WO2011077371A1

WO2011077371A1 - Method for enhancing the expression of hpv l1

Info

Publication number: WO2011077371A1
Application number: PCT/IB2010/055979
Authority: WO
Inventors: Edward Peter Rybicki; Inga Isabel Hitzeroth
Original assignee: University Of Cape Town
Priority date: 2009-12-22
Filing date: 2010-12-21
Publication date: 2011-06-30

Abstract

A method of enhancing the expression of human papillomavirus (HPV) L1 in various expression systems, in particular by producing a chimaeric polypeptide with improved expression levels relative to the native polypeptide, is described. The expression system may be an insect, animal, plant or yeast system. Furthermore, chimaeric nucleotide and polypeptide sequences are described, and expression vectors comprising chimaeric nucleotide sequences. A pharmaceutical composition comprising a chimaeric HPV L1 made according to the invention and a method of treating or preventing an HPV infection or cervical cancer in a subject is also described.

Description

10 055979

METHOD FOR ENHANCING THE EXPRESS!QN OF HPV L1

BACKGROUND OF THE INVENTION

This invention relates to a method for enhancing the expression of HPV L1 in various expression systems, in particular by producing a chimaeric polypeptide with improved expression levels.

Papillomaviruses (PVs) are small double-stranded DNA viruses that infect many different species such as humans, dogs, cattle, horses, rabbits, non-human primates, mice, sheep and birds. They are extremely species specific. Human papillomaviruses (HPV) are known to cause warts, and have also been associated with certain cancers in humans (zur Hausen, 2009). They are divided into high and low-risk HPV types, where the high-risk types such as HPV 16, 18, 33 and 58 infect the genital epithelium and can produce lesions which progress to invasive cervical cancer. Specific high-risk types of HPV are causally associated with cervical cancer and cancer of the cervix is the most common cancer in South African women (18.2% of all cancers reported in 1997) and the second most prevalent cancer in women worldwide. HPV 16 is the most prevalent high-risk HPV type found to be associated with cervical cancer (Maclean et al., 2005). Although the incidence of HPV 16 is lower in South Africa than that reported in Europe and the USA, it is still the predominant HPV type found in the South African population (Williamson et al., 1994).

Although cervical screening programs have resulted in a drop in the number of cervical cancer cases in the developed world, this is not the case in Africa, where screening programmes are either inadequate or non-existent. Moreover, genital warts and lesions caused by low-risk HPVs are also extremely common, and can cause serious morbidity and reduction in quality of life, as well as increasing the risk of oesophageal cancer. Therefore, in most parts of Africa, the only hope of reducing papillomavira) disease is a successful HPV vaccination campaign. Additionally, women who are already infected and have developed cancer or precancerous lesions also need to be treated. Therefore, the development of a therapeutic vaccine against HPV that will allow regression of cancer in patients already infected with the virus and that also provides protection against future exposure is desirable.

The most successful HPV prophylactic vaccine candidates to date - from GlaxoSmithKline and Merck - are based on L1 major capsid protein virus-like particles (VLPs) produced by recombinant baculovirus in insect cells, and by recombinant yeast, respectively. These VLPs are almost indistinguishable from native virions in morphology, and induce effectively identical immune responses (for review see Maclean et al., 2005).

In animal and human studies, VLP vaccines have been well tolerated and have induced high titres of neutralising antibodies as well as protecting against papillomaviral infection and especially disease (Harper et al., 2004; Brown et al., 2004). However, these are expensive vaccines, and are moreover very type-specific: a mixture of several types will be needed to protect against the majority of circulating high-risk types alone.

Chimaeric HPV L1/L2 polypeptides have been described, but these have often not led to good cross-protection between HPV types or have produced poor expression yields (Jochmus et al. 1999, Pastrana et al. 2005; Varsani et al. 2003a). Another problem with chimaeric proteins is that different immune responses are obtained with chimaeras compared to peptides alone (Slupetzky et al. 2007; Kondo et al. 2006).

One of the problems encountered so far is the low yield of VLPs or capsomers produced in plants and other expression systems such as insect cells and yeast (Varsani et al. 2003b; Maclean et al. 2007). Low expression levels of proteins will in turn have an effect on costs and ease of production of vaccines. A main consideration in vaccine development is the level of production in the various systems to determine whether a particular production system will be commercially viable. In plants, a yield of 25mg/kg plant material or 1 % of total soluble protein is considered to be at the lower end of a viable yield (Rybicki 2009). An ideal HPV vaccine would be affordable, safe, and stable and would protect from and/or clear lesions caused by the major oncogenic HPV types. Human and animal studies have shown that many of these criteria can be met with VLPs. However, affordability and vaccine stability will be a problem in developing countries, given the requirement for a cold chain and the predicted expense of current vaccine candidates.

SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided a method of producing a chimaeric HPV L1 polypeptide with increased expression levels relative to a L1 polypeptide, the method comprising the steps of:

(i) codon optimising a first nucleotide sequence encoding a papillomavirus peptide or polypeptide which has been shown to elicit an immune response in humans or animals;

(ii) inserting the first codon optimised nucleotide sequence into a second codon optimised nucleotide sequence encoding an HPV L1 polypeptide or a portion thereof, the insertion site of the first codon optimised nucleotide sequence being at or about amino acid position 414 of the HPV L1 polypeptide, so as to form a chimaeric codon optimised HPV L1 nucleotide sequence;

(iii) introducing the chimaeric codon optimised HPV L1 nucleotide sequence into a cell; and

(iv) expressing a chimaeric HPV L1 polypeptide from the codon optimised chimaeric HPV L1 nucleotide sequence in the cell.

The first peptide or polypeptide may be an HPV or BPV L2 peptide, such as the peptides selected from any one of SEQ ID NOs: 3 to 6. Alternatively, the first peptide or polypeptide may be a human or mouse PV E7 peptide, such as the peptides selected from either one of SEQ ID NOs: 7 or 8.

The codon optimised nucleotide sequence encoding the first peptide or polypeptide may be any one of SEQ ID NOs: 9 to 14, 32 or 47. The second codon optimised HPV L1 nucleotide sequence may be modified to be nuclear localisation signal deficient.

The method may further comprise the step of inserting a third codon optimised nucleotide sequence into the second codon optimised HPV L1 nucleotide sequence at or about any one of amino acid positions 430 to 434 of the HPV L1 polypeptide, the third codon optimised nucleotide sequence encoding a third immunogenic papillomavirus peptide or polypeptide.

Preferably, the first and third peptides or polypeptides are 10 amino acids or more in length.

The third codon optimised nucleotide sequence may be either a mouse or human E7 epitope, and in particular may be either of SEQ ID NOs: 13, 14, or 32 or a sequence encoding either of SEQ ID NOs: 7 or 8.

The first codon optimised nucleotide sequence and optionally also the third codon optimised nucleotide sequence may replace the nucleotides of the codon optimised HPV L1 nucleotide sequence at the point of insertion.

The expressed chimaeric HPV L1 polypeptide may be recovered from the cell. The cell may be an insect, animal, plant or yeast cell.

More particularly, the codon optimised nucleotide sequences may be human or plant codon optimised for expressing the chimaeric HPV L1 polypeptide in a plant cell, human codon optimised for expressing the chimaeric HPV L1 polypeptide in an insect cell, or yeast codon optimised for expressing the chimaeric HPV L1 polypeptide in a yeast cell.

The HPV L1 polypeptide may be a HPV-16 L1 polypeptide, such as the one provided in SEQ ID NO: 1 and the codon optimised HPV L1 nucleotide sequence may be SEQ ID NO: 2.

The chimaeric HPV L1 polypeptide sequence may be selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 and 22. The chimaeric codon optimised HPV L1 nucleotide sequence may be selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30 and 31.

The method may further comprise, following step (ii), introducing the codon optimised chimaeric HPV L1 nucleotide sequence into an expression vector and in step (iii) introducing the expression vector comprising the chimaeric codon optimised HPV L1 nucleotide sequence into the cell.

The expression vector may be adapted for expression of polypeptides in plant, insect, animal, such as BHKs, CHO, HEK 293s, or yeast cells.

The expression vector may be adapted to target a component of a plant cell, such as a plant chloroplast, endoplasmic reticulum, vacuole, or apoplast.

The method, when used in a plant expression system, may further include in step (iii) introducing into a plant cell a suppressor protein adapted to inhibit post- transcriptional gene silencing in a plant. Preferably, the suppressor protein is the NSs protein of the tomato spotted wilt virus or the p19 of tomato bushy stunt virus.

The chimaeric HPV L1 polypeptide may assemble into a virus-like particle, capsomer or pentamer. Preferably, the chimaeric HPV L1 polypeptide can assemble into a capsomer or a pentamer.

The chimaeric HPV L1 polypeptide may be an immunogenic polypeptide for eliciting a neutralising antibody and/or CTL response in a subject. Preferably, the chimaeric HPV L1 polypeptide is able to elicit a cross-protective response in the subject.

According to a further aspect of the invention, there is provided a chimaeric HPV L1 nucleotide sequence comprising a codon optimised nucleotide sequence encoding an HPV L1 polypeptide into which a first codon optimised nucleotide sequence encoding a heterologous papillomavirus peptide has been inserted at or about amino acid position 414 of the HPV L1 polypeptide.

The codon optimised chimaeric HPV L1 nucleotide sequence may additionally include a second codon optimised nucleotide sequence encoding a heterologous papillomavirus peptide inserted at or about any one of amino acid positions 430 to 434 of the HPV L1 polypeptide.

The codon optimised HPV L1 , first and/or second nucleotide sequences may be human, plant or yeast codon optimised.

The first codon optimised nucleotide sequence and optionally also the second codon optimised nucleotide sequence may replace the nucleotides of the codon optimised HPV L1 nucleotide sequence at the point of insertion.

The codon optimised chimaeric HPV L1 nucleotide sequence may be modified to be nuclear localisation signal deficient.

The codon optimised chimaeric HPV L1 nucleotide sequence may be any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30 and 31 , or a sequence which has at least 95% identity thereto.

According to a further aspect of the invention, there is provided an expression vector comprising the codon optimised chimaeric HPV L1 nucleotide sequence as described above.

The expression vector may be adapted for expression of polypeptides in plant, insect, animal or yeast cells.

According to a further aspect of the invention, there is provided a chimaeric HPV L1 polypeptide produced according to the invention.

The chimaeric HPV L1 polypeptide may be used for eliciting a neutralising antibody and/or CTL response in a subject to which it is administered. Preferably, the chimaeric HPV L1 polypeptide elicits a cross-protective response against other HPV types in the subject. The chimaeric HPV L1 polypetide may assemble into a capsomer or pentamer or may assemble into virus-like particles.

The chimaeric HPV L1 polypeptide may be selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 and 22, or a sequence which has at least 95% identity thereto.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a chimaeric HPV L1 polypeptide expressed by the codon optimised nucleotide sequence as described above, and a pharmaceutically acceptable carrier.

The composition may also include an adjuvant.

According to a further aspect of the invention, there is provided the use of a codon optimised chimaeric HPV L1 nucleotide or a chimaeric HPV L1 polypeptide expressed by the codon optimised nucleotide as described above in the manufacture of a medicament for use in a method of preventing and/or treating HPV infection and/or cervical cancer in a subject.

According to a further aspect of the invention, there is provided a method for preventing and/or treating an HPV infection and/or cervical cancer in a subject, the method comprising the step of administering a prophylactically or therapeutically effective amount of a chimaeric HPV L1 polypetide produced as described above or a pharmaceutical composition as described above to the subject.

The subject is preferably a human.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a schematic representation of the HPV-16 L1 protein showing points of insertion of L2 and E7 peptides on the L1 protein.

Figure 2 shows the two plasmids used in creation of the human codon optimised chimaeras. Figure 3 shows the results of PCR to check chimaeric input DNA and purity of each viral infectant.

Figure 4 shows a direct comparison of expression levels of human codon optimised chimaeras in insect cells by western blot analysis and detection with HPV- 16 H16:J4 and antimouse antibody conjugated with alkaline phophatase.

Figure 5 shows the concentration of chimaeric proteins expressed in insect cells.

Figure 6 shows the Agrobacterium plant expression vectors pTRAc (cytoplasmic-targeting) and pTRAkc-rbcs1-cTP (chloroplast-targeting). P35SS: CaMV 35S promoter containing duplicated transcriptional enhancer, CHS: chalcone synthase 5' untranslated region, pA35S: CaMV 35S polyadenylation signal for foreign gene expression, SAR: tobacco Rb7 scaffold attachment regions flanking the expression cassette; LB/RB: left and right borders for T-DNA integration, ColElori: E. coll origin of replication, RK2ori: Agrobacterium origin of replication, bla: ampicillin/carbenicillin-resistance bla gene. In addition, the pTRAkc-rbcs1-cTP vector contains npt II: kanamycin-resistant npt II gene, Pnos/pAnos: promoter/polyadenylation signal of the nopaline synthase gene and rbcs1-cTP: Solanum tuberosum chloroplast-transit peptide sequence of the Rubisco small- subunit gene rbcS1.

Figure 7 shows chloroplast-targeted human codon optimised L1/L2 chimaeras 3-9 days post-infiltration, either with (+) or without (-) co-expression of the NSs silencing suppressor. The arrow indicates the position of the L1/L2 chimaera (-56 kDa).

Figure 8 shows cytoplamic expression of human codon optimised L1/L2 chimaeras and human codon optimised HPV L1 protein (hl_1 ) utilising pTRAc or self- replicating vector pRIC3.

Figure 9 shows maximum protein yields obtained for the human codon optimised chimaeras and hl_1 targeted to the cytoplasm when co-expressed with the NSs silencing suppressor and utilizing pTRAc and pRIC3. Figure 10 shows cytoplamic expression of human codon optimised L1/L2 (108- 120), also called SAF and hl_1 compared to chloroplast targeted human codon optimised SAF and hl_1.

Figure 11 shows transmission electron micrographs of the human codon optimised L1/L2 chimaeras 5 days post-infiltration at 75.000X magnification. Positive control: insect cell-derived HPV-16 L1 VLPs & capsomers shown in the L1/L2 BPV (1-88) inset. Negative control: crude NSs-infiltrated plant extract. Scale bar = 100 nm.

Figure 12 shows the protein and codon optimised DNA sequences of the HPV L1 polypeptide (SEQ ID NO: 1) and polynucleotide (SEQ ID NO: 2), respectively, used herein.

Figure 13 shows the amino acid and codon optimised nucleotide sequences of the L2 and E7 peptides (SEQ ID NOs: 3 to 14, 32 and 47) which were inserted into the L1 sequence.

Figure 14 shows the protein sequences of the chimaeras produced according to the invention (SEQ ID NOs: 15 to 22).

Figure 15 shows the codon optimised nucleotide sequences of the chimaeras produced according to the invention (SEQ ID NOs: 23 to 31).

Figure 16 shows the Hansenula polymorpha expression vector and cloning strategy for SAF.

Figure 17 shows the Pichia pastoris expression vector and cloning strategy for SAF.

Figure 18A shows yeast colony PCR to confirm integrations of SAF into H. polymorpha genome; Lane 1 λ Pstl DNA marker; Lane 2 positive control (1.6 Kb); Lane 3 negative control; Lanes 4 - 13 recombinant colonies. Figure 18B shows yeast colony PCR to confirm integrations of SAF into P. patoris genome. Lane 1 λ Pstl DNA marker; Lanes 2 - 8 recombinant colonies, Lane 9 positive control (1.7 Kb), Lane 10 negative control.

Figure 18C shows southern hybridization of H. polymorpha containing various copies integrations of the yeast optimised pHIPX4-SAF. Lane 1 , untransformed wild type H. polymorpha; lane 2-5, L-multicopy intergrants; lane 6, S-single copy intergrant.

Figure 19A shows western blot analysis of intracellular SAF produced by H. polymorpha during small-scale expression in shake-flasks. Lane 1 , prestained marker (Fermentas); lane 2, wild type H. polymorpha (24 h); lane 3, single copy (24 h); lane 4 and 5, multicopy (24 h); lane 6, wild type H. polymorpha (48 h); lane 7, single copy (48 h); lane 8 and 9, multicopy (48 h); lane 10, wild type H. polymorpha (72 h); lane 11 , single copy (72 h); lane 12 and 13, multicopy (72 h).

Figure 19B shows western blot analysis of intracellular SAF (KMSAF3) produced by P. pastoris KM71 during small-scale expression in shake-flasks. Lane 1 , prestained marker (Fermentas); lane 2, 24h post methanol induction; lane 3, 48h post methanol induction; lane 4, 72h post methanol induction.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for increasing the production of HPV L1 polypeptides, compared to native polypeptides, in expression systems such as insect, animal, yeast and plant systems. The polypeptides can be used in prophylactic or therapeutic compositions, including vaccines, for preventing and/or treating HPV infection or cervical cancer.

As used herein, the terms "peptide" and "polypeptide" are used interchangeably to refer to a sequence of amino acids and "protein" refers to a full-length protein.

One of the problems encountered in the prior art is the low yield of VLPs or capsomers produced in plants and other expression systems, such as insect cells and yeast. Furthermore, given the diversity of HPV types, cross-protection against multiple types by a single vaccine would be highly beneficial. The applicant describes herein the creation of a number of codon optimised L1/L2 chimaeras, L1/E7 chimaeras and L1/L2/E7 chimaeras, to determine their ability to cross-protect against other HPV types. The applicant has shown that insertion of 10 or more codon optimised amino acids at the postion 414 of HPV-L1 significantly increases the expression level of the protein in insect, plant and yeast cells. This appears to also be the case for an insertion of 10 or more codon optimised amino acids in the region of position 433.

According to the invention, expression of HPV L1 polypeptides can be increased by producing codon optimised chimaeric nucleotide sequences comprising a HPV L1 nucleotide sequence into which a first heterologous nucleotide sequence has been inserted at or about amino acid position 414 of the HPV L1 polypeptide encoded by the HPV L1 nucleotide sequence. The heterologous papillomavirus sequence is typically an immunogenic human or bovine L2 epitope or an immunogenic human or mouse E7 peptide. The applicant has found that if both of these sequences are human codon optimised, expression is greatly increased compared to the wild-type sequence when expressed in plants and insect cells. The same applies when these sequence are yeast codon optimised and expressed in yeast cells.

The codon optimised HPV L1 sequence can be further modified to be nuclear localisation signal deficient.

A second codon optimised heterologous nucleotide sequence may be inserted into the codon optimised HPV L1 sequence at or about any one of amino acid positions 430 to 434 of the HPV L1 polypeptide which the nucleotide sequence encodes, the second codon optimised nucleotide sequence also encoding an immunogenic papillomavirus peptide or polypeptide.

Examples are provided below where the first codon optimised heterologous nucleotide sequence is selected from SEQ ID NOs: 9 to 14, 32 or 47, encoding peptides of SEQ ID NOs: 3 to 8 respectively, and the second codon optimised heterologous sequence is either of SEQ ID NOs: 13, 14 or 32, encoding either of peptides of SEQ ID NOs: 7 or 8, respectively, or sequences which have at least 80%, more preferably 90%, and even more preferably 95% identity thereto. The first and second codon optimised heterologous sequences can be either simply inserted into the codon optimised HPV L1 sequence or can be inserted into the codon optimised HPV L1 sequence by replacing an equivalent number of HPV L1 codons at the point of insertion.

The codon optimised HPV L1 nucleotide sequence can be SEQ ID NO: 2 encoding an HPV L1 polypeptide of SEQ ID NO: 1 , or sequences which have at least 80%, more preferably 90%, and even more preferably 95% identity thereto.

Examples of chimaeric codon optimised HPV L1 nucleotide sequences that can be produced according to the invention are SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30 and 31 , and examples of chimaeric HPV L1 polypeptide sequences are SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 and 22, or sequences which have at least 80%, more preferably 90%, and even more preferably 95% identity thereto. In one embodiment, the chimaeric HPV L1 polypeptide is SEQ ID No: 15.

The chimaeric codon optimised HPV L1 nucleotide sequence may be inserted into an expression vector which can then be introduced into the plant, insect, animal, or yeast cell.

In the case of expression in plants, the expression vector can be adapted to target a component of a plant cell, such as a plant chloroplast, endoplasmic reticulum, vacuole, or apoplast. A suppressor protein adapted to inhibit post-transcriptional gene silencing, such as the NSs protein of the tomato spotted wilt virus or the p19 of tomato bushy stunt virus, can also be introduced into the plant cell.

Virus-like particles, capsomers or pentamers can be formed by the chimaeric HPV L1 polypeptides.

The chimaeric HPV L1 polypeptides are preferably immunogenic polypeptides for eliciting a neutralising antibody and/or CTL response in a subject, and even more preferably, they are able to elicit a cross-protective response in the subject.

The invention is described in more detail below with reference to the following non- limiting examples. Examples

Creation of codon optimised chimaeras

Nucleotide sequences that had been optimised for human codon usage encoding epitopes of HPV-16 L2 (Kawana et al. 1999), BPV L2, mouse E7 and human E7 (Torrens et al. 2005) were inserted into a human codon optimised HPV-16 L1 sequence to show that chimaeric constructs according to the invention can result in better expression than expression of native L1 amino acid sequences. Furthermore, yeast codon optimised nucleotide sequences encoding epitopes of HPV-16 L2 were inserted into a yeast codon optimised HPV-16 L1 sequence to show that the chimaeric construct according to the invention can result in better expression than expression of the native L1 amino acid sequence. However, the invention is not intended to be limited to these specific peptides, epitopes or sequences.

The L2 epitopes selected were shown elsewhere to provide protection against other HPV types. This is summarised in the table below:

Table 1 : L2 epitopes

Therefore, by addition of any one of these epitopes to the L1 protein, it could be possible to create a vaccine that will protect against more than one type of HPV.

The codon optimised L2 sequences that were used are as follows:

HPV16 L2 (108-120)

Human optimised

DNA: CTGGTGGAGGAGACCAGCTTCATCGACGCCGGAGCCCCCGC (SEQ ID NO. 9)

Yeast optimized DNA: TTGGTTGAAGAAACCTCTTTCATCGACGCTGGTGCTCCAGC (SEQ ID NO. 47) aa: LVEETSFIDAGAP (SEQ ID NO. 3)

HPV16 L2 (56-81)

DNA: GGCGGCCTGGGCATCGGCACCGGCAGCGGCACCGGGGGCAGGACCG

GCTACATCCCCCTGGGCACCAGACCCCCCACC (SEQ ID NO. 10) aa: GGLGIGTGSGTGGRTGYIPLGTRPPT (SEQ ID NO. 4)

HPV16L2 (17-36)

DNA: CAGCTGTACAAGACCTGCAAGCAGGCCGGCACCTGCCCCCCTGACATCATC CCCAAGGTG (SEQ ID NO. 11)

aa: QLYKTCKQAGTCPPDIIPKV (SEQ ID NO. 5)

BPVL2 (1-88)

DNA:

ATGAGCGCCCGGAAGCGGGTGAAGCGGGCCAGCGCCTACGACCTGTACCGGA CCTGCAAGCAGGCCGGCACCTGCCCCCCTGACGTGATCCCCAAGGTGGAGGG CGACACAATCGCCGACAAGATCCTGAAGTTCGGCGGCCTGGCCATCTACCTG GGCGGCCTGGGCATTGGCACCTGGTCCACCGGCAGAGTGGCCGCTGGAGGAA GCCCTAGATACACCCCCCTGCGGACCGCCGGCAGCACAAGCAGCCTGGCCAG CATCTGATGA (SEQ ID NO. 12)

aa :

MSARKRVKRASAYDLYRTCKQAGTCPPDVIPKVEGDTIADKILKFGGLAIYL GGLGIGTWSTGRVAAGGSPRYTPLRTAGSTSSLASI (SEQ ID NO. 6)

The E7 peptide was selected for introduction into a L1-E7 or L1/L2-E7 chimaera so as to obtain polymeric presentation of several E7 peptides, which is anticipated will lead to a vaccine with both prophylactic and therapeutic responses.

The sequence of human CTL epitope for HLA-A2 that was used is TLGIVCPI (SEQ ID NO. 7) (position 86-93 of E7/HPV-16 protein sequence). For preclinical studies in the mouse model, a CTL epitope RAHYNIVTF (SEQ ID NO. 8) (position 49-57 of E7/HPV-16 protein sequence) was used.

The following human codon optimised chimaeras were created:

1. HPV16 L1/L2 (108-120) referred to as SAF (SEQ ID NOs: 15 and 23);

2. HPV16 L1/L2(56-81 ) (SEQ ID NOs: 16 and 24);

3. HPV16L1/L2 (17-36) (SEQ ID NOs: 17 and 25); 4. HPV16L1/ BPVL2 (1-88) (SEQ ID NOs: 18 and 26);

5. HPV16L1 + E7(mouse epitope) denoted as L1/E7/M (SEQ ID NOs: 19 and 27);

6. HPV16L1 + E7(human epitope) denoted as L1/E7/H (SEQ ID NOs: 20 and 28);

7. HPV16L1/L2 + E7(mouse epitope) denoted as L1/L2/E7/M (SEQ ID NOs:

21 and 29); and

8. HPV16L1/L2 + E7(human epitope) denoted as L1/L2/E7H (SEQ ID NOs:

22 and 30).

The human codon optimised HPV-16 L1/L2 (108-120) nucleotide sequence (SEQ ID NO: 23) was made by Geneart with relevant cloning sites at the 5' and 3' ends. Internally, there was a Pstl site at the F position (amino acids 406 and 407 LQ), which allowed the applicant to clone different nucleotide sequences into that position (Figure 15).

In addition, seven human codon optimised nucleotide sequences were made by Geneart from the F position to the end of gene, in order to insert human codon optimised L2 or E7 nucleotide sequences (SEQ ID NOs: 9 to 14 and 32) (Figure 13), and these were cloned into the L1 sequence above by use of Pst\ and Xho\, thereby replacing the existing sequence from the Pst\ site onwards.

Human codon optimised HPV-16 L1/L2 (108-120) was cloned into pGA4 with HindW and Xho\ (also called SAF-MOD). This clone was modified by inserting the relevant epitopes into the E- or F-loops (F starts at nucleotide position 1239 and amino acid 414; E loop starts at nucleotide position 1296 and amino acid at position 433) by replacing the whole Pst\-Xho\ fragment on the C-terminus with the L2 or E7 sequences. These latter fragments were cloned from PCR4-TOPO-Blunt with Pst\ and Xho\ (Figure 2). Genes were sequenced to confirm the sequence of the new chimaeras.

Example 1 : Expression in insect cells:

Cloning into insect cell expression vectors

The human codon optimised chimaeras were cloned from the pGA4 plasmid into the pFastBacDual under the control of P_PH. SAF-MOD was cloned into pFastBacDual using EcoRI and the orientation was checked. All the other human codon optimised chimeric nucleotide sequences were excised with EcoRI and Xho\ and cloned into PFastBacDual digested with EcoRI and Sa/I. Sail and Xho\ are compatible, allowing this insertion.

Insect cell production of HPV-16 Chimaeras via baculovirus expression

All work on the baculovirus-produced HPV-16 chimaeras was performed via the Bac- to-Bac baculovirus-derived expression system (Invitrogen). Viral stocks for HPV-16 chimaeras were obtained, plaque purified and titred. Each chimaera was verified ustilising PCR, which will only amplify one specific chimaera (Table 2 and Figure 3).

The basic protocol for all the PCR reactions was as follows:

95 °C for 2min

X25

72 °C for 3 minutes

Table 2: Primers used in PCR and sequencing of the human codon optimised

HPV chimaeras

Primer Chimaera Primer name Primer sequence Product target construct size (bp)

Vector All chimaeras pTRAc Fwd b'-CA I I I OA I I I GGAGAGGACACG-3' -1800

(SEQ ID NO: 33)

pTRAc Rvs 5'-GAACTACTCACACATTATTCTGG- 3'(SEQ ID NO: 34)

Chimaera All L1/L2 Mod New Fwd 5'-CGACGACCTGTACATCAAGG- chimaeras 3'(SEQ ID NO: 35)

L1/L2(108-120) VEET Rvs 5'-GATGAAGCTGGTCTCCTCC- 410

3'(SEQ ID NO: 36)

L1/L2(56-81 ) SAF2 Rvs 5'-GGATGTAGCCGGTCCTGC-3'(SEQ 442

ID NO: 37)

L1/L2( 17-36) QLYK Rvs 5 -ACCTTGGGGATGATGTCAGG-3' 444

(SEQ ID NO: 38)

L1/L2 BPV(1-88) SALIBPV Rvs 5'-TATCTAGGGCTTCCTCCAGC-3' 559

(SEQ ID NO: 39) Chimaera L1/E7M RAHY Fwd 5'-CCACTACAACATCGTGACCTTC-3' 224

(SEQ ID NO: 40)

L1/L2/E7M 167

L1/E7H TLGI Fwd 5'-CTGGGCATCGTGCCCTATC-3' 227

(SEQ ID NO: 41)

L1/L2/E7H 176

All E7 chimaeras EndMod Rvs b'-CA I UAUAGC I I CCG M I CH CC-3'

(SEQ ID NO: 42)

Production of human codon optimised HPV-16 L1 and chimaeric human codon optimised SAF was optimised using several insect cell lines, namely Sf21 , Sf9, High Five and TniPro (a derivative of High Five cells) insect cells, that were grown in serum free medium as well as in medium with added serum as shaker cultures or in monolayers. The multiplicity of infection, time of infection and time of harvest were also optimised in these cultures.

All other human codon optimised chimaeras were produced in Sf-9 cells, infected at a multiplicity of infection of . The density of the cells at the time of infection was 1 x 10⁶cells/millilitre.

Chimaera purification

Cells were resuspended in Dulbecco's PBS with protease inhibitor at a cell density (as measured before infection) in a range of 1x10⁶-50x10⁶ cells per ml. The cells were sonicated for 3 times at 20 second intervals. Cells were then pelleted and the supernatant mixed with a solution of Optiprep^® in Dulbecco's PBS with protease inhibitor at a final concentration of 24%. The solutions were centrifuged in the SW55Ti rotor at a speed of 116000xg for about 16 hours. The lower band was extracted.

Purification of baculovirus produced SAF and HPV-16 L1 VLPs

The human codon optimised SAF chimaeric pentamers and human codon optimised L1 VLPs were partially purified and concentrated by microfiltration, ultrafiltration and affinity/ion exchange chromatography. A heparin-based affinity column and Poros ion exchange column was used for capturing and partially purifying HPV-16 L1 protein and the chimaeric SAF. Before purification on the affinity columns, the cell lysate from the baculovirus culture was clarified by filtering through a 0.45μι^η microfiltration membrane. The L1 protein was then enriched on a Hollowfiber cross- flow filtration system that removed more than 80% of unwanted protein whilst the majority of the L1 in the starting material was retained using a membrane with a nominal molecular weight cut-off (NMWC) of 300kDa. Differential precipitation of HPV-16 and SAF proteins using polyethylene glycol or ammonium sulphate was investigated.

Example 2: Expression of HPV chimaeras in tobacco plants

The human codon optimised HPV-16 L1/L2, L1/E7 and L1/L2/E7 chimaera sequences were excised from pGA4 vectors and directionally subcloned into pTRAc, and pTRAkc-rbcs1-cTP provided by Rainer Fischer (Fraunhofer Institute for Molecular Biology and Applied Ecology, Germany) using the Aflll - BspHI and Xhol or Mlul and Xhol restriction sites that flanked the chimaeric genes (Figures 2 and 6). The chimaeras were also cloned into pRIC3 utilising Hindlll and Xhol. The plasmid constructs used in this study are shown in Table 3. DH5-a chemically competent E. coli cells (E.cloni™, Lucigen) were transformed with the pTRAc-rbcs1-cTP chimaera constructs and recombinants were selected using ampicillin-resistance (100pg/ml).

Table 3: Agrobacterium expression constructs

Expression

Vector Chimaera Construct name Target

pTRAc-L1/L2(108- pTRAc L1/L2(108-120) 120) Cytoplasm

L1/L2(56-81) pTRAc-L1/L2(56-81)

L1/L2(17-36) pTRAc-L1/L2( 17-36)

pRIC L1/L2(108-120) pRIC-L1/L2(108-120) Cytoplasm

L1/L2(56-81) pRIC-L1/L2(56-81)

L1/L2(17-36) pRIC-L1/L2(17-36)

pTRAkc-rbcsl- cTP L1/L2(108-120) cTP-L1/L2(108-120) Chloroplast

L1/L2(56-81) cTP-L1/L2(56-81)

L1/L2(17-36) cTP-L1/L2(17-36)

L1/L2(BPV 1- 88) CTP-L1/L2 BPV(1-88)

L1/E7(49-57) CTP-L1/E7M

L1/L2(108- CTP-L1/L2/E7M 120)/E7(49-57)

L1/E7(86-93) CTP-L1/E7H

L1/L2(108-

120)/E7(86-93) CTP-L1/L2//E7H

Colonies were screened for the presence of the HPV-16 chimaera by colony PCR using vector-specific primers flanking the multiple cloning site (Table 2). PCR was performed using GoTaq Flexi DNA Polymerase kit (Promega) as per the manufacturer's instructions using 1 μΜ of each primer in a final magnesium chloride concentration of 3mM. The PCR profile consisted of an initial denaturation step at 95°C for 3 min, followed by 25 cycles at 95°C for 30s, 59°C for 30s and 72°C for 3 min, and a final elongation step at 72°C for 3 min. PCR products were separated on a 0.8% TBE agarose gel and detected using ethidium bromide staining.

Recombinant human codon optimised clones were further verified by restriction enzyme digestion using EcoR1 and Xhol. An additional colony PCR reaction using individual chimaera-specific primers was done to verify that each clone contained the correct chimaera insert (Table 2). The PCR profile for the human codon optimised L1/L2 chimaeras consisted of an initial denaturation step at 95°C for 2 min, followed by 25 cycles at 95°C for 30s, 55°C for 20s and 72°C for 30s, and a final elongation step at 72°C for 3 min. The PCR profile for the human codon optimised E7 chimaeras was similar, except that an annealing temperature of 61 °C was used. Human codon optimised clones were also sequenced using vector-specific primers to verify the presence of the chimaera nucleotide sequence insert.

Agrobacterium transformation

Agrobacterium tumefaciens GV3101 ::pMP90RK cells were made electrocompetent using the method described by Shen and Ford, 1989. Transformation of Agrobacterium was performed as described by Maclean et al. (2007) and clones were screened by antibiotic selection (50 pg/ml carbenicillin, 50 Mg/ml rifampicin and 30 g/ml kanamycin). Successful transformation was confirmed by colony PCR (using vector-specific and chimaera-specific primers) and restriction enzyme digestion of back-transformed £ coli clones.

Aqroinfiltration A. tumefaciens recombinant chimaera cultures and A. tumefaciens LBA4404 cultures containing the pBIN-NSs plasmid (Takeda et al. 2002) encoding the TSWD NSs silencing suppressor were prepared for infiltration as described by Maclean ef al. (2007). The Agrobacterium suspensions were diluted in infiltration media to give a final OD₆oo of 0.25 for individual Agrobacterium chimaera strains, and a combined OD₆₀₀ of 0.5 for the constructs co-infiltrated with A. tumefaciens LBA4404 (pBIN- NSs). The strains were incubated at 22°C for 2 hours to allow for expression of the vir genes prior to infiltration. Six-week old N. benthamiana tobacco leaves were agroinfiltrated by injecting the bacterial suspension into the abaxial air spaces from the ventral side of the leaf (Maclean ef al., 2007). The plants were grown under conditions of 16h light, 8h dark at 22°C for the desired time period and separate plants were used for each construct. Each agroinfiltration experiment was performed in duplicate.

Detection of chimaera expression in plants

Leaf disks, cut using the cap of an eppendorf tube, were harvested from agroinfiltrated leaves and ground in 250μΙ per disk high-salt PBS (0.5M NaCI) extraction buffer containing protease inhibitor (EDTA-fee Complete Protease Inhibitor; Roche). The crude extract was clarified twice by centrifugation at 13,000 rpm for 5 min and stored at -20°C.

For western blot analysis, the plant extracts were incubated at 85°C for 2 min in loading buffer (Sambrook et al., 1989), separated by a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane by semi-dry electroblotting. HPV-16 L1 protein was detected with either monoclonal (mAb) CamVirl anti-HPV-16 L1 antibody (1 :10000, Abeam, Cambridge) which binds to the aa 230-236 L1 linear epitope (Maclean et al, 1990), or H16.J4 (1 :2500) which binds to the aa 261-280 L1 linear epitope (Christensen ef al., 1996), followed by secondary goat-anti-mouse- alkaline phosphatase conjugate (1 :10000; Sigma). Membranes were developed with Nitro blue tetrazolium chloride/5-broma-4-chloro-3-indoyl phosphate (NBT/BCIP tablets; Roche). A comparison of the relative chimaera concentrations was acheived by measuring the density of the bands detected on anti-HPV-16 L1 western blots using GeneTools (SYNGENE).

Protein quantification The HPV-16 chimaeras extracted from N. benthamiana were quantified by capture ELISA using a modified polyvinyl alcohol (PVA)-blocking ELISA method (Studentsov et al., 2002). Briefly, a 96-well Maxisorp microtitre plate was coated with mAb H16.J4 (1 :2000) overnight at 4°C and blocked with PVA. Plant extract was added to the wells and incubated for 1 hr at 37°C. This was followed by a washing step and the addition of rabbit anti-HPV-16 polyclonal serum (1 :1000). The plate was incubated overnight at 4°C and HPV-16 L1 protein was detected with swine anti- rabbit horseradish peroxidase (HRP) conjugate (1 :5000; DAKO; Denmark) and 1.2- phenylenediamine dihydrochloride substrate (OPD; DAKO; Denmark). Insect cell- derived HPV-16 VLPs of a known concentration were used as standards and each sample was analysed in triplicate. Total soluble protein (TSP) for each crude leaf extract was determined using the Lowry protein assay (Biorad) as per the manufacturer's instructions. The concentration of the HPV chimaeras (pg/ml) was expressed as a percentage of TSP in order to account for differences in leaf tissue mass and protein extraction efficiency.

Immunocapture electron microscopy

Crude protein extracted from N. benthamiana five days post-infiltration (dpi) was analysed using immunocapture electron microscopy. The extract was immunotrapped with H16.V5 antibody (1 :1000) on glow-discharged carbon-coated copper grids, stained with 2% uranyl acetate and viewed using a JOEL 200 EX or a LEO 912 transmission electron microscope.

Chimaera expression and extraction

Five N. benthamiana plants were co-infiltrated by injection with the Agrobacterium GV3101 strain containing the human codon optimised HPV-16 L1/L2(108-120) construct and A. tumefaciens LBA4404 (pBIN-NSs). At seven dpi, the infiltrated leaves were weighed and ground in liquid nitrogen using a mortar and pestle. High- salt PBS (0.5M NaCI) was added at a ratio of 1 :15 (w/v) and samples were homogenized at 24000 rpm on ice. The homogenate was filtered through cheesecloth, protease inhibitor was added and the crude extract was clarified by centrifugation at 10 000 rpm for 5 min.

Example 3: Expression of HPV chimaera in yeasts The chimaera HPV-16 L1/L2 (108-120), or yeast (SAF), was double codon optimized for both yeasts P. pastoris and H. polymorpha and synthesized by Geneart (SEQ ID NO: 31 ). The relevant cloning sites Hind\\\, EcoR\ were incorparated at the 5'- end as well as BamHI, Xho\ at the 3' end to facilitate cloning into the relevant expression plasmids.

Yeast SAF, was sub-cloned into pHIPX4-HNBESX using HindW and BamHI for integration into H. polymorpha and in parallel sub-cloned into pBLHIS-IX using EcoRI and Xho\ for integration into P. pastoris (Figure 16, 17).

Human codon-optimized HPV-16 L1/L2 (108-120), or human SAF, was sub-cloned into the H. polymorpha expression plasmid using HindlW and BamHI. For expression in P. pastoris the cassette was cloned using ircoRI and the correct orientation confirmed.

Confirmation of yeast integration

The yeast expression plasmids were linearized and transformed into the respective hosts and recombinant colonies were selected following incubation on auxotrophic selective plates. PCR was used to screen the recombinants and confirm integration of the cassettes.

The basic protocol for all the PCR reactions was as follows:

Initial denaturation: 95 °C for 2min

28 cycles of: Denaturation - 95 °C for 30sec

Annealing - 52°C for 30 sec

Elongation - 72°C for 90 sec

Final elongation: 72°C for 3 minutes

Table 4: Primers used in PCR of the yeast codon optimised HPV chimaeras in yeast

Primer Sequence Primer Target Amplicon target gene size pHIPX4-F 5'-CAGTGCACGGTGGTGACATC- H. SAF 1626 bp

3' (SEQ ID NO: 43) polymorpha

pHIPX4-R 5'-GTCATGGCGTAGGAAGGCTG- genome L1 1637 bp

3 (SEQ ID NO: 44)

5'- AOX 5'- P. pastoris SAF 1809 bp

GACTGGTTCCAATTGACAAGC- genome

3 (SEQ ID NO: 45) 3'- AOX 5'- L1 1859 bp

GCAAATGGCATTCTGACATCC- 3'(SEQ ID NO: 46)

To confirm correct integration within the yeast genome, Southern blot hybridization was performed. A 845 bp probe of the methanol inducible promoter was labeled using DIG-dUTP. The probes were visualized on the hybridization membrane using NBT/BCIP-T substrates and recombinant colonies displaying the predetermined banding patterns were kept for expression analysis.

Expression in H. polvmorpha cells

Recombinant H. polymorpha colonies were inoculated into minimal media containing glucose and incubated overnight at 37°C with shaking at 200 rpm to provide aeration. Cultures were then diluted into fresh, pre-warmed minimal media containing glucose to an OD₆₀₀ of 0.1 and incubated while shaking at 200 rpm until an OD₆₀o between 1.0 to 1.5 was reached. Mid-exponential cells were diluted into minimal media containing 0.5% [v/v] methanol to an OD₆₀o of 0.1 , so as to induce expression. After 72 h of induction, samples were centrifuged to pellet the cells, the supernatants removed and the pellets were stored at -80°C until ready to assay using SDS-PAGE, western blotting and densitometry. In addition, OD₆₀₀ of cultures were measured using a Pharmacia LKB Ultrospec III spectrophotometer and wet and dry weights (mg/ml) were recorded. H. polymorpha transformed with pHIPX4-HNBESX and untransformed H. polymorpha served as negative controls for human and yeast codon optimised SAF expression.

Expression in P. pastoris cells

P. pastoris KM71 recombinants were cultured and protein expression induced according to Invitrogen's Easyselect™ Pichia Expression Kit Instruction Manual. Cultures were incubated at 28-30°C while shaking at 200 rpm until the culture reached an OD₆₀o of between 2 to 6. After culturing overnight, KM71 cells were centrifuged at 3000 ^χ g for 5 min and the cell pellet was resuspended in 50 ml buffered methanol-complex medium (BMMY) in a 500 ml baffled flask covered with a cotton wool plug to allow 0₂ diffusion. Methanol was supplemented to a final concentration of 1% [v/v] every 24 h to maintain induction. After 72 h of induction, samples were centrifuged to pellet the cells, the supernatants removed and the pellets were stored at -80°C until ready to assay using SDS-PAGE, western blotting and densitometry. In addition, OD₆₀₀ of cultures were measured using a Pharmacia LKB Ultrospec III spectrophotometer and wet and dry weights (mg/ml) were recorded. P. pastoris KM71 and GS115 transformed with pBLHIS-IX and untransformed P. pastoris served as negative controls for human and yeast codon optimised SAF expression.

Western blot analysis

To determine whether recombinant yeast colonies produced the SAF proteins, the yeast cell pellets were thawed and 60 mg of each recombinant were lysed to liberate the proteins. This was done using breaking buffer and equal volume glass beads as outlined in the Invitrogen's Easyselect™ Pichia Expression Kit Instruction Manual. Following extraction of the proteins, an equal volume of SDS-PAGE sample buffer (Sambrook et al., 1989) was added to the samples and boiled for 5 min to allow for denaturing and coating with SDS. This was followed by SDS-PAGE gel electrophoresis and transfer of the proteins to a nitrocellulose by semi-dry electroblotting. For detection, monoclonal antibody CAMVIR-1 was used (1 :15000, Santa Cruise Biotechnology, California) which binds to the aa 230-236 L1 linear epitope (Maclean er al, 1990), followed by secondary goat-anti-mouse-alkaline phosphatase conjugate (1 :5000; Santa Cruise Biotechnology, California). Membranes were developed with Nitro blue tetrazolium chloride/5-broma-4-chloro-3- indoyl phosphate (NBT/BCIP; Fermentas). Protein production levels were accessed using the Cervarix vaccine as a positive control and comparing the density profiles of known concentrations of the Cervarix HPV16L1 protein to that human and yeast codon optimised SAF. Levels of yeast SAF were then compared to that obtained for humanised L1 (hL1).

Results

Expression in insect cells

Each chimaera was verified by PCR to check purity of each viral infectant (Figure 3). Viral stocks were titered and insect cells infected with chimaeras at MOI of 1. Cell lysates were run on acrylamide gels and western blots probed with HPV16 H16:J4 anti L1 antibodies revealed that human codon optimised SAF yielded most protein (Figure 4). Purification of pentamers and VLPs utilizing optiprep allowed determination of yield. The yield varied depending on insertion at F position in L1. The human codon optimised 108-120 L2 sequence and 17-36 sequence revealed highest yields with 50 and 44pg/ml yield, respectively. Other amino acid replacements, such as human codon optimised L2 56-81 or BPV L2 1-88 caused a decrease in expression of the resulting protein (Figure 5).

It is also significant that the chimaeric vaccine candidate, human codon optimised SAF, showed better yields than unmodified L1 in insect cells.

The highest human codon optimised SAF yield of approximately 250mg/L was obtained utilising a bioreactor by infecting at an MOI 1.0, TOI 1.0 x 10⁶ cells/ml after 96 h. A very similar yield of 218 mg/L was obtained by infecting at MOI 0.5. Under these conditions hl_1 was expressed to 200mg/L.

On smaller scale expression of human codon optimised SAF was about 10 fold lower. Levels of human codon optimised SAF expression in insect cells was 23pg/ml in 4 days or 21 pg/106 cells. Human codon optimised chimaera L2(56 -81)and L2(17- 36) expressed 35 pg/ml and 135 pg/ml respectively and the other 3 human codon optimised chimaeras expressed only 1.5 - 14pg/ml. In contrast hL1 expression was ~5pg/ml about 4 fold lower than human codon optimised SAF in the same setup.

Construction of HPV-16 chimaera plasmids in plant expression vectors

The 8 human codon optimised HPV-16 chimaera constructs were successfully cloned into the plant expression vectors pTAc, pTRAkc-rbcs1-cTP and pRIC3 and transformed into E. coli and Agrobacterim GV3101. Recombinant clones were screened by colony PCR with vector-specific primers and all the constructs gave amplification products of the expected size (data not shown). Construct clones were also verified by means of EcoRI/Xhol restriction enzyme digests (data not shown). As the chimaera inserts were all ~1.5 kb in size and produced similar restriction enzyme patterns, colony PCR using human codon optimised chimaera-specific primers was used to further verify the presence of the correct chimaera sequence in the vector and all constructs gave specific chimaera amplification products of the expected size (data not shown). In addition, sequencing analysis confirmed that the human codon optimised HPV-16 chimaera E. coli clones contained the intact HPV chimaeric sequence and the correct L2 or E7 epitope.

Agrobacterium-mediated transient expression of human codon optimised HPV-16 L1 and chimaeras in N. benthamiana Chloroplast-targeted hl_1 yielded from 230mg to 533mg of L1/kg plant material, representing 12% or 17.1% of TSP, respectively. This is a highly significant increase in yield compared with N. tabacum cv. Xanthi plants transformed with hl_1 and plant- codon-optimised L1 in a non-targeting transformation vector, where hl_1 expression was less than 1 % TSP. The yields obtained in our latest experiments are >100 000- fold higher than transgenic tobacco expression of native HPV-16 L1 achieved in previous work by the applicant. One unexpected result was the steep decline in yield of VLPs with successive generations of plants, presumably due to silencing, which is occasionally a problem with transgenic plants.

The figure of 12% total soluble protein for hL1 in transgenic plants should allow much easier production than was originally anticipated: the yield is well above (ΊΟχ) the "commercially viable" limit of 25 mg/kg considered as the lower end of a viable yield by Large Scale Biology Corp. in their established manufacturing processes.

Chloroplast- and cytoplasm-targeted HPV-16 L1/L2 chimaera expression

The N. benthamiana transient expression profile of the human codon optimised L1/L2 chimaera constructs in pTRAkc-rbcs1-cTP, pTRAc and pRIC3 was evaluated in a 1-9 day post-infiltration (dpi) time trial. The Agrobacterium GV3101 pTRAkc-rbcs1-cTP human codon optimised L1/L2 chimaera strains were infiltrated either with (+) or without (-) A. tumefaciens LBA4404 (pBIN-NSs) to determine the effect of the NSs silencing suppressor on transient protein expression and accumulation. As a positive control, N. benthamiana plants were co-infiltrated with pBIN-NSs and a %GC- modified HPV-16 L1/L2 (108-120) chimaera construct in pTRAkc-rbcs1-cTP which is highly expressed in plants. A plant infiltrated with Agrobacterium LBA4404 (pBIN- NSs) was used as a negative control for the plant's response to infiltration and the presence of the NSs protein.

Anti-HPV-16 L1 western blot time trial expression profiles for human codon optimised HPV-16 L1/L2(108-120), L1/L2(56-81 ), L1/L2(17-36) and L1/L2 BPV(1 -88) expressed in the chloroplast are shown in Figure 7. The pBIN-NSs-infiltrated plant extract 5 dpi was used as a negative control. Two positive controls were included; the 5 dpi N benthamiana-expressed %GC-modified L1/L2(108-120) chimaera and insect cell- expressed HPV-16 l_1/L2(108-120). The 5 dpi plant extracts were chosen as previous studies have indicated that HPV plant-expressed protein in crude leaf extracts 5 dpi usually have the highest protein levels. All time trials were done in duplicate and western blot analysis and TSP assays were performed on both time trial samples.

The human codon optimised chimaeras are all -56 kDa in size and were analysed using a plant-derived and insect-cell derived L1/L2(108-120) chimaera control to confirm the specificity of the antibody. Previous studies with insect cell-derived HPV- 16 L1/L2(108-120) chimaeras have shown that L1/L2(108-120) runs higher than other L1/L2 chimaeras on western blots (see Slupetzky ei a/., 2007), however DNAMAN analysis: confirms the chimaera size as 56 kDa. The NSs-infiltrated plant extract (negative control) was not detected by western blot analysis thus confirming native plant proteins and NSs proteins do not react with the anti-HPV antibodies.

To further determine expression levels of the human codon optimised L1/L2 chimeras, they were compared when targeted to the chloroplast (pTRAc-rbcs-cTP) or when targeted to the cytoplasm utilizing two different expression vectors, pTRAc and pRIC3 (Figure8). When the product is targeted to the cytoplasm it becomes very clear that hL1 expression levels are reduced compared to the human codon optimised SAF or human codon optimised L1/L2(17-36) (Figure 8, Table 5). Expression levels of the human codon optimised L1/E7 and human codon optimised L1/L2/E7 chimaeras were much lower than the human codon optimised L1/L2 chimaeras both in cytoplasm and chloroplast.

Both human codon optimised SAF and hL1 expression are enhanced when the product is targeted to the chloroplast (Figure 8 and 10). All other human codon optimised L1/L2 chimaeras expressed better when accumulated in the chloropast then when expressed in the cytoplam of plants (Table 5).

Direct comparison of hl_1 and human codon optimised SAF expression revealed that human codon optimised SAF expression is about 7 fold more compared to hl_1 when targeted to the cytoplasm (Table 5, Fig 8, 9 and 10). The human codon optimised L1/L2 (17-36) chimaera expression levels were also about four fold more than hl_1 and in contrast the human codon optimised L1/L2 (56-81) had only slightly increased expression levels when compared to hl_1.

When the products were targeted to the chloroplast, the yield of human codon optimised SAF and the other human codon optimised L1/L2 chimaeras is not increased, which indicates that the increase detected might due to differences at the transcription and translation level, but when the protein is stored in an organelle, such as the chloroplast, this difference can be overcome.

Table 5: Comparison of human codon optimised HPV chimaera yields (%TSP) using different plant expression vectors*

*Values calculated for 2 replicate timetrials In previous experiments the expression levels of the human codon optimised chimaeras in the chloroplast were 2057 mg/kg plant material for L1/L2 (108-120), 3455 mg/kg for L1/L2 (17-36) and 710 mg/kg for L1/L2 (56-81 ). This represents a 10-fold increase to the hl_1 production in plants, which averaged about 533mg/kg plant material. The other human codon optimised chimaeras L1/L2 BPV (1-88) and the L1/E7 and L1/L2/E7 all were expressed to about 30mg/kg plant material or less.

Electron microscopy revealed that human codon optimised L1/L2 BPV (1-88) formed VLPs in plants, whereas the other human codon optimised L1/L2 chimaeras formed only capsomers (Figure 11).

Construction of HPV-16 chimaera plasmids in yeast expression vectors

The yeast optimized construct HPV16 L1/L2 (108-120), referred to as yeast SAF, were successfully sub-cloned into yeast expression vectors, pBLHIS-IX and pHIPX4- HNBESX. Subsequent to transformation in £. coli, recombinant clones were screened by colony PCR with vector-specific primers and amplification products of the expected 1.6 kb size were identified. Recombinant vectors pBLHIS-IX-SAF and pHIPX4-HNBESX-SAF were transformed into P. pastoris and H. polymorpha, respectively and positive yeast transformants were identified by means of yeast colony PCR (Figure 18 A, B).

In the case of H. polymorpha, Southern blot hybridization was also used to confirm that integration occurred at the correct genomic locus and as an indicator of relative plasmid copy number (Figure 18 C).

Yeast HPV-16 SAF expression

Following transformation of P. pastoris KM71 and GS115, several putative recombinant colonies were screened for expression of yeast SAF. One P. pastoris KM71 strain, KMSAF3, was identified for cultivation. One single copy integrant (S) and one multi copy integrant (L) identified during Southern hybridization of H. polymorpha was selected for cultivation. Shake-flask cultivations were performed and the presence of the yeast SAF protein was detected using western blots. Levels of yeast SAF in H. polymorpha were higher in L5 than S5 (Figure 19 A, B).

Table 6: Expression levels of yeast SAF3

Yeast strain Shake-flask cultivation Fermentation levels levels mg.l^"1 mg.l^"1 Yeast optimized constructs:

KMSAF3 139 Not done

HpSAFL5 38 133

Human optimized constructs:

X33L1 ND 1x10^"4

HpL1 ND Not done

HpSAF Not done Not done

Hp - H. polymorphs; KM and X33 - P. pastoris strains; ND - none detected

Summary of expression for insect, plant and yeast systems

Insect cells

In insect cells, production yields of human codon optimised SAF (L1/L2 (108-120)) were up to levels of 250 mg/L to 1g/L in the High Five derivative cells, TniPro cells, cultivated in ESF-AF animal-free medium, whereas wildtype L1 was only expressed to 40 mg/L. The human codon optimised L1 expressed about 4 fold better than the wild type L1. Thus, human codon optimisation allowed significantly greater expression, but the chimaeric protein expressed considerably better than the native polypeptide. Neither of these findings, but more particularly the second, is obvious in the light of any predictions from theory or from previous work. Other insertions, such as L2 17-36 and E7M, at the same site (aa 414 of the L1 polypeptide) also increased expression levels to those similar to SAF, which was unexpected.

Plant cells

HPV chimaeras were successfully expressed in Nicotiana benthamiana.

Expression of the human codon optimised L1/L2 chimaeras was optimized by co- expression with NSs and protein targeting to the chloroplast.

Plant expression systems have potential for the production of low-cost human codon optimised L1/L2 HPV-16 chimaera candidate vaccines. The yield of all these chimaeras is well above the "commercially viable" limit of 25 mg/kg considered as the lower end of a viable yield.

Expression of the human codon optimised L1/L2 BPV(1-88), L1/E7 and L1/L2/E7 chimaeras was less successful and requires further optimisation. The large difference in expression levels depending on which sequence is inserted, and where, could not have been predicted. When targeted to the cytoplasm, human codon optimised L1/L2 (108-120), also called human SAF, expression levels were 200mg/kg and hL1 levels were about seven fold lower at 30mg/ml. Human codon optimised L1/L2 (17-36) were 110mg/kg and human codon optimised L1/L2 (56-81 ) were 40mg/kg.

The human codon optimised L1/L2 chimaeras expressed up to 10 times higher levels when targeted to the chloroplast than did the hi_1. Chloroplast-targeted hl_1 yielded up to 530 mg of L1/kg plant material, whereas human SAF, human codon optimised L1/L2 (17-36) and human codon optimised L1/L2 (56-81) yielded 800 - 5000 mg of protein/kg plant material. Nevertheless, this represents 19.9, 14.7 and 4.96% of TSP compared to hl_1 , which represented about 17%TSP, indicating that the increase in expression detected might due to differences at the transcription and translation level, but that when the protein is stored in an organelle, such as the chloroplast, this difference can be overcome.

These findings reinforce the empirical nature of the expression of L1 in plant cells: it was not predictable that humanised genes would express better than native genes; neither was it obvious that insertion of some (but not all) peptides would further increase expression levels, or that different proteins would accumulate or express better when localised to chloroplasts or the cytoplasm.

In yeast the expression levels were increased from non-detectable levels of hl_1 to 30 -140mg/L for yeast codon optimised SAF, indicating a 10 - 100 fold increase in production.

Conclusion

The results obtained by the applicant were unexpected and surprising, and showed that by changing 10 amino acids or more at the F position of L1 and codon optimising the sequence, the yield in protein produced in different systems, i.e. insect cells and plants, can be enhanced by up to 100 fold utilising this approach. It was also demonstrated that a similar strategy used in yeast, but with yeast codon optimisation, enhanced expression by 10 to 100 fold. This in turn, will reduce costs of vaccine production considerably and could allow the development of second generation vaccines that will protect against more than one type of HPV with one polypeptide.

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Claims

A method of producing a chimaeric HPV L1 polypeptide with increased expression levels relative to a L1 polypeptide, the method comprising the steps of:

(i) codon optimising a first nucleotide sequence encoding a first papillomavirus peptide or polypeptide which has been shown to elicit an immune response in humans or animals;

The method according to claim 1 , wherein the first peptide or polypeptide is an HPV or BPV L2 peptide.

The method according to claim 2, wherein the HPV or BPV L2 peptide is selected from any one of SEQ ID NOs: 3 to 6.

The method according to claim 1 , wherein the first peptide or polypeptide is a human or mouse papillomavirus E7 peptide.

The method according to claim 4, wherein the human or mouse papillomavirus E7 peptide is selected from either one of SEQ ID NOs: 7 or 8.

The method according to claim 1 , wherein the codon optimised nucleotide sequence encoding the first peptide or polypeptide is any one of SEQ ID NOs: 9 to 14, 32 or 47.

7. The method according to any one of claims 1 to 6, wherein the second codon optimised HPV L1 nucleotide sequence is modified to be nuclear localisation signal deficient.

8. The method according to any one of claims 1 to 7, further comprising a step of inserting a third codon optimised nucleotide sequence into the second codon optimised HPV L1 nucleotide sequence at or about any one of amino acid positions 430 to 434 of the HPV L1 polypeptide, the third codon

• optimised nucleotide sequence encoding a third immunogenic papillomavirus peptide or polypeptide.

9. The method according to any one of claims 1 to 8, wherein the first and/or third HPV peptide or polypeptide are at least 10 amino acids in length.

10. The method according to either claim 8 or 9, wherein the third codon optimised nucleotide sequence is either a mouse or human E7 epitope sequence.

11. The method according to claim 10, wherein the mouse or human E7 epitope nucleotide sequence is any one of SEQ ID NOs: 13, 14, 32, or a sequence encoding either of SEQ ID NOs: 7 or 8.

12. The method according to any one of claims 1 to 11 , wherein the first codon optimised nucleotide sequence and optionally also the third codon optimised nucleotide sequence replace the nucleotides of the codon optimised HPV L1 nucleotide sequence at the point of insertion.

13. The method according to any one of claims 1 to 12, wherein the expressed chimaeric HPV L1 polypeptide is recovered from the cell.

14. The method according to any one of claims 1 to 13, wherein the cell is an insect, animal, plant or yeast cell.

15. The method according to any one of claims 1 to 14, wherein the codon optimised nucleotide sequences are human codon optimised for expressing the chimaeric HPV L1 polypeptide in an insect cell, human or plant codon optimised for expressing the chimaenc HPV L1 polypeptide in a plant cell, or yeast codon optimised for expressing the chimaeric HPV L1 polypeptide in a yeast cell.

16. The method according to any one of claims 1 to 15, wherein the HPV L1 polypeptide is a HPV-16 L1 polypeptide.

17. The method according to claim 16, wherein the HPV-16 L1 polypeptide is as set out in SEQ ID NO: 1 and the HPV-16 L1 polypeptide is encoded by a codon optimised HPV-16 L1 nucleotide sequence as set out in SEQ ID NO: 2.

18. The method according to any one of claims 1 to 17, wherein the chimaeric HPV L1 polypeptide sequence is selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 and 22.

19. The method according to any one of claims 1 to 18, wherein the chimaeric codon optimised HPV L1 nucleotide sequence is selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30 and 31.

20. The method according to any one of claims 1 to 19, further comprising, following step (ii), introducing the codon optimised chimaeric HPV L1 nucleotide sequence into an expression vector and in step (iii) introducing the expression vector comprising the chimaeric codon optimised HPV L1 nucleotide sequence into the cell.

21. The method according to claim 20, wherein the expression vector is adapted for expression of polypeptides in plant, insect, animal or yeast cells.

22. The method according to claim 21 , wherein the expression vector is adapted to target a component of a plant cell.

23. The method according to claim 22, wherein the plant cell component targeted is a chloroplast. - 37 -

24. The method according to any one of claims 21 to 23, further including in step (iii), introducing into the plant cell a suppressor protein adapted to inhibit post- transcriptional gene silencing in a plant.

25. The method according to claim 24, wherein the suppressor protein is the NSs protein of the tomato spotted wilt virus or the p19 of tomato bushy stunt virus.

26. The method according to any one of claims 1 to 25, wherein the chimaeric HPV L1 polypeptide assembles into a virus-like particle, capsomer or pentamer.

27. The method according to any one of claims 1 to 26, wherein the chimaeric HPV L1 polypeptide assembles into a capsomer or a pentamer.

28. The method according to any one of claims 1 to 27, wherein the chimaeric HPV L1 polypeptide is an immunogenic polypeptide for eliciting a neutralising antibody and/or CTL response in a subject.

29. The method according to claim 28 wherein the chimaeric HPV L1 polypeptide is for eliciting a cross-protective response in the subject.

30. A chimaeric HPV L1 nucleotide sequence comprising a codon optimised nucleotide sequence encoding an HPV L1 polypeptide into which a first codon optimised nucleotide sequence encoding a heterologous papillomavirus peptide has been inserted at or about amino acid position 414 of the HPV L1 polypeptide.

31. The chimaeric HPV L1 nucleotide sequence according to claim 30, further comprising a second codon optimised nucleotide sequence encoding a heterologous papillomavirus peptide inserted at or about any one of amino acid positions 430 to 434 of the HPV L1 polypeptide.

32. The chimaeric HPV L1 nucleotide sequence according to either claim 30 or claim 31 , wherein the first and/or second codon optimised nucleotide sequence encodes an HPV or BPV L2 peptide.

33. The chimaeric HPV L1 nucleotide sequence according to either claim 30 or claim 31 , wherein the first and/or second codon optimised nucleotide sequence encodes either a human or mouse PV E7 peptide.

34. The chimaeric HPV L1 nucleotide sequence according to claim 33, wherein the second codon optimised nucleotide sequence is either a mouse or human E7 epitope nucleotide sequence.

35. The chimaeric HPV L1 nucleotide sequence according to any one of claims 30 to 34, wherein the codon optimised HPV L1 , first and/or second nucleotide sequences are human, plant or yeast codon optimised.

36. The chimaeric HPV L1 nucleotide sequence according to any one of claims 30 to 35, wherein the first codon optimised nucleotide sequence and optionally also the second codon optimised nucleotide sequence replace the nucleotides of the codon optimised HPV L1 nucleotide sequence at the point of insertion.

37. The chimaeric HPV L1 nucleotide sequence according to any one of claims 30 to 36, wherein the codon optimised chimaeric HPV L1 nucleotide sequence is modified to be nuclear localisation signal deficient.

38. The chimaeric HPV L1 nucleotide sequence according to claim 32, wherein the HPV or BPV L2 peptide is selected from any one of SEQ ID NOs: 3 to 6, or a sequence which has at least 95% identity thereto.

39. The chimaeric HPV L1 nucleotide sequence according to either claim 33 or 34, wherein the human or mouse PV E7 peptide is selected from either one of SEQ ID NOs: 7 or 8, or a sequence which has at least 95% identity thereto.

40. The chimaeric HPV L1 nucleotide sequence according to any one of claims 30 to 39, wherein the first and/or second codon optimised sequence is any one of those as set out in SEQ ID NOs: 9 to 14, 32, 47, or a sequence which has at least 95% identity thereto.

41. The chimaeric HPV L1 nucleotide sequence according to any one of claims 30 to 40, wherein the codon optimised chimaeric HPV L1 nucleotide sequence is as set out in any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30 or 31 , or a sequence which has at least 95% identity thereto.

42. An expression vector comprising the codon optimised chimaeric HPV L1 nucleotide sequence according to any one of claims 30 to 41.

43. The expression vector according to claim 42, which is adapted for expression of polypeptides in plant, insect, animal or yeast cells.

44. The expression vector according to claim 43, which is adapted to target a plant cell component.

45. The expression vector according to claim 44, wherein the targeted plant cell component is a chloroplast.

46. A chimaeric HPV L1 polypeptide produced by the method of any one of claims 1 to 29.

47. The chimaeric HPV L1 polypeptide according to claim 46, for eliciting a neutralising antibody and/or CTL response in a subject to which it is administered.

48. The chimaeric HPV L1 polypeptide according to claim 47, which elicits a cross-protective response in the subject.

49. The chimaeric HPV L1 polypeptide according to any one of claims 46 to 48, which is a capsomer, pentamer, or assembles into virus-like particles.

50. The chimaeric HPV L1 polypeptide according to any one of claims 46 to 49, which is selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21 and 22, or a sequence which has at least 95% identity thereto.

51. A pharmaceutical composition comprising a chimaeric HPV L1 polypeptide according to any one of claims 46 to 50, or a chimaeric HPV L1 polypeptide encoded by the chimaeric HPV L1 nucleotide sequence according to any of of claims 30 to 41 , and a pharmaceutically acceptable carrier.

52. The pharmaceutical composition according to claim 51 , further comprising an adjuvant.

53. Use of a chimaeric HPV L1 polypeptide according to any one of claims 46 to 50, or a chimaeric HPV L1 nucleotide sequence according to any of of claims 30 to 41 in the manufacture of a medicament for use in a method of preventing and/or treating HPV infection and/or cervical cancer in a subject.

54. A method for preventing and/or treating an HPV infection and/or cervical cancer in a subject, the method comprising the step of administering a prophylactical!y or therapeutically effective amount of a chimaeric HPV L1 polypeptide according to any one of claims 46 to 50, or a chimaeric HPV L1 polypeptide encoded by the chimaeric HPV L1 nucleotide sequence according to any of of claims 30 to 41 , or pharmaceutical composition according to either claim 51 or 52 to the subject.

55. The method according to claim 54 wherein the subject is a human.