Genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia LC16m8

Abstract

A highly attenuated LC16m8 (m8) smallpox vaccine has been licensed in Japan because of its extremely low neurovirulence profile, which is comparable to that of replication incompetent strains of vaccinia virus. From 1973 to 1975, m8 was administrated to >100,000 infants where it induced levels of immunity similar to that of the originating Lister strain, without any serious side effects. Recently, we observed that m8 reverts spontaneously to large plaque forming clones that possess virulence equivalent to that of LC16mO, a parental virus strain of m8. Here, we report that the B5R gene is responsible for the reversion, and that we could construct a more genetically stable virus by deleting B5R from m8. The protective immunogenicity of the vaccine candidate proved to be equivalent to that of the U.S.-licensed product Dryvax, and much superior to modified vaccinia Ankara in a mouse model. Furthermore, the vaccine strain never elicited any symptoms in severe combined immunodeficiency disease mice, even at a dose 1,000-fold greater than that used in the immune protection experiments, which is in contrast to the lethal pathogenicity induced by Dryvax inoculation of severe combined immunodeficiency disease mice. Our results suggest that this vaccine strain is a good candidate as a suitable smallpox vaccine and a vector virus, and that B5R is not essential for protective immunity against smallpox.B5R gene ͉ reversion ͉ Lister strain ͉ extracellular enveloped virion

Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies

“…Kretzschmar et al recently reported that the frequency of death associated with vaccination might be higher than previously believed [10]. To diminish these health risks, attenuated VACV viruses, such as modified vaccinia Ankara (MVA) and LC8m16 have been developed [11][12][13][14]. However, failed to induce protective immunity in immuno-compromised animals, possibly due to host defects in B-cell antibody class switching [15].…”

LC16m8, a Highly Attenuated Vaccinia Virus Vaccine Lacking Expression of the Membrane Protein B5R, Protects Monkeys from Monkeypox

“…LC16m8 lacks expression of the full-length membrane protein B5R, one of the most immunogenic proteins, because of a frameshift mutation in the membrane protein gene B5R (19,35). It is expected that LC16m8 does not pass through the EEV stage in the viral life cycle, because the membrane protein B5R is essential in packaging the intracellular mature virion with the trans-Golgi membrane or endosomal cisternae to form intracellular enveloped virions (13,32,34) and this protein is also involved in the release of cell-associated enveloped virions from the cell surface to form EEV in cooperation with proteins A36R and A33R (17,29).…”

An Attenuated LC16m8 Smallpox Vaccine: Analysis of Full-Genome Sequence and Induction of Immune Protection

“…Nevertheless, mice immunized with these vaccines were 100% protected against pathogenic WR challenge as early as 3 weeks after vaccination. Moreover, m8 with the whole B5R gene deleted protected mice from lethal WR challenge (32). These findings suggest that many viral antigens other than B5R are also involved in protective immunity to EEV.…”

“…However, a single nucleotide insertion just upstream of the m8 B5R mutation site has recently been reported to restore the ORF to the parental mO phenotype after repeated (10 or more) virus passages. Although the repaired viruses were a marginal population, attenuation that is achieved by a deletion of the whole B5R gene prevented the reversion of m8-to mO-type viruses (32), which have, however, much lower virulence than LO and NYBH (24,25,39). In turn, the genetic manipulation of m8 to replace genes related to protective immunity, but not to pathogenicity, with the counterpart genes of VAR may make m8 more efficacious.…”

“…Today, no vaccines under development or in human trials can be tested for protective efficacy against smallpox by infection of humans with the causative virus, VAR. However, a pathogenic vaccinia virus WR strain provides an alternative small-animal model suited for evaluating protective immunization (2,32,50,51). VV has rather low infectivity for mice, but WR is an exception, because it is adapted to mice by repeated passages in the mouse brain (27).…”