Sequence and immunogenicity of a clinically approved novel measles virus vaccine vector

Abstract

The measles virus vaccine (MVbv) is a clinically certified and well-tolerated vaccine strain that has been given both parenterally and mucosally. It has been extensively used in children and has proven to be safe and effective in eliciting protective immunity. This specific strain was therefore chosen to generate a measles viral vector. The genome of the commercial MVbv vaccine strain was isolated, sequenced and a plasmid, p(+)MVb, enabling transcription of the viral antigenome and rescue of MVb, was constructed. Phylogenic and phenotypic analysis revealed that MVbv and the rescued MVb constitute another evolutionary branch within the hitherto classified measles vaccines. Plasmid p(+)MVb was modified by insertion of artificial MV-type transcription units (ATUs) for the generation of recombinant viruses (rMVb) expressing additional proteins. Replication characteristics and immunogenicity of rMVb vectors were similar to the parental MVbv and to other vaccine strains. The expression of the additional proteins was stable over 10 serial virus transfers, which corresponds to an amplification greater than 10 ( 20) . The excellent safety record and its efficient application as aerosol may add to the usefulness of the derived vectors.

Keywords: live-attenuated vaccines; recombinant measles virus; viral vectors.

Figure 1. Phylogenetic analysis of MVbv. Sequences of different MV strains were aligned with ClustalW and a phylogram was generated by the neighbor-joining method using the parental MV Edmonston sequence as an out-group. The scale (0.0002) indicates the number of substitutions per nucleotide.

Figure 2. Comparison of the cloned MVb with different MV vaccine strains and a recombinant virus. (A) Growth kinetic analyses. Comparison of the propagation kinetics of the standard MVbv, cloned MVb and the MVEZ virus on MRC-5. Cell-free virus preparations from infected cells were collected at daily intervals and virus titers were determined by plaque assay. (B) Phenotypic assessment of various MV vaccine strains (MVbv, MVb, MVEZ, MVsch, rMVb and standard MV). Vero cells were infected, incubated at different temperatures, and the phenotype was evaluated according to the cytopathic effect. (C) Transgenic mice expressing human CD46 were immunized i.m with 1 × 10⁵ pfu rMVb MVbv, MVEZ and MVsch. Measles end point titers are shown on a logarithmic scale.

Figure 3. Transgene expression. Expression of the transgene was assessed by (A) immunofluorescence. Recombinant MV expressing HIV env (see M&M) was passaged 10 time on MRC5 cells, the resultant virus shed from passage 1 and 10 were analyzed by immunofluorescence, Dapi staining and phase contrast (Phc). Only positive expressing HIV env were formed by passaged viruses, and only negative syncytia by the control MVb (> 100 syncytia were counted). Images show typical syncytia formed by passaged viruses. (B) Antigen expression by rMVb was analyzed by western blots against HIV-env and measles N and P protein are shown. Note: mature HIV env (gp-140) is detected in the medium because it is a secreted protein. In the cell, different forms of the protein are revealed due to various maturation stages of the glycoprotein.

Figure 4. Induction of humoral immune responses upon intranasal application. To test the ability of rMVb vectors to induce a humoral immune response against HIV-env and MV-specific antigens, respectively, rMVb2-HIV-env was applied at 1 × 10⁵ pfu i.n; a UV-inactivated preparation was used as control. Anti-measles and anti-HIV-env antibody titers, respectively, are represented as in Figure 2C.