CCF25587338S
THE GOAL OF DR. JIANRONG LIÂ S LABORATORY IN THIS U01 PROJECT IS TO DEVELOP MEASLES VIRUS (MEV)/MUMPS VIRUS (MUV)-BASED KSHV VACCINE CANDIDATES AND TEST THEIR IMMUNOGENICITY OF THESE VACCINE CANDIDATES IN IFNAR-/- MICE (AIM 3). THE AIM 3 HAS 4 TASKS: 1. RECOVER AND CHARACTERIZE RMEV AND RMUV EXPRESSING FULL-LENGTH, SOLUBLE, AND STABILIZED K8.1, GB, AND GH/GL OF KSHV. THE FULL-LENGTH, SOLUBLE [LACKING THE TRANSMEMBRANE/CYTOPLASMIC TAIL (TM/CT)], STABILIZED FORM (E.G. GB-FOLDON TRIMER) OF K8.1, GB, AND GH/GL OF KSHV WILL BE INSERTED INTO THE P-M GENE JUNCTION OF THE MEV, RMUV-JL1, AND RMUV-JL2 GENOME. RECOMBINANT MEV AND MUV EXPRESSING INDIVIDUAL KSHV WILL BE RECOVERED AND CHARACTERIZED. ALL RECOMBINANT VIRUSES WILL BE PLAQUE PURIFIED AND SEQUENCED, VIRUS GROWTH, GENETIC STABILITY, AND PROTEIN EXPRESSION WILL BE CHARACTERIZED. 2. RECOVER AND CHARACTERIZE RMEV AND RMUV CO-EXPRESSING TWO OR THREE KSHV GLYCOPROTEINS. WE WILL GENERATE MEV, MUV-JL1, AND MUV-JL2 EXPRESSING 2 OR 3 KSHV GLYCOPROTEINS: K8.1+GB, K8.1+GH/GL, GB+GH/GL, AND K8.1+GB-GH/GL. ALL RECOMBINANT VIRUSES WILL BE PLAQUE PURIFIED AND SEQUENCED, VIRUS GROWTH, GENETIC STABILITY, AND PROTEIN EXPRESSION WILL BE CHARACTERIZED. 3. GENERATE RMEV AND RMUV EXPRESSING ONE, TWO, OR THREE MEMBRANE-BOUND KSHV GLYCOPROTEINS. TO GENERATE RMEV AND RMUV EXPRESSING MEMBRANE-BOUND KSHV GLYCOPROTEINS, THE ECTODOMAIN OF K8.1, GB, AND GH WILL BE FUSED TO THE TM/CT OF THE MEV F PROTEIN (FOR MEV CONSTRUCTS) OR THE MUV F PROTEIN (FOR MUV CONSTRUCT). IN ADDITION, THE COMBINATION OF 2 OR 3 MEMBRANE-BOUND GLYCOPROTEINS INCLUDING K8.1+GB, K8.1+GH/GL, GB+GH/GL, AND K8.1+GB+GH/GL WILL ALSO BE BUILT INTO MEV AND MUV, ALLOWING FOR THE INCORPORATION OF 2 OR 3 KSHV GLYCOPROTEINS ON MEV AND MUV VIRIONS. ALL RECOMBINANT VIRUSES WILL BE RECOVERED AND CHARACTERIZED AS DESCRIBED ABOVE. 4. QUANTIFY THE IMMUNOGENICITY OF RMUV AND RMEV EXPRESSING THE DIFFERENT FORMS OF KSHV GLYCOPROTEINS IN IFNAR1-/- MICE. FOUR-WEEK-OLD FEMALE AND MALE IFNAR1-/- MICE WILL BE IMMUNIZED SUBCUTANEOUSLY WITH 105 PFU OF EACH RMUV OR RMEV-BASED KSHV VACCINE CANDIDATE AND BOOSTED WITH THE SAME VACCINE CANDIDATE AT THE SAME DOSE FOUR WEEKS LATER. WE WILL COLLECT SERUM RETRO-ORBITALLY BEFORE THE INITIAL IMMUNIZATION, 4 WEEKS AFTER THE FIRST IMMUNIZATION AND 4 WEEKS AFTER THE SECOND IMMUNIZATION. SERA WILL BE USED FOR QUANTIFYING KSHV-SPECIFIC IGG ANTIBODY AND NAB, AND SPLENOCYTES WILL BE ISOLATED FOR QUANTIFYING ANTIGEN-SPECIFIC CD4 AND CD8 T CELL RESPONSES. 5. DETERMINE THE BEST FORMULATION OF MMR-K VACCINES. EQUAL AMOUNTS (E.G., 105 PFU) OF RMEV, RMUV-JL1, AND RMUV-JL2 EXPRESSING KSHV GLYCOPROTEINS WILL BE MIXED TO GENERATE VARIOUS MMR-K VACCINES. FOUR-WEEK-OLD FEMALE AND MALE IFNAR1-/- MICE (N=10) WILL BE IMMUNIZED I.N. WITH 3Ã 105 PFU OF MMR-K VACCINE CANDIDATES AND BOOSTED WITH THE SAME VACCINE CANDIDATE (3Ã 105 PFU) FOUR WEEKS LATER. MONOVALENT VACCINE CANDIDATES WILL BE USED AS CONTROLS. WE WILL COLLECT SERUM AT 4 WEEKS AFTER THE FIRST IMMUNIZATION, AND 4 WEEKS AFTER THE SECOND IMMUNIZATION. ALL SERA WILL BE TESTED FOR SERUM IGG ANTIBODIES AND NABS, AND SPLENOCYTES WILL BE ISOLATED FOR QUANTIFYING ANTIGEN-SPECIFIC CD4 AND CD8 T CELL RESPONSES. DELIVERABLE: WE WILL IDENTIFY THE MOST IMMUNOGENIC FORM OF K8.1, GB, AND GH/GL IN THE MEV AND MUV VECTORS. WE WILL IDENTIFY THE TOP 3 MMR-K VACCINE CANDIDATES, WHICH ARE CAPABLE OF INDUCING STRONG KSHV-SPECIFIC HUMORAL AND T CELL IMMUNE RESPONSES.
The Ohio State University
Cooperative Agreement U01CA294881
$203.6k 1/10/25