Mucosal Immunization And The Use Of Plga As Carrier

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Kalaivani.M

Kalaivani. M

Immunization is one of the most important cost effective weapons for protecting individuals and the community from infectious diseases. It provides complete protection against infectious disease.The introduction of vaccines into medical practice at the beginning of the past century has had an extraordinary impact on human health and welfare and represents an unparalleled success story in modern medicine. Vaccines are considered to be the safest and most effective medical intervention currently available. In conjunction with the introduction of antibiotics and modern hygiene practices, vaccines have contributed enormously to a steady decline in the mortality and morbidity caused by infectious diseases.29

Successful immunization means complete/maximum protection against infection with minimum number of doses. But currently available vaccines needs atleast 2-3 doses for maximum immunity. Immunisation with available vaccine is difficult for massive immunization in developing countries due to inadequate compliance, need of trained professional and cost of sterility for parentral administration. The number of doses  can be reduced by developing controlled release antigen delivery systems and by improving immune responses of weak antigen.The current global scenario calls for a more-efficacious, acceptable, cost-effective and reliable method of immunization.5,7

Mucosal immunization and its advantages :

The most attractive route of vaccination is mucosal immunization, becauseof its high patient compliance, ease of administration, applicabilityto mass vaccination. It is also an effective means of immunisation due to local and systemic induction of immunity.38  Mucosal immunity generated by mucosal vaccines can serve as a strong first line defense against the pathogens infecting through the mucosal lining.8Therefore, local protection at mucosal surface as well as systemic defense is beneficial. For mucosal vaccination particulate drug delivery systems are studied widely. 3,4,23 Microparticles smaller than 10 micrometer acts as carrier for antigen and are transported directly to peyer’s patch in gut associated lymphoid tissue(GALT).15,28

Site of mucosal immunization

The mucosal immune system provides a first line defense against numerous infectious agents. Intestinal Peyer's patch (PP) and Nasal-associated lymphoid tissue (NALT) are considered as a site of induction and regulation of mucosal immune responses. PP and NALT possesses a unique antigen-sampling cell population "M cells." M cells are group of cells at the basal membrane site where T cells, B cells, macrophages or dendritic cells are situated. The antigen uptake by M cells does not result in the degradation of the antigen, but rather in the delivery of the intact antigen to the underlying antigen-presenting cells such as macrophages and dendritic cells.

Locally produced secretory IgA is considered to be most important among humoral immune factors, which constitutes over 80% of all antibodies produced in mucosa associated tissues.Most environmental pathogens enters the body through mucosa associated lymphoid tissue(MALT) after ingestion or inhalation, which are respectively taken up by Gut-associated lymphoid tissue(GALT) or Nasal-associated lymphoid tissue(NALT) or Bronchus associated lymphoid tissue (BALT).41

Gut-associated lymphoid tissue is the largest lymphoid organ in the body and is in close proximity with a vast array of antigen in the gut lumen. It is significantly different from the systemic immune system. Gut-associated lymphoid tissue contains specialised immune cells, such as intraepithelial lymphocytes (IELs) and Ag-presenting epithelial cells. The gut immune system has evolved to protect epithelial surfaces and the underlying tissues from potentially harmful environmental agents and microbial organisms. The organisation of GALT is characteristic and is composed of Peyer’s patches, lamina propria lymphocytes (LPLs) and IELs. Peyer’s patches contain five or more lymphoid follicles and are found predominantly in the terminal ileum. The centre of the follicle consists of B-lymphocytes surrounded by mantles of mixed cellularity. The interfollicular region contains T lymphocytes. The mucosa overlying the Peyer’s patches is composed of specialised epithelial cells called M cells,which are responsible for uptake of viruses, bacteria and particulate antigens smaller than 10micrometer.15 They differ from the rest of the epithelial cells by the absence of mucus, short microvilli, small cytoplasmic vesicles and few lysosomes.48 The mucous that covers normal epithelial cells acts as a physical barrier preventing attachment of luminal antigens. Absence of mucous overlying the M cells allows the ready attachment of luminal particulate antigen to M cell surfaces. The attachment of antigen and their subsequent endocytosis by M cells appears to operate selectively.

Intestinal lumen showing M-cells

Intestinal lumen showing M-cells

Figure 1:Intestinal lumen showing M-cells and their mechanism in uptake of antigen to antigen presenting cells(macrophage,lymphocytes,dendritic cells).28

The live oral polio vaccine was the first mucosal vaccine accepted for general use. Since then, similar vaccines have been developed against typhoid fever, cold-adapted influenza vaccines, cholera and rotavirus infection in their study on Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) proved that orally vaccinated mice showed higher levels of early CNS-infiltration of B cells producing anti-TMEV antibody as well as virus-specific CD4(+) and CD8(+) T cells in the CNS compared to intraperitonally immunized mice. Not only for viral vaccine ,efficacy of oral vaccination was proved in bacterial vaccines also.14

The nature of antigen exposed to NALT determines the type of immune response. Soluble antigen penetrates the whole nasal epithelium and reaches the superficial cervical lymph nodes and induces systemic immune response (IgG). Particulate antigens are easily removed by cilia in NALT but viable pathogens are able to escape from this method of excretion  and are taken up by M-cells, which are morphologically and functionally similar to M-cells in gut. After antigens are transported to NALT mucosal immune responses (IgA) are elicited.37

PLGA (Polylactic-Co-Glycolic Acid ) as a carrier in immunization

As an alternative to parenteral administration of vaccines, mocosal vaccines offer significant logistical advantages. Successful oral immunization, however, requires that vaccine antigens be protected from gastric secretions.2However, vaccines are peptide in nature and degradation of most proteins and peptides by digestive enzymes or proteolytic enzymes in the gut renders this route of delivery system ineffective, because the degraded proteins and peptides do not interact specifically with the epithelium of MALT, or induce strong immune responses. Also the hydrophilicity of certain vaccines increases its solubility  in biological fluids inturn it reduces the half life of vaccine and lowers the  bioavailability because of impermeability to mucosa. The most promising potential approaches to protect proteins and peptides from degradation in gut, to reduce hydrophilicity of certain vaccines and to increase the bioavailability of vaccines are the association of vaccines with biodegradable microparticles and to target vaccines to the antigen presenting cells in the epithelium of gut.38

 Polymer use for such purpose should posses ideal characters like ;

Ødepot formation at the site of administration,

ØShould able to present the antigen to antigen presenting cells(APC),

Øact as immunostimulant,

Øshould be ideal and safe,

Øshould elicit early, high and long lasting immune response,

Øshould be stable and chemically reproducible.

In recent years biodegradable polymer  have received much attention for the purposes of controlled drug delivery due to;

Øreduction in the number of doses needed for primary immunization

Øimproved efficiency of targeting antigen presenting cells

Ødecreasing side effects

Øoptimizing therapeutic response etc.

These studies on immunoadjuvants or polymers are a long way from rendering such vaccines strong and efficient for human use.various attempts have been madeto demonstrate the efficacy of biodegradable and biocompatible polymers carrier ( eg ;microspheres as particulate delivery systems) in order to inducesystemic and local immune responses following oral immunization.44,45 A wide range of polymeric materialshave been explored for their ability to enhance the immunogenicityof oral vaccines, such as poly (DL-lactide-co-glycolic acid)(PLGA),24,51 starch,57chitosanand other cationic polymers26,55 Of these, PLGA has already been approved as a component of number of drug delivery systems and has a long history of safeuse in humans35,46 and it have been widely explored in several immunological studies due to their biodegradability, biocompatibility, reproducibilty and slow release characteristics in vivo. 54

PLGA or poly(lactic-co-glycolic acid) is an Food and Drug Administration (FDA) approved copolymer. PLGA is synthesized by means of random ring-opening co-polymerization of two different monomers, the cyclic dimers (1,4-dioxane-2,5-diones) of glycolic acid and lactic acid.

Structure of poly

Figure:2 Structure of poly(lactic-co-glycolic acid)

Advantages with the use of PLGA:

PLGA has been successful as a biodegradable polymer because it undergoes hydrolysis in the body to produce the original monomers, lactic acid and glycolic acid. These two monomers under normal physiological metabolic pathways in the body. Since the body effectively deals with the two monomers, there is very minimal systemic toxicity associated with using PLGA for drug delivery or biomaterial applications.

Efficacy of PLGA microspheres in intranasal immunization was proved. In this study Mice were intranasally administered with enterotoxigenic E.coli colonization factor CS6 encapsulated in poly (DL-lactide-co-glycolide) microspheres (CS6-PLG) and immune responses measured were compared to that of similarly administered native CS6 and CS6 plus mutant heat-labile enterotoxin mucosal adjuvant (CS6+mLT). The study proved that the encapsulation of CS6 in PLG-microspheres showed improved immune response11.

A variety of vaccine antigens have been encapsulated in microspheres usually composed of poly (lactic/glycolic) acid (PLGA) due to their degradation rate ranges from months to years and is a function of the polymer molecular weight, the ratio of polylactic acid to polyglycolic acid residues and the size of microsphere.56 The size of microparticle should also be considered for oral delivery, microparticles less than 10 µmare readily taken up by intestinal M cells, macrophages andother antigen-presenting cells (APCs), leadsto antigen presentation in regional inductive immune sites.9,34,46 Several products using PLGA for parenteral applications are currently on the international market.39

 The above said concept had been proved in the study by encapsulating tetanus toxoid (TT) and Haemophilus influenzae type b capsular polysaccharide conjugated to TT (Hib-T) inside PLGA microspheres and found that single dose of these micro-encapsulated vaccines elicited high antibody levels which persisted for several months. 21 The efficiency of  PLGA microsphere as a potential oral delivery system for antigen was proved by delivering an immunogenic model protein IgY  and bovine serum albumin and reported enhanced immune response.25,53

New development in Mucosal Vaccines:

Tetanus toxoid:

Tetanus is a rare serious, and preventable disease that affects the  muscles and nervous system . It often arises from a skin wound that becomes contaminated by the bacterium Clostridium tetani, which is often found in soil. It is not contagious. Once the bacteria are in the body, they produce a neurotoxin (a protein that acts as a poison to the body's nervous system) known as tetanospasmin that causes muscle spasms. The toxin first affects nerves controlling the muscles near the wound. It can travel to other parts of the body through the bloodstream and lymph system. As it circulates more widely, the toxin interferes with the normal activity of nerves throughout the body, leading to generalized muscle spasms. Without treatment, tetanus can be fatal. Tetanus often begins with muscle spasms in the jaw (called trismus), accompanied by difficulty swallowing and stiffness or pain in the muscles of the neck, shoulders, or back. These spasms can spread to the muscles of the abdomen, upper arms, and thighs.There are two ways to prevent tetanus: getting vaccinated against it as part of routine immunization programs; or, after an injury that could cause tetanus, receiving a shot as post-exposure tetanus prophylaxis and are administered by injection.

More recently in a study the potential of novel tetanus toxoid (TT) loaded nanoparticles (NP)administered through oral (p.o.), nasal (i.n.) and intraperitoneal (i.p) was evaluated. The authors immunized Six week-old female Balb/c mice with TT nanoparticle by oral (p.o.), nasal (i.n.) and intraperitoneal (i.p.) .As polymer a novel polyester, sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide), SB(43)-PVAL-g-PLGA was used. Their analysis of blood samples collected at 4 and 6 weeks after immunization proved that both  p.o. and i.n. administration of TT associated NP increased serum titers up to 3 x 10(3) (IgG) and 2 x 10(3) (IgA).The authors also observed that small nanoparticle induced significantly higher titers then larger ones after oral administration and the intermediate nanoparticle induced antibodies after nasal application.31

Salmonella typhi:

Typhoid fever, also known as enteric fever, is an illness caused by the bacterium Salmonella typhi. Common worldwide, it is transmitted by ingestion of food or water contaminated with feces from an infected person.18   The bacteria then multiply in the blood stream of the infected person and are absorbed into the digestive tract and eliminated with the waste. People with typhoid fever typically have a sustained fever as high as 40° C (104° F). They may also feel weak, or have gastroenteritis, headache, diarrhea and anorexia (loss of appetite). In some cases, patients have a rash of flat, rose-colored spots. Typhoid can be treated with antibiotics like ciprofloxacin but it can be prevented by vaccines. Typhoid vaccine also available in oral formulation, which is  Live Typhoid Vaccine and it needs four doses for complete protection.

Encapsulation of vaccines in biodegradable microspheres provides high potential of immunization against bacterial infections.The protective immunity elicited by intragastric vaccination with phosphorylcholine (PC) encapsulated in poly(DL-lactide-co-glycolide) (DL-PLG) microspheres against Salmonella typhimurium(phosphorylcholine bearing bacteria) in a mouse model of invasive intestinal infection was tested.4 The results of the study highlighted the potential of antigen encapsulation in DL-PLG microspheres for eliciting protective immunity against invasive intestinal bacterial diseases and the researchers suggested that a similar strategy could be used against diseases caused by other PC-bearing microorganisms. It was also proved the same in the study by using biodegradable poly(DL-lactide-co-glycolide) (PLGA) microparticles to deliver both bacterial phosphorylcholine (PC) and dietary antigen beta lactoglobulin (BLG) by mucosal route.16

Bordetella pertusis:

Pertussis, also known as whooping cough, is a highly contagious disease caused by the bacterium Bordetella pertussis; it derived its name from a characteristic severe hacking cough followed by a high-pitched intake of breath that sounds like "whoop"; a similar, milder disease is caused by B. parapertussis.17Worldwide, there are 30–50 million pertussis cases and about 300,000 deaths per year. Despite generally high coverage with the DTP and DTaP vaccines, pertussis is one of the leading causes of vaccine-preventable deaths world-wide.

The immunogenicity and protective efficacy of systemically and orally delivered pertussis antigens entrapped in either microparticle poly-lactide-co-glycolide (PLG) or nanoparticle PLG formulations were evaluated in a murine respiratory challenge model for infection with Bordetella pertussis.13 The results demonstrate that immunization with two parenteral doses of 1 microg or three oral doses of 100 microg of pertussis toxoid (PTd) and filamentous haemagglutinin (FHA) encapsulated in PLG conferred a high level of protection against B. pertussis challenge. In another study fimbriae from Bordetella pertussis have been encapsulated in poly(lactide-co-glycolide) microparticles of a size appropriate for uptake by the immune inductive tissues of the gastrointestinal tract proved that only orally immunized animals mounted measurable immune responses in external secretions.30

Helicobactor vaccine:

Helicobacter pylori is a major cause of chronic antral gastritis and peptic ulcer diseases. Many researchers have examined the possibility of immunologically-mediated prevention of H. pylori infection using an oral vaccine. The  induction of  mucosal and systemic immune responses by oral immunization with H. pylori lysate-loaded poly(D, L-lactide-coglycolide)[PLG] nanoparticles was investigated. From this study the authors showed that prepared H. pylori-PLG nanoparticles were spherical, nonporous particles with a mean diameter of less than 1 microm. The multiple oral immunization with H. pylori-PLG nanoparticles induced significantly H.pylori-specific mucosal IgA response as well as serum IgG responses. The serum antibody subclasses elicited were predominantly IgG1 and IgG2b and they concluded that oral immunization of H. pylori-PLG nanoparticles induced the H. pylori-specific mucosal and systemic responses in mice and enhanced Th2-type responses.34

Diphtheria Toxoid:

Diphtheria is an upper respiratory tract illness characterized by sore throat, low-grade fever, and an adherent membrane (a pseudomembrane) on the tonsil(s), pharynx, and/or nose. A milder form of diphtheria can be restricted to the skin. It is caused by Corynebacterium diphtheriae, a facultatively anaerobic Gram-positive bacterium, Which can be prevented by DTP vaccine.

The efficacy of PLGA microsphere was also proved by encapsulating diphtheria toxoid (DT) in Poly-(epsilon-caprolactone) (PCL), a poly(lactide-co-glycolide) (PLGA)-PCL blend and co-polymer nanoparticles. 52

Hepatitis vaccine:

Hepatitis B Virus(HBV) infection often referred as silent killer causes acute as well as chronic necroinflammatory liver disease and it may develop into serious complications such as liver cirrhosis etc. There are about 300 million chronic HBV carrier worldwide. It can be prevented by hepatitis B vaccine.

Oral hepatitis B vaccine formulation was prepared successfully by  encapsulation of immunogenic peptide representing residues 127-145 of the immunodominant B-cell epitope of hepatitis B surface antigen (HBsAg) in poly(D,L-lactide co-glycolide) (PLG) microparticles. In their study the researchers found that Single oral immunization of mice with B-cell epitope loaded PLG microparticles (BCEM)led to significant induction of specific serum IgG and IgM anti-HB antibodies and also found that the  rapid production of antibodies against HBsAg as a result of secondary immune response the same orally immunized mice. It was also proved the efficacy of PLGA microparticles in their research on development of HBs Ag microspheres and they also targeted the nanoparticle to antigen-presenting M cells in peyer’s patch of gut and found that noval ligand-coupled antigen would be potential antigen carrier for oral targeted delivery of antigen to antigen presenting cells.49

The efficacy of HBs Ag loaded PLGA microsphere was also proved intranasally in the study on development of surface-modified DL-lactide/glycolide copolymer(PLGA) microsphere for chitosan for nasal immunisation was also studied. From their study the authors concluded that surface-modified DL-lactide/glycolide copolymer(PLGA) microsphere had demonstrated great potential as a nasal delivery system for vaccine where humoral, cellular as well as mucosal responses are desirable.27

Influenza vaccine:

Influenza, commonly known as flu, is an infectious disease of birds and mammals caused by a RNA virus of the family Orthomyxoviridae (the influenza viruses). In humans, common symptoms of influenza infection are fever, sore throat, muscle pains, severe headache, coughing, and weakness and fatigue. In more serious cases, influenza causes pneumonia, which can be fatal, particularly in young children and the elderly. Vaccinations against influenza are most commonly given to high-risk humans in industrialized countries and to farmed poultry. The most common human vaccine is the trivalent flu vaccine that contains purified and inactivated material from three viral strains.

The comparative immune response of  biodegradable microparticles loaded with influenza viral vaccine using subcutaneous and oral routes. Influenza viral vaccine was entrapped in poly(d,l-lactide-co-glycolide) (PLG) and poly(isobutylcyanoacrylate) (PIBCA) microparticles was investigated. Stability and immunogenicity of entrapped antigen were retained .In their study the authors immunized mice with microparticle loaded antigen and monitered  IgG levels in blood and IgA levels in saliva and gastric secretions and reported that IgG levels were higher if administered by subcutaneous primed by oral route compared to oral primed by subcutaneous route or subcutaneous or oral route. The IgA level in saliva and gastric secretions were also found to be higher when subcutaneous immunization was given followed by oral booster than oral priming followed by subcutaneous booster.12

DNA vaccines:

DNA vaccines was found to be  lack of adequate potency in humans due to inefficient delivery of DNA vaccines to antigen-presenting cells. One effective means of facilitating delivery of DNA vaccines to APCs is through the use of microparticles.44 More recently, it was shown that PLGA-encapsulated plasmid DNAelicited systemic and mucosal antibodies to the encoded antigen,as well as CTL responses after oral delivery in non-primateand primate models.24,35,50

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    About Authors:

    Kalaivani.M

    Kalaivani. M(Assistant professor IIMT College of Pharmacy Greater Noida)

    M. Zaffer Ahmad

    M. Zaffer Ahmad (Senior Lecturer IIMT College of Pharmacy, Greater Noida)

    Sanjar Alam

    Sanjar Alam(Research Scholar Jamia Hamdard, New Delhi)

    Sohail Akhte

    Sohail Akhter(Research Scholar Jamia Hamdard, New Delhi)