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Modulation of the immune response using Rapana thomasiana hemocyanin
International Immunopharmacology (2008) 8, 1033–1038
w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Modulation of the immune response using Rapana thomasiana hemocyanin Andrey Tchorbanov a,⁎, Krassimira Idakieva b,⁎, Nikolina Mihaylova a , Lyuba Doumanova a a b
Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev-Str. 26, 1113 Sofia, Bulgaria Institute of Organic Chemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev-Str., bl. 9, 1113 Sofia, Bulgaria
Received 31 January 2008; received in revised form 7 March 2008; accepted 12 March 2008
KEYWORDS Adjuvant; Protein-carrier; Hemocyanin; Rapana thomasiana; Gastropod
Abstract We have investigated the non-specific immunostimulatory and specific immunomodulatory effects of hemocyanin from marine gastropod Rapana thomasiana (RtH). The purified RtH, its structural subunits RtH1 and RtH2 and a construct with influenza virus hemagglutinin intersubunit peptide (IP) were used in immunization protocols of Balb/c mice. Antibody formation against RtH, RtH1, RtH2, RtH-IP as well as anti-RtH IgG antibody isotypes were determined by ELISA. The immune homology between both subunits and the whole RtH molecule was investigated by cross-blotting technique. The retaining of the B-cell epitope of IP, coupled to the RtH was recognised by Western blot. The results obtained demonstrate that the immunization with RtH or its subunits in experimental models resulted in strong immune response in vivo. Common epitope of influenza A virus hemagglutinin jointed to RtH results in generation of molecule with increased immunogenicity. Our results are the first demonstration that RtH and/or its subunits could be used in different immunization protocols as an adjuvant or as a protein-carrier. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Recent approaches in the practical immunology involve the use of different highly immunogenic molecules for nonspecific immunomodulation (as adjuvants) or for active specific immunostimulation (as protein carrier covalently bound to specific antigen). Numerous compounds are under ⁎ Corresponding authors. A. Tchorbanov is to be contacted at fax: +359 2 8700109. K. Idakieva, fax: +359 2 8700225. E-mail addresses: [email protected] (A. Tchorbanov), [email protected] (K. Idakieva). 1567-5769/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2008.03.008
evaluation such immunological reagents [1–3]. Among them are the hemocyanins (Hcs)—extracellular type-3 copper proteins occurring freely dissolved in the hemolymph of many arthropod and mollusc species [4,5]. Hcs, due to their xenogenic nature and large molecular size (4 to 8 MDa), are strongly immunogenic. The keyhole limpet hemocyanin (KLH) of the marine gastropod Megathura crenulata is the most widely used as an adjuvant and a carrier for vaccines and antigens [6,7]. Hc from another marine gastropod Haliotis tuberculata, is considered to be a possible substitute for KLH as an immunostimulant [8]. Recently, Hc obtained from the Chilean gastropod Concholepas Concholepas was
1034 reported to possess adjuvant immunostimulatory effect, as well as a significant antitumor activity against mouse bladder carcinoma cells [9,10]. In our previous studies on the hemocyanin isolated from Rapana thomasiana (RtH)—a marine gastropod living along the west coast of the Black Sea—we demonstrated that hemocyanin molecules are built from two structural subunits, RtH1 and RtH2 [11]. It has been shown that the structural subunits RtH1 and RtH2 are closely related and both correspond immunologically to KLH2, one of the two hemocyanin isoforms of the gastropod M. crenulata [12]. Based on this structural similarity, a logical conclusion is that the hemocyanin of R. thomasiana will have strong adjuvant immunostimulatory effect as well as it would be possible to use RtH as a protein carrier. To our knowledge, till now it is not known whether RtH and its subunits, RtH1 and RtH2, possess such immunologic properties. The aim of the present work is to investigate in mouse experimental model the adjuvant properties of RtH and its two subunits and also to use it as a carrier of a viral peptide (common epitope of influenza A virus hemagglutinin).
2. Materials and methods 2.1. Isolation of R. thomasiana hemocyanin Living marine snails R. thomasiana were caught near the Bulgarian coast of Black Sea (Varna) and stored in sea water. The hemolymph was collected by bleeding through several diagonal slits made on the foot muscle of the mollusc and filtered through gauze. Phenylmethanesulphonyl fluoride (PMSF, 1 mM) was added to the crude material to avoid possible proteolysis of the hemolymph. Hemocytes and other cells were removed by centrifugation at 5000 × g for 30 min at 4 °C and the native hemocyanin was isolated from freshly obtained hemolymph by pelleting in an ultracentrifuge (Beckman LM-80, rotor Ti 45) at 180 000×g for 4 h at 4 °C and stored in the presence of 20% sucrose (w/v) at − 20 °C until use. RtH was further purified by gel filtration chromatography on a Sepharose 4B column, equilibrated and eluted with 50 mM PBS, pH 7.2. The purity of the isolated Hc was controlled by SDS- and native PAGE as described in [11,16].
2.2. Isolation of Rapana hemocyanin structural subunits Purified RtH was dissociated into individual subunits by dialysis overnight at 4 °C, against 130 mM glycine–NaOH, pH 9.6, containing 10 mM EDTA. Both structural subunits, RtH1 and RtH2, were isolated by anion-exchange chromatography of dissociated RtH on DEAE-Sepharose CL-6B column (Pharmacia, Uppsala, Sweden) as described previously [11]. They were purified additionally by rechromatography on the same column. The purity of the isolated subunits was controlled by SDS-PAGE. Protein concentration was determined spectrophotometrically using the −1 absorption coefficient A0.1% ml cm− 1. 278 = 1.36 mg Hc solutions were passed once through a purification column to remove endotoxin contaminations (Detoxigel column, Pierce). The level of the remaining endotoxin was determinated by Limilus Amebocyte Lysate coatest gel (LAL) (Chromogenix AB, Molndal, Sweden).
2.3. Construction of conjugated molecules The hemagglutinin intersubunit peptide (IP) (containing T and B cell epitopes) from the influenza virus strain A/PR/8/34 was used to make the construct. The synthesis of Ac-(coding region HA317-341)NH-(CH2)6-NH2 was carried out using a Fmoc-based manual solid
A. Tchorbanov et al. phase peptide synthesis protocols on 2-Cl-Trt resin. The peptides were purified (≥ 95% purity) by reversed-phase sample displacement chromatography [13] on a series of 3 × 50 mm Nucleodur 100-5 C18 columns (Macherey-Nagel, Germany). The coupling of the hemocyanin to the peptides was carried out using the classical EDC (1-ethyl-3(3′-dimethylaminopropyl)carbodiimide.HCl, Fluka AG, Buchs, Switzerland) cross-linking technique [14]. During the synthesis of the peptides a spacer (H2N(CH2)6H2N) was added to their C-end with the aim to allow the peptide to form its own structure. To avoid cross-linking of the hemocyanin molecules during the conjugation step their concentration was kept low. The hemocyanin (in concentration 0.1 mg/ml in sterile 0.1 M sodium phosphate buffer, pH 6.0) was mixed with a 20-fold molar excess of the peptide (dissolved in 10% (v/v) N, N-dimethylformamide (SigmaAldrich, Taufkirchen, Germany)) in the same buffer to 0.02 mg/ml final concentration. The reaction was started by the addition of carbodiimide at 60-fold molar excess over the hemocyanin. The reaction mixture was stirred overnight at 4 °C, dialyzed against PBS and concentrated by ultrafiltration. HPLC analyses using a model 1046A fluorescent detector (Hewlett Packard), operating at excitation and emission wavelengths of 280 nm and of 340 nm, respectively were performed. The other construct (Bovine serum albumin (BSA)-IP) was prepared as mentioned above for coating of plates in ELISA measuring of anti-IP antibodies formation.
2.4. Animals Groups of Balb/c mice were obtained from Iffa-Credo, L'Arbresle, France (Charles River Company) and bred under specific-pathogenfree conditions in our animal facility. Female mice aged 8 to 10 weeks were used for immunization.
2.5. Immunization protocols The protocols used were approved by the Animal Care Commission at the Institute of Microbiology in accordance with International lows. Groups of mice (6 to 8 mice each) were injected i.p. with phosphate buffered saline (PBS) as a control or with different doses (250, 100 or 40 μg per mouse) of RtH or with its two subunits (RtH1 or RtH2) each. Other groups of mice were administered with 10 μg per mouse of influenza HA317-342 (IP) alone or with 100, 40 or 16 μg per mouse of a hybrid molecule RtH–HA317-342 IP peptide (RtH-IP), all diluted in 0.1 ml sterile PBS. Control animals was injected with PBS only or with IP peptide emulsified in an equal volume of Freund's complete adjuvant (CFA, Sigma). Mice were immunized 21 days later with the same doses of PBS, RtH, RtH1, RtH2, RtH–IP or IP (without adjuvant) as described above. The reimmunization of animals treated with (HA317-342) IP emulsified in CFA was done with IP emulsified in an incomplete Freund's adjuvant. Each next treatment was administered 14 days later. The mice were bled before immunizations and collected sera were kept frozen at −20 °C before testing for antibodies.
2.6. Enzyme linked immunosorbent assay (ELISA) 2.6.1. ELISA for anti-RtH, anti-RtH1 and anti-RtH2 IgG antibodies RtH diluted to 20 μg/ml in coating buffer (NaHCO3, pH-9.6) was used for coating of microplates (Falcon, USA) by incubation overnight at 4 °C. After washing with PBS/0.05% Tween 20 and blocking with 1% BSA, serum samples diluted 1:100 for measuring of IgG antibodies were added and incubated for 1 h at 37 °C. The plates were then washed and incubated for 1 h at 37 °C with alkaline phosphataselabelled goat anti-mouse IgG (Pharmingen BD, San Diego, USA). After washing Sigma 104 phosphatase substrate was added and the absorbance was measured at 405 nm. The obtained ELISA results were presented as optical density (OD), corresponding to the titer of anti-Hc IgG antibodies.
Modulation of the immune response using Rapana thomasiana hemocyanin 2.6.2. ELISA for anti-RtH IgG antibody isotypes The ELISA was performed as described above. After the serum incubation the plates were washed and incubated for 1 h at 37 °C with alkaline phosphatase-labelled goat anti-mouse IgG subclasses antibodies (Pharmingen BD, San Diego, USA). After washing Sigma 104 phosphatase substrate was added and the absorbance was measured at 405 nm. The obtained ELISA results were presented as OD, corresponding to the titer of the anti-RtH IgG antibody isotypes. 2.6.3. ELISA for anti-IP antibodies The construct BSA-IP diluted to 50 μg/ml in coating buffer (NaHCO3, pH-9.6) was used for coating of microplates (Falcon, USA) by incubation overnight at 4 °C. Further, the ELISA was performed as described above. The ELISA results obtained were presented as OD, corresponding to the titer of anti-IP IgG antibodies.
2.7. Measurement of cross-reactivity between the RtH subunits
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means between animal groups. A value of p b 0.05 was considered to be statistically significant.
3. Results and discussion 3.1. Preparation of R. thomasiana hemocyanin Numerous compounds are under evaluation as immunological adjuvants (and peptide-carriers) to improve the immune response. Initially, they should be well purified and characterized. The purification of RtH from the hemolymph yielded a highly pure protein preparation as assessed by gel filtration chromatography on a Sepharose 4B column and PAGE. The evaluation of purity and chemical characterization of the whole hemocyanin molecule and its two subunits by means of SDS- and native PAGE, and N-terminal sequence analysis was described in details in our previously works [11,16]. The final Hc solutions
Each subunit and intact RtH was incubated with mouse sera, obtained after immunization with whole RtH or separated subunits. Cross-blotting technique described previously was used [15]. Briefly, a nitrocellulose membrane (0.45 μm, from Sartorius, Germany) was inserted in a mini blotter cassette (Miniblotter 28 SL, Immunetics, Camridge, MA, USA). Serial dilutions of RtH, RtH1 or RtH2 were added to the individual slots for 60 min at RT. After extensive washing, the membrane was blocked with TBS/Tween 20 (0.2%) overnight at 4 °C. The membrane was returned to the mini blotter cassette in a direction perpendicular to that of the imprints of the slots containing RtH, RtH1 or RtH2 during the first incubation step. It was further incubated for 60 min at RT with serial dilutions of sera obtained after the last immunizations with RtH, RtH1 or RtH2. Each antigen dilution was cross-blotted with each serum dilution. After washing, the membrane was incubated for 1 h with alkaline phosphatase-labelled goat anti-mouse IgG and developed using the nitroblue tetrazolium/bromo-chloro-indolylphosphate chromogenic substrate (Sigma-Aldrich, Taufkirchen, Germany).
2.8. Data analysis For quantitation of the cross-blot reactivities, the densitometric profiles of the lanes corresponding to them were taken. The background defined by the secondary antibody was subtracted from the densitometric profile.
2.9. SDS-PAGE and Western blotting The constructed conjugates (RtH-IP and BSA-IP) were subjected to SDS-PAGE and Western blotting analysis. SDS-PAGE was performed using 10% gels and a MiniProtean II system (BioRad, Richmond, CA) in the presence of 0.1% SDS. After the electrophoresis, the proteins were transferred to a nitrocellulose membrane (0.45 μm, from Sartorius, Germany) using a MiniTrans Blot device (from BioRad) in a buffer containing 48 mM Tris and 110 mM glycine in the presence of 20% (v/v) methanol. The membranes were blocked overnight in a TBS buffer, containing 0.4% Tween 20 and were incubated for 1 h with the IP-specific rat standard polyclonal IgG. After washing, the membranes were incubated in an optimal dilution of a goat anti-rat IgG antibody, conjugated to peroxidase (from Sigma). The reaction was visualized using sodium nitroprusside and o-dianisidin-dihydrochloride-(3, 3′-dimethoxybenzidine, from Sigma).
2.10. Statistical analysis All ELISA samples were triplicated. Values in the figures were expressed as mean ± SD. The Student's t-test was used to compare
Figure 1 Anti-RtH IgG antibodies levels after i.p. immunization of the experimental animals with different doses (250, 100 or 40 μg per mouse) of RtH (A), RtH1 (B) or RtH2 (C). The mice were bled before each immunization and the sera analyses were performed by ELISA.
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A. Tchorbanov et al. 3.2. Adjuvant activity of RtH and its subunits
Figure 2 The immunization with RtH induces both Th1 and Th2 types of immune response. Levels of anti-RtH IgG antibody isotypes in the mice sera after multiple immunization of the experimental animals with 100 μg of RtH, RtH1 or RtH2 performed by ELISA. The obtained results were presented as OD, corresponding to the titer of the anti-RtH IgG antibody isotypes. contained only 0.48 EU/microgram protein, respectively less than 0.05 EU in the quantity for immunizations. Purified sterile RtH, RtH1 and RtH2 were used for further study of their immunological properties.
Many different protocols were used to determine the optimal scheme of immunization. The blood was collected by retroorbital puncture and mouse sera were prepared. The results obtained from the testing of the sera after each immunization for specific IgG antibodies by ELISA showed that the groups immunized with high doses of RtH or its subunits (250 and 100 μg per mouse) produced high levels of IgG antibodies even after the first reimmunization (week 5) (Fig. 1A–C). In the groups immunized with 40 μg per mouse high levels of IgG antibody synthesis was developed 2 weeks later after the next reimmunization. At the end point of the experiment after the last immunization all mice treated with the different doses of RtH, RtH1 or RtH2 had high titer of antiRtH IgG antibodies. The immune response to the RtH1 subunit was found to be higher than that of the RtH2 subunit (Fig. 1B–C). Administration of 100 μg per mouse for three times immunizations is enough and can ensure very strong adjuvant activity. Our data correlate to the data for using of KLH in the same types of experiments. The results obtained demonstrate that RtH and its subunits are able to induce strong humoral immune response and could be used as adjuvant.
Figure 3 Cross-blot analysis of the cross-reactivity between RtH and its subunits. The membrane in squares A, B and C (left part of the picture) was loaded with falling dilutions of RtH, squares D, E and F with falling dilutions of RtH1 and squares G, H and I with falling dilutions of RtH2. The squares A, D and G were incubated with falling dilutions of sera obtained after immunization with RtH; squares B, E and H with falling dilutions of sera obtained after immunization with RtH1 and squares C, F and I with falling dilutions of sera obtained after immunization with RtH2. After the incubation with alkaline phosphatase-labelled goat anti-mouse IgG and developed using the nitroblue tetrazolium/bromo-chloro-indolyl-phosphate chromogenic substrate the membrane was scanned. The densitometric profiles of the cross-blot results (right part) were performed as intensity of binding (relative units). One of five representative similar results was shown.
Modulation of the immune response using Rapana thomasiana hemocyanin
Figure 4 Western blot analysis of the conjugates RtH-IP and BSA-IP. After the coupling of the IP peptide to the RtH or BSA, the peptide retains its ability to be recognized by anti-IP antibodies. 10 μg samples from BSA (lane 1); BSA-IP (lane 2), RtH (lane 3) or the RtH-IP (lane 4) were subjected to SDS-PAGE (under nonreducing conditions using a 10% gel) and transferred to a nitrocellulose membrane. The latter was blocked, incubated with the IP-specific rat standard polyclonal IgG antibody, washed, incubated further with a goat anti-rat IgG antibody, conjugated to peroxidase and developed. The IP-specific antibody recognizes an IP in the conjugates BSA-IP (lane 2) and RtHIP (lane 4), but does not react with non-conjugated BSA (lane 1) and RtH (lane 3).
3.3. Anti-hemocyanin IgG subclass determination To investigate whether the anti-hemocyanin response corresponds to the Th1/Th2 type of immune response we studied the IgG subclass profile of RtH specific antibodies. The determination of different IgG subclasses showed that the immunization of the experimental animals with RtH, RtH1 or RtH2 induced mainly synthesis of IgG1 and IgG2a isotypes (Fig. 2). The levels of IgG2b and IgG3 anti-RtH antibodies were much lower. The ratio between IgG subclasses was not dependent on the immunization doses—250, 100 or 40 μg per mouse (data not shown). We did not observe any correlation between the IgG subclasses and administered doses in the experimental scheme. Our data suggest that immunization with RtH induces both Th1 and Th2 type of immune response. Compared to the results, obtained after immunizations with 100 μg/mouse of KLH we found a difference in the ratio IgG1/IgG2a [10]. Beside, the levels of IgG1 and IgG2a in both cases are much stronger than other subclasses of IgG antibodies.
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its molecular weight [17]. It is well known, that the most applications use Hcs from different sources as carriers of haptens and peptides for production of monoclonal antibodies and as carriers of vaccines against infectious diseases. Despite, with the increasing use of sophisticated small antigenic fragments, containing defined B and T cell epitopes, distinct adjuvants and vaccine preparations are desirable. We have prepared a conjugate between RtH and hemagglutinin intersubunit peptide (IP) from the influenza virus strain A/PR/8/34 [18,19], containing both B and T cell epitopes, using chemical coupling of the hemocyanin to the peptide, according to the classical crosslinking technique. The R. thomasiana hemocyanin is a big protein molecule holding different active parts on its surface. There are many free carboxyl groups on the RtH molecules and some of them are available for interaction with the reactive H2N-group of the peptide spacer. The spacer (H2N(CH2)6H2N) was added to their C-end with the aim to allow the peptide to form its own structure and to distant it form the big RtH molecule. The recognition of the Influenza peptide (IP) by the IP-specific polyclonal antibody in a Western blot (Fig. 4) confirmed that the peptides coupled to the RtH molecules retained their B-cell recognizing epitope. The latter is available for interaction with IP-specific antigen receptors on B lymphocytes. Sera collected after multiple immunizations of mice with different doses of RtH-IP were tested for anti-IP IgG antibodies by ELISA (Fig. 5). The administration of 100 μg per mouse RtH-IP conjugate and IP + CFA, resulted in similar antibody titers with a peak on the 9 weeks–2 weeks after the last immunization. Treatment with lower doses of the conjugate did not result in high antibody level. The increase of the doses above 100 μg per mouse also did not lead to better result (data not shown). Anti-IP antibody titers were low when IP was administrated alone. Comparatively, the most used concentration of KLH for immunization of different antigens for monoclonal antibody development is 100 μg per mouse. The number of doses and the schedule for immunization with protein-carrier vaccines is of great
3.4. Cross-reactivity between RtH and its subunits The immune homology between RtH1 and RtH2 subunits and to whole RtH molecule as well was investigated using a cross blot method. The serum obtained after immunization with whole RtH recognized RtH itself, but also both subunits (Fig. 3). The sera, obtained after treatment of the animals with the separated subunits also recognized itself, but with high activity bound to the intact molecule and to the other subunit. Interestingly, the sera from RtH2 treated mice recognized stronger the whole RtH that RtH2 itself. These results suggest that the cross-reactivity between both subunits is related to the epitope-similarity between single polypeptide chains. The planed future DNA sequencing of RtH could confirm that similarity.
3.5. RtH as a protein-carrier Several strategies have been utilized to increase the immunogenicity of small and weak antigens, mainly through increase of
Figure 5 Serum anti-IP antibody titers in mice injected i.p. with PBS or with different doses (100, 40 or 16 μg per mouse) of the conjugate RtH-IP, or with 10 μg of IP alone or 10 μg of IP in CFA performed by ELISA. The experimental animals were boosted in the same way after 21 days and two times later every 14 days. The results are presented as OD. Data represent mean ± SD from three determinations of individual sera collected at different intervals (n = 6–8). The experiments were repeated twice. ⁎⁎⁎p b 0.001 vs the IP group.
1038 importance for the induction of significant humoral immunity. The antibody response after a single immunization is known to be low as compared to that provoked by conventional virus vaccines or to that seen after natural infection [20,21]. More powerful antibody production is achieved after repeated administration of a conjugate RtH-IP. Our results showed that repeated immunization with an influenza vaccine without adjuvant, did not lead to a significant antibody production [18]. The conventional vaccine provoked high antibody titers if it was administered in Freund's complete adjuvant (CFA) and it might be concluded that at least partially, the effect was due to the adjuvant. Vaccination with RtH-IP appears to be very effective at inducing a strong humoral response. The conventional vaccine in CFA induced high antiinfluenza cytotoxic immunity. Exogenous antigens can prime CD8 + cytotoxic T cells when administered in CFA [22–24]. The immunization with protein antigens (ovalbumin or insulin) in CFA has been shown to generate specific cytotoxic T lymphocytes (CTLs) with different phenotypes [25]. Hemocyanins used as hapten-carriers stimulate major histocompatibility complex class I CD8 and also class II CD4 T-cell responses [10]. These data suggest the idea that we can expect also a CTL activity after immunization with RtH-IP. In conclusion, the results obtained demonstrate for the first time that RtH and/or its subunits could be used in any immunization protocol as adjuvants or as protein-carriers of small none-immunogenic molecules for increasing its immunogenicity. Our investigations are the first step in developing and elucidating the properties of new whole RtH and its subunits as an acceptable compound needed for adjuvanticity in the future.
Acknowledgement This work was supported by research grant TK-X-1611 from the National Science Fund of the Ministry of Education and Science, Bulgaria.
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w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Modulation of the immune response using Rapana thomasiana hemocyanin Andrey Tchorbanov a,⁎, Krassimira Idakieva b,⁎, Nikolina Mihaylova a , Lyuba Doumanova a a b
Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev-Str. 26, 1113 Sofia, Bulgaria Institute of Organic Chemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev-Str., bl. 9, 1113 Sofia, Bulgaria
Received 31 January 2008; received in revised form 7 March 2008; accepted 12 March 2008
KEYWORDS Adjuvant; Protein-carrier; Hemocyanin; Rapana thomasiana; Gastropod
Abstract We have investigated the non-specific immunostimulatory and specific immunomodulatory effects of hemocyanin from marine gastropod Rapana thomasiana (RtH). The purified RtH, its structural subunits RtH1 and RtH2 and a construct with influenza virus hemagglutinin intersubunit peptide (IP) were used in immunization protocols of Balb/c mice. Antibody formation against RtH, RtH1, RtH2, RtH-IP as well as anti-RtH IgG antibody isotypes were determined by ELISA. The immune homology between both subunits and the whole RtH molecule was investigated by cross-blotting technique. The retaining of the B-cell epitope of IP, coupled to the RtH was recognised by Western blot. The results obtained demonstrate that the immunization with RtH or its subunits in experimental models resulted in strong immune response in vivo. Common epitope of influenza A virus hemagglutinin jointed to RtH results in generation of molecule with increased immunogenicity. Our results are the first demonstration that RtH and/or its subunits could be used in different immunization protocols as an adjuvant or as a protein-carrier. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Recent approaches in the practical immunology involve the use of different highly immunogenic molecules for nonspecific immunomodulation (as adjuvants) or for active specific immunostimulation (as protein carrier covalently bound to specific antigen). Numerous compounds are under ⁎ Corresponding authors. A. Tchorbanov is to be contacted at fax: +359 2 8700109. K. Idakieva, fax: +359 2 8700225. E-mail addresses: [email protected] (A. Tchorbanov), [email protected] (K. Idakieva). 1567-5769/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2008.03.008
evaluation such immunological reagents [1–3]. Among them are the hemocyanins (Hcs)—extracellular type-3 copper proteins occurring freely dissolved in the hemolymph of many arthropod and mollusc species [4,5]. Hcs, due to their xenogenic nature and large molecular size (4 to 8 MDa), are strongly immunogenic. The keyhole limpet hemocyanin (KLH) of the marine gastropod Megathura crenulata is the most widely used as an adjuvant and a carrier for vaccines and antigens [6,7]. Hc from another marine gastropod Haliotis tuberculata, is considered to be a possible substitute for KLH as an immunostimulant [8]. Recently, Hc obtained from the Chilean gastropod Concholepas Concholepas was
1034 reported to possess adjuvant immunostimulatory effect, as well as a significant antitumor activity against mouse bladder carcinoma cells [9,10]. In our previous studies on the hemocyanin isolated from Rapana thomasiana (RtH)—a marine gastropod living along the west coast of the Black Sea—we demonstrated that hemocyanin molecules are built from two structural subunits, RtH1 and RtH2 [11]. It has been shown that the structural subunits RtH1 and RtH2 are closely related and both correspond immunologically to KLH2, one of the two hemocyanin isoforms of the gastropod M. crenulata [12]. Based on this structural similarity, a logical conclusion is that the hemocyanin of R. thomasiana will have strong adjuvant immunostimulatory effect as well as it would be possible to use RtH as a protein carrier. To our knowledge, till now it is not known whether RtH and its subunits, RtH1 and RtH2, possess such immunologic properties. The aim of the present work is to investigate in mouse experimental model the adjuvant properties of RtH and its two subunits and also to use it as a carrier of a viral peptide (common epitope of influenza A virus hemagglutinin).
2. Materials and methods 2.1. Isolation of R. thomasiana hemocyanin Living marine snails R. thomasiana were caught near the Bulgarian coast of Black Sea (Varna) and stored in sea water. The hemolymph was collected by bleeding through several diagonal slits made on the foot muscle of the mollusc and filtered through gauze. Phenylmethanesulphonyl fluoride (PMSF, 1 mM) was added to the crude material to avoid possible proteolysis of the hemolymph. Hemocytes and other cells were removed by centrifugation at 5000 × g for 30 min at 4 °C and the native hemocyanin was isolated from freshly obtained hemolymph by pelleting in an ultracentrifuge (Beckman LM-80, rotor Ti 45) at 180 000×g for 4 h at 4 °C and stored in the presence of 20% sucrose (w/v) at − 20 °C until use. RtH was further purified by gel filtration chromatography on a Sepharose 4B column, equilibrated and eluted with 50 mM PBS, pH 7.2. The purity of the isolated Hc was controlled by SDS- and native PAGE as described in [11,16].
2.2. Isolation of Rapana hemocyanin structural subunits Purified RtH was dissociated into individual subunits by dialysis overnight at 4 °C, against 130 mM glycine–NaOH, pH 9.6, containing 10 mM EDTA. Both structural subunits, RtH1 and RtH2, were isolated by anion-exchange chromatography of dissociated RtH on DEAE-Sepharose CL-6B column (Pharmacia, Uppsala, Sweden) as described previously [11]. They were purified additionally by rechromatography on the same column. The purity of the isolated subunits was controlled by SDS-PAGE. Protein concentration was determined spectrophotometrically using the −1 absorption coefficient A0.1% ml cm− 1. 278 = 1.36 mg Hc solutions were passed once through a purification column to remove endotoxin contaminations (Detoxigel column, Pierce). The level of the remaining endotoxin was determinated by Limilus Amebocyte Lysate coatest gel (LAL) (Chromogenix AB, Molndal, Sweden).
2.3. Construction of conjugated molecules The hemagglutinin intersubunit peptide (IP) (containing T and B cell epitopes) from the influenza virus strain A/PR/8/34 was used to make the construct. The synthesis of Ac-(coding region HA317-341)NH-(CH2)6-NH2 was carried out using a Fmoc-based manual solid
A. Tchorbanov et al. phase peptide synthesis protocols on 2-Cl-Trt resin. The peptides were purified (≥ 95% purity) by reversed-phase sample displacement chromatography [13] on a series of 3 × 50 mm Nucleodur 100-5 C18 columns (Macherey-Nagel, Germany). The coupling of the hemocyanin to the peptides was carried out using the classical EDC (1-ethyl-3(3′-dimethylaminopropyl)carbodiimide.HCl, Fluka AG, Buchs, Switzerland) cross-linking technique [14]. During the synthesis of the peptides a spacer (H2N(CH2)6H2N) was added to their C-end with the aim to allow the peptide to form its own structure. To avoid cross-linking of the hemocyanin molecules during the conjugation step their concentration was kept low. The hemocyanin (in concentration 0.1 mg/ml in sterile 0.1 M sodium phosphate buffer, pH 6.0) was mixed with a 20-fold molar excess of the peptide (dissolved in 10% (v/v) N, N-dimethylformamide (SigmaAldrich, Taufkirchen, Germany)) in the same buffer to 0.02 mg/ml final concentration. The reaction was started by the addition of carbodiimide at 60-fold molar excess over the hemocyanin. The reaction mixture was stirred overnight at 4 °C, dialyzed against PBS and concentrated by ultrafiltration. HPLC analyses using a model 1046A fluorescent detector (Hewlett Packard), operating at excitation and emission wavelengths of 280 nm and of 340 nm, respectively were performed. The other construct (Bovine serum albumin (BSA)-IP) was prepared as mentioned above for coating of plates in ELISA measuring of anti-IP antibodies formation.
2.4. Animals Groups of Balb/c mice were obtained from Iffa-Credo, L'Arbresle, France (Charles River Company) and bred under specific-pathogenfree conditions in our animal facility. Female mice aged 8 to 10 weeks were used for immunization.
2.5. Immunization protocols The protocols used were approved by the Animal Care Commission at the Institute of Microbiology in accordance with International lows. Groups of mice (6 to 8 mice each) were injected i.p. with phosphate buffered saline (PBS) as a control or with different doses (250, 100 or 40 μg per mouse) of RtH or with its two subunits (RtH1 or RtH2) each. Other groups of mice were administered with 10 μg per mouse of influenza HA317-342 (IP) alone or with 100, 40 or 16 μg per mouse of a hybrid molecule RtH–HA317-342 IP peptide (RtH-IP), all diluted in 0.1 ml sterile PBS. Control animals was injected with PBS only or with IP peptide emulsified in an equal volume of Freund's complete adjuvant (CFA, Sigma). Mice were immunized 21 days later with the same doses of PBS, RtH, RtH1, RtH2, RtH–IP or IP (without adjuvant) as described above. The reimmunization of animals treated with (HA317-342) IP emulsified in CFA was done with IP emulsified in an incomplete Freund's adjuvant. Each next treatment was administered 14 days later. The mice were bled before immunizations and collected sera were kept frozen at −20 °C before testing for antibodies.
2.6. Enzyme linked immunosorbent assay (ELISA) 2.6.1. ELISA for anti-RtH, anti-RtH1 and anti-RtH2 IgG antibodies RtH diluted to 20 μg/ml in coating buffer (NaHCO3, pH-9.6) was used for coating of microplates (Falcon, USA) by incubation overnight at 4 °C. After washing with PBS/0.05% Tween 20 and blocking with 1% BSA, serum samples diluted 1:100 for measuring of IgG antibodies were added and incubated for 1 h at 37 °C. The plates were then washed and incubated for 1 h at 37 °C with alkaline phosphataselabelled goat anti-mouse IgG (Pharmingen BD, San Diego, USA). After washing Sigma 104 phosphatase substrate was added and the absorbance was measured at 405 nm. The obtained ELISA results were presented as optical density (OD), corresponding to the titer of anti-Hc IgG antibodies.
Modulation of the immune response using Rapana thomasiana hemocyanin 2.6.2. ELISA for anti-RtH IgG antibody isotypes The ELISA was performed as described above. After the serum incubation the plates were washed and incubated for 1 h at 37 °C with alkaline phosphatase-labelled goat anti-mouse IgG subclasses antibodies (Pharmingen BD, San Diego, USA). After washing Sigma 104 phosphatase substrate was added and the absorbance was measured at 405 nm. The obtained ELISA results were presented as OD, corresponding to the titer of the anti-RtH IgG antibody isotypes. 2.6.3. ELISA for anti-IP antibodies The construct BSA-IP diluted to 50 μg/ml in coating buffer (NaHCO3, pH-9.6) was used for coating of microplates (Falcon, USA) by incubation overnight at 4 °C. Further, the ELISA was performed as described above. The ELISA results obtained were presented as OD, corresponding to the titer of anti-IP IgG antibodies.
2.7. Measurement of cross-reactivity between the RtH subunits
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means between animal groups. A value of p b 0.05 was considered to be statistically significant.
3. Results and discussion 3.1. Preparation of R. thomasiana hemocyanin Numerous compounds are under evaluation as immunological adjuvants (and peptide-carriers) to improve the immune response. Initially, they should be well purified and characterized. The purification of RtH from the hemolymph yielded a highly pure protein preparation as assessed by gel filtration chromatography on a Sepharose 4B column and PAGE. The evaluation of purity and chemical characterization of the whole hemocyanin molecule and its two subunits by means of SDS- and native PAGE, and N-terminal sequence analysis was described in details in our previously works [11,16]. The final Hc solutions
Each subunit and intact RtH was incubated with mouse sera, obtained after immunization with whole RtH or separated subunits. Cross-blotting technique described previously was used [15]. Briefly, a nitrocellulose membrane (0.45 μm, from Sartorius, Germany) was inserted in a mini blotter cassette (Miniblotter 28 SL, Immunetics, Camridge, MA, USA). Serial dilutions of RtH, RtH1 or RtH2 were added to the individual slots for 60 min at RT. After extensive washing, the membrane was blocked with TBS/Tween 20 (0.2%) overnight at 4 °C. The membrane was returned to the mini blotter cassette in a direction perpendicular to that of the imprints of the slots containing RtH, RtH1 or RtH2 during the first incubation step. It was further incubated for 60 min at RT with serial dilutions of sera obtained after the last immunizations with RtH, RtH1 or RtH2. Each antigen dilution was cross-blotted with each serum dilution. After washing, the membrane was incubated for 1 h with alkaline phosphatase-labelled goat anti-mouse IgG and developed using the nitroblue tetrazolium/bromo-chloro-indolylphosphate chromogenic substrate (Sigma-Aldrich, Taufkirchen, Germany).
2.8. Data analysis For quantitation of the cross-blot reactivities, the densitometric profiles of the lanes corresponding to them were taken. The background defined by the secondary antibody was subtracted from the densitometric profile.
2.9. SDS-PAGE and Western blotting The constructed conjugates (RtH-IP and BSA-IP) were subjected to SDS-PAGE and Western blotting analysis. SDS-PAGE was performed using 10% gels and a MiniProtean II system (BioRad, Richmond, CA) in the presence of 0.1% SDS. After the electrophoresis, the proteins were transferred to a nitrocellulose membrane (0.45 μm, from Sartorius, Germany) using a MiniTrans Blot device (from BioRad) in a buffer containing 48 mM Tris and 110 mM glycine in the presence of 20% (v/v) methanol. The membranes were blocked overnight in a TBS buffer, containing 0.4% Tween 20 and were incubated for 1 h with the IP-specific rat standard polyclonal IgG. After washing, the membranes were incubated in an optimal dilution of a goat anti-rat IgG antibody, conjugated to peroxidase (from Sigma). The reaction was visualized using sodium nitroprusside and o-dianisidin-dihydrochloride-(3, 3′-dimethoxybenzidine, from Sigma).
2.10. Statistical analysis All ELISA samples were triplicated. Values in the figures were expressed as mean ± SD. The Student's t-test was used to compare
Figure 1 Anti-RtH IgG antibodies levels after i.p. immunization of the experimental animals with different doses (250, 100 or 40 μg per mouse) of RtH (A), RtH1 (B) or RtH2 (C). The mice were bled before each immunization and the sera analyses were performed by ELISA.
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A. Tchorbanov et al. 3.2. Adjuvant activity of RtH and its subunits
Figure 2 The immunization with RtH induces both Th1 and Th2 types of immune response. Levels of anti-RtH IgG antibody isotypes in the mice sera after multiple immunization of the experimental animals with 100 μg of RtH, RtH1 or RtH2 performed by ELISA. The obtained results were presented as OD, corresponding to the titer of the anti-RtH IgG antibody isotypes. contained only 0.48 EU/microgram protein, respectively less than 0.05 EU in the quantity for immunizations. Purified sterile RtH, RtH1 and RtH2 were used for further study of their immunological properties.
Many different protocols were used to determine the optimal scheme of immunization. The blood was collected by retroorbital puncture and mouse sera were prepared. The results obtained from the testing of the sera after each immunization for specific IgG antibodies by ELISA showed that the groups immunized with high doses of RtH or its subunits (250 and 100 μg per mouse) produced high levels of IgG antibodies even after the first reimmunization (week 5) (Fig. 1A–C). In the groups immunized with 40 μg per mouse high levels of IgG antibody synthesis was developed 2 weeks later after the next reimmunization. At the end point of the experiment after the last immunization all mice treated with the different doses of RtH, RtH1 or RtH2 had high titer of antiRtH IgG antibodies. The immune response to the RtH1 subunit was found to be higher than that of the RtH2 subunit (Fig. 1B–C). Administration of 100 μg per mouse for three times immunizations is enough and can ensure very strong adjuvant activity. Our data correlate to the data for using of KLH in the same types of experiments. The results obtained demonstrate that RtH and its subunits are able to induce strong humoral immune response and could be used as adjuvant.
Figure 3 Cross-blot analysis of the cross-reactivity between RtH and its subunits. The membrane in squares A, B and C (left part of the picture) was loaded with falling dilutions of RtH, squares D, E and F with falling dilutions of RtH1 and squares G, H and I with falling dilutions of RtH2. The squares A, D and G were incubated with falling dilutions of sera obtained after immunization with RtH; squares B, E and H with falling dilutions of sera obtained after immunization with RtH1 and squares C, F and I with falling dilutions of sera obtained after immunization with RtH2. After the incubation with alkaline phosphatase-labelled goat anti-mouse IgG and developed using the nitroblue tetrazolium/bromo-chloro-indolyl-phosphate chromogenic substrate the membrane was scanned. The densitometric profiles of the cross-blot results (right part) were performed as intensity of binding (relative units). One of five representative similar results was shown.
Modulation of the immune response using Rapana thomasiana hemocyanin
Figure 4 Western blot analysis of the conjugates RtH-IP and BSA-IP. After the coupling of the IP peptide to the RtH or BSA, the peptide retains its ability to be recognized by anti-IP antibodies. 10 μg samples from BSA (lane 1); BSA-IP (lane 2), RtH (lane 3) or the RtH-IP (lane 4) were subjected to SDS-PAGE (under nonreducing conditions using a 10% gel) and transferred to a nitrocellulose membrane. The latter was blocked, incubated with the IP-specific rat standard polyclonal IgG antibody, washed, incubated further with a goat anti-rat IgG antibody, conjugated to peroxidase and developed. The IP-specific antibody recognizes an IP in the conjugates BSA-IP (lane 2) and RtHIP (lane 4), but does not react with non-conjugated BSA (lane 1) and RtH (lane 3).
3.3. Anti-hemocyanin IgG subclass determination To investigate whether the anti-hemocyanin response corresponds to the Th1/Th2 type of immune response we studied the IgG subclass profile of RtH specific antibodies. The determination of different IgG subclasses showed that the immunization of the experimental animals with RtH, RtH1 or RtH2 induced mainly synthesis of IgG1 and IgG2a isotypes (Fig. 2). The levels of IgG2b and IgG3 anti-RtH antibodies were much lower. The ratio between IgG subclasses was not dependent on the immunization doses—250, 100 or 40 μg per mouse (data not shown). We did not observe any correlation between the IgG subclasses and administered doses in the experimental scheme. Our data suggest that immunization with RtH induces both Th1 and Th2 type of immune response. Compared to the results, obtained after immunizations with 100 μg/mouse of KLH we found a difference in the ratio IgG1/IgG2a [10]. Beside, the levels of IgG1 and IgG2a in both cases are much stronger than other subclasses of IgG antibodies.
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its molecular weight [17]. It is well known, that the most applications use Hcs from different sources as carriers of haptens and peptides for production of monoclonal antibodies and as carriers of vaccines against infectious diseases. Despite, with the increasing use of sophisticated small antigenic fragments, containing defined B and T cell epitopes, distinct adjuvants and vaccine preparations are desirable. We have prepared a conjugate between RtH and hemagglutinin intersubunit peptide (IP) from the influenza virus strain A/PR/8/34 [18,19], containing both B and T cell epitopes, using chemical coupling of the hemocyanin to the peptide, according to the classical crosslinking technique. The R. thomasiana hemocyanin is a big protein molecule holding different active parts on its surface. There are many free carboxyl groups on the RtH molecules and some of them are available for interaction with the reactive H2N-group of the peptide spacer. The spacer (H2N(CH2)6H2N) was added to their C-end with the aim to allow the peptide to form its own structure and to distant it form the big RtH molecule. The recognition of the Influenza peptide (IP) by the IP-specific polyclonal antibody in a Western blot (Fig. 4) confirmed that the peptides coupled to the RtH molecules retained their B-cell recognizing epitope. The latter is available for interaction with IP-specific antigen receptors on B lymphocytes. Sera collected after multiple immunizations of mice with different doses of RtH-IP were tested for anti-IP IgG antibodies by ELISA (Fig. 5). The administration of 100 μg per mouse RtH-IP conjugate and IP + CFA, resulted in similar antibody titers with a peak on the 9 weeks–2 weeks after the last immunization. Treatment with lower doses of the conjugate did not result in high antibody level. The increase of the doses above 100 μg per mouse also did not lead to better result (data not shown). Anti-IP antibody titers were low when IP was administrated alone. Comparatively, the most used concentration of KLH for immunization of different antigens for monoclonal antibody development is 100 μg per mouse. The number of doses and the schedule for immunization with protein-carrier vaccines is of great
3.4. Cross-reactivity between RtH and its subunits The immune homology between RtH1 and RtH2 subunits and to whole RtH molecule as well was investigated using a cross blot method. The serum obtained after immunization with whole RtH recognized RtH itself, but also both subunits (Fig. 3). The sera, obtained after treatment of the animals with the separated subunits also recognized itself, but with high activity bound to the intact molecule and to the other subunit. Interestingly, the sera from RtH2 treated mice recognized stronger the whole RtH that RtH2 itself. These results suggest that the cross-reactivity between both subunits is related to the epitope-similarity between single polypeptide chains. The planed future DNA sequencing of RtH could confirm that similarity.
3.5. RtH as a protein-carrier Several strategies have been utilized to increase the immunogenicity of small and weak antigens, mainly through increase of
Figure 5 Serum anti-IP antibody titers in mice injected i.p. with PBS or with different doses (100, 40 or 16 μg per mouse) of the conjugate RtH-IP, or with 10 μg of IP alone or 10 μg of IP in CFA performed by ELISA. The experimental animals were boosted in the same way after 21 days and two times later every 14 days. The results are presented as OD. Data represent mean ± SD from three determinations of individual sera collected at different intervals (n = 6–8). The experiments were repeated twice. ⁎⁎⁎p b 0.001 vs the IP group.
1038 importance for the induction of significant humoral immunity. The antibody response after a single immunization is known to be low as compared to that provoked by conventional virus vaccines or to that seen after natural infection [20,21]. More powerful antibody production is achieved after repeated administration of a conjugate RtH-IP. Our results showed that repeated immunization with an influenza vaccine without adjuvant, did not lead to a significant antibody production [18]. The conventional vaccine provoked high antibody titers if it was administered in Freund's complete adjuvant (CFA) and it might be concluded that at least partially, the effect was due to the adjuvant. Vaccination with RtH-IP appears to be very effective at inducing a strong humoral response. The conventional vaccine in CFA induced high antiinfluenza cytotoxic immunity. Exogenous antigens can prime CD8 + cytotoxic T cells when administered in CFA [22–24]. The immunization with protein antigens (ovalbumin or insulin) in CFA has been shown to generate specific cytotoxic T lymphocytes (CTLs) with different phenotypes [25]. Hemocyanins used as hapten-carriers stimulate major histocompatibility complex class I CD8 and also class II CD4 T-cell responses [10]. These data suggest the idea that we can expect also a CTL activity after immunization with RtH-IP. In conclusion, the results obtained demonstrate for the first time that RtH and/or its subunits could be used in any immunization protocol as adjuvants or as protein-carriers of small none-immunogenic molecules for increasing its immunogenicity. Our investigations are the first step in developing and elucidating the properties of new whole RtH and its subunits as an acceptable compound needed for adjuvanticity in the future.
Acknowledgement This work was supported by research grant TK-X-1611 from the National Science Fund of the Ministry of Education and Science, Bulgaria.
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