Recombinant dimeric envelope vaccine against flaviviral...

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof

Reexamination Certificate

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C435S069100, C435S069700, C530S350000, C424S192100

Reexamination Certificate

active

06749857

ABSTRACT:

TECHNICAL FIELD
This invention relates to protection against and diagnosis of flaviviral infection. More specifically, the invention concerns recombinantly produced dimers of truncated flaviviral envelope protein secreted as mature proteins from eucaryotic cells and which induce high titer virus neutralizing antibodies believed to be important in protection against flaviviral infection and which are useful in diagnosis of infection by the virus.
BACKGROUND ART
The four serotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4) belong to the family Flaviviridae which also includes the Japanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE), West Nile virus (WN), and the family prototype, Yellow fever virus (YF). Flaviviruses are small, enveloped viruses containing a single, positive-strand, genomic RNA. The envelope of flaviviruses is derived from the host cell membrane and is decorated with virally-encoded transmembrane proteins membrane (M) and envelope (E). While mature E protein and the precursor to M, prM, are glycosylated, the much smaller mature M protein is not. The E glycoprotein, which is the largest viral structural protein, contains functional domains responsible for cell surface attachment and intraendosomal fusion activities. It is also a major target of the host immune system, inducing virus neutralizing antibodies, protective immunity, as well as antibodies which inhibit hemagglutination.
Dengue viruses are transmitted to man by mosquitoes of the genus Aedes, primarily
A. aegypti
and
A. albopictus.
The viruses cause an illness manifested by high fever, headache, aching muscles and joints, and rash. Some cases, typically in children, result in a more severe forms of infection, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), marked by severe hemorrhage, vascular permeability, or both, leading to shock. Without diagnosis and prompt medical intervention, the sudden onset and rapid progression of DHF/DSS can be fatal.
Flaviviruses are the most significant group of arthropod-transmitted viruses in terms of global morbidity and mortality with an estimated one hundred million cases of dengue fever occurring annually (Halstead, 1988). With the global increase in population and urbanization especially throughout the tropics, and the lack of sustained mosquito control measures, the mosquito vectors of flavivirus have distributed throughout the tropics, subtropics, and some temperate areas, bringing the risk of flaviviral infection to over half the world's population. Modern jet travel and human emigration have facilitated global distribution of dengue serotypes, such that now multiple serotypes of dengue are endemic in many regions. Accompanying this in the last 15 years has been an increase in the frequency of dengue epidemics and the incidence of DHF/DSS. For example, in Southeast Asia, DHF/DSS is a leading cause of hospitalization and death among children (Hayes and Gubler, 1992).
The flaviviral genome is a single strand, positive-sense RNA molecule, approximately 10,500 nucleotides in length containing short 5′ and 3′ untranslated regions, a single long open reading frame, a 5′ cap, and a nonpolyadenylated 3′ terminus. The complete nucleotide sequence of numerous flaviviral genomes, including all four DEN serotypes and YF virus have been reported (Fu et al., 1992; Deubel et al, 1986; Hahn et al, 1988; Osatomi et al., 1990; Zhao et al, 1986; Mackow et al., 1987; Rice et al., 1985). The ten gene products encoded by the single open reading frame are translated as a polyprotein organized in the order, capsid (C), premembrane/membrane (prM/M), envelope (E), nonstructural protein (NS) 1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5 (Chambers, et al. 1990). Processing of the encoded polyprotein is initiated cotranslationally, and full maturation requires both host and virally-encoded proteases. The sites of proteolytic cleavage in the YF virus have been determined by comparing the nucleotide sequence and the amino terminal sequences of the viral proteins. Subsequent to initial processing of the polyprotein, prM is converted to M during viral release (Wengler, G. et al.,
J Virol (
1989) 63:2521-2526) and anchored C is processed during virus maturation (Nowak et al.,
Virology
(1987)
156
:
127
-
137
).
While all dengue viruses are antigenically related, antigenic distinctions exist which define the four dengue virus serotypes. Infection of an individual with one serotype does not apparently provide long-term immunity against the other serotypes. In fact, secondary infections with heterologous serotypes are becoming increasingly prevalent as multiple serotypes co-circulate in a geographic area. In general, primary infections elicit mostly IgM antibodies directed against type-specific determinants. On the other hand, secondary infection by a heterologous serotype is characterized by IgG antibodies that are flavivirus crossreactive. Dengue virus vaccine development is complicated by the observation that immunity acquired by infection with one serotype may in fact enhance pathogenicity by dengue virus of other types. Halstead (1982) demonstrated that anti-dengue antibodies can augment virus infectivity in vitro, and proposes that serotype crossreactive, non-neutralizing antibodies to E enhance infection in vivo, resulting in DHF/DSS (Halstead, 1981). This viewpoint is not however, universally accepted (Rosen, 1989). For example, Kurane et al (1991) proposed that dengue serotype-cross-reactive CD4
+
CD8

cytotoxic T cells (CTLs) specific for NS
3
may contribute to the pathogenesis of DHF/DSS by producing IFN-&ggr; and by lysing dengue virus-infected monocytes. Recent evidence demonstrating that CTLs specific for E are not serotype-crossreactive may suggest that use of E subunit vaccines would not induce the potentially harmful cross-reactive CTL response (Livingston et al, 1994). Regardless of the mechanism for enhanced pathogenicity of a secondary, heterologous dengue viral infection, strategies employing a tetravalent vaccine should avoid such complications. Helpful reviews of the nature of the flaviviral diseases, the history of attempts to develop suitable vaccines, and structural features of flaviviruses in general as well as the molecular structural features of the envelope protein of flaviviruses are available (Halstead 1988; Brandt 1990; Chambers et al., 1990; Mandl et al., 1989; Henchal and Putnak, 1990; Putnak 1994; Rey et al., 1995).
Although many approaches to dengue virus vaccines have been pursued, there is no acceptable vaccine currently available. Until recently, the low titer of dengue virus grown in culture has made a killed vaccine impractical, and candidate live-attenuated dengue virus vaccine strains tested to date have proven unsatisfactory (see, e.g., Eckels et al, 1984; Bancroft et al, 1984; McKee et al, 1987), although live attenuated candidate vaccine strains continue to be developed and tested (Hoke et al, 1990; Bhamarapravati et al, 1987). The construction of several full-length infectious flavivirus clones (Rice et al., 1989; Lai et al., 1991; Sumiyoshi et al., 1992) has facilitated studies aimed at identifying the determinants of virulence in flaviviruses (Bray and Lai, 1991; Chen et al., 1995; Kawano et al., 1993). However, these studies are in preliminary stages and little information on virulence has been obtained. A similar approach to vaccine development in the poliovirus system, while extremely informative, has taken years.
In the absence of effective live attenuated or killed flavivirus vaccines, a significant effort has been invested in the development of recombinant, flaviviral subunit or viral-vectored vaccines. Many of the vaccine efforts which use a recombinant DNA approach have focused on the E glycoprotein. This glycoprotein is a logical choice for a subunit vaccine as it is exposed on the surface of the virus and is believed to be responsible for eliciting protective immunity as monoclonal antibodies directed against purified flaviviral E proteins are neutralizing in

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