Langat Virus Strain TP21/DEN4 (LGT/DEN4) Chimera Is Attenuated, Immunogenic And Protects Rhesus Monkeys Against LGT TP21 Challenge. Rhesus monkeys (Macacca mulatta) were inoculated subcutaneously (SC) with 1000, 100,000 or 10,000,000 PFU of TP21/DEN4 chimera, 100,000 or 10,000,000 PFU of wild-type LGT TP21 or 100,000 PFU of DEN4. Monkeys inoculated with TP21 developed viremia that lasted 1 to 5 days. Significantly, viremia was not detected in monkeys that received 1000 or 100,000 PFU of chimera. Monkeys inoculated with 1000 or 100,000 PFU of chimera developed a moderate to high titer of TP21 and TBEV neutralizing antibodies. On day 43 post-immunization, each of the immunized monkeys was challenged by the SC route with 100,000 PFU of LGT TP21. During the 12 days post-challenge viremia was not detected in monkeys previously immunized with chimeric virus or its TP21 parent, whereas all of the monkeys previously inoculated with DEN4 developed viremia lasting 2 to 4 days. This study indicated that, even at the lowest dose (1000 PFU), the chimera induced an immune response to LGT preM and E structural proteins that completely protected animals from TP21 virus challenge. We were unable to evaluate protective efficacy of the chimeric vaccine by direct challenge of the monkeys with the highly virulent TBEV, because a challenge inoculum that consistently causes fatal encephalitis in monkeys was not available in the laboratory of either of our collaborators in Moscow or Frederick, Maryland (Fort Detrick). However, in collaboration with the US Army laboratory (USAMRIID) at Fort Detrick, we evaluated the ability of serum from vaccinated monkeys to passively protect mice from TBEV challenge. Groups of female BALB/c mice were injected SC with 100 microliters of pooled sera from a group of 4 immunized monkeys and mice were subsequently challenged IP with 100 IP LD50 of TBEV, strain Sofjin. Sixty percent protection from the TBEV challenge was provided by sera from the monkeys that were immunized with 10,000,000 PFU of the chimera or 100,000 PFU of TP21. These monkeys had developed the highest neutralizing antibody response to both LGT and TBEV. None of the mice inoculated with pooled sera from the other groups of monkeys survived the TBEV challenge. In another experiment, groups of mice received 100 microliters of pooled sera twice, 16 hrs before TBEV challenge and 6 days after TBEV challenge. All of the mice that received sera from monkeys inoculated with 100,000 PFU of TP21 and 80% of mice that received sera from monkeys 10,000,000 PFU of chimera remained healthy after TBEV challenge. None of the mice in the other groups survived TBEV challenge. These data are encouraging and suggest that the chimeric vaccine may offer protective immunity to primates. High level of attenuation for mice and monkeys, and satisfactory immunogenicity and protective efficacy of the chimeric LGT TP21/DEN4 virus makes this chimera a promising vaccine candidate that should be evaluated in humans.
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