Development and immunological assessment of VLP-based immunogens exposing the membrane-proximal region of the HIV-1 gp41 protein
© Benen et al.; licensee BioMed Central 2014
Received: 22 February 2014
Accepted: 11 August 2014
Published: 27 August 2014
The membrane-proximal external region (MPER) of HIV-1 gp41 is particularly conserved and target for the potent broadly neutralizing monoclonal antibodies (bnMAbs) 2F5, 4E10 and 10E8. Epitope focusing and stabilization present promising strategies to enhance the quality of immune responses to specific epitopes.
The aim of this work was to design and evaluate novel immunogens based on the gp41 MPER with the potential to elicit cross-clade neutralizing antibodies. For that purpose, gp41 was truncated N-terminally in order to dispose immunodominant, non-neutralizing sites and enhance the exposure of conserved regions. To stabilize a trimeric conformation, heterologous GCN4 and HA2 zipper domains were fused based on an in silico “best-fit” model to the protein’s amino terminus. Cell surface exposure of resulting proteins and their selective binding to bnMAbs 2F5 and 4E10 could be shown by cytometric analyses. Incorporation into VLPs and preservation of antigenic structures were verified by electron microscopy, and the oligomeric state was successfully stabilized by zipper domains. These gp41 immunogens were evaluated for antigenicity in an immunization study in rabbits primed with homologous DNA expression plasmids and boosted with virus-like particle (VLP) proteins. Low titers of anti-MPER antibodies were measured by IgG ELISA, and low neutralizing activity could be detected against a clade C and B viral isolate in sera.
Thus, although neutralizing titers were very moderate, induction of cross-clade neutralizing antibodies seems possible following immunization with MPER-focusing immunogens. However, further refinement of MPER presentation and immunogenicity is clearly needed to induce substantial neutralization responses to these epitopes.
KeywordsHIV-1 gp41 VLPs Vaccine Immunization study Neutralizing antibodies
The development of a preventive vaccine against human immunodeficiency virus type 1 (HIV-1) has been pursued by many research groups around the globe using a variety of approaches . Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of an effective immune response. Eliciting such antibodies by vaccination has proven to be difficult due to the substantial genetic variation of HIV-1 and its ability to evade host immune defenses by different mechanisms . Although bnAbs alone may not be able to fully control HIV-1 replication once the infection has been established, they can protect at low titers if they are present in immunologically relevant sites, as has been shown in several passive immunization experiments in non-human primate studies .
Besides the surface protein gp120, the transmembrane protein gp41 is target for bnAbs during infection , rendering this protein a worthwhile target for vaccine design. According to the currently accepted model for HIV-1 fusion , following engagement of host cellular receptors, the viral envelope trimer undergoes conformational changes and inserts the amino-terminal fusion peptide (FP) of gp41 into the host cell membrane. The fusion mechanism involves two helical regions of gp41, the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), which are associated to form a pre-hairpin intermediate. In this conformation, the gp41 molecules are extended forming a trimer of CHR helices and a trimer of NHR helices that bridge both the viral and cellular membranes. It subsequently collapses into a trimer-of-hairpins structure that pulls both membranes into tight apposition and induces membrane fusion. This structure consists of the six-helix bundle, in which three CHR peptides pack in an antiparallel manner against a central three-stranded coiled-coil formed by the NHR regions .
Three of the identified monoclonal Abs against gp41, denoted 2F5, 4E10 and 10E8, bind to adjacent epitopes located in the membrane-proximal external region (MPER) . The MPER is indispensable for membrane fusion and viral entry, and is highly conserved among HIV-1 groups. It is accessible for IgGs already in the native envelope conformation, but thought to have higher accessibility during the fusion process . Some residues may be buried within the membrane in pre-fusion and intermediate conformations . Although 2F5 and 4E10 bind to linear core epitopes in the MPER, a correct three-dimensional display of their epitopes and proximal residues in the ectodomain might enhance the recognition . Additionally to binding to the gp41 protein, both 2F5 and 4E10 can interact with membranes through hydrophobic residues in their heavy chain CDR3 regions. A model has been suggested, in which 4E10 first binds to the membrane, then recognizes its protein epitope and locks conserved residues by an induced-fit mechanism . Removal of the membrane component decreases binding and neutralization of these special antibodies . Taken together, these results suggest displaying the MPER in a native three-dimensional context, embedded into a membrane, for optimal mimicry of the natural epitopes in an immunogen.
Unfortunately, the MPER is weakly immunogenic, compared to the loop region of gp41. Past immunization studies demonstrated that MPER-containing proteins, fusion intermediate-mimicking proteins expressed on HIV-1 VLPs and chimeric proteins all failed to elicit MPER-specific neutralizing antibodies ,. The antibody response was directed towards immunodominant, but non-neutralizing epitopes in these studies. Others were able to elicit high titers of MPER-targeting Abs by a soluble 6-helix bundle immunogen, however, neutralizing activity could only be detected after purification of IgG and not in unfractionated sera . Thus, the problem of eliciting high titers of anti-MPER Abs with broadly neutralizing activity remains unsolved. A reduction of gp41 to its neutralizing epitopes, yet expanding those sites with additional residues responsible for membrane embedment and formation of the full epitopes, seems to be a promising strategy.
The objective of this work was to design and evaluate novel immunogens based on HIV-1 gp41 MPER which combine the above mentioned criteria. For this purpose, variants of truncated gp41 proteins consisting of CHR, MPER and TM regions were designed and characterized. Heterologous peptide sequences were fused for intracellular trafficking and efficient trimerization. The immunogens were designed to strongly expose the MPER with binding sites to known bnMAbs in a natural conformation and membrane environment. Plasmid DNA and pseudotyped virus-like particles (VLPs) were chosen as immunization vehicles in a rabbit model. After a DNA prime and VLP boost regimen, the presence of anti-MPER Abs and neutralizing activity in unfractionated sera was analyzed.
Software-assisted design of gp41 derivatives
An N- and C-terminally truncated gp41 (“gp41CTM”) was designed according to Lenz et al. . Wild-type sequences for synthesis of viral gene fragments were obtained from GenBank and optimized for human codon usage (Life Technologies). Molecular clones of viral isolates HIV-1 89.6 (subtype B, accession number U39362), HIV-1 96ZM651 (subtype C, AF286224) and SIVmac239 (M33262) served as templates with aa 629–724 of 89.6 and 96ZM651 (HXB2 reference numbering) and aa 639–740 of SIVmac239 (SIVmac239 numbering). Membrane incorporation and orientation of all constructs was checked by the use of the transmembrane prediction TMHMM Server v. 2.0 . A human tissue plasminogen activator (TPA) leader sequence was fused N-terminally. Correct cleavage of the leader sequence was predicted by SignalP 3.0 software . A Kozak consensus sequence (GCCGCCACC) was inserted 5’ of the start codon for enhanced transcription. The sequence for an HA-tag from Influenza virus Hemagglutinin HA1 protein (YPYDVPDYA) was codon-optimized and inserted C-terminally by primer extension PCR for recognition of recombinant proteins (primers 3E8 & 2H4/2H5/2H6, see Additional file 1: Table S1 for primer sequences). The topology of all gene variants was checked with Phobius Prediction Server .
Trimer-stabilized constructs and molecular visualization
Two zipper domains were inserted between the TPA leader and gp41-derived sequences by fusion PCR. One motif was derived from the HA2 protein (amino acids 372–417, primers 3H4 & 3H5/3H6/3H7) of human Influenza virus strain H3 followed by a linker composed of amino acids Gly-Ser-Thr . A second domain was derived from the GCN4 protein of Saccharomyces cerevisiae (primers 3B6 & 3B7/3B8/3B9). Sequences for GCN4 and H3 zippers were derived from Genbank (IDs: CY002064, BK006939) and codon-optimized. To confirm the correct gene fusion position of zippers to gp41 fragments, molecular models were built using Modeller and evaluated by calculating the DOPE score (Discrete Optimized Protein Energy . Resulting atomic models were visualized using PyMOL Molecular Graphics System.
SDS-PAGE, Western Blots and Slot Blots
SDS-PAGE was done with acrylamide concentrations of 12.5%. Western Blot transfers of proteins from SDS gels were done in a semi-dry system (Serva, Heidelberg, Germany) according to manufacturer’s instructions. For slot blots, protein solutions were loaded onto a Bio-Dot SF (Biorad) apparatus including 5 layers of pre-wetted Whatman Chromatography paper (Whatman International Ltd, Maidstone, UK). For both methods, nitrocellulose membranes with a pore size of 0.2 μm were applied (Millipore, Bedford, USA) and blocked in TBS (150 mM NaCl, 50 mM Tris/HCl, pH 7.4) containing 5% skim milk powder over night at 4°C. For antibody staining, blots were incubated for 1 h in TTBS (TBS + 0.3% (v/v) Tween-20) with primary antibody, washed four times for 15 min in TTBS, incubated for 1 h in TTBS with secondary antibody, washed again four times for 15 min in TTBS, and subjected to either ECL (2.5 mM luminol, 0.4 mM coumaric acid, 0.1 M TrisHCl pH 8.5 plus equal volume of 0.018% H2O2, 0.1 M TrisHCl pH 8.5) or alkaline phosphatase (AP) staining solution (5 mM TrisHCl pH 9.5, 5 mM NaCl, 2.5 mM MgCl2 plus 1/50 volume of NBT/BCIP stock (Roche)). AP reactions were stopped with excess of dH2O. ECL reactions were measured in a ChemiluxPro device (Intas, Göttingen, Germany). Intensity of bands was quantified with the aid of Gel-Pro Analyzer software (Media Cybernetics, Bethesda, USA). Human monoclonal antibodies (MAbs) 2F5 and 4E10 (diluted to 5 μg/ml each) served for detection of gp41 derivatives, the HA-tag was recognized by rat MAb 3F10 (“anti-HA High Affinity”, 0.1 μg/ml, Roche). Pr55/p24 was detected with mouse MAb M13/5 (cell culture supernatant, 1:500, ). Polyclonal HRP- or AP-coupled anti-human-IgG, anti-rat-IgG, and anti-mouse-IgG antibodies (all 1:2,000, all from Dako, Glostrup, Denmark) served as secondary antibodies.
The amount of Pr55 or p24 protein in lysates was quantified with the aid of an Enzyme-linked Immunosorbent Assay (ELISA) using MAb M01 (1:1000, Polymun, Vienna, Austria) as coating antibody . An ELISA with gp41-derived peptides was used to quantify anti-gp41 immunoglobulins in animal sera by the end-point dilution method in duplicates . Peptides spanning the MPER (EQNEKDLLALDSWNNLWNWFDITKWLWYIK) and CHR regions (MQWDREISNYTNTIYRLLEDSQSQQEQNEK, both from Pepscan Presto BV, Lelystad, Netherlands) were used for coating at 100 ng/ml on Nunc Maxisorp plates (Thermo Fisher Scientific, Waltham, USA). HRP-coupled polyclonal anti-rabbit-IgG and anti-human-IgG served as secondary antibodies (1:4,000, Dako). Washing was done with the aid of a high-throughput microplate washing device (MAP-C2 workstation, Titertek Instruments Inc., Huntsville, USA).
HEK 293 T (ATCC Microbiology Collections) were grown in Gibco Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Invitrogen). FreeStyle 293 F cells (Invitrogen) were grown in Gibco FreeStyle 293 expression medium (Invitrogen) supplemented with 50 U/ml penicillin and 0.05 mg/ml streptomycin at 125 rpm and handled according to the manufacturer’s instructions. HEK293T cells were seeded in 6-well plates (4x105 cells in 2 ml medium). 24 h after seeding, medium was replaced by 1 ml of DMEM without supplements. Cells were transfected with the respective expression vector(s) according to the poly-ethylenimine (PEI) transfection method using a total of 2 μg DNA and 8 μl of a 1 mg/ml PEI in H2O solution ad 50 μl DMEM without supplements. Components were mixed, left standing for 10 min at room temperature and added drop-wise to the cell suspension. 4 h later, transfection medium was replaced with 2 ml of supplemented growth medium. For smaller (96-well) or larger (10 cm/15 cm Petri dishes) assays, the number of cells, the amount of DNA and PEI, and the volume of medium was down- or up-scaled, based on the vessel surface area. 293 F cells were adjusted to 1 million cells/ml in fresh FreeStyle medium and 30 ml of cell suspension were transferred to 125 ml flasks. 37.5 μg of DNA and 150 μl of PEI ad 1.8 ml DMEM without supplements were mixed for transfection of one flask. 4 h later, transfection medium was replaced with 30 ml of FreeStyle medium supplemented with 100 U/ml penicillin and 0.1 mg/ml streptomycin.
Cells were detached 48 h p.tr from wells in PBE buffer (PBS + 1% FCS, 1 g/l NaN3, 2 mM EDTA), pelleted at 300 g for 3 min, washed in PBE, incubated with the indicated fluorochrome-labeled antibodies (3 μg/ml 2F5 or 4E10, 5 μg/ml anti-HA-MAb 3F10) for 1 h at room temperature and washed 2 times with cold PBE. 2F5 and 4E10 antibodies were labeled with AlexaFluor-647 (Protein Labeling Kit, Invitrogen) according to the manufacturer’s instructions. A mean of 6 fluorochromes was attached to each IgG molecule, as measured by photometric analysis (NanoDrop software v3.7.1). anti-HA-antibody was purchased as conjugate with Fluorescein (Roche). Apoptosis staining was done by the use of 7-AAD and Annexin-V-APC antibody (both diluted 1:100, both BD Biosciences). Finally, cells were subjected to cytometric analysis in FACS Canto II (BD, with FACS Diva software v6.0).
After purification through a sucrose cushion, VLPs were loaded onto a 10 ml 10-50% sucrose gradient and ultra-centrifuged at 100,000 g for 2.5 h. 20 fractions of 0.5 ml each were collected. For Western Blot analysis, particles were pelleted by trichloroacetic acid precipitation. A quarter of volume of 100% (w/v) TCA was added to the samples, incubated for 10 min at 4°C and spun at 14,000 g and 4°C for 5 min. The pellet was washed two times with cold acetone, dried at 95°C for 5 min and resuspended in SDS sample buffer. For EM experiments, fractions 13–17 were pooled, diluted in PBS ad 12 ml, ultra-centrifuged at 100,000 g for 1.5 h and resuspended in PBS.
Sucrose-cushion purified VLPs were incubated with primary antibody 4E10 for 1 h at 37°C and further purified on a sucrose gradient. Pooled and resuspended VLPs were adsorbed to grids after fixation with 2% glutaraldehyde. Grids were washed in TBS (Leica EM IGL), blocked with 3% gelatine in TBS for 1 h at RT, and incubated with an anti-human IgG immuno-gold conjugate (particle size: 10 nm; Aurion, Wageningen, Netherlands) for 1 h at room temperature. After three washes in TBS, grids were contrasted with phosphoric tungstic acid and examined in an electron microscope (Zeiss EM 10C/CR).
VLPs for immunization purposes were produced in 293 F cells with the use of a codon-optimized, Rev-independent gene for Gag(IIIB) . For pseudotyped VLPs, plasmids encoding for Gag and Env were mixed in a ratio of 2:1 in a co-transfection assay. VLPs were harvested 72 h p.tr., cleared by centrifugation at 3,000 g for 15 min, loaded onto a 30% sucrose in PBS cushion (5 ml for 30 ml of supernatant) and ultra-centrifuged at 100,000 g for 2 h. The pellet was resuspended overnight in PBS and stored at −80°C, with an aliquot analyzed by SDS gel electrophoresis and Coomassie staining for quality control. Both VLPs and DNA for immunization purposes were checked for low endotoxin levels with aid of Limulus Amebocyte Lysate QCL-1000 assay (Lonza Group Ltd, Basel, Switzerland), with a result of <20 EU/ml.
The animal study was carried out in strict accordance with the UK Animals (Scientific Procedure) Act 1986, and the protocol was approved by the local Ethical and Welfare Committee of the University of Cambridge and the UK Home Office (Project license no. 80/2238). All efforts were made to minimize suffering. New Zealand rabbits (Harlan UK Ltd, Belton, Leicestershire, UK) were kept under pathogen-free conditions and pre-bled by ear vein lancing at the age of 12 weeks. Animals were grouped into 4 groups of 6 animals each and immunized at weeks 0 and 4 with 500 μg of plasmid DNA by i.m. saline injection in quadriceps muscles, followed by i.m. booster immunizations at weeks 12 and 16 with 100 μg of VLPs (Gag protein, analyzed in Coomassie gel), adjuvanted with 1% Carbopol 974 adjuvant. The integrity of pseudotyped VLPs in Carbopol was checked by sucrose gradient ultracentrifugation and following Western Blot analysis. Rabbits were terminally bled by cardiac puncture under isoflurane anesthesia and then euthanized. Serum was obtained by incubation of blood for 1 h at 37°C, 1 h on ice, centrifugation at 20,000 g for 15 min, and complement inactivation by incubation of supernatant at 56°C for 30 min. Unfortunately 2 animals from group 2 and 3 and one animal from group 1 had to be euthanized prior to finalization of the immunization protocol for reasons not related to the study and were therefore not included in data analysis. Statistical analysis was performed based on the correct number of animals in each group.
Removal of anti-cell antibodies
HEK293F cells were stacked by centrifugation at 300 g for 3 min, supernatant was aspirated and cells were stored at 4°C for up to 5 days until usage. Stacked cells were dissolved in an equal volume of animal serum and shaken 3 times for 4 h at 800 rpm at room temperature, until the signal of anti-cell antibodies was below two times the background signal in cytometric analysis.
Virus neutralization assays were performed in the laboratory of D.C. Montefiori (Duke University School of Medicine, Durham, USA), according to the published protocol . Assays were done with Env-pseudotyped viruses in TZM-bl cells for isolate MW965.26 and with Env.IMC.LucR viruses in A3R5 cells (CEM human lymphoblastoid cell origin) for isolate SF162. Dilutions mediating inhibition of infection by 50% (ID50) were subtracted by ID50 of pre-bleed sera or in any case subtracted by a minimum of 20 (detection limit).
Significance of anti-gp41 responses in ELISA and neutralizing activity in neutralization assays was determined using the Wilcoxon Two Sample Test. Each group was compared to its respective pre-bleeds to assess a potential neutralizing activity, and all groups were compared to each other to detect inter-group differences in neutralizing activity.
Design of gp41 constructs with enhanced MPER exposure
Membrane exposure and selective recognition by bnMAbs
Incorporation of oligomeric gp41 derivatives into VLPs
Induction of anti-gp41 antibodies
Neutralizing capacity of unfractionated immune sera
Discussion and conclusions
Previous attempts to elicit nAbs against the 2F5 , and the 4E10 epitope , have largely failed, emphasizing the challenges associated with generating a focused response to MPER-neutralizing epitopes. However, an approach that combines the exposure of neutralizing sites on the immunogen fixed in a favorable structure following truncation of unwanted or immunodominant epitopes and presented in a membrane environment may be effective. Therefore, this work focused on the creation and immunological evaluation of novel gp41-derived immunogens, which meet the above mentioned requirements of such an MPER-based immunogen.
Various steps were undertaken to improve exposure of conserved regions, to stabilize relevant conformations and to focus B cell responses: (i) gp41 was truncated N-terminally to eliminate dispensable sites; (ii) the CHR was included to promote proper trimerization and MPER folding and (iii) the gp41 C-terminus was truncated to allow for efficient incorporation into VLPs  and to support an optimal configuration for 4E10 epitope exposure .
Cytometric analyses validated membrane topology and binding to bnMAbs 2F5 and 4E10. Signals for 2F5 and 4E10 epitopes as well as the C-terminally fused HA tag were low, but significant and specific, indicating a low level of presentation, yet proper membrane incorporation. The recognition of the HA-tag located in the antigenic region of the “Kennedy sequence” in the cytoplasmic tail supports the hypothesis of additional membrane-spanning domains leading to exposure of the tag –. Electron microscopy experiments of VLPs stained with 4E10 clearly demonstrated the gp41 derivatives to be presented on the surface of VLPs. Higher molecular weight bands of trimer-stabilized envelopes (Figure 3) upon assessment of VLPs by Western Blot analysis indicated a high stability of dimeric and trimeric assemblies. According to previous finding by Lenz et al. , the observed shift towards di- and trimeric conformations following N-terminal fusion of the zipper domains (gp41CTM) may be a result of GCN induced trimer formation, trimer stability or both. Generally, trimeric conformations are considered superior to monomeric gp41 derivatives as (i) they present and stabilize discontinuous epitopes and (ii) occlude immunodominant epitopes disclosed by the monomeric protein . Taken together, the in silico models of designed gp41 variants could be implemented into proteins with the desired biochemical specifications.
For immunogenicity studies a DNA prime and VLP boost regimen of rabbits was chosen as a positive effect of DNA priming on antibody titers had been observed by others studying immunogenicity of MPER-containing proteins . Inter-individual variations in gp41 specific IgG responses within groups were notable, which had also been observed before . gp41 specific antibody titers induced by any gp41 DNA prime/VLP boost combination were generally weak compared to titers determined in the gp145 control group. As reactivities against MPER and CHR were similarly low in groups receiving gp41CTM and trimer-stabilized derivatives, the low reactivity may be attributed to low protein expression and exposition on the VLP surface. This limitation may be overcome by using heterologous transmembrane domains from e.g. MMV or Influenza virus, which have been previously shown to mediate enhanced incorporation and display of truncated Env proteins . Moderate neutralization found in animals immunized with VLPs presenting gp145 on their surface may benefit from (i) increased numbers of presented Envelopes and (ii) from the additional epitopes such as e.g. V3 and others presented by the gp120 moiety of gp145.
Sera were pre-incubated several times with non-transduced HEK293T production cells to remove anti-cell antibodies from immune sera prior to neutralization assays. Figure 5a demonstrates as exemplified by sera obtained from rabbit 10 that the pre-incubation procedure leads to a significant reduction of anti-cell antibodies below the level detected in the corresponding prebleed serum. Whereas Figure 5b (lower panel) demonstrates for 2 MPER specific MAbs, 2F5 and 4E10, that the pre-incubation procedure reduces the binding of these MAbs to a 30-mer MPER-peptide, the upper panel depicting the reactivity of selected antisera before and after the pre-incubation procedure does not provide any evidence for removal of MPER-specific antibodies. However, despite these ELISA binding data we cannot completely rule out the possibility that there was a subspecies of 2F5- or 4E10-like antibodies, which was not captured by the 30-mer linear MPER-peptide but may have been depleted by pre-incubation procedure. As a consequence, future immunization studies engrafting the MPER on VLPs should emphasize the production of VLPs in cell-lines derived from the homologous species to be immunized.
Neutralization in our study was observed only in sera which proved to contain ELISA reactive antibodies. However, whereas a previous study claimed that trimer stabilization of VLP-exposed MPER peptides exhibit a beneficial effect on the formation of neutralizing antibody titers , our constructs induced comparable neutralizing titers irrespective of the absence or presence of the fused trimerization domain (Figure 4). As the gp41CTM core construct used in this study comprised a more extended CHR portion (55 residues) compared to the more truncated forms (42 and 24 residues) used by others , the longer CHR per se may already be sufficient to support or stabilize gp41CTM trimerization and proper display in vivo.
Recent studies underscored the importance of the membrane environment to properly present MPER immunogens to B cells. Consistently it was demonstrated that these approaches can induce a low titer of neutralizing antibodies, yet highlight the difficulty of achieving high titers ,. Some other immunogen designs where the MPER had been fused to various heterologous carriers elicited strong peptide specific reactivities, yet had not been successful in showing HIV-1 neutralizing activity , The gp41 constructs presented here were antigenic for 4E10 and induced a basic level of neutralization, which needs to be further enhanced - a common phenomenon among recent gp41 immunization studies –. Finally, a study by Bomsel et al. showed that not only neutralization, but also transcytosis-blocking properties and antibody-dependent cellular-cytotoxicity may protect from SHIV infection after animals had been immunized with virosomes containing an MPER peptide . Thus, assessment of IgG neutralizing activity of sera in vitro may not reflect protection at mucosal sites in situ. The evaluation of functional parameters other than neutralization, such as ADCC, ADVI and transcytosis blocking, may be of value in future MPER targeting studies.
Design of hemagglutinin zipper H3 was inspired by previous work of W. Weissenhorn and A. Hinz from Unit of Virus Host Cell Interactions (UVHCI), Grenoble. Neutralization tests were performed by D. Montefiori and his group at Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC. Antibodies for p24 ELISA were kindly provided by Hermann Katinger, Polymun, Vienna. This study was funded by CAVD VDC Grant (PI Robin Weiss) to R.W.
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