Recombinant chimeric horsepox virus (TNX-801) is attenuated relative to vaccinia virus strains in both in vitro and in vivo models.

Recombinant chimeric horsepox virus (TNX-801) is a preclinical vaccine in development against mpox and smallpox. In this report, we investigated the potential phenotypic differences in in vitro and in vivo models between TNX-801 and older vaccinia virus (VACV)-based vaccine strains (VACV-Lis and VACV-NYCBH) used in the eradication of smallpox as well as VACV-WR, VACV-IHD, and MVA. TNX-801 displayed a small plaque phenotype (~1-2 mm) in BSC-40 and Vero-E6 cells. Multi-step replication kinetics in immortalized nonhuman primate cell lines, and human primary cells from dermal and respiratory tracts yielded >10- to 100-fold lower infectious titers than the VACV strains. In addition, the infectious particle-to-genome copy ratio data suggests that TNX-801 genome packaging is ~10- to 100-fold less efficient than the VACV strains and the potential mechanism of TNX-801 attenuation is at the packaging/egress stage. Lastly, the susceptibility to VACV and TNX-801 infection of three new immunocompromised murine models (C56BL/6 Ifnar^-/-, C56BL/6 Ifngr^-/-, and C56BL/6 Ifnar^-/-/Ifngr^-/-) was investigated. VACV strains were able to produce severe disease including decrease in body weight and temperature, as well as lethality in murine models via the intraperitoneal or intranasal routes. In contrast to VACV strains, TNX-801 was unable to produce any disease in murine models. These data demonstrate that TNX-801 is >10- to 1,000-fold more attenuated compared to older VACV-based smallpox vaccine strains in human primary cell lines and immunocompromised mice.

Importance: Variola and monkeypox viruses are medically important pathogens that can cause fatal human disease. The two FDA-approved vaccines, ACAM-2000 and JYNNEOS, have important advantages and disadvantages. ACAM-2000 offers durable immunity; however, it has high adverse event rates. In contrast, JYNNEOS has a safer profile but requires two doses 4-weeks apart to achieve comparable immunity. Consequently, there is a need for vaccines offering durable immunity via single immunization with minimal adverse events. TNX-801 is a preclinical stage vaccine that can stimulate potent immunity via a single dose and provides protection against lethal mpox disease in the nonhuman primate model. Here, we show that TNX-801 is >10- to 1,000-fold attenuated in in vitro and in vivo models including human primary cells and immunocompromised murine models than vaccine strains utilized in smallpox eradication. The natural attenuation of TNX-801 and its ability to induce protective immunity via a single vaccination are promising and warrants further development.