Hawaii Biotechnology Group, Inc.

John M Ivy

David Edward Clements

Michael McDonell

Beth-Ann G Coller

Eilen Nakano

W Dorsey Stuart

Gary S Bignami

Iain D Peters

Kent Harada

Kenneth Koo

The present invention discloses and claims vaccines containing, as an active ingredient, a secreted recombinantly produced dimeric form of truncated flaviviral envelope protein. The vaccines are capable of eliciting the production of neutralizing antibodies against flaviviruses. The dimeric forms of truncated flaviviral envelope protein are formed 1) by directly linking two tandem copies of 80% E in a head to tail fashion via a flexible tether; 2) via the formation of a leucine zipper domain through the homodimeric association of two leucine zipper helices each fused to the carboxy terminus of an 80% E molecule; or 3) via the formation of a non-covalently associated four-helix bundle domain formed upon association of two helix-turn-helix moieties each attached to the carboxy terminus of an 80% E molecule. All products are expressed as a polyprotein including prM and the modified 80% E products are secreted from Schneider 2 cells using the human tissue plasminogen activator secretion signal sequence (tPA ). Secreted products are generally more easily purified than those expressed intracellularly, facilitating vaccine production. One embodiment of the present invention is directed to a vaccine for protection of a subject against infection by dengue virus. The vaccine contains, as active ingredient, the dimeric form of truncated envelope protein of a dengue virus serotype. The dimeric truncated E is secreted as a recombinantly produced protein from eucaryotic cells. The vaccine may further contain portions of additional dengue virus serotype dimeric E proteins similarly produced. Another embodiment of the present invention is directed to methods to utilize the dimeric form of truncated dengue envelope protein for diagnosis of infection in individuals at risk for the disease. The diagnostic contains, as active ingredient, the dimeric form of truncated envelope protein of a dengue virus serotype. The dimeric truncated E is secreted as a recombinantly produced protein from eucaryotic cells. The diagnostic may further contain portions of additional dengue virus serotype dimeric E proteins similarly produced.

Recombinant dimeric envelope vaccine against flaviviral infection

A vaccine contains at least one Drosophila cell-secreted, recombinantly-produced form of a truncated Flavivirus envelope glycoprotein, as an active ingredient, and an adjuvant, as a critical component of the vaccine. The adjuvant is an immunomodulating agent having an iscom-like structure and comprising within the iscom-like structure at least one lipid and at least one saponin, and a pharmaceutically acceptable vehicle. Such a vaccine protects a subject against infection by a Flavivirus.

Recombinant envelope vaccine against flavivirus infection

A recombinant DNA encoding coffee bean &agr;-galactosidase permits the production of purified forms of this protein. The protein is useful in converting human Type B red blood cells into cells physiologically similar to Type O red blood cells. The availability of this enzyme permits more effective conversion than use of &agr;-galactosidase from other sources.

DNA encoding recombinant coffee bean alpha-galactosidase

The Flaviviridae comprise a number of medically important pathogens that cause significant morbidity in humans including the dengue (DEN) virus, Japanese encephalitis (JE) virus, tick-borne encephalitis virus (TBE), and yellow fever virus (YF). Flaviviruses are generally transmitted to vertebrates by chronically infected mosquito or tick vectors. The viral particle which is enveloped by host cell membranes, comprises a single positive strand genomic RNA and the structural capsid (CA), membrane (M), and envelope (E) proteins. The E and M proteins are found on the surface of the virion where they are anchored in the membrane. Mature E is glycosylated and contains functional domains responsible for cell surface attachment and intraendosomal fusion activities. Problems have arisen in the art with respect to producing recombinant forms of the E glycoprotein that retain their native configuration and attendant properties associated therewith (i.e., ability to induce neutralizing antibody responses). To date, recombinantly produced E glycoproteins have suffered from a number of limitations including improper glycosylation, folding, and disulfide bond formation. The claimed invention has addressed these concerns by providing secreted recombinant forms of the E glycoprotein that are highly immunogenic and appear to retain their native configuration. Carboxy-terminally truncated forms of E containing the amino terminal 395 amino acids and a suitable secretion signal sequence were generated in Drosophila melanogaster Schneider cell lines. Immunogenic compositions comprising these recombinant envelope glycoproteins were capable of inducing protective, neutralizing antibody responses when administered to a suitable host.

Subunit immonogenic composition against dengue infection

The Flaviviridae comprise a number of medically important pathogens that cause significant morbidity in humans including the dengue (DEN) virus, Japanese encephalitis (JE) virus, tick-borne encephalitis virus (TBE), and yellow fever virus (YF). Flaviviruses are generally transmitted to vertebrates by chronically infected mosquito or tick vectors. The viral particle which is enveloped by host cell membranes, comprises a single positive strand genomic RNA and the structural capsid (CA), membrane (M), and envelope (E) proteins. The E and M proteins are found on the surface of the virion where they are anchored in the membrane. Mature E is glycosylated and contains functional domains responsible for cell surface attachment and intraendosomal fusion activities. Problems have arisen in the art with respect to producing recombinant forms of the E glycoprotein that retain their native configuration and attendant properties associated therewith (i.e., ability to induce neutralizing antibody responses). To date, recombinantly produced E glycoproteins have suffered from a number of limitations including improper glycosylation, folding, and disulfide bond formation. The claimed invention has addressed these concerns by providing secreted recombinant forms of the E glycoprotein that are highly immunogenic and appear to retain their native configuration. Carboxy-terminally truncated forms of E containing the amino terminal 395 amino acids and a suitable secretion signal sequence were generated in Drosophila melanogaster Schneider cell lines. The recombinant proteins produced by this expression system should prove useful, inter alia, as immunogens and diagnostic reagents.

Methods of preparing carboxy-terminally truncated recombinant flavivirus

Neurospora crassa transformations are employed for production of heterologous proteins, where DNA constructs are prepared using a foreign gene under transcriptional and translational regulatory regions functional in Neurospora. A functional signal sequence is provided for secretion of the heterologous gene product. An auxotrophic Neurospora host is employed and transformed by the combination of the subject construct and a complementing gene. The resulting transformants provide for efficient secretion of the heterologous product.

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College Station, TX, United States of America

John M Ivy