Tetravalent neutralizing antibody response against four dengue serotypes by a single chimeric dengue envelope antigen
Introduction
Dengue viruses are transmitted through bites of Aedes mosquitoes. Four virus serotypes (dengue-1 to dengue-4) of the genus Flavivirus can produce a spectrum of clinical illness, ranging from an uncomplicated but debilitating dengue fever (DF) to sometimes severe and fatal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Infection with one serotype results in a long-lasting protective immunity against the homotypic virus, but only confers short lived cross-protective immunity to heterologous serotypes [1]. Therefore, people living in endemic areas can have multiple infections, with secondary infections posing a risk for the most severe manifestation of the disease, DHF/DSS. With an estimate of up to 100 million human infections each year and several hundred thousand cases of DHF/DSS [2], [3], dengue viruses have become one of the most important arthropod-borne viruses from a medical and public health perspective.
There is currently no specific treatment for dengue diseases, but modern supportive intensive care can reduce the fatality rate of DHF/DSS patients from approximately 20% in an untreated group to 0.1%. Vaccination could provide a promising approach for controlling dengue virus infections. Although the development of dengue vaccines has been ongoing for the past 50 years [4], no licensed dengue vaccine is available yet. The major challenge is to generate a vaccine that induces protective immunity against all four serotypes of dengue viruses. Several dengue vaccine programs have focused on manufacturing four live attenuated viruses, which are administered as a tetravalent mixture. Promising preclinical results have been achieved using this approach, but formulation complexities and competition among serotypes leading to non-uniform immune responses hamper the speed of clinical development [5], [6], [7]. Protective immunity was also achieved in animal models using inactivated dengue-2 viruses or recombinant dengue-2 E proteins as subunit vaccines against homotypic dengue-2 viruses [8], [9]. Chimeric dengue-2/1, -2/3 and -2/4 virus constructs using the backbone of attenuated dengue-2 virus have been shown to retain the attenuation and elicit specific immune responses to dengue-1, -3 and -4, respectively [10]. Considerable advances have also been made in developing chimeric dengue-yellow fever viruses [11]. These chimeric dengue/dengue or dengue/yellow fever viruses could provide a basis for live attenuated tetravalent vaccine. However, manufacturing four vaccine candidates and formulation complexities pose a significant challenge to this approach as well.
DNA vaccines may offer an attractive alternative or additional platform. Feasibility of DNA vaccines is supported by induction of both humoral and cellular immune responses in several human clinical trials [12], [13], [14] and by a recent report that a West Nile virus DNA vaccine is under consideration for USDA approval for use in horses [15]. A dengue-2 DNA vaccine expressing the viral pre-membrane (prM) protein and the amino terminal 92% of envelope protein was shown to elicit neutralizing and protective antibody responses in mice challenged with live dengue-2 virus [16]. A dengue-1 construct expressing prM and full-length envelope was shown to produce virus-like particles in transfected cells, induce neutralizing antibodies in mice [17], and protect rhesus macaques and Aotus monkeys when challenged with the corresponding live dengue-1 virus [18], [19]. However, progress in this area has been slow, and is attributed to the need for developing 4 individual vaccines, which then must be formulated optimally to produce the tetravalent vaccine. It would be highly beneficial to develop a single vaccine that combines attributes from all four dengue serotypes and elicits protective immune responses to each of them.
We have addressed these challenges by using directed molecular evolution by DNA shuffling and screening to generate chimeric dengue antigens, which combine neutralizing epitopes from all four dengue virus E proteins. Several studies have been conducted to identify the location and functional importance of neutralizing antibody epitopes for the dengue-2 E antigen [20], [21], [22], [23], but currently available information regarding the structure, localization and sequences of the antigenic epitopes exposed on the virion surfaces of all four dengue serotypes is insufficient to enable rational design of broadly protective, multivalent recombinant dengue antigens. DNA shuffling and screening was therefore a practical tool to search for tetravalent dengue vaccine candidates, since it does not require an understanding of the number and location of specific neutralizing epitopes but simply relies on a functional screen for desired improvements. Shuffling technologies have previously been used for a wide-range of applications and have been successful, for example, in evolving interferon-α, interleukin-12, co-stimulatory molecules and viruses [24], [25], [26], [27], [28]. In the present study, we describe directed molecular evolution of dengue envelope genes to generate a single chimeric antigen that induces neutralizing antibodies against all four dengue serotypes in mice when administered as a DNA vaccine.
Section snippets
Cells, antigens, and antibodies
HEK293 cells (ATCC) were grown in DMEM medium, supplemented with 10% fetal bovine serum (FBS). Dengue antigens used for ELISA and ELISpot assays included recombinant dengue-3 and dengue-4 antigens, and dengue-1, -2, -3, and -4 virus infected vero cell lysates, dengue virus infected insect cell lysates (Immunology Consultants Laboratory, Oregon), and vero cell derived purified dengue-2 virus (Microbix Biosystems, Canada.). The recombinant dengue-3 and dengue-4 antigens, comprising the viral prM
Construction and screening of shuffled antigen libraries
Chimeric antigen libraries were constructed by DNA shuffling using four codon-optimized sequences encoding wild-type dengue-1–4, prM15/tE (15 amino acid leader sequence of prM and C-terminal truncated E) proteins. Truncated E proteins were used as shuffling templates because of previous studies demonstrating improved secretion of dengue E from which the C-terminal hydrophobic sequences were deleted [32]. Individual shuffled clones from two libraries were screened for tetravalent antigen
Discussion
A safe and effective dengue vaccine must protect against all four dengue serotypes. No licensed vaccine is currently available for dengue, but mixtures of four individual wild-type antigens in different immunization formats are currently in both preclinical and clinical studies. We have aimed to facilitate dengue vaccine development by using DNA shuffling and screening to construct a single recombinant dengue antigen capable of eliciting tetravalent immune responses.
We were able to select seven
Acknowledgements
We would like to thank Ms. Heather Kelly for excellent technical assistance. We would also like to thank Dr. Robert Shope at University of Texas Medical Branch at Galveston for support and expert advice. Part of this work was performed under a Cooperative Research and Development Agreement between Maxygen, Inc. and the Naval Medical Research Center. The opinions and assertions contained herein are not to be considered official or to reflect the views of the U.S. Navy or the Naval Service at
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2014, Advances in Virus ResearchCitation Excerpt :Danko et al. found formulation with the adjuvant Vaxfectin® enhanced the neutralizing antibody response in monkeys immunized with a tetravalent DNA vaccine (Danko, Beckett, & Porter, 2011), and a phase 1 study of the tetravalent DNA vaccine (TVDV) given with Vaxfectin® began in 2011 (Clinicaltrials.gov NCT01502358). In parallel, DNA shuffling and screening technologies were utilized to develop a single recombinant antigen containing epitopes from all four DENV serotypes (Apt et al., 2006). Three chimeric clones (one containing truncated E and two expressing full-length prM/E) induced neutralizing antibodies against all four serotypes and protected mice from lethal i.c. DENV2 challenge.