Recombinant human parainfluenza virus type 2 vaccine candidates containing a 3′ genomic promoter mutation and L polymerase mutations are attenuated and protective in non-human primates

Previously, we identified several attenuating mutations in the L polymerase protein of human parainfluenza virus type 2 (HPIV2) and genetically stabilized those mutations using reverse genetics [Nolan SM, Surman S, Amaro-CarambotE, Collins PL, Murphy BR, Skiadopoulos MH. Live-attenuated intranasal parainfluenza virus type 2 vaccine candidates developed by reverse genetics containing L polymerase protein mutations imported from heterologous paramyxoviruses. Vaccine 2005;39(23):4765-74]. Here we describe the discovery of an attenuating mutation at nucleotide 15 (15 (T -> C)) in the 3' genomic promoter that was also present in the previously characterized mutants. We evaluated the properties of this promoter mutation alone and in various combinations with the L polymerase mutations. Amino acid substitutions at L protein positions 460 (460A or 460P) or 948 (948L), or deletion of amino acids 1724 and 1725 (A 1724), each conferred a temperature sensitivity (ts) phenotype whereas the 15 (T -> C) Mutation did not. The 460A and 948L mutations each contributed to restricted replication in the lower respiratory tract of African green monkeys, but the A 1724 mutation increased attenuation only in certain combinations with other mutations. We constructed two highly attenuated viruses, rV94(15C)/46OA/948L and rV94(15C)/948L/Delta 1724, that were immunogenic and protective against challenge with wild-type HPTV2 in African green monkeys and, therefore, appear to be suitable for evaluation in humans. (c) 2007 Elsevier Ltd. All rights reserved.