Can we develop an effective vaccine producing mucosal immunity against strep throat?

Britni, Jesus, Ashley, and Katie

A Mucosal Immune Response

A mucus cell secreting mucus Disease causing antigens, such as Streptococcus pyogenes which causes strep throat, most often enter the body through the mucosal surfaces of the throat and nose. In this way, the mucosal immune response is the primary barrier of defense preventing the disease from ever causing illness. The mucosal immune system consists of tissues, lymphoid cells, constitutive cells, and effector cells which prevent infection of the mucous membrane surface. Therefore, a vaccine which elicits an immune response by strengthening the mucosal immunity can be an effective way of targeting the pathogen before any infection occurs1).

Mucosal vaccines must produce very specific immune responses, while carefully not interfering with other immune processes. The most important function of the mucosal immune system is the synthesis of IgA and IgG, which is capable of being secreted across the mucosal layer. The mucosal immune system also figures largely in CD8+ Cytotoxic T lymphocyte responses and TH1 and TH2-type CD4+ responses. However, the production of mucosal vaccines is difficult. The actual amount of mucosal antibody is difficult to measure and tedious to quantitate. Several strategies for the delivery of a mucosal immunization, such as mucosal adjuvants and attenuated viral vectors, are being explored as effective strategies2).

While Streptococcus pyogenes may not cause any terminal illnesses, it may potentially be used to find the mechanism necessary to find a strong response. In the future, a mucosal immune reaction may be able to stop the spread of HIV by eliciting a similar response. A vaccine capable of producing increased mucosal immunity would prevent the infection from ever occuring and eliminating the symptoms associated with the disease and boosting the immune system. The design and implementation of a mucosal vaccine has several obstacles, but unlimited potential3).

Background

Strep

upload.wikimedia.org_wikipedia_commons_thumb_6_61_streptococcus_pyogenes_01.jpg_240px-streptococcus_pyogenes_01.jpgStreptococcus pyogenes is a spherical gram-positive bacterium, grows in long chains (seen in the picture on the left), and causes Group A streptococcal infections. S. pyogenes exhibits group A antigen on its cell wall and β-hemolysis when it is cultured on a blood agar plate. β-hemolysis is the complete disruption of erythrocytes and the release of hemoglobin, which is seen as areas around the colonies which are lightened and transparent. S. pyogenes is also called GAS, Group A Streptococcus. GAS is first recognized by the immune system at the mucosal layer.

The serotype (cell surface antigens) for S. pyogenes is based on its M protein, a virulence factor, as well as its T antigen. Four of the twenty T antigens have been found to be pili, which are used by S. pyogenes to attach to host cells.

S. pyogenes has many virulence factors which allows it to attach to host tissues, evade the immune system response, and spread by penetrating host tissue layers. The bacterium has a carbohydrate capsule that is composed of hyaluronic acid, which protects it from phagocytosis by neutrophils. The capsule along with other factors embedded in the cell wall (M protein, lipoteichoic acid, and protein F) facilitates attachment to host cells. M proteins inhibit opsonization by the alternative complement pathway by binding to host complement regulators. M protein can also prevent opsonization by binding to fibrinogen.

S. pyogenes secretes other proteins, of which many are virulence factors:

Streptolysin O and S: These toxins are the basis of the bacterium's β-hemolytic property. Streptolysin O is a cell poison that affects many types of cells including neutrophils, platelets, and sub-cellular organelles. This factor causes an immune response and antibody production. Antistreptolysin O can be used to detect a recent infection. Streptolysin is also cardiotoxic. Streptolysin S is a cytolytic factor that creates the zone of β-hemolysis around the colonies on blood agar.

Streptokinase: This factor enzymatically activates plasminogen, which is a proteolytic enzyme, into plasmin which then digests fibrin and other proteins.

Hyaluronidase: It was thought that hyaluronidase facilitated the spread of bacteria through connective tissues by breaking down hyaluronic acid (a key component of connective tissue). However, very few S. pyogenes are able to secrete active hyaluronidase due to gene mutations that encode the enzyme. In addition, it does not appear that hyaluronidase is needed for the bacteria to spread. At this time, the role of hyaluronidase in pathogenesis is unknown.

Streptodornase: Many strains of S. pyogenes secrete up to four different DNases, which are called streptodornases. These protect the bacteria from being trapped in neutrophil extracellular traps (NETs). They do this by digesting the NET's web of DNA, which have neutrophil serine proteases bound to it that kill the bacteria.

C5a Peptidase: This cleaves a neutrophil chemotoxin, C5a, which is produced by the complement system. C5a peptidase helps to control the influx of neutrophils early on in infection while the bacteria are colonizing host tissue.

GAS is often found in the throat and on the skin. People may carry this bacterium and show no signs of illness. Most infections caused by GAS are mild and include pharyngitis (strep throat) and impetigo (a skin rash). There are some severe, often life-threatening diseases caused by GAS. This happens when the bacteria get into parts of the body where the bacteria are usually not found, such as the blood, muscle, or lungs. Some of the diseases caused by “invasive GAS disease” are necrotizing fasciitis and streptococcal toxic shock syndrome.

In order to diagnose strep throat, a throat swab is performed. A Gram stain can be performed to show Gram positive cocci in a chain formation. An ELISA can also be performed to determine whether specific antigens are present. In addition, a culture can be grown on blood agar to show hemolysis.

Penicillin is used to treat strep infections. So far, S. pyogenes shows no resistance to the drug, although it has started to show some tolerance to it. Macrolides, chloramphenicol, and tetracyclines can be used on isolated strains tested for drug sensitivity, however, there is more antibiotic resistant concerns with these.4)

Mucosal Immunity

drawn by Ashley

Mucosal immunity is the form of protective immunity that acts at mucosal surfaces of the gastrointestinal and respiratory tracts to prevent colonization by ingested and inhaled microbes. There are mucosa-associated lymphoid tissues (MALT), such as tonsils and Peyer's Patches, that act to prevent infection. The mucosal layers are separated from the outside world by epithelial barriers. This layer of epithelium serves as the first line of defense against microbes. Epithelia and their associated glands, such as salivary glands, produce innate defenses including mucins and antimicrobial proteins. If pathogens breach the epithelial layer, mucosal tissues are the sites of intense immunological activity. Epithelial cells detect when dangerous microbial components such as PAMPs are present through pattern recognition receptors such as TLRs. They send cytokine and chemokine signals to underlying mucosal cells such as macrophages and DCs to trigger innate responses and promote adaptive immune responses. Epithelial cells are able to regulate these responses so that undesirable responses are not activated by normal flora that could lead to mucosal inflammation.5) A picture of immune responses at the mucosal surface is seen above.

IgA

A very important characteristic of mucosal immunity is the local production and secretion of dimeric IgA (shown at the right). IgA is produced in the lamina propria where mucosal lymphoid tissues are located.6) These antibodies are resistant to degradation by proteases produced both by mucosal cells and microbes. This is due to its dimerization and its high degree of glycosylation during its synthesis in mucosal plasma cells, and its association with a glycosylated fragment derived from the epithelial polymeric immunoglobulin receptor (pIgR) that mediates transport of dimeric IgA across epithelial cells to the lumen.7)

IgA is the major isotype produced in mucosal tissues because B cells migrate to these tissues, where cytokines that promote switching to IgA (LPS, TGFβ, IL-4, and IL-10) are made. IgA is the main antibody isotype in mucosal tissue because it can be actively secreted through mucosal epithelia, and thus is why mucosal tissues are the major sites of IgA production.8)

Secreted IgA has many roles in mucosal defense. One is to promote entrapment of antigens in the mucus, which prevents the pathogen from directly contacting the mucosal surface. sIgA is also able to neutralize microbes by binding to surface molecules, thus hindering epithelial attachment. IgA is also found in interstitial fluids of mucosal tissues underneath the epithelium. IgA found here is able to transport pathogens that have breached the epithelial barrier back into the lumen through pIgR or by mediating ADCC (antibody-dependent cell-mediated cytotoxicity) that leads to the destruction of local infected cells.9)

IgG

IgG can be secreted by mucosal plasma cells, however, IgG is susceptible to to degradation by bacterial proteases. Even so, intact IgG in the mucosal tissues (in the lamina propria) are able to neutralize pathogens that enter the mucosa and prevent spread.10)

M cells

Epithelium differentiates into follicular-associated epithelium, FAE. The FAE produces chemokines, CCL20 and CCL9, that attract lymphocytes and DCs that express chemokine receptors, CCR6 and CCR1. The FAE contains microfold, M, cells, which are specialized for endocytosis and rapid transepithelial transport of pathogen. M cells form intraepithelial pockets that contain B cells, T cells, and DCs. Pathogens are transported to these regions to be phagocytosed and processed. DCs are also able to migrate into the narrow spaces between epithelium where they can obtain samples of antigens from the luminal compartment. DCs that have captured pathogens can potentially interact with lymphocytes to stimulate a memory response. They can also exit the mucosa to present antigens to naïve T cells.11)

Homing

DCs that recognize antigens present them to B and T cells. This activates them, thus up-regulating the expression of tissue-specific adhesins and chemokine receptors. The chemokine receptors function as homing receptors that guide lymphocytes to the mucosa through recognition of endothelial counter-receptors in the mucosal vasculature. This response can be general, and the secretion of specific IgA antibodies can occur at multiple mucosal tissues. This response can also be specific so that the immune response is focused at that site where the antigen was initially encountered.12)

Effector Mechanisms

Other effector mechanisms participate in order to destroy infection. Inflammation at the site can occur, as well as the recruitment of phagocytes to destroy ingested microbes. IgG can be found in lamina propria in order to neutralize, opsonize, or stimulate complement activation. ADCC may occur in which NK cells target IgG coated cells in order to lyse the cells. T helper cells are able to activate macrophages to phagocytose and kill pathogens. CTLs are able to recognize and kill infected cells.13)

Mucosal vaccines

Mucosal vaccines

Mucosal vaccines are becoming increasingly important in combating infectious diseases whose pathogens infect the mucosa. Creating an effective mucosal response is mostly induced by the administration of a vaccine onto mucosal surfaces. Generating a mucosal response via mucosal vaccine has also shown to prime a systemic response when exposed later to a systemic vaccination 14). To produce a successful mucosal vaccine several factors must be taken into account. Effective mucosal vaccines must be protected from physical elimination and enzymatic digestion, they should target mucosal inductive sites, and stimulate the innate immune system to generate effective adaptive immunity 15). There are several characteristics of a mucosal vaccine that would induce an effective mucosal response.

Multimeric

Having multiple antigen sites associated rather than soluble antigen results in a better signal. This allows the cross-linking of B-cell receptors resulting in a better B-cell response.

Particulate

Particulates are attached to a larger molecule (such as a bead) which makes them more likely to stick to the mucosa or to be transported by M cells. If they were soluble antigen they may be broken down before they even reach the mucosa. Some vaccines that have some theoretical advantage for mucosal delivery is particulate vaccines. M cells are accessible to microparticles and thus can actively transport them into Peyer’s patches. These vaccines can also be readily be taken up by mucosal Dendritic Cells thus providing antigen depots.16)

Adherent

Another promising avenue for mucosal vaccines is the bacterial adhesins. Mucosal antibodies to these proteins block the pathogen’s ability to penetrate the mucosal barriers. Adhesins are very attractive options because theses proteins are highly conserved due to their association with conserved host receptor proteins. One current vaccine target is the plius-associated adhesion FimH from uropathogenic E. coli binding to manse-oligosaccharides. Thus mucosally administered vaccines containing FimH are currently in clinical trials to assess their efficacy. A recently approved acellular pertussis vaccine contains these adhesins which happen to be the filamentous hemagglutinin and pertactin, which recognize sulphated sugars on glycoconjugates and the integrin-binding protein motif Arg-Gly-Asp, respectively. Thus the adhesion-specific immunity may be a successful approach for generating mucosal protection against pathogens. This approach of preventing attachment and entry into the cells has been well utilized for viruses, and is a relatively new approach toward combating bacteria, but has enormous potential for mucosal vaccines.17)

PAMP recognition

The addition of a PAMP or the attachment of a PAMP to the antigen would stimulate an innate immune response which could then help activate an adaptive response giving an overall immunity 18). Virus-like particles and small vesicles that are derived from bacterial outer-membrane components are very promising as mucosal vaccines because of their size which is best for uptake by M cells and dendritic cells, their surface mimic those of mucosal pathogens, and they can activate an innate immune response. The only set back is that a rather large amount of virus–like particles or small vesicles would be needed for mucosal immunization because microparticles tend to become trapped in mucus, thus only a small fraction of the vaccine would be likely to enter the mucosal inductive sites.

Mucosal Response Vaccines

Although mucosal application of vaccines is attractive for many reasons, only a few mucosal vaccines, mostly oral, have been approved for human use. These mucosal vaccines include poliovirus, Salmonella typhi, and the tetravalent rotavirus vaccine that has been recently approved which are made up of reassorted rhesus-human rotaviruses. The human approved mucosal vaccines involve live attenuated pathogens and thus oral poliovirus vaccine is recommended after receiving the injected inactivated virus because a limited number of polio cases occur after immunization with the live attenuated virus. Administration of this vaccine can be seen below as the polio vaccine is being administered to a young child. Another oral vaccine is the typhoid fever vaccine that consists of attenuated S. typhi strain Ty21a. However, there are advanced clinical trials of a new cold-adapted influenza virus vaccine which is administered nasally which has been show to generate protective immune responses. The reason why live pathogens are so effective as mucosal vaccines is in part due to their adaptation to survive in lumenal environments and to efficiently invade organized mucosal lymphoid tissues. Two of the most effective oral vaccines (mentioned before), live attenuated poliovirus and live attenuated S. typhi are derived from pathogens that have preferentially adhere to M cells and exploit M-cell transport to invade mucosal lymphoid tissue in the intestine. These vaccines can be slightly protected from degradation by oral delivery in enteric-coated gelatin capsules or by inclusion in coploymeric micropartides, liposomes, or proteosomes. The antigen uptake and immune responses have been shown to increase when the vaccine antigens on the mucosal surface are delivered in adherent gel-forming polymers. Another thing that enhances mucosal immune responses is the coupling of the antigen with proteins that happen to be adherent to epithelial surfaces.19)

Oral vaccines are not the only approach that is being tested to provide mucosal immunity. A great deal of research has been done using DNA vaccines. In fact, plasmid DNA has been used in several clinical trials to induce protection against several pathogens. These pathogens include hepatitius B virus, herpes simplex virus, HIV, malaria, and influenza. However, these attempts have not induced the production of antibodies and CTLs in the mucosal compartment. Limited success has been made in inducing mucosal immunity in laboratory animals with DNA vaccines by using cationic lipids, as well as immunostimulatory CpG dinucleotide motifs. However, clinical trials on humans are yet to be reported.20)

It would be ideal to develop a vaccine that would provide both humoral and cell-mediated protection at both the mucosal surface and throughout the body. Thus nasal vaccination has drawn some attention for its potential to create this protection. Nasal vaccines administration has been shown to induce specific mucosal IgA antibody responses in the salivary glands, upper and lower respiratory tracts, male and female genital tracts, and the small and large intestines in humans. These nasal vaccines can also induce CTLs in distant mucosal tissues that include the female genital tract. These vaccines along with particular live viral vectors have also been shown to generate systemic antiviral CTLs and IgG at concentrations that very similar to the concentrations induced by parenteral vaccination routes. However, nasal vaccinations do not work effectively for all species nor do they induce all of the systemic responses more effectively than other vaccinations. For example, rectal immunization in mice with a non-living peptidebased vaccine was more effective than nasal vaccines at inducing systemic CTLs. This implies that determining what mucosal vaccination to use requires consideration of the species, the nature of the vaccine, and the site of challenge. There are also some other difficulties in developing a good mucosal vaccine. Mucosal vaccines that are administered orally or deposited directly on mucosal surfaces are being diluted in mucosal secretions, captured in mucus gels, attacked by proteases and nucleases, and excluded by epithelial barriers. Due to all of these factors a large does of the vaccine is needed and is not know how much of the vaccine actually crosses the mucosa. Thus mucosal vaccines will have a higher degree of effectiveness if they can mimic successfully mucosal pathogens and that they are multimeric, particulate, adhere to mucosal surfaces, efficiently stimulate innate responses, and evoke adaptive immune responses that correlate with the target pathogen.21)

Thus live attenuated pathogens as mucosal vaccines have been shown to be the most effective mucosal vaccines because they can activate multiple innate responses and the importance of innate immunity in the development of the adaptive immune responses. However, these vaccines present several safety and acceptability issues that can reflect innate immune responses and inflammation. For example, in nasal vaccines there is a possibility of retrograde transport to the brain through olfactory nerves. Therefore, a vaccine as effective as using live attenuated pathogens has to be developed so that the vaccine is also shown to be safe with minimal health risks.22)

Strep Throat Vaccine

There are several routes that can be used to administer a mucosal vaccine such as the oral, nasal, rectal and vaginal routes. The nasal route provides the broadest mucosal immune response and results in an antibody response in the upper airway mucosa and regional secretions 23). In the case of Streptococcus pyogenes a nasal vaccine would prove to be the most effective due to the site of infection of the organism.

The most effective way to provide a strong and natural response to a vaccine would be to use the closest thing possible to the actual organism without causing disease in the individual. A live attenuated vaccine uses the living pathogen with an alteration that prevents the disease when administered in the vaccine 24). Streptococcus pyogenes produces a hemolytic exotoxins which causes red blood cells to lyse. The genes that code for the exotoxins could be deleted in the vaccine which would eliminate this adverse effect of the infection. S. pyogenes contains a surface protein antigen M which helps the bacterium resist phagocytosis, multiply rapidly in human tissues, helps the bacterium adhere to host cell surfaces and initiate disease. Excluding the M surface protein from the bacterium in our vaccine would help to lessen the ability for the organism to cause disease. The organism still needs to be able to attach to the mucosa for an effective immune response and can do so using lipoteichoic acid (LTA) which is present on the cell wall. LTA also helps the organism to resist phagocytosis, proliferate, and begin to invade the local tissues. The biological activity of LTA would allow the mucosal immune system to react to the invader without as sever effects as if the M surface protein was still present 25).

After adhering to the mucosa S. pyogenes could either be detected directly by a dendritic cell extending through the epithelial or it could be transported through the mucosa and epithelial by M cells within the epithelial lining. From that point a mucosal immune response would occur which could also stimulate an innate immune response leading to humoral and cell-mediated responses as shown in the diagram above 26).

Challenges

There are risks associated with using a live attenuated vaccine. There is a chance that some people will still get the disease. In this case most individuals will get Strep at least once in their lives anyway and in most cases the disease is not life threatening. It might be better to get the disease once and obtain immunity than to not get the vaccine and have the potential to contract the disease several times in a lifetime 27).

The mucosa is exposed to a variety of microorganisms taken into the body through breathing and eating. It is also exposed to the organisms that are regularly found in this area, the normal flora. The epithelial lining regulates how much the mucosal immune system can respond so it does not trigger a response to the organisms that the area is normally exposed to 28). This presents a challenge for vaccine dosage. If the concentration of the vaccine is not high enough the mucosal immunity will not recognize it as a threat due to regulation and no immunity would develop. Finding the correct concentration is a challenge and is difficult to measure because the vaccine can be diluted in mucosal secretions, captured in the mucus, attacked by proteases and nucleases, and can be excluded by epithelial bariers 29).

Having an organism concentration that is too high could also be a problem. Activated T-cells that are specific to S. pyogenes antigens may also cross-react with heart valve antigens leading to Rheumatic Fever. It has been shown that S. pyogenes that are rich in M surface proteins seem to be more involved in leading to Rheumatic Fever. The exclusion of the M surface proteins in our vaccine may help to lessen the risk of complications 30).

The Efficacy of Mucosal Vaccine to Strep

A picture of strep infected tonsils While the illnesses caused by Streptococcus pyogenes are generally mild, there are severe complications which can result from infection. A vaccine capable of producing increased mucosal immune response could eliminate the bacterial threat before infection could take place. By targeting mucosal immunity there is a possibility of eliciting a variety a responses which would lead to increased immunity, not just protection against strep. The role of IgA as a mechanism of transporting microbes out of the epithelium and its neutralization of pathogens are integral to human health. The gastrointestinal and respiratory tracts are easy targets for pathogen entry, which could be protected by mucosal immunity. The regulatory roles of the mucosal immune cells and tissues can also be stimulated using a vaccine. The addition of a PAMP would cause the overall immune response to increase, also possible to stimulate using a mucosal vaccine. The mucosal vaccine has challenges to both creation and effective delivery which make it difficult to produce immune responses comprehensive enough to prevent disease. However, successful mucosal vaccines are possible, most likely using a live attenuated virus delivered through the nasal passage. Novel strategies to produce mucosal immunity, such as adhesins, are being created increasing the possibility of finding an effective method. New discoveries in mucosal immune function lead to the belief it will be possible to create vaccines for many pathogens using mucosal immunity. As the knowledge of mucosal immune system increases, vaccine design will become easier and effective. Therefore, it is possible to create an effective vaccine to create mucosal immune response capable of protecting against strep.

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2) , 3) Neutra, M. R., and P. A. Kozlowski. 2006. Mucosal vaccines: the promise and the challenge. Nat. Rev. Immunol. 6:148-158
5) , 7) , 9) , 10) , 11) , 12) , 13) Neutra, M.R. and P.A. Kozlowski. 2006. Mucosal vaccines: the promise and the challenge. Nature. 6:148-158.
6) Abbas, A.D. and A.H. Lichtman. 2008. Basic Immunology. pp. 167. Saunders Elsevier, Philadelphia.
8) Holmgren, J. and C. Czerkinsky. 2005. Mucosal immunity and vaccines. Nature Med Suppl. 11:45-53.
14) Neutra, M.R. and P.A. Kozlowski. 2006. Mucosal vaccines: the promise and the challenge. Nature. 6:148-158.
15) , 18) Holmgren, J. and C. Czerkinsky. 2005. Mucosal immunity and vaccines. Nature Med Suppl. 11:45-53.
16) Neutra, M. R., and P. A. Kozlowski. 2006. Mucosal vaccines: the promise and the challenge. Nat. Rev. Immunol. 6:148-158.
21) , 22) Neutra, M. R., and P. A. Kozlowski. 2006. Mucosal vaccines: the promise and the challenge. Nat. Rev. Immunol. 6:148-158.
23) , 28) Holmgren, J., and C. Czerkinsky. 2005. Mucosal immunity and vaccines. Nature Medicine 11:545-553
24) Neutra, M. R., and P. A. Kozlowski 2006. Mucosal vaccines: the promise and the challenge. Nature Reviews 6:149-158
26) , 27) , 29) Neutra, M. R., and P. A. Kozlowski 2006. Mucosal vaccines: the promise and the challenge. Nature Reviews 6:149-158
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