Enhanced antibody response to ovalbumin coupled to poly-dispersed acid functionalized single walled carbon nanotubes

Journal Pre-proof Enhanced antibody response to ovalbumin coupled to poly-dispersed acid functionalized single walled carbon nanotubes Taru S. Dutt, Rajiv K. Saxena

PII:

S0165-2478(19)30469-9

DOI:

https://doi.org/10.1016/j.imlet.2019.11.003

Reference:

IMLET 6393

To appear in:

Immunology Letters

Received Date:

10 September 2019

Revised Date:

21 October 2019

Accepted Date:

9 November 2019

Please cite this article as: Dutt TS, Saxena RK, Enhanced antibody response to ovalbumin coupled to poly-dispersed acid functionalized single walled carbon nanotubes, Immunology Letters (2019), doi: https://doi.org/10.1016/j.imlet.2019.11.003

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Enhanced antibody response to ovalbumin coupled to poly-dispersed acid functionalized single walled carbon nanotubes

Short title: Antibody response to ovalbumin couples to carbon nanotubes

Taru S. Dutt and Rajiv K. Saxena

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Faculty of Life Sciences and Biotechnology, South Asian University, Akbar Bhawan, Chanakyapuri, New Delhi 110021 (India)

Correspondence:

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Prof. RK Saxena,

Phone: 91-9910048241

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Chanakyapuri, New Delhi 110021 (India)

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Faculty of Life Sciences and Biotechnology, South Asian University, Akbar Bhawan

Email: [email protected]

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Ovalbumin was covalently coupled to poly-dispersed acid-functionalized single walled carbon nanotubes (AF-SWCNTs) Mice immunized subcutaneously with ovalbumin - AF-SWCNT complex induced significantly higher antibody response as compared to the mice immunized with free ovalbumin Antibody response generated by ovalbumin - AF-SWCNT complex was comparable to that induced by ovalbumin along with Freund’s adjuvant. We suggest immunization with antigens coupled to AF-SWCNTs can be explored for improving the antibody response

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Highlights

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Abstract

Activated mouse B cells as compared to the resting B cells are known to internalize substantially more acid-functionalized single walled carbon nanotubes (AF-SWCNTs). It was hypothesized that the antigens coupled to AF-SWCNTs would also be taken up more efficiently by B cells. Further the enhanced uptake of the antigen by B cells may facilitate antigen presentation by B cells resulting in a better antibody response. Aim of this study was to test this hypothesis. Ovalbumin was chemically coupled to AF-SWCNTs that yielded a coupled product that had 0.08% of all carbon atoms in AF-SWCNTs occupied by ovalbumin. Coupling of ovalbumin to

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AF-SWCNTs was confirmed by staining the product with anti-ovalbumin antibodies. B cells incubated with ovalbumin-AF-SWCNT internalized more ovalbumin than the B cells incubated with free ovalbumin. Groups of mice were immunized subcutaneously with (a) free ovalbumin,

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(b) free ovalbumin and AF-SWCNTs at two different subcutaneous sites respectively on mice, and (c) ovalbumin-AF-SWCNT coupled product. In each case a primary immunization was

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followed by three weekly booster doses. It was found that the anti-ovalbumin antibody response assessed by ELISA, was highest in the group where ovalbumin coupled to AF-SWCNTs was

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used for immunization (p<0.001). Antibody response in ovalbumin-AF-SWCNT group was comparable to the group where ovalbumin was used for immunization using complete and

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incomplete Freund’s adjuvant (primary and secondary immunizations respectively). We propose that AF-SWCNTs could be explored as an adjuvant to improve the antibody response especially

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in vaccine development.

1. Introduction:

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Structurally, carbon nanotubes (CNTs) are rolled up graphite sheets with a diameter of about 2 nanometers, with variable lengths[1]. This allotrope of carbon may not occur in nature but is now manufactured in huge quantities as it has found numerous commercial applications [2–6]. CNTs are sturdy chemical entities that do not degrade easily in nature. Further CNTs efficiently interact with cells and alter cellular functions [7–13]. Introduction of this new unprecedented and unique material in environment has raised issues about its short and long term health effects in humans and its potential interference in physiology of animals, plants and microbes [14–22].

Several biomedical applications of CNTs have been proposed including their use in drug targeting [23–27]. Since all carbon atoms in CNTs are on surface and can be linked chemically to desired ligands, huge amounts of drugs and ligands may be attached to CNTs [28]. Functionalized carbon nanotubes are being examined as scaffolding for multiple ligands that can be covalently attached to CNTs for subsequent delivery to cells in vitro and in vivo. Multiple antigens like peptides derived from protein antigens and other molecules that may enhance the immune response may be coupled to CNTs and used for preparing efficient vaccines [29–33].

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An immune response requires the antigen to be processed and presented by antigen presenting cells (APCs) to T helper cells and subsequently T helper dependent activation and proliferation

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of specific B cells to get antibody secreting plasma cells [34,35]. Many types of APCs are known that include dendritic cells, macrophages and B cells. APCs must internalize protein antigens and

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present peptides derived from these protein antigens in association with MHC molecules to T helper cells [36]. Anything that would enhance the antigen internalization by the APCs may

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therefore facilitate antigen presentation. We have recently demonstrated that B cells upon activation internalize large amounts of poly-dispersed acid-functionalized single walled carbon nanotubes (AF-SWCNTs) as compared to resting B cells [37]. Since B cells are efficient APCs,

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we hypothesized that protein antigens covalently coupled to AF-SWCNTs, may be more efficiently internalized by B cells thereby facilitating antigen presentation. Better antigen

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presentation may result in better antibody response. In the present study we have tested this hypothesis by immunizing mice with a known protein antigen ovalbumin either free or covalently coupled to AF-SWCNTs. Antibody titers developed to free and AF-SWCNT-coupled ovalbumin were compared. Our results indicate that AF-SWCNT coupled ovalbumin elicited a significantly better immune response as compared to free ovalbumin. These results indicate that

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AF-SWCNTs may be explored as an adjuvant for improved immune responses to various vaccines.

2. Materials and Methods

2.1 Animals: Inbred C57BL/6 mice (8-15 weeks old, 20-25g body weight) were used throughout the study. Animals were maintained in the animal house facility at South Asian University (SAU), New Delhi, and obtained from the National Institute of Nutrition (NIN), Hyderabad.

Animals were housed in positive pressure air-conditioned units (25 C, 50% relative humidity) and kept on a 12-hour light/dark cycle. Water and chow were provided ad libitum. All the experimental protocols were conducted strictly in compliance with the guidelines notified by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India (www.envfor.nic.in/divisions/awd/

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cpcsea laboratory.pdf). The study was duly approved by SAU Institutional Animal Ethics Committee (IAEC project approval code: SAU/IAEC/2016/02).

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2.2 Reagents and other supplies: Single walled carbon nanotubes (Cat# 775535, > 95 % carbon

(NHS),

1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide

(EDAC),

2-(N-morpholino)

(MES), Complete Freund’s adjuvant (CFA) and Incomplete Freund’s

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ethanesulfonic acid

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purity) were procured from Sigma-Aldrich (St. Louis, MO, USA), N-Hydroxysuccinimide

Adjuvant (IFA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Albumin from

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chicken egg white (ovalbumin), 3,3',5,5'-Tetramethylbenzidine (TMB), Fluorescein isothiocynate (FITC) and casein was from sigma Aldrich (India). RPMI complete medium with 2mM

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glutamine, 1 mM sodium pyruvate, 4.5 g glucose/liter, 10 mM HEPES, 1.5 g/liter sodium bicarbonate and 20µg/ml gentamycin and Fetal Bovine Serum (FBS) were from Gibco (Carlsbad, CA, USA), Centricon 3 kDa centrifugal filter device was obtained from Millipore

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(Billerica, MA, USA) and 45 kDa dialysis tubing was from spectra laboratories (Milpitas, CA). Anti-mouse CD16/32 (Fc Block), FITC/PE anti-mouse CD19, FITC/PE/APC anti mouse F4/80 and 7AAD were purchased from eBiosciences (San Diego, CA, USA). Anti-mouse IgG-HRP conjugated secondary antibody, anti-mouse IgG FITC conjugated secondary antibody and Bicinchoninic acid (BCA) protein estimation kit was procured from Thermo Fisher Scientific (Waltham, MA, USA).

2.3. Acid functionalization of Single-walled carbon nanotubes: Acid-functionalized singlewalled carbon nanotubes (AF-SWCNTs) were prepared as described before [12,38]. Briefly, SWCNTs (20 mg) were suspended in 1:1 ratio of concentrated H2SO4 and HNO3 (20 ml) and heated in a high pressure vessels of volume 100 ml and placed in a microwave ovan (pressure 20 ± 2 psi, power of 450W, temperature 135-150 C for 3 minutes). Suspension was cooled, diluted

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three times with water and dialyzed against deionized water. Dialyzed suspension was lyophilized and weighed. Detailed characterization of AF-SWCNTs including size and charge

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distribution, BET surface area, and electron microscopic features, have been reported earlier

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[37].

2.4. Coupling of ovalbumin to AF-SWCNTs: Ovalbumin was covalently conjugated to AF-

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SWCNTs by utilizing diimmide-activation amidation of carboxylic acid groups. AF-SWCNTs (100 µg) were incubated with EDAC (100 mM) and NHS (650 mM) in 100 µl MES buffer (100

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mM MES prepared in deionized water, pH adjusted to 6.0) for 2 hours at room temperature. Unbound EDAC and NHS were removed by using 3 kDa Centricon filter units (centrifugation at

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10000 rpm for 10 minutes four times) and AF-SWCNTs retained in the upper chamber were collected (~70µl). Activated AF-SWCNTs were further incubated overnight with 300 µl of ovalbumin (1, 5 and 10 mg/ml in MES buffer). Samples were centrifuged, and pelleted

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ovalbumin conjugated nanotubes were collected. Concentrations of ovalbumin before and after conjugation reaction were estimated using Bicinchoninic acid protein estimation kit (Thermo Fisher, cat #23225).

Amount of ovalbumin tagged to AF-SWCNTs was determined by

subtracting the amount of free ovalbumin in supernatant after coupling reaction from initial amount of ovalbumin taken for conjugation.

2.5. Immunization of mice: Four groups of six C57BL/6 male mice each (8-15 weeks old, 2025 g body weight) were immunized subcutaneously with PBS alone (control), ovalbumin (20 µg/mouse for primary immunization, 5 µg/mouse for booster dose), AF-SWCNTs (8 µg/mouse for primary immunization 2 µg/mouse for booster dose) and ovalbumin coupled AF-SWCNTs (20 µg ovalbumin coupled to 8 µg AF-SWCNTs for primary immunization/mice and 5 µg

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ovalbumin coupled to 2 µg AF-SWCNTs for booster immunizations). An additional positive control group of mice was immunized with ovalbumin (20 µg/mouse emulsified with complete

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Freund's adjuvant for primary immunization and 5 µg ovalbumin emulsified with incomplete Freund's adjuvant for subsequent booster immunizations). Blood samples were obtained by

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cardiac puncture 5 days after the fourth immunization dose, serum was prepared (All anti-sera

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were stored at -80 C till used) and ELISA was performed to check antibody titer obtained at different dilutions (1:500, 1:2000, 1:8000 and 1:32000).

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2.6. ELISA: ELISA was performed as described before [39]. In brief, 96 well polystyrene plates were coated with ovalbumin (500 ng/100 µl/well) prepared in 0.2 M carbonate-bicarbonate

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buffer (pH-9.2) and incubated overnight at 4 C. Coated wells were washed five times with 200 µl wash buffer [Phosphate buffer saline containing 0.05% Tween 20 (PBST)] and then incubated with 200 µl blocking buffer (1% casein prepared in PBST) for 2 hours. Wells were washed five

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times with PBST to remove excess blocking buffer. Dilutions of serum were prepared of control and immunized mice (1:500, 1:2000, 1:8000 and 1:32000 d/iluted in PBST containing 0.1 % casein) and 100 µl of serum were added to the ovalbumin coated wells. Plates were incubated for 2 hours at room temperature and washed five times with PBST. Anti-mouse IgG HRPconjugated secondary antibody (100µl, 1:1000 dilution prepared in PBST) were added and incubated at room temperature for 1 hour. After the plates has been washed as described,

colorimetric signal was generated by addition of TMB substrate, 100 µl/well (prepared in phosphate-citrate buffer, 0.05M, pH-5.0 and added 2 µl of 30% H2O2 to every 10 ml of TMB solution immediately prior to use). The reaction was stopped by adding 100 µl of 1 N H2SO4 in assay wells. Absorbance was read at 450 nm using Biotek synergy HT microplate reader (Winooski, VT). Mean absorbance values were plotted against anti-sera dilutions using sigma

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plot version 11 (Systat software, USA) and two-way ANOVA was performed to find statistical significance between groups. Comparison of antibody titers in different groups of anti-sera could

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be made by converting each ELISA curve to linear equations (y=mx+c) and computing serum

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dilutions for a fixed ELISA absorbance.

2.7. Flow cytometry: For evaluating tagging of ovalbumin to AF-SWCNT, 5µg/100µl AF-

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SWCNT or ovalbumin (1, 5 and 10 mg/ml) tagged AF-SWCNT preparations were incubated with 100 µl anti-ovalbumin antibody produced in mice (1:500 dilution prepared in 1% casein in

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PBST) for 30 minutes. Samples were washed thrice with PBST to remove unbound primary antibody by centrifugation at 10000 rpm for 10 minutes. Supernatant was removed and 100 µl

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anti mouse FITC conjugated secondary antibody (1:1000 dilution prepared in PBST) were added. Samples were again washed three times and resuspended the tagged and untagged AFSWCNTs in 500 µl PBS. Data were acquired to evaluate percent ovalbumin positive AF-

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SWCNTs using FACS Verse flow cytometer. To evaluate the uptake of ovalbumin and ovalbumin coupled AF-SWCNTs by B cells and macrophages, splenocytes (2 x 106 cells/ml) were cultured in RPMI1640 medium with 10% FCS, with or without ovalbumin (14 μg/ml) or ovalbumin coupled AF-SWCNTs (19 µg/ml complex containing, 5 µg AF-SWCNT and 14 µg coupled ovalbumin) in a 1.5 ml microcentrifuge tube for 4 and 24 h. Cells were harvested, washed and stained with PE anti-mouse CD19 and APC

anti mouse F4/80 antibody to stain B cells and macrophages respectively. Cells were washed thrice to remove unbound antibody and were fixed using 4% paraformaldehyde and were permeabilized using PBS containing 0.1% triton-X-100. Non-specific sites were blocked using mouse serum (100 µl of 10% mouse serum prepared in PBS) and were incubated with antiovalbumin anti-sera tagged with FITC (1:250 dilution, 100 µl) for 1 h. Cells were washed thrice,

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resuspended in 500 μl PBS and were analyzed using FACS aria III. 2.8. Fluorescein Isothiocyanate (FITC) conjugation of mouse sera: Mouse sera were labelled

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with FITC as described by McKinney et al. [40]. Protein estimation of control mouse serum and

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anti-ovalbumin anti-serum performed using BCA protein estimation kit. Sera were incubated with FITC (400 µg per mg of serum protein in 500 mM carbonate-bicarbonate, pH-9.5 for 1 hour

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at room temperature in dark). Unbound dye was removed by washing 5 times using 3 kDa centricon. The values of moles of FITC attached to a mole of protein (IgG) was calculated by the

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method described elsewhere [41]. The values for control sera and anti-ovalbumin anti-sera were

3. Results

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10.5 and 10.4 moles of dye per mole of protein respectively.

3.1. Coupling of ovalbumin to AF-SWCNTs:

Chemically, CNTs can be written as (CH)n.

Each carbon atom can theoretically be carboxylated to get (C-COOH)n. However, the protocol

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used to acid-functionalize the CNTs may not carboxylate all carbon atoms and may also introduce sulfonate groups instead of COOH groups. Using the COOH group, proteins with free amino groups may be covalently linked to AF-SWCNTs by standard chemical protocols [42]. We coupled ovalbumin (molecular weight 44.3 kDa, containing 20 Lysine residues with free NH2 groups) to AF-SWCNTs by using the protocol described in methods and depicted in Figure

1A. In order to get an idea of the nature and extent of coupling of ovalbumin to AF-SWCNTs, we estimated the amount of ovalbumin that got attached to a unit weight of AF-SWCNTs. Knowing the number of carbon atoms in the AF-SWCNTs taken for coupling reaction and the number of

ovalbumin molecules that got bound with AF-SWCNTs (calculated from the

molecular weight of ovalbumin and Avogadro’s number), we could calculate the percentage of

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carbon atoms on AF-SWCNTs that were occupied by ovalbumin. Results in Figure 1B show that the percentage of carbon atoms occupied by ovalbumin was dependent on the initial

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concentration of ovalbumin used in coupling reaction. Optimal value of 0.08% occupancy of carbon atoms was obtained when 5 mg/ml ovalbumin was used for coupling reaction. While this

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percentage of occupancy of carbon atoms may appear low, consideration of C-C bond length in

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AF-SWCNTs (0.145 nm) and the size of ovalbumin molecule (7 nm x 4.5 nm x 5 nm) would indicate that at this level of occupancy of ovalbumin, virtually whole surface of CNTs would be After coupling with ovalbumin, the average size of the coupled

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covered by ovalbumin.

molecules increased from 244 nm to 300 nm and zeta potential changed from -45.3 mV to +15.9

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mV for the coupled product (Zeta sizer data in supplementary Figure S1). The coupling of the ovalbumin to CNTs could be due to one or more lysine residues on the molecule and it was important to assess if the bound ovalbumin had retained its secondary

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structure and was not denatured in the process. We examined the binding of coupled ovalbumin with anti-ovalbumin antibody coupled to FITC and analyzed the product by flow cytometry. Results in Figure 2 show the staining of three preparations of AF-SWCNTs (prepared by using different concentrations of ovalbumin, see Figure 1B) with anti-ovalbumin antibodies. Results in Figure 2 show that best staining was obtained with the AF-SWCNT-Ovalbumin product with 0.08% ovalbumin occupancy (Figure 2C). These results indicate that the secondary structure of

bound ovalbumin was sufficiently intact to be recognized by the anti-ovalbumin antibody. This coupled product was further used for immunizing the mice. 3.2. Uptake of free and AF-SWCNT coupled Ovalbumin by B cells and macrophages. B cells and macrophages are important antigen-presenting cells which present antigen to effector and memory T cells resulting in a prolonged immune response. Mouse spleen cells were

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incubated with free ovalbumin or AF-SWCNT coupled ovalbumin, washed and stained with

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anti-CD19 and anti-F4/80 antibodies to gate B cells and macrophages in flow cytometry. Cells were then permeabilized using triton-X100 and counter stained with anti-ovalbumin antibody-

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FITC. Results in Figure 3 show the comparison of relative levels of internalized free or coupled ovalbumin in B cells and macrophages. For B cells, uptake of ovalbumin coupled AF-SWCNTs

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was similar to ovalbumin at 4 hours (5% ovalbumin positive cells). However, it increased to

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around 14% after 24 hours of incubation (p<0.01). On the other hand, uptake of ovalbumin coupled AF-SWCNTs by macrophages was comparable to the uptake of free ovalbumin and did

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not change significantly with time (Figure 3).

3.3. Comparison of antibody response in mice to free and AF-SWCNT coupled ovalbumin. Four groups of 6 C57BL/6 mice each were immunized with (A) ovalbumin alone, (B) ovalbumin coupled to AF-SWCNTs, and (C) uncoupled AF-SWCNTs along with free ovalbumin. Control

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group of mice (group D) received only saline, and in three other groups of mice, amount of ovalbumin per immunization dose was identical (20 µg for primary immunization and 5 µg for booster doses). Mice were sacrificed five days after the completion of the immunization schedule (four weekly immunizations), and anti-sera prepared for determining anti-ovalbumin titers estimated by ELISA. Results in Figure 4A show that control mice given only the vehicle had no

anti-ovalbumin titers. Anti-sera from the other three groups of mice had significant titers of antiovalbumin antibodies. Mice immunized with ovalbumin and free AF-SWCNTs had significantly better antibody titer than the group immunized with ovalbumin alone (ANOVA, p<0.02), but highest antibody titers were obtained when ovalbumin coupled with AF-SWCNTs was used for immunization (ANOVA p<0.001, comparison with ovalbumin alone and ovalbumin and AF-

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SWCNTs injected separately). These results indicate that AF-SWCNTs as such may have an adjuvant effect on antibody titers when used along with free antigens, but best titers may be

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induced when the immunogen ovalbumin was covalently coupled to AF-SWCNTs. It was also essential to compare the adjuvant effect of AF-SWCNTs with the complete Freund’s adjuvant

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(CFA) and incomplete Freund’s adjuvant (IFA), since, it is one of the most effective

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immunopotentiators. Results in Figure 4B show that the use of CFA(for primary immunization), and IFA (during booster doses), also resulted in significant boosting of anti-ovalbumin antibody

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response. A comparison of the boosting effects of AF-SWCNTs and FA (Figures 4A and 4B) show that the boosting effect of the two agents was comparable (no significant difference

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between ANOVA values) indicating that the adjuvant effects of CFA+ IFA and AF-SWCNTs were similar (Table 1). A quantitative comparison of antibody titers in anti-sera raised in different groups of mice could be made by comparing the anti-serum dilutions that gave a fixed ELISA absorbance. For an ELISA absorbance of 0.5, the serum dilutions calculated from the

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ELISA curves were 1:1428 (for ovalbumin alone group of mice), 1:5000 (for free ovalbumin + AF-SWCNTs group), 1:11111 (ovalbumin coupled to AF-SWCNTs group) and 1:10000 (ovalbumin + CFA/IFA group). These results clearly indicate that ovalbumin coupled to AFSWCNTs resulted in best antibody titer that was comparable with the anti-sera raised by using Freund's adjuvant.

4. Discussion We have previously shown that a variety of cells including erythrocytes, epithelial cells and different types of immune cells internalize poly dispersed acid functionalized single-walled carbon nanotubes (AF-SWCNTs) [11–13,43,44]. Interestingly, activated T and B lymphocytes internalize substantially more AF-SWCNTs than resting T and B cells [37,45]. We therefore

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hypothesized that if specific protein antigens are attached to AF-SWCNTs, the uptake of antigens in APCs like the B cells may be facilitated leading to a better antigen presentation and

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hence a better antibody response. An important issue of toxicity of AF-SWCNTs should be

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considered here since we have also shown that enhanced uptake of AF-SWCNTs result in toxicity towards activated T and B cells [37,45]. In our immunization protocols in the present

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study, we injected the ovalbumin coupled AF-SWCNT subcutaneously and the dose was minimal (8 µg per immunization) and it is likely that at this dose, the internalization of AF-

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SWCNTs may be sufficiently low and may cause toxicity. We could demonstrate a significant adjuvant effect of coupling the antigen to AF-SWCNTs and a significantly higher antibody

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response, comparable to that seen with Freund’s Adjuvant. Many side effects of using complete and incomplete Freund’s adjuvant are known [46–48]. We found a sustained localized inflammatory response when FA was used in immunizattion. No such inflammation was noticed

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when AF-SWCNT coupled antigen was administered (data not shown). There is a body of literature focused upon the in vivo toxicity of carbon nanotubes and in general it appears that while the administration of large amounts of nanotubes may be associated with some toxicity, functionalization of nanotubes may considerable lower the toxic effect [15,49– 51]. We therefore suggest that in cases where very small doses of AF-SWCNTs coupled with antigen are administered subcutaneously, generalized toxicity may not occur. However this issue

would require further testing. Further, it is encouraging that the use of AF-SWCNT coupled antigens gave a robust antibody response that was comparable with the use of FA. Few technical issues require further comments. Our calculations show that virtually all AFSWCNT surfaces may be covered with ovalbumin in the ovalbumin-AF-SWCNT complex, and that is not consistent with 33% staining with anti-ovalbumin antibodies (Figure 2C). We however

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observed that the product of Ovalbumin AF-SWCNT coupling was considerably aggregated. Average size of these aggregates was larger and net charge of these complexes also turned

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positive (Supplementary Figure 1). Particle size distribution in these complexes would be wide

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and our use of flow cytometry to see the binding of ovalbumin with AF-SWCNTs may not be an ideal system to record the fluorescence of particles of all sizes. In flow cytometry intensity of particles over a certain

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fluorescence per particle is crucial and ovalbumin-AF-SWCNT

threshold size would only be recorded. This explain that why only 33% positivity was observed

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when ovalbumin AF-SWCNT was stained with FITC tagged anti-ovalbumin antibodies (Figure 2C). Binding of anti-ovalbumin antibody with the ovalbumin-AF-SWCNT complex however

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confirmed that the coupled ovalbumin retained its secondary structure, which would be important in sensitization of B cells in vivo. Enhanced uptake of the ovalbumin-AF-SWCNT complex in B cells was consistent with our hypothesis that attachment with particles may

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increase the internalization of the complex by B cells. Our study suggests that AF-SWCNTs may act as a potent adjuvant for an antibody response if the antigen is covalently coupled to it. Further, since multiple antigens can be attached to AFSWCNTs, this approach may be further explored for designing vaccines. Only limited adjuvants have been approved for testing in humans and it is worth examining if the toxicity and side

effects of using tiny amounts of AF-SWCNTs is sufficiently low for it to be used in human system. Acknowledgment: Research funding from the Department of Science and Technology, Government of India, and

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fellowship support to TSD from ICMR are gratefully acknowledged.

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Legend to figures Figure 1: Coupling of ovalbumin to AF-SWCNTs. AF-SWCNTs (100 µg) was suspended in MES buffer and incubated with 100 mM 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) for 2 h, dialyzed using 3 kDa MWCO centricon at 10000 rpm for 10 minutes and

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washed 4 times with MES buffer. Different concentrations of ovalbumin prepared in MES buffer

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(1, 5, 10 and 20 mg/ml) were added and incubated overnight at room temperature. Centrifuged the tubes at 10000 rpm for 10 minutes to settle all the tagged AF-SWCNTs and performed

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protein estimation of unbound ovalbumin present in supernatant using BCA protein estimation

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kit. Panel A represents the chemistry involved ovalbumin coupling. Panel B represent the

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percentage of carbon atoms in AF-SWCNTs that were tagged with ovalbumin.

Figure 2: Percentage ovalbumin positive AF-SWCNTs. Earlier prepared ovalbumin tagged AF-SWCNTs (5 µg/100 µl) were incubated with 100 µl of anti-ovalbumin antibody produced in mice [dilution 1:500 prepared in 1% casein in PBS + 0.01% Triton X-100 (PBST)] for 30

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minutes. Same amount of untagged AF-SWCNTs were taken as control for setting gates. Washing was done twice with PBST to remove unbound primary antibody and anti-mouse IgG secondary antibody (1:1000 dilution prepared in PBST) were added and incubated for 30 minutes. Samples were washed, resuspended in 500 µl PBS and acquired using FACSverse. Panel A represents AF-SWCNTs without ovalbumin coupling (gating control). Panels B, C and

D shows AF-SWCNTs coupled to 1, 5 and 10 mg/ml starting concentration of ovalbumin respectively. Values within bracket represents Mean ± SEM of three replicate experiments, and

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outside bracket values are representative of displayed histogram.

Figure 3: Comparison of uptake of ovalbumin and ovalbumin coupled AF-SWCNTs by B

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lymphocytes and macrophages. Splenocytes (2 x 106 cells/ml) were cultured incubated with ovalbumin (14 μg/ml) and ovalbumin coupled AF-SWCNTs (19 µg/ml complex containing, 5

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µg AF-SWCNT and 14 µg coupled ovalbumin) in a 1.5 ml polypropylene microcentrifuge tube

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for 4 and 24 h. Cells were harvested, stained with anti-mouse CD19 or F4/80 antibodies and intracellular staining was performed as described in materials and methods and analyzed using

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FACS aria III. Left panel shows percentage of B cells and right panel shows percentage of macrophages stained for intracellular ovalbumin. Values here represents mean ± SEM of

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triplicates. A two-way ANOVA was used for determining the statistical significance. Sigma plot software was used to derive the F and p values. For B cells, p value between A and B = 0.03, between A and C = 0.02, between B and C = 0.03. For macrophages, p value between A

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and B = 0.003, between A and C = 0.001, between B and C = 0.06.

Figure 4: Antibody response to free and AF-SWCNT coupled ovalbumin. Panel A: Groups of 5 mice each were immunized with free ovalbumin (20 µg/mouse for primary immunization

and 5 µg/ml for booster doses), free ovalbumin (20 µg/ml for primary immunization and 5 µg/ml for booster doses) and AF-SWCNTs (8 µg/mouse for primary immunization and booster 2 µg/mouse) on separate sites and ovalbumin chemically coupled with AF-SWCNTs (20 µg ovalbumin coupled to 8 µg AF-SWCNTs for primary immunization/mice and 5 µg ovalbumin coupled to 2 µg AF-SWCNTs for booster immunizations). Five days after the last booster dose,

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blood samples were collected, and sera derived from these samples. Anti-ovalbumin antibody titer was assessed using ELISA. Panel B: Groups of 5 mice were immunized with free

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ovalbumin and ovalbumin emulsified with Complete Freund’s Adjuvant (for primary

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mean of ELISA ODs for five antisera in each group.

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immunization) or Incomplete Freund’s adjuvant (for booster doses). Each data point represents

A: Ovalbumin alone B: Ovalbumin + Freund’s adjuvant A and B

A and C

A and D

C and D

B and D

F value

48.23

7.084

47.37

14.93

2.53

P value

0.000012

0.0136

0.000014

0.0007

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C: Ovalbumin and AF-SWCNTs separately

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ANOVA

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D: Ovalbumin coupled AF-SWCNTs

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Table 1: Level of significance between different groups by ANOVA

0.87