This post was reviewed by Dr. Brett Finlay, one of our subject matter experts.
The paper we’re demystifying today can be found here if you want to follow along.
As you may know, the race for a vaccine against SARS-CoV-2, the virus that causes COVID-19, is on. According to one COVID19 vaccine tracker, there are at least 28 vaccine trials active around the world. Finding a vaccine for this virus is critical because it is one of the only ways that we can start to return to ‘normal,’ whatever that normal may be. Today’s post talks about a new vaccine candidate for COVID19.
This study was published in Nature Communications on May 20, 2020. It contains pre-clinical data for a new DNA vaccine designed by Inovio Pharmaceuticals. Pre-clinical means that all of this data was collected from cell and animal trials and is done as a required first step before clinical testing. It also means that no human trials had been conducted at the time of publication. This pre-clinical dataset shows that Inovio’s new vaccine candidate has some success in animal and cell models. These findings mean that Inovio has good cause to move the vaccine candidate into further trials.
If you want a refresher on vaccines, now may be a good time to read over Deanna’s sidebar. In this sidebar, she explains how vaccines work and she takes you through how a few different types of vaccines work.
DNA Vaccines, an Overview:
The vaccine candidate being studied in this paper is a type of vaccine called a synthetic DNA-based vaccine. Synthetic DNA vaccines have many advantages. Thanks to modern molecular biology techniques it’s easy to quickly design and generate many different candidates, cutting down the lag time between design and preclinical testing. Beyond that, these types of vaccines are very easy to scale up for manufacturing. They are also highly temperature stable and don’t require a temperature-controlled delivery process (a cold chain). Another advantage of DNA vaccines is that plasmid DNA can induce innate immune responses, which can then boost the adaptive immune response against the antigens that get expressed (read more here). This can be beneficial for vaccine development. But how do DNA vaccines work?
This awesome explanation goes over how a DNA vaccine works.
A quick recap of how viruses infect cells: the virus binds to a surface receptor and releases its genetic material into the cell interior. Since the genetic material is nicely dressed up (like the Trojan horse), the cells fall for the trick and use that genetic material to make new viruses.
Remember that a virus’ genetic material is an instruction manual to make new viruses. Scientists can open up that instruction manual and tear out the pages with instructions on how to make a highly antigenic part of the virus. Antigenic means that this particular part of the virus is very likely to trigger an immune response.
So now you have a mental image of a person in a lab coat holding a sheaf of mangled pages from an instruction manual. Let’s adjust that image a bit- what actually happens is that the scientists find the portion of the virus genetic sequence that codes for a particular antigen (an antigen is a part of a virus or other pathogen that can cause an immune response). They can then make synthetic DNA that has this same genetic sequence.
This synthetic DNA sequence is put into an expression vector, which is a larger carrier DNA sequence. The expression vector, along with the genetic sequence for the antigen, makes a plasmid.
This plasmid is then introduced into cells or test animals. Now here’s what happens: the plasmid gets into the cells and the cell sees the pretty plasmid DNA and falls for the trap. This time, however, when the cell copies the DNA, makes it into RNA and then uses that RNA to build things, it only builds the one piece of the virus that the small snip of chosen genetic material coded for. In this case, no infectious virus particles are made. This means that a DNA based vaccine doesn’t cause you to be infected by the virus. What is made, however, is a very identifiable part of the virus that can’t get you sick but can still scare the immune system enough to raise an alarm.
As we said earlier, the plasmid DNA itself can activate an innate immune response. This might actually boost the downstream adaptive immune response to the antigen that is made from the instructions in the plasmid DNA.
Imagine you have a highly secure compound (your cell). The guards patrolling this compound (the immune cells) are operating on high alert and will raise the alarm at a moment’s notice. Now imagine that our scientists are secret agents trying to raise the alarm in the compound so that the guards will be ready to fend off an upcoming attack (the virus). The scientists could use their catapult to throw an entire enemy into the compound, but that might be dangerous. On the other hand, the scientists could just throw an enemy uniform (an antigen) into the compound. This is still suspicious enough to raise the alarm but doesn’t endanger anyone in the compound.
The Paper:
Inovio Pharmaceuticals is no stranger to synthetic DNA-based vaccines. The company has used the same approach in the past to make synthetic DNA vaccine candidates against the MERS coronavirus and Zika. Both of these vaccine candidates are currently in clinical testing. A lot of early research showed that the spike protein of the SARS-CoV-2 virus is an ideal vaccine target. Thanks to the quick pace of SARS-CoV-2 scientific research, the sequence of the SARS-CoV-2 spike protein was published very early on. The researchers realized that the SARS-CoV-2 spike protein has a very similar ‘shape’ to the MERS-CoV-2 spike protein. This allowed the research team to build on their previous MERS vaccine design to make a SARS-CoV-2 vaccine candidate.
Making the Vaccine:
The first thing the researchers did was design their DNA vaccine constructs. They did this by taking four published sequences of the SARS-CoV-2 spike protein from GISAID. GISAID stands for Global Initiative on Sharing All Influenza Data. It provides public access to an enormous databank of genetic sequence data on influenza viruses. When a research group manages to gene sequence a virus or part of a virus, they can put a text file containing that sequence onto GISAID. These sequences can then be used by other research groups. Recently, over 37,000 SARS-CoV-2 sequences were shared internationally using GISSAID.
Three of the SARS-CoV-2 spike protein DNA sequences from GISAID matched each other perfectly. A fourth DNA sequence was a little different (only a 98.6% match). They took these two sequences added some other DNA bits to improve expression. Then they put the whole thing through a top-secret algorithm. This ensured that when they put the optimized synthetic DNA into cells, two things would happen:
- The cells would read this DNA over and over again, and make LOTS of spike protein
- The immune response would be very strongly activated (a 5 alarm fire, if you will)
Once they had their fully optimized DNA sequence, they put it into an expression vector. They now had two different plasmids. One that contained the spike protein sequence from the 3 perfectly matched candidates (let’s call this plasmid 1). The other contained the sequence from the outlier candidate (plasmid 2).
Testing the vaccine:
In Cells:
The researchers got both of their test plasmids into COS-7 and HEK293 cells. They confirmed that the cells were expressing the SARS-CoV-2 S RNA and were making the spike protein itself. This was the case for both plasmid 1 and plasmid 2.
In mice and guinea pigs:
While the researchers were going through their study some new research came out on the spike protein. This new research showed a spike protein sequence that was a 99% match to their first plasmid. If you recall, plasmid 1 was made with the sequence from those 3 perfectly matched candidates. Thanks to this new data, they scrapped plasmid 2 and proceeded with plasmid 1. The researchers ended up calling plasmid 1 INO-4800.
The researchers administered INO-4800 to mice and collected blood serum. They tested how reactive the serum was against several different SARS-CoV and SARS-CoV-2 antigens. If serum is highly reactive, that indicates that antibodies against SARS-CoV-2 antigens are being produced in the animal.
From this experiment, they found that the mice were producing IgG antibodies that bound to the SARS-CoV-2 S antigens. They also measured very little cross-reactivity to SARS-CoV S antigens. This means that the IgG antibodies being produced are very specific to the SARS-CoV-2 S protein. They found a significant antibody response 14 days after immunization.
That experiment only studied whether the mice were producing antibodies capable of binding the spike protein. They also tested whether the mice were producing neutralizing antibodies by performing a neutralization assay. Neutralizing antibodies are valuable because they swarm virus particles and prevent the virus from getting into cells. The researchers found that serum from the immunized mice was capable of neutralizing the SARS-CoV-2 virus. This implies that the mice were producing neutralizing antibodies
The researchers also tested for the presence of binding and neutralizing antibodies in immunized guinea pigs. Once again, they found binding antibodies present in the blood serum of immunized guinea pigs by day 14 post-immunization. They also found that their vaccine caused the production of SARS-CoV-2 neutralizing antibodies in guinea pigs. The main difference between the mouse and guinea pig studies is that the guinea pigs got the plasmid through the clinically established method of vaccine delivery. In contrast, the way the mice got the vaccine wasn’t quite the same as how humans would eventually receive it.
After this, they tested whether immunization with INO-4800 would cause the animals’ cells to produce antibodies that can block the virus spike protein from binding with the host receptor (ACE2). They evaluated this in immunized mice and immunized guinea pigs. Both immunized mice and immunized guinea pigs produced antibodies that were capable of blocking the SARS-CoV-2 spike protein from binding to the ACE2 receptor.
Are the antibodies in the lungs?
Having antibodies at the lung mucosa might be protective against lower respiratory disease. The researchers wanted to know whether SARS-CoV-2 antibodies would be found in the lungs of their immunized mice and guinea pigs. The mice received 2 doses of the vaccine, on days 0 and 14 of the experiment. The guinea pigs received 3 doses of the vaccine, on days 0, 14 and 21.
The researchers collected fluid washed through the lower respiratory system. Then they investigated the fluid for SARS-CoV-2 specific antibodies. They found that there were indeed SARS-CoV-2 specific antibodies in the lungs following immunization.
Mouse T cell response
The researchers used several methods to check the T cell response and cross-reactive T cell response in immunized mice. For a review of what T cell responses are and why they matter, check out this sidebar. They found that there were strong T cell responses against SARS-CoV-2. There were also lower (but still detectable) cross-reactive T cell responses against SARS-CoV. There were no detectable cross-reactive T cell responses against MERS-CoV. They successfully detected T cell responses against SARS-CoV-2 S protein epitopes (parts of the spike protein that the immune system recognizes).
Discussing the results:
The data in this paper shows that INO-4800 expresses well in cell and animal models. It also seems to encourage a strong immune response. The researchers detected T cell and antibody-driven immune responses in mice. In serum samples taken from guinea pigs that received the vaccine through a clinically approved method, they saw SARS-CoV-2 binding antibodies and blocking of S protein binding to ACE2. They also found neutralizing antibodies in serum taken from both immunized mice and immunized guinea pigs. The researchers also detected a T cell response as early as 7 days after vaccination. This is important because quick cellular responses can reduce the amount of virus spreading through the body (viral load), and work to slow the spread of SARS-CoV-2.
As exciting as these results are, it’s important to note that this data is pre-clinical. This paper serves as an awesome proof-of-concept showing that INO-4800 is worth further study. That being said, there are no guarantees that the success seen in these cell and animal models will be extended into human trials. Before the INO-4800 hits our clinics and hospitals, it will have to go through several phases of rigorous clinical trials to ensure its safety and effectiveness in humans.
Getting a shot to protect against SARS-CoV-2 might’ve seemed like a moonshot a few months ago. However, with the rapid progression of science and research, we have a lot more shots on goal. Let’s see which one hits its target.
