Utilization of recombinant influenza viruses and modified vaccinia ankara virus (mva) with genes that encode for the toxoplasma gondii sag1 and sag2 surface proteins, as vaccines against toxoplasmosis

Abstract

The present invention concerns to recombinant influenza viruses and modified Vaccinia Ankara viruses (MVA), and to a process for construction of recombinant influenza viruses and modified vaccinia Ankara viruses (MVA) with genes that encode for the T. gondii parasite SAGI (MVA) and SAG2 (MVA and influenza) proteins, by means of a homologous recombination technique between two transfer vectors (for construction of MVA virus) and reverse genetics (for construction of influenza virus). Additionally, the present invention describes a vaccine composition using recombinant influenza viruses and modified vaccinia Ankara viruses (MVA), or recombinant adenoviruses and modified vaccinia Ankara viruses (MVA), for immunization against infections caused by the T. gondii parasite.

1 . A process of construction of recombinant Modified Vaccinia Ankara (MVA) viruses carrying an antigen of a  Toxoplasma gondii  protozoa parasite, said process comprising the following steps:
 (a) cloning of  T. gondii  genes that encode SAG1 or SAG2 proteins in a transfer vector to provide a plasmid;   (b) transfection of the plasmid obtained in step (a) and infection by a wild MVA virus;   (c) generation of the recombinant MVA viruses by homologous recombination;   (d) cloning and amplification of the recombinant MVA viruses; and   (e) characterization of an expression of proteins of interest.   
     
     
         2 . Recombinant Modified Vaccinia Ankara (MVA) viruses carrying an antigen of the  Toxoplasma gondii  protozoa parasite, wherein said MVA virus is obtained according to the process claimed in  claim 1 . 
     
     
         3 . A process of construction of recombinant influenza viruses of any subtype carrying a viral segment that became bicistronic by duplication of a 3′ non-encoding region and of nucleotides of a 5′ encoding region, carrying an antigen of a  Toxoplasma gondii  protozoa parasite ( T. gondii ), comprising the following steps:
 (a) cloning of a  T. gondii  gene in a transfer plasmid carrying a bicistronic segment of the influenza virus; 
 (b) co-transfection of the transfer plasmid of the bicistronic segment carrying an encoding sequence of the  T. gondii  antigen, transfer plasmids of remaining influenza virus segments, and plasmids encoding influenza virus replication proteins; 
 (c) generation of the recombinant influenza viruses by reverse genetics; and 
 (d) cloning and amplification of the recombinant influenza viruses. 
 
     
     
         4 . Recombinant influenza viruses of any subtype carrying a viral segment that became bicistronic by duplication of the 3′ non-encoding region and of nucleotides of the 5′ encoding region, carrying an antigen of  T. gondii , wherein the recombinant influenza viruses are obtained according to the process claimed in  claim 3 . 
     
     
         5 . Viruses in accordance with the  claim 4 , wherein the viral segment is neuraminidase. 
     
     
         6 . A vaccinal composition against Toxoplasmosis, comprising:
 an initial immunization dose adapted for any inoculation route and comprising a recombinant adenovirus from any serotype, and   a booster dose adapted for any inoculation route and comprising an MVA virus.   
     
     
         7 . A vaccinal composition in accordance with  claim 6 , wherein the recombinant adenovirus serotype is a HuAd5 serotype. 
     
     
         8 . A vaccinal composition against Toxoplasmosis, comprising:
 an initial immunization dose adapted for any inoculation route and comprising a recombinant MVA virus, and   a booster dose adapted for any inoculation route and comprising a recombinant adenovirus of any serotype.   
     
     
         9 . A vaccinal composition against Toxoplasmosis, comprising:
 an initial immunization dose adapted for any inoculation route and comprising a recombinant influenza virus of any subtype, and   a booster dose adapted for any inoculation route and comprising a MVA virus.   
     
     
         10 . The composition in accordance with  claim 9 , wherein the recombinant influenza virus subtype is the H1N1 subtype or the H3N2 subtype. 
     
     
         11 . A vaccinal composition against Toxoplasmosis, comprising:
 an initial immunization dose adapted for any inoculation route and comprising a recombinant MVA virus, and   a booster dose adapted for any inoculation route and comprising a recombinant influenza virus of any subtype.   
     
     
         12 . The composition in accordance with  claim 11 , wherein the recombinant influenza virus subtype is the H1N1 subtype or the H3N2 subtype.