Many current vaccines were developed years ago using what are now considered outdated technologies. In spite of being phenomenally successful, these technologies have proved unable to make progress against some major human diseases such as tuberculosis, malaria, or human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). In addition, they cannot be developed rapidly enough to control explosive outbreaks of diseases such as Ebola or Zika, or even new strains of influenza. Over the past few decades, however, the development of vaccines has been transformed by the introduction of innovative technologies. As a result, the possibilities of developing new effective vaccines against these diseases are increasingly plausible. It is important to point out, however, that veterinary vaccine production is governed by the marketplace. An innovative and effective technology that works in humans and appeals to scientists may not be sufficiently economical to produce nor to satisfy animal vaccine needs. Nevertheless, two new technologies are in the process of transforming veterinary vaccinology. These are the development of DNA-plasmid vaccines and of reverse vaccinology. In conventional vaccines, a modified live virus is injected into an animal. The virus infects cells and then uses its genome to encode viral antigens. These antigens are processed and fragments expressed on the cell surface. The expressed viral proteins are recognized as foreign by B and T cells, and the animal responds by mounting antibody or cell-mediated immune responses. An alternative way to trigger these responses is to inject a DNA plasmid (a piece of circular DNA, usually from Escherichia coli that acts as a vector) containing the genes encoding the specific protein antigen of interest (Fig. 6.1). When this DNA plasmid enters a cell nucleus in a recipient animal, it will be transcribed into mRNA. The mRNA is transported to the cell cytoplasm and translated into protein. The recipient cells will therefore synthesize and express the foreign antigen. As with virus-encoded proteins, this endogenous antigen will be processed, bound to MHC class I molecules, and expressed on the cell surface. It will then prime both B and T cell responses and trigger cytotoxic T cell responses and antibody production. The plasmid, however, unlike viral vectors, cannot replicate in mammalian cells and it encodes only the target antigen. There are several DNA-plasmid vaccines approved for use in animals and numerous others are in various stages of development for humans. Although yet to reach significant commercial production, DNA vaccines appear to be ideal for organisms that are difficult or dangerous to grow in the laboratory. They are very stable at room temperature and relatively easy to manufacture to consistent specifications. DNA vaccines are often more effective than recombinant proteins and avoid the need for complex vectors. The DNA sequence of a plasmid can be easily modified to manipulate the type 1 T helper to type 2 T helper (Th1/ Th2) ratio in the induced response. Likewise the DNA can be combined with genes for cytokines, and also adjuvants, polymeric carriers, or viral vectors (Table 6.1). TABLE 6.1 ■ MHC, Major histocompatibility complex; Th1, type 1 helper T cell. The selected DNA sequence from the organism of interest is isolated and inserted into a bacterial plasmid usually obtained from E. coli. In addition to the genes encoding the vaccine antigen or antigens of interest, the plasmid must also contain a strong promoter sequence upstream (usually from another virus like Rous sarcoma virus or cytomegalovirus), in addition to a transcriptional termination/polyadenylation sequence downstream. When the plasmid enters the cell nucleus, the promoter causes transcription of the mRNA of the antigen gene. The polyadenylation sequence is required for the mRNA to leave the nucleus and enter the cytoplasm. Plasmids may be modified in many ways to improve antigen yield and immunogenicity. Some plasmids may be engineered to express more than one antigen or perhaps an antigen plus an immunostimulatory protein separated by a spacer. Alternatively, a mixture of two plasmids, one expressing the antigen gene and one expressing a cytokine gene, may be administered at the same time. Protein expression may also be optimized by changing the plasmid codon usage. (Pathogens often have a somewhat different codon usage than do eukaryotic cells, so this can be modified to match the target species and so increase transcription efficiency.) Genetically engineered plasmids are usually suspended in saline and injected intramuscularly into an animal where they are taken up by the skeletal muscle cells. This is the limiting step for these vaccines because the physical barriers of the cell wall and the nuclear membrane interfere with plasmid uptake while DNA in the extracellular space is rapidly degraded. Several approaches have been taken to overcome this problem. For example, if the muscle cells are first damaged by mycotoxins or by the use of hypertonic saline solution, this may assist penetration into cells. Plasmid penetration is also enhanced by the use of “adjuvants” such as lipid complexes, microcapsules, or nonionic copolymers. Aluminum phosphate seems especially effective in improving DNA vaccine efficiency. DNA vaccines may also be injected subcutaneously where the plasmids are deposited in extracellular spaces. They can then be carried to draining lymph nodes where they are probably taken up by antigen presenting cells. Once it enters a cell, like a virus, the DNA plasmid is transcribed into messenger RNA and translated into the endogenous target protein. The newly generated protein then undergoes appropriate processing, is bound to MHC class I molecules, and presented to the antigen-presenting cells. These carry the peptide to the draining lymph node where it is presented to T and B cells. This leads to the production not only of neutralizing antibodies but also of cytotoxic T cells because the antigen is endogenous. These expressed peptides have an authentic tertiary structure and posttranslational modifications such as glycosylation. The immune response to these peptides is also enhanced because the bacterial DNA in the plasmid itself contains unmethylated CpG motifs that are recognized by toll-like receptor 9 (TLR9). As a result, they initiate innate immune responses, activate dendritic cells, and eventually promote a strong Th1 response. This type of DNA-plasmid has been used to protect horses against West Nile virus infection (West-Nile Innovator, Ft Dodge). The commercial vaccine consists of a plasmid engineered to express high levels of the virus envelope (E) and premembrane (prM) proteins. In addition, the plasmid contains gene promoters and marker genes (Fig. 6.2). Upon injection with a biodegradable oil adjuvant, this plasmid enters cells and causes them to express the West Nile virus proteins. Other DNA vaccines have been licensed to prevent infectious hematopoietic necrosis and salmonid pancreas diseases in farmed Atlantic salmon (Chapter 21), and also melanomas in dogs (Chapter 23). This approach has also been applied experimentally to produce DNA vaccines against avian influenza, lymphocytic choriomeningitis, canine and feline rabies, canine parvovirus, bovine viral diarrhea, feline immunodeficiency virus, feline leukemia virus, pseudorabies, foot-and-mouth disease virus, bovine herpesvirus-1, and Newcastle disease. Although theoretically producing a response similar to that induced by attenuated live viral vaccines, these nucleic acid vaccines are ideally suited to protect against organisms that are difficult or dangerous to grow in the laboratory. Some DNA vaccines can also induce immunity even in the presence of very high titers of maternal antibodies. Although the maternal antibodies can block serological responses, the development of memory responses is not impaired. DNA-plasmid vaccination is well tolerated and after intramuscular injection most of the plasmids remain at the injection site for several weeks. Some may be transiently detected in the blood and other organs. Antibody responses after DNA vaccination increase slowly but are unusually prolonged when compared with a single protein injection. It may take as long as 12 weeks to reach peak antibody titers in mice. DNA plasmid vaccines must get inside target cells. This can be achieved by intramuscular injection of saline solutions although they require quite a lot of DNA (10 μg–1 mg). Much of the DNA in saline goes into the intracellular spaces and is therefore “wasted.” Muscle cells may also take up the plasmids by phagocytosis or perhaps through specific receptors. Although intramuscular injection is very inefficient because the transfection rate is low (about 1%–5% of myofibrils in the vicinity of an intramuscular injection site), the expression of the transfected gene can persist for at least two months. Its encoded peptides are either treated as endogenous antigens and displayed on the cell surface or secreted and presented to antigen-processing cells. It may be that the skeletal muscle cells are not the most important pathways for antigen processing. The injected plasmid may simply be carried into the draining lymph node where it is taken up by dendritic cells. This processed antigen preferentially stimulates a Th1 response associated with IFN-γ production. It is possible to modify the DNA to bias the T cell response toward type 1 or type 2 responses simply by inserting appropriate cytokine genes. Plasmid entry into cells can also be achieved by “shooting” the DNA plasmids directly through the skin adsorbed onto microscopic gold or tungsten nanoparticles. These are fired by a “gene gun” using compressed helium (Fig. 6.3). The “gene gun” delivers the plasmid-coated particles directly to the cell cytoplasm. Saline injections promote a Th1 response. By bypassing TLR9, the gene gun technique preferentially stimulates Th2 responses. The use of a gene gun is more efficient than injection because some of this DNA is taken up by dendritic cells bypassing more conventional endocytosis mechanisms, and it therefore minimizes degradation. Another way to overcome the problems associated with getting DNA plasmids inside cells is electroporation. This combines an intramuscular injection with the local application of a pulsed electric field of 50 to 1000 v through needle electrodes for a few micro- or milliseconds. After this electrical pulse the muscle cell membranes become temporarily permeable and open pores that allow the plasmids to enter the cell. For intradermal electroporation, the electrodes are placed on the skin and the pulses stimulate the skin dendritic cells to take up the plasmids. These very short pulses are well tolerated (by humans) and cause minimal erythema and irritation. It is difficult to see how this method could be applied in a practical manner to many domestic animal species. Most dendritic cells only express the plasmids for a few days whereas the muscle cells do so for several months. Thus the intramuscular route is preferred. The major impediment to the development of DNA vaccines is their low immunogenicity because of the relatively low transfection efficiency of the plasmid. In other words, large amounts of DNA are injected and very little antigen is expressed. Up to several milligrams of DNA must be injected to induce a strong immune response. They also only work for protein antigens. The purification required to separate the plasmid from contaminating cellular DNA and RNA is expensive. One way to solve the efficacy problem is to enhance plasmid uptake by using different routes and methods of administration such as gene guns or electroporation. Alternatively, the plasmids may be delivered in association with regulatory cytokine genes, such as those for GM-CSF, IFN-γ, IL-2, and IL-12. The plasmids may also be adsorbed onto cationic microparticles that are then phagocytosed by antigen presenting cells. It has long been normal practice to use exactly the same vaccine for boosting an immune response as was employed when priming an animal. This approach has many advantages, not the least of which is simplicity in manufacturing and regulating vaccine production. There is, however, no reason why different forms of a vaccine should not be used for priming and for boosting if this will induce an optimal response. This approach is known as a heterologous prime-boost strategy. Under some circumstances this may result in significantly improved vaccine effectiveness. The heterologous prime-boost approach is somewhat empirical, and researchers may simply test numerous vaccine combinations to determine which combination yields the best results. Prime-boosting has been investigated in efforts to improve the effectiveness of DNA plasmid vaccines, because it is clear that DNA-plasmid vaccines alone may not be sufficient to trigger a protective immune response. However greatly improved results may be obtained by boosting with a conventional or virus vectored vaccine. Combinations may involve priming with one DNA plasmid, but boosting with either another plasmid, perhaps in another vector, or with recombinant protein antigens. These strategies may increase the avidity and persistence of antibody responses and also increase cytotoxic T cell responses. It should be noted that no DNA-plasmid vaccines for human use have yet been approved. They have not been as immunogenic in humans as in laboratory animals. Primates need ten times as much DNA as rodents do because a human serum protein, amyloid P, binds plasmids and inhibits their transfection. Although DNA-plasmid vaccines do not currently have great advantages over conventional protein vaccines, they may provide the solution to diseases not currently well controlled by conventional antigens such as malaria and tuberculosis. Messenger RNA is produced by transcription of DNA and translated into proteins. Thus when it enters cells it triggers protein expression. This mRNA is only transiently expressed, and as a result is potentially safer than persistent DNA. RNA can be synthesized so that it incorporates open reading frames that encode proteins combined with sequences at both termini that regulate translation and protein expression. Whereas conventional vaccines require large and expensive production facilities, RNA synthesis is relatively simple. It can be readily produced by a standardized process reducing both cost and time. Only information about the RNA sequence is required and there is no need to handle dangerous pathogens. RNA is relatively stable as long as it is not exposed to RNase. RNA is also self-adjuvanting in that it is a potent stimulator of interferon production. It should also be pointed out that RNA, unlike DNA, does not need to get into the cell nucleus. It is sufficient for it to cross the cell membrane into the cytoplasm. In fact, naked mRNA is spontaneously taken up by many cell types and expressed within minutes. Its stability can be further enhanced by chemical changes or incorporation into nanoparticles such as dendrimers (highly branched proteins). RNA can be delivered to cells in two forms, conventional mRNA or self-amplifying mRNAs also called replicons. Both types of RNA are safe and well tolerated and induce antigen-specific immune responses. Conventional mRNA vaccines have largely been developed for use in human cancers. They encode tumor specific antigens that can trigger a protective immune response. Most of the infectious disease focus has however been on self-amplifying vectors—replicons, derived from alphaviruses because these are highly effective and require a much lower dose of RNA to induce a protective immune response. Replicons are defined as nucleic acids that contain the instructions for their own replication. Alphaviruses are positive-sense, single-stranded RNA viruses. Examples of alphaviruses include Eastern, Western, and Venezuelan encephalitis viruses, Sindbis virus, and Semliki Forest virus. All can be used as vaccine vectors because their genome is relatively simple, and they generate large quantities of RNA in the cytoplasm of infected cells. Their 5’ end encodes nonstructural proteins that are responsible for this cytoplasmic RNA replication. Their 3’ end encodes structural proteins plus a promoter region (Fig. 6.4). If foreign genes of interest are inserted in place of the alphavirus structural genes, the viral RNA will continue to replicate and translate large amounts of the foreign proteins. Because of their very efficient promoters, the newly synthesized proteins may account for 10% to 20% of the total cell protein. It is also possible to incorporate genes for viral capsids or glycoproteins into the replicon so that virus-like particles form and effectively package and protect the RNA. They are not, however, able to replicate or produce progeny virus. If more promoter sites are inserted downstream, several different genes can be expressed and are able to generate multivalent vaccines.
Nucleic acid vaccines and reverse vaccinology
DNA-plasmid vaccines
Conventional Vaccines
DNA-Plasmid Vaccines
Not limited to protein antigens
Very safe
No risk of inducing anti-DNA antibodies
Presented by both MHC class I and II molecules
Less risk of tolerance induction
Can be polarized to Th1 or Th2 responses
No risk of affecting the genes controlling cell growth
Relatively easy and cheap to produce
Difficult to polarize responses to Th1 or Th2
No need for adjuvants
More strongly immunogenic
Correct antigen folding
Low doses required
Persistent response
Immunity develops rapidly
Very specific for an antigen
Vaccine production
Vaccine administration
Cell entry
Gene guns
Electroporation
Problems
Prime-boost strategies
RNA vaccines
Alphavirus replicons
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Nucleic acid vaccines and reverse vaccinology
A Comparison of the Advantages of Conventional and DNA-Plasmid Based Vaccines
