Materials and Methods
The gene encoding the H5 hemagglutinin from H5N1 (Vietnam) subtype avian influenza virus (accession number: EF541402.1) was
codon-optimized for expression in Tetrahymena and introduced into the macronuclear genome, either on high-copy number rDNA vectors, or in the somatic MTT1 metallothionein
gene locus by homologous recombination.16 In both instances, the transgene was under the control of the endogenous MTT1 promoter.17 For the constitutively secreted gene product, the region encoding 48 amino acids at the C-terminus of the protein was deleted.
For targeting to mucocysts, the truncated H5 gene was tagged with a sequence encoding one of several granule lattice proteins
of Tetrahymena. The gene encoding the IAG4B[G1] i-antigen gene of Ichthyophthirius multifiliis (accession number: AAD31283) was introduced into cells, either at the b-tubulin 1 (BTU1) locus under MTT1 promoter control,
or on high copy number rDNA vectors as above. For the truncated gene product, the region encoding the C-terminal 19 amino
acids of the IAG48[G1] gene was removed. Cells were transformed biolistically and positive transformants selected by growth
in paromomycin.18 Cells were grown to ~5 x 105 cells/mL in Neff medium and treated with 2 μg/mL CdCl2 for varying periods of time to induce expression of the transgenes. In the case of granule-targeted H5, cells were washed
and mucocyst discharge induced by adding dibucaine to a final concentration of 2 mM.14 The PRISM matrix was then harvested for further analysis by centrifugation at 6,000g for 5 min and transferred to fresh tubes.
To prepare monoclonal antibodies (MAbs) recognizing H5, BALB/c mice were injected intraperitoneally with killed reassortant
A/Vietnam/1203/2004 X PR8 (H5N1; 105 HAU) mixed with Freund's incomplete adjuvant (FIA). One to four months later, mice were
injected intravenously with 6–9 x 104 HAU virus in phosphate-buffered saline (PBS). After three days, mice were euthanized and spleens were collected. Lymphocytes
were harvested and fused to SP2/0-AG14 mouse myeloma cells using conventional methods.19 Mice were purchased from Harlan (Indianapolis, IN) and housed in the James A. Baker Institute vivarium according to the
guidelines of the American Association of Laboratory Animal Care. Hybrid cells secreting antibodies specific for virus were
identified by testing supernatants in hemagglutination inhibition (HI) assays, according to Barret and Inglis.20 Supernatants from positive wells were tested in ELISA for binding to virus using a peroxidase-conjugated anti-mouse IgG
as previously described.21 Supernatants were tested for binding to allantoic fluid (1:5 dilution) as a negative control. Positive hybrid cells were
cloned by limiting dilution on mouse peritoneal exudate cells. Cloning was repeated until 100% of single colony wells tested
positive in ELISA and HI. Antibodies were isotyped by modifying the ELISA, adding rat anti-mouse isotype reagents detected
with peroxidase conjugated goat antibodies specific for rat kappa chain (BD Pharmingen, San Diego, CA).
Western blotting of whole cell lysates and cell fractions was carried out using standard protocols22 and signals generated using Super signal West Pico Luminol/Enhancer Solution and Stable Peroxide Solution, mixed 1:1 (Pierce
Biotechnologies), before image capture either on film (Kodak BioMAX MS) or with a CCD camera (Chemigenius; BioRad). Primary
antibodies in all cases were dilutions of hybridoma culture supernatant fractions. Expression was quantitated by densitometry
using internal software on the Chemigenius.