The DNA vaccine combination (pComb) and the pNP-only plasmid vaccine used in this study have been described previously (18
). Briefly, consensus sequences were generated by aligning multiple primary sequences (obtained from the Los Alamos National Laboratory influenza sequence database) and choosing the most common amino acid at each position to generate a sequence not necessarily found in nature (synthetic) but which retained characteristics of the component sequences chosen. Sequences were then optimized for codon usage, RNA structure, and GC content and were synthesized. Synthetic genes were then subcloned into a pVax expression vector (Invitrogen). For the NHP studies, DNA preparations were made at VGX Pharmaceuticals, Inc. (The Woodlands, TX), as previously described (11
) and formulated at 10 mg/ml in water plus 1% (wt/wt) poly-l
-glutamate sodium salt.
The high-pathogenicity avian influenza virus strain A/Vietnam/1203/2004 (H5N1), which was used for the monkey challenge, was obtained from the Centers for Disease Control and Prevention (Atlanta, GA). Stock virus was expanded in the allantoic cavities of 10-day-old embryonated chicken eggs at 37°C for 24 h and stored at −70°C. The 50% egg infective dose of this virus stock was 108.7/ml. All experiments with the high-pathogenicity avian influenza virus were conducted in the Bioqual, Inc., biosafety level 3+ containment facility (Rockville, MD), approved for use by the U.S. Department of Agriculture and the CDC. The low-pathogenicity avian influenza virus H5N1-PR8 reassortant strains used for the hemagglutination inhibition (HAI) assay were obtained from the CDC or from NIBSC, Hertfordshire, United Kingdom. Experiments with the low-pathogenicity avian influenza virus strains were conducted in a biosafety level 2+ containment facility approved for use by the USDA and the CDC. Seed stocks were expanded in 10-day-old embryonated chicken eggs at 35 to 37°C for 48 h and stored at −70°C.
Rhesus macaques were housed at Bioqual, Inc., in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care. Animals were allowed to acclimate for at least 30 days in quarantine prior to any immunization. Four groups of five rhesus macaques were immunized at weeks 0, 4, and 8 with 1 mg/construct (at a concentration of 10 mg/ml) of pVax (intramuscularly [i.m.]); pH5HA, pNP, and pN1NA (i.m.); pH5HA, pNP, and pN1NA (intradermally [i.d.]); and pNP (i.m.). DNA was delivered into the quadriceps muscle (i.m.) or skin (i.d.), followed by in vivo electroporation. Square-wave pulses were used in all experiments and administered by using an adaptive constant current electroporator, the Cellectra device (VGX Pharmaceuticals, Inc., The Woodlands, TX). Two types of arrays were used. The electrode array used for i.m. electroporation is a circular array (1-cm diameter) of five equally spaced 21-gauge solid stainless steel needle electrodes mounted on a nonconductive material. All i.m. electroporation immunizations were performed at 0.5 A, with 3 pulses at 52 ms/pulse and 1-s intervals between pulses. The i.d. microelectrode array consisted of three 26-gauge solid stainless steel needle electrodes, 3 mm in length, placed in an isosceles triangle formation (the two long sides are 5 mm in length, and the short side is 3 mm in length) and mounted on nonconductive material. All i.d. electroporation immunizations were performed at 0.2 A, with 2 pulses at 52 ms/pulse and 1-s intervals between pulses, followed by a 3-s rest period and another 2 pulses under identical conditions (2 by 2 pulse pattern).
Animals were bled every 2 weeks. Ten milliliters of blood was collected in EDTA tubes, and peripheral blood mononuclear cells were isolated by standard Ficoll-Hypaque centrifugation and resuspension in complete culture medium (RPMI 1640 with 2 mM/liter l-glutamine, 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 55 μM/liter β-mercaptoethanol). Red blood cells were lysed with ammonium chloride-potassium (ACK) lysis buffer (Cambrex BioScience, East Rutherford, NJ).
Sera were treated with receptor-destroying enzyme by diluting 1 part serum with 3 parts enzyme and were incubated overnight in a 37°C water bath. The enzyme was inactivated by a 30-min incubation at 56°C, followed by the addition of 6 parts phosphate-buffered saline for a final dilution of 1/10. HAI assays were performed in V-bottomed 96-well microtiter plates, using 4 hemagglutination units of virus and 1% horse red blood cells as previously described (27
). Viruses used for the HAI assay are reassortant strains obtained from either the influenza branch of the CDC (Atlanta, GA) [clade 1, A/Vietnam/1203/2004 (H5N1)/PR8-IBCDC-RG; clade 2.1, A/Indonesia/05/2005 (H5N1)/PR8-IBCDC-RG2; and clade 2.3.4, Anhui/01/2005/PR8-IBCDC-RG5] or from NIBSC (United Kingdom) (clade 2.2, A/turkey/Turkey/1/2005/NIBRG-23 [reference strain]).
Neutralizing antibody activity was analyzed in a microneutralization assay based on the methods of the pandemic influenza virus reference laboratories of the CDC (24
). All sera were treated with a receptor-destroying enzyme overnight, followed by heat inactivation. Low-pathogenicity H5N1 viruses (A/Vietnam/1203/2004 [SJCRH, clade 1]; A/Indonesia/5/2005 [PR8-IBCDC-RG2, clade 2.1]; A/turkey/Turkey/1/2005 [NIBRG-23, clade 2.2]; and A/Anhui/1/2005 [IBCDC-RG5, clade 2.3.4]), generated by reverse genetics, were obtained from St. Jude's Hospital (Memphis, TN), the CDC, and NIBSC.
Primates were sedated as previously described for the challenge and placed in dorsal recumbency, and the mouth was opened manually. The epiglottis was opened with a laryngoscope, and a syringe containing 2 ml of sterile, buffered, physiological saline used for washing was attached to a sterile tube 2 to 3 mm in diameter and inserted into the epiglottis. Two milliliters of sterile saline solution was pushed through the tubing and then collected and aliquoted into two vials.
Enzyme-linked immunospot (ELISPOT) assays were conducted as previously described (17
). Briefly, ELISPOT 96-well plates (Millipore, Billerica, MA) were coated with anti-human (clone GZ-4; Mabtech, Cincinnati, OH) gamma interferon (IFN-γ) capture antibody and incubated overnight at 4°C. The following day, plates were washed with phosphate-buffered saline and blocked for 2 h with R10. Peripheral blood mononuclear cells from each group were added to each well and stimulated overnight at 37°C in the presence of R10 peptide (negative control) or specific peptide antigens (10 μg/ml) (Invitrogen, Carlsbad, CA). The peptide pools consisted of 15-mer peptides overlapping by 11 amino acids. After 24 h of stimulation, the cells were washed and incubated for 24 h at 4°C with anti-human (clone 7-B6-1; Mabtech) IFN-γ capture antibody. The plates were washed, streptavidin-alkaline phosphatase (R&D Systems, Minneapolis, MN) was added to each well, and the mixtures were incubated for 2 h at room temperature. The plates were washed, and BCIP (5-bromo-4-chloro-3-indolylphosphate)/Nitro Blue Tetrazolium chromogen (R&D Systems, Minneapolis, MN) was added. The plates were then rinsed with distilled water and dried. Spots were counted by an automated ELISPOT reader (Cellular Technology Limited, Shaker Heights, OH).
Virus (A/Vietnam/1203/2004) was diluted in L-15 tissue culture medium to a concentration of 1 × 106 50% egg infectious dose per ml. One milliliter of the virus dilution was inoculated into the trachea and 0.5 ml into each nostril. Animals were sedated with ketamine hydrochloride (10 mg/kg i.m.). For the intratracheal inoculation, the animal was placed in dorsal recumbency, and the mouth was opened manually. The epiglottis was opened with a large laryngoscope, and the small end of a sterile French rubber feeding tube (size 10, cut to 4 in. in length) was inserted into the glottis with the inoculation syringe attached. Once in place, the inoculum (1 ml) was injected into the trachea. The catheter was left in place while the inoculum syringe was removed and replaced with a syringe containing 2 ml of sterile, buffered physiological saline used for washing the catheter to ensure that all of the inoculum had been introduced into the trachea. For the i.n. inoculation, 0.5 ml inoculum was administered with a sterile luer-tip syringe introduced approximately 3 to 5 mm into each nostril. Body weights, temperature, and food intake were measured, and clinical observations were recorded.
Determination of viral titers.
For RNA isolation, tracheal lavage samples were spun down at 10,000 × g for 1 h, liquid was poured off, and 1 ml of RNA Stat-60 (IsoTex Diagnostics, Friendswood, TX) was added. Samples were then incubated at room temperature for 5 min and resuspended in 250 μl of chloroform by vortexing. The samples were spun down at 10,000 × g for 1 h, the aqueous top layer was removed, 0.5 ml of isopropanol and 10 μl of tRNA (10 μg/ml) were added, and the mixture was precipitated overnight at −20°C. Samples were spun down for 1 h, washed with cold 75% ethanol, and spun again for another hour. RNA was resuspended in 30 μl RNase-free water. For real-time PCR, 10% RNA was added to TaqMan reagents (Applied Biosystems, Foster City, CA) along with primers and probe (listed below) and amplified in a 7700 sequence detection system (Applied Biosystems). Briefly, the sample was reverse transcribed at 48°C for 30 min, held at 95°C for 10 min, and then run for 40 cycles at 95°C for 15 s and at 60°C for 1 min. The signal was compared to a standard curve of known concentrations of RNA, from 106 copies/ml down to 1 copy/ml, and multiplied by 10, giving a detection range from 20 to 107 copies/ml. All samples were run in triplicate. The primers and probe were designed to bind to a highly conserved region on the nucleoprotein gene. The primer sequences were VIETA-U, 5′-CGT CTC AAG GCA CCA AAC G-3′, and VIETA-D, 5′-GTA GAA CCT CCC AAT GCC AC-3′. The probe sequence was VIETA-P, FAM-GGA ACG CCA GAA TGC TAC TGA GAT CAG GGC-TAMRA, where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine.
Early deaths did not occur among the animals in the study; all animals survived to the scheduled necropsy time point at 10 days after challenge. Animals were euthanized via sodium pentobarbital overdose (>100 mg/kg, intravenously). At termination, a complete gross necropsy was conducted by testing facility personnel. Tissues were preserved in 10% neutral buffered formalin and sent to Pathology Associates International's Frederick, MD, facility for processing and histopathological evaluation. Appropriate tissues were trimmed, processed, embedded in paraffin, sectioned at approximately 5 μm, and stained with hematoxylin and eosin. The resulting glass slides were examined by a board-certified veterinary pathologist, and all microscopic pathology findings were directly entered into a Microsoft Office Excel (version 2003 SP2) spreadsheet. Histopathological evaluations were performed by the undersigned veterinary pathologist on the livers, spleens, tracheas, tonsils, hearts, and lungs from the 20 macaques. For each animal, at least 5 fields were analyzed; the analyzer was blinded to animal group. The sections were visualized, using a Zeiss Axioplan 2 microscope with a 10× objective. Digital images of the slides were captured, using a CoolSnap digital color camera (Roper Scientific, Tucson, AZ) equipped with MetaMorph software (Universal Imaging Corporation, Downington, PA).
Statistical analysis of the data was performed using Microsoft Excel or GraphPad Prism software. Data analysis was carried out with treatment comparisons using the Wilcoxon signed-rank test or one-way analysis of variance, where statistically significant results were defined as having a P value of less than 0.05. The viral load comparison between the groups at each time point was performed using the unpaired Student's t test, where statistically significant results were defined as having a P value of less than 0.05. Correlations were established by using the Pearson test.