http://dx.doi.org/10.4110/in.2011.11.5.268 ORIGINAL ARTICLE pISSN 1598-2629 eISSN 2092-6685 Distinct Humoral and Cellular Immunity Induced by Alternating Prime-boost Vaccination Using Plasmid DNA and Live Viral Vector Vaccines Expressing the E Protein of Dengue Virus Type 2 Junu A. George and Seong Kug Eo* College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju 561-756, Korea Background: Dengue virus, which belongs to the Flavivirus significantly enhanced by booster vaccination with the DNA genus of the Flaviviridae family, causes fatal dengue hemor- vaccine. Therefore, our observation will provide valuable in- rhagic fever (DHF) and dengue shock syndrome (DSS) with formation for the establishment of optimal prime-boost vac- infection risk of 2.5 billion people worldwide. However, ap- cination against DenV. proved vaccines are still not available. Here, we explored the [Immune Network 2011;11(5):268-280] immune responses induced by alternating prime-boost vacci- nation using DNA vaccine, adenovirus, and vaccinia virus ex- INTRODUCTION pressing E protein of dengue virus type 2 (DenV2). Methods: Following immunization with DNA vaccine (pDE), adenovirus Dengue virus (DenV) has four serotypes (DenV1, DenV2, (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E DenV3, and DenV4), and belongs to the Flavivirus genus of protein-specific IgG and its isotypes were determined by the Flaviviridae family transmitted to humans by the mosquito conventional ELISA. Intracellular CD154 and cytokine stain- Aedes aegypti (1-3). DenV poses a significant public health ing was used for enumerating CD4＋ T cells specific for E threat to 2.5 billion people at the risk of infection (1-3). protein. E protein-specific CD8＋ T cell responses were eval- uated by in vivo CTL killing activity and intracellular IFN-γ Around 100 million cases of DenV infections occur annually, staining. Results: Among three constructs, VV-E induced the producing symptoms ranging from mild fever to severe hem- most potent IgG responses, Th1-type cytokine production by orrhagic, potentially fatal fever (1-3). Dengue hemorrhagic fe- stimulated CD4＋ T cells, and the CD8＋ T cell response. ver (DHF) and dengue shock syndrome (DSS), which re- Furthermore, when the three constructs were used for alter- portedly affect about 500,000 people per year, are potentially nating prime-boost vaccination, the results revealed a differ- fatal diseases (1-3). These diseases are spreading from trop- ent pattern of CD4＋ and CD8＋ T cell responses. i) Priming ical to subtropical areas of the world by global warming, in- with VV-E induced higher E-specific IgG level but it was de- creasing travel activity, and uncontrolled urbanization (1-3). creased rapidly. ii) Strong CD8＋ T cell responses specific Despite global morbidity and mortality, the pathogenesis of for E protein were induced when VV-E was used for the pri- diseases caused by DenV infection is poorly understood. ming step, and such CD8＋ T cell responses were sig- Even though several factors such as viral virulence, age and nificantly boosted with pDE. iii) Priming with rAd-E induced genetic predisposition of the patient are implicated, the most stronger CD4＋ T cell responses which subsequently boost- ed with pDE to a greater extent than VV-E and rAd-E. important factor is considered to be sequential infection by Conclusion: These results indicate that priming with live viral different serotypes in an endemic area (4,5). While approved vector vaccines could induce different patterns of E pro- vaccines remain unavailable, several approaches to develop tein-specific CD4＋ and CD8＋ T cell responses which were a dengue vaccine have been evaluated. These include the tra- Received on September 16, 2011. Revised on September 28, 2011. Accepted on October 4, 2011. CC This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribu- tion, and reproduction in any medium, provided the original work is properly cited. *Corresponding Author. Tel: 82-63-270-3882; Fax: 82-63-270-3780; E-mail: [email protected] Keywords: Dengue virus type 2, E protein, DNA vaccine, Recombinant adenovirus, Vaccinia virus, Prime-boost vaccination 268 IMMUNE NETWORK http://www.ksimm.or.kr Volume 11 Number 5 October 2011 Immunity Induced by Alternating Prime-boost Vaccination against DenV2 Junu A. George, et al. ditional live attenuated vaccines (6-9), recombinant subunit a variety of cells, making them ideal vaccine carriers (32,33). vaccines produced using several different host systems In contrast, vaccinia virus-vectored recombinants show unre- (10-13), chimeric virus such as yellow fever vaccine vector stricted replication in immune-compromised individuals (34). YF17D-based ChimeriVax (14,15) and RepliVax (16), and Here, we explored the immune responses induced by alter- DNA vaccine (17,18). nating prime-boost vaccination using these different viral vec- All four dengue serotypes have co-circulated in most en- tor vaccines expressing E protein of DenV2, which is a major demic countries at various times, thereby causing concurrent immunogen involved in conferring protective immunity and/or sequential infection by multiple serotypes (19). against dengue infection. We found that vaccinia virus ex- Furthermore, there is the potential for antibody-dependent pressing E protein produced better responses compared to enhancement (ADE) associated with non-neutralizing cross- adenovirus at equivalent antigen doses, and these viral vec- reactive antibodies arising from immunization with mono- tors elicited different patterns of CD4＋ and CD8＋ T cell re- valent dengue virus vaccines (19). Therefore, a dengue virus sponses against E protein when used for alternating prime- vaccine should elicit protective immunity simultaneously to all boost vaccination. Therefore, humoral and cellular immune four serotypes. The current approach to making a tetravalent responses induced by alternating prime-boost vaccination us- dengue vaccine is to create monovalent vaccine candidates, ing live viral vector vaccine, vaccinia and adenovirus, and and then mix these to obtain a tetravalent formulation DNA vaccine are discussed. (20-22). The application of this approach to live, replicating virus vaccine has revealed the potential for viral interference MATERIALS AND METHODS in some instances (22,23). Considering viral interference in vaccination with live viral vector vaccine, multiple prime- Animals and ethics statement boost vaccinations with alternating vaccine vehicles using Female BALB/c (H-2d) mice, 5 to 6 weeks of age, were pur- DNA vaccine expressing the same antigen may become an chased from Samtako (Osan, Korea). The mice were main- effective strategy for eliciting robust immune responses to the tained in the animal facility at Chonbuk National University target antigen (24). Notably, the prime-boost protocol, in under standard conditions. All experimental procedures and which antigen-encoding DNA vaccine is administered first, animal management procedures were undertaken in accord- followed by a boost with live viral vector expressing the same ance with the requirement of the Animal Care and Ethics antigen, has elicited effective protective immunity in both Committees of Chonbuk National University. The animal fa- mouse and primate models of several infectious diseases cility at Chonbuk National University is fully accredited by the (25,26). However, some experiments claimed that priming National Association of Laboratory Animal Care. with live viral vector vaccine and boosting with DNA vaccine induces superior immune responses against encoded antigens Plasmid DNA preparation (27,28), which suggest that optimal prime-boost protocol to Plasmid DNA encoding E protein of dengue virus type 2 induce effective immunity may be dependent on several fac- (DenV2) under the control of the cytomegalovirus (CMV) pro- tors such as encoded antigens, animal species, and properties moter (pDE) was constructed by inserting cDNA of the E pro- of vaccine vectors. tein gene into pCI-neo vector (Promega, Madison, WI). For Various viral vectors expressing foreign antigen, such as immunization, plasmid DNA was purified by polyethylene vaccinia virus, adenovirus or Fowlpox have been used for glycol (PEG) precipitation as described previously (35). prime-boost vaccination. DNA- and vaccinia-based vaccines Briefly, cellular proteins were precipitated with one volume for a pre-erythrocytic malaria antigen that were delivered in of 7.5 M ammonium acetate followed by isopropanol precip- a prime-boost protocol induced 5- to 10-fold greater T-cell itation of the supernatant. After PEG precipitation, the plas- responses than each vaccine alone (29). In addition, gene- mid was extracted three times with phenol-chloroform and based vectors, such as replication-incompetent adenovirus, precipitated with pure ethanol. The DNA quality was checked have proven particularly effective in eliciting enhanced cellular by electrophoresis on a 1% agarose gel. The plasmid DNA and humoral immunities compared to either agent alone concentration was measured using a NanoDrop 2000 spec- (30,31). Replication-incompetent adenovirus has the ability to trophotometer (Thermo Fisher Scientitific, Pittsburgh, PA). efficiently deliver antigens and express them at high levels in The amount of endotoxin was determined by the Limulus IMMUNE