Construction and characterization of a second-generation pseudoinfectious West Nile virus vaccine propagated using a new cultivation system

Summary

Safer vaccines are needed to prevent flavivirus diseases. To help develop these products we have produced a pseudoinfectious West Nile virus (WNV) lacking a functional C gene which we have named RepliVAX WN. Here we demonstrate that RepliVAX WN can be safely propagated at high titer in BHK cells and vaccine-certified Vero cells engineered to stably express the C protein needed to trans-complement RepliVAX WN growth. Using these BHK cells we selected a better growing mutant RepliVAX WN population and used this to generate a second-generation RepliVAX WN (RepliVAX WN.2). RepliVAX WN.2 grown in these C-expressing cell lines safely elicit strong protective immunity against WNV disease in mice and hamsters. Taken together, these results indicate the clinical utility of RepliVAX WN.2 as a vaccine candidate against West Nile encephalitis.

Introduction

West Nile virus (WNV) is a positive-sense, single-stranded RNA virus belonging to the Flavivirus genus of the Flaviviridae family. Medically important members of this genus include dengue virus, yellow fever virus (YFV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) [1]. Since its introduction into the US, WNV has spread throughout the hemisphere and become endemic in North America. Although WNV infection is often asymptomatic, it can produce human disease ranging from febrile illness to fatal encephalitis. To date there have been over 25,000 human cases reported in the United States [2].

The WNV genome consists of a single open reading frame encoding a polyprotein that is cleaved into three structural and seven non-structural proteins. The structural proteins C, M (produced in cells as a precursor, prM), and E make up the viral particle, while the non-structural (NS) proteins are required for genome replication and polyprotein processing [1]. WNV virions consist of a C-containing nucleocapsid surrounded by a host-derived membrane containing M and E. During flavivirus infection subviral particles (SVPs) composed of M and E but lacking nucleocapsid are released with mature virions. Expression of prM and E in the absence of C produces SVPs [3], [4], and several vaccine candidates, including subunit [5], [6], [7], [8], [9], DNA [10], [11], [12], RNA [13] and live-vectors [3], [14], [15], [16], [17], [18] have been developed based on SVPs. Animal studies have demonstrated that these candidates elicit protective immune responses that include both humoral and cell-mediated activities [7], [15]. Among these candidates, DNA vaccines suffer from poor immunogenicity [10], [11], [12], subunit vaccines are difficult to produce in large quantities [5], [7], [9], and live-vectored vaccines have proven ineffective in the face of pre-existing vector immunity [18].

Currently there are few vaccines available to prevent flavivirus diseases. The YFV-17D live-attenuated vaccine (LAV), although extremely effective, has recently been associated with a number of severe adverse events including the development of viscerotropic YF disease [19], [20], [21], [22]. Moreover, this vaccine is not recommended for use in infants, pregnant women, or immunocompromised individuals. A promising line of research into new vaccines has focused on genetically derived chimeras of YFV-17D (ChimeriVax) encoding prM and E of other flaviviruses [23], [24], [25]. Other vaccine candidates have been produced by using a similar chimerization strategy to insert prM and E genes into an attenuated dengue type 4 backbone [26], [27], [28]. Despite the fact that ChimeriVax-WN has been successfully evaluated in non-human primates [29], the 17D backbone in ChimeriVax presents the same potential hazards associated with the YFV vaccine, and it is unclear if chimeric viruses will display unwanted pathogenic characteristics. Licensed inactivated viral vaccines (INV) exist to prevent JE and TBE, but these INVs are expensive and require multiple vaccinations [30]. Recently, the JEV INV was removed from the universal vaccination campaign in Japan due to concerns over adverse reactions [31], [32].

A promising line of research has focused on development of attenuated flaviviruses containing large in-frame deletions within the C gene. Specifically, C-deleted genomes of TBEV have been shown to be replicationally competent, capable of producing SVPs, and unable to spread between cells, making them a useful RNA vaccine [13], [33]. Utilizing a similar strategy we have developed a trans-encapsidation system to package C-deleted genomes, producing a novel WN vaccine candidate, RepliVAX WN [34]. Unlike the RNA vaccines described above, RepliVAX WN is a special type of LAV that can be produced in cell lines expressing the missing C [34], [35]. When RepliVAX WN infects normal cells, it cannot produce infectious progeny due to the lack of the C protein, however these cells produce SVPs that have demonstrated ability to protect animals from flavivirus infection (see above) and RepliVAX WN efficiently protects mice against West Nile encephalitis (WNE) [34].

Here we describe an improved system for growth of RepliVAX, and development of an improved second-generation RepliVAX WN (RepliVAX WN.2). Our new propagation system consists of cell lines based on either baby hamster kidney (BHK) cells or vaccine-certified Vero cells expressing a form of the WNV C protein engineered for long-term stability and an inability to recombine with the RepliVAX genome. RepliVAX WN.2 produced in these cells was more potent than our original RepliVAX WN and efficiently protected two different animals from WNE.