US20150297714A1 - Novel mucosal adjuvants and delivery systems - Google Patents

Novel mucosal adjuvants and delivery systems Download PDF

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Publication number
US20150297714A1
US20150297714A1 US14/439,536 US201314439536A US2015297714A1 US 20150297714 A1 US20150297714 A1 US 20150297714A1 US 201314439536 A US201314439536 A US 201314439536A US 2015297714 A1 US2015297714 A1 US 2015297714A1
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Prior art keywords
chitosan
vaccine
antigen
adjuvant
canceled
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US14/439,536
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Inventor
Billy M. Hargis
Neil R. Pumford
Marion Morgan
Srichaitanya Shivaramaiah
Guillermo Tellez
Amanda Wolfenden
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University of Arkansas
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University of Arkansas
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Priority to US14/439,536 priority Critical patent/US20150297714A1/en
Assigned to THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS reassignment THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HARGIS, BILLY M., MORGAN, Marion, PUMFORD, NEIL R., TELLEZ, GUILLERMO, WOLFENDEN, Amanda, SHIVARAMAIAH, Srichaitanya
Publication of US20150297714A1 publication Critical patent/US20150297714A1/en
Abandoned legal-status Critical Current

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Definitions

  • An adjuvant is a pharmacological or immunological agent that modifies the effect of other agents, such as a drug or vaccine.
  • Adjuvants are often included in vaccines to enhance the recipient's immune response to a supplied antigen, while keeping the injected foreign, material to a minimum.
  • Adjuvants do not in themselves confer immunity. Adjuvants can act in various ways in presenting an antigen to the immune system. Adjuvants can act as a depot for the antigen, presenting the antigen over a long period of time, thus maximizing the immune response before the body clears the antigen. Examples of depot type adjuvants are oil emulsions, like Freund's adjuvant. Adjuvants can also act as an irritant which causes the body to recruit and amplify its immune response.
  • the tetanus, diphtheria, and pertussis vaccine for example, contains minute quantities of toxins produced by each of the target bacteria, but also contains aluminum hydroxide, Aluminum salts are common adjuvants in vaccines sold in the United States and have been used in vaccines for over 70 years.
  • Chitosan is a linear polysaccharide composed of randomly distributed ⁇ -(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is made by treating shrimp and other crustacean shells with the alkali sodium hydroxide. Chitosan has been used as a carrier for both oral and subcutaneous vaccines with some success.
  • novel chitosan-based adjuvant formulations which are shown to perform better as adjuvants than the traditionally used Alum adjuvants.
  • the chitosan-based adjuvants provided herein were effective at stimulating an IgA response.
  • adjuvants comprising the adjuvants, methods of making the adjuvants and methods of using the adjuvants and vaccine formulations.
  • chitosan and an antigen may be cross-linked using an aldehyde.
  • a composition comprising 0.5% to 2% of an aldehyde cross-linked chitosan and an antigen is provided. The final concentration of aldehyde in a vaccine composition is less than 0.5%.
  • an adjuvant composition comprising a carbohydrate linked to chitosan to form a Schiff base.
  • the adjuvant may be combined with an antigen.
  • the carbohydrate may be mannose.
  • Vaccine formulations may include the adjuvants provided herein and an antigen.
  • the antigens may be proteins or microbial in nature, suitable microbes include bacteria, yeast, or other fungi, eukaryotic parasites and viruses and may be attenuated, recombinant, killed or otherwise inactivated.
  • chitosan is dissolved in a solution of acetic acid, and an antigen is added to the dissolved chitosan. Finally the antigen and chitosan are combined with an aldehyde such that the final concentration of the aldehyde is between 0.02% and 0.5%. Tris may be added to the adjuvant to quench tree aldehydes and result in a more stable adjuvant.
  • methods of enhancing the immune response of a subject to an antigen include administering a vaccine formulation comprising an antigen and a chitosan-based adjuvant disclosed herein to the subject.
  • the chitosan-based adjuvant may be an aldehyde cross-linked chitosan or a carbohydrate-linked chitosan.
  • FIG. 1 is a graph showing the anti- ⁇ -galactosidase IgG antibody response in turkeys following primary vaccination and boost with the indicated vaccine-adjuvant formulations. Different letters indicate significant differences (p ⁇ 0.05).
  • FIG. 2 is a graph showing the Clostridium septicum IgG antibody response in turkeys following primary vaccination and boost with the indicated vaccine-adjuvant formulations. Different letters indicate significant differences (p ⁇ 0.05).
  • FIG. 3 is a set of graphs showing the IgG ( FIG. 3A ) and IgA ( FIG. 3B ) antibody response in chickens at various time points after vaccination and boost with the indicated Bacillus -vectored avian influenza vaccine-adjuvant formulations.
  • FIG. 4 is a graph showing the IgG antibody levels against Salmonella following primary vaccination and boost with the indicated vaccine-adjuvant formulations measured using a competitive ELISA. Different letters indicate significant differences (p ⁇ 0.05).
  • FIG. 5 is a graph showing the IgA antibody levels against Salmonella following primary vaccination and boost with the indicated vaccine-adjuvant formulations measured using a competitive ELISA, Different letters indicate significant differences (p ⁇ 0.05).
  • FIG. 6 is a set of graphs showing the IgG ( FIG. 6A ) and IgA ( FIG. 6B ) antibody levels against Salmonella following primary vaccination and boost with the indicated vaccine-adjuvant formulations measured using a competitive ELISA. Different letters indicate significant differences (p ⁇ 0 . 05 ).
  • FIG. 7 is a graph showing the percent recovery of Salmonella in the liver and spleen (L/S) or cecal tonsils (CT) on day 22 after primary vaccination (day 3 after challenge).
  • the vaccination protocol was the same as that used in FIG. 6 and a * indicates P ⁇ 0.05.
  • FIG. 8 is a graph showing the IgA antibody level against Salmonella at Day 22 following primary vaccination and boost (Day 12) with the indicated vaccine-adjuvant formulations via the indicated routes of administration as measured using a competitive ELISA. Different letters indicate significant differences (p ⁇ 0.05).
  • FIG. 9 is a graph showing the IgG immune response to Salmonella after vaccination of chicks with the indicated vaccine-adjuvant formulations as measured by a competitive ELISA. Different letters indicate significant differences (p ⁇ 0 . 05 ).
  • FIG. 10 is a graph showing the IgA immune response to Salmonella after vaccination, of chicks with the indicated vaccine-adjuvant formulations as measured by competitive ELISA. Different letters indicate significant differences (p ⁇ 0.05).
  • FIG. 11 is a graph showing the IgG immune response to Bordetella avium after a single parenteral vaccination of turkeys with the indicated vaccine-adjuvant formulations. Different letters indicate significant differences (p ⁇ 0.05).
  • FIG. 12 is a graph showing the IgG immune response to Bordetella avium after subcutaneous vaccination with the indicated vaccine-adjuvant formulation of day-of-hatch turkeys followed by a drinking water administration, of the same vaccine-adjuvant combination on day 14. The response was measured at day 21. Different letters indicate significant differences (p ⁇ 0.05).
  • adjuvants which include chitosan, vaccine formulations comprising the adjuvants, methods of making the adjuvants and methods of using the adjuvants and vaccine formulations.
  • the base molecule involves chitosan, which is a deacetylated form of chitin, the exoskeleton of many invertebrate animals (shrimp, crabs, insects, etc.).
  • Chitosan is considered a generally recognized as safe (GRAS) compound and is used for weight loss, cholesterol reduction, insomnia, and kidney function improvement.
  • Chitosan is also used as an adjuvant used, with various mucosal vaccines (Jabbal-Gill et al., 2012), but the chitosans described herein are new and function better than traditional chitosan as shown in the Examples.
  • Chitosan-protein cross-linked with formaldehyde and carbohydrate-linked chitosan provide a unique adjuvant for oral or parenteral delivery of vaccine antigens.
  • Chitosan has been used as a carrier for both oral and subcutaneous vaccines.
  • the antigen is covalently bound to the chitosan by treatment with formaldehyde.
  • the adjuvant system is improved by addition of a carbohydrate (mannose, fucose, and galactose) linked to the chitosan, allowing targeting of the mannose receptors on the antigen presenting cells, thus enhancing the immune response to the chitosan-antigen complex.
  • Both the chitosan-protein cross-linked with formaldehyde and the mannosylated-chitosan protein complex give a robust immune response by both parenteral and oral (or other mucosal) delivery routes, which is unique for inactivated vaccines.
  • an adjuvant composition comprising a carbohydrate linked to chitosan to form a Schiff base.
  • the adjuvant may be combined with an antigen.
  • the carbohydrate may be mannose, mannobiose, glucose, galactose or fructose.
  • Other suitable carbohydrates may be used. Without being limited by theory, the carbohydrate is added to the chitosan for the purpose of targeting the chitosan to receptors for these carbohydrates on the surface of antigen presenting cells.
  • the carbohydrate-chitosan used herein is made as described more fully in the Examples below.
  • Our method is based on Jayasree (Jayasree et al., 2011) using an open ring carbohydrate with an available carbonyl group that reacts with the amino group on chitosan to form a Schiff-base.
  • This Schiff-base can be stabilized by reduction with sodium cyanoborohydride (NaCNBH 4 ).
  • NaCNBH 4 sodium cyanoborohydride
  • the non-reduced form of the mannosylated chitosan does not require the addition of a toxic chemical (NaCNBH 4 ).
  • a toxic chemical NaCNBH 4
  • the carbohydrate suitably mannose (10 ⁇ M)
  • chitosan (0.2-2%) is dissolved in 1.5% acetic acid.
  • the dissolved mannose and the dissolved chitosan are then combined and incubated at room temperature to allow the amine group on the chitosan to react with the carbonyl on the sugar to produce a Schiff base.
  • Reduction of the Schiff base is not necessary for the adjuvant to function and indeed the Examples show the non-reduced Schiff base is a better adjuvant (see FIG. 6 ).
  • the Schiff base may be reduced.
  • chitosan and an antigen may be cross-linked using an aldehyde.
  • a composition comprising 0.5% to 2% of an aldehyde cross-linked chitosan and an antigen.
  • the final vaccine formulation suitably contains 0,5 to 1.5% chitosan.
  • the adjuvant may contain 0.5% to 3% chitosan, suitably 0.5% to 2% chitosan, suitably 0.5% to 1.5% chitosan, suitably 0.5% to 1.2% chitosan.
  • the final concentration of aldehyde in a vaccine composition is suitably less than 0.5%.
  • the maximum concentration of aldehyde is based on the maximum level of residual aldehyde allowed in vaccines.
  • a higher level of an aldehyde may be used for cross-linking the chitosan, but the final vaccine formulation suitably contains less than 0.5% aldehyde.
  • formaldehyde was used as the aldehyde to cross-link the chitosan.
  • Other aldehydes such as formalin, glutaraldehyde, acetaldehyde, propionaldehyde, or butyraldehyde, may also be used.
  • the aldehydes cross-link the chitosan amino groups with those on other chitosan molecules or on the antigens.
  • Methods of making a vaccine formulation comprising an aldehyde cross-linked chitosan and an antigen is also provided herein.
  • the methods include dissolving chitosan in a solution of acetic acid.
  • the carbohydrate-linked chitosan may also be used as the chitosan in this method.
  • the acetic acid is used at 1.5% final concentration in water or 15 mL of acetic acid dissolved in 1 L of water.
  • the amount of chitosan is between 0.5% and 2%, suitably between 0.5% and 1.5%.
  • An antigen is added to the dissolved chitosan at the appropriate level.
  • the amount and form of the antigens used in the vaccine formulations can be determined by those of skill in the art.
  • the antigen and chitosan are combined with the aldehyde such that the final concentration of the aldehyde is between 0.02% and 0.5%.
  • the aldehyde is capable of chemically cross-linking the chitosan to other chitosan molecules and the chitosan to the antigen.
  • Tris-HCl can be added to quench free aldehydes.
  • the Tris can be added to a final concentration of 0.5 g/L.
  • Either adjuvant composition disclosed herein may be combined with enhancing molecules including but not limited to saponin, toll-like receptors, the B subunit of a bacterial toxin, bacterial toxins, tetanus toxoid, CpG motifs, liposomes or monophosphoryl lipid A.
  • enhancing molecules include but not limited to saponin, toll-like receptors, the B subunit of a bacterial toxin, bacterial toxins, tetanus toxoid, CpG motifs, liposomes or monophosphoryl lipid A.
  • the enhancing molecules act as further stimulators of the immune system and enhance the immune response generated after administration of the vaccine formulation to a subject.
  • the vaccine formulations provided herein comprise the chitosan-based adjuvants described herein and antigens.
  • the antigens may be any antigens available to those of skill in the art. Antigens such as proteins, synthetic peptides, peptides conjugated to earners, or microbes may be used in the vaccines.
  • Microbes include bacteria, yeast, parasites, fungi, viruses, helminthes or other disease causing organisms. Microbes include live, dead, attenuated, recombinant, or inactivated organisms.
  • microbes examples include, but are not limited to Salmonella, Escherichia, Shigella, Bordetella, Clostridium, Mycoplasma, Staphylococcus, Streptococcus, Bacillus, Influenza, and Eimeria.
  • Microbes may be inactivated or killed prior to use by treatment with heat, methanol or other fixatives such as formaldehyde or other aldehydes.
  • the aldehydes can be quenched by subsequent addition of Tris-HCl to a final, concentration of 0.5 g/L.
  • Suitable antigens may also include peptide antigens such as Influenza M2e, Hemaglutinin, Neuraminidase, or nuclear proteins; Eimeria TRAP or MPP; Clostridium sialidase, SagA, alpha-toxin, NetB toxin, or iron transport protein. Examples of other peptide antigens can be found at least in U.S. application Ser. Nos. 12/441,851; 12/740,631; 12/740,608; 13/574,504; and 13/702,827, all of which are incorporated herein by reference in their entireties.
  • the chitosan based adjuvants may be used to increase the immune response to vaccines already available or to newly developed vaccines or autogenous vaccines.
  • modified chitosan when modified chitosan is co-administered with inactive vaccine by the parenteral route, we see an immune response that is superior to the immune response observed with other adjuvants, such as alum, with a minimal injection-site reaction.
  • Many adjuvants work by causing an inflammatory response at the site of injection or delaying absorption from the injection site, or both.
  • One of the down sides to traditional adjuvants is that they often cause some reaction, soreness, and in some cases they cause persistent lesions that cause downgrading or trimming of meat-producing animals at slaughter.
  • the modified chitosan may reduce these concerns associated other vaccine adjuvants. It is cheap to produce and easy to make into commercial vaccines.
  • killed vaccines There are huge advantages to killed vaccines in that they can be produced quickly with very low risk of causing infection and disease, they cannot genetically change back into the disease-causing parent type, and they have much lower regulatory issues for these reasons. Also, there are a large and ever-growing number of orphan diseases which are not sufficiently common for a vaccine company to develop a regulated/licensed vaccine, and there are provisions in US law (and many other countries) for producing “autogenous” vaccines specifically made from the pathogen of interest, killed, and used on the source flocks (or animal or human populations). In developing countries orphan diseases occur that require vaccines that are not affordable or that are technically not possible to produce locally or quickly enough to deal with an outbreak. The adjuvants provided herein are affordable and technologically straightforward to produce. They can be readily combined with a killed or inactivated microbe to generate a vaccine.
  • the systemic response to killed vaccines can be improved by incorporation of the altered chitosan as an adjuvant for injection.
  • We can prevent some diseases through oral administration of killed vaccines with this adjuvant platform.
  • This adjuvant platform when administered orally, may be targeted to stimulate systemic and/or mucosal responses—meaning that it has many of the advantages of live vaccines, but avoiding the issues of live vaccines described above.
  • a pharmaceutically acceptable carder is any carrier suitable for in vivo administration.
  • pharmaceutically acceptable carriers suitable for use in the compositions include, but are not limited to, water, buffered solutions, glucose solutions, oil-based or bacterial culture fluids. Additional components of the compositions may suitably include, for example, excipients such as stabilizers, preservatives, diluents, emulsifiers and lubricants.
  • Examples of pharmaceutically acceptable carriers or diluents include stabilizers such as carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing agents such as bovine serum or skimmed milk and buffers (e.g., phosphate buffer). Especially when such stabilizers are added to the compositions, the composition, is suitable for freeze-drying or spray-drying. The composition may also be emulsified.
  • carbohydrates e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran
  • proteins such as albumin or casein
  • protein-containing agents such as bovine serum or skimmed milk
  • buffers e.g., phosphate buffer
  • compositions described herein may also be combined with other pharmaceutical compositions and these compositions may be administered in any order, at the same time or as part of a unitary composition.
  • the two compositions may be administered such that one is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more.
  • An effective amount or a therapeutically effective amount of the vaccine formulations as used herein means the amount of the composition that, when administered to a subject for enhancing the immune response of the subject to the targeted disease is capable of increasing the immune response, such as the cell-mediated or antibody mediated immune response to limit the morbidity or mortality associated with infection or exposure to the targeted disease.
  • the immune response is enhanced to a level such that administration is sufficient to effect a treatment or block disease related, morbidity or mortality.
  • the therapeutically effective amount will vary depending on the vaccine, formulation or composition, the disease and its severity and the age, weight, physical condition and responsiveness of the subject.
  • the level of antibody produced in response to vaccination may be increased by two fold, three fold, four fold or more by inclusion of the adjuvant described herein as compared to administration of the same antigen without an adjuvant or with alum as an adjuvant.
  • the increased immune response may be an IgA response, or an IgG response.
  • the adjuvant may also lead to a reduction in the morbidity or mortality associated with subsequent infection.
  • use of the adjuvants described herein in combination with an antigen may lead to a reduction in the rate of subsequent infection or the severity of subsequent infection with the microbe to which the antigen elicits an immune response as compared to vaccination with the antigen alone or vaccination with the antigen and a distinct adjuvant
  • the severity of the infection may be measured by the ability of a microorganism to invade tissues beyond the site of introduction, replicate and/or persist within the organism over time, or cause morbidity or mortality.
  • the vaccinated animals may be subsequently infected with a pathogen. In such cases, the growth of the pathogen in the subject after challenge is reduced by at least 1 log 10 , 2 log 10 or even 3 log 10 in subjects administered the vaccine as compared to subjects administered a control.
  • compositions described herein may be administered by any means known to those skilled in the art, including, but not limited to, oral, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, nasopharyngeal, or transmucosal absorption.
  • the compounds may be formulated as an ingestable, sprayable or injectable formulation.
  • oral administration may entail addition to the drinking water, spraying on food, spraying on the animals (such as chickens or turkeys that will ingest the vaccine in the spray when they preen their feathers).
  • the subjects may be mammals, including humans, cows, pigs, cats, dogs or other domesticated animals or non-mammals such as poultry, i.e., chickens or turkeys.
  • the specific dosage administered and timing of administration in any given case will be adjusted in accordance with the formulation being administered, the disease being targeted, the risk of exposure, the condition of the subject, and other relevant medical factors that may modify the response of the subject or feasibility of providing the formulation to the subject.
  • the specific dose for a subject depends on type of subject, age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the vaccine is targeted.
  • the initial vaccination and the boost may be administered by different means. For example, an initial vaccination via a subcutaneous mute can be boosted by inclusion of the adjuvant-antigen complex in the drinking water or food.
  • the percentage of chitosan in the vaccine formulations is generally between 0.2 and 2%, suitably 0.5-1.5%.
  • the total, amount of chitosan administered may be from less than 1 mg per vaccination to 100 mg, suitably, 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 50, 75 or 100 mg of chitosan. In the Examples, 2-5 mg chitosan was used per dose.
  • the microbe When combined with a microbial antigen, the microbe may be included at between 1 ⁇ 10 6 to 1 ⁇ 10 9 microbes per dose. In the Examples, 1 ⁇ 10 7 to 1 ⁇ 10 8 microbes were used per dose.
  • An antigen may be included at 10 ⁇ g to 10 mg per dose. In the Examples, 100 ⁇ g per dose was used.
  • the immune response to the ⁇ -Galactosidase was determined using serum in an ELISA for ⁇ -galactosidase and the results are shown in FIG. 1 .
  • Levels of the immune response are reported as sample to positive control ratios of absorbance in an indirect ELISA. Higher S/P ratios indicate higher anti- ⁇ -galactosidase antibody titers.
  • a common commercial adjuvant used currently is 15% alum and there was a good immune response when this was used.
  • the CS bacterin with aluminum hydroxide adjuvant induced IgG levels that were approximately 30% lower than IgG levels induced by CS with chitosan compared by S/P ratios, 0.27 and 0.4, respectively. (See FIG. 2 ).
  • injection site lesions are less pronounced at 72 hours (or later) due to chitosan administration whereas alum always produces local inflammation and granulomas, often progressing to encapsulated scar tissue.
  • BSBB Bacillus Wild Type
  • BSAI Bacillus vectored avian influenza vaccine
  • BSAI Bacillus vectored avian influenza vaccine
  • Each vaccine was administered at 10 6 cfu/chick in 0.25 ml or 0.25 ml saline.
  • BSAI live, BSAI inactivated and lyophilized were given a booster vaccination of the same treatment they received at day 0 and all other groups did not receive the 2 nd vaccine dose.
  • the adjuvant was further enhanced through a series of experiments designed to improve the chitosan-based adjuvant by addition of potential enhancing molecules or alternative delivery strategies.
  • Immunostimulatory compounds can potentially improve responses when used with adjuvants and several have been investigated previously; see reviews (Guy, 2007; Mutwiri et al., 2011).
  • Potential adjuvants include saponins, bacterial components, compounds that interact with the innate immune system such as Toll-like receptors, nucleic acids such, as the CpG motif, viruses, emulsions including liposomes, or a combination of any of these components.
  • TERT Tetanus toxoid
  • LTB heat-labile enterotoxin B summit
  • CTB Cholera toxin B subunit
  • Other compounds shown to enhance the immune system empirically through innate chemical properties include saponin and monophosphoryl lipid A (MPLA). Using mannose or other sugars to target binding to macrophage receptors may enhance immune function. Combinations of different adjuvants may act synergistically such as with IL-12 or other cytokines to stimulate the immune response.
  • MPLA monophosphoryl lipid A
  • the first experiment to improve the adjuvant compared the formaldehyde-cross-linked chitosan adjuvant, which consists of an antigen of interest cross-linked with 0.5% chitosan, using formaldehyde to generate the data shown in FIGS. 1-3 above.
  • This adjuvant system was then used as the control or baseline for selection of the best combinations of selected candidate immune enhancing molecules.
  • the test immunogen was a Salmonella enteritidis (SE) bacterin grown to 10 8 cfu/ml and inactivated with formaldehyde.
  • test immunogen Salmonella bacterin with chitosan was 4 ⁇ 10 7 cfu/ml with a final dose of 1 ⁇ 10 7 cfu per bird
  • TT tetanus toxoid
  • LTB heat-labile enterotoxin B subunit
  • mannosylated chitosan administered in either the drinking water or feed.
  • the heat-labile enterotoxin from E. coli has been shown to be a powerful immunostimmulatory molecule but is very toxic and is, therefore, not suitable as an adjuvant.
  • the heat-labile enterotoxin consists of two subunits, a central core LTA and five subunits of LTB (da Hora et al., 2011).
  • the LTB sub-unit retains the Immune adjuvant properties and yet is non-toxic. Therefore, this is a safe potential adjuvant component
  • Mannose and some other carbohydrates are ligands for receptors that activate macrophages.
  • the mannosylated chitosan was prepared by a method similar to that described previously by Yalpani and Hall (1980 and 1985) and Jayasree et al., (2011) without the addition of the zinc. Briefly, two molar equivalents of mannose in one volume of 0.1 M sodium acetate were heated at 60° C. for two hours. The solution was then added to two volumes of one molar equivalent of 2% chitosan in 0.15% acetic acid and allowed to react for 10 min at room temperature to produce 1.5% mannosylated chitosan. The SE bacterin was then added to 1.5% mannosylated chitosan in a two to one ratio. The Schiff-bases formed were then reduced with sodium cyanoborohydrate (NaCNBH 4 ).
  • Day-of-hatch broiler chicks were primed with 0.25 ml of the indicated preparations subcutaneously as outlined above. These groups were primed the same as the chitosan only group. Chicks were boosted by oral gavage at 12 days of age except for the drinking water and TPP groups which were boosted in water at 1:128 or in the feed at 0.5% (wt/wt) for 8 hours, respectively.
  • Antibody levels on day 22 in serum (IgG) and ileal, mucosal (IgA) were determined with a competitive ELISA kit (IDEXX). Decreased absorbance levels or sample to control ratios indicate higher levels of antibodies that recognize the SE flagellin coated plates.
  • the chitosan adjuvant was superior to alum in producing a robust immune response.
  • each of the chitosan based adjuvants was able to produce a robust response to the SE bacterin, with significantly higher levels of both IgG and IgA as compared to chitosan alone administered subcutaneously ( FIGS. 4 and 5 , respectively).
  • the TT and dry powder TPP groups had a significantly higher immune response than the sq primed with sq boost chitosan adjuvant ( FIGS. 4 and 5 ).
  • Mannosylated chitosan treatment group was added in this experiment. Also added in this experiment was another treatment group that was the similar to the mannosylated chitosan treatment group, which was shown to be an excellent adjuvant in the previous experiment (Mannosylated chitosan version 1, Man-C V1), but this group was not reduced with NaCNBH 4 (Man-C V2).
  • the SE flagellin competitive ELISA again showed that the birds vaccinated subcutaneously with 0.5% chitosan (C) for both the primary and boost had higher levels of immunoglobulins in the serum ( FIG. 6A ).
  • the birds vaccinated with 0.5% chitosan with CTB (C+CTB), Man-C V2 and Chitosan with saponin (C+saponin) gave the best IgG response ( FIG. 6A ),
  • the Man-C V2 gave numerically the best IgA response ( FIG. 6B ). All three of these treatment groups were significantly different from the benchmark group (0.5% chitosan vaccinated sq for both the primary and boost) ( FIG. 6 ).
  • the birds were challenged at day 19 with live Salmonella at 5 ⁇ 10 7 cfu/chick.
  • Chitosan with CTB, both versions of mannosylated chitosan, chitosan in the drinking water boost, and chitosan plus saponin significantly decreased Salmonella to below detectable limits in the liver/spleen (L/S) when compared to the negative (saline vaccinated) control ( FIG. 7 ; p ⁇ 0.05).
  • the levels of Salmonella were significantly reduced using either of the two versions of mannosylated chitosan and in the group boosted with 0.5% chitosan diluted 1:128 in the drinking water.
  • the significant decrease in the mannosylated chitosan groups indicate that the direct targeting of the macrophage with a ligand for the mannose receptor increases the effectiveness of the chitosan adjuvant.
  • the best route and adjuvant combination for vaccination was investigated.
  • Day-of-hatch chicks were administered 0.25 mL of either saline or the vaccine with the respective adjuvant mixture as indicated in FIG. 8 .
  • the adjuvants compared include the formaldehyde-cross-linked chitosan, the reduced mannosylated chitosan (Man C V1), the non-reduced mannosylated chitosan (Man C V2), and each adjuvant was combined with antigen and administered, at day-of-hatch either subcutaneously or in the drinking water. Birds were boosted with a second administration of the same adjuvant-antigen combination either subcutaneously, in the drinking water or via oral gavage.
  • the groups that were vaccinated in the drinking water (DW) were diluted 1:128 in the water. Those boosted by oral gavage were given 0.25 ml. Mucosal IgA response was measured using a competitive SE flagellin ELISA assay (IDEXX) as described above.
  • IDEXX competitive SE flagellin ELISA assay
  • the numerically lowest group was the mannosylated chitosan V2 delivered sq in the primary vaccination and a 1:128 dilution in DW for the boost.
  • This group had significantly higher levels of IgA in the ileum compared to 0.5% chitosan with sq primary vaccination and either sq or DW boost. No significant differences were observed between the reduced and non-reduced forms of the mannosylated chitosan or when the boost was given via the drinking water or oral gavage.
  • the mannosylated chitosan was then compared to and combined with a commercially available mineral oil based adjuvant.
  • Salmonella enteritidis (SE) bacterin grown to 10 8 cfu/ml and inactivated with formaldehyde was used as the antigen.
  • SE Salmonella enteritidis
  • the Salmonella bacterin was mixed with chitosan, mannosylated chitosan, the mineral oil adjuvant, a combination of chitosan and the mineral oil adjuvant or PBS at 4 ⁇ 10 7 cfu/ml with a final dose of 1 ⁇ 10 7 cfu per bird in a 2:1 ratio.
  • Day-of-hatch broiler chicks were primed with 0.25 ml of the indicated preparations subcutaneously as outlined above. Chicks were boosted by oral gavage at 12 days of age. Antibody levels on day 22 in serum (IgG) and ileal mucosal (IgA) were determined with a competitive ELISA kit (IDEXX) and results are shown in FIGS. 9 and 10 , respectively. Decreased absorbance levels of sample to control ratios indicate higher levels of antibodies that recognize the SE flagellin coated plates. The mannosylated chitosan vaccination and boost protocol produced significantly increased IgG and IgA levels as compared to each of the other groups.
  • the final product of chitosan without mannose can range from a minimum final concentration of 0.5% chitosan and maximal final concentration of 2% chitosan in the vaccine formulation.
  • Chitosan is dissolved in a solution containing 15 ml of glacial acetic acid per L deionized water at the appropriate concentration (1.5% acetic acid in water).
  • a solution containing 15 ml of glacial acetic acid per L deionized water at the appropriate concentration (1.5% acetic acid in water).
  • Typically for broth cultures 2 volumes of culture are mixed with one volume of 1.5% chitosan (0.5% chitosan in the final vaccine formulation).
  • Other antigens are diluted as minimal as possible giving a final concentration of up to 1.5% chitosan.
  • the formaldehyde is then added to the antigen-dissolved chitosan mixture such that the final concentration is 0.2% formaldehyde or 0.008 M formaldehyde. In the Examples above, a 37% solution of formaldehyde is used. Tris-HCl can be added to a final concentration 0.5 g/L.

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US11382962B2 (en) 2016-05-03 2022-07-12 The Board Of Trustees Of The University Of Arkansas Yeast vaccine vector including immunostimulatory and antigenic polypeptides and methods of using the same

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