Attenuation of Vaccinia Virus

Abstract

Since 1980, in the post-smallpox vaccination era the human population has become increasingly susceptible compared to a generation ago to not only the variola (smallpox) virus, but also other zoonotic orthopoxviruses. The need for safer vaccines against orthopoxviruses is even greater now. The Lister vaccine strain (LIVP) of vaccinia virus was used as a parental virus for generating a recombinant 1421ABJCN clone defective in five virulence genes encoding hemagglutinin (A56R), the IFN-γ-binding protein (B8R), thymidine kinase (J2R), the complement-binding protein (C3L), and the Bcl-2-like inhibitor of apoptosis (N1L). We found that disruption of these loci does not affect replication in mammalian cell cultures. The isogenic recombinant strain 1421ABJCN exhibits a reduced inflammatory response and attenuated neurovirulence relative to LIVP. Virus titers of 1421ABJCN were 3 lg lower versus the parent VACV LIVP when administered by the intracerebral route in new-born mice. In a subcutaneous mouse model, 1421ABJCN displayed levels of VACV-neutralizing antibodies comparable to those of LIVP and conferred protective immunity against lethal challenge by the ectromelia virus. The VACV mutant holds promise as a safe live vaccine strain for preventing smallpox and other orthopoxvirus infections.

Keywords: attenuation; protection; vaccine; vaccinia virus; virulence genes.

Figures

Fig. 1

Schematic diagram illustrating the generation of VACV B8R mutants (see text for details)

Fig. 2

Schematic diagram illustrating the amplification of PCR fragments flanking the deleted sequences in the VACV genome. A. Map of the VACV genome with the location of the C3L, N1L, J2R, A56R, and B8Rvirulence genes (indicated with arrows); B. Schematic of amplified regions (shaded area) flanking a sequence stretch upstream (L-flank) and downstream (R-flank) of the deleted genes (grey block arrows). The zigzag arrows stand for the position of primer pairs so that a deletion in one gene does not affect the adjacent genes and amplified DNA fragments are flanked by the restriction sites

Fig. 3

The strategy to introduce mutations in each of the VACV virulence genes. The order of sequential inactivation with plasmids targeting the genomic sites of choice is shown

Fig. 4

Verification of deletions/insertions by PCR. PCR products formed from DNA of the parent clone 14 VACV LIVP and 1421ABJCN with disrupted virulence genes (see text for details). M – molecular size marker, the lengths are given on the left . Lanes A, B, C, N, J contain VACV DNA fragments amplified from the genes A56R, B8R, C3L, N1L, and J2R, respectively

Fig. 5

Growth curves for VACV mutants in CV-1 cells

Fig. 6

Time-course of mortality following intracerebral infection of newborn mice with 1421ABJCN and LIVP VACV

Fig. 7

Levels of serum-neutralizing activity to VACV following double subcutaneous immunization of mice with 1421ABJCN and LIVP at different doses. Neutralizing titers were expressed in -log10 of the highest dilution which gives 50% neutralization of VACV ± standard deviation

Fig. 8

Time-course of mortality after double subcutaneous immunization of mice with 1421ABJCN or LIVP VACV at a dose of 106 pfu/mouse, followed by challenge with ECTV at a dose of 10 LD50/mouse