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As IgA are immunoglobulins associated with secretion and mucosis membranes, I am interested whether after intramuscular vaccination with Hepatitis A vaccine IgA antibodies will be produced by the immune system before an actual infection?
Also, would they be secreted into breast milk, intestine lumen etc. before the infection coming in a traditional (gestational) way?
Or does it take to have the antigen presented at the mucosis mambrane to start IgA production or secretion?
Antibodies are produced after exposure to cognate antigen.
Remember that B cells start off expressing membrane IgM and/or IgD. IgA requires class switching recombination at the B-cell receptor locus to the IgA isotype. Further differentiation of the B cells to antibody-secreting plasma cells results in soluble IgA production and immunologic memory to the antigen. Whether you'll get IgA, IgE or IgG antibodies against the vaccine will largely depend on the signals they get in the muscle, however (Figure 1). I think IgG is largely a result, since the tissue antiviral response is often interferon gamma (Figure 2).
The importance of intramuscular vaccination is outlined here, just in case it wasn't apparent.
Figure 1. Activated helper T cells that can recognize the same antigen mediate the class switch by providing co-stimulatory signals and cytokines. Source
Figure 2. Class-switching continued. Source: Parham, The Immune System, 4th Ed.
Frontiers in Microbiology
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Several different types of antigen are used in vaccines
Some vaccines contain the killed whole microorganism that the vaccine is designed to protect against. The virus or bacterium is grown in a laboratory and killed by heat or chemicals so that it is no longer infectious. 34 The injectable polio vaccine and inactivated hepatitis A vaccine are examples of this type.
Other vaccines contain only components of the pathogen as their antigens. These components can be prepared by purifying them from the whole bacterium or virus, or by genetically engineering them. 35–37 Engineered vaccines include the human papillomavirus vaccine, which protects against cervical cancer, and the hepatitis B virus vaccine.
In some vaccines, components of the pathogen are linked with proteins to create an antigen that can generate a stronger response—this allows even 6-week-old babies to make significant amounts of antibodies, which they otherwise could not do until they are older. 38 These vaccines are called conjugate vaccines and include those against Haemophilus influenzae type b (Hib) infection, meningococcal and pneumococcal disease.
Another group of vaccines is based on the toxin produced by the pathogen that causes the disease symptoms. The toxin is chemically treated to make it harmless. The antibodies produced against it can still neutralise the toxin and prevent disease symptoms from developing. Examples of this type include tetanus and diphtheria vaccines.
The hepatitis A virus (HAV) is transmitted via the fecal-oral route, usually through direct person-to-person contact or consumption of contaminated food or water (1,2). HAV infection is clinically indistinguishable from other types of acute viral hepatitis, and the illness is usually mild and self-limited when healthy persons are infected (1,2). Disease severity increases in persons who are older or immunocompromised, have chronic liver disease, or have other underlying health conditions (2&ndash4). Infection with HAV has not been found to cause chronic infection, although prolonged or relapsing hepatitis A has been reported (5).
Recommendations for hepatitis A (HepA) vaccine were introduced incrementally in the United States. In 1996, the Advisory Committee on Immunization Practices (ACIP) recommended routine vaccination of children aged &ge2 years who lived in communities with high rates of HAV infection, for populations at increased risk for HAV infection or the adverse consequences of infection, and in outbreak settings (6). In 1999, ACIP expanded the recommendations to include routine vaccination for the following groups: 1) children aged &ge2 years in 11 states (Alaska, Arizona, California, Idaho, Nevada, New Mexico, Oklahoma, Oregon, South Dakota, Utah, and Washington) with average incidence rates that were at least twice the national average during 1987&ndash1997 (i.e., &ge20 cases per 100,000 population) and 2) consideration of routine vaccination of children aged &ge2 years in six states (Arkansas, Colorado, Missouri, Montana, Texas, and Wyoming) where average incidence rates were greater than, but less than twice, the national average (i.e., &ge10 but <20 cases per 100,000 population) (7). In 2006, ACIP recommended routine HepA vaccination of all children aged 12&ndash23 months (8). These recommendations resulted in a 95.5% decrease in reported hepatitis A cases during 1996&ndash2011 (9). Small increases in cases occurred in 2013 and 2016 attributed to foodborne outbreaks associated with contaminated food (10,11). Beginning in 2016, greater increases in the number of reported cases occurred across the United States, primarily from widespread outbreaks of hepatitis A from person-to-person transmission (12). Low adult HepA vaccination coverage and high population susceptibility to HAV infection allow outbreaks to continue to occur (13,14). This report supplants and summarizes previously published recommendations from ACIP for the prevention of HAV infection in the United States. The recommendations can be used by health care providers to update the current practice for providing HepA vaccines for preexposure and postexposure prophylaxis.
A. Organisms Causing Nosocomial Infections
Escherichia. coli, Enterococcus, Staphylococcus aureus, and Pseudomonas are responsible for one half of all nosocomial infections.
B. Factors Fostering Nosocomial Infections
1. immunocompromised patients people with AIDS, organ transplant recipients (they take immunosuppressants so that the organ will not be rejected by their body), the elderly, cancer patients, patients taking steroids (ex. those with asthma).
2. invasive medical procedures - ex. blood drawing, i.v.'s, urinary catheters, endoscopes, implants, coronary bypass surgery, hemodialysis, gynecological equipment, tooth extractions, injections
3. antibiotic resistance - many bacteria found in hospitals have developed antibiotic resistance .
C. Types of Nosocomial Infection: (From most common to least common)
1. UTI's (urinary tract infections) - usually E. coli, Proteus, Klebsiella, Enterobacter can be from catheterization more commonly results from improper hygiene (wiping the wrong way).
2. surgical wound infections - most commonly Staphylococcus aureus & enterics at least 10% of surgery patients develop an infection despite scrubbing, etc.!
3. respiratory tract (ex. pneumonia) - include Streptococcus, Staphylococcus, Pseudomonas aeruginosa, enterics.
4. skin infections - particularly in newborns (usually Staphylococcus aureus ) & burn victims (usually
D. Nosocomial Infection Control
1. hospitals hire hospital epidemiologists.
2. once an epidemic is recognized, take cultures from hospital workers.
4. patient isolation reverse isolation separates infection-prone patients from sources of infection (ex. the boy in the plastic bubble).
4. enforce CDC program.
5. treat every patient as if they are infected with AIDS.
IV. PUBLIC HEALTH: PREVENTING DISEASE - PROPHYLAXIS
Public health deals with disease prophylaxis (prevention) 2 methods of prophylaxis:
1.) decrease or eliminate the reservoir or interrupt disease transmission.
2.) immunization - artificially augments the body's natural immune defenses.
A. Decrease or Eliminate the Reservoir or Interrupt Disease Transmission
1. Clean Water - diseases such as cholera, typhoid fever, & diarrhea can be spread when human sewage contaminates the water supply.
2. Clean Food - pasteurization, boiling, adequate cooking, refrigeration prevent food poisoning, trichinosis (roundworm), salmonellosis, tapeworm infection, etc.
3. Personal Cleanliness - hand washing of #1 importance.
4. Insect Control - to decrease mosquito populations early programs drained swamps, screened living areas, used mosquito netting, used insecticides such as DDT (until it was found to be carcinogenic to humans!) now efforts concentrate on educating the public to remove stagnant water biological control is also used - ex. Gambusia, the mosquito fish, was introduced to the U.S. this fish feeds on mosquito larvae
5. Prevention of STD's - public education, limit sexual exposure, use of condoms.
6. Prevention of Respiratory Diseases - isolate infected individuals, wear face masks most effective way is immunization.
1. Active Immunization (= Immunization or Vaccination)
a. Active Immunization Defined - a person's own immune system is stimulated, memory cells are produced to protect against future natural infection.
b. Vaccine Defined - an agent containing antigen capable of inducing active immunity without causing disease vaccines must be safe & immunogenic (stimulate an immune response strong enough to confer protection against natural infection) vaccines can be given orally, subcutaneously (below skin), or intramuscularly some stimulate both Ab & cell mediated immune responses, other stimulate primarily Ab mediated immunity.
c. Types of Active Vaccines
1.) attenuated Live, weakened viruses or bacteria virus is cultivated in the lab until it loses its virulence the organism is then injected into a human and allowed to multiply may cause a limited infection, usually without serious illness provides strong & long-lasting immunity. Ex. tuberculosis (b), oral Sabin polio (v), mumps (v), measles (v), rubella (German measles) (v). The latter 3 are referred to as MMR. The fairly new chicken pox vaccine is also attenuated. This type of vaccine is not recommended for those who are immunocompromised.
2.) inactivated (killed) - By heat or chemical agents such as formalin, phenol, or acetone process can destroy the Ag's that stimulate immunity (ex. heat denaturation of protein Ag's) inactivated microorganisms can't multiply in host so vaccine dose must contain enough Ag to produce a protective immunologic reaction usually requires a booster Ex. pertussis (b), typhoid fever (b), rabies (v), Haemophilus influenzae type B (b) (causes meningitis), injectable Salk polio (v) (sometime referred to as IPV inactivated polio vaccine), cholera (b), viral influenza
Haemophilus influenzae type B - combined polysaccharide Ag with a protein to make it more powerful (polysaccharides are weak stimulants of Ab production) called a protein conjugate vaccine.
3.) genetically engineered Genetic engineering & recombinant DNA technology have allowed us to use bacteria to produce the protein antigens found in the capsids of certain viruses and the cell envelopes of bacteria. Scientists determine the genetic code for these antigens & insert the gene into the chromosome of bacterial cells. The bacteria produce the antigens coded for on the inserted genes when they go through their regular process of protein synthesis. These antigens can then be injected as a vaccine (your body doesn't care if the protein antigens are in the real viral capsid or if they were made by a bacterium they are the same proteins & your body's immune system will respond to these antigens in the same way). These vaccines do not pose the same risks as inactivated and attenuated viruses! Examples:
Pertussis (b) the inactivated vaccine contains many Ag's that contribute to the frequent undesirable side effects of this vaccine an acellular, genetically engineered vaccine has recently been licensed it has fewer side effects, but may not stimulate vigorous immunity.
Hepatitis B (v) - originally produced from recovering viruses from the serum of infected patients, which could have other diseases like AIDS (made people nervous) now a genetically engineered vaccine.
4.) toxoids - for diseases caused by exotoxins rather than the microorganisms themselves, vaccines are made of toxoids (toxins that have been modified by heat or chemical agents to render them harmless) toxoids stimulate the production of Ab's called antitoxins ex. tetanus (b), diphtheria (b).
a. Defined - Ab's from an immune person or animal are transferred to a patient like an Ab transfusion!
1. gamma globulin , a collection of Ab's from the pooled serum of many different donors
2. special preparations contain high titers of specific Ab's ex. varicella zoster (v), (chickenpox & shingles), tetanus (b), mumps (v), measles (v), hepatitis A & B (v), rabies (v), pertussis (b)
c. Advantages - even severely immunosuppressed patients can be protected & protection is immediate.
d. Disadvantages - protection lasts only as long as the Ab molecules survive in the recipient months if from a human, only weeks if from an animal also a risk of serum sickness.
Serum sickness - Occurs when proteins from animal serum are used in medical therapy ex. horse antiserum is used in the treatment of venomous snake bites. Small concentrations of venom are injected into the horse to get it to produce antibody against the toxin. Patients then receive an infusion of these horse antibodies to bind to the snake venom antigen in their blood. The patients may produce antibodies against the horse antibodies, forming large complexes that are deposited in the tissues.
3. Boosters Immunity is not always life-long. Booster shots boost immunity by greatly increasing the numbers of antibody.
V. NOTIFIABLE DISEASES
These are infectious diseases that are potentially harmful to the publics health and must be reported by physicians to the CDC. Make sure you can list some of these!
In this study, we comprehensively characterized the immune system of young and older subjects across multiple technological platforms and identified new candidate traits that associate with and partially predict the humoral response to vaccination in both a young and an old cohort.
We first characterized the reactivity of influenza-specific antibodies recognizing HA peptides from baseline serum samples with the aims of (1) examining the HA-linear epitope specificities in the pre-vaccine antibodies and (2) finding the minimal set of pre-existing antibodies that could potentially explain the negative effect of baseline HAI titers on the response to the influenza vaccine observed here and elsewhere ( Beyer et al, 1996 He et al, 2008 ). We found a set of four peptides, the reactivities of which correlated with the pre-vaccination HAI titer. In two of these, robust activity negatively correlated with the HAI response to influenza vaccination. Thus, our system can rapidly identify GRs from PRs based on their pre-vaccination antibody repertoire against HA protein regions with known neutralization activity ( Khurana et al, 2009 ), as conserved sequences are characteristic for neutralization epitopes on HA across different influenza virus strains ( Yamashita et al, 2010 ). The negative effect of pre-existing antibodies on the response to vaccination has been suggested to be due to pre-existing flu-specific memory CD4+ T cells that inhibit antigen-presentation by dendritic cells, thereby suppressing the subsequent CD4+ T-cell help to B cells ( He et al, 2008 ). Therefore, it is possible that such memory CD4+ T cells are able to maintain a background T-cell-dependent antibody production represented by the reactivities against the linear epitopes we found here. Further studies are needed to clarify the mechanisms underlying these observations.
We identified both negative and positive peptide predictors of the HAI response, many of which were age-associated. These may partially explain the overall poor response observed in aging. However, neither of the reactivities to peptides associated with pre-vaccine HAI changed with age, although the levels of baseline HAI titers were lower in older subjects. This could be due to the limitations of the assay that (1) does not detect conformational epitopes and (2) does not allow for the identification of antibodies that depend on glycosylation for binding. Several previous studies have compared the pre-GMT (baseline titers) with influenza in elderly versus young individuals with no general consensus ( Clark et al, 2009 Vajo et al, 2010 Chen et al, 2011 Ehrlich et al, 2012 ). These discrepancies have been examined in detail by Sasaki et al (2011) who used plasmablast-derived polyclonal antibodies and found greater influenza-specific broad reactivity of antibodies from older versus young vaccine recipients. Thus, the pre-GMT in the elderly is more dependent on their previous exposure history than that of young subjects. This is relevant for vaccinology, as it suggests differential strategies in young versus older subjects based on the historical findings of circulating strains.
Additional predictors of vaccine response included various immune features connected with apoptosis function. Consistent with this, apoptosis-deficient (lpr) mice exhibited poor serologic responses to the influenza vaccine. It is known that the lpr mice have defects in T-cell and B-cell development. Thus, our results might not be only due to apoptosis defects in these mice (although this is suggested by the apoptosis gene modules and sFasL), but these other factors. However, there is no agreement as to whether the germinal center (GC) formation and B-cell responses in these mice are normal ( Smith et al, 1995 Takahashi et al, 2001 ). In particular, our results argue against a previous study that reported memory and antibody-forming cell populations appear to be normal in lpr mice ( Smith et al, 1995 ). However, more recent studies have shown that these mice have defects in clonal selection and the establishment of the memory B-cell repertoire ( Takahashi et al, 2001 ).
Apoptosis has a fundamental role in lymphocyte development and in the termination of the immune response. In the GC reaction, an optimal apoptotic machinery ensures the survival of only high-affinity plasmablasts ( Smith et al, 2000 Takahashi et al, 1999 ), which we found to decrease with age in a recent study ( Sasaki et al, 2011 ). In addition, a functional Fas-mediated pro-apoptotic program is required for clearance of reactive T and B cells after an immune response takes place ( Nagata, 1999 ), as well as for augmentation and maintenance of naive cells, which ultimately results in an optimal balance between the naive and memory cell pools ( Zhou et al, 1995 ). When an immune response ends, the expanded clones of effector cells must be reduced in size. This allows the immune system to cope with new influenza virus challenges, such as those originating from antigenic drift and/or found in a new influenza vaccine preparation. Thus, the appropriate regulation of apoptosis and rapid removal of reactive memory cells may improve immune responses to newly encountered antigenic challenges as observed in the study by Haynes et al (2005) , in which new functional CD4+ naive T cells developed after CD4+ T-cell depletion in aged and young mice, restoring optimal responses to antigen ( Haynes et al, 2005 ). This has a number of implications for vaccination and suggests possible ways to overcome, at least partially, the immune system defects observed here. This would also suggest competition and a restricted immunological niche largely occupied by memory cells that, unless cross-reactive, cannot respond optimally to novel antigens.
We also find an age-dependent reduction in the gene modules associated with apoptosis. However, at the level of the serum cytokine sFasL, there was no difference between young and older subjects. This indicates that aging results in the accumulation of factors contributing to less cell renewal and complements previous observations of decreased susceptibility to apoptosis in cells from older subjects ( Zhou et al, 1995 Salvioli et al, 2001 Hsu and Mountz, 2010 ). Indeed, Hsu et al (2006) showed that T cells from nonagenarians have much higher levels of Fas and increased activation-induced cell death than subjects 65 to 89 years old ( Hsu et al, 2006 ), suggesting that apoptosis is beneficial in maintaining good immune health and attaining a longer life span. Although the role of apoptosis in the maintenance of immune tolerance has been well established, to the best of our knowledge, its association with a robust antibody response to a vaccine has not been shown previously.
The accumulation of memory cells in those older subjects who do not respond to vaccination could also be due to chronic antigenic stimulation. Thus, it has been postulated that the constant activation of clonal T-cell populations may lead to increasing numbers of memory cells filling the immunological space and compromising responses to newly encountered antigens ( De Martinis et al, 2005 ). Our results indicate that the increasing size of the memory compartment in older individuals that do not respond well could result from a loss of pro-apoptotic activity and increased levels of cell survival factors. Indeed, recent investigations have shown that memory CD4+ T cells are more resistant to apoptosis than naive CD4+ T cells ( Grayson et al, 2002 Jaleco et al, 2003 ) and, at the gene expression level, memory CD4+ T cells express lower levels of pro-apoptotic-related genes and higher levels of cell survival and proliferation-related genes ( Liu et al, 2001 ).
Our study also identifies a link between cell survival and proliferation, and defective antibody responses. We found that the CD40L gene clustered in a module enriched for proliferation genes (module PROL), which is a negative indicator of the vaccine response. Although molecules in the gene module PROL (e.g., CD40L, CD320) are critical for plasmablast growth and survival, results from different studies suggest that elevated proliferation signals from these molecules have a detrimental effect. For example, heightened CD40 signaling causes B cells to shunt into an extrafollicular plasma cell fate and this prevents the generation of long-lived bone-marrow plasma cells. This has consequences for the B-cell response, including the premature termination of the humoral immunity and the disruption of GC formation in vivo ( Erickson et al, 2002 ). Therefore, it is appealing to hypothesize that weaker responses to the vaccine in individuals with augmented CD40L expression result from the preferential generation of extrafollicullar plasma cells that compromise the accumulation of somatic mutations in the GC B cells, dampening late-appearing high-affinity antibodies, as demonstrated in transgenic CD40L mice ( Kishi et al, 2010 ). An interesting finding in support of this hypothesis is the impairment in GC formation found in old mice with poor responses to vaccine ( Eaton et al, 2004 ). Further studies in humans could potentially address this.
Our results also suggest the presence of other genes in the PROL module with similar functions to that of CD40L, which could contribute to the diminished antibody responses. For example, the gene CD320 has been shown to participate in GC differentiation by directing B cells to mature into plasma cells ( Zhang et al, 2001 ).
The large variation in the responses to vaccines in humans creates a number of challenges. We have utilized a ‘systems biology’ approach that allows us to embrace this variation and to integrate diverse measurements in the same individuals to generate hypothesis on how a system functions. Systems biology approaches, which have been extensively applied in the study of metabolism networks and genetics, have only recently been utilized in vaccinology ( Gaucher et al, 2008 Querec et al, 2009 Nakaya et al, 2011 ). For instance, a recent study by Nakaya et al (2011) has been successful in finding features of the innate immune response, 3 and 7 days after influenza vaccination, which partially predict the subsequent antibody response in young, healthy adults. One of the genes the authors found in their predictive signatures and confirmed in a mouse model, encoding for the Ca2+/calmodulin kinase IV (CaMKIV), was inversely correlated with the HAI response to the influenza vaccine. This kinase has been implicated in the regulation of CRE-dependent transcription ( Sato et al, 2006 ) and, consistent with our findings, a recent study has shown the requirement of CaMKIV for the accumulation of the anti-apoptotic molecules Bcl-2 and Bcl-x and the survival of DCs ( Illario et al, 2008 ). Despite the fact that we did not find CaMKIV in our predictive gene modules, we hypothesize that CaMKIV could also prevent apoptosis in other immune cells, resulting in an overall low cell renewal and poor immune response. The study of Nakaya et al (2011) is therefore complementary to the work described here, which identifies biomarkers before vaccination in different compartments of the human immune system, and across age groups. Being able to predict whether or not an individual will respond productively to a given vaccine is important clinically, because if the prognosis is poor, one might choose not to vaccinate or choose a more potent formulation. For example, in seasonal influenza vaccines, there is a 4 × formulation that has been reported to be more efficacious than the standard one used here ( DiazGranados et al, 2013 ), and adjuvanted influenza vaccines, although not yet licensed in the United States, are available in many other countries.
In summary, we have used immune aging as a model for impaired immunity and identified biomarkers that point to what factors have the greatest role in the response to a seasonal influenza vaccine, now recommended annually for all persons older than 6 months of age with few exceptions. An important feature of our approach is that each individual is profiled for many facets of the immune system. This enables us to observe within single individuals or across groups of individuals the relationship between multiple factors as well as the effects these factors have on the immune response. Ultimately, this methodology, applied to further studies of influenza or other vaccines and infections, should enable us to gain a more complete understanding of immune system function and dysfunction, as well as identifying key variables and mechanisms of immunological health ( Davis, 2008 ).
Next to clean water, vaccines have had the most important significance in terms of prevention and treatment of diseases, becoming the most successful medical invention in the past Century. Vaccines have evolved significantly since the development of first successful smallpox vaccine in 1796. Live-attenuated and whole-inactivated vaccines form the basis of most vaccines approved for clinical use. With attenuated or inactivated virus, there is a constant risk of reversion of the killed and/or attenuated pathogen in these vaccines to its virulence form, leading to a pressing need for safer vaccines. Advances in the field of biotechnology and recombinant DNA technology in the early 1980s allowed for engineering of subunit protein antigens. These synthetic antigens offered several prominent advantages over conventional vaccines, such as improved safety, higher yield, improved stability, and lower cost of production[1, 3]. Higher purity and safety of these antigens came with a drawback of being less potent and immunogenic compared to attenuated/inactivated vaccines.
Thus, a need to provide an additional or &lsquoexternal&rsquo innate immune activation along with subunit antigens was evident. This led to addition of immunostimulatory components to the vaccine antigen, called adjuvants (in Latin, it means &lsquoto help&rsquo), to enhance immunogenicity of the immunogen . Over the past two decades,research on vaccine adjuvants has grown remarkably, with several new generation of adjuvants being included in licensed vaccine products against infectious diseases such as malaria, influenza, shingles and many more.
Classification of Vaccine Adjuvants
Vaccine adjuvants have evolved in the past Century from only Alum and emulsion based adjuvants administered clinically between 1920s-2000s to next generation adjuvants that include combination of first generation and newer molecular specific adjuvants . Figure 1 illustrates a timeline of key milestones in the development of adjuvants in vaccine products. Vaccine adjuvants are of a diverse family, and hence, cannot be defined using a single integrated structure. They comprise of several naturally occurring or synthetic materials that boost the immunological effect of the antigen. This in return provides advantages such as, antigen dose reduction, cutback on the number of vaccinations, improving the quality of immune response, and in some cases potentially increasing the stability of the final vaccine product .
Figure 1.Key developments and milestones in vaccine adjuvantsand a timeline for approval and marketingof the vaccines containing adjuvants. Top of the arrow indicatesthe licensure of vaccines and the bottom of the arrow shows key stages in clinical development.
There are several vaccine adjuvants that are approved and used in commercial productsand other currently in clinical development (Table 1). These vaccine adjuvants can be broadly classified into two main classes based on their mechanism of interaction with innate immune system &ndash First class are adjuvants that effectively present the antigen to the innate immune cells at the site of injection to facilitate rapid uptake of the antigen for enhanced immune response . These first generation are also called as particulate adjuvants more so because of their physicochemical properties and particle size that would mimic &lsquopathogens&rsquo and act as efficient delivery vehicles for antigen also providing for added antigen stability . Alum salts, emulsions, liposomes are examples of this class of adjuvants. Alum salts have been used as adjuvants from as early as the 1930s and are considered as the &lsquogold standard&rsquo in the field of adjuvants. Second class would be immune potentiators that provide specific innate immune signals through Pattern Recognition Receptors (PRRs) such as Toll-like Receptors (TLRs). These immune potentiators represent an array of molecules from natural products, to semi-synthetic and synthetic molecules. For most instances, these molecules when injected alone manifest unwanted, and thus undesired, pharmacological effects. The newer generation adjuvants are essentially combination adjuvants that consist of a particulate/delivery vehicle carrying antigen and or immune potentiator.
Table 1. Specific examples of adjuvants used in commercial vaccines as well as those in clinical development
C. Mechanismsof Adjuvant Action
Several factors affect the adjuvant immune action once administered, such as antigen-adjuvant interaction, mechanism of adjuvant action, choice of adjuvant, toxicity or local/systemic reaction of the adjuvant. Different adjuvants have several proven or proposed mechanisms of action (Figure 2) to elicit cellular and/or humoral immune response. Thus, prediction of an immune response post adjuvant administration becomes rather difficult. Janeway  described adjuvant mechanism of action as &lsquoimmunologists dirty little secret&rsquo. Schijns  has systematically summarised several theories explaining critical pathways of adjuvanticity that gives an overall understanding of key immunological routes paving a path for novel adjuvant development. These theories explain the general mechanism, however, no adjuvant follows a single pathway of enhancing the immunogenicity. The mechanism of adjuvant action remains a &lsquotreasure&rsquo that keeps unfolding with advances in research and analytical tools for characterisation. In general, purified vaccine antigens require co-localisation at injection site to facilitate pick up by Antigen Presenting Cells (APCs). Depending on the type of antigen, and adjuvant, spatial and temporal availability of antigen and/or immune potentiator changes and whicheventually iscrucial to achieve desired immune response of sufficient magnitude and quality. Combination of adjuvants (addressed in a later section) that provide synergy in two or more pathways of adjuvant action could provide for a more potent adjuvant. In the next few sections, we have described the current status of vaccine adjuvants prominently used in licensed vaccines.
Figure 2.Putative mechanism of action of adjuvants after an intramuscular or subcutaneous injection. Reprinted with permission from Nature Publishing group Nature Medicine. Reed, S.G., M.T. Orr, and C.B. Fox, Key roles of adjuvants in modern vaccines. Nat Med, 2013. 19(12): p. 1597-608. © 2013
Specific Types of Adjuvants
Around 1920s, in attempt to purify tetanus and diphtheria vaccine antigens, aluminum salts (referred to as &lsquoAlum&rsquo in this paper) were added to precipitate them from the growth media. Later, it was discovered that these precipitated antigens were more immunogenic than the soluble ones. Since then, Alum-based adjuvants have been used extensively in clinical vaccines providing for a vast database for safety and efficacy of vaccine adjuvants, as well as providing for a gold standard for comparison for all future novel adjuvants. Ironically, very little is known about molecular and cellular mechanism of adjuvant action for Alum, although several theories have only recently started to surface . It was initially hypothesized that Alum enhances the recruitment of immune cells to injection site and hence uptake of antigens by APCs via Nlrp3 inflammasome pathway [14-16] which was debated by several scientists most prominent of which was work from Franchi. et al showing that although Alum produced higher humoral response via nlrp3 inflammasome pathway, its mechanism of action did not depend on it . In general, Alum promotes a strong Th2 adaptive immune response which relates to higher IgG1 antibody, thus producing enhanced humoral immune response. Other mechanisms for adjuvant action of Alum include release of DNA from cell death causing Danger-associated Molecule Pattern (DAMP) recognition, and enhanced phagocytosis by antigen presenting cells . We believe there will be more work on unraveling mechanism of Alum which will significantly help in novel adjuvant research however, certain pathways like recruitment of immune cells, enhanced antigen presentation, induction of danger signals for innate immune activation and eliciting abundant Th2 response . Alum proved as a successful vaccine adjuvant for several antigens, however, it manifested poor efficacy with certain intracellular pathogens such as influenza virus. Although, Alum alone is still used in some licensed vaccines, in the past 2 decades, advances in adjuvant research has allowed for more potent vaccine adjuvants to be used for more virulent pathogens. Alum has now been used extensively as a delivery adjuvant, to co-deliver antigen and immune potentiating molecules  such as monophosphoryl lipid A MPL (AS04 adjuvant used in Cervarix). We will discuss such adjuvants in our later sections. Alum salts, being suspensions in buffers, aren&rsquot inherently the most stable formulations. Additionally, Alum cannot be frozen as it causes significant aggregation of Alum particles, rendering them less effective in adsorbing antigen and/or immunepotentiator [20, 21]. To avoid potential freezing, Alum based vaccines are transported and stored in a very narrow temperature range. Promising proof-of-concept studies have shown the feasibility of lyophilising Alum formulations and could potentially be an alternative to stabilize Alum formulations and create a single vial vaccine making it independent of cold chain distribution [22, 23].
Emulsion adjuvants have also been around for as long as Alum in the form of Freund&rsquos complete (FCA) and Freund&rsquos incomplete (FIA) adjuvants made with mineral oil. The non-degradable mineral oil content of these adjuvants proved to produce detrimental systemic effects when administered in humans [24, 25]. Around 1980s, effort to create oil-in-water emulsion adjuvants with biodegradable oils in attempt to reduce the toxicity and maintain the adjuvanticity began. Squalene oil was abundantly used in several emulsionssuch as MF59 (developed by Novartis Vaccines) being the first squalene oil emulsion adjuvant approved for human use in Europe for seasonal flu vaccine, Fluad . MF59 has been shown to be well-tolerated and safe with millions of doses already administered [26, 27]. Composed of squalene oil and two surfactants - Span85 and Polysorbate80 - MF59 does not interact with antigen or create a depot effect like Alum. Although the exact mechanism of action is still unknown, when injected, MF59 is known to create an immune competent environment  at the site of injection which leads to an influx of antigen presenting cells eventually leading to increased uptake of the antigen. The target APC population for MF59 is monocytes and neutrophils both of which aid the translocation of antigen to draining lymph nodes where MF59 additionally primes the adaptive immune response [28-30]. AS03 (a GSK emulsion adjuvant) is another emulsion adjuvant used with pandemic (Pandemrix, GSK) and seasonal flu (Arepanrix) vaccines. AS03 is known to have an additional immunomodulatory component &alpha-tocopherol, which is known to create local and transient inflammatory response enhancing recruitment of monocytes and macrophages to injection site. Morel, et al., have shown that using a hepatitis B antigen, AS03 showed significantly higher titers compared to AS03 without &alpha-tocopherol . However, the exact mechanism of &alpha-tocopherol in providing immune-stimulation is unknown. One of the recent papers  sheds light on the downregulation of lipid-metabolism related genes in the draining lymph node which were associated with alteration of Endoplasmic Reticulum (ER) morphology. It was discovered that IRE1-&alpha, an ER stress sensor kinase is a sensor for metabolic changes induced by AS03 in monocytes, that may help in immunomodulatory function of AS03. Finally, both MF59 and AS03 have proven to be immunogenic in children, provide cross protection against mismatched influenza virus strains, and allow for antigen sparing which is important especially in pandemic setting . Emulsion adjuvants have also been targeted to deliver immune potentiators to enhance the potency of the adjuvant and thus, immunogenicity of the antigen [35, 36].
Other Lipid-based Adjuvants
Liposomes are spherical nanoparticles comprising of a phospholipid bilayer with an aqueous core and are used in vaccines for delivery of both antigen and additional immune potentiators. Based on the choice of the phospholipids and other components, liposomes can be used as a carrier by encapsulating in the core, incorporation in the bilayer, or by adsorbing on the surface. CAF01 is one of the oldest cationic liposome-containing adjuvants with Dimethyldioctadecylammonium (DDA) and Trehalose Dibehenate (TDB) is now in development for Tuberculosis (TB) vaccine [37, 38]. Recent formulation advances has allowed for a successful single vial vaccine development using CAF01 and H56, which is a multistage TB antigen by spray drying the two to obtain a single powder that can reconstituted at the time of administration. Another prominent lipid-based adjuvant is ISCOMATRIX  comprising of Immunostimulating Complexes(ISCOMS) and QuilA adjuvant (now replaced with refined Saponin preparations). These liposomal adjuvants offer strong CD4+ T-helper cell 1 type immune response as well as high CD8+ immune response  More recently, a combination adjuvant from GSK, AS01 a liposome delivering two synergistically acting immune potentiators &ndash QS21 and MPL, was developed for Shingrix, a shingles vaccine in the United States.
PRRs like TLRs  were found to be activated via Pathogen-asssociated Molecular Pattern (PAMPs) provided by TLR agonists (TLRa). These TLRa molecules are typically present in older vaccines, however resurfaced much later for their immune potentiation activity. This has allowed a better understanding of how adjuvants work, and how preferred immune responses can be induced . One of the first molecules to be identified as a TLRa was Monophosphoryl Lipid A (MPL-A) a lipopolysaccharide from gram negative bacteria. Unfortunately, lipopolysaccharides molecules like MPL from gram negative bacteria , are typically large and complex molecules, which bring significant formulation challenges. Recognition of the receptor systems such as TLRs involved in adjuvant immune potentiation pathways has allowed the discovery of Small Molecule Immune Potentiators (SMIPs) [46, 47]. These SMIPs have been used in cancer immunotherapy such as Resiquimod and Imiquimod, applied topically to melanoma cells. Thus, SMIPs can be discovered by applied this knowledge with existing synthetic chemistry to create synthetic small molecules that also offer ease in formulation . Encouragingly, SMIPs have been shown to be more potent than the large biologic molecules [49, 50] and as most immune potentiators, they also have potential for inducing unwanted systemic inflammatory responses when allowed to diffuse away from the site of injection. Hence, a formulation approach is necessary to efficiently deliver the SMIP to local immune cells, while restricting the ability of the SMIP to diffuse from the site of injection . This perceptively led to next generation adjuvants involving delivery vehicles/adjuvants that limit these immune potentiators spatially and temporally to allow for desired adjuvant action.
These are &ldquonext&rdquo generation adjuvants that essentially utilize the knowledge from existing adjuvants, mainly the immunological pathways and immune system activation, to create novel combinations that enhance the immunogenicity of the antigen in general [51, 52]. Depending on the adjuvant combination, the overall immune response can be shifted. For example, Alum when used alone promotes a Th2 dominant immune response, however when used to deliver MPL (as in AS04) the there is significant increase in antibody titers as well as the cellular response is skewed towards Th1 . Another one of GSK&rsquos adjuvant AS02 uses the emulsion AS03 to deliver MPL and was shown in a Malaria vaccine antigen study to be more potent than Alum alone, AS03 as well as AS04 . This tells us that based on the inherent disease-targeting antigen and desired immune response, the combination of adjuvants can vary and provide the expected outcome. AS01 is a classic example for not two but three adjuvants &ndash liposomes, MPL and QS21, are delivered along with the antigen. These adjuvants are presumed to act via separate pathways to provide an enhanced immune response. Recently, AS01 was licensed to use in Shingrix in the elderly, a vaccine for Shingles. AS01 has shown significantly higher cellular immune response compared to AS02 when administered with a tuberculosis antigen M72. The frequency of polyfunctional Th1 cells was particularly significantly higher for AS01 groups compared to AS02. In another scenario, Alum poses as the &ldquobest delivery candidate&rdquo for immune potentiators for several reasons such as vast database of proven safety and efficacy profile, easy to characterize and optimize the formulation [54, 55] and fairly easy to manipulate the molecule and Alum for adsorption purposes. As previously mentioned, Wu,et al., used the rational design to develop a synthetic small molecule immune potentiator and carefully modified to include the phosphate group for efficient adsorption to Alum facilitating the final Alum/TLR7 adjuvant. Other combination vaccines include E6020 which is TLR4a with MF59. This adjuvant when compared with MF59 alone, administered with meningitis B (MenB) antigens, showed higher bactericidal and serum titers . A recent study with emulsion adjuvants shows the importance of formulation in stabilizing a lipophilic TLR7 agonist and facilitating its formulation with squalene based emulsions to obtain Adjuvant Nanoemulsion (ANE) . These ANE formulations showed higher titers and favorable pharmacokinetic profile of SMIP compared to TLR7 SMIP or ANE alone. Other example of emulsion combination adjuvant is GLA-SE from IDRI, which includes Stable Emulsion (SE) delivering a TLR4a Glucopyranosyllipid Adjuvant (GLA). It has also been successfully shown that GLA-SE can be lyophilized with tuberculosis antigen  to obtain a single vial vaccine.
Future Considerations in the Development of Adjuvants
Plethora of published research with adjuvants thus far clearly point at two major considerations: first, the type of immune response elicited by the adjuvants majorly depends upon the co-administered antigen and second, evaluate the need for an adjuvant with the antigen, and then based on the desired immune response, use most appropriate adjuvant to improve vaccine immunogenicity. There are several theories on the mechanisms of currently approved adjuvants. These adjuvants, however, have naturally-derived components that are difficult to characterize and standardize, such as squalene oil (from shark liver) in oil-in-water emulsions such as MF59, AS03, and GLA-SE. Additionally, QS21 which has an active fraction of Quillaja Saponaria tree bark. The focus now should be on finding the synthetic alternatives for these components with inspiration drawn from SMIPs that have shown comparable or even better immune responses compared to naturally derived TLRa. Furthermore, use of high throughput techniques can facilitate the adjuvant research mainly finding novel molecules as well as combinations for targeting TLRs and newer targets such as Stimulator of Interferon Genes (STING), RIG-1-like Receptors (RLRs), and Nod-like Receptors (NLRs). Along with the discovery of novel immune potentiators, as seen with the summary above, formulation of these immune potentiators will play a pivotal role in shaping the immune response. Another key approach in adjuvant discovery should be the idea of simplicity meaning one should ask if the adjuvant is really needed in the vaccine, and if yes, would Alum alone generate the desired immune response. Thus, one should carefully map the desired immune response and use the existing knowledge for further adjuvant/vaccine development.
The development of novel vaccine adjuvants has been rapid in the past two decades and we believe that it will only be uphill from here. There are several unanswered questions  which would take several years to be remedied, however, we&rsquore optimistic that the as the underlying immunological principles of adjuvant action unfold, the development of better vaccine adjuvants is evident. Understanding how adjuvanted vaccines affect the immune system in general, can help answer questions about the possible adverse events and the risk/benefit profile for the adjuvant. Advancement in vaccine development programs of mechanism of action studies and systems biology analyses can significantly advocate critical decision making for adjuvant design and selection. Finally, it is imperative to truthfully communicate the vast adjuvant knowledge to mitigate skepticism regarding vaccines and vaccine adjuvants and highlight the possible advantages over safety concerns.
The authors deeply appreciate the assistance of Dr. Derek O&rsquoHagan at GSK Vaccines for discussions and assistance with the review of this article.
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18. Marichal, T., et al., DNA released from dying host cells mediates aluminum adjuvant activity. Nature Medicine, 2011. 17: p. 996.
19. Garcon, N. and M. Van Mechelen, Recent clinical experience with vaccines using MPL- and QS-21-containing adjuvant systems. Expert Rev Vaccines, 2011. 10(4): p. 471-86.
20. S., S.M., et al., Influence of formulation pH and suspension state on freezing-induced agglomeration of aluminum adjuvants. Journal of Pharmaceutical Sciences, 2012. 101(3): p. 1050-1062.
21. Braun, L.J., et al., Development of a freeze-stable formulation for vaccines containing aluminum salt adjuvants. Vaccine, 2009. 27(1): p. 72-79.
22. Hassett, K.J., et al., Glassy-State Stabilization of a Dominant Negative Inhibitor Anthrax Vaccine Containing Aluminum Hydroxide and Glycopyranoside Lipid A Adjuvants. Journal of Pharmaceutical Sciences, 2015. 104(2): p. 627-639.
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26. O'Hagan, D.T., MF59 is a safe and potent vaccine adjuvant that enhances protection against influenza virus infection. Expert Rev Vaccines, 2007. 6(5): p. 699-710.
27. Schultze, V., et al., Safety of MF59&trade adjuvant. Vaccine, 2008. 26(26): p. 3209-3222.
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29. Seubert, A., et al., Adjuvanticity of the oil-in-water emulsion MF59 is independent of Nlrp3 inflammasome but requires the adaptor protein MyD88. Proc Natl Acad Sci U S A, 2011. 108(27): p. 11169-74.
30. Seubert, A., et al., The Adjuvants Aluminum Hydroxide and MF59 Induce Monocyte and Granulocyte Chemoattractants and Enhance Monocyte Differentiation toward Dendritic Cells. The Journal of Immunology, 2008. 180(8): p. 5402-5412.
31. Garcon, N., D.W. Vaughn, and A.M. Didierlaurent, Development and evaluation of AS03, an Adjuvant System containing alpha-tocopherol and squalene in an oil-in-water emulsion. Expert Rev Vaccines, 2012. 11(3): p. 349-66.
32. Morel, S., et al., Adjuvant System AS03 containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity. Vaccine, 2011. 29(13): p. 2461-73.
33. Givord, C., et al., Activation of the endoplasmic reticulum stress sensor IRE1&alpha by the vaccine adjuvant AS03 contributes to its immunostimulatory properties. npj Vaccines, 2018. 3(1): p. 20.
34. Wilkins, A.L., et al., AS03- and MF59-Adjuvanted Influenza Vaccines in Children. Frontiers in Immunology, 2017. 8: p. 1760.
35. Baudner, B.C., et al., MF59 Emulsion Is an Effective Delivery System for a Synthetic TLR4 Agonist (E6020). Pharmaceutical Research, 2009. 26(6): p. 1477-1485.
36. Lodaya, R.N., et al., Stable Nanoemulsions for the Delivery of Small Molecule Immune Potentiators. Journal of Pharmaceutical Sciences.
37. Christensen, D., et al., Cationic liposomes as vaccine adjuvants. Expert Review of Vaccines, 2011. 10(4): p. 513-521.
38. van Dissel, J.T., et al., A novel liposomal adjuvant system, CAF01, promotes long-lived Mycobacterium tuberculosis-specific T-cell responses in human. Vaccine, 2014. 32(52): p. 7098-7107.
39. Thakur, A., et al., Immunological and physical evaluation of the multistage tuberculosis subunit vaccine candidate H56/CAF01 formulated as a spray-dried powder. Vaccine, 2018. 36(23): p. 3331-3339.
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45. Wang, Y., et al., TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS. Proc Natl Acad Sci U S A, 2016. 113(7): p. E884-93.
46. Testerman, T.L., et al., Cytokine induction by the immunomodulators imiquimod and S-27609. J Leukoc Biol, 1995. 58(3): p. 365-72.
47. Wagner, T.L., et al., Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod. Cell Immunol, 1999. 191(1): p. 10-9.
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50. Wu, T.Y.-H., Strategies for designing synthetic immune agonists. Immunology, 2016. 148(4): p. 315-25.
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Rushit N Lodaya is a doctoral candidate in the Department of Pharmaceutical Sciences at Northeastern University, Boston, MA under a Doctoral Fellowship from GSK Vaccine, Rockville, MD. He completed his undergraduate degree in Pharmacy from Mumbai University with outstanding Pharmacy student in Maharashtra state award.
Braz J Med Biol Res, December 2012, Volume 45(12) 1102-1111
Recombinant vaccines and the development of new vaccine strategies
I.P. Nascimento and L.C.C. Leite
Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brasil
Correspondence and Footnotes
Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.
Key words: Human vaccines Vectors for immunization Safety Recombinant vaccines
Most current vaccines owe their success to their ability to target pathogens that have low antigenic variability and for which protection depends on antibody-mediated immunity. This is the case for polio, tetanus, diphtheria, measles, and hepatitis B, among others ( Table 1) (1-3). As a consequence, vaccines capable of generating neutralizing or opsonizing antibodies against these pathogens were successful.
On the other hand, important cell-mediated immunity against intracellular pathogens (which in most cases leads to chronic infections) is much more difficult to obtain using current vaccine strategies. The live attenuated pathogen vaccines, which are capable of eliciting this type of response, although not often, may offer potential risks that cannot be overlooked, such as virulence in susceptible hosts and potential reversal of attenuation.
Recombinant vaccines rely on the capacity of one or multiple defined antigens to induce immunity against the pathogen, when administered in the presence of adjuvants or when expressed by plasmids or harmless bacterial/viral vectors. Recombinant protein vaccines permit the avoidance of several potential concerns raised by vaccines based on purified macromolecules, such as the risk of co-purification of undesired contaminants or reversal of the toxoids to their toxigenic forms, if considering diphtheria or tetanus toxoid vaccines, for example. Another fundamental issue overcome by this technology is the complexity involved in obtaining sufficient quantities of purified antigenic components.
However, one of the main challenges in the development of these new strategies of immunization consists of designing vaccines that elicit the appropriate kind of immune response to confer immunity mainly to intracellular pathogens and especially to those that establish chronic, often lifelong infections. For this, the knowledge of the biology of highly conserved antigens involved in pathogenesis and of the immune mechanisms that should be elicited for protection must be obtained to rationally design vaccine strategies that can overcome the low protective immunity naturally generated by infection (reviewed in Ref. 4).
Substantial efforts have been made towards the identification of protective antigens, which have been selected by several rational and experimental approaches (5,6). However, the use of these antigens as vaccines goes beyond their discovery. The development of efficient vaccines will require the combination of diverse strategies, such as different delivery systems/adjuvants, to present the antigen in a manner that can elicit an adequate and efficient immune response against these antigens. The use of novel biotechnological tools has provided a new arsenal of strategies and possibilities to the field of vaccinology. Here we review some of these strategies being currently used and discuss their potential for the generation of new human vaccines, as well as the challenges that remain to be solved for their development and use (5).
Recombinant vaccine strategies
Several genes from different etiologic agents have been cloned, expressed and purified to be tested as vaccines. There are a variety of expression systems for antigenic protein components, such as bacteria, yeast, mammalian cells and insect cells, in which the DNA encoding the antigenic determinant can be inserted and expressed. However, several factors must be taken into account before selecting the system for antigen expression. The level of expression obtained using each specific expression vector and promoter, the selection marker of choice, the presence or absence of post-translational modification by the recombinant vector, among others, are essential features that interfere in the efficacy of production of recombinant antigens as vaccines. Bacterial expression systems are the most used due to the ease of handling and to their capacity for high level expression. However, for antigens in which post-translational modifications (e.g., glycosylation) are necessary, the use of mammalian or insect cells should be considered (7,8).
Recombinant protein vaccines
Most of the vaccines under investigation today are based on highly purified recombinant proteins or subunits of pathogens (9). The classical example of recombinant protein vaccines currently in use in humans is the vaccine against hepatitis B ( Table 1) (10). Hepatitis B virus (HBV) infection is a chronic liver disease occurring worldwide. HBV presents a marked tropism for human liver cells, partially due to a specific receptor that is expressed on the surface of infected cells. The current vaccines are produced by expressing the hepatitis B surface antigen (HBsAg) in yeast cells. The HBsAg assembles into virus-like particles (VLPs), which are extremely immunogenic, making the HBV vaccine a very efficacious vaccine. The yeast expression system may secrete the antigen into the culture supernatant that can facilitate its purification (11,12). Furthermore, yeast cells offer some of the eukaryotic cellular machinery responsible for the post-translational modification of proteins, being capable of rendering proteins glycosylated. The technology of production of the HBV vaccine has been transferred to several manufacturers and the prices have decreased due to competition, which has rendered this vaccine affordable to most developing countries.
A more recently developed example of recombinant vaccine is the vaccine against human papillomaviruses (HPVs) (13) ( Table 1). HPV is one of the most common sexually transmitted diseases and this infection is associated with many types of mucocutaneous diseases in humans, including cervical, vulva, and vaginal cancers, and genital warts. There are two vaccines in use against HPV, which have both been developed based on VLPs derived from HPV-6, -11, -16, and/or -18 subtypes. These vaccines utilize the L1 recombinant proteins of each subtype, produced either in yeast or in an insect-cell system. The L1 is the major capsid protein and its expression in vitro results in the assembly of VLPs. The vaccines are given in a three-dose regimen, using aluminum potassium sulfate as adjuvant, which induces high titers of virus-neutralizing serum antibodies (13). These vaccines are proprietary and extremely expensive, and therefore will have limited accessibility for low-income countries for some time.
Even though vaccines based on recombinant proteins offer several advantages when compared with traditional vaccines, such as safety and production cost, most of them present weak or poor immunogenicity when given alone, and thereby require the use of adjuvants to elicit a protective and long-lasting immune response (14). The successful use of recombinant proteins as vaccines, including hepatitis B and, more recently, HPV, was possible due to the use of aluminium salt as adjuvant (9,13). Therefore, the investigation of new adjuvants is an extremely important field in vaccinology. The main difficulties for the development of new adjuvants involve understanding their molecular complexity and the mechanisms by which they operate to stimulate or induce the immune response. For example, the mechanism of action of the aluminum salts, which are the most commonly used adjuvants in human and animal vaccines worldwide, remains unknown. However, Richard Flavell’s group (15) recently suggested that they would activate an intracellular innate immune response system called Nalp3 inflammosome. An alternative path for antigen presentation has been the use of live vectors, such as bacteria and viruses, in which their natural adjuvant properties are explored. Formulation and safety, among other concerns, are also important aspects to be considered (14).
Live recombinant vaccines using bacterial or viral vectors
As a result of advances in the fields of molecular biology and genetic engineering it is now possible to create live recombinant vectors capable of delivering heterologous antigens by the introduction of antigen-encoding genes. The idea behind this approach is to use the capacity of infection and the immunological properties of the live vector to elicit an immune response against its own proteins, as well as towards the heterologous protein being presented (16). A number of bacteria (such as Salmonella typhi (17) and bacille Calmette-Guérin (BCG) (18) and viruses (such as vaccinia and adenovirus) (19) have been investigated as live recombinant vector vaccines. In general, these approaches have advantages that are intrinsic to the pathogen itself, such as mimicry of a natural infection, their capacity of stimulating both CD4 + and CD8 + T-cell subsets, and, in some cases, the possibility to be administered orally.
The use of live-attenuated bacterial vaccines is not novel. However, their utilization as carriers or delivery vehicles for heterologous antigen expression represents a technology with broad applicability that may have a significant impact on vaccine development. Significant advances in molecular biology have enabled precise deletions of genes encoding important virulence factors, as well as the introduction of recombinant DNA into avirulent yet immunogenic vaccine strains. Bacterial vectors have many advantages that make them attractive systems for heterologous antigen presentation. They can elicit humoral and/or cellular immune responses and can be administered orally, thereby eliciting mucosal immunity. Most are antibiotic-sensitive strains, which allow antibiotic treatment if any adverse reaction occurs. In general, they display very favorable cost-effectiveness (9).
Several bacteria have been used as vectors, such as Mycobacterium bovis BCG (18), Listeria monocytogenes (20), Salmonellae spp (17) and Shigellae spp (21), all of which have been shown to be capable of eliciting immune responses against important viral, bacterial, protozoan, and metazoan pathogens in animal models (9). Although such bacterial vectors present similar features, they have distinct characteristics that should be considered before making a choice for any one of them. For instance, while Listeria elicits strong antigen-specific T helper (Th)1-driven CD8+ T cells, BCG and Salmonella induce immune responses with mixed Th1/Th2 patterns (9,22).
Among these bacterial vectors, M. bovis BCG and S. typhi are the most representative of the current status of this approach, as it can be seen by the numerous and assorted papers that have been published using both vectors (18,23). BCG offers several features that render it an attractive vaccine vector. It is safe and has been administered to over 3 billion individuals with minimum side effects, it can be administrated soon after birth, it is a potent adjuvant, and it provides the possibility of generating T cell-mediated immunity against the cloned heterologous antigen. This last feature is considered to be an essential element of a successful vaccine against intracellular pathogens. Several examples of recombinant BCG (rBCG) expressing foreign antigens from diverse pathogens have been described, such as malaria, tuberculosis (TB), HIV, leishmania, pertussis, and others. These were demonstrated to induce both humoral and cellular immune responses and, in some cases, protection against challenge with the infective microorganism (18,23). Much work has been done on rBCG expressing HIV antigens, in which different antigens have been found to elicit specific antibodies, production of interferon (IFN)-γ, as well as to induce T helper cells and cytotoxic T lymphocytes (CTLs), thus demonstrating the ability of different strains of rBCG-HIV to produce both humoral and cellular immune responses against HIV antigens (18,24,25). Another example of promising results involves rBCG expressing the non-toxic subunit 1 of pertussis toxin (rBCG-S1PT). This strain was shown to induce a cellular immune response in adult and neonate mice that protected them against a lethal challenge with virulent B. pertussis (26). An antibiotic-free strain has been constructed by autotrophic complementation to be investigated in clinical trials (27).
Recently, many studies have focused on the use of rBCG as a means of increasing the protection against TB (28,29). Recombinant BCG expressing important M. tuberculosis antigens, such as Ag85A, have been shown to induce better immune responses than those elicited by standard BCG in animal models and, as a consequence, these strains are under evaluation in clinical trials (30). In fact, rBCG-Ag85A was the first rBCG vaccine to be used in a clinical trial against TB. The idea was to improve the BCG vaccine by overexpressing an immunodominant antigen that had been demonstrated to be protective. Another BCG-based vaccine that is also in clinical trials involves a more sophisticated approach in technical terms. In this case, a BCG mutant deficient in the urease gene was used to express the Listeriolysin O gene from L. monocytogenes (29). This rBCG::ΔureC-llo+ mutant has the advantage of being less virulent than the wild-type BCG, a characteristic that may be advantageous when considering immunocompromised individuals. In this vaccine, Listeriolysin expression could lead to disruption of the phagosome membrane, allowing BCG antigens to escape into the cytosol, thereby potentially increasing presentation to CD8 + T cells and protection (31,32). Another approach has been the construction of strains of rBCG expressing cytokines involved in antimycobacterial immunity, such as IFN-γ and interleukin (IL)-2, which have been used as a means to enhance the immune response against TB (33), although concerns with their potential cytotoxicity have been raised. It is a consensus that a Th1 immune response is important for protection against TB and the production of IFN-γ by specific T cells is an important factor for protection (34), even though there is evidence of the absence of correlation between IFN-γ levels and the degree of protection (35).
TB vaccines based on the attenuation of M. tuberculosis have also been developed (36,37). However, this strategy has occasionally rendered M. tuberculosis strains less immunogenic than wild-type BCG itself, perhaps due to the deletion of important regions responsible for inducing the appropriate immune responses (29). In general, similar approaches could be applicable to recombinant S. typhimurium, Vibrio cholerae, Listeria monocytogenes, and Shigella (16,17). Another important class of presentation systems for heterologous antigens is based on viral vectors. The use of viral vectors in vaccine development has been recently reviewed (19,38,39). Vaccines based on viral vectors represent an important strategy against infectious diseases caused by intracellular pathogens, partially due to the fact that they localize in the same compartment that may mimic a natural viral infection. By delivering antigens within the host cells, processing and antigen presentation via major histocompatibility complex (MHC) class I molecules on the surface of infected cells can occur, facilitating the induction of cellular immune responses, which are known to be important in the control of intracellular infections. There are a wide variety of viral vectors under investigation as vaccine delivery vehicles. Some characteristics are desirable for a virus to be used as a delivery vehicle, such as: 1) the capacity to receive large fragments of foreign genes along with regulatory sequences, which could replace a segment of the viral genome not essential to the virus 2) to be genetically stable 3) to be capable of growing to higher titers and allow purification 4) to lack persistence or genomic integration in the host, and 5) most importantly, not to induce disease or show toxicity (19,40).
Numerous viral vectors are available for vaccine development, such as vaccinia, modified vaccinia virus Ankara (MVA), adenovirus (Ad), adeno-associated virus (AAV), retrovirus/lentivirus, alphavirus, herpes virus, and many others (19,41). There are many differences between the viral vectors available. They can be classified according to the virion type (DNA or RNA), particle size, transgene capacity, and cell tropism (40,41). Viral vectors can be replicating or non-replicating viruses the replication-defective viruses being the most tested in clinical trials, partly due to their higher safety. However, some groups are focusing on the use of replicating vectors in clinical trials as they are more likely to elicit stronger cellular and mucosal immune responses, as well as antibodies against the expressed proteins, depending on their cell tropism and sites of replication (40).
Several studies have demonstrated that recombinant viral vectors encoding genes from important pathogens, (such as malaria, HIV and TB) are able to induce both humoral and cell-mediated immune responses against their expressed antigens in immunized animals and, in some cases, may even protect the animals from lethal challenge (19,41). Co-expression of immunomodulating cytokines in viral vectors has also been used in order to enhance their immunogenicity, also with the above-cited restrictions (42).
This strategy has been used extensively in the development of vaccines against HIV. Similar to other viral vaccines or viral vector-based vaccines developed, a vaccine against HIV infection could be devised based on its attenuation. However, due to the possible risk of reversion or recombinant events, which can lead to a pathogenic HIV phenotype, vaccines based on HIV virus attenuation have been avoided. Therefore, live recombinant viral vectors such as Ad and MVA have been proposed as safer and less concern raising approaches. Ad and MVA are among the most promising live viral vector systems and, besides having been tested as vaccines against HIV (19), are currently being used in clinical trials against other important infectious diseases such as TB (43) and malaria (41).
Adenoviruses are non-enveloped icosahedral viruses containing a linear double-stranded DNA in their genome, which can infect and replicate in different locations in the body, such as the respiratory tract and the urinary bladder. There are over 50 subtypes of human Ad, with Ad serotype 5 (Ad5) being the best characterized and most used in several vaccination trials. Ad5 is a stable, non-replicating virus, characteristics that contribute to its safe application. This virus allows the insertion of large segments of foreign DNA (
8 kb) into its genome and, in addition, it can be obtained in high titers and easily purified. Replication-competent adenovirus vectors are also under development as vaccine carriers for HIV. The advantages of this type of adenoviruses vector are the lower doses necessary for inducing immune responses and longer persistence in the host, which may be associated with a more prolonged immune response. However, contrary to the non-replicating type, replication-competent adenovirus vectors present lower cloning capacity, limited to
3-4 kb. Noteworthy, both systems elicit a potent and long-lasting immune response carrying the same gene inserts (44).
Antigens from HIV such as gag, pol, env, and nef have been expressed in adenovirus vectors, particularly Ad5, showing promising results in diverse animal models and in phase I trials (45). Ad5 expressing the simian immunodeficiency virus (SIV) gag protein was able to attenuate the viral infection in monkeys after a challenge with a pathogenic HIV-SIV hybrid virus (SHIV) (46). However, the same results were not observed in phase II trials in humans (47). The candidate MRK Ad5 HIV-1 clade B gag/pol/nef vaccine from Merck & Co., Inc. (USA) has been considered to be the most promising vaccine against HIV-1 to date however, the clinical trial of this vaccine was interrupted after it was demonstrated not to be protective against HIV infection. Moreover, an increase was observed in the rate of HIV infection in vaccinees that had pre-existing immunity to Ad5 (48). New immunization strategies have been developed to overcome this problem, including the use of other adenovirus serotypes and heterologous vector prime-boost regimens (reviewed in Ref. 45).
Similar to Ad5 and other viral vaccine vectors, MVA has also been used as a vaccine platform in the development of HIV and TB vaccines, as well as for other infections. MVA is an attenuated strain derived from vaccinia virus, which was obtained after 570 passages in chicken embryo fibroblasts. This process resulted in several deletions, making the MVA strain unable to replicate in mammalian cells and inefficient in evading the immune system of the host. In addition, this process modified the host range of the virus. Studies using MVA-based recombinant vaccines in animal models have shown them to be immunogenic and protective against several infectious agents, including HIV, SIV, TB, and malaria (40,49).
Even though promising results have been obtained using vaccines based on viral vectors in recent clinical trials, the use of this technique alone has not been shown to be sufficient to induce a protective immune response. Other approaches are therefore under investigation to be used in combination with this technique. A promising technique that will be discussed in more detail below is called heterologous prime boost. It combines the use of two methods of immunization sequentially, for example, first an immunization with viral vectors, followed by a recombinant protein or live bacterial vaccines.
The direct injection of a naked DNA plasmid into muscle as a vaccine system with the ability to induce an immune response and protection after challenge is now well established, since this approach has been used to express numerous antigens from different pathogens with promising results (50-52). A DNA vaccine (or genetic vaccine as it is also called) consists of a plasmid containing: 1) one origin of replication of Escherichia coli, for the amplification of the plasmid 2) a strong promoter, generally from cytomegalovirus 3) multiple cloning sites, in which one can insert the gene to be expressed, and 4) an antibiotic as selection marker (50,51). The idea behind the DNA vaccine system is that the antigen can be expressed directly by the cells of the host in a way similar to that occurring during viral infection. As a result, the antigens can be processed as proteins synthesized in the cytoplasm, and the fragmented peptides presented to the immune system by class I MHC molecules. In addition, if the protein is exported or secreted, it can be processed by class II MHC molecules and, as a result, mount a specific antibody response (50-52).
Initially, DNA vaccines were administrated either by intramuscular (im) injection or using a DNA particle delivery system called Gene Gun (53). Unlike im injection, which requires micrograms of plasmid DNA and several doses, the Gene Gun system requires nanogram levels of plasmid DNA to induce the same level of immune response. However, the type of immune response induced in response to the same antigen by the two systems was shown to be distinct. While im injection raised predominantly a Th1 response, Gene Gun immunization induced a mixed Th1/Th2 or a Th2 shifted profile. These findings are particularly important in vaccine design, as it is desirable to establish previously the kind of immune response required for protection against a specific pathogen (54). DNA vaccines have several properties that could represent advantages over other immunization procedures: there is no risk of infection, contrary to attenuated vaccines they elicit both humoral and cell-mediated immunity, and they are capable of inducing long-lived immune responses and increased cytotoxic T-cell responses. In addition, DNA vaccines avoid problems associated with producing recombinant protein vaccines, such as inadequate folding of target molecules or high purification cost of recombinant proteins. Although DNA vaccines present many advantages, some concerns regarding suitability and capability should be investigated, such as the possibility of production of anti-DNA antibodies, integration of DNA plasmid into the cell genome (now considered a remote possibility), and low efficiency of transfection of the cells in vivo (55).
DNA vaccines have been used to express antigens from many different pathogens, such as influenza, HIV, malaria, TB, and leishmaniasis, leading to the induction of immune responses against these etiologic agents in several animal models, and in some cases to protection (54,56). However, DNA vaccines have been shown to be less immunogenic in non-human primates and humans, even though they have been demonstrated to be safe and well tolerated (55).
To increase the effectiveness of these vaccines some approaches have been designed that constitute a second generation of DNA vaccines: plasmid alterations that augment the gene expression, as well as systems that co-express cytokines or other molecules capable of enhancing the immune responses, are some of these new strategies. Among the molecules used for co-expression are genes that induce apoptosis and genes encoding ligands for Toll-like receptors (TLRs) (55,57). Other important approaches that have been developed consist of the formulation of DNA in ways that can protect the DNA from degradation or facilitate its uptake into cells. One good example is the DNA encapsulation into microparticles or the use of live vectors such as viruses or bacteria to protect and facilitate delivery of DNA into specific cells (55,57). The uptake of DNA into cells can also be improved using in vivo electroporation, a technique by which small amounts of electric current applied in vivo are used to cause the localized formation of pores in cells, which allow more DNA to enter the target cells (58). However, widespread use of electroporation in vaccination campaigns is difficult to envisage. Despite the relative success in improving the immunogenicity induced by DNA vaccines, the precise cellular mechanisms by which a DNA vaccine works in the body are still not totally elucidated. Again, since DNA vaccines alone have been shown not to be sufficient to induce a strong immune response, strategies such as prime boost have been used to improve the immune response for the development of efficient vaccines against a variety of infectious diseases.
The prime-boost approach
Current vaccination traditionally known to be effective requires immunization of an individual with two or more doses and this consists of a "prime-boost regime". As the vaccines used in the prime and boost consist of the same formulation, such regime is called homologous prime-boost. On the other hand, an immunization regime involving different formulations used sequentially in more than one administration will be called heterologous prime-boost. Research results accumulated over the past decade have shown that heterologous immunization can be more effective than homologous immunization, especially against intracellular pathogens, the infectious agents of higher complexity that are currently considered to be more challenging for vaccine development (59).
The heterologous prime-boost or simply "prime-boost" immunization, as it is commonly called, is a strategy, which involves the administration of the same antigens but formulated in different ways, either as purified antigens or recombinant protein in the presence of appropriate adjuvants, as live recombinant viral or bacterial vectors or DNA vaccines. This approach has opened new venues for vaccine development, and appears to be able to induce a more adequate and efficient immune response against intracellular pathogens. The idea behind the heterologous prime-boost immunization is to combine both humoral and cellular immunity, potentially elicited by each delivery system individually, in an attempt to enhance and modify the immune response induced against a specific antigen. For example, subunit vaccines will usually induce a predominant humoral immune response, while recombinant live vector vaccines and DNA vaccines are effective delivery systems for eliciting cell-mediated immunity (CMI) (59).
The great potential of this strategy has been well demonstrated in the context of HIV vaccine development. Monkeys (Macaca fascicularis) primed with the recombinant vaccinia virus expressing SIVmne gp160 antigen and boosted with the recombinant gp160 protein were protected against an intravenous challenge with SIVmne virus. These results were considered among the most promising obtained in the early effort of HIV vaccine development (60). On the other hand, the combination of DNA vaccines with other immunization approaches has also proven to induce greatly increased immunogenicity. Mice primed with a DNA vaccine encoding the hemagglutinin gene of influenza and boosted two weeks later with a recombinant viral vector Fowl poxvirus (FPV) expressing the same antigen were able to produce high levels of anti-hemagglutinin serum antibodies, predominantly of the IgG2a isotype, unlike animals immunized with each vector alone (61).
Since these seminal investigations, several groups have obtained good results using either similar combinations or alternative protocols (62). Many different combinations of heterologous prime-boost will be possible: DNA vaccine-recombinant protein live recombinant bacteria/virus-recombinant protein live recombinant bacterial/virus-DNA vaccine (and vice versa). However, in spite of some positive results, in general prime-boost immunization protocols initiating with recombinant vectors followed by recombinant protein have produced disappointing results (63). Interestingly, the order of the prime and boost has been shown to alter the immune response obtained. In a prime-boost strategy of immunization against malaria, mice immunized with consecutive DNA and MVA vectors encoding antigens from Plasmodium berghei have been shown to be protected against challenge with P. berghei sporozoites, and such protection was associated with high levels of peptide-specific IFN-γ-secreting CD8 + T cells. However, reversal of the order of the immunization or substitution of the viral vector resulted in failure of protection (64). This result showed the importance of using DNA as a priming vehicle and attenuated virus as a booster.
Prime-boost strategies have been applied for the development of vaccines against important infectious diseases such as HIV, TB, and malaria, demonstrating promising results even in clinical trials. In the last HIV clinical trial using a combination of two earlier vaccines that had previously failed, researchers found that the prime-boost combo reduced by 31% the risk of contracting HIV (65). Unfortunately, they have also shown that the observed protection was limited to 1 year. In spite of this short-lived protection, the authors believe this result is encouraging and that a new and safer HIV vaccine will soon be available. Presently, clinical trials are ongoing to further assess this line of research (66).
The exact mechanism underlying the efficacy of the heterologous prime-boost vaccination is still poorly understood, being likely that several distinct mechanisms participate in the success of this approach. One mechanism proposed suggests that the different characteristics of the vectors are important. A second advantage of a heterologous prime-boost is the fact that the use of different immunization strategies results in reduced induction of anti-vector immunity. A third, and possibly the most relevant mechanism, is due to immunodominance. During priming immunization, T cells will be induced against the most immunodominant epitopes of the antigen. Upon heterologous boosting, which shares only the relevant antigen with the prime immunization, the immune response will focus preferentially on the expansion of immunodominant T cells induced by priming (67,68) live recombinant vectors, such as MVA and adenovirus, seem to be especially efficient in boosting pre-existing memory immune responses, especially primed T-cell responses (65,66,69).
A number of studies have shown that at least one plasmid vector (consisting of DNA vaccine) or a recombinant viral vector should be included as a component of the prime-boost vaccination in order to elicit a potent cell-mediated immunity (59,64,70). Although DNA vaccines so far have shown low immunogenicity when used alone, they have also proven to act as strong priming vehicles, while viral vectors seem to be much more effective when used as boosters. As a consequence, DNA prime-viral vector boost regimes have become the main scheme of choice to induce T cell-mediated immune responses (59,64,70).
One possible mechanism to explain the success of these prime-boost regimes relies on the induction of high-avidity T cells. Mice immunized with DNA prime/live vector boost protocols expressed high frequencies of high-avidity T cells and were capable of eliminating target cells expressing 10- to 100-fold less immunogenic peptide than mice vaccinated with either vector alone (70). Other features characteristic of the vaccine vectors used in prime-boost immunization may as well be essential for their ability to induce increased CMI ( Table 2). The presence of cytosine-phosphodiester bond-guanine (CpG) motifs in the plasmid of the DNA vector has also been shown to strongly stimulate the production of IL-12, the main inducer cytokine of Th1 cells. The use of non-replicating DNA vaccines followed by live vectors may result in an immune response focused almost exclusively on the encoded antigen. The efficient presentation of the encoded antigen by MHC class I and class II molecules will result in efficient induction of CD4 + T and CD8 + T cells (70). The types of antigens and the types of vectors used, the order of vector administration, the routes and interval between priming and boosting vaccinations, among other factors, should be taken into account to determine the effectiveness of the prime-boost strategies ( Table 1). Further investigation of the mechanism of action of this promising strategy will allow its optimization, and eventually lead to improved vaccines.
We have seen here that the prevention of important infectious diseases such as HIV, TB and malaria, among others, continues to be a challenge for the vaccinology field in the 21st century. Furthermore, it is most likely that vaccines for such pathogens will not become available by following the classical approaches of successful traditional vaccines.
Nonetheless, considerable advances in the fields of immunology, molecular biology, recombinant DNA, microbiology, genomics, bioinformatics, and related areas have provided novel insights to help elucidate important pathogenic mechanisms involved in these infectious diseases and in pathogen interaction with the host. Altogether, these advances have led to the development of several new vaccine strategies with promising results. It seems now clear that an integrated approach will be necessary to foster continued progress in the immunology field, which probably constitutes the limiting factor for the development of new vaccines.
It is also important to realize that the challenges of vaccine development are not limited to the discovery of safe and effective antigens, adjuvants and delivery systems. The balance between cost, benefits and risk should certainly be evaluated before translating a vaccine candidate to the clinic. Millions of children worldwide die from infectious diseases, despite currently available vaccines. Thus, social, political and economic policies are not less important issues and cannot be overlooked.
Applications of polymeric adjuvants in studying autoimmune responses and vaccination against infectious diseases
Polymers as an adjuvant are capable of enhancing the vaccine potential against various infectious diseases and also are being used to study the actual autoimmune responses using self-antigen(s) without involving any major immune deviation. Several natural polysaccharides and their derivatives originating from microbes and plants have been tested for their adjuvant potential. Similarly, numerous synthetic polymers including polyelectrolytes, polyesters, polyanhydrides, non-ionic block copolymers and external stimuli responsive polymers have demonstrated adjuvant capacity using different antigens. Adjuvant potential of these polymers mainly depends on their solubility, molecular weight, degree of branching and the conformation of polymeric backbone. These polymers have the ability not only to activate humoral but also cellular immune responses in the host. The depot effect, which involves slow release of antigen over a long duration of time, using different forms (particulate, solution and gel) of polymers, and enhances the co-stimulatory signals for optimal immune activation, is the underlying principle of their adjuvant properties. Possibly, polymers may also interact and activate various toll-like receptors and inflammasomes, thus involving several innate immune system players in the ensuing immune response. Biocompatibility, biodegradability, easy production and purification, and non-toxic properties of most of the polymers make them attractive candidates for substituting conventional adjuvants that have undesirable effects in the host.
Over recent decades, material science research has been expanding exponentially to design and develop novel biomaterials for biomedical applications, and polymers have proved to be promising candidates. The easy and controllable synthesis of polymers in various formats with good mechanical properties, biocompatibility and biodegradability makes them more valuable in the biomedical field and in tissue engineering as scaffolds to grow mammalian cells for regeneration of damaged organs. Use of polymers in immunology as an adjuvant could be a useful substitute for conventional bacterial adjuvants. In immunology, an adjuvant is defined as the substance normally used with a weak antigen to enhance its immunogenic properties via activation of innate and adaptive immune responses. Adjuvants, such as polymers that are immunologically inert but capable of inducing an immune response when given with an antigen, have several advantages. Generally, they act as depot carriers through slow release of the antigen, thereby modulating the ensuing immune responses. Polymers mixed with an antigen can follow different signalling pathways. For example, the polymer–antigen can be phagocytosed and processed through proteasomes, activation of inflammasome pathway via secretion of IL-1β cytokine, ligand for toll-like receptor(s) or directly interact with B cells. Alternatively, processed antigens can be presented by antigen-presenting cells via major histocompatibility complex (MHC) molecules to naive T cells, which in turn can become activated and release various cytokines leading to enhanced T and B cell interactions. The activated B cells in turn can undergo differentiation into antibody-secreting plasma cells [1,2] (figure 1), and the antibodies thus produced can activate the downstream events of the effector phase of an immune response involving various chemokines, cytokines, proteases and effector cell populations such as neutrophils, macrophages, osteoclasts, mast cells and eosinophils .
Figure 1. Schematic diagram of possible mode of action of polymeric adjuvants. TLR, toll-like receptor TCR, T cell receptor IL, interleukin.
Conventionally used adjuvants are alum compounds , Freund's adjuvants (complete Freund's adjuvant, CFA and incomplete Freund adjuvant, IFA) , diphosphoryl lipid A , muramyldipeptide, monocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor  and oligodeoxynucleotides containing CpG motifs . Among them, Freund's adjuvants are frequently used and well characterized, involving slow release of injected antigen and the pathogen-associated molecular patterns of the mycobacteria present in this adjuvant interacts with the pathogen recognition receptors such as toll-like receptors (TLRs), leading to the activation and proliferation of T cells . The mycobacterium component of CFA specifically activates the Th1 pathway, resulting in the initiation of the delayed-type hypersensitivity reaction against the injected antigen . Moreover, some systemic effects were also observed in the blood system characterized by proliferation of Mac-11 + immature myeloid cells. While, IFA does not have this mycobacterium content, it activates the Th2 pathway with a strong bias towards the humoral immune response . However, the paraffin oil present in Freund's adjuvant has been shown to be non-degradable and cause toxicity problems . In recent years, polymers have proved to be promising candidates to replace such conventional adjuvants with their biocompatible and biodegradable properties. These polymers, together with an antigen, can activate the immune response effectively and have thus been used as an adjuvant in vaccination strategies and in the induction of autoimmune responses.
2. Polymers as vaccine adjuvants
Vaccination is the most effective way to control and prevent diseases. But multiple doses of conventional vaccines for the activation of the desired immune responses are very difficult and challenging, especially in developing countries. Interestingly, polymers in vaccine formulations could improve the delivery of antigens and thus reduce the booster doses of vaccines required for the activation of an appropriate immune response. For many decades, various biodegradable natural and synthetic polymers have been used for antigen delivery for the controlled release of vaccines over longer periods of time and as an adjuvant for enhancing immunogenicity of weak vaccines (table 1). In this review, we will discuss the application of polymers as adjuvants in vaccination.
Table 1. Polymeric adjuvants used in vaccination.
2.1. Natural polymers
Numerous polysaccharides originated from plant and microbes have been tested for their adjuvant potential in vaccination. In this scenario, various derivatives of dextran have shown to have immunological properties. The sulphate-derivatized form of dextran sulphate was shown to have anti-inflammatory properties in vivo and it was used in the induction of inflammatory colitis in mice . Another derivative, diethylaminoethyl dextran (DEAED), is polycationic in nature and has been used as a vaccine adjuvant. DEAED, together with Venezuelan equine encephalomyelitis virus antigen, induced primary antibody responses after vaccination of rhesus monkeys . In another study, adjuvant properties of DEAED were proved in mice immunized with whole-cell cholera vaccine . Another derivative of dextran, acetylated dextran, in the form of microparticles was proven to be an activator of toll-like receptor pathways and to induce inflammatory cytokines .
As an adjuvant, lentinan, another polysaccharide composed of β-1,3-glucohexaose monomer units with β-1,6 branching has been demonstrated in a vaccination study. Lentinan activated macrophages with an increasing respiratory burst activity and IL-6 production after lethal influenza virus infection . It also enhanced antigen presentation through a dendritic cell vaccine procedure, leading to the activation of T and natural killer cell populations with a concomitant production of cytokines by splenocytes . The sulphated derivative of lentinan was proven to be a powerful adjuvant in chickens immunized with Newcastle disease vaccine with an increased serum antibody titre and proliferation of lymphocytes . Moreover, lentinan suppresses tumouricidal activity of macrophages non-specifically unlike other adjuvants such as lipopolysaccharide (LPS) .
Inulin, another polysaccharide, is a linear chain polymer linked through a β(2–1) glycosidic bond. Adjuvancity of inulin was linked to the activation of the complement cascades . Structurally, inulin occurred in various isoforms α, β, γ and δ. Out of these, γ inulin was proven to be a powerful adjuvant in immune activation . More recently, δ inulin has been characterized as a more potent adjuvant than the γ isoform, enhancing both humoral and cellular immune responses when administrated together with an antigen . The T cell memory immune response was also strong as evidenced by a higher cellular proliferation rate. In addition, enhanced production of antigen-specific antibodies was also noticed . Furthermore, δ inulin has been effective with Japanese encephalitis  and HIV vaccine antigens . Its mechanism of action is mainly through interactions with APCs, including monocytes, macrophages and dendritic cells. However, unlike other adjuvants, it is unable to activate the pro-inflammatory NF-κB pathway. But a positive aspect of inulin adjuvants is that like many other carbohydrate compounds, they are well tolerated by the body and cause minimal reactogenicity .
In this direction, mannan polysaccharide is also known as a vaccine adjuvant that mainly binds to mannose receptors and leads to the activation of the complement pathway with enhanced phagocytosis of the antigen [54,55]. In addition, it also operates via the inflammasome pathway through secretion of IL1β and as a ligand for TLR4 . The reduced form of mannan (mannose) is known for the activation of the Th2 pathway when administrated with an antigen, while oxidized mannan in known to activate the Th1 pathway . Mannan coupled with a recombinant protein antigen was shown to enhance higher production of specific IgGs compared with cholera toxin . The acetylated derivative of mannan (Acemannan) has also shown anti-viral and anti-tumour activities. It has the capacity to induce maturation of DCs through upregulation of MHC class II molecules and production of IL-12 . Interestingly, mannan has been shown to induce not only transient arthritis in naive BALB/c mice but also Th17-mediated chronic arthritis in ZAP-70 mutated (SKG) mice via activation of innate immunity through toll-like receptors and dectin-1 . However, C5a receptor (C5aR) deficiency in SKG mice inhibited the differentiation/expansion of Th17 cells after mannan or β-glucan treatment, and consequently suppressed the development of arthritis . Recently, we have also found in naive mice that mannan can induce inflammation that is dependent on MHC class II but not on complement factor 5 (Khmaladze et al. 2012, unpublished data).
Subsequently, another natural polymer, chitosan, was shown to exhibit a range of immunological properties, including macrophage activation, cytokine production and enhanced antibody synthesis when co-delivered with an antigen. Similar to mannan, chitosan interacts with dectin-1 and toll-like receptors. Mostly, chitosan has been used for antigen delivery in the form of particles [60–62]. Recently, inactivated influenza virus vaccine entrapped in chitosan particles was found to increase the antibody titre two- to 10-fold more after intramuscular immunization in mice than the controls . In other study, chitosan polymer has been used as an adjuvant in the form of particles for delivery of HBsAg antigen with an enhancement of immunogenicity ninefold more compared with naive mice . Owing to the mucoadhesive property of chitosan, the N-trimethylchloride derivative of chitosan was used to demonstrate enhanced immunogenicity and protective efficiency through increased absorption of proteins at the mucosal surfaces by opening tight junctions . In few studies, chitosan particles have been used in combination with alginate for delivery of HBsAg with CpG adjuvant via the intranasal route, which induced significant antibody responses . Furthermore, chitosan as an adjuvant enhanced protection against Helicobacter pylori through induction of Th2 responses . Similarly, trimethyl and mono-N-carboxymethyl chitosan-based nanoparticles encapsulated with tetanus toxoid antigen enhanced both mucosal and systemic immune responses in response to vaccination via the nasal route . To prove this concept, Verheul et al.  fabricated trimethylchitosan particles with hyaluronic acid and found improved adjuvant potential compared with trimethylchitosan particles alone for nasal and intradermal vaccinations.
Poly-γ-glutamic acid (PGA) is another polypeptide that is composed of gamma-linked glutamic acid units with an alpha carboxylate side group, produced by Bacillus bacteria. PGA with l -phenylalanine ethyl ester has shown immunologic activity in the form of particulates. Ovalbumin-entrapped γ-PGA nanoparticles have activated human monocyte-derived dendritic cells and enhanced the production of inflammatory cytokines and chemokines . Moreover, it was involved in the upregulation of co-stimulatory molecules, leading to better T-cell priming. In another study, γ-PGA particles induced innate immune cells in vitro and produced antigen-specific immune responses through TLR4 and MyD88 signalling pathways . Recently, γ-PGA and benzalkonium-chloride-based anionic complexes were successfully encapsulated with ovalbumin antigen that were shown to be efficiently taken by dendritic cell line, DC 2.4. Subcutaneous administration of these complexes induced antibodies with activation of both Th1 and Th2 pathways .
2.2. Synthetic polymers
Synthetic polymers as an adjuvant have several benefits over conventional metal-based adjuvants such as alum compounds. Polymers in the micro/nano-size range can be directly internalized via the lymphatic system and retained for a longer period of time, which helps to maintain a significant level of immune response and thus reduce multiple doses of antigen required for activation of the immune system. They are biocompatible, easy to degrade and non-toxic to animals, unlike alum compounds.
2.2.1. Multiphase emulsions
Among the various adjuvants available for vaccination, adjuvants based on emulsion such as Freund's adjuvant, montanide ISA 51 have the advantage of being easy to manufacture at low cost. Generally, these adjuvants are defined as water-in-oil emulsion with dispersed antigen . However, preparation time, injection difficulties and localized toxicities at the injection site are considerable problems with these emulsions [59,69]. To improve injectiblity, a new method consisting of re-dispersion of these emulsions in an aqueous phase using the hydrophilic emulsifier Tween-80 (polyoxyethylene sorbitan monooleate) has been demonstrated . The adjuvancity of Tween-80 proved more immunogenic than controls however, Tween-80 has been restricted in use owing to its lipophilic toxicity and extra reactivity problems [71,72]. To reduce the toxicity problems, a synthetic block polymer with low molecular weight has been suggested as a good alternative to surfactants . For example, TiterMax is squalene-based water-in-oil emulsion containing polyoxyethylene–polyoxypropylene–polyoxyethylene (POE–POP–POE) polymer . However, mild toxicity has also been reported with the use of the TiterMax adjuvant owing to its poor biodegradation properties . To further improve this adjuvant, recently Huang et al. have proposed multiphase emulsion based on the polymeric emulsifiers poly(ethylene glycol)-blockpolylactide (PEG-b-PLA), poly(ethylene glycol)-block-poly(ɛ-caprolactone) (PEG-b-PCL) and poly(ethylene glycol)-block-poly(lactide-co-ɛ-caprolactone) (PEG-b-PLACL) in the antigen phase of oily ISA51-adjuvant-based vaccines. Good biodegradability and biocompatibility of these polymers makes them promising candidates for vaccines .
Polyphosphazenes are one of most frequently used polymers as an adjuvant, which consists of alternate nitrogen and phosphorus elements in the backbone with organic side groups. Polyphosphazene-based polymers generally form stable complexes with an antigen and thus produce more potent adjuvant preparations [77,78]. Their synthetic flexibility and susceptibility towards hydrolysis produces a promising matrix for adjuvants in vaccine formulations. Among polyphosphazene, poly(di(sodium carboxylatophenoxy)phosphazene, PCPP) was demonstrated for its adjuvant activity with influenza and other retrovirus-based vaccines [27,28]. Recently, PCPP has been injected with influenza virus X31 antigen subcutaneously and strong B and T cell responses were observed . In another study, the adjuvant effect of PCPP was studied in combination with CpG oligodinucleotides via the subcutaneous route . Recently, Shim and co-workers  demonstrated the adjuvant potential of PCPP via the mucosal route. Interestingly, PCPP has a film-forming nature and is thus able to encapsulate the antigen efficiently even under mild conditions. It is a water-soluble molecule, which can be easily formulated with proteins and has the capacity to dissolve in a highly hydrated environment. Owing to this dual functionality of PCPP (as an adjuvant and film-forming nature), it is one of the most promising materials available for use in vaccine delivery .
Very recently, polyelectrolyte multilayer capsules (PMLC) have been demonstrated as a carrier for antigen delivery [83–85]. PMLC can easily be self-assembled under aqueous conditions without the aid of any other chemicals, which may denature the antigen. Usually, PMLC are fabricated in three steps: encapsulation of antigen in porous microtemplates  followed by layer-by-layer coating of polymers  and removal of microtemplates through decomposition into low-molecular-weight components. These porous multilayer capsules rapidly rupture during cellular uptake and permit rapid intracellular release of the encapsulated antigen. De Geest et al.  have fabricated PMLC composed of dextran sulphate and poly- l -arginine encapsulated antigen, which was rapidly phagocytozed by dendritic cells, resulting in better antigen presentation to CD4 + and CD8 + cells. Subsequently, these capsules were demonstrated as carriers for an antigen in pulmonary vaccination. PMLC are efficiently uptaken by alveolar macrophages, followed by initiation of Th17 immune responses, which is considered a central part in mucosal immune responses. Furthermore, PMLC-based immunization was also shown to provide protective immunity against B16 melanoma and influenza infection. High doses of PMLC activate the NALP inflammasome pathway.
Similarly, a cationic lipopolymer, liposome–polyethylene glycol–polyethyleneimine complex (LPPC), was also evaluated for adjuvant and immunomodulation properties. LPPC strongly adsorbs the antigens over the surface and enhances the immune responses. It was found that this polymer–antigen complex activates immune responses through enhanced uptake and presentation of antigens, cell surface receptor expression and release of pro-inflammatory cytokines. When, LPPC was given with LPS or CpG oligodeoxynucleotides, a strong level of IgA antibody responses was observed .
Water-soluble, non-toxic and synthetic polyelectrolytes were found to be efficient immunomodulators . A more prominent increase in antibody responses was achieved by complexing an outer membrane protein of Salmonella, porin, with the heteropolymer, polyoxydonium . Similarly, Salmonella O and H antigens complexed with polyelectrolytes have been shown to induce several-fold higher antibody responses against respective antigens .
Polyanhydrides are a novel class of biodegradable polymers that have been extensively used in protein/antigen delivery in vaccinations [91,92]. In recent years, this polymer has been approved by the FDA for human use owing to its tissue compatibility and controlled biodegradation properties . Degradation of this polymer occurs through surface erosion mechanisms, resulting in controlled release of antigens. Normally, degradation can be extended up to one month depending on the polymer chemistry [74,94]. Furthermore, controlled release of antigen activates the immune system requiring only a single injection, which potentially improves patient compliance . Intrinsic adjuvant activity of polyanhydrides has been reported that was independent of the type of antigen delivered. Recently, Torres et al.  reported adjuvant activity of various polyanhydrides, including sebacic anhydride, 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG). A specific combination of CPH and CPTEG copolymer microparticles has been used as vaccine adjuvants and carriers for antigen. These particles were efficiently phagocytosed by APCs and localized in phagolysosomes. Expression of surface markers CD86, CD40 and MHC class II on dendritic cells was significantly increased after phagocytosis of microparticles. Moreover, the presence of polyanhydride particles induced specific proliferation of CD4 + and CD8 + T-cells. Recently, molecular design of amphiphilic polyanhydride nanoparticles has been proposed for the activation of APCs mimicking pathogens . The dose–response effect of poly(methyl vinyl ether-co-maleic anhydride) nanoparticles was studied in the stimulation of TLRs. These particles were also involved in the activation of the complement pathway .
2.2.5. Non-ionic block copolymers
Non-ionic block copolymers containing polyethylene (POE) and polypropylene (POP) blocks are known as poloxamers/pluronics . Experiments have proven the capability of low molecular weight pluronic polymers (3 kDa) with 90 per cent POP and 10 per cent POE in the activation of macrophages. Intraperitoneal injection of these copolymers in mice induced high-level expression of MHC class II molecules, which is a direct indication of macrophage activation [100,101]. Although the exact mechanism of action of the block copolymers has not been delineated, these polymers are known to work through the activation of the alternative pathway of the complement system . It is of interest to note that certain complement proteins are also involved in the activation of macrophages . These copolymers are also known for the stabilization interactions of protein antigens at the interface of oil- and water-based emulsions . One of the major limitations of these copolymers is their poor solubility in aqueous formulations owing to their low molecular weight (2000–5000 Da), making it necessary to use them in oil-based emulsions only. To improve the solubility, Todd et al.  synthesized a larger molecular weight block copolymer CRL1005 (9000 Da), which was compatible in aqueous formulations. The CRL1005 polymeric adjuvant has a higher POP content, around 95 per cent compared with 80–90% POP in small-molecular-weight copolymers. These large polymers are soluble in water at 18°C but form aggregates at body temperature.
Recently, an interesting form of block copolymers known as polymersomes have been demonstrated in vaccine delivery for the activation of immune responses [104,105]. Generally, polymersomes are composed of amphiphilic block copolymers, which after self-assembly form polymeric shells. Polymersomes composed of POE glycol-polybutadiene block copolymer nanoparticles functionalized with Tat peptide of HIV enhanced the cellular uptake by dendritic cells . Similarly, Quer et al. synthesized poly(g-benzyl- l -glutamate)-K (PBLG50-K) polymersomes and tested them as an adjuvant for influenza haemagglutinin (HA) antigen . For targeted delivery to antigen-presenting cells, specifically to dendritic cells, POP-sulphide-based nanoparticles have been designed. These nanoparticles can be easily taken up in to lymphatic system and retained in lymph nodes for a longer period of time. A total of 40–50% nodal resident dendritic cells and other antigen-presenting cells internalized these nanoparticles . The advantage of using polysulphide-based particles is that they are stimuli-responsive and can encapsulate hydrophobic drugs efficiently .
Polymethacrylic-acid-based nanoparticles were shown to be effective as adjuvants for inactivated HIV-2 antigen compared with several other conventional adjuvants, including alum and Freund's adjuvant . They have been reported to enhance immune responses against different antigens, including influenza virus and bovine serum albumin . The optimum methylmethacrylic acid concentration for enhancing antibody response against influenza virus was 2 per cent w/v. Above this concentration, immune responses declined. This can be explained better by antigen protection at higher concentrations of polymer. Furthermore, antigen concentration also affects the antibody response. Higher antigenic doses induced better antibody responses than lower amounts of antigen . Cytokine induction and the adjuvant effect of PMMA particles was also demonstrated by Lou et al.  in DNA vaccination. In this vaccination procedure, TNF-α production was enhanced when macrophages were exposed to PMMA particles. They have synthesized a number of PMMA particles for DNA vaccination, and different levels of tumour protection effect were observed, which was dependent on the size and charge of the particles. Kreuter & Liehl  also tested the adjuvant potential of polymethylmethacrylate and polyacrylamide copolymer-based nanocapsules using inactivated influenza virus vaccine with an increased vaccine potential against lethal influenza infection. Besides protection, polymers were also effective in the induction of antibody responses. Recently, cationic poly(2-aminoethylmethacrylate) polymers with controlled chain length and defined molecular weight were synthesized and characterized for delivery of DNA vaccine to dendritic cells . In another study, pH-sensitive polydiethylaminoethyl methacrylate core polyaminoethyl methacrylate shell nanoparticles were designed for targeted delivery of membrane impermeable molecules to dendritic cells . Cytosolic delivery of antigens by these particles decreased the dose required for the activation of immune responses, significantly .
The adjuvant potential of PLGA using the particulate system [38,39] has been well documented. This polymer enhanced the uptake of delivered antigens by APCs [114,115]. Besides antigen presentation, numerous antigens in the form of proteins, peptides, viruses or DNA can easily be encapsulated in the form of nanoparticles . In such vaccines, controlled release of antigen(s) for a longer period of time can effectively activate the immune responses, thereby avoiding booster doses required for the induction of protective immunity . PLGA particles can also act as the delivery system for more than one type of antigen or combinations of antigen–adjuvant formulations in the same particle . Furthermore, PLGA microparticles can retain the antigens in local lymph nodes and protect them from proteolytic degradation, which ensures longer retention of antigen. It has been shown that low doses of antigen are effectively delivered using PLGA particles for strong induction of T cell responses . In addition, these particles can also be used for delivering exogenous antigens via MHC class I complexes to CD8 + cells . An additional advantage of PLGA nanoparticles is their functionalization with ligands such as antibodies, proteins and polysaccharides binding to membrane receptors of the cells, which is relatively easier than other polymers . Uptake of PLGA particles depend on the characteristic features of the delivered antigen and also on the size, shape, charge and nature of the particle surface . Out of these properties, size is one of the critical factors that decides the uptake of antigen loaded nanoparticles. The optimum dimensions of nanoparticles are yet to be confirmed, but 20–100 nm is enough for internalization. Apart from the antigen uptake via APCs, peripheral lymph nodes can also directly internalize these particles [43,123]. Recently, PLGA was mixed with alginate to improve the encapsulation efficiency. The incorporated alginate also elicited higher humoral and cellular response after immunization with two malarial synthetic peptides, SPf66 and S3, in BALB/c mice. Furthermore, PLGA with alginate modified with RGD peptide have been shown to enhance immunogenicity by cell-specific targeting . PLGA microparticles coated with protamine significantly enhanced the immunogenicity of weak antigen in comparison with uncoated particles. The protamine moiety facilitates better cell penetration of these particles .
The efficacy of PLGA-based microspheres also has been tested for immunotherapy against experimental tumours. The mice vaccinated with PLGA-MS loaded ovalbumin and CpG oligodeoxynucleotides elicited a strong cytotoxic T-lymphocyte response . In another study, the efficacy of PLGA particles with poly- l -arginine was compared with polyelectrolyte microcapsules in the induction of Th1/Th2 responses .
Poly-ε-caprolactone (PEC) is another biocompatible and biodegradable synthetic polymer, which is used as an adjuvant. Unlike PLGA polymer, PEC degrades very slowly and does not generate an acidic environment after its degradation. The good biodegradability and low acidic pH makes PEC a potential adjuvant candidate and carrier for different vaccines. The adjuvant property of PEC-based microparticles was demonstrated with an antigen of Schistosoma mansoni for the induction of immunity after a single administration . Compared with PLGA, PEC particles encapsulated with a similar antigen induced immune responses for a longer duration after oral or nasal antigen administration . Better adjuvant properties of PEC were again proven in the delivery of antigenic extract (HS) from Brucella ovis either subcutaneously or per orally in BALB/c mice. Moreover, PEC particles with HS extract were found to interact more strongly with mucosal and Peyer's patches than PLGA. PEC-induced immune responses are of the Th1-type, while PLGA-induced Th2 responses are characterized by significant production of IL-4 levels. Furthermore, PEC induced higher release of IFN-γ and IL-2 cytokines than PLGA . PEC particles were also proven to be efficient carriers for Diphtheria toxoid in mucosal vaccines compared to PLGA, and the higher uptake of PEC particles is due to the more hydrophobic nature of the PEC polymer .
Another biocompatible polymer polyvinylpyrrolidone (PVP) was used as an adjuvant in a few studies. PVP was used successfully in entrapping an antigen extracted from Aspergillus fumigates and was capable of significantly inducing IgG levels compared to free antigen. Interestingly, the IgE response to PVP particles loaded with antigen was lower than the free antigen .
2.2.10. Cationic polymers
In general, cationic polymers, polyethylamines, were used in DNA delivery and immunization of various antigens via different routes. The approaches for DNA delivery usually involve injection of DNA either in solution or with cationic polymers/lipids. Efficient delivery of DNA to APCs in such a way could enhance the efficacy of nucleic acid and produce a strong immune response . Such cationic polymer-based microparticles can also be synthesized via the incorporation of DNA non-covalently .
3. Polymers in the induction of autoimmune responses
Adjuvant potential of polymers in the induction of autoimmunity, mainly for the development of diseases models, has recently been explored (table 2). Animal models of autoimmune diseases have similarities to human disease phenotypes and are thus essential to delineate the disease pathways, genetics and, for developing and testing drug targets. Autoimmune disease models are developed mainly by disrupting the immune homeostasis either via immunization with autoantigens, spontaneous or induced genetic mutations or by using surgical procedures. Autoimmune diseases can be induced by immunization with polymers and autoantigens successfully. LPS is the first polymer that has been used in the activation of immune responses, which depend on time and dose of administration along with an antigen . β-Glucans and zymosan are other natural polymers isolated form microbes that have been proven to be potential adjuvants for the development of autoimmune diseases [142,143]. Parallel to natural polymers, a few synthetic block copolymers of POP and POE has also been demonstrated for their adjuvant capacity in the development of myasthenia gravis, when injected along with torpedo acetylcholine receptor . Recently, we have developed a synthetic polymer-based poly-N-isopropylacrylamide (PNiPAAm) adjuvant for the development of collagen-induced arthritis. When collagen type II (CII) purified from rat chondrosarcoma was injected with PNiPAAm, it efficiently activated the immune response in several different mouse strains and subsequently induced arthritis [137,138]. Most importantly, PNiPAAm was found to induce the immune responses independent of toll-like receptors.
Table 2. Polymeric adjuvants used in autoimmunity.
3.1. Natural polymers
In adjuvant research, few natural polymers proved to be efficient adjuvants for the induction of autoimmune responses. LPS is the first natural polymer that has been used as an adjuvant and its immunomodulation activity was first reported by Claman , who used LPS in conjunction with a T-dependent antigen. Recently, the adjuvant properties of LPS have been comprehensively characterized. LPS is a component of the cell wall of Gram-negative bacteria and used widely as the B cell mitogen . It is composed of O antigen (polysaccharides) and lipid A moiety linked by a trisaccharide unit . The mode of action of LPS is not fully understood yet, but lipid A is responsible for mitogenic activity and toxicity. In earlier experiments, it was demonstrated that LPS in conjunction with sheep red blood cells was capable of inducing significant antibody responses . The mode of action of LPS is mainly via the humoral immune response to various T-independent antigens. Enhancement of antibody responses was observed when LPS and antigen were injected at the same time, and it was mainly dependent on the amount of antigen injected. Two- or threefold antibody increase was observed at the optimal amount of antigen, while the effect was strongest up to 40-fold when a low dose of antigen was given . The effect was almost suppressed or absent when LPS was given 1–2 days after antigen injection . Co-oral [131,149] administration of LPS along with CII enhanced both CII-specific antibody production and T-cell responses leading to a more chronic arthritis.
In this respect, β-glucans, another natural polysaccharide extracted from Candida albicans, has been tested as an adjuvant in the induction of experimental arthritis. β-Glucan is the polymer of d -glucose monomeric units linked via β-type glycosidic linkage and mainly occurs in variety of microbes. β-Glucan as an adjuvant has immunologic properties, which depend on solubility, molecular weight, degree of branching and conformation of the polymer [150–152]. Additionally, β-glucan has the ability to activate leukocytes, production of reactive oxygen species (ROS) and TNF-α cytokine [132,153]. Mice injected with β-glucan polymer are able to produce autoantibodies . Hida and co-workers studied autoimmunity of the particulate form of oxidized β-glucans isolated from C. albicans in DBA/1 mice . In vitro, these glucans activated the alternative complement pathway, enhanced vascular permeability and induced a higher level of IL-6 production by macrophages . Furthermore, they have tested adjuvant activity of a water-soluble β-glucan fraction released from C. albicans and this fraction accelerated cytokine synthesis and activated immune cells leading to autoimmune arthritis [150,151]. Similarly, the adjuvant potential of β-glucan polymer isolated from a pathogenic fungus Laminaria digitata was tested in the spontaneous SKG model of autoimmune arthritis. These mice were unable to develop spontaneous arthritis in a specific pathogen-free environment but were able to develop arthritis after an injection of β-glucans intraperitoneally .
Furthermore, zymosan has also been demonstrated to activate autoimmune responses. Zymosan is a polysaccharide extracted from the cell wall of the yeast, Saccharomyces cerevisiae and it mainly contains β-1,3-glucan residues. As an adjuvant, zymosan binds to the TLR-2 receptor and activates the NF-κB signalling pathway, thereby inducing inflammatory cytokine production [153,154]. β-Glucan derived from zymosan and their brief oxidation product act as a powerful adjuvant (carbohydrate adjuvant) for collagen-induced arthritis and at first, Keystone et al.  studied the arthritogenicity of zymosan independently. Induction of arthritis was mediated via activation of the alternative complement pathway and macrophages, through binding to the membrane receptor dectin-1.
In this direction, the potential effect of lipomannan as an adjuvant has been analysed in collagen-antibody-induced arthritis, a model widely used to study the effector phase of arthritis without involving the priming phase. Lipomannan enhanced arthritis in the presence of functional phagocytes, which produced more ROS when injected with a cocktail of anti-collagen antibodies binding to well-defined CII epitopes by mainly interacting with TLR2, and the disease phenotype was mainly from the activity of granulocytes [134,135].
3.2. Synthetic polymers
Similarly, the adjuvant potential of synthetic polymers was studied using animal models of auotimmune diseases. Shenoy and Christadoss used TiterMax non-ionic block copolymer as an adjuvant with torpedo acetylcholine receptor antigen in C57BL/6 mice to induce autoimmune myasthenia gravis disease, characterized by weakness in muscles with electrophysiological defects with a significant increase in serum IgG levels . Non-ionic block polymers are a copolymer of hydrophobic POP and hydrophilic POE blocks . Block copolymers with high hydrophobicity were showing greater adjuvant potential, which could be changed by altering hydrophobicity and hydrophilicity content of the copolymer. These copolymers act as surface-active agents and form a stable water-in-oil emulsion, which act as a depot for slow release of antigen through the polymer.
Very recently, the adjuvant potential of temperature responsive PNiPAAm has been studied in the development of murine rheumatoid arthritis. PNiPAAm was synthesized through free radical polymerization (having a molecular weight of 120 kDa) and purified through repeated cycles of cooling and heating. This polymer, when injected with CII showed gelation properties, which led to clear precipitation and subsequent slow release of antigen (figure 2). More importantly, the polymer alone was unable to induce an immune response and only mice immunized with the polymer mixed with CII induced antigen-specific autoimmunity leading to polyarthritis. All the mice immunized with PNiPAAm-CII developed an antibody response significantly comprising of all the major IgG subclasses and an antigen-specific recall immune response was also observed using lymphocyte proliferation assay. All polymer immunized arthritic mice had massive infiltration of effector cells, including macrophages, neutrophils, eosinophils and osteoclasts, with extensive damage to the joint architecture. CII mixed with the polymer retained its native confirmation, which is a requirement for arthritis induction. CII mixed with a high-molecular-weight form of moderately hydrophobic PNiPAAm induced a significantly higher anti-CII response compared with covalently linked CII and polymer. Interestingly, all the polymer-CII immunized TLR deficient mice developed anti-CII antibodies, demonstrating adjuvancity of PNiPAAm could be independent of TLR pathways. This polymer PNiPAAm grafted with gelatine has been also used as a scaffold to support growth of primary chondrocytes for regeneration of cartilage .
Figure 2. Schematic of reversible behaviour of temperature responsive poly-N-isopropylacrylamide (PNiPAAm). Below the lower critical solution temperature, PNiPAAm-antigen mixture is in the soluble state and it becomes a precipitate above this temperature.
Despite the use of polymers in animal models, autoimmunity has also been reported owing to polymer implants. Lately, a new autoimmune syndrome induced by adjuvants has been reported. A well-known example is silicone polymer implantation . Patients with silicone implants were developing a high level of anti-silicone antibodies in the surrounding tissues , and the presence of autoantibodies was demonstrated via immuno-fluorescence staining in the capsular tissue . In another study, the presence of autoantibodies, including rheumatoid factor and anti-dsDNA antibodies, was observed in experimental mice after silicone implantation, leading to the development of autoimmune diseases . Lidar and co-workers extensively studied autoimmunity in symptomatic women, who had silicone breast implants compared with asymptomatic women  and a 20 per cent increase in IgG titre was observed, and most of the antibodies were against dsDNA, ss-DNA, silicone and collagen type II. Also, chronic fatigue syndrome was triggered in patients with hepatitis B vaccination after silicone implantation . Similarly, various autoimmune diseases including RA, systemic lupus erythematosus and systemic sclerosis were observed in several patients who had undergone silicone implantation . At the cellular level, various phenotypic and functional alterations of T-cell subsets, B-cell activation, autoantibodies directed against endothelial and nuclear antigens were observed as a result of the implanted silicone polymer .
In conclusion, polymers as an adjuvant have the ability to enhance the immunogenicity of antigens in vaccination as well as in the induction of autoimmune responses. They can induce immune responses efficiently through different signalling mechanisms (toll-like receptors, inflammasome pathways) and offer the possibility of substituting bacterial-based adjuvants, which may cause toxicity and undesired activation of the immune system. Polymers have the ability to induce both humoral and cellular immune responses, when given along with an antigen, although they are not capable of eliciting any immune response on their own. Adjuvant potential of polymers depends on the solubility, molecular weight, degree of branching and conformation of the polymer used. Usually, they work on the principle of depot generation for slow release of the antigen for a longer period of time and act as an immuno-modulator via strong antigen presentation. Thus, polymeric adjuvants are highly valuable for better vaccination strategies and in studying basic pathogenic mechanisms involved in autoimmune diseases.
Vaccine Safety Forum: Summaries of Two Workshops (1997)
discussed a selected number of these populations. The final section presents a preliminary research agenda of possible or putative links in these areas.
IMMUNE RESPONSE TO VACCINE ANTIGENS 13
Infection-causing microbes and the vaccines designed to combat them have portions of proteins called antigens. These antigens stimulate a number of cells in the immune system, including macrophages, T cells, and B cells. An immune response begins when macrophages ingest antigens such as proteins entering the body and digest them into antigen fragments. A molecule called MHC (major histocompatibility complex) carries certain of these fragments to the surface of the cell, where they are displayed but they are still locked into the cleft of the MHC molecule. These displayed antigen fragments are recognized by T cells, which stimulate B cells to secrete antibodies to the fragments as well as prompt other immune defenses. According to Berkower, studies suggest that because T cells only recognize antigen fragments from proteins predigested by macrophages, they cannot distinguish between a specific antigen fragment that comes from an infecting microbe and the same antigen fragment that comes from a vaccine (Chisari and Ferrari, 1995).
Stimulated immune cells secrete a variety of chemical substances called cytokines, which determine which class of antibodies are generated. The cytokine interleukin 4, for example, can prompt B cells to secrete immunoglobin E (IgE) antibodies, which trigger allergic reactions. Other cytokines cause B cells to preferentially secrete IgG, which is mainly found in the blood, or IgA, most of which is found in body fluids.
Different MHC molecules bind to different antigen fragments the set of MHC molecules and the genes that commandeer their production vary widely from one individual to another. Therefore, although the immune systems of two people may respond to the same protein in a vaccine, their T cells may respond to different portions of that protein. This diversity fosters differences in responses to vaccine antigens.
There are at least nine chemically distinct classes of immunoglobulins, and the balance of the various types of cytokines that stimulate antibody secretion determines the final response to a vaccine. Consequently, vaccine responses can differ between individuals because the same vaccine stimulates different individuals to generate different amounts of the various cytokines. Vaccine responses might not only differ in the short term, but they could also vary in the
This section is based on information presented by Ira Berkower, Hira. Nakhasi, Henry McFarland, and Burton Waisbren.
long term if they trigger a predominantly IgE response. This response could trigger an allergic reaction to future immunizations with the same antigens (IOM, 1994a).
To prevent the body's immune system from destroying its own tissues in what is known as an autoimmune response, immature T cells that react against self-antigens are thought to be destroyed in the thymus gland, creating what is known as central tolerance. Peripheral tolerance might also occur, whereby those T cells that could potentially react to self-antigens and that are not destroyed in the thymus are somehow prevented from causing an autoimmune reaction. Although studies suggest that peripheral tolerance exists, at least in experimental animals, the mechanism for the process is not yet known. If peripheral tolerance exists in people, an autoimmune response might occur in response to vaccination if the vaccine somehow disrupts that peripheral tolerance or if peripheral tolerance is not strong on the day of vaccination (Miller et al., 1989).
Some people have suggested that vaccines can stimulate autoimmune reactions if some of the antigen fragments in vaccines resemble a person's self-antigens. However, it is unclear why an immune system that is tolerant of its own self-antigens would respond to a self-antigen mimic in a vaccine. Berkower speculated that vaccines might counter peripheral tolerance and foster an autoimmune reaction if they contain molecular mimics of self-antigens that are usually not exposed to T cells, because peripheral tolerance seems to depend on the continuous presence of an antigen.
Nakhasi suggested that an autoimmune response might be instigated by a vaccine or by natural infection if the microbial antigens bind to self-antigens in infected cells and change the antigens' shape such that they are no longer tolerated and can elicit an immune response.
According to McFarland, researchers suspect that molecular mimicry, which could possibly lead to an autoimmune disorder, might be occurring between self-antigens and antigens from microbes or vaccines if the two antigens share much of the same chemical structure. Recent studies suggest, however, that they need to have a similar structure only in the narrow region that binds to the T-cell receptor (Vogt et al., 1994 Wucherpfennig et al., 1994). In addition, the amino acids in this region do not have to be identical rather studies suggest that they must have the same basic chemical and charge properties (Vogt et al., 1994 Wucherpfennig et al., 1994 Vergelli et al., 1996).
Some researchers have hypothesized that autoimmune diseases may be stimulated by viruses (Fujinami et al., 1985 Westall and Root-Bernstein, 1983). Westall and Root-Bernstein have postulated that this may occur if three criteria are met. The first one is that antigens are present that have molecular structure similar to self antigens found in certain human tissue (Westall and Root-
Bernstein, 1983). The second one is that an adjuvant derived from walls of the bacterial cells used in the vaccine's manufacture is present. The third one is that antigens exhibiting complimentarity are present. When these three criteria are present an antigenic challenge to the host evokes autoimmunity. This hypothesis has been named Multiple-Antigen-Mediated-Autoimmunity (MAMA) Syndrome. Root-Bernstein has presented evidence that it exists in AIDS patients who develop encephalomyelitis (Sela and Arnon, 1992). Waisbren suggested that the MAMA Syndrome might be a mechanism that induces autoimmunity in susceptible individuals after vaccination with virus components that are also present in human tissue. These antigens may be associated with other complimentary viral antigens and bacterial cell wall components present in the patient who has been vaccinated.
Premature Infants 14
Each year about 300,000 premature infants are born in the United States, and most of these survive past infancy (Ventura et al., 1996). Preliminary studies suggest that premature large birthweight infants have the same or less risk of experiencing the usual adverse effects reported for full-term infants following vaccination with HBV or DPT (Bernbaum et al., 1989 Koblan et al., 1988 Ramsay et al., 1995 Losonsky, unpublished data). However, recent data with DPT immunization in very low birth weight infants suggest that moderate to severe adverse reactions can occur following routine vaccination with DPT in infants born less than 1,000 grams (Sudhakarow et al., 1996). Routine immunization of extremely premature infants is associated with significant adverse events. According to Losonsky, larger studies that cover the full range of vaccines given to infants need to be done to assess the risk of common adverse events in premature infants.
Current vaccine schedules are based largely on the immune responses seen in full-term rather than premature infants. The human immune system matures throughout fetal life and up until about 2 years of age, however, so even full-term infants do not develop an adequate protective immune response to most vaccines administered in the first weeks of life. Losonsky expressed concern that premature infants given vaccines at the standard times may not develop adequate immunity to the diseases against which the vaccines are designed to protect. There is also speculation that some premature infants might develop immunologic tolerance to the viral or bacterial antigens in vaccines such that
This section is based on information presented by Genevieve Losonsky.
they do not develop an immune response to subsequent vaccines given later in life.
According to Losonsky, 98 percent of full-term infants achieve protective immune responses after receiving the third dose of HBV (Losonsky, unpublished data). A study of HBV given to premature infants, however, found that only 54 percent of infants with birth weights of less than 1,000 grams and 70 percent of infants with birth weights of 1,000 to 1,500 grams attained protective immune responses after receiving the third dose of the vaccine. Thirteen of the premature infants with little or no detectable antibody responses to the three doses of HBV were given a fourth dose of the vaccine at 9 to 12 months of age. Only 46 percent of these revaccinated infants achieved protective antibody levels (Losonsky, unpublished data).
These data suggest, Losonsky noted, the possibility that tolerance develops in some premature infants. Researchers are following those infants who did not respond to the fourth HBV dose to see if their lack of response is permanent. Other studies suggest that the immune systems of premature infants given DPT and oral polo vaccine at standard times respond as well as those of full-term infants (Koblin et al., 1988 Conway et al., 1993 D'Angio et al., 1995). However, a decreased immune response to some enhanced-potency inactivated polio-oral polio vaccine combinations and to certain Haemophilus influenzae type b conjugate vaccines was seen in premature infants. The response depended on gestational age and weight at the time of the first immunization (Munoz et al., 1995 D'Angio et al., 1995).
According to Losonsky, these findings suggest that vaccine schedules, dosages, and combinations for preterm infants may have to differ from those for full-term infants. She added that the best schedule and dosage for each vaccine cannot be predicted on theoretical grounds but can only be determined by further study. Care must be taken, she said, not to leave premature infants vaccinated but unprotected from disease. A representative of the National Institute of Allergy and Infectious Diseases (NIAID) pointed out, however, that the risk of inducing tolerance to vaccination for a specific disease must be balanced with the risk of the infant dying or suffering long-term consequences from natural infection. Premature infants, for example, are more likely to have respiratory abnormalities that may put them at greater risk for complications from the respiratory disease pertussis (whooping cough).