Adjuvant compositions based on phycobiliproteins

Abstract

Disclosed herein is a method for enhancing immune response of a vaccine. The method includes preparing a Phycocyanin compound. The Phycocyanin compound includes at least one of C-phycocyanin (C-PC), Phycoerythrin (PE), Allophycocyanin (APC), phycocyanobilin (PCB), and combinations thereof. The method further includes administering the prepared Phycocyanin along with a vaccine composition to a living body.

Description

ADJUVANT COMPOSITIONS BASED ON PHYCOBILIPROTEINS TECHNICAL FIELD

[0001] The present disclosure generally relates to vaccine and adjuvants compositions, andparticularly, to adjuvant formulations for enhancing immune response of vaccines and methods of producing and administrating thereof. BACKGROUND

[0002] Vaccination is considered to be one of the greatest achievements of modern medicine.According to the World Health Organization (WHO), before COVID-19 pandemic, vaccinations save approximately 2.5 million lives each year. Numerous studies and efforts have been made over time to increase the effectiveness of vaccines, including the use of various substances known as adjuvants as one of the primary components of vaccines.

[0003] In 1920, Gaston Ramon, a veterinarian working at the Pasteur Institute, first used theterm “adjuvant.” He demonstrated that modifying the inactivated diphtheria toxin with various chemicals led to inflammation at the injection site and boosted antibody expression in response to the vaccine. Numerous additional adjuvant vaccinations have been introduced since the 1920s.

[0004] Since the immunological capacity of a subunit of the whole microorganism, despite itshigh immunity, is much lower than the whole microorganism, the need for adjuvant, especially in subunit vaccines, has grown more obvious in light of the significance and rising trend of recombinant vaccine production. Qualities that make a substance a potential adjuvant include being safe (in terms of having minimal adverse effects), neutral, and degradable, inducing a long-lasting immunological response, having a defined half-life, being economically advantageous, and having a minimal impact on the environment. Type of adjuvant employed, in addition to structure and quantity of an antigen, is crucial in design and production of a vaccine to elicit an adequate and desirable immune response. Moreover, formulation, vaccine administration method, administration time, volume of each vaccine dose, antigen structure, host type, interspecies variety, and target organism's immune system are additional crucial aspects to consider when selecting an adjuvant along with an antigen. The most frequently used adjuvant overall is alum, which can stimulate potent humoral immunological reactions. This frequent adjuvant has a number of drawbacks, including the fact that it does not boost cellularimmunity or induced poorly and causes side effects like fever, chills, and neurological issues. However, a portion of the population, known as the non-responsive population, does not always respond well to vaccinations. For instance, with the vaccine against COVID-19, 7.5% of people between the ages of 18 and 59 and 11.7% of people over the age of 60 do not respond. Additionally, 10%-15% of persons who receive the hepatitis B vaccine do not generate a good immune response; therefore, it is possible that by altering the formulation of this type of vaccine, particularly by altering the adjuvant, the efficacy can be boosted in this population. Accordingly, adjuvants may play a crucial role in vaccination so that an adjuvant may be as important as the antigen in vaccine composition.

[0005] Therefore, there is a need in the art for new adjuvants that have higher efficiency andless side effects. Moreover, there is a need in the art for substances that enhance immune response of vaccines. Specifically, there is a need in the art for vaccine compositions adequate and effective for all groups of population. SUMMARY

[0006] This summary is intended to provide an overview of the subject matter of the presentdisclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0007] In one general aspect, the present disclosure describes a method for enhancing immuneresponse of a vaccine. In an exemplary embodiment, the method may include preparing aPhycocyanin and administering the prepared Phycocyanin along with a vaccine composition toa living body. In an exemplary embodiment, preparing the Phycocyanin may include preparingat least one compound of phycobiliprotein (PBP) family. In an exemplary embodiment, the atleast one compound of PBP family may include at least one of C-phycocyanin (C-PC),Phycoerythrin (PE), Allophycocyanin (APC), phycocyanobilin (PCB), and combinationsthereof.

[0008] In an exemplary embodiment, administering the prepared Phycocyanin along with thevaccine composition may include administering a dosage in a range of 0.1 µg to 100 mg of theprepared Phycocyanin per dose of the vaccine composition. In an exemplary embodiment,administering the prepared Phycocyanin along with the vaccine composition may includeadministering a dosage in a range of 100 µg to 500 µg of the prepared Phycocyanin per doseof the vaccine composition.

[0009] In an exemplary embodiment, preparing the Phycocyanin may include culturing an algain a salt medium under specific environmental conditions and purifying the Phycocyanin fromthe cultured alga. In an exemplary embodiment, culturing the alga in the salt medium underspecific environmental conditions may include culturing Arthrospira platensis (Spirulina) inthe salt medium under specific environmental conditions.

[0010] In an exemplary embodiment, culturing the alga in the salt medium under specificenvironmental conditions may include applying changes in the salt medium while culturing thealga therein while culturing the alga in the salt medium. In an exemplary embodiment, applyingchanges in the salt medium while culturing the alga therein may include at least one of enriching the salt medium with a plurality of micronutrients, changing a light wavelength of a light emitting to the salt medium, ceasing light emitting to the salt medium, applying food deficiency in the salt medium, and combinations thereof.

[0011] In an exemplary embodiment, enriching the salt medium with the plurality ofmicronutrients may include increasing an amount of at least one of iron ion, zinc ion,magnesium ion, and combinations thereof up to 100 times in the salt medium. In an exemplaryembodiment, culturing the alga in the salt medium under specific environmental conditionsmay include culturing the alga in the salt medium exposed to a light beam with a wavelengthin a range 350 nm to 500 nm.

[0012] In an exemplary embodiment, purifying the Phycocyanin from the cultured Spirulinamay include forming a mixture by mixing the cultured alga with a first portion of a buffer solution, freeze-thawing the mixture iteratively, sonicating the mixture at a sonication intensity in a range of 20 kHz to 50 kHz for a time period in a range of 2 minutes to 20 minutes, centrifuging the sonicated mixture at a range of 15000 rpm to 20000 rpm for a time period in a range of 15 minutes to 30 minutes, collecting a supernatant portion of the centrifuged mixture,and extra purifying the supernatant portion.

[0013] In an exemplary embodiment, forming the mixture may include mixing the culturedalga with a 0.05 M buffer solution of Na-phosphate at a weight ratio of 1:1 (the cultured alga: Na-phosphate buffer solution).

[0014] In an exemplary embodiment, extra purifying the supernatant portion may includeprecipitating a pellet of the supernatant portion using an ammonium sulfate ((NH4)2SO4)solution, recovering the pellet by centrifuging at 27,000 rpm for 15 min at 4 °C, dissolving the pellet in a second portion of the buffer solution, dialyzing the pellet dissolved in the second portion of the buffer solution against the buffer solution using a dialysis membrane, and filtering an extract obtained from the dialysis membrane.

[0015] In an exemplary embodiment, preparing the Phycocyanin may further include obtainingPCB from the purified Phycocyanin by cleaving the purified Phycocyanin. In an exemplary embodiment, cleaving the purified Phycocyanin may include at least one of methanolysis of the purified Phycocyanin, ethanolysis of the purified Phycocyanin, enzymic hydrolysis of the purified Phycocyanin, acidic hydrolysis of the purified Phycocyanin, and combinations thereof.

[0016] In an exemplary embodiment, administering the prepared Phycocyanin along with thevaccine composition may include at least one of introducing the prepared Phycocyanin and the vaccine composition separately from each other to the living body, introducing the prepared Phycocyanin and the vaccine composition in combination with each other to the living body, and combinations thereof.

[0017] In an exemplary embodiment, introducing the prepared Phycocyanin and the vaccinecomposition separately from each other to the living body may include administering thevaccine composition to the living body by at least one of intramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof andadministering the prepared Phycocyanin to the living body by at least one of intramuscularinjection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof.

[0018] In an exemplary embodiment, administering the vaccine composition to the living bodyand administering the prepared Phycocyanin to the living body may be done simultaneously.In an exemplary embodiment, administering the vaccine composition to the living body andadministering the prepared Phycocyanin to the living body may be done with a time interval ina range of a few seconds to 24 hours or more.

[0019] In an exemplary embodiment, introducing the prepared Phycocyanin and the vaccinecomposition in combination with each other to the living body may include adding the preparedPhycocyanin to the vaccine composition and administering the vaccine composition containingthe prepared Phycocyanin to the living body by at least one of intramuscular injection,subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof.

[0020] In another general aspect, the present disclosure describes a vaccine composition. In anexemplary embodiment, the vaccine composition may include a Phycocyanin compound andan antigen. In an exemplary embodiment, the Phycocyanin compound may include at least oneof C-phycocyanin (C-PC), Phycoerythrin (PE), Allophycocyanin (APC), phycocyanobilin(PCB), Biliproteins, and combinations thereof. In an exemplary embodiment, the Phycocyanincompound may be present in the vaccine composition at a range of 0.1 µg to 100 mg per doseof the vaccine composition. In an exemplary embodiment, the Phycocyanin compound may bepresent in the vaccine composition at a range of 100 µg to 500 µg per dose of the vaccine composition. In an exemplary embodiment, the antigen may include at least one of a viral antigen, a bacterial antigen, a cancer antigen, and combinations thereof. In an exemplary embodiment, the vaccine composition may include an injectable vaccine composition or an oral vaccine composition administered by at least one of intramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof.

[0021] Other exemplary systems, methods, features and advantages of the implementationswill be, or will become, apparent to one of ordinary skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, methods, features and advantages be included within this description and this summary, be within the scope of the implementations, and be protected by the claims herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawing figures depict one or more implementations in accord with the presentteachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements.

[0023] FIG. 1 schematically shows a tertiary structure unit of C-phycocyanin (C-PC) fromSpirulina platensis which includes alpha subunits and beta subunits, consistent with one ormore exemplary embodiments of the present disclosure.

[0024] FIG. 2 shows a flowchart of an exemplary method for enhancing immune response ofa vaccine, consistent with one or more exemplary embodiments of the present disclosure.

[0025] FIG. 3 shows a flowchart of an exemplary method for preparing an exemplaryPhycocyanin, consistent with one or more exemplary embodiments of the present disclosure.

[0026] FIG. 4 shows a flowchart of an exemplary method for purifying an exemplaryPhycocyanin from an exemplary cultured and harvested alga, consistent with one or more exemplary embodiments of the present disclosure.

[0027] FIG. 5 shows comparison of average level of hepatitis B surface antibody (HBsAb)expression in immune response to hepatitis B surface antigen (HBsAg) in different groups receiving the phycocyanobilin (PCB) injectable adjuvant, consistent with one or more exemplary embodiments of the present disclosure.

[0028] FIG. 6 shows comparison of average level of expression of interferon gamma (IFN-γ)in different groups receiving the injectable adjuvant, consistent with one or more exemplary embodiments of the present disclosure.

[0029] FIG. 7 shows comparison of average level of interleukin 4 (IL-4) expression indifferent groups receiving the injectable adjuvant, consistent with one or more exemplary embodiments of the present disclosure.

[0030] FIG. 8 shows comparison of average level of ALT changes in different groupsreceiving injectable adjuvant, consistent with one or more exemplary embodiments of the present disclosure.

[0031] FIG. 9 shows comparison of average level of AST changes in different groupsreceiving injectable adjuvant, consistent with one or more exemplary embodiments of the present disclosure.

[0032] FIG. 10 shows hepatitis B surface antibody (HBsAb) expression rate in response to theoral adjuvants, consistent with one or more exemplary embodiments of the present disclosure.

[0033] FIG. 11 shows interferon gamma (IFN-γ) expression level in the oral adjuvantconsumption form, consistent with one or more exemplary embodiments of the present disclosure.

[0034] FIG.12 shows interleukin 4 (IL-4) expression level in oral adjuvant consumption form,consistent with one or more exemplary embodiments of the present disclosure.

[0035] FIG. 13 shows comparison of average level of ALT changes in different groupsreceiving oral adjuvant, consistent with one or more exemplary embodiments of the present disclosure.

[0036] FIG. 14 shows comparison of average level of AST changes in different groupsreceiving oral adjuvant, consistent with one or more exemplary embodiments of the present disclosure. DETAILED DESCRIPTION

[0037] In the following detailed description, numerous specific details are set forth by way ofexamples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0038] The following detailed description is presented to enable a person skilled in the art tomake and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0039] Herein, an exemplary method is disclosed to enhance immune response of a vaccine.In an exemplary embodiment, an exemplary method may include preparing a Phycocyanin, forexample, at least one of C-phycocyanin (C-PC), phycocyanobilin (PCB), and combinationsthereof and administering exemplary prepared Phycocyanin along with a vaccine compositionto a living body. As used herein, a “Phycocyanin” may refer to several phycobiliproteins (PBP), such as phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), andphycoerythrocyanin (PEC), specially, C-phycocyanin (C-PC) and / or phycocyanobilin (PCB)compounds. In an exemplary embodiment, exemplary Phycocyanin may be prepared from analga, such as Arthrospira platensis (Spirulina platensis). As used herein, “Arthrospiraplatensis” is a species of Spirulina, which is a kind of microalgal that is classified undercyanobacteria phylum. For simplicity, “Arthrospira platensis” is called “Spirulina” herein.Spirulina may have high protein concentration, vitamins, essential fatty acids, and essentialamino acids. In addition, 55%-70% of Spirulina’s dry weight may be composed of protein.Phycocyanins or phycobiliproteins (PBPs) may include proteins originating from cyanobacteria. For example, C-phycocyanin (C-PC) is a kind of PBP. In an exemplaryembodiment of the present disclosure, C-PC may be extracted from Arthrospira platensis(Spirulina) and may be used as an adjuvant for a vaccine composition. Herein, an “adjuvant”may refer to a vaccine component that enhance adaptive immune responses to antigens. In an exemplary embodiment, C-PC may be investigated to be a novel adjuvant in light of its qualities and significance, including anticancer activity, pain relief, and anti-inflammatory effects, in addition to its FDA approval (GRAS category). In detail, C-PC is a protein purified from Spirulina (Arthrospira platensis) that includes a bilin part and a protein part. The bilin part of C-PC that may be cleaved of its protein part is called Phycocyanobilin (PCB). In another exemplary embodiment of the present disclosure, PCB may be separated from C-PC and maybe used as an adjuvant for a vaccine composition. FIG.1 schematically shows tertiary structureunit 100 of C-phycocyanin (C-PC) from Spirulina platensis which includes alpha subunits 102and beta subunits 104, consistent with one or more exemplary embodiments of the presentdisclosure. As it is demonstrated in FIG.1, core part of PBPs (C-PC) is phycocyanobilin (PCB)106 (or bilins (chromophores)).

[0040] FIG.2 shows a flowchart of a method 200 for enhancing immune response of a vaccine,consistent with one or more exemplary embodiments of the present disclosure. In an exemplaryembodiment, method 200 may include preparing a Phycocyanin (step 202) and administeringan exemplary prepared Phycocyanin along with a vaccine composition to a living body (step204). In an exemplary embodiment, an exemplary Phycocyanin may include a compound ofphycobiliprotein (PBP) family. In an exemplary embodiment, an exemplary Phycocyanin mayinclude at least one of C-phycocyanin (C-PC), Phycoerythrin (PE), Allophycocyanin (APC),and combinations thereof. In an exemplary embodiment, an exemplary Phycocyanin mayinclude phycocyanobilin (PCB).

[0041] In further detail with respect to step 202, step 202 may include preparing an exemplaryPhycocyanin. In an exemplary embodiment, preparing an exemplary Phycocyanin may includepreparing at least one of C-phycocyanin (C-PC), Phycoerythrin (PE), Allophycocyanin (APC),and combinations thereof. In an exemplary embodiment, preparing an exemplary Phycocyaninmay include preparing at least one of C-phycocyanin (C-PC), phycocyanobilin (PCB), andcombinations thereof. FIG. 3 shows a flowchart of a method 300 for preparing an exemplaryPhycocyanin, consistent with one or more exemplary embodiments of the present disclosure.In an exemplary embodiment, method 200 of preparing an exemplary Phycocyanin mayinclude culturing an alga in a salt medium under specific environmental conditions (step 302)and purifying an exemplary Phycocyanin from an exemplary cultured and harvested alga (step304).

[0042] In further detail with respect to step 302, step 302 may include culturing an exemplaryalga in an exemplary salt medium under specific environmental conditions. In an exemplaryembodiment, step 302 may include culturing Arthrospira platensis (Spirulina) in an exemplarysalt medium under specific environmental conditions. In an exemplary embodiment, step 302of culturing an exemplary alga in an exemplary salt medium under specific environmental conditions may include culturing an exemplary alga at a specific range of pH, temperature, light intensity which an exemplary salt medium of culturing an exemplary alga exposed to, air circulation within an exemplary salt medium of culturing an exemplary alga, etc.

[0043] In an exemplary embodiment, step 302 of culturing an exemplary alga in an exemplarysalt medium under specific environmental conditions may include culturing an exemplary algaat a pH range of 8 to 11. In an exemplary embodiment, step 302 of culturing an exemplary algain an exemplary salt medium under specific environmental conditions may include culturing an exemplary alga at a temperature range of 25 °C to 32 °C. In an exemplary embodiment, step302 of culturing an exemplary alga in an exemplary salt medium under specific environmentalconditions may include culturing an exemplary alga at a light intensity of 1000 lux to 3000 lux.In an exemplary embodiment, step 302 of culturing an exemplary alga in an exemplary saltmedium under specific environmental conditions may include culturing an exemplary alga with aeration (air circulation) of an exemplary salt medium for a time period in a range of 5 minutes to 20 minutes once a day or more.

[0044] In an exemplary embodiment, step 302 of culturing an exemplary alga in an exemplarysalt medium under specific environmental conditions may include motivating culturing an exemplary alga in an exemplary salt medium by applying significant changes in nutrients of an exemplary salt medium and irradiated light to an exemplary salt medium while culturing anexemplary alga therein. In an exemplary embodiment, culturing an exemplary alga in anexemplary salt medium under specific environmental conditions may include enriching anexemplary salt medium with a plurality of micronutrients, changing a light wavelength of a light emitting to the salt medium, ceasing light emitting to the salt medium, applying food deficiency in the salt medium, and combinations thereof while culturing an exemplary alga inan exemplary salt medium. In an exemplary embodiment, enriching an exemplary salt mediumwith an exemplary plurality of micronutrients may include increasing an amount of at least one of iron ion, zinc ion, magnesium ion, and combinations thereof up to 100 times in an exemplarysalt medium while culturing an exemplary alga therein. In an exemplary embodiment, culturingan exemplary alga in an exemplary salt medium under specific environmental conditions mayinclude culturing an exemplary alga in an exemplary salt medium exposed to a light beam witha wavelength in a range 350 nm to 500 nm. In an exemplary embodiment, culturing anexemplary alga in an exemplary salt medium under specific environmental conditions mayinclude culturing an exemplary alga in an exemplary salt medium exposed to blue light with awavelength in a range 450 nm to 495 nm.

[0045] Regarding step 304, an exemplary Phycocyanin may be prepared and purified from anexemplary cultured and harvested alga obtained in step 302. In an exemplary embodiment, step304 may include preparing and purifying C-PC from an exemplary cultured Spirulina. FIG. 4shows a flowchart of a method 400 for purifying an exemplary Phycocyanin from an exemplarycultured and harvested alga, consistent with one or more exemplary embodiments of the presentdisclosure. In an exemplary embodiment, method 400 of purifying an exemplary Phycocyaninfrom an exemplary cultured and harvested alga (step 304) may include forming a mixture bymixing an exemplary cultured and harvested alga with a buffer solution (step 402), freeze-thawing an exemplary mixture iteratively (step 404), sonicating an exemplary mixture at asonication intensity in a range of 20 kHz to 50 kHz for a time period in a range of 2 minutes to20 minutes (step 406), centrifuging an exemplary sonicated mixture at a range of 15000 rpmto 20000 rpm for a time period in a range of 15 minutes to 30 minutes (step 408), collecting asupernatant portion of an exemplary centrifuged mixture (step 410), and extra purifying anexemplary supernatant portion (step 412).

[0046] In an exemplary embodiment, step 402 of mixing an exemplary cultured and harvestedalga, for example, Arthrospira platensis (Spirulina), with a first portion of an exemplary buffersolution may include mixing an exemplary cultured alga with a sodium phosphate buffer solution. In an exemplary embodiment, mixing an exemplary cultured alga with an exemplary buffer solution may include mixing an exemplary cultured and harvested alga with a 0.05 Mbuffer solution of Na-phosphate at a weight ratio of 1:1 (an exemplary cultured alga: Na- phosphate buffer solution). In an exemplary embodiment, an exemplary buffer solution may be prepared by mixing about 2.12 cc of a 1 M solution of monosodium phosphate (NaH2PO) and about 2.89 cc of a 1 M solution of sodium diphosphate (Na4O7P2), and adding distilled water to obtain a 100 cc mixture.

[0047] In an exemplary embodiment, step 404 of freeze-thawing an exemplary mixtureiteratively may include freeze-thawing an exemplary mixture for several times, for example, three times.

[0048] In an exemplary embodiment, step 410 of collecting an exemplary supernatant portionof an exemplary centrifuged mixture may include resting an exemplary centrifuged mixture; allowing for completely separation of an exemplary supernatant portion from a precipitated portion, and removing an exemplary supernatant portion from top of an exemplary precipitated portion. In an exemplary embodiment, an exemplary supernatant portion may include purified Phycocyanin extracted from an exemplary cultured alga. In an exemplary embodiment, an exemplary supernatant portion may include purified C-PC extracted from exemplary cultured Arthrospira platensis (Spirulina).

[0049] In an exemplary embodiment, step 412 of extra purifying an exemplary supernatantportion may include precipitating a pellet of an exemplary supernatant portion by mixing anexemplary supernatant portion with an ammonium sulfate ((NH4)2SO4) solution, recovering an exemplary pellet by centrifuging an exemplary obtained mixture at 27,000 rpm for 15 min at 4 °C, dissolving an exemplary pellet in a second portion of an exemplary buffer solution, dialyzing an exemplary pellet dissolved in an exemplary second portion of an exemplary buffer solution against an exemplary buffer solution using a dialysis membrane, and filtering an extract obtained from an exemplary dialysis membrane. In an exemplary embodiment, precipitating an exemplary pellet of an exemplary supernatant portion may include mixing an exemplary supernatant portion with a 65% solution of ammonium sulfate ((NH4)2SO4) andkeeping an exemplary obtained mixture overnight at 4 °C. In an exemplary embodiment,dissolving an exemplary pellet in an exemplary second portion of an exemplary buffer solution may include dissolving an exemplary pellet in 10 ml of an exemplary buffer solution, resultingin forming an ammonium sulfate extract (ASE). In an exemplary embodiment, dialyzing anexemplary pellet dissolved in an exemplary second portion of an exemplary buffer solution may include dialyzing 10 ml of exemplary ASE against 1000 ml of an exemplary buffersolution firstly at room temperature and then at 4 °C overnight. In an exemplary embodiment,filtering an extract obtained from an exemplary dialysis membrane may include filtering anextract obtained from an exemplary dialysis membrane through a 0.45 µm filter paper.

[0050] In an exemplary embodiment, an exemplary purified Phycocyanin as protein part of anexemplary alga obtained in step 304 may be used as an adjuvant in a vaccine composition oralong with an exemplary vaccine composition before, during, or after administering an exemplary vaccine composition into a living body (e.g., a human or an animal). In an exemplary embodiment, an exemplary purified Phycocyanin (e.g., each of C-PC, PE, and APC)as protein part of Arthrospira platensis (Spirulina) obtained in step 304 may be used as anexemplary adjuvant in an exemplary vaccine composition or along with an exemplary vaccine composition before, during, or after administering an exemplary vaccine composition into an exemplary living body (e.g., a human or an animal). In another exemplary embodiment, PCB as bilin (chromophore) part of an exemplary purified Phycocyanin (e.g., each of C-PC, PE, and APC) may be prepared by cleaving of an exemplary purified Phycocyanin, and may be added to an exemplary vaccine composition or may be used along with an exemplary vaccine composition before, during, or after administering vaccine composition into an exemplary living body (e.g., a human or an animal) as an exemplary adjuvant.

[0051] Referring again to FIG.3, method 300 for preparing a Phycocyanin (step 202 of method200) may further include obtaining PCB by cleaving an exemplary purified Phycocyanin (step306). In an exemplary embodiment, step 306 of cleaving an exemplary purified Phycocyaninmay include at least one of methanolysis of an exemplary purified Phycocyanin, ethanolysis ofan exemplary purified Phycocyanin, enzymic hydrolysis of an exemplary purifiedPhycocyanin, acidic hydrolysis of an exemplary purified Phycocyanin, and combinationsthereof.

[0052] Referring back to FIG.2 in further detail with respect to step 204, step 204 may includeadministering exemplary prepared Phycocyanin along with an exemplary vaccine compositionto a living body (e.g., a human or an animal). In an exemplary embodiment, exemplary preparedPhycocyanin may be administered to an exemplary living body in oral form or by injection. Inan exemplary embodiment, exemplary prepared Phycocyanin may be administered to anexemplary living body in combination with an exemplary vaccine composition or separately from an exemplary vaccine composition. In an exemplary embodiment, administeringexemplary prepared Phycocyanin along with an exemplary vaccine composition may includeat least one of introducing an exemplary prepared Phycocyanin and an exemplary vaccinecomposition separately from each other to an exemplary living body, introducing an exemplaryprepared Phycocyanin and an exemplary vaccine composition in combination with each otherto an exemplary living body, and combinations thereof.

[0053] In an exemplary embodiment, step 204 of administering exemplary preparedPhycocyanin along with an exemplary vaccine composition may include administering adosage in a range of 0.1 micrograms (µg) to 100 milligrams (mg) of exemplary preparedPhycocyanin per dose of an exemplary vaccine composition. In an exemplary embodiment,administering exemplary prepared Phycocyanin along with an exemplary vaccine compositionmay include administering a dosage in a range of 100 µg to 500 µg of exemplary preparedPhycocyanin per dose of an exemplary vaccine composition.

[0054] In an exemplary embodiment, administering exemplary prepared Phycocyanin alongwith an exemplary vaccine composition may include administering exemplary preparedPhycocyanin along with one of a hepatitis B vaccine, a COVID-19 vaccine, a Humanpapillomavirus vaccine, etc.

[0055] In an exemplary embodiment, step 204 of administering exemplary preparedPhycocyanin along with an exemplary vaccine composition may include administering anexemplary vaccine composition to an exemplary living body and administering exemplaryprepared Phycocyanin to an exemplary living body separately from each other. In an exemplaryembodiment, administering an exemplary vaccine composition and administering exemplaryprepared Phycocyanin to an exemplary living body may be done simultaneously or with a timeinterval in a range of a few seconds to 24 hours or more. In an exemplary embodiment, administering an exemplary vaccine composition to an exemplary living body may be done by at least one of intramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof. In an exemplary embodiment, administeringexemplary prepared Phycocyanin to an exemplary living body may be done by at least one ofintramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof.

[0056] In another exemplary embodiment, step 204 of administering exemplary preparedPhycocyanin along with an exemplary vaccine composition may include adding exemplaryprepared Phycocyanin to an exemplary vaccine composition and administering an exemplaryvaccine composition containing exemplary added at least one of Phycocyanin to an exemplaryliving body. In an exemplary embodiment, an exemplary vaccine composition containingexemplary added Phycocyanin may be administered into an exemplary living body by at leastone of intramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof.

[0057] In another general aspect, the present disclosure may describe a vaccine composition.In an exemplary embodiment, an exemplary vaccine composition may include a portion or whole of a phycocyanin of phycobiliprotein (PBP) family and an antigen. In an exemplary embodiment, an exemplary portion or whole of an exemplary phycocyanin may include at least one of C-phycocyanin (C-PC), Phycoerythrin (PE), Allophycocyanin (APC), phycocyanobilin (PCB), and combinations thereof. In an exemplary embodiment, an exemplary vaccinecomposition may include at least one of Spirulina extract, C-phycocyanin (C-PC),phycocyanobilin (PCB), and combinations thereof and an antigen.

[0058] In an exemplary embodiment, an exemplary Phycocyanin may be present in anexemplary vaccine composition at a range of 0.1 µg to 100 mg per dose of an exemplaryvaccine composition. In an exemplary embodiment, an exemplary Phycocyanin may be presentin an exemplary vaccine composition at a range of 100 µg to 500 µg per dose of an exemplary vaccine composition.

[0059] In an exemplary embodiment, an exemplary antigen may include at least one of a viralantigen, a bacterial antigen, a cancer antigen, and combinations thereof. In an exemplary embodiment, an exemplary antigen may include at least one of HBsAg, RBD COVID-19 Ag, and combinations thereof. In an exemplary embodiment, an exemplary vaccine composition may include an injectable vaccine composition or an oral vaccine composition. In an exemplary embodiment, an exemplary vaccine composition may be administered by at least one of intramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof.

[0060] EXAMPLE 1: Preparation of C-PC and PCB

[0061] In this example, a protein part of Arthrospira platensis (Spirulina) (C-PC) and its bilinpart (PCB) were prepared and purified to be used as vaccine adjuvants. Herein, C-PC and PCBwere prepared and purified via processes similar to methods 200 and 300 describedhereinabove. Firstly, Arthrospira platensis (Spirulina) was cultured using salt medium (SM)and under specific environmental conditions, including a pH range of 8-11, light intensity of 1000-3000 lux, temperature range of 25-32 °C, and aeration (circulation) of 15 minutes twice daily. To purify C-PC, five times freeze-thawing in the presence of 0.05 M Na-phosphate buffer (pH = 7.0) was conducted. Then, sonicating at 48000 Hz for about 5 min at room temperature by ultrasonic bath was done. Then, centrifuging at a speed of 14000 rpm, a temperature of 4°Cfor about 20 min was carried out. Finally, precipitated plate was removed and supernatant wasused as the purified C-PC.

[0062] To separate bilin part of the purified C-PC via a methanolysis process, the dried andpurified C-PC was dissolved in 100% methanol, the obtained emulsion was allowed to sit for 15 minutes, and then spun at 3000 rpm for 15 minutes. To get rid of unbound tetrapyrroles and contaminants, the obtained two-phase mixture was rinsed numerous times. The supernatant was discarded. In a 500-mL distillation flask attached to a condenser, 100 mL of an alcoholic reagent (methanol) was blended with each 1 gr of C-PC. Stirring was done in a flask submerged in a silicone oil bath at a temperature 10°C above the boiling point of the reagent. Increasing the reaction temperature increases the reaction rate, and the optimum temperature was around seventy-five degrees, that is, ten degrees higher than the boiling temperature of methanol, and at temperatures higher than that, not only the efficiency did not increase, but it also caused the efficiency to decrease. The procedure was terminated after 16 hours by submerging the flask in an ice bath for 15 minutes. In the conventional reflux method, the appropriate reaction time to achieve higher efficiency is 16 hours, and longer reaction time has no effect on increasing efficiency. The supernatant is then filtered through a filter paper. The obtained deep blue solution is poured into the separating funnel and distilled water is added to the solution in a ratio of 2:1 and then a few milliliters of chloroform was added to the above solution and the top lid of the funnel was closed then thoroughly mixed, and the chloroform phase was separated. Using a freeze dryer, the chloroform phase containing PCB at the bottom of the funnel was separated, evaporated, and dried.

[0063] EXAMPLE 2: Animal testing phase

[0064] In this example, adjuvant capability of Spirulina extract, c-phycocyanin (C-PC), andphycocyanobilin (PCB), were tested along with hepatitis B surface antigen (HBsAg) in comparison with common vaccine, including hepatitis B surface antigen (HBsAg) and alum.The vaccine groups received the common vaccine and adjuvants 3 times with a 2-week interval, administered orally or by injection. Inbred BALB / c, 6-7 weeks male mice weighing 20-25 grams were housed separately in polycarbonate cages without restricting food or water access. In addition, all mice were exposed to constant temperatures (22.2 ° C) and humidity (55%). They were under 12 hours fast before treatment. The animals fasted for 12 hours before vaccination, and they fasted from water for 3 hours. The impact of adjuvant on promotion of the immune response, namely the expression of hepatitis B antibody, was examined and compared to the negative control (distilled water) and positive control (common vaccine). The common vaccine was administered intraperitoneally. Prepared adjuvants (C-PC and PCB) wereadministered to the test groups orally, intravenously, or both. Tables 1 and 2 list all pertinentinformation, abbreviations, and classifications of animal types and administered doses for injected and orally administered adjuvants, respectively. The humoral immune reactions were assessed 2 weeks after the final treatment (third dose). The average results of the groups’ dataare also presented in Tables 1 and 2. Measured factors include interleukin 4 (IL-4), interferongamma (IFN-γ), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Levels of ALT and AST were measured by biochemical method using an autoanalyzer, and other immunological factors including IL4, IFN-γ and HBs Ab were evaluated using ELISA Technique.

[0065] Table 1. Average results of studied factors in injectable adjuvant consumptionConsumption dose Average range of the studied factorsGroup Abbre BILB / Adjuvants Vaccin HBs IL-4 IFN-γ ALT AST name viation cNo. e Ab (pg / ml) (pg / ml) (U / l) (U / l) (IU / ml) common I.PCB+3 500µg10 µg 1349 11.0800 25.4100 70.66 263. HBsAg Vac. injectable .666 67 6667 vaccine + PCB used 7 PCB beside vaccine Control–I.C - 3 - - 132.2.6433 10.7043 86.33 292. (without 9333 33 3333 treatment) Control + I.C + 3 - 10 µg 11188.2333 19.3333 47.00 171. (common .666 00 6667 HBsAg 7 vaccine) Total 15 - 888.886.42 17.5282 67.0667 237.2

[0066] Table 2. Average results of studied factors in oral adjuvant consumptionGroup name AbbrBILConsumption dose Average range of the studied factorseviat B / cNAdjuvants Vaccine HBsAbIL-4 IFN-γ ALT AST ion o. (IU / ml) (pg / (pg / ml) (U / l) (U / l) ml) Spirulina / ArtO.SE 4 1000 µg - 33.00 146.760.58 136.5 304. hrospira oral SE 800 82 0 75 platensis 2 times 0 extraction weekly for 8 weeks Spirulina / O.SE6 1000 µg 10 µg 493.33 370.821.9662.17 102.Arthrospira + oral SE 244 08 17 platensis Vac 2 times 4 extraction + weekly for common HBs 8 weeks vaccine C-PC O. C- 6 500 µg oral - 52.00 143.397.25 106.0 293. PC C-PC 133 49 0 50 2 times 3 weekly for 8 weeks C-PC + O.C-4 500 µg oral 10 µg 424.75 382.778.8272.50 210.common HBs PC + C-PC / 2 133 35 00 vaccine Vac times 3 weekly for 8 weeks C-1(sterile O.C-3 - - 17.00 105.395.2959.00 114.w 355 41 67Total / mean 32 - - 199.69 268.832.2794.63 208.300 94 94 0

[0067] In complementary to Table 1, FIGs. 5, 6 and 7 demonstrate the effect of the PCBadjuvant effect as an injectable form of C-PC on HBsAb, IFN-γ and IL-4, respectively. FIG. 5shows comparison of average level of hepatitis B surface antibody (HBsAb) expression in immune response to hepatitis B surface antigen (HBsAg) in different groups receiving the phycocyanobilin (PCB) injectable adjuvant, consistent with one or more exemplaryembodiments of the present disclosure. It may be seen from FIG. 5 that the amount ofexpressed hepatitis B surface antibody (HBsAb) varied significantly between the different groups. Comparing the test group with the positive controls, the I.PCB + Vac. group showed a significantly higher level of antibody expression. Based on

[0068] FIG. 6 shows comparison of average level of expression of interferon gamma (IFN-γ)in different groups receiving the injectable adjuvant, consistent with one or more exemplaryembodiments of the present disclosure. FIG. 6 demonstrates that the level of IFN-γ factor inthe I.PCB+ Vac. group was significantly higher from the others. Based on Table 1, theimprovement rate of the IFN-γ factor was approximately 2.5 times higher than that of the I.C- group.

[0069] FIG. 7 shows comparison of average level of interleukin 4 (IL-4) expression indifferent groups receiving the injectable adjuvant, consistent with one or more exemplaryembodiments of the present disclosure. According to FIG. 7, results of the effect on theinterleukin 4 (IL-4) factor shows that the I.C+group has a significant difference compared to the I.C–group. The groups were classified into 3 categories in terms of IL-4 expression, including class A of I.C -; class B of I.C+, and class C of I.C+and I.PCB + Vac. The fact that all groups receiving the vaccine with the prepared adjuvant PCB were placed in category C indicated the beneficial effect of the prepared PCB adjuvant. Similar to HBsAb and IFN-γ factors, the highest level of IL-4 stimulation was observed in the I.PCB+Vac. group. Such increase in expression compared to I.C- and I.C+(common vaccine) is about 4.2 times and 1.35 times, respectively.

[0070] In addition, FIG. 8 shows comparison of average level of alanine transaminase (ALT)changes in different groups receiving injectable adjuvant, consistent with one or moreexemplary embodiments of the present disclosure. The average level of ALT is about 86.33 forgroup IC-, 47.00 for group IC+, and 70.67 for group I.PCB +Vac. Moreover, FIG. 9 showscomparison of average level of aspartate aminotransferase (AST) changes in different groups receiving injectable adjuvant, consistent with one or more exemplary embodiments of thepresent disclosure. The average level of AST is about 292.33 for group IC-, 171.67 for groupIC+, and 263.67 for group I.PCB +Vac.

[0071] In supplementary to Table 2, FIGs. 10, 11 and 12 demonstrate the effect of the S.E(Spirulina extraction) and purified C-PC (C-phycocyanin) as different oral forms of adjuvanton HBsAb, IFN-γ and IL-4, respectively. FIG.10 shows hepatitis B surface antibody (HBsAb)expression rate in response to the oral adjuvants, consistent with one or more exemplaryembodiments of the present disclosure. Based on FIG. 11, all the groups without receiving thevaccine, including O.C- w, O.C-, O.SE (oral consumption of Spirulina extraction), and O.C-PC, are classified in category A. Both positive control groups receiving vaccine alone(including O.C+ w and O.C+) are classified in category B. Both groups received Spirulinaextract and pure adjuvant composition (ie, O.SE + Vac. and O.C-PC + Vac.) are classified incategory C. Significant increase in HBsAb expression in the O.SE + Vac. and O.C-PC + Vac.groups are at least 2.3 and 2 times the ratio of the positive control groups (O.C+w and O.C+), respectively.

[0072] FIG. 11 shows interferon gamma (IFN-γ) expression level in the oral adjuvantconsumption form, consistent with one or more exemplary embodiments of the presentdisclosure. FIG. 12 shows interleukin 4 (IL-4) expression level in oral adjuvant consumptionform, consistent with one or more exemplary embodiments of the present disclosure.According to FIGs. 11 and 12, the impact of C-PC on IFN-γ and IL-4 factors was investigated,and it was observed that the groups that received the new adjuvant with the vaccines (O.C-PC + Vac. and O.SE + Vac) differed significantly from the negative control groups (O.C- w and O.C-).

[0073] FIG. 13 shows comparison of average level of ALT changes in different groupsreceiving oral adjuvant, consistent with one or more exemplary embodiments of the presentdisclosure. The average level of ALT is about 136.50 for group O.SE, 62.17 for groupO.SE+Vac., 106.00 for group O.C-PC, 72.50 for group O.C-PC+Vac., 59.00 for group O.C-w,212.00 for group O.C+w, 58.00 for group O.C-, and 65.33 for group O.C+. FIG. 14 showscomparison of average level of AST changes in different groups receiving oral adjuvant,consistent with one or more exemplary embodiments of the present disclosure. The averagelevel of AST is about 304.75 for group O.SE, 102.17 for group O.SE+Vac., 293.50 for group O.C-PC, 210.00 for group O.C-PC+Vac., 114.67 for group O.C-w, 278.33 for group O.C+w,155.67 for group O.C-, and 202.33 for group O.C+. Although there were significant differencesbetween the groups that received the oral adjuvant, these changes did not follow a particular pattern that would suggest the harmful effects of C-PC. While the injection groups tested herein, however, did not experience any alterations in ALT or AST. Industrial Applicability

[0074] Herein, a set of compounds called Phycocyanins and their derivatives were purified,synthesized or isolated as cyclic components, and incorporated in vaccines compositions as adjuvants for enhancing immune response to exemplary vaccines. Both oral and injectable forms of these adjuvants were prepared and are usable in the form of naked or capsule and linked or separate. An expression of antibody in response to exemplary antigens can increased up to two times compared to control groups that only may receive conventional adjuvants along with an exemplary vaccine. This level of improving humoral immune response indicates a capability of this group of compounds for use in adjuvant position in several vaccines.

[0075] While the foregoing has described what may be considered to be the best mode and / orother examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0076] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes,and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0077] The scope of protection is limited solely by the claims that now follow. That scope isintended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0078] Except as stated immediately above, nothing that has been stated or illustrated isintended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0079] It will be understood that the terms and expressions used herein have the ordinarymeaning as is accorded to such terms and expressions with respect to their correspondingrespective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0080] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain thenature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0081] While various implementations have been described, the description is intended to beexemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and / or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

WHAT IS CLAIMED IS:

1. A method for enhancing immune response of a vaccine, the method comprising:preparing a Phycocyanin; and administering the prepared Phycocyanin along with a vaccine composition to a living body.

2. The method of claim 1, wherein preparing the Phycocyanin comprises preparing at leastone compound of phycobiliprotein (PBP) family, the at least one compound of PBP family comprising at least one of C-phycocyanin (C-PC), Phycoerythrin (PE), Allophycocyanin (APC), phycocyanobilin (PCB), and combinations thereof.

3. The method of claim 1, wherein administering the prepared Phycocyanin along withthe vaccine composition comprises administering a dosage in a range of 0.1 µg to 100 mg of the prepared Phycocyanin per dose of the vaccine composition.

4. The method of claim 3, wherein administering the prepared Phycocyanin along withthe vaccine composition comprises administering a dosage in a range of 100 µg to 500 µg of the prepared Phycocyanin per dose of the vaccine composition.

5. The method of claim 1, wherein preparing the Phycocyanin comprises:culturing an alga in a salt medium under specific environmental conditions; and purifying the Phycocyanin from the cultured alga.

6. The method of claim 5, wherein culturing the alga in the salt medium under specificenvironmental conditions comprises culturing Arthrospira platensis (Spirulina) in the saltmedium under specific environmental conditions.

7. The method of claim 5, wherein culturing the alga in the salt medium under specificenvironmental conditions comprises applying changes in the salt medium while culturing the alga therein, applying changes comprising at least one of enriching the salt medium with a plurality of micronutrients, changing a light wavelength of a light emitting to the salt medium, 1ceasing light emitting to the salt medium, applying food deficiency in the salt medium, and combinations thereof.

8. The method of claim 7, wherein enriching the salt medium with the plurality ofmicronutrients comprises increasing an amount of at least one of iron ion, zinc ion, magnesium ion, and combinations thereof up to 100 times in the salt medium.

9. The method of claim 7, wherein culturing the alga in the salt medium under specificenvironmental conditions comprises culturing the alga in the salt medium exposed to a lightbeam with a wavelength in a range 350 nm to 500 nm.

10. The method of claim 5, wherein purifying the Phycocyanin from the cultured algacomprises: forming a mixture by mixing the cultured alga with a first portion of a buffer solution; freeze-thawing the mixture iteratively; sonicating the mixture at a sonication intensity in a range of 20 kHz to 50 kHz for a time period in a range of 2 minutes to 20 minutes; centrifuging the sonicated mixture at a range of 15000 rpm to 20000 rpm for a time period in a range of 15 minutes to 30 minutes; collecting a supernatant portion of the centrifuged mixture; and extra purifying the supernatant portion, comprising: precipitating a pellet of the supernatant portion using an ammonium sulfate ((NH4)2SO4) solution; recovering the pellet by centrifuging at 27,000 rpm for 15 min at 4 °C; dissolving the pellet in a second portion of the buffer solution; dialyzing the pellet dissolved in the second portion of the buffer solution against the buffer solution using a dialysis membrane; and filtering an extract obtained from the dialysis membrane.

211. The method of claim 9, wherein forming the mixture comprises mixing the culturedalga with a 0.05 M buffer solution of Na-phosphate at a weight ratio of 1:1 (the cultured alga: Na-phosphate buffer solution).

12. The method of claim 5, wherein preparing the Phycocyanin further comprises obtainingPCB from the purified Phycocyanin by cleaving the purified Phycocyanin, cleaving the purified Phycocyanin comprising at least one of methanolysis of the purified Phycocyanin, ethanolysis of the purified Phycocyanin, enzymic hydrolysis of the purified Phycocyanin, acidic hydrolysis of the purified Phycocyanin, and combinations thereof.

13. The method of claim 1, wherein administering the prepared Phycocyanin along withthe vaccine composition comprises at least one of introducing the prepared Phycocyanin and the vaccine composition separately from each other to the living body, introducing the prepared Phycocyanin and the vaccine composition in combination with each other to the living body, and combinations thereof.

14. The method of claim 12, wherein introducing the prepared Phycocyanin and the vaccinecomposition separately from each other to the living body comprises: administering the vaccine composition to the living body by at least one of intramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof; and administering the prepared Phycocyanin to the living body by at least one of intramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof.

15. The method of claim 13, wherein administering the vaccine composition to the livingbody and administering the prepared Phycocyanin to the living body are done simultaneously.

316. The method of claim 13, wherein administering the vaccine composition to the livingbody and administering the prepared Phycocyanins to the living body are done with a time interval in a range of a few seconds to 24 hours or more.

17. The method of claim 12, wherein introducing the prepared Phycocyanin and the vaccinecomposition in combination with each other to the living body comprises: adding the prepared Phycocyanin to the vaccine composition; and administering the vaccine composition comprising the prepared Phycocyanin to the living body by at least one of intramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof.

18. A vaccine composition, comprising:a portion or whole of a phycocyanin of phycobiliprotein (PBP) family, the portion or whole of the phycocyanin comprising at least one of C-phycocyanin (C-PC), Phycoerythrin (PE), Allophycocyanin (APC), phycocyanobilin (PCB), and combinations thereof; and an antigen, comprising at least one of a viral antigen, a bacterial antigen, a cancer antigen, and combinations thereof.

19. The vaccine composition of claim 19, wherein the Phycocyanin compound is presentin the vaccine composition at a range of 100 µg to 500 µg per dose of the vaccine composition.

20. The vaccine composition of claim 17, wherein the vaccine composition comprises aninjectable vaccine composition or an oral vaccine composition administered by at least one of intramuscular injection, subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and combinations thereof. 4

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