Chitosan as an adjuvant for poliovaccine
Abstract
The use of inactivated poliomyelitis vaccine is very important for eradicating poliomyelitis. However, this vaccine is not available readily in underdeveloped countries due to the high cost. Adjuvants can improve the immunogenicity of a vaccine and reduce the antigen dose required for vaccination, thus lowering the cost of the vaccine. Chitosan glutamate solution and a chitosan sulfate micro/nanoparticle suspension were tested as adjuvants for Imovax-inactivated poliovaccine and for inactivated monovalent poliovirus type 1, 2, and 3 vaccines obtained by inactivation of the attenuated Sabin poliovirus strains. Inactivated vaccines admixed with either chitosan glutamate or chitosan sulfate micro/nanoparticles and administered to mice showed significantly enhanced immunogenicity to poliovirus type 1, 2, and 3 strains compared to the respective vaccines administered without chitosan. Chitosan preparations increased the immunogenicity of 1:2 and 1:4 diluted inactivated Sabin strain preparations in mice 8- to 16-fold, so that the neutralizing antibody titers after vaccination with adjuvanted diluted vaccine were equal to those obtained after vaccination with undiluted vaccine administered without chitosan. Neutralizing antibodies could be detected in sera of rats vaccinated with undiluted, 1:10, and 1:100 diluted Imovax vaccine admixed with chitosan sulfate micro/nanoparticles, although in the control group, vaccination only with the undiluted vaccine resulted in antibody production. These results show that the chitosan glutamate solution and chitosan sulfate micro/nanoparticle suspension can significantly improve the immunogenicity of various poliovaccines, and reduce the effective antigen dose. J. Med. Virol. 83:847–852, 2011. © 2011 Wiley-Liss, Inc.
INTRODUCTION
The World Health Organization Polio Eradication Initiative was established in 1988 [World Health Assembly, 1988]. Today, more than 20 years later, significant success has been achieved in this field. Circulation of the virulent wild-type poliovirus strains has ceased in most countries, and no cases of poliomyelitis caused by wild-type viruses have been reported in years. In 2007, only 1,315 cases of poliomyelitis were reported worldwide, compared to approximately 350,000 cases of poliomyelitis registered annually before the WHO program started [WHO, 2009a]. However, virulent poliovirus strains continue to circulate in India, Nigeria, Pakistan, and Afghanistan. For instance, in 2008, 801, 559, 188, and 31 poliomyelitis cases were reported in Nigeria, India, Pakistan, and Afghanistan, respectively [WHO, 2009a]. In 2008, a virulent poliovirus strain spread from Nigeria to a number of neighboring countries, causing 1,655 cases of poliomyelitis in 16 African countries [WHO, 2009a]. From January 1 to November of 2009, 1,337 cases of poliomyelitis were registered worldwide in 23 countries [WHO, 2010]. In the four countries endemic for poliomyelitis, the number of cases in 2009 fell slightly in Nigeria (to 651 cases) from 2008, but not in India, Pakistan, or Afghanistan [WHO, 2009b]. In 2010, more than 400 cases of poliomyelitis were reported in Tajikistan and spread to neighboring countries. All these data indicate that it is crucial to proceed with mass vaccination against poliomyelitis even in countries in which the virulent poliovirus strains no longer circulate, since the spread of poliovirus from endemic to polio-free regions cannot be excluded.
Live poliovaccine is used for vaccinating against poliomyelitis in most countries. The major drawback in the use of this vaccine is the vaccine-associated poliomyelitis that can occur after vaccination. Vaccine-associated poliomyelitis is caused by revertants of attenuated vaccine strains. Although vaccine-associated disease is an extremely rare adverse event, many countries in which the wild-type virulent poliovirus strains do not circulate have started using inactivated vaccine. However, in most of the economically underdeveloped countries, the use of inactivated vaccine is not possible due to its relatively high cost.
Recent research on adjuvants for inactivated vaccines (for instance, influenza vaccines) has shown that adjuvants significantly improve vaccine immunogenicity and protective efficacy and permit a reduction in the number of revaccinations and the required antigen dose [Gupta and Siber, 1995; Atmar et al., 2006; Aguilar and Rodriguez, 2007; Leroux-Roels et al., 2008; Banzhoff et al., 2009]. It is likely that adjuvants would enhance the immunogenicity of inactivated poliovaccine and reduce the antigen dose and the number of immunizations needed, lowering significantly the cost of using this preparation. To date, few investigations have examined adjuvant usage with inactivated poliovaccine (e.g., CpG-oligodezoxinucleotides and vitamin D3 adjuvants) [Ivanov et al., 2006; Yang et al., 2008].
Using chitosan as an adjuvant can enhance significantly immunogenicity and protective efficacy of inactivated influenza vaccine, and reduces the antigen dose needed for vaccination and the number of immunizations [Ghendon et al., 2008, 2009]. Chitosan is a linear polysaccharide consisting of randomly located β-1-4 linked D-glucosamine and N-acetyl-D-glucosamine residues obtained by deacetylation of chitin, the structural base of the exoskeletons of crustaceans, insects, and fungi. Chitosan is a non-allergenic, biodegradable, and biocompatible substance widely used in medicine, pharmaceutics, and food industry [Illum, 1998; Illum et al., 2001; Singla and Chawla, 2001].
This study aimed to investigate the adjuvant capacity of chitosan administered with inactivated poliovaccine.
MATERIALS AND METHODS
Chitosan
Two preparations of chitosan were investigated: 0.5% chitosan glutamate solution (300 kDa, 85% deacetylation), and 0.5% suspension of chitosan sulfate micro/nanoparticles. A 1% chitosan glutamate solution in 0.2 M glutamate buffer was added to an equal volume of vaccine (0.5% final chitosan concentration).
To prepare chitosan micro/nanoparticles, hydroperite (urea hydrogen peroxide; cat. # 289132-1006; Sigma–Aldrich, St. Louis, MO) was added to a 1.2% aqueous solution of autoclave-sterilized chitosan acetate to 3% final H2O2 concentration, and the resulting solution was stored for 36 hr at room temperature in a loosely closed vessel. The required volume of 0.5 M Na2SO4 was added to a final concentration of 0.05 M. The solution was mixed vigorously and stored for 30–60 min at room temperature until pellets formed. The obtained microparticles were washed five times with 0.05 M Na2SO4 by centrifugation (5 min at 3,000 rpm). pH of the resulting suspension of micro/nanoparticles was approximately 4.0–4.5. The resulting preparation was dried under vacuum. One percent suspension of micro/nanoparticles was obtained by adding 0.3 volumes of 0.2 M phosphate buffer (pH 7.2) and 0.05 volumes of 3 M NaCl to 1 volume of suspension with acidic pH. The resulting substance had a pH of 6.4, and was mixed with poliovaccine in equal volumes. The final concentration of micro/nanoparticles in the vaccine mixture was 0.5%. Chitosan glutamate and chitosan sulfate were produced in the Laboratory of Genetic Therapy of Viral Diseases (Research Institute for Vaccines and Sera, Moscow, Russia). The size of micro/nanoparticles of chitosan was evaluated with Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK).
Poliovaccine
A trivalent Imovax Polio-inactivated poliovaccine (Sanofi Pasteur, Lyon, France) was used. The vaccine contained 40 U of the D antigen of type 1, 8 U of type 2, and 32 U of type 3 poliovirus strains. In some experiments, inactivated preparations of attenuated poliovirus type 1, 2, or 3 Sabin strains (family Picornaviridae, genus Enterovirus, species Poliovirus) were used as monovaccines. To this end, attenuated poliovirus was propagated in Vero cells, purified by ultracentrifugation, and inactivated with formalin (0.05% at 36°C for 48 hr) that was removed from the preparation using Na2SO3 (0.5%). Initial viral titers before inactivation were 108.0/0.1 ml tissue culture infection dose (TCID50) for the Sabin type 1 strain, and 107.0/0.1 ml TCID50 for the Sabin type 2 and 3 strains.
Immunization
Groups of 4–6 female Balb/c mice (12–14 g) were vaccinated intramuscularly 2 or 3 times with 0.2 ml of vaccine admixed with an equal volume of 1% chitosan glutamate, 1% chitosan sulfate micro/nanoparticle suspension, or saline solution (for the control group). The second vaccination was performed 21 days after the first, and the third vaccination was performed 10 days after the second. After the second and the third vaccinations, mice were fully anesthetized with ether and bled.
Groups of five female Wistar rats (200 g) were vaccinated with undiluted vaccine, or with vaccine diluted 1:10 or 1:100. Immunization was performed according to WHO guidelines for the safe production and quality control of inactivated poliomyelitis vaccines manufactured from wild polioviruses [WHO, 2002]. Vaccine admixed with an equal volume of 1% chitosan sulfate micro/nanoparticle suspension was administered to rats vaccinated once intramuscularly (1 ml per rat). Three weeks after vaccination, rats were fully anesthetized with ether and bled.
All animal experiments were performed in compliance with relevant laws and institutional guidelines, and in accordance with the ethical standards of the Declaration of Helsinki. The Ethical Committee of the Mechnikov Research Institute of Vaccines and Sera approved these experiments.
Neutralizing Antibody Detection
Mouse and rat sera samples were heated at 56°C for 30 min and diluted 1:10. Serial twofold dilutions of 1:10 diluted sera samples were prepared. Neutralization reactions were performed separately with every serotype of the virus when attenuated Sabin strains were used. Reactions were performed in Vero cells according to the protocol recommended by WHO [2004].
RESULTS
Neutralizing Antibodies After Immunization of Mice With Inactivated Poliovirus Sabin Strains
The first series of experiments aimed to investigate neutralizing antibody formation after two-dose and three-dose immunizations of mice with monovalent inactivated preparations of attenuated poliovirus type 1, 2, or 3 strains admixed with chitosan glutamate solution. The data obtained (Table I) show that addition of chitosan glutamate solution (final concentration 0.5%) to the inactivated preparations of poliovirus Sabin strains led to a 32-, 8-, and 16-fold increase in neutralizing antibody titers after the second vaccine dose for Sabin type 1, type 2, and type 3 strains, respectively. Addition of chitosan sulfate micro/nanoparticles as an adjuvant increased neutralizing antibody titers 16-, 4-, and 8-fold for Sabin type 1, 2, and 3 strains, respectively. After three doses of immunization with inactivated Sabin poliovirus strains alone, antibody titers for type 1 and 3 poliovirus strains were virtually the same, and a twofold increase in antibody titers was detected for the type 2 strain. Addition of chitosan glutamate or chitosan sulfate micro/nanoparticle suspension enhanced antibody titers similarly to the two-dose vaccinations above.
| Antigen | Adjuvant | Neutralizing antibody titers | |
|---|---|---|---|
| After two vaccine doses | After three vaccine doses | ||
| Type 1 | Saline solution (control) | 160a | 160 |
| Chitosan glutamate solution | 5,120 | 5,120 | |
| Chitosan sulfate micro/nanoparticles | 2,560 | 2,560 | |
| Type 2 | Saline solution (control) | 320 | 640 |
| Chitosan glutamate solution | 2,560 | 5,120 | |
| Chitosan sulfate micro/nanoparticles | 1,280 | 2,560 | |
| Type 3 | Saline solution (control) | 640 | 640 |
| Chitosan glutamate solution | 10,240 | 10,240 | |
| Chitosan sulfate micro/nanoparticles | 5,120 | 5,120 | |
- Mice were vaccinated intramuscularly two or three times. Inactivated vaccine preparations were admixed with saline solution (control), chitosan glutamate solution, or chitosan sulfate micro/nanoparticle suspension. Neutralization reactions were performed in Vero cells. One hundred tissue culture infection dose (TCID50) of the corresponding Sabin strain were used for neutralization.
- a Neutralizing antibody titers (sera dilutions).
Similar data were obtained when antibody titers were measured by enzyme-linked immunosorbent assay (ELISA; data not shown).
Reducing Antigen Dose
In further experiments, the dose of antigen (inactivated poliovirus Sabin strain type 1) was reduced 2- or 4-fold. The data (Table II) show that, for the usual antigen dose, chitosan glutamate solution or chitosan sulfate micro/nanoparticle suspension induced, respectively, a 16- or 8-fold increase in neutralizing antibody titers compared to the control (vaccine admixed with saline solution). The same was observed when antigen dose was two times lower than usual. It should be noted that even when antigen dose was four times lower than usual, neutralizing antibody titers in mice vaccinated with vaccine admixed with chitosan glutamate solution were higher than or equal to those of mice vaccinated with undiluted vaccine preparation administered without chitosan.
| Antigen dose | Adjuvant | Neutralizing antibody titers |
|---|---|---|
| Undiluted preparation | Saline solution (control) | 160 |
| Chitosan glutamate solution | 2,560 | |
| Chitosan sulfate micro/nanoparticles | 1,280 | |
| Twofold dilution | Saline solution (control) | 160 |
| Chitosan glutamate solution | 2,560 | |
| Chitosan sulfate micro/nanoparticles | 1,280 | |
| Fourfold dilution | Saline solution (control) | 20 |
| Chitosan glutamate solution | 320 | |
| Chitosan sulfate micro/nanoparticles | 160 |
- Mice were vaccinated twice with the preparation of inactivated Sabin poliovirus type 1 in different dilutions (undiluted, 1:2, and 1:4 dilutions). Chitosan glutamate solution or chitosan sulfate micro/nanoparticles were used as adjuvants. Saline solution was admixed with the vaccine in the control group.
Neutralizing Antibodies After Immunization of Mice With a Trivalent Poliovaccine
The next series of experiments examined the adjuvant activity of chitosan glutamate solution and chitosan sulfate micro/nanoparticle with the inactivated trivalent Imovax. The results (Table III) show that the immunogenicity of the investigated vaccine alone after two vaccination doses was relatively low. Nevertheless, when chitosan glutamate solution was used as an adjuvant, neutralizing antibody titers increased 5-fold for type 1, 10-fold for type 2, and 8-fold for type 3 poliovirus strains. When a chitosan sulfate micro/nanoparticle suspension was used, antibody titers increased 10, 40, and 16 times, respectively, for poliovirus type 1, 2, and 3 strains.
| Preparation added to vaccine | Inactivated trivalent Imovax poliovaccine | ||
|---|---|---|---|
| Type 1 | Type 2 | Type 3 | |
| Saline solution (control) | 4a | 4 | 20 |
| Chitosan glutamate solution | 20 | 40 | 160 |
| Chitosan sulfate micro/nanoparticle suspension | 40 | 160 | 320 |
- Mice were vaccinated twice intramuscularly.
- Inactivated vaccine was admixed with saline solution (control), chitosan glutamate solution, or chitosan sulfate micro/nanoparticle suspension. Neutralization reactions were performed in Vero cells. One hundred TCID50 of the Sabin strain of the corresponding serotype were used for neutralization.
- a Neutralizing antibody titers (sera dilutions).
Neutralizing Antibodies After Vaccinating White Rats With Dilutions of the Inactivated Poliovaccine
WHO Guidelines for the safe production and quality control of inactivated poliomyelitis vaccine manufactured from wild polioviruses suggest determining vaccine immunogenicity through one-dose vaccinations of rats [WHO, 2002]. Rats were vaccinated once intramuscularly with the Imovax vaccine. Rats were bled 3 weeks after vaccination, and neutralizing antibody titers were determined separately with Sabin poliovirus strains of three different serotypes. After vaccinating rats with undiluted vaccine admixed with saline solution (control group), neutralizing antibodies to Sabin type 1 poliovirus strain (at titers 1:10 and higher) were detected in only one animal (Table IV). However, when the chitosan sulfate micro/nanoparticle suspension was added to the vaccine, antibodies against poliovirus type 1 could be detected in all animals in the group when undiluted vaccine was used (with a mean titer of 1:46), in three out of five animals vaccinated with vaccine diluted 1:10 (mean titer 1:20), and in one out of five animals vaccinated with vaccine diluted 1:100 (mean titer 1:8). Thus, this chitosan preparation enhanced the immunogenicity of the poliovirus type 1 component of Imovax-inactivated poliovaccine significantly. When poliovirus type 2 was analyzed, all rats in the control group vaccinated with undiluted vaccine admixed with saline solution developed neutralizing antibodies (mean titer 1:104). After adding chitosan sulfate micro/nanoparticle suspension to the vaccine, antibodies were detected in all rats vaccinated with the undiluted vaccine (mean titer 1:264), in three out of five rats vaccinated with vaccine diluted 1:10 (mean titer 1:44), and in four out of five rats vaccinated with vaccine diluted 1:100 (mean titer 1:32). In experiments with type 3 poliovirus, similar data were obtained. Thus, addition of chitosan preparations increased vaccine immunogenicity more than 100 times.
| Virus serotype | Adjuvant used | Vaccine dilutions | Neutralizing antibody titers in sera samples from individual rats | Mean antibody titers in 5 rats | ||||
|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | ||||
| Type 1 | Vaccine + saline solution | Undiluted | <10 | <10 | <10 | 20 | <10 | 4 |
| 1:10 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| 1:100 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| 1:1,000 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| Vaccine + chitosan sulfate micro/nanoparticles | Undiluted | 20 | 10 | 40 | 80 | 80 | 46 | |
| 1:10 | <10 | 10 | 80 | 10 | <10 | 20 | ||
| 1:100 | <10 | <10 | <10 | 40 | <10 | 8 | ||
| 1:1,000 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| Type 2 | Vaccine + saline solution | Undiluted | 40 | 80 | 160 | 160 | 80 | 104 |
| 1:10 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| 1:100 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| 1:1,000 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| Vaccine + chitosan sulfate micro/nanoparticles | Undiluted | 40 | 320 | 320 | 320 | 320 | 264 | |
| 1:10 | <10 | 160 | 20 | 40 | <10 | 44 | ||
| 1:100 | 40 | 80 | 20 | 20 | <10 | 32 | ||
| 1:1,000 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| Type 3 | Vaccine + saline solution | Undiluted | <10 | <10 | <10 | <10 | 40 | 8 |
| 1:10 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| 1:100 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| 1:1,000 | <10 | <10 | <10 | <10 | <10 | <10 | ||
| Vaccine + chitosan sulfate micro/nanoparticles | Undiluted | 40 | 40 | 320 | 320 | 160 | 176 | |
| 1:10 | 40 | 40 | 40 | 80 | 20 | 44 | ||
| 1:100 | 40 | 40 | 40 | 20 | <10 | 24 | ||
| 1:1,000 | <10 | <10 | <10 | <10 | <10 | <10 | ||
- Rats were vaccinated once with different dilutions of Imovax vaccine admixed with saline solution (control) or chitosan sulfate micro/nanoparticles. Animals were bled 21 days after vaccination. Sera samples were diluted 1:10, and serial twofold dilutions were prepared from the 1:10 dilution. Neutralization reactions were performed in Vero cells using 100 TCID50 of the Sabin strain of the corresponding serotype.
DISCUSSION
Many countries in which circulation of wild-type poliovirus strains has virtually ceased had started to use inactivated poliovaccine. The use of this vaccine excludes the possibility of vaccine-associated paralytic poliomyelitis, as well as the problem of live poliovirus vaccine strains interfering with other intestinal viruses. However, inactivated vaccine is much more expensive than live vaccine, and, therefore, changing vaccine type would be difficult in underdeveloped countries.
It has been shown that effective adjuvants used with inactivated vaccines, like the inactivated influenza vaccine, can enhance the immunogenicity and protective efficacy of the vaccine and reduce antigen dose and number of immunizations without affecting the immunogenicity of the preparation [Aguilar and Rodriguez, 2007; Ghendon et al., 2008, 2009; Leroux-Roels et al., 2008].
There are few studies devoted to adjuvant usage for inactivated poliovaccine. One of these studies revealed that adding CpG-oligodezoxynucleotides, alone or with alum, to inactivated vaccine prepared from attenuated Sabin poliovirus strains enhances significantly humoral and cellular immunity. Immunity enhancement was detected even with reduced antigen doses [Yang et al., 2008]. In another study, 1.25-dihydrovitamin D3 was used as an adjuvant. After two-dose or three-dose intraperitoneal vaccinations of mice with an inactivated poliovaccine, a significant enhancement of antiviral IgG and IgA antibody titers was observed in blood and saliva, respectively [Ivanov et al., 2006].
Experiments on mice performed in this study showed that vaccinating these animals with chitosan preparations admixed with inactivated monovalent poliovaccine prepared from poliovirus Sabin type 1, 2, or 3 strains induced a significant increase in neutralizing antibody titers compared to vaccine alone. It should be noted that high antibody titers were detected after only two vaccination doses. High antibody titers were detected even when the antigen dose was four times lower than usual.
Experiments on rats are recommended by WHO as one of the methods for evaluating immunogenicity of inactivated vaccines [WHO, 2002]. It was shown that after one-dose vaccinations of rats with different dilutions of inactivated trivalent Imovax poliovaccine, neutralizing antibodies could be detected in sera of rats vaccinated with 1:100 diluted vaccine admixed with chitosan micro/nanoparticles.
It should be noted that the immunogenicity of different virus serotypes in the Imovax vaccine did not comply with the quantity of D antigen present in the vaccine. This is especially true of serotype 1 D antigen, which proved to be the least immunogenic. This peculiarity could be due to the fact that, in neutralization reactions, the strains used were not the wild strains included in the Imovax vaccine, but the Sabin attenuated strains. Antigenic specificity of Sabin strains may differ slightly from that of the wild-type strains. In experiments with monovaccines prepared from the same Sabin strains that were used in neutralization reactions, antibody titers were relatively high.
Since wild-type poliovirus strains no longer circulate, it was recently discussed that the safer Sabin strains should be used for producing inactivated vaccine instead of virulent strains. This would exclude the possibility of transmission of wild-type strains to the population. However, preliminary data show that inactivated poliovaccines prepared on the base of Sabin strains possess but low immunogenicity. The present study shows that inactivated Sabin strain poliovirus type 1, 2, or 3, admixed with different chitosan derivatives, are highly immunogenic.
It has been shown previously [Ghendon et al., 2009] that adding chitosan glutamate solution to inactivated influenza vaccine administered to mice stimulated proliferation of immune cells containing CD3, CD3/NK, I-AK, and H-2Db markers. It was also demonstrated that adding chitosan preparations to inactivated Sabin poliovirus strains or to Imovax-inactivated poliovaccine stimulated proliferation of CD3, NK, CD3/NK, CD8, and γδT lymphocytes (Markushin et al., 2011).
CONCLUSIONS
The data show that adding chitosan glutamate or chitosan sulfate micro/nanoparticles to inactivated poliovaccine significantly enhances the immunogenicity of this preparation, and induces high neutralizing antibody titers even with reduced antigen doses and fewer immunizations. This would allow reduction of the cost of inactivated poliovaccine and to make the vaccine more available in economically disadvantaged countries.
