L1O2 antigen constructs
LINE-1.3 (L19088) (46
) was used for human constructs (hL1O2). A L1O2 consensus sequence of eight hot L1 elements identified by similarity to L1.3 (47
) was used to probe the mouse genome for similar intact L1O2 genes (O88913, O88914, O88915, Q792I9, Q91Z88, Q91Z89, Q9QUI2, Q9QWY0, Q9QWY2 & Q9QWY3), which were used to produce a mL1O2 consensus sequence. Amino acid substitutions D205A in the endonuclease domain, and D702A in the reverse transcriptase (RT) domain were made in both hL1O2 and mL1O2 by site-directed mutagenesis to prevent enzymatic functions. Full-length (FL) L1O2 as well as L1O2 fragment 1 (Fr1) covering amino acids 1–400, including the endonuclease domain; Fr2 covering amino acids 401–800 including the RT domain; and Fr3 covering amino acids 801–1275, were synthesized for both hL1O2 and mL1O2. Alignments of L1O2 were made with the draft rhesus genome sequence, but large gaps were found in the RT domain and the endonuclease domain was missing. Investigation of the translated nucleotide database for rhesus macaques using tBLASTn identified 47 nucleotide sequences for rhesus L1O2. A consensus sequence of these had a predicted amino acid sequence identity of 92% (96% positivity according to the NCBI BLOSUM62 scoring matrix) with hL1O2. We opted to use the hL1O2 sequence in macaque vaccination studies on the grounds of its known provenance and high conservation with the expected rhesus form.
SERV-K antigen constructs
tBLASTn and a library of HERV-K(HML-2) RT peptide sequences were used to screen build 36.3 of the Celera genome assembly. PERL scripts were used to extract, de-fragment and align matches corresponding to proviral insertions containing both LTRs. The alignments constructed were edited manually in Se-Al (http://tree.bio.ed.ac.uk/software/seal/
) to construct consensus ORFs. A second round of screening was then performed, in which the consensus gag
ORFs were used to BLAST search the human genome. Reading frames that approximated the expected size were considered potentially intact and were manually inspected in Se-Al. Phylogenetic screening of the macaque genome identified an ERV family closely related to HML-2 (48
). Five intact (or nearly intact) SERV-K (HML-2) gag
genes (NC_007858.1, NC_007864.1, NC_007864.1_2, NC_007875.1 and NC_007876.1) and four intact (or nearly intact) env
genes (NC_007868, NC_007858, NC_007862 and AC200900_BAC) were identified in the macaque genome, and used to construct consensus SERV-K (HML-2) gag
Genes were synthesized in their native (L1O2) or codon-optimized form (ERV-K), at GeneArt, and cloned into pPJV7563 as described previously (49
). DNA vaccines were precipitated onto gold beads as described (50
). The control vaccine plasmid was vector backbone only. For rhesus macaque experiments, the antigen plasmids were co-precipitated onto gold beads at a 9:1 ratio together with pPJV7563-encoded rhesus GM-CSF as described (49
). For recombinant adenovirus serotype 5 (rAd5) production, the genes were cloned into pShuttle-CMV and recombined with the Ad5 genome using the AdEasy™ system (Q-Biogene Inc, Carlsbad, CA). Control rAd5 vectors encoded eGFP. Final production and purification of rAd5 vaccines was performed by ViraQuest Inc. (North Liberty, IA).
Anti-HERV-K Env mouse mAb HERM-1811-5 was obtained from Austral Biologics (San Ramon, CA). Antibodies capable of binding human, macaque and mouse L1O2, or both HERV- and SERV-K Gag, were derived by affinity-purification from peptide-KLH hyper-immunized New Zealand White rabbit serum by Lampire (Pipersville, PA) for hL1O2, and Cambridge Research Biochemicals, (Billingham,UK) for HERV-K Gag. Surface-accessible, immunogen peptides were selected using Protean software from DNASTAR-Lasergene 6. For hL1O2 an anti-RT 781–800 FKENYKPLLKEIKEETNKWK peptide (90% conserved with the predicted macaque sequence) was selected. For HERV-K Gag, peptides were selected from p15: 229–250 ENKTQPPVAYQYWPPAELQYR (92% conserved with the SERV-K p15), and capsid (CA) 337–360 KSFSIKLLKDLKEGVKQYGPNS (96% conserved with the SERV-K CA). Peptide synthesis and conjugations for hL1O2 were performed by New England Peptide; and for HERV-K Gag by Cambridge Research Biochemicals. Antibodies were validated for antigen specificity and cross-species reactivity by ELISA, western blot, and immunoprecipitation followed by in-gel digestion and MALDI-TOF MS to demonstrate antigen specific pull-down from transfected cell lysates.
In-gel digestion and MALDI-TOF MS
Bands of interest in Bis-Tris gels were excised, cut into small pieces and the proteins reduced, alkylated and digested with trypsin (Promega, Madison, WI) overnight at 37°C. The supernatant was removed and two extractions with 60 % acetonitrile, 0.1 % TFA were performed. All three fractions were combined and their volume reduced to 5 µL in a speed-vac. 15 µL of 0.1 % TFA was added to each sample and the peptides were purified using C18 ZipTips (Millipore, Bedford, MA) according to the manufacturer’s instructions. 1 µL of the eluted peptide solution was mixed with 1 µL of a saturated solution of α-cyano-4- hydroxycinnamic acid (in 50 % acetonitrile/ 0.1 % TFA) and spotted onto a stainless steel MALDI plate. Spectra were acquired on a Bruker Ultraflex II MALDI mass spectrometer in reflector mode. After internal calibration using two trypsin autolysis peaks (m/z = 842.50 and 2211.09 Da) experimental peptide masses were matched against mass lists generated by in silico digestion of the antigen sequences.
Tissue arrays were created for humans mice, rhesus and cynomolgus macaques. One or more representative sections from each pivotal organ (with a minimum of one section from each paired organ) were collected and processed from healthy individuals. The tissue list comprised of the following: gastrointestinal tract (tongue, salivary glands, stomach, duodenum, jejunum, ileum, cecum and colon); endocrine organs (thyroid, pancreas, adrenal and thyroid); skeletal muscle (gastrocnemius in mice and quadriceps in macaques); cardiovascular system (heart, aorta and mesenteric arteries); skin; lymphoid organs (tonsil, spleen, lymph node and thymus); bone marrow; central (cerebrum including hippocampus and hypothalamus, cerebellum, medulla, pituitary and spinal cord) and peripheral (sciatic nerve) nervous system; respiratory tract (nasal mucosa, trachea, bronchus and lung); adipose tissue; urinary tract (urinary bladder and kidney); eye (retina); and liver for both sexes. The reproductive tract tissue list was: female (ovary, uterus, cervix, vagina and mammary gland) and male (testis, prostate, seminal vesicle, epididymis and mammary gland).
Processing for Histopathology and Immunohistochemistry
Tissue sections or cell cultures were fixed in 10% neutral buffered formalin, embedded in paraffin, cut into 4 or 5µm sections, and mounted onto Superfrost plus slides (Fisher Scientific, Pittsburg, PA). Immunohistochemistry was performed by loading onto the Ventana XT autostainer (Roche Diagnostics Indianapolis, IN). The system dewaxes the slides, pre-treats them with a Tris/EDTA pH 8.0 antigen retrieval system (Ventana mCC1) for 16 min, blocks endogenous peroxidise and then stains with primary antibody for 1 h at room temperature, and subsequently a biotinylated donkey anti-species (Jackson ImmunoResearch Laboratories Inc, Westgrove, PA) for 30 min before incubation with streptavidin HRP and DAB substrate (Roche Diagnostics). A hematoxylin counterstain (Merck KGaA, Darmstadt, Germany) was applied according to manufacturer’s instructions. After removal of the slides from the Ventana system they were dehydrated, treated with xylene to clear and mounted using DPX. Primary antibodies were validated on fixed eGFP- or antigen-transfected HEK 293 cells. In order to maximize the likelihood of staining at physiological expression levels in tissues, the antibodies were used at the highest concentration that gave isotype control-like staining of the eGFP-transfected cells, but an intense staining of the antigen-transfected positive control cells. The specificity of staining with peptide-specific antibodies was tested by pre-incubation of the antibody with increasing concentrations of the cognate or control irrelevant peptides for 30 min prior to application of the entire mixture to the slide. All images were captured using the NanoZoomer 2.0 (Hamamatsu, Japan) and analyzed by drug safety and ACVP certified pathologists with extensive immunohistochemistry experience. The intensity (scored on a 4-point scale from minimal to marked intensity), distribution (nuclear, cytoplasmic or cell membrane associated), and characteristics (granular, punctuate or diffuse), of the staining as well as the proportion of cells stained (approximate percentage) were recorded. The staining was considered specific to ERE expression if it differed in intensity (usually 3–4), distribution and/or characteristics from the isotype control; and could be inhibited only by the immunogen peptide. Staining consistent with common artefacts (e.g. necrotic cells) was discounted.
Female Balb/C (18–20g body weight) were purchased from Charles River Canada (Montreal, QC). All mouse experiments were conducted under the guidelines of Pfizer Canada Inc., Animal Care Committees and under the requirements of the Canadian Council on Animal Care (CCAC). Indian rhesus macaques (Macaca mulatta,
16 male and 8 female, median weight 7.78 kg, median age 8 years) were housed at the Wisconsin National Primate Research Center. The University of Wisconsin Institutional Animal Care and Use Committee reviewed and approved all study protocols, which were in accordance with the US Department of Health and Human Services Guide for the Care and Use of Laboratory Animals. Animals used in this study were typed for the MHC-I alleles Mamu-A*01
, and Mamu
using sequence-specific primers (51
). We excluded Mamu-B*17+
animals from this study because these alleles are associated with spontaneous control of SIV replication.
Mice: DNA vaccination was given using the PMED technology which delivers DNA directly into the cells of the epidermis (53
). Mice were randomly assigned to groups (n = 10) and the abdomen was shaved 20 min prior to DNA vaccination. 2 µg pDNA was administered to the abdomen by the PMED ND10 device, or rAd5 (108
virus particles in 50 µL PBS per dose) was i.m. injected to the tibialis anterior. Mice were primed with pDNA followed by boosts with pDNA or rAd5 in 4-week-intervals and examined two weeks following the final boost. At the end of the vaccination phase following termination, the mice underwent necropsy examination. The following tissues based either on biological importance and/or ERE expression were then collected and processed as described above for histopathological evaluation: lungs, heart, kidneys, liver, urinary bladder, pancreas, mesentery, adrenal glands, thymus, lymph node, brain, skeletal muscle (gastrocnemius/soleus block), haired skin and injection site (skeletal muscle). Rhesus macaques: animals were prepared for vaccinations as described (49
). A total of six actuations of the PMED ND10 device were given spread bilaterally into the epidermis of the inguinal lymph node regions to vaccine group 1 and the controls at weeks 0, 6 and 12, and to vaccine group 2 at weeks 8, 14 and 20. At week 33 vaccine group 2 was given 12 actuations using the PMED X15 device spread over both inguinal lymph node regions and the lower abdomen as a final boost. The total DNA doses were 3.6 or 7.2 µg of each of three antigen encoding plasmids and 1.8 or 3.6µg of RhGM-CSF (co-formulated onto the same gold beads) per dosing session for the ND10 and X15 immunizations respectively. Four weeks after the final vaccination, the macaques were challenged by the intrarectal route with SIVsmE660 once per week until they had detectable plasma viremia, or by the intravenous route if they remained uninfected, as described (54
) (and Sheppard et al
manuscript in preparation). 10 to 12 weeks following infection the macaques were euthanized with Beuthanasia-D (up to 177 mg/k IV) (manufactured by Schering-Plough Animal Health Corp., Union NJ) and were necropsied. The following tissues were collected and prepared as described above: cerebrum, cerebellum, brain stem, spinal cord, pituitary gland, stomach, duodenum, jejunum, ileum, cecum, colon, pancreas, liver, gallbladder, lung, kidneys, thyroid glands, trachea, esophagus, ascending aorta, adrenal glands, axillary lymph nodes, inguinal lymph nodes, mesenteric lymph nodes, mandibular salivary glands, spleen, tongue, skeletal muscle, urinary bladder, diaphragm, eyes with optic nerve, bone (stifle), bone marrow, thymus (if present), mammary gland, haired skin, ovary/testis, prostate, seminal vesicles, uterus/cervix, vagina, heart and any lesions noted during gross examination.
Clinical chemistry, haematology and urinalysis
The complete blood count, differential and reticulocyte parameters were measured using whole blood (K2 EDTA) on the Siemens Advia 120 hematology analyzer. Standard clinical chemistry parameters were measured in serum on the Siemens Advia 2400 chemistry analyzer. Serum insulin was measured using the Siemens Advia Centaur automated immunoassay platform. Glucagon was measured in plasma (K2 EDTA and aprotonin) using the BioPlex Luminex suspension array system. Urinalysis was performed using the Clinitek Atlas Chemistry analyzer. Additionally, urine creatinine and N-acetylglucosamide were also performed on the Advia 2400. Microscopic analysis of urine sediment was performed on all urinalysis samples using light microscopy.
For mL1O2, peptides were synthesized by New England Peptide Inc (Gardner, MA). Two sets were obtained: 15-mer peptides overlapping by 10 residues; and predicted MHC class I (H-2d
) restricted epitope peptides of 9 amino acids generated using the MHC-I processing method at IEDB (ANN). Seventeen predicted peptides were derived from Fr1; 18 from Fr2; and 27 peptides from Fr3. For the macaque studies, 15-mers overlapping by 11 amino acids spanning the entire protein sequence of hL1O2 (JPT, Berlin, Germany), SERV-K Gag and Env (both from Pepscan, Lelystad, The Netherlands) were obtained. 9-mer peptides predicted to bind the Mamu-A*01 and –A*02 MHC-I molecules for the same proteins were predicted using the MHCPathway Macaque algorithm (www.mamu.liai.org
) and obtained from Genescript (Piscataway, NJ). Peptides were divided into pools of no more than 10 peptides.
T cell cytotoxicity assay
CTL assay was conducted using Cr- release assay as described previously (56
) with modifications. Briefly, splenocytes (3×107
cells) from individual mice were cultured in complete RPMI1640 medium supplemented with recombinant mouse IL-2 (10U/ml) and a pool of predicted 9-mer peptides in the context of H-2d
(10 µg/ml each) at 37°C for 5 days ex vivo
. Target cells P815 (H-2d
) were pulsed with and without peptides and then labeled with sodium chromate (Perkin Elmer, Waltham, MA). Unpulsed target cells were used as negative controls. Various effector: target ratios were incubated for 4 h. Data presented as percentage of specific lysis (mean ± S.D.).
T cell enrichment
Splenocytes (2×107 cells) were enriched for either CD4+ or CD8+ T cells by negative selection using magnetic beads following the manufacturer’s protocol (Milltenyi Biotechnoies Inc., Auburn, CA). Flow through cells were collected and counted. The purity of T cells as assessed by Flow cytometry analysis using FlowJo Software V8.1 (Tree Star Inc., Ashland, OR) was consistently over 93%.
Intracellular cytokine staining
All antibodies were purchased from BD Biosciences (San Jose, CA). We incubated 1 × 106 PBMC with 10 µg of Brefeldin A (Sigma–Aldrich, ST. Louis, MO) per ml for 6 h at 37 °C in 100 µL of R10 [RPMI 1640 containing 10% fetal calf serum, 2 mM l-Glutamine, 10 U/ml Penicillin G, 10 µg/ml Streptomycin, and 0.025 µg/ml Amphotericin B (HYClone, Logan, UT)] to prevent protein transport from the Golgi apparatus with or without peptide stimulation with 10 µM peptide. For experiments with CD107a, we added the anti-CD107a at the beginning of the assay and added Monensin according to the manufacturer’s directions with the BFA. After the incubation period we washed and stained the cells for selected surface markers (CD8 and CD4) and fixed overnight in 2% paraformaldehyde at 4 °C. On the following day, we permeabilized the cells with 0.1% saponin in PBS containing 10% FCS (HyClone) and stained them for intracellular expression of CD3 and the cytokines gamma-interferon (IFN-γ) and TNF-α. After staining, we washed the cells with PBS containing 10% FCS and fixed in 2% paraformaldehyde for at least 30 min at 4 °C. We collected 1–3 × 105 events within the lymphocyte gate with FASCDiva 6.0 software on a custom made BD LSR II flow cytometer (BD Biosciences). We analyzed the data with FlowJo v.8.7.3. (Treestar Inc.).
For murine studies, the frequency of IFN-γ-secreting cells in whole splenocytes, or enriched CD4+ or CD8+ T cells was determined using ELISPOT assay as previously described (57
) with modifications. Syngenic naive splenocytes were incubated with a pool of 9-mers or two pools of 15-mers derived from designated fraction of mL1O2, at a concentration of 10µg/ml each followed by X-ray (22Gy) irradiation. The irradiated cells were co-cultured with effectors for overnight at 37°C. Following IFN-γ spot development, spots were enumerated on a Zeiss Reader using KS ELISPOT 4.5 software by ZellNet Consulting Inc., (Fort Lee, NJ). For rhesus macaque ELISPOTs fresh peripheral blood mononuclear cells (PBMC) isolated from EDTA-anticoagulated blood were used for the detection of IFN-γ-secreting cells as previously described using both predicted MHC-binding peptides and overlapping 15-mer peptides (55
Recombinant SERV-K Gag CA, HERV-K Env transmembrane (TM) and surface unit (SU) protein ELISAs were performed in 384-well plates. Proteins were diluted to 2µg/ml in PBS and the plates coated overnight at 4°C before washing, blocking for 1h with 1% (w/v) BSA/PBS, and application of a eight-point 0.5 Log10 dilution series of rhesus macaque sera diluted from a top concentration of 1:10 (v/v) in 1% (w/v) BSA/PBS. After extensive washing, the binding of antigen-specific IgG was measured using HRP-conjugated anti-human IgG (Southern Biotech, Birmingham, AL). The reciprocal titer was reported as the intercept of the curve with an arbitrary cut-off value of OD450nm = 1 (based on a reader range of 0 – 4), intersecting the linear portion of the curves of positive samples in all cases and excluding weakly- or non-reactive samples.
Protein expression and antigen-lysate preparation
SERV-K CA consensus residues 282–554 (with N-terminal His-tag) was expressed in E. coli (T7 express, Merck KGaA). Soluble protein was purified from cell lysates by affinity chromatography using a 5ml HisTrap FF column on an Äkta Xpress (GE Healthcare, Port Washington, NY). HERV-K Env SU and TM were expressed at 10–25L wave-bag scale in transiently-transfected HEK293 cells (Invitrogen) and were harvested 5 days post-transfection. Secreted protein was purified via N- and C-terminal His-tags by overnight incubation with 5ml resin (HisSelect, Sigma). Lysates containing full-length SERV-K Gag and Env, and hL1O2 Fr2 for use in western blots were expressed by transient transfection of 106 HEK293 cells with 5µg of the appropriate expression plasmid followed by lysis via sonication in lysis buffer (sterile PBS/0.05% Tween 20 plus cOmplete Mini protease inhibitor cocktail [Roche Diagnostics]) at 48 h post transfection.
All statistical analyses were performed using GraphPad Prism software (v5.01, GraphPad Software, San Diego, CA) and were vetted by a statistician. Groups were compared using Kruskal-Wallis test followed by two-tailed, unpaired Mann-Whitney tests, except in the case of ELISA titer data, which was log-transformed and analysed by ANOVA and unpaired Student’s t-test.