2.5. Therapeutic Evaluation of CHIKV-EIG in Immunocompromised Mice

A total of thirty (30) 7–8-week-old gender-balanced A129 IFNαβR^−/− mice were obtained from the colony maintained at the University of Texas Medical Branch (Galveston, TX, USA). IFNαβR^−/− mice are an immunocompromised strain homozygous for the type 1 interferon receptor knock-out mutation that has been used extensively to study CHIKV pathogenesis and the efficacy of anti-CHIKV vaccines and therapeutics [21,24,62,63,64,65,66,67]. Mice were infected intradermally (i.d) in the right rear footpad with 10³ PFU of CHIKV strain 99,659. Treatment groups were administered CHIKV-EIG intra-peritoneally (i.p.) daily for five consecutive days, beginning 5 h or 1 day after infection (post-exposure), at a dose of 100 mg/kg/day in a final volume of 100 μL. Vehicle control animals were administered 100 μL PBS i.p. daily for five consecutive days, beginning 1 day post-infection (dpi). Animals were monitored daily for clinical signs of infection, body weight loss, and survival. Severely ill animals were closely monitored, and moribund animals were euthanized according to the test facility procedures. The primary endpoint for efficacy was survival at 21 dpi. Retro-orbital blood samples were collected from surviving female animals at 1, 3, and 9 dpi and surviving male animals at 2, 4, and 9 dpi for infectious virus quantification. Terminal serum samples from all animals were also collected by cardiac puncture for infectious virus quantification and anti-CHIKV antibody titers by PRNT₈₀ assay.

Viral load and antibody assessment in serum samples were performed as described above. Anti-CHIKV antibody titers in terminal serum samples from all animals surviving to the end of the study were assayed using PRNT₈₀, following methods described above [24].

Group sizes were calculated to provide at least 80% power when α = 0.05 for the primary endpoints of survival at 21 dpi, assuming no control animal survival and at least 60% survival in treated animals with no correction for multiple comparisons.