Physicians may soon be armed with guns. Not weapons loaded with metal bullets but gene guns which hold nucleotide-based vaccines. Medical researchers at the National Institute of Allergy and Infectious Diseases (NIAID) have already started using this new ‘gun’ to deliver DNA vaccine in a Phase I clinical trial involving 120 individuals (clinical trial number NCT02840487).
Novel vaccine delivery methods like the gene gun prevent needle-stick injuries and problems associated with needle phobia facilitating progress toward wide vaccine coverage. Thus, the use of nucleotide-based vaccines in the clinic, especially DNA and mRNA vaccines along with novel delivery methods is no longer elusive.
Most contemporary vaccines are produced from weakened forms of live viruses and bacteria. Alternatively, dead microorganisms or specific parts of viruses and bacteria are used (NHS 2016). The use of live viruses and bacteria in vaccines raises the possibility of reactivation if the microorganism reverts to its virulent form. This limits the use of live vaccines to individuals with competent immune systems. For instance, the MMR vaccination which protects against measles, mumps and rubella virus is not suitable for pregnant women and people with an impaired immune system (CDC 2017). As nucleotide-based vaccines do not contain any live viruses or bacteria these considerations need not apply.
Figure 1. Central dogma of molecular biology
To understand the functioning principles and production of nucleotide-based vaccines, we first have to look at the central dogma of molecular biology. Proteins are assembled according to genetic information in all living organisms. It all starts with enzymes in our cell nucleus that act as copying machines. These enzymes use DNA which are molecules arranged like spiral ladders as the master copy. Once the enzyme attaches itself to the DNA, many Xeroxes of DNA are produced which are called mRNA. Subsequently, mRNA are read by our cell’s protein factory in the cytoplasm to build proteins.
Nucleotide-based vaccines work at this fundamental level to achieve prolonged immunity. Once delivered, DNA in the vaccine enters our cell nucleus and instructs our cells to produce the same proteins contained within the live virus or bacteria. Similar to the response triggered by an actual infection, these proteins assembled within our own cells prime the immune system enabling it to trigger an effective response against the relevant virus or bacteria in the future. In reality, the recipient has not been exposed to the dangerous microorganism! The activation of immune system without direct exposure is especially important in endemic viral infections with poor treatment options such as the Zika virus. In the period since November 2016 when Zika virus ceased to be a public health emergency according to the World Health Organization (WHO), several nucleotide-based vaccines for Zika have been developed and a number have already begun clinical trial.
One of these novel nucleotide-based vaccines is the plasmid-DNA vaccine produced by researchers at NIAID. They found that a single injection of 50μg of DNA vaccine into mice and two 1mg injections of the same vaccine into monkeys caused a sufficient rise in Zika-specific antibodies. Neither group of animals succumbed to Zika infection when exposed to the live virus, unlike unvaccinated controls demonstrating that Zika DNA vaccines may provide immunity in animal models (Dowd et al. 2016).
Figure 2. Structure of Zika virus, source: http://www.virology.ws/2016/01/28/zika-virus/
In order to produce DNA vaccines, mRNA from live Zika virus coding for a protein which is able to activate the immune system was isolated. Genes coding for pre-membrane proteins and the E-proteins found on the outermost membrane of Zika virus (see image above) were able to fit the brief. Next, the isolated mRNA was used to produce complementary DNA (cDNA) by reverse transcription. Referring back to the central dogma, this is similar to using the Xerox copy to make the master copy. With the help of some ‘cutting-edge’ enzymes, the cDNA was cut and pasted into a plasmid which is a short circular strand of DNA found in bacteria. This process is called transformation (see image below). Once successfully transformed plasmids have been identified using specific markers, they are industrially amplified and manufactured into vaccines which can be loaded into the ‘gun’.
Figure 3. Process of DNA vaccine production
However, plasmid-DNA vaccines have a drawback; since DNA is transcribed into RNA within the nucleus of cells, the vaccine DNA must enter the recipient’s cell nucleus which carries its own difficulties. Another nucleotide-based vaccine candidate by a different group of researchers at NIAID does not face this problem. These are the mRNA vaccines which bypass the need for cDNA carrying viral protein sequence (Pardi et al. 2017). This type of vaccine is unique because it serves as a blueprint for viral protein production without entering the nucleus. However, their preferential activity in cytoplasm leads to a relatively shorter lifespan since they are broken down rapidly. Hence, the group are investigating on adaptations to the molecular structure to improve the stability of this agent. While the plasmid-DNA vaccine was delivered using the ‘gun’, the researchers working on the mRNA vaccine developed a distinct method of delivery using an injection containing enclosed lipid nanoparticles carrying the mRNA vaccine which can unfold once it reaches the cells in our body.
Unsurprisingly, the scientists working with Zika mRNA vaccine have found that this mode of vaccination is superior to its counterpart, the DNA vaccine due to its ability to bypass the need to access the cell’s nucleus to produce its desired effect. The comparison is valid because both the mRNA and the DNA vaccines tested coded for the same E-proteins and pre-membrane viral proteins. A single 50μg injection of mRNA vaccine given to monkeys induced at least 50 times greater levels of Zika-specific antibodies compared to one injection of DNA vaccine delivered by gene gun in previous trials (Pardi et al. 2017).
Ultimately, these successful trials suggest that it won’t be long before nucleotide-based vaccines become available to the public. Already this novel vaccination method has been deployed in the veterinary field, with horses receiving West Nile virus DNA vaccine (CDC 2005). So the next time you go for a routine medical consultation, don’t be surprised when the doctor pulls out a ‘gun’ because it’s time to get shot with nucleotides.
Subashan Vadibeler is an MBBS student at the University of Malaya
CDC. (2005). “CDC and Fort Dodge Animal Health Achieve First Licensed DNA Vaccine.” 2017, from https://www.cdc.gov/media/pressrel/r050718.htm.
CDC. (2017). “Vaccine Recommendations and Guidelines of the ACIP “, 2017, from https://www.cdc.gov/vaccines/hcp/acip-recs/general-recs/contraindications.html.
Dowd, K. A., et al. (2016). “Rapid development of a DNA vaccine for Zika virus.” Science 354(6309): 237-240.
NHS. (2016). “Vaccination ingredients.” 2017, from http://www.nhs.uk/Conditions/vaccinations/Pages/vaccine-ingredients.aspx.
Pardi, N., et al. (2017). “Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination.” Nature 543(7644): 248-251.