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).