Splenocytes were negatively selected for CD4, CD8, or total T cells using EasySep isolation kits (Catalog Nos
Splenocytes were negatively selected for CD4, CD8, or total T cells using EasySep isolation kits (Catalog Nos. replication, latency, or persistence in immunocompetent hosts. It stimulates robust innate immunity, differentiates virus-specific memory T cells, and elicits neutralizing antibodies. A single vaccination affords durable protection that blocks the establishment of latency following challenge with the wild type MHV-68 for Nrp2 at least six months post-vaccination. These results provide a framework for effective vaccination against cancer-associated herpesviruses through the elimination of latency and key immune evasion mechanisms from the pathogen. for the insertion of translational stop codons as these genes are dispensable for viral replication and are conserved among MHV-68, KSHV, and EBV. We also inactivated K3, a viral inhibitor of the MHC class I antigen presentation pathway, by truncation to increase the immunogenicity of the vaccine virus19,20. We hypothesized that removal of these four immune evasion genes would increase immunogenicity while attenuating replication of the vaccine virus by inducing a robust IFN response and presenting all viral epitopes. A critical safety component of our design is eliminating the latency of the vaccine virus. Viral latency is directly linked to tumorigenesis of -herpesviruses. Moreover, latent infection also increases the possibility of viral DNA integration into host chromosomes21. In KSHV and MHV-68, the biphasic life cycle is regulated by RTA, the replication and transcription activator, and by LANA, the latency associated nuclear antigen. The latter is required for latency establishment22C24 while the former upregulates lytic genes25C27. We previously showed that abolishing LANA expression combined with constitutive RTA expression results in a latency-deficient virus28. Here, we replaced the latency locus comprising ORF72, ORF73 (LANA), ORF74, and M11 with constitutively expressed RTA driven by the phosphoglycerate kinase 1 (PGK) promoter in a two-tiered approach to prevent persistent infection. Deletion of the latency locus, constitutive RTA expression, and the removal of immune evasion genes created a live attenuated -herpesvirus vaccine named DIP PT-2385 (deficient in immune evasion and persistence) (Fig. ?(Fig.1a1a). Open in a separate window Fig. 1 Construction of DIP virus and its replication properties in vitro.a Schematic representation of mutations introduced in the MHV-68 genome to generate the DIP vaccine. Red lines indicate insertion of translation stop codons into ORF10, ORF36, and ORF54. The open red tetragon indicates deletion of the coding sequence in K3. PT-2385 The latency locus was replaced by the RTA cassette (arrowhead) constitutively driven by the PGK promoter. b Growth curves of the WT and DIP viruses in 3T3 cells using MOI?=?0.01 and measured by plaque assay to quantify virion production. c NIH 3T3 cells were either mock treated or treated with 100 U mL?1 IFN- for 24?h then infected with either WT or DIP virus at MOI?=?0.01 for 72?h. Virion production was quantified with plaque assays. All experiments were performed in triplicate and statistical significance was analyzed by a two-tailed Students GS500 harboring a BAC containing the WT MHV-68 genome PT-2385 was used to construct recombinant MHV-68 by allelic exchange with conjugation-competent GS111 containing the suicide shuttle plasmid pGS28474C76. For each recombinant MHV-68, an overlap extension PCR was used to construct the unique shuttle plasmid pGS284 harboring the desired mutation and a ~500-bp flanking region. Sequences upstream of the desired mutation (A fragments) were amplified by AF and AR primers. The downstream sequences (B fragments) were amplified by BF and BR primers using wild type MHV-68 virion DNA as the template. The A and B fragments had 20-bp overlapping sequences. For the subsequent PCR reaction, the A and B fragments were used as templates and amplified by AF and BR primers. The final PCR products were digested with the appropriate enzymes and cloned into pGS284. To screen for the correct mutation, restriction enzyme digestion was performed on the PCR products obtained using the AF and BR primers on the BAC MHV-68 clones. Sequential allelic exchanges were.