Title of Invention	COMBINED MEASLES-HUMAN PAPILLOMA VACCINE FOR THERAPEUTIC AND PROPHYLACTIC USE
Abstract	The present invention relates to combined vaccines against measles and human papilloma virus (HPV).In particular, the invention relates to recombinant measles virus vectors containing heterologous nucleic acid encoding single or several antigens derived from HPV, preferably, the major capside antigen Li, the minor capside antigen L2, the early gene E6 and the early gene E7 oncoproteins of HPV type 16, and optionally of types 18, 6 and 11. In a first embodiment, prophylactic vaccines are generated expressing HPV antigens, preferably Li and/or L2 such that they induce a potent long-lasting immune response in mammals, preferably humans, to protect against HPV and MV infection. In another embodiment, therapeutic vaccines are generated expressing E6 and E7 proteins, and optionally LI and L2, such that they induced strong immune responses will resolve persistent HPV infections at early or late stages, including HPV-induced cervical carcinoma. In a preferred embodiment, the combined vaccines are easy to produce on a large scale and can be distributed at low cost.

Title of Invention

COMBINED MEASLES-HUMAN PAPILLOMA VACCINE FOR THERAPEUTIC AND PROPHYLACTIC USE

Abstract

The present invention relates to combined vaccines against measles and human papilloma virus (HPV).In particular, the invention relates to recombinant measles virus vectors containing heterologous nucleic acid encoding single or several antigens derived from HPV, preferably, the major capside antigen Li, the minor capside antigen L2, the early gene E6 and the early gene E7 oncoproteins of HPV type 16, and optionally of types 18, 6 and 11. In a first embodiment, prophylactic vaccines are generated expressing HPV antigens, preferably Li and/or L2 such that they induce a potent long-lasting immune response in mammals, preferably humans, to protect against HPV and MV infection. In another embodiment, therapeutic vaccines are generated expressing E6 and E7 proteins, and optionally LI and L2, such that they induced strong immune responses will resolve persistent HPV infections at early or late stages, including HPV-induced cervical carcinoma. In a preferred embodiment, the combined vaccines are easy to produce on a large scale and can be distributed at low cost.

Full Text	FORM 2 THE PATENTS ACT, 1970 (39 of 1970) & The Patent Rules, 2003 PROVISIONAL SPECIFICATION (See section 10 and rule 13) TITLE OF THE INVENTION "COMBINED MEASLES-HUMAN PAPILLOMA VACCINE FOR THERAPEUTIC AND PROPHYLACTIC USE" We, CADILA HEALTHCARE LIMITED, a company incorporated under the Companies Act, 1956, of Zydus Tower, Satellite Cross Roads, Ahmedabad-380015, Gujarat, India. : The following specification describes the invention Zydus-Vac-002 Technical Field of the Invention The present invention relates to combined vaccines against measles and human papilloma virus (HPV).In particular, the invention relates to recombinant measles virus vectors containing heterologous nucleic acid encoding single or several 5 antigens derived from HPV, preferably, the major capside antigen LI, the minor capside antigen L2, the early gene E6 and the early gene E7 oncoproteins of HPV type 16, and optionally of types 18, 6 and 11 such that they induce a potent long-lasting immune response in mammals, preferably humans, to protect against HPV and MV infection. Background information 10 HPVs belong to a large family of small double-stranded DNA viruses that infect squamous epithelia. (For a-recent comprehensive review on papillomaviruses see Howley, PM and Lowy. DR (2007) in: Fields Virology, fifth edition), eds.-in-chief Knipe, DM. &. Howley, P. M. Lippincott Williams & Wilkins, Philadelphia PA 19106, USA, pp.2299-2354 To date, more than 100 genotypes have been described, among which at least 35 types infect the genital tract. Although most of the HPV types produce benign lesions, a small subset of genotypes is strongly associated with the development of high- 15 grade squamous intraepithelial lesions and cervical cancer. This subset has been identified as "high risk" and it is estimated that HPV-16 accounts for approximately 60% of cervical cancers, with HPV-18 adding another 10%-20%. Other high-risk types include types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73. Low-risk HPVs, such as HPV-6 and HPV-11, cause benign genital warts (90% begnin condylomata accuminata are HPV6 or 11 positive). Bivalent vaccines 16/18 eliminating the most common high-risk types may permit to overcome also the low-risk types. An ideal vaccine 20 would protect against other HPV types through use of antigens from different types and/or antigens containing conserved regions. The HPV genome encodes eight proteins. The late LI and L2 genes code for capsid proteins; the early proteins El and E2 are responsible for viral replication and transcription, and E4 is involved in virus release from infected cells. The integration of high risk type HPV viral DNA into host genome results in a loss of El or E2 mediated transcriptional 25 control and consequently in an over-expression of the E6 and E7 proteins responsible of the malignant transformation process. Structural protein LI from high risk types represents an optimal target for prophylactic vaccines. On the other hand, E6 and E7 proteins are obvious therapeutic targets. There are actually several prophylactic HPV vaccine formulations based upon the major viral capsid protein LI, either as a monomer, or as a virus like particle (VLP) from HPV 16 and 18 types and in some cases additional types 30 (W094/00152, W094/20137, WO93/02184 and WO94/05792). VLPs may additionally comprise L2 proteins, for example, L2 based vaccines described in WO93/00436. These vaccines are highly immunogenic and appear safe; however their high cost does not permit generalized access to populations at risk and their HPV type specificity represents another limitation. Therefore, a remaining need exists to develop additional improved vaccines against HPV which should be inexpensive. Moreover vaccinations with antigen mainly induce an antibody specific response that is of little or no benefit 35 on established HPV infection and related-disease. Approaches involving recombinant viral vector vaccines are under development (Poxvirus, Adenovirus, Alphavirus, Poliovirus e Herpes Virus). Adenovirus based vaccine is described, for example, in US2007269409 (WO2004044176) which encodes the E6 or E7 protein of HPV. The adenovirus based vaccine is able to generate long term immunity; however, integration of HPV DNA into the host genome remains possible and may represent a safety 40 limitation. In view of the above shortcomings the use of measles, virus as a vector to express HPV antigens represents an original strategy to develop a prophylactic combined HPV-measles vaccine as well as a therapeutic HPV vaccine. Zydus-Vac-002 Immunisation vectors based on measles virus Measles virus (MV) is a member of the family Paramyxoviridae. The non segmented genome of MV has an anti-message polarity which results in a genomic RNA which, when purified, is not translated either in vivo or in vitro and is not infectious. 5 MV is a major cause of acute febrile illness in infants and young children. According to estimates of the World Health Organisation (WHO), one million young children die every year from measles. This high toll arises primarily in developing countries, but in recent years also industrialised countries such as the USA have been affected again by measles epidemics, primarily due to incomplete adherence to immunisation programs. At present, several live attenuated MV vaccine strains are in use (including the Schwarz, Moraten and Edmonston-Zagreb strains), almost all derived from 10 the original Edmonston strain by multiple passage in non human cells. MV vaccine is proven to be one of the safest, most stable, and effective human vaccines developed so far. Produced on a large scale in many countries and distributed at low cost through the Extended Program on Immunization (EPI) of WHO, this vaccine induces life-long immunity after a single injection and boosting is effective. Protection is mediated both by antibodies and by CD4 and CD8 T cells. Persistence of antibodies and CD8 cells has been shown for as long as 25 years after vaccination. 15 The recombinant measles virus nucleotide sequence must comprise a replicon having a total number of nucleotides which is a mutiple of six. The «rule of six» is expressed in the fact that the total number of nucleotides present in the recombinant cDNA finally amount to a total number of nucleotides which is a multiple of six, a rule which allows efficient replication of genome RNA of the measles virus. The heterologous DNA is cloned in the MV vector within an Additional Transcription Unit (ATU) inserted in the 20 cDNA corresponding to the antigenomic RNA of measles virus. The location of the ATU can vary along said cDNA: it is however located in such a site that it will benefit from the expression gradient of the measles virus. Therefore, the ATU or any insertion site suitable for cloning of the heterologous DNA sequence can be spread along the cDNA, with a preferred embodiment for an insertion site and especially in an ATU, present in the N-terminal portion of the sequence and especially within the region upstream from the L-gene of the measles virus and advantageously upstream from the M gene 25 of said virus and more preferably upstream from the N gene of said virus. The technology permits to produce rescued viruses containing and stably expressing foreign genes suitable for use as combined MV vaccines. As a proof of concept, MV has been used to express antigens derived from SIV, HIV, hepatitis B, mumps, West Nile (WN) Virus and SARSCoV. In most of these studies, recombinant MVs that express heterologous antigens appeared to induce specific humoral neutralizing antibodies in a transgenic mouse model and were 30 shown to induce cellular immune responses to some proteins. At the present, clinical trials with any recombinant vaccine candidate based on MV are only in the planning stage however experimental results support the hypothesis that MV combined vaccines should be as efficient in eliciting long-lasting immune protection against other pathogenic agents as against the vector virus itself. In fact, in the case of MV expressing WNV gpE, a complete protection up to six months has been documented in monkey, and MV expressing a Dengue antigen induced long term production of neutralizing 35 antibodies. Moreover, in transgenic mice and macaques, rescued recombinant MV was capable of inducing specific antibody responses to heterologous antigen in the presence of pre-existing immunity against MV. Rescued live recombinant MV vaccines are easily produced on a large scale in most countries and can be distributed at low cost. Regarding safety, MV replicates exclusively in the cytoplasm, ruling out the possibility of integration into host DNA. These characteristics make rescued recombinant MV vaccine an attractive candidate to be used as a multivalent 40 vaccination vector for HPV antigens. Adult populations, even already MV immunized individuals, may however also benefit from MV recombinant immunization because re-administering MV virus under the recombinant form of the present invention may result in a boost of anti-MV antibodies 3 Zydus-Vac-002 So far, no approach has been developed to produce a vaccine able to induce immunity against MV combined with immunity against HPV. The invention relates in particular to the preparation of recombinant measles viruses, bearing heterologous nucleic acid encoding antigens from HPV. 5 Detailed description of the invention The object of the invention is the production of combined measles-HPV vaccines from a recombinant Measles vector capable of containing stably integrated nucleotide sequences which code for LI, L2, E6 and/or E7 protein from different HPV types. 10 The invention includes the rescue of recombinant MV-HPV viruses which are capable of infection, replication and expression of LI, L'2, E6 or E7 protein in susceptible transgenic mice, monkeys and human host. Furthermore, the invention includes the construction of multivalent recombinant measles-HPV vectors, in which two different antigens are simultaneously cloned and expressed in the same vector, conferring immunity against both of them. 15 Moreover, the invention relates to the combination of different recombinant measles-HPV viruses, each carrying and expressing a gene from a different HPV type, in order to elicit immune response in the host, directed against the different HPV types. Furthermore, the invention comprises a method to produce a vaccine containing such recombinant viruses. The invention finally relates to a vaccine capable to induce a potent and lifelong immune response against HPV 20 and measles virus in human and to prevent from infection and/or treat diseases associated with infection. DESCRIPTION OF THE FIGURES Fig. la. Complete nucleotide sequence of p(+)MV2EZ-GFP (19774 bp). The sequence can be described as follows with reference to the position of the nucleotides: 25 - 592-608 T7 promoter 609-17335 MV Edmoston Zagreb antigenome 4049-4054 Mlu\ restriction site 4060-4065 BssHII restriction site 4066-4782 Green Fluorescent Protein (GFP) ORF 30 - 4786-4791 BssHU restriction site 4798-4803 AatII restriction site 17336-17561 HDV ribozyme and T7 terminator Fig. lb. Complete nucleotide sequence of p(+)MV3EZ-GFP (19774 bp). The sequence can be described as follows with 35 reference to the position of the nucleotides: 4 Zydus-Vac-002 592-608 T7 promoter 609-17335 MV Edmoston Zagreb antigenome 9851-9856 MM restriction s:te 9862-9867 BssHIIrestriction site 5 - 9868-10584 Green Fluorescent Protein (GFP) ORF 10588-10593 BssHII restriction site 10600-10605 Aatll restriction site 17336-17561 HDV ribozyme and T7 terminator Fig. 2a. ANL1TE: this is the HPV16-L1 sequence ORF (1518 bp) cloned by the inventors. The sequence can be described 10 as follows with reference to the position of the nucleotides: 1-3 Start codon 4-1515 HPV-L1 ORF 1516-1518 STOP codon Fig. 2b. HPV16-L2 sequence ORF. The sequence can be described as follows with reference to the position of the 15 nucleotides: 1 -3 Start codon 4-1419 HPV-L2 0RF 1420-1422 STOP codon Fig. 2c. HPV16-E6 sequence ORF. The sequence can be described as follows with reference to the position of the 20 nucleotides: 1 -3 Start codon 4-474 HPV-E6 ORF 475-477 STOP codon 25 Fig. 2d. HPV16-E7 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides: 1 -3 Start codon 4-294 HPV-E7 ORF 295-297 STOP codon 30 Fig. 2e. HPV18-L1 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides: 1 -3 Start codon 4-1704 HPV-L1 ORF Zydus-Vac-002 1705-1707 STOP codon Fig. 2f. HPV18-L2 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides: 1 -3 Start codon 5 - 4-1386 HPV-L2 0RF 1387-1389 STOPcodon Fig. 2g. HPV18-E6 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides: 1 -3 Start codon 10 - 4-474 HPV-E6 0RF 475-477 STOP codon Fig. 2h. HPV18-E7 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides: I -3 Start codon 15 - 4-315 HPV-E7 0RF 316-318 STOP codon Fig. 2i. HPV6-L1 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides: 20 - 1-3 Start codon 4-1500 HPV-L1 ORF 1501-1503 STOP codon Fig. 2j. HPV6-L2 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides: 25 - 1-3 Start codon 4-1377 HPV-L2 0RF 1378-1380 STOPcodon Fig. 2k. HPV6-E6 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides: 30 - 1-3 Start codon 4-450 HPV-E6 ORF 451-453 STOPcodon 6 Zydus-Vac-002 ' Fig. 21. HPV6-E7 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides: I -3 Start codon 4-294 HPV-E7 ORF 5 - 295-297 STOP codon The examples describe the invention. Example 1: construction of recombinant p(+)MV2EZ-HPV and p(+)MV3EZ-HPV plasmids All cloning procedures were basically as described in Sambrook et a/. (1989). 10 Ad the restriction enzymes were from New England BioLabs; the oligonucleotides PCR primers were from Invitrogen. The LI sequence is amplified by PCR, and directly cloned into the definitive MV vectors, obtaining two recombinant MV- HPV16-L1 plasmids: p(+)MV2EZ-HPV-Ll and p(+)MV3EZ-HPV-Ll. PCR amplification is carried out using the proof-reading Pfu DNA polymerase (Stratagene). DNA sequences of the synthetic oligonucleotides primers are given in upper case for the MV nucleotides and in lower case for non-MV 15 nucleotides; sequences of relevant restriction endonucleases recognition sites are underlined. The following primers are used: FOR-LI 5' - ttggcgegccATGAGCCTGTGGCTGCCC - 3'; REV-LI 5' atgacgtcTCACAGCTTCCTCTTCTTCCTC - 3'. For-Ll contains an overhang (in lower case) with BssHll restriction site (gcgcgc), after 3-bp long-protection site (ttg). Rev-Ll contains an overhang (in lower case) with AatW restriction site (gacgtc). 20 The PCR-HPV16-LI (1536 bp) which is obtained is cloned in the p(+)MVEZ vector between genes for P and M protein or between H and L, after digestion with B55HII + AatW. Ligations are performed overnight at 16°C in an equimolar ratio respect to the pre-digested MeV2EZ and MeV3EZ vectors BssHW + AatW (19 Kb in length), using one unit of T4 DNA Ligase, obtaining, respectively, p(+)MV2EZ-Ll (20561 bp) and p(+)MV3EZ- LI (20561 bp). In figures 1 and 2 are listed, respectively, the sequences of the vectors p(+)MV2EZ-GFP and p(+)MV3EZ-GFP. 25 XL10 Gold chemical competent cell are then transformed with all ligation volume, following a standard transformation protocol (Sambrook et al. 1989), plated and selected on LB-Agar plates for ampicillin resistance. Colonies are screened by DNA plasmid preparation (QIAGEN, mini- midi and maxi kit) and restriction enzymes digestion. The right clones are sent to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, thereby showing 100% identity. Nucleotide sequence encoding for HPV16-L1 is presented in figure 2a. 30 All the other antigens L2, E6, and E7 from HPV 16, and LI, L2, E6, and E7 from HPV 18 and 6 types, are cloned as above detailed. The ORFs sequences are listed in figure 2 (2b to 21). List of the recombinant MV-HPV plasmids from HPV 16, 18, and 6 types: 35 p(+)MV2EZ-HPV16-Ll plasmid p(+)MV3EZ-HPV16-Ll plasmid p(+)MV2EZ-HPV16-L2 plasmid p(+)MV3EZ-HPV16-L2 plasmid p(+)MV2EZ-HPVI6-E6 plasmid 7 Zydus-Vac-002 ' p(+)MV3EZ-HPV16-E6 plasmid p(+)MV2EZ-HPV16-E7 plasmid p(+)MV3EZ-HPV16-E7 plasmid p(+)MV2EZ-HPV18-Ll plasmid 5 p(+)MV3EZ-HPV18-Ll plasmid p(+)MV2EZ-HPV18-L2 plasmid p(+)MV3EZ-HPV18-L2 plasmid p(+)MV2EZ-HPV 18-E6 plasmid p(+)MV3EZ-HPVl 8-E6 plasmid 10 p(+)MV2EZ-HPV 18-E7 plasmid p(+)MV3EZ-HPV6-E7 plasmid p(+)MV2EZ-HPV6-Ll plasmid p(+)MV3EZ-HPV6-Ll plasmid p(+)MV2EZ-HPV6-L2 plasmid 15 p(+)MV3EZ-HPV6-L2 plasmid p(+)MV2EZ-HPV6-E6 plasmid p(+)MV3EZ-HPV6-E6 plasmid p(+)MV2EZ-HPV6-E7 plasmid p(+)MV3EZ-HPV6-E7 plasmid 20 Example 2: cells and viruses Cells are maintained as monolayers in Dulbecco's Modified Eagles Medium (DMEM), supplemented with 5% Foetal Calf Serum (FCS) for Vero cells (African green monkey kidney) and with 10% FCS and 1% penicillin/streptomycin for 293T cells (human embryonic kidney); DMEM supplemented with Glutamax (F12) and 10% 25 FCS forMRC-5 (human foetal fibroblast); DMEM supplemented with 10% FCS and 1.2 mg/ml of G 418 for 293-3-46. To grow MV virus stocks reaching titers of about 107 pfu/ml, recombinant viruses and the vaccine strain Edmoston Zagreb are propagated in MRC-5 cells: plaque purification is carried out by transferring a syncythium to 35 mm MRC-5 cell culture which is expanded first to a 10 cm dish, and afterwards to a 175 cm flask. Virus stocks are made from 175cm2 cultures when syncythia formation is about 90% pronounced. Medium corresponding to the so-called "free-cell 30 virus fraction" is collected, freeze and thaw three times and spin down to avoid cell debris. The medium is then stored at -80°C. Cells, which correspond to the so-called "cell-associated virus fraction", are scraped into 3 ml of OPT1MEM (Gibco BRL) followed by three rounds freezing and thawing, spin down and the cleared supernatant stored at -80CC. Example 3: transfection of plasmids and rescue of MV viruses 8 Zydus-Vac-002 Recombinant measles-HPV vaccine viruses are obtained using the 293-3-46 helper cell (human embryonic kidney cells), stably expressing the measles N and P proteins as well as the T7 RNA polymerase. The viral RNA polymerase (large protein, L) is expressed by co-transfecting the cells with 15 ng of the plasmid peMCLa. Calcium-phosphate method is used for transfection. 5 293T-3-46 cells are seeded into a 35mm well to reach ~ 50-70% confluence when being transfected. 4 h before transfection, the medium is replaced with 3 ml DMEM containing 10% FCS. All recombinant plasmids are prepared according to the Q1AGEN plasmid preparation kit. The kit for the Ca2+phosphate coprecipitation of DNA is from Invitrogen. Cells are co-transfected with the plasmids in the following final concentration: pEMCLa 25 ng and the 10 recombinant p(+)MV2EZ-Ll plasmid 5 µg. All plasmids, diluted in H,0, are added in an Eppendorf tube containing 2M CaCl2, the mix is added to another Eppendorf tube containing HEPES buffer under shaking conditions, and is incubated for 30 min at room temperature (RT). Thus, the co-precipitates are added dropwise to the culture and the transfection is carried out at 37 °C and 5% C02 for about 18h. Then, the transfection medium is replaced with 3ml of DMEM containing 10% FCS. 15 To allow syncytia formation to progress more easily, almost confluent cell monolayers of each 35mm well are then transferred to a 10 cm dish. Each syncytium is taken up in 300 ul of transfection medium and put in a sterile Eppendorf tube containing 700 µ1 of OPT1MEM, freeze and thaw for three rounds, and stored at -80°C. Example 4: virus titration by plaque assay Serial 10-times dilutions of virus preparations are carried out using OPTIMEM to a final volume of 0.5 ml. Each 20 dilution is added on 35 mm Vero cell cultures. After 1 h of virus adsorption, the inoculum is removed and the infected cells are overlaid with 2ml of DMEM containing 5% FCS and 1% low melting point agarose (LMP agarose). After 5 days of incubation at 37°C and 5% C02, cultures are fixed with 1ml of 10% TCA for 1 h, then UV cross-linked for 30 min. After removal of the agarose overlay, cell monolayers are stained with crystal violet dissolved in 4% ethanol, washed with water and the plaques are counted under the inverted microscope. 25 Example 5: MRC-5 virus serial passages of recombinant viruses Rescued viruses are serially passaged 10-times on MRC5 cells, seeded into 10 cm diameter plates, that are infected with the standard and the recombinant MV viruses at MOI of 0-01 PFU/cells. After monolayer is full infected, 1% surnatant of each culture is used to infect the subsequent MRC5 cells monolayer. To test transgene expression and stability, viruses from passage 1, 5, and 10 are used for further characterisation of expression by Western blot and 30 immunofluorescence. Isolation of VLPs Monolayer Vero cells grown are infected at 0,1 MOI with recombinant virus MV2EZ-L1 or negative control virus MVEZ and incubated at 37°. 1 hour after viral adsorption medium is substituted by DMEM containing 5%FCS and incubated at 37° for 48 hours to obtain 90% syncytia. Medium from infected cells are collected, centrifugated and submitted to 35 centrifugation on a 40% (w/v) saccharose layer to separate proteins from particles at 110,00 x g for 2.5 h at 4°. Pellet is solubilised in cesium chloride 27% (w/w) in PBS and analysed on density gradient centrifugation in cesium chloride 27% (w/w) in PBS for 20h at 141,000g at 4°. 9 Zydus-Vac-002 The immunogenic power of the rescued recombinant MV-HPV viruses described is proved by immunisation tests performed on transgenic mice CD46, susceptible for MV infections. The presence HPVLl-specific antibodies in the sera of immunised CD46 mice iss determined by ELISA assay and by neutralization assay. . 5 Example 6: Production of a combined measles-malaria vaccine The working seed of the described recombinant measles-malaria virus is incubated on MRC5 cell monolayer in 1750cm2 roller bottles at 35°C for ten days. The cells are monitored every day for status of health and confluence. On day ten at highest level of syncytia formation, the supernatant is pumped in a steel cylinder for storage in liquid nitrogen. The same 10 procedure is repeated two days later. After performing of all the tests (virus titer, genome stability, virus safety, cell safety, chemical analysis, sterility and others), the harvests is thawed up and mixed with stabilizer containing gelatine, sorbitol, amminoacids and other sugars to final dilution of 105. With a automated filling machine small lyo bottles (F3) are inoculated with 0.5ml each. A specially calculated lyophilisation program is used to guarantee maximal survival of the product during the freeze-drying process. 15 Dated this the 24th day of May 2008 20

Full Text

FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patent Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION
"COMBINED MEASLES-HUMAN PAPILLOMA VACCINE FOR THERAPEUTIC AND PROPHYLACTIC USE"
We, CADILA HEALTHCARE LIMITED, a company incorporated under the Companies Act, 1956, of Zydus Tower, Satellite Cross Roads, Ahmedabad-380015, Gujarat, India.
: The following specification describes the invention

Zydus-Vac-002
Technical Field of the Invention
The present invention relates to combined vaccines against measles and human papilloma virus (HPV).In particular, the invention relates to recombinant measles virus vectors containing heterologous nucleic acid encoding single or several 5 antigens derived from HPV, preferably, the major capside antigen LI, the minor capside antigen L2, the early gene E6 and the early gene E7 oncoproteins of HPV type 16, and optionally of types 18, 6 and 11 such that they induce a potent long-lasting immune response in mammals, preferably humans, to protect against HPV and MV infection.
Background information
10 HPVs belong to a large family of small double-stranded DNA viruses that infect squamous epithelia. (For a-recent comprehensive review on papillomaviruses see Howley, PM and Lowy. DR (2007) in: Fields Virology, fifth edition), eds.-in-chief Knipe, DM. &. Howley, P. M. Lippincott Williams & Wilkins, Philadelphia PA 19106, USA, pp.2299-2354 To date, more than 100 genotypes have been described, among which at least 35 types infect the genital tract. Although most of the HPV types produce benign lesions, a small subset of genotypes is strongly associated with the development of high-
15 grade squamous intraepithelial lesions and cervical cancer. This subset has been identified as "high risk" and it is estimated that HPV-16 accounts for approximately 60% of cervical cancers, with HPV-18 adding another 10%-20%. Other high-risk types include types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73. Low-risk HPVs, such as HPV-6 and HPV-11, cause benign genital warts (90% begnin condylomata accuminata are HPV6 or 11 positive). Bivalent vaccines 16/18 eliminating the most common high-risk types may permit to overcome also the low-risk types. An ideal vaccine
20 would protect against other HPV types through use of antigens from different types and/or antigens containing conserved regions.
The HPV genome encodes eight proteins. The late LI and L2 genes code for capsid proteins; the early proteins El and E2 are responsible for viral replication and transcription, and E4 is involved in virus release from infected cells. The integration of high risk type HPV viral DNA into host genome results in a loss of El or E2 mediated transcriptional
25 control and consequently in an over-expression of the E6 and E7 proteins responsible of the malignant transformation process. Structural protein LI from high risk types represents an optimal target for prophylactic vaccines. On the other hand, E6 and E7 proteins are obvious therapeutic targets.
There are actually several prophylactic HPV vaccine formulations based upon the major viral capsid protein LI, either as a monomer, or as a virus like particle (VLP) from HPV 16 and 18 types and in some cases additional types
30 (W094/00152, W094/20137, WO93/02184 and WO94/05792). VLPs may additionally comprise L2 proteins, for example, L2 based vaccines described in WO93/00436. These vaccines are highly immunogenic and appear safe; however their high cost does not permit generalized access to populations at risk and their HPV type specificity represents another limitation. Therefore, a remaining need exists to develop additional improved vaccines against HPV which should be inexpensive. Moreover vaccinations with antigen mainly induce an antibody specific response that is of little or no benefit
35 on established HPV infection and related-disease.
Approaches involving recombinant viral vector vaccines are under development (Poxvirus, Adenovirus, Alphavirus, Poliovirus e Herpes Virus). Adenovirus based vaccine is described, for example, in US2007269409 (WO2004044176) which encodes the E6 or E7 protein of HPV. The adenovirus based vaccine is able to generate long term immunity; however, integration of HPV DNA into the host genome remains possible and may represent a safety
40 limitation. In view of the above shortcomings the use of measles, virus as a vector to express HPV antigens represents an original strategy to develop a prophylactic combined HPV-measles vaccine as well as a therapeutic HPV vaccine.

Zydus-Vac-002
Immunisation vectors based on measles virus
Measles virus (MV) is a member of the family Paramyxoviridae. The non segmented genome of MV has an anti-message polarity which results in a genomic RNA which, when purified, is not translated either in vivo or in vitro and is not infectious. 5 MV is a major cause of acute febrile illness in infants and young children. According to estimates of the World Health Organisation (WHO), one million young children die every year from measles. This high toll arises primarily in developing countries, but in recent years also industrialised countries such as the USA have been affected again by measles epidemics, primarily due to incomplete adherence to immunisation programs. At present, several live attenuated MV vaccine strains are in use (including the Schwarz, Moraten and Edmonston-Zagreb strains), almost all derived from
10 the original Edmonston strain by multiple passage in non human cells. MV vaccine is proven to be one of the safest, most stable, and effective human vaccines developed so far. Produced on a large scale in many countries and distributed at low cost through the Extended Program on Immunization (EPI) of WHO, this vaccine induces life-long immunity after a single injection and boosting is effective. Protection is mediated both by antibodies and by CD4 and CD8 T cells. Persistence of antibodies and CD8 cells has been shown for as long as 25 years after vaccination.
15 The recombinant measles virus nucleotide sequence must comprise a replicon having a total number of
nucleotides which is a mutiple of six. The «rule of six» is expressed in the fact that the total number of nucleotides present in the recombinant cDNA finally amount to a total number of nucleotides which is a multiple of six, a rule which allows efficient replication of genome RNA of the measles virus.
The heterologous DNA is cloned in the MV vector within an Additional Transcription Unit (ATU) inserted in the
20 cDNA corresponding to the antigenomic RNA of measles virus. The location of the ATU can vary along said cDNA: it is however located in such a site that it will benefit from the expression gradient of the measles virus. Therefore, the ATU or any insertion site suitable for cloning of the heterologous DNA sequence can be spread along the cDNA, with a preferred embodiment for an insertion site and especially in an ATU, present in the N-terminal portion of the sequence and especially within the region upstream from the L-gene of the measles virus and advantageously upstream from the M gene
25 of said virus and more preferably upstream from the N gene of said virus.
The technology permits to produce rescued viruses containing and stably expressing foreign genes suitable for use as combined MV vaccines. As a proof of concept, MV has been used to express antigens derived from SIV, HIV, hepatitis B, mumps, West Nile (WN) Virus and SARSCoV. In most of these studies, recombinant MVs that express heterologous antigens appeared to induce specific humoral neutralizing antibodies in a transgenic mouse model and were
30 shown to induce cellular immune responses to some proteins. At the present, clinical trials with any recombinant vaccine candidate based on MV are only in the planning stage however experimental results support the hypothesis that MV combined vaccines should be as efficient in eliciting long-lasting immune protection against other pathogenic agents as against the vector virus itself. In fact, in the case of MV expressing WNV gpE, a complete protection up to six months has been documented in monkey, and MV expressing a Dengue antigen induced long term production of neutralizing
35 antibodies. Moreover, in transgenic mice and macaques, rescued recombinant MV was capable of inducing specific antibody responses to heterologous antigen in the presence of pre-existing immunity against MV.
Rescued live recombinant MV vaccines are easily produced on a large scale in most countries and can be distributed at low cost. Regarding safety, MV replicates exclusively in the cytoplasm, ruling out the possibility of integration into host DNA. These characteristics make rescued recombinant MV vaccine an attractive candidate to be used as a multivalent
40 vaccination vector for HPV antigens. Adult populations, even already MV immunized individuals, may however also benefit from MV recombinant immunization because re-administering MV virus under the recombinant form of the present invention may result in a boost of anti-MV antibodies

3

Zydus-Vac-002
So far, no approach has been developed to produce a vaccine able to induce immunity against MV combined with immunity against HPV.
The invention relates in particular to the preparation of recombinant measles viruses, bearing heterologous nucleic acid encoding antigens from HPV. 5
Detailed description of the invention
The object of the invention is the production of combined measles-HPV vaccines from a recombinant Measles vector capable of containing stably integrated nucleotide sequences which code for LI, L2, E6 and/or E7 protein from different HPV types.
10 The invention includes the rescue of recombinant MV-HPV viruses which are capable of infection, replication
and expression of LI, L'2, E6 or E7 protein in susceptible transgenic mice, monkeys and human host.
Furthermore, the invention includes the construction of multivalent recombinant measles-HPV vectors, in which two different antigens are simultaneously cloned and expressed in the same vector, conferring immunity against both of them.
15 Moreover, the invention relates to the combination of different recombinant measles-HPV viruses, each carrying
and expressing a gene from a different HPV type, in order to elicit immune response in the host, directed against the different HPV types.
Furthermore, the invention comprises a method to produce a vaccine containing such recombinant viruses.
The invention finally relates to a vaccine capable to induce a potent and lifelong immune response against HPV 20 and measles virus in human and to prevent from infection and/or treat diseases associated with infection.
DESCRIPTION OF THE FIGURES
Fig. la. Complete nucleotide sequence of p(+)MV2EZ-GFP (19774 bp). The sequence can be described as follows with reference to the position of the nucleotides:
25 - 592-608 T7 promoter
609-17335 MV Edmoston Zagreb antigenome
4049-4054 Mlu\ restriction site
4060-4065 BssHII restriction site
4066-4782 Green Fluorescent Protein (GFP) ORF
30 - 4786-4791 BssHU restriction site
4798-4803 AatII restriction site
17336-17561 HDV ribozyme and T7 terminator
Fig. lb. Complete nucleotide sequence of p(+)MV3EZ-GFP (19774 bp). The sequence can be described as follows with 35 reference to the position of the nucleotides:
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592-608 T7 promoter
609-17335 MV Edmoston Zagreb antigenome
9851-9856 MM restriction s:te
9862-9867 BssHIIrestriction site
5 - 9868-10584 Green Fluorescent Protein (GFP) ORF
10588-10593 BssHII restriction site
10600-10605 Aatll restriction site
17336-17561 HDV ribozyme and T7 terminator
Fig. 2a. ANL1TE: this is the HPV16-L1 sequence ORF (1518 bp) cloned by the inventors. The sequence can be described 10 as follows with reference to the position of the nucleotides:

1-3 Start codon
4-1515 HPV-L1 ORF
1516-1518 STOP codon
Fig. 2b. HPV16-L2 sequence ORF. The sequence can be described as follows with reference to the position of the 15 nucleotides:
1 -3 Start codon
4-1419 HPV-L2 0RF
1420-1422 STOP codon
Fig. 2c. HPV16-E6 sequence ORF. The sequence can be described as follows with reference to the position of the 20 nucleotides:
1 -3 Start codon
4-474 HPV-E6 ORF
475-477 STOP codon
25 Fig. 2d. HPV16-E7 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides:
1 -3 Start codon
4-294 HPV-E7 ORF
295-297 STOP codon
30 Fig. 2e. HPV18-L1 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides:
1 -3 Start codon
4-1704 HPV-L1 ORF

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1705-1707 STOP codon
Fig. 2f. HPV18-L2 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides:
1 -3 Start codon
5 - 4-1386 HPV-L2 0RF
1387-1389 STOPcodon
Fig. 2g. HPV18-E6 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides:
1 -3 Start codon
10 - 4-474 HPV-E6 0RF
475-477 STOP codon
Fig. 2h. HPV18-E7 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides:
I -3 Start codon
15 - 4-315 HPV-E7 0RF
316-318 STOP codon
Fig. 2i. HPV6-L1 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides:
20 - 1-3 Start codon
4-1500 HPV-L1 ORF
1501-1503 STOP codon
Fig. 2j. HPV6-L2 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides:
25 - 1-3 Start codon
4-1377 HPV-L2 0RF
1378-1380 STOPcodon
Fig. 2k. HPV6-E6 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides:
30 - 1-3 Start codon
4-450 HPV-E6 ORF
451-453 STOPcodon
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' Fig. 21. HPV6-E7 sequence ORF. The sequence can be described as follows with reference to the position of the nucleotides:
I -3 Start codon
4-294 HPV-E7 ORF
5 - 295-297 STOP codon
The examples describe the invention.
Example 1: construction of recombinant p(+)MV2EZ-HPV and p(+)MV3EZ-HPV plasmids
All cloning procedures were basically as described in Sambrook et a/. (1989). 10 Ad the restriction enzymes were from New England BioLabs; the oligonucleotides PCR primers were from Invitrogen.
The LI sequence is amplified by PCR, and directly cloned into the definitive MV vectors, obtaining two recombinant MV-
HPV16-L1 plasmids: p(+)MV2EZ-HPV-Ll and p(+)MV3EZ-HPV-Ll.
PCR amplification is carried out using the proof-reading Pfu DNA polymerase (Stratagene). DNA sequences of the
synthetic oligonucleotides primers are given in upper case for the MV nucleotides and in lower case for non-MV 15 nucleotides; sequences of relevant restriction endonucleases recognition sites are underlined. The following primers are
used: FOR-LI 5' - ttggcgegccATGAGCCTGTGGCTGCCC - 3'; REV-LI 5'
atgacgtcTCACAGCTTCCTCTTCTTCCTC - 3'.
For-Ll contains an overhang (in lower case) with BssHll restriction site (gcgcgc), after 3-bp long-protection site (ttg).
Rev-Ll contains an overhang (in lower case) with AatW restriction site (gacgtc). 20 The PCR-HPV16-LI (1536 bp) which is obtained is cloned in the p(+)MVEZ vector between genes for P and M protein or
between H and L, after digestion with B55HII + AatW. Ligations are performed overnight at 16°C in an equimolar ratio
respect to the pre-digested MeV2EZ and MeV3EZ vectors BssHW + AatW (19 Kb in length), using one unit of T4 DNA
Ligase, obtaining, respectively, p(+)MV2EZ-Ll (20561 bp) and p(+)MV3EZ- LI (20561 bp). In figures 1 and 2 are listed,
respectively, the sequences of the vectors p(+)MV2EZ-GFP and p(+)MV3EZ-GFP. 25 XL10 Gold chemical competent cell are then transformed with all ligation volume, following a standard transformation
protocol (Sambrook et al. 1989), plated and selected on LB-Agar plates for ampicillin resistance. Colonies are screened by
DNA plasmid preparation (QIAGEN, mini- midi and maxi kit) and restriction enzymes digestion. The right clones are sent
to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, thereby showing
100% identity. Nucleotide sequence encoding for HPV16-L1 is presented in figure 2a. 30 All the other antigens L2, E6, and E7 from HPV 16, and LI, L2, E6, and E7 from HPV 18 and 6 types, are cloned as above
detailed.
The ORFs sequences are listed in figure 2 (2b to 21).
List of the recombinant MV-HPV plasmids from HPV 16, 18, and 6 types: 35 p(+)MV2EZ-HPV16-Ll plasmid
p(+)MV3EZ-HPV16-Ll plasmid
p(+)MV2EZ-HPV16-L2 plasmid
p(+)MV3EZ-HPV16-L2 plasmid
p(+)MV2EZ-HPVI6-E6 plasmid
7

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' p(+)MV3EZ-HPV16-E6 plasmid
p(+)MV2EZ-HPV16-E7 plasmid
p(+)MV3EZ-HPV16-E7 plasmid
p(+)MV2EZ-HPV18-Ll plasmid 5 p(+)MV3EZ-HPV18-Ll plasmid
p(+)MV2EZ-HPV18-L2 plasmid
p(+)MV3EZ-HPV18-L2 plasmid
p(+)MV2EZ-HPV 18-E6 plasmid
p(+)MV3EZ-HPVl 8-E6 plasmid 10 p(+)MV2EZ-HPV 18-E7 plasmid
p(+)MV3EZ-HPV6-E7 plasmid
p(+)MV2EZ-HPV6-Ll plasmid
p(+)MV3EZ-HPV6-Ll plasmid
p(+)MV2EZ-HPV6-L2 plasmid 15 p(+)MV3EZ-HPV6-L2 plasmid
p(+)MV2EZ-HPV6-E6 plasmid
p(+)MV3EZ-HPV6-E6 plasmid
p(+)MV2EZ-HPV6-E7 plasmid
p(+)MV3EZ-HPV6-E7 plasmid 20
Example 2: cells and viruses
Cells are maintained as monolayers in Dulbecco's Modified Eagles Medium (DMEM), supplemented with 5% Foetal Calf Serum (FCS) for Vero cells (African green monkey kidney) and with 10% FCS and 1% penicillin/streptomycin for 293T cells (human embryonic kidney); DMEM supplemented with Glutamax (F12) and 10% 25 FCS forMRC-5 (human foetal fibroblast); DMEM supplemented with 10% FCS and 1.2 mg/ml of G 418 for 293-3-46.
To grow MV virus stocks reaching titers of about 107 pfu/ml, recombinant viruses and the vaccine strain Edmoston Zagreb are propagated in MRC-5 cells: plaque purification is carried out by transferring a syncythium to 35 mm MRC-5 cell culture which is expanded first to a 10 cm dish, and afterwards to a 175 cm flask. Virus stocks are made from 175cm2 cultures when syncythia formation is about 90% pronounced. Medium corresponding to the so-called "free-cell 30 virus fraction" is collected, freeze and thaw three times and spin down to avoid cell debris. The medium is then stored at -80°C. Cells, which correspond to the so-called "cell-associated virus fraction", are scraped into 3 ml of OPT1MEM (Gibco BRL) followed by three rounds freezing and thawing, spin down and the cleared supernatant stored at -80CC.
Example 3: transfection of plasmids and rescue of MV viruses
8

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Recombinant measles-HPV vaccine viruses are obtained using the 293-3-46 helper cell (human embryonic kidney cells), stably expressing the measles N and P proteins as well as the T7 RNA polymerase. The viral RNA polymerase (large protein, L) is expressed by co-transfecting the cells with 15 ng of the plasmid peMCLa. Calcium-phosphate method is used for transfection.
5 293T-3-46 cells are seeded into a 35mm well to reach ~ 50-70% confluence when being transfected. 4 h before
transfection, the medium is replaced with 3 ml DMEM containing 10% FCS. All recombinant plasmids are prepared according to the Q1AGEN plasmid preparation kit. The kit for the Ca2+phosphate coprecipitation of DNA is from Invitrogen.
Cells are co-transfected with the plasmids in the following final concentration: pEMCLa 25 ng and the 10 recombinant p(+)MV2EZ-Ll plasmid 5 µg. All plasmids, diluted in H,0, are added in an Eppendorf tube containing 2M CaCl2, the mix is added to another Eppendorf tube containing HEPES buffer under shaking conditions, and is incubated for 30 min at room temperature (RT). Thus, the co-precipitates are added dropwise to the culture and the transfection is carried out at 37 °C and 5% C02 for about 18h. Then, the transfection medium is replaced with 3ml of DMEM containing 10% FCS.
15 To allow syncytia formation to progress more easily, almost confluent cell monolayers of each 35mm well are
then transferred to a 10 cm dish. Each syncytium is taken up in 300 ul of transfection medium and put in a sterile Eppendorf tube containing 700 µ1 of OPT1MEM, freeze and thaw for three rounds, and stored at -80°C.
Example 4: virus titration by plaque assay
Serial 10-times dilutions of virus preparations are carried out using OPTIMEM to a final volume of 0.5 ml. Each 20 dilution is added on 35 mm Vero cell cultures. After 1 h of virus adsorption, the inoculum is removed and the infected cells are overlaid with 2ml of DMEM containing 5% FCS and 1% low melting point agarose (LMP agarose). After 5 days of incubation at 37°C and 5% C02, cultures are fixed with 1ml of 10% TCA for 1 h, then UV cross-linked for 30 min. After removal of the agarose overlay, cell monolayers are stained with crystal violet dissolved in 4% ethanol, washed with water and the plaques are counted under the inverted microscope.
25 Example 5: MRC-5 virus serial passages of recombinant viruses
Rescued viruses are serially passaged 10-times on MRC5 cells, seeded into 10 cm diameter plates, that are
infected with the standard and the recombinant MV viruses at MOI of 0-01 PFU/cells. After monolayer is full infected, 1%
surnatant of each culture is used to infect the subsequent MRC5 cells monolayer. To test transgene expression and
stability, viruses from passage 1, 5, and 10 are used for further characterisation of expression by Western blot and
30 immunofluorescence.
Isolation of VLPs
Monolayer Vero cells grown are infected at 0,1 MOI with recombinant virus MV2EZ-L1 or negative control virus MVEZ and incubated at 37°. 1 hour after viral adsorption medium is substituted by DMEM containing 5%FCS and incubated at 37° for 48 hours to obtain 90% syncytia. Medium from infected cells are collected, centrifugated and submitted to 35 centrifugation on a 40% (w/v) saccharose layer to separate proteins from particles at 110,00 x g for 2.5 h at 4°. Pellet is solubilised in cesium chloride 27% (w/w) in PBS and analysed on density gradient centrifugation in cesium chloride 27% (w/w) in PBS for 20h at 141,000g at 4°.
9

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The immunogenic power of the rescued recombinant MV-HPV viruses described is proved by immunisation tests performed on transgenic mice CD46, susceptible for MV infections.
The presence HPVLl-specific antibodies in the sera of immunised CD46 mice iss determined by ELISA assay and by neutralization assay.
. 5
Example 6: Production of a combined measles-malaria vaccine
The working seed of the described recombinant measles-malaria virus is incubated on MRC5 cell monolayer in 1750cm2 roller bottles at 35°C for ten days. The cells are monitored every day for status of health and confluence. On day ten at highest level of syncytia formation, the supernatant is pumped in a steel cylinder for storage in liquid nitrogen. The same 10 procedure is repeated two days later. After performing of all the tests (virus titer, genome stability, virus safety, cell safety, chemical analysis, sterility and others), the harvests is thawed up and mixed with stabilizer containing gelatine, sorbitol, amminoacids and other sugars to final dilution of 105. With a automated filling machine small lyo bottles (F3) are inoculated with 0.5ml each. A specially calculated lyophilisation program is used to guarantee maximal survival of the product during the freeze-drying process.
15

Dated this the 24th day of May 2008
20

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http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=HCkGFNeq86DUeNMF3uEorw==&loc=vsnutRQWHdTHa1EUofPtPQ==

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Patent Number

272803

Indian Patent Application Number

1113/MUM/2008

PG Journal Number

18/2016

Publication Date

29-Apr-2016

Grant Date

27-Apr-2016

Date of Filing

26-May-2008

Name of Patentee

CADILA HEALTHCARE LIMITED

Applicant Address

ZYDUS TOWER, SATELLITE CROSS ROADS, AHMEDABAD 380015, GUJARAT, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SANJEEV KUMAR MENDIRETTA	ZYDUS TOWER, SATELLITE CROSSROADS, AHMEDABAD-380015, GUJARAT, INDIA.

PCT International Classification Number

A61K9/08; A61K39/12; A61K47/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA