protein (for example, at least

5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250,

275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550 or 570 consecutive amino acid residues of said reference protein).

In one embodiment, an antigenic fragment comprises (or consists of) SEQ ID NO: 6. In one embodiment, an antigenic fragment comprises (or consist of) at least 10 consecutive amino acid residues from the sequence of SEQ ID NO: 6 (for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 91 amino acids of SEQ ID NO: 6, wherein said fragment has at least 70% sequence homology over a corresponding peptide sequence of (contiguous) amino acids of the reference protein.

In one embodiment, an antigenic fragment comprises (or consists of) SEQ ID NO: 7. In one embodiment, an antigenic fragment comprises (or consist of) at least 10 consecutive amino acid residues from the sequence of SEQ ID NO: 7 (for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 91 amino acids of SEQ ID NO: 7, wherein said fragment has at least 70% sequence homology over a corresponding peptide sequence of (contiguous) amino acids of the reference protein.

In one embodiment, an antigenic fragment comprises (or consists of) SEQ ID NO: 7 and SEQ ID NO: 8. In one embodiment, an antigenic fragment comprises (or consist of) at least 10 consecutive amino acid residues from the sequence of SEQ ID NO: 7 (for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 91 amino acids of SEQ ID NO: 7, and at least 10 consecutive amino acid residues from the sequence of SEQ ID NO: 8 (for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 91 amino acids of SEQ ID NO: 8 wherein said fragment has at least 70% sequence homology over a corresponding peptide sequence of (contiguous) amino acids of the reference protein.

An antigenic fragment of a reference protein may have a common antigenic cross- reactivity and/or substantially the same in vivo biological activity as the reference protein. For example, an antibody capable of binding to an antigenic fragment of a reference protein would also be capable of binding to the reference protein itself. By way of further example, the reference protein and the antigenic fragment thereof may share a common ability to induce a“recall response” of a T lymphocyte (e.g. CD4+, CD8+, effector T cell or memory T cell such as a TEM or TCM), which has been previously exposed to an antigenic component of a Lassa virus infection.

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4.

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4. In a further embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4.

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 8. In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 8. In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 8.

SEQ ID NO: 8 is the nucleic acid sequence that encodes the polypeptide sequence of SEQ ID NO: 6:

CAAATCCACACAGCAGAAAAGTGTCTTAAGAGTTGGAACCTTAAGTTCAG ACGATCTACTAATCCTGGCCGCTGATTTAGAAAAACTGAAATCAAAAGTC ACC AGAAC AGAAAGGCCTTT GAGTTC AGGAGTTT AT ATGGGGAATTT GAG TTCACAACAGCTTGATCAAAGGAGAGCCCTTTTGAACATGATTGGCATGA CTGGAGT AAGT GGAGGGGGAAAGGGT GCC AGTGATGGC ATT GT GAGAGT TT GGGAT GT C A A A A AT GC AGAGTT A (SEQ ID NO: 8).

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 9. In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 9. In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 9.

SEQ ID NO: 9 is the nucleic acid sequence that encodes the polypeptide sequence of SEQ ID NO: 7. CATCTCTGGTTACAATTTCAGTTTGGGTGCTGCTGTCAAAGCAGGGGCCTG CAT GC TT GAT GGT GGT A AC AT GTT AGAGACT ATT A AGGTTT C AC CTC AGAC CAT GG AT GGT ATC TT G A AGT C A AT C T T G A A AGTT A AG A AG AGT C T GGG A A TGTTTGTATCAGACACACCGGGTGAAAGGAACCCTTATGAGAACATCCTA TACAAGATCTGTCTCTCAGGAGACGGATGGCCCTATATTGCATCAAGGAC CTC GATT GT GGGA AGAGC AT GG (SEQ ID NO: 9).

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 8 and a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92,

94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 9. In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 8 and a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 9. In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 95% (such as at least

95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 8 and a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 9.

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 73% (such as at least 73, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4; wherein said nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 80% (such as at least 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 8 and 9. In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 80% (such as at least 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4; wherein said nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 8 and 9.

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4; wherein said nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 8 and 9.

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 73% (such as at least 73, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4; wherein said nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises a nucleic acid sequence having at least 80% (such as at least 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 8 and a nucleic acid sequence having at least 80% (such as at least 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 9.

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 80% (such as at least 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4; wherein said nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises a nucleic acid sequence having at least 90% (such as at least 90, 92,

94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 8 and a nucleic acid sequence having at least 90% (such as at least 90, 92, 94,

95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 9.

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4; wherein said nucleic acid sequence encoding a Lassa virus NP or antigenic fragment thereof comprises a nucleic acid sequence having least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 8 and a nucleic acid sequence having least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 9.

The present inventors have found that the Lassa virus NPs encoded by the nucleic acid sequences of SEQ ID NOs: 1 and 4 can be used to generate effective immune responses in individuals against Lassa virus. In particular, the inventors have found that a highly effective immune response against Lassa virus is obtained when Lassa virus NP is delivered to the subject using a bacterial vector or a viral vector, such as a non-replicating poxvirus vector or an adenovirus vector.

Vectors are tools which can be used as vectors for the delivery of genetic material into a target cell. By way of example, viral vectors serve as antigen delivery vehicles and also have the power to activate the innate immune system through binding cell surface molecules that recognise viral elements. A recombinant viral vector can be produced that carries nucleic acid encoding a given antigen. The viral vector can then be used to deliver the nucleic acid to a target cell, where the encoded antigen is produced and then presented to the immune system by the target cell’s own molecular machinery. As“non-self’, the produced antigen generates an adaptive immune response in the target subject. Advantageously, vectors of the invention have been demonstrated herein to provide a protective immune response. Viral vectors suitable for use in the present invention include poxvirus vectors (such as non-replicating poxvirus vectors), adenovirus vectors, and influenza virus vectors.

In certain embodiments, a“viral vector” may be a virus-like particle (VLP). VLPs are lipid enveloped particles which contain viral proteins. Certain viral proteins have an inherent ability to self-assemble, and in this process bud out from cellular membranes as independent membrane-enveloped particles. VLPs are simple to purify and can, for example, be used to present viral antigens. VLPs are therefore suitable for use in immunogenic compositions, such as described below. In certain embodiments, the viral vector is not a virus-like particle.

Bacterial vectors can also be used as antigen delivery vehicles. A recombinant bacterial vector can be produced that carries nucleic acid encoding a given antigen. The recombinant bacterial vector may express the antigen on its surface. Following administration to a subject, the bacterial vector colonises antigen-presenting cells (e.g. dendritic cells or macrophages). An antigen-specific immune response is induced. The immune response may be a cellular (T cell) immune response, or may comprise both humoral (e.g. B cell) and cellular (T cell) immune responses. Examples of bacteria suitable for use as recombinant bacterial vectors include Escherichia coli , Shigella , Salmonella (e.g. S. typhimurium), and Listeria bacteria. In one embodiment, the vector of the invention is a bacterial vector, wherein the bacterium is a Gram-negative bacterium. In one embodiment, the vector of the invention is a bacterial vector selected from an Escherichia coli vector, a Shigella vector, a Salmonella vector and a Listeria vector.

Without wishing to be bound by any one particular theory, the inventors believe that antigen delivery using the vectors of the invention stimulates, amongst other responses, a T cell response in the subject. Thus, the inventors believe that one way in which the present invention provides for protection against Lassa virus infection is by stimulating T cell responses and the cell-mediated immunity system. In addition, humoral (antibody) based protection can also be achieved.

A viral vector of the invention may be a non-replicating viral vector. As used herein, a non-replicating viral vector is a viral vector which lacks the ability to productively replicate following infection of a target cell. Thus, the ability of a non replicating viral vector to produce copies of itself following infection of a target cell (such as a human target cell in an individual undergoing vaccination with a non replicating viral vector) is highly reduced or absent. Such a viral vector may also be referred to as attenuated or replication-deficient. The cause can be loss/deletion of genes essential for replication in the target cell. Thus, a non-replicating viral vector cannot effectively produce copies of itself following infection of a target cell. Non replicating viral vectors may therefore advantageously have an improved safety profile as compared to replication-competent viral vectors. A non-replicating viral vector may retain the ability to replicate in cells that are not target cells, allowing viral vector production. By way of example, a non-replicating viral vector (e.g. a non replicating poxvirus vector) may lack the ability to productively replicate in a target cell such as a mammalian cell (e.g. a human cell), but retain the ability to replicate (and hence allow vector production) in an avian cell (e.g. a chick embryo fibroblast, or CEF, cell).

A viral vector of the invention may be a non-replicating poxvirus vector. Thus, in one embodiment, the viral vector encoding a Lassa virus NP or antigenic fragment thereof is a non-replicating poxvirus vector.

In one embodiment, the non-replicating poxvirus vector is selected from: a Modified Vaccinia virus Ankara (MV A) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector. MVA and NYVAC are both attenuated derivatives of vaccinia virus. Compared to vaccinia virus, MVA lacks approximately 26 of the approximately 200 open reading frames.

In one embodiment, the non-replicating poxvirus vector is a FPV vector.

In a preferred embodiment, the non-replicating poxvirus vector is an MVA vector. A viral vector of the invention may be an adenovirus vector. Thus, in one embodiment, the viral vector encoding a Lassa virus NP or antigenic fragment thereof is an adenovirus vector.

In one embodiment, the adenovirus vector is a non-replicating adenovirus vector (wherein non-replicating is defined as above). Adenoviruses can be rendered non replicating by deletion of the El or both the El and E3 gene regions. Alternatively, an adenovirus may be rendered non-replicating by alteration of the El or of the El and E3 gene regions such that said gene regions are rendered non-functional. For example, a non-replicating adenovirus may lack a functional El region or may lack functional El and E3 gene regions. In this way the adenoviruses are rendered replication incompetent in most mammalian cell lines and do not replicate in immunised mammals. Most preferably, both El and E3 gene region deletions are present in the adenovirus, thus allowing a greater size of transgene to be inserted. This is particularly important to allow larger antigens to be expressed, or when multiple antigens are to be expressed in a single vector, or when a large promoter sequence, such as the CMV promoter, is used. Deletion of the E3 as well as the El region is particularly favoured for recombinant Ad5 vectors. Optionally, the E4 region can also be engineered.

In one embodiment, the adenovirus vector is selected from: a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.

In one embodiment, the expression cassette comprising the nucleic acid sequence encoding a Lassa virus NP (or antigenic fragment thereof) is less than 9kb (such as less than 9. 8.5, 8.0, 7.5, 7.0, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8,

4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9,

2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

In one embodiment, the expression cassette comprising the nucleic acid sequence encoding a Lassa virus NP (or antigenic fragment thereof) is less than 8kb (such as less than 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4,

6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5,

4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6,

2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

In one embodiment, the expression cassette comprising the nucleic acid sequence encoding a Lassa virus NP (or antigenic fragment thereof) is less than 7kb (such as less than 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4,

5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5,

3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6,

1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

In one embodiment, the expression cassette comprising the nucleic acid sequence encoding a Lassa virus NP (or antigenic fragment thereof) is less than 6kb (such as less than 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3,

4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4,

2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

In one embodiment, the expression cassette comprising the nucleic acid sequence encoding a Lassa virus NP (or antigenic fragment thereof) is less than 5kb (such as less than 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4,

3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5,

1.4, 1.3, 1.2, 1.1, 1.0 kb).

In one embodiment, the expression cassette comprising the nucleic acid sequence encoding a Lassa virus NP (or antigenic fragment thereof) is less than 4.5kb (such as less than 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 kb).

In one embodiment, wherein the vector is a viral vector, the virus (i.e. viral vector) is not a pseudotyped virus. Thus, in one embodiment, the envelope of the viral vector does not comprise foreign glycoproteins (i.e. glycoproteins that are not native to said viral vector). In one embodiment, wherein the vector is a viral vector, the vector is not a retrovirus vector.

In one embodiment, wherein the vector is a viral vector, the vector is not a murine leukaemia virus (MLV) vector (for example, a Moloney murine leukaemia virus (MoMLV) vector).

In one embodiment, wherein the vector is a viral vector, the vector is not a Newcastle disease virus (NDV) vector.

In one embodiment, wherein the vector is an adenovirus vector, the adenovirus is not a human adenovirus serotype 5 (AdHu5).

Thus, in one embodiment, wherein the vector is a viral vector, the vector is not a retrovirus vector, a Newcastle disease virus vector, or a human adenovirus serotype 5 vector.

In one embodiment, the vector does not comprise a nucleic acid encoding a Lassa virus glycoprotein (GP).

In one embodiment, the vector does not comprise a nucleic acid encoding an epitope of a Lassa virus glycoprotein (GP).

In one embodiment, the vector does not comprise a nucleic acid encoding a Lassa virus matrix protein (Z).

In one embodiment, the vector does not comprise a nucleic acid encoding an epitope of a Lassa virus matrix protein (Z).

In one embodiment, the vector comprises a nucleic acid encoding a Lassa virus NP and a nucleic acid encoding a Lassa virus GP, but does not comprise a nucleic acid encoding a Lassa virus matrix protein (Z). In one embodiment, the vector comprises a nucleic acid encoding a Lassa virus NP and a nucleic acid encoding a Lassa virus GP, but does not comprise a nucleic acid encoding an epitope of a Lassa virus matrix protein (Z).

In one embodiment, the vector comprises a nucleic acid encoding a Lassa virus NP and a nucleic acid encoding a Lassa virus GP, wherein the nucleic acid encoding a Lassa virus GP comprises one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) stabilising mutations as compared to wild-type Lassa virus GP. In one embodiment, said vector does not comprise a nucleic acid encoding a Lassa virus matrix protein (Z). In one embodiment, said vector does not comprise a nucleic acid encoding an epitope of a Lassa virus matrix protein (Z).

In one embodiment, the vector does not comprise a nucleic acid encoding a Lassa virus matrix protein (Z) or a Lassa virus glycoprotein (GP).

In one embodiment, the vector does not comprise a nucleic acid encoding an epitope of a Lassa virus matrix protein (Z) or an epitope of a Lassa virus glycoprotein (GP).

In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding a Lassa Virus NP or antigenic fragment thereof does not comprise a nucleic acid encoding a Lassa virus glycoprotein (GP).

In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding a Lassa Virus NP or antigenic fragment thereof does not comprise a nucleic acid encoding an epitope of a Lassa virus glycoprotein (GP).

In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding a Lassa Virus NP or antigenic fragment thereof does not comprise a nucleic acid encoding a Lassa virus matrix protein (Z). In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding a Lassa Virus NP or antigenic fragment thereof does not comprise a nucleic acid encoding an epitope of a Lassa virus matrix protein (Z).

In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding a Lassa Virus NP or antigenic fragment thereof does not comprise a nucleic acid encoding a Lassa virus glycoprotein (GP) or a Lassa virus matrix protein (Z).

In one embodiment, wherein the vector is a non-replicating poxvirus vector (such as an MVA vector), the nucleic acid sequence encoding a Lassa Virus NP or antigenic fragment thereof does not comprise a nucleic acid encoding an epitope of a Lassa virus glycoprotein (GP) or an epitope of a Lassa virus matrix protein (Z).

In one embodiment, the Lassa virus nucleoprotein or antigenic fragment thereof is the only Lassa virus nucleic acid sequence in the vector.

In one embodiment, wherein the vector is an adenovirus vector, the vector is stable, expresses a Lassa virus NP product, and induces a protective immune response in a subject.

The nucleic acid sequences as described above may comprise a nucleic acid sequence encoding a Lassa virus NP wherein said NP comprises a fusion protein. The fusion protein may comprise a Lassa virus NP polypeptide fused to one or more further polypeptides, for example an epitope tag, another antigen, or a protein that increases immunogenicity (e.g. a flagellin). In embodiments wherein the vector is a non replicating poxvirus vector (such as an MVA vector), and the NP comprises a fusion protein, said fusion protein typically does not comprise Lassa virus glycoprotein (GP) and/or Lassa virus matrix protein (Z).

In one embodiment, the nucleic acid sequence encoding a Lassa virus NP (as described above) further encodes a Tissue Plasminogen Activator (tPA) signal sequence, and/or a V5 fusion protein sequence. In certain embodiments, the presence of a tPA signal sequence can provide for increased immunogenicity; the presence of a V5 fusion protein sequence can provide for identification of expressed protein by immunolabelling.

In one embodiment, the vector (as described above) further comprises a nucleic acid sequence encoding an adjuvant (for example, a cholera toxin, an E. coli lethal toxin, or a flagellin).

A bacterial vector of the invention may be generated by the use of any technique for manipulating and generating recombinant bacteria known in the art.

In another aspect, the invention provides a nucleic acid sequence encoding a viral vector, as described above. Thus, the nucleic acid sequence may encode a non replicating poxvirus vector as described above. Alternatively, the nucleic acid sequence may encode an adenovirus vector as described above.

The nucleic acid sequence encoding a viral vector (as described above) may be generated by the use of any technique for manipulating and generating recombinant nucleic acid known in the art.

In one aspect, the invention provides a method of making a viral vector (as described above), comprising providing a nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding a vector (as described above); transfecting a host cell with the nucleic acid; culturing the host cell under conditions suitable for the propagation of the vector; and obtaining the vector from the host cell.

As used herein,“transfecting” may mean any non-viral method of introducing nucleic acid into a cell. The nucleic acid may be any nucleic acid suitable for transfecting a host cell. Thus, in one embodiment, the nucleic acid is a plasmid. The host cell may be any cell in which a vector (e.g. a non-replicating poxvirus vector or an adenovirus vector, as described above) may be grown. As used herein,“culturing the host cell under conditions suitable for the propagation of the vector” means using any cell culture conditions and techniques known in the art which are suitable for the chosen host cell, and which enable the vector to be produced in the host cell. As used herein, “obtaining the vector”, means using any technique known in the art that is suitable for separating the vector from the host cell. Thus, the host cells may be lysed to release the vector. The vector may subsequently be isolated and purified using any suitable method or methods known in the art.

In one aspect, the invention provides a host cell comprising a nucleic acid sequence encoding a viral vector, as described above. The host cell may be any cell in which a viral vector (e.g. a non-replicating poxvirus vector or an adenovirus vector, as described above) may be grown or propagated. In one embodiment, the host cell is selected from: a 293 cell (also known as a HEK, or human embryonic kidney, cell), a CHO cell (Chinese Hamster Ovary), a CCL81.1 cell, a Vero cell, a HELA cell, a Per.C6 cell, a BHK cell (Baby Hamster Kidney), a primary CEF cell (Chick Embryo Fibroblast), a duck embryo fibroblast cell, a DF-l cell, or a rat IEC-6 cell.

The present invention also provides compositions comprising vectors as described above.

In one aspect, the invention provides a composition comprising a vector (as described above) and a pharmaceutically-acceptable carrier.

Substances suitable for use as pharmaceutically-acceptable carriers are known in the art. Non-limiting examples of pharmaceutically-acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long term storage. Examples of buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 7.4).

In addition to a pharmaceutically-acceptable carrier, the composition of the invention can be further combined with one or more of a salt, excipient, diluent, adjuvant, immunoregulatory agent and/or antimicrobial compound. The composition may be formulated as a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

In one embodiment, the composition (as described above) further comprises at least one Lassa virus NP antigen (i.e. an antigen present in the composition in the form of a polypeptide). Thus, the composition may comprise both vector and polypeptide. In one embodiment, the polypeptide antigen is a Lassa virus NP. In one embodiment, the presence of a polypeptide antigen means that, following administration of the composition to a subject, an improved simultaneous T cell and antibody response can be achieved. In one embodiment, the T cell and antibody response achieved surpasses that achieved when either a vector or a polypeptide antigen is used alone.

In one embodiment, the polypeptide antigen is not bonded to the vector. In one embodiment, the polypeptide antigen is a separate component to the vector. In one embodiment, the polypeptide antigen is provided separately from the vector.

In one embodiment, the polypeptide antigen is a variant of the antigen encoded by the vector. In one embodiment, the polypeptide antigen is a fragment of the antigen encoded by the vector. In one embodiment, the polypeptide antigen comprises at least part of a polypeptide sequence encoded by a nucleic acid sequence of the vector. Thus, the polypeptide antigen may correspond to at least part of the antigen encoded by the vector.

In one embodiment, the polypeptide antigen is a Lassa virus NP comprising (or consisting of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 2 and 5. In one embodiment, the polypeptide antigen is a Lassa virus NP comprising (or consisting of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 6 and 7.

In one embodiment, the polypeptide antigen is a Lassa virus NP comprising an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the amino acid of SEQ ID NOs: 6 and an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the amino acid of SEQ ID NO: 7.

The polypeptide antigen may be the same as (or similar to) that encoded by a nucleic acid sequence of the vector of the composition. Thus, administration of the composition comprising a vector and a polypeptide antigen may be used to achieve an enhanced immune response against a single antigen, wherein said enhanced immune response comprises a combined T cell and an antibody response, as described above.

In one embodiment, a composition of the invention (as described above) further comprises at least one naked DNA (i.e. a DNA molecule that is separate from, and not part of, the viral vector of the invention) encoding a Lassa virus NP or antigenic fragment thereof. In one embodiment, the naked DNA comprises (or consists of) a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4. In one embodiment, the naked DNA encodes a Lassa virus NP comprising (or consisting of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 2 and 5.

In one embodiment, a composition of the invention (as described above) further comprises an adjuvant. Non-limiting examples of adjuvants suitable for use with compositions of the present invention include aluminium phosphate, aluminium hydroxide, and related compounds; monophosphoryl lipid A, and related compounds; outer membrane vesicles from bacteria; oil-in-water emulsions such as MF59; liposomal adjuvants, such as virosomes, Freund’s adjuvant and related mixtures; poly- lactid-co-glycolid acid (PLGA) particles; cholera toxin; E. coli lethal toxin; and flagellin.

The vectors and compositions of the invention (as described above) can be employed as vaccines. Thus, a composition of the invention may be a vaccine composition.

As used herein, a vaccine is a formulation that, when administered to an animal subject such as a mammal (e.g. a human, bovine, porcine, ovine, caprine, equine, cervine, canine or feline subject; in particular a human subject), stimulates a protective immune response against an infectious disease. The immune response may be a humoral and/or a cell-mediated immune response. Thus, the vaccine may stimulate B cells and/or T cells.

The term “vaccine” is herein used interchangeably with the terms “therapeutic/prophylactic composition”,“immunogenic composition”,“formulation”, “antigenic composition”, or“medicament”.

In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in medicine.

In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in a method of inducing an immune response in a subject. The immune response may be against a Lassa virus antigen (e.g. a Lassa virus NP) and/or a Lassa virus infection. Thus, the vectors and compositions of the invention can be used to induce an immune response in a subject against a Lassa virus NP (for example, as immunogenic compositions or as vaccines).

In one embodiment, the immune response comprises a T cell response.

In one embodiment, the method of inducing an immune response in a subject comprises administering to a subject an effective amount of a vector (as described above) or a composition (as described above). In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in a method of preventing or treating a Lassa virus infection in a subject.

As used herein, the term“preventing” includes preventing the initiation of Lassa virus infection and/or reducing the severity of intensity of a Lassa virus infection. Thus, “preventing” encompasses vaccination.

As used herein, the term “treating” embraces therapeutic and preventative/prophylactic measures (including post-exposure prophylaxis) and includes post-infection therapy and amelioration of a Lassa virus infection.

Each of the above-described methods can comprise the step of administering to a subject an effective amount, such as a therapeutically effective amount, of a vector or a compound of the invention.

In this regard, as used herein, an effective amount is a dosage or amount that is sufficient to achieve a desired biological outcome. As used herein, a therapeutically effective amount is an amount which is effective, upon single or multiple dose administration to a subject (such as a mammalian subject, in particular a human subject) for treating, preventing, suppressing curing, delaying, reducing the severity of, ameliorating at least one symptom of a disorder or recurring disorder, or prolonging the survival of the subject beyond that expected in the absence of such treatment.

Accordingly, the quantity of active ingredient to be administered depends on the subject to be treated, capacity of the subject’s immune system to generate a protective immune response, and the degree of protection required. Precise amounts of active ingredient required to be administered may depend on the judgement of the practitioner and may be particular to each subject.

Administration to the subject can comprise administering to the subject a vector (as described above) or a composition (as described above) wherein the composition is sequentially administered multiple times (for example, wherein the composition is administered two, three or four times). Thus, in one embodiment, the subject is administered a vector (as described above) or a composition (as described above) and is then administered the same vector or composition (or a substantially similar vector or composition) again at a different time.

In one embodiment, administration to a subject comprises administering a vector (as described above) or a composition (as described above) to a subject, wherein said composition is administered substantially prior to, simultaneously with, or subsequent to, another immunogenic composition.

Prior, simultaneous and sequential administration regimes are discussed in more detail below.

In certain embodiments, the above-described methods further comprise the administration to the subject of a second vector, wherein the second vector comprises a nucleic acid sequence encoding a Lassa virus NP. Preferably, the second vector is a vector of the invention as described above (such as a viral vector, for example a non replicating poxvirus vector or an adenovirus vector as described above).

In one embodiment, the first and second vectors encode the same Lassa virus NP(s). In one embodiment, the first and second vectors encode different Lassa virus antigens.

In one embodiment, the first and second vectors are of the same vector type. In one embodiment, the first and second vectors are of different vector types. In one embodiment, the first vector is an adenovirus vector (as described above) and the second vector is a non-replicating poxvirus vector (as described above). In one embodiment, the first vector is a non-replicating poxvirus vector (as described above) and the second vector is an adenovirus vector (as described above).

In one embodiment, the first and second vectors are administered sequentially, in any order. Thus, the first (“1”) and second (“2”) vectors may be administered to a subject in the order 1-2, or in the order 2-1. As used herein, “administered sequentially” has the meaning of “sequential administration”, as defined below. Thus, the first and second vectors are administered at (substantially) different times, one after the other.

In one embodiment, the first and second vectors are administered as part of a prime- boost administration protocol. Thus, the first vector may be administered to a subject as the“prime” and the second vector subsequently administered to the same subject as the“boost”. Prime-boost protocols are discussed below.

In one embodiment, each of the above-described methods further comprises the step of administration to the subject of a Lassa virus polypeptide antigen. In one embodiment, the Lassa virus polypeptide antigen is a Lassa virus NP (or antigenic fragment thereof) as described above. In one embodiment, the Lassa virus polypeptide antigen is a Lassa virus NP comprising an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 2 and 5.

In one embodiment, the polypeptide antigen is administered separately from the administration of a vector; preferably the polypeptide antigen and a vector are administered sequentially. In one embodiment, the vector (“V”) and the polypeptide antigen (“P”) may be administered in the order V-P, or in the order P-V.

In one embodiment, each of the above-described methods further comprises the step of administration to the subject of a naked DNA encoding a Lassa virus NP or antigenic fragment thereof. In one embodiment, the naked DNA comprises (or consists of) a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4. In one embodiment, the naked DNA encodes a Lassa virus NP comprising (or consisting of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 2 and 5. In one embodiment, the naked DNA is administered separately from the administration of a vector; preferably the naked DNA and a vector are administered sequentially. In one embodiment, the vector (“V”) and the naked DNA (“D”) may be administered in the order V-D, or in the order D-V.

In one embodiment, a naked DNA (as described above) is administered to a subject as part of a prime-boost protocol.

Heterologous prime-boosting approaches can improve immune responses, by allowing repeated vaccinations without increasing anti-vector immunity. A Lassa virus NP or an antigenic fragment thereof can be serially delivered via different vectors (as described above) or naked DNA vectors (as described above). In any heterologous prime-boost vaccination regime, NP-specific antibody response is increased, NP- specific T-cell response is increased, and/or clinical illness is reduced, as compared to use of a single vector. Suitable combinations of vectors include but are not limited to:

DNA prime, MVA boost

DNA prime, Fowlpox boost

Fowlpox prime, MVA boost

MVA prime, Fowlpox boost

DNA prime, Fowlpox boost, MVA boost

MVA prime, Adenovirus boost

As used herein, the term polypeptide embraces peptides and proteins.

In certain embodiments, the above-described methods further comprise the administration to the subject of an adjuvant. Adjuvant may be administered with one, two, three, or all four of: a first vector, a second vector, a polypeptide antigen, and a naked DNA.

The immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention may be given in a single dose schedule (i.e. the full dose is given at substantially one time). Alternatively, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention may be given in a multiple dose schedule.

A multiple dose schedule is one in which a primary course of treatment (e.g. vaccination) may be with 1-6 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example (for human subjects), at 1-4 months for a second dose, and if needed, a subsequent dose(s) after a further 1-4 months.

The dosage regimen will be determined, at least in part, by the need of the individual and be dependent upon the judgment of the practitioner (e.g. doctor or veterinarian).

Simultaneous administration means administration at (substantially) the same time.

Sequential administration of two or more compositions/therapeutic agents/vaccines means that the compositions/therapeutic agents/vaccines are administered at (substantially) different times, one after the other.

For example, sequential administration may encompass administration of two or more compositions/therapeutic agents/vaccines at different times, wherein the different times are separated by a number of days (for example, at least 1, 2, 5, 10, 15, 20, 30, 60, 90, 100, 150 or 200 days).

For example, in one embodiment, the vaccine of the present invention may be administered as part of a‘prime-boost’ vaccination regime.

In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention can be administered to a subject such as a mammal (e.g. a human, bovine, porcine, ovine, caprine, equine, cervine, ursine, canine or feline subject) in conjunction with (simultaneously or sequentially) one or more immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (e.g. IL-2, IL-12), and/or cytokines (e.g. IFNy) The immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) may contain 5% to 95% of active ingredient, such as at least 10% or 25% of active ingredient, or at least 40% of active ingredient or at least 50, 55, 60, 70 or 75% active ingredient.

The immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.

Administration of immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) is generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes. The administration may be by parenteral administration; for example, a subcutaneous or intramuscular injection.

Accordingly, immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may alternatively be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules.

The active ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and/or pH buffering agents. Generally, the carrier is a pharmaceutically-acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long term storage.

Examples of buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 6.5 and 7.5).

Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.

Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

It may be desired to direct the compositions of the present invention (as described above) to the respiratory system of a subject. Efficient transmission of a therapeutic/prophylactic composition or medicament to the site of infection in the lungs may be achieved by oral or intra-nasal administration.

Formulations for intranasal administration may be in the form of nasal droplets or a nasal spray. An intranasal formulation may comprise droplets having approximate diameters in the range of 100-5000 pm, such as 500-4000 pm, 1000-3000 pm or 100- 1000 pm. Alternatively, in terms of volume, the droplets may be in the range of about 0.001-100 pi, such as 0.1-50 pl or 1.0-25 pi, or such as 0.001-1 pi. Alternatively, the therapeutic/prophylactic formulation or medicament may be an aerosol formulation. The aerosol formulation may take the form of a powder, suspension or solution. The size of aerosol particles is relevant to the delivery capability of an aerosol. Smaller particles may travel further down the respiratory airway towards the alveoli than would larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution may be selected to target a particular section of the respiratory airway, for example the alveoli. In the case of aerosol delivery of the medicament, the particles may have diameters in the approximate range of 0.1-50 pm, preferably 1-25 pm, more preferably 1-5 pm.

Aerosol particles may be for delivery using a nebulizer (e.g. via the mouth) or nasal spray. An aerosol formulation may optionally contain a propellant and/or surfactant.

In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention comprise a pharmaceutically acceptable carrier, and optionally one or more of a salt, excipient, diluent and/ or adjuvant.

In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention may comprise one or more immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (e.g. IL-2, IL- 12), and/or cytokines (e.g. IFNY).

The present invention encompasses polypeptides that are substantially homologous to polypeptides based on any one of the polypeptide antigens identified in this application (including fragments thereof). The terms “sequence identity” and “sequence homology” are considered synonymous in this specification.

By way of example, a polypeptide of interest may comprise an amino acid sequence having at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity with the amino acid sequence of a reference polypeptide. There are many established algorithms available to align two amino acid sequences. Typically, one sequence acts as a reference sequence, to which test sequences may be compared. The sequence comparison algorithm calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of amino acid sequences for comparison may be conducted, for example, by computer implemented algorithms (e.g. GAP, BESTFIT, FASTA or TFASTA), or BLAST and BLAST 2.0 algorithms.

The BLOSUM62 table shown below is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992; incorporated herein by reference). Amino acids are indicated by the standard one- letter codes. The percent identity is calculated as:

Total number of identical matches

_ x 100

[length of the longer sequence plus the number of gaps

Introduced into the longer sequence in order to align the two sequences]

BLOSUM62 table

A R N D C Q E G H I L K M F P S T W Y V

A 4

R-l 5

N-206

D-2-2 1 6

C 0 -3 -3 -3 9

Q-l 1 00-3 5

E -1 002 -425

G 0 -20 -1 -3 -2 -26

H -20 1 -1 -3 00-28

1-1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4

K -l 2 0 -1 -3 1 1 -2 -1 -3 -2 5

M -l -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5

F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6

P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4

T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5

W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11

Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7

V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

In a homology comparison, the identity may exist over a region of the sequences that is at least 10 amino acid residues in length (e.g. at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,

450, 475, 500, 525, 550 or 570 amino acid residues in length - e.g. up to the entire length of the reference sequence.

Substantially homologous polypeptides have one or more amino acid substitutions, deletions, or additions. In many embodiments, those changes are of a minor nature, for example, involving only conservative amino acid substitutions. Conservative substitutions are those made by replacing one amino acid with another amino acid within the following groups: Basic: arginine, lysine, histidine; Acidic: glutamic acid, aspartic acid; Polar: glutamine, asparagine; Hydrophobic: leucine, isoleucine, valine; Aromatic: phenylalanine, tryptophan, tyrosine; Small: glycine, alanine, serine, threonine, methionine. Substantially homologous polypeptides also encompass those comprising other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of 1 to about 30 amino acids (such as 1-10, or 1-5 amino acids); and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

As used herein, the terms“nucleic acid sequence” and“polynucleotide” are used interchangeably and do not imply any length restriction. As used herein, the terms “nucleic acid” and“nucleotide” are used interchangeably. The terms“nucleic acid sequence” and “polynucleotide” embrace DNA (including cDNA) and RNA sequences.

The polynucleotide sequences of the present invention include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.

The polynucleotides of the present invention may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.

The polynucleotides of the present invention may also be produced by chemical synthesis, e.g. by the phosphoramidite method or the tri-ester method, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

When applied to a nucleic acid sequence, the term“isolated” in the context of the present invention denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment. In view of the degeneracy of the genetic code, considerable sequence variation is possible among the polynucleotides of the present invention. Degenerate codons encompassing all possible codons for a given amino acid are set forth below:

Amino Acid Codons Degenerate Codon

Cys TGC TGT TGY

Ser AGC AGT TCA TCC TCG TCT WSN

Thr ACA ACC ACG ACT ACN

Pro CCA CCC CCG CCT CCN

Ala GCA GCC GCG GCT GCN

Gly GGA GGC GGG GGT GGN

Asn AAC AAT AAY

Asp GAC GAT GAY

Glu GAA GAG GAR

Gln CAA CAG CAR

His CAC CAT CAY

Arg AGA AGG CGA CGC CGG CGT MGN

Lys AAA AAG AAR

Met ATG ATG

He ATA ATC ATT ATH

Leu CTA CTC CTG CTT TTA TTG YTN

Val GTA GTC GTG GTT GTN

Phe TTC TTT TTY

Tyr TAC TAT TAY

Trp TGG TGG

Ter TAA TAG TGA TRR

Asn/ Asp RAY

Glu/ Gln SAR

Any NNN

One of ordinary skill in the art will appreciate that flexibility exists when determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention.

A“variant” nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or fragment thereof is“substantially homologous” (or“substantially identical”) to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 99% of the nucleotide bases. Methods for homology determination of nucleic acid sequences are known in the art.

Alternatively, a“variant” nucleic acid sequence is substantially homologous with (or substantially identical to) a reference sequence (or a fragment thereof) if the“variant” and the reference sequence they are capable of hybridizing under stringent (e.g. highly stringent) hybridization conditions. Nucleic acid sequence hybridization will be affected by such conditions as salt concentration (e.g. NaCl), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30°C, typically in excess of 37°C and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.

Methods of determining nucleic acid percentage sequence identity are known in the art. By way of example, when assessing nucleic acid sequence identity, a sequence having a defined number of contiguous nucleotides may be aligned with a nucleic acid sequence (having the same number of contiguous nucleotides) from the corresponding portion of a nucleic acid sequence of the present invention. Tools known in the art for determining nucleic acid percentage sequence identity include Nucleotide BLAST. One of ordinary skill in the art appreciates that different species exhibit“preferential codon usage”. As used herein, the term“preferential codon usage” refers to codons that are most frequently used in cells of a certain species, thus favouring one or a few representatives of the possible codons encoding each amino acid. For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells ACC is the most commonly used codon; in other species, different Thr codons may be preferential. Preferential codons for a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species.

Thus, in one embodiment of the invention, the nucleic acid sequence is codon optimized for expression in a host cell.

A“fragment” of a polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of said full-length polynucleotide. By way of example, a“fragment” of a polynucleotide of interest may comprise (or consist of) at least 30 consecutive nucleotides from the sequence of said polynucleotide (e.g. at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, or 1710 consecutive nucleic acid residues of said polynucleotide). A fragment may include at least one antigenic determinant and/or may encode at least one antigenic epitope of the corresponding polypeptide of interest and/or may have a common antigenic cross-reactivity and/or substantially the same in vivo biological activity as the polypeptide of interest.

Figure legends

Figure 1A-B. Example MVA vector construction. Figure 1A provides a schematic representation of cassette “MVALassaNP”. Figure IB provides a schematic representation of plasmid l6ADHWFP_2037865QAD_MVALassaNP (pMVALassaNP). Figure 2. Agarose gel confirming the presence of the MVALassaNP construct. Flank to flank primers (SEQ ID NOs: 20 and 22) cover the entire insert and run from the MVA flanking regions at either end of the vaccine insert yielding an expected amplification product size of 32l8bp. Contents of wells are as follows (numbered left to right): 1. Ladder; 2.“Passage 1” Pl; 3. P2; 4. P3; 5. P4; 6. P4; 7. P4; 8. P4; 9. P4; 10. P4; 11. P4; 12. P4; 13. MVA WT; 14. LNP positive control; 15-28: correspond to wells 1-14 but with GFP to flank primers (SEQ ID NOs: 22 and 21), yielding an expected amplification product size of 2330bp).

Figure 3. Western blot confirming expression of the V5 tag located downstream from the NP. The expected size of the protein (the NP + linker and V5 tag) is 65kDa. Contents of wells are as follows (numbered left to right): 1. Ladder; 2.“Passage 1” Pl; 3. P2; 4. P3; 5. P4; 6. P4; 7. P4; 8. P4; 9. P4; 10. P4; 11. P4.

Figure 4. Clinical scores (% daily weight gain) during the immunisation study.

Figure 5. Total ELISPOT response from vaccinated and unvaccinated mice: Statistically significant differences between groups depicted by ***.

Figure 6. Splenocyte IFN-g ELISPOT re-stimulation responses to individual peptide pools i) black bars indicate mice vaccinated with a single dose of MVALassaNP; ii) white bars indicate mice vaccinated with a prime and boost regime iii) checked bars indicate MVA wild-type prime and boost vaccinated mice; and iv) diagonal striped bars indicate PBS control mice.

Figure 7: IgG response to Lassa virus NP in mouse sera. Absorbance readings provide a readout of antibody binding activity to recombinant Lassa virus NP.

Figure 8. Percentage weight compared to day of challenge in guinea pigs challenged with Lassa virus and previously immunised with MVALassaNP vaccine or control.

Figure 9. Change in temperature compared to day of challenge in guinea pigs challenged with Lassa virus and previously immunised with MVALassaNP vaccine or control. Figure 10. Clinical score in guinea pigs challenged with Lassa virus and previously immunised with MVALassaNP vaccine or control.

Examples

Example 1. Preparation of an example M A-NP (nucleoprotein) vector

The inventors conducted high fidelity sequencing of the GA391 Nigeria genome, and the reference nucleic acid sequence identified for Lassa virus nucleoprotein is represented by (SEQ ID NO: 1).

A cassette for MVA-Lassa NP (denoted“MVALassaNP”) was generated to contain a Pl l promotor, Green Fluorescence Protein (GFP) and MH5 promotor followed by a kozak sequence upstream of the NP sequence of SEQ ID NO: 1. Downstream of the NP sequence was adjacent to a 24 residue linker sequence followed by a V5 epitope and stop codon. A schematic representation of MVALassaNP is provided in Fig. 1(A).

The cassette was inserted into a Sfil/Sfil cloning site of plasmid pMK-RQ to produce plasmid l6ADFtWFP_2037865QAD_MVAlassaNP (pMVALassaNP). A schematic representation of pMVALassaNP is provided in Fig. 1(B), and the nucleic acid sequence of pMVALassaNP is provided in SEQ ID NO: 10.

GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAGT

A A A AC AT GGCGAGGA A AT A A ATC AT AT A A A A A AT GATTTC AT GATT A A AC

CAT GTT GT GA A A A AGT C A AG A AC GTT C AC ATT GGC GGAC A AT C T A A A A AC

AATACAGTGATTGCAGATTTGCCATATATGGATAATGCGGTATCCGATGT

AT GCA ATT C AC T GT AT A A A A AG A AT GT AT C A AG A AT ATCC AG ATTTGC T A

ATTTGATAAAGATAGATGACGATGACAAGACTCCTACTGGTGTATATAAT

TATTTTAAACCTAAAGATGCCATTCCTGTTATTATATCCATAGGAAAGGAT

AGAGATGTTTGTGAACTATTAATCTCATCTGATAAAGCGTGTGCGTGTATA

GAGTTAAATTCATATAAAGTAGCCATTCTTCCCATGGATGTTTCCTTTTTTA

CCAAAGGAAATGCATCATTGATTATTCTCCTGTTTGATTTCTCTATCGATG

CGGCACCTCTCTTAAGAAGTGTAACCGATAATAATGTTATTATATCTAGAC

ACCAGCGTCTACATGACGAGCTTCCGAGTTCCAATTGGTTCAAGTTTTACA

TAAGTATAAAGTCCGACTATTGTTCTATATTATATATGGTTGTTGATGGAT

CTGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAATATTAGCAAA AATATATTAGACAATACTACAATTAACGATGAGTGTAGATGCTGTTATTTT

GAACC AC AGATT AGGATTCTT GAT AGAGATGAGAT GCTC A ATGGAT CATC

GTGTGATATGAACAGACATTGTATTATGATGAATTTACCTGATGTAGGCG

AATTTGGATCTAGTATGTTGGGGAAATATGAACCTGACATGATTAAGATT

GCTCTTTCGGTGGCTGGGTACCAGGCGCGCCTTTCATTTTGTTTTTTTCTAT

GCTATAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCA

TCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCC

GGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCAT

CTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCT

GACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGC

ACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACC

ATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTT

CGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA

AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG

CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGA

ACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGAC

CACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGA

CAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA

AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACT

CTCGGCATGGACGAGCTGTACAAGTAAGAGCTCCGGCCCGCTCGAGGCCG

CTGGTACCCAACCTAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTT

GTGTTAAATTGAAAGCGAGAAATAATCATAAATAAGCCCGGTGCCACCAT

GAGTGCTTCCAAGGAAGTGAGGTCATTCTTGTGGACTCAATCCCTAAGAA

GGGAACTATCTGGCTACTGTTCCAACATAAAGTTGCAGGTAGTTAAAGAC

GCTCAAGCTCTCCTTCATGGTCTTGACTTCTCCGAGGTCAGTAATGTTCAG

AGATTGATGCGCAAGCAGAAAAGAGATGATGGCGACCTAAAACGACTGA

GAGATCTCAATCAAGCAGTCAACAATCTTGTTGAGCTCAAATCCACACAG

C AGAAAAGT GTCTT AAGAGTTGGAACCTT AAGTTC AGACGATCT ACT AAT

CCTGGCCGCTGATTT AGAAAAACTGAAAT C A AAAGT C ACC AGAAC AGAAA

GGCCTTTGAGTTCAGGAGTTTATATGGGGAATTTGAGTTCACAACAGCTTG

ATCAAAGGAGAGCCCTTTTGAACATGATTGGCATGACTGGAGTAAGTGGA

GGGGGAAAGGGT GCC AGTGAT GGC ATTGT GAGAGTTT GGGAT GTC AAAA

ATGCAGAGTTACTCAACAATCAGTTCGGAACAATGCCAAGCCTAACTTTA

GC AT GCC T GAC C A A AC AGGGGC A AGT GGACC T G A AT GAT GCC GTT C A AGC

TTTGACAGATTTAGGGCTGATTTACACAGCCAAATACCCCAACTCATCTGA

TCTCGACAGATTGTCTCAGAGTCATCCAATTCTGAATATGATTGACACTAA

GAAAAGTTCACTCAACATCTCTGGTTACAATTTCAGTTTGGGTGCTGCTGT

C AAAGC AGGGGCCTGC AT GCTT GAT GGT GGT AAC AT GTT AG AG ACT ATT A

AGGTTTCACCTCAGACCATGGATGGTATCTTGAAGTCAATCTTGAAAGTTA

AGAAGAGTCTGGGAATGTTTGTATCAGACACACCGGGTGAAAGGAACCCT

TATGAGAACATCCTATACAAGATCTGTCTCTCAGGAGACGGATGGCCCTA

TATTGCATCAAGGACCTCGATTGTGGGAAGAGCATGGGAAAATACTGTGG

TGGACCTTGAGCAAGACAACAAGCCCCAGAAAATTGGAAATGGGGGGTC

CAACAAGTCATTACAGTCTGCAGGCTTTGCTGCAGGATTAACTTACTCTCA

GTTGATGACTCTCAAAGATTTCAAGTGCTTCAACTTGATTCCCAACGCAAA

AAC C T GG AT GG AT ATT G A AGG A AG AC C AG A AG AC C C AGT T GAG AT AGC C

CTTTATCAACCGAGCTCGGGTTGCTATGTACATTTCTTTAGGGAGCCAACA

GATTTGAAGCAATTCAAACAAGATGCAAAGTATTCACATGGTATTGATGT

GACTGATTTGTTTGCTGCCCAACCTGGGTTAACCAGTGCAGTGATAGAAG

CCCTTCCTCGGAACATGGTCATCACTTGCCAAGGATCAGAGGATATCAGA A A ACTCC TT GAGTC AC A AGGGAGGAGAGAC AT A A A AC T GATTGAC AT C AC

TCTTAGTAAAGCAGATTCAAGAAAGTTTGAGAATGCTGTTTGGGATCAAT

T C A AGGAT C T AT GT C AC AT GC AC ACTGGGGT AGTTGT GGAGA A A A AGA AG

AGAGGT GGT AAAGAGGAAAT AACTCCTC ATTGTGC ACTGATGGATTGC AT

TATGTTTGATGCAGCAGTTTCAGGAGGACTTGATGCAAAAGTCCTGAGAG

CTGTGCTCCCTAGAGACATGGTGTTCAGAACTTCAACACCTAAAGTCGTCC

T GgatctagagggcccgcggttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgtaaC T C GAC

CTGC AGGGAAAGTTTT AT AGGT AGTTGAT AGAAC AAAAT AC AT AATTTTG

TAAAAATAAATCACTTTTTATACTAATATGACACGATTACCAATACTTTTG

TTACTAATATCATTAGTATACGCTACACCTTTTCCTCAGACATCTAAAAAA

ATAGGTGATGATGCAACTTTATCATGTAATCGAAATAATACAAATGACTA

CGTTGTTATGAGTGCTTGGTATAAGGAGCCCAATTCCATTATTCTTTTAGC

TGCTAAAAGCGACGTCTTGTATTTTGATAATTATACCAAGGAT AAAAT ATC

TTACGACTCTCCATACGATGATCTAGTTACAACTATCACAATTAAATCATT

GACTGCTAGAGATGCCGGTACTTATGTATGTGCATTCTTTATGACATCGCC

T AC AAAT GAC ACTGAT AAAGT AGATT AT GAAGAAT ACTCC AC AGAGTTGA

TTGTAAATACAGATAGTGAATCGACTATAGACATAATACTATCTGGATCT

ACACATTCACCGGAAACTAGTT (SEQ ID NO: 10) pMVALassaNP comprises:

Dellll Left flank:

GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAGT

A A A AC AT GGCGAGGA A AT A A ATC AT AT A A A A A AT GATTTC AT GATT A A AC

CAT GTT GT GA A A A AGT C A AG A AC GTT C AC ATT GGC GGAC A AT C T A A A A AC

AATACAGTGATTGCAGATTTGCCATATATGGATAATGCGGTATCCGATGT

AT GCA ATT C AC T GT AT A A A A AG A AT GT AT C A AG A AT ATCC AG ATTTGC T A

ATTTGATAAAGATAGATGACGATGACAAGACTCCTACTGGTGTATATAAT

TATTTTAAACCTAAAGATGCCATTCCTGTTATTATATCCATAGGAAAGGAT

AGAGATGTTTGTGAACTATTAATCTCATCTGATAAAGCGTGTGCGTGTATA

GAGTTAAATTCATATAAAGTAGCCATTCTTCCCATGGATGTTTCCTTTTTTA

CCAAAGGAAATGCATCATTGATTATTCTCCTGTTTGATTTCTCTATCGATG

CGGCACCTCTCTTAAGAAGTGTAACCGATAATAATGTTATTATATCTAGAC

ACCAGCGTCTACATGACGAGCTTCCGAGTTCCAATTGGTTCAAGTTTTACA

TAAGTATAAAGTCCGACTATTGTTCTATATTATATATGGTTGTTGATGGAT

CTGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAATATTAGCAAA

AATATATTAGACAATACTACAATTAACGATGAGTGTAGATGCTGTTATTTT

GAACC AC AGATT AGGATTCTT GAT AGAGATGAGAT GCTC A ATGGAT CATC

GTGTGATATGAACAGACATTGTATTATGATGAATTTACCTGATGTAGGCG

AATTTGGATCTAGTATGTTGGGGAAATATGAACCTGACATGATTAAGATT

GCTCTTTCGGTGGCTGGGTACCAGGCGCGCC (SEQ ID NO: 11) pH :

TTTCATTTTGTTTTTTTCTATGCTATAA (SEQ ID NO: 12)

GFP:

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT

CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG

GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCT

ACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGAC

TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC

TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG

GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG

GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA

ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTC

AAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTA

CCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC

ACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC

GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGG

CAT GGACGAGC T GT AC A AGT A A (SEQ ID NO: 13)

First spacer:

GAGCTCCGGCCCGCTCGAGGCCGCTGGTACCCAACCT (SEQ ID NO: 14)

MH5 promoter:

AAAAATT GAAAAT AAAT AC AAAGGTTCTTGAGGGTT GT GTT AAATT GAAA GCGAGAAATAATCATAAATAA (SEQ ID NO: 15)

Kozak sequence:

GCCCGGTGCCACC (SEQ ID NO: 16)

Second spacer:

gatctagagggcccgcggttcgaa (SEQ ID NO: 17)

Lassa virus NP (SEQ ID NO: 1)

V5 tag:

ggtaagcctatccctaaccctctcctcggtctcgattctacg (SEQ ID NO: 18)

STOP codon:

taa

Dellll Left flank:

CTCGACCTGCAGGGAAAGTTTTATAGGTAGTTGATAGAACAAAATACATAA TTTT GTAAAAAT AAAT CACTTTTT AT ACT AAT AT G ACACG ATT ACCAAT ACT TTT GTT ACT AAT AT CATT AGT AT ACGCT ACACCTTTT CCT CAG ACAT CT AA AAAAAT AGGT GAT GATGCAACTTT AT CAT GT AAT CGAAAT AAT ACAAAT GA CTACGTTGTTATG AGTG CTT G GTAT AAG G AG CCCAATT CCATT ATT CTTTT AG CT G CT AAAAG CG ACGT CTT GT ATTTT GAT AATT AT ACCAAG G AT AAAAT AT CTT ACG ACT CT CCAT ACG AT GAT CT AGTT ACAACT AT CACAATT AAAT C ATT GACTGCT AGAGATGCCGGTACTT ATGTAT GTGCATT CTTT AT GACAT CGCCT ACAAAT G ACACT GAT AAAGT AG ATT AT G AAG AAT ACT CCACAG AG TT GATT GTAAAT ACAG AT AGT G AAT CG ACT AT AGACAT AAT ACT AT CT GG A T CT ACACATT CACCGG AAACT AGTT (SEQ ID NO: 19)

The plasmid DNA was purified from transformed bacteria (E.coli K12 DH10B™ T1R) and concentration determined by UV spectroscopy by GeneArt (Thermofisher). BHK-21 cells were infected with MVA 1974 at a multiplicity of infection of 0.05. Infected cells were transfected with pMVALassaNP using lipofectamine (Life Technologies) as directed by the manufacturer. The resulting recombinant MVALassaNP was serially plaque-purified 4 times in Chick Embryo Fibroblast (“CEF”) cells, based on GFP expression. MVALassaNP was amplified on CEF cells, purified by sucrose cushion centrifugation and titrated by plaque assay on CEF cells prior to in vivo use. Plaques were visualised using GFP fluorescence and by immunostaining with rabbit anti-vaccinia antibody (AbD Serotec, UK) and Vectastain Universal ABC-AP kit (Vector laboratories, USA). Genomic DNA from infected cells was extracted using Wizard SV genomic DNA purification system (Promega, USA) and used as a template in PCR with KAPA2G Fast HotStart PCR Kit (KAPABiosystems, USA) for genotype analysis.

Polymerase chain reaction (PCR) confirmed presence of the MVALassaNP construct. One set of primers was designed specifically to check for the GFP to the MVA flanking region with an expected size of 2330bp - this is shown in Fig. 2.

The passaged picks align to a positive control (the original received plasmid from Geneart). A second set of primers were designed to identify the entire insert, from both MVA flanking regions. The results indicate presence of pure recombinant MVA (MVA containing the insert) from passage 3 onwards. Again the original plasmid was used as a positive control and all passaged picks from passage 3 have the same expected size product as the positive control. An MVA wild-type control was also run to show the expected product size without the MVALassa insert and this can be seen in passage 1 and 2.

Primer details are as follows:

SEQ ID NO: 20: CGGCACCTCTCTTAAGAAGT (Fwd targets Del III Left flank) SEQ ID NO: 21: GTGTAGCGTATACTAATGATATTAG (Rev targets Del III Right flank)

SEQ ID NO: 22: GGAGTACAACTACAACAGCCACAACG (Fwd targets GFP) The GFP Fwd primer binds to the GFP sequence and, when used in combination with the Rev Del III Right flank primer, covers the GFP through the nucleoprotein to the right MVA flank, and specifically identifies presence of the NP gene.

Detection of protein expression

CEF cells were infected with MVALassaNP at a multiplicity of infection of 0.05 and incubated at 37°C in Modified Eagle Medium (MEM) supplemented with 2% FBS (Sigma-Aldrich. UK). The medium was removed after 48 hours once good GFP fluorescence and CPE was observed microscopically. Cells were lysed with lx LDS Nupage® reducing sample buffer (Nupage® LDS sample buffer containing lx Nupage® sample reducing buffer) (Thermofisher, UK), transferred to Eppendorf tubes and heated at 70°C for 10 minutes. Uninfected cells were treated in the same manner as a negative control. MVALassaNP lysates were subjected to SDS-PAGE on a 4-12% Bis-Tris gel (Life technologies) and proteins transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked using 5% milk powder (Merck Millipore), then incubated in the presence of a primary antibody (Rabbit anti-V5 polyclonal (Invitrogen) at 1/1000 in PBS-0.05%Tween) for 1-2 hours rocking, before washing in PBS containing 0.05% Tween-20 (Sigma-Aldrich) 3 times. Membranes were incubated in the presence of a HRP-conjugated secondary antibody (anti-rabbit IgG peroxidase (Sigma-Aldrich) at 1/1000 in PBS-0.05%Tween) for 1 hour rocking and washed as before. Protein expression was determined by detection of bound antibody using Pierce ECL WB substrate kit (Thermofisher) according to the manufacturer’s instructions and visualised in a Chemi-Illuminescent Imager (Syngene). Molecular weights were determined using molecular ladder MagicMark XP Western Protein Standard (Invitrogen) as a reference.

Western blot analysis (see Fig. 3) confirms expression of the V5 tag located downstream from the NP. The expected size of the protein (the NP + linker and V5 tag) is 65kDa and the protein sequence is provided in SEQ ID NO: 23. Expression is observed from the passage 1 through to the passage 4. The band of interest is located at the expected size of the protein which again suggests good expression.

KREIIINKPGATMSASKEVRSFLWTQSLRRELSGYCSNIKLQVVKDAQALLHG LDF SE V SN V QRLMRKQKRDDGDLKRLRDLN Q A VNNL YELK S TQ QK S VLRV G TL S SDDLLIL A ADLEKLK SK VTRTERPL S S GVYMGNL S S QQLDQRR ALLNMIG

MTGVSGGGKGASDGIVRVWDVKNAELLNNQFGTMPSLTLACLTKQGQVDL

ND A V Q ALTDLGLI YT AK YPN S SDLDRL S Q SHPILNMIDTKK S SLNI S GYNF SLG

AAVKAGACMLDGGNMLETIK V SPQTMDGILKSILKVKKSLGMF V SDTPGER

NPYENILYKICLSGDGWPYIASRTSIVGRAWENTVVDLEQDNKPQKIGNGGSN

KSLQSAGFAAGLTYSQLMTLKDFKCFNLIPNAKTWMDIEGRPEDPVEIALYQP

SSGCYVHFFREPTDLKQFKQDAKYSHGIDVTDLFAAQPGLTSAVIEALPRNM

VITCQGSEDIRKLLESQGRRDIKLIDITLSKADSRKFENAVWDQFKDLCHMHT

GVVVEKKKRGGKEEITPHCALMDCIMFDAAVSGGLDAKVLRAVLPRDMVFR

T S TPK VVLDLEGPRFEGKPIPNPLLGLD S T (SEQ ID NO: 23).

The antigenic Lassa virus NP region of SEQ ID NO: 23 corresponds to SEQ ID NO: 2

The inventors note that short amino acid sequence KREIIIN (SEQ ID NO: 24) corresponds to the translated 3’ terminus of the MH5 promoter; and short amino acid sequence KPGAT (SEQ ID NO: 25) corresponds to the translated 3’ terminus of the MH5 promoter and Kozak sequence. SEQ ID NOs: 24 and 25 are bi-products of translation and are not considered to contribute to the advantageous technical effects provided by the invention. Amino acid sequence GKPIPNPLLGLDST (SEQ ID NO:

26) corresponds to the V5 tag; and amino acid sequence DLEGPRFE (SEQ ID NO:

27) corresponds to the translated second spacer.

Example 2. MVALassaNP Immunogenicity in Balb/c mice

24 female 5-8 week old Balb-C mice were randomly divided into 4 groups and ear tagged prior to vaccinations.

Group 1 received a single vaccine shot of MVALassaNP in endotoxin free phosphate buffered saline (PBS) at 1 x 10⁷ plaque forming units (pfu) per animal on day 14.

Group 2 received a two dose vaccination of MVALassaNP in endotoxin free PBS at 1 x 10⁷ pfu per animal on days 0 and 14.

Group 3 received a two dose vaccination of MV A 1974 (wild-type) in endotoxin free PBS at 1 x 10⁷ pfu per animal on days 0 and 14. Group 4 received a two dose vaccination of endotoxin free PBS as a negative control on days 0 and 14.

All mice were injected intramuscularly into the caudal thigh. IOOmI was administered at each vaccination (50m1 into each thigh). Animal weights were recorded daily throughout the study. Animals were euthanised and spleen tissue and blood collected on day 28 after the primary vaccination. All efforts were made to minimise animal suffering. These studies were approved by the ethical review process of PHE, Porton Down, UK and the Home Office, UK via project licence number 30/2993. Work was performed in accordance with the Animals (Scientific procedures) Act 1986 and the Home Office (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989).

Throughout the study, no clinical signs were observed with regards to the vaccinations and all mice gained weight as expected (see Fig. 4). One mouse in Group 4 developed a lump that was not at the injection site and thought to be unrelated to any injections received. As the mouse exhibited no other symptoms and showed no signs of distress (even following palpation of the lump) it was kept in the study for the duration. Group 2 (prime/boost) appeared to lose weight on day 2 compared to the other groups but then gained weight throughout the study as expected. By the end of the study all the groups observed a similar % weight gain. These clinical data demonstrate that the mice tolerated the vaccine without adverse effects.

To determine the T-cell responses in immunised animals, an interferon-gamma ELISPOT assay was used to measure frequencies of responsive T-cells after stimulation with Lassa virus specific peptides.

Spleens from test animals were collected aseptically, homogenised, and red blood cells lysed. Splenocytes were resuspended in RPMI medium (Sigma-Aldrich) supplemented with 5% FBS, 2 mM L-Glutamine, 100 U penicillin & 0.1 mg/ml streptomycin, 50 mM 2-mercaptoethanol and 25 mM HEPES solution (Sigma- Aldrich). Splenocytes were assessed for antigen recall response via IFN-g ELISPOT (Mabtech, Sweden), performed as per the manufacturer’s instructions. Cells were seeded in PVDF microtitre plates at 2 x l0e6 per well and re-stimulated with peptide pools (JPT, Berlin).

Peptides spanning the Lassa NP protein sequence were 15 residues long, with an overlap of 10 residues between peptides. 140 peptides were produced in total that were tested in seven peptide pools. They were applied to cells at a final concentration of 2.5 mg/ml per peptide, with 20 peptides per pool. Plates were developed after 18 hours at 37°C, 5% C0₂ in a humidified incubator. Spots were counted visually on an automated ELISPOT reader (Cellular Technologies Limited, USA). Background values from wells containing cells and medium but no peptides were subtracted and data presented as response to individual pools or summed across the target protein. Results were expressed as spot forming units (SFU) per 10⁶ cells. Wells that had too many spots to count were recorded as“TNTC” (too numerous to count) and given an arbitrary value of 100-200 greater than the highest countable value.

The PMA/Ionomycin wells were recorded as TNTC for all mice in all groups. All mice from groups 1 to 3 (MVALassaNP Prime/Boost, MVALassaNP Prime and MVA-WT respectively) had TNTC spots for a vaccinia peptide mix with the PBS group remaining negative when stimulated with the vaccinia peptides.

The MVA-WT group and PBS group (groups 3&4) were negative when stimulated with all LassaNP pools. In the prime/boost and prime groups, an IFN-g response was detected to 2 distinct regions of the NP (corresponding to pools 2 and 4).

Peptide pool 2 corresponds to positions 81-171 of SEQ ID NO: 2 (or SEQ ID NO: 5), and is represented by SEQ ID NO: 6:

K S T Q QK S VLR V GTL S SDDLLIL A ADLEKLK SK VTRTERPL S S G V YMGNL S S Q Q LDQRRALLNMIGMTGVSGGGKGASDGIVRVWDVKNAEL (SEQ ID NO: 6)

Peptide pool 4 corresponds to positions 241-331 of SEQ ID NO: 2 (or SEQ ID NO: 5), and is represented by SEQ ID NO: 7: ISGYNF SLGAAVKAGACMLDGGNMLETIKV SPQTMDGILKSILKVKKSLGMF V SDTPGERNPYENILYKICLSGDGWP YIASRTSIV GRAW (SEQ ID NO: 7)

The inventors found that T-cell (IFN-g) stimulation significantly increased in respect of SEQ ID NOs: 6 and 7.

A 2-way ANOVA was used to analyse the data and statistically significant differences were observed between the prime and prime boost vaccinated groups of mice in pool 2 (P<0.000l) and 4 (P<0.000l) suggesting a prime/boost vaccination regime may provide better immunity compared to a prime vaccination only. However, even a single dose of MVALassaNP had a significantly increased response (P<0.000l) compared to the control groups of wild type-MVA and PBS. There were small responses detected to pools 1,3, 5, 6 and 7 (particularly groups 1, 6 and 7) for the prime/boost and prime groups compared to the control groups however, these responses were not found to be of statistical significance, under the conditions tested. Total ELISPOT responses from vaccinated and unvaccinated mice are provided in Fig. 5; and Fig. 6 shows ELISPOT responses to individual peptide pools.

To measure the antibody responses in immunised mice, ELISA analysis was undertaken to assess binding of antibodies to Lassa virus specific protein. Recombinant Lassa NP as a crude lysate (Native Antigen Company, ETC) was diluted in 0.2M carbonate-bicarbonate buffer pH 9.4 (Thermo Scientific) and used to coat Maxisorp 96-well plates (Nunc, Denmark) at l0pg/ml in lOOpl. Plates were incubated at 4°C overnight, then washed with PBS + 0.0l%Tween-20 (Sigma-Aldrich) and blocked with lOOpl of 5% Milk powder (Merck, Millipore) in PBS+0.0l% Tween-20 at 37°C for 1 hour, before re-washing in PBS+0.0l% Tween-20. Samples were diluted 1 :50 in 5% milk powder in PBS+0.0l% Tween-20 buffer, added to the plates in triplicate (lOOpl per well) and incubated at 37°C for 1 hour. Normal mouse serum (Sigma-Aldrich) and a polyclonal Anti-Lassa virus hyper immune mouse ascetic fluid sample (BEI Resources, EISA) were used as positive and negative control samples respectively. Plates were washed with PBS + 0.0l%Tween-20 and lOOpl of a polyclonal anti-mouse HRP conjugate (Sigma-Aldrich) at a 1 :20,000 dilution in 5% milk PBS + 0.0l%Tween-20 was added to each well. Following a further 1 hour incubation at 37°C, plates were washed with PBS + 0.0l%Tween-20 and lOOpl of TMB substrate (Surmodics) added to each well then incubated at 20°C for 1 hour. The reaction was stopped by addition of IOOmI of Stop solution (Surmodics) prepared according to the manufacturer’s instructions and plates read at 450nm using a molecular devices plate reader and Softmax Pro version 5.2 software (Molecular Devices). Background absorbance values were subtracted from the sample values and results reported as Absorbance (450nm) at a 1 :50 dilution. Data was illustrated and analysed using Graph Pad Prism 7 (see Fig. 7).

The MVA-WT and the PBS control groups showed very little absorbance with values similar to those in the blank wells. The normal mouse serum observed an absorbance of 0.28. The mean of the normal mouse serum +3 Standard Deviations (0.36) was used as a positive/negative cut off. All sera observing an OD greater than 0.36 were deemed as positive and therefore to have sero-converted. Table 1 shows the mean OD of each serum sample - those highlighted are deemed to have seroconverted and those un-highlighted have not.

Table 1. showing individual results from each mouse (average OD values at a 1:50 dilution). Values highlighted have shown sero-conversion with an OD above the cut-off of 3 standard deviations greater than the average OD for normal mouse serum. Values that are not highlighted have not sero-converted.

All mice in the MVA-WT and PBS control group had ODs below the 0.36 cut off and the response of all mice in both the prime and the prime/boost vaccinated groups were greater than the cut-off. The prime only group recorded an average absorbance of -0.75 and the prime/boost an average OD of - 1.2. The difference between the response in the prime/boost group and the prime only group was significant (<0.0001 using a one way ANOVA with multiple comparisons) as was the difference between the prime only group and the control groups. Example 3. Efficacy testing

40 female Dunkin-Hartley guinea-pigs at a weight of 220-3 OOg were randomly divided into 4 groups prior to ear tagging and microchipping for identification, weight monitoring and temperature monitoring.

Group 1 received a single vaccine shot of MVALassaNP in endotoxin free PBS at 2 x 10⁷ pfu per animal on day 14.

Group 2 received a two dose vaccination of MVALassaNP in endotoxin free PBS at 2 x 10⁷ pfu per animal on days 0 and 14.

Group 3 received a two dose vaccination of MV A 1974 (wild-type) in endotoxin free PBS at 2 x 10⁷ pfu per animal on days 0 and 14.

Group 4 received a two dose vaccination of endotoxin free PBS as a negative control on days 0 and 14.

All animals were injected intramuscularly into the caudal thigh - 200m1 was administered into the caudal thigh at each vaccination. All animals were anaesthetised and challenged with 200m1 of Lassa virus 1 x 10³ TCID₅₀ delivered subcutaneously 38 days post primary vaccination.

Animal weights and temperatures were recorded daily throughout the study and clinical scores recorded twice daily post challenge. Clinical signs were assigned a numerical value based on the severity: normal = 0, ruffled fur = 2, hunching = 3, lethargy = 3, laboured breathing = 5, animal culled = 5.

All efforts were made to minimise suffering and any animal that reached a humane end point (body weight <20% or <10% with moderate clinical signs) was removed from the study early. All remaining animals were euthanised and tissues collected on day 21 post challenge. Liver and spleen were removed for histology and viral load analysis. These studies were approved by the ethical review process of PHE, Porton Down, UK and the Home Office, UK via project licence number 30/3247. Work was performed in accordance with the Animals (Scientific procedures) Act 1986 and the Home Office (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989).

A 2 way ANOVA (Tukey multiple comparison test) identified statistically significant differences in weights between vaccinated groups and control groups from day 11 onwards. Unvaccinated groups started to lose weight from 5 days post challenge. The weights remained consistent in vaccinated groups with all guinea pigs gaining weight as expected for the duration of the study (see Fig. 8).

A 2 way ANOVA (Tukey multiple comparison test) identified statistically significant differences in temperature between vaccinated groups and control groups from day 11. The body temperature of the animals in the vaccinated groups remained consistent for the duration of the study (see Fig. 9).

No clinical signs were observed throughout the study in the vaccinated groups. In the MVA wild-type and control groups all animals had clinical signs and some animals met humane endpoints (see Fig. 10).

Example 4: Preparation of an example Adenovirus vector

A non-replicating adenovirus is engineered to express a Lassa virus NP or partial fragment thereof. The genetic sequence for the Lassa virus NP is inserted into the genome of the adenovirus vector. Expression of the Lassa virus NP is indicated by reactivity between a NP-specific antibody and products from the adenovirus by Western blotting or ELISA as follows:

Cellular lysate of cells infected with the recombinant adenovirus, subjected to SDS- PAGE and Western blotting with an antibody specific for the Lassa virus NP, show a specific reactivity compared to negative controls. Alternatively, products from cells infected with the recombinant adenovirus are used to coat an ELISA plate. Lassa virus-specific antibodies bind to the coating and are detected via a chemical reaction.

Example 5: Lassa virus vaccine provides cross-strain protection

A vaccine expressing the Lassa virus NP gene or functional fragment thereof, in an adenovirus or non-replicating poxvirus vector, is delivered via a parenteral route into mice that are susceptible to disease caused by Lassa virus. They are challenged with a lethal dose of Lassa virus, from a strain other than that on which the vaccine is based. The challenged animals show no or mild clinical signs of illness, and do not require euthanasia. Control animals which received the same challenge dose of Lassa virus, but did not receive the vaccine, show severe signs of illness, reach humane clinical endpoints and require euthanasia.

Example 6. Preparation and efficacy of a recombinant Influenza virus vector

Reverse genetics are used to construct a recombinant influenza virus that carries a protective epitope of Lassa virus NP in the neuraminidase stalk. Lassa virus-specific cytotoxic T lymphocytes (CTLs) are induced in mice after intranasal or parenteral administration. These CTLs provide a reduction in viral load and clinical illness after challenge with Lassa virus.

Example 7. Preparation and efficacy of a recombinant bacterial vector

The Lassa virus NP gene, or functional fragment thereof, is expressed on the surface of genetically attenuated, gram-negative bacteria. After intranasal or parenteral administration to mice, the bacterial vector colonises antigen-presenting cells (e.g. dendritic cells or macrophages). A humoral and cellular Lassa virus-specific immune response is induced. These immune responses provide a reduction in viral load and clinical illness after challenge with Lassa virus.

Claims

1. A viral vector or bacterial vector, said vector comprising a nucleic acid sequence encoding a Lassa virus nucleoprotein or antigenic fragment thereof; wherein said vector is capable of inducing an immune response in a subject.

2. The vector of claim 1, wherein the nucleic acid sequence encoding a Lassa virus nucleoprotein or antigenic fragment thereof comprises a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1 and 4.

3. The vector of claim 1 or claim 2, wherein the vector is a viral vector.

4. The vector of claim 3, wherein the vector is a non-replicating poxvirus vector.

5. The vector of claim 4, wherein the non-replicating poxvirus vector is selected from: a Modified Vaccinia virus Ankara (MV A) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector.

6. The vector of claim 4 or claim 5, wherein the non-replicating poxvirus vector is an MVA vector.

7. The vector of claim 4 or claim 5, wherein the non-replicating poxvirus vector is a fowlpox vector.

8. The vector of claim 3, wherein the vector is an adenovirus vector.

9. The vector of claim 8, wherein the adenovirus vector is a non-replicating adenovirus vector.

10. The vector of claim 8 or claim 9, wherein the adenovirus vector is selected from: a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.

11. The vector of any preceding claim, wherein the Lassa virus nucleoprotein or antigenic fragment thereof comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2 and 5.

12. A nucleic acid sequence encoding a viral vector according to any one of claims 1-11.

13. A method of making a viral vector, comprising:

providing a nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding a vector according to any one of claims 1-11;

transfecting a host cell with the nucleic acid;

culturing the host cell under conditions suitable for the propagation of the vector; and

obtaining the vector from the host cell.

14. A host cell comprising the nucleic acid sequence of claim 12.

15. A composition comprising a vector according to any one of claims 1-11, and a pharmaceutically-acceptable carrier.

16. The composition of claim 15, further comprising an adjuvant.

17. A vector according to any one of claims 1-11 or a composition according to claim 15 or claim 16, for use in medicine.

18. A vector according to any one of claims 1-11 or a composition according to claim 15 or claim 16, for use in a method of inducing an immune response in a subject.

19. The vector for use according to claim 18, wherein the immune response comprises a T cell response.

20. A vector according to any one of claims 1-11, or a composition according to claim 15 or claim 16, for use in a method of preventing or treating a Lassa virus infection in a subject.