2.6. Passive Transfer Studies

Pooled serum containing 750 µg of total IgG (quantified by IgG ELISA, Invitrogen, Carlsbad, CA) harvested from intramuscularly ΔgD-2 or control (VD60) vaccinated C57Bl/6 mice was inoculated intraperitoneally into naïve WT or C1q-knockout BL/6 mice 24 h prior to skin challenge with HSV-2(4674) (5 × 10 PFU/mouse [13] ), as previously described [18]. Mice were then monitored daily for epithelial and neurological disease and scored as follows: (1) erythema at inoculation site; (2) spread to distant site, zosteriform lesions, edema; (3) ulcerations, epidermal spread, limb paresis; (4) hind limb paralysis and (5) death. Mice were euthanized at a score of 4 and assigned a score of 5 the following day. At the time of euthanasia (when mice succumbed or day 14 post-challenge), sacral nerve tissue was collected and DNA isolated using the Qiagen Blood and Tissue DNA isolation kit (Qiagen, Hilden, Germany). A total of 10 ng of DNA per sample was loaded, and primers and probes specific for HSV polymerase were used to quantify HSV DNA (forward primer sequence, 5′-GGCCAGGCGCTTGTTGGTGTA-3′; reverse primer sequence, 5′-ATCACCGACCCGGAGAGGGA-3′; probe sequence, 5′-CCGCCGAACTGAGCAGACACCCGC-3′). Mouse β actin was used as a loading control (Applied Biosystems, Foster City, CA, USA), and qPCR was run in an Applied Biosystems QuantStudio 7 Flex.