MXPA97002336A - Vac compositions - Google Patents

Abstract

The present invention relates to a vaccine composition adapted for mucosal administration, and in particular, for intranasal administration, this composition comprises one or more antigens of influenza viruses, such as the antigens of the influenza virus of the hemagglutinin and neuroaminidase, and an effective amount of an adjuvant of a chitosan. The compositions can be used to immunize a host against influenza infection, this chitosan serves to enhance the stimulation of a mucosal immune protective response of IgA and a systemic immune protective response of the

Description

VACCINE COMPOSITIONS The invention relates to a vaccine composition for intranasal administration, comprising antigens of influenza or influenza viruses and a mucosal adjuvant. The invention also relates to a method for immunizing a patient against influenza, for administering this composition to this patient, and to a method for increasing the immunogenicity of an influenza viral antigen, when administered intranasally, by its administration. joint with the adjuvant. In a further aspect, the invention provides the use of an influenza viral antigen in combination with a chitosan thereby obtaining a vaccine composition for intranasal administration to immunize a patient against influenza. Current influenza vaccines consist of an inactivated whole virus, a fragmented virus (divided vaccines) or purified preparations of the glycoprotein hemagglutinin (HA) subunit and membrane neuroaminidase (NA) vaccines. The hemagglutinin and neuroa inidase are the antigens to which the protective antibody responses are directed, with the hemagglutinin being the main protective antigen. It is estimated that the efficiency of these vaccines, administered parenterally, varies greatly. These vaccines are believed to act primarily by extracting antibodies from the circulating anti-hemagglutinin IgG, which transudate in the lower respiratory tract. M. L. Clemens et al., In J. Clinical Microbiolcgy 24, 157-160, 1986, have previously reported that both secreted IgA and serum IgG participate in immunity to influenza virus. Also, in mice, a number of published studies have shown the importance of respiratory IgA for protection against influenza infection. It has also been found that an advantage of the stimulus of a local IgA response to influenza is that, often, it has a wider specificity than the serum response and thus can provide a cross protection against viruses possessing hemagglutinin molecules. different from those present in the vaccine. Therefore, influenza vaccines that produce both local secretory and serum anti-hemagglutinin responses must provide superior immunity to current vaccines. However, parenteral (intramuscular, subcutaneous, etc.) vaccine is not effective in eliciting local production of antibodies, if there has not been previous mucosal challenge (eg, infection). In order to stimulate the mucosal immune system, the vaccine should be applied topically to a mucosal surface.

The mucosal administration of influenza vaccine will have a number of advantages over traditional parenteral immunization regimens. Mainly among these are the stimulation of the local mucosal immune system of the respiratory tract and the probability that the admission regimens of vaccines will be increased because they will avoid the fear and discomfort associated with the injections. Therefore, a number of attempts have been made to develop mucosal influenza vaccines. However, one drawback is that inactivated vaccines are often poorly immunogenic when they are delivered mucosally. In order to overcome this problem, different approaches to improving the immunogenicity of influenza vaccines administered orally or intranasally have included the use of the B subunit of cholera toxin (CTB) as an adjuvant, the vaccine encapsulation. in a variety of microspheres and the use of live attenuated strains. However, until now no practical resources have been developed to increase the immunogenicity of mucosally administered influenza vaccines. It has now been found by the applicants, that by administering the antigens of influenza hemagglutinin and neuroaminidase, together with a chitosan derivative, in an intranasal formulation, it is possible to achieve good IgG and IgA responses.

Chitosans are derivatives of chitin or poly-N-acetyl-D-glucosamine, in which the largest proportion of N-acetyl groups have been removed through hydrolysis. Chitosans have previously been used in pharmaceutical formulations and are described in EP-A-0460020, as agents that increase mucosal absorption. However, this patent EP-A-0460020 does not disclose or suggest that chitosan can provide an adjuvant effect when administered in a vaccine composition. The present applicants have now found that if chitosan is incorporated into intranasal vaccine compositions containing the neuraminidase antigens and influenza virus hemagglutinin, good local systemic and immunological responses are produced. : Therefore, in a first aspect of the invention, a vaccine composition adapted for mucosal administration is provided; this composition comprises one or more antigens of the influenza virus and an effective adjuvant amount of the chitosan. The vaccine composition is preferably adapted for intranasal administration. Preferably, the composition contains influenza virus antigens of both hemagglutinin and neuraminidase.

In a preferred embodiment, the invention provides a vaccine composition adapted for intranasal administration; this composition comprises the purified antigens of influenza virus, hemagglutinin and neuraminidase; and an effective amount of adjuvant of a chitosan. It is preferred that the purified antigens of hemagglutinin and neuroaminidase be present in the form of rosettes. These rosettes are preferably particles with a radius in the range of 10 to 25 nanometers. It is preferred that the rosettes be substantially free of lipids and, likewise, it is preferred that the preparation of purified antigens of the hemagglutinin and the neuraminidase, as a whole, be substantially free of lipids. An example of a hemagglutinin / neuraminidase preparation, suitable for use in the compositions of the present invention is the product "Fluvirin", manufactured and sold by Evans, Medical Limited, of Speke, Merseyside, United Kingdom, and also see S Renfrey and A. Watts, in Vaccine 1994, Volume 12, Number 8, pages 747-752. The compositions may contain influenza virus antigens from a single viral strain, or from a plurality of strains For example, the composition may contain antigens taken from up to three or more viral strains. The composition may contain antigens from one or more strains of influenza A, together with antigens from one or more strains of influenza B. Preferably, chitosan is soluble in water.Chitosan may, advantageously, be a deacetylated chitin, which is at least 80% deacetylated Preferably, the chitosan is at least deacetylated by 85% and more preferably by 88 to 90% A particular deacetylated chitosan is the "Sea Cure +" chitosan glutamate, available from Protan Biopolymer A / S, from Drammen, Norway In a further aspect, the invention provides a method to immunize a host against infection with influenza, this method comprises administering to a surface ie host mucosal (preferably in intranasal form), a vaccine composition comprising influenza virus antigens, such as the purified antigens of hemagglutinin and neuraminidase, together with an effective amount of a chitosan adjuvant, as defined here prev. In yet another aspect, the invention provides a method of increasing the immunological response of influenza virus antigens, such as purified hemagglutinin and neuraminidase, (for example when administered intranasally), by coadministration with a chitosan, as defined above. The compositions of the invention and, in particular, intranasal compositions, can be formulated as liquids or dry powders, for administration as aerosols or drops. Compositions for administration as nasal drops may contain one or more excipients of the type usually included in these compositions, for example, preservatives, viscosity adjusting agents, lean tonicity adjusting agents, regulating agents and the like. In order to ensure that the chitosan remains soluble in the aqueous medium and also to ensure that the hemagglutinin is not adversely affected by too acidic pH, a solution (eg, for nasal administration) preferably has a pH in the range of 5.5. to 6.5, more preferably a pH of about 6. The present invention also considers the provision of elements for distributing intranasal formulations of influenza virus antigens, such as a purified surface antigen and chitosan. A dispensing device may, for example, take the form of an aerosol delivery system and may be arranged to distribute only a single dose, or a multiplicity of doses. The vaccine will be administered to the patient in an effective amount to stimulate a protective immune response in the patient. For example, the vaccine can be administered to humans in one or more doses, each dose with 1 to 250 micrograms and, more preferably, 5 to 50 micrograms, of protein, prepared from each virus strain. For example, when formulations of hemagglutinin and neuraminidase are formulated from three strains of virus, for example 2 x Influenza A and 1 x Influenza B, a total dose of the viral protein administered may be in the range of 15-150 micrograms. BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the anti-hemagglutinin response of serum IgG in mice immunized with PSA. Drop bar represents the geometric mean titration of four mice. The error bars represent 1 standard error of the average. The value of the cut is 50, which is the lower limit of detection. Figure 2 illustrates the anti-hemagglutinin response of nasal IgA in mice immunized with purified surface antigen (PSA). As with Figure 1, each bar represents the geometric average title of four mice and the error bars represent 1 standard error of the average. Figures 3a and 3b illustrate the determination of the nasal and pulmonary anti-haemagglutinin secretion cells of mice immunized with the purified surface antigen, using the ELISPOT. Figure 3a uses a logarithmic scale, while Figure 3 uses a linear scale.

EXAMPLE 1 Preparation of the purified surface antigen formulation of influenza B / chitosan 1A qutatamate. A 1% solution of chitosan glutamate, a deacetylated medium viscosity chitin, having approximately 11% residual N-acetyl groups, was prepared by dissolving the chitosan glutamate in 0.8% sodium chloride. The grade of the chitosan glutamate used was "Sea Cure + 210", available from Protan Biopolymer A / S, from Drammen, Norway. IB. The purified surface antigen of Influenza (PSA), which contains both Influenza A and Influenza B proteins, commercially available from Evans Medical Limited, of Speke, Merseyside, United Kingdom, under the trademark of "Fluvirin", was elaborated in a phosphate saline solution to give a protein concentration of about 1 mg / ml. PSA consists almost entirely of spike protein hemagglutinin (HA), although it contains some neuraminidase. 1 C. A 1: 1 mixture of the chitosan glutamate solution and the PSA solution was prepared to give an intranasal vaccine composition, containing 0.5% chitosan glutamate (11% acetylated), 0.8% NaCl, 0.1% PSA and phosphate buffer to give a solution with a pH of 6. ID. Control solutions, which contain the same concentrations of PSA, but without chitosan glutamate, and the same concentrations of chitosan glutamate, but without PSA, were also prepared. In the addition, a composition comprising the same concentration of PSA adsorbed on the known adjuvant alhydrogel (aluminum hydroxide) was prepared. The PSA was adsorbed on the alhydrogel overnight at 42c.

EXAMPLE 2 Immunization Studies of Mice 2A. The four compositions, prepared as described in Example 1, were administered to groups of twelve adult mice (6-8 weeks) female BALB / c, as follows: Group 1. 20 μl (10 μl per nostril) of a solution of PSA / chitosan administered intranasally. PSA dose = 10 μg.

Group 2. 20 μl of the PSA administered intranasally (total PSA dose = 10 μg). Group 3. 200 μl of PSA / alhydrogel, administered subcutaneously (PSA dose = 10 μg). Group 4. 20 μl of chitosan solution, administered intranasally. Group 5. 20 μl of PSA (10 μl per nostril) administered daily for three days. (Groups of four mice used for this study). 2B. The immunization procedure was carried out three times at one month intervals, with the exception of Group 5, where the mice were immunized with three successive daily doses. The immunization and the sample regimen are illustrated in Table 1.

TABLE 1 At each sampling point, four mice from each group were bled terminally by cardiac puncture, their heads were removed and their nasal passages were washed with 1 ml of PBS + 1% of bovine serum albumin. Group 5 contained only four mice, so the blood obtained by a puncture in the tail for the first two samples and the nasal washes were performed only at the third sampling point.

Antibody Assays In all trials, the total influenza vaccine (WIV) was used as the antigen. Although the WIV is only ~ 50% HA, the trials were thought to be primarily measuring the anti-HA antibodies. This assumption was confirmed by substituting the PSA (~ 100% HA) for the WIV and some trials were repeated. The results were similar with any antigen. HA specific serum IgG and nasal IgA antibodies were measured by the Enzyme Linked Immunosorbent Assay (ELISA). After correcting for the background, the individual optical density (OD) dilution curves were projected and the titration values were determined. The titration was determined as the dilution of the serum that gives a reading of the DO of 0.2 or the dilution of the nasal wash that gives a reading of the DO of 0.1 As when taking the nasal washes in the third sample, the lymphocytes were isolated from the mucous membranes of the nasal cavity and the lungs and the analysis of the local immunological response by ELISPOT.

Result »l. Respiration of serum anti-HA serum Purified Surface Antigen (Figure 1 and Table 2) As expected, a good serum response was produced by subcutaneous immunization (S \ C) with PSA + Alhydrogel. All animals tested were seroconverted after primary immunization and the geometric mean titre (GMT) was good. The response increased after each pulse, the GMT after the third dose was very high (~ 800,000). In contrast, the response of serum to PSA administered only intranasally was poor: only two of the four mice were seroconverted after the first dose, none of the mice tested had serum HA antibodies after the second dose) (these are mice separated from those tested after the first immunization) and although all the animals tested were seroconverted after the third dose, the GMT was lower than that of the animals that received a dose of PSA + Alhydrogel. Chitosan increased the response of serum PSA administered intranasally; after the third vaccine, the antibody response in the mice that received the PSA + chitosan was 360 times higher than that of the mice that received the PSA only I \ N. The magnitude of the serum response in the PSA + chitosan mice was very similar to that of the di. immunized mice S \ C; in fact, there is no statistical difference in the GMT of the two groups at any point in the sample (Test Students t p> 0.01). Some mice were immunized three times on successive days with PSA administered only intranasally, to study whether this regimen has advantages over the regimen once a month. Although all the mice in this group had detectable serum antibodies 21 days after the first dose and the GMT at this point in time was higher than in mice that had received a single dose of PSA intranasally, the number of seropositive mice decreased during the course of the study, although the GMT did not (in this group, the same mice were sampled at each point of time). At the final time point, the GMT of the mice in the monthly regimen was of the order of magnitude higher than the mice in the daily regimen.

TABLE 2 Anti-HA response of serum IgG in mice immunized with PSA a Positive number / Tested number b Geometric metric titration. 2. Anti-HA response of the nasal wash IgA Purified Surface Antigen (Figure 1 and Table 3) The PSA + Alhydrogel administered subcutaneously was very poor in inducing the nasal IgA response, which is consistent with our previous findings and those of others. PSA alone, given intranasally was also a poor mucosal immunogen, although it was slightly better than subcutaneous immunization in terms of the number of animals that responded. The addition of chitosan greatly boosted the IgA response, although the response was low after the first dose, HA specific IgA could be detected in three of the four mice. The IgA response was greatly boosted in these mice by the second immunizacilon. The final immunization had little effect, in fact, l | - > s average specific IgA levels decreased slightly.

TABLE 3 Anti-HA nasal response of Igß in mice immunized with PSA Post-Dose Group 1 Post-Dose 2 Post-Dose 3 Conversion GMT Conversion GMT Conversion GMT Mucosal Mucosal Mucosal PSA + Chitosarium 3/4 2.26 4/4 282.81 4/4 184.47 PSA l / N 0/4 < 1 1/4 1.20 3/4 2.31 PSA S / C 1/4 < 1 0/4 < 1 2/4 1.32 PSA 3 dosils 0/4 < 1 daily a Positive number / Proven number b Geometric average titration.

Answer »to Chitosan Only Sera and nasal lavage fluid from the control mice immunized with chitosan alone were negative in all trials.

Response of the secretion cells of local anti-HA antibody (ASO in nasal and pulmonary tissues) Lymphocytes of the nasal mucosa and lung parenchyma were isolated from groups of four mice at the third sampling point. were grouped and tested for anti-influenza IgA antibodies, IgG and IgM, which secrete the cells, using the ELISPOT assay. The results are shown in Figures 3a and 3b. The HG-specific antibodies that secrete B cells were detectable in the nasal and lung tissues of all groups. There was a much larger number of these cells in the PSA + chitosan group and this is more evident when the results are projected on a linear scale (Figure 3b). In all cases, except subcutaneously immunized mice, the cells secreting the IgA antibody predominated in the nasal cavity, while either IgG or IgM predominated in the lungs. The magnitude of the response is similar in the lungs and the nose of the PSA + chitosan mice. The aforementioned examples are merely exemplary of the present invention and do not attempt, in any way, to limit the scope of the invention, which is defined only by the appended claims.

Claims (7)

1. CLAIMS 1. A vaccine composition, adapted for nasal administration ,; this composition comprises an antigen of the influenza virus and an effective amount of chitosan adjuvant, this chitosan is a chiatin deacetyleide, which is deacetylated at least in 80%.
2. 2. A vaccine composition, according to claim 1, wherein the chitosan is deacetylated at at least 1 85%.
3. 3. A vaccine composition, according to claim 2, wherein the chitosan is deacetylated at 88 to 90%
4. 4. A vaccine composition, according to any of the preceding claims, which is adapted for intranasal administration .
5. 5. A vaccine composition, according to any of the preceding claims, which contains influenza virus antigens of both hemagglutinin and neuraminidase.
6. 6. A vaccine composition, according to claim 1, which is adapted for nasal administration, this composition comprises influenza virus antigens purified from hemagglutinin and neuraminidase and an effective amount of chitosan adjuvant.
7. 7. A vaccine composition, according to claim 5 or claim 6, wherein the flu of the hemajglutinin and the neuraminidase are present in the form of rosettes, having a radius in the range of 10 to 25 nanomemes. A vaccine composition , according to any of the preceding claims, in which the chitosan is soluble in water. A vaccine composition, according to any of the preceding claims, wherein the composition has a pH in the range of 5.5 to 6.5 LO. A vaccine composition, according to claim 9, wherein the pH is about 6.0. Ll. A pharmaceutical product, comprising a dispensing device, adapted to deliver a composition intranasally, in combination with a vaccine composition, as defined in any of the preceding claims. L2. A pharmaceutical product, according to claim 11, in which the dispensing device is an aerosol delivery system. L3. A method for immunizing a host against infection -in influenza, this method comprises administering to a mucosal host surface, a vaccine composition comprising an influenza virus antigen, such as a mixture of purified antigens of hemagglutinin. and neuraminidase, together with an effective amount of adjuvant of a chitosan, as defined in any. of claims 1 to 12. 14. A method for increasing a mucosal immune protective response of the IgA and a systemic immune response of the IgG, by administration to a surface! mucosal of a patient, a vaccine composition that purchased an influenza virus antigen; such as a mixture of purified antigens of hemagglutinin and neuraminidase, and an effective amount of adjuvant of a chitosan, as defined in any of claims 1 to 12. 15. A method for increasing the immune response of antigens of the influenza, such as hemagglutinin and neuraminidase, when administered intranasally, by administration in conjunction with a chitosan, as defined in any of claims 1 to 12. 16. The use of a chitosan, as defined in any , of the preceding claims, for the manufacture of an intranasal adjuvant composition for enhancing the immunogenicity of influenza virus antigens, such as purified hemagglutinin and neuraminidase, when administered intranasally.