Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350

Mok, Hoyin; Cheng, Xing; Xu, Qi; Zengel, James R; Parhy, Bandita; Zhao, Jackie; Wang, C. Kathy; Jin, Hong

Antigenomic cDNA was synthesized by DNA2.0 (Menlo Park, CA) based on the sequence of live attenuated measles virus Edmonston-Zagreb strain (NCBI DNA accession number F266290). The T7 RNA polymerase promoter sequence was added to the 5' end of the antigenomic cDNA and the hepatitis delta virus ribozyme and a T7 RNA polymerase terminator sequences were added to the 3' end of the antigenomic cDNA. Sequence at nucleotides 5502 (Sca I) and 9343 (Mlu I) was modified to facilitate cDNA cloning. The three amino acid residues at positions 3682, 3862 and 10555 (3682 K R , 3862 S N , 10555 K R ) that were different from the EZ vaccine strains were corrected by sitedirected mutagenesis. The MV antigenomic cDNA was cloned into pUC18 vector and denoted as pEZ (Fig. 1) . Supporting plasmids encoding measles N, P and L genes were cloned into pCITE plasmids under the control of T7 promoter as previously described and were designed pN, pP and pL respectively [30] . (1) . Construction of measles vaccine vectors encoding ecto domains of RSV F and EBV gp350 and protein expression in virus-infected Vero cells. (A) RSV F ectodomain and EBV gp350 ectodomains were inserted in the 1 st and 3 rd transcription units of the measles Zagreb strain genome. Insertion was performed in the non-coding region 3' proximally of the MV N gene or between P and M genes of pEZ under the control of additional measles transcriptional units containing gene stop (GE)-gene start (GS) sequences. *RSV F gene (aa. 1-524) was inserted into EcoRV site using blunt ligation. # EBV gp350 full length (aa. 1-960), EBV gp350 truncated (aa. 1-470) or RSV F gene was inserted between EcoRV and AfeI site. (B) Vero cells were infected at an MOI of 3.0 with measles viruses encoding soluble RSV F (sF1 or sF3) or soluble EBV gp350 (gp350 or gp350 tr). After 18 h, western blots were used to detect presence of measles virus nucleoprotein in infected Vero cell lysates or RSV F protein, EBV gp350 protein in the supernatant. Black arrowheads indicate the predicted molecular masses of the proteins. Relative intensity of the MV N protein bands was expressed as the intensity of the band relative to the rEZ control. Construction of measles virus vectored antigenomic cDNA. The ectodomain of RSV F and EBV gp350 ectodomains were inserted into the non-coding region 3' proximal of the N gene or in the junction between the P and M genes of pEZ (Fig. 1) . Each inserted gene contained measles virus transcriptional unit consisting of the sequences of N gene stop (GE-N) and P gene start (GS-P). To insert the RSV F gene into the promoter proximal location, a pUC-SnaBI/SacII (pUC-S/S) subclone that contained the T7 promoter and MV sequence of 1 to 2044 with a silent mutation at nt 821 to knockout EcoRV enzyme site was generated. A gene cassette containing the ectodomain of RSV-F (aa 1-524) followed by GE-N and GS-P sequence were inserted into EcoRV site at nt 99. The genomic DNA was in multiples of 6 nucleotides (the rule of six). The SnaB I-Sac II fragment was swiped back to pEZ antigenomic cDNA (Fig. 1A ) and the resulting cDNA was denoted as pEZ-RSVsF1.