Rhesus macaques (Macaca mulatta) were housed at the New England Primate Research Center in accordance with standards of the Association for Assessment and Accreditation of Laboratory Animal Care and the Harvard Medical School Animal Care and Use Committee. The Harvard Medical Area Standing Committee on Animals approved the animal experiments. Procurement of rhesus macaque blood was conducted according to the principles of relevant national and international guidelines. The animals used in this study were healthy at the time of blood draw.
Cell culture and antibodies.
HEK293T and CEMx174 cells were obtained from the American Type Culture Collection. C8166 secreted alkaline phosphatase (C8166-SEAP) cells were generated previously (39
). HEK293T cells were maintained in complete Dulbecco's modified Eagle medium (DMEM) (DMEM supplemented with 10% fetal bovine sera, 100 U/ml penicillin G sodium, and 100 μg/ml streptomycin; Gibco BRL, Rockville, MD). CEMx174 and C8166-SEAP cells were maintained in complete RPMI medium (RPMI medium supplemented with 10% fetal bovine sera, 100 U penicillin G sodium, and 100 μg/ml streptomycin; Gibco). Cell lines expressing each rhesus monoclonal antibody (RhMAb) were maintained in RPMI medium supplemented with 20% fetal bovine sera and 100 μg/ml Primocin (InvivoGen, San Diego, CA).
Viral stocks were generated by transient transfection of a full-length SIV proviral vector in HEK293T cells. Briefly, 1.5 × 106 cells were plated in a T75 flask 24 h prior to transfection. For each viral stock produced, 5 μg of proviral DNA was transfected by the calcium phosphate method (Promega Corporation, Madison, WI). The medium was replaced 16 h after transfection. Two days later, the cell culture supernatant was passed through a 0.45-μm polyethersulfone syringe filter. Virus production was measured with a capsid protein (p27) antigen capture assay (Advanced Bioscience Laboratories, Inc., Kensington, MD).
Replication of SIVmac239 in the presence of HHA or GNA.
CEMx174 cells at a density of 1 × 106 cells per milliliter were infected with 30 ng/ml capsid protein (p27) of SIVmac239. The cultures were divided 24 h after infection, and the medium was replaced with medium that contained GNA or HHA at 0, 0.1, 1, 10, 100, or 200 μg/ml. Every 3 to 4 days, the cell cultures were split one to two with medium that contained the same concentration of either GNA or HHA. SIV p27 released into the supernatant was measured by antigen capture (Advanced Bioscience Laboratories, Inc.).
GNA and HHA selection of SIVmac239.
CEMx174 cells at a density of 1 × 106 cells per milliliter were infected with 30 ng/ml capsid protein (p27) of SIVmac239. The cultures were divided 24 h after infection, and the medium was replaced with medium that contained GNA or HHA at 0, 10, 100, or 200 μg/ml. Every 3 to 4 days, the cell cultures were split one to two with medium that contained the same concentration of either GNA or HHA. SIV p27 released into the supernatant was measured by antigen capture (Advanced Bioscience Laboratories, Inc.). For the second passage, CEMx174 cells were infected with 30 ng p27 of virus that grew from GNA at 100 μg/ml, GNA at 200 μg/ml, or HHA at 10 μg/ml. The medium was changed 24 h later to that which contained both GNA (100 μg/ml) and HHA (25 μg/ml). Every 3 to 4 days, the cell cultures were split one to two with medium that contained the same concentration of GNA and HHA. SIV p27 released into the supernatant was measured. Virus that grew from selection at passage 2 was used for a new infection (passage 3). The medium was changed 24 h after infection to medium that contained twice the concentration of GNA and HHA than that used in the previous passage. Virus was passaged in this manner until passage 6, when the selection medium used contained GNA at 1 mg/ml and HHA at 0.5 mg/ml. Virus was then passaged 3 more times in the presence of GNA (1 mg/ml) and HHA (0.5 mg/ml). The GNA/HHA-selected virus was passaged 9 times in the presence of lectin. As a control, SIVmac239 was passaged in CEMx174 cells concurrently with the lectin-selected population.
Isolation of RNA and env from the GNA/HHA-resistant population.
Viral RNA was isolated from culture supernatant using the MagMAX viral RNA isolation kit (Applied Biosystems, Foster City, CA). Briefly, 400 μl of culture supernatant containing SIV was incubated with 800 μl of a guanidinium thiocyanate-based lysis/binding solution and 20 μl of paramagnetic beads with a nucleic acid binding surface. The beads were washed three times before viral RNA was eluted. The envelope gene (env) sequence was amplified from isolated viral RNA using the SuperScript III One-Step reverse transcription-PCR (RT-PCR) system (Invitrogen, Carlsbad, CA). Primers used to amplify env were as follows: forward primer, 5′-GACGAGCGCTCTTCATGCATTTCAGAGG-3′; reverse primer, 5′-AAGCTTGCATGCTATAACACATGC-3′. The PCR product was then cloned with the TOPO XL PCR cloning kit (Invitrogen). Twenty env clones were sequenced (Retrogen Inc., San Diego, CA) and then aligned with the SIVmac239 sequence.
Construction of GNA/HHA-based SIV variant clones.
Mutant derivatives of the SIVmac239 proviral vector that introduced a glutamine codon in place of an asparagine codon were constructed in the following manner: (i) the codon at nucleotides (nt) 7333 to 7335 was changed from AAT to CAG, (ii) the codon at nt 7981 to 7983 was changed from AAC to CAA, (iii) the codon at nt 8029 to 8031 was changed from AAC to CAG, and (iv) the codon at nt 8476 to 8478 was changed from AAT to CAA. In addition, the codon that encoded asparagine at nt 7981 to 7983 was changed from AAC to TCA to encode serine and the glycine codon (GGA) at nt 8026 to 8028 was changed to GAG to encode glutamic acid. The nucleotide number corresponds to the original published sequence of SIVmac239 (47
). To introduce changes to the desired codons, the 3′ half of the SIVmac239 genome was used as a template for PCR site-directed mutagenesis. For each single mutant, complementary and reverse-oriented mutagenic primers containing one of the above-listed nucleotide changes compared to the SIVmac239 sequence were designed. Multiple-round PCR using PfuUltra II Fusion HS DNA polymerase (Stratagene, Cedar Creek, TX) incorporated the mutation from the primers into the 3′ SIVmac239 plasmid. Double mutants were generated by sequential PCRs. Following amplification, the entire coding region of the 3′ half of the SIVmac239 genome was sequenced to confirm that only the intended changes were introduced. Sequence-confirmed variants were then digested with SphI and XhoI and ligated to the 5′ half of SIVmac239. Full-length clones were confirmed by restriction digestion.
SIVmac239 and each GNA/HHA-based virus stock were used to infect an immortalized human T-cell line (C8166-45) with a stably integrated, Tat-inducible SEAP gene as described previously (39
). Briefly, 25 ng p27 for each virus was serially diluted 2-fold in RPMI complete medium. Virus from each dilution (100 μl) was added to 5,000 C8166-SEAP cells in 100 μl medium. Three days later, SIV infection of C8166-SEAP cells was determined by measuring the production of SEAP in the culture medium. SEAP activity in the supernatant was measured using a Phospha-Light assay system (Applied Biosystems).
Replication of SIV in rhesus PBMCs.
Rhesus macaque peripheral blood mononuclear cells (PBMCs) were isolated from fresh citrate-containing blood of a rhesus macaque using lymphocyte separation medium (LSM; MP Biomedicals, LCC, Solon, OH). PBMCs were resuspended at 1 × 106 cells per milliliter in RPMI medium supplemented with 20% fetal bovine serum and were activated for 72 h with 1 μg/ml of phytohemagglutinin (Sigma, St. Louis, MO). For analysis of viral replication in culture, activated PMBCs were washed two times with RPMI medium supplemented with 20% fetal bovine serum (FBS) and 10% interleukin-2. Cells at a density of 1 × 106 cells per milliliter were infected with SIVmac239 containing 30 ng/ml capsid protein (p27). The culture was divided in half 24 h later. PBMCs from one half of the culture were resuspended with RPMI medium supplemented with 20% FBS and 10% interleukin-2. PBMCs from the other half of the culture were resuspended with RPMI medium supplemented with 20% FBS, 10% interleukin-2, 1 mg/ml GNA, and 0.5 mg/ml HHA. One-half of the culture supernatant was replaced every 3 to 4 days, and the concentration of p27 capsid protein in the supernatant was measured using a SIV p27 antigen capture assay (Advanced BioScience Laboratories, Inc.).
c.o. SIV gp120 expression vectors.
The codon-optimized (c.o.) SIVmac239 Env expression vector (50
) was modified using PCR mutagenesis to introduce two stop codons after the arginine codon at nt 8176 to 8178 in the SIVmac239 genome. These changes were made in the c.o. expression vector with the forward primer 5′-GAACAAGCGGTGATAATTCGTCCTGG-3′ together with a reverse-oriented mutagenic primer (c.o.SIVmac239 gp120). In addition the SIVN2 variant was made using PCR mutagenesis in the context of the c.o.SIVmac239 gp120 expression vector. c.o.PBj14 clone 6.6 was synthesized (GenScript, Piscataway, NJ) based on the genomic sequence (accession number L09212.1). SIVsmE543.3 gp120 was cloned from the c.o.SIVsmE543.3 expression vector, provided by Vanessa Hirsch. For both SIVsmPBj14 6.6 and SIVsmE543.3, env
was amplified with primers that contained the appropriate restriction site. env
was then cloned via the NheI and ApaI cut sites into the cytomegalovirus (CMV)-driven mammalian expression vector pcDNA3.1(+) (Invitrogen).
ELISA protein production.
The c.o. SIV gp120 constructs and the c.o. HIV-2 gp120 constructs were used to transfect HEK293T cells using the GenJet method (SignaGen Laboratories, Ijamsville, MD). The medium was replaced 24 h later with serum-free DMEM. Two days later, culture supernatant was passed through a 0.45-μm polyethersulfone syringe filter. Filtered supernatant was incubated with concanavalin A-agarose beads (Sigma) for 24 h at 4°C. The beads were washed sequentially with 10 ml phosphate-buffered saline (PBS). The last wash was determined when the eluent reached an optical density less than 0.03. Then, gp120 was eluted from the beads with 18 ml of 0.75 M methyl manno-pyranoside in PBS. The samples were concentrated using Vivaspin20 columns with a 50-kDa cutoff (Fisher, Pittsburgh, PA). Each sample was dialyzed against PBS. Following dialysis, protein was recovered and total protein was determined with a bicinchoninic acid assay (Pierce, Rockford, IL). Recombinant histidine-tagged SIVmac239 gp120, SIVmac239 gp140, SIVsmE660 gp120, SIVsmE660 gp140, HIV-1 Bal gp120, HIV-1 JRCSF gp120, HIV-1 p1006_11.C3.1601 gp120, HIV-1 consensus B gp120, HIV-1 consensus A2 gp120, HIV-1 consensus C gp120, SIVcpzEK505 gp120, and SIVcpzMB66 gp120 produced from HEK293 cells were purchased from Immune Technology Corp., New York, NY.
A normalized amount of total protein was loaded into six wells of a high-protein-binding 96-well plate (Fisher). After two washes with 0.05% Tween 20 in PBS, the wells were blocked for 1 h at 37°C with carbohydrate-free blocking reagent (Vector Laboratories, Burlingame, CA). Wells were washed 5 times with 0.05% Tween 20 in PBS. Then, 50 μl of GNA-horseradish peroxidase (HRP) (US Biological, Swapscott, MA) diluted 1,000-fold in carbohydrate-free blocking buffer was added to four of the test wells. For the case of SIV and HIV proteins purchased from Immune Technology Corp., 50 μl of His-HRP antibody (Abcam, Cambridge, MA) was diluted 1:100 and added to duplicate wells of each sample as a normalization control. SIVgp120 proteins that did not include the His tag were normalized with polyclonal sera from SIVmac239-positive rhesus macaques. Serum IgGs were detected with anti-rhesus-HRP antibody (Southern Biotech, Birmingham, AL). Following a 1-hour incubation with the secondary antibody, wells were washed 10 times with 0.05% Tween 20 in PBS, and then 50 μl of fresh soluble TMB (3,3′,5,5′-tetramethylbenzidine; EMD Chemicals, Gibbstown, NJ) was added to each well. Wells were allowed to develop for 30 min before the reaction was stopped. Plates were read at 450 nm on an MRX Revelation (Dynex Technologies, Chantilly, VA).
Statistical significance of GNA binding.
Prism software (GraphPad Software, Inc., La Jolla, CA) was used to run an unpaired t test to calculate the significance of the difference in GNA binding capacities between SIVmac/sm and HIV-1. The unpaired t test method tests the null hypothesis that the population means relating to two independent random samples from an approximately normal distribution are equal.
Sensitivity of the infection of GNA/HHA-based viral variants SIVmac239 and SIV316 to neutralization by sera from SIVmac239-infected rhesus macaques, soluble CD4 (sCD4), and monoclonal antibodies that bind SIV Env was measured using C8166-SEAP cells as previously described (39
). Briefly, virus equivalent to 2 ng capsid protein (p27) for SIVmac239 and each GNA/HHA-based SIV variant and 5 ng of SIV316 was used to infect C8166-SEAP cells in the absence and presence of one of the following neutralizing agents: a pool of sera collected from four rhesus macaques infected with SIVmac239 for greater than 6 months, sCD4, and monoclonal antibodies that bind SIV Env. To perform neutralization assays, each virus was set up in a 96-well plate as follows: three wells in the second column contained 100 μl of RPMI complete medium, three wells in the third column contained 25 μl of complete medium, and three wells in each of columns 4 through 11 contained 25 μl of each of 8 dilutions of the neutralizing agent. Sera from SIVmac239-infected rhesus macaques were pooled and then heat inactivated at 56°C for 30 min before use in neutralization assays. Each virus in a total volume of 75 μl was added to each of the wells containing 25 μl of either medium or diluted sera, and the plate was incubated in 5% CO2
at 37°C for 1 h. After the 1-h incubation, 5,000 C8166-SEAP cells in a volume of 100 μl were added to each well in columns 2 through 11 of the 96-well plate. SEAP activity in the culture supernatant was measured 3 and 5 days later using the chemiluminescent Phospha-Light assay system (Applied Biosystems) and detected using a Victor V multilabel counter (Perkin-Elmer; Waltham, MA). Neutralization activity for all antibodies and serum samples was reported as a percentage of SEAP activity. The percentage of SEAP activity was calculated as follows: SEAP activity from cells alone (column 2) was averaged and subtracted from that for each of the wells in columns 3 through 11. Then the counts from each column were divided by those for column 3 and multiplied by 100.