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Immune stimulating activity of two new chitosan containing adjuvant formulations
Vaccine 19 (2001) 661 – 668 www.elsevier.com/locate/vaccine
Immune stimulating activity of two new chitosan containing adjuvant formulations Peter G. Seferian, Mitzi L. Martinez * Zonagen Inc., 2408 Timberloch Place, B-4, The Woodlands, TX 77380, USA Received 30 August 1999; received in revised form 18 May 2000; accepted 30 May 2000
Abstract Recombinant proteins have potential as both human and veterinary vaccine antigens, but they are often weakly immunogenic and immunization with recombinant proteins may not elicit a significant immune response that recognizes the native protein. This report describes the immune stimulating activity of two new adjuvant formulations, a zinc-chitosan particle formulation designed to bind to histidine tagged recombinant proteins; and an emulsion formulation containing chitosan. BALB/c mice vaccinated with formulations comprising recombinant beta-human chorionic gonadotropin (bhCG) and each adjuvant had prolonged high titer antibodies that recognized both the recombinant bhCG and native hCG. bhCG is an established target for immunocontraceptive vaccines and a potential target for tumor immunotherapy. Isotype analysis of these antibodies revealed an IgG1 response in mice immunized with zinc-chitosan particles and a mixed IgG1, IgG2a and IgG2b response with the emulsion. These chitosan based adjuvant formulations were effective in sensitizing mice and guinea pigs for antigen specific DTH responses, indicating that these adjuvants stimulate both B and T lymphocytes. The ability of these adjuvants to stimulate significant responses with a poorly immunogenic recombinant protein suggests that they may have potential in developing vaccines based on synthetic peptides and subunit antigens. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Adjuvant; Chitosan; Histidine
1. Introduction Adjuvants are substances which enhance the ability of antigens to elicit an immune response [1,2]. Recombinant proteins are potentially better-defined and safer components of both veterinary and human vaccines. However, recombinant proteins are often weakly immunogenic and require adjuvants that are superior to aluminum to make them effective vaccines. The importance of effective adjuvants is demonstrated by the fact that there are many adjuvant systems being developed [3]. This report describes the immune response elicited by the injection of recombinant beta-human chorionic gonadotropin (rbhCG), a well established target for immunocontraceptive vaccination and a potential target for tumor immunotherapy [4], in conjunction with two adjuvant formulations containing chitosan. * Corresponding author. Tel.: +1-281-3675892; fax: + 1-2813638796. E-mail address: [email protected] (M.L. Martinez).
Chitosan suspensions or microparticles have been reported to have immune stimulating activity such as increasing accumulation and activation of macrophage and polymorphonuclear cells, suppressing tumor growth, promoting resistance to infections by microorganisms, inducing cytokines, augmenting antibody responses and enhancing delayed type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses [5–14]. Chitosan also has utility in transmucosal delivery of drugs and peptides [15]. Chitin is a naturally occurring polymer of N-acetylD-glucosamine present in crustaceans, insects, mushrooms and microorganisms. Chitosan, derived by the deacetylation of chitin, is a polymer of D-glucosamine and N-acetyl-D-glucosamine. Compared with other natural polysaccharides, chitosan has high biodegradability, low toxicity and is neither an irritant nor allergen in humans [16–18]. This paper describes the ability of two new adjuvant formulations containing chitosan to stimulate antibody responses and prime for DTH. One formulation results in a zinc-chitosan particle (ZCP)
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that binds antigen and is particularly useful with recombinant proteins purified by immobilized metal affinity chromatography such as those containing a histidine tag [19]. The other formulation results in an emulsion containing chitosan (ECC) and antigen. Both the formulations enhance and prolong the immune stimulating activity of chitosan. The immune stimulating activity of these adjuvant formulations could not be attributed to the reported adjuvant activity of chitosan alone. Specifically, in combination with recombinant proteins these formulations were found to be effective in stimulating high titer antibody responses to both the recombinant antigen given and the native protein from which it was derived. Vaccination with recombinant proteins in both the formulations also primed T-cell mediated DTH responses.
2. Materials and methods
2.1. Animals and antigens Female BALB/c mice and Hartley guinea pigs were obtained from Harlan, rabbits were obtained from Covance and all animals were maintained as recommended in the Guide for the Care and Use of Laboratory Animals (NRC, 1996). Ovalbumin (OVA: fraction VI) and native human chorionic gonadotropin (hCG) were obtained from Sigma Chemicals (St Louis, MO). Histidine-tagged rbhCG was expressed in yeast cells from a proprietary construct.
2.2. Adju6ants and immunizations The ECC [20] was developed to elicit strong and sustained immune responses with many different types of antigens, even with a single injection. To prepare 1 ml of vaccine, 420 ml of phosphate buffered saline (PBS) containing antigen was mixed with 340 ml of a sterile 2% chitosan (Sigma) in 2% acetic acid solution (Mallinkrodt Chemical, Paris, KY). As the chitosanantigen mixture was neutralized by the addition of NaOH (Sigma), antigen and chitosan form a fine gelatinous precipitate. The pH of the precipitated solution was 6.8–7.2, as determined by spotting onto pH indicator strips (Baxter, Deerfield, IL). One hundred and fifty microliters of a 2.5:1 solution containing squalene (Sigma) and Pluronic® L121 (BASF Corp., Parsippany, NJ) was added and upon vortex mixing formed a cloudy emulsion. Mice were immunized with 25 mg of rbhCG in 200 ml of this emulsion by intraperitoneal injection on days 0 and 21. Guinea pigs were immunized by intramuscular injection of 35 mg of OVA in 300 ml on days 0 and 21. ECC has been referred to as ImmuMax SR [21].
The ZCP [22] were designed to facilitate immunization with recombinant proteins containing a histidine tag [19]. To prepare this adjuvant a 2% chitosan in 2% acetic acid solution was added to a 10% zinc acetate (Sigma) solution and the mixture rocked end to end for 4 h at room temperature. This results in a fine white precipitate, which was washed several times with PBS. To prepare injections, the antigen was placed in PBS, pH 8, to optimize the histidine tag charge and therefore its binding to the adjuvant particles. For each 25 mg of antigen to be injected, 100 ml of zinc-chitosan particle slurry was transferred to a small sterile tube. The final ratio of adjuvant to antigen was 1 mg of ZCP particles (wet weight) for every 1 mg of protein. The adjuvant particles were sedimented by centrifugation and the supernatant removed. The adjuvant pellet was washed twice by suspension in PBS (pH 8), centrifugation and supernatant removal. Antigen solution was used to resuspend the washed adjuvant pellet and mixed for 2 h at room temperature. The adjuvant–antigen preparation was sedimented by centrifugation and the supernatant removed and saved for protein analysis. The adjuvant–antigen pellet was washed with PBS and suspended in the desired volume of PBS (pH 7.4) prior to injection. The amount of protein bound to the ZCP was determined using a Coomassie® Plus protein assay (Pierce, Rockford, IL) of the antigen-containing supernatant protein concentration before and after binding. Mice were immunized with 25 mg of rbhCG in 100 ml of adjuvant–antigen complex by intramuscular injection. Rabbits received intramuscular injections containing 250 mg of rbhCG in 500 ml of the adjuvant– antigen complex. Several different adjuvant formulations were prepared to compare the activity of ZCP and ECC adjuvant formulations to the adjuvant activity reported for chitosan alone and the combination of squalene and Pluronic® L121. Chitosan particles were prepared by the neutralization of a 2% chitosan in 2% acetic acid solution with NaOH. This resulted in a gelatinous precipitation of chitosan particles that were washed several times with PBS. Chitosan particles were then suspended in PBS containing rbhCG. To prepare 1 ml of vaccine formulated with the same concentration of squalene and Pluronic® L121 as is found in ECC adjuvant, 150 ml of a 2.5:1 solution containing squalene and Pluronic® L121 was added to 1050 ml of PBS containing 150 mg of rbhCG. Mice were immunized with 25 mg of rbhCG in 200 ml of this emulsion by intraperitoneal injection. The traditional research adjuvant combination of antigen formulated in complete Freund’s adjuvant (CFA) followed by booster immunizations using antigen in incomplete Freund’s Adjuvant (IFA) was used as a reference standard for the adjuvant activity of ZCP and ECC. CFA or IFA (Sigma) was combined with an
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equal volume of PBS containing rbhCG. This mixture was whipped into a stable emulsion by sonification with a Branson Sonifier 250 (Branson Ultrasonics Corp., Danbury, CT). The stability of these emulsions was confirmed by floating an aliquot on saline. Mice were immunized with 25 mg of rbhCG in 200 ml of CFA or IFA emulsion by intraperitoneal injection.
2.3. Antibody assay (ELISA) Antibody titers were determined in an antibody capture enzyme-linked immunosorbent assay (ELISA) [23]. Briefly, 96-well assay plates (Falcon c 3912) were coated with rbhCG (1 mg/ml) or native hCG (10 U/ml). Plates were blocked with 2% nonfat dry milk (NFDM) to inhibit nonspecific binding. Blocked plates were emptied and washed with PBS containing 0.05% Tween 20 (PBS-T: Sigma) and emptied prior to use. Sample and control sera were serially diluted in PBS-T containing 2% NFDM prior to application. Plates were covered and incubated for 2 h at 37°C, emptied and washed four times with PBS-T. Bound antibody was detected using biotin labeled rabbit anti-mouse IgG (H+ L) antibody (Zymed, S. San Francisco, CA). Bound antibody class and subclass analyses were done using biotin labeled rabbit anti-mouse IgM, IgG1, IgG2a or IgG2b specific reagents (Zymed). The presence of bound antibody was detected using horseradish peroxidase conjugated streptavidin (Zymed) and 800 mg/ml O-phenylenediamine (Sigma) substrate solution. Plates were developed in the dark at room temperature for 10 min and the absorbence at 450 nm was read on an Emax plate reader (Molecular Devices). Endpoint titers were determined as the reciprocal dilution whose absorbence exceeded twice the absorbence obtained using unimmunized negative control sera. This method has been reported to be conservative in its estimation of titer [24]. Using this cutoff, the endpoint titer obtained for a commercial IgG1 monoclonal anti-human chorionic go-
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nadotropin, b subunit specific antibody (PE-4 clone, Lot No. 37F-4963-1: Sigma) was 6400.
2.4. Bioneutalization of HCG induced o6ulation Fifty units of hCG (Sigma) was administered by subcutaneous injection to four female New Zealand white rabbits immunized with rbhCG formulated with ZCP and four non-immunized rabbits. Blood was drawn for serum three and seven days following administration of hCG. Rabbit serum progesterone levels were determined by RIA (Diagnostic Products Corp.) as an indication of ovulation.
2.5. Elicitation of delayed type hypersensiti6ity (DTH) Vaccinated mice and guinea pigs were tested for antigen specific DTH responses by injection of 10 or 50 ml, respectively, of priming antigen into the right ear pinna and an equal amount of control antigen in the left ear. Ear thickness was measured as described [25], just prior to injection, and 24 and 48 h post-vaccination. Percent ear swelling was calculated as follows: [(Post− Pre)/Pre] ×100, where Post is post-injection ear pinna thickness and Pre is pre-elicitation ear pinna thickness. Mice were tested for elicitation of DTH 318 days after primary immunization and guinea pigs were tested 47 days after primary immunization.
2.6. Statistical analysis ELISA results are shown as geometric mean titers. DTH response results are expressed as the arithmetic mean9 standard deviation (S.D.). Comparison of means was done using a two-tailed t-test and statistical significance was determined using StatGraphics Plus, version 2.1.
3. Results
3.1. Effect of adju6ant formulations containing chitosan on antibody responses elicited by recombinant protein
Fig. 1. The antibody response of BALB/c mice (n = 10) to recombinant protein elicited by injection of recombinant bhCG formulated with PBS (open squares), ZCP (closed triangles) or ECC (closed circles) on days 0 and 21. Both adjuvant formulations stimulated statistically (P B 0.05) greater antibody titers than PBS at points labeled a.
BALB/c mice were vaccinated on days 0 and 21 with 25 mg of rbhCG formulated with no adjuvant (PBS), ZCP or ECC. Serum was obtained every 3 weeks and the anti-rbhCG was assayed in an antibody capture ELISA (Fig. 1). The IgG antibody responses elicited by rbhCG formulated with either adjuvant were 2 logs (or more) greater than the antibody response elicited in the absence of either adjuvant. In addition, antibody responses stimulated with ZCP or the ECC were significant for up to 181 days. Serum from these mice was also assayed for antibody binding to native hCG in an antibody capture ELISA
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Fig. 2. The antibody response of BALB/c mice (n= 10) to native protein elicited by injection of recombinant bhCG formulated with PBS (open squares), ZCP (closed triangles) or ECC (closed circles) on days 0 and 21. Both adjuvant formulations stimulated statistically (PB0.05) greater antibody titers than PBS at points labeled a. Only the higher adjuvant stimulated titer is statistically (PB 0.05) greater than the antibody titer stimulated with PBS at points labeled b.
Fig. 3. The range and geometric mean titers of antibody response of BALB/c mice (n =6) to recombinant protein at 8 weeks following injection of recombinant bhCG with either PBS, at the same time as ZCP, but in an opposite leg (OppLegs), chitosan particles, ZCP, ECC or the squalene: Pluronic® L121 phase of ECC (Oil phase). Formulation of rbhCG with either ZCP or ECC elicited antibody responses that were statistically (P B 0.05) greater than antibody titers elicited with other formulations, points labeled a. Formulation of rbhCG in squalene:Pluronic® L121 elicited antibody titers statistically (PB 0.05) greater than the antibody titer stimulated with PBS, points labeled b.
(Fig. 2). Significantly, mice immunized with rbhCG and either ZCP or the ECC also had high titer antibodies that bound the native hCG. The use of these adjuvants, when injecting recombinant protein enhanced the antibody response to native protein by 2 logs. The specificity of this response was confirmed by polyacrylamide gel electrophoresis and western blotting (data not shown). The duration of the anti-native hCG response stimulated by each adjuvant formulation differed. The ECC adjuvant formulation stimulated an anti-native hCG response that was slow to rise but was sustained. In comparison, the ZCP adjuvant formulation stimulated an anti-native hCG response that rose rapidly, but was not as well sustained.
3.2. Comparison of adju6ant formulations containing chitosan and their components It has been reported that chitosan particles them-
selves have adjuvant activity [5–14]. Both squalene and Pluronic® L121 are also known to have adjuvant activity alone and in combination [26–28]. To determine whether the adjuvant activity observed with ZCP and ECC could be attributed to the chitosan alone, or in the case of ECC, to the squalene and Pluronic® L121, mice were vaccinated with rbhCG formulated with either PBS, chitosan particles, ZCP, ECC or the oil phase of ECC (squalene and Pluronic® L121). In addition, because there have been reports that the immune stimulating activity of chitosan is a generalized, non-specific stimulation of the immune response [13], a sixth group of mice received ZCP in one leg and antigen in the other. The assumption was that if ZCP stimulated a generalized increase in the immune response, its physical relationship to the antigen would be irrelevant to its ability to enhance the immune response elicited by the antigen. Five groups, each containing five BALB/c mice, were vaccinated on days 0 and 21 with 25 mg of rbhCG formulated with either PBS, chitosan particles, ZCP, ECC or squalene and Pluronic® L121 at the same concentration as used in ECC. A sixth group of five mice was vaccinated by intramuscular injection with 25 mg of rbhCG in PBS and at the same time but in an opposite leg (OppLegs) with ZCP particles. Serum obtained 8 weeks after initial vaccination was assayed in an antibody capture ELISA (Fig. 3). Unexpectedly, vaccination with rbhCG formulated with chitosan particles did not significantly increase the antibody response to rbhCG as compared with that obtained following vaccinations with rbhCG formulated without adjuvant (PBS). In contrast, as expected, vaccination with formulations combining rbhCG with squalene and Pluronic® L121 elicited antibody levels that were significantly greater than those elicited by vaccination with rbhCG in PBS. Vaccination with rbhCG with both chitosan containing adjuvant formulations, ZCP and ECC, elicited significantly greater antibody responses than did rbhCG formulated with squalene and Pluronic® L121. The injection of ZCP and rbhCG in opposite legs at the same time did not elicit an increased antibody response. This suggests that, at least for this chitosan containing adjuvant formulation, that the physical association of the chitosan and antigen is important to its adjuvant activity.
3.3. Comparison of adju6ant formulations containing chitosan and CFA/IFA Having established the adjuvant activity of these adjuvant formulations containing chitosan, they were compared with the combination of CFA/IFA. To directly compare the ability of formulation of rbhCG
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with ZCP or ECC to elicit an anti-native hCG response to that elicited by formulation of rbhCG in CFA/IFA, a three-immunization protocol was chosen. Ten mice in each group were immunized on days 0, 28 and 42 with rbhCG in either ZCP, ECC or CFA/IFA and sera samples were assayed for binding to native hCG. The anti native hCG antibody response elicited by the injection of rbhCG in combination with CFA/IFA peaked more quickly than those elicited by rbhCG formulated in ECC or ZCP. This was evidenced by statistically greater antibody titers seen in the CFA/IFA group on days 77–133 of the experiment (Fig. 4). However, with time the CFA/IFA induced native hCG titers decline. The anti-native hCG antibody response elicited by vaccination with rbhCG formulated with ZCP do not peak as do those induced by CFA/IFA. ZCP induced titers undergo a slow and steady rise but were equivalent to CFA/IFA induced titers from day 151 onward, with no statistical differences between the titers induced by the two adjuvant groups for the remainder of the observation period. In contrast, the anti-native hCG antibody response elicited by rbhCG formulated with ECC requires more time than CFA/IFA induced titers to rise, but they continue rising. Antibody titers elicited by rbhCG formulation in ECC were stabilized at a level equivalent to those of the CFA/IFA and the ZCP groups between days 151 and 234 but are sustained while the titers in the CFA/IFA group and the ZCP group decline. The ECC induced native hCG antibody titers then become statistically greater than those elicited by the other adjuvant groups from day 234 onward.
3.4. Functional acti6ity of antibodies elicited with an adju6ant containing chitosan In an infectious disease model, functional immunity equals protection. With immunocontraceptive vaccines, protection equals infertility. When working with hormones, functional immunity manifests itself as bioneutralization of the hormone’s induced effects. The
Fig. 4. The antibody response of BALB/c mice (n= 10) to native hCG elicited by injection of recombinant bhCG formulated with complete/incomplete Freund’s adjuvant (closed squares), ZCP (closed triangles) or ECC (closed circles) on days 0, 28 and 42. Statistically (P B0.05) greater antibody titers are labeled a.
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administration of hCG by injection to female rabbits induces them to ovulate. Ovulation is accompanied by a rise in serum progesterone levels that occurs between three and 12 days following induction. Progesterone levels greater than 1 ng/ml are considered indicative of ovulation. Four female rabbits immunized with rbhCG formulated with ZCP had antibodies to native rbhCG with geometric mean titer of 23 600. Average serum progesterone levels for immunized rabbits were 0.51 ng/ml on the third day after treatment and 0.72 ng/ml on the seventh day. In contrast, serum progesterone levels for four unimmunized rabbits averaged 3.23 and 6.41 ng/ ml on the third and seventh day, respectively. This indicates that the antibody elicited by vaccination with recombinant protein and ZCP had neutralized the biological activity of the native hCG.
3.5. IgG isotypes elicited using adju6ants containing chitosan and recombinant protein Isotype analysis of the BALB/c mouse antibody responses to both recombinant and native protein elicited by vaccination with rbhCG, in either adjuvant formulation was determined by ELISA with isotype specific reagents. There were no significant IgM responses detected following the day 21-booster injection in the serum of mice immunized using either adjuvant. The IgG subclasses of antibody to both recombinant and native protein in serum collected on day 147 of the response were assayed and are presented as a percent of the total response (Table 1). The injection of rbhCG and ZCP elicited IgG1 antibody responses to both native and recombinant protein. In contrast, the injection of rbhCG and the ECC elicited mixed subclass antibody responses containing IgG2a, IgG2b and IgG1.
3.6. Ability of chitosan containing adju6ants and recombinant protein to establish DTH responses DTH is a classical method used to assess in vivo T cell mediated immunity. To investigate the ability of the chitosan-containing adjuvant formulations to prime for DTH responses, mice and guinea pigs were tested by intradermal injection of the ear pinna (Table 2). BALB/ c mice immunized with rbhCG and ZCP on days 0 and 21 were tested on day 318. The DTH response was determined by injection of rbhCG or OVA into the pinna of opposite ears. Mice immunized with rbhCG without adjuvant had less than a 10% mean ear swelling in response to injection with rbhCG (data not shown). Mice immunized with rbhCG and ZCP had far greater ear swelling in response to rbhCG than to the OVA control antigen (Table 2). This indicates that the use of the ZCP adjuvant formulation had enhanced the specific DTH responses to recombinant protein.
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Table 1 Isotype profile of antibodies elicited by injection of recombinant proteins with chitosan-containing adjuvants Immunoglobulin isotype
Adjuvant formulation Particulate
Emulsion
Antibody response to
Antibody response to
Recombinant bhCG (%) IgG1 IgG2a IgG2b
99 1 B1
Native hCG (%) 99 1 B1
To determine the ability of the ECC formulation to prime for DTH responses, guinea pigs immunized with OVA and the ECC on days 0 and 28 were tested on day 47 for their ability to respond in a DTH test using OVA and a control antigen, BSA. The mean percent ear swelling was much greater to the immunizing antigen (OVA) than to the control (BSA).
4. Discussion This report documents the ability of both a zinc-chitosan particle (ZCP) formulation and an emulsion containing chitosan (ECC) formulation to elicit antibody responses that are more than 100 times those elicited by recombinant protein without adjuvant. Importantly, the use of either adjuvant formulation also significantly enhanced the stimulation of antibody that bound native protein. The adjuvant activity of ZCP and ECC could not be attributed to either chitosan alone or the squalene and Pluronic® L121 present in the ECC. When used with a second booster injection, the anti-native protein antibody titers elicited by recombinant protein formulated with ZCP or ECC were similar to those elicited using Freund’s adjuvant, an effective adjuvant limited to research use by toxicity. The antibody responses stimulated by two injections of antigen with these adjuvant formulations were long lived, with significant titers to recombinant lasting throughout the experiment. The ability of antibodies elicited by vaccination with rbhCG formulated with ZCP to abrogate the anticipated biological effects following injection of hCG indicates that this adjuvant can be used to elicit functional antibodies that are capable of in vivo bioneutralization. Similarly, ECC has been used to elicit functional immunity, preventing the vertical transmission of the parasite Neospora caninum in a mouse model [21]. Production of immunoglobulin isotypes is associated with T-helper (Th) responses. Th1 cells produce IFN-g and IL-2, promoting the production of IgG2a. Th2 cells produce IL-4, IL-5 and IL-10, promoting the produc-
Recombinant bhCG (%)
Native hCG (%)
81 17 2
91 6 2
tion of IgG1 and IgE. Protective immune responses may depend upon the induction of a Th1 or Th2 response [29], but protection can be provided by more than one mechanism [30]. Isotype analysis of the day 147 antibody responses revealed an IgG1 response in BALB/c mice immunized with ZCP. The antibody response of mice immunized with ECC was more mixed with IgG2a, IgG2b and IgG1 antibodies present. These results must be interpreted with some caution because there is a Th2 bias in BALB/c mice and the nature of the response can be influenced by the nature of the antigen [31]. Both the chitosan containing adjuvant formulations were able to prime animals for DTH responses, suggesting some T-cell stimulatory activity. On-going experiments characterizing the cytokines stimulated by these adjuvants should add further insight into whether a Th1 or Th2 response is being stimulated.
Table 2 DTH responses induced by immunization using chitosan-containing adjuvants Species
Adjuvant
Miceb Particulate (n =15) Guinea pigs Emulsion (n= 6)
Mean swelling (%) in ears elicited witha Priming antigen
Control antigen
P-value
94 9 29
16 916
B0.0001
54 9 29
12 9 9
0.008
Mice were immunized on days 0 and 21 with recombinant bhCG and zinc-chitosan particles, on day 318 DTH responses were assayed by injection of bhCG and OVA. Guinea pigs were immunized on days 0 and 28 with OVA and the emulsion containing chitosan. On day 47 their DTH responses were assayed by injection of OVA and BSA. b Mean% swelling9 S.D. of ear pinna 24 h post injection. The P-value was calculated using a t-test to compare mean swelling response elicited by priming antigens with that elicited by control antigens. Animals immunized with antigen without adjuvant had less than a 10% mean ear swelling in response to injection of antigen. a
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Both the ZCP and ECC adjuvant formulations capitalize on the ability of chitosan to stimulate the immune response via multiple mechanisms as well as the ability of chitosan to bind and slowly release antigen [14]. In addition, chitosan microparticles, such as those present in these preparations, stimulate antigen presenting cells and the release of cytokines [5–14]. Particulate adjuvants elicit efficient immune responses and facilitate antigen uptake and translocation to draining lymph nodes via dendritic cells [32]. Chitosan has also been reported to bind to CD14, the lipopolysaccaride receptor [12]. Chitosan may also stimulate innate immunity through interaction of its N-acetyl-glucosamine subunits and mannose-binding protein, an acute phase protein, that can activate complement, act as opsonin and link the innate and adaptive immune systems [33]. Chitosan is a polycation which may account for the ability of chitosan to facilitate transmucosal delivery of small molecules as well as antigens [15]. Cationized proteins have been shown to adhere to slightly anionic cell membranes and be better retained by the reticuloendothelial system [34]. The ZCP formulation uses zinc to enhance its affinity for histidine tagged recombinant proteins. It also capitalizes on the immune stimulating properties of zinc [35]. Part of the adjuvant activity of ZCP is likely to be similar to that of aluminum salts, with the association of antigen and adjuvant affecting both the rate and route of antigen delivery. This hypothesis is supported by the finding that, in BALB/c mice, antibody of the IgG1 isotype predominated the response elicited by recombinant protein formulated with ZCP. This is similar to reports that antigen formulated in aluminum salts elicit an IgG1, Th2 type, antibody response in mice [36]. Aqueous emulsions containing squalene or squalane can be efficacious adjuvants [28]. Hunter’s group demonstrated the adjuvant activity of certain nonionic block copolymers such as Pluronic® L121 (poloxamer 401) [37]. Pluronic® L121 is a spreading agent, poorly soluble in water and adheres to the surface of oil droplets suspended in an aqueous media, where it can increase the concentration of protein at the interface [38]. Pluronic® L121 activates complement [39], which facilitates the localization of antigen to follicular dendritic cells (FDC). In germinal centers, B cells that undergo stimulation from antigen bearing FDC, differentiate into either the small resting B-cells of classical memory or into long-lived antibody producing plasmacytes, concentrated in the bone marrow [40]. The long-lived, high titer antibody responses elicited by antigen formulated in ZCP and ECC adjuvants suggest that they stimulate the production of plasmacytes. This property is somewhat unique and suggests that ZCP and ECC adjuvant for-
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mulations may be particularly useful in situations where a prolonged, protective and pre-existing antibody response is required. Examples of this include vaccines directed at toxins or intracellular pathogens that can infect host cells long before classical immunologic memory could respond. Such pathogens would include viruses and parasites, like malaria, where the window of opportunity to prevent infection is narrow, measured in minutes rather than days. The ECC formulation combines the activity of chitosan with the adjuvant activity of squalene and Pluronic® L121. Adjuvant formulations containing metabolizable oils and block copolymers are known [26–28]. In their mature form preformed adjuvant emulsions are added to antigen. This facilitates their use and prevents potential epitope denaturation during emulsion formation. ECC is an oil in water emulsion. As described here it is an easy to form temporary emulsion in which the oil coats the chitosan and antigen microparticles. Unbound antigen may also be stabilized at the interface between the oil and aqueous phases by Pluronic® L121. It is likely that ECC presents antigen in several different contexts and that this in combination with the multiple ways in which chitosan stimulates both the innate and adaptive immune system, accounts for its high degree of adjuvant activity. In summary, this report describes the immune stimulating activity of a zinc-chitosan particle adjuvant formulation and an emulsion formulation containing chitosan. BALB/c mice immunized with recombinant bhCG formulated with either adjuvant had prolonged high titer antibodies that recognized both the recombinant bhCG and native hCG. The adjuvant activity of these adjuvant formulations compared favorably to CFA/IFA. Isotype analysis of these antibodies revealed an IgG1 response in BALB/c mice immunized with zinc-chitosan particles and a mixed IgG1, IgG2a and IgG2b response with the emulsion. These chitosan based adjuvant formulations were effective in sensitizing mice and guinea pigs for antigen specific DTH responses, indicating that these adjuvants stimulate both B and T lymphocytes. The ability of these adjuvants to stimulate significant responses with a poorly immunogenic recombinant protein suggests that they may have potential in developing vaccines based on synthetic peptides and subunit antigens.
Acknowledgements We thank C Newby, for her technical assistance, GP Singh, and PJ Kelleher, for scientific discussions, CA Seid, for the recombinant protein and KT Hsu, for adjuvant manufacturing expertise.
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References [1] Eisen HN. Immunology: antibody formation. In: Davis BD, Dulbecco R, Eisen HN, Ginsberg HS, Wood WB, editors. Microbiology. Maryland: Harper & Row, 1973:481. [2] Gupta RK, Siber GR. Adjuvants for human vaccines-current status, problems and future prospects. Vaccine 1995;13:1263 – 76. [3] Vogel FR, Powell MF. A compendium of vaccine adjuvants and excipients. In: Powell MF, Newman MJ, editors. Vaccine design: the subunit and adjuvant approach. New York: Plenum Press, 1995:141 – 228. [4] Snyder LL, Woo DV, Triozzi PL, et al. Synthetic hormone/ growth factor subunit vaccine with application to antifertility and cancer. In: Powell MF, Newman MJ, editors. Vaccine design: the subunit and adjuvant approach. New York: Plenum Press, 1995:907 – 30. [5] Nishimura K, Nishimura S, Nishi N, et al. Adjuvant activity of chitin derivatives in mice and guinea-pigs. Vaccine 1985;3:379 – 84. [6] Azuma I. Synthetic immunoadjuvants: application to non-specific host stimulation and potentiation of vaccine immunogenicity. Vaccine 1992;10:1000–6. [7] Muzzarelli R, Baldassarre V, Conti F, et al. Biological activity of chitosan: ultrastructural study. Biomaterials 1988;9:247–52. [8] Iida J, Une T, Ishihara C, et al. Stimulation of non-specific host resistance against Sendai virus and Escherichia coli infections by chitin derivatives in mice. Vaccine 1987;5:270–4. [9] Peluso G, Petillo O, Ranieri M, et al. Chitosan-mediated stimulation of macrophage function. Biomaterials 1994;15:1215 – 20. [10] Usami Y, Okamoto Y, Minami S, et al. Chitin and chitosan induce migration of bovine polymorphonuclear cells. J Vet Med Sci 1994;56:761 – 2. [11] Shimbata Y, Foster LA, Metzger WJ, Myrvik QN. Alveolar macrophage priming by intravenous administration of chitin particles, polymers of N-acetyl-D-glucosamine, in mice. Infect Immun 1997;65:1734–41. [12] Otterlei M, Varum KM, Ryan L, Espevik T. Characterization of binding and TNF-a-inducing ability of chitosans on monocytes: the involvement of CD14. Vaccine 1994;12:825–32. [13] Marcinkiewicz J, Polewska A, Knapczyk J. Immunoadjuvant properties of chitosan. Archivum Immunologiae Et Therapiae Experimentalis 1991;39:127–32. [14] Calvo P, Remunan-Lopez C, Vila-Jato JL, Alonso MJ. Chitosan and chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers and vaccines. Pharm Res 1997;14:1431 – 6. [15] Illum L. Chitosan and its use as a pharmaceutical excipient. Pharm Res 1998;15:1326–31. [16] Hirano S, Seino H, Akiyama Y, et al. Biocompatibility of chitosan by oral and intravenous administration. Polym Mater Sci Eng 1988;59:897–901. [17] Knapczyk J, Krowczynski L, Marchut E, et al. Some biomedical properties of chitosan. In: Skjak-Braek G, Anthonsen T, Sandford PA, editors. Chitin and chitosan. New York: Elsevier, 1989:605 – 16. [18] Tokura S. Structure and chemical modification of chitin and chitosan. In: Skjak-Braek G, Anthonsen T, Sandford PA, editors. Chitin and chitosan. New York: Elsevier, 1989:45– 50. [19] Smith MC, Furman TC, Ingolia TD, Pidgeon C. Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins. J Biol Chem 1988;263:7211–5.
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[20] Podolski JS, Martinez ML. Chitosan induced immunopotentiation. United States Patent 5,980,912. Issued 9 November 1999, www.uspto.gov. [21] Liddell S, Jenkins MC, Collica CM, et al. Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination. J Parasitol 1999;85:1072 – 5. [22] Podolski JS, Hsu K, Singh G. Chitosan induced immunopotentiation. United States Patent 5,912,000. Issued June 15, 1999, www.uspto.gov. [23] Seferian PG, Rodkey LS. Multivalent antibody induces active antibody synthesis. Vacc Res 1992;1:373 – 82. [24] Frey A, Di Canzio J, Zurakowski D. A statistically defined endpoint titer determination method for immunoassays. J Immunol Methods 1998;221:35 – 41. [25] Ambrose MW, Wyde PR. Parainfluenza virus type 3 (PIV3)-specific and non-virus specific delayed type hypersensitivity responses in cotton rats given different PIV3 antigen preparations. Vaccine 1993;11:336 – 42. [26] Newman MJ, Actor JK, Balusubramanian M, et al. Use of nonionic block copolymers in vaccines and therapeutics. Crit Rev Ther Drug Carrier Syst 1998;15:89 – 142. [27] Allison AC. The mode of action of immunological adjuvants, In: Brown F, Haaheim LR, editors. Modulation of the immune response to vaccine antigens. Dev Biol Stand Basel, Karger, 1998;92:3 – 11. [28] Allison AC. Squalene and squalane emulsions as adjuvants. Methods 1999;19:87 – 93. [29] Mosmann TR, Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 1996;17:138 – 46. [30] Garvy BA, Wiley JA, Gigliotti F, Harmsen AG. Protection against Pneumocystis carnii pneumonia by antibodies from either T helper 1 or T helper 2 responses. Infect Immun 1997;65:5052– 6. [31] McInerney TL, Brennan FR, Jones TD, Dimmock NJ. Analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying HIV-1 peptide. Vaccine 1999;17:1359 – 68. [32] Dupuis M, Murphy TJ, Higgins D, et al. Dendritic cells internalize vaccine adjuvant after intramuscular injection. Cell Immunol 1998;186:18 – 27. [33] Stahl PD, Ezekowitz RA. The mannose receptor is a pattern recognition receptor involved in host defense. Curr Opin Immunol 1998;10:50 – 5. [34] Apple RJ, Domen PL, Muckerheide A, et al. Cationization of proten antigens. IV. Increased antigen uptake by antigen-presenting cells. J Immunol 1988;140:3290 – 5. [35] Wellinghausen N, Kirchner H, Rink L. The immunobiology of zinc. Immunol Today 1997;18:519 – 21. [36] Gupta RK, Rost BE, Relyveld E, et al. Adjuvant properties of aluminum and calcium compounds. In: Powell MF, Newman MJ, editors. Vaccine design: the subunit and adjuvant approach. New York: Plenum Press, 1995:229 – 76. [37] Hunter RL, Strickland F, Kedzy F. Studies on the adjuvant activity of nonionic block polymer surfactants, I. The role of hydrophile-lipophile balance. J Immunol 1981;127:1244–50. [38] Hunter RL, Bennett B. The adjuvant activity of nonionic block polymer surfactants, III. Characterization of selected biologically active surfaces. Scand J Immunol 1986;23:287 – 300. [39] Hunter RL, Bennett B. The adjuvant activity of nonionic block polymer surfactants, II. Antibody formation and inflamation related to the structure of triblock and octablock copolymers. J Immunol 1984;133:3167 – 75. [40] Przylepa J, Himes C, Kelsoe G. Lymphocyte development and selection in germinal centers. Curr Top Microbiol Immunol 1998;229:85 – 104.
Immune stimulating activity of two new chitosan containing adjuvant formulations Peter G. Seferian, Mitzi L. Martinez * Zonagen Inc., 2408 Timberloch Place, B-4, The Woodlands, TX 77380, USA Received 30 August 1999; received in revised form 18 May 2000; accepted 30 May 2000
Abstract Recombinant proteins have potential as both human and veterinary vaccine antigens, but they are often weakly immunogenic and immunization with recombinant proteins may not elicit a significant immune response that recognizes the native protein. This report describes the immune stimulating activity of two new adjuvant formulations, a zinc-chitosan particle formulation designed to bind to histidine tagged recombinant proteins; and an emulsion formulation containing chitosan. BALB/c mice vaccinated with formulations comprising recombinant beta-human chorionic gonadotropin (bhCG) and each adjuvant had prolonged high titer antibodies that recognized both the recombinant bhCG and native hCG. bhCG is an established target for immunocontraceptive vaccines and a potential target for tumor immunotherapy. Isotype analysis of these antibodies revealed an IgG1 response in mice immunized with zinc-chitosan particles and a mixed IgG1, IgG2a and IgG2b response with the emulsion. These chitosan based adjuvant formulations were effective in sensitizing mice and guinea pigs for antigen specific DTH responses, indicating that these adjuvants stimulate both B and T lymphocytes. The ability of these adjuvants to stimulate significant responses with a poorly immunogenic recombinant protein suggests that they may have potential in developing vaccines based on synthetic peptides and subunit antigens. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Adjuvant; Chitosan; Histidine
1. Introduction Adjuvants are substances which enhance the ability of antigens to elicit an immune response [1,2]. Recombinant proteins are potentially better-defined and safer components of both veterinary and human vaccines. However, recombinant proteins are often weakly immunogenic and require adjuvants that are superior to aluminum to make them effective vaccines. The importance of effective adjuvants is demonstrated by the fact that there are many adjuvant systems being developed [3]. This report describes the immune response elicited by the injection of recombinant beta-human chorionic gonadotropin (rbhCG), a well established target for immunocontraceptive vaccination and a potential target for tumor immunotherapy [4], in conjunction with two adjuvant formulations containing chitosan. * Corresponding author. Tel.: +1-281-3675892; fax: + 1-2813638796. E-mail address: [email protected] (M.L. Martinez).
Chitosan suspensions or microparticles have been reported to have immune stimulating activity such as increasing accumulation and activation of macrophage and polymorphonuclear cells, suppressing tumor growth, promoting resistance to infections by microorganisms, inducing cytokines, augmenting antibody responses and enhancing delayed type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses [5–14]. Chitosan also has utility in transmucosal delivery of drugs and peptides [15]. Chitin is a naturally occurring polymer of N-acetylD-glucosamine present in crustaceans, insects, mushrooms and microorganisms. Chitosan, derived by the deacetylation of chitin, is a polymer of D-glucosamine and N-acetyl-D-glucosamine. Compared with other natural polysaccharides, chitosan has high biodegradability, low toxicity and is neither an irritant nor allergen in humans [16–18]. This paper describes the ability of two new adjuvant formulations containing chitosan to stimulate antibody responses and prime for DTH. One formulation results in a zinc-chitosan particle (ZCP)
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that binds antigen and is particularly useful with recombinant proteins purified by immobilized metal affinity chromatography such as those containing a histidine tag [19]. The other formulation results in an emulsion containing chitosan (ECC) and antigen. Both the formulations enhance and prolong the immune stimulating activity of chitosan. The immune stimulating activity of these adjuvant formulations could not be attributed to the reported adjuvant activity of chitosan alone. Specifically, in combination with recombinant proteins these formulations were found to be effective in stimulating high titer antibody responses to both the recombinant antigen given and the native protein from which it was derived. Vaccination with recombinant proteins in both the formulations also primed T-cell mediated DTH responses.
2. Materials and methods
2.1. Animals and antigens Female BALB/c mice and Hartley guinea pigs were obtained from Harlan, rabbits were obtained from Covance and all animals were maintained as recommended in the Guide for the Care and Use of Laboratory Animals (NRC, 1996). Ovalbumin (OVA: fraction VI) and native human chorionic gonadotropin (hCG) were obtained from Sigma Chemicals (St Louis, MO). Histidine-tagged rbhCG was expressed in yeast cells from a proprietary construct.
2.2. Adju6ants and immunizations The ECC [20] was developed to elicit strong and sustained immune responses with many different types of antigens, even with a single injection. To prepare 1 ml of vaccine, 420 ml of phosphate buffered saline (PBS) containing antigen was mixed with 340 ml of a sterile 2% chitosan (Sigma) in 2% acetic acid solution (Mallinkrodt Chemical, Paris, KY). As the chitosanantigen mixture was neutralized by the addition of NaOH (Sigma), antigen and chitosan form a fine gelatinous precipitate. The pH of the precipitated solution was 6.8–7.2, as determined by spotting onto pH indicator strips (Baxter, Deerfield, IL). One hundred and fifty microliters of a 2.5:1 solution containing squalene (Sigma) and Pluronic® L121 (BASF Corp., Parsippany, NJ) was added and upon vortex mixing formed a cloudy emulsion. Mice were immunized with 25 mg of rbhCG in 200 ml of this emulsion by intraperitoneal injection on days 0 and 21. Guinea pigs were immunized by intramuscular injection of 35 mg of OVA in 300 ml on days 0 and 21. ECC has been referred to as ImmuMax SR [21].
The ZCP [22] were designed to facilitate immunization with recombinant proteins containing a histidine tag [19]. To prepare this adjuvant a 2% chitosan in 2% acetic acid solution was added to a 10% zinc acetate (Sigma) solution and the mixture rocked end to end for 4 h at room temperature. This results in a fine white precipitate, which was washed several times with PBS. To prepare injections, the antigen was placed in PBS, pH 8, to optimize the histidine tag charge and therefore its binding to the adjuvant particles. For each 25 mg of antigen to be injected, 100 ml of zinc-chitosan particle slurry was transferred to a small sterile tube. The final ratio of adjuvant to antigen was 1 mg of ZCP particles (wet weight) for every 1 mg of protein. The adjuvant particles were sedimented by centrifugation and the supernatant removed. The adjuvant pellet was washed twice by suspension in PBS (pH 8), centrifugation and supernatant removal. Antigen solution was used to resuspend the washed adjuvant pellet and mixed for 2 h at room temperature. The adjuvant–antigen preparation was sedimented by centrifugation and the supernatant removed and saved for protein analysis. The adjuvant–antigen pellet was washed with PBS and suspended in the desired volume of PBS (pH 7.4) prior to injection. The amount of protein bound to the ZCP was determined using a Coomassie® Plus protein assay (Pierce, Rockford, IL) of the antigen-containing supernatant protein concentration before and after binding. Mice were immunized with 25 mg of rbhCG in 100 ml of adjuvant–antigen complex by intramuscular injection. Rabbits received intramuscular injections containing 250 mg of rbhCG in 500 ml of the adjuvant– antigen complex. Several different adjuvant formulations were prepared to compare the activity of ZCP and ECC adjuvant formulations to the adjuvant activity reported for chitosan alone and the combination of squalene and Pluronic® L121. Chitosan particles were prepared by the neutralization of a 2% chitosan in 2% acetic acid solution with NaOH. This resulted in a gelatinous precipitation of chitosan particles that were washed several times with PBS. Chitosan particles were then suspended in PBS containing rbhCG. To prepare 1 ml of vaccine formulated with the same concentration of squalene and Pluronic® L121 as is found in ECC adjuvant, 150 ml of a 2.5:1 solution containing squalene and Pluronic® L121 was added to 1050 ml of PBS containing 150 mg of rbhCG. Mice were immunized with 25 mg of rbhCG in 200 ml of this emulsion by intraperitoneal injection. The traditional research adjuvant combination of antigen formulated in complete Freund’s adjuvant (CFA) followed by booster immunizations using antigen in incomplete Freund’s Adjuvant (IFA) was used as a reference standard for the adjuvant activity of ZCP and ECC. CFA or IFA (Sigma) was combined with an
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equal volume of PBS containing rbhCG. This mixture was whipped into a stable emulsion by sonification with a Branson Sonifier 250 (Branson Ultrasonics Corp., Danbury, CT). The stability of these emulsions was confirmed by floating an aliquot on saline. Mice were immunized with 25 mg of rbhCG in 200 ml of CFA or IFA emulsion by intraperitoneal injection.
2.3. Antibody assay (ELISA) Antibody titers were determined in an antibody capture enzyme-linked immunosorbent assay (ELISA) [23]. Briefly, 96-well assay plates (Falcon c 3912) were coated with rbhCG (1 mg/ml) or native hCG (10 U/ml). Plates were blocked with 2% nonfat dry milk (NFDM) to inhibit nonspecific binding. Blocked plates were emptied and washed with PBS containing 0.05% Tween 20 (PBS-T: Sigma) and emptied prior to use. Sample and control sera were serially diluted in PBS-T containing 2% NFDM prior to application. Plates were covered and incubated for 2 h at 37°C, emptied and washed four times with PBS-T. Bound antibody was detected using biotin labeled rabbit anti-mouse IgG (H+ L) antibody (Zymed, S. San Francisco, CA). Bound antibody class and subclass analyses were done using biotin labeled rabbit anti-mouse IgM, IgG1, IgG2a or IgG2b specific reagents (Zymed). The presence of bound antibody was detected using horseradish peroxidase conjugated streptavidin (Zymed) and 800 mg/ml O-phenylenediamine (Sigma) substrate solution. Plates were developed in the dark at room temperature for 10 min and the absorbence at 450 nm was read on an Emax plate reader (Molecular Devices). Endpoint titers were determined as the reciprocal dilution whose absorbence exceeded twice the absorbence obtained using unimmunized negative control sera. This method has been reported to be conservative in its estimation of titer [24]. Using this cutoff, the endpoint titer obtained for a commercial IgG1 monoclonal anti-human chorionic go-
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nadotropin, b subunit specific antibody (PE-4 clone, Lot No. 37F-4963-1: Sigma) was 6400.
2.4. Bioneutalization of HCG induced o6ulation Fifty units of hCG (Sigma) was administered by subcutaneous injection to four female New Zealand white rabbits immunized with rbhCG formulated with ZCP and four non-immunized rabbits. Blood was drawn for serum three and seven days following administration of hCG. Rabbit serum progesterone levels were determined by RIA (Diagnostic Products Corp.) as an indication of ovulation.
2.5. Elicitation of delayed type hypersensiti6ity (DTH) Vaccinated mice and guinea pigs were tested for antigen specific DTH responses by injection of 10 or 50 ml, respectively, of priming antigen into the right ear pinna and an equal amount of control antigen in the left ear. Ear thickness was measured as described [25], just prior to injection, and 24 and 48 h post-vaccination. Percent ear swelling was calculated as follows: [(Post− Pre)/Pre] ×100, where Post is post-injection ear pinna thickness and Pre is pre-elicitation ear pinna thickness. Mice were tested for elicitation of DTH 318 days after primary immunization and guinea pigs were tested 47 days after primary immunization.
2.6. Statistical analysis ELISA results are shown as geometric mean titers. DTH response results are expressed as the arithmetic mean9 standard deviation (S.D.). Comparison of means was done using a two-tailed t-test and statistical significance was determined using StatGraphics Plus, version 2.1.
3. Results
3.1. Effect of adju6ant formulations containing chitosan on antibody responses elicited by recombinant protein
Fig. 1. The antibody response of BALB/c mice (n = 10) to recombinant protein elicited by injection of recombinant bhCG formulated with PBS (open squares), ZCP (closed triangles) or ECC (closed circles) on days 0 and 21. Both adjuvant formulations stimulated statistically (P B 0.05) greater antibody titers than PBS at points labeled a.
BALB/c mice were vaccinated on days 0 and 21 with 25 mg of rbhCG formulated with no adjuvant (PBS), ZCP or ECC. Serum was obtained every 3 weeks and the anti-rbhCG was assayed in an antibody capture ELISA (Fig. 1). The IgG antibody responses elicited by rbhCG formulated with either adjuvant were 2 logs (or more) greater than the antibody response elicited in the absence of either adjuvant. In addition, antibody responses stimulated with ZCP or the ECC were significant for up to 181 days. Serum from these mice was also assayed for antibody binding to native hCG in an antibody capture ELISA
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Fig. 2. The antibody response of BALB/c mice (n= 10) to native protein elicited by injection of recombinant bhCG formulated with PBS (open squares), ZCP (closed triangles) or ECC (closed circles) on days 0 and 21. Both adjuvant formulations stimulated statistically (PB0.05) greater antibody titers than PBS at points labeled a. Only the higher adjuvant stimulated titer is statistically (PB 0.05) greater than the antibody titer stimulated with PBS at points labeled b.
Fig. 3. The range and geometric mean titers of antibody response of BALB/c mice (n =6) to recombinant protein at 8 weeks following injection of recombinant bhCG with either PBS, at the same time as ZCP, but in an opposite leg (OppLegs), chitosan particles, ZCP, ECC or the squalene: Pluronic® L121 phase of ECC (Oil phase). Formulation of rbhCG with either ZCP or ECC elicited antibody responses that were statistically (P B 0.05) greater than antibody titers elicited with other formulations, points labeled a. Formulation of rbhCG in squalene:Pluronic® L121 elicited antibody titers statistically (PB 0.05) greater than the antibody titer stimulated with PBS, points labeled b.
(Fig. 2). Significantly, mice immunized with rbhCG and either ZCP or the ECC also had high titer antibodies that bound the native hCG. The use of these adjuvants, when injecting recombinant protein enhanced the antibody response to native protein by 2 logs. The specificity of this response was confirmed by polyacrylamide gel electrophoresis and western blotting (data not shown). The duration of the anti-native hCG response stimulated by each adjuvant formulation differed. The ECC adjuvant formulation stimulated an anti-native hCG response that was slow to rise but was sustained. In comparison, the ZCP adjuvant formulation stimulated an anti-native hCG response that rose rapidly, but was not as well sustained.
3.2. Comparison of adju6ant formulations containing chitosan and their components It has been reported that chitosan particles them-
selves have adjuvant activity [5–14]. Both squalene and Pluronic® L121 are also known to have adjuvant activity alone and in combination [26–28]. To determine whether the adjuvant activity observed with ZCP and ECC could be attributed to the chitosan alone, or in the case of ECC, to the squalene and Pluronic® L121, mice were vaccinated with rbhCG formulated with either PBS, chitosan particles, ZCP, ECC or the oil phase of ECC (squalene and Pluronic® L121). In addition, because there have been reports that the immune stimulating activity of chitosan is a generalized, non-specific stimulation of the immune response [13], a sixth group of mice received ZCP in one leg and antigen in the other. The assumption was that if ZCP stimulated a generalized increase in the immune response, its physical relationship to the antigen would be irrelevant to its ability to enhance the immune response elicited by the antigen. Five groups, each containing five BALB/c mice, were vaccinated on days 0 and 21 with 25 mg of rbhCG formulated with either PBS, chitosan particles, ZCP, ECC or squalene and Pluronic® L121 at the same concentration as used in ECC. A sixth group of five mice was vaccinated by intramuscular injection with 25 mg of rbhCG in PBS and at the same time but in an opposite leg (OppLegs) with ZCP particles. Serum obtained 8 weeks after initial vaccination was assayed in an antibody capture ELISA (Fig. 3). Unexpectedly, vaccination with rbhCG formulated with chitosan particles did not significantly increase the antibody response to rbhCG as compared with that obtained following vaccinations with rbhCG formulated without adjuvant (PBS). In contrast, as expected, vaccination with formulations combining rbhCG with squalene and Pluronic® L121 elicited antibody levels that were significantly greater than those elicited by vaccination with rbhCG in PBS. Vaccination with rbhCG with both chitosan containing adjuvant formulations, ZCP and ECC, elicited significantly greater antibody responses than did rbhCG formulated with squalene and Pluronic® L121. The injection of ZCP and rbhCG in opposite legs at the same time did not elicit an increased antibody response. This suggests that, at least for this chitosan containing adjuvant formulation, that the physical association of the chitosan and antigen is important to its adjuvant activity.
3.3. Comparison of adju6ant formulations containing chitosan and CFA/IFA Having established the adjuvant activity of these adjuvant formulations containing chitosan, they were compared with the combination of CFA/IFA. To directly compare the ability of formulation of rbhCG
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with ZCP or ECC to elicit an anti-native hCG response to that elicited by formulation of rbhCG in CFA/IFA, a three-immunization protocol was chosen. Ten mice in each group were immunized on days 0, 28 and 42 with rbhCG in either ZCP, ECC or CFA/IFA and sera samples were assayed for binding to native hCG. The anti native hCG antibody response elicited by the injection of rbhCG in combination with CFA/IFA peaked more quickly than those elicited by rbhCG formulated in ECC or ZCP. This was evidenced by statistically greater antibody titers seen in the CFA/IFA group on days 77–133 of the experiment (Fig. 4). However, with time the CFA/IFA induced native hCG titers decline. The anti-native hCG antibody response elicited by vaccination with rbhCG formulated with ZCP do not peak as do those induced by CFA/IFA. ZCP induced titers undergo a slow and steady rise but were equivalent to CFA/IFA induced titers from day 151 onward, with no statistical differences between the titers induced by the two adjuvant groups for the remainder of the observation period. In contrast, the anti-native hCG antibody response elicited by rbhCG formulated with ECC requires more time than CFA/IFA induced titers to rise, but they continue rising. Antibody titers elicited by rbhCG formulation in ECC were stabilized at a level equivalent to those of the CFA/IFA and the ZCP groups between days 151 and 234 but are sustained while the titers in the CFA/IFA group and the ZCP group decline. The ECC induced native hCG antibody titers then become statistically greater than those elicited by the other adjuvant groups from day 234 onward.
3.4. Functional acti6ity of antibodies elicited with an adju6ant containing chitosan In an infectious disease model, functional immunity equals protection. With immunocontraceptive vaccines, protection equals infertility. When working with hormones, functional immunity manifests itself as bioneutralization of the hormone’s induced effects. The
Fig. 4. The antibody response of BALB/c mice (n= 10) to native hCG elicited by injection of recombinant bhCG formulated with complete/incomplete Freund’s adjuvant (closed squares), ZCP (closed triangles) or ECC (closed circles) on days 0, 28 and 42. Statistically (P B0.05) greater antibody titers are labeled a.
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administration of hCG by injection to female rabbits induces them to ovulate. Ovulation is accompanied by a rise in serum progesterone levels that occurs between three and 12 days following induction. Progesterone levels greater than 1 ng/ml are considered indicative of ovulation. Four female rabbits immunized with rbhCG formulated with ZCP had antibodies to native rbhCG with geometric mean titer of 23 600. Average serum progesterone levels for immunized rabbits were 0.51 ng/ml on the third day after treatment and 0.72 ng/ml on the seventh day. In contrast, serum progesterone levels for four unimmunized rabbits averaged 3.23 and 6.41 ng/ ml on the third and seventh day, respectively. This indicates that the antibody elicited by vaccination with recombinant protein and ZCP had neutralized the biological activity of the native hCG.
3.5. IgG isotypes elicited using adju6ants containing chitosan and recombinant protein Isotype analysis of the BALB/c mouse antibody responses to both recombinant and native protein elicited by vaccination with rbhCG, in either adjuvant formulation was determined by ELISA with isotype specific reagents. There were no significant IgM responses detected following the day 21-booster injection in the serum of mice immunized using either adjuvant. The IgG subclasses of antibody to both recombinant and native protein in serum collected on day 147 of the response were assayed and are presented as a percent of the total response (Table 1). The injection of rbhCG and ZCP elicited IgG1 antibody responses to both native and recombinant protein. In contrast, the injection of rbhCG and the ECC elicited mixed subclass antibody responses containing IgG2a, IgG2b and IgG1.
3.6. Ability of chitosan containing adju6ants and recombinant protein to establish DTH responses DTH is a classical method used to assess in vivo T cell mediated immunity. To investigate the ability of the chitosan-containing adjuvant formulations to prime for DTH responses, mice and guinea pigs were tested by intradermal injection of the ear pinna (Table 2). BALB/ c mice immunized with rbhCG and ZCP on days 0 and 21 were tested on day 318. The DTH response was determined by injection of rbhCG or OVA into the pinna of opposite ears. Mice immunized with rbhCG without adjuvant had less than a 10% mean ear swelling in response to injection with rbhCG (data not shown). Mice immunized with rbhCG and ZCP had far greater ear swelling in response to rbhCG than to the OVA control antigen (Table 2). This indicates that the use of the ZCP adjuvant formulation had enhanced the specific DTH responses to recombinant protein.
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Table 1 Isotype profile of antibodies elicited by injection of recombinant proteins with chitosan-containing adjuvants Immunoglobulin isotype
Adjuvant formulation Particulate
Emulsion
Antibody response to
Antibody response to
Recombinant bhCG (%) IgG1 IgG2a IgG2b
99 1 B1
Native hCG (%) 99 1 B1
To determine the ability of the ECC formulation to prime for DTH responses, guinea pigs immunized with OVA and the ECC on days 0 and 28 were tested on day 47 for their ability to respond in a DTH test using OVA and a control antigen, BSA. The mean percent ear swelling was much greater to the immunizing antigen (OVA) than to the control (BSA).
4. Discussion This report documents the ability of both a zinc-chitosan particle (ZCP) formulation and an emulsion containing chitosan (ECC) formulation to elicit antibody responses that are more than 100 times those elicited by recombinant protein without adjuvant. Importantly, the use of either adjuvant formulation also significantly enhanced the stimulation of antibody that bound native protein. The adjuvant activity of ZCP and ECC could not be attributed to either chitosan alone or the squalene and Pluronic® L121 present in the ECC. When used with a second booster injection, the anti-native protein antibody titers elicited by recombinant protein formulated with ZCP or ECC were similar to those elicited using Freund’s adjuvant, an effective adjuvant limited to research use by toxicity. The antibody responses stimulated by two injections of antigen with these adjuvant formulations were long lived, with significant titers to recombinant lasting throughout the experiment. The ability of antibodies elicited by vaccination with rbhCG formulated with ZCP to abrogate the anticipated biological effects following injection of hCG indicates that this adjuvant can be used to elicit functional antibodies that are capable of in vivo bioneutralization. Similarly, ECC has been used to elicit functional immunity, preventing the vertical transmission of the parasite Neospora caninum in a mouse model [21]. Production of immunoglobulin isotypes is associated with T-helper (Th) responses. Th1 cells produce IFN-g and IL-2, promoting the production of IgG2a. Th2 cells produce IL-4, IL-5 and IL-10, promoting the produc-
Recombinant bhCG (%)
Native hCG (%)
81 17 2
91 6 2
tion of IgG1 and IgE. Protective immune responses may depend upon the induction of a Th1 or Th2 response [29], but protection can be provided by more than one mechanism [30]. Isotype analysis of the day 147 antibody responses revealed an IgG1 response in BALB/c mice immunized with ZCP. The antibody response of mice immunized with ECC was more mixed with IgG2a, IgG2b and IgG1 antibodies present. These results must be interpreted with some caution because there is a Th2 bias in BALB/c mice and the nature of the response can be influenced by the nature of the antigen [31]. Both the chitosan containing adjuvant formulations were able to prime animals for DTH responses, suggesting some T-cell stimulatory activity. On-going experiments characterizing the cytokines stimulated by these adjuvants should add further insight into whether a Th1 or Th2 response is being stimulated.
Table 2 DTH responses induced by immunization using chitosan-containing adjuvants Species
Adjuvant
Miceb Particulate (n =15) Guinea pigs Emulsion (n= 6)
Mean swelling (%) in ears elicited witha Priming antigen
Control antigen
P-value
94 9 29
16 916
B0.0001
54 9 29
12 9 9
0.008
Mice were immunized on days 0 and 21 with recombinant bhCG and zinc-chitosan particles, on day 318 DTH responses were assayed by injection of bhCG and OVA. Guinea pigs were immunized on days 0 and 28 with OVA and the emulsion containing chitosan. On day 47 their DTH responses were assayed by injection of OVA and BSA. b Mean% swelling9 S.D. of ear pinna 24 h post injection. The P-value was calculated using a t-test to compare mean swelling response elicited by priming antigens with that elicited by control antigens. Animals immunized with antigen without adjuvant had less than a 10% mean ear swelling in response to injection of antigen. a
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Both the ZCP and ECC adjuvant formulations capitalize on the ability of chitosan to stimulate the immune response via multiple mechanisms as well as the ability of chitosan to bind and slowly release antigen [14]. In addition, chitosan microparticles, such as those present in these preparations, stimulate antigen presenting cells and the release of cytokines [5–14]. Particulate adjuvants elicit efficient immune responses and facilitate antigen uptake and translocation to draining lymph nodes via dendritic cells [32]. Chitosan has also been reported to bind to CD14, the lipopolysaccaride receptor [12]. Chitosan may also stimulate innate immunity through interaction of its N-acetyl-glucosamine subunits and mannose-binding protein, an acute phase protein, that can activate complement, act as opsonin and link the innate and adaptive immune systems [33]. Chitosan is a polycation which may account for the ability of chitosan to facilitate transmucosal delivery of small molecules as well as antigens [15]. Cationized proteins have been shown to adhere to slightly anionic cell membranes and be better retained by the reticuloendothelial system [34]. The ZCP formulation uses zinc to enhance its affinity for histidine tagged recombinant proteins. It also capitalizes on the immune stimulating properties of zinc [35]. Part of the adjuvant activity of ZCP is likely to be similar to that of aluminum salts, with the association of antigen and adjuvant affecting both the rate and route of antigen delivery. This hypothesis is supported by the finding that, in BALB/c mice, antibody of the IgG1 isotype predominated the response elicited by recombinant protein formulated with ZCP. This is similar to reports that antigen formulated in aluminum salts elicit an IgG1, Th2 type, antibody response in mice [36]. Aqueous emulsions containing squalene or squalane can be efficacious adjuvants [28]. Hunter’s group demonstrated the adjuvant activity of certain nonionic block copolymers such as Pluronic® L121 (poloxamer 401) [37]. Pluronic® L121 is a spreading agent, poorly soluble in water and adheres to the surface of oil droplets suspended in an aqueous media, where it can increase the concentration of protein at the interface [38]. Pluronic® L121 activates complement [39], which facilitates the localization of antigen to follicular dendritic cells (FDC). In germinal centers, B cells that undergo stimulation from antigen bearing FDC, differentiate into either the small resting B-cells of classical memory or into long-lived antibody producing plasmacytes, concentrated in the bone marrow [40]. The long-lived, high titer antibody responses elicited by antigen formulated in ZCP and ECC adjuvants suggest that they stimulate the production of plasmacytes. This property is somewhat unique and suggests that ZCP and ECC adjuvant for-
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mulations may be particularly useful in situations where a prolonged, protective and pre-existing antibody response is required. Examples of this include vaccines directed at toxins or intracellular pathogens that can infect host cells long before classical immunologic memory could respond. Such pathogens would include viruses and parasites, like malaria, where the window of opportunity to prevent infection is narrow, measured in minutes rather than days. The ECC formulation combines the activity of chitosan with the adjuvant activity of squalene and Pluronic® L121. Adjuvant formulations containing metabolizable oils and block copolymers are known [26–28]. In their mature form preformed adjuvant emulsions are added to antigen. This facilitates their use and prevents potential epitope denaturation during emulsion formation. ECC is an oil in water emulsion. As described here it is an easy to form temporary emulsion in which the oil coats the chitosan and antigen microparticles. Unbound antigen may also be stabilized at the interface between the oil and aqueous phases by Pluronic® L121. It is likely that ECC presents antigen in several different contexts and that this in combination with the multiple ways in which chitosan stimulates both the innate and adaptive immune system, accounts for its high degree of adjuvant activity. In summary, this report describes the immune stimulating activity of a zinc-chitosan particle adjuvant formulation and an emulsion formulation containing chitosan. BALB/c mice immunized with recombinant bhCG formulated with either adjuvant had prolonged high titer antibodies that recognized both the recombinant bhCG and native hCG. The adjuvant activity of these adjuvant formulations compared favorably to CFA/IFA. Isotype analysis of these antibodies revealed an IgG1 response in BALB/c mice immunized with zinc-chitosan particles and a mixed IgG1, IgG2a and IgG2b response with the emulsion. These chitosan based adjuvant formulations were effective in sensitizing mice and guinea pigs for antigen specific DTH responses, indicating that these adjuvants stimulate both B and T lymphocytes. The ability of these adjuvants to stimulate significant responses with a poorly immunogenic recombinant protein suggests that they may have potential in developing vaccines based on synthetic peptides and subunit antigens.
Acknowledgements We thank C Newby, for her technical assistance, GP Singh, and PJ Kelleher, for scientific discussions, CA Seid, for the recombinant protein and KT Hsu, for adjuvant manufacturing expertise.
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