Microbiol Immunol 2014; 58: 126–134 doi: 10.1111/1348-0421.12125
ORIGINAL ARTICLE
Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection-enhancing antibody response Fithriyah Sjatha1,2, Miwa Kuwahara3, T. Mirawati Sudiro2, Masanori Kameoka1,3 and Eiji Konishi1,4 1
Department of Vaccinology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, 2Department of Microbiology, Faculty of Medicine, University of Indonesia, 16 Jalan Pegangsaan Timur, Jakarta, Indonesia, 3Department of International Health, Kobe University Graduate School of Health Sciences, Kobe, Japan and 4BIKEN Endowed Department of Dengue Vaccine Development, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
ABSTRACT Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection-enhancing activities at sub-neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM-E) genebased expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM-E gene backbone showed high neutralizing antibody titers with less DENV infection-enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy. Key words
Antibody-dependent enhancement, chimera, dengue, vaccine.
Dengue virus, a member of the genus Flavivirus, is the causative agent of dengue fever and dengue hemorrhagic fever, an estimated 50 million infections occurring annually (1). Although DENV infection is a global public health concern, no approved vaccines or antiviral substances are currently available (2). Several research groups are currently trying to establish an effective DENV vaccine by using various strategies to induce antiDENV neutralizing antibodies, which mediate the most effective anti-DENV immune response (3–6). The pathogenic mechanism of DENV infection has not been fully elucidated. Difference in disease severity upon secondary exposure to homotypic (protection) and
heterotypic (deterioration) infection is the primary characteristic distinguishing dengue fever from dengue hemorrhagic fever (7). Antibody dependent enhancement (ADE) is the proposed mechanism for deterioration induced by secondary DENV infection; this is suggested by the observation that pre-existing neutralizing antibodies that have been induced by primary infection crossprotectively neutralize viruses during some post-infection periods only. In addition, the Fc portion of the antibody molecule may facilitate viral uptake to Fc receptorbearing cells, eventually enhancing viral propagation (8). Intriguingly, ADE activity has been detected in both polyclonal and monoclonal antibody states (9).
Correspondence Fithriyah Sjatha, Department of Vaccinology, Center for Infectious Diseases, Graduate School of Medicine, Kobe University, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel: þ81 78 382 5502; fax: þ81 78 382 5519; email: [email protected] Received 25 September 2013; revised 26 November 2013; accepted 10 December 2013. List of Abbreviations: ADE, antibody-dependent enhancement; DENV, dengue virus; DENV-1, DENV type-1; DI, domain I; DII, domain II; DIII, domain III; E, envelope; JEV, Japanese encephalitis virus; pcD1ME, pcDNA3-based expression plasmid DENV1 Mochizuki; pcJEME, pcDNA3-based expression plasmid for prM and E proteins of JEV Nakayama; prM, premembrane.
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Being the outer part of the virion, flavivirus E protein is highly exposed to the immune system (10). This protein is composed of three domains—DI, DII and DIII—each of which have specific locations and functions (11). DI contains predominantly type-specific, non-neutralizing epitopes on its eight-stranded b-barrel, whereas DII contains cross-reactive epitopes, which elicit both neutralizing and non-neutralizing antibody responses, and a highly conserved internal fusion peptide (12–13). DIII possesses an Ig-like structure and contains the major typespecific potent neutralizing epitopes (14–16). Although DIII is the main target of most neutralizing murine monoclonal antibodies during viral neutralization, antibodies against this domain are not significantly responsible for viral clearance and neutralization in human serum. The function of DIII and its correlation with neutralizing activity remain to be elucidated (17–19). Chimerization of two viruses has been widely employed for developing vaccines against flaviviruses (20–21). In this context, chimerization of DENV and JEV has been tested and found to show promise as a vaccine candidate (22–24). Sharing almost 50% amino acid similarity in their E proteins, both viruses show significant cross-reactivity in serological assays, with undefined clinical implications (25–26). Until now; however, there have been no epidemiological reports of antibodies induced by JEV infection or vaccination enhancing uptake of viruses from the same or other JEV serocomplexes such as DENV (27–29). Meanwhile, in immunized mice with a prM-E DNA-based vaccine, greater antigen expression and neutralizing antibody response were shown by JEV than by DENV (30). In this study, we constructed and evaluated a pcDNA3-based expression plasmid containing the DIII of DENV1 Mochizuki strain on the prM-E gene backbone of JEV Nakayama strain as a chimeric DNA vaccine, designated as pcJEDIME. Evidence for poor antigen expression and antibody induction in mice immunized with DENV1 Mochizuki prM-E DNA vaccine (30) led us to chimerize this virus with JEV. We found that the chimeric DNA vaccine induced potent neutralizing antibody responses with reduced ADE activity in vaccinated mouse serum, suggesting its application as a promising strategy for DENV vaccine development.
MATERIALS AND METHODS Viruses, cells and antibodies The Nakayama strain of JEV (30), Mochizuki strain of DENV1, NGC strain of DENV2, H87 strain of DENV3, and H241 strain of DENV4 (31) were propagated in C6/ 36 cells. Vero, K562 and CHO-K1 cells were maintained © 2013 The Societies and Wiley Publishing Asia Pty Ltd
as described previously (32–33). A mAb recognizing DIII of DENV1 Mochizuki, D1-I-15C12 (15C12, Yamanaka et al., 2013), and rabbit polyclonal anti-JEV and anti-DENV1 antibodies were prepared in our laboratory (31). In addition, the anti-flavivirus antigen antibody D1–4G2–4-15 (4G2) was obtained from the American Type Culture Collection (Manassas, VA, USA). Plasmids pcDNA3-based expression plasmids for prM and E proteins of JEV Nakayama (30) (pcJEME) and DENV1 Mochizuki (31) (pcD1ME) were used in this study. Chimeric genes with DIII substitutions of JEV and DENV1 prM-E genes were constructed by an overlapping PCR method using primers containing a silent mutation for the AflII restriction enzyme site; these constructs were then inserted into pcDNA3 (Fig. 1a).
Fig. 1. Schematic drawing of plasmid constructs showing the chimeric junction, and time course of antigen expression. (a) The conserved amino acid motif GHLKCRLKMDKL in the upstream region of domain III of JEV Nakayama (closed bar) and DENV1 Mochizuki (open bar) strains was chosen as the overlap region (shaded gray region) with introduction of an Af lII restriction site (underlined). Primers used are 91.67% and 77.78% identical to JEV Nakayama and DENV1 Mochizuki strains, respectively. (b) Precipitated post-transfected CHO-K1 supernatants were subjected to ELISA to measure extracellular prM-E antigen titers using polyclonal anti-JEV (RaJEV) and polyclonal anti-DENV1 (RaDENV1) as detected by flavivirus cross-reactive mAb 4G2. Positive absorbance was defined as absorbance higher than that obtained from precipitated supernatant of non-transfected cells during the same incubation period. Each data point represents mean and SDs (indicated by error bars) obtained from three independent experiments.
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Plasmids were confirmed by sequencing analysis and purified using a Qiagen plasmid purification kit (Qiagen, Hilden, Germany).
were adjusted based on absorbances of positive controls. Positive absorbances were defined as absorbances higher than the mean plus three times the SD of non-transfected precipitated supernatant absorbance.
Mouse experiments Groups of six 4-week-old male C3H/He mice (Japan SLC, Shizuoka, Japan) and ICR mice (CLEA Japan, Tokyo, Japan) were immunized three times at 4 week intervals with 100 mg of DNA plasmid using a springpowered needle-free jet injector (ShimaJET; Shimadzu, Kyoto, Japan). The first immunization was designated as week 0. Mice were retro-orbitally bled at 3-week intervals after the first immunization until week 12. Murine sera obtained from the same group of mice at each time interval were pooled and subjected to neutralization and ADE tests and IgG isotyping. All animal experiments were approved by the Institutional Animal Care and Use Committee of Kobe University (Permission number A120504) and conducted according to Kobe University Animal Experimentation regulations. Neutralization testing Neutralizing activity of immunized murine serum against JEV and DENV1 was evaluated by a neutralization test using Vero cells, as previously described (31). Plaques were visualized by immunostaining as previously described (32). Neutralizing antibody titers are expressed as the maximum serum dilution yielding 90% and 70% reductions in plaque formation by JEV and DENV1, respectively. Assay for the balance between neutralizing and enhancing activities Normalized sera (described below) derived from each group of mice were tested to evaluate the balance between neutralizing and enhancing activities of immunized serum against DENV1–4 using K562 cells, as previously described (34). Evaluation of extracellular antigen expression CHO-K1 cells were transfected with 1 mg of plasmid DNA using lipofectamine-plus reagent (Invitrogen, Carlsbad, CA, USA). Sample preparation and ELISA were conducted as in a previous study (35), except for the use of rabbit polyclonal anti-JEV or anti-DENV1 as the capture antibody and flavivirus cross-reactive mAb 4G2 as the detector. To minimize inter-plate variation, JEV or DENV1 viral antigen was included as a constant positive control during the assay. Measured sample absorbances 128
ELISA for measuring antibody isotype titers An ELISA to measure the amount of each IgG subclass was carried out essentially as described previously (35), except for an initial 1:100 serial murine serum dilution and the use of alkaline phosphatase conjugated goat anti-mouse IgG1/IgG2a/IgG2b/IgG3 as a secondary antibody. Experiments were performed in duplicate. Antibody titers are expressed as the maximum serum dilution showing an absorbance value greater than absorbance values obtained with normal murine serum. ELISA for normalization of anti-DENV1 antibody titer among serum samples An ELISA to standardize titers of anti-DENV1 Mochizuki antibody among groups of sera was performed essentially as previously described (35), except for the use of rabbit polyclonal anti-DENV1 as a capture antigen, DENV1 Mochizuki as an antigen, and an initial 1:100 serially diluted murine serum. For each group, diluted serum giving similar absorbance values was used as the starting serum dilution for the ADE test (see the Section headed ‘‘Assay for the balance between neutralizing and enhancing activities above).
RESULTS Neutralizing antibody responses in immunized mice Prior to immunization, extracellular antigen expression was confirmed by transfecting CHO-K1 cells with each vaccine construct (Fig. 1b). As detected by flavivirus cross-reactive mAb 4G2 (36), pcJEME consistently produced the highest extracellular antigen titers in both ELISA systems, followed by pcJED1ME, pcD1JEME and pcD1ME. Replacement of DIII in pcJEME with DIII of DENV1, generating chimeric pcJED1ME, increased extracellular antigen expression compared with the original DENV1 construct (pcD1ME). To evaluate the ability of chimeric JEV and DENV1 prM-E genes to reproducibly induce neutralizing antibody responses, C3H/He and ICR mice were immunized with each vaccine construct. In order to test reproducibility, both C3H/He and ICR mice were
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Chimeric dengue-JEV DNA vaccine
Balance of neutralizing and enhancing antibodies induced in serum of immunized mice Because the Vero cell-based neutralization test only enables examination of neutralizing activity, enhancing activity was evaluated using an FcgR-bearing, semiadherent K562 cell-based neutralization/ADE test. Because each group of vaccinated mice induced different anti-DENV1 antibody titers, antibody titers of pooled serum collected from all groups were normalized by ELISA using DENV1 Mochizuki as an antigen. Serum dilutions giving equivalent absorbances in the ELISA system were used as initial dilutions for the K562 cellbased neutralization/ADE test (Fig. 3). Serum of pcJEME-immunized mice showed no neutralizing or enhancing activity against DENV1 Mochizuki at any
Fig. 2. Time course of neutralizing activities against JEV Nakayama and DENV1 Mochizuki strains induced in immunized murine serum. (a) Sera of C3H/He and (b) ICR mice immunized with pcJEME, pcD1ME, pcJED1ME and pcD1JEME were studied for their neutralizing activities on replication of JEV and DENV1 strains in Vero cells.
used to confirm that different mice breeds did not cause discrepancies in the results. Neutralizing antibody titers for DENV1 Mochizuki and JEV Nakayama strains were examined using a Vero cell-based neutralization test. Results obtained using C3H/He and ICR mice are shown in Figure 2a and 2b, respectively. In general, neutralizing antibody titers increased over time, different titers being observed in different groups of mice. The original vaccine construct only triggered a neutralizing antibody response against its viral antigen; interestingly, however, both chimeric pcJED1ME and pcD1JEME induced neutralizing antibody responses against both viral antigens. The strongest neutralizing antibody response against DENV1 was induced by chimeric pcJED1ME, followed by pcD1ME, pcD1JEME and pcJEME. In addition, the strongest neutralizing antibody response against JEV was induced by pcJEME, followed by chimeric pcJED1ME, pcD1JEME and pcD1ME. Neutralizing antibody titers were positively correlated with the titers of viral antigens expressed by DNA vaccine constructs (Figs. 1b and 2). © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Fig. 3. Time course of neutralizing and enhancing activities against DENV1 Mochizuki strain induced in immunized murine serum. Sera from mice immunized with pcJEME, pcD1ME, pcJED1ME and pcD1JEME were studied. The same mice sera collected and used for the neutralization test were also used in this assay (a) C3H/He; (b) ICR. The experiment was performed in the absence or presence of complement. Dotted lines indicate cut-off values for differentiating enhancing/neutralizing activities from non-enhancing/neutralizing activities (average 2SD obtained with eight negative controls lacking antibodies). Each data point represents the mean of two individual experiments and SDs (indicated by bars). C(), absence of complement; C(þ), presence of complement.
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serum dilution. In contrast, sera from all other groups of mice displayed both neutralizing and enhancing activity; that is, neutralizing activity was detected at low (101and 102-fold) dilutions, followed slowly by a switch to enhancing activity at intermediate (103- and 104-fold) dilutions and then a further switch to neither neutralizing nor enhancing activity at high (105-fold) dilutions. However, in the presence of complement in the K562 cell-based neutralization/ADE test, enhancing activity observed at intermediate serum dilutions was generally replaced by neutralizing activity. The highest neutralizing activity was induced by pcJED1ME, followed by pcD1ME and pcD1JEME. These results are consistent with those obtained in the Vero cell-based neutralizing test. Interestingly, almost no enhancing activity was detected in serum of pcJED1MEimmunized mice in the presence of complement, indicating that substituting DI and DII of the E protein is an effective strategy for reducing DENV1 infectionenhancing activity in immunized serum. However, the pcD1JEME group, which showed the lowest neutralizing activity in both Vero and K562 tests, showed the highest enhancing activity among all groups. This enhancing activity was not fully abolished even in the presence of complement, indicating that DI and DII of the DENV1 E protein mainly induce enhancing, rather than neutralizing, antibody responses. Balance of neutralizing and enhancing activity of immunized mice sera against heterologous viral serotypes Enhancing activity of induced antibody not only correlates with insufficient antibody to fully neutralize viral antigen but also with the condition of heterologous viral infection. Thus, neutralizing and enhancing activities of immunized mice sera were also tested against heterologous DENV serotypes in the presence or absence of complement; the results are shown in Figure 4. The same sera collection and conditions as for the neutralizing-enhancing antibody activity assay against DENV1 (Fig. 3) were used in this test. Sera from chimeric pcJED1ME mice did not show any enhancing activity against heterologous DENV2, DENV3 or DENV4 viral infection and showed neutralizing activity in high serum concentrations (101-fold) only in sera collected 12 weeks after the first immunization. Similar results were also found with pcJEMEimmunized mice sera. In contrast, sera from mice immunized with pcD1ME or chimeric pcD1JEME showed neutralizing activity in high concentrations of sera (101-fold) and gradually shifted into enhancing activity as the sera dilution increased (102- to 103130
fold). No activity was detected in the higher sera dilutions (104- to 108-fold), even in the sera collected 12 weeks after the first immunization. As also shown by homologous assays (Fig. 3), enhancing antibody activity was reduced when complement was added to the assay system. IgG isotype profiling in immunized mice Because each isotype has a different binding affinity to complement (37), there could be a correlation between antibody activities and IgG isotypes induced in immunized mice. To examine this possibility, IgG isotypes in immunized serum were profiled as shown in Figure 5. IgG2 was the main subclass induced in immunized mice, followed by IgG1 and IgG3. The highest antibody titers against JEV were induced by pcJEME, followed by pcJED1ME, pcD1JEME and pcD1ME. In contrast, the highest antibody titers against DENV1 were induced by pcJED1ME, followed by pcD1ME, pcD1JEME and pcJEME.
DISCUSSION Dengue virus consists of four distinct serotypes. Because neutralizing antibody response against one serotype does not give full protection against the others, development of a vaccine against DENV is more challenging than for other flaviviruses. An effective DENV vaccine would induce a potent neutralizing antibody response that is well balanced and simultaneously active against all four DENV serotypes; establishment of a vaccine strategy that provides the required amounts of antigens among the four serotypes is thus needed. Unlike conventional vaccine approaches, the use of DNA encoding a viral antigen in an expression plasmid provides a new approach to DENV vaccine development that features minimal immune bias and continuous and stable production of antigen to induce antiviral immune responses. Our previous studies have demonstrated the ability of DENV and JEV DNA-based vaccines to induce neutralizing antibodies in a mouse model system (31–33). In this study, we successfully constructed chimeric JEV and DENV prM-E genes, in which we replaced the DIII of JEV with that of DENV, in a prM-E expression plasmid with no lag or overlap nucleotides. The viral chimerization technique has been widely employed in vaccine development; chimerization between DENV and JEV has itself been used to generate chimeric progeny with unique characteristics derived from parental viruses (23–25). Because the chimerization technique can potentially generate viral progeny or chimeric antigens that possess © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Chimeric dengue-JEV DNA vaccine
Fig. 4. Neutralizing and enhancing activities of immunize murine serum against heterologous dengue serotypes. Sera from immunized mice of pcJEME, pcD1ME, pcJED1ME and pcD1JEME were studied. The same mice sera collected and used for neutralization and enhancingneutralizing antibody balance tests against DENV1 were used in this assay. (a) C3H/He mice sera against DENV2; (b) ICR mice sera against DENV2; (c) C3H/HE mice sera against DENV3; (d) ICR mice sera against DENV3; (e) C3H/HE mice sera against DENV4; and (f) ICR mice sera against DENV4. The experiment was performed in the absence or presence of complement in duplicate. Dotted lines indicate cut-off values for differentiating enhancing/neutralizing activities from non-enhancing/neutralizing activities (average 2SD obtained with eight negative controls lacking antibodies). C(), absence of complement; C(þ), presence of complement.
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Fig. 5. ELISA titer of IgG isotypes in immunized murine serum. The same mice sera collected and used for neutralization and ADE tests were also used in this assay (a) C3H/He; (b) ICR, tested against JEV Nakayama and DENV1 Mochizuki viruses. A positive titer was defined as the maximum serum dilution showing an absorbance above that obtained with non-immunized serum.
characteristics of both parental viruses, improvements with respect to these characteristics are expected in viral or antigenic progeny. Results are variable for one such characteristic, expression yield: some chimeras have lower yields than parental viruses (22, 24, 38), whereas others have similar or higher yields (39). Our chimeric plasmid induces greater expression than occurs with the original DENV1 construct. The highest stably expressed extracellular protein titers have been obtained from a chimeric DNA vaccine containing a JEV-derived prM signal sequence, which is known to induce significantly increased E protein expression (23). This strong antigen production in turn positively affects antibody induction titers in mice, thus fulfilling the stoichiometric antibody requirement for viral neutralization (40). In addition, the construct may be further improved (41–44) and used as part of a new strategy to potentially strengthen antigen expression. Epitope mapping of the E protein with mouse mAbs or human immune serum has demonstrated that the 132
antibody against DIII has strong neutralization capability with type-specific activity, whereas antibodies against DI and DII have lower neutralization potency but are broadly cross-reactive against various DENV serotypes (44–48). With respect to the E protein-specific response, the proportion of DI/DII to DIII antibodies showed considerable variability. Our strategy of substituting DIII in the JEV prM-E gene backbone with that of DENV generated an interesting antibody profile, less enhancing activity being detected in immunized mouse serum with the chimeric construct. Antibody induction by DIII itself (chimeric pcJED1ME) was strongly neutralizing against DENV1, with less enhancing activity. In addition, incorporation of prM and DI/DII (original pcD1ME) provided moderate neutralizing and enhancing activity, whereas elimination of DIII (chimeric pcD1JEME) resulted in the lowest neutralizing activity and strongest enhancing activity against DENV1. These results demonstrate the importance of DIII in inducing neutralizing activity against DENV1 while simultaneously reducing enhancing activity. Consequently, incorporation of JEV prM, DI and DII indeed induced detectable neutralizing antibodies against JEV but, as shown in previous studies, they were not cross-reactive against DENV1 (28–29). Secondary exposure to a heterologous viral serotype can result in more severe disease because all dengue viral serotypes can be co-circulated and pre-existing nonneutralizing antibody against one serotype is unable to fully abolish heterologous viral infection, but rather enhances viral uptake through the ADE mechanism. An effective dengue vaccine would address the possibility of ADE activity from induced antibody. One characteristic of antibody responses against DIII of E proteins is that they are serotype-specific, which makes them less crossreactive in neutralizing or enhancing viral infection; therefore, the DIII of E protein may have an advantage as a vaccine antigen (49–50). As also shown in previous studies, dengue vaccines using DIII provide high serotype specificity with less cross-neutralizing capacity against heterologous viral serotypes (51–53). With almost no detectable enhancing activity, as shown in this study, they may fulfill the requirement for reduced antibody-enhancing activity. Not only antigen, but also the isotypic profile of antibodies regarding their affinity to complement, affect antibody responses in mouse sera. Because mouse IgG2 has stronger binding affinity to complement than IgG1, when complement is present in the assay system, the enhancing activity of IgG2 isotype may switch to neutralizing activity. It is generally known that genebased vaccines tend to induce Th1 immune responses, which induce IgG2a isotype, whereas protein-based © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Chimeric dengue-JEV DNA vaccine
vaccines tend to induce Th2 immune responses and induce IgG1 isotype. Thus, the type of vaccine and immunization route could be an alternative approach to developing an effective dengue vaccine. Finally, Crill et al. observed a correlation between antibody titers against DIII and neutralization potency of immunized serum samples (54). In contrast, other researchers have found only small amounts of antibody against DIII in human serum samples (18–19, 55), and depleting it with recombinant DIII protein does not significantly reduce the neutralizing activity of serum samples. Further studies are needed to explain these contradictory findings. Nevertheless, our present study illustrates the advantages of a chimeric DNA vaccine consisting of DENV DIII on a JEV prM-E gene backbone for increasing antigen expression and inducing strong neutralizing antibody responses against DENV with reduced ADE activity.
ACKNOWLEDGMENTS This study was supported by the Science and Technology Research Partnership for Sustainable Development (SATREPS) of the Japan Science and Technology Agency/Japan International Cooperation Agency (JST/ JICA). This work is part of PhD thesis by Fithriyah Sjatha.
DISCLOSURE The authors have no conflict of interests to declare.
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24. Chambers T.J., Jiang X., Droll D.A., Liang Y., Wold W.S.M., Nickels J. (2006) Chimeric Japanese encephalitis virus/dengue 2 virus infectious clone: biological properties, immunogenicity and protection against dengue encephalistis in mice. J Virol 87: 3131–40. 25. Heinz F.X., Stiasny K. (2012) Flavivirus and their antigenic structure. J Clin Virol 55: 289–95. 26. Mansfield K.L., Horton D.L., Johnson N., Li L., Barrett A.D.T., Smith D.J., Galbraith S.E., Solomon T., Fooks A.R. (2011) Flavivirus-induced antibody cross-reactivity. J Virol 92: 2821–29. 27. Lobigs M., Diamond M.S. (2011) Feasibility of cross-protective vaccination against flaviviruses of the Japanese encephalitis serocomplex. Expert Rev Vaccines 11: 177–87. 28. Putvana S., Yokasan S., Chayyodin T., Bhamarapravati N., Halstead S.B. (1984) Absence of dengue 2 infection enhancement in human sera containing Japanese encephalitis antibodies. Am J Trop Med Hyg 33: 288–94. 29. Anderson K.B., Gibbons R.V., Thomas R.J., Rothman A.L., Nisalak A., Berkelman R.L., Libraty D.H., Endy T.P. (2011) Preexisting Japanese encephalitis virus neutralizing antibodies and increased symptomatic dengue illness in a school-based cohort in Thailand. PLoS Negl Trop Dis 5: e1311. 30. Konishi E., Terazawa A., Fujii A. (2003) Evidence for antigen production in muscles by dengue and Japanese encephalitis DNA vaccines and relation to their immunogenicity in mice. Vaccine 21: 3713–20. 31. Konishi E., Kosugi S., Imoto J. (2006) Dengue tetravalent DNA vaccine inducing neutralizing antibody and anamnestic responses to four serotypes in mice. Vaccine 24: 2200–7. 32. Konishi E., Fujii A. (2002) Dengue type 2 virus subviral extracellullar particles produced by a stably transfected mammalian cell line and their evaluation for a subunit vaccine. Vaccine 20: 1058–67. 33. Yamanaka A., Kosugi S., Konishi E. (2008) Infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type 2 and 4 viruses are controlled by complement levels. J Virol 82: 927–37. 34. Konishi E., Tabuchi Y., Yamanaka A. (2010) A simple assay system for infection-enhancing and -neutralizing antibodies to dengue type 2 virus using layers of semi-adherent K562 cells. J Virol Methods 163: 360–7. 35. Kuwahara M., Konishi E. (2010) Evaluation of extracellular subviral particles of dengue virus type 2 and Japanese encephalitis virus produced by Spodoptera frugiperda cells for use as vaccine and diagnostic antigens. Clin Vaccine Immunol 17: 1560–6. 36. Serafin I.L., Aaskov J.G. (2001) Identification of epitopes on the envelope (E) protein dengue 2 and dengue 3 viruses using monoclonal antibodies. Arch Virol 146: 2469–79. 37. Mehlhop E., Ansarah-Robinho C., Jhonson S., Engle M., Freemont D.H., Pierson T.C., Diamond M.S. (2007) Complement protein C1q inhibits antibody-dependent enhancement of flavivirus infection in an IgG subclass-specific manner. Cell Host Microbes 2: 417–26. 38. Tang W.F., Eshita Y., Tadano M., Morita K., Makino Y. (2005) Molecular basis for adaptation of a chimeric dengue type-4/Japanese encephalitis virus to Vero cells. Microb Immunol 49: 2019–27. 39. Caufour P.S., Motta M.C.A., Yamamura A.M., Vasquez 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. 40. Dowd K.A., Pierson T.C. (2011) Antibody-mediated neutralization of flaviviruses: a reductionist view. Virology 411: 306–11. 41. Hsieh S.C., Liu I.J., King C.C., Chang G.J., Wang W.K. (2008) A strong endoplasmic reticulum retention signal in the stem-anchor
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ORIGINAL ARTICLE
Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection-enhancing antibody response Fithriyah Sjatha1,2, Miwa Kuwahara3, T. Mirawati Sudiro2, Masanori Kameoka1,3 and Eiji Konishi1,4 1
Department of Vaccinology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, 2Department of Microbiology, Faculty of Medicine, University of Indonesia, 16 Jalan Pegangsaan Timur, Jakarta, Indonesia, 3Department of International Health, Kobe University Graduate School of Health Sciences, Kobe, Japan and 4BIKEN Endowed Department of Dengue Vaccine Development, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
ABSTRACT Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection-enhancing activities at sub-neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM-E) genebased expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM-E gene backbone showed high neutralizing antibody titers with less DENV infection-enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy. Key words
Antibody-dependent enhancement, chimera, dengue, vaccine.
Dengue virus, a member of the genus Flavivirus, is the causative agent of dengue fever and dengue hemorrhagic fever, an estimated 50 million infections occurring annually (1). Although DENV infection is a global public health concern, no approved vaccines or antiviral substances are currently available (2). Several research groups are currently trying to establish an effective DENV vaccine by using various strategies to induce antiDENV neutralizing antibodies, which mediate the most effective anti-DENV immune response (3–6). The pathogenic mechanism of DENV infection has not been fully elucidated. Difference in disease severity upon secondary exposure to homotypic (protection) and
heterotypic (deterioration) infection is the primary characteristic distinguishing dengue fever from dengue hemorrhagic fever (7). Antibody dependent enhancement (ADE) is the proposed mechanism for deterioration induced by secondary DENV infection; this is suggested by the observation that pre-existing neutralizing antibodies that have been induced by primary infection crossprotectively neutralize viruses during some post-infection periods only. In addition, the Fc portion of the antibody molecule may facilitate viral uptake to Fc receptorbearing cells, eventually enhancing viral propagation (8). Intriguingly, ADE activity has been detected in both polyclonal and monoclonal antibody states (9).
Correspondence Fithriyah Sjatha, Department of Vaccinology, Center for Infectious Diseases, Graduate School of Medicine, Kobe University, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel: þ81 78 382 5502; fax: þ81 78 382 5519; email: [email protected] Received 25 September 2013; revised 26 November 2013; accepted 10 December 2013. List of Abbreviations: ADE, antibody-dependent enhancement; DENV, dengue virus; DENV-1, DENV type-1; DI, domain I; DII, domain II; DIII, domain III; E, envelope; JEV, Japanese encephalitis virus; pcD1ME, pcDNA3-based expression plasmid DENV1 Mochizuki; pcJEME, pcDNA3-based expression plasmid for prM and E proteins of JEV Nakayama; prM, premembrane.
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Being the outer part of the virion, flavivirus E protein is highly exposed to the immune system (10). This protein is composed of three domains—DI, DII and DIII—each of which have specific locations and functions (11). DI contains predominantly type-specific, non-neutralizing epitopes on its eight-stranded b-barrel, whereas DII contains cross-reactive epitopes, which elicit both neutralizing and non-neutralizing antibody responses, and a highly conserved internal fusion peptide (12–13). DIII possesses an Ig-like structure and contains the major typespecific potent neutralizing epitopes (14–16). Although DIII is the main target of most neutralizing murine monoclonal antibodies during viral neutralization, antibodies against this domain are not significantly responsible for viral clearance and neutralization in human serum. The function of DIII and its correlation with neutralizing activity remain to be elucidated (17–19). Chimerization of two viruses has been widely employed for developing vaccines against flaviviruses (20–21). In this context, chimerization of DENV and JEV has been tested and found to show promise as a vaccine candidate (22–24). Sharing almost 50% amino acid similarity in their E proteins, both viruses show significant cross-reactivity in serological assays, with undefined clinical implications (25–26). Until now; however, there have been no epidemiological reports of antibodies induced by JEV infection or vaccination enhancing uptake of viruses from the same or other JEV serocomplexes such as DENV (27–29). Meanwhile, in immunized mice with a prM-E DNA-based vaccine, greater antigen expression and neutralizing antibody response were shown by JEV than by DENV (30). In this study, we constructed and evaluated a pcDNA3-based expression plasmid containing the DIII of DENV1 Mochizuki strain on the prM-E gene backbone of JEV Nakayama strain as a chimeric DNA vaccine, designated as pcJEDIME. Evidence for poor antigen expression and antibody induction in mice immunized with DENV1 Mochizuki prM-E DNA vaccine (30) led us to chimerize this virus with JEV. We found that the chimeric DNA vaccine induced potent neutralizing antibody responses with reduced ADE activity in vaccinated mouse serum, suggesting its application as a promising strategy for DENV vaccine development.
MATERIALS AND METHODS Viruses, cells and antibodies The Nakayama strain of JEV (30), Mochizuki strain of DENV1, NGC strain of DENV2, H87 strain of DENV3, and H241 strain of DENV4 (31) were propagated in C6/ 36 cells. Vero, K562 and CHO-K1 cells were maintained © 2013 The Societies and Wiley Publishing Asia Pty Ltd
as described previously (32–33). A mAb recognizing DIII of DENV1 Mochizuki, D1-I-15C12 (15C12, Yamanaka et al., 2013), and rabbit polyclonal anti-JEV and anti-DENV1 antibodies were prepared in our laboratory (31). In addition, the anti-flavivirus antigen antibody D1–4G2–4-15 (4G2) was obtained from the American Type Culture Collection (Manassas, VA, USA). Plasmids pcDNA3-based expression plasmids for prM and E proteins of JEV Nakayama (30) (pcJEME) and DENV1 Mochizuki (31) (pcD1ME) were used in this study. Chimeric genes with DIII substitutions of JEV and DENV1 prM-E genes were constructed by an overlapping PCR method using primers containing a silent mutation for the AflII restriction enzyme site; these constructs were then inserted into pcDNA3 (Fig. 1a).
Fig. 1. Schematic drawing of plasmid constructs showing the chimeric junction, and time course of antigen expression. (a) The conserved amino acid motif GHLKCRLKMDKL in the upstream region of domain III of JEV Nakayama (closed bar) and DENV1 Mochizuki (open bar) strains was chosen as the overlap region (shaded gray region) with introduction of an Af lII restriction site (underlined). Primers used are 91.67% and 77.78% identical to JEV Nakayama and DENV1 Mochizuki strains, respectively. (b) Precipitated post-transfected CHO-K1 supernatants were subjected to ELISA to measure extracellular prM-E antigen titers using polyclonal anti-JEV (RaJEV) and polyclonal anti-DENV1 (RaDENV1) as detected by flavivirus cross-reactive mAb 4G2. Positive absorbance was defined as absorbance higher than that obtained from precipitated supernatant of non-transfected cells during the same incubation period. Each data point represents mean and SDs (indicated by error bars) obtained from three independent experiments.
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Plasmids were confirmed by sequencing analysis and purified using a Qiagen plasmid purification kit (Qiagen, Hilden, Germany).
were adjusted based on absorbances of positive controls. Positive absorbances were defined as absorbances higher than the mean plus three times the SD of non-transfected precipitated supernatant absorbance.
Mouse experiments Groups of six 4-week-old male C3H/He mice (Japan SLC, Shizuoka, Japan) and ICR mice (CLEA Japan, Tokyo, Japan) were immunized three times at 4 week intervals with 100 mg of DNA plasmid using a springpowered needle-free jet injector (ShimaJET; Shimadzu, Kyoto, Japan). The first immunization was designated as week 0. Mice were retro-orbitally bled at 3-week intervals after the first immunization until week 12. Murine sera obtained from the same group of mice at each time interval were pooled and subjected to neutralization and ADE tests and IgG isotyping. All animal experiments were approved by the Institutional Animal Care and Use Committee of Kobe University (Permission number A120504) and conducted according to Kobe University Animal Experimentation regulations. Neutralization testing Neutralizing activity of immunized murine serum against JEV and DENV1 was evaluated by a neutralization test using Vero cells, as previously described (31). Plaques were visualized by immunostaining as previously described (32). Neutralizing antibody titers are expressed as the maximum serum dilution yielding 90% and 70% reductions in plaque formation by JEV and DENV1, respectively. Assay for the balance between neutralizing and enhancing activities Normalized sera (described below) derived from each group of mice were tested to evaluate the balance between neutralizing and enhancing activities of immunized serum against DENV1–4 using K562 cells, as previously described (34). Evaluation of extracellular antigen expression CHO-K1 cells were transfected with 1 mg of plasmid DNA using lipofectamine-plus reagent (Invitrogen, Carlsbad, CA, USA). Sample preparation and ELISA were conducted as in a previous study (35), except for the use of rabbit polyclonal anti-JEV or anti-DENV1 as the capture antibody and flavivirus cross-reactive mAb 4G2 as the detector. To minimize inter-plate variation, JEV or DENV1 viral antigen was included as a constant positive control during the assay. Measured sample absorbances 128
ELISA for measuring antibody isotype titers An ELISA to measure the amount of each IgG subclass was carried out essentially as described previously (35), except for an initial 1:100 serial murine serum dilution and the use of alkaline phosphatase conjugated goat anti-mouse IgG1/IgG2a/IgG2b/IgG3 as a secondary antibody. Experiments were performed in duplicate. Antibody titers are expressed as the maximum serum dilution showing an absorbance value greater than absorbance values obtained with normal murine serum. ELISA for normalization of anti-DENV1 antibody titer among serum samples An ELISA to standardize titers of anti-DENV1 Mochizuki antibody among groups of sera was performed essentially as previously described (35), except for the use of rabbit polyclonal anti-DENV1 as a capture antigen, DENV1 Mochizuki as an antigen, and an initial 1:100 serially diluted murine serum. For each group, diluted serum giving similar absorbance values was used as the starting serum dilution for the ADE test (see the Section headed ‘‘Assay for the balance between neutralizing and enhancing activities above).
RESULTS Neutralizing antibody responses in immunized mice Prior to immunization, extracellular antigen expression was confirmed by transfecting CHO-K1 cells with each vaccine construct (Fig. 1b). As detected by flavivirus cross-reactive mAb 4G2 (36), pcJEME consistently produced the highest extracellular antigen titers in both ELISA systems, followed by pcJED1ME, pcD1JEME and pcD1ME. Replacement of DIII in pcJEME with DIII of DENV1, generating chimeric pcJED1ME, increased extracellular antigen expression compared with the original DENV1 construct (pcD1ME). To evaluate the ability of chimeric JEV and DENV1 prM-E genes to reproducibly induce neutralizing antibody responses, C3H/He and ICR mice were immunized with each vaccine construct. In order to test reproducibility, both C3H/He and ICR mice were
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Balance of neutralizing and enhancing antibodies induced in serum of immunized mice Because the Vero cell-based neutralization test only enables examination of neutralizing activity, enhancing activity was evaluated using an FcgR-bearing, semiadherent K562 cell-based neutralization/ADE test. Because each group of vaccinated mice induced different anti-DENV1 antibody titers, antibody titers of pooled serum collected from all groups were normalized by ELISA using DENV1 Mochizuki as an antigen. Serum dilutions giving equivalent absorbances in the ELISA system were used as initial dilutions for the K562 cellbased neutralization/ADE test (Fig. 3). Serum of pcJEME-immunized mice showed no neutralizing or enhancing activity against DENV1 Mochizuki at any
Fig. 2. Time course of neutralizing activities against JEV Nakayama and DENV1 Mochizuki strains induced in immunized murine serum. (a) Sera of C3H/He and (b) ICR mice immunized with pcJEME, pcD1ME, pcJED1ME and pcD1JEME were studied for their neutralizing activities on replication of JEV and DENV1 strains in Vero cells.
used to confirm that different mice breeds did not cause discrepancies in the results. Neutralizing antibody titers for DENV1 Mochizuki and JEV Nakayama strains were examined using a Vero cell-based neutralization test. Results obtained using C3H/He and ICR mice are shown in Figure 2a and 2b, respectively. In general, neutralizing antibody titers increased over time, different titers being observed in different groups of mice. The original vaccine construct only triggered a neutralizing antibody response against its viral antigen; interestingly, however, both chimeric pcJED1ME and pcD1JEME induced neutralizing antibody responses against both viral antigens. The strongest neutralizing antibody response against DENV1 was induced by chimeric pcJED1ME, followed by pcD1ME, pcD1JEME and pcJEME. In addition, the strongest neutralizing antibody response against JEV was induced by pcJEME, followed by chimeric pcJED1ME, pcD1JEME and pcD1ME. Neutralizing antibody titers were positively correlated with the titers of viral antigens expressed by DNA vaccine constructs (Figs. 1b and 2). © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Fig. 3. Time course of neutralizing and enhancing activities against DENV1 Mochizuki strain induced in immunized murine serum. Sera from mice immunized with pcJEME, pcD1ME, pcJED1ME and pcD1JEME were studied. The same mice sera collected and used for the neutralization test were also used in this assay (a) C3H/He; (b) ICR. The experiment was performed in the absence or presence of complement. Dotted lines indicate cut-off values for differentiating enhancing/neutralizing activities from non-enhancing/neutralizing activities (average 2SD obtained with eight negative controls lacking antibodies). Each data point represents the mean of two individual experiments and SDs (indicated by bars). C(), absence of complement; C(þ), presence of complement.
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serum dilution. In contrast, sera from all other groups of mice displayed both neutralizing and enhancing activity; that is, neutralizing activity was detected at low (101and 102-fold) dilutions, followed slowly by a switch to enhancing activity at intermediate (103- and 104-fold) dilutions and then a further switch to neither neutralizing nor enhancing activity at high (105-fold) dilutions. However, in the presence of complement in the K562 cell-based neutralization/ADE test, enhancing activity observed at intermediate serum dilutions was generally replaced by neutralizing activity. The highest neutralizing activity was induced by pcJED1ME, followed by pcD1ME and pcD1JEME. These results are consistent with those obtained in the Vero cell-based neutralizing test. Interestingly, almost no enhancing activity was detected in serum of pcJED1MEimmunized mice in the presence of complement, indicating that substituting DI and DII of the E protein is an effective strategy for reducing DENV1 infectionenhancing activity in immunized serum. However, the pcD1JEME group, which showed the lowest neutralizing activity in both Vero and K562 tests, showed the highest enhancing activity among all groups. This enhancing activity was not fully abolished even in the presence of complement, indicating that DI and DII of the DENV1 E protein mainly induce enhancing, rather than neutralizing, antibody responses. Balance of neutralizing and enhancing activity of immunized mice sera against heterologous viral serotypes Enhancing activity of induced antibody not only correlates with insufficient antibody to fully neutralize viral antigen but also with the condition of heterologous viral infection. Thus, neutralizing and enhancing activities of immunized mice sera were also tested against heterologous DENV serotypes in the presence or absence of complement; the results are shown in Figure 4. The same sera collection and conditions as for the neutralizing-enhancing antibody activity assay against DENV1 (Fig. 3) were used in this test. Sera from chimeric pcJED1ME mice did not show any enhancing activity against heterologous DENV2, DENV3 or DENV4 viral infection and showed neutralizing activity in high serum concentrations (101-fold) only in sera collected 12 weeks after the first immunization. Similar results were also found with pcJEMEimmunized mice sera. In contrast, sera from mice immunized with pcD1ME or chimeric pcD1JEME showed neutralizing activity in high concentrations of sera (101-fold) and gradually shifted into enhancing activity as the sera dilution increased (102- to 103130
fold). No activity was detected in the higher sera dilutions (104- to 108-fold), even in the sera collected 12 weeks after the first immunization. As also shown by homologous assays (Fig. 3), enhancing antibody activity was reduced when complement was added to the assay system. IgG isotype profiling in immunized mice Because each isotype has a different binding affinity to complement (37), there could be a correlation between antibody activities and IgG isotypes induced in immunized mice. To examine this possibility, IgG isotypes in immunized serum were profiled as shown in Figure 5. IgG2 was the main subclass induced in immunized mice, followed by IgG1 and IgG3. The highest antibody titers against JEV were induced by pcJEME, followed by pcJED1ME, pcD1JEME and pcD1ME. In contrast, the highest antibody titers against DENV1 were induced by pcJED1ME, followed by pcD1ME, pcD1JEME and pcJEME.
DISCUSSION Dengue virus consists of four distinct serotypes. Because neutralizing antibody response against one serotype does not give full protection against the others, development of a vaccine against DENV is more challenging than for other flaviviruses. An effective DENV vaccine would induce a potent neutralizing antibody response that is well balanced and simultaneously active against all four DENV serotypes; establishment of a vaccine strategy that provides the required amounts of antigens among the four serotypes is thus needed. Unlike conventional vaccine approaches, the use of DNA encoding a viral antigen in an expression plasmid provides a new approach to DENV vaccine development that features minimal immune bias and continuous and stable production of antigen to induce antiviral immune responses. Our previous studies have demonstrated the ability of DENV and JEV DNA-based vaccines to induce neutralizing antibodies in a mouse model system (31–33). In this study, we successfully constructed chimeric JEV and DENV prM-E genes, in which we replaced the DIII of JEV with that of DENV, in a prM-E expression plasmid with no lag or overlap nucleotides. The viral chimerization technique has been widely employed in vaccine development; chimerization between DENV and JEV has itself been used to generate chimeric progeny with unique characteristics derived from parental viruses (23–25). Because the chimerization technique can potentially generate viral progeny or chimeric antigens that possess © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Chimeric dengue-JEV DNA vaccine
Fig. 4. Neutralizing and enhancing activities of immunize murine serum against heterologous dengue serotypes. Sera from immunized mice of pcJEME, pcD1ME, pcJED1ME and pcD1JEME were studied. The same mice sera collected and used for neutralization and enhancingneutralizing antibody balance tests against DENV1 were used in this assay. (a) C3H/He mice sera against DENV2; (b) ICR mice sera against DENV2; (c) C3H/HE mice sera against DENV3; (d) ICR mice sera against DENV3; (e) C3H/HE mice sera against DENV4; and (f) ICR mice sera against DENV4. The experiment was performed in the absence or presence of complement in duplicate. Dotted lines indicate cut-off values for differentiating enhancing/neutralizing activities from non-enhancing/neutralizing activities (average 2SD obtained with eight negative controls lacking antibodies). C(), absence of complement; C(þ), presence of complement.
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Fig. 5. ELISA titer of IgG isotypes in immunized murine serum. The same mice sera collected and used for neutralization and ADE tests were also used in this assay (a) C3H/He; (b) ICR, tested against JEV Nakayama and DENV1 Mochizuki viruses. A positive titer was defined as the maximum serum dilution showing an absorbance above that obtained with non-immunized serum.
characteristics of both parental viruses, improvements with respect to these characteristics are expected in viral or antigenic progeny. Results are variable for one such characteristic, expression yield: some chimeras have lower yields than parental viruses (22, 24, 38), whereas others have similar or higher yields (39). Our chimeric plasmid induces greater expression than occurs with the original DENV1 construct. The highest stably expressed extracellular protein titers have been obtained from a chimeric DNA vaccine containing a JEV-derived prM signal sequence, which is known to induce significantly increased E protein expression (23). This strong antigen production in turn positively affects antibody induction titers in mice, thus fulfilling the stoichiometric antibody requirement for viral neutralization (40). In addition, the construct may be further improved (41–44) and used as part of a new strategy to potentially strengthen antigen expression. Epitope mapping of the E protein with mouse mAbs or human immune serum has demonstrated that the 132
antibody against DIII has strong neutralization capability with type-specific activity, whereas antibodies against DI and DII have lower neutralization potency but are broadly cross-reactive against various DENV serotypes (44–48). With respect to the E protein-specific response, the proportion of DI/DII to DIII antibodies showed considerable variability. Our strategy of substituting DIII in the JEV prM-E gene backbone with that of DENV generated an interesting antibody profile, less enhancing activity being detected in immunized mouse serum with the chimeric construct. Antibody induction by DIII itself (chimeric pcJED1ME) was strongly neutralizing against DENV1, with less enhancing activity. In addition, incorporation of prM and DI/DII (original pcD1ME) provided moderate neutralizing and enhancing activity, whereas elimination of DIII (chimeric pcD1JEME) resulted in the lowest neutralizing activity and strongest enhancing activity against DENV1. These results demonstrate the importance of DIII in inducing neutralizing activity against DENV1 while simultaneously reducing enhancing activity. Consequently, incorporation of JEV prM, DI and DII indeed induced detectable neutralizing antibodies against JEV but, as shown in previous studies, they were not cross-reactive against DENV1 (28–29). Secondary exposure to a heterologous viral serotype can result in more severe disease because all dengue viral serotypes can be co-circulated and pre-existing nonneutralizing antibody against one serotype is unable to fully abolish heterologous viral infection, but rather enhances viral uptake through the ADE mechanism. An effective dengue vaccine would address the possibility of ADE activity from induced antibody. One characteristic of antibody responses against DIII of E proteins is that they are serotype-specific, which makes them less crossreactive in neutralizing or enhancing viral infection; therefore, the DIII of E protein may have an advantage as a vaccine antigen (49–50). As also shown in previous studies, dengue vaccines using DIII provide high serotype specificity with less cross-neutralizing capacity against heterologous viral serotypes (51–53). With almost no detectable enhancing activity, as shown in this study, they may fulfill the requirement for reduced antibody-enhancing activity. Not only antigen, but also the isotypic profile of antibodies regarding their affinity to complement, affect antibody responses in mouse sera. Because mouse IgG2 has stronger binding affinity to complement than IgG1, when complement is present in the assay system, the enhancing activity of IgG2 isotype may switch to neutralizing activity. It is generally known that genebased vaccines tend to induce Th1 immune responses, which induce IgG2a isotype, whereas protein-based © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Chimeric dengue-JEV DNA vaccine
vaccines tend to induce Th2 immune responses and induce IgG1 isotype. Thus, the type of vaccine and immunization route could be an alternative approach to developing an effective dengue vaccine. Finally, Crill et al. observed a correlation between antibody titers against DIII and neutralization potency of immunized serum samples (54). In contrast, other researchers have found only small amounts of antibody against DIII in human serum samples (18–19, 55), and depleting it with recombinant DIII protein does not significantly reduce the neutralizing activity of serum samples. Further studies are needed to explain these contradictory findings. Nevertheless, our present study illustrates the advantages of a chimeric DNA vaccine consisting of DENV DIII on a JEV prM-E gene backbone for increasing antigen expression and inducing strong neutralizing antibody responses against DENV with reduced ADE activity.
ACKNOWLEDGMENTS This study was supported by the Science and Technology Research Partnership for Sustainable Development (SATREPS) of the Japan Science and Technology Agency/Japan International Cooperation Agency (JST/ JICA). This work is part of PhD thesis by Fithriyah Sjatha.
DISCLOSURE The authors have no conflict of interests to declare.
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