Early detection of flaviviruses using the NS1 glycoprotein

Abstract

The invention concerns a method for early detection of a flavivirus-induced infection, comprising the detection of the flavivirus non-structural glycoprotein NS1 in a biological sample during the clinical phase of the infection, by an immunological method using at least two identical or different antibodies, the first antibody consisting of polyclonal or monoclonal antibodies pre-selected for their high affinity for said NS1 protein hexameric in shape.

1 - 20 . (canceled) 
     
     
         21 . A method for purifying an NS1 protein of a flavivirus, wherein the NS1 protein is in hexameric form, from a culture supernatant either of infected mammalian cells or of mammalian cells transfected with a recombined plasmid, comprising:
 (A) expressing the NS1 protein or a fragment of the NS1 protein from an NS1 gene or a fragment of the flaviviral genome, wherein the fragments are capable of expressing the NS1 protein prior to the purification of the NS1 protein;   (B) treating the NS1 protein with a precipitating agent;   (C) centrifuging the treated NS1 protein; and,   (D) separating a soluble form of the NS1 protein from a microparticulate form of NS1 protein.   
     
     
         22 . The method for purifying NS1 protein as claimed in  claim 27 , wherein the flavivirus is a dengue virus. 
     
     
         23 . The method for purifying NS1 protein as claimed in  claim 28 , wherein the flavivirus is dengue virus serotype 1. 
     
     
         24 . An immunogenic composition, comprising as the active principle, an NS1 protein of a flavivirus, wherein the NS1 protein is in hexameric form, optionally associated with other proteins, and at least one pharmaceutical vehicle. 
     
     
         25 . The immunogenic composition as claimed in claim  30 , wherein the composition further comprises at least one mixture of NS1 proteins in hexameric form of a dengue virus serotype. 
     
     
         26 . A method for preparing an immunogenic composition capable of inducing the production of antibodies in vivo comprising combining an NS1 protein in hexameric form, or an NS1 protein expressed from a system for the expression of NS1 protein in hexameric form, with at least one pharmaceutical vehicle. 
     
     
         27 . A method for manufacturing a medicinal product capable of inducing passive immunization comprising producing at least one monoclonal anti-NS1 antibody having a high affinity for NS1 protein in hexameric form, purifying the monoclonal antibody, and modifying the monoclonal antibody by selecting for Fab fragments or humanizing the monoclonal antibody, wherein the hexameric form is nondegraded. 
     
     
         28 . An immunogenic composition, comprising an active principle and a pharmaceutical vehicle, wherein the active principle is either a polynucleotide capable of expressing all or part of an NS1 protein of a dengue virus of any serotype, or the active principle is an expression system comprising at least one promoter capable of expressing, in a host into which it is injected, a DNA encoding an NS1 protein of a dengue virus of any serotype. 
     
     
         29 . A method for expressing a polynucleotide encoding an NS1 protein of a dengue virus, comprising associating a polynucleotide of SEQ ID No. 1 with a promoter for said polynucleotide, and expressing the polynucleotide in a eukaryotic cell.