Chimeric Dengue 2 PDK-53/West Nile NY99 Viruses Re
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病菌学杂志 2005年第12期
Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Fort Collins, Colorado
ABSTRACT
Chimeric dengue serotype 2/West Nile (D2/WN) viruses expressing prM-E of WN NY99 virus in the genetic background of wild-type D2 16681 virus and two candidate D2 PDK-53 vaccine variants (PDK53-E and PDK53-V) were engineered. The viability of the D2/WN viruses required incorporation of the WN virus-specific signal sequence for prM. Introduction of two mutations at M-58 and E-191 in the chimeric cDNA clones further improved the viability of the chimeras constructed in all three D2 carriers. Two D2/WN chimeras (D2/WN-E2 and -V2) engineered in the backbone of the PDK53-E and -V viruses retained all of the PDK-53 vaccine characteristic phenotypic markers of attenuation and were immunogenic in mice and protected mice from a high-dose 107 PFU challenge with wild-type WN NY99 virus. This report further supports application of the genetic background of the D2 PDK-53 virus as a carrier for development of live-attenuated, chimeric flavivirus vaccines in general and the development of a chimeric D2/WN vaccine virus against WN disease in particular.
INTRODUCTION
West Nile (WN) virus, a member of the Flavivirus genus, is a mosquito-borne virus of the Japanese encephalitis (JE) serocomplex. The JE serocomplex contains viruses that cause central nervous system infections, such as JE virus in Asia; St. Louis encephalitis virus in the Americas; Rocio virus in Brazil; Murray Valley encephalitis virus in Australia, New Guinea, and New Zealand; and Kunjin virus (reclassified as subtype WN recently) in Australia (44). Before the mid 1990s, WN virus caused sporadic outbreaks of illness, ranging from fever to occasional encephalitis, in Africa, the Middle East, and Western Asia. However, since 1996, WN encephalitis in humans has been reported more frequently in Europe, the Middle East, northern and western Africa, and Russia (33). In 1999, WN virus first emerged in the western hemisphere in New York City and surrounding areas, where the virus caused the deaths of seven humans and numerous birds and horses (15, 33). Since then, WN virus has spread throughout most of the continental United States, with more than 4,156 reported human cases and 284 deaths in 2002 (37) and 9,862 cases with 264 deaths in 2003 (12). WN virus activity in humans, birds, and horses has been documented in Canada, the Caribbean, and Central America. The rapid spread of WN virus suggests it may pose a significant public health problem in future years (15). There is no licensed human WN vaccine available to protect at-risk populations from WN illness.
Dengue (DEN) viruses are also human pathogens that are transmitted by mosquitoes. These viruses cause illness in millions of people every year throughout tropical regions of the world. Flaviviruses of the DEN serocomplex are classified into four serotypes, DEN 1 to DEN 4 (D1 to D4). Flaviviruses contain a single-stranded positive-sense genomic RNA of approximately 11 kb with the genomic organization 5'NCR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'NCR (whereNCR is noncoding region, C is capsid, prM is premembrane, E is envelope, and NS is nonstructural protein). One of the most promising D2 vaccine candidates, strain PDK-53, was derived by passage of the wild-type D2 16681 virus 53 times in primary dog kidney (PDK) cells (48). To study the attenuation loci of the candidate D2 PDK-53 vaccine virus, as well as to develop effective tetravalent DEN vaccines against all four DEN serotypes, we constructed infectious cDNA clones of the D2 viruses (9, 26) and used them to engineer chimeric DEN viruses containing the prM-E genes of D1, D3, or D4 virus in the D2 genetic backbones (18, 19). The uncloned D2 PDK-53 vaccine virus contains a mixture of two genotypic variants (26), designated PDK53-E and PDK53-V (18, 19) 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 (26). The PDK53-E variant contains eight of the nine mutations of the PDK-53 vaccine, and the NS3-250-Glu of its parental D2 16681 virus. Our results showed that the phenotypic markers associated with the attenuation of PDK-53 virus, including small plaque size and temperature sensitivity in mammalian cell culture, limited replication in mosquito cells, and attenuation of neurovirulence in newborn mice, are determined by mutations 5'NCR-57 C to T, NS1-53 Gly to Asp, and NS3-250 Glu to Val (9). Because these loci reside outside of the structural region of the genome, chimeric viruses expressing structural genes of heterologous flaviviruses within the context of the PDK-53 background might retain the attenuation markers of the D2 PDK-53 virus. We demonstrated that this held true for the intertypic D2/D1, D2/D3, and D2/D4 chimeric viruses that contain the prM-E genes of the wild-type D1, D3, and D4 viruses, respectively, in the D2 PDK53-E and PDK53-V genetic backbones (19). These chimeric viruses elicited appropriate DEN serotype-specific neutralizing antibodies in mice. Chimeric D2/D1 viruses provided protection against wild-type D1 virus in mice and elicited anti-D1 neutralizing antibodies with low viremias in monkeys (10, 19). D2 PDK-53-vectored chimeric DEN viruses appear to be potential candidates for a tetravalent vaccine against DEN viruses.
More recently, we have investigated the potential of using this well-studied D2 PDK-53 vaccine virus as a vector for expressing the prM-E genes of other flaviviruses. Here, we report the construction of viable D2/WN chimeras in which the structural prM-E gene region of the D2 backbone was replaced with prM-E from distantly related WN virus strain NY99. We investigated the in vitro phenotypes of these chimeric D2/WN viruses, their neurovirulence/attenuation for mice, and their immunogenicity in mice. Our goal was to determine the effectiveness of the D2 PDK-53 virus-specific mutations in controlling the attenuated phenotypes of chimeric viruses expressing the prM-E gene region of a heterologous flavivirus, other than a heterologous DEN virus, in the PDK-53 background. An attenuated D2/WN virus may be a potential vaccine candidate against WN disease.
MATERIALS AND METHODS
Viruses and cell cultures. Wild-type D2 16681 and WN NY99-35262 viruses were available at the Division of the Vector-Borne Infectious Disease, Centers for Disease Control and Prevention. D2 16681 virus was originally recovered from the serum of a patient with dengue hemorrhagic fever/dengue shock syndrome in Thailand (17). The D2 16681 virus was passaged several times in grivet monkey kidney BS-C-1 cells, six times in rhesus macaque monkey, twice in Toxorhynchites amboinensis mosquitoes, and then 53 times in PDK cells at the Center for Vaccine Development, Mahidol University, to derive the candidate PDK-53 vaccine virus (17, 48). The D2 16681-P48 (wild-type 16681 virus), D2 PDK53-E48 (PDK-53 NS3-250-E variant), and D2 PDK53-V48 (PDK-53 NS3-250-V variant) infectious cDNA clones and viruses have been previously constructed and described (18, 19). WN NY99-35262 virus was originally isolated in 1999 from a Chilean flamingo at the Bronx zoo, New York (29).
Viruses were grown in Vero, LLC-MK2, and C6/36 cells as described previously (18). Virus plaque titrations were performed under double agarose overlay in six-well plates of confluent Vero cells (18). For titration of D2 and chimeric D2/WN viruses, the second agarose overlay containing neutral red vital stain was added 7 days after infection, and plaques were counted 8 to 11 days after infection. For WN viral titration, the second overlay stain was added at 1 to 2 days after infection, and plaques were counted 3 to 5 days after infection.
Construction of chimeric D2/WN plasmids. Three previously constructed D2 infectious clones, pD2-16681-P48, pD2-PDK53-E48, and pD2-PDK53-V48 (18, 19), were used to derive the chimeric D2/WN viruses. Our initial chimeric D2/WN infectious clones (D2/WN-P0, -E0, and -V0 in Fig. 1A and Table 1) contained the same junction sites as our previous chimeric D2/1, D2/3, and D2/4 constructs (19). The cDNA fragment containing the prM-E genes of WN virus was amplified by reverse transcriptase PCR (RT-PCR) from WN NY99-35262 viral RNA with primers WN-M (5'-GGAGCAGTTACCCTCTCTACGCGTCAAGGGAAGGTGATG-3'; underlined MluI site followed by WN virus sequence near the 5' end of the prM gene) and cWN-E (5'-GAAGAGCAGAACTCCGCCGGCTGCGAGAAACGTGAGAGCTATGG-3'; underlined NgoMIV site followed by complementary sequence near the 3' end of the E gene of WN virus). The amplified WN virus-specific prM-E cDNA fragment was cloned into the MluI-NgoMIV sites of our previously constructed pD2I/D3-P and -E intermediate plasmids (19) to replace the prM-E fragment of the D3 16562 virus. These D2/WN intermediate clones were ligated in vitro with the 3'-end D2 clones (backbones 16681-P48, PDK53-E48, and PDK53-V48), as described previously for the chimeric D2/3 plasmids (19), to produce the full genome length chimeric D2/WN viral cDNAs prior to transcription of chimeric D2/WN viral RNA.
To improve the viability of chimeric D2/WN viruses derived from cloned cDNA, we later modified the chimeric D2/WN clones by including part of the signal sequence (SS) for the WN prM protein (D2/WN-P1, -E1, and -V1 in Fig. 1B and Table 1). A unique SstII restriction site was introduced by site-directed mutagenesis (QuickChange site-directed mutagenesis kit; Stratagene) upstream from the 5' terminus of the prM gene in the pD2I/D3-P and pD2I/D3-E intermediate clones (19). This SstII site was engineered to make only silent mutations at the new 5' chimeric junction of each D2/WN clone (Fig. 1B). The cDNA fragment encoding part of the WN virus-specific SS at the carboxyl end of C, prM, and the amino-terminal 487 amino acids (aa) of E was amplified with primers WN-452.SAG (5'-AATTCAACGCGTACATCCGCGGGCACCGGAATTGCAGTCATGATTGGCCTGATGGC-3'; underlined SstII site followed by WN virus sequence 45 nucleotides (nt) upstream of the prM gene) and cWN-E (as described above). The D2/WN chimeras contained the carboxyl-terminal 15 aa of E from D2 virus. This amplified cDNA was cloned into the SstII-NgoMIV sites of pD2I/D3-P and pD2I/D3-E to make the intermediate clones pD2I/WN-P-SA and pD2/WN-E-SA, respectively. These intermediate clones were ligated with the 3'-end D2 clones to produce the full genome length chimeric D2/WN-P1, D2/WN-E1, and D2/WN-V1 cDNAs required for transcription of genomic viral RNA. In a third set of chimeric D2/WN clones, we engineered two extra amino acid mutations, M-58 M to L and E-191 E to A, to further facilitate the viability and stability of the chimeric D2/WN viruses derived in Vero or LLC-MK2 cells (D2/WN-P2, -E2, and -V2 in Fig. 1B and Table 1). All plasmids were amplified in Escherichia coli XL1-Blue cells and sequenced. One silent mutation at nt 1428 (CT) was identified in all clones. After ligation, the full genome length cDNAs were cut with XbaI to produce the linearized 3' end of the viral cDNA required for viral genomic RNA transcription (18).
Recovery of recombinant viruses. Vero, LLC-MK2, or C6/36 cells were transfected with transcribed recombinant viral RNA as described previously (18). Viral proteins expressed in the transfected cells were analyzed by indirect immunofluorescence assay (IFA). Virus-infected cells were fixed in cold acetone for 30 min. WN E-specific monoclonal antibody (MAb) 3.67 (kindly provided by Roy Hall of the University of Queensland, Queensland, Australia), D2 E-specific MAb 3H5, and a polyclonal mouse hyperimmune ascitic fluid against D2 New Guinea C virus were used in the assay. Antibody binding was detected with fluorescein-labeled goat anti-mouse antibody. Viruses were harvested from transfected cell cultures when the cells were over 30% IFA positive with the polyclonal mouse ascitic fluid against D2 New Guinea C virus, usually 6 to 11 days after transfection. The harvested viruses were then passaged once in Vero or LLC-MK2 cells to obtain working seeds of virus. For transfected cultures that failed to develop 30% IFA-positive cells, medium was harvested on day 14 and then passaged in Vero or LLC-MK2 cells to further evaluate the viability of the chimeras.
Replication phenotypes of chimeric viruses in cell culture. For optimal comparison, plaque phenotypes of the wild-type WN virus, D2 backbone virus controls, and chimeric D2/WN viruses were measured in the same test and using the same batch of Vero cells in six-well plates. Except for WN viral plaques, mean plaque diameters were calculated from 12 plaques for each virus at 10 days after infection. Only a few well-isolated, large WN viral plaques were measured at 10 days.
Viral growth curves were performed in 75-cm2 flasks of LLC-MK2 or C6/36 cells at a multiplicity of infection of approximately 0.001 PFU per cell. After adsorption of virus for 2 h, 30 ml of minimal essential medium (LLC-MK2) or overlay nutrient medium (C6/36 cells) (34), each containing 5% fetal bovine serum and penicillin-streptomycin, was added, and the cultures were incubated in 5% CO2 at 37°C (LLC-MK2) or 28°C (C6/36). Aliquots of culture medium were harvested at 24- to 48-h intervals, adjusted to 15 to 20% fetal bovine serum, and stored at –80°C prior to plaque titration of virus.
Temperature sensitivity was tested in LLC-MK2 cells. Following virus adsorption for 2 h at 37°C, one set of cultures was incubated for 7 days at 37°C, the other at 39°C. Aliquots of culture medium were harvested at days 4 to 7 postinfection for the D2 control viruses and D2/WN chimeras and from days 1 to 5 postinfection for the WN NY99 virus.
Sequencing of viral genomes. The genomes of all the working seeds of the chimeric viruses were fully sequenced, except for about 24 bases at the extreme 5' and 3' termini of the genome. Viral genomic RNA was extracted from virus working seed by using the QIAmp viral RNA kit (QIAGEN). For each recombinant virus, six overlapping cDNA fragments which covered the entire viral genome were amplified by RT-PCR. The cDNAs were sequenced by using the CEQ 8000 sequencer (Beckman Coulter). Primers used for RT-PCR and sequencing will be provided upon request. They were based on the published sequences of WN NY99-35262 (GenBank accession no. AF196835) and D2 16681 (GenBank accession no. U87411).
Neurovirulence in suckling mice. Litters of newborn (less than 1 day old) outbred NIH Swiss Webster mice (colony maintained at the Centers for Disease Control and Prevention [CDC]) were inoculated intracranially with 20 μl of diluent containing 104 or 5,000 PFU of virus. Inoculated mice were observed daily for 4 weeks. A fatal endpoint was evidenced by moribund status, paralysis, or death.
Immunogenicity and protection in adult mice. Neutralizing antibody responses were tested in 3- to 4-week-old NIH Swiss Webster mice (CDC colony). Mice were inoculated intraperitoneally (i.p.) with 104 PFU of virus. Several groups of immunized mice were boosted with an equivalent dose of the same virus 4 weeks later. Mice were bled at 26 days after primary immunization and 12 days after boosting. To study protection, immunized mice were challenged i.p. with a lethal dose (105 or 107 PFU) of WN NY99-35262 virus at 4 to 6 weeks after primary immunization or 2 weeks after boosting. The surviving mice were bled again 21 to 28 days after challenge. The sera were tested for neutralizing antibodies against D2 16681 and WN NY99 viruses.
Neutralization assays. The plaque reduction neutralization test (PRNT) was performed in six-well plates of Vero cells as described previously (18). The mouse sera were heat inactivated (56°C for 30 min), and the tests were performed without addition of exogenous complement. Titrations of the input D2 16681 and WN NY99-35262 viruses were included in each assay. The neutralizing antibody titer was identified as the highest serum dilution, in serial twofold dilution series, that reduced the number of input virus plaques in the test by at least 50%.
RESULTS
Construction and recovery of chimeric D2/WN viruses. The first construction strategy used our originally engineered MluI junction site at nt position 451 (19) (Fig. 1A). From the three D2/WN cDNA clones, D2/WN-P0, -E0, and -V0, we recovered only low-titered (fewer than 10 PFU/ml) D2/WN-P0 virus from transfected C6/36 cells and no virus from transfected LLC-MK2 cells. After five passages in LLC-MK2 cells, the D2/WN-P0 virus titer increased to 2.6 x 105 PFU/ml. Genomic sequencing of the high-titer C6/36-0/LLC-MK2-5 seed revealed two new mutations in the E gene (WN-E-248 Thr to Met and D2-E-492 Met to Val) and one nucleotide insertion in the 3'NCR (extra A preceding nt –10337) that were not present in the corresponding cDNA plasmids. IFA of C6/36 and LLC-MK2 cells transfected with D2/WN-P0 RNA showed 15 to 20% and <1% cells positive to WN MAb 3.67 at day 14 posttransfection, respectively. No detectable virus was rescued from C6/36 or LLC-MK2 cells transfected with transcribed D2/WN-E0 or -V0 RNA (Table 1). These cultures showed less than 1% IFA-positive cells at 14 days of incubation.
The second strategy of D2/WN construction utilized the newly engineered SstII site at nt 398 of the D2 cDNA, immediately downstream of the potential NS2B-3 protease cleavage site between the C and prM proteins (Fig. 1B). This strategy included a portion of the WN viral SS of prM, which is a transmembrane signal located between C and prM. All D2/WN chimeras retained the originally engineered NgoMIV junction site located in the transmembrane 2 domain of the translated D2 E protein (19). Using this new strategy, we constructed D2/WN-P1, -E1, and -V1 chimeras in the backbones of D2 16681-P48, PDK53-E48, and PDK53-V48, respectively (Table 1). D2/WN-P1 virus was successfully rescued from both transfected C6/36 cells (high titers of 105 to 106 PFU/ml) and LLC-MK2 cells (103 to 105 PFU/ml). The C6/36-0/LLC-MK2-1 D2/WN-P1 virus acquired no new mutation, suggesting this virus was more viable and stable in cell cultures than the D2/WN-P0 virus.
D2/WN-E1 virus (in the PDK53-E backbone) was recovered from transfected LLC-MK2, Vero, and BHK-21 cells at titers ranging from 1 to 104 PFU/ml (Table 1). After one to three Vero passages of the recovered D2/WN-E1 viruses, titers increased to above 106 or 107 PFU/ml. Sequence analyses of two independently derived D2/WN-E1 viral seeds, clone 112 and clone 40, revealed evolving mutations that were not present in their corresponding cDNA. Mutations NS2A-22 Met to Val/Met mix and NS2B-93 Gln to Gln/Arg occurred in clone 112, while different mutations E-208 Thr to Ile/Thr and NS2A-36 Val to Val/Phe occurred in clone 40. We were not able to recover detectable viable chimeric D2/WN-V1 virus (in the PDK53-V backbone) from transfected LLC-MK2, Vero, or BHK-21 cells. We did not attempt to transfect C6/36 cells with either D2/WN-E1 or -V1 transcribed viral RNA (Table 1), because chimeras constructed in the D2 PDK53-E or -V backbone were not expected to replicate efficiently in the C6/36 cells (18, 19).
To further improve the viability and stability of the D2/WN chimeras in cell culture, we incorporated mutations E-191 Glu to Ala and M-58 Met to Leu (as well as a cDNA clone-derived nt –1905 A-to-G silent mutation) in the cDNA to construct D2/WN-P2, -E2, and -V2. These mutations occurred in a rescued chimeric D2/WN virus which exhibited improved fitness in Vero cells (data not shown). We derived D2/WN-P2, -E2, and -V2 viruses from transfected Vero cells. The viability of D2/WN-V2 virus was especially encouraging, because it was the first viable chimeric D2/WN-V virus in the PDK53-V backbone to be rescued (Table 1). The E-191 and M-58 mutations also improved the stability of the D2/WN viruses derived from backbones D2 16681 and PDK53-E. The titers of the clone-derived D2/WN-P2 and -E2 viruses were greater than 106 PFU/ml after a single amplifying passage in Vero cells, compared to 105 PFU/ml for the D2/WN-P1 and -E1 viruses. The genome sequence of D2/WN-P2 virus was identical to the sequence of its cDNA. However, the D2/WN-E2 and -V2 viral genomes contained new mutations that were acquired during transfection and passage in cell cultures. The D2/WN-E2 working viral seed contained an NS2A-22 Met-to-Met/Val mutation, while two mutations, NS2A-22 Met to Val and nt 10661 (based on the D2 3'NCR) C to T, were found in the D2/WN-V2 working seed. Interestingly, an identical NS2A-22 mutation was also identified in the D2/WN-E1 clone 112 virus described above.
Plaque sizes of D2/WN chimeras in Vero cells. Among the D2 backbone viruses, 16681-P48 generated the largest plaques, followed by the PDK53-E48 and then the PDK53-V48 virus (Fig. 2). The relative plaque sizes of the D2 backbone viruses in Vero cells were similar to our previous results with LLC-MK2 cells (19). However, all the viruses produced smaller plaques in Vero cells than in LLC-MK2 cells. The chimeric D2/WN viruses produced plaques that averaged 0.4 to 1.8 mm in diameter. Their plaques were much smaller than those of the prM-E donor WN virus (greater than 16 mm average diameter) and were more similar in size to their backbone D2 viruses. Interestingly, the D2/WN-P1 and -P2 plaques were smaller than the plaques of the D2/WN-E1 (clone 112 and 40) and -E2 viruses, and also smaller than plaques of their backbone D2 16681-P48 virus. The D2/WN-V2 virus produced plaques that were smaller than those of D2/WN-E2 virus, which corresponded to the relative sizes of their backbone D2 PDK53-V48 and -E48 viruses, respectively.
Growth of D2/WN chimeras in LLC-MK2 and C6/36 cells. WN NY99 virus grew much faster and to higher titers in LLC-MK2 cells than the D2 and chimeric D2/WN viruses (Fig. 3A). WN virus reached a peak titer of over 109 PFU/ml at 3 days postinfection, at which time a significant cytopathic effect was evident. The D2 16681-P48 virus replicated somewhat more efficiently than the D2 PDK53-E48 and -V48 viruses. The D2 16681-P48 virus usually reaches a peak titer of 106 to 107 PFU/ml within 6 to 8 days after infection, while PDK53-E48 and -V48 viruses typically reach peak titers of 105 to 107 PFU/ml between 7 and 10 days postinfection (Fig. 3A) (18, 19). Replication of the chimeric D2/WN-P2, -E2, and -V2 viruses, using the D2 backbone replication machinery, was similar to that of the D2 PDK53-E48 and -V48 viruses in LLC-MK2 cells (Fig. 3A).
In C6/36 cells, the WN NY99 virus again replicated much faster with a higher peak titer (1010 PFU/ml) than the D2 and D2/WN chimeras (Fig. 3B). D2 16681-P48 virus typically grows to 107 to 108 PFU/ml in C6/36 cells (18, 19), replicating much more efficiently than the PDK53-E48 and -V48 viruses, which usually reach peak titers of 104 to 105 PFU/ml (Fig. 3B) (18, 19). The D2/WN -P2 virus replicated with an initial efficiency that was similar to that of its D2 backbone 16681-P48 virus (Fig. 3B), although the peak titer of D2 16681-P48 virus was about 18-fold greater than that of D2/WN-P2 virus at 11 days postinfection. Replication of the chimeric D2/WN-E2 and -V2 viruses was very inefficient in the C6/36 cells, with peak titers of only 102 to 103 PFU/ml (Fig. 3B).
Temperature sensitivities of chimeric D2/WN viruses in LLC-MK2 cells. The WN virus was not temperature sensitive at 39°C in LLC-MK2 cells. D2 16681-P48 and D2/WN-P2 viruses demonstrated similar levels of temperature sensitivity, as indicated by a maximal reduction in viral titers of 1.9 log PFU/ml at 39°C. D2/WN-E2 and -V2 viruses showed up to 2.2- and 2.0-log reductions at 39°C, respectively. As reported previously (18, 19), D2 PDK53-E48 virus was more temperature sensitive (up to 3-log reduction at 39°C) than 16681-P48 virus but less sensitive than the PDK53-V48 virus, which suffered an up to 3.7-log reduction in titer at 39°C (Fig. 4).
Neurovirulence in newborn mice. Previously, we tested the D2 16681-P48, PDK53-E48, and PDK53-V48 viruses and chimeric DEN viruses in newborn ICR mice (9, 18, 19). During the present study, the CDC mouse colony switched to NIH Swiss Webster mice, which we found to be more sensitive to D2 virus. Therefore, the neurovirulence experiments in this report were performed in newborn NIH Swiss Webster mice. D2 16681-P48, PDK53-E48, and PDK53-V48 viruses consistently caused 100%, 62.5%, and 6.25% mortality in the newborn Swiss mice, respectively (Fig. 5).
The WN NY99 virus was highly neurovirulent for the newborn Swiss Webster mice (100% mortality within 6 days of challenge). Due to low seed titers of the D2/WN-P1, -E1 clone 112, and -E1 clone 40 viruses, we challenged mice with 5,000 PFU of these three viruses instead of the 104-PFU dose that we used for the other viruses reported in Fig. 5. Inoculation of mice with 5,000 PFU or 104 PFU of WN NY99, D2 16681, or D2 PDK53-E48 virus resulted in equivalent mortality rates for each virus in newborn Swiss mice (data not shown). Therefore, it is unlikely that the twofold difference in challenge dose would affect comparative interpretation of results. The D2/WN-E1 (clone 112 and 40), -E2, and -V2 viruses were less virulent than the WN NY99 and D2 16681-P48 viruses, and they were at least as attenuated as their backbone D2 PDK53-E48 or -V48 virus in these mice. Interestingly, the D2/WN-P1 and -P2 viruses, which expressed prM-E from highly virulent WN NY99 in the D2 backbone of 16681-P48 virus, caused no mortality in these mice.
Immunogenicity and protective efficacies of D2/WN chimeras in mice. All chimeric D2/WN viruses tested in this study induced neutralizing antibodies against WN NY99 virus after primary i.p. immunization (Tables 2 and 3). Anti-D2 16681 PRNT titers of pooled sera from each D2/WN group were not detectable (titer less than 1:10). The pooled sera of the D2 16681-P48 group also had a nondetectable primary PRNT titer against D2 16681 virus but had a low PRNT titer of 1:20 against D2 16681 virus after a boost (data not shown). The control phosphate-buffered saline (PBS) and D2 16681-P48 groups had no background PRNT titers against WN NY99 virus (Table 2 and 3). The D2/WN-P2 virus induced individual primary PRNT titers against WN NY99 virus, ranging from 1:20 to 1:1,280 in 87.5% of the mice (geometric mean titers [GMTs] of 1:160 and 1:119; Tables 2 and 3, respectively). After boosting with the D2/WN-P2 virus, the seroconversion rate increased to 100% with a PRNT GMT of 1:1,902 and individual titers ranging from 1:640 to 1:5,120 (Table 3). Chimeric D2/WN-E2 virus induced WN neutralizing titers ranging from 1:10 to 1:320 (GMT = 1:40) in 75 to 87.5% of the mice after primary inoculation (Tables 2 and 3) and from 1:20 to 1:5,120 (GMT = 1:580) in all mice after a boost (Table 3). D2/WN-V2 virus induced WN neutralizing titers ranging 1:10 to 1:1,280 (GMT = 1:108 and 1:269 in Tables 2 and 3, respectively) in 87.5 to 100% of mice, and boosted titers ranging from 1:640 to 1:40,960 (GMT = 1:4695) in 100% of mice (Table 3). The D2/WN-E1 viruses, clones 112 and 40, were also immunogenic in these mice, and they induced a primary WN neutralizing GMT of 1:93 in 87.5% of mice and a GMT of 1:22 in 100% of mice, respectively (Table 2).
The D2/WN-P2, -E2, and -V2 viruses protected most mice against an i.p. challenge of 105 PFU of WN NY99 virus (about 104 median intraperitoneal lethal doses [IPLD50]) according to a previous report (42) after one immunization (Table 2). Only one mouse died in the D2/WN-E2 group, while all mice in the D2/WN-P2 and -V2 groups survived a WN virus challenge. All mice in the PBS and D2 16681-P48 control groups succumbed to a WN virus challenge. When the WN challenge dose was increased to 107 PFU (about 106 IPLD50), one immunizing dose of the D2/WN chimeras was still able to provide at least partial protection against a WN virus challenge. Two (25%) of the eight mice in the D2/WN-P2 group, four (57%) of seven mice in the D2/WN-E2 group, and one (12.5%) of the eight mice in the D2/WN-V2 group died from the extremely high-dose challenge (data not shown in Table 2), while none survived in the PBS or D2 16681-P48 control group. Chimeric D2/WN-E1 viruses, clones 112 and 40, provided similar partial protection against the 107 PFU WN challenge after just one immunization (Table 2). All mice were fully protected against the high 107 PFU WN NY99 challenge after a booster dose of the D2/WN-P2, -E2, or -V2 virus (Table 3). All surviving vaccinated mice exhibited no symptoms of illness. We tested the high 107-PFU challenge dose of WN virus to ensure 100% death in the PBS control groups. In earlier experiments, although 100% mortality was observed in most of the PBS control groups challenged with 105 PFU of WN NY99 virus, some groups only suffered 50 to 87.5% mortality. Mock-immunized mice surviving the 105-PFU WN virus challenge showed illness between days 7 and 10 after a challenge and then recovered soon without any significant symptoms, and they all had very high neutralizing antibody titers against WN virus (data not shown), apparently ruling out challenge failure as a cause of such inconsistent mortality rates.
DISCUSSION
The 5'NCR-57, NS1-53, and NS3-250 loci are the major attenuating determinants of the candidate D2 PDK-53 vaccine virus (9). Their location outside of the structural gene region makes the PDK-53 background ideal as a potential vector for chimeric flavivirus vaccine development. Both D2 PDK53-E and PDK53-V backbones have been used to develop intertypic D2/1, D2/3, and D2/4 viruses that maintain the phenotypic markers of the D2 PDK-53 vaccine virus (18, 19). In this study, the potential of the PDK53 variants to serve as vectors for chimeric D2/WN vaccine viruses was explored. WN and DEN viruses belong to two different serocomplexes (JE complex and DEN complex), and their mosquito vector preferences are different (Aedes for DEN and Culex for WN).
Viability- and fitness-enhancing amino acid substitutions of the chimeric D2/WN viruses. The initial D2/WN chimeras, which utilized our original MluI-prM splicing site (19), were either not viable or were very unstable in cell culture. The flavivirus C and prM proteins are separated by an internal SS of 14 to 22 aa which directs translocation of prM into the lumen of the endoplasmic reticulum (31, 45). The internal SS of DEN (14 aa) and WN (18 aa) are flanked by upstream viral NS2B-3 protease and C-terminal cellular signalase cleavage sites (Fig. 1). Our first constructs, D2/WN-P0, -E0, and -V0, were spliced at prM-6 of the D2 virus and contained both NS2B-3 and signalase cleavage sites from the D2 backbone (Fig. 1A). When we changed our strategy to include the C-terminal 15 aa of the WN viral SS to replace the C-terminal 11-aa SS of D2 virus (Fig. 1B), we recovered viable D2/WN-P1 and -E1 viruses but not D2/WN-V1 virus. The new constructs retained the NS2B-3 cleavage site and the downstream 3 aa (SAG) of the D2 backbone virus, but the 15 C-terminal aa of the 18-aa SS were derived from the SS of WN virus. Clearly, the WN virus-specific SS and signalase cleavage site improved the viability of D2/WN-P1 and -E1. Coordination of the two cleavages at the C-prM junction is critical to the assembly of flaviviruses (1, 2, 30, 32, 45). Signalase cleavage of prM is inefficient until the cytosolic C protein is proteolytically removed by the virus-specific NS2B-3 protease (1, 45). Coordination of cytosolic and luminal cleavages at the C-prM junction may be important for efficient maturation of nucleocapsid (NC) during virus assembly (32). Uncoupling of the cleavages has resulted in reduced incorporation of NC into budding membranes and augmented release of NC-free virus-like particles (32). It is likely that our modified splicing strategy improved the coordination between the two C-prM cleavages and NC assembly in the D2/WN chimeras. It is also possible that the modified D2/WN SS provided more optimal polypeptide conformation surrounding the signalase cleavage site to promote proper cleavage at the N terminus of prM. Other reports have also shown that different compositions of the SS can profoundly affect the viability of the yellow fever virus and chimeric WN/DEN4 virus (30, 42).
It is interesting that our first construct, D2/WN-P0, was viable in C6/36 cells but not in mammalian cells. We reported a similar phenomenon for a D2/4-P chimera that had the same chimeric junction at D2 prM-6 (19). The transient existence of a C-prM precursor has been shown in flavivirus-infected C6/36 cells, but not in Vero cells (36), indicating cleavage of the C protein at the NS2B-3 cleavage site may be delayed in C6/36 cells. Such a delay may have contributed to the more efficient replication of our chimeric D2/WN-P0 and D2/4-P constructs in C6/36 cells than in mammalian cells. Engineered mutations, including one at the N-terminal end of the SS, greatly enhanced viabilities of chimeric D2/4 viruses in mammalian cells (19). In this report, viabilities of the D2/WN chimeras were enhanced in both C6/36 and mammalian cells by including the SS of prM from WN virus.
We further optimized the fitness of the D2/WN chimeras in Vero cells by incorporating M-58 Met-to-Leu and E-191 Glu-to-Ala mutations into the D2/WN-P2, -E2, and -V2 cDNAs. The permissive mechanism of these mutations is unclear, but they permitted the D2/WN-P2, -E2, and -V2 chimeras to grow to high titers in mammalian cells. The mutations were especially important for the D2/WN-V2 virus, which otherwise was not viable in Vero, BHK-21, or LLC-MK2 cells. The prominent effect of the mutations for the D2/WN-V2 virus may have resulted from the slower replication of chimeras based on the PDK53-V backbone (9, 18, 19). The M-58 and E-191 mutations, however, did not change the fitness of the D2/WN chimeras in C6/36 cells, which was evident from the efficient growth observed for both D2/WN-P1 and -P2 chimeras in C6/36 cells (Table 1). Despite these mutations, the D2/WN-E2 and -V2 viruses, based on the PDK-53 background, still replicated poorly in C6/36 cells. Crippled replication in C6/36 cells is one of the phenotypic attenuation markers exhibited by the D2 PDK-53 vaccine virus (Fig. 3).
Replication phenotypes of chimeric D2/WN viruses in cell cultures. The mutations M-58 and E-191 permitted the D2/WN-P2 virus to replicate better and produce larger plaques than the D2/WN-P1 virus in Vero cells. Direct comparisons of plaque sizes among the D2/WN-E1 clone 112, D2/WN-E1 clone 40, and D2/WN-E2 viruses were not straightforward because these viruses acquired different additional mutations during derivation. Our previous studies showed that chimeric DEN viruses engineered in the PDK53-E and -V backbones had smaller plaques than those constructed in the 16681 backbone (19). However, in this study D2/WN-E1 (clones 112 and 40) and D2/WN-E2 (in the PDK53-E backbone) produced larger plaques relative to their counterparts in the 16681 backbone, D2/WN-P1 and D2/WN-P2, respectively. The additional mutations acquired during production of the D2/WN-E1 and -E2 viruses probably contributed to the larger plaque sizes of the viruses. All of the chimeric D2/WN viruses exhibited greatly reduced plaque sizes in Vero cells, relative to the plaques of the prM-E donor WN NY99 virus. Their plaque sizes and growth curves were more similar to those of the D2 backbone viruses, whose replication machinery largely controlled the phenotypes of the chimeric viruses.
The D2/WN-P2 virus produced smaller plaques and grew to somewhat lower titers in LLC-MK2 cells than its backbone D2 16681-P48 virus, indicating some degree of incompatibility between the inserted WN prM-E and D2 background genes. The growth curves of the D2/WN-E2 and -V2 viruses were very similar to, and their plaque size larger than, those of their backbone D2 PDK53-E48 and -V48 viruses, suggesting the likelihood that their additional acquired mutations, such as the common NS2A-22 Met-to-Val mutation, further improved the fitness of these D2/WN chimeras in mammalian cells. The chimeric D2/WN-P2, -E2, and -V2 virus seeds produced in Vero cells had high titers of 106 to 107 PFU/ml, which is encouraging, because Vero cells are licensed for vaccine virus production.
Reduced replication of the candidate D2 PDK-53 vaccine virus in Aedes aegypti mosquitoes and C6/36 (A. albopictus) cells is a significant biological attenuation marker of the PDK-53 virus (9, 25, 26). PDK-53 virus is not transmitted by A. aegypti mosquitoes (25), reducing the potential for secondary transmission of the vaccine virus and providing an important safety feature for the vaccine. We and others have reported this low-replication phenotype in C6/36 cells for different DEN vaccine candidates (11, 19, 21, 22, 46). The crippled C6/36 replication phenotype of D2 PDK-53 virus is encoded by both the 5'NCR-57 C-to-T and the NS1-53 Gly-to-Asp mutations (9). In this study, chimeric D2/WN-P2 virus and its backbone 16681-P48 virus replicated to similar high titers in C6/36 cells, indicating good compatibility between the heterologous genes for replication in C6/36 cells. The chimeric D2/WN-E2 and -V2 viruses, both of which possessed the 5'NCR-57 and NS1-53 mutations, retained the crippled C6/36 replication phenotype of D2 PDK-53 virus (Fig. 3B). Based on analyses of several DEN vaccine candidates (21, 22, 25, 26), the poor C6/36 replication phenotype of DEN vaccine viruses likely reflects limited replication, dissemination, and transmission in mosquitoes.
The temperature sensitivity phenotype of D2 PDK-53 virus has been attributed to a synergism between the NS1-53-Asp and NS3-250-Val loci (9). The PDK53-V variant, containing both loci, is more temperature sensitive than the PDK53-E variant, and both variants are more temperature sensitive than the 16681 virus (Fig. 4) (9, 19). Unlike the prM-E donor WN NY99 virus, which was not temperature sensitive at 39°C, the D2/WN-P2, -E2, and -V2 viruses exhibited temperature sensitivities that were equivalent to or slightly greater than that of the D2 16681-P48 virus. The D2/WN-V2 virus did not show a more prominent temperature sensitivity phenotype than the D2/WN-E2 virus. These results are consistent with those observed previously for DEN chimeras (19).
Attenuation, immunogenicity, and protective efficacy of the chimeric D2/WN viruses in mice. Most studies have identified major determinants of mouse virulence/attenuation in the flavivirus E protein (3, 13, 20, 24). However, attenuation of the D2 PDK-53 virus in newborn ICR mice is determined mainly by the 5'NCR-57 and NS1-53 loci (9, 18). In this study, the more D2-susceptible newborn Swiss mice further identified a contribution of the NS3-250-Val locus to this attenuation phenotype (Fig. 5). Both D2/WN-P1 and -P2 viruses, which contained the expected recombinant viral genome sequences, were attenuated for the Swiss mice, whereas the WN NY99 donor and D2 16681-P48 carrier viruses were uniformly fatal. The recombination of heterologous genes in flavivirus chimeras per se may contribute significantly to the attenuation phenotype of the resulting chimeric virus, as has been observed previously (39, 41, 42). Comparing results between the D2/WN-P1 and -P2 chimeras, it is apparent that the engineered M-58 and E-191 mutations in the D2/WN-P2 virus did not affect the attenuated phenotype of the D2/WN-P2 virus. Unexpectedly, the D2/WN-E1 clone 112 and 40, D2/WN-E2, and D2/WN-V2 viruses were more neurovirulent than the D2/WN-P1 and -P2 viruses. This may be due to the additional mutations acquired in the D2/WN-E and -V viruses, such as the NS2A-22 mutation that occurred in three of these viruses. Such mutations may promote the replication efficiency of these chimeras not only in mammalian cells but also in mice. Nonetheless, all of the D2/WN-E and -V viruses tested were at least as attenuated as their PDK53-E48 and -V48 carrier viruses in the newborn Swiss mice, and the degree of the attenuation correlated well with the PDK53-E or -V backbone of each virus.
All of the chimeric D2/WN viruses tested induced neutralizing antibodies against WN NY99 virus in mice (Tables 2 and 3). Most mice receiving a primary immunization with chimeric D2/WN virus and all mice receiving a booster dose of D2/WN virus were protected from a severe i.p. WN viral challenge. D2/WN virus-immunized mice that survived a WN NY99 virus challenge all had greater PRNT titers than were present before challenge, indicating that the D2/WN chimeras provided efficient priming immunity against WN virus.
Application of the D2 PDK-53 virus as a carrier for flavivirus vaccine development. Advanced recombinant genetic technologies have recently shown potential for the engineering of safe and effective live attenuated chimeric flavivirus vaccines that may provide a safe balance between attenuation and induction of protective immunity (4, 5, 8, 16, 18, 19, 27, 28, 35, 38, 40). Such recombinants include chimeric yellow fever 17D/WN (4, 5) and DEN-4/WN (40, 42) viruses. In human trials the candidate D2 PDK-53 vaccine virus has proved to be safe, to elicit neutralizing antibodies for at least 2 years, and to induce significant memory T-cell responses (6, 7, 14, 47). Our previous results for chimeric DEN viruses suggested that D2 PDK-53 virus is an ideal carrier for chimeric DEN vaccine development (19). D2 PDK-53 virus is one of the few attenuated viruses for which attenuation determinants have been thoroughly studied at the molecular level (9) and have been tested safely and effectively in human trials (6, 23, 43). The well-studied attenuation loci of the PDK-53 genetic background should permit more straightforward quality and safety verification during vaccine development and use. Any possible reversion to the wild type of a vaccine production lot or in a vaccinee can be quickly identified by advanced molecular technologies. In this study, the chimeric D2/WN-E2 and -V2 viruses retained the attenuated phenotypic characteristics of the D2 PDK-53 vaccine virus, were immunogenic in mice, and protected mice from severe WN viral challenge. Although the D2/WN-P2 virus was also attenuated and immunogenic in mice, it still retained certain wild-type characteristics, such as efficient growth in C6/36 cells and less temperature sensitivity in LLC-MK2 cells. These results provide a basis for further evaluation of the chimeric D2/WN viruses in nonhuman primates and horses as potential vaccine candidates against WN virus and further support utilization of the PDK-53 virus as a chimeric carrier for flavivirus vaccine development.
ACKNOWLEDGMENTS
We thank Rhett Mays, Becky Rose, Kiyotaka Tsuchiya, Steven Gire, and Lindsey Doyle for technical support at different stages of this project. We also thank John Roehrig and David Withum for comments on the manuscript.
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ABSTRACT
Chimeric dengue serotype 2/West Nile (D2/WN) viruses expressing prM-E of WN NY99 virus in the genetic background of wild-type D2 16681 virus and two candidate D2 PDK-53 vaccine variants (PDK53-E and PDK53-V) were engineered. The viability of the D2/WN viruses required incorporation of the WN virus-specific signal sequence for prM. Introduction of two mutations at M-58 and E-191 in the chimeric cDNA clones further improved the viability of the chimeras constructed in all three D2 carriers. Two D2/WN chimeras (D2/WN-E2 and -V2) engineered in the backbone of the PDK53-E and -V viruses retained all of the PDK-53 vaccine characteristic phenotypic markers of attenuation and were immunogenic in mice and protected mice from a high-dose 107 PFU challenge with wild-type WN NY99 virus. This report further supports application of the genetic background of the D2 PDK-53 virus as a carrier for development of live-attenuated, chimeric flavivirus vaccines in general and the development of a chimeric D2/WN vaccine virus against WN disease in particular.
INTRODUCTION
West Nile (WN) virus, a member of the Flavivirus genus, is a mosquito-borne virus of the Japanese encephalitis (JE) serocomplex. The JE serocomplex contains viruses that cause central nervous system infections, such as JE virus in Asia; St. Louis encephalitis virus in the Americas; Rocio virus in Brazil; Murray Valley encephalitis virus in Australia, New Guinea, and New Zealand; and Kunjin virus (reclassified as subtype WN recently) in Australia (44). Before the mid 1990s, WN virus caused sporadic outbreaks of illness, ranging from fever to occasional encephalitis, in Africa, the Middle East, and Western Asia. However, since 1996, WN encephalitis in humans has been reported more frequently in Europe, the Middle East, northern and western Africa, and Russia (33). In 1999, WN virus first emerged in the western hemisphere in New York City and surrounding areas, where the virus caused the deaths of seven humans and numerous birds and horses (15, 33). Since then, WN virus has spread throughout most of the continental United States, with more than 4,156 reported human cases and 284 deaths in 2002 (37) and 9,862 cases with 264 deaths in 2003 (12). WN virus activity in humans, birds, and horses has been documented in Canada, the Caribbean, and Central America. The rapid spread of WN virus suggests it may pose a significant public health problem in future years (15). There is no licensed human WN vaccine available to protect at-risk populations from WN illness.
Dengue (DEN) viruses are also human pathogens that are transmitted by mosquitoes. These viruses cause illness in millions of people every year throughout tropical regions of the world. Flaviviruses of the DEN serocomplex are classified into four serotypes, DEN 1 to DEN 4 (D1 to D4). Flaviviruses contain a single-stranded positive-sense genomic RNA of approximately 11 kb with the genomic organization 5'NCR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'NCR (whereNCR is noncoding region, C is capsid, prM is premembrane, E is envelope, and NS is nonstructural protein). One of the most promising D2 vaccine candidates, strain PDK-53, was derived by passage of the wild-type D2 16681 virus 53 times in primary dog kidney (PDK) cells (48). To study the attenuation loci of the candidate D2 PDK-53 vaccine virus, as well as to develop effective tetravalent DEN vaccines against all four DEN serotypes, we constructed infectious cDNA clones of the D2 viruses (9, 26) and used them to engineer chimeric DEN viruses containing the prM-E genes of D1, D3, or D4 virus in the D2 genetic backbones (18, 19). The uncloned D2 PDK-53 vaccine virus contains a mixture of two genotypic variants (26), designated PDK53-E and PDK53-V (18, 19) 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 (26). The PDK53-E variant contains eight of the nine mutations of the PDK-53 vaccine, and the NS3-250-Glu of its parental D2 16681 virus. Our results showed that the phenotypic markers associated with the attenuation of PDK-53 virus, including small plaque size and temperature sensitivity in mammalian cell culture, limited replication in mosquito cells, and attenuation of neurovirulence in newborn mice, are determined by mutations 5'NCR-57 C to T, NS1-53 Gly to Asp, and NS3-250 Glu to Val (9). Because these loci reside outside of the structural region of the genome, chimeric viruses expressing structural genes of heterologous flaviviruses within the context of the PDK-53 background might retain the attenuation markers of the D2 PDK-53 virus. We demonstrated that this held true for the intertypic D2/D1, D2/D3, and D2/D4 chimeric viruses that contain the prM-E genes of the wild-type D1, D3, and D4 viruses, respectively, in the D2 PDK53-E and PDK53-V genetic backbones (19). These chimeric viruses elicited appropriate DEN serotype-specific neutralizing antibodies in mice. Chimeric D2/D1 viruses provided protection against wild-type D1 virus in mice and elicited anti-D1 neutralizing antibodies with low viremias in monkeys (10, 19). D2 PDK-53-vectored chimeric DEN viruses appear to be potential candidates for a tetravalent vaccine against DEN viruses.
More recently, we have investigated the potential of using this well-studied D2 PDK-53 vaccine virus as a vector for expressing the prM-E genes of other flaviviruses. Here, we report the construction of viable D2/WN chimeras in which the structural prM-E gene region of the D2 backbone was replaced with prM-E from distantly related WN virus strain NY99. We investigated the in vitro phenotypes of these chimeric D2/WN viruses, their neurovirulence/attenuation for mice, and their immunogenicity in mice. Our goal was to determine the effectiveness of the D2 PDK-53 virus-specific mutations in controlling the attenuated phenotypes of chimeric viruses expressing the prM-E gene region of a heterologous flavivirus, other than a heterologous DEN virus, in the PDK-53 background. An attenuated D2/WN virus may be a potential vaccine candidate against WN disease.
MATERIALS AND METHODS
Viruses and cell cultures. Wild-type D2 16681 and WN NY99-35262 viruses were available at the Division of the Vector-Borne Infectious Disease, Centers for Disease Control and Prevention. D2 16681 virus was originally recovered from the serum of a patient with dengue hemorrhagic fever/dengue shock syndrome in Thailand (17). The D2 16681 virus was passaged several times in grivet monkey kidney BS-C-1 cells, six times in rhesus macaque monkey, twice in Toxorhynchites amboinensis mosquitoes, and then 53 times in PDK cells at the Center for Vaccine Development, Mahidol University, to derive the candidate PDK-53 vaccine virus (17, 48). The D2 16681-P48 (wild-type 16681 virus), D2 PDK53-E48 (PDK-53 NS3-250-E variant), and D2 PDK53-V48 (PDK-53 NS3-250-V variant) infectious cDNA clones and viruses have been previously constructed and described (18, 19). WN NY99-35262 virus was originally isolated in 1999 from a Chilean flamingo at the Bronx zoo, New York (29).
Viruses were grown in Vero, LLC-MK2, and C6/36 cells as described previously (18). Virus plaque titrations were performed under double agarose overlay in six-well plates of confluent Vero cells (18). For titration of D2 and chimeric D2/WN viruses, the second agarose overlay containing neutral red vital stain was added 7 days after infection, and plaques were counted 8 to 11 days after infection. For WN viral titration, the second overlay stain was added at 1 to 2 days after infection, and plaques were counted 3 to 5 days after infection.
Construction of chimeric D2/WN plasmids. Three previously constructed D2 infectious clones, pD2-16681-P48, pD2-PDK53-E48, and pD2-PDK53-V48 (18, 19), were used to derive the chimeric D2/WN viruses. Our initial chimeric D2/WN infectious clones (D2/WN-P0, -E0, and -V0 in Fig. 1A and Table 1) contained the same junction sites as our previous chimeric D2/1, D2/3, and D2/4 constructs (19). The cDNA fragment containing the prM-E genes of WN virus was amplified by reverse transcriptase PCR (RT-PCR) from WN NY99-35262 viral RNA with primers WN-M (5'-GGAGCAGTTACCCTCTCTACGCGTCAAGGGAAGGTGATG-3'; underlined MluI site followed by WN virus sequence near the 5' end of the prM gene) and cWN-E (5'-GAAGAGCAGAACTCCGCCGGCTGCGAGAAACGTGAGAGCTATGG-3'; underlined NgoMIV site followed by complementary sequence near the 3' end of the E gene of WN virus). The amplified WN virus-specific prM-E cDNA fragment was cloned into the MluI-NgoMIV sites of our previously constructed pD2I/D3-P and -E intermediate plasmids (19) to replace the prM-E fragment of the D3 16562 virus. These D2/WN intermediate clones were ligated in vitro with the 3'-end D2 clones (backbones 16681-P48, PDK53-E48, and PDK53-V48), as described previously for the chimeric D2/3 plasmids (19), to produce the full genome length chimeric D2/WN viral cDNAs prior to transcription of chimeric D2/WN viral RNA.
To improve the viability of chimeric D2/WN viruses derived from cloned cDNA, we later modified the chimeric D2/WN clones by including part of the signal sequence (SS) for the WN prM protein (D2/WN-P1, -E1, and -V1 in Fig. 1B and Table 1). A unique SstII restriction site was introduced by site-directed mutagenesis (QuickChange site-directed mutagenesis kit; Stratagene) upstream from the 5' terminus of the prM gene in the pD2I/D3-P and pD2I/D3-E intermediate clones (19). This SstII site was engineered to make only silent mutations at the new 5' chimeric junction of each D2/WN clone (Fig. 1B). The cDNA fragment encoding part of the WN virus-specific SS at the carboxyl end of C, prM, and the amino-terminal 487 amino acids (aa) of E was amplified with primers WN-452.SAG (5'-AATTCAACGCGTACATCCGCGGGCACCGGAATTGCAGTCATGATTGGCCTGATGGC-3'; underlined SstII site followed by WN virus sequence 45 nucleotides (nt) upstream of the prM gene) and cWN-E (as described above). The D2/WN chimeras contained the carboxyl-terminal 15 aa of E from D2 virus. This amplified cDNA was cloned into the SstII-NgoMIV sites of pD2I/D3-P and pD2I/D3-E to make the intermediate clones pD2I/WN-P-SA and pD2/WN-E-SA, respectively. These intermediate clones were ligated with the 3'-end D2 clones to produce the full genome length chimeric D2/WN-P1, D2/WN-E1, and D2/WN-V1 cDNAs required for transcription of genomic viral RNA. In a third set of chimeric D2/WN clones, we engineered two extra amino acid mutations, M-58 M to L and E-191 E to A, to further facilitate the viability and stability of the chimeric D2/WN viruses derived in Vero or LLC-MK2 cells (D2/WN-P2, -E2, and -V2 in Fig. 1B and Table 1). All plasmids were amplified in Escherichia coli XL1-Blue cells and sequenced. One silent mutation at nt 1428 (CT) was identified in all clones. After ligation, the full genome length cDNAs were cut with XbaI to produce the linearized 3' end of the viral cDNA required for viral genomic RNA transcription (18).
Recovery of recombinant viruses. Vero, LLC-MK2, or C6/36 cells were transfected with transcribed recombinant viral RNA as described previously (18). Viral proteins expressed in the transfected cells were analyzed by indirect immunofluorescence assay (IFA). Virus-infected cells were fixed in cold acetone for 30 min. WN E-specific monoclonal antibody (MAb) 3.67 (kindly provided by Roy Hall of the University of Queensland, Queensland, Australia), D2 E-specific MAb 3H5, and a polyclonal mouse hyperimmune ascitic fluid against D2 New Guinea C virus were used in the assay. Antibody binding was detected with fluorescein-labeled goat anti-mouse antibody. Viruses were harvested from transfected cell cultures when the cells were over 30% IFA positive with the polyclonal mouse ascitic fluid against D2 New Guinea C virus, usually 6 to 11 days after transfection. The harvested viruses were then passaged once in Vero or LLC-MK2 cells to obtain working seeds of virus. For transfected cultures that failed to develop 30% IFA-positive cells, medium was harvested on day 14 and then passaged in Vero or LLC-MK2 cells to further evaluate the viability of the chimeras.
Replication phenotypes of chimeric viruses in cell culture. For optimal comparison, plaque phenotypes of the wild-type WN virus, D2 backbone virus controls, and chimeric D2/WN viruses were measured in the same test and using the same batch of Vero cells in six-well plates. Except for WN viral plaques, mean plaque diameters were calculated from 12 plaques for each virus at 10 days after infection. Only a few well-isolated, large WN viral plaques were measured at 10 days.
Viral growth curves were performed in 75-cm2 flasks of LLC-MK2 or C6/36 cells at a multiplicity of infection of approximately 0.001 PFU per cell. After adsorption of virus for 2 h, 30 ml of minimal essential medium (LLC-MK2) or overlay nutrient medium (C6/36 cells) (34), each containing 5% fetal bovine serum and penicillin-streptomycin, was added, and the cultures were incubated in 5% CO2 at 37°C (LLC-MK2) or 28°C (C6/36). Aliquots of culture medium were harvested at 24- to 48-h intervals, adjusted to 15 to 20% fetal bovine serum, and stored at –80°C prior to plaque titration of virus.
Temperature sensitivity was tested in LLC-MK2 cells. Following virus adsorption for 2 h at 37°C, one set of cultures was incubated for 7 days at 37°C, the other at 39°C. Aliquots of culture medium were harvested at days 4 to 7 postinfection for the D2 control viruses and D2/WN chimeras and from days 1 to 5 postinfection for the WN NY99 virus.
Sequencing of viral genomes. The genomes of all the working seeds of the chimeric viruses were fully sequenced, except for about 24 bases at the extreme 5' and 3' termini of the genome. Viral genomic RNA was extracted from virus working seed by using the QIAmp viral RNA kit (QIAGEN). For each recombinant virus, six overlapping cDNA fragments which covered the entire viral genome were amplified by RT-PCR. The cDNAs were sequenced by using the CEQ 8000 sequencer (Beckman Coulter). Primers used for RT-PCR and sequencing will be provided upon request. They were based on the published sequences of WN NY99-35262 (GenBank accession no. AF196835) and D2 16681 (GenBank accession no. U87411).
Neurovirulence in suckling mice. Litters of newborn (less than 1 day old) outbred NIH Swiss Webster mice (colony maintained at the Centers for Disease Control and Prevention [CDC]) were inoculated intracranially with 20 μl of diluent containing 104 or 5,000 PFU of virus. Inoculated mice were observed daily for 4 weeks. A fatal endpoint was evidenced by moribund status, paralysis, or death.
Immunogenicity and protection in adult mice. Neutralizing antibody responses were tested in 3- to 4-week-old NIH Swiss Webster mice (CDC colony). Mice were inoculated intraperitoneally (i.p.) with 104 PFU of virus. Several groups of immunized mice were boosted with an equivalent dose of the same virus 4 weeks later. Mice were bled at 26 days after primary immunization and 12 days after boosting. To study protection, immunized mice were challenged i.p. with a lethal dose (105 or 107 PFU) of WN NY99-35262 virus at 4 to 6 weeks after primary immunization or 2 weeks after boosting. The surviving mice were bled again 21 to 28 days after challenge. The sera were tested for neutralizing antibodies against D2 16681 and WN NY99 viruses.
Neutralization assays. The plaque reduction neutralization test (PRNT) was performed in six-well plates of Vero cells as described previously (18). The mouse sera were heat inactivated (56°C for 30 min), and the tests were performed without addition of exogenous complement. Titrations of the input D2 16681 and WN NY99-35262 viruses were included in each assay. The neutralizing antibody titer was identified as the highest serum dilution, in serial twofold dilution series, that reduced the number of input virus plaques in the test by at least 50%.
RESULTS
Construction and recovery of chimeric D2/WN viruses. The first construction strategy used our originally engineered MluI junction site at nt position 451 (19) (Fig. 1A). From the three D2/WN cDNA clones, D2/WN-P0, -E0, and -V0, we recovered only low-titered (fewer than 10 PFU/ml) D2/WN-P0 virus from transfected C6/36 cells and no virus from transfected LLC-MK2 cells. After five passages in LLC-MK2 cells, the D2/WN-P0 virus titer increased to 2.6 x 105 PFU/ml. Genomic sequencing of the high-titer C6/36-0/LLC-MK2-5 seed revealed two new mutations in the E gene (WN-E-248 Thr to Met and D2-E-492 Met to Val) and one nucleotide insertion in the 3'NCR (extra A preceding nt –10337) that were not present in the corresponding cDNA plasmids. IFA of C6/36 and LLC-MK2 cells transfected with D2/WN-P0 RNA showed 15 to 20% and <1% cells positive to WN MAb 3.67 at day 14 posttransfection, respectively. No detectable virus was rescued from C6/36 or LLC-MK2 cells transfected with transcribed D2/WN-E0 or -V0 RNA (Table 1). These cultures showed less than 1% IFA-positive cells at 14 days of incubation.
The second strategy of D2/WN construction utilized the newly engineered SstII site at nt 398 of the D2 cDNA, immediately downstream of the potential NS2B-3 protease cleavage site between the C and prM proteins (Fig. 1B). This strategy included a portion of the WN viral SS of prM, which is a transmembrane signal located between C and prM. All D2/WN chimeras retained the originally engineered NgoMIV junction site located in the transmembrane 2 domain of the translated D2 E protein (19). Using this new strategy, we constructed D2/WN-P1, -E1, and -V1 chimeras in the backbones of D2 16681-P48, PDK53-E48, and PDK53-V48, respectively (Table 1). D2/WN-P1 virus was successfully rescued from both transfected C6/36 cells (high titers of 105 to 106 PFU/ml) and LLC-MK2 cells (103 to 105 PFU/ml). The C6/36-0/LLC-MK2-1 D2/WN-P1 virus acquired no new mutation, suggesting this virus was more viable and stable in cell cultures than the D2/WN-P0 virus.
D2/WN-E1 virus (in the PDK53-E backbone) was recovered from transfected LLC-MK2, Vero, and BHK-21 cells at titers ranging from 1 to 104 PFU/ml (Table 1). After one to three Vero passages of the recovered D2/WN-E1 viruses, titers increased to above 106 or 107 PFU/ml. Sequence analyses of two independently derived D2/WN-E1 viral seeds, clone 112 and clone 40, revealed evolving mutations that were not present in their corresponding cDNA. Mutations NS2A-22 Met to Val/Met mix and NS2B-93 Gln to Gln/Arg occurred in clone 112, while different mutations E-208 Thr to Ile/Thr and NS2A-36 Val to Val/Phe occurred in clone 40. We were not able to recover detectable viable chimeric D2/WN-V1 virus (in the PDK53-V backbone) from transfected LLC-MK2, Vero, or BHK-21 cells. We did not attempt to transfect C6/36 cells with either D2/WN-E1 or -V1 transcribed viral RNA (Table 1), because chimeras constructed in the D2 PDK53-E or -V backbone were not expected to replicate efficiently in the C6/36 cells (18, 19).
To further improve the viability and stability of the D2/WN chimeras in cell culture, we incorporated mutations E-191 Glu to Ala and M-58 Met to Leu (as well as a cDNA clone-derived nt –1905 A-to-G silent mutation) in the cDNA to construct D2/WN-P2, -E2, and -V2. These mutations occurred in a rescued chimeric D2/WN virus which exhibited improved fitness in Vero cells (data not shown). We derived D2/WN-P2, -E2, and -V2 viruses from transfected Vero cells. The viability of D2/WN-V2 virus was especially encouraging, because it was the first viable chimeric D2/WN-V virus in the PDK53-V backbone to be rescued (Table 1). The E-191 and M-58 mutations also improved the stability of the D2/WN viruses derived from backbones D2 16681 and PDK53-E. The titers of the clone-derived D2/WN-P2 and -E2 viruses were greater than 106 PFU/ml after a single amplifying passage in Vero cells, compared to 105 PFU/ml for the D2/WN-P1 and -E1 viruses. The genome sequence of D2/WN-P2 virus was identical to the sequence of its cDNA. However, the D2/WN-E2 and -V2 viral genomes contained new mutations that were acquired during transfection and passage in cell cultures. The D2/WN-E2 working viral seed contained an NS2A-22 Met-to-Met/Val mutation, while two mutations, NS2A-22 Met to Val and nt 10661 (based on the D2 3'NCR) C to T, were found in the D2/WN-V2 working seed. Interestingly, an identical NS2A-22 mutation was also identified in the D2/WN-E1 clone 112 virus described above.
Plaque sizes of D2/WN chimeras in Vero cells. Among the D2 backbone viruses, 16681-P48 generated the largest plaques, followed by the PDK53-E48 and then the PDK53-V48 virus (Fig. 2). The relative plaque sizes of the D2 backbone viruses in Vero cells were similar to our previous results with LLC-MK2 cells (19). However, all the viruses produced smaller plaques in Vero cells than in LLC-MK2 cells. The chimeric D2/WN viruses produced plaques that averaged 0.4 to 1.8 mm in diameter. Their plaques were much smaller than those of the prM-E donor WN virus (greater than 16 mm average diameter) and were more similar in size to their backbone D2 viruses. Interestingly, the D2/WN-P1 and -P2 plaques were smaller than the plaques of the D2/WN-E1 (clone 112 and 40) and -E2 viruses, and also smaller than plaques of their backbone D2 16681-P48 virus. The D2/WN-V2 virus produced plaques that were smaller than those of D2/WN-E2 virus, which corresponded to the relative sizes of their backbone D2 PDK53-V48 and -E48 viruses, respectively.
Growth of D2/WN chimeras in LLC-MK2 and C6/36 cells. WN NY99 virus grew much faster and to higher titers in LLC-MK2 cells than the D2 and chimeric D2/WN viruses (Fig. 3A). WN virus reached a peak titer of over 109 PFU/ml at 3 days postinfection, at which time a significant cytopathic effect was evident. The D2 16681-P48 virus replicated somewhat more efficiently than the D2 PDK53-E48 and -V48 viruses. The D2 16681-P48 virus usually reaches a peak titer of 106 to 107 PFU/ml within 6 to 8 days after infection, while PDK53-E48 and -V48 viruses typically reach peak titers of 105 to 107 PFU/ml between 7 and 10 days postinfection (Fig. 3A) (18, 19). Replication of the chimeric D2/WN-P2, -E2, and -V2 viruses, using the D2 backbone replication machinery, was similar to that of the D2 PDK53-E48 and -V48 viruses in LLC-MK2 cells (Fig. 3A).
In C6/36 cells, the WN NY99 virus again replicated much faster with a higher peak titer (1010 PFU/ml) than the D2 and D2/WN chimeras (Fig. 3B). D2 16681-P48 virus typically grows to 107 to 108 PFU/ml in C6/36 cells (18, 19), replicating much more efficiently than the PDK53-E48 and -V48 viruses, which usually reach peak titers of 104 to 105 PFU/ml (Fig. 3B) (18, 19). The D2/WN -P2 virus replicated with an initial efficiency that was similar to that of its D2 backbone 16681-P48 virus (Fig. 3B), although the peak titer of D2 16681-P48 virus was about 18-fold greater than that of D2/WN-P2 virus at 11 days postinfection. Replication of the chimeric D2/WN-E2 and -V2 viruses was very inefficient in the C6/36 cells, with peak titers of only 102 to 103 PFU/ml (Fig. 3B).
Temperature sensitivities of chimeric D2/WN viruses in LLC-MK2 cells. The WN virus was not temperature sensitive at 39°C in LLC-MK2 cells. D2 16681-P48 and D2/WN-P2 viruses demonstrated similar levels of temperature sensitivity, as indicated by a maximal reduction in viral titers of 1.9 log PFU/ml at 39°C. D2/WN-E2 and -V2 viruses showed up to 2.2- and 2.0-log reductions at 39°C, respectively. As reported previously (18, 19), D2 PDK53-E48 virus was more temperature sensitive (up to 3-log reduction at 39°C) than 16681-P48 virus but less sensitive than the PDK53-V48 virus, which suffered an up to 3.7-log reduction in titer at 39°C (Fig. 4).
Neurovirulence in newborn mice. Previously, we tested the D2 16681-P48, PDK53-E48, and PDK53-V48 viruses and chimeric DEN viruses in newborn ICR mice (9, 18, 19). During the present study, the CDC mouse colony switched to NIH Swiss Webster mice, which we found to be more sensitive to D2 virus. Therefore, the neurovirulence experiments in this report were performed in newborn NIH Swiss Webster mice. D2 16681-P48, PDK53-E48, and PDK53-V48 viruses consistently caused 100%, 62.5%, and 6.25% mortality in the newborn Swiss mice, respectively (Fig. 5).
The WN NY99 virus was highly neurovirulent for the newborn Swiss Webster mice (100% mortality within 6 days of challenge). Due to low seed titers of the D2/WN-P1, -E1 clone 112, and -E1 clone 40 viruses, we challenged mice with 5,000 PFU of these three viruses instead of the 104-PFU dose that we used for the other viruses reported in Fig. 5. Inoculation of mice with 5,000 PFU or 104 PFU of WN NY99, D2 16681, or D2 PDK53-E48 virus resulted in equivalent mortality rates for each virus in newborn Swiss mice (data not shown). Therefore, it is unlikely that the twofold difference in challenge dose would affect comparative interpretation of results. The D2/WN-E1 (clone 112 and 40), -E2, and -V2 viruses were less virulent than the WN NY99 and D2 16681-P48 viruses, and they were at least as attenuated as their backbone D2 PDK53-E48 or -V48 virus in these mice. Interestingly, the D2/WN-P1 and -P2 viruses, which expressed prM-E from highly virulent WN NY99 in the D2 backbone of 16681-P48 virus, caused no mortality in these mice.
Immunogenicity and protective efficacies of D2/WN chimeras in mice. All chimeric D2/WN viruses tested in this study induced neutralizing antibodies against WN NY99 virus after primary i.p. immunization (Tables 2 and 3). Anti-D2 16681 PRNT titers of pooled sera from each D2/WN group were not detectable (titer less than 1:10). The pooled sera of the D2 16681-P48 group also had a nondetectable primary PRNT titer against D2 16681 virus but had a low PRNT titer of 1:20 against D2 16681 virus after a boost (data not shown). The control phosphate-buffered saline (PBS) and D2 16681-P48 groups had no background PRNT titers against WN NY99 virus (Table 2 and 3). The D2/WN-P2 virus induced individual primary PRNT titers against WN NY99 virus, ranging from 1:20 to 1:1,280 in 87.5% of the mice (geometric mean titers [GMTs] of 1:160 and 1:119; Tables 2 and 3, respectively). After boosting with the D2/WN-P2 virus, the seroconversion rate increased to 100% with a PRNT GMT of 1:1,902 and individual titers ranging from 1:640 to 1:5,120 (Table 3). Chimeric D2/WN-E2 virus induced WN neutralizing titers ranging from 1:10 to 1:320 (GMT = 1:40) in 75 to 87.5% of the mice after primary inoculation (Tables 2 and 3) and from 1:20 to 1:5,120 (GMT = 1:580) in all mice after a boost (Table 3). D2/WN-V2 virus induced WN neutralizing titers ranging 1:10 to 1:1,280 (GMT = 1:108 and 1:269 in Tables 2 and 3, respectively) in 87.5 to 100% of mice, and boosted titers ranging from 1:640 to 1:40,960 (GMT = 1:4695) in 100% of mice (Table 3). The D2/WN-E1 viruses, clones 112 and 40, were also immunogenic in these mice, and they induced a primary WN neutralizing GMT of 1:93 in 87.5% of mice and a GMT of 1:22 in 100% of mice, respectively (Table 2).
The D2/WN-P2, -E2, and -V2 viruses protected most mice against an i.p. challenge of 105 PFU of WN NY99 virus (about 104 median intraperitoneal lethal doses [IPLD50]) according to a previous report (42) after one immunization (Table 2). Only one mouse died in the D2/WN-E2 group, while all mice in the D2/WN-P2 and -V2 groups survived a WN virus challenge. All mice in the PBS and D2 16681-P48 control groups succumbed to a WN virus challenge. When the WN challenge dose was increased to 107 PFU (about 106 IPLD50), one immunizing dose of the D2/WN chimeras was still able to provide at least partial protection against a WN virus challenge. Two (25%) of the eight mice in the D2/WN-P2 group, four (57%) of seven mice in the D2/WN-E2 group, and one (12.5%) of the eight mice in the D2/WN-V2 group died from the extremely high-dose challenge (data not shown in Table 2), while none survived in the PBS or D2 16681-P48 control group. Chimeric D2/WN-E1 viruses, clones 112 and 40, provided similar partial protection against the 107 PFU WN challenge after just one immunization (Table 2). All mice were fully protected against the high 107 PFU WN NY99 challenge after a booster dose of the D2/WN-P2, -E2, or -V2 virus (Table 3). All surviving vaccinated mice exhibited no symptoms of illness. We tested the high 107-PFU challenge dose of WN virus to ensure 100% death in the PBS control groups. In earlier experiments, although 100% mortality was observed in most of the PBS control groups challenged with 105 PFU of WN NY99 virus, some groups only suffered 50 to 87.5% mortality. Mock-immunized mice surviving the 105-PFU WN virus challenge showed illness between days 7 and 10 after a challenge and then recovered soon without any significant symptoms, and they all had very high neutralizing antibody titers against WN virus (data not shown), apparently ruling out challenge failure as a cause of such inconsistent mortality rates.
DISCUSSION
The 5'NCR-57, NS1-53, and NS3-250 loci are the major attenuating determinants of the candidate D2 PDK-53 vaccine virus (9). Their location outside of the structural gene region makes the PDK-53 background ideal as a potential vector for chimeric flavivirus vaccine development. Both D2 PDK53-E and PDK53-V backbones have been used to develop intertypic D2/1, D2/3, and D2/4 viruses that maintain the phenotypic markers of the D2 PDK-53 vaccine virus (18, 19). In this study, the potential of the PDK53 variants to serve as vectors for chimeric D2/WN vaccine viruses was explored. WN and DEN viruses belong to two different serocomplexes (JE complex and DEN complex), and their mosquito vector preferences are different (Aedes for DEN and Culex for WN).
Viability- and fitness-enhancing amino acid substitutions of the chimeric D2/WN viruses. The initial D2/WN chimeras, which utilized our original MluI-prM splicing site (19), were either not viable or were very unstable in cell culture. The flavivirus C and prM proteins are separated by an internal SS of 14 to 22 aa which directs translocation of prM into the lumen of the endoplasmic reticulum (31, 45). The internal SS of DEN (14 aa) and WN (18 aa) are flanked by upstream viral NS2B-3 protease and C-terminal cellular signalase cleavage sites (Fig. 1). Our first constructs, D2/WN-P0, -E0, and -V0, were spliced at prM-6 of the D2 virus and contained both NS2B-3 and signalase cleavage sites from the D2 backbone (Fig. 1A). When we changed our strategy to include the C-terminal 15 aa of the WN viral SS to replace the C-terminal 11-aa SS of D2 virus (Fig. 1B), we recovered viable D2/WN-P1 and -E1 viruses but not D2/WN-V1 virus. The new constructs retained the NS2B-3 cleavage site and the downstream 3 aa (SAG) of the D2 backbone virus, but the 15 C-terminal aa of the 18-aa SS were derived from the SS of WN virus. Clearly, the WN virus-specific SS and signalase cleavage site improved the viability of D2/WN-P1 and -E1. Coordination of the two cleavages at the C-prM junction is critical to the assembly of flaviviruses (1, 2, 30, 32, 45). Signalase cleavage of prM is inefficient until the cytosolic C protein is proteolytically removed by the virus-specific NS2B-3 protease (1, 45). Coordination of cytosolic and luminal cleavages at the C-prM junction may be important for efficient maturation of nucleocapsid (NC) during virus assembly (32). Uncoupling of the cleavages has resulted in reduced incorporation of NC into budding membranes and augmented release of NC-free virus-like particles (32). It is likely that our modified splicing strategy improved the coordination between the two C-prM cleavages and NC assembly in the D2/WN chimeras. It is also possible that the modified D2/WN SS provided more optimal polypeptide conformation surrounding the signalase cleavage site to promote proper cleavage at the N terminus of prM. Other reports have also shown that different compositions of the SS can profoundly affect the viability of the yellow fever virus and chimeric WN/DEN4 virus (30, 42).
It is interesting that our first construct, D2/WN-P0, was viable in C6/36 cells but not in mammalian cells. We reported a similar phenomenon for a D2/4-P chimera that had the same chimeric junction at D2 prM-6 (19). The transient existence of a C-prM precursor has been shown in flavivirus-infected C6/36 cells, but not in Vero cells (36), indicating cleavage of the C protein at the NS2B-3 cleavage site may be delayed in C6/36 cells. Such a delay may have contributed to the more efficient replication of our chimeric D2/WN-P0 and D2/4-P constructs in C6/36 cells than in mammalian cells. Engineered mutations, including one at the N-terminal end of the SS, greatly enhanced viabilities of chimeric D2/4 viruses in mammalian cells (19). In this report, viabilities of the D2/WN chimeras were enhanced in both C6/36 and mammalian cells by including the SS of prM from WN virus.
We further optimized the fitness of the D2/WN chimeras in Vero cells by incorporating M-58 Met-to-Leu and E-191 Glu-to-Ala mutations into the D2/WN-P2, -E2, and -V2 cDNAs. The permissive mechanism of these mutations is unclear, but they permitted the D2/WN-P2, -E2, and -V2 chimeras to grow to high titers in mammalian cells. The mutations were especially important for the D2/WN-V2 virus, which otherwise was not viable in Vero, BHK-21, or LLC-MK2 cells. The prominent effect of the mutations for the D2/WN-V2 virus may have resulted from the slower replication of chimeras based on the PDK53-V backbone (9, 18, 19). The M-58 and E-191 mutations, however, did not change the fitness of the D2/WN chimeras in C6/36 cells, which was evident from the efficient growth observed for both D2/WN-P1 and -P2 chimeras in C6/36 cells (Table 1). Despite these mutations, the D2/WN-E2 and -V2 viruses, based on the PDK-53 background, still replicated poorly in C6/36 cells. Crippled replication in C6/36 cells is one of the phenotypic attenuation markers exhibited by the D2 PDK-53 vaccine virus (Fig. 3).
Replication phenotypes of chimeric D2/WN viruses in cell cultures. The mutations M-58 and E-191 permitted the D2/WN-P2 virus to replicate better and produce larger plaques than the D2/WN-P1 virus in Vero cells. Direct comparisons of plaque sizes among the D2/WN-E1 clone 112, D2/WN-E1 clone 40, and D2/WN-E2 viruses were not straightforward because these viruses acquired different additional mutations during derivation. Our previous studies showed that chimeric DEN viruses engineered in the PDK53-E and -V backbones had smaller plaques than those constructed in the 16681 backbone (19). However, in this study D2/WN-E1 (clones 112 and 40) and D2/WN-E2 (in the PDK53-E backbone) produced larger plaques relative to their counterparts in the 16681 backbone, D2/WN-P1 and D2/WN-P2, respectively. The additional mutations acquired during production of the D2/WN-E1 and -E2 viruses probably contributed to the larger plaque sizes of the viruses. All of the chimeric D2/WN viruses exhibited greatly reduced plaque sizes in Vero cells, relative to the plaques of the prM-E donor WN NY99 virus. Their plaque sizes and growth curves were more similar to those of the D2 backbone viruses, whose replication machinery largely controlled the phenotypes of the chimeric viruses.
The D2/WN-P2 virus produced smaller plaques and grew to somewhat lower titers in LLC-MK2 cells than its backbone D2 16681-P48 virus, indicating some degree of incompatibility between the inserted WN prM-E and D2 background genes. The growth curves of the D2/WN-E2 and -V2 viruses were very similar to, and their plaque size larger than, those of their backbone D2 PDK53-E48 and -V48 viruses, suggesting the likelihood that their additional acquired mutations, such as the common NS2A-22 Met-to-Val mutation, further improved the fitness of these D2/WN chimeras in mammalian cells. The chimeric D2/WN-P2, -E2, and -V2 virus seeds produced in Vero cells had high titers of 106 to 107 PFU/ml, which is encouraging, because Vero cells are licensed for vaccine virus production.
Reduced replication of the candidate D2 PDK-53 vaccine virus in Aedes aegypti mosquitoes and C6/36 (A. albopictus) cells is a significant biological attenuation marker of the PDK-53 virus (9, 25, 26). PDK-53 virus is not transmitted by A. aegypti mosquitoes (25), reducing the potential for secondary transmission of the vaccine virus and providing an important safety feature for the vaccine. We and others have reported this low-replication phenotype in C6/36 cells for different DEN vaccine candidates (11, 19, 21, 22, 46). The crippled C6/36 replication phenotype of D2 PDK-53 virus is encoded by both the 5'NCR-57 C-to-T and the NS1-53 Gly-to-Asp mutations (9). In this study, chimeric D2/WN-P2 virus and its backbone 16681-P48 virus replicated to similar high titers in C6/36 cells, indicating good compatibility between the heterologous genes for replication in C6/36 cells. The chimeric D2/WN-E2 and -V2 viruses, both of which possessed the 5'NCR-57 and NS1-53 mutations, retained the crippled C6/36 replication phenotype of D2 PDK-53 virus (Fig. 3B). Based on analyses of several DEN vaccine candidates (21, 22, 25, 26), the poor C6/36 replication phenotype of DEN vaccine viruses likely reflects limited replication, dissemination, and transmission in mosquitoes.
The temperature sensitivity phenotype of D2 PDK-53 virus has been attributed to a synergism between the NS1-53-Asp and NS3-250-Val loci (9). The PDK53-V variant, containing both loci, is more temperature sensitive than the PDK53-E variant, and both variants are more temperature sensitive than the 16681 virus (Fig. 4) (9, 19). Unlike the prM-E donor WN NY99 virus, which was not temperature sensitive at 39°C, the D2/WN-P2, -E2, and -V2 viruses exhibited temperature sensitivities that were equivalent to or slightly greater than that of the D2 16681-P48 virus. The D2/WN-V2 virus did not show a more prominent temperature sensitivity phenotype than the D2/WN-E2 virus. These results are consistent with those observed previously for DEN chimeras (19).
Attenuation, immunogenicity, and protective efficacy of the chimeric D2/WN viruses in mice. Most studies have identified major determinants of mouse virulence/attenuation in the flavivirus E protein (3, 13, 20, 24). However, attenuation of the D2 PDK-53 virus in newborn ICR mice is determined mainly by the 5'NCR-57 and NS1-53 loci (9, 18). In this study, the more D2-susceptible newborn Swiss mice further identified a contribution of the NS3-250-Val locus to this attenuation phenotype (Fig. 5). Both D2/WN-P1 and -P2 viruses, which contained the expected recombinant viral genome sequences, were attenuated for the Swiss mice, whereas the WN NY99 donor and D2 16681-P48 carrier viruses were uniformly fatal. The recombination of heterologous genes in flavivirus chimeras per se may contribute significantly to the attenuation phenotype of the resulting chimeric virus, as has been observed previously (39, 41, 42). Comparing results between the D2/WN-P1 and -P2 chimeras, it is apparent that the engineered M-58 and E-191 mutations in the D2/WN-P2 virus did not affect the attenuated phenotype of the D2/WN-P2 virus. Unexpectedly, the D2/WN-E1 clone 112 and 40, D2/WN-E2, and D2/WN-V2 viruses were more neurovirulent than the D2/WN-P1 and -P2 viruses. This may be due to the additional mutations acquired in the D2/WN-E and -V viruses, such as the NS2A-22 mutation that occurred in three of these viruses. Such mutations may promote the replication efficiency of these chimeras not only in mammalian cells but also in mice. Nonetheless, all of the D2/WN-E and -V viruses tested were at least as attenuated as their PDK53-E48 and -V48 carrier viruses in the newborn Swiss mice, and the degree of the attenuation correlated well with the PDK53-E or -V backbone of each virus.
All of the chimeric D2/WN viruses tested induced neutralizing antibodies against WN NY99 virus in mice (Tables 2 and 3). Most mice receiving a primary immunization with chimeric D2/WN virus and all mice receiving a booster dose of D2/WN virus were protected from a severe i.p. WN viral challenge. D2/WN virus-immunized mice that survived a WN NY99 virus challenge all had greater PRNT titers than were present before challenge, indicating that the D2/WN chimeras provided efficient priming immunity against WN virus.
Application of the D2 PDK-53 virus as a carrier for flavivirus vaccine development. Advanced recombinant genetic technologies have recently shown potential for the engineering of safe and effective live attenuated chimeric flavivirus vaccines that may provide a safe balance between attenuation and induction of protective immunity (4, 5, 8, 16, 18, 19, 27, 28, 35, 38, 40). Such recombinants include chimeric yellow fever 17D/WN (4, 5) and DEN-4/WN (40, 42) viruses. In human trials the candidate D2 PDK-53 vaccine virus has proved to be safe, to elicit neutralizing antibodies for at least 2 years, and to induce significant memory T-cell responses (6, 7, 14, 47). Our previous results for chimeric DEN viruses suggested that D2 PDK-53 virus is an ideal carrier for chimeric DEN vaccine development (19). D2 PDK-53 virus is one of the few attenuated viruses for which attenuation determinants have been thoroughly studied at the molecular level (9) and have been tested safely and effectively in human trials (6, 23, 43). The well-studied attenuation loci of the PDK-53 genetic background should permit more straightforward quality and safety verification during vaccine development and use. Any possible reversion to the wild type of a vaccine production lot or in a vaccinee can be quickly identified by advanced molecular technologies. In this study, the chimeric D2/WN-E2 and -V2 viruses retained the attenuated phenotypic characteristics of the D2 PDK-53 vaccine virus, were immunogenic in mice, and protected mice from severe WN viral challenge. Although the D2/WN-P2 virus was also attenuated and immunogenic in mice, it still retained certain wild-type characteristics, such as efficient growth in C6/36 cells and less temperature sensitivity in LLC-MK2 cells. These results provide a basis for further evaluation of the chimeric D2/WN viruses in nonhuman primates and horses as potential vaccine candidates against WN virus and further support utilization of the PDK-53 virus as a chimeric carrier for flavivirus vaccine development.
ACKNOWLEDGMENTS
We thank Rhett Mays, Becky Rose, Kiyotaka Tsuchiya, Steven Gire, and Lindsey Doyle for technical support at different stages of this project. We also thank John Roehrig and David Withum for comments on the manuscript.
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