Introduction

Since the discovery of the smallpox vaccine by Dr. Edward Jenner in the nineteenth century, advances in vaccination technology have helped eradicate numerous infectious diseases. However, there are currently no effective vaccines available for infectious agents such as the human immunodeficiency virus, malaria Plasmodium, tuberculosis bacteria, and dengue virus. For domesticated animals, a host of pathogens continue to threaten their health and welfare. Vaccination involves artificial induction of immunity by exposing individuals to modified pathogenic or toxic components. Significant improvements have been made to increase vaccine safety while maintaining effectiveness, or antigenicity, of the vaccine components. Advances in immunology and molecular biology have also paved the way for developing sophisticated forms of vaccine.

This chapter will cover the principles of the various forms of vaccine and introduce recent advances for each vaccine category. Live and attenuated vaccines, generally eliciting strong immune responses, will be discussed first. Inactivated or killed vaccines, with better safety profile, will then be introduced. Recombinant DNA technology has allowed the development of subunit vaccines, and production of these protein vaccines through bacterial and insect systems will be covered. Finally, since DNA encoding various antigens has been used as a form of vaccine with notable success, the principles of DNA vaccine and the challenges of development will be discussed. Except for live and attenuated vaccines, the other forms of vaccine usually require adjuvants to enhance immune responses. The appropriate adjuvants for each vaccine category will be covered under each section, while encapsulation technology as an adjuvant form will be discussed separately.

Live Vaccine

Live vaccines contain low virulent or attenuated microorganisms. They are altered so that they cannot cause disease but retained their ability to induce protective immunity.

As the live organism can still infect target cells, these vaccines can replicate and are thought to better simulate natural infections and produce a broad immune response including both cellular and humoral immunity and generally do not require an adjuvant to be effective.

Attenuated microorganisms including particularly bacteria and viruses may be found naturally, or they may be attenuated via passage of the virus through a foreign host such as tissue culture, embryonated eggs, and live animals. But some viruses are difficult to passage in nonhost materials. Reverse genetics can be used to rapidly develop a live-attenuated vaccine based on currently circulating virus. For example, NDV genotype VII is difficult to attenuate. Recurrent outbreaks of ND in vaccinated birds is due to an antigenic mismatch between the genotype II commercial vaccine strains and the genotype VII virulent strains that are presently circulating. The reverse genetics NDV genotype VII vaccine significantly reduced virus shedding from vaccinated birds when compared to B1 vaccine (Xiao et al. 2012). Live vaccine can produce a broad immune response including cellular immunity, humeral immunity system, and local immunity.

Live vaccines provide stronger, longer-lasting, and more rapid protection, and thus boosters are required less frequently. They are low cost and may require only one dose to be effective. Additionally, live vaccines can easily be administered by various routes, such as injection, drinkable water, or inoculation into the nasal cavity or eyes.

However, they still have a residual virulence or a risk of reversion to virulent wild types. As an example, in 1996, a porcine respiratory and reproductive syndrome (PRRS) vaccine program was initiated in Denmark for the control and prevention of PRRS (Botner et al. 1997; Grebennikova et al. 2004). Following the implementation of the vaccination program, epizootics of PRRS-like disease were reported in some of the vaccinated herds and PRRSV was isolated from clinically affected swine. In some cases, genetic analysis indicated the PRRSV was closely related to the vaccine virus (North American genotype) and not the Danish field viruses (European genotype) (Madsen et al. 1998). In conclusion, live vaccines have played a major and effective role in animal disease control, but are not without drawbacks.

Inactivated Vaccine

An inactivated vaccine (or killed vaccine) consists of microorganisms killed using methods and agents such as chemicals (formalin, binary ethyleneimine), heat, radioactivity, or antibiotics. The inactivated microorganisms are destroyed and cannot replicate in a vaccinated host, but the capsid and outer membrane proteins are intact enough to be recognized by the immune system. Inactivated vaccines are exogenous antigens, which activate CD4 + helper T cells to induce immune response. It cannot revert to virulent forms capable of causing diseases, but incomplete inactivation can result in intact and infectious particles.

Inactivated microorganisms generally provide a weaker immune response than live microorganisms. In particular, immune response elicited by inactivated vaccines lacks cell-mediated immunity. For this reason, inactivated vaccines are administered with adjuvants, and booster shots are required to elicit sufficient response and a long-term immunity. It is important to note that field virulent microorganisms should be used as vaccine antigen to provide good immune protection.

Subunit Vaccine

Subunit vaccine became a reality in human and animals due to the DNA recombination technique. The subunit vaccine is produced from specific subunit protein of a pathogen and therefore has less risk of adverse effects than whole pathogen vaccines. While there are many subunit vaccines used in animal (Kushnir et al. 2012), the human hepatitis B virus surface protein (HBV S protein) was the first subunit protein in humans (Kniskern et al. 1989). The primary advantage of subunit vaccine is safety. It is safe to use even in immunocompromised humans. The vaccine contains only the important immunogen or epitope and not the genetic material of pathogens. On the other hand, subunit vaccines must be combined with an adjuvant, and repeated immunization is required to enhance humoral and cellular immune responses. Subunit vaccine can be expressed by Escherichia coli, yeast, baculovirus, plant, and mammalian cell systems.

Bacterially Expressed Subunit Vaccine

Bacterial expression systems are excellent candidates for mass production. There are many bacterial species used for foreign genes expression. The bacteria E. coli is extensively used for expression in subunit proteins development. See Table 1 for the comparison of the advantages and disadvantages between prokaryotic and eukaryotic systems. There are several advantages in using bacterial expression for subunit vaccine. The production cost of the bacterial system is lower than other systems and the production equipment and processes are easy to handle. The low cost is the main reason for choosing the E. coli expression system, especially when the vaccine is being developed for veterinarian use. Moreover, with E. coli, the protein overproduction system is available and easy to control. The lac promoter is a common overexpress system induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the resulting expressed protein is abundant. However, protein overexpression will kill the bacteria or the protein might become included in E. coli’s cytoplasm. In an overexpressed system, the bacterial-related secretion, when fused with target genes, can reduce inclusion body formation. Many signaling peptides have been used in the E. coli system to increase protein solubility. Unfortunately, there is not a universal signaling sequence for all recombinant proteins. Selecting an optimal signal sequence is important for the efficient secretion of recombinant proteins. The selection is done through trial and error (Choi and Lee 2004).

Table 1 The advantage and disadvantage between different protein expression systems
Full size table

The primarily disadvantage of E. coli expression system for subunit vaccine is that there are few posttranslational modification systems of proteins in E. coli. The bacteria protein glycosylation is rare and relatively few bacterial glycoproteins are known. In E. coli, only a few glycoproteins have been described (Sherlock et al. 2006; Benz and Schmidt 2001). Glycosylation is very important for protein’s tertiary and quaternary structure. The B cell epitope conformation is different between pathogens and the expressed un-glycosylated subunit protein. Because of the tertiary and quaternary structure, un-glycosylated proteins cannot induce property neutralization antibody against the pathogens. In a research, the author engineered a synthetic pathway in E. coli for the production of eukaryotic trimannosyl chitobiose glycans. The pathway also allowed for the transfer of glycans to specific asparagine residues in the target proteins (Valderrama-Rincon et al. 2012). These genetically modified bacteria could then produce glycosylated protein for subunit vaccine development. The gene codon usage bias is different between eukaryotic and prokaryotic systems. There are many rare codons in E. coli and it is difficult to express a eukaryotic gene. According to the bioinformatics and computational biology, many statistical methods have been proposed and used to analyze codon usage bias in E. coli (Comeron and Aguade 1998).

Several studies have demonstrated that subunit vaccines require repeated vaccination in order to establish and prolong sufficient immune response against viral, bacterial, and protozoal infections. The adjuvant is the key factor in subunit vaccine efficiency. The word “adjuvant” is derived from the Latin word “adjuvare,” which means “to help.” There are many kinds of adjuvants. Aluminum salts have frequently been incorporated as an adjuvant in vaccines licensed for use in the wild. Aluminum hydroxide, Al(OH)3, has been approved by the FDA as a vaccine adjuvant for more than 70 years. It is used to boost immune response to vaccines by adsorbing the antigen. Oil-based adjuvants are commonly used in many commercial vaccines. Their major purpose is to hold the antigens at the injection site and release them slowly into the bloodstream and lymph node for longer-lasting immunity. Microparticle encapsulation also prolongs antigen release to enhance the immune response and will be discussed later in this chapter. Recently, some bacterial toxins were expressed as fusion protein with the main antigen and used to increase the immune response. The E. coli heat labile toxin (HT), Clostridium perfringens exotoxin (CPE), and Shiga-like toxin (Stx) were fused to the antigen to target the immune cell. The fusion increased the antibody level, T cell responses, and cytokines concentrate. Due to the adjuvant, same level of immune response can be achieved with a lower antigen dosage and fewer vaccinations. After taking into account the advantages and disadvantages of subunit vaccines and also the boost by an adjuvant, E. coli- expressed protein remains a good candidate for vaccine development.

Insect Cell-Expressed Subunit Vaccine

Baculoviruses are a diverse group of large, rod-shaped DNA viruses capable of infecting more than 500 insect species. Their double-stranded, circular genomes are 80–180 kbp in size depending on the viral species. Among the numerous baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and, to a lesser extent, the Bombyx mori nucleopolyhedrovirus (BmNPV) are most commonly utilized. The former infects insect-cell lines for protein expression, and the latter infects the silkworm for the production of recombinant protein. Both viruses can express foreign genes under the control of highly expressed, very late promoters, including polyhedrin (polh) and p10 promoters. Protein production in the BEVS is easily achieved by infecting cultured insect cells. The most common insect-cell lines include Sf-9 and Sf-21, derived from Spodoptera frugiperda and BTI-TN-5B1-4 (commercially known as High-Five™). Other cell lines in use include Tni PRO (Expression Systems) and Express SF (Protein Sciences). These insect cells grow optimally at 27 °C and do not require CO2, making cell cultures feasible for most laboratories. The cells can grow in serum-free media and the cultures can easily be scaled up. Various serum-free media, desired for biopharmaceutical protein production, have been developed for Sf-9 (e.g., Ex-Cell 420, SF-900 II, and HyQ-SFX-Insect) and High-Five™ cells (e.g., Express Five®). Furthermore, these cell lines can be easily cultivated and infected in the monolayer or suspension cultures, making the scaling up from bench to industrial feasible (Ikonomou et al. 2003). The parameters in the expression and the process scale-up depend on various bioreactor types (e.g., stirred tank reactor, airlift reactor, packed-bed reactor, etc.), the biological parameters (e.g., cell density upon infection, dissolved oxygen in media, multiplicity of infection (MOI) of infection, harvest timing, etc.), and the environmental parameters (e.g., shear stress, agitation speed, etc.). These conditions influencing the production yield have been extensively studied and reviewed (O’Reilly and Luckow 1992; Taticek and Shuler 1995).

Compared to other expression systems, BEVS has a number of advantages:

  1. (i)

    The strong p10 or polyhedrin promoter enables high levels of expression of heterologous genes compared to mammalian cell expression systems.

  2. (ii)

    Baculoviruses are insect pathogens and are unable to replicate in vertebrates, including humans, and is therefore safer to work with than mammalian viruses. The lepidopteran insect cells used are also free of human pathogens.

  3. (iii)

    Recombinant baculoviruses can be generated rapidly within 1 week. Several commercially available BES kits (e.g., BaculoGold™; FlashBAC, BacPAK™, Bac-to-Bac®) use the conventional method of homologous recombination in vivo or in E. coli.

  4. (iv)

    The large baculoviral genome makes it possible to insert multiple gene expression cassettes and then to express multiple proteins simultaneously in a single infection.

  5. (v)

    Insect cells permit multiple posttranslational modifications such as folding, glycosylation, phosphorylation, acylation, proteolytic cleavage, and so on. These modifications are similar or identical to those occurring in mammalian cells. Thanks to these attributes, BEVS has become one of the most popular systems for routine production of recombinant proteins (Hitchman et al. 2009).

Currently, a number of biologicals, including interferons, antigens, and vaccines produced in BEVS, are already approved or are in various phases of clinical trials (Hitchman et al. 2009). The proteins produced are used for functional studies, vaccine preparations, or diagnostics. The first commercialized vaccine produced in the baculovirus expression system is directed against the classic swine fever or hog cholera (Intervet). Among these products was GlaxoSmithKline’s Cervarix™, a human papillomavirus vaccine that contained BEVS-produced virus capsid protein. Cervarix™ prevents cervical cancer and was approved for human use in the States in 2009 (Aucoin et al. 2010). In addition, a trivalent recombinant hemagglutinin (HA) vaccine (FluBlok®, Protein Sciences) exhibited high efficacy against seasonal influenza for human adults and was approved by the FDA in January 2013. There were also several BEVS-derived and licensed subunit vaccines for veterinary use. Porcilis® Pesti (Intervet) and Bayovac® CSF E2 (Bayer) are the first two subunit vaccines for the classic swine fever, which is one of the most important porcine contagious diseases. Porcilis® PCV(Merck) and CircoFLEX®(B. Ingelheim) are the second veterinary vaccines for PCV2 produced by BEVS (Cox 2012).

The disadvantages of the baculovirus expression system are as follows:

There are drawbacks associated with the BEVS. First, polyhedron and p10 are late promoters, and baculovirus infection results in cell death and lysis a few days postinfection. Protein production reaches a maximum near the death of the infected cells, so some posttranslational modification machinery might be suppressed at that time. Hence, the quantity and quality of the expressed protein could be suboptimal then. In order to overcome the drawback, early baculovirus promoters (e.g., IE-1) were used in either transiently or stably transformed cells systems. These systems provide a more suitable environment for protein production, but the expression level is lower than those obtained with the lytic baculovirus system.

The second disadvantage of BEVS is its inefficiency in properly processing large inactive precursor proteins, such as peptide hormones, matrix metalloproteases, and fusogenic viral envelope glycoproteins.

Thirdly, although BEVS has become a well-established platform for the production of recombinant glycoproteins, the glycoproteins produced by insect cells have a clearly different N-glycans than those produced by mammalian cells. In particular, insect cells lack sufficient processes such as sialyltransferase activities and metabolic enzymes such as CMP-sialic acid (Hooker et al. 1999). These are necessary for generating complex-type proteins. N-Glycans from insect cells are not usually processed into sialylate recombinant N-glycoproteins. Some have attempted to “mammalianize” or “humanize” the N-glycosylation capacity of insect cells (Jarvis et al. 2004).

Another limitation of BEVS is that up to 40 % of the genomic DNA, which also includes the inserted foreign gene, might be autonomously deleted during the baculovirus propagation (Kool et al. 1991). This problem becomes important in large-scale biotechnological applications since defective baculovirus might be formed and accumulated in a continuous production or scale-up of AcMNPV.

Baculovirus Transduction of Mammalian Cells

For a long time, the baculovirus was thought to be capable of infecting only insect and invertebrate cells, not mammalian cells. In 1983, however, the virus was found capable of entering mammalian cells. Subsequently, Hofmann et al. showed that recombinant baculoviruses harboring a mammalian expression cassette were able to transduce human hepatocytes (Hofmann et al. 1995). They also efficiently expressed the reporter genes in the mammalian cells under the control of cytomegalovirus (CMV) promoter. Since then, numerous cell types of human, rodent, porcine, bovine, fish, and chicken have been found permissive to baculovirus transduction (Hu 2006). Recombinant baculoviruses containing mammalian system promoters (e.g., CMV or Rous sarcoma virus (RSV) promoter) can perform high-level transgene expression after delivering foreign genes into mammalian cells. The efficient transduction of baculovirus into many cell types has made baculovirus a promising vector for in vivo gene delivery. It will also contribute to the study of gene functions, the development of non-replicative vector vaccines, and the increase in gene therapy strategies.

DNA Vaccine

In 1990, Wolff injected animals with vectors that contained CAT or lactase gene, resulting in the expression of CAT protein and lactase for a short period (Wolff et al. 1990). Subsequently, in 1993, Ulmer and others performed intramuscular injection of a vaccine containing viral DNA. The vaccine proved successful against the flu virus by simultaneously generating humoral and cellular immune responses (Ulmer et al. 1993). DNA vaccine is a new technique that combines an existing vaccine with plasmid DNA or RNA of specific pathogen antigen gene within the bacterial plasmid. After the injection of the immune plasmid DNA, the host cells produced antigen protein and induced humoral and cellular immune responses that protected the host (Wolff et al. 1990; McDonnell and Askari 1996). This type of vaccine is also known as the nucleic acid vaccine, the DNA vaccine, the gene vaccine, or the third-generation vaccine.

DNA Vaccine

Vaccines train the immune system to identify pathogens by treating exogenous protein as an immunogen. DNA vaccines primarily leverage mammalian expression vectors. The muscle cells express protein antigens after immunization. Subsequently, the antigen-presenting cells (APC) activate helper T cells (Th) through phagocytosis of the protein antigens. The resulting release of cytokines stimulates B cells to produce antibodies. This process is known as humoral immunity. Alternatively, cell-mediated immunity occurs when the cytotoxic T lymphocytes (CTLs) destroy the pathogens directly. Once cleared of pathogens, some B cells and T cells turn into memory cells. In a future invasion by the same pathogen, the pathogen is immediately identified and the immune response triggered.

In recent years, studies have pointed out that some DNA vaccines such as the ones for hepatitis B virus or influenza can cause a very strong immune response (Ulmer et al. 1993; Chow et al. 1998). The results of this study showed that animals vaccinated with one dose of the DNA vaccine can produce persistent immune response but not all antigens produce the same effect. Currently, the mechanism with which DNA vaccine induces immune response is not known. However, there are three hypotheses (Gurunathan et al. 2000).

The first hypothesis describes the immune response as follows. An intramuscular injection or gene gun immunization of the DNA vaccine into muscle cells or skin cells turns them into antigen-presenting cells. Antigenic protein and proteasome are produced through intracellular transcription and translation. Proteolytic enzymes then turn proteasome into digested peptides. Small fragments of these peptides bind to major histocompatibility complex class I (MHC class I) molecules in the endoplasmic reticulum and get sent to the Golgi complex. They are then transferred to the cell surface, forming the MHC class I-peptides complexes. At this time, cytotoxic T cells use T cell receptor (TCR), combined with MHC class I-peptides complex, to activate an immune response. This process is similar to the response induced by a viral infection. However, there are some unanswered questions posed by this hypothesis. For example, in order to activate the T cells, the MHC class I-peptides complex binding of TCR by itself is not sufficient. The antigen-presenting cell surface of B7-1 and B7-2 binding of CD28 T cells to transmit secondary signal is also needed. However, skin cells or muscle cells do not express B7-1 or B7-2 molecules. Furthermore, the cell surface does not have MHC class II molecules and therefore cannot activate T helper cells.

The second hypothesis theorizes that the DNA enters the antigen-presenting cells directly. Examples of these types of cells are the macrophage and dendritic cells in muscle tissue and the Langerhans cells in the skin. The antigen-presenting cells express the antigen. The antigen then combines with MHC class I or MHC class II molecules and gets sent to the cell surface in order to activate T cells for the immune response.

The final hypothesis begins with the muscle or skin cells translating the antigenic proteins by themselves. The intracellular materials bind to heat shock protein (HSP) and then get secreted out of the cell. The antigen-presenting cells engulf the protein antigen to form lysosome through phagocytosis. In an acidic environment, antigens are digested into peptides by enzymes. Endosomal lysosome is formed through endosome and lysosome fusion. The endosome of MHC class II molecules then binds to the pathogen peptides, forming MHC class II-peptides complex. The complex is then transferred to the cell membrane. The antigen-presenting cells activate CD4+ T cells to provide the signal. They also activate B cells to secrete antibodies and cytokine, which in turn activates CD8+ T cells to eliminate pathogen. For antigen proteins without phagocytosis, they are recognized by the membrane-bound IgM on B cell surface, triggering the first signal. When the cells are released, hormones stimulate B cells to transmit the second signal and also activate them to produce antibodies. When Iwasaki and other scholars initiate intramuscular and gene gun immunization, they find that the activated T cells caused CTL response by the bone marrow. The responses were all derived from antigen-presenting cells rather than the immune position. At this point, the second and third hypotheses remain under investigation, while the first hypothesis was disproved.

Influence Factors of DNA Vaccines

Studies have indicated that some DNA virus vaccine for viruses such as the common flu virus or the hepatitis B virus (HBV) can elicit a good immune response. However, some DNA vaccines do not offer protective immunity. The effectiveness of DNA vaccines is determined by several factors.

The first factor is DNA’s ability to enter cells. Although some type of cells, such as muscle or skin cells, are easy to enter, others are not so. The biggest challenge is the cell membrane. Since both the DNA and the membrane surface are negatively charged, they repel each other. The plasmid DNA has a hard time entering the membrane and requires mechanical or chemical methods to get past it.

The second factor concerns antigen expression and intracellular DNA characteristics. Factors affecting gene expression include gene promoter, gene enhancer, intron, etc. Currently, the most commonly used vector is the cytomegalovirus immediate-early promoter (pCMV). Antigen variety and transgenic cells result in promoters having different specificity and induced transcription efficiency. For example, pCMV in muscle cells has a higher expression of the antigen protein (Cheng et al. 1993). Additionally, promoter of plasmids can only be induced and expressed on specific type of cells.

Finally, the effectiveness of DNA vaccines can be enhanced with adjuvants. Adjuvants such as CpG motif, cytokine, and chemokine can be added to DNA vaccines to increase immune response.

Advantages of DNA Vaccines

DNA vaccines can make use of plasmid RNA or DNA for immunization. The advantage of using RNA is that there is no danger posed by embedded chromosomes. However, the disadvantage is that the vaccine will not be stable and the expression of antigen is brief. As a result, RNA vaccines are rarely used in recent vaccines. Conversely, DNA vaccines have proven effective in the study of various diseases. DNA vaccines have been studied extensively in the context of infectious diseases, cancer, allergies, and autoimmune diseases. Advantages of DNA vaccines include the elicitation of cellular and humoral immune responses and natural antigen expression. The absence of purified proteins distress improves cross protection and avoids the possibility of toxic recovery and virus strains mutation. The vaccine can also be combined with various genes under the same vector and become a multivalent vaccine. DNA vaccines are more effective and their quality easier to control. Lastly, vast amounts of plasmid DNA can be produced quickly.

DNA Vaccine Safety

Although DNA vaccines in clinical applications have many advantages, safety is still the most important consideration. The use of DNA vaccines may lead to these three types of unintended effects.

The first consideration is whether the plasmid DNA will cause anti-DNA antibody response related autoimmune disorders such as systemic lupus erythematosus. While the first phase of clinical testing did not show any anti-DNA antibody response, this is a possibility that we have to watch out for.

The second unintended effect is the increased risk of cancer. When a retrovirus or plasmid DNA enters the host cell chromosome, it might lead to gene mutations or alter the proto-oncogene and tumor-suppressor gene expression. This increases the risk of cancer. In 1992, Wolff and other scholars find that after the plasmid DNA enters into the cells, it forms a complete ring and is stable inside the nucleus (Wolff et al. 1992). The DNA is not inserted into the host cell chromosome. For DNA vaccine development, this is good news.

The third unintended effect is that the plasmid DNA might be expressed in a host over the long term, causing intolerance or toxicity. Since vaccination is usually applied after birth to a baby whose immune system is not yet mature, it may be mistakenly targeted by the baby’s immune system, leading to antigen tolerance. Once tolerance occurs, additional vaccination no longer produces protection. Studies have been conducted on a day-old mouse injected with DNA influenza vaccine. The mouse turns into an adult mouse; a booster shot still produces memory response, proving that antigen tolerance does not occur (Hassett et al. 2000). Although tolerance is not observed in newborn mice, we need to look for it in human trials. Additionally, long-term antigen expression, such as cytokine or chemokine gene expression, might cause toxicity.

DNA Vaccine Preparation

DNA vaccines are created by cloning the pathogen gene into the selected vector, and then the constructed vector is injected into animals. After animals have been vaccinated, protein is expressed in vivo, thereby achieving immunization.

Plasmids of DNA Vaccine

Plasmids are the most used vectors for DNA vaccines. The vectors are usually double-stranded loop from microorganisms with antigenic genes. The plasmid’s promoter should be sufficiently strong as this is a prerequisite for DNA vaccines. Different promoters have different protein expression capability. High and stable expression promoters, such as the cytomegalovirus (CMV) or the Rous sarcoma virus (RSV) promoter, are currently the most often used DNA vaccine promoters. Additionally, plasmids must have replication origin and the resistance gene. A large number of copies can be made from plasmids.

Viral Vectors

Viral vectors are obtained from infected and invaded host cells. Recombinant vectors are embedded in viral gene vectors and injected into the host. To facilitate entry into the host cell, DNA segments were used. Recombinant vectors do not normally cause disease onset in the host and at most cause low morbidity rate. When the viral vector infects human or animals, DNA is inserted into cells, followed by the expression of the unit antigen and the triggering of immune response. The most frequently used viral vectors are retroviruses such as the adeno-associated virus. Infectious viral vectors are difficult to prepare and purify. In addition, the viral vector may also induce the immune response of the host so its clinical applications are limited. However, since viral vectors are used in DNA vaccines and gene therapy, it is still worth attention.

Bacterial Vector

Using genetic engineering technology, we can insert genes that express specific antigens into the genome of bacteria that can grow in the cells of human or animals. The host is then infected with the bacteria. The type of bacteria selected for infection does not cause significant pathogenic disease. Upon infection, DNA is inserted into the cell and its expression induces an immune response.

DNA Vaccines via Gene Gun

Gene guns are most frequently used in the mammalian DNA vaccination. Its principal is similar to that of an air gun that uses high-pressure helium gas to shoot DNA-coated gold particles into the target tissue for transfection. In a study, gene gun immunization with DNA vaccines in poultry elicited good immune effect (Kodihalli et al. 2000). However, the gene gun method is expensive. In 1993, Fynan and others compared different application methods in chickens: intramuscularly (IM), intravenously (IV), intratracheally (IT), intrabursally (IB), intraperitoneally (IP), subcutaneously (SC), and intraocularly (IO). The specificity of the IgA antibody titer in chicken tears and bile was higher in IO vaccination versus IM vaccination. However, the specificity of IgG antibody titers was significantly higher in the IM vaccination (Fynan et al. 1993).

DNA Vaccine Doses

With a gene gun vaccination, protection can be conferred with as little as 0.25 μg of plasmids. For intramuscular application, 100 μg should be used. The dose of vaccination correlates with antibody production. Antibodies, however, do not increase significantly beyond 100 μg when applied intramuscularly.

Future Development of DNA Vaccine

Recently, many scholars have committed to the development of DNA vaccines. Vaccines are under development for the human immunodeficiency virus, malaria, tuberculosis, measles virus, B-type hepatitis, C-type hepatitis virus, and dengue fever. Vaccines are also under development for allergies, autoimmune diseases, and tumor (Gurunathan et al. 2000). DNA vaccines are being developed for animals such as cattle, sheep, pigs, dogs, fish, and shrimp. DNA vaccines are currently in clinical trials and will become widely used in the future.

CpG Adjuvant

The Immunostimulatory Effects of CpG ODNs

Adjuvants are important components of vaccine formulations. Effective adjuvants line innate and adaptive immunity by signaling through pathogen recognition receptors. Synthetic cytosine-phosphate-guanine (CpG) and oligodeoxynucleotides (ODNs) have been shown to have potentials as adjuvants for vaccines. Experiments have shown that the stimulatory effects of CpG ODNs can be influenced by several factors, including the number of motifs, the space between motifs, the flanking bases, the presence of motifs other than CpG (poly-G), and also the backbone (Hartmann and Krieg 2000). In addition, the effective motifs of CpG ODNs are species specific. For example, the optimal motif for mice, GACGTT, showed very weak activities in human cells. The most optimal motif for humans, GTCGTT, has been shown to elicit different levels of lymphocyte proliferation in many species of domestic animals, including chickens. A recent study demonstrated that a CpG ODN containing three copies of GACGTT motifs enhanced antibody production and lymphocyte proliferation in SPF chickens. The same CpG ODN, when being used as the adjuvant, also increased the protection of a Newcastle disease virus vaccine. In other words, it appears that both GACGTT and GTCGTT can interact with chicken cells.

CpG Adjuvant in Waterfowls Vaccine

In one study, the immunostimulatory effects of CpG ODNs on waterfowls, such as ducks and geese, were investigated (Lee et al. 2010). Two types of CpG ODNs (each containing three copies of GTCGTT or GACGTT motifs, respectively) were selected to test their effects on duck lymphocyte proliferation. Recombinant parvovirus VP2 (rVP2) was formulated with different types of adjuvant, including aluminum adjuvant and CpG oligodeoxynucleotides (ODNs), and the immunological responses in ducks were examined. Compared with the control group, the production of rVP2-specific antibodies, the expression of cytokines in peripheral blood mononuclear cells (PBMC) stimulated by rVP2, and the percentage of CD4+/CD8+ cells in PBMC were all significantly increased in ducks immunized with rVP2. The rVP2 was formulated with CpG ODNs containing three copies of GACGTT motif. The result revealed that the motif GACGTT was more stimulatory to duck lymphocytes and might be used to improve the efficacy of vaccines for ducks (Lee et al. 2010).

CpG Adjuvant in Bovine Vaccine

In another study, the adjuvant activity of CpG ODNs was studied in cattle vaccinated with a model antigen, keyhole limpet hemocyanin (KLH) (Chu et al. 2011). Results showed that the CpG ODNs containing three copies of GACGTT motif resulted in the highest lymphocyte stimulation index. Additionally, CpG ODN significantly increased the mRNA expression of interferon (IFN)-γ, interleukin (IL)-12, and IL-21 in bovine PBMC. Production of KLH-specific antibodies in the serum and in the milk whey was enhanced. CpG ODNs regulated cytokine gene expression in bovine PBMC and enhanced the production of opsonic antibodies and the secretion of IFN-γ. These preliminary data are still inconclusive. Application of this CpG ODN as an adjuvant requires further investigations and may have positive effects on vaccines for dairy cows (Chu et al. 2011).

Microparticle Vaccine

Significant information obtained recently indicates that future investigations on vaccine development will have to include adjuvants for enhancing the protective immune responses against pathogenic infections in animals and humans (Schijns and Lavelle 2011). Different adjuvants capable of improving immunity and protection have been described in numerous studies. However, the safety concern of an adjuvant is still a crucial issue in adjuvant development (Tomljenovic and Shaw 2011). Therefore, vaccine antigens formulated with safe and potent adjuvants that promised to induce “appropriate” immune responses seem more likely to be approved for use.

Microparticles derived from different polymers, including polylactide-co-glycolide (PLG), alginate, starch, and other carbohydrate polymers, can be designed as carriers for proteins or drugs (Heegaard et al. 2011). Particularly, in the last 10 years, biodegradable and biocompatible PLG polymers, approved by the US Food and Drug Administration (FDA), have become safe and potent adjuvants. This adjuvant delivery system encapsulates vaccine antigens for the development of controlled-release microparticle vaccines (Jain et al. 2011). Through hydrolysis, the biodegradable microparticles that are made from PLG polymers break down into biocompatible metabolites and also lactic and glycolic acids. These produce little inflammatory activity and are excreted from the body via natural metabolic pathways (Eldridge et al. 1991). In addition, PLG polymers have been extensively used as sutures and drug carriers for many years due to their biodegradability and biocompatibility. Different forms of PLG polymers can be obtained according to the ratio of lactide to glycolide used for the polymerization (Eldridge et al. 1991). PLG polymers are soluble in some organic solvents, such as dichloromethane and ethyl acetate.

PLG polymers provide a number of practical advantages in acting as vaccine adjuvants or delivery systems. Adjuvant effects of the PLG microencapsulation can protect antigens from unfavorable degradation, allow the sustained and extended release of antigens over a long period (Lim et al. 2009), and enhance antigen uptake by antigen-presenting (APC) cells. The APC cells include macrophages and dendritic cells in specific lymphoid regions. These effects in turn reinforce the antigen immunogenicity to favorably generate strong immune responses, particularly Th1 cell-mediated immunity. In other words, microparticle vaccines made from PLG polymers may fulfill the need for induction of a functional cell-mediated immune response. The response is required in order to eliminate intracellular pathogens in host cells. In addition, antigen-loaded PLG microparticles can enhance the antigen uptake by APC cells that migrate to other lymphoid compartments, such as the spleen and mesenteric lymph nodes. The cells then stimulate immunocompetent cells to induce antigen-specific immunity (Singh and O’Hagan 2003). These APC cells containing microparticles have been demonstrated to travel to specialized mucosal lymphoid compartments, known as mucosa-associated lymphoid tissues (MALTs). These sites stimulate potent immunity following intranasal vaccination. Thus, antigen from PLG MPs may also fulfill the need for the development of an effective mucosal vaccine that would induce strong immunity via the intranasal route of administration (McNeela and Lavelle 2012).

The sustained antigen release appears to substantially enhance antigen-specific immune responses, achieving long-term protection (Lim et al. 2009). The release of an antigen from biodegradable PLG microparticles is governed by the degradation rate of the PLG copolymer. The rate is influenced by the polymer molecular weight, polymer hydrophilicity, the ratio of lactide/glycolide, and the processing conditions employed during microparticle preparation. The processing conditions include the type of organic solvents used, the acidity, and the temperature. Sustained antigen release of antigen-loaded PLG microparticles has been achieved in the development of various potent microparticle vaccines. In two previous studies, long-lasting effect of the PLG encapsulation was possible and anti-SAG1 and anti-SAG1/2 immunity against Toxoplasma gondii was maintained (Chuang et al. 2013a, b). The microencapsulated proteins, SAG1 and SAG1/2, were released in vitro in a triphasic programmed manner consisted of an initial burst release of protein through fast diffusion, then a gradual release by slow diffusion, and finally a fast release of protein. The pattern mimics the multi-injection of an antigen during a conventional vaccination process (Rajkannan et al. 2006). Therefore, antigen-loaded PLG microparticles capable of sustaining triphasic release of an antigen can be designed as a single-dose vaccine without the need for booster doses. Additionally, for future studies, the design of a single-dose vaccine should consider the following factors: the protein load in PLG microparticles, the rate of protein release, the antigen amount for immunization, as well as the immunization route used (Gupta et al. 1998).

According to previous studies, a strong cell-mediated immune response elicited by PLG microparticles appears to be largely a consequence of their uptake into antigen-presenting cells and the subsequent delivery of these microparticle-containing APCs to specific lymphoid compartments (Grebennikova et al. 2004; Choi and Lee 2004; Sherlock et al. 2006). The particle size used for vaccination in animals is an important parameter in enhancing the uptake of antigen-presenting cells (Sherlock et al. 2006). Particles smaller than 10 μm in diameter are appropriate for direct uptake by antigen-presenting cells, such as macrophages and dendritic cells (Sherlock et al. 2006). The proper size thus stimulates APCs to facilitate the microparticle uptake. Following the uptake, the APCs then effectively process and present the epitopes of microencapsulated antigen to T lymphocytes, especially Th1 and Tc, thereby inducing strong antigen-specific Th1 cell-mediated immunity (Men et al. 1999). Therefore, better uptake and delivery of antigen-loaded PLG microparticles by APCs can lead to a more effective inducement of cell-mediated immune responses (Jain et al. 2011).

Antigenicity retention following encapsulation and during the release of antigen from microparticles is critical. Although a number of proteins have been successfully entrapped in PLG microparticles without losing structural integrity, immunogenicity, or bioactivity, antigen denaturation due to organic solvent exposure in the encapsulation process is still a concern (Heegaard et al. 2011). Numerous studies have explored different encapsulation procedures to decrease contact between the antigen and the organic solvent containing the PLG polymers (Ye et al. 2010).

As mentioned previously, PLG polymers serve both as a potent adjuvant and as a delivery system, allowing antigen to gain access to specific lymphoid compartments. Future applications of the PLG adjuvant are likely to include the development of a more site-specific delivery system for both mucosal and systemic administration. Mucosal surfaces, such as the gastrointestinal tract, are principal sites of entry for many pathogens. Therefore, the development of effective mucosal vaccines formulated with potent adjuvants such as PLG polymers promises to elicit long-lasting protective immunity. It is also a critical step forward in the enduring control of mucosal infections. The use of antigen-loaded polymeric PLG MPs capable of sustaining the antigen release is a significant platform for the induction of long-lasting mucosal immunity and, consequently, long-lasting protection in animals (McNeela and Lavelle 2012).

Conclusion and Future Directions

Live and attenuated vaccines remain the most effective at eliciting robust immune responses, but reversion to virulence poses great safety concern. Since mutations are inherent for living pathogens, the development of nonliving, exogenous protein components as vaccines provides a safe alternative. However, protein components do not provide sufficient “danger” signals required for strong immune response; therefore, adjuvants are generally needed. DNA vaccines can induce endogenous expression of immunogenic components, but the risk of chromosome integration and the optimal site of antigen expression still need to be addressed. While contributing enormously to the control of most infectious diseases, vaccines may not be the silver bullet due to the evolution of pathogens and the nature of host-pathogen interaction. Nevertheless, continued effort in vaccine development and advances in immunology and disease pathogenesis will allow the most efficient use of vaccines for disease control.

Cross-References

Basic Chemistry of Botulinum Neurotoxins Relevant to Vaccines, Diagnostics, and Countermeasures

Immunoneutralization of Abrin

Immunosensors: Using Antibodies to Develop Biosensors for Detecting Pathogens and Their Toxins