Construction and Characterization of a Single-Cycle Chimeric Flavivirus Vaccine Candidate That Protects Mice against Lethal Challenge with Dengue Virus Type 2

Abstract

We have previously described a novel flavivirus vaccine technology based on a single-cycle, capsid (C) gene-deleted flavivirus called RepliVAX. RepliVAX can be propagated in cells that express high levels of C but undergoes only a single cycle of infection in vaccinated hosts. Here we report that we have adapted our RepliVAX technology to produce a dengue vaccine by replacing the prM/E genes of RepliVAX WN (a West Nile virus [WNV] RepliVAX) with the same genes of dengue virus type 2 (DENV2). Our first RepliVAX construct for dengue virus (RepliVAX D2) replicated poorly in WNV C-expressing cells. However, addition of mutations in prM and E that were selected during blind passage of a RepliVAX D2 derivative was used to produce a second-generation RepliVAX D2 (designated D2.2) that displayed acceptable growth in WNV C-expressing cells. RepliVAX D2.2 grew better in DENV2 C-expressing cells than WNV C-expressing cells, but after several passages in DENV2 C-expressing cells it acquired further mutations that permitted efficient growth in WNV C-expressing cells. We tested the potency and efficacy of RepliVAX D2.2 in a well-described immunodeficient mouse model for dengue (strain AG129; lacking the receptors for both type I and type II interferons). These mice produced dose-dependent DENV2-neutralizing antibody responses when vaccinated with RepliVAX D2.2. When challenged with 240 50% lethal doses of DENV2, mice given a single inoculation of RepliVAX D2.2 survived significantly longer than sham-vaccinated animals, although some of these severely immunocompromised mice eventually died from the challenge. Taken together these studies indicate that the RepliVAX technology shows promise for use in the development of vaccines that can be used to prevent dengue.Dengue viruses (DENV) are the etiologic agents of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. The viruses are transmitted to humans by Aedes spp. mosquitoes. DENV infections are a serious cause of morbidity and mortality in most tropical and subtropical areas of the world (12). Dengue cases are estimated to occur in up to 100 million individuals annually, and there are over 2.5 billion people living in areas at risk for infection, making dengue the most important arbovirus disease in the world. DENV belongs to the Flavivirus genus in the family Flaviviridae and exists as four antigenically distinct serotypes (DENV1 to -4) (4). The four serotypes of DENV do not confer cross-protective immunity, and epidemiological evidence indicates that immunity to one serotype of DENV increases the chance of a more severe disease upon infection with a second serotype by about 10-fold (26).DENV are single-stranded, positive-sense RNA viruses and have an 11-kb genome characterized by a single open reading frame encoding three structural proteins (C, prM/M, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) and untranslated regions at its 5Ј and 3Ј termini (5Ј untranslated region [UTR] and 3ЈUTR). Viral RNA replication occurs in the ...

Extension of Flavivirus Protein C Differentially Affects Early RNA Synthesis and Growth in Mammalian and Arthropod Host Cells

“…Thus, we showed that the NS2B/3 cleavage at the C terminus of protein C is not required when protein C is present on the same polyprotein as the other viral proteins. This is in good agreement with previous work that obviated the need for NS2B/3 cleavage of protein C by supplying protein C from separate translation units (10,13,27,36,40,42). The viruses in such experiments replicated to high titers; therefore, it seems likely that interference with the NS2B/3 cleavage on protein C has only a minor effect on the efficiency of viral replication.…”

“…Nevertheless, previous investigations of the C-terminal region of TBEV protein C revealed remarkable functional flexibility in the exact NS2B/3 cleavage position, as well as the cleavage substrate (35). In addition, several groups have also been able to supply protein C in trans to allow single-round infectious particles of several flaviviruses to be generated (10,13,27,36,40).…”

Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique

“…Another advantage of the replicons are the different ways in which they can be used as immunizing: DNA, RNA or defective particles (Varnavski et al 2000, Aberle et al 2005, Suzuki et al 2009.…”

Functional RNA Delivery Targeted to Dendritic Cells By Synthetic Nanoparticles

“…Frey et al [112] and Suter et al [113] employed VRP based on the classical swine fever virus genome, demonstrating that a single inoculation was efficient at inducing both humoral and cellular immunity. Flavivirus replicons, such as those derived from West Nile fever virus, have been modified to deliver genes of dengue virus type 2 [114]. Again, a single inoculation was sufficient to induce anti-dengue-virus neutralizing antibodies and the animals showed increased resistance to challenge infection.…”