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

Abstract

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-Carambot E, 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 (Delta1724), 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 Delta1724 mutation increased attenuation only in certain combinations with other mutations. We constructed two highly attenuated viruses, rV94(15C)/460A/948L and rV94(15C)/948L/Delta1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys and, therefore, appear to be suitable for evaluation in humans.

Figures

Figure 1

Nucleotide assignment at position 15 in the 3’ genomic promoter region of recombinant and biologically derived HPIV2 strains. A.) Sequence analysis of HPIV2 isolates recovered from AGMs inoculated with biologically derived V94. Electropherograms show the sequence of nucleotides 12-18 for each indicated isolate. The predominant nucleotide at each position is listed, and nucleotide 15 is indicated with a dot. The first electropherogram shows the sequence of the biologically derived V94 HPIV2, the virus used to infect the AGMs. The remaining four electropherograms to the right show the sequences of HPIV2 isolated from the TL samples of four different AGMs on day four after IN and IT inoculation with V94. Compare nucleotide position 15 of the V94 input virus (far left) with that of isolates from AGMs. B.) The sequence of nucleotides 1-30 is shown for the biologically derived viruses V98 and V94 (at the ninth passage in Vero cells and at an additional passage in LLC-MK2 cells), recombinant V94, and the laboratory strain Greer. Bold letters indicate the nucleotide assignment at position 15 for each virus.