Answer by Tirumalai Kamala:
At its simplest, a DNA vaccine is just a bacterial plasmid, containing one or more genes from a parasite, a bacterium, a virus or a tumor cell, coupled to a mammalian promoter for driving protein expression inside our body. Once inside our cells, the plasmid doesn’t (or shouldn’t) replicate but its gene(s) are transcribed and translated into protein(s), which in turn are targeted by our immune responses through mechanisms that we don’t fully understand.
Where did the idea of DNA Vaccines come from?
Foreign DNA is transcribed
- In 1950, chromatin derived from a tumor cell transfers malignancy to a normal cell (1). In 1960, naked DNA could be delivered into mammalian cells in vivo (2).
- Hepatitis viral DNA causes hepatitis when injected into animals (3, 4).
- In 1983, protein is expressed in vivo when a plasmid containing its gene is injected into rats (5) or in mouse muscle (6).
Foreign DNA is immunogenic
- In 1963, DNA or RNA from liver could induce Type I interferons from chicken cells (7).
- In 1965, mycobacterial ribosomes and RNA are immunogenic (8).
- In 1984, DNA from BCG has anti-tumor activity (9).
- A 1992 study injects plasmids containing human Growth Hormone (hGH) gene into the ears of mice (10). A few weeks later, many, not all, mice have high circulating antibodies against hGH. Shortly after, in other studies, DNA plasmids encoding flu antigens protect mice against lethal flu (11, 12).
Foreign DNA could be a vaccine? What has happened since? Lots, some useful, much not.
From Reference 13
How are DNA vaccines made?
Constructing a DNA vaccine entails many choices, choices tempered by regulatory guidelines (14, 15, 16). What antigens? Wild type or mutated genes? Membrane-bound, intracellular or secreted antigens? What type of plasmid (and promoters, enhancers, and introns)? Delivered how? Where? Skin or muscle? Dose? Adjuvants? Boosted how and how often? Each of these choices greatly influence the effectiveness of the resulting DNA vaccine, effectiveness alluding to strength and type of immune responses, anything from neutralizing antibodies to cytotoxic CD8 killer cells.
For illustration purposes, let’s look at this graphic that describes the steps necessary in constructing a DNA vaccine for West Nile virus.
How are DNA vaccines delivered? (20, 23, 24, 25)
Originally they were injected directly into muscle. This resulted in variable and less immunogenic uptake. An innovation called the gene gun shot DNA coated onto gold micro particles into the skin using pressurized gas. Improved uptake but not by much. Then came electroporation. Here simple electrodes generate electric pulses across the cell membrane to transiently induce pores in the cell membrane (cell electropermeabilization), promoting cellular uptake of DNA (17,18). A variation of the gene gun approach, the Particle-Mediated Epidermal Delivery (PMED) directs delivery into skin, primarily into keratinocytes (19). This method also improves uptake (20). DNA tattooing is a newer delivery method (21, 22), highly immunogenic possibly due to skin damage by tattoo needles.
Advantages of DNA Vaccines (20, 23, 24,25, 26)
- Easier, cheaper, safer. Let’s consider a bit. Targets of vaccines are usually microbes or tumors capable of terrible disease and/or mortality. With DNA vaccines, we don’t work directly with a dangerous microbe or tumor. We don’t have to culture it, inactivate it or purify proteins from it. We just choose microbial or tumor genes likely to drive strong and specific immune responses and clone them into a plasmid.
- More natural. Almost counter-intuitively so compared to their closest counterpart, sub-unit vaccines. Typically, non-living vaccines require lengthy and tedious purification of the constituent proteins, processes that inherently introduce changes that could change, subvert or minimize immune responses against these proteins’ natural counterparts. With DNA vaccines, our body’s own cells synthesize these proteins, replicating their native structures and essential post-translational modifications, likely mimicking their natural capacity to induce immunity.
- Much easier to change (mutate). This is an advantage with viruses that rapidly drift, i.e. frequently change antigen coding sequences, e.g. flu.
- More practical and realistic. Plasmid DNA is stable at room temperature hugely reducing costs for storage and shipping. Most other types of vaccines need to be kept and shipped cold. Refrigerators and refrigerated trucks, the so-called cold chain, are too expensive to be norm the world over.
Disadvantages of DNA vaccines (20, 23, 24,25, 26)
- Plasmid DNA could integrate into the cell’s DNA. Depending on the site of such integration, at least three adverse outcomes are possible. Either the integration could turn on an oncogene, turn off a tumor suppressor gene, or induce chromosomal instability. How does plasmid DNA integration compare with spontaneous gene mutation frequencies? The standard accepted value is 2X10-6 spontaneous gene-inactivating mutations per gene (20, 27). While there is no known human study that did such a comparison, mouse, rabbit and guinea pig model studies confirmed DNA vaccine genomic integration but at rates much lower than this standard value (28, 29, 30, 31, 32, 33, 34)
- Not useful for polysaccharide vaccines. Genes are translated into proteins. DNA vaccines aren’t useful for microbes where immune responses against polysaccharides are necessary and sufficient to prevent disease. For example, against pneumococcal and meningococcal infections.
- Risk of auto-immunity. If the target of the immune responses against anti-DNA vaccine included common elements between it and our own DNA, it could trigger auto-immunity. However, while circulating anti-double stranded DNA antibodies increased in mice following DNA vaccination (35), same was not seen in humans (36).
DNA vaccines in humans: Disappointing results thus far.
(20, 23, 24, 37, 50, 71)
Nearly 20 years and counting, human clinical trials of DNA vaccines for flu, hepatitis B, HIV, malaria and cancer ended with largely disappointing results. Vaccines that worked well at earlier stages, inducing strong immune responses in mouse models, just didn’t work as well in humans. Why? Insufficient expression? In hindsight, maybe these vaccines were optimized for mouse models, not for humans? At a minimum, they were safe when injected into humans, with local injection site reaction being the most commonly reported side effect (38,39, 40,41). What about autoimmunity? Published reports suggest not (36).
How do DNA vaccines drive immunity?
This is still a mystery. Let’s first make explicit the nature of this mystery. A DNA vaccine is quite simply gene(s) encoding protein(s) derived from microbes or from tumor cells. How could proteins derived from these genes drive immunity? After all, our cells make all kinds of proteins all the time. In health, we don’t make immune responses against them…unless those proteins were made in the context of death or damage, i.e. something that activated the so-called innate arm of immunity. After all, immunity is so powerful, plenty of checks and balances exist for getting it started and sustained. So what could be happening with DNA vaccines? Considered in this light, there are at least three targets.
- One, the bacterial DNA plasmid with its abundant unmethylated CpG motifs, itself a likely target of mammalian immunity.
- Two, location, i.e. double-stranded DNA in the cytoplasm. Surely, that’s abnormal?
- Three, if one and two trigger immune activation of a cell, surely the protein product(s) of such DNA would automatically feed into the ongoing immunity?
Now, let’s consider the players involved.
- Professional antigen-Presenting Cells (pAPCs). Why are they important? pAPCs are necessary for a strong CD4 T cell response. Without CD4 T cell help, B cell and CD8 T cell responses are weak and transient. Current DNA vaccines are designed to specifically target and stimulate APCs (42) but they aren’t delivered into the body to do so ( 3). For practical and not scientific reasons, we choose to give most experimental DNA vaccines intramuscularly, i.e. inject them inside muscle cells. Muscle tissue is considered lacking in pAPCs and myocytes considered poor at driving immune responses. Less straightforward approach? Yes. Unnecessary complication? Yes. Immunological surprise? Undeniably so. At least in a mouse model, DNA vaccines encoding antigens driven by myocyte-specific promoters were similarly immunogenic compared to other, more common promoters (44). At the very least, such studies demonstrate that even when we deliver DNA vaccines into sites that current paradigms predict are unfavorable for immunity, robust immunity can and does ensue.
- TLR9. Bacterial DNA has abundant unmethylated CpG motifs. Such motifs induce strong immune responses by triggering activation of molecules such as TLR9. However, mice lacking TLR9 respond similarly to DNA vaccines (45, 46, 47, 48, 49, 50).
- Double-stranded DNA in cytoplasm: cause for alarm? When we inject DNA vaccines, we directly deliver double-stranded DNA into a cell’s cytoplasm. The nucleus is the normal location for DNA. Surely the cytoplasm is an abnormal location for double-stranded DNA? Surely that must happen only when a cell is infected, distressed, damaged or dying? Surely, over evolutionary time, we must have evolved systems to sense double-stranded DNA in the cytoplasm? We are still hunting for such mystery cytoplasmic DNA sensor(s) (51, 52). Recent mouse model studies suggests we may be close with molecules such as TBK1, IRF3 and STING (53, 54, 55, 56). Caveat being mouse studies. If past is any guide to present and future, mouse model immune responses are slightly to radically different from those in humans. As good as wishing on a shooting star.
From Reference 50
From Reference 57
4. We are still unclear how much of the immune response is against the antigen(s) encoded by the plasmid versus the plasmid itself. Maybe once inside the cytoplasm, DNA vaccines induce changes similar to those induced byor certain viruses (58)? Such instability or imbalance inside the host cells could in turn induce metabolic changes (59) such as local accumulation of uric acid, which in turn could trigger immune responses (50, 60).
5. Cells that take up DNA vaccines could turn over faster, with more rapid cell death (61), leading to extracellular DNA release which would trigger immune responses.
Approved DNA vaccines
Notwithstanding disappointing human data and the mystery about how they drive immunity, several veterinary DNA vaccines have been approved.
From Reference 24
- West Nile Virus, Horses, USA (62, 63).
- Infectious hematopoietic necrosis virus, Salmon, Canada (64, 65).
- Melanoma, Dogs, USA (66, 67).
- Growth hormone releasing hormone, Pigs, Australia (68, 69, 70).
Where are DNA vaccines today? In a disappointing place, at least for humans. Full circle with so-called heterologous prime-boost vaccination protocols with DNA vaccine prime followed by viral vector boost (13, 24, 71). All the advantages I pointed out in the beginning? Down the drain because a viral vector boost negates them all. My reading of the literature suggests that tremendous effort was expended in designing DNA vaccines that seem optimal for mouse and other non-human pre-clinical models. When the success of those models didn’t translate into equally successful human data, does that not suggest at the very least that such models don’t predict human immunity, at least not very well? Why not focus instead on improving DNA vaccine design exclusively for humans, using in vitro human models to guide the path? Maybe we would end up with a human DNA vaccine that worked by itself, either injected once or boosted, greater likelihood of regulatory approval, and without added bells and whistles that increase cost, artifice and difficulty.
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