Chitosan as an adjuvant for parenterally administered inactivated influenza vaccines
- First Online:
- Cite this article as:
- Ghendon, Y., Markushin, S., Krivtsov, G. et al. Arch Virol (2008) 153: 831. doi:10.1007/s00705-008-0047-4
- 172 Views
The addition of 0.5% of a chitosan derivative to inactivated influenza vaccines injected parenterally resulted in a four or six to tenfold increase in antibody titres after a single-dose or two-dose intramuscular immunization of mice, respectively, in comparison with antibody titres after immunization without chitosan. Chitosan-adjuvanted vaccines enhanced antibody titers against drift variants of A- and B-type human influenza viruses four to six times compared with the vaccines without chitosan. Inactivated avian influenza virus A/H5N2 admixed with chitosan, when administered to mice challenged afterwards with the same virus, showed higher immunogenicity and protective efficacy compared with the antigen without chitosan.
Inactivated influenza vaccines administered parenterally have been successfully used for influenza prophylaxis for more than 50 years [2, 20]. However, the majority of the existing inactivated vaccines are less effective in the elderly [6, 14] and are unable to protect the vaccines from influenza virus drift variants [1, 3].
Aluminium salts were previously used as adjuvants in order to enhance the immunogenicity of inactivated influenza vaccines. However, the only adjuvant that has been used recently for influenza vaccines is MF-59. MF-59-adjuvanted vaccine has proved to be more immunogenic compared with a nonadjuvanted vaccine in the elderly. However, addition of MF-59 increased the reactogenicity of the vaccine [16, 17].
Some recent reports describe the cationic polysaccharide chitosan as an effective mucosa adhesive adjuvant for a number of intranasally administered inactivated viral vaccines, including influenza vaccine .
In this study, we investigated a chitosan derivative as an adjuvant for inactivated influenza vaccines administered parenterally. The results obtained confirm that this preparation significantly enhances the immunogenicity of inactivated influenza vaccines and induces the production of antibodies against influenza virus drift variants.
Materials and methods
We used trivalent split vaccines Vaxigrip for epidemical seasons 2003/04 and 2005/06, MG subunit vaccines (Russia) for the 2005/06 season and subunit vaccine Influvac for the 2005/06 season. Purified and inactivated strain A/Mallard duck/Pennsylvania/1021/84(H5N2) was used in experiments with avian influenza virus. The latter was adapted to mice and kindly provided by Dr. Smirnov .
We used influenza virus strain A/New Caledonia/20/99 as an A/H1N1 strain recommended by WHO for the 2003/04, 2004/05 and 2005/06 influenza seasons; strain A/Panama/2007/99 (analogous to A/Moscow/10/99 strain), recommended by WHO for the 2003/04 influenza season, strain A/Wyoming/3/2003 (analogous to A/Fujian/411/2002 strain), recommended by WHO for the 2004/05 influenza season, and strain A/New York/55/2004 (analogous to A/California/7/2003 strain), recommended by WHO for the 2005/06 influenza season, as A/H3N2 strains. Strain B/Beijing/243/97 (analogous to B/Hong Kong/330/2001), recommended by WHO for the 2003/04 influenza season and B/Jangsu/10/2003 strain (analogous to B/Shanghai/361/2002), recommended by WHO for the 2004/05 and 2005/06 influenza seasons, were used as influenza B strains. In addition, a mouse-adapted avian influenza virus, A/Mallard duck/Pennsylvania/1021/84(H5N2), was used.
We used a 1% solution of a chitosan derivative (deacetylated by 85%) in 0.2 M glutamate buffer.
Equal quantities of 1% chitosan in glutamate buffer solution or of glutamate buffer solution alone were added to inactivated influenza vaccines (split and subunit). Each experimental group consisted of four to six Balb/c mice (6–8 weeks old). Mice were vaccinated intramuscularly with 0.2 ml of a preparation containing 3 μg of each vaccine component and 0.5% chitosan or buffer. In the experiments with inactivated avian H5N2 influenza virus, the preparation contained 3 μg hemagglutinin in 0.2 ml and 0.5% chitosan or buffer. Mice were vaccinated once or twice intramuscularly. After primary vaccine administration, mice received a booster immunisation dose with the same composition of antigens on day 21. Mice were anesthetized with ether and bled on the twenty-first day after the first vaccination and 10 days after the second one. In experiments with the mouse-adapted inactivated avian influenza virus, mice were lightly anesthetized with ether and challenged intranasally with 50 μl of different LD50 doses of H5N2 virus on the twenty-first day after the first and on the tenth day after the second vaccination. The survival rates of these mice were observed for 10 days following vaccination.
Analysis of hemagglutination-inhibiting and neutralizing antibodies
Serum was heat-inactivated for 30 min at 56°C. Initial serum dilutions were 1:10, and then twofold dilutions of each sample were made. Serologic responses were measured according to standard techniques . In the neutralization test, twofold dilutions of the initially diluted samples were prepared. Each twofold dilution (25 μl) was incubated with 100 EID50 of virus (25 μl) for 1 h at room temperature (24°C). Following incubation, two chick embryos were inoculated with each dilution of every sample and were incubated at 36°C for 48 h. The allantoic fluid was tested for hemagglutination with 0.5% chicken erythrocytes.
Three to four independent experiments were performed and the data were analyzed for statistical significance by Student test. Results with P < 0.05 were considered significant.
Analysis of different chitosan concentrations
Influence of chitosan on production of antibodies against three serotypes of influenza viruses
Antibody titres did not increase in the experiment in which vaccine and chitosan solution were injected into different limbs of the mice (Fig. 2).
Antibody response after single-dose vaccination
Antibodies against influenza virus drift variants
In the experiments with Vaxigrip vaccine, recommended for the 2005/06 season, after vaccination without chitosan, titres of hemagglutination-inhibiting antibodies against drift variants A/Wyoming/3/2003 (recommended for the 2004/05 season) and against A/Panama/2007/99 (2003/04) were three and four times lower, respectively, compared with the A/H3N2 vaccine strain (A/New York/55/2004). Titres against influenza B drift variant virus B/Beijing/243/97 (2003/04 season) were three times lower than those against the vaccine influenza B strain (B/Jangsu/10/2003) (P < 0.05). In the neutralization test, antibody titres against A/H3N2 and influenza type B drift variants were three times lower than those obtained with the original vaccine virus (P < 0.05). However, sera of mice vaccinated with chitosan-containing vaccine revealed four to six and four-fold increases in hemagglutination-inhibiting antibody titres against A/H3N2 and type B influenza virus drift variants, respectively, compared with the vaccine administered without chitosan (P < 0.05). Neutralizing antibody titres against drift variants of A/H3N2 and type B influenza viruses were eight and four times higher, respectively, in comparison with murine sera obtained from mice vaccinated without chitosan (P < 0.05). The data obtained show that chitosan-adjuvanted inactivated influenza vaccines in comparison with vaccines without chitosan enhanced antibody titres, not only to the homologous strain but also to drift variants of influenza virus.
Experiments with avian influenza virus A/H5N2 (A/Mallard duck/Pennsylvania/10218/84)
Protection of mice vaccinated with inactivated avian influenza virus A/H5N2 with and without chitosan
Groups of mice
Challenge after the first vaccination infection dose (LD50)
Challenge after the second vaccination infection dose (LD50)
Vaccination with 0.5% chitosan
Vaccination without chitosan
Nonvaccinated mice (control)
Because of the threat of an influenza pandemic, there is growing interest in improving immunogenicity and protective effectiveness of the existing influenza vaccines. WHO noted that an increase in the quantity of antigens in a vaccine is nonrealistic because it would conflict with a rapid increase in the quantities of produced vaccines. Therefore, WHO recommends the investigation of new adjuvants capable of enhancing vaccine immunogenicity and effectiveness without increasing the antigen dose .
One of the first adjuvants used for inactivated influenza vaccines was aluminium salts. However, a number of studies did not reveal any significant advantages of vaccines with such an adjuvant when human vaccination is considered [18, 20]. The interest in this adjuvant has recently increased, especially in the case of the single-dose vaccination [7, 13]. At present, an MF-59-adjuvanted vaccine is being produced. It is characterized by high immunogenicity, especially in elderly subjects [16, 17].
Currently, mucosa adhesive adjuvants are the subject of great interest due to their ability to induce a local as well as a systemic antibody response against influenza when admixed with inactivated influenza vaccines and administered intranasally. Chitosan is an adjuvant from this group. Experiments performed on animals confirm that chitosan-adjuvanted intranasally administered vaccines induce immunity to influenza, whooping cough and diphtheria . It should be noted, however, that when mass vaccination of people with an intranasal influenza vaccine adjuvanted with mutant E. coli toxin was performed  there were some complications, such as Bells palsy syndrome .
Our investigations were aimed at studying chitosan as an adjuvant for inactivated influenza vaccines administered parenterally. The data obtained show that chitosan significantly enhances the immunogenicity of the vaccines administered to mice intramuscularly. Our results revealed that the use of chitosan induces relatively high antibody titres even after a single-dose vaccination.
It is known that protection of inactivated influenza vaccines against human influenza virus drift variants is very weak [1, 3, 11]. In our study, antibody titres against influenza virus drift variants after parenteral vaccination with vaccines admixed with chitosan were much higher than after vaccination without chitosan. We have no data that chitosan acts to broaden the spectrum of antibody reactivity. But taking into account that chitosan-adjuvanted vaccine enhances the antibody titre to drift variants, it is possible to suggest that protection against influenza virus drift variants could be better with chitosan-adjuvanted vaccines than with vaccines without chitosan.
Recently, the possibility of a pandemic caused by avian influenza (e.g., with A/H5 virus) has been a subject of great anxiety. The results obtained in the present study with the mouse-adapted A/H5N2 duck influenza virus show that vaccination of mice with chitosan-adjuvanted inactivated virus enhances immunogenicity and protective effectiveness of the inactivated virus.
The data obtained have allowed us to assume that the chitosan derivative investigated in the present study can become a promising adjuvant for parenterally administered inactivated influenza vaccines.
The authors are deeply grateful to Dr. Smirnov, who kindly provided us with the mouse-adapted avian influenza virus A/H5N2 strain that was needed for our investigations.