Natural and cross-inducible anti-SIV antibodies in Mauritian cynomolgus macaques

RESEARCH ARTICLE Natural and cross-inducible anti-SIV antibodies in Mauritian cynomolgus macaques Hongzhao Li 1 , Mikaela Nykoluk 2 , Lin Li 1 , Lewis R. Liu 1 , Robert W. Omange 1 , Geoff Soule 2 , Lukas T. Schroeder 1 , Nikki Toledo 1 , Mohammad Abul Kashem 1 , Jorge F. Correia-Pinto 3 , Binhua Liang 2,4 , Nancy Schultz-Darken 5 , Maria J. Alonso 3 , James B. Whitney 6,7 , Francis A. Plummer 1,2 , Ma Luo 1,2 * 1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada, 2National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada, 3CIMUS Research Institute, University of Santiago de Compostela, Santiago de Compostela, La Coruña, Spain, 4Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada, 5Wisconsin National Primate Research Center, Madison, Wisconsin, United States of America, 6Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America, 7Ragon Institute of MGH, MIT, and Harvard, Cambridge, Massachusetts, United States of America *[email protected]c.ca,[email protected] Abstract Cynomolgus macaques are an increasingly important nonhuman primate model for HIV vaccine research. SIV-free animals without pre-existing anti-SIV immune responses are generally needed to evaluate the effect of vaccine-induced immune responses against the vaccine epitopes. Here, in order to select such animals for vaccine studies, we screened 108 naïve female Mauritian cynomolgus macaques for natural (baseline) antibodies to SIV antigens using a Bio-Plex multiplex system. The antigens included twelve 20mer peptides overlapping the twelve SIV protease cleavage sites (-10/+10), respectively (PCS peptides), and three non-PCS Gag or Env peptides. Natural antibodies to SIV antigens were detected in subsets of monkeys. The antibody reactivity to SIV was further confirmed by Western blot using purified recombinant SIV Gag and Env proteins. As expected, the immunization of monkeys with PCS antigens elicited anti-PCS antibodies. However, unexpectedly, antibodies to non-PCS peptides were also induced, as shown by both Bio-Plex and Western blot analyses, while the non-PCS peptides do not share sequence homology with PCS peptides. The presence of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques should be considered in animal selection, experimental design and result interpretation, for their best use in HIV vaccine research. Introduction Simian immunodeficiency virus (SIV) infection of nonhuman primates (NHPs) is currently the best animal model to test HIV vaccine strategies or study HIV pathogenesis [1–11]. PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 1 / 20 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Li H, Nykoluk M, Li L, Liu LR, Omange RW, Soule G, et al. (2017) Natural and crossinducible anti-SIV antibodies in Mauritian cynomolgus macaques. PLoS ONE 12(10): e0186079. https://doi.org/10.1371/journal. pone.0186079 Editor: Cristian Apetrei, University of Pittsburgh Centre for Vaccine Research, UNITED STATES Received: August 3, 2017 Accepted: September 25, 2017 Published: October 5, 2017 Copyright: ©2017 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by the following funding awarded to ML: a National Institute of Allergy and Infectious Diseases grant, with grant number: R01AI111805 and URL: https:// www.niaid.nih.gov/; a Canadian Institutes of Health Research/Canadian HIV Vaccine Initiative Bridging grant (no grant number available), with URL: http:// www.cihr-irsc.gc.ca/e/42458.html; and funding Traditionally, rhesus macaques (Macaca mulatta) are the favorite choice among NHPs in HIV vaccine studies [1–5]. A wealth of knowledge has been accumulated for this species regarding SIV-host interaction, viral and cellular dynamics following SIV infection, genetics and physiology [6,7]. However, the availability of rhesus macaques has been greatly reduced due to a ban of their export from India and most other south Asian countries [6,12]. Cynomolgus macaques (Macaca fascicularis) have become by far the most internationally traded NHP for laboratory experiments [6]. In comparison to rhesus macaques, several characteristics make cynomolgus macaques a particularly useful animal model for HIV vaccine research, apart from their availability. SIV infection of cynomolgus macaques leads to a disease pattern that closely mimics that of human HIV infection, with lower peak and set-point viral loads and slower disease progression typical of human AIDS [6]. The largest laboratory supply of cynomolgus macaques is available from the island of Mauritius. The Mauritian cynomolgus macaques descended from a small group of founder animals and are characterized by high genetic homogeneity with much fewer MHC haplotypes and alleles [6,7,13–15]. This helps reduce outbred variability between animals and thus reduces the number of animals needed to achieve statistical power, making them practical for HIV vaccine studies [7]. Commonly, vaccine studies are carried out in specific pathogen-free animals to rule out the impact of on-going infection or pre-existing immune responses in order to soley evaluate the vaccine efficacy absent of confounding variables. This may require screening larger numbers of animals than those used in the vaccine experiments. Published reports from HIV vaccine studies using NHPs generally failed to provide details of the cohort screening. However, such information is valuable and can serve to guide future vaccine projects. A practical concern in vaccine studies is that a large number of animals are required to achieve statistical power. The information of expected frequency of animals with pre-existing infection or immune responses is important for estimating the starting number of animals to be screened. Here, we report the levels and frequencies of natural antibodies to SIV antigens among a population of 108 Mauritian cynomolgus macaques. These SIV antigens include peptides surrounding the twelve protease cleavage sites [16] (PCS peptides) and three non-PCS Gag or Env peptides of SIVmac239 [17–19]. In addition, we observed that vaccination of Mauritian cynomolgus macaques with PCS antigens not only elicited antibodies to the PCS peptides, but also crossinduced antibodies to non-PCS peptides, while the non-PCS peptides share no sequence homology with the PCS peptides. This suggests that a vaccine could elicit immune responses targeting SIV antigens other than those directly from the vaccine. These novel antibody responses need to be taken into consideration in HIV vaccine projects using Mauritian cynomolgus macaques. Materials and methods Experimental animals and ethics statement 108 female Mauritian cynomolgus macaques (Macaca facicularis) from Bioculture (Mauritius) Ltd were involved in this study, including groups of 94 colony-bred animals and 14 capturebred animals. From the former group 94 animals only their plasma samples were purchased for in vitro antibody analysis, without any physical involvement of these animals in this study. Only the latter group 14 animals were physically involved in the animal work of this study and were used for immunization and viral challenge experiments. The immunization and viral challenge experiments are detailed below as part of Materials and Methods. The animal work was conducted in accordance with Canadian Council on Animal Care guidelines and the Animal Use Document was approved by the Canadian Sciences Centre for Human and Animal Novel cynomolgus anti-SIV antibodies PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 2 / 20 from National Microbiology Laboratory of Canada (no grant number available), with URL: https:// www.nml-lnm.gc.ca/index-eng.htm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Health Animal Care Committee (protocol number: H-12-014R2). The humane care of animals was performed as previously described [20]: "Animals were double housed in standard nonhuman primate cages, received standard primate feed as well as fresh fruit and enrichment daily, and had continual access to water. Temperature (19–24˚C), humidity (45–60%) and light (approximately 323 lux) were monitored and maintained within recommended limits, the light/dark cycle was maintained at 12 hour split. Environmental enrichment was provided. Animals were observed twice daily by the PI, a co-investigator, or the veterinary staff for signs of clinical illness”. The cages meet CCAC guidelines for primate housing. The dimensions of the one-over-one primate cages used measure at 38 inches wide, 49 inches deep and 101 inches high. Anesthesia was administered to alleviate suffering by injection with Ketamine Hydrochloride at a dosage of 10 mg/kg (i.m., using 23–25 gauge, 3/8-1 inch needles) prior to phlebotomy or intravaginal viral challenge. Animals were sacrificed at the end of the study or in situations of multiple systemic consequences from simian AIDS, such as wasting (weight loss), diarrhea, generalized lymphadenopathy, pneumonia, encephalitis, vascular thrombosis and secondary infections coupled with complete anorexia for more than two days resulting in acute weight loss of more than 20%. Following induction of deep anesthesia with Ketamine Hydrochloride at a dosage of 25–50 mg/kg (using 23–25 gauge, 3/8-1 inch needles), euthanasia was performed by terminal bleeding (femoral and/or intracardiac exsanguination). Sedation was maintained by inhalation of Isoflurane 2.5–3.5% by mask in 100% oxygen. The SIV antigens During HIV or SIV replication, each of the 12 protease cleavage reactions is essential for the production of a functional viral particle [16]. A novel vaccine strategy targeting the protease cleavage sites (PCS) has been suggested by our studies [16,21] and is being evaluated using Mauritian cynomolgus macaque SIV infection model. As part of the ongoing work, we used PCS peptide antigens (along with non-PCS peptides) to screen for potentially pre-existing natural antibody responses in Mauritian cynomolgus macaques, while no natural immune response screen study has been reported in these animals. Specifically, the SIV antigens used in this study were twelve 20mer peptides overlapping the twelve PCS (-10/+10) and three nonPCS Gag or Env peptides, derived from SIVmac239 [17–19] (Table 1). The sequences of all these peptides were confirmed to be specific for SIV by NCBI protein BLAST and are conserved among multiple SIV strains (Data not shown). No sequence homology was shared between PCS versus non-PCS peptides (Figure A in S1 File). Bio-Plex multiplexed antibody assay Plasma IgG antibodies to SIV antigens were quantified by largely following the previously published protocols [22,23] with slight modifications. Briefly, 20 μg of antigen peptide (synthesized by Genscript, Piscataway, NJ) was coupled to 1.25 x 10 6 Bio-Plex Pro™Magnetic COOH Beads (Bio-Rad) using a Bio-Plex Amine Coupling Kit (Bio-Rad). 50 μl plasma (1:80 diluted) was incubated with 2,500 beads/antigen type. SIV-specific IgG was detected with phycoerythrin-labelled mouse anti-monkey IgG (Southern Biotech, Birmingham, AL) at 5 μg/ml. Bead fluorescence intensities were acquired on the Bio-Plex 200 system (Bio-Rad) and converted to concentrations based on estimation using a PCS2 monoclonal antibody (National Microbiology Laboratory, Canada) as standard. Viral challenge and antiretroviral drug treatment (ARV) Five macaques (previously immunized with a vesicular stomatitis virus vaccine vector, rVSVwt) were intravaginally challenged with 1000 TCID 50 SIVmac251 (Desrosiers 2010-Day Novel cynomolgus anti-SIV antibodies PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 3 / 20 8 viral stock, provided by Drs. Jon Warren, and Nancy Miller, Vaccine Research Program, NIH). The challenge was repeatedly carried out (ranging two to four times) in each animal until positive plasma viral load (VL) was detected. The VL was then monitored weekly throughout the experiment. Daily antiretroviral drug treatment with a combination of FTC (50mg/kg body weight), PMPA (20mg/kg) and raltegravir (10mg/kg) was initiated 25 weeks after SIVmac251 infection. Plasma IgG antibodies to SIV peptides were quantified by Bio-Plex multiplexed antibody assay for time points of weeks 0 and 5 post ARV initiation. Viral load assay This was conducted following a previously published protocol [20]. Indirect enzyme-linked immunosorbent assay (ELISA) quantification of antibodies to vesicular stomatitis virus (VSV) and Zaire Ebola virus (ZEBOV) 96-well ELISA plates were coated with 100 μl of 1 μg/ml purified VSV or ZEBOV [24] (National Microbiology Laboratory, Canada) as capture antigens at 4˚C overnight. Plate washing along the ELISA procedure was performed with PBS containing 0.1% Tween20 and blocking was with 5% skim milk in PBS containing 0.1% Tween20. Monkey plasmas diluted at 1:100 and a following HRP-conjugated goat anti-human IgG secondary antibody (Kirkegaard & Perry Laboratories, catalog number 074–1006) diluted at 1:2000 were used, respectively, both at 37˚C for 1 hour. After a final incubation with a TMB substrate solution (ThermoFisher Scientific, catalog number 34028) at 37˚C for 30 minutes, optical density (OD) values were read at 650 nm. MHC genotyping The Cynomolgus macaque MHC haplotype typing was conducted by Wisconsin Nonhuman Primate Research Centre Genetics Services [25,26]. Table 1. SIV antigen peptides used in antibody screening. PCS or non-PCS SIV protease cleavage location Sequence PCS1 p15(MA)/p27(CA) APSSGRGGNY/PVQQIGGNYV PCS2 p27(CA)/p2 GGPGQKARLM/AEALKEALAP PCS3 p2/p8(NC) LAPVPIPFAA/AQQRGPRKPI PCS4 p8(NC)/p1 MAKCPDRQAG/FLGLGPWGKK PCS5 p1/p6gag GPWGKKPRNF/PMAQVHQGLM PCS6 Ncgag-pol/TFP YGQMPRQTGG/FFRPWSMGKE PCS7 TFP/p6gag-pol WSMGKEAPQF/PHGSSASGAD PCS8 p6gag-pol/p10(PR) LQGGDRGFAA/PQFSLWRRPV PCS9 p10(PR)/p66(RT/RNase) LTALGMSLNF/PIAKVEPVKV PCS10 p51(RT)/p15(RNase) KDPIEGEETY/YTDGSCNKQS PCS11 p66(RT/RNase)/p31(IN) LVSQGIRQVL/FLEKIEPAQE PCS12 Nef NQGQYMNTPW/RNPAEEREKL SIVgag No cleavage VGDHQAAMQIIRDIINEEAADWDL SIVenv1 No cleavage NVTESFDAWNNTVTEQAIEDVWQLFETSIRPCVKLSP SIVenv2 No cleavage RVTAIEKYLKDQAQLNAWGCAFRQVCHTTVPWPNA https://doi.org/10.1371/journal.pone.0186079.t001 Novel cynomolgus anti-SIV antibodies PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 4 / 20 Construction of recombinant VSV vectors encoding the nucleotide sequences of the 12 protease cleavage sites (rVSVpcs vectors) The sequence of Simian immunodeficiency virus strain SIVmac239 was retrieved from the Los Alamos National Laboratory HIV database (http://www.hiv.lanl.gov). The nucleotide sequences encoding 20 amino acids (10 amino acids flanking each side of the cleavage site) overlapping each of the 12 PCS [MA(p15)/CA(p27), CA(p27)/p2, p2/NC(p8), NC(p8)/p1, p1/p6gag, NCgag-pol/TFP, TFP/NCgag-pol, p6gag-pol/Protease(p10), Protease(p10)/ RT(p66), RT(p51)/ RNase(p15), RNase(p15)/integrase (p31) and Nef] (Table 1) were synthesized and cloned in a Blue Heron pUC(-)MCS plasmid (BlueHeron Biotechnology, Bothell, WA, USA). Each PCS sequence was flanked by an upstream MluI restriction site (AAACGCGT), Kozak sequence (GCCACC), start codon, and downstream stop codon and AvrII restriction site (CCTAGGTT). The PCS coding fragment was then sub-cloned into a modified Vesicular Stomatitis Virus (VSV) vector, a gift to Gary Kobinger from John Rose, Yale University School of Medicine. The modified VSV plasmid (pATX VSV-G) expresses the positive-strand RNA complement of the VSV genome and can tolerate the addition of four foreign genes at four multiple cloning sites (MCS #1–4). MCS#3 was identified to promote the highest expression levels of luciferase and EGFP reporter genes by luciferase assays and FACS analysis (Wong 2011, personal communications). The pATX VSV-G vector contained in order: bacteriophage T7 promoter (T7P), the VSV leader, nucleoprotein (N), phosphoprotein (P), matrixprotein (M), glycoprotein (G) (in MCS#4), polymerase (L), ampicillin resistance gene (AmpR), hepatitis delta virus ribozyme and the T7 terminator sequence. Sub-cloning of PCS coding sequences into MCS#3 of pATX VSV-G was performed using the following procedure. pATX-VSV-G vector and Blue Heron pUC(-)MCS plasmids containing each of the twelve PCS were double digested with AvrII and MluI (New England BioLabs, ON, Canada). 20 ul of each digested product was electrophoresed in a 1% low-melt agarose gel (Invitrogen, CA, USA) and stained with SYBR 1 Gold (Invitrogen, CA, USA) for 15 minutes. The linearized pATX VSV-G vector band was excised and purified using the QIAquick Gel Extraction kit (Qiagen, USA). The PCS DNA band from each digested Blue Heron pUC(-)MCS plasmid was excised and purified using the QIAEX II Agarose Gel Extraction kit (Qiagen, USA). Each of the 12 PCS fragments was cloned into the digested pATX VSV-G vector by ligation with T4 DNA ligase (Invitrogen, CA, USA). Ligated plasmids were transformed into One Shot 1 Top10 cells (Invitrogen, CA, USA) and purified following the Endofree Plasmid Maxi purification kit (Qiagen, USA). All recombinant VSV plasmids were verified by sequencing (DNA Core Facility, National Microbiology Laboratory, Canada) using BigDye Terminator Cycle Sequencing Ready Reaction kits (Applied Biosystems, Foster City, CA) and Prism 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). rVSVpcs virus generation HEK293T and VeroE6 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml), L glutamine (2mM) (Invitrogen, CA, USA). Cells were transfected with rVSVpcs plasmid (2 μg) and support plasmids (2 μg of T7, 0.5 μg of N, 0.3 μg of L and 1.3 μg of P) with Lipofectamine 2000 (Invitrogen, CA, USA) according to manufacturer’s protocols. Cells were incubated at 37˚C/5% CO 2 until the transfected cells displayed cytopathetic effect compared to negative control cells. Rescued rVSVs were then passaged on VeroE6 cells to obtain a virus stock. Virus stock was purified and concentrated by ultracentrifugation through a 20% sucrose cushion in a Beckman XPN-80 ultracentrifuge at 27,000 RPM, 4˚C for 2 hours. The final pellet was resuspended in DMEM and stored at -80˚C. Purified rVSVpcs stock was plaque titrated with VeroE6 cells. Novel cynomolgus anti-SIV antibodies PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 5 / 20 Packaging of PCS peptides into nanoparticles (NANOpcs) Twelve 20mer peptides overlapping the PCS of SIVmac239 were synthesized (GenScript) for nanopackaging. The peptides were associated to a nanoparticle system formed by chitosan (CS) and dextran sulfate (DS). Due to their opposite charges, it is possible to form nanoparticles by a simple ionic interaction process under mild conditions. Briefly, the CS:DS nanoparticles were formed spontaneously upon addition of 0.825 ml of an aqueous DS solution (1.875 mg/ml) to the same volume of an aqueous CS solution (0.625 mg/ml) under magnetic stirring. For peptide encapsulation, the peptide was incorporated in the anionic (DS solution) or in the cationic phase (CS solution) according to its isoelectric point (pI). Peptides with pI lower than 7, were dissolved in the anionic phase while the rest of the peptides were dissolved in the cationic phase. The peptide theoretical loading in nanoparticles was 9.6%. Each one of the peptides was encapsulated separately, and then, a pool of loaded nanoparticles, containing 50 μg of each peptide, was prepared in individual vials for a single administration. For an improved conservation, the nanoparticles suspension in each vial, were incorporated in a cryoprotectant solution (trehalose 5%, w/v) and submitted to a freeze-thaw cycle. The vials were frozen at -80˚C overnight and then submitted to two sequential drying steps in a lyophilizer. The primary drying was carried out at -35˚C (40 h) under high vacuum, and then the second drying step (8 h), in which the temperature gradually raised until +20˚C, forming an uniform dry cake. To evaluate the effect of this operation over the formulation characteristics, loaded nanoparticles with each peptide, were freeze-dried with the same protocol and afterwards reconstituted with ultrapure water in order to analyze their size and polydispersity index (PdI). The nanoparticles diameter and PdI were evaluated by diffraction laser spectroscopy and its surface charge by electrophoretic mobility using a Zetasizer Nano ZS90 (Malvern Instruments, UK). The morphology of the particles was evaluated by transmission electronic microscopy (TEM). Ultrapure CS hydrochloride salt, having a molecular weight of around 125 kDa and an acetylation degree of 14% (Protasan UP Cl 113), was purchased from Novamatrix (Norway). The DS with a molecular weight of about 14,500 Da was obtained from Sigma–Aldrich (Madrid, Spain). Immunization: PCS vaccination Fourteen monkeys were immunized (i.m., right hind leg). Each of 8 monkeys in the vaccine group received a total of 1.2×10 7 plaque forming units (pfu) of pooled rVSVpcs viral particles (1×10 6 pfu for each of the 12 rVSVpcs). Each of the 6 monkeys in the control group received 1.2×10 7 pfu of rVSV wild type (rVSVwt). The monkeys were boosted at week 5, with PCS peptides packaged in nanoparticles (NANOpcs) for vaccine group and water (NANO vehicle) for control group, and boosted again at week 9, with rVSVpcs/NANOpcs for vaccine group and rVSVwt/water for control group. Enrichment of SIV peptide-specific antibodies SIV peptides (synthesized by Genscript, Piscataway, NJ) were coupled to CarboxyLink coupling gel (ThermoFisher Scientific, Rockford, IL; Catalog 20266) and used to enrich SIV peptide-specific antibodies from crude monkey plasma. Total plasma antibodies were purified using Pierce protein A/G agarose beads (ThermoFisher Scientific, Catalog 20422). Western blot SDS-PAGE was conducted following the NuPAGE Bis-Tris mini gel electrophoresis protocol (Thermo Fisher Scientific, Waltham, MA). Purified recombinant SIV proteins (NIH AIDS Novel cynomolgus anti-SIV antibodies PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 6 / 20 Reagent Program), SIVmac251 Gag (Catalog 1845) and SIVmac239 Env (Catalog 2322) were diluted in 1×NuPAGE LDS sample buffer (Thermo Fisher Scientific, Catalog NP0008) containing 1×NuPAGE reducing agent (Thermo Fisher Scientific, NP0009) and heated at 70˚C for 10 minutes. 10 μl molecular weight marker, Precision Plus protein Dual Color Standards (Bio-Rad, 161–0374), Magic Mark XP Western Standard (Thermo Fisher Scientific, LC5602) or Spectra™Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific, 26634), or 1 μg SIV protein in 15 μl sample buffer was loaded onto a NuPAGE 4–12% Bis-Tris 1.0mm×10well gel (Thermo Fisher Scientific, NP0321BOX) assembled in a mini gel tank (Thermo Fisher Scientific, A25977). The buffer chambers were filled with 1×NuPAGE MES SDS running buffer (Thermo Fisher Scientific, NP0002). NuPAGE™Antioxidant (Thermo Fisher Scientific, NP0005) was added to the running buffer of the upper (cathode) chamber at 400×dilution. The electrophoresis was run at 200V for 35 minutes. Blotting was performed using iBlot gel transfer device (Thermo Fisher Scientific, IB1001) and iBlot gel transfer nitrocellulose mini stacks (Thermo Fisher Scientific, IB301002), according to the supplier’s protocol. Blotting membranes were rinsed with wash buffer (PBS containing 0.1% Tween 20) and then blocked with 5% skim milk in wash buffer at room temperature with shaking at 100 rpm. 3×wash of 5 minutes at room temperature with shaking was performed after blocking and between/after antibody incubation steps. Two types of primary antibodies were used. To confirm if natural SIV peptide-specific antibodies recognize authentic SIV proteins, antibodies enriched from monkey plasma (as described above) were diluted at 1 μg/ml in antibody buffer (wash buffer containing 0.5% skim milk). Control antibodies were protein A/G-purified total Ig from SIV antibody-negative monkey plasma. To confirm if PCS vaccination-induced monkey antibodies recognize authentic SIV proteins, monkey crude plasmas from time points of baseline or 1 week after PCS vaccination were diluted at 1:125 in antibody buffer. In both cases, the diluted primary antibodies were incubated with membranes at 4˚C with shaking overnight. The secondary antibody, goat anti monkey IgG-HRP (Santa Cruz Biotechnology, sc-2458) was diluted at 1:2000 in antibody buffer and incubated with membrane for 1 hour at room temperature with shaking. Chemiluminescent detection was based on Pierce ECL Western blotting substrate (Thermo Fisher Scientific, 32209) and carried out on a ChemiDoc XRS instrument using Quantity One 4.6.9 software (Bio-Rad). The application setting was chemi Hi sensitivity, no light 2×gain, 2×2 Bin and manual exposure for 5 minutes. To control sample loading, membranes were stripped in Restore™PLUS Western blot stripping buffer (Thermo Fisher Scientific, 46428) for 15 minutes at room temperature with shaking and re-probed with standard SIV antibodies. The standard SIV antibodies were a mixture of multiple clones of NIH AIDS Reagent Program mouse monoclonal antibodies specific for SIV Gag or Env proteins. For Gag, the antibodies include catalog numbers of 2320 and 2321, which were each 1:4,000 diluted. For Env, the antibodies include catalog numbers of 4669, 2695, 2319, 2696, 12224, 2317, 4670, 1403, 12223 and 1812, which were each 1: 20,000 diluted. The secondary antibody in combination was goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2005), used at 1:5,000 dilution. To quantify vaccine-induced changes in the levels of monkey plasma IgG antibodies to SIV Gag or Env proteins, the SIV antibody levels were defined by normalizing the band intensities in the initial monkey antibody blots to their counterparts in the re-probed, mouse antibody blots. Statistical analysis Statistical analysis was performed using GraphPad Prism statistical software. Continuous variables were compared with Student’s ttest; categorical variables were compared with Fisher’s exact test. Novel cynomolgus anti-SIV antibodies PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 7 / 20 Results Natural antibodies to novel SIV antigens are detected in naïve Mauritian cynomolgus macaque populations To prepare for a preclinical evaluation of a novel HIV vaccine strategy targeting the 12 viral protease cleavage sites (PCS) using an intravaginal challenge model, we screened 108 randomly selected, naïve female Mauritian cynomolgus macaques for potential natural antibodies against SIV antigens, including 12 PCS peptides (our vaccine of interest) and 3 non-PCS Gag and Env peptides (See Materials and methods for details). The macaques, 14 capture-bred and 94 colony-bred, had not been used in any previous SIV challenge or vaccination study before the antibody screening. Using a Bio-Plex multiplexed antibody assay, we found that subsets of animals had higher than background levels of antibodies (Fig 1 and Table A in S1 File). Since these monkeys had not been previously infected with SIV or exposed to SIV antigens that would induce SIV antibodies, we carefully validated the results obtained with the Bio-Plex method. We used two other methods to verify whether these are anti-SIV antibodies. We first tested whether these antibodies recognize authentic SIV proteins by Western blot analysis using affinity-purified SIV peptide-specific antibodies from monkey plasma samples. The results showed that these antibodies indeed recognized purified recombinant SIV Gag and Env proteins (Fig 2). Next, we characterized antibody responses to these SIV peptide antigens in the scenario of SIV infection (Fig 3). Five monkeys were experimentally infected with SIVmac251, followed by treatment with anti-retroviral drugs (ARV) FTC, PMPA and raltegravir [27,28]. We found that the levels of antibodies to PCS and non-PCS peptides were high before ARV treatment, but were significantly decreased after ARV (Fig 3A) and the decrease of antibodies to the SIV peptide antigens corresponded to the decrease of SIV viral load (Fig 3B). To exclude the possibility that the reduction of anti-SIV antibody responses might be due to a general impairment by ARV in the host antibody production function, we evaluated antivesicular stomatitis virus (VSV) antibodies in these animals as they had previously been immunized with a VSV vector (rVSVwt). We found that their anti-VSV antibody responses were not inhibited by ARV (Fig 3C). Thus, a potential non-specific effect of ARV was ruled out. Together, these results confirmed that the antibodies to PCS and non-PCS peptide antigens are indeed anti-SIV antibodies. Possible factors influencing the level of natural antibodies to SIV antigens Having observed the natural anti-SIV antibodies in Mauritian cynomolgus macaues, including capture bred and colony bred animals, we asked whether environment or host factors influence the level of these antibodies. While these antibodies were detected in both the capture bred and colony bred monkeys and the antibody variability showed similar patterns, the capture bred monkeys had higher levels of antibodies to PCS1, PCS7, PCS11 and SIVgag (Fig 4A). This suggests that environmental factors may influence the magnitude of natural antibodies to some SIV antigens. In addition, we observed host-specific patterns when the antibody levels to all the SIV peptides were examined in individual monkeys (Table A in S1 File). Among important host factors underlying immune responses and susceptibility or resistance to HIV/SIV infection, the major histocompatibility complex (MHC) plays an important role in the initiation and regulation of immune responses based on their ability to bind and present viral epitopes [7,15,19,29]. We analyzed the MHC haplotypes of the 108 monkeys (Table A in S1 File) and correlated MHC haplotypes with levels of natural antibodies to each of the SIV peptides (Fig 4B and Table B in S1 File). This was based on a relatively small number of monkeys Novel cynomolgus anti-SIV antibodies PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 8 / 20 available for some of the MHC haplotypes (Table B in S1 File). MHC haplotype M2 at MHC I or II was significantly correlated with higher natural antibody responses to PCS2 (Fig 4B and 4C, Table B in S1 File). In addition, MHC II of M1 haplotype showed trends of lower antibody responses to PCS1 (p = 0.0883), PCS2 (p = 0.0833), PCS3 (p = 0.0934) and SIVgag (p = 0.0829) (Table B in S1 File). Moreover, Monkeys with M2 haplotype at MHC I and MHC II trended to have higher antibody responses to PCS10 (p = 0.0772) and PCS12 (p = 0.0691), respectively (Table B in S1 File). These data suggest that host MHC haplotypes may differentially affect natural antibody responses to SIV antigens. We expect that larger sample sizes may provide more insight into the correlations between MHC haplotype and antibody responses. Cross induction of non-PCS antibodies by PCS vaccination In a pilot experiment to evaluate the immunogenicity of the PCS peptides as a novel vaccine candidate (Figs 5and 6), we observed that, besides their natural occurrence, anti-SIV antibodies could also be nonspecifically induced by vaccination with the vaccine targeting PCS (Fig 6). In the vaccine group (Figs 5A and 6A), eight Mauritian cynomolgus macaques were immunized with PCS peptides delivered by recombinant vesicular stomatitis virus (rVSVpcs) and nanoparticles (NANOpcs). In the control group (Figs 5B and 6B), six animals were immunized Fig 1. Natural antibodies to SIV peptide antigens in Mauritian cynomolgus macaques. 108 naïve healthy female Mauritian cynomolgus macaques maintained in SIV-free facilities were analyzed by Bio-Plex for plasma IgG antibodies to SIV peptide antigens (Ag). Each black dot indicates one monkey. Red lines represent medians of antibody levels. Ab hi : antibody level 2 ng/ml. Frequency of Ab hi monkeys was listed for each Ag type. https://doi.org/10.1371/journal.pone.0186079.g001 Novel cynomolgus anti-SIV antibodies PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 9 / 20 large molecules would be perceived as “on-target”. In our case of testing novel vaccine candidates based on short peptides, we analyzed antibody responses focusing on the short peptides, and included additional non-target peptide antigens in the analysis, making it easier to observe an “off-target” effect. In conclusion, our findings of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques provide useful new information for HIV vaccine study using this increasingly important NHP model. The presence of these novel immune responses should be considered to achieve better animal selection, experimental design and result interpretation. Supporting information S1 File. A single PDF file including Figures A and B and Tables A and B. (PDF) Acknowledgments We would like to thank the VTS staff at Canadian Science Centre for Human and Animal Health, Christine De Graff, Julie Kubay, Michelle French, Stephanie Kucas, Kimberly Azaransky, Carissa EmburyHyatt and Valerie Smid, and the staff of the Mass Spectrometry and Proteomics Core Facility at the National Microbiology Laboratory, Dr. Garrett Westmacott, Dr. Christopher C. R. Grant, and Stuart McCorrister, for tremendous technical support. We would also like to thank Dr. Jon Warren and Dr. Nancy Miller, NIH Vaccine Research Program, for providing the SIVmac251 Desrosiers” 2010-Day 8 viral stock. We recognize Dr. Stuart Shapiro, NIH Vaccine Research Program, and Dr. Matthew Gilmour, National Microbiology Laboratory of Canada, for important support and discussion. This work was supported by an NIH grant (R01AI111805), a CIHR/CHVI bridging grant and funding from National Microbiology Laboratory of Canada. Author Contributions Conceptualization: Hongzhao Li, Maria J. Alonso, Francis A. Plummer, Ma Luo. Table 3. Pearson correlation analysis of Non-PCS vs PCS antibody levels in Table 2. Correlation coefficient r value p value SIVgag SIVenv1 SIVenv2 SIVgag SIVenv1 SIVenv2 PCS1 0.3415 -0.1181 0.0027 PCS1 0.0557 0.5196 0.9882 PCS2 0.9117 0.9128 0.9625 PCS2 4.08E-13 3.37E-13 1.53E-18 PCS3 0.9282 0.9071 0.9551 PCS3 2.05E-14 8.41E-13 2.16E-17 PCS4 0.5915 0.4065 0.3547 PCS4 0.0004 0.0210 0.0464 PCS5 0.6766 0.6375 0.6955 PCS5 2.12E-05 8.70E-05 9.92E-06 PCS6 0.9134 0.9299 0.9537 PCS6 3.07E-13 1.43E-14 3.32E-17 PCS7 0.4429 -0.0210 0.1236 PCS7 0.0111 0.9092 0.5003 PCS8 0.9198 0.9166 0.9551 PCS8 1.01E-13 1.77E-13 2.12E-17 PCS9 0.9327 0.8959 0.9495 PCS9 7.95E-15 4.29E-12 1.20E-16 PCS10 0.9208 0.9149 0.9383 PCS10 8.49E-14 2.37E-13 2.27E-15 PCS11 0.3770 0.4033 0.4483 PCS11 0.0334 0.0221 0.0101 PCS12 0.9254 0.9003 0.9530 PCS12 3.57E-14 2.33E-12 4.20E-17 https://doi.org/10.1371/journal.pone.0186079.t003 Novel cynomolgus anti-SIV antibodies PLOS ONE | https://doi.org/10.1371/journal.pone.0186079 October 5, 2017 16 / 20 Data curation: Hongzhao Li, Mikaela Nykoluk, Lin Li, Lewis R. Liu, Robert W. Omange, Geoff Soule, Lukas T. Schroeder, Nikki Toledo, Mohammad Abul Kashem, Jorge F. Correia-Pinto. Formal analysis: Hongzhao Li, Mikaela Nykoluk, Lin Li, Lewis R. Liu, Robert W. Omange, Geoff Soule, Lukas T. Schroeder, Nikki Toledo, Mohammad Abul Kashem, Jorge F. Correia-Pinto, Binhua Liang, Ma Luo. Funding acquisition: Binhua Liang, Nancy Schultz-Darken, Maria J. Alonso, James B. Whitney, Francis A. Plummer, Ma Luo. Investigation: Hongzhao Li, Mikaela Nykoluk, Lin Li, Lewis R. Liu, Robert W. Omange, Geoff Soule, Lukas T. Schroeder, Nikki Toledo, Mohammad Abul Kashem, Jorge F. CorreiaPinto, Binhua Liang, Nancy Schultz-Darken, Maria J. Alonso, James B. Whitney, Ma Luo. Methodology: Hongzhao Li, Mikaela Nykoluk, Lin Li, Lewis R. Liu, Robert W. Omange, Geoff Soule, Lukas T. Schroeder, Nikki Toledo, Mohammad Abul Kashem, Jorge F. CorreiaPinto, Binhua Liang, Nancy Schultz-Darken, Maria J. Alonso, James B. Whitney, Ma Luo. Project administration: Binhua Liang, Nancy Schultz-Darken, Maria J. Alonso, James B. Whitney, Francis A. Plummer, Ma Luo. Resources: Nancy Schultz-Darken, Maria J. Alonso, James B. Whitney, Francis A. Plummer, Ma Luo. Software: Lewis R. Liu, Binhua Liang. Supervision: Binhua Liang, Nancy Schultz-Darken, Maria J. Alonso, James B. Whitney, Francis A. Plummer, Ma Luo. Validation: Hongzhao Li, Mikaela Nykoluk, Lin Li, Lewis R. Liu, Robert W. Omange, Geoff Soule, Lukas T. Schroeder, Nikki Toledo, Mohammad Abul Kashem, Jorge F. CorreiaPinto, Binhua Liang. Visualization: Hongzhao Li. Writing – original draft: Hongzhao Li. Writing – review & editing: Hongzhao Li, Lin Li, Lewis R. Liu, Robert W. Omange, Geoff Soule, Lukas T. Schroeder, Nikki Toledo, Mohammad Abul Kashem, Jorge F. CorreiaPinto, Binhua Liang, Nancy Schultz-Darken, Maria J. Alonso, James B. Whitney, Francis A. Plummer, Ma Luo. References 1. Lu S, Arthos J, Montefiori DC, Yasutomi Y, Manson K, Mustafa F, et al. 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