CHARACTERIZATION OF A QUADRUPLE GLYCOPROTEIN-DELETED PSEUDORABIES VIRUS MUTANT FOR USE AS A BIOLOGICALLY SAFE LIVE VIRUS-VACCINE

Herpesvirus envelope glycoproteins play important roles in mediating i nfection initiation and also represent major immunogens. We recently s howed that a pseudorabies virus (PrV) mutant lacking the essential gly coprotein gD (gp50), after phenotypic complementation by propagation o n genetically engineered PrV gD-expressing cell lines was able to infe ct primary target cells and spread exclusively by means of direct cell -to-cell transmission. Virions released from non-complementing cells t hat lacked gD were not infectious because of a defect in penetration a nd so free infectious virions did not arise after infection of animals by phenotypically complemented gD-negative PrV. This formed the basis for the development of novel non-spreading live herpesvirus vaccines. However, the gD-negative PrV mutant still retained a residual level o f virulence, which prevented its use as vaccine, and the need to propa gate the gDnegative PrV mutant on trans-complementing cell lines resul ted in the appearance of wild-type revertants, rescued by the resident gene in the cell line. To overcome these problems we isolated a PrV m utant designated PrV(376) that, in addition to gD, also lacked the non -essential glycoproteins gG, gI and gE. PrV(376), because of the lack of gD, was also dependent on gD-expressing cells for productive replic ation. Non-complementing cells infected by phenotypically gD-complemen ted PrV(376) produced non-infectious particles lacking glycoproteins g D and gE as shown by immunoelectron microscopy. Owing to the absence o f any homologous sequences between the viral genome and the viral gene s resident in the complementing cell line, rescue by homologous recomb ination was impossible. In cell culture, plaques of PrV(376) were sign ificantly smaller than those of either wild-type, or gD- or gE-deleted mutants, indicating an additive or synergistic effect of the combined deletion on viral cell-to-cell spread capability. Intranasal or intra muscular infection of pigs with phenotypically gD-complemented PrV(376 ) showed a complete attenuation of viral virulence, with an expected l ack of shedding of infectious virus. The PrV(376)-vaccinated pigs exhi bited a significant level of protection against challenge infection, m easured by survival and weight loss. In summary, PrV(376) represents a novel type of herpesvirus vaccine that combines innocuity, efficacy a nd biological safety.