ReviewPreclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses
Introduction
Several members of the Flavivirus genus are serious threats to human and/or animal health. Among them, Dengue virus (DENV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) represent a major and growing medical problem [1].
First, the number of DENV infections in endemic areas has continued to increase over the past two decades, and more than 100 million dengue infections resulting in 24,000 deaths are now estimated annually with children bearing the bulk of the disease burden. There are four serotypes of DENV and infection by each of them can lead to clinical manifestations ranging from self-limiting dengue fever to severe dengue hemorrhagic fever (DHF) and fatal dengue shock syndrome (DSS). Over one hundred countries are affected with over three billion people at risk (for a review see [2]). This increased incidence and extended geographical reach of dengue have made the development of an effective vaccine an international health priority.
WNV is a zoonotic pathogen which first appeared in the Western Hemisphere in 1999 in New York City, before spreading rapidly across North America, causing disease in wild birds, horses, and humans. The virus has also been isolated in the West Indies and neutralizing antibody-positive birds and horses have been identified in Mexico, Jamaica, and the Dominican Republic; West Nile virus also appeared recently in South America, including Brazil and Argentina. The spectrum of disease extends from a mild febrile exanthem to fatal encephalitis; neuroinvasive disease occurs in ∼30% of reported WNV cases. Around 30,000 cases and more than 1000 deaths have been attributed to WNV infection since its introduction [3], [4]. There is no effective drug treatment against this infection. While the introduction of vaccines for horses represents a potent tool to control the veterinary disease, a human vaccine against WNV may represents an important approach to the prevention and control of this emerging disease (for a review see [5]), although recent epidemiological data show a sharp decline in the number WNV cases in the past years.
Finally, JEV is the main cause of viral encephalitis in Asia and thus constitutes a major threat for approximately three billion people living in endemic regions. JEV has also spread to previously unaffected areas of Australia [6], [7]. It causes an estimated 50,000 cases of disease and 10,000 deaths per year, mostly in children [8]. Acute encephalitis occurs in about 1–20 cases/1000 infections, being lethal in 29% of cases. A high proportion of survivors have neurological and psychiatric sequelae. Different vaccines are available: mouse brain-derived inactivated vaccine, and cell-culture derived, inactivated or live vaccines, yet none has been prequalified by WHO at this time (for a review see [9], [10]). The introduction of inactivated JE vaccines in Japan in the mid-1950s lead to a dramatic drop of reported cases but raised concerns regarding adverse events (AEs) following immunization (Acute Disseminated EncephaloMyelitis, ADEM, reported in 1 per 50,000–1,000,000 cases). An inactivated-vaccine produced on primary hamster kidney (PHK) cells has also been used in China from the 1970s to the 1990s, before being replaced by the live-attenuated vaccine SA14-14-2. This vaccine has been successfully used for massive immunization campaigns in China and India, in which it has been given to >100 million children and provides 80–96% protection after a single dose. First attempts made in the 1980s to adapt this virus in a more acceptable cell substrate for vaccine production have lead to a decrease immunogenicity and development of such a vaccine was discontinued [11]. The SA14-14-2 strain has been adapted to growth on Vero for use as a two dose inactivated vaccine (IXIARO®, Intercell, Vienna) which has recently been approved by the US FDA for adult travelers and military personnel.
While a live-attenuated vaccine exists since decades against the prototype flavivirus, i.e. the YFV, there is thus still a need for vaccines against dengue or West Nile diseases, and for improved vaccines against JEV which protect after a single dose.
Several vaccine candidates against these diseases exist or are being developed, including live, vectored and recombinant preparations (for reviews see [5], [12], [13], [14], [15], [16]). Sanofi Pasteur has developed chimeric vaccines using the ChimeriVax technology, originated at St Louis University, where the first YFV 17D-based chimera was constructed in 1999 by Chambers et al for Japanese encephalitis virus [17] and further developed as a vaccine virus by Guirakhoo et al. [18] at Acambis inc., now a part of Sanofi Pasteur. The first YFV 17D/dengue chimera was constructed by Guirakhoo et al in 2000 [19]. These vaccines, and later on other chimeric flaviviruses, were constructed by replacing the genes for YF vaccine (YFV 17D 204) Premembrane (prM) and Envelope (E) proteins, with those of heterologous flaviviruses [17], [18], [19], [20], [21], [22], [23], [24], [25], [26] (see Fig. 1).
Characteristics of different ChimerVax-based vaccines are summarized in Table 1. PrM and E genes were either derived from wild type (wt) viruses without modification (e.g. dengue and veterinary West Nile vaccines), or from empirically derived attenuated vaccine (e.g. JEV strain SA14-14-2 for JEV vaccine) or by introduction of specific attenuating mutations into the WT E by site directed mutagenesis (e.g. NY99 strain for development of a human West Nile vaccine). In contrast to neurotropic flaviviruses such as TBEV and JEV, where residues involved in virulence had been previously identified to lie within the envelope proteins, no such residues were known for non-neurotropic viruses such as dengue. Therefore for construction of chimeric vaccine viruses for non-neurotropic viruses such as dengue, it was envisioned that the wild type E sequences linked to the YFV 17D backbone should be sufficient for attenuation of the chimeric viruses. This was indeed turned out to be true as was shown by preclinical and clinical testing (see below).
All chimeric vaccines are immunogenic and safe in humans, and are either waiting registration (JEV) or are undergoing large-scale efficacy studies (DEN) [27]. The particular – live attenuated and chimeric – nature of theses vaccines required however an extensive preclinical and clinical characterization, from the early stages of research to clinical development. In fact, the licensure of new vaccines is more challenging now than it was more than half a century ago, in particular in the case of live-attenuated vaccines, despite decades of proven efficacy and good to excellent safety record of long-established vaccines such as live polio and YF vaccines. The situation is even more complex for chimeric vaccine candidates, which are genetically modified organisms (GMO), and they have to comply with additional and specific regulations. The different tools, assays and strategies developed to address these points will be presented in this review, focusing to some extent on dengue vaccines, which generated diverse and in-depth evaluation because of their particular complexity. As presented in Fig. 2, we will first address vaccine characterization (phenotypic and genetic properties/stability), followed by preclinical in vitro or animal evaluation, and then clinical testing. Finally, we will address the theoretical issues linked to the live GMO nature of the ChimeriVax-based vaccines.
Section snippets
Phenotypic and genotypic characterization of chimeric vaccine candidates
Compared to live-attenuated vaccines obtained by empirical passages, ChimeriVax-based vaccines are rationally designed, starting from cDNA to plaque-purified clonal vaccines. Further scale-up of viral vaccine production from laboratory scale to GMP manufacture requires multiple rounds of virus amplification, which have to be strictly controlled in order to avoid the loss of critical attenuation properties. The seed lot system (primary or master seed lot [MSL] and secondary/working seed lot
Preclinical evaluation of safety and immunogenicity
Before entering clinical trials, all candidate vaccines must be tested for safety and immunogenicity in preclinical studies. Such evaluations can be conducted both in vitro on primary or transformed cells – including human cells – and in vivo in animal models, in particular non-human primates (NHP). In the case of arboviral vaccines, studies with potential vectors also have to be documented. Results obtained in these models can be correlated with safety and immunogenicity of the candidate
Clinical evaluation
The three different chimeric vaccine candidates against DENV, JEV and WNV have now been tested extensively in clinical trials, in volunteers ranging from children to the elderly. All results obtained so far demonstrate their excellent safety profile, with low/absent viremia of short duration [41], [62]. Satisfactory immunogenicity is obtained after a single dose (JEV, WNV) or after 2–3 doses (DENV). The following paragraphs will describe the main findings and conclusions obtained from these
Evaluation of theoretical safety/GMO issues
It was important to address the theoretical issues linked to both the live and GMO nature of ChimeriVax-based vaccines throughout their development. The four most frequently raised concerns were specifically addressed: transmission by arthropod vectors, reversion to virulence, recombination with a circulating virus, and risks of viscerotropism. A summary of each of these potential risks follows and the main points are summarized in Table 3.
Dengue-specific issue: antibody-dependent enhancement (ADE)
As stated previously, the most often proposed markers of protection are seroneutralizing antibodies, as measured for instance by the PRNT50, although titres required for protection are yet to be determined [59], [111]. Cross-protection between DENV serotypes is limited and antibody dependent enhancement (ADE) has been proposed to constitute one mechanism leading to severe dengue disease, e.g. DHF or DSS [65], [66]: pre-existing non-neutralizing heterotypic antibodies, instead of being
Conclusions
The ChimeriVax candidates developed against DENV, WNV and JEV demonstrated satisfactory safety and immunogenicity characteristics in in vitro and in vivo pre-clinical tests. We tried however to consider potential issues linked to these chimeric vaccines, unlikely as their occurrences may be. The utilization of the infectious clone and recombinant technologies facilitated their rapid evaluation.
Clinical data generated in humans with the chimeric dengue vaccines demonstrated expected safety and
Acknowledgements
The authors would like to acknowledge Simon Jones for editorial assistance and Claude Meric for critical reading of the manuscript.
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