Three decades have passed since the global eradication of smallpox (variola). This eradication was made possible by the development of effective vaccinia virus vaccines (VVs), such as strains Lister and Dryvax. Unfortunately, we now face the potential threat of bioterrorism with variola virus, the causative agent of variola. This threat has led to the production and stockpiling of vaccinia virus-based vaccines in several countries. Human monkeypox (MPX), infection of humans with monkeypox virus (MPXV), is endemic to central and western Africa (18
), and the first human MPX outbreaks outside Africa were reported in the United States in 2004 (6
). Most human MPX patients in this outbreak acquired the virus from prairie dogs (Cynomys
spp.) that became ill after contact with various exotic rodents shipped from Ghana, Africa (30
). Therefore, VVs are still of great importance, although variola has already been eradicated.
LC16m8, a highly attenuated VV strain, was developed in the early 1970s by multiple passages in cell culture through a temperature-sensitive and low-virulence strain, LC16mO, from the original Lister strain (Elstree) (11
). LC16m8 forms smaller plaques than Lister in the chicken chorioallantoic membrane. LC16m8 is temperature sensitive, as demonstrated by the fact that LC16m8 does not grow well in primary rabbit kidney (PRK) cells cultured at 41°C, while Lister grows efficiently (36
). The fact that LC16m8 grows efficiently in PRK cells but not in African green monkey kidney (Vero) cells, while the parental strain Lister grows well in both cell lines, suggests that LC16m8 has a narrow host cell range, growing in a cell-selective manner (36
We recently determined the complete genome sequences of LC16m8, the parental LC16mO strain, and the original Lister strain (GenBank accession no. AY678275, AY678277, and AY678276, respectively) (24
). It was revealed that there was a single nucleotide deletion of guanosine (G) at the 274th position from the initiation codon in the membrane protein gene B5R
(GenBank accession no. M55434 and AY678275) that generated a premature termination codon and truncated the B5R membrane protein of LC16m8 extracellular enveloped virions (EEV) at amino acid position 93. LC16m8 may possess nearly all the open reading frames corresponding to the VV strains Copenhagen and Lister except for the membrane protein B5R. Because Lister had no history of virus cloning, nucleotide polymorphisms were observed at more than 1,000 sites in the whole genome, indicating that it is difficult to make a simple comparison between the nucleotide sequences of LC16m8 and Lister. However, alignments of the EEV-related membrane proteins in LC16m8 and Lister indicated that there were only 1, 1, 1, and 2 amino acid differences in the EEV-related membrane proteins A36R, F13L, A56R, and A33R, respectively, and that the EEV-related membrane protein A34R of LC16m8 was identical to that of Lister. Although the genetic background responsible for the temperature sensitivity has not been elucidated, it has been confirmed that mutation in the membrane protein gene B5R
is responsible for small-plaque formation and cell-selective growth of this strain (35
LC16m8 has very low neurovirulence in animal models (11
). More than 100,000 people were vaccinated with LC16m8 in Japan, but no LC16m8-associated adverse events such as serious complications and/or death were reported. The currently available VVs, such as strains Lister and Dryvax, are known to be efficacious. However, severe adverse events, such as encephalitis, encephalopathy, progressive and generalized vaccinia, ocular vaccinia virus infections, and cardiac dysfunction, have been reported for recipients and are of great concern (1
). These observations suggest that LC16m8 is safer than the currently used VVs derived from bovine skin (20
). Thus, LC16m8 is considered a potentially useful replacement for the currently available VVs.
Unfortunately, the protective efficacy of LC16m8 against variola has not been evaluated, because variola had already been eradicated at the time of its development. 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
). 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
) 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
). Furthermore, the membrane protein B5R induces protective neutralizing antibodies to EEV in vaccinia (8
). Recently, it was reported that LC16m8 induced protective immunity to vaccinia virus challenge in mice and rabbits (19
). However, the protective efficacy of LC16m8 against variola and human MPX has not been confirmed in humans.
If LC16m8 is as efficacious as Lister and Dryvax, it will be of great benefit to humans, because it is expected to induce much less severe VV-associated adverse events. A nonhuman-primate model for MPXV infections is expected to mimic natural variola virus infection in humans. In the present study, the protective efficacy of LC16m8 against MPX was evaluated in comparison with that of Lister VV in cynomolgus monkeys (Macaca fascicularis). The present study was performed to examine the protective efficacy of LC16m8 against variola in humans.