TECHNICAL FIELD

The present disclosure relates broadly to antigen binding protein that recognises yellow fever virus.

BACKGROUND

Yellow Fever Virus (YFV) is a mosquito-borne virus that is endemic in South America and Africa. There are approximately 80,000-200,000 YFV cases worldwide per year with a case fatality rate of 20-60%.

The yellow fever virus is an enveloped virus containing a single-stranded, positive strand RNA. The viral RNA encodes for three structural proteins (Core, prM, and E) and seven non-structural proteins. The host cellular receptors important for interacting with YFV envelope glycoprotein E to facilitate viral entry into cells remains unknown.

Three to six days after being bitten by an infected mosquito, an infected person will display flu-like symptoms. Approximately 20-60% of these infected individuals will progress to a more toxic phase of the disease that could lead to multiorgan dysfunction and even death.

As treatment for YFV infection is currently not available, recent outbreaks of YFV in non-endemic regions highlight the urgency for developing treatments for YFV infection. As such, there is a need to provide an agent that may control, prevent and/or treat yellow fever viral infection.

SUMMARY

In one aspect, there is provided an antigen binding protein that specifically binds to yellow fever virus, wherein the antigen binding protein comprising a CDRL1 sequence selected from the group comprising:

QSFGSSY (CDRL1 ; SEQ ID NO: 1),

SGHSSYA (CDRL1 ; SEQ ID NO: 4), and; a CDRL2 sequence selected from the group comprising: GAS (CDRL2; SEQ ID NO: 2),

LNSDGSH (CDRL2; SEQ ID NO: 5), and a CDRL3 sequence selected from the group comprising:

QQYGISPHD (CDRL3; SEQ ID NO: 3), QTWGTGTVV (CDRL3; SEQ ID NO: 6), and a CDRH1 sequence selected from the group comprising:

GYTFTDYY (CDRH1 ; SEQ ID NO: 7),

GYTFTSYD (CDRH1 ; SEQ ID NO: 10), and a CDRH2 sequence selected from the group comprising:

INPDSGVT (CDRH2; SEQ ID NO: 8),

ISAYNGKT (CDRH2; SEQ ID NO: 11), and a CDRH3 sequence selected from the group comprising:

ARSHTTTNYFDTVGYLNWLDS (CDRH3; SEQ ID NO: 9), ARDRTKGRFGVVISNFDY (CDRH3; SEQ ID NO: 12), or a sequence at least 75% identical thereto and/or having two or three amino acids substitutions.

In some examples, the light chain variable region (VL) has CDRs selected from the group consisting of:

QSFGSSY (CDRL1 ; SEQ ID NO: 1), GAS (CDRL2; SEQ ID NO: 2), and QQYGISPHD (CDRL3; SEQ ID NO: 3), or

SGHSSYA (CDRL1 ; SEQ ID NO: 4), LNSDGSH (CDRL2; SEQ ID NO: 5), and QTWGTGTVV (CDRL3; SEQ ID NO: 6), and a heavy chain variable region (VH) has CDRs selected from the group consisting of:

GYTFTDYY (CDRH1 ; SEQ ID NO: 7), INPDSGVT (CDRH2; SEQ ID NO: 8), and ARSHTTTNYFDTVGYLNWLDS (CDRH3; SEQ ID NO: 9), GYTFTSYD (CDRH1 ; SEQ ID NO: 10), ISAYNGKT (CDRH2; SEQ ID NO: 11), and ARDRTKGRFGVVISNFDY (CDRH3; SEQ ID NO: 12), or a sequence at least 75% identical thereto and/or having two or three amino acids substitutions.

In some examples, the antigen binding protein neutralizes yellow fever virus infection, and/or binds to yellow fever virus envelope protein, and/or inhibits yellow fever virus replication. In some examples, the antigen binding protein binds wild type yellow fever and/or yellow fever vaccine strain, optionally yellow fever vaccine strain YFV-17D.

In some examples, the antigen binding protein comprises a light chain variable region comprising the sequence

EIVLTQSPGTLSLSPGERATLSCRASQSFGSSYLAWYQQKPGQAPRLLIYGAS

RRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGISPHDFGGGTKVEIK (F3C6K4 VL lgG1 , SEQ ID NO: 13), or

QLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAWHQQQPEKGPRYLMKLNS DGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCQTWGTGTWFGGGTKLT VL (F6H1 L4 VL lgG1 , SEQ ID NO: 14), or a sequence at least 75% identical thereto and/or having six or seven amino acids substitutions.

In some examples, the antigen binding protein comprises a heavy chain variable region comprising the sequence

EVQLVQSGAEVKKPGASVTVSCKASGYTFTDYYIHWVRQAPGQGLEWMGWI NPDSGVTKYAQRFQGRVTMTRDTSISSSISTAYLALSRLRSDDTAVYYCARSHTTTNY FDTVGYLNWLDSWGQGTRVTVSS (F3C6K4 VH lgG1 , SEQ ID NO: 15), or

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDISWVRQAPGQGLEWMGWI SAYNGKTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDRTKGRFGV VISNFDYWGQGTLVTVSS (F6H1 L4 VH lgG1 , SEQ ID NO: 16), or a sequence at least 75% identical thereto and/or having six or seven amino acids substitutions.

In some examples, the antigen binding protein comprises a light chain variable region (VL) having one or more CDR region encoded by a nucleotide sequence selected from the group consisting of

CAGAGTTTTGGCAGCAGTTAC (F3C6K4 CDRL1 ; SEQ ID NO: 17),

GGTGCATCC (F3C6K4 CDRL2; SEQ ID NO: 18),

CAGCAGTATGGTATCTCACCTCACGAT (F3C6K4 CDRL3; SEQ ID NO: 19), AGTGGGCACAGCAGCTACGCC (F6H1 L4 CDRL1 ; SEQ ID NO: 20), CTTAACAGTGATGGCAGCCAC (F6H1 L4 CDRL2; SEQ ID NO: 21), and CAGACCTGGGGCACTGGCACCGTGGTA (F6H1 L4 CDRL3; SEQ ID NO: 22), or a sequence at least 75% identical thereto. In some examples, the antigen binding protein comprises a heavy chain variable region (VH) having one or more CDR region encoded by a nucleotide sequence selected from the group consisting of

GGATACACCTTCACCGACTACTAT (F3C6K4 CDRH1; SEQ ID NO: 23), ATCAACCCTGACAGTGGTGTTACA (F3C6K4 CDRH2; SEQ ID NO: 24), GCGAGAAGCCACACGACCACAAATTACTTTGATACTGTTGGTTATCTGAAC TGGCTCGACTCC (F3C6K4 CDRH3; SEQ ID NO: 25),

GGTTACACCTTTACCAGCTATGAT (F6H1 L4 CDRH1 ; SEQ ID NO: 26), ATCAGCGCTTACAATGGTAAGACA (F6H1 L4 CDRH1 ; SEQ ID NO: 27), and GCGAGAGATAGGACAAAGGGACGTTTTGGAGTGGTTATTTCTAACTTTGAC TAG (F6H1 L4 CDRH3; SEQ ID NO: 28), or a sequence at least 75% identical thereto.

In some examples, the antigen binding protein comprises a light chain variable region encoded by a nucleotide sequence comprising the sequence

GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTTTTGGCAGCAGTTACTTAGCC TGGTACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCA GGAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACT TCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAG CAGTATGGTATCTCACCTCACGATTTCGGCGGAGGGACCAAGGTGGAGATCAAA (F3C6K4 VL lgG1 , SEQ ID NO: 29), or

CAGCTTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTC GGTCAAGCTCACCTGCACTCTGAGCAGTGGGCACAGCAGCTACGCCATCGCATG GCATCAGCAGCAGCCAGAGAAGGGCCCTCGGTACTTGATGAAGCTTAACAGTGAT GGCAGCCACAGCAAGGGGGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCT GGGGCTGAGCGCTACCTCACCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGAC TATTACTGTCAGACCTGGGGCACTGGCACCGTGGTATTCGGCGGAGGGACCAAG CTGACCGTCCTA (F6H1 L4 VL lgG1 , SEQ ID NO: 30), or a sequence at least 65% identical thereto.

In some examples, the antigen binding protein comprises a heavy chain variable region comprising the sequence

GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT CAGTGACGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGACTACTATATACA CTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTTGGATCAACCC TGACAGTGGTGTTACAAAATATGCACAGAGGTTTCAGGGCAGGGTCACCATGACC AGGGACACGTCCATCAGCTCATCCATCAGCACAGCCTACCTGGCCCTGAGCAGG CTGAGGTCTGACGACACGGCCGTGTACTACTGTGCGAGAAGCCACACGACCACA AATTACTTTGATACTGTTGGTTATCTGAACTGGCTCGACTCCTGGGGCCAGGGAA

CCCGGGTCACCGTCTCCTCA (F3C6K4 VH lgG1 , SEQ ID NO: 31), or

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCT CAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGATATCAGC TGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCT TACAATGGTAAGACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCA CAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACG ACACGGCCGTATATTACTGTGCGAGAGATAGGACAAAGGGACGTTTTGGAGTGGT TATTTCTAACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (F6H1 L4 VH lgG1 , SEQ ID NO: 32), or a sequence at least 65% identical thereto.

In some examples, the antigen binding protein has at least an EC50 of about 1 ng/ml to about 4000 ng/ml.

In some examples, the antigen binding protein is a monoclonal antibody.

In some examples, the antigen binding protein is a monoclonal humanized antibody.

In some examples, the antigen binding protein is of a IgG class, optionally the antigen binding protein is of the lgG1 subtype.

In another aspect, there is provided a polynucleotide encoding the antigen binding protein as described herein.

In yet another aspect, there is provided, a vector comprising a nucleic acid sequence encoding the antigen binding protein as described herein.

In yet another aspect, there is provided a host cell comprising the vector as described herein.

In yet another aspect, there is provided a composition comprising the antigen binding protein as described herein.

In yet another aspect, there is provided a pharmaceutical composition comprising the antigen binding protein as described herein and suitable pharmaceutical composition thereof.

In yet another aspect, there is provided the antigen binding protein or composition as described herein for use in therapy/medicine/vaccine. In yet another aspect, there is provided the use of the antigen binding protein as described herein in the manufacture of a medicament for preventing and/or treating a yellow fever virus infection.

In yet another aspect, there is provided a method of preventing and/or treating a yellow fever virus infection in a subject in need thereof, the method comprises administering to the subject an antigen binding protein as described herein or composition as described herein.

In yet another aspect, there is provided a method of detecting a yellow fever virus infection in a subject in need thereof, the method comprising contacting the antigen binding protein as described herein or composition as described herein to a sample obtained from the subject.

In yet another aspect, there is provided a kit comprising the antigen binding protein as described herein.

DEFINITIONS

As used herein, the term “antibody” includes intact antibodies as well as binding fragments thereof. In some examples, fragments include separate heavy chains, light chains, Fab, Fab’, F(ab’)2, F(ab)c, Fv, and single domain antibodies (such as VHH antibodies, and the like). Single domain antibodies in which one chain is separated from its natural partners are sometimes known as Dabs. In some examples, constant regions or parts of constant regions may or may not be present in single domain antibodies.

In some examples, the term "antibody" may also include a multispecific antibody. In some examples, the term “antibody” may be a bispecific antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.

The term "epitope" refers to a site on an antigen to which an antibody bind. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. When an epitope is said to be within a range of amino acid residues in a protein, the range is inclusive of the residues defining its borders. Certain residues within the range contribute to the epitope, whereas others may not. The residues that form the epitope may or may not be contiguous with one another. Similarly, when an antibody binds to an epitope found within a particular range of amino acids, the antibody need not contact all the amino acids residues within the range, and the residues of the epitope that are contacted by the antibody may or may not be contiguous with one another. Methods of determining spatial conformation of epitopes are known in the art and may include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

The term “neutralizing” with respect to an antigen binding protein refers to the ability to prevent virus binding, or to inhibit virus entry or to prevent virus from infecting a susceptible cell or prevent formation of viral particles.

As used herein, the term “sequence identity” refers to the percentage sequence identities that are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.

The term “EC50,” as used herein, refers to the concentration of an antibody or an antigen-binding protein/portion thereof, which induces a response, either in an in vivo or an in vitro assay, such as neutralization of yellow fever virus (e.g., blocking yellow fever virus infection of a host cell) as is described herein, which is 50% of the maximal response (i.e. , halfway between the maximal response and the baseline).

A "vector" is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable host cell where e.g. synthesis of the encoded polypeptide can take place. Typically and preferably, a vector is a nucleic acid that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate a desired nucleic acid sequence (e.g., a nucleic acid of the invention). Expression vectors typically contain one or more of the following components (if they are not already provided by the nucleic acid molecules): a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. In various examples, the term “treating", "treat" and “therapy”, and synonyms thereof refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a medical condition, which includes but is not limited to diseases (such as yellow fever virus infections), symptoms and disorders. A medical condition also includes a body’s response to a disease or disorder, e.g. inflammation. Those in need of such treatment include those already with a medical condition as well as those prone to getting the medical condition or those in whom a medical condition is to be prevented.

In some examples, the term “preventing” and/or “reducing the severity of symptoms” or “treating” refer to process of delaying the onset, reducing the severity of flu-like symptoms, reducing viral load (viremia), reducing and/or preventing weight loss, reducing and/or preventing nausea, reducing and/or preventing vomiting hemorrhage, preventing death, inhibiting deterioration, inhibiting further deterioration, and/or ameliorating at least one sign or symptom of yellow fever virus infection. In some examples, the term “reducing the severity of symptoms” refers to prevention of weight loss, viremia, multiple-organ failure and/or death.

A “biological sample” may be a solid biological sample or a liquid biological sample. Examples of a “solid biological sample” include a tissue specimen or a biopsy. Examples of a “fluid biological sample” or “liquid biological sample” include blood, serum, plasma, sputum, lavage fluid (for example peritoneal lavage), cerebrospinal fluid, urine, semen, sweat, tears, saliva, and the like. As used herein, the term “blood”, “plasma” and “serum” encompass fractions or processed portions thereof. Similarly, where a sample is taken from a biopsy, swab, smear, etc., the “sample” encompasses a processed fraction or portion derived from the biopsy, swab, smear, etc.

In some examples, the “sample” may refer to non-biological sample such as swab of a surface, liquid sample from water bodies (e.g. reservoir, etc), and the like.

In various examples, the term “subject” as used herein includes patients and nonpatients. The term “patient” refers to individuals suffering or are likely to suffer from a medical condition such as a yellow fever virus infection, while “non-patients” refer to individuals not suffering and are likely to not suffer from the medical condition. “Nonpatients” include healthy individuals, non-diseased individuals and/or an individual free from the medical condition. The term “subject” includes humans and animals. Animals may include, but is not limited to, mammals (for example non-human primates, canine, murine, Laporidae, and the like), and the like. “Murine” refers to any mammal from the family Muridae, such as mouse, rat, and the like. “Laporidae” refers to any mammal from the family of “Laporidae”, such as rabbits, hare, and the like.

The terms "coupled" or "connected" as used in this description are intended to cover both directly connected or connected through one or more intermediate means, unless otherwise stated.

The term "associated with", used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other, or element A may contain element B or vice versa.

The term "adjacent" used herein when referring to two elements refers to one element being in close proximity to another element and may be but is not limited to the elements contacting each other or may further include the elements being separated by one or more further elements disposed therebetween.

The term "and/or", e.g., "X and/or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

As used herein, the term “substantially” refers to less than limit of detection, i.e. in an amount that is less than what can be detected by acceptable methods known in the art. Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements/components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of +/- 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1% of the disclosed value. Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1% to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1 % to 5% is intended to have specifically disclosed the ranges 1.00% to 5.00% and also 1.0% to 5.0% and all their intermediate values (such as 1.01%, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1%, 1.2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth/breadth of a range.

Additionally, when describing some embodiments, the disclosure may have disclosed a method and/or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and/or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features/characteristics discussed herein, one or more of these features/characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

DESCRIPTION OF EMBODIMENTS

Exemplary, non-limiting embodiments of antigen binding proteins against yellow fever virus are disclosed hereinafter. A highly potent, live-attenuated YFV vaccine (YFV-17D) is available to control and prevent future outbreaks. YFV-17D was developed by passaging the virulent Asibi strain YFV-17D more than 240 times in various tissues. The resulting YFV-17D differs from the original Asibi virus in 32 amino acids, and 12 of these amino acids are in the Envelope (E) protein. Attenuation of the wild-type virulent virus to YFV- 17D also allows the handling of the virus in a biosafety level 2 facility. Due to the effectiveness of the live-attenuated YFV vaccine in containing outbreaks, the inventors hypothesized that highly potent neutralizing antibodies are generated in vaccinated individual. As such, the present disclosure identified therapeutic neutralizing antibodies against YFV from individuals who are vaccinated with YFV- 17D.

In one aspect, there is provided an antigen binding protein that specifically binds to yellow fever virus, wherein the antigen binding protein comprises a CDRL1 sequence selected from the group comprising: QSFGSSY (CDRL1 ; SEQ ID NO: 1), SGHSSYA (CDRL1 ; SEQ ID NO: 4), and; a CDRL2 sequence selected from the group comprising: GAS (CDRL2; SEQ ID NO: 2), LNSDGSH (CDRL2; SEQ ID NO: 5), and a CDRL3 sequence selected from the group comprising: QQYGISPHD (CDRL3; SEQ ID NO: 3), QTWGTGTVV (CDRL3; SEQ ID NO: 6), and a CDRH1 sequence selected from the group comprising: GYTFTDYY (CDRH1 ; SEQ ID NO: 7), GYTFTSYD (CDRH1 ; SEQ ID NO: 10), and a CDRH2 sequence selected from the group comprising: INPDSGVT (CDRH2; SEQ ID NO: 8), ISAYNGKT (CDRH2; SEQ ID NO: 11), and a CDRH3 sequence selected from the group comprising: ARSHTTTNYFDTVGYLNWLDS (CDRH3; SEQ ID NO: 9), ARDRTKGRFGVVISNFDY (CDRH3; SEQ ID NO: 12), or a sequence at least 75% identical thereto and/or having two or three amino acids substitutions.

In some examples, the antigen binding protein as disclosed herein specifically binds to yellow fever virus. In some examples, the antigen binding protein neutralizes yellow fever virus infection, and/or binds to yellow fever virus envelope protein, and/or inhibits yellow fever virus replication.

In some examples, the antigen binding protein as described herein does not bind to soluble yellow fever virus envelope protein. In some examples, the antigen binding protein as described herein does not bind to soluble YFV-17D envelope protein.

In some examples, the antigen binding protein requires the involvement of yellow fever virus envelope protein epitope E55 and/or D270. In some examples, the antigen binding protein may bind to wild type yellow fever and/or yellow fever vaccine strain.

In some examples, the yellow fever virus strain is the YFV-17D strain. In some examples, the YFV-17D envelope protein sequence comprises the following sequence: AHCIGITDRDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDISLETVAIDRPAEVRKV CYNAVLTHVKINDKCPSTGEAHLAEENEGDNACKRTYSDRGWGNGCGLFGKGSIVA CAKFTCAKSMSLFEVDQTKIQYVI RAQLH VGAKQEN WNTDI KTLKFDALSGSQEVEFI GYGKATLECQVQTAVDFGNSYIAEMETESWIVDRQWAQDLTLPWQSGSGGVWREM HHLVEFEPPHAATIRVLALGNQEGSLKTALTGAMRVTKDTNDNNLYKLHGGHVSCRV KLSALTLKGTSYKICTDKMFFVKNPTDTGHGTWMQVKVSKGAPCRIPVIVADDLTAAI NKGILVTVNPIASTNDDEVLIEVNPPFGDSYIIVGRGDSRLTYQWHKEGSSIGKLFTQT MKGVERLAVMGDTAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNWITKVIMGAV LIWVGINTRNMTMSMSMILVGVIMMFLSLGVGA (SEQ ID NO: 34).

In some examples, the YFV-17D envelope protein may be encoded by the following nucleotide sequence: GCTCACTGCATTGGAATTACTGACAGGGATTTCATTGAGGGGGTGCATGGAGGAA CTTGGGTTTCAGCTACCCTGGAGCAAGACAAGTGTGTCACTGTTATGGCCCCTGA CAAGCCTTCATTGGACATCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAG GTGAGGAAAGTGTGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTG CCCCAGCACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTG CAAGCGCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGGAAA GGGAGCATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAGTTTGTTTG AGGTTGATCAGACCAAAATTCAGTATGTCATCAGAGCACAATTGCATGTAGGGGC CAAGCAGGAAAATTGGAATACCGACATTAAGACTCTCAAGTTTGATGCCCTGTCAG GCTCCCAGGAAGTCGAGTTCATTGGGTATGGAAAAGCTACACTGGAATGCCAGGT GCAAACTGCGGTGGACTTTGGTAACAGTTACATCGCTGAGATGGAAACAGAGAGC TGGATAGTGGACAGACAGTGGGCCCAGGACTTGACCCTGCCATGGCAGAGTGGA AGTGGCGGGGTGTGGAGAGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATG CCGCCACTATCAGAGTACTGGCCCTGGGAAACCAGGAAGGCTCCTTGAAAACAG CTCTTACTGGCGCAATGAGGGTTACAAAGGACACAAATGACAACAACCTTTACAAA CTACATGGTGGACATGTTTCTTGCAGAGTGAAATTGTCAGCTTTGACACTCAAGGG GACATCCTACAAAATATGCACTGACAAAATGTTTTTTGTCAAGAACCCAACTGACA CTGGCCATGGCACTGTTGTGATGCAGGTGAAAGTGTCAAAAGGAGCCCCCTGCA GGATTCCAGTGATAGTAGCTGATGATCTTACAGCGGCAATCAATAAAGGCATTTTG GTTACAGTTAACCCCATCGCCTCAACCAATGATGATGAAGTGCTGATTGAGGTGA ACCCACCTTTTGGAGACAGCTACATTATCGTTGGGAGAGGAGATTCACGTCTCAC TTACCAGTGGCACAAAGAGGGAAGCTCAATAGGAAAGTTGTTCACTCAGACCATG AAAGGCGTGGAACGCCTGGCCGTCATGGGAGACACCGCCTGGGATTTCAGCTCC GCTGGAGGGTTCTTCACTTCGGTTGGGAAAGGAATTCATACGGTGTTTGGCTCTG CCTTTCAGGGGCTATTTGGCGGCTTGAACTGGATAACAAAGGTCATCATGGGGGC GGTACTTATATGGGTTGGCATCAACACAAGAAACATGACAATGTCCATGAGCATGA TCTTGGTAGGAGTGATCATGATGTTTTTGTCTCTAGGAGTTGGGGCG (SEQ ID NO: 33).

In some examples, the antigen binding protein binds yellow fever vaccine strain YFV-17D.

In some examples, the antigen binding protein is a neutralizing antigen binding protein.

In some examples, the neutralizing ability of the antigen binding protein may be determined using methods known in the art. For example, the neutralizing ability of the antigen binding protein may be analyzed by focus reduction neutralization test (FRNT) or neutralization assay by flow cytometry.

In some examples, the antigen binding protein comprises a light chain variable region (VL) having one or more CDRs selected from the group consisting of QSFGSSY (F3C6K4 CDRL1; SEQ ID NO: 1), GAS (F3C6K4 CDRL2; SEQ ID NO: 2), QQYGISPHD (F3C6K4 CDRL3; SEQ ID NO: 3), SGHSSYA (F6H1 L4 CDRL1; SEQ ID NO: 4), LNSDGSH (F6H1 L4 CDRL2; SEQ ID NO: 5), and QTWGTGTVV (F6H1 L4 CDRL3; SEQ ID NO: 6), or a sequence at least 75% identical thereto and/or having two or three amino acids substitutions.

In some examples, the antigen binding protein comprises a light chain variable region comprising a CDR1 selected from SEQ ID NO: 1 or 4, and/or a CDR2 selected from SEQ ID NO: 2 or 5, and/or a CDR3 selected from SEQ ID NO: 3 or 6.

In some examples, the antigen binding protein comprises a light chain variable region comprising a CDR1 selected from SEQ ID NO: 1, and/or a CDR2 selected from SEQ ID NO: 2, and/or a CDR3 selected from SEQ ID NO: 3.

In some examples, the antigen binding protein comprises a light chain variable region comprising a CDR1 selected from SEQ ID NO: 4, and/or a CDR2 selected from SEQ ID NO: 5, and/or a CDR3 selected from SEQ ID NO: 6. In some examples, the antigen binding protein comprises a heavy chain variable region (VH) having one or more CDRs selected from the group consisting of

GYTFTDYY (F3C6K4 CDRH1 ; SEQ ID NO: 7),

INPDSGVT (F3C6K4 CDRH2; SEQ ID NO: 8),

ARSHTTTNYFDTVGYLNWLDS (F3C6K4 CDRH3; SEQ ID NO: 9), GYTFTSYD (F6H1 L4 CDRH1 ; SEQ ID NO: 10), ISAYNGKT (F6H1 L4 CDRH2; SEQ ID NO: 11), and ARDRTKGRFGVVISNFDY (F6H1 L4 CDRH3; SEQ ID NO: 12), or a sequence at least 75% identical thereto and/or having two or three amino acids substitutions.

In some examples, the antigen binding protein comprises a heavy chain variable region comprising a CDR1 selected from SEQ ID NO: 7 or 10, and/or a CDR2 selected from SEQ ID NO: 8 or 11 , and/or a CDR3 selected from SEQ ID NO: 9 or 12.

In some examples, the antigen binding protein comprises a heavy chain variable region comprising a CDR1 selected from SEQ ID NO: 7, and/or a CDR2 selected from SEQ ID NO: 8, and/or a CDR3 selected from SEQ ID NO: 9.

In some examples, the antigen binding protein comprises a heavy chain variable region comprising a CDR1 selected from SEQ ID NO: 10, and/or a CDR2 selected from SEQ I D NO: 11 , and/or a CDR3 selected from SEQ I D NO: 12.

In some examples, the antigen binding protein comprises a light chain variable region comprising the sequence

EIVLTQSPGTLSLSPGERATLSCRASQSFGSSYLAWYQQKPGQAPRLLIYGAS RRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGISPHDFGGGTKVEIK (F3C6K4 VL lgG1 , SEQ ID NO: 13), or

QLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAWHQQQPEKGPRYLMKLNS DGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCQTWGTGTWFGGGTKLT VL (F6H1 L4 VL lgG1 , SEQ ID NO: 14), or a sequence at least 75% identical thereto and/or having six or seven amino acids substitutions.

In some examples, the antigen binding protein comprises a heavy chain variable region comprising the sequence

EVQLVQSGAEVKKPGASVTVSCKASGYTFTDYYIHWVRQAPGQGLEWMGWI NPDSGVTKYAQRFQGRVTMTRDTSISSSISTAYLALSRLRSDDTAVYYCARSHTTTNY FDTVGYLNWLDSWGQGTRVTVSS (F3C6K4 VH lgG1 , SEQ ID NO: 15), or QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDISWVRQAPGQGLEWMGWI SAYNGKTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDRTKGRFGV VISNFDYWGQGTLVTVSS (F6H1 L4 VH lgG1 , SEQ ID NO: 16), or a sequence at least 75% identical thereto and/or having six or seven amino acids substitutions.

In some examples, the antigen binding protein comprises a light chain variable region (VL) having one or more CDR region encoded by a nucleotide sequence selected from the group consisting of

CAGAGTTTTGGCAGCAGTTAC (F3C6K4 CDRL1 ; SEQ ID NO: 17),

GGTGCATCC (F3C6K4 CDRL2; SEQ ID NO: 18),

CAGCAGTATGGTATCTCACCTCACGAT (F3C6K4 CDRL3; SEQ ID NO: 19), AGTGGGCACAGCAGCTACGCC (F6H1 L4 CDRL1 ; SEQ ID NO: 20), CTTAACAGTGATGGCAGCCAC (F6H1 L4 CDRL2; SEQ ID NO: 21), and CAGACCTGGGGCACTGGCACCGTGGTA (F6H1 L4 CDRL3; SEQ ID NO: 22), or a sequence at least 75% identical thereto.

In some examples, the antigen binding protein comprises a heavy chain variable region (VH) having one or more CDR region encoded by a nucleotide sequence selected from the group consisting of

GGATACACCTTCACCGACTACTAT (F3C6K4 CDRH1 ; SEQ ID NO: 23), ATCAACCCTGACAGTGGTGTTACA (F3C6K4 CDRH2; SEQ ID NO: 24), GCGAGAAGCCACACGACCACAAATTACTTTGATACTGTTGGTTATCTGAAC TGGCTCGACTCC (F3C6K4 CDRH3; SEQ ID NO: 25),

GGTTACACCTTTACCAGCTATGAT (F6H1 L4 CDRH1 ; SEQ ID NO: 26), ATCAGCGCTTACAATGGTAAGACA (F6H1 L4 CDRH1 ; SEQ ID NO: 27), and GCGAGAGATAGGACAAAGGGACGTTTTGGAGTGGTTATTTCTAACTTTGAC TAG (F6H1 L4 CDRH3; SEQ ID NO: 28), or a sequence at least 75% identical thereto.

In some examples, the antigen binding protein comprises a light chain variable region encoded by a nucleotide sequence comprising the sequence

GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTTTTGGCAGCAGTTACTTAGCC TGGTACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCA GGAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACT TCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAG CAGTATGGTATCTCACCTCACGATTTCGGCGGAGGGACCAAGGTGGAGATCAAA (F3C6K4 VL lgG1 , SEQ ID NO: 29), or

CAGCTTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTC GGTCAAGCTCACCTGCACTCTGAGCAGTGGGCACAGCAGCTACGCCATCGCATG GCATCAGCAGCAGCCAGAGAAGGGCCCTCGGTACTTGATGAAGCTTAACAGTGAT GGCAGCCACAGCAAGGGGGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCT GGGGCTGAGCGCTACCTCACCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGAC TATTACTGTCAGACCTGGGGCACTGGCACCGTGGTATTCGGCGGAGGGACCAAG CTGACCGTCCTA (F6H1 L4 VL lgG1 , SEQ ID NO: 30), or a sequence at least 65% identical thereto.

In some examples, the antigen binding protein comprises a heavy chain variable region comprising the sequence

GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT CAGTGACGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGACTACTATATACA CTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTTGGATCAACCC TGACAGTGGTGTTACAAAATATGCACAGAGGTTTCAGGGCAGGGTCACCATGACC AGGGACACGTCCATCAGCTCATCCATCAGCACAGCCTACCTGGCCCTGAGCAGG CTGAGGTCTGACGACACGGCCGTGTACTACTGTGCGAGAAGCCACACGACCACA AATTACTTTGATACTGTTGGTTATCTGAACTGGCTCGACTCCTGGGGCCAGGGAA CCCGGGTCACCGTCTCCTCA (F3C6K4 VH lgG1 , SEQ ID NO: 31), or

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCT CAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGATATCAGC TGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCT TACAATGGTAAGACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCA CAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACG ACACGGCCGTATATTACTGTGCGAGAGATAGGACAAAGGGACGTTTTGGAGTGGT TATTTCTAACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (F6H1 L4 VH lgG1 , SEQ ID NO: 32), or a sequence at least 65% identical thereto.

In some examples, the antigen binding protein comprises a sequence or an amino acid region or is encoded by a nucleotide region sharing at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% with a sequence as described herein. In some examples, the antigen binding protein comprises a sequence or an amino acid region or is encoded by a nucleotide region that differs by about one, about two, about three, about four, about five, about six, about seven, about eight, about nine, about ten or more amino acids or nucleobase with a sequence as described herein.

In some examples, the antigen binding protein has at least an EC50 of about 1 ng/ml to about 4000 ng/ml, 4000 ng/ml, 3950 ng/ml, 3900 ng/ml, 3850 ng/ml, 3800 ng/ml, 3750 ng/ml, 3700 ng/ml, 3650 ng/ml, 3600 ng/ml, 3550 ng/ml, 3500 ng/ml, 3450 ng/ml,

3400 ng/ml, 3350 ng/ml, 3300 ng/ml, 3250 ng/ml, 3200 ng/ml, 3150 ng/ml, 3100 ng/ml,

3050 ng/ml, 3000 ng/ml, 2950 ng/ml, 2900 ng/ml, 2850 ng/ml, 2800 ng/ml, 2750 ng/ml,

2700 ng/ml, 2650 ng/ml, 2600 ng/ml, 2550 ng/ml, 2500 ng/ml, 2450 ng/ml, 2400 ng/ml,

2350 ng/ml, 2300 ng/ml, 2250 ng/ml, 2200 ng/ml, 2150 ng/ml, 2100 ng/ml, 2050 ng/ml,

2000 ng/ml, 1950 ng/ml, 1900 ng/ml, 1850 ng/ml, 1800 ng/ml, 1750 ng/ml, 1700 ng/ml,

1650 ng/ml, 1600 ng/ml, 1550 ng/ml, 1500 ng/ml, 1450 ng/ml, 1400 ng/ml, 1350 ng/ml,

1300 ng/ml, 1250 ng/ml, 1200 ng/ml, 1150 ng/ml, 1100 ng/ml, 1050 ng/ml, 1000 ng/ml,

950 ng/ml, 900 ng/ml, 850 ng/ml, 800 ng/ml, 750 ng/ml, 700 ng/ml, 650 ng/ml, 600 ng/ml, 550 ng/ml, 500 ng/ml, 400 ng/ml, 350 ng/ml, 300 ng/ml, 250 ng/ml, 200 ng/ml, 150 ng/ml, 100 ng/ml, 50 ng/ml, 40 ng/ml, 30 ng/ml, 20 ng/ml, 10 ng/ml, or 9 ng/ml, or 8 ng/ml, or 7 ng/ml, or 6 ng/ml, or 5 ng/ml, or 4 ng/ml, or 3 ng/ml, or 2 ng/ml, or 1 ng/ml. In some examples, the antigen binding protein has an EC50 of about 3 ng/ml, 30 ng/ml, 50 ng/ml, 100 ng/ml, 150 ng/ml, 200 ng/ml, 250 ng/ml, 300 ng/ml, 350 ng/ml, or 400 ng/ml.

In some examples, the EC50 is measured in vitro. For example, the EC50 of about 1 ng/ml to about 4000 ng/ml is based on focus reduction neutralization tests or neutralization tests by flow cytometry and when analysed on BHK-DC-SIGN cells and/or Vero cells.

In some examples, the antigen binding protein is an antibody.

In some examples, the antigen binding protein is a monoclonal antibody.

Also considered as part of the present disclosure is humanized antibodies. Therefore, in some examples, there is provided humanized CDR grafted yellow fever virus - binding antibodies or binding fragments thereof that may comprise constant regions of human origin. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided into the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (subtypes), e.g. lgG1 , lgG2, lgG3, and lgG4, IgAI, and lgA2. Therefore, human IgG constant region domains may be used, especially of the IgG 1 and lgG3 isotypes when the antibody molecule is intended for therapeutic uses and antibody effector functions are required. Alternatively, lgG2 and lgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required.

In some examples, the antigen binding protein is a monoclonal humanized antibody.

In some examples, the antigen binding protein is of a IgG class, optionally the antigen binding protein is of the lgG1 subtype.

It will be appreciated that sequence amendments of these constant region domains may also be used. For example, one or more amino acid, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 amino acid substitutions, additions and/or deletions may also be made to the antibody constant domains without significantly altering the ability of the antibody to bind to a yellow fever virus epitope.

Antibodies as disclosed herein typically bind to their designated target with an association constant of at least 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , or 10 ¹⁰ M' ¹ . Such binding is specific binding in that it is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an antibody binds one and only one target.

The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region, means a light chain variable region without the light chain signal peptide. The carboxyterminal portion of each chain defines a constant region primarily responsible for effector function. A constant region can include any or all of a CH 1 region, hinge region, CH2 region and CH3 region.

Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon. The heavy chains of an antibody define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids.

The mature variable regions of each light/heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same. The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest. Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number.

In another aspect, there is provided a polynucleotide encoding for the antibodies or antigen binding protein as described herein.

In yet another aspect, there is provided a vector comprising a nucleic acid sequence encoding the antigen binding protein as described herein.

Vectors are typically selected to be functional in the host cell in which the vector will be used (the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur. The vector as described herein may be an expression vector and/or a cloning vector.

In some examples, the vector is selected from the group consisting of a plasmid, a viral particle, a phage, a baculovirus, a yeast plasmid, a lipid based vehicle, a polymer microsphere, a liposome, and a cell based vehicle, a colloidal gold particle, lipopolysaccharide, polypeptide, polysaccharide, a viral vehicle, an adenovirus, a retrovirus, a lentivirus, an adeno-associated viruses, a herpesvirus, a vaccinia virus, a foamy virus, a cytomegalovirus, a Semliki forest virus, a poxvirus, a pseudorabies virus, an RNA virus vector, a DNA virus vector and a vector derived from a combination of a plasmid and a phage DNA, further optionally wherein said polynucleotide is operatively linked to an expression control sequence(s) to direct peptide synthesis, even further optionally wherein the vector comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

In yet another aspect, there is provided a host cell comprising the vector as described herein. In some examples, the host cell may comprise a cloning or expression vectors configured to express the antigen binding protein as disclosed herein.

In some examples, the host cell comprises cloning or expression vectors as described above and/or nucleic acid sequences encoding for the antigen binding protein, antibodies and binding fragments thereof as described above.

The host cell can be any type of cell capable of being transformed or transfected with the nucleic acid or vector so as to produce a yellow fever virus (such as YFV-17D)- binding antibody or binding fragment/protein thereof encoded thereby. The host cell comprising the nucleic acid or vector can be used to produce the yellow fever virus (such as YFV-17D)-binding antibody or binding fragment/protein thereof, or a portion thereof (e.g., a heavy chain sequence, or a light chain sequence encoded by the nucleic acid or vector). After introducing the nucleic acid or vector into the cell, the cell is cultured under conditions suitable for expression of the encoded sequence. The antibody, antigen binding protein, or fragment, or portion of the antibody then can be isolated from the cell.

The host cells may be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast cell, an insect cell, or a vertebrate cell). The host cell, when cultured under appropriate conditions, expresses an antibody or binding fragment thereof which can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). Selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity, such as glycosylation or phosphorylation, and ease of folding into a biologically active molecule. Selection of the host cell will depend in part on whether the antibody or binding fragment thereof is to be post-transcriptionally modified (e.g., glycosylated and/or phosphorylated). The host cell may comprise a bacterial cell, a yeast cell, an animal cell e.g. a mammalian cell and/or a plant cell.

Suitable mammalian host cells include CHO, myeloma or hybridoma cells. Many are available from the American Type Culture Collection (ATCC), Manassas, Va. Examples include mammalian cells, such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), 3T3 cells (ATCC No. CCL92), or PER.C6 cells. Other cell types of use in expressing antibodies include lymphocytic cell lines, e.g. NSO myeloma cells and SP2 cells, COS cells. In yet another aspect, there is provided a method of producing a yellow fever virus-binding antigen binding protein comprising culturing the host cell as described herein, optionally isolating said antigen binding protein thereof.

In yet another aspect, there is provided a composition comprising the antigen binding protein as described herein.

In yet another aspect, there is provided a pharmaceutical composition comprising the antigen binding protein as described herein and suitable pharmaceutical composition thereof.

In some examples, the composition or pharmaceutical composition as descrbed herein is a prophylactic and/or therapeutic composition.

In some examples, pharmaceutical compositions comprise a therapeutically or prophylactically effective amount of a yellow fever virus (such as YFV-17D)-binding antigen binding protein or fragment thereof in admixture with a suitable carrier, e.g., a pharmaceutically acceptable agent. Diagnostic compositions comprise a diagnostically effective amount of a yellow fever virus (such as YFV-17D)-binding antigen binding protein or fragment thereof in admixture with a suitable carrier, e.g., a diagnostically acceptable agent.

Pharmaceutically acceptable agents for use in the present pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.

In yet another aspect, there is provided the antigen binding protein or composition as described herein for use in therapy/medicine/vaccine.

In some examples, the composition or pharmaceutical composition as described herein may further comprising an excipient and/or stabilizers.

In yet another aspect, there is provided a antigen binding protein, composition, or pharmaceutical as described herein for use in preventing and/or treating a yellow fever virus infection.

In yet another aspect, there is provided the use of the antigen binding protein as described herein in the manufacture of a medicament for preventing and/or treating a yellow fever virus infection.

In yet another aspect, there is provided a method of preventing and/or treating a yellow fever virus infection in a subject in need thereof, the method comprises administering to the subject an antigen binding protein or composition or pharmaceutical composition as described herein.

In yet another aspect, there is provided a method of preventing and/or reducing the severity of symptoms caused by a yellow fever virus infection in a subject in need thereof, the method comprises administering to the subject an antigen binding protein or composition or pharmaceutical composition as described herein.

In some examples, the antigen binding protein or composition as described herein reduces viremia to at least below 100 copies RNA/pl, or about 1 to 100 copies RNA/pl, or at least below 90 copies RNA/pl, or at least below 80 copies RNA/pl, or at least below 70 copies RNA/pl, or at least below 60 copies RNA/pl, or at least below 50 copies RNA/pl, or at least below 40 copies RNA/pl, or at least below 30 copies RNA/pl, or at least below 20 copies RNA/pl, or at least below 10 copies RNA/pl, or at least below 1 copy RNA/pl, or at least below log 1 copy RNA/pl, or substantially no copies detected.

In yet another aspect, there is provided a method of detecting a yellow fever virus infection in a subject in need thereof, the method comprising contacting the antigen binding protein or composition as described herein to a sample obtained from the subject.

In some examples, the sample may be a biological sample obtained from a biological subject, including a sample of biological tissue or fluid origin obtained in vivo or in in vitro.

In some examples, the antibody or composition or pharmaceutical composition may be administered to the subject through one or more routes of administration including, but not limited to, topical, intravascular, intravenous, oral, subcutaneous, intraarterial, intrathecal, intraperitoneal, intranasal, intradermal, intramuscular, and the like.

In yet another aspect, there is provided a kit comprising the antigen binding protein as described herein.

In some examples, the kit is for specifically detecting a yellow fever virus.

In some examples, the detection is on a non-biological and/or in a biological sample.

In some examples, non-biological sample may include, but is not limited to, comprise surfaces of items (such as surgical items, non-surgical items, general surfaces, and the like.

In yet another aspect, there is provided an antibody, a method, a kit, a product, and/or an expression vector as described herein. DETAILED DESCRIPTION OF FIGURES

Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to anti-yellow fever virus antigen binding protein may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

Fig. 1 shows F3C6K4 and F6H1 L4 neutralized infection of YFV-17D by (A) focus reduction neutralization test (FRNT) and (B) neutralization test. (C) Only F3C6K4 interacted to soluble YFV-17D Envelope protein by ELISA.

Fig. 2 shows prophylactic treatment of mice with F3C6K4 at 24 hour before YFV- 17D challenge protected mice from YFV-17D infection. (A) Study plan to describe the animal study. Mice were treated with 1 , 10 or 100 pg of antibodies 24 hour prior to YFV- 17D challenge. The mice were bled on day 3 and 5 post-YFV-17D challenge. Prophylactic treatment of F3C6K4 (A) reduced mortality, (B) prevented weight loss, and (C) reduced viremia in a dose-dependent manner.

Fig. 3 shows prophylactic treatment of mice with F6H1 L4 at 24 hour before YFV- 17D challenged protected mice from YFV-17D infection. (A) Study plan to describe the animal study. Mice were treated with 1 , 10 or 100 pg of antibodies 24 hour prior to YFV- 17D challenge. The mice were bled on day 3 and 5 post-YFV-17D challenge. Prophylactic treatment of F6H1 L4 (A) reduced mortality, (B) prevented weight loss, and (C) reduced viremia in a dose-dependent manner.

Fig. 4 shows therapeutic treatment of mice with F3C6K4 at 24, 48, or 72 hour post YFV-17D challenge protected mice from infection. (A) Study plan to describe the animal study. Mice were treated with 100 pg of antibodies at 24, 48, or 72 hour after YFV-17D challenge. The mice were bled on day 3 and 5 post-YFV-17D challenge. Treatment of mice with F3C6K4 (A) reduced mortality, (B) prevented weight loss, and (C) reduced viremia in a dose-dependent manner.

Fig. 5 shows the amino acid sequences of various anti-yellow fever virus antibody. CDRs are highlighted with underlined (CDR1), underlined and italics (CDR2) or underlined and bold (CDR3). Fig. 6 shows the nucleotide sequences of various anti-yellow fever virus antibody. CDRs are highlighted with underlined (CDR1), underlined and italics (CDR2) or underlined and bold (CDR3).

Fig. 7 shows the nucleotide sequence of the YFV-17D Envelope protein.

Fig. 8 shows the amino acid sequence of the YFV-17D Envelope protein.

Fig. 9 shows that escape viruses are more resistant to neutralisation by antibodies. The inventors have identified mutation at E55G of the envelope protein in the F3C6K4 escape mutant, and a D270G mutation in the envelope protein of F6H1 L4 escape mutant.

Experimental Data

METHODS AND MATERIALS

Isolation of antibodies against YFV-17D from human volunteers

Experiments involving whole blood from volunteers who were vaccinated with YFV-17D vaccine were approved by the Singhealth Centralized Institutional Review Board (CIRB Ref: 2018/3047) and A*STAR Institutional Review Board (CIRB Ref: 2017/2806).

Whole-blood from volunteers who were vaccinated with YFV-17D vaccine was collected in EDTA-vacutainer tube. The peripheral blood mononuclear cells (PBMCs) were isolated from the blood by Ficoll-paque Plus (GE Healthcare) purification. The buffy coat were harvested, washed with PBS-supplemented with 1% (w/v) BSA (PBS-BSA), treated with ACK lysing buffer for 5 minutes at RT, and then washed twice with PBS-BSA. The resulting PBMC were used immediately or resuspended in heat-inactivated fetal bovine serum (HI-FBS) supplemented with 10% (v/v) DMSO and then stored at < -70°C.

Two different approaches were used to isolate antibodies against YFV-17D from vaccinated volunteers. For the first approach, the light and heavy chain were sequenced from single YFV-specific B cells and then cloned into pTT5. The antibodies were expressed and then screened for neutralization activities. PBMCs were washed once with PBS, stained with Near-IR fluorescent reactive dye (Invitrogen) for 20 min on ice, and then washed with staining buffer (PBS supplemented with 1mM EDTA and 2% HI- FBS). The cells were stained with Human TruStain FcX (Biolegend) to block the Fc receptors for 10 min on ice, washed, and then stained with YFV-17D labelled with Alexa Fluor 488 and Alexa Fluor 647 for 20 min on ice. After the cells were washed, they were incubated with an antibody cocktail consisting of anti-lgD-PE-Cy7 (Biolegend), anti- CD38-PerCP-Cy5.5 (BD), anti-CD-27-PE (BD), anti-lgG-BV510 (BD), and anti-CD19- BV421 (BD) for 20 min on ice. Plasmablasts (CD19+lgG+lgD-CD27+CD38+) and memory B cells (CD19+lgG+lgD-CD27+CD38-) that bound to YFV-17D labeled with Alex Fluor 488 and 647 were sorted, the full-length paired B-cell receptors sequences from these cells were analyzed using Chromium Single cell immune profiling kit (10X Genomics). The paired heavy and light chain sequences were synthesized (Genscripts) and cloned into pTT5 mammalian expression vector expression system as described below.

The second approach was modified from Huang and coworkers (Huang, Doria-Rose et al. 2013) and involved sorting and culturing single YFV-17D specific B cell into 96-well plates, screening the supernatant of the cultured B cells for YFV-17D neutralizing activities, and then cloning the light and heavy chains from the cultured B cells. Briefly, PBMCs were stained with live-dead Aqua fluorescent reactive dye (Invitrogen) for 20 min on ice, washed, then incubated with Alexa Fluor® 488-labeled formaldehyde-inactivated purified YFV-17D for 20 min on ice. After washing, the cells were incubated with an antibody cocktail containing anti-CD3-V450 (BD), anti-CD19-PE (Biolegend), anti-IgD- PE-Cy7 (Biolegend), and anti-lgM-APC/Cy7 (Biolegend) for 20 min on ice. After washing, CD19+lgD-lgM-CD3- cells that interacted with the formaldehyde-inactivated YFV-17D were sorted into Complete Iscove’s modified Dulbecco’s medium (IM DM) supplemented with Glutamax, 10%HI-FBS, and Penicillin/Streptomycin [Complete IMDM], The sorted B cells (4 cells/well) were mixed with gamma-irradiated L929-CD40L cells (5x10 ³ cells/well), 100 U/rnL IL-2, and 100pg/mL recombinant IL-21 (Merck) in Complete IMDM, and the mixture was seeded 100 pL/well into a 96-well plate. After 13-14 days incubation at 37°C, 5%CO2 for, the culture supernatant was harvested and screened for the presence of neutralizing antibodies against YFV-17D using a micro-neutralization focus forming assay. RNA-inhibiting RT-PCR catch buffer (10mM Tris pH 8.0 containing Rnasin® Ribonuclease inhibitor (Promega)) was added into the well containing B cells (20pL/well). The culture plates were flash frozen on dry ice, and then stored at < -70°C. The heavy and light antibody chains from B-cells that produced neutralizing antibodies against YFV-17D were sequenced and cloned into pTT5 vector.

Ig cloning, expression, purification, and sequencing

Methods to clone and express the antibodies have been described previously (Appanna, Kg et al. 2016). Human IgG heavy and light chains were amplified from mRNA of single B cells using One-step RT-PCR (Qiagen), and the resulting RT-PCR products were used for nested PCR with primers that included restriction sites. The PCR products were cloned into the pTT5 expression vector (National Research Council of Canada). The plasmids that express the heavy and light chains were co-transfected into HEK293-6E cells using 293fectin ^TM (Thermo Fisher Scientific) as per the manufacturer’s instructions. Five days after transfection at 37°C, 5% CO2 at 125rpm, antibodies were purified from clarified culture supernatant using Protein G Agarose beads, the antibodies were eluted from the beads, and buffer exchanged into PBS, sterile filtered before use.

Cells and viruses

Aedes albopictus (C6/36), African green monkey kidney cells (Vero), and Baby hamster kidney cells expressing DC-SIGN (BHK-21-DC-SIGN) were maintained in RPMI-1640 supplemented with 5% heat- inactivated fetal bovine serum (HI-FBS). C6/36 were maintained at 28°C and Vero and BHK-21-DC-SIGN cells were maintained at 37°C, 5% CO2. Yellow fever virus YFV-17D (isolate YFV_EHI, accession: MF289572) was obtained from the Environmental Health Institute, Singapore and propagated in C6/36 cells. The virus used in this study contained G to A mutation at nucleotide 1949, leading to an E326K in the envelope protein.

Purification, formalin-inactivation, and labeling of virus

YFV-17D produced from C6/36 cells were harvested, clarified at 500xg for 10 min, and then concentrated using 100kDa MWCO filter unit (Amicon). The virus was purified using lodixanol discontinuous gradients. Discontinuous gradient was prepared using various percentage (w/v) of lodixanol solutions (15, 20, 25, 30, 35, 40, 45, and 55%) prepared in HNE buffer (5mM HEPES, 150mM NaCI, 0.1mM EDTA at pH7.4). The concentrated virus solution was overlay onto the gradient, and the gradient was subjected to centrifugation at 125,000xg for 2h at 4°C without brake. The purified virus bands were harvested, and then stored at < -70°C. To generate inactivated virus, the purified virus was incubated with 0.05% formaldehyde at 28°C for 10 days as described previously (Appaiahgari and Vrati 2004, Fan, Chiu et al. 2015).

To label the purified virus or inactivated virus with fluorescent probes, the purified virus was buffer exchanged into PBS using a 30kDa MWCO filter unit (Amicon) and the protein concentration of the purified virus was determined by Micro BCA ^TM Protein Assay kit (Thermo Fisher Scientific) as per the manufacturer’s instructions. The virus was labeled with Alexa Fluor® 647 or Alexa Fluor® 488, and then fluorescently labeled virus was purified from the free fluorescence dye as per manufacturer’s instructions (Thermo Fisher Scientific). Focus reduction neutralization test (FRNT)

BHK-DC-SIGN cells (1x10 ⁵ cells/well) were seeded in 24-well plates overnight at 37°C, 5%CC>2. YFV-17D (240 FFU/mL, 300 pL) was mixed with 300 pL of antibodies at various concentrations for 1 h at 37°C. Medium from BHK-DC-SIGN cells were removed, and the virus-antibody mixture (250 pL/well) was added to the cell monolayer for 2h at 37°C, and then 500 pL/well of overlay medium (RPMI supplemented with 0.8% (w/v) Methylcellulose (Aquacide II, Calbiochem) and 5%HI-FBS) was added to the cells for 3 days at 37°C, 5%CO2. The overlay medium was removed, the monolayer was treated with 3.7% formaldehyde for 30 min at RT, washed 3 times with water, followed by treatment with 1 % (w/v) TritonX for 20 min at RT. After 3 washes with water, the monolayer was washed once with PBS-1 %HI-FBS, 4G2 antibody (1 pg/mL) was added to the wells for 1h at RT, washed, and then horseradish peroxidase conjugated goat antimouse antibodies (1/2000 dilution; Dako) were added for 30 min at RT. After the monolayers were washed 3 times with water, TrueBlue™ peroxidase substrate (KPL) was added for 10 min, the monolayers were washed, and the foci were manually counted. PRNT50 is defined as the concentration of antibody that results in a reduction of plaques by 50%, determined by applying a three-parameter non-linear curve fit in GraphPad Prism.

Micro-neutralization focus forming assay

BHK-DC-SIGN cells (2x10 ⁴ cells/well) were seeded in 96-well plates overnight at 37°C, 5%CO2. Culture supernatant from B cell were transferred to 96-well II bottom plate, subjected to UV-inactivation for 10 minutes, and then 30 pL of YFV (-100 FFU) was added into the B cell culture. After 1 h incubation at 37°C, the culture medium from BHK- DC-SIGN was removed, and then 50 pL/well of the virus-B cell culture supernatant was added to the BHK-DC-SIGN monolayer for 2h at 37°C, and then 200 pL/well of overlay medium was added to the cells for 48h at 37°C, 5%CO2. The overlay medium was removed, the monolayer was treated with 3.7% formaldehyde (100 pL/well) for 30 min at RT, washed 3 times with water, followed by treatment with 1% (w/v) TritonX for 20 min at RT. After 3 washes with water, the monolayer was washed once with PBS-1 %HI-FBS, 4G2 antibody (1 pg/mL) was added to the wells for 1 h at RT, washed, and then horseradish peroxidase conjugated goat anti-mouse antibodies (1/2000 dilution; Dako) were added for 30 min at RT. After the monolayers were washed 3 times with water, TrueBlue™ peroxidase substrate (KPL) was added for 10 min, the monolayers were washed, and the wells were observed using a microscope. Neutralizing test

Vero cells (5x10 ⁴ cells/well) were seeded in 48-well plates overnight at 37°C, 5%CC>2. Next day, 2-fold serial dilutions of antibody solutions were mixed with equal volume of YFV-17D (10 ⁶ FFU/mL) for 1 h at 37°C, 5%CC>2. Medium was removed from the cells, 100 pL/well of virus-antibody mixture was added to the cells for 2h at 37°C, 5%CC>2. Viral inoculum was removed, and 250 pL/well of RPMI-10%HI-FBS was added to the cells at 37°C, 5%CC>2. After 2 days incubation, the cells were harvested with Trypsin-EDTA, fixed with 3.7% formaldehyde for 20 minutes, washed with permeabilization buffer (PBS containing 2%HI-FBS, 5mM EDTA, 0.1 % (w/v) sodium azide, and 0.1% (w/v) saponin), and then incubated with permeabilization buffer for 20min on ice. The cells were subjected to centrifugation at 1000xg for 4 min, supernatant discarded, and then incubated with AF647-conjugated 4G2 antibody for 1 h on ice. The cells were washed, resuspended in FACS buffer (PBS supplemented with 2% HI-FBS, 5mM EDTA and 0.1% (w/v) sodium azide). Samples were analyzed by flow cytometry.

Animal study

All animal studies were approved by the BRC (Biological Resource Centre), A*STAR Institutional Animal Care and USE Committee under rules and regulations of the National Advisory Committee for Laboratory Animal Research (NACLAR). Male and female AG129 mice (B&K Universal, UK) that were 6-7 weeks old were used for all animal studies. Prophylactic studies were performed by injecting various concentrations of antibodies into the mice via the intravenous route, and then the mice were infected with 10 ⁵ FFU of YFV-17D intraperitoneally. For therapeutic studies, mice were infected intraperitoneally with 10 ⁵ FFU of YFV-17D, and then treated with various concentrations of purified antibodies via the intravenous route at different time points post-infection. For all animal studies, the mice were weighed daily for up to 21 days and serially bled on day 3 and 5. Mice that had 10% body weight loss within 24 h, or 20% body weight loss within 48 h, or moribund were sacrificed due to humane purposes.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Primers and probes used for qRT-PCR have been previously described (Thibodeaux, Garbino et al. 2012). Each reaction mixture contained 1 pl of extracted RNA; primers and probes were used at a final concentration of 0.5 pM for 8280F (forward primer) and 8354R (reverse primer) and 0.2 pM for 8308pr (probes). Amplification was performed in an using TaqMan® RNA-to-Ct™ 1-step kit (Applied Biosystem) under the following conditions: 50°C for 30 minutes, 95°C for 12.5 minutes, followed by 45 cycles of 94°C for 15 s, 55°C for 1 minutes. The RNA control was prepared as described previously (Calvert, Dixon et al. 2016). Viral RNA was extracted using High Pure Viral RNA kit (Roche) and then reverse transcribed into cDNA using SuperScript® III First-Strand Synthesis System (Invitrogen) using the random hexamers following the manufacturer’s instructions. A PCR fragment containing T7 promoter sequence followed by nucleotide sequence 7944 to 8589 of YFV-17D was generated and then purified. Control RNA was prepared by in vitro transcription of the PCR fragment using MAXI script™ T7 Transcription kit (Thermo Fisher Scientific), purified, quantified by spectrophotometry and then the copy/pL were calculated.

Viral RNA from the plasma was extracted using Abgenix Viral RNA and DNA kits (Aitbiotech). The viral RNA was quantify using qRT-PCR as described above, and copy/pL in the plasma samples were interpolated from the standard curve.

ELISA

MaxiSorp Immuno plates (Thermo Fisher Scientific) were coated with purified anti-V5 Tag antibody (poly29038, BioLegend) at 2 pg/mL in coating buffer (0.1M NaHCCh) overnight at 4°C. The plates were washed with PBST (PBS supplemented with 0.05% Tween 20), blocked with 200 pL/well PBS-3% (w/v) skim milk for 2 h at RT, and then recombinant YFV-17D Envelope protein (5 pg/mL in PBS-3% (w/v) skim milk) was added to the wells for 1h at RT. The wells were washed, and various concentrations of antibodies were added to the wells for 1 h at RT. After 3 washes with PBS-T, the bound human antibodies were detected by the addition of HRP-conjugated anti-human IgG (1/5000) for 1 h at RT, washed, followed by the addition of 3, 3’, 5, 5’- Tetramethylbenzidine (TMB) Liquid substrate system for ELISA (Sigma). 1M HCI was added to the wells to stop the enzyme reaction and the absorbance was measured at 450 nm.

Selection for escape mutants

C6/36 cell (9x10 ⁵ cells/well) were seeded into a 6-well plate at 28°C overnight. YFV-17D (2x10 ⁴ FFU in 600 pL) were mixed with 600 pL of antibody solution for 1 h at 37°C. Culture supernatant was removed from C6/36 cells, 1mL/well of virus-antibody solution was added to the cells for 1 h at 28°C, the inoculum was removed, and RPMI-10% Hl- FBS containing antibody solution was added to the cells for 5-6 days at 28°C. The culture supernatant was harvested and clarified, 600 pL of the culture supernatant was mixed with 600 pL of antibody solution for 1 h at 37°C, and then 1mL/well of virus-antibody solution was added to fresh monolayer of cells for 1 h at 28°C. The inoculum was removed, and RPMI-10% HI-FBS containing antibody solution was added to the cells for 5-6 days at 28°C. The cycle was repeated a total of 5 times to generate an escape mutant.

Sequencing of the escape mutant viruses

Viral RNA was extracted from culture supernatant using Abgenix Viral RNA and DNA kits. cDNA was synthesized from the extracted RNA using First-Strand cDNA Synthesis kit (Thermo Fisher Scientific) using random hexamers as per manufacturer’s instructions. PCR was performed using Taq PCR Mastermix (Qiagen) and then the sequence of the viral RNA was determined by Sanger sequencing method (1 ^st Base, Axil Scientific).

RESULTS

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of volunteers who were vaccinated with YFV-17D. Two different approaches were then used to identify neutralizing antibodies against YFV-17D. The first approach involved sorting the plasmablasts (CD19+lgG+lgD-CD27+CD38+) and memory B cells (CD19+lgG+lgD-CD27+CD38-) that interacted with YFV-17D, and analyzing the full length paired B-cell receptors sequences from these cells using the Chromium Single cell immune profiling kit (10X Genomics). The paired heavy and light chain sequences were synthesized and cloned into the antibody expression system, expressed and tested for their ability to neutralize YFV-17D using a focus forming assay.

The second approach involved sorting for CD19+lgD-lgM-CD3- cells that interacted with the formaldehyde inactivated YFV-17D and then culturing these cells on a monolayer of feeder cells. The culture supernatant from the cultured B cells was screened for the presence of neutralizing antibodies against YFV-17D using a microneutralization focus forming assay. The heavy and light antibody chains from wells containing neutralizing antibodies against YFV-17D were sequenced and cloned into the antibody expression system.

From the two different approaches, two neutralizing antibodies: F3C6K4 and F6H1 L4 were identified. Both antibodies neutralized YFV-17D infection on BHK-DC- SIGN using an FRNT and Vero cells using a neutralization test in a dose-dependent manner (Figure 1A, 1 B). By ELISA, it was found that F3C6K4, but not F6H1 L4 bound to recombinant YFV-17D Envelope protein (Figure 1C). In addition, an antibody of this invention (e.g. F6H1 L4) did not bind to soluble YFV-17D Env protein but neutralized infection. As such, this demonstrate that F6H1 L4 binds to a conformational epitope that is distinct from an antibody known in the art.

Next, the inventors examined the potential use of these antibodies as prophylactic treatment for YFV-17D infection in a mouse model. AG129 mice were treated with 1 , 10, or 100 pg of antibodies intravascularly one day before they were infected intraperitoneally with YFV-17D. Three and five days post-inoculation, the mice were serially bled. The survival of these mice was monitored for 21 days. Treatment of mice with 10 and 100 pg of F3C6K4 and 100 pg of F6H1 L4 survived infection, with minimal weight loss and below the detection level of viral genome in their blood (Figure 2 and 3). Therefore, Figure 2 and 3 show treatment of mice with 100 pg of the antibodies of this invention 1 day after viral infection protected 100% of YFV-17D infection and completely inhibited viral replication. This data demonstrates that the antibodies of this invention are more superior than antibodies known in the art.

Finally, the inventors determined if F3C6K4 could be used as therapeutic treatment for YFV-17D infection. Mice were infected with YFV-17D, and then 100 pg of antibodies were administered at 24-, 48-, or 72-hour post virus inoculation. All mice that were treated with F3C6K4 at 24-hour post-infection survived infection, 89% of the mice treated with F3C6K4 at 48-hour post-infection survived infection, and 62% of the mice survived if they were treated with F3C6K4 at 72-hour post-infection (Figure 4).

By selecting for mutant viruses that escape neutralization by the antibodies, the inventors have identified an E55G mutation in mutant virus that escaped neutralization by F3C6K4, and a D270G mutation in the mutant virus that escaped neutralization by F6H1 L4. These results suggest that amino acid 55 and 270 on YFV Envelope proteins are involved in binding to the antibodies. These amino acids are conserved in the wild type virus, suggesting that the antibodies will wild type YFV infection (Figure 12).

APPLICATIONS

Embodiments of the antibodies disclosed herein could be used as prophylactic and therapeutic antibodies against yellow fever infection.

Advantageously, the antibodies as described herein are capable of protecting subjects (such as mice in Experimental Section) of YFV-17D infection. In particular, the antibodies are shown to be capable of neutralising infection. Even more advantageously, the antibodies as described herein are capable of inhibiting viral replication (reduce viremia) to a level less than the limit of detection level (100 copies RNA/pl).

It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.