We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci The viral structural proteins, C, prM/M, and E, and the nonstructural proteins, NS1 to NS5, are translated as a single polyprotein and processed by cellular and viral proteases (12,49).Transmitted by Aedes aegypti mosquitoes to humans, DEN viruses cause tens of millions of cases, ranging from dengue fever to the sometimes fatal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), in tropical and subtropical regions of the world every year (42). Epidemiologic studies have shown that individuals who experience a secondary infection with a DEN virus serotype that differs from that of the previous infection are at higher risk of developing DHF/DSS (21). Therefore, an efficacious tetravalent vaccine is needed to provide solid and long-term immunity against all four DEN virus serotypes. Four parental DEN virus serotypes (DEN-1 16007, DEN-2 16681, DEN-3 16562, and DEN-4 1036) were passaged in cell cultures to obtain attenuated vaccine candidates at Mahidol University, Bangkok, Thailand (51). Human clinical trials have been conducted in Thailand and the United States (4-6, 17, 48). These attenuated viruses are currently the most promising DEN virus vaccine candidates in terms of immunogenicity and safety in humans. The Mahidol vaccine candidates DEN-1 PDK-13, DEN-2 PDK-53, DEN-3 PGMK-30/FRhL-3, and DEN-4 PDK-48 viruses have 50% minimum infectious dose values of 10 4 , 5, 3,500, and 150 PFU, respectively, in humans (4). The candidate DEN-2 PDK-53 virus vaccine, which has the lowest infectious dose in humans, is strongly immunogenic and has produced no untoward clinical symptoms. The DEN-1 PDK-13 virus vaccine, on the other hand, has a high infectious dose and has resulted in minimal reactogenicity with lower seroconversion rate in human trials (4). While only one immunization with DEN-2 PDK-53 virus was required to achieve 100% seroconversion, a DEN-1 PDK-13 virus booster was needed to achieve the same seroconversion rate.An understanding of the attenuation markers of the candidate DEN-2 PDK-53 virus vaccine should permit engineering of improved DEN virus vaccines. For this purpose, infectious cDNA clones of DEN-2 16681 and PDK-53 viruses (25), as well as recombinant DEN-2 16681/PDK-53 viruses (10), have been constructed. The uncloned PDK-53 virus vaccine contains a mixture of two genotypic variants (25), designated PDK53-E and PDK53-V in this report. The PDK53-V variant contains all nine PDK-53 virus vaccine-specific nucleotide mutations, including the Glu-to-Val mutation at amino acid position NS3-250. The PDK53-E variant contains eight of the nine mutations of the PDK-53 vaccine and the NS3-250-...
Plasmid vectors containing Japanese encephalitis virus (JEV) premembrane (prM) and envelope (E) genes were constructed that expressed prM and E proteins under the control of a cytomegalovirus immediate-early gene promoter. COS-1 cells transformed with this plasmid vector (JE-4B clone) secreted JEV-specific extracellular particles (EPs) into the culture media. Groups of outbred ICR mice were given one or two doses of recombinant plasmid DNA or two doses of the commercial vaccine JEVAX. All mice that received one or two doses of DNA vaccine maintained JEV-specific antibodies 18 months after initial immunization. JEVAX induced 100% seroconversion in 3-week-old mice; however, none of the 3-day-old mice had enzyme-linked immunosorbent assay titers higher than 1:400. Female mice immunized with this DNA vaccine developed plaque reduction neutralization antibody titers of between 1:20 and 1:160 and provided 45 to 100% passive protection to their progeny following intraperitoneal challenge with 5,000 PFU of virulent JEV strain SA14. Seven-week-old adult mice that had received a single dose of JEV DNA vaccine when 3 days of age were completely protected from a 50,000-PFU JEV intraperitoneal challenge. These results demonstrate that a recombinant plasmid DNA which produced JEV EPs in vitro is an effective vaccine.
We have constructed a series of plasmids encoding premembrane (prM) and envelope (E) protein genes of dengue virus type 2 (DEN-2). These plasmids included an authentic DEN-2 prM-E construct (pCBD2-14-6), and two chimeric constructs, 90% DEN-2 E-10% Japanese encephalitis (JE) virus E (pCB9D2-1J-4-3) and 80% DEN-2 E-20% JE E (pCB8D2-2J-2-9-1). Monoclonal antibody (MAb) reactivity indicated that all three plasmids expressed authentic DEN-2 virus E protein epitopes representative of flavivirus domains 1, 2, and 3. However, only the pCB8D2-2J-2-9-1 construct secreted high levels of prM, M (membrane), and E proteins into the culture fluid of plasmid-transformed COS-1 cells. The major portion of the prM and E proteins expressed by COS-1 cells transformed by pCBD2-14-6 or pCB9D2-4-3 plasmids remained membrane-bound. The results supported the notion that an unidentified membrane retention sequence is located between E-397 and E-436 of DEN-2 virus E protein. Replacing the carboxyl-terminal 20% of DEN-2 E (397-450) with the corresponding JE sequence had no effect on anti-DEN-2 MAb reactivity, indicating that this region is antigenically inert, although it is required for antigen secretion. Plasmid pCBD2-2J-2-9-1, which expressed secreted forms of prM/M and E that have the potential to form subviral particles, was superior to other constructs in stimulating an antibody response. Ninety percent neutralization titers ranging from 1:40 to >1:1000 were observed in seven of nine serum specimens from pCB8D2-2J-2-9-1-immunized mice. Eleven of twelve 2-day-old neonatal mice, derived from a pCB8D2-2J-2-9-1 immunized female mouse, survived intraperitoneal challenge of DEN-2 New Guinea C virus.
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