Patent Grant
9623098
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 L1, 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 L1 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 L1 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.
Syndromes Induced by Human Papilloma Virus
Every year approximately half a million new cervical cancer cases are registered worldwide, particularly in developing countries, representing the second most common cause of mortality in women. Human Papillomaviruses (HPVs) are the primary etiologic agent of cervical carcinoma; HPV DNA can be found in more than 95% of these cancers (1). Since 1998, prevention and treatment strategy mainly rely on structured screening programs to detect and ablate pre-invasive disease. However, use of HPV testing is limited by social issues and currently the main obstacle is its high cost. Thus the development of vaccines that prevent HPV infection represent an important opportunity to prevent cervical cancer whilst a therapeutic immunization would be valuable in treating pre-malignant and malignant disease.
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, P M and Lowy, D R (2007) in: Fields Virology, fifth edition), eds.-in-chief Knipe, D. M. &. 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-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 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 L1 and L2 genes code for capsid proteins; the early proteins E1 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 E1 or E2 mediated transcriptional control and consequently in an over-expression of the E6 and E7 proteins responsible of the malignant transformation process (2). Structural protein L1 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 L1, either as a monomer, or as a virus like particle (VLP) from HPV 16 and 18 types and in some cases additional types (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 on established HPV infection and related-disease.
The development of therapeutic vaccine relies not only on production of neutralising antibodies, but principally on the induction of specific cellular immune responses, that are key components for clearance of established infection. Thus, therapeutic vaccines are required to include some antigenic determinants derived from early HPV proteins rather than the late proteins. The early genes of the high-risk HPV types (E6 and E7) encode the main transforming proteins. These genes are capable of immortalization of epithelial cells and are thought to play a role in the initiation of the oncogenic process. The protein products of these early genes interfere with the normal function of tumour suppressor genes. HPV E6 is able to interact with p53, leading to its dysfunction, thereby impairing its ability to block the cell cycle when DNA errors occur. E6 also keeps the telomerase length above its critical point, protecting the cell from apoptosis. HPV E7 binds to retinoblastoma protein (pRb) and activates genes that start the cell cycle, leading to tissue proliferation. E6 and E7 proteins represent good targets and various approaches of HPV therapeutic vaccines have been described based on E6. In the last few years a number of peptide/protein-based or genetic immunization strategies have been described for the induction of HPV specific CTL activity. For a review of progress in the development of vaccines against HPV see ref (3). Attempts were made with DNA vaccines (plasmid DNA encoding HPV proteins) known to promote primarily a cellular response. Despite the fact that DNA vaccines work well in mouse models, numerous clinical trials have failed to provide proof of principle in man. Major drawbacks associated with a peptide-based approach include the problem of MHC-polymorphism and the risk of inducing T cell tolerance rather than T cell activation. Due to the induction of specific T cell tolerance, vaccination with a tumour-specific peptide has been shown to result in an enhanced outgrowth of the tumour. Immunization with larger proteins would overcome these problems, but this requires an efficient in vivo expression system and/or safe adjuvants for priming an efficient cellular immune response. 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 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.
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. Transcription and replication of non-segmented (−) strand RNA viruses and their assembly as virus particles have been studied and reported (4). Transcription and replication of measles virus do not involve the nucleus of the infected cells but rather take place in the cytoplasm of said infected cells. The genome of the measles virus comprises genes encoding six major structural proteins from the six genes (designated N, P, M, F, H and L) and additional two-non structural proteins from the P gene. 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 (5). At present, several live attenuated MV vaccine strains are in use (including the Schwarz, Moraten and Edmonston-Zagreb strains), almost all derived from the original Edmonston strain (6) 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 (4, 7) 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 (7).
Martin Billeter and colleagues established an original and efficient reverse genetics procedure to generate non-segmented negative-strand RNA viruses from cloned deoxyribonucleic acid (cDNA) derived from Edmonston strain MV, as described in 8 and WO 97/06270, a scheme of the organization of the antigenomic p(+)MVEZ of measles virus is represented in
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 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 of said virus and more preferably upstream from the N gene of said virus.
The advantageous immunological properties of the recombinant measles viruses can be shown in an animal model which is chosen among animals susceptible to measles viruses, and wherein the humoral and/or cellular immune response against the heterologous antigen and/or against the measles virus is determined. Among such animals suitable to be used as model for the characterization of the immune response, the skilled person can especially use transgenic mice expressing CD46 specific receptor for MV, or in monkeys.
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 (9-12). In most of these studies, recombinant MVs that express heterologous antigens appeared to induce specific humoral neutralizing antibodies in a transgenic mouse model (13) and were shown to induce cellular immune responses to some proteins (9, 11). 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 monkeys (14), and MV expressing a Dengue antigen induced long term production of neutralizing antibodies (15). 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 (9, 11, 16).
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 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 (11)
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.
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 L1, L2, E6 and/or E7 protein from different HPV types.
The invention includes the rescue of recombinant MV-HPV viruses which are capable of infection, replication and expression of L1, L2, 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.
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.
Moreover the invention also relates to the use of interleukin or interleukin-2 as adjuvent in order to increase the response of combined vaccine.
The invention finally relates to a vaccine capable to induce a potent and lifelong immune response against HPV and measles virus in human and to prevent from infection and/or treat diseases associated with infection.
The examples describe the invention.
All cloning procedures were basically as described in Sambrook et al. (1989).
All the restriction enzymes were from New England BioLabs; the oligonucleotides PCR primers were from Invitrogen.
The L1 sequence has been amplified by PCR, and direct cloned into the definitive MV vectors, obtaining two recombinant MV-HPV16-L1 plasmids: p(+)MV2EZ-HPV-L1 and p(+)MV3EZ-HPV-L1.
PCR amplification was 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 nucleotides; sequences of relevant restriction endonucleases recognition sites are underlined. The following primers have been used: FOR-L1 5′-ttggcgcgccATGAGCCTGTGGCTGCCC-3′; REV-L1 5′-atgacgtcTCACAGCTTCCTCTTCTTCCTC-3′.
For-L1 contains an overhang (in lower case) with BssHII restriction site (gcgcgc), after 3-bp long-protection site (ttg).
Rev-L1 contains an overhang (in lower case) with AatII restriction site (gacgtc).
The obtained PCR-HPV16-L1 (1536 bp) has been cloned in the p(+)MVEZ vector (
XL10 Gold chemical competent cell were 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 were screened by DNA plasmid preparation (QIAGEN, mini-midi and maxi kit) and restriction enzymes digestion. The right clones were sent to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, showed 100% identity. Nucleotide sequence encoding for HPV16-L1 is presented in
All the other antigens L2, E6, and E7 from HPV16, and L1, L2, E6, and E7 from HPV 18 and 6 types, have been cloned as above detailed.
The ORFs sequences are listed in
List of the Recombinant MV-HPV Plasmids from HPV 16, 18, and 6 Types:
Cells were 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% FCS for MRC-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 were propagated in MRC-5 cells: plaque purification was carried out by transferring a syncythium to 35 mm MRC-5 cell culture which was expanded first to a 10 cm dish, and afterwards to a 175 cm flask. Virus stocks were made from 175 cm2 cultures when syncythia formation was about 90% pronounced. Medium corresponding to the so-called “free-cell virus fraction” was collected, freeze and thaw three times and spin down to avoid cell debris. The medium was then stored at −80° C. Cells, which correspond to the so-called “cell-associated virus fraction”, were scraped into 3 ml of OPTIMEM (Gibco BRL) followed by three rounds freezing and thawing, spin down and the cleared surnatant stored at −80° C.
Recombinant measles-HPV vaccine viruses have been 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) was expressed by co-transfecting the cells with 15 ng of the plasmid peMCLa. Calcium-phosphate method was used for transfection.
293T-3-46 cells were seeded into a 35 mm well to reach ˜50-70% confluence when being transfected. 4 h before transfection, the medium was replaced with 3 ml DMEM containing 10% FCS. All recombinant plasmids were prepared according to the QIAGEN plasmid preparation kit. The kit for the Ca2+phosphate coprecipitation of DNA was from Invitrogen.
Cells were co-transfected with the plasmids in the follows final concentration: pEMCLa 25 ng and the recombinant p(+)MV2EZ-L1 plasmid 5 μg. All plasmids, diluted in H2O, were added in a Eppendorf tube containing 2M CaCl2, the mix was added to another Eppendorf tube containing HEPES buffer under shaking conditions, and was incubated 30 min at room temperature (RT). Thus, the co-precipitates were added dropwise to the culture and the transfection was carried out at 37° C. and 5% CO2 for about 18 h. Then, the transfection medium was replaced with 3 ml of DMEM containing 10% FCS.
First syncytia appeared 3-4 days after transfection when the cells were still subconfluent as shown in
Serial 10-times dilutions of virus preparations were carried out using OPTIMEM to a final volume of 0.5 ml. Each dilution was added on 35 mm Vero cell cultures. After 1 h of virus adsorption, the inoculum was removed and the infected cells were overlaid with 2 ml of DMEM containing 5% FCS and 1% low melting point agarose (LMP agarose). After 5 days of incubation at 37° C. and 5% CO2, cultures were fixed with 1 ml of 10% TCA for 1 h, then UV cross-linked for 30 min. After removal of the agarose overlay, cell monolayers were stained with crystal violet dissolved in 4% ethanol, washed with water and the plaques were counted under the inverted microscope (
Rescued viruses were serially passaged 10-times on MRC5 cells, seeded into 10 cm diameter plates, that were infected with the standard and the recombinant MV viruses at MOI of 0.01 PFU/cells. After monolayer was full infected, 1% surnatant of each culture was used to infect the subsequent MRC5 cells monolayer. To test transgene expression and stability, viruses from passage 1, 5, and 10 were used for further characterisation of expression by Western blot and immunofluorescence.
MRC5 cells seeded on 35 mm dish (1-5×105) were monitored for 90% confluence and infected with cleared virus suspension from cell-associated virus fraction at 0.05 MOI, including MVEZ as control. Samples, corresponding to the so-called “free-cell virus fraction” and to the so-called “cell-associated virus fraction”, were collected daily for one week and titrated. From Growth curve comparison of it is interesting that the replication of MV2EZ-L1 was only slightly impaired: the recombinant virus reached peak titers of 6.12×106 TCID50 s/ml 48 hpi, whereas MVEZ gave final titers of 6.8×106 TCID50 s/ml 36 hpi (
To analyse the expression either MV and HPV antigen, immunofluorescence, Western blot and isolation of VLP were carried out.
Immunofluorescence
Vero cells were seeded on 24 mm×24 mm glass cover slips in 35 mm wells, cultured overnight and infected with rescued recombinant virus MV2EZ-L1 or with negative control virus MVEZ at 0.05b M.O.I. 48 hours after infection cells on coverslips were fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.1% TX-100, washed with blocking solution (PBS containing 1% BSA) for 1 h, and stained with the specific HPV-L1 mouse monoclonal antibody (Biogenesis) and by FITCH conjugated goat anti-mouse secondary antibody.
All syncytia of rescued MV2EZ-L1 showed positive signals (
Western Blot
For Western blot, Vero cells seeded on 35 mm dish (1-5×105) were monitored the next day for 90% confluence and infected with cleared virus suspension from cell-associated virus fraction, using 0.05 MOI (Multiplicity Of Infection), including MVEZ as control. When about 80% syncythia formation was observed, proteins from medium and from cells were analysed. Cells were first washed with PBS and then scraped in 1 ml PBS and collected in an Eppendorf tube, and centrifuge at 2000 RPM/4 min. Cells were then lysed 5 min/RT with 70 μl of lysis buffer (1% NP-40, 50 mM Tris pH 8, 150 mM NaCl) supplemented with protease inhibitor cocktail (Complete Mini, Roche, 1 836 153). Surnatants were cleared by centrifuge at 13000 RPM/5 min, and transferred into a new tube: 30 □l of 4× loading buffer (Invitrogen) were added; samples were mixed and boiled at 95° C./2 min, spun down and stored at −20° C.
An SDS-PAGE migration was performed, running a NuPAGE 12% Bis-acrylamide gel in reducing conditions, using 1× Running Buffer, for 50 min at 200V (start 100-125 mA, end 60-80 mA). Then, semi-dry method was used to transfer separated cell-proteins to Nitrocellulose Membrane, at 14V/1h30.
Mouse monoclonal antibody against HPV-L1 (Biogenesis) was used as first antibody. The second antibody was a goat anti-mouse antibody coupled to horse-radish peroxidase allowing the visualization of the bands by the enhanced chemiluminescence kit (ECLTM, Amersham LifeScience).
The anti-HPVL1 antibody reacted with a protein of approximately 55 kDa released in the culture medium of MV2EZ-L1-infected cells, whereas no such protein was detected in culture medium of MVEZ-infected cells (
Isolation of VLPs
Monolayer Vero cells grown were 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 was substituted by DMEM containing 5% FCS and incubated at 37° for 48 hours to obtain 90% syncytia. Medium from infected cells has been collected, centrifugated and submitted to centrifugation on a 40% (w/v) saccharose layer to separate proteins from particles at 110,00×g for 2.5 h at 4°. Pellet was successively solubilised in cesium chloride 27% (w/w) in PBS and analysed on density gradient centrifugation in cesium chloride 27% (w/w) in PBS for 20 h at 141,000 g at 4°. Gradient fractions were analyzed for the presence of HPV-L1 by SDS-page electrophoresis and western blot (
The immunogenic power of the rescued recombinant MV-HPV viruses described was proved by immunisation tests performed on transgenic mice CD46, susceptible for MV infections. The animals were kept under optimal hygienic conditions and were immunized at 6-8 weeks of age. Immunisation was performed intra-peritoneal using 105 PFU of each recombinant MV-HPV in two injections, at 0 and 4 weeks. Non-infected mice as well as mice immunized with PBS served as control. UV inactivated MV was used as a control to determine the effect of virus replication on activation of immune responses.
The presence of MV-specific antibodies in the sera from the immunised CD46 mice (at least 6 per group) was determined by ELISA using 96-microwell plates, coated with Measles virus EIA bulk (ATCC VR-24), for IgG antibody detection. Protein was diluted 0.6 μg/ml with 0.05 M carbonate buffer (pH 9.4), and 100 μl per well was added to 96-well-microtiter plates. The plates were incubated overnight at 4° C., washed with PBS/0.05% Tween 20 (PT) (ph 7.4), incubated with PT (0.1 ml/well)-10% BSA for 60 min at 37° C., and washed again with PT. Serial 2-folds dilutions of the tested sera were added (100 μl/well), and the plates were incubated for 60 min at 37° C. The plates were washed with PT and were incubated with 100 μl of goat anti-mouse IgG HRP diluted 1:2000 in PBS-0.05% Tween 20 for 30 min at 37° C. The plates were washed with PT and incubated with 100 μl OPD (o-Phenylendiamin, Fluka 78411). The reaction was stopped after 3-4 min. Plates were read on a MicroElisa Reader at a wave length of 490 nm. Readings higher than three-folds negative controls were scored as positive reaction.
The presence HPVL1-specific antibodies in the sera of immunised CD46 mice was determined by ELISA assay and by neutralization assay.
Briefly, for the Elisa assay, 96-microwell plates were coated with HPVL1 antigen, diluted with carbonate buffer pH 9.4 at a concentration of 2-50 ng/well. The plates were incubated overnight at 4° C., washed with PBS/0.05% Tween 20 (PT). Subsequently, unspecific interaction were blocked with 10% defatted milk dissolved in PT for 1 hour at 37° C. and wells were washed again with PT. The plates were consecutively incubated with various dilutions of mouse sera (starting at 1:200, followed by serial two-fold dilutions), peroxidase-conjugate goat anti-mouse IgG and with OPD substrate. Optical density values were measured at 490 nm. Values above the cut-off background level (mean value of sera from MV immunised mice multiplied by a factor of 2.1) were considered positive. Titres were depicted as reciprocal end-dilutions.
To assay for neutralizing antibodies twofold serum dilutions were incubated with 50 PFU of recombinant for 1 h at 37° C. and plated in duplicate onto 104 Vero cells/well (96-well plate). Five days later titers were determined microscopically. The endpoint titer was calculated as the highest serum dilution tested that reduced the number of PFU by at least 50%
The specific immune responses to HPV-L1 is shown in table I. The titers in both the Elisa and neutralization assays are similar to those observed in women or mice after three injections of VLPs. Moreover, the very similar titers observed in the two assays indicate that most of the L1 expressed is conformationally correct.
It is known from literature that after a certain number of passages with Paramyxoviruses, and in particular with measles virus, an accumulation of defective interfering particles (DIs) will occur (23, 24). It has been described that these DIs develop various defects: negative impact on vaccine safety, negative influence on virus yields in production, genome instability and suppression of immune reaction after vaccination. In order to avoid such DIs with our new recombinant viruses, we have applicated the method of plaque purification as described in example 7 with the exception that we use MRC5 cell instead of Vero cell. After the formation of clear, well defined syncytia we aspirated under the microscope with a micropipette such material for further passaging in a fresh MRC5 tissue colture.
The end point dilution technique was applicated in microplates: in all wells a fresh monolayer with MRC5 cells had just developed. The virus suspension containing recombinant measles-malaria viruses was prepared in two fold dilutions. From the well of the latest monolayer where a syncytia was detected the supernatant was aspirated with a pipette. The supernatant was mixed with a suspension containing MRC5 cells. This mixture was incubated at 4° C. for 1 hour. Finally, it was transferred in a small Costar flask and incubated at 35° C.+5% CO2 and harvested for purify recombinant measles-malaria virus after ten days.
The working seed of the described recombinant measles-malaria virus has been incubated on MRC5 cell monolayer in 1750 cm2 roller bottles at 35° C. for ten days. The cells have been monitored every day for status of health and confluence. On day ten at highest level of syncytia formation, the supernatant was pumped in a steel cylinder for storage in liquid nitrogen. The same procedure was 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 have been 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) have been inoculated with 0.5 ml each. A specially calculated lyophilisation program is used to guarantee maximal survival of the product during the freeze-drying process.
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| Number | Date | Country | Kind |
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| 1113/MUM2008 | May 2008 | IN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
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| PCT/IN2009/000302 | 5/26/2009 | WO | 00 | 2/10/2011 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2010/079505 | 7/15/2010 | WO | A |
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| 2004076619 | Sep 2004 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20110129493 A1 | Jun 2011 | US |
