Tick-borne Langat/Mosquito-Borne dengue flavivirus chimera, a candidate live attenuated vaccine for protection against disease caused by members of the tick-borne encephalitis virus complex: Evaluation in rhesus monkeys and in mosquitoes

Langat virus (LGT), strain TP21, a naturally avirulent tick-borne flaviviru s, was used to construct a chimeric candidate virus vaccine which contained LGT genes for premembrane (preM) and envelope (E) glycoprotein and all oth er sequences derived from dengue type 4 virus (DEN4). The live virus vaccin e was developed to provide resistance to the highly virulent, closely relat ed tick-borne flaviviruses that share protective E epitopes among themselve s and with LGT. Toward that end the chimera, initially recovered in mosquit o cells, was adapted to grow to high titer in qualified simian Vero cells. When inoculated intraperitoneally (i.p.), the Vero cell-adapted LGT TP21/DE N4 chimera remained completely attenuated for SCID mice. Significantly, the chimera protected immunocompetent mice against the most virulent tick-born e encephalitis virus (TBEV). Subsequently, rhesus monkeys were immunized in groups of 4 with 10(5) or 10(7) PFU of LGT strain TP21, with 105 PFU of DE N4, or with 10(3),10(5), or 10(7) PFU of the chimera. Each of the monkeys i noculated with DEN4 or LGT TP21 became viremic, and the duration of viremia ranged from 1 to 5 days. In contrast, viremia was detected in only 1 of 12 monkeys inoculated with the LGT TP21/DEN4 chimera; in this instance the le vel of viremia was at the limit of detection. All monkeys immunized with th e chimera or LGT TP21 virus developed a moderate to high level of neutraliz ing antibodies against LGT TP21 as well as TBEV and were completely protect ed against subsequent LGT TP21 challenge, whereas monkeys previously immuni zed with DEN4 virus became viremic when challenged with LGT TP21. These obs ervations suggest that the chimera is attenuated, immunogenic, and able to induce a protective immune response. Furthermore, passive transfer of serum from monkeys immunized with chimera conferred significant protection to mi ce subsequently challenged with 100 i.p. 50% lethal doses of the highly vir ulent TBEV. The issue of transmissibility of the chimera by mosquitoes was addressed by inoculating a nonhematophagous mosquito, Toxorhynchites splend ens, intrathoracically with the chimera or its DEN4 or LGT parent. Neither the LGT TP21/DEN4 vaccine candidate nor the wild-type LGT TP21 virus was ab le to infect this mosquito species, which is highly permissive for dengue v iruses. Certain properties of the chimera, notably its attenuation for monk eys, its immunogenicity, and its failure to infect a highly permissive mosq uito host, make it a promising vaccine candidate for use in immunization ag ainst severe disease caused by many tick-borne flaviviruses.