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ABSTRACT |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Members of the JE serocomplex can be distinguished by the neutralization test that is the most specific serologic test to retrospectively diagnose and confirm human cases and infections in avians.1 Since both SLE and WN viruses are Biosafety level 3 (BSL-3) agents, there is considerable practical difficulty in performing neutralization assays, which require infectious virus, for diagnostic and epidemiologic purposes due to biocontainment constraints. We have used our proprietary ChimeriVax technology to engineer attenuated chimeric viruses that can be safely used in the neutralization test under BSL-2 containment and laboratory practices.4–10 The technology uses the yellow fever (YF) 17D vaccine virus as a vector in which the prM-E envelope protein genes are replaced with those from a heterologous flavivirus resulting in a chimeric virus with antigenic specificity of the envelope of the heterologous virus. A YF/WN chimera (ChimeriVax-WN) has been constructed previously.5 Here we describe the construction of two YF/SLE chimeras encoding the prM-E genes of SLE viruses that are genetically and biologically distinct. The YF/SLE viruses were constructed using the prM-E envelope protein genes from either the virulent MSI-7 (isolated in the United States) or naturally attenuated CorAn9124 (isolated in Argentina) wild-type SLE virus strains. The virulence differences between these SLE virus strains have been previously described.11 In addition to their potential as diagnostic reagents, the YF/SLE chimeras are considered candidate vaccines for SLE.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Construction of chimeras.
The prM-E genes (plus short flanking stretches) of the MSI-7 and CorAn9124 parents were sequenced by the consensus method using RNA extracted directly from the original infected mouse brain tissue.7,13 The first-strand cDNA used for sequencing was also used as the template for polymerase chain reaction amplification of specific regions of SLE virus for construction of plasmids shown in Figure 1B
. Plasmids p5'3'/SLE/CO and p5'3'/SLE/MSI (the CorAn9124 and MSI-7 versions, respectively) were constructed by replacing the dengue 3 (DEN 3) virus-specific part in 5'3'/Den3/
Xho,7 with an SLE-specific fragment containing the prM gene and the first 552 nucleotides of the E gene, ending with an artificially introduced Bss HII site necessary for in vitro ligation. The Bss HII site does not change the amino acid sequence of the E protein. In the process, an extra Xho I site present at nucleotide 259 of the CorAn9124 prM gene was ablated by silent oligonucleotide-directed mutagenesis. Plasmids pCL/E/CO and pCL/E/MSI (the CorAn9124 and MSI-7 versions, respectively) were obtained by cloning the 3' portion of the SLE virus E gene in a modified low-copy number vector pCL1921 (pCL/MCS). They contain a corresponding strain-specific Bss HII-Nar I fragment encompassing the last 1,011 nucleotides of the E gene. The artificial Nar I site at the end of the gene results in a Q to G amino acid change at the penultimate residue of the E protein (as in most other previously constructed chimeras).5–8,14 These plasmids were grown in MC1061 recA– (a gift from T. Chambers, St. Louis University School of Medicine, St. Louis, MO) or Sure cells (Stratagene, Cedar Creek, TX). Several clones of each plasmid were sequenced to find ones representing the consensus sequence of each SLE virus strain. To generate a DNA template for in vitro transcription, three-fragment ligation was done as shown in Figure 1B and previously described for ChimeriVax-DEN1 and -DEN3.7,8 The Bss HII-Nar I fragment of pCL/E/CO (or MSI) and the Nar I-Bsp EI fragment from the YFM5.2/SA14-14-2 plasmid, which contained most of the YF virus-specific NS genes,14 were ligated with the large Bss HII-Bsp EI portion of the p5'3'/SLE/CO (or MSI) plasmid. Ligation products were linearized with Xho I, extracted with phenol-chloroform, and then used for in vitro transcription with SP6 RNA polymerase. The YF/SLE chimeras (YF/CorAn and YF/MSI) were produced by Lipofectin-mediated transfection of Vero cells with the in vitro RNA transcripts. The prM-E regions of both chimeras were sequenced at the P2 level, and no unexpected mutations were observed.
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Patient sera. Serum specimens were obtained from the historical collection of the Division of Vector-Borne Infectious Diseases of CDC, which consisted of specimens sent to this division for arboviral diagnostic testing. Sera were selected on the basis of positive results to either WN virus or SLE virus infection, or YF vaccination in previously performed serologic tests.
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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).17 Most differences were silent nucleotide heterogeneities. Seven nucleotide heterogeneities resulted in amino acid heterogeneities. A few differences were clear nucleotide changes. Most likely, these differences were due to the fact that the previously reported sequence was determined on virus propagated in SW-13 cells, which was plaque purified, using cloned cDNA, rather than on a heterogenous virus population. To verify the precise location of the E protein genes, we aligned the SLE virus sequences with other flaviviruses and found that the N terminus of prM should be two residues downstream from the previously predicted N terminus,17 following the Ser-Ser residues (Figure 1A
). The difference between the early published alignment and our alignment is obviously due to the use of different algorithms. Both Clustal (Figure 1A) and Jotun Hein methods invariably positioned the N terminus of SLE virus as shown in Figure 1A, regardless of alignment parameters (different residue weight tables). However, the positions of gaps in the upstream signal peptide differed depending on the method and on which viruses were included into alignment, indicating that precise alignment of hydrophobic stretches is difficult.
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. This strategy overcomes difficulties sometimes associated with the standard two-fragment strategy because of the toxicity for Escherichia coli of one of the used plasmids (of the YFM5.2 series containing both intermediate fragments in Figure 1B as one fragment) as we previously described for ChimeriVax-DEN1 and DEN3.7,8 Although we were able to also generate the YF/MSI chimera using the two-fragment approach, attempts to clone a CorAn9124-specific YFM5.2 plasmid analog failed. Following transfection of cells with in vitro RNA transcripts the two chimeras were recovered. These viruses grew to high titers of ~7 and 8 log10 pfu/mL for YF/CorAn and YF/MSI (~100 times higher compared with the wild-type parents), respectively, produced a CPE, and formed distinct plaques in Vero cells. The CPE caused by the chimeras was more pronounced compared with the wild-type parents, possibly due to their high level of replication.
Attenuation of YF/SLE virus chimeras in mice.
Neurovirulence of the two chimeras was compared in two experiments with that of the wild-type parental SLE viruses and YF 17D in 3–4-week-old outbred mice by the IC inoculation of graded doses of the viruses (Table 2). The wild-type CorAn9124 virus was highly neurovirulent, with the mortality rate of 87.5–100% across all doses of virus tested (10–105 pfu/dose) and a short average survival time (AST) ranging from 6.7 to 9.3 days. Mortality observed at the lowest dose was higher than 50%, therefore, the IC median lethal dose (LD50) could not be precisely calculated, but was less than 1 log10 pfu. Neurovirulence of YF 17D virus was also high with an IC LD50 of 1.65 log10 pfu, which is similar to the value determined previously,6 but lower than that of wild-type CorAn9124. The AST of mice inoculated with YF 17D was 1.1–3.4 days longer compared with corresponding dose groups that received wild-type CorAn9124. The YF/CorAn chimera was highly attenuated as compared with both its SLE and YF 17D parents. Mortality ratios caused by this chimera did not exceed 25% for any of the tested doses (up to 5 log10 pfu). Thus, the IC LD50 of the chimera is > 5 log10 pfu. The AST of the few non-survivors observed after inoculation of the chimera was dramatically increased by 6.6–10.8 days compared with the corresponding doses of YF 17D, and by more than that (9–10.8 days) compared with wild-type CorAn9124 virus. The difference in mortality caused by YF/CorAn was statistically significant (Table 2) when compared with the same doses of YF 17D (except for the 101 dose, which is irrelevant because mortality caused by YF 17D at this dose was low), and wild type CorAn9124 virus.
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). The IC LD50 was 0.55 pfu, and the AST was shorter than that of CorAn9124. The YF/MSI chimera had a significantly higher IC LD50 of 2.74 log10 pfu (550 pfu), and appeared to be more attenuated than YF 17D, but less attenuated than YF/CorAn. For YF/MSI, the difference in mortality was statistically significant when compared with wild-type MSI-7, but not YF 17D (Table 2).
The ability of the parental viruses and chimeras to cause encephalitis following peripheral inoculation of 3–4-week-old mice (SC and/or IP) was examined (Table 3). Both the wild-type CorAn9124 parent and the YF/CorAn chimera, as well as YF 17D, were avirulent after SC (and IP for CorAn9124) inoculation of all tested doses. We did not additionally examine the IP route for the YF 17D and YF/CorAn viruses because many previous studies have established the lack of neuroinvasiveness of adult mice by YF 17D after IP inoculation,18,19 and because the CorAn9124 parent caused no mortality by this route. Testing of virulence of the YF/CorAn virus (selected for use as a diagnostic reagent) by the olfactory or pulmonary routes was unnecessary since it was significantly less virulent than a licensed human vaccine (YF 17D) when inoculated directly into the brain and because its wild-type parent is known to be naturally attenuated for mice and primates.11 Similarly, the YF/MSI chimera was avirulent at the tested IP dose of 2.6 x 107 pfu (undiluted virus stock). In contrast, the wild-type MSI-7 virus caused partial mortality at IP doses as low as 20 pfu.
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) were challenged at 4.5 weeks following the first inoculation by the IC route with 4,000 pfu of the highly virulent wild-type MSI-7 virus (a very high dose compared with previous studies).20,21 Mice initially inoculated (immunized) with YF/CorAn at SC doses of 8 x 105 and 1 x 104 pfu were partially resistant to challenge (two of eight and two of seven challenged animals survived, respectively). Eighty percent (four of five) of mice immunized IP with 2.6 x 107 pfu of the YF/MSI chimera survived, while all five sham-immunized control animals died. Pooled (within groups) sera from immunized mice and post-challenge survivors contained detectable SLE virus-specific neutralizing antibodies (PRNT50 titers of up to 1:32 and 1:128, respectively). Both YF/CorAn and YF/MSI chimeras appeared to be equally well neutralized by the mouse sera against either virus, indicating that each of them can be used to diagnose infections by geographically distant SLE strains.
YF/SLE and YF/WN chimeras as reagents for specific serodiagnosis of human SLE and WN cases.
Patient sera from serologically confirmed WN, SLE, and YF cases (five, five, and two cases, respectively) were tested for virus neutralizing activity against the YF/WN and YF/CorAn chimeras, wild-type WN and SLE viruses (strains Eg101 and TBH-28, respectively), and YF 17D in a standard PRNT90 test performed at CDC (Table 4). Although the PRNT90 titers were generally lower (up to four times) for the chimeric viruses than the wild-type viruses, they more specifically distinguished between WN fever and SLE cases. The YF 17D control was specifically neutralized by the sera from YF cases only.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Prior to construction of the chimeras, we determined the consensus sequence of the prM-E region (plus short flanking stretches) of the parental MSI-7 and CorAn9124 SLE strains. We found 15 amino acid differences between these strains: seven in the prM protein of which two were in the M portion, and eight in the E protein (see GenBank AY289617 and AY289618). The two strains differed in terms of their growth properties in Vero cells (higher peak titers for MSI-7 compared with CorAn9124), which correlated with those of the chimeras (higher peak titers for YF/MSI compared with YF/CorAn). In addition the YF/MSI chimera was more neurovirulent in mice than YF/CorAn. Therefore it is likely that some of the amino acid differences are determinants of the unequal virulence profiles of these strains. Of the eight amino acid differences in the E protein, the most intriguing is an Ala residue at E-316 in MSI-7, at which CorAn9124 contains Thr. Wild-type JE and WN viruses also contain Ala at this position, while the attenuated JE SA14-14-2 vaccine virus contains Val (residue E-315 in these viruses).10,22 The E-316 Ala residue in MSI-7 could be an important factor contributing to the high virulence of this strain. Wild-type JE and the SA14-14-2 viruses differ at 10 amino acids in the E protein. Of these, most wild-type JE virus-specific residues were also found in wild-type WN virus.10 After the YF/WN chimera was initially constructed, it was further attenuated by replacing some of these amino acids in the chimera with SA14-14-2 specific residues resulting in the ChimeriVax-WN vaccine candidate.5 If necessary, the same approach could be used to further attenuate the YF/SLE chimeras because both the MSI and CorAn variants contain six of the 10 residues coinciding with wild-type JE virus and three more that differ from both wild-type JE and SA14-14-2 viruses. We also aligned our MSI-7 and CorAn9124 sequences with complete or partial prM-E protein sequences of 21 other SLE viral strains available from GenBank representing both high and low virulence strains,11 and found no common residues that could be responsible for the high and low virulence profiles. By aligning the SLE virus sequences with those of other flaviviruses, we more precisely determined the start site of the prM protein compared with that previously predicted for MSI-717 (Figure 1A). According to our data, the signalase cleavage resulting in the N terminus of prM should occur after the Leu-Ala-Ser-Ser stretch, two residues downstream from the previously proposed cleavage site.
The two YF/SLE chimeras were produced by the recently developed three-fragment ligation method,7,8 and we were able to also produce the YF/MSI version by the standard two-fragment ligation method (see Results). In both chimeras, the CYF/prMSLE junction was in accordance with our alignment (Figure 1A), with the prM sequence starting with Leu-Gln-Leu. We attempted to produce a YF/MSI chimera using the previously predicted N-terminus of prM (Ser-Ser-Leu-Gln-Leu). If such a chimera were non-viable, this would constitute experimental proof of the correctness of our alignment. Another possibility was that such a chimera would be viable, but defective in its replication due to a less efficient signalase cleavage at the junction, which could be a promising approach for stable flavivirus attenuation. Surprisingly, the Ser-Ser chimera was viable and did not differ in plaque morphology and the rate of replication in cell culture. It was not significantly attenuated in mice as examined by the IC route compared with the Leu-Gln-Leu version. Thus, the presence of the extra Ser-Ser residues at the CYF/prMSLE junction did not affect virus growth demonstrating high structural flexibility of the signalase cleavage site. However, this result could not serve as experimental proof of the correctness of our alignment.
Both chimeras were attenuated compared with the parental SLE viral strains in a sensitive mouse neurovirulence test, as shown by higher survival rates and longer AST of animals following IC inoculation. This was an expected effect of chimerization with YF 17D, since the chimerization process is inherently attenuating.4 The YF/CorAn chimera was less neurovirulent than YF/MSI, which correlated with the unequal virulence profiles of the CorAn9124 and MSI-7 viruses in our studies and those previously described for these viruses.11 Thus, strain-specific determinants in the envelope play a role as was noted previously for some other YF 17D chimeras.14 Judged by the IC LD50 values, the YF/CorAn chimera is > 10,000 and > 2,222 times less neurovirulent than wild-type CorAn9124 and YF 17D, respectively; the YF/MSI chimera is 1,000 and 13.75 times less neurovirulent than MSI-7 and YF 17D, respectively. Undoubtedly, each of these viruses can replicate efficiently in the brain, but with less severe symptoms in the case of chimeras. The ChimeriVax-JE vaccine virus grew to more than 6 log10 pfu/gram of mouse brain tissue without causing disease.6 The chimerization also ablated the ability of the highly neuroinvasive MSI-7 virus to cause encephalitis following peripheral inoculation. The YF/MSI chimera caused no mortality following IP inoculation of 2.6 x 107 pfu/mouse. In contrast, the MSI-7 virus was partially lethal at IP doses as low as 20 pfu. A similar effect of chimerization with a non-encephalitogenic DEN 4 virus on neuroinvasiveness of tick-borne encephalitis virus has been documented.23 As expected, the CorAn9124 parent and thus the YF/CorAn chimera, as well as YF 17D virus, were avirulent after peripheral inoculation. Based on these data, the YF/CorAn chimera (but not YF/MSI) was downgraded to a BSL-2 level by the Acambis Institutional Biosafety Committee, according to current guidelines, and the data were reviewed and approved by CDC. The genetic stability and maintenance of the highly attenuated phenotype of YF 17D-based chimeras during prolonged propagation in cell culture has been demonstrated for ChimeriVax vaccines.4–10 For serodiagnosis, working stocks of YF/CorAn will be made from seed virus distributed by CDC, and will be no more than two passages away from the seed virus to preserve the attenuated phenotype.
Definitive serologic diagnosis of WN virus or SLE virus infection depends on the use of the neutralization test. While flavivirus-specific IgG is highly cross-reactive in the hemagglutination-inhibition test and enzyme-linked immunosorbent assays (ELISAs), the IgM ELISA is more specific and can be used for rapid serodiagnosis. However, the specificity of the IgM-based ELISA remains a problem for closely related flaviviruses.24 The cross-reactions between the two endemic flaviviruses causing encephalitis in the United States (SLE and WN viruses) are problematic. Diagnosis by virus isolation or detection and identification of viral genomes by a reverse transcriptase-polymerase chain reaction can provide a specific diagnosis. However, the latter methods are severely limited by the short duration of postinfection viremia, which generally occurs before onset of clinical signs.
As a proof of principle, in this study we used PRNTs to demonstrate that sera from small cohorts of confirmed clinical cases of WN and SLE could be serologically differentiated using the YF/SLE (the CorAn version) and YF/WN (ChimeriVax-WN) chimeric viruses (Table 4). Data for larger groups of cases will be accumulated and reported elsewhere. These chimeras can be safely handled at the BSL-2 level. Currently, they are being distributed to State Health Department Laboratories and others engaged in diagnosis and epidemiologic surveillance of WN and SLE. Our preliminary data also indicate that the constructed YF/SLE chimeras have vaccine potential. If a vaccine against SLE becomes a necessity, the development of these chimeras as vaccine candidates will be continued.
Received February 2, 2004. Accepted for publication May 30, 2004.
Acknowledgments: We are grateful to Francis A. Ennis and John Cruz (Center for Infectious Disease and Vaccine Research, University of Massachusetts, Worcester, MA) for providing us access to UMASS BSL-3 facilities for virus and animal experiments and assistance.
Disclosure: Some of the authors of this paper wish to disclose that they are employees of Acambis Inc. and may hold stock in this company. This statement is made in the interest of full disclosure and not because the authors consider this to be a conflict of interest.
Authors’ addresses: Konstantin V. Pugachev, Farshad Guirakhoo, Fred Mitchell, Simeon W. Ocran, Megan Parsons, Dennis W. Trent, and Thomas P. Monath, Acambis Inc., 38 Sidney Street, Cambridge, MA 02139, Telephone: 617-761-4200; Fax: 617-494-1741; E-mails: konstantin.pugachev{at}acambis.com, farshad.guirakhoo{at}acambis.com, fred.mitchell{at}acambis.com, simeon.ocran{at}acambis.com, megan.parsons{at}acambis.com, dennis.trent{at}acambis.com, and tom.monath{at}acambis.com. Barbara W. Johnson, Olga L. Kosoy, Robert S. Lanciotti, and John T. Roehrig, Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80522, E-mails: bfj9{at}cdc.gov, oak3{at}cdc.gov, rsl2{at}cdc.gov, and jtr1{at}cdc.gov.
Reprint requests: Konstantin V. Pugachev, Acambis Inc., 38 Sidney Street, Cambridge, MA 02139.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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