CA2237529C - Vaccine compositions for intranasal administration comprising chitosan and use thereof - Google Patents

Vaccine compositions for intranasal administration comprising chitosan and use thereof Download PDF

Info

Publication number
CA2237529C
CA2237529C CA002237529A CA2237529A CA2237529C CA 2237529 C CA2237529 C CA 2237529C CA 002237529 A CA002237529 A CA 002237529A CA 2237529 A CA2237529 A CA 2237529A CA 2237529 C CA2237529 C CA 2237529C
Authority
CA
Canada
Prior art keywords
antigen
chitosan
vaccine composition
composition according
fha
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CA002237529A
Other languages
French (fr)
Other versions
CA2237529A1 (en
Inventor
Lisbeth Illum
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kyowa Kirin Services Ltd
Original Assignee
Archimedes Development Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Archimedes Development Ltd filed Critical Archimedes Development Ltd
Publication of CA2237529A1 publication Critical patent/CA2237529A1/en
Application granted granted Critical
Publication of CA2237529C publication Critical patent/CA2237529C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/54Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541Mucosal route
    • A61K2039/543Mucosal route intranasal
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55583Polysaccharides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588Adjuvants of undefined constitution

Abstract

There is provided vaccine compositions for intranasal administration, which compositions comprise one or more antigens and an effective adjuvant amount of a chitosan.

Description

Vaccine compositions for intranasaT administration comprising chitosan and use thereof This invention relates to vaccine compositions for intranasal administration, which compositions comprise one or more antigens and an effective adjuvant. The invention further relates to a method of immunising a mammal against diseases by administering such compositions to the mammal, methods of enhancing the immunogenicity of intranasally administered antigens, and uses of antigens in combination with an adjuvant for the manufacture of a vaccine composition for io intranasal administration to immunise a mammal against specific diseases.
Vaccines are preparations of antigenic materials, administered to recipients with a view to enhancing resistance to infection by inducing active immunity to specific microorganisms, for example bacteria or viruses.
Vaccines, which may be as single or mixed component vaccines, are presented in a variety of forms. For example, current influenza vaccines consist of either inactivated whole virus, disrupted virus (split vaccines) or purified preparations of antigenic proteins.

Vaccines are typically administered parenterally via injections. Traditional parenteral immunisation regimes are known to have a number of drawbacks. For example, many individuals possess a natural fear of injections and may experience psychological discomfort as a result.
Furthermore, many individuals find injections physically uncomfortable.
Moreover, parenteral vaccination (e.g. intramuscular, sub-cutaneous etc.) is not an effective means of eliciting local antibody production if there has been no previous local exposure (e.g. by way of infection).

An effective local and/or topical administration regime is therefore
2 desirable.
In the case of some diseases, it would be advantageous to stimulate the mucosal immune system. In order to do this, the vaccine must be applied topically to a mucosal surface. Thus, in certain cases (e.g. in the case of infections of the upper respiratory tract), it would be beneficial to obtain more effective stimulation of the local mucosal immune system of the respiratory tract.

io Accordingly, a number of attempts have been made to develop mucosal vaccines. One drawback, however, is that inactivated vaccines are often poorly immunogenic when given mucosally. In order to overcome this problem, different approaches to improving the immunogenicity of vaccines given orally or intranasally have included the use of adjuvants (see below), encapsulation of the vaccine in a variety of microspheres, and the use of live attenuated strains.

Certain adjuvants have been shown, when co-administered with vaccine antigens, to further boost the effectiveness of vaccine compositions by 2o stimulating the immune response (see e.g. Hibberd et al, Ann. Intern.
Med., 110, 955 (1989)). Examples of adjuvants which have been shown to be effective include interferon alpha, Klebsiella pneumoniae, glycoprotein and interleukin-2.

Chitosans are derivatives of chitin or poly-N-acetyl-D-glucosamine in which the greater proportion of the N-acetyl groups have been removed through hydrolysis.

European Patent Application 460 020 discloses pharmaceutical formulations including chitosans as mucosal absorption enhancers. That
3 the chitosan could provide an adjuvant effect when administered in a vaccine composition is neither disclosed nor suggested.

We have now found surprisingly that, upon intranasal co-administration, chitosan enhances the immune response of antigens and thus provides an adjuvant effect.

Accordingly, in a first aspect of the invention, there is provided a vaccine composition adapted for intranasal administration, which composition comprises antigen and an effective adjuvant amount of a chitosan (hereinafter referred to as "the compositions according to the invention").
The term "effective adjuvant amount" will be well understood by those skilled in the art, and includes an amount of a chitosan which is capable of stimulating the immune response to nasally administered antigens, i.e.
an amount that increases the immune response of a nasally administered antigen composition, as measured in terms of the IgA levels in the nasal washings. Suitably effective increases in IgA levels include by more than 5%, preferably by more than 25%, and in particular by more than 50%, as compared to the same antigen composition without any adjuvant.

Preferred concentrations of the chitosan in the compositions according to the invention are in the range 0.02 to 10%, more preferably 0.1 to 5%
and particularly 0.25 to 2%.

We have found that, by administration of an antigen together with a particular chitosan derivative in an intranasal composition, it is possible ' to achieve an immune (e.g IgG and IgA) response. We have also found that, if a chitosan is incorporated into intranasal vaccine compositions containing an antigen, good systemic and local immune responses are
4 produced. In particular, we have found that the intranasal administration of the compositions according to the invention enhances both a protective IgA mucosal immune response and an IgG systemic immune response.

Thus, the invention further provides a method of enhancing a protective IgA mucosal immune response and an IgG systemic immune response by administering intranasally to a mammal a vaccine composition comprising an antigen and an effective adjuvant amount of a chitosan.

The antigen may be provided as a sub-unit of a cell wall protein or polysaccharide, or as DNA which produces the antigen in the cells after introduction of the DNA (e.g. by transfection). Strictly speaking, the DNA is not itself an "antigen" but it encodes the antigen and is termed antigen herein.

The antigen may further be provided in a purified or an unpurified form.
However, we prefer the antigen to be provided in a purified form.

The invention may be applied to antigens including proteins from 2o pathogens, recombinant proteins, peptides, polysaccharides, glycoproteins, lipopolysaccharides and DNA molecules (polynucleotides).

The following list of antigens is provided by means of illustration and is not meant to be exclusive: influenza virus antigens (such as haemagglutinin and neuraminidase antigens), Bordetella pertussis antigens (such as pertussis toxin, filamentous haemagglutinin, pertactin), human papilloma virus (HPV) antigens, Helicobacter pylori antigens, rabies antigens, tick-borne encephalitis (TBE) antigens, meningococcal antigens (such as capsular polysaccharides of serogroup A, B, C, Y and W-135), tetanus antigens (such as tetanus toxoid), diphtheria antigens (such as diphtheria toxoid), pneumococcal antigens (such as Streptococcus pneumoniae type 3 capsular polysaccharide), tuberculosis antigens, human immunodeficiency virus (HIV) antigens (such as GP-120, GP-160), cholera antigens (such as cholera toxin B subunit), staphylococcal antigen
5 (such as staphylococcal enterotoxin B), shigella antigens (such as shigella polysaccharides), vesicular stomatitis virus antigen (such as vesicular stomatitis virus glycoprotein), cytomegalovirus (CMV) antigens, hepatitis andgens (such as hepatitis A(HAV), B (HBV), C (HCV), D (HDV) and G (HGV) virus antigens), respiratory syncytial virus (RSV) antigens, 1o herpes simplex antigens, or combinations thereof (e.g. combinations of diphtheria, pertussis and tetanus (DPT)). Suitable antigens also include those delivered for induction of tolerance, such as retinal antigens.
Preferred antigens include Bordetella pertussis antigens, meningococcal 1s antigens, tetanus antigens, diphtheria antigens, pneumococcal antigens, tuberculosis antigens and RSV antigens.

According to a further aspect of the invention, we prefer that the antigen is not an influenza virus antigen.

Preferably, the chitosan is water-soluble, and may advantageously be produced from chitin by deacetylation to a degree of greater than 40%, preferably between 50 % and 90 %, and more preferably between 70 % and 95 %, deacetylation.
Particular deacetylated chitosans which may be mentioned include the "Sea Cure+" chitosan glutamate available from Protan Biopolymer A/S, Drammen, Norway.

The molecular weight of the chitosan may be between 10 kD and 500 kD,
6 PCT/GB96/03019 preferably between 50 kD and 300 kD and more preferably between 100 kD and 300 kD.

The compositions according to the invention may be used in the s immunisation of a host against diseases, for example as described in the tests below.

According to a further aspect of the invention, there is provided a method of immunising a host against infection by disease, which method comprises administering intranasally to the host a vaccine composition comprising antigen together with an effective adjuvant amount of a chitosan as hereinbefore defined.

Moreover, according to a further aspect of the invention, there is provided a method of enhancing the immune response of an intranasally administered antigen, which method comprises co-administration of said antigen and a chitosan as hereinbefore defined.

The intranasal compositions according to the invention can be formulated 2o as liquids or dry powders, for administration as aerosols, drops or insufflations.

We prefer that the compositions according to the invention are formulated as dry powders or in the form of microspheres.

Compositions for administration as nasal drops may contain one or more excipients of the type usually included in such compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents and the like.
7 In order to ensure that the chitosan remains soluble in an aqueous medium, and to ensure also that the antigen is not adversely affected by too acidic a pH, a solution for intranasal administration preferably has a pH in the range 5.5 to 6.5, most preferably approximately pH 6.
The present invention also provides a means for dispensing the intranasal compositions of purified surface antigen and chitosan. A dispensing device may, for example, take the form of an aerosol delivery system, and may be arranged to dispense only a single dose, or a multiplicity of doses.
The vaccine will be administered to the patient in an amount effective to stimulate a protective immune response in the patient. For example, the vaccine may be administered to humans in one or more doses, each dose containing 1-250 micrograms and more preferably 2-50 micrograms of protein or polysaccharide antigen prepared from each viral or bacterial strain. For example, where haemagglutinin and neuraminidase preparations are prepared from three virus strains, e.g. 2 x Influenza A
and 1 x Influenza B, a total dose of viral protein administered may be in the range 15-150 micrograms. Where Bordetella pertussis antigens are employed, a total dose of bacterial protein administered as FHA, pertussis toxin (toxoid) or pertactin, either individually or in combination may be in the range 5-150 micrograms.

The invention is illustrated, but in no way limited, by the following examples. The studies described clearly indicate that chitosan used as an adjuvant for mucosal vaccination, has the potential for enhancing both systemic and mucosal humoral responses to a number of antigens.
8 Brief Description of the Figures Figure 1 illustrates the serum IgG anti-haemagglutinin response in mice immunised with purified surface antigen of influenza (PSA). Each bar represents the geometric mean titre of four mice. The error bars represent 1 standard error of the mean. The cut-off value is 50 which is the lower limit of detection.

Figure 2 illustrates the nasal IgA anti-haemagglutinin response in mice immunised with purified surface antigen (PSA). As with Figure 1, each bar represents the geometric mean titre of four mice, and the error bars represent 1 standard error or mean.

Figures 3a and 3b illustrate the determination of nasal and pulmonary anti-haemagglutinin secreting cells of mice immunised with purified surface antigen (PSA), using ELISPOT. Figure 3a uses a log scale whilst Figure 3b uses a linear scale.

Figures 4a, 4b and 4c illustrate the anti-Bordetella pertussis filamentous haemagglutinin (anti-FHA) serum IgG response, the anti-FHA secretory IgA response in lung lavage and the anti-FHA secretory IgA response, respectively, in nasal wash in mice immunised with FHA.

Figure 5a, 5b and 5c illustrate the anti-FHA and anti-Pertussis toxin (toxoid; anti-PT) serum IgG response, lung lavage secretory IgA response and the nasal wash secretory IgA response, respectively, in mice immunised with FHA and PT.
9 Example 1 Preparation of influenza B purified surface antigen/chitosan glutamate composition 1A. A solution of 1% chitosan glutamate, a medium viscosity deacetylated chitin having approximately 11 % residual N-acetyl groups, was prepared by dissolving the chitosan glutamate in 0.8% sodium chloride. The grade of chitosan glutamate used was "Sea Cure} 210", available from Protan Biopolymer A/S, Drammen, Norway.

1B. Influenza purified surface antigen (PSA) containing both Influenza. A
and Influenza B protein, commercially available from Evans Medical Limited, Speke, Merseyside, United Kingdom, under the Trade Mark "Fluvirin", was made up in phosphate buffered saline to give a protein concentration of approximately 1 mg/m1. The PSA consists almost entirely of the spike protein haemagglutinin (HA), although it does contain some neuraminidase.

1C. 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 % NaCI, 0.05 % PSA and phosphate buffer to give a solution pH of 6.

1D. Control solutions containing the same concentrations of PSA but not chitosan glutamate, and the same concentrations of chitosan glutamate but no PSA, were also prepared. In addition, a composition comprising the same concentration of PSA adsorbed on to the known adjuvant Alhydrogel (aluminium hydroxide) was prepared. The PSA was adsorbed on to the Alhydrogel overnight at 40 C.

Example 2 Mice Immunisation Studies 2A. The four compositions prepared as described in Example I were 5 administered to groups of twelve adult (6-8 weeks) female BALB/c mice as follows:

Group 1: 20 l (10 gl per nostril) PSA/chitosan solution administered intranasally. PSA dose = 10 g.
lo Group 2: 20 l PSA administered intranasally (total PSA dose = 10 ltg) =
Group 3: 200 l PSA/Alhydrogel administered subcutaneously (PSA
dose = 10 fcg) Group 4: 20 l chitosan solution administered intranasally.
Group 5: 20 /cl PSA (10 l per nostril) administered daily for three days. (Groups of four mice employed for this study).

2B. The immunisation procedure was carried out three times at monthly intervals, with the exception of Group 5 where the mice were immunised with three successive daily doses. The immunisation and sampling regime is shown in Table 1.

Table 1 Immunisation Day Sample Day 3 57 3 71 +72 Il Immunisation and sampling regime At each sampling point four mice from each group were terminally bled by cardiac puncture, their heads were removed and their nasal passages lavaged with I ml PBS + 1% bovine serum albumin. Group 5 contained four mice only so blood was obtained by tail puncture for the first two samples and nasal washes were only performed at the third sampling point.

Antibody assays io In all assays whole influenza vaccine (WIV) was used as antigen.
Although WIV is only about 50% HA the assays were thought to be measuring primarily anti-HA antibodies. This assumption was confirmed by substituting PSA (100% HA) for WIV and repeating some assays. The results were similar with either antigen. HA-specific serum IgG and nasal IgA antibodies were measured by Enzyme Linked Immunosorbant Assay (ELISA). After correcting for background, the individual optical density (OD) dilution curves were plotted and the titre values determined. The titre was determined as the dilution of serum that gave an OD reading of 0.2 or the dilution of nasal wash that gave an OD reading of 0.1.

As well as taking nasal washes at the third sample, lymphocytes were isolated from the mucous membranes of the nasal cavity and the lungs and the local immune response analysed by ELISPOT.

Results l. Serum anti-HA serum response Purified Surface Antigen (Figure 1 and Table 2):
3o As expected a good serum response was elicited by subcutaneous (SIC) immunisation with PSA + Alhydrogel. All the animals tested had seroconverted after the primary immunisation and the geometric mean titre (GMT) was good. The response increased after each boost, the GMT after the third dose was very high (about 800,000). In contrast the serum response to PSA alone administered intranasally was poor: only two of four mice had seroconverted after the first dose, none of the mice tested had serum HA antibodies after the second dose (these are separate mice from those tested after the first immunisation) and although all animals tested had seroconverted after the third dose the GMT was lower than that of animals receiving one dose of PSA + Alhydrogel. Chitosan enhanced the serum response of intranasally administered PSA; after the third vaccination the antibody response in mice that received PSA + chitosan was 360-fold greater than that of mice receiving PSA alone I/N. The magnitude of the serum response in the PSA + chitosan mice was very similar to that of SIC immunised mice; in fact there was no statistical difference in the GMT's of the two groups at any sampling point (Student's t-Test p > 0.01).

Some mice were immunised three times on successive days with PSA
2o alone administered intranasally to study whether this regime had advantages over the once monthly regime. Although all the mice in this group had detectable serum antibodies 21 days after the first dose and the GMT at this time point was greater than in mice that had received a single dose of PSA intranasally, the number of mice seropositive decreased during the course of the study although the GMT did not (in this group the same mice were sampled at each time point). At the final time point the GMT of the mice on the monthly regime was an order of magnitude greater than mice on the daily regime.

Table 2 Serum IgG anti-HA response in PSA immunised mice Group Post-Dose 1 Post-Dose 2 Post-Dose 3 Sero- GMT Sero- GMT Sero- GMT
Convn.' Convn. Convn.

PSA + 4/4 557 4/4 40504 4/4 653113 Chitosan PSA I/N 2/4 67 0/4 <50 4/4 1818 PSA 3 DaiIy 4/4 182 3/4 229 2/4 180 Doses ' No. positive/No. tested 2. Nasal wash IgA anti-HA response Purified Surface Antigen (Figure 2 and Table 3):
PSA + Alhydrogel given subcutaneously was very poor at inducing a 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 siightly better than subcutaneous immunisation in terms of the number of animals responding. Adding chitosan greatly boosted the IgA response, although the response was low after the first dose, HA-specific IgA could be detected in three out of four mice. The IgA response was boosted greatly in these mice by the second immunisation. The final immunisation had little effect; in fact the mean specific IgA levels had decreased slightly.

Table 3 Nasal IgA anti-HA response in PSA immunised mice Group Post-Dose 1 Post-Dose 2 Post-Dose 3 =
Mucosal GMT Mucosal GMT Mucosal GMT
Convn.a Convn. Convn.
PSA + 3/4 2.26 4/4 282.81 4/4 184.47 Chitosan PSA I/N 0/4 < 1 1/4 1.20 3/4 2.31 PSA S/C 0/4 < 1 0/4 < 1 2/4 1.32 PSA 3 Daily 0/4 <1 Doses ' No. positive/No. tested Responses to Chitosan AIone The sera and nasal lavage fluid from the control mice immunised with chitosan alone were negative in all the assays.

Local anti-HA antibody secreting cell response (ASC) in nasal and pulmonary tissues Lymphocytes were isolated from the nasal mucosa and lung parenchyma of groups of four mice at the third sampling point. Lymphocytes from individual mice were pooled and assayed for cells secreting IgA, IgG and IgM anti-flu antibodies using ELISPOT. The results are shown in Figures 3a and 3b.

B cells secreting HA-specific antibodies were detectable in the nasal and lung tissue of all groups. There were far greater numbers of such cells in the PSA + chitosan group and this is most apparent when the results are plotted on a linear scale (Figure 3b). In all cases, except subcutaneously immunised mice, IgA antibody secreting cells (ASC) predominated in the nasal cavity whereas either IgG or IgM predominated in the lungs. The 5 magnitude of the response is similar in the lungs and nose of PSA +
chitosan mice.

Example 3 Preparation of Bordetella pertussis filamentous haemagglutinin/
io chitosan glutamate composition 3A. A solution of 1% (10 mg/ml) chitosan glutamate (CSN) was prepared as described in Example 1 above.

15 3B Bordetella pertussis filamentous haemagglutinin (FHA), obtained from the National Institute for Biological Sciences, South Mimms, Potters Bar, London, United Kingdom, was made up in phosphate buffered saline (PBS) to give a protein concentration of approximately 1 mg/ml.

2o 3C A 1:1 mixture of the CSN solution and the FHA solution was prepared to give an intranasal vaccine containing 0.5% CSN, 0.5 mg/ml FHA, 0.8% sodium chloride and phosphate buffer, pH 6Ø

3D Control solution for intranasal vaccination containing the same concentration of FHA but not CSN was also prepared. In addition, a mixture containing approximately 0.1 mg/ml of FHA adsorbed onto the known adjuvant Alhydrogei (aluminium hydroxide) was prepared. The FHA was adsorbed on to Alhydrogel by stirring the mixture for 30 minutes at room temperature.

Mice Immunisation Studies 3E The three compositions prepared as described above (sections 3C
and 3D) were administered to groups of 13 adult male Balb/c mice as follows:

Group 1: 20 I (10 Icl per nostril) FHA solution administered intranasally. (FHA dose = 10 Fcg) Group 2: 20 l (10 1 per nostril) FHA/CSN solution administered intranasally. (FHA dose = 10 g and chitosan = 100 99) Group 3: 50 l FHA/Alhydrogel mixture administered subcutaneously.
(FHA dose =5 tcg and Alhydrogel =0.25 mg) Immunisation and sampling regime 3D The immunisation procedure was carried out three times at monthly intervals. The immunisation and sampling regime is shown in Table 4.
3E Four mice from each group at sampling points 1 and 2 and five mice 2o from each group at sampling point 3 were terminally bled by cardiac puncture. After collection of blood samples the animals were killed with an intravenous overdose of pentobarbitone sodium and the nasal passages and the lungs were respectively lavaged with 1 ml PBS containing 1 1 bovine serum albumin.

Ta 1 4 Immunisation Day Sample Day 11 Antibody anal,ysis 3F Anti-FHA IgG antibodies in the serum samples and anti-FHA
secretory IgA antibodies in the nasal wash and lung lavage samples were measured by Enzyme Linked Immunosorbant Assay (ELISA). To compare the antibody response elicited by the different compositions in the individual animals, certain values were assigned to the control positive serum and the control positive lung lavage samples, used for preparing the standard curves during the analysis of the test samples. The control positive serum and the control positive lung lavage were given values of 106 IgG Eq. units/ml and 104 IgA Eq. units/ml respectively.

Results 3G The serum IgG concentrations (Table 5 and Figure 4a) show that the nasally administered FHA/chitosan composition elicited a steady increase in response on days 22, 43 and 70. In comparison, the nasally administered FHA solution also elicited some response by Day 70, but this was only one fifth of the corresponding response produced by FHA/chitosan solution. As expected, the subcutaneously administered FHA/Alhydrogel composition elicited a high secondary response (Day 43).

This response was seven times as much as the corresponding response produced by the FHA/chitosan nasal composition. However, the response to subcutaneous administration decreased by Day 70 and this was only three times as much as the corresponding value for the FHA/chitosan nasal composition. 3H The secretory IgA concentrations in the lung lavages (Table 5, Figure 4b) clearly show that the FHA/chitosan nasal composition elicited the most response both on days 43 and 70 as compared to the responses produced by either the nasally administered FHA solution or the subcutaneously administered FHA/Alhydrogel composition. On Day 43 the response was 100 and 250 times as much as the responses elicited by the nasal FHA solution and the subcutaneous FHA/Alhydrogel composition respectively.

31 The secretory IgA concentrations in the nasal washes (Table 5 and Figure 4c) also show that the EHA/chitosan nasal composition elicited a steady increase in response on days 43 and 70. In comparison the subcutaneous administration produced no response at all, whereas the nasal FHA solution produced some response on Day 70, which was only a third of the response produced by the FHA/chitosan nasal composition.
SUBSTITUTE SHEET (RULE 26) ~

F( Q .y.

a =~ ~

~ ~ N et N N Y1 ~" a ~-1 =!-1 =~~G!b ~+ N

..~.~

8 ~
T N %n M 2'~

G U ~ N +[~ ~v_~j~ i+i~N
A
OR
00 M p~ v cc N
M N d st M <!' 00 p C OQ t+ 1+
~..~ ~~-= b oM0 M C'" ,~~= 4C w ~
N iD a M ~ Q~ ~
~
Ct~ W? '1=~+ 2 a ~ ~q'~ @ y a !~ Q b~ W tw U W ;C ~ b N~ O.~ M_ G FL G
U C M~O f~ CK O w !Q
. C tL." a .~
aa~a 8 I ~~ q ~= ef' ~7' v~ Q' Q~ ~/1 bp G. Q.
s E
cc 0 0 0 ~+[ u"o S. .iZ
u u E a N > cn > > 0 v OA N O 7'.' z Cn E c 43 1 p cn Cn rn SUBSTITUTE SHEET (RULE 26) Example 4 Preparation of Bordetella pertussis filamentous haemagglutinin/pertussis toxin (toxoid) /chitosan glutamate composition s 4A. A solution of 2% (20 mg/ml) chitosan glutamate (CSN) was prepared by dissolving 200 mg chitosan in 10 ml water.

4B Bordetella pertussis filamentous haemagglutinin (FHA), obtained from CAMR, Salisbury, Wiltshire, United Kingdom, was concentrated
10 using an Aquacide II column and made up in phosphate buffered saline (PBS) to give a protein concentration of 267 tcg/ml. Pertussis toxin (PT) , non-toxic mutant, obtained from IRIS, Siena, ItaIy was concentrated using an Aquacide II column and made up in 0.5 M sodium chloride to give a final concentration of 267 ltg/ml. Equal volumes of these two antigen 15 solutions were mixed to give a solution containing each antigen at a final protein concentration of 133 g/ml.

4C A 1:3 mixture of 2% CSN solution and FHA/PT solution was prepared to give an intranasal vaccine containing 0.5% CSN, 20 approximately 100 g/ml FHA, 100 p,g/ml PT, 0.8 % sodium chloride and phosphate buffer.

4D A control solution for intranasal vaccination containing the same concentration of FHA and PT but not CSN was prepared. A negative control solution for intranasal vaccination containing 0.5% CSN but neither FHA nor PT was also prepared by diluting one part 2% CSN
solution with three parts PBS containing 0.0014 M potassium chloride and 0.3185 M sodium chloride. In addition, a mixture containing approximately 10 g/mI FHA and 10 g/ml PT adsorbed onto the known adjuvant Alhydrogel (200 --- g/ml) was prepared. The antigens were SUBSTITUTE SHEET (RULE 26) adsorbed on to Alhydrogel by stirring the mixture for 30 min at room temperature.

Mice Immunisation Studies 4E The four compositions prepared as described above (sections 3C
and 3D) were administered to groups of 10 adult female Balb/c mice as follows:

1o Group 1: 20 I (10 l per nostril) FHA/PT/CSN solution administered intranasally. (FHA dose = 2/.cg, PT dose =2 g, chitosan dose = 100 g) Group 2: 20 l (10 {tl per nostril) PT/FHA solution administered intranasally. (FHA dose = 10 iug, PT dose = 2/cg) Group 3: 20 Al (10 ItI per nostril) CSN solution administered intranasally (CSN dose = 100 -- g) Group 4: 200 l FHA/PT/Alhydrogel mixture administered intraperitoneally. (FHA dose = 2 g, PT dose =2 g, Alhydrogel =40 ,ug) Immunisation and sampling regime 4D The immunisation procedure was carried out twice at a monthly interval. The immunisation and sampling regime is shown in Table 6.
4E Five mice from each group at sampling points 1 and 2 were terminally bled by cardiac puncture. After collection of blood samples the animals were killed with an intravenous overdose of pentobarbitone sodium and the nasal passages and the lungs were respectively lavaged with I ml PBS containing 1% bovine serum albumin.

SUBSTITUTE SHEET (RULE 26) Table 6 Immunisation Day Sample Day Antibody analysis 4F Anti-FHA IgG, anti-PT IgG antibodies in the serum samples and anti-FHA secretory IgA, anti-PT secretory IgA antibodies in the nasal wash and lung lavage samples were measured by Enzyme Linked Immunosorbant Assay (ELISA). The test samples were appropriately diluted with the sample buffer to four different concentrations and analysed in duplicate for each antibody. Titration curves were produced, the y-axis value was fixed at 2.5 times the background value and the interpolation value for each sample was calculated using the Kineticalc programme KC3. The geometric mean titration (GMT) value for each sample was then obtained by calculating the inverse of the interpolation value.

Results 4G The GMT values for both anti-FHA and anti-PT serum IgG
- antibodies (Tables 7, 8 and Figure 5a) show that the intranasally administered FHA/PT/chitosan composition (Group 1) elicited a primary systemic response and a considerably enhanced secondary response. In comparison, the nasally administered FHA/PT solution (Group 2) also elicited some primary systemic response and an enhanced secondary SUBSTITUTE SHEET (RULE 26) response. However the secondary responses to both FHA and PT
produced by Group 1 were approximately 5 fold higher than the corresponding responses produced by Group 2.

4H As expected, the intraperitoneally administered FHA/PT solution (Group 4) elicited high primary and secondary responses to FHA and PT
(Tables 7, 8 and Figure 5A). However, the secondary responses to FHA
and PT were approximately 30 and 10 fold higher than the primary responses respectively. The negative control group serum samples (Group 1o 3) were found to be negative both for anti-FHA and anti-PT IgG
antibodies (Tables 7, 8).

41 The GMT values for secretory IgA antibodies in the lung lavages (Tables 7, 8 and Figure 5b) clearly show that both the nasal compositions FHA/PT/chitosan (Group 1) and FHA/PT (Group 2) elicited a secondary response to FHA and PT but neither antigen produced a primary response.
The secondary responses to PT produced by Group 1 and Group 2 were similar, whereas this response to FHA produced by Group I was approximately 15 fold higher than the response to FHA produced by Group 2.

4J The GMT values for secretory IgA antibodies in the nasal washes (Tables 7, 8 and Figure 5c) also show that both the nasal compositions in Group 1 and Group 2 elicited a secondary response to both the antigens, but only PT produced a small primary response. The secondary response to FHA produced by Group I was almost 15 fold higher than that produced by Group 2, whereas the corresponding response to PT produced by Group 1 was only twice as much as that produced by Group 2.

4K The lung lavage and nasal wash samples from the chitosan control SUBST{TUTE SHEET (RULE 26) group (Group 3) were found to be negative both for anti-FHA and anti-PT
IgA antibodies (Tables 7, 8). The intraperitoneally administered FHA/PT/Alhydrogel (Group 4) was also found to produce no mucosal response (IgA antibodies) to either antigen in the lung lavage or the nasal washes.

SUBSTITUTE SHEET (RULE 26) o 0 0 o q ~ a o 0 O O O O Cn r'~'=, C~i O C
~Y (C
~ > >
=~ b O O O ~~ ~ ~ b b O C O C

Zs =~ ~!1 V'1 Vl V' "'~ Yl V1 Yl Yl > O 0 O O > O O O O
=~ O O O YO O O O O
C. O b F? vl b b (% i/]

~] O O O O Q N O C ~y fn C O C O C G p -t~A ~S?p 00QQJJ w > ~ > ~
O C
Cis O
v~ C C. C c-4 O C
CO = OA C =i"n c~C ,~., pppp 00 q O
D
a'~ h N vl ~t 1-5 =~ N v'1 t/1 4A ~
> O O O O > 0 0 0 O
O O O O O O O b p b O O et N O O ~=
y to) V

K
~D O ~ p h O N p o ~ t/} o O~ IiQai=
~> N %

~ I
Nr o= tn h N Y1 ~t =T' h Yl Y=f ~f1 > O O O O G>i O Q O O
~ p O O O O O O
~ M O st vl ~t O tn Cn C%
O
.G
C = C"
vv O ~

'L7 U r., C V
~7 O. G. 0. [1. 7 O. Q. ~ GL [3. 0 ~ (sa. ~s sa cLi ~2 SUBSTITUTE SHEET (RULE 26) v1 a7 p C
~ ~o o p ~ ~t N O O Cn O. ~D O O
a a ic cn CV C C7 CaQ, 'Y p p z zo tn In In h o a o 0 , o 0 0 0 0 & o ~

-Ir cn o o v, ~n o 0 cn Go q o vi o o '~ " o o a.
VI O O C C p N ~
ar ~,,,~f ~ U V
pp~ ~ a ~~. O C O O U~~. ~ y~

~t V't vl '7 tf~ Vl eP1 M
~ O 0 O O ~ O O O O ~
O O ~ O O O
O O O h et O O
~ t) K
' o ~i; M p Qp~ p%
~
th coM Ifif M ~ ~C tn N
~ p ~ 'je~ N pppp p 00 6i 4 V .~. oo --r cn y Yl Yl V-~ 'it ~ N v1 V1 h ~
O O 0 O =~ 0 0 O Q ~v o ~ o 0 0 tn v1 p tn h p In ~ y A
o c Ki '~ C. O 7d W
OOOCCC~~!f [[[rrr ~~~---rrr(((...rrr~'i M ~ ~
LX CC ~ ~
a (7 C? ~ C7 C~ ~ C7 C~ ~ t? (.7 v~

SUBSTITUTE SHEET (RULE 26)

Claims (26)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A vaccine composition adapted for intranasal adminstration, which composition comprises antigen and an effective adjuvant amount of a chitosan, provided that the antigen is not an influenza virus antigen.
2. A vaccine composition according to claim 1, wherein the antigen is a protein from a pathogen, a recombinant protein, a glycoprotein, a peptide, a polysaccharide, a lipopolysaccharide or a polynucleotide.
3. A vaccine composition according to claim 1, wherein the antigen is a whole cell or a virus.
4. A vaccine composition according to claim 1, wherein the antigen is provided as DNA which encodes the antigen.
5. A vaccine composition according to claim 1 or claim 2, wherein the antigen is a Bordetella pertussis antigen, a meningococcal antigen, a tetanus antigen, a diphtheria antigen, a pneumococcal antigen, a tuberculosis antigen or a RSV
antigen.
6. A vaccine composition according to claim 1 or claim 2, wherein the antigen is one which is delivered for induction of tolerance.
7. A vaccine composition according to any one of claims 1 to 6, wherein the antigen is present in a purified form.
8. A vaccine composition according to any one of claims 1 to 7, wherein the concentration of the chitosan is in the range 0.02 to 10%.
9. A vaccine composition as claimed in claim 8, wherein the concentration of the chitosan is in the range 0.1 to 5%.
10. A vaccine composition as claimed in claim 9, wherein the concentration of the chitosan is in the range 0.25 to 2%.
11. A vaccine composition according to any one of claims 1 to 10, wherein the chitosan is water soluble.
12. A vaccine composition according to any one of claims 1 to 11, wherein the chitosan is produced from chitin by deacetylation to a degree of greater than 40%
deacetylation.
13. A vaccine composition as claimed in claim 12, wherein the degree of deacetylation is between 50% and 90%.
14. A vaccine composition as claimed in claim 12, wherein the degree of deacetylation is between 70% and 95%.
15. A vaccine composition according to any one of claims 1 to 14, wherein the molecular weight of the chitosan is between 10 kD and 500 kD.
16. A vaccine composition as claimed in claim 15, wherein the molecular weight of the chitosan is between 50 kD and 300 kD
17. A vaccine composition as claimed in claim 16, wherein the molecular weight of the chitosan is between 100 kD and 300 kD.
18. A vaccine composition according to any one of claims 1 to 17, wherein the composition has a pH in the range 5.5 to 6.5.
19. A vaccine composition as claimed in claim 18, wherein the pH is approximately pH6.
20. A vaccine composition according to any one of claims 1 to 19, which is formulated as a dry powder or in the form of microspheres.
21. A pharmaceutical product comprising a dispensing device adapted to deliver a composition intranasally, in combination with a vaccine composition as defined in any one of claims 1 to 20.
22. A pharmaceutical product according to claim 21, wherein the dispensing device is an aerosol delivery system.
23. Use of a vaccine composition comprising an antigen together with an effective adjuvant amount of a chitosan in the manufacture of a medicament for immunising a host against infection with a pathogen, provided that the antigen is not an influenza virus antigen.
24. Use of a vaccine composition comprising an antigen and an effective adjuvant amount of a chitosan in the manufacture of a medicament for enhancing a protective IgA mucosal immune response and an IgG systemic response, provided that the antigen is not an influenza virus antigen.
25. Use of a composition comprising an antigen and a chitosan in the manufacture of a medicament for enhancing the immune response of an intranasally administered antigen, provided that the antigen is not an influenza virus antigen.
26. The use of a chitosan for the manufacture of an intranasal adjuvant composition for enhancing the immunogenicity of an intranasally administered antigen, provided that the antigen is not an influenza virus antigen.
CA002237529A 1995-12-07 1996-12-09 Vaccine compositions for intranasal administration comprising chitosan and use thereof Expired - Fee Related CA2237529C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9525083.3A GB9525083D0 (en) 1995-12-07 1995-12-07 Vaccine compositions
GB9525083.3 1995-12-07
PCT/GB1996/003019 WO1997020576A1 (en) 1995-12-07 1996-12-09 Vaccine compositions for intranasal administration comprising chitosan and use thereof

Publications (2)

Publication Number Publication Date
CA2237529A1 CA2237529A1 (en) 1997-06-12
CA2237529C true CA2237529C (en) 2008-10-14

Family

ID=10785093

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002237529A Expired - Fee Related CA2237529C (en) 1995-12-07 1996-12-09 Vaccine compositions for intranasal administration comprising chitosan and use thereof

Country Status (13)

Country Link
US (2) US6391318B1 (en)
EP (1) EP0865297B1 (en)
JP (1) JP4063876B2 (en)
AT (1) ATE227134T1 (en)
CA (1) CA2237529C (en)
DE (1) DE69624726T2 (en)
DK (1) DK0865297T3 (en)
ES (1) ES2186809T3 (en)
GB (2) GB9525083D0 (en)
NO (1) NO323084B1 (en)
NZ (1) NZ323973A (en)
PT (1) PT865297E (en)
WO (1) WO1997020576A1 (en)

Families Citing this family (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9419979D0 (en) * 1994-10-04 1994-11-16 Medeva Holdings Bv Vaccine compositions
US5993852A (en) * 1997-08-29 1999-11-30 Pharmaderm Laboratories Ltd. Biphasic lipid vesicle composition for transdermal administration of an immunogen
GB9725084D0 (en) 1997-11-28 1998-01-28 Medeva Europ Ltd Vaccine compositions
US7022683B1 (en) * 1998-05-13 2006-04-04 Carrington Laboratories, Inc. Pharmacological compositions comprising pectins having high molecular weights and low degrees of methoxylation
CA2366908A1 (en) * 1999-03-24 2000-09-28 The Secretary Of State For Defence Vaccine composition
GB9914412D0 (en) * 1999-06-22 1999-08-18 Worrall Eric E Method for the preservation of viruses,bacteria and biomolecules
US20030206958A1 (en) * 2000-12-22 2003-11-06 Cattaneo Maurizio V. Chitosan biopolymer for the topical delivery of active agents
WO2001098206A1 (en) * 2000-06-22 2001-12-27 Rxkinetix, Inc. Delivery vehicle composition and methods for delivering antigens and other drugs
WO2002034287A2 (en) * 2000-10-27 2002-05-02 Pharmexa A/S Therapeutic vaccine formulations containing chitosan
US7494669B2 (en) * 2001-02-28 2009-02-24 Carrington Laboratories, Inc. Delivery of physiological agents with in-situ gels comprising anionic polysaccharides
US6777000B2 (en) * 2001-02-28 2004-08-17 Carrington Laboratories, Inc. In-situ gel formation of pectin
JP2005501845A (en) 2001-08-16 2005-01-20 メディカル リサーチ カウンシル Chitin microparticles and their medical uses
CA2466020A1 (en) 2001-09-28 2003-04-10 University Of South Florida Rsv gene expression vaccine
CA2466982A1 (en) * 2001-11-19 2003-05-30 Becton, Dickinson And Company Pharmaceutical compositions in particulate form
DE60328481D1 (en) * 2002-05-14 2009-09-03 Novartis Vaccines & Diagnostic SLEEP-CAPACITIVE VACCINE CONTAINING THE ADJUVANZ CHITOSAN AND MENIGOKOKKENANTIGENE
BR0310042A (en) * 2002-05-14 2005-04-05 Chiron Srl Mucosal combination vaccines for bacterial meningitis
GB0302218D0 (en) 2003-01-30 2003-03-05 Chiron Sri Vaccine formulation & Mucosal delivery
EP1539208A2 (en) * 2002-06-28 2005-06-15 Nastech Pharmaceutical Company Inc. Compositions and methods for modulating physiology of epithelial junctional adhesion molecules for enhanced mucosal delivery of therapeutic compounds
JP2006516609A (en) * 2003-01-30 2006-07-06 カイロン ソチエタ ア レスポンサビリタ リミタータ Mucosal meningococcal vaccine
CA2514667C (en) * 2003-01-30 2013-01-08 Chiron Corporation Adjuvanted influenza vaccine
US20070116767A1 (en) 2003-02-14 2007-05-24 Mohapatra Shyam S Chitosan-microparticles for ifn gene delivery
WO2004076664A2 (en) * 2003-02-21 2004-09-10 University Of South Florida Vectors for regulating gene expression
CN100575955C (en) * 2003-04-30 2009-12-30 纳斯泰克制药公司 Close albumen do not express label as metastases
US20040248804A1 (en) * 2003-05-01 2004-12-09 Khurshid Iqbal Nasal administration of the LH-RH analog Leuprolide
ES2425221T3 (en) * 2003-05-30 2013-10-14 Amylin Pharmaceuticals, Llc New methods and compositions for enhanced transmucosal delivery of peptides and proteins
GB0315632D0 (en) * 2003-07-04 2003-08-13 West Pharm Serv Drug Res Ltd Pharmaceutical formulations
WO2005056021A1 (en) 2003-12-04 2005-06-23 University Of South Florida Polynucleotides for reducing respiratory syncytial virus gene expression
US7968122B2 (en) * 2003-12-10 2011-06-28 Adventrx Pharmaceuticals, Inc. Anti-viral pharmaceutical compositions
US20050129679A1 (en) * 2003-12-15 2005-06-16 Nastech Pharmaceutical Company Inc. Method for opening tight junctions
US7740883B2 (en) * 2004-03-28 2010-06-22 University Of Debrecen Nanoparticles from chitosan
JP2007536259A (en) * 2004-05-06 2007-12-13 イヴレア ファーマスーティカルズ インコーポレイテッド Particles for active drug delivery
US20090069226A1 (en) * 2004-05-28 2009-03-12 Amylin Pharmaceuticals, Inc. Transmucosal delivery of peptides and proteins
US20050283004A1 (en) * 2004-06-18 2005-12-22 Hopax Chemicals Manufacturing Co., Ltd. Alkylsulfonated polyaminosaccharides
BRPI0515301A (en) * 2004-09-15 2008-07-15 Otsuka Pharma Co Ltd permeate composition and method of perfecting permeate absorption
WO2007086923A2 (en) * 2005-05-23 2007-08-02 University Of South Florida Controlled and sustained gene transfer mediated by thiol-modified polymers
US20080184618A1 (en) * 2005-08-03 2008-08-07 Amcol International Virus-Interacting Layered Phyllosilicates and Methods of Use
US20100272769A1 (en) * 2005-08-03 2010-10-28 Amcol International Virus-, Bacteria-, and Fungi-Interacting Layered Phyllosilicates and Methods of Use
US20070031512A1 (en) * 2005-08-03 2007-02-08 Amcol International Corporation Virus-interacting layered phyllosilicates and methods of inactivating viruses
US8536324B2 (en) * 2005-10-21 2013-09-17 University Of South Florida Method of drug delivery by carbon nanotube-chitosan nanocomplexes
WO2007059605A1 (en) * 2005-11-04 2007-05-31 Bio Syntech Canada Inc. Composition and method for efficient delivery of nucleic acids to cells using chitosan
CA2635603C (en) 2005-11-30 2016-01-19 Endo Pharmaceuticals Inc. Treatment of xerostomia
CN102164618A (en) * 2006-03-30 2011-08-24 恩根尼公司 Non-viral compositions and methods for transfecting gut cells in vivo
AU2007262845B2 (en) 2006-06-20 2013-07-25 Mucovax Inc. Compositions comprising chitin microparticles and their medical uses
PL2601970T3 (en) 2006-09-29 2017-07-31 Takeda Vaccines, Inc. Norovirus vaccine formulations
EP2112923A1 (en) 2007-01-22 2009-11-04 Targacept Inc. Intranasal, buccal, and sublingual administration of metanicotine analogs
US20080182819A1 (en) * 2007-01-29 2008-07-31 Act Iii Licensing, Llc Chitosan compositions and methods for using chitosan compositions in the treatment of health disorders
WO2008147807A2 (en) 2007-05-23 2008-12-04 Amcol International Corporation Cholesterol-interacting layered phyllosilicates and methods of reducing hypercholesteremia in a mammal
JP2010539192A (en) 2007-09-18 2010-12-16 リゴサイト ファーマスーティカルズ,インコーポレイテッド Methods for conferring a protective immune response against norovirus
US20100266636A1 (en) 2007-09-18 2010-10-21 Ligocyte Pharmaceuticals, Inc. Method of conferring a protective immune response to norovirus
BRPI0905725B8 (en) * 2008-01-11 2021-05-25 Univ Emory vaccine and pharmaceutical packaging
WO2009108867A1 (en) 2008-02-27 2009-09-03 Amcol International Corporation Methods of treating cardiovascular disorders associated with atherosclerosis
CN104911154A (en) 2008-08-08 2015-09-16 武田疫苗股份有限公司 Virus-like particles comprising composite capsid amino acid sequences for enhanced cross reactivity
CN102405058B (en) * 2009-04-15 2015-11-25 法国诗华大药厂 For restructuring avian herpetoviruses carrier and the vaccine of immunity inoculation aquatic bird species
ES2926487T3 (en) 2011-07-11 2022-10-26 Takeda Vaccines Inc Parenteral Norovirus Vaccine Formulations
DK2841097T3 (en) 2012-04-24 2022-11-28 Ohio State Innovation Foundation COMPOSITIONS AND METHODS FOR THE TREATMENT AND PREVENTION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
US10279028B2 (en) 2012-04-24 2019-05-07 Ohio State Innovation Foundation Compositions and methods for treating and preventing porcine reproductive and respiratory syndrome
CN108715607B (en) * 2018-04-12 2021-12-10 华南农业大学 Recombinant cholera toxin B subunit protein, PEDV inactivated vaccine, preparation and application
CN109172817B (en) * 2018-08-16 2021-10-26 华中农业大学 Preparation method and application of chitosan microspheres of attenuated live vaccine of porcine epidemic diarrhea virus
EP3698773A1 (en) 2019-02-21 2020-08-26 Università degli Studi di Parma Composition and manufacturing of powders containing nanoadjuvants for mucosal vaccination

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1261264A (en) * 1984-11-29 1989-09-26 Shigeo Suzuki Immunopotentiating agents and method
US4950740A (en) * 1987-03-17 1990-08-21 Cetus Corporation Recombinant diphtheria vaccines
GB8904370D0 (en) 1989-02-25 1989-04-12 Cosmas Damian Ltd Liquid delivery compositions
US5554388A (en) * 1989-02-25 1996-09-10 Danbiosyst Uk Limited Systemic drug delivery compositions comprising a polycationi substance
GB9202464D0 (en) * 1992-02-05 1992-03-18 Danbiosyst Uk Composition for nasal administration
US5912000A (en) * 1994-09-23 1999-06-15 Zonagen, Inc. Chitosan induced immunopotentiation
JP3365635B2 (en) 1994-09-23 2003-01-14 ゾナジェン,インコーポレイテッド Chitosan-induced immune enhancement
GB9419979D0 (en) 1994-10-04 1994-11-16 Medeva Holdings Bv Vaccine compositions
JP2001523216A (en) * 1995-06-26 2001-11-20 リサーチ.トライアングル.ファーマシューティカルズ Novel adjuvant composition and vaccine formulation comprising the same
GB9522351D0 (en) * 1995-11-01 1996-01-03 Medeva Holdings Bv Vaccine compositions

Also Published As

Publication number Publication date
NO323084B1 (en) 2006-12-27
AU1102597A (en) 1997-06-27
GB9525083D0 (en) 1996-02-07
NO982497L (en) 1998-06-02
EP0865297B1 (en) 2002-11-06
CA2237529A1 (en) 1997-06-12
GB2322801A (en) 1998-09-09
US6391318B1 (en) 2002-05-21
PT865297E (en) 2003-03-31
EP0865297A1 (en) 1998-09-23
ATE227134T1 (en) 2002-11-15
GB9811810D0 (en) 1998-07-29
US20030039665A1 (en) 2003-02-27
JP2000501412A (en) 2000-02-08
DK0865297T3 (en) 2003-03-03
NZ323973A (en) 2004-12-24
DE69624726T2 (en) 2003-09-18
AU705452B2 (en) 1999-05-20
US7323183B2 (en) 2008-01-29
NO982497D0 (en) 1998-06-02
ES2186809T3 (en) 2003-05-16
JP4063876B2 (en) 2008-03-19
WO1997020576A1 (en) 1997-06-12
GB2322801B (en) 2000-01-19
DE69624726D1 (en) 2002-12-12

Similar Documents

Publication Publication Date Title
CA2237529C (en) Vaccine compositions for intranasal administration comprising chitosan and use thereof
CA2201598C (en) Vaccine compositions
JP4137640B2 (en) Novel proteosome-liposaccharide vaccine adjuvant
CA2236538C (en) Influenza vaccine compositions
MXPA97002336A (en) Vac compositions
JP2003526676A (en) Adjuvants for vaccines
JP2023091085A (en) mucosal adjuvant
JP2002529427A (en) Pharmaceutical agent formulations and administration devices
AU705452C (en) Vaccine compositions for intranasal administration comprising chitosan and use thereof
EP0721782B1 (en) Influenza vaccine containing pertussis toxin
AU754675B2 (en) Influenza vaccine compositions

Legal Events

Date Code Title Description
EEER Examination request
MKLA Lapsed

Effective date: 20161209