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Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention (CDC), Public Health Service, US Department of Health and Human Services, PO Box 2087, Fort Collins, CO 80522, USA
Correspondence
Amanda E. Calvert
zpz0{at}cdc.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Monath, 1994). Currently, no vaccine exists against any of the four serotypes of DEN virus (DEN1–4). The production of a DEN virus vaccine is difficult, because infection with one serotype does not elicit long-term cross-protective immunity against the other serotypes and may, in fact, increase the severity of disease, causing DHF or dengue shock syndrome (DSS). This phenomenon of antibody-dependent enhancement (ADE) is thought to be mediated by immune complexes of non-neutralizing antibody and virus that attach to Fc receptor-bearing cells and increase virus infection directly or elicit the production of a variety of chemokines that modify the disease process (Halstead & O'Rourke, 1977; Littaua et al., 1990). A successful DEN vaccine will need to be tetravalent and capable of eliciting virus-neutralizing antibodies against all four serotypes to minimize the effects of non-neutralizing, virus-enhancing antibodies that may result in DHF or DSS.
Whilst a variety of experimental vaccine candidates have been investigated, most of these are either live-attenuated viruses or subunit vaccines utilizing the pre-membrane (prM) and envelope (E) proteins to produce high levels of virus-neutralizing antibody. The E protein, the major virion surface protein, is responsible for virus attachment to animal cells and virus-mediated cell-membrane fusion (Roehrig, 2003). Utilizing the E protein as a subunit vaccine is logical. However, including the E protein in any vaccine candidate could lead to the production of virus-enhancing antibodies that result in ADE as virus-neutralizing antibody wanes. In this case, regular reimmunization may be necessary to maintain protective levels of virus-neutralizing antibody.
The non-structural (NS) proteins, particularly NS1, are immunogenic and can elicit high-titre antibody. NS1, a glycoprotein, is also expressed on the surface of virus-infected cells. NS1 is not incorporated into the virion and therefore does not elicit virus-enhancing antibodies (Roehrig, 2003). The mechanism by which NS1 elicits protection is not fully understood; however, antibodies to NS1 bind surface-expressed NS1 and induce complement-mediated cytolysis or antibody-dependent cellular cytotoxicity (Jacobs et al., 1994; Schlesinger et al., 1993). Immunization with recombinant or purified NS1 has been shown to be protective against flavivirus challenge (Falgout et al., 1990; Hall et al., 1996; Jacobs et al., 1992; Lin et al., 1998; Qu et al., 1993).
Until recently, comparing the relative protective capacity of the E protein and the NS proteins following virus immunization has been difficult, largely because there has been no system available for direct comparisons. Animals vaccinated with live-attenuated viruses elicit both anti-E and anti-NS protein antibodies, and protection is largely mediated by the anti-E protein virus-neutralizing antibodies. Protection of animals immunized individually with either purified E protein or NS1 protein is also difficult to compare, because neither antigen is administered in the context of replicating virus. Differences in protein immunogenicity might be overcome by increasing the dose of one or the other protein in the administered vaccine.
Chimeric flaviviruses are excellent tools to compare protective immunity elicited by virus structural or NS proteins during virus replication. A wide variety of heterotypic chimeric flaviviruses, containing a backbone from one flavivirus that has had its surface proteins replaced with the surface proteins from another flavivirus, have been shown to replicate and induce protective immunity in mice and/or non-human primates (Arroyo et al., 2001; Bray et al., 1996; Caufour et al., 2001; Chambers et al., 2003; Guirakhoo et al., 2000; Huang et al., 2003, 2005; Johnson & Roehrig, 1999; Pletnev et al., 2001, 2003; van der Most et al., 2000).
In earlier studies, we demonstrated that adult AG129 mice were susceptible to a peripheral infection with mouse-adapted DEN2 virus strain New Guinea C (NGC) (Johnson & Roehrig, 1999). These mice produced neutralizing antibody when immunized with either DEN2 wild-type 16681 virus or its vaccine strain PDK-53, both of which protected the animals against lethal DEN2 NGC virus challenge (Johnson & Roehrig, 1999). In similar studies, the alpha/beta interferon (IFN-
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)-receptor pathway was found to play an essential role in inhibiting initial virus replication in extraneural sites and controlling subsequent viral spread to the central nervous system (CNS) (Shresta et al., 2004). However, this interferon pathway is not required for protection from disease (Johnson & Roehrig, 1999; Shresta et al., 2004).
Here, we have used infectious cDNA clones of DEN2 16681 virus to produce fully infectious chimeric viruses that have had the DEN2 prM and E proteins replaced by the prM and E proteins of West Nile virus (WN virus), strain NY99. By inoculating DEN2 NGC virus-susceptible AG129 mice with these chimeric viruses and challenging them with the NGC virus, we have been able to determine directly protective effects contributed by the DEN2 viral NS proteins following immunization with live virus in this interferon-deficient mouse model.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). WN virus strain NY99 35262, originally isolated from a Chilean flamingo at the Bronx Zoo (NY, USA) in 1999, was obtained from the Arbovirus Diseases Branch Diagnostic and Reference Activity, CDC. The virus was passaged once in suckling mouse brain and twice in C6/36 cells. Stock virus was grown in C6/36 cells to a titre of 1·5x109 p.f.u. ml–1. The DEN2 mouse-adapted NGC virus has been passaged numerous times in suckling mouse brain to increase its neurovirulence (a kind gift from Kenneth Eckels, Walter Reed Army Medical Center, Washington DC, USA). A stock of this virus was grown in C6/36 cells and titrated at 2·5x106 p.f.u. ml–1.
Construction and characterization of the chimeric D2/WN viruses has been described previously (Huang et al., 2005). These chimeric viruses were derived from infectious cDNA clones pD2/WN-P1 and pD2/WN-E1-112. The D2/WN-P1 chimera contains the prM-E genes of WN NY99 virus in the wild-type DEN2 16681 genomic background, whilst the D2/WN-E1-112 chimera contains the WN NY99 prM-E genes in the genomic background of the candidate DEN2 PDK-53 vaccine virus. Genomic sequence analysis revealed the expected sequence for the D2/WN-P1 chimera. However, two mutations resulted in amino acid substitutions at NS2A-22 (Met to Val/Met mix) and NS2B-93 (Gln to Gln/Arg mix) in the D2/WN-E1-112 chimera (Huang et al., 2005).
Mouse experiments.
AG129 mice deficient in IFN-/ and -
responses (van den Broek et al., 1995) were bred in-house. The mice were handled as specified by institutional guidelines for care and use in accordance with the Institutional Animal Care and Use Committee recommendations. Both of the D2/WN chimeras, WN NY99 virus and DEN2 NGC virus were evaluated for neuroinvasiveness in 5–8-week-old AG129 mice. Seven groups of mice (n=5) were inoculated intraperitoneally (i.p.) with 10-fold dilutions of each virus (0·01–10 000 p.f.u.). Animals were monitored for 4 weeks after inoculation and 50 % intraperitoneal lethal-dose end points (i.p. LD50) were calculated (Reed & Muench, 1938).
For immunogenicity studies, 3–5-week-old AG129 mice (n=10) were inoculated i.p. with 1000 p.f.u. in 0·1 ml D2/WN-E1-112 or D2-WN-P1 chimeric virus. Mice were boosted 2 weeks later with an equivalent dose. Naïve mice were inoculated in the same manner with medium. Mice were bled 2 weeks after each vaccination. Challenge was performed by i.p. inoculation with 100 p.f.u. WN NY99 virus or 1000 p.f.u. DEN2 NGC virus, diluted in medium, at 14 days after the booster immunization. Challenged mice that showed signs of morbidity were euthanized. Sera were collected from surviving mice for antibody analysis.
Production of virus cell lysate.
C6/36 cell monolayers were grown in 150 cm2 culture flasks. Confluent monolayers were inoculated with either WN NY99 virus or DEN2 16681 virus at an m.o.i. of 0·1. WN and DEN2 virus-infected cells were harvested on day 4 or 7 post-infection, respectively. Cell lysate was produced as described previously (Blitvich et al., 2003). Control antigen was prepared similarly with uninfected C6/36 cells.
Virus purification.
WN NY99 and DEN2 16681 viruses were grown in C6/36 cells and purified on glycerol tartrate gradients as described by Obijeski et al. (1976). These purified stocks were used in subsequent serological assays.
Standardization of viral proteins for use in serological assays.
DEN2 and WN E proteins were quantified for ELISA by end-point titration in an antigen-capture ELISA to standardize the amounts of the E protein present in virus-infected cell lysate and purified virus. WN virus antigen was captured with monoclonal antibody (mAb) 3.91D and detected with mAb 6B6C-1 and goat anti-mouse Ab conjugated to alkaline phosphatase. As indicated by the ELISA end-point titrations of WN antigens, purified WN virus was used at a concentration of 0·07 µg per well and WN viral lysate was diluted to 1 : 400 in the experimental ELISA. DEN2 virus antigen was captured with mAb 4G2 and detected with mAb 3H5 and goat anti-mouse Ab conjugated to alkaline phosphatase. In the experimental assays, DEN2 purified virus was used at a concentration of 0·03 µg per well and DEN2 viral lysate was diluted to 1 : 100.
DEN2 antigen was quantified for Western blotting by running dilutions of lysate on a 4–12 % Bis/Tris SDS-PAGE gel and staining with anti-E protein mAb 1A6A-8 to DEN2 Jamaica strain 1409. E protein bands from lysate were detected with a Bio-Rad densitometer and compared in order to quantify relative amounts of E protein. DEN2 viral lysate antigen was used at a dilution of 1 : 10.
ELISA.
The inner 60 wells of a 96-well microtitre plate (Immulon 2HB; Thermo Lab Systems) were coated with viral lysate or purified virus at an appropriate dilution in carbonate/bicarbonate buffer (50 mM sodium carbonate, 50 mM sodium bicarbonate, pH 9·6) and incubated overnight at 4 °C. Plates were washed five times with PBS/0·1 % Tween wash buffer with an automatic plate washer. Non-specific binding sites were blocked with 3 % goat serum in PBS (100 µl per well) for 1 h at 37 °C. Blocking solution was discarded and sera or mAb were added in twofold serial dilutions (50 µl per well) and incubated at 37 °C for 1 h. Plates were washed five times before the addition of goat anti-mouse antibody conjugated to alkaline phosphatase (50 µl per well), diluted 1 : 100 in PBS. After an incubation period of 1 h at 37 °C, plates were washed again five times. Alkaline phosphatase substrate (Sigma) was added to each well of the plate (100 µl per well) and incubated in the dark at room temperature for 30 min. OD405 was read on an automatic plate reader. Test samples for each dilution of sera were analysed in duplicate. End points were determined as an OD405 reading of at least twice that of the mean OD405 from normal sera reacting to antigen. Titres were expressed as the geometric mean of the reciprocal of the end points.
Plaque-reduction neutralization test (PRNT).
The PRNT was performed in six-well plates of Vero cells as described previously (Huang et al., 2005). The mouse sera were heat-inactivated (56 °C for 30 min) and the tests were performed without addition of exogenous complement. End-point titres were expressed as the geometric mean of the serum dilution yielding at least 80 % reduction in the number of plaques (PRNT80).
Immunoblotting.
DEN2 16681 viral proteins from the lysate of virus-infected C6/36 cells were separated by SDS-PAGE on 4–12 % Bis/Tris gels (Invitrogen). Proteins were blotted electrophoretically from the gels onto nitrocellulose membranes and washed for 15 min in PBS/0·1 % Tween wash buffer. Non-specific binding sites were blocked with 3 % goat serum in PBS overnight at 4 °C. Sera taken from mice vaccinated with the D2/WN-E1-112 or D2/WN-P1 chimera were pooled and diluted 1 : 50 in PBS. Anti-DEN2 E protein mAb 1A6A-8, anti-DEN2 NS1 mAb 9A9 (generously provided by Jacob Schlesinger, University of Rochester Medical Center, NY, USA) and anti-DEN2 NGC mouse hyperimmune ascitic fluid were also tested. Diluted sera were incubated with the membrane for 1 h with gentle rocking. Membranes were washed again in PBS/0·1 % Tween wash buffer three times for 5 min each. Goat anti-mouse antibody conjugated to alkaline phosphatase was diluted 1 : 200 and incubated on the membrane for 1 h with gentle rocking. Membranes were washed as described previously and BCIP/NBT phosphatase substrate (KPL) was added to the membrane until a colour change appeared. The reaction was stopped by the addition of water.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). As shown previously (Johnson & Roehrig, 1999), neuroadapted DEN2 NGC virus was also highly virulent, with an i.p. LD50 of 2·1 p.f.u. Mice receiving the D2/WN-P1 or D2/WN-E1-112 chimeric virus (0·01–105 p.f.u.) did not exhibit morbidity or mortality (Fig. 1). Thus, the D2/WN chimeras were highly attenuated in AG129 mice. Mice were bled at 4 weeks after viral inoculation to assess the antibody response to the D2/WN chimeras. Mice inoculated with more than 10 p.f.u. D2/WN chimeric virus produced measurable amounts of ELISA antibody to WN and DEN2 virus, low neutralizing antibody to WN virus and no detectable neutralizing antibody to DEN2 virus (data not shown).
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; Fig. 2). Three sets of sera were pooled for these analyses: (i) sera obtained 2 weeks after the primary immunization, (ii) sera obtained 2 weeks after the secondary immunization and (iii) sera obtained 47 days after challenge of boosted mice with DEN2 NGC virus.
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1·7 log10) against gradient-purified DEN2 16681 virus, but were significant (2·03–2·18 log10) against the DEN2 16681 virus-infected C6/36 cell lysate, following primary or secondary immunization of AG129 mice with each of the D2/WN chimeras (Table 1
). Correspondingly, pooled D2/WN-immune sera reacted strongly with DEN2 NS1, but minimally with DEN2 E protein, by Western blotting (Fig. 2, lanes 1–2) and these sera showed minimal or no neutralizing activity against DEN2 virus (log10 titres of 1·0 in Table 1) following primary or secondary immunization. The identity of the DEN2 NS1 band in the Western blot was confirmed by detection with anti-DEN2 NS1-specific mAb 9A9 (Fig. 2, lane 4). A band of about 92 kDa appeared to be a non-viral protein in the C6/36 lysate, because sera reacted with this protein in the uninfected C6/36 lysate control (Fig. 2, control lane 7). Although this band was approximately the expected size of dimeric NS1, the band was not detected by DEN2 NS1-specific mAb 9A9 (Fig. 2, lane 4). The broader E protein band in Fig. 2, lane 5, might be explained by a probably higher concentration of DEN2 E-specific antibody in mAb 1A6A-8, relative to sera obtained from D2/WN-immunized AG129 mice (Fig. 2, lanes 3 and 6). In contrast to the non-structural protein specificity of anti-DEN2 antibodies, D2/WN-immune AG129 sera recognized the WN E protein. After the primary immunization, D2/WN-P1- and D2/WN-E1-112-immune mouse sera reacted to somewhat higher titres against gradient-purified WN virus (log10 titres of 3·72 and 3·35, respectively) than against WN-infected C6/36 cell lysate (log10 titres of 3·05 and 2·99, respectively) by ELISA. Following secondary immunization, ELISA antibody titres versus gradient-purified WN virus (D2/WN-P1, 3·54 log10; D2/WN-E1-112, 3·48 log10) and WN viral lysate (D2/WN-P1, 3·51 log10; D2/WN-E1-112, 3·44 log10) were equivalent. The WN E protein would be expected to be present in both purified WN virus and lysates of WN virus-infected C6/36 cells. The D2/WN chimeric viruses elicited significant levels of neutralizing antibody against WN virus after the primary (D2/WN-P1, log10 titre 2·32; D2/WN-E1-112, log10 titre 2·04) and the secondary (D2/WN-P1, log10 titre 2·41; D2/WN-E1-112, log10 titre 2·04) immunizations. These data demonstrated that the D2/WN chimeras elicited WN E protein-reactive neutralizing antibodies in AG129 mice. Pooled D2/WN-immune AG129 mouse sera also reacted with the WN E protein by Western blot analysis (data not shown).
These results indicated that AG129 mice immunized with the D2/WN chimeric viruses developed antiviral antibodies that were specific for the E and prM proteins of WN virus and the non-structural proteins (primarily NS1, as indicated by Western blot analysis) of the DEN2 16681 chimeric vector.
Protective efficacy of D2/WN chimeric viruses
Two weeks after the secondary vaccination, immunized and control (BA-1) mice were challenged i.p. with WN NY99 or DEN2 NGC virus. No D2/WN-immunized mice (0/20) survived WN virus challenge and only 15 % (3/20), all in the D2/WN-E1-112 group, survived challenge with DEN2 virus (Table 2). However, mean survival times (MSTs) of the D2/WN chimera-vaccinated mice challenged with WN virus or DEN2 virus were all significantly longer than those of the BA-1 control group (P<0·011). Relative to the non-vaccinated control mice, immunization with the D2/WN-E1-112 or D2/WN-P1 chimera increased mouse MST by 2 or 4 days following WN viral challenge, or by 14 or 11 days following DEN2 NGC viral challenge, respectively (Table 2). High ELISA antibody titres to WN (
4·03) and DEN2 (2·99 or 3·50 for purified virus or virus-infected cell lysate, respectively) viral antigen were detected in the sera from the three D2/WN-E1-112 mice that survived DEN2 NGC virus challenge (Table 1). High levels of neutralizing antibodies to WN virus (2·51) and moderate levels of neutralizing antibodies to DEN2 virus (1·68) were produced after DEN2 virus challenge in these three mice (Table 1). Western blot analysis revealed antibody responses to the E protein as well as the NS1 protein of DEN2 virus following DEN2 virus challenge (Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, - and - responses, are susceptible to a peripheral challenge with DEN virus. These mice serve as a reliable model for DEN virus vaccination-challenge studies (Johnson & Roehrig, 1999). AG129 mice demonstrated 100 % mortality when challenged with the neuroadapted NGC strain of DEN2 virus or the Mochizuki strain of DEN1 virus (Johnson & Roehrig, 1999; Huang et al., 2003). By utilizing this small-animal model, we showed previously that vaccination with wild-type DEN2 16681, its PDK-53 vaccine derivative or chimeric DEN2/DEN1 virus elicited high levels of virus-neutralizing antibody and protected all animals from subsequent i.p. challenge with lethal DEN2 NGC or DEN1 Mochizuki virus (Johnson & Roehrig, 1999; Huang et al., 2003). Here, we have used D2/WN chimeric viruses to evaluate their ability to (i) replicate in AG129 interferon-deficient mice, (ii) produce antiviral- and neutralizing-antibody responses to DEN and WN viruses and (iii) induce a protective antibody response against peripheral challenge with DEN2 NGC virus. Both the wild-type DEN2 16681 virus-vectored D2/WN-P1 chimera and the attenuated DEN2 PDK-53 virus-vectored D2/WN-E1-112 chimera were non-pathogenic in AG129 mice and elicited high anti-WN virus antibody titres. Not surprisingly, the D2/WN chimeric vaccines elicited little anti-DEN E protein antibody, high levels of anti-DEN NS1 antibody and no or very low levels of DEN2 virus-neutralizing antibodies. Whilst the MST of mice immunized with D2/WN-P1 or D2/WN-E1-112 chimera increased by 11 and 14 days, respectively, over non-vaccinated controls, only 15 % (three of 20, all three in the D2/WN-E1-112 group) of the D2/WN-immunized mice survived DEN2 NGC virus challenge after two vaccinations. These results indicated that, when tested in replicating chimeric viruses containing the heterologous WN prM-E genes, the DEN2 non-structural proteins were able to significantly increase the MSTs of DEN2 NGC virus-challenged AG129 mice. However, this protection was not as effective as the complete protection afforded by the DEN2 virus-specific E protein following immunization of AG129 mice with DEN2 16681 or PDK-53 virus (Johnson & Roehrig, 1999).
Recently, we showed that the D2/WN chimeras based on the DEN2 PDK-53 vaccine retain the attenuated phenotypic characteristics of the candidate PDK-53 vaccine virus, and induce in Swiss Webster mice a protective immune response against WN NY99 virus challenge (Huang et al., 2005). We have also reported previously that chimeric DEN2/DEN1 viruses containing prM-E of the DEN1 virus in the DEN2 genomic background were able to protect AG129 mice against lethal DEN1 Mochizuki virus challenge (Huang et al., 2003). In the present study, the attenuated D2/WN viruses induced ELISA antibodies and neutralizing antibodies against WN virus. However, this response was not sufficient to protect the immunized AG129 mice against peripheral challenge with the neurotropic WN virus. This may be due to the major role that IFN-/ plays in controlling virus replication. Whilst AG129 mice have normal-functioning immune systems, it is evident that they are unable to clear overloading or long-term viral infections. G129 mice lacking the IFN- receptor were able to clear a lymphocytic choriomeningitis virus (LCMV) infection, whereas A129 mice lacking the IFN-/ receptor were unable to do so even after surviving viral infection for 20 days (van den Broek et al., 1995). Similarly, IFN-/ receptor-deficient mice infected with Semliki Forest virus (SFV) died within 4–10 days, whilst wild-type and IFN- receptor-deficient mice survived SFV infection (Muller et al., 1994).
IFN-/ also plays a major role in regulating the cytotoxic T-cell response. This may be an important component for clearance of overwhelming virus load and exhaustion of cytotoxic T-lymphocyte (CTL) precursors (van den Broek et al., 1995). This has been shown to occur in AG129 mice with slow-replicating strains of LCMV. The CTLs and CTL precursors were depleted quickly in AG129 mice when infected with the LCMV strain ARM. This led to lifelong viral persistence in the mice because of overwhelming virus replication and exhaustion of the CTLs, resulting in a lack of virus-specific CTL precursors (van den Broek et al., 1995). This indicates that the IFN-/ receptor is essential for antiviral clearance, especially in highly virulent encephalitic and meningitic viruses such as SFV and LCMV, and may be the reason that AG129 mice succumbed to WN virus challenge in our study. From this study and previous reports, WN NY99 virus replicates more robustly in AG129 mice, resulting in significantly lower i.p. LD50 values, than either DEN2 NGC or DEN1 Mochizuki virus (Johnson & Roehrig, 1999; Huang et al., 2003).
CTLs are primarily generated against the non-structural protein 3 (NS3) of flaviviruses (Lobigs et al., 1994). Spaulding et al. (1999) demonstrated that a primary dengue infection induces flavivirus cross-reactive NS3-specific CTLs. These cross-reactive CTLs may play a protective role in limiting virus replication, but they may also contribute to an increased risk of DHF during secondary infections. Children suffering from DHF were found to have higher levels of soluble CD8+ in their serum than children with dengue fever (Kurane et al., 1991). Although CTL responses are important in protective immunity, antibody-mediated protection, especially neutralizing antibody, plays a key role in vaccine-induced immunity and may be a more relevant focus than the CTL response. The CTL response of these mice to the D2/WN chimeras is of interest and, whilst it is beyond the scope of this study, it should be considered for future investigations.
In earlier studies, we showed that AG129 mice produce neutralizing antibody to DEN2 virus and are protected completely against DEN2 NGC peripheral virus challenge when immunized with the candidate DEN2 PDK-53 vaccine strain (Johnson & Roehrig, 1999). Shresta et al. (2004) showed that the IFN-/ receptor-mediated activity limited the initial DEN2 replication in extraneural sites and controlled viral spread into the CNS. They also showed that the IFN- pathway later acted to restrict virus replication in the periphery and eliminate DEN virus from the CNS (Shresta et al., 2004). We hypothesize that AG129 mice in this study may not have been able to clear a DEN2 virus infection after vaccination, due to a lack of neutralizing antibody to DEN2 virus and the absence of a functioning IFN response to clear DEN2 virus from extraneural sites and the CNS.
NS1, a major immunogen in flavivirus infection, is an attractive choice for vaccination studies because it avoids the risk of incorporating anti-virion antibodies that might lead to ADE. NS1 is secreted from and expressed on the surface of infected cells and induces complement-mediated cytolysis for clearance of the virus infection (Schlesinger et al., 1993). NS1 has been expressed in a variety of ways to induce protective immunity in mice. Studies expressing NS1 along with the structural proteins in recombinant viruses, such as Vaccinia virus and baculoviruses, have shown that NS1 does not produce a high-titre antibody response to DEN virus (Bray et al., 1989; Zhang et al., 1988; Zhao et al., 1987). However, immunization with recombinant or purified NS1 has been shown to provide protection against flavivirus challenge (Falgout et al., 1990; Hall et al., 1996; Jacobs et al., 1992; Qu et al., 1993). Other studies have shown that the amino acid sequence context of flaviviral NS1 is vital to its correct expression and secretion. DNA constructs expressing NS1 and portions of NS2A do not secrete NS1 as well as constructs expressing NS1 alone, and do not induce protective immunity in mice (Konishi et al., 1991; Lin et al., 1998; Pugachev et al., 1995).
In this study, the DEN2 virus-specific non-structural proteins, including NS1, were expressed in a replicating chimeric D2/WN virus system to determine the degree of protection against DEN2 NGC virus challenge conferred by the non-structural proteins of DEN2 virus alone. The results showed that, whilst some increase in the MSTs was afforded to D2/WN-immunized, DEN2 virus-challenged AG129 mice, the DEN2 non-structural proteins alone were not adequate to induce protective immunity in these mice. Even though a few mice survived DEN-2 virus challenge and MSTs increased without pre-existing neutralizing antibody to DEN-2 virus, the anti-NS1 antibody response mounted against DEN-2 virus was not sufficient to protect mice from lethal challenge. These results are not consistent with other studies that have found anti-NS1 antibodies to be sufficient to protect mice from lethal virus challenge following direct immunization with NS1. However, given that the previous studies were performed in mice with a functioning interferon response, such comparisons might not be valid. Further investigation into the immunogenic characteristics of NS1 should be conducted before recommending its use as the sole component of a vaccine against DEN viral infection.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Blitvich, B. J., Marlenee, N. L., Hall, R. A., Calisher, C. H., Bowen, R. A., Roehrig, J. T., Komar, N., Langevin, S. A. & Beaty, B. J. (2003). Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to West Nile virus in multiple avian species. J Clin Microbiol 41, 1041–1047.
Bray, M., Zhao, B., Markoff, L., Eckels, K. H., Chanock, R. M. & Lai, C.-J. (1989). Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis. J Virol 63, 2853–2856.
Bray, M., Men, R. & Lai, C.-J. (1996). Monkeys immunized with intertypic chimeric dengue viruses are protected against wild-type virus challenge. J Virol 70, 4162–4166.[Abstract]
Caufour, P. S., Motta, M. C. A., Yamamura, A. M. Y., Vazquez, S., Ferreira, I. I., Jabor, A. V., Bonaldo, M. C., Freire, M. S. & Galler, R. (2001). Construction, characterization and immunogenicity of recombinant yellow fever 17D-dengue type 2 viruses. Virus Res 79, 1–14.[CrossRef][Medline]
Chambers, T. J., Liang, Y., Droll, D. A., Schlesinger, J. J., Davidson, A. D., Wright, P. J. & Jiang, X. (2003). Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization, immunogenicity, and protection against dengue encephalitis in the mouse model. J Virol 77, 3655–3668.
Falgout, B., Bray, M., Schlesinger, J. J. & Lai, C.-J. (1990). Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis. J Virol 64, 4356–4363.
Gubler, D. J. (1998). Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11, 480–496.
Guirakhoo, F., Weltzin, R., Chambers, T. J. & 7 other authors (2000). Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates. J Virol 74, 5477–5485.
Hall, R. A., Brand, T. N. H., Lobigs, M., Sangster, M. Y., Howard, M. J. & Mackenzie, J. S. (1996). Protective immune responses to the E and NS1 proteins of Murray Valley encephalitis virus in hybrids of flavivirus-resistant mice. J Gen Virol 77, 1287–1294.
Halstead, S. B. & O'Rourke, E. J. (1977). Dengue virus and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J Exp Med 146, 201–217.
Huang, C. Y.-H., Butrapet, S., Pierro, D. J., Chang, G.-J. J., Hunt, A. R., Bhamarapravati, N., Gubler, D. J. & Kinney, R. M. (2000). Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine. J Virol 74, 3020–3028.
Huang, C. Y.-H., Butrapet, S., Tsuchyia, K. R., Bhamarapravati, N., Gubler, D. J. & Kinney, R. M. (2003). Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development. J Virol 77, 11436–11447.
Huang, C. Y.-H., Silengo, S. J., Whiteman, M. C. & Kinney, R. M. (2005). Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus. J Virol 79, 7300–7310.
Jacobs, S. C., Stephenson, J. R. & Wilkinson, G. W. G. (1992). High-level expression of the tick-borne encephalitis virus NS1 protein by using an adenovirus-based vector: protection elicited in a murine model. J Virol 66, 2086–2095.
Jacobs, S. C., Stephenson, J. R. & Wilkinson, G. W. G. (1994). Protection elicited by a replication-defective adenovirus vector expressing the tick-borne encephalitis virus non-structural glycoprotein NS1. J Gen Virol 75, 2399–2402.
Johnson, A. J. & Roehrig, J. T. (1999). New mouse model for dengue virus vaccine testing. J Virol 73, 783–786.
Konishi, E., Pincus, S., Fonseca, B. A. L., Shope, R. E., Paoletti, E. & Mason, P. W. (1991). Comparison of protective immunity elicited by recombinant vaccinia viruses that synthesize E or NS1 of Japanese encephalitis virus. Virology 185, 401–410.[CrossRef][Medline]
Kurane, I., Innis, B. L., Nimmannitya, S., Nisalak, A., Meager, A., Janus, J. & Ennis, F. A. (1991). Activation of T lymphocytes in dengue virus infections: high levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon- in sera of children with dengue. J Clin Invest 88, 1473–1480.[Medline]
Lin, Y.-L., Chen, L.-K., Liao, C.-L., Yeh, C.-T., Ma, S.-H., Chen, J.-L., Huang, Y.-L., Chen, S.-S. & Chiang, H.-Y. (1998). DNA immunization with Japanese encephalitis virus nonstructural protein NS1 elicits protective immunity in mice. J Virol 72, 191–200.
Littaua, R., Kurane, I. & Ennis, F. A. (1990). Human IgG Fc receptor II mediates antibody-dependent enhancement of dengue virus infection. J Immunol 144, 3183–3186.[Abstract]
Lobigs, M., Arthur, C. E., Müllbacher, A. & Blanden, R. V. (1994). The flavivirus nonstructural protein NS3 is a dominant source of cytotoxic T cell peptide determinants. Virology 202, 195–201.[CrossRef][Medline]
Monath, T. P. (1994). Dengue: the risk to developed and developing countries. Proc Natl Acad Sci U S A 91, 2395–2400.
Muller, U., Steinhoff, U., Reis, L. F., Hemmi, S., Pavlovic, J., Zinkernagel, R. M. & Aguet, M. (1994). Functional role of type I and type II interferons in antiviral defense. Science 264, 1918–1921.
Obijeski, J. F., Bishop, D. H. L., Murphy, F. A. & Palmer, E. L. (1976). Structural proteins of La Crosse virus. J Virol 19, 985–997.
Pletnev, A. G., Bray, M., Hanley, K. A., Speicher, J. & Elkins, R. (2001). Tick-borne langat/mosquito-borne dengue flavivirus chimera, a candidate live attenuated vaccine for protection against disease caused by members of the tick-borne encephalitis virus complex: evaluation in rhesus monkeys and in mosquitoes. J Virol 75, 8259–8267.
Pletnev, A. G., Claire, M. S., Elkins, R., Speicher, J., Murphy, B. R. & Chanock, R. M. (2003). Molecularly engineered live-attenuated chimeric West Nile/dengue virus vaccines protect rhesus monkeys from West Nile virus. Virology 314, 190–195.[CrossRef][Medline]
Pugachev, K. V., Mason, P. W., Shope, R. E. & Frey, T. K. (1995). Double-subgenomic Sindbis virus recombinants expressing immunogenic proteins of Japanese encephalitis virus induce significant protection in mice against lethal JEV infection. Virology 212, 587–594.[CrossRef][Medline]
Qu, X., Chen, W., Maguire, T. & Austin, F. (1993). Immunoreactivity and protective effects in mice of a recombinant dengue 2 Tonga virus NS1 protein produced in a baculovirus expression system. J Gen Virol 74, 89–97.
Reed, L. J. & Muench, H. (1938). A simple method of estimating fifty percent endpoints. Am J Hyg 27, 493–497.
Roehrig, J. T. (2003). Antigenic structure of flavivirus protein. Adv Virus Res 59, 141–175.[CrossRef][Medline]
Schlesinger, J. J., Foltzer, M. & Chapman, S. (1993). The Fc portion of antibody to yellow fever virus NS1 is a determinant of protection against YF encephalitis in mice. Virology 192, 132–141.[CrossRef][Medline]
Shresta, S., Kyle, J. L., Snider, H. M., Basavapatna, M., Beatty, P. R. & Harris, E. (2004). Interferon-dependent immunity is essential for resistance to primary dengue virus infection in mice, whereas T- and B-cell-dependent immunity are less critical. J Virol 78, 2701–2710.
Spaulding, A., Kurane, I., Ennis, F. A. & Rothman, A. L. (1999). Analysis of murine CD8+ T-cell clones specific for the dengue virus NS3 protein: flavivirus cross-reactivity and influence of infecting serotype. J Virol 73, 398–403.
van den Broek, M. F., Muller, U., Huang, S., Aguet, M. & Zinkernagel, R. M. (1995). Antiviral defense in mice lacking both alpha/beta and gamma interferon receptors. J Virol 69, 4792–4796.[Abstract]
van der Most, R. G., Murali-Krishna, K., Ahmed, R. & Strauss, J. H. (2000). Chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific CD8 T-cell response. J Virol 74, 8094–8101.
Zhang, Y.-M., Hayes, E. P., McCarty, T. C., Dubois, D. R., Summers, P. L., Eckels, K. H., Chanock, R. M. & Lai, C.-J. (1988). Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis. J Virol 62, 3027–3031.
Zhao, B. T., Prince, G., Horswood, R., Eckels, K., Summers, P., Chanock, R. & Lai, C.-J. (1987). Expression of dengue virus structural proteins and nonstructural protein NS1 by a recombinant vaccinia virus. J Virol 61, 4019–4022.
Received 10 June 2005;
accepted 6 November 2005.
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