Patent Application
20120269853
The present invention relates to an attenuated dengue virus vaccine which comprises infectious mutants of wild type dengue virus
Dengue is a vector-borne virus, transmitted to humans via infected Aedes mosquitoes in tropical and sub-tropical areas. The severity of the disease varies from asymptomatic infections, to a febrile fever, or potentially life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The WHO reports that two-fifths of the world's population is at risk of dengue infection, with up to 100 million cases of infections each year resulting in hundreds of thousands of cases of DHF and DSS. The virus is now endemic in more than 100 countries, affecting South-East Asia and the Western Pacific significantly, in some countries becoming the leading cause of hospitalization and death among children, with a mortality rate of up to 25,000 annually. There are dramatic increases in incidence and disease severity, attributed in part to geographic expansion of the vector by many means; the Aedes aegypti and A. albopictus mosquitoes, leading to the increased co-circulation of all dengue 1-4 serotypes in urban areas.
To date, no licensed vaccine is available. This has brought together many groups including: The Pediatric Dengue Vaccine Initiative (funded by Bill and Melinda Gates Foundation), the WHO, the US military, as well as industry and governments in many different countries to collaborate in the hopes of accelerating the development of a successful vaccine. For dengue virus, attenuation was first achieved by Sabin in 1945 by passaging the virus (DENY-1) in mouse brains (Sabin A B (1952) Research on dengue during World War II. Am J Trop Med Hyg 1(1): 30-50). However, the degree of attenuation seemed to vary depending on the strain of the virus, as many human volunteers developed a reaction in the form of a rash. This complication was addressed by Halstead and Marchette with the discovery that dengue virus could be propagated and attenuated by serial dilutions in primary dog kidney (PDK) cells (Halstead S B et al., (2003) Biologic properties of dengue viruses following serial passage in primary dog kidney cells: studies at the University of Hawaii. Am J Trop Med Hyg 69(6 Suppl): 5-11). When attenuated by serial passage in cell cultures, the molecular specifics are often unknown. Now, attenuation is often obtained by introducing genetic mutations into the genome of the virus, which interfere with the virus's ability to replicate. Recurring problems have prevented many vaccine models from advancing past clinical phases. Difficulties lie in achieving optimal attenuation of each of the four DENY serotypes, which are needed to provide a minimal level of reactogenicity and maximum immunogenicity.
The present invention provides a dengue virus vaccine which comprises attenuated infectious mutants of wild type dengue virus, wherein the mutants have a mutation at E-E345K site in envelope protein. The attenuated infectious mutants of wild type dengue virus mutants are selected from the group consisting of dengue virus serotype 1, dengue virus serotype 2, dengue virus serotype 3 and dengue virus serotype 4. In the preferred embodiment, the mutants comprise SEQ ID NO: 3 or SEQ ID NO: 4. In the present invention, the mutants are propagated in MRC-5 cells.
The present invention provides a dengue virus vaccine which comprises attenuated infectious mutants of wild type dengue virus, wherein the mutants have a mutation at E-E345K site in envelope protein. The mutants mentioned in this specification are selected from the group consisting of dengue virus serotype 1, dengue virus serotype 2, dengue virus serotype 3 and dengue virus serotype 4. The mutants have a backbone of dengue virus serotype 4 strain 2A or dengue virus serotype 4 strain 2AΔ30, wherein prM and E genes can be replaced with dengue virus serotype 1, 2 and 3 to create mutants of all types of dengue virus. In the preferred embodiment, the mutants comprise SEQ ID NO: 3 or SEQ ID NO: 4. Furthermore, the attenuated dengue virus mutants are propagated in MRC-5 cells.
In present invention, target mutagenesis at Glu345Lys (E345K) in envelope protein gene was constructed in two infectious cDNA clones of a recombinant version of wild type virus DEN-4 2A and its derived 3′ NCR deletion mutant vaccine candidate virus DEN-4 2AΔ30. Using PCR-mediated site-directed mutagenesis method, the infectious cDNA clone-derived Glu345-Lys mutants of DEN-4 2A and DEN-4 2AΔ30 were passaged in Vero cells and MRC-5 cells for five consecutive times. Passage numbers are limited to less than 10 since live-attenuated vaccines require limited passage levels from the seed virus to prevent unsafe virus reversion. Single point mutation of E-Glu345Lys was found to revert to Glu345 when the virus was passaged in Vero cells. However, single point mutation E-Glu345Lys of DEN-4 2A and DEN-4 2AΔ30 were found stably existed when passaged in MRC-5 cells. The E-Glu345Lys substitution predicted using molecular modeling showed the increase of positive charges on the surface of E protein. The immunogenicity and virulence of these recombinant mutant viruses were further analyzed in mice. Virulence attenuation inducing by adaptation mutation implicated important information to the development of live-attenuated dengue vaccine.
In present invention, PCR-mediated site-directed target mutagenesis technology was used to construct two infectious clones, DEN-4 2A E-E345K and DEN-4 2AΔ30 E-E345K, and then passaged the mutant viruses derived from these clones in Vero and MRC-5 cells for consecutive 5 passages. The E-E345K mutation consistently presented in viruses recovered from MRC-5 cells, but it didn't present in viruses recovered from Vero cells (
Vero, Vero E6, and MRC-5 cells were all obtained from the Bioresource Collection and Research Center (BCRC) of the Food Industrial Research and Development Institute, Hsinchu, Taiwan. Vero cells (African green monkey kidney cells) were derived from ATCC CCL-81, and its BCRC number is 60013. Vero E6 cells (African green monkey kidney cells) were a clone of VERO 76 cells (ATCC CRL-1587) and were cloned by the dilution method into microtiter plates in 1979 by P. J. Price. The BCRC number of Vero E6 cells is 60476 (derived from ATCC CRL-1586). Vero E6 cells are more sensitive to several hemorrhagic fever viruses. MRC-5 cells (human embryonal lung fibroblasts) were derived from ATCC CCL-171, and its BCRC number is 60023. Vero, Vero E6, and MRC-5 cells were grown in Dulbecco's Modified Essential Medium (DMEM) (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/ml of penicillin G sodium-streptomycin (Invitrogen).
Stock viruses were prepared from supernatants of infected C6/36 cells grown in Hank's MEM medium (Gibco-BRL) plus supplements 6 days post-infection at 28° C. The plasmids of DEN-4 2A and its 3′NCR deletion mutant DEN-4 2AΔ30 contained the full-length genomic sequences. The plasmids were first linearized by cleavage with restriction enzyme Kpn I and then added to a transcription reaction mixture (Promega kit) containing m7G(5′)ppp(5′)G (Merck) for cap addition at the RNA 5′-end. After incubation at 37° C. for 1.5 hour, the RNA product was purified with TRIzol reagent (Invitrogen) according to manufacturer's instructions. Prior to RNA transfection, subconfluent Vero cells and MRC-5 cells in a 6-well plate were rinsed once with serum-free medium and then covered with 0.3 ml of DMEM medium per well. The transfection mixture was prepared by adding 4 μl of DMRIE-C reagent (Invitrogen) to 1 ml of DMEM, then mixing with 10 μg of the RNA product. The transfection mixture was added directly to cell monolayer. After 18 hours incubation at 37° C., either DMEM with 10% FBS or M-VSFM medium was added to the well. Eight days after transfection, culture supernatants were collected. All virus stocks were stored at −80° C. freezer for further analysis. The virus titer was then determined by plaque assay on a Vero-E6 cell line. To prepare high titers of DENY, virus supernatant was concentrated with a Centriplus device (10-kDa cutoff) (Amicon; Millipore) by 2,800 rpm centrifugation for 30 minutes before the plaque assay. The virus titer could reach 109 PFU/ml after concentration. The inoculum was prepared by diluting virus stocks in phosphate-buffered saline (PBS) immediately before inoculation. In order to confirm the homogenous virus population for our study, biological clones of each passage regimens were generated by one round of plaque purification in Vero cells or MRC-5 cells. Medium 199 (Gibco) containing 3% FBS was used for plaque purifications done in six-well culture plates by the agarose overlay method with neutral red staining described previously. The agarose plug was carefully removed without disturbing the monolayer. Each of the selected clones was then propagated once in Vero cells or MRC-5 cells in order to confirm the viability of each clone.
Target Mutagenesis, Construction of DEN-4 2A E345K, DEN-4 2AΔ30 E345K, DEN-4 2A E327G and DEN-4 2AΔ30 E327G Infectious cDNA Clones, and Recovery of Mutant Viruses
The clone-derived virus, DEN-4 2A and DEN-4 2AΔ30, exhibit the same phenotypes as the DENV-4 vaccine candidate strain 814669 virus and its less virulent 3′ NCR deletion mutant virus and was used as wt control. Target mutagenesis generating the mutant cDNA clones were performed by using overlapped PCR method. To construct DEN-4 2A E345K, DEN-4 2AΔ30 E345K, DEN-4 2A E327G and DEN-4 2AΔ30 E327G infectious cDNA clones, PCR fragments containing corresponding mutations were amplified by two rounds of PCR reactions. The first round was done by using primer pairs NsiI-f: 5′-TTTAAGGTTCCTCATGCCAAT-3′ (SEQ ID NO:7) and E345K-r: 5′-AACCACTTTTTTCTTGTTTACAT-3′ (SEQ ID NO:8), and E345K-f: 5′-ATGTAAACAAGAAAAAA (this changed E gene amino acid no. 345 from Glu to Lys; from GAA to AAA) GTGGTT-3′ (SEQ ID NO:9) and StuI-r 5′-CAACATGATGAGGGCTCGTA-3′ (SEQ ID NO:10) for construction of DEN-4 2A E345K and DEN-4 2AΔ30 E345K; primer pairs NsiI-f 5′-TTTAAGGTTCCTCATGCCAAT-3′ (SEQ ID NO:7) and E327G-r: 5′-CCAGCACCTCCATACTTGAC-3′ (SEQ ID NO: 11), and E327G-f: 5′-GTCAAGTATGGA (this changed E gene amino acid no. 327 from Glu to Gly; from GAA to GGA) GGTGCTG-3′ (SEQ ID NO:12) and StuI-r 5′-CAACATGATGAGGGCTCGTA-3′ (SEQ ID NO:10) for construction of DEN-4 2A E327G and DEN-4 2AΔ30 E327G. The infectious cDNA clones of parental viruses DEN-4 2A and DEN-4 2AΔ30 were used as templates in PCR reactions. The second round was done by using the same primer pairs NsiI-f: 5′-TTTAAGGTTCCTCATGCCAAT-3′ and StuI-r 5′-CAACATGATGAGGGCTCGTA-3′. The 2,003 bp NsiI-StuI PCR fragments were cloned into the pJET1.2/blunt cloning vector (Fermentas Life Sciences Corp.) for amplification. To introduce changes at E protein residues 345 or 327, a 2 kb region flanked by NsiI and StuI restriction enzyme sites in DEN-4 2A and DEN-4 2AΔ30 infectious clones were replaced with NsiI-StuI fragments derived from confirmed clones, which contained E345K or E327G mutations. The mutant DEN-4 2A E345K, DEN-4 2AΔ30 E345K, DEN-4 2A E327G and DEN-4 2AΔ30 E327G infectious clones were confirmed by sequencing analysis. The mutant plasmids were first linearized by cleavage with restriction enzyme KpnI and then added to a transcription reaction mixture, transcribed using SP6 RNA polymerase within the RiboMAX™ large scale RNA production system (Promega Corp.). Full-length RNA transcripts were further capped with m7G(5′)ppp(5′)G at the RNA 5′-end by using Script Cap Capping enzyme (EPICENTRE Corp.). After incubation at 37° C. for 1 hour, the RNA product was purified with TRIzol LS reagent (Invitrogen Corp.) according to manufacturer's instructions. Prior to RNA transfection, subconfluent Vero cells and MRC-5 cells in a 6-well plate were rinsed once with serum-free medium and then covered with 0.3 ml of DMEM medium per well. The transfection mixture was prepared by adding 4 n1 of DMRIE-C reagent (Invitrogen) to 1 ml of DMEM, then mixing with 10 μg of the RNA product. The transfection mixture was added directly to cell monolayer. After 18 hours incubation at 37° C., either DMEM+10% FBS or M-VSFM medium were added to the well. Eight days after transfection, culture supernatants were collected. All virus stocks were stored at −80° C. freezer for further analysis. The virus titer was then determined by plaque assay on a Vero-E6 cell line. To prepare high titers of DENY, virus supernatant was concentrated with a Centriplus device (10-kDa cutoff) (Amicon; Millipore) by centrifugation before the plaque assay. The virus titer could reach 109 PFU/ml after concentration. The inoculum was prepared by diluting virus stocks in Hank's balanced salt solution (Invitrogen) containing 0.4% bovine serum albumin fraction V (Gibco) (HBSS-0.4% BSA fraction V) immediately before inoculation.
The number of cells attached to the microcarriers was determined by nuclei staining. Briefly, a 1-ml sample of the microcarrier culture was taken and centrifuged at 200 g for 5 min to remove the supernatant. The pellets were treated with 1 ml 0.1 M citric acid [containing 0.1% (w/v) crystal violet] and incubated at 37° C. for 1 hour. The released nuclei were counted in a hemocytometer. The virus titer was measured by 10-fold serial dilutions of the culture supernatant in duplicate infections of Vero-E6 cell monolayers in a 6-well plate. After 1 hour incubation at 37° C., 4 ml of medium containing 1× Eagle's Minimum Essential Medium (EMEM) (Invitrogen), 1.1% methylcellulose, and 100 U/ml of penicillin G sodium-Streptomycin (4 ml/well) was added to each well. Virus plaques were stained with 1% crystal violet dye six days after incubation. The infectivity titer in plaque forming units (PFU) per ml was determined
DNA fragments were synthesized from DEN-4 RNA by RT-PCR using Platinum® Pfx DNA polymerase with forward and reverse primers WE1 and W02R. The DNA products were purified by Gel/PCR DNA fragments extraction kit (Geneaid, Taiwan). The nucleotide sequences of each fragment were determined by Mission Biotech Inc., Taipei, Taiwan. Sequences were aligned in the WE1/W02R region (1,653-bp) using the program Lasergene version 6.00 to generate the consensus sequence for each of the three fragments. The mean diversity of amino acids was determined as the number of amino acid substitutions divided by the total number amino acids sequenced.
Molecular modeling showed the mutations of Glu345Lys and Glu327Gly which were predicted by SWISS-MODEL based on the DEN-4 E DIII structural model determined by the nuclear magnetic resonance (NMR) spectroscopic method (Protein Data Bank code 2H0P). Surface mapping of the electrostatic field of DENV-4 E DIII was display using the software PyMOL (version 0.99, Delano Scientific). Blue and red colors denote positive and negative charges.
Heparin-Sepharose and control protein A-Sepharose beads (Pharmacia, Uppsala, Sweden) were suspended in phosphate-buffered saline (30%, wt/vol) and equilibrated before use by pelleting and washing three times in HBSS-BSA plus 10 mM HEPES (pH 8.0). 105 PFU parental DEN-4 2A, DEN-4 2AΔ30, DEN-4 2A harboring a E Glu345-Lys mutation (DEN-4 E345K), DEN-4 2AΔ30 harboring a E Glu345-Lys mutation (DEN-4 2AΔ30 E345K), DEN-4 harboring a Glu327-Gly mutation (DEN-4 E327G), and DEN-4 2AΔ30 harboring a Glu327-Gly mutation (DEN-4 2AΔ30 E327G) viruses diluted in 100 μl Hank's balanced salt solution (Invitrogen) containing 0.2% bovine serum albumin (Gibco) (HBSS-BSA) plus 100 μl of HBSS-BSA with or without Sepharose beads were mixed in Eppendorf tubes and held at 4° C. for 6 h with repeated mixing. Virus-bead mixtures were then centrifuged for 5 min at 6,000×g at 4° C. to pellet the Sepharose beads, and infectious titers in supernatants were determined by focus-forming assay (6) on Vero cells or MRC-5 cells.
To determine the binding ability of clone-derived mutated viruses, serial dilution of the virus samples were directly incubated with Vero cells. Confluent monolayers of Vero cells in 96-well plates were rinsed with PBS and then fixed by adding 10% formaldehyde overnight prior to blocking with 5% skim milk in TBST. The mutated viruses were incubated with fixed Vero cells monolayers for 1 h at room temperature. After being washed with TBST, bound viruses were quantified by ELISA analysis with mAb HB-114. The bound antibodies were detected after incubation with the anti-mouse IgG conjugated to peroxidase (KPL) for 1 h at room temperature. The ELISA products were developed with a chromogen solution containing ABTS and hydrogen peroxide and then the A405 was measured. For binding inhibition assays by GAGs, each mutated viruses were preincubated with HBSS, and GAGs, including heparin, heparin sulfate, chondroitin sulfates A, B, and C, and hyaluronic acid (Sigma Chemical Co., St. Louis, Mo.), at room temperature for 1 h. The GAG-virus mixture was added into fixed Vero cell monolayers for additional 1 h incubation at room temperature. After being washed with TBST, bound viruses were quantified by ELISA analysis.
Neutralizing antibody responses were tested in 3-week-old ICR mice (colony maintained at BioLASCO Taiwan CO., Ltd.). They were inoculated intraperitoneally with 250 μl of diluent HBSS-0.4% BSA fraction V (as mock) or diluent containing 104 PFU of viruses and were boosted with the same amount of viruses 3 weeks later. Mice were bled 2 days prior to the boost and 3 weeks after boosting.
Mouse serum samples were tested for neutralizing antibodies by serum dilution-plaque reduction neutralization test (PRNT) without addition of complement. Seventy-five PFU of virus was incubated with equal volumes of serial twofold dilutions of heat-inactivated (56° C. for 30 min) mouse serum specimens overnight at 4° C. Six-well plates of Vero cells were inoculated with the serum-virus mixtures and incubated at 37° C. in a 5% CO2 incubator for 1.5 h. Plates were then treated as described for the plaque titration protocol. Back titrations of the input DEN-4 2A and DEN-4 2AΔ30 virus were included in quadruplicate in each assay. The neutralizing antibody titer was identified as the highest serum dilution that reduced the number of virus plaques in the test by 50% or greater. The 50% neutralization inhibition dose (ID50) that is the geometric reciprocal of the serum dilution yielding 50% reduction in the virus titer was obtained using the software, ID50 version 5.0 (John L. Spouge, National Center for Biotechnology Information, Bethesda, Md., USA).
Litters of newborn (less than 1 day old) outbred white ICR mice (BioLASCO Taiwan Co., Ltd) were inoculated intracranially with 30 μl of diluent (Mock) as diluent or diluent containing 104 PFU of DEN-4 2A E345K (MRC-5)-P4, DEN-4 2A (Vero)-P4, DEN-4 2A E327G (Vero)-P4, DEN-4 2AΔ30 E345K (MRC-5)-P4, DEN-4 2AΔ30 (Vero)-P4 and DEN-4 2AΔ30 E327G (MRC-5)-P4. The diluent was HBSS-0.4% BSA fraction V (GIBCO, U.S.A.). They were observed daily for 18 days and the survival rate of each experimental group was evidenced by moribund status, paralysis, or death.
The examples below are non-limiting and are merely representative of various aspects and features of the present invention.
Target mutagenesis of E-Glu345Lys (E-E345K) and E-Glu327Gly (E-E327G) on two infectious cDNA clones, DEN-4 2A and DEN-4 2AΔ30 was conducted. RNA transcripts were obtained by incubating the cDNAs with SP6 RNA polymerase and rNTPs for 2 h and then the in vitro transcribed RNAs were capped with GTP and 5′-Cap capping enzyme for 1 h at 37° C. (
The E-E345K and E-E327G mutant viruses derived from DEN-4 2A and DEN-4 2AΔ30 clones were investigated in Vero cells and MRC-5 cells. The E-E345K mutant virus derived from the DEN-4 2A clone was only able to grow in MRC-5 cells. No virus titer was detected in the E-E345K mutant virus in Vero cells (
Since the virus genome may change during cell passages through adaptive selection, and mutations may affect cell tropism and virus virulence. The neurovirulence of the E-E345K and E-E327G mutant viruses derived from DEN-4 2A and DEN-4 2AΔ30 clones was further investigated. The virulence of these recombinant mutant viruses were analyzed in newborn ICR mice. The E-E345K mutant virus derived from the DEN-4 2A clone showed less virulent compared to its wild type DEN-4 2A virus and E-E327G mutant virus in newborn ICR mice. However, DEN-4 2A E-E345K mutant virus still killed 41.7% of the mice, compared to DEN-4 2A E-E327G mutant virus and DEN-4 2A virus killed 68.8% and 92% of the mice, respectively (
Based on the DEN-4 structural model determined by nuclear magnetic resonance (NMR) spectroscopic method (Protein Data Bank code 2H0P) (Volk D E et al., (2007) Solution structure of the envelope protein domain III of dengue-4 virus. Virology 364:147-154), Glu345 in DEN-4 E is located within the C loop at the lower central region of DIII and is nearby on the virion surface. Glu327 in DEN-4 E is located within the BC loop at the upper lateral ridge of DIII and is accessible on the virion surface, based on the DEN-2 structural model determined by cryo-electron microscopy (cryo-EM) (Protein Data Bank code 1THD). Molecular modeling predicted showed that both mutations of E-Glu345Lys and E-Glu327Gly are predicted to increase the net positive charge at the local area, as shown by surface mapping of the electrostatic field generated by the PyMOL software (version 0.99, Delano Scientific, CA, USA) using the DEN-4 DIII NMR reconstruction. The transition of the mutation E-Glu345Lys showed more net positive charge generated than that of the mutation of E-Glu327Gly. Heparin binding assay was carried to examine the binding of the parent and both DEN-4 mutated viruses to heparin-Sepharose beads. The results showed that the fraction of the mutant containing either the E-Glu345Lys and E-Glu327Gly retained by heparin beads was significantly higher than that of the parental DEN-4 2A and its 3′UTR 30 nucleotides deletion derivative DEN-4 2AΔ30 viruses (
