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. 2011 Dec 19;9(2):184–190. doi: 10.1038/cmi.2011.55

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

Chang-Zheng Li 1,2,3,7, Zhong-Kun Liang 1,7, Zhen-Rui Chen 1, Hai-Bo Lou 1, Ye Zhou 2, Zhe-Huan Zhang 1, Fei Yu 4, Shuwen Liu 4, Yuanping Zhou 1, Shuguang Wu 4, Wenling Zheng 2, Wanlong Tan 1, Shibo Jiang 5, Chen Zhou 4,6
PMCID: PMC4002807  PMID: 22179672

Abstract

Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.

Keywords: antibody display, antibody screening, full-length antibody, HBsAg-specific antibody, mammalian display

Introduction

The hepatitis B virus (HBV) causes one of the most common infections in the world.1, 2 Two billion people have been infected with the virus, and about 350 million people are suffering from chronic HBV infection. More than one million people are estimated to die every year with liver failure, cirrhosis and hepatocellular carcinoma associated with HBV infection. Widely used, vaccination has been effective in preventing HBV infection.3, 4 Hepatitis B immunoglobulin (HBIG) is also important in the management of HBV infection.5, 6 Since vertical transmission is responsible for about 40%–50% of HBV carriers,5 newborns of mothers who are HBV surface antigen (HBsAg)-positive are generally required to receive HBIG and hepatitis B vaccine within 24 h following delivery and complete the whole regimen of recommended vaccination injections.7 Liver transplantation is an effective method for end-stage liver disease management, and many people with liver diseases related to HBV receive liver transplantation. However, liver transplantation cannot eliminate HBV infection in the blood or in other organs.8 With the inhibition of immunity, HBV reinfection occurs frequently, and it is more aggressive than before liver transplantation. Under these conditions, acute liver failure, fibrous cholestasis hepatitis and liver cirrhosis can follow with 50% mortality after 2 years.9, 10 Combination therapy with HBIG and lamivudine was shown to be an effective management therapy.11 Medical staff having contact with the blood or blood fluids of HBV carriers should receive HBIG immediately, if the titers of their hepatitis B surface antigen-specific antibody (anti-HBs) are lower than 10 mIU/ml.7

HBIG is commonly prepared from the blood plasma of anti-HBs-positive persons through natural infection or vaccination. Being a human plasma-derived product, HBIG is inconvenient for large-scale acquisition. Also, there are risks associated with blood-borne infectious pathogens, such as hepatitis C virus and human immunodeficiency virus.

So far, scientists have applied multiple strategies in the effort to find a way to produce HBIG safely and more effectively. A variety of HBV donor-specific phage display antibody libraries have been constructed in order to screen, select and develop HBsAg-specific monoclonal antibody therapeutics.12, 13 Using phage display technology, the complex structure of full-length antibodies have previously necessitated a cumbersome ‘divide and conquer' approach whereby antibodies are first dissected into fragments that can be engineered in microorganisms, followed by ‘stitching back' the optimized fragments into the full-length form for production in mammalian cells.14 Most recently, mammalian display promises to enable rapid and direct screening of full-length antibodies. Furthermore, since the selected antibody can be directly produced in mammalian cells, scientific research is further benefited by savings in both time and money. However, most of these systems display multiple copies of antibodies with different specificities on a single cell surface, making it difficult to identify and isolate antibodies with a desired property. These limitations significantly hamper the application of mammalian display technology in the development of therapeutic antibodies.15 Some researchers have tried to overcome these limitations by sufficiently diluting the antibody gene library plasmids with non-library plasmids prior to transfection. Under well-controlled transfection conditions, the majority of the transfectants display one kind of antibody on the cell surface.16 Nevertheless, the dilution ratio and transfection efficiency were not very easily controlled.

To overcome these challenges, we have successfully developed a platform in which only one specific antibody can be displayed on each mammalian cell surface.15 Coupled with fluorescence-activated cell sorting (FACS) and two-color staining, antibody with antigen specificity can be selected, and the affinity can be analyzed directly by detecting fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA).15 Using the vector pDGB-HC-TM and the enzymes BsmBI and SfiI, we have also developed a method to construct large full-length antibody libraries with sizes of 1010–1011 within 2–8 weeks.17, 18, 19, 20 Using the one-step four-way ligation method and the vector pDGB4, we can construct highly efficient antibody libraries with sufficient stability to display full-length antibodies on mammalian cell surfaces. When coupled with the Flp recombinase-mediated integration (Flp-In)21 system, each cell displays only one kind of antibody molecule,15, 22, 23 which may be helpful for the screening of high-affinity antibodies with biological function.

Here we report the construction of a fully human, full-length mammalian cell surface-displayed antibody library of a healthy volunteer and the successful identification of HBsAg-specific antibodies from the library.

Materials and methods

Reagents and cell lines

Primers were synthesized by Invitrogen (San Diego, CA, USA). T4 DNA ligase and restriction enzymes were purchased from Fermentas (Hanover, MD, USA). Ready-to-use Taq DNA polymerase (2× Master Mix) came from Promega (San Luis Obispo, CA, USA). Cell transfection reagent was kindly provided by Dgen Biotech Ltd (Hong Kong, China). The Flp-In system, including vector pOG44, Flp-In Chinese hamster ovary (FCHO) cell line and related cell maintenance media, was bought from Invitrogen (Carlsbad, CA, USA). Antibody reagents were bought from BD Pharmingen (San Diego, CA, USA). FCHO cells and 293-T cells (ATCC, Manassas, VA, USA) were maintained in Ham's F12 or Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cell dissociation buffer was purchased from Invitrogen. Vector pDGB-HC-TM and vector pDGB4 had already been constructed as described previously.19, 22

Miniprep, maxiprep and gel extraction

Using the kits bought from Axygen (Union City, NJ, USA), experiments were performed according to the manufacturer's instructions.

DNA digestion and fragment purification

Vector DNA was isolated from overnight cultures of Escherichia coli bacteria. DNA fragments were separated through electrophoresis in 1% agarose gel after digestion with proper restriction enzymes. The target DNA fragments were later isolated as described previously.24

Isolation of human peripheral blood mononuclear cells and amplification of antibody genes

Fifty milliliters of peripheral blood were obtained from a healthy volunteer who had recovered from hepatitis B virus infection. The protocol for collection of blood for this study was approved by Medical Ethics Committee of Nanfang Hospital and the volunteer signed the written informed consent before blood collection. ELISA analysis of his blood serum was performed by Nanfang Hospital and showed positive result of anti-HBsAg antibody. Peripheral blood mononuclear cells (PBMCs) were collected by gradient centrifugation at 400g for 20 min in a swing-bucket rotor, and PBMC layer was carefully transferred to a new tube and washed twice using phosphate-buffered saline. Total RNA of PBMCs was isolated using RNesay Mini Kit according to manufacturer's instructions. Using the RNA as template, the antibody genes were amplified by two-step reverse transcription (RT)-PCR. RT was carried out with 500 nM specific primer following Promega's instructions. The RT products were used as template in PCR amplification. In a 50 µl tube, 200 nM forward and reverse primers were mixed with 2 µl of RT products and 25 µl of 2× Master Mix.19 Amplification conditions were as follows: 94 °C for 5 min to denature the template, followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C and extension at 72 °C for 1 min per 1 kb length of DNA to be amplified, ending with 7 min of extension at 72 °C. PCR products were separated by electrophoresis on a 1% agarose gel and purified. The targeted fragments were digested by proper restriction enzymes according to experimental needs, purified by electrophoresis in another agarose gel, and then used in ligation.

Vector ligation and transformation

About 100 ng of total vector and insert fragments were mixed in a total volume of 10 µl with one unit of T4 DNA ligase. After ligation for at least 2 h, 1 µl of ligation mixture was used in transformation with 50 µl of bacteria-competent cells by standard procedure. The proper amount of bacterial solutions was plated on LB-ampicillin plate and cultured at 37 °C overnight. The colonies were counted, and the transformation efficiencies and library sizes were calculated.

Transfection and selection

Transfection was performed according to experimental needs with either FCHO cells or 293-T cells. Typically, transient transfection was performed in a 12-well plate, unless otherwise stated. The day before transfection, 4×105 cells were seeded in each well. The transfection reagent (5 µg) and DNA (2 µg) were separately diluted in 100 µl of Dulbecco's modified Eagle's medium each and then mixed well. The mixture was incubated for 30 min at room temperature and directly added into each well without changing the culture medium; then cells were commonly incubated at 37 °C. Six hours later, the medium was changed with fresh culture medium. At 48–72 hours post-transfection, the antibody expression was analyzed by FACS. For stable transfection using a T-75 flask, 8×106 cells were seeded the day before transfection. Thirty-five micrograms of vector DNA in total (20 µg of antibody library plus 15 µg of pOG44) and 105 µg of transfection reagent were separately diluted in 1 ml of Dulbecco's modified Eagle's medium each and then mixed. The mixture was incubated at room temperature for 30 min and directly added into the flask without changing the culture medium. After 24 hours of incubation at 37 °C, most of the cells were split with a ratio of 1∶10 and small amount of cell suspension was used for serial dilution of 1∶100, 1∶1000 and 1∶10 000. Hygromycin B was added in the medium 24 hours post-split to a concentration of 500 µg/ml. After selection for 10–12 days, the hygromycin B-resistant cell clones were collected as the stable cell library for FACS analysis and selection of specific antibodies. The cell clones in the flask of proper dilution were counted and the total library size was calculated accordingly.

Preparation of FITC fluorescence-labeled HBsAg

Genetic HBsAg protein was purchased from Keyuezhongkai (Beijing, China). Fluoro Tag FITC Conjugation Kit was purchased from Sigma-Aldrich (St Louis, MO, USA). HBsAg protein was prepared in 0.1 M carbonate–bicarbonate buffer at a concentration of 5.0 mg/ml. One milligram of the protein was used for FITC labeling according to manufacturer's instructions. The concentration of FITC fluorescence-labeled HBsAg (FITC-H) was 1.8 mg/ml, and the fluorescein/protein molar ratio (F/P) was 1.18∶1, which means that there were 1.18 FITC fluorescein molecules on each HBsAg molecule.

FACS analysis and selection of cell library

FCHO cells or 293-T cells were dissociated by cell dissociation buffer, followed by one wash using staining buffer (2% fetal bovine serum in phosphate-buffered saline). Then, cells were stained on ice by PE fluorescence-labeled anti-human kappa chain antibody (PE-K) only or FITC-H added, according to experimental needs. After staining for 30 min, cells were washed and resuspended in staining buffer for FACS analysis. The FACS data were analyzed using FCS Express V3 (De Novo Software, Los Angeles, CA, USA). FSE, FSB and RBA were calculated as described previously.15 Single cells with specific binding to HBsAg were sorted into a 96-well plate for further analysis, as needed.

Results

Construction and analysis of primary heavy chain (HC) and light chain (LC) libraries

The primary fully human, full-length antibody gene libraries were constructed as described previously.19 Using total RNA isolated from donor's PBMC as template, the full-length kappa LC repertoire and variable domain of HC (VH) repertoire were amplified by two-step RT-PCR procedure. PCR primers were designed according to the sequence information of V-base as described (http://vbase.mrc-cpe.cam.ac.uk/) and contained comparable restriction endonuclease-recognizing sequences at the 5′-ends.19, 20 The forward primers, from 5′- to 3′-, contain restriction endonuclease-recognizing sequences, Kozak sequence (CCACC), start codon and gene-specific sequences. The reverse primers contain restriction endonuclease-recognizing sequences and gene-specific sequence. After PCR amplification, the fragments were digested by proper restriction endonuclease. After insertion of VH and LC into vector pDGB-HC-TM separately, an HC (IgG1) library and a kappa chain library with sizes of 1.02×106 and 1.78×105 were constructed, respectively. The combinatory library size is 1.82×1011 ((1.02×106)×(1.78×105)).

To assess the quality of the library, 10 clones each were randomly chosen from HC and LC libraries for sequence analysis. The results showed that six HC clones had correct reading frame, coding for six unique VH (Figure 1a) and that nine LC clones have correct reading frame, coding for eight unique LCκ (Figure 1b). According to the sequence results, the combinatory library diversity is 8.71×1010.

Figure 1.

Figure 1

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Analysis of primary antibody library. (a) Alignment analysis of amino acid sequences of VH regions of HC clones. (b) Alignment analysis of amino acid sequences of LCκ regions of LC clones. (c) FACS analysis of antibody expression of HC and LC libraries. 293T cells were transiently cotransfected with HC and LC libraries. After labeling with PE-K, the cells were analyzed by FACS. (I) Parent 293T cells; (II) 293T cells cotransfected by HC and LC libraries. FACS, fluorescence-activated cell sorting; HC, heavy chain; LC, light chain; PE-K, PE fluorescence-labeled anti-human Kappa chain antibody; VH, variable domain of heavy chain.

After cotransfection of these two libraries into 293T cells, the antibody expression was analyzed by FACS. Cells were stained by PE-K. Results showed that 75.23% of the cells had detectable antibody expression on the cell surface (Figure 1c).

Construction and screening of secondary fully human, full-length antibody mammalian display library

The secondary fully human, full-length antibody gene library was constructed by four-way ligation.22 The gene fragments from primary HC library (BsmBI digested) and primary LC library (SfiI digested) were simultaneously cloned into vector pDGB4 by four-way ligation. After transformation, the number of bacterial clones was counted and transformation efficiency was calculated as described previously.19, 22 The size of the secondary library was 2.20×105 with a background of 0.40%.

By stable transfection of the secondary library into FCHO cells, a stable antibody display library was constructed with a size of 4.32×105, calculated by counting the cell clones. FACS analysis showed that 40.12% of FCHO cells in the library could display detectable full-length antibodies on cell surfaces (Figure 2a).

Figure 2.

Figure 2

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FACS analysis and screening of secondary antibody library. (a) FACS analysis of full-length antibody expression on FCHO cell surfaces of secondary antibody display library. FCHO cells were stably transfected with secondary display antibody library. Cells of stable antibody display library were labeled with PE-K and FITC-H and analyzed by FACS. (I) FCHO cells transfected with parent vector pDGB4, but not labeled with PE-K; (II) FCHO cells stably transfected with pDGB4 and labeled with PE-K; (III) FCHO cells stably transfected with secondary full-length antibody display library and labeled with PE-K. (b) FACS analysis of anti-HBsAg antibody expression on FCHO cell surface of secondary antibody display library. FCHO cells stably transfected by secondary antibody display library were labeled with PE-K plus FITC-H and analyzed by FACS. (I) FCHO cells stably transfected with pDGB4; (II) FCHO cells stably transfected with secondary antibody display library; 1.52% double-positive cells were sorted out for further analysis. (c) FACS analysis of anti-HBsAg antibody expression on FCHO cell surface after the first two rounds of cell pool sorting. FCHO cells sorted from secondary antibody display library were labeled with PE-K plus FITC-H and analyzed by FACS. (I) FCHO cells stably transfected with secondary antibody display library; (II) FCHO cells sorted from the first round of sorting and expanded; 0.10% double-positive cells were single-cell sorted for further analysis. FACS, fluorescence-activated cell sorting; FCHO, Flp-In Chinese hamster ovary; FITC-H, FITC fluorescence-labeled HBsAg; HBsAg, hepatitis B virus surface antigen; PE-K, PE fluorescence-labeled anti-human Kappa chain antibody.

The FITC-conjugated HBsAg (FITC-H) was prepared as described in the section on ‘Materials and methods'. After labeling with PE-K plus FITC-H, the FCHO cell library was analyzed by FACS. In Figure 2b, 1.52% of the cells, which are PE-K and FITC-H double-positive, were sorted out and expanded for a second round of FACS analysis and sorting. After two rounds of FACS screening, the percentage of PE and FITC double- positive cells rose to 10.31% (Figure 2c).

As shown in Figure 2c, 0.10% double-positive cells were sorted into two 96-well plates for single-cell cloning and 65 sorted cell clones were successfully grown out. The cells were labeled with PE-K plus FITC-H and analyzed by FACS. The results showed that cells displaying non-specific antibodies exhibited only PE-K or/and FITC-G signals but no FITC-H signal (Figure 3b–d), indicating that the cells express full-length antibodies which has no binding ability to HBsAg and there are no other molecules on cell surface which can bind to HBsAg specifically or non-specifically. The results of double staining of clones #1–#8 with PE-K plus FITC-H indicated that eight single-cell clones displayed full-length HBsAg-specific antibodies on their cell surfaces (Figure 3e-l) because PE-K could bind to antibodies during cell staining only when the kappa chains were assembled with paired heavy chains and expressed on cell surfaces.

Figure 3.

Figure 3

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FACS analysis of anti-HBsAg antibody expression on single-cell clones. FACS-sorted single-cell clones were expanded and analyzed by FACS. (a–d) Cell clone displayed non-specific antibodies: (a) no fluorescence label; (b) labeled with PE-K; (c) labeled with PE-K plus FITC-conjugated mouse anti-human IgG antibodies (FITC-G); (d) labeled with PE-K plus FITC-H. (e–l) cell clones (#1–#8) displayed HBsAg-specific antibodies and labeled with PE-K plus FITC-H. FACS, fluorescence-activated cell sorting; FITC-H, FITC fluorescence-labeled HBsAg; HBsAg, hepatitis B virus surface antigen; PE-K, PE fluorescence-labeled anti-human Kappa chain antibody.

Sequences and binding ability of selected clones

Total RNAs were isolated from the 8 FCHO cell clones. VH and LC were amplified by RT-PCR and cloned into pDGB4. Sequence analysis shows that #1, #2, #3, #5, #6 and #7 cell clones coded the same amino-acid sequence, while clones #4 and #8 coded two unique amino acid sequences (Figure 4). BLAST analysis of amino-acid sequences through GeneBank database showed that the amino-acid sequences of the three clones (#1, #4 and #8) were unique.

Figure 4.

Figure 4

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Amino-acid sequences of eight cell clones. (a) Alignment of amino-acid sequences of VH regions of eight cell clones. (b) Alignment of amino-acid sequences of LCκ regions of eight cell clones. LC, light chain; VH, variable domain of heavy chain.

To confirm that the antibodies cloned bind to HBsAg, the plasmid DNAs of clones #1, #4 and #8 were transiently transfected into FCHO cells. FACS analysis of the cell clones double-stained by PE-K and FITC-H demonstrated the HBsAg-specific binding of these antibodies (Figure 5). Clone #1 shows the highest RBA (Table 1).

Figure 5.

Figure 5

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FACS analysis of the binding of HBsAg to anti-HBsAg antibody on cell surface. FCHO cells were transiently transfected with plasmid DNA of antibody gene clones, labeled with PE-K plus FITC-H, and analyzed by FACS. (a) FCHO cells transfected with pDGB4; (b) FCHO cells transfected with pDGB4 and labeled with PE-K; (c) FCHO cells transfected with pDGB4 and labeled with PE-K and FITC-H; (d–f) FCHO cells transfected with vector DNA of clones #1, #4 and #8 and labeled with PE-K and FITC-H. FACS, fluorescence-activated cell sorting; FCHO, Flp-In Chinese hamster ovary; FITC-H, FITC fluorescence-labeled HBsAg; HBsAg, hepatitis B virus surface antigen; PE-K, PE fluorescence-labeled anti-human Kappa chain antibody.

Table 1. The binding ability of three monoclonal antibodies with HBsAg.

Monoclonal antibody FSE FSB RBA
#1 18.21 3.22 0.18
#4 10.80 0.58 0.05
#8 15.19 2.40 0.16
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Abbreviations: FSB, fluorescent strength of binding; FSE, fluorescent strength of expression; HBsAg, hepatitis B virus surface antigen; RBA, relative binding ability.

Data were calculated from the FACS results of Figure 5d–f. FSE=% of PE-positive cells×mean of fluorescent strength of expression signal. FSB=% of FITC-positive cells×mean of fluorescent strength of binding signal. RBA=FSB/FSE.

Discussion

A key step in the development of antibody therapy is the identification of target antigen-specific antibodies. Hybridoma25, 26, 27 technology is commonly used in screening and selecting these antibodies. In most situations, they are nonhuman antibodies, which need to be humanized for further development as therapeutics. Phage display28, 29 is a newly developed antibody screening technology, and it is now the most widely used antibody display technology in developing therapeutic antibodies. However, because of the non-full-length display and the non-mammalian property, the development of identified antibody fragments is challenged by high production costs and a low rate of success.

To address these issues, we have utilized a newly developed mammalian display technology to identify HBsAg-specific antibodies. Using total RNA of a natively immunized donor as template, the antibody repertoire was amplified by RT-PCR and fully human, full-length antibody display libraries were constructed. Three unique HBsAg-specific antibodies were identified from the established FCHO cell library. Because they are fully human, full-length antibodies, they can be easily expressed from CHO cells, with no further need to humanize or convert fragments to full length, if they are proved to be candidates for further development of therapeutic antibodies.

It has been reported that the Flp-In single integration-based mammalian display system can be successfully used in affinity maturation by mammalian display technology15 and is a more powerful platform in identification of antigen-specific antibodies compared to the multiple integration system used by other researchers.16, 30 Using this system, cell clones expressing antigen-specific antibodies can be efficiently screened and selected by single-cell sorting after only brief enrichment.

In the present report, separate primary HC and LC libraries were constructed with the goal of preserving the highest diversity of HC and LC, thus permitting us to reserve the original materials in stock for further manipulation should we, at any time, fail to construct a single-integration, dual-expression cassette, full-length antibody display FCHO library. According to the sequence analysis of single DNA clones, 6/10 of heavy chain clones (Figure 1a) and 9/10 of light chain clones (Figure 1b) are expressible. Theoretically, 54% of the DNA clones in the secondary DNA library would be expressible. In other words, 54% of the FCHO cells, after stable single integration, will express full-length antibodies. Results show that about 40% of the cells express detectable full-length antibodies on FCHO cell surfaces, which roughly agrees with theoretical calculation, considering that not every expressible DNA clone will express and display enough antibody molecules on the cell surface to be detectable after integration into the cell genome.

The secondary library has a size of 2.2×105 in bacteria transformants and 4.32×105 in FCHO cell clones. Because 6/10 of HC clones (Figure 1a) and 8/10 of LC clones (Figure 1b) are unique, the diversity of the secondary library is about 1×105, and the cell library has coverage of four times the diversity. The results demonstrate that an immunized antibody library of 105 in diversity is large enough for the identification of target antigen-specific antibodies. For antibodies with some specific functions, such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, a library with more diversity may be required. The primary libraries provide the potential to meet these needs because our primary library has a combinatory library size of 1011 and a combinatory diversity of more than 1010.

Coupled with FACS, we successfully screened eight cell clones which could stably display antibody binding to HBsAg. Sequence analysis results revealed three unique antibodies among these eight cell clones. Antibody from cell #1 has the highest RBA among the three antibodies. To our knowledge, it is the first time to directly identify antigen-specific full-length human antibodies by the Flp-In single integration-based mammalian display platform15 from mammalian cell surface displayed full-length antibody library constructed using materials from immunized personal. These results confirm that our antibody mammalian display and screening platform can be successfully used in specific antibody screening and will be very helpful in the development of therapeutic antibodies.

Acknowledgments

This work was supported by Dgen Biotech Ltd (DGB12012008), National Science and Technology Major Projects for ‘Major New Drugs Innovation and Development' (2011ZX09506–004) and Guangdong Medical Research Foundation (A2011363). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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