This post was reviewed by one of our faculty experts: Dr. Scott Covey of UBC Biochemistry
The paper we’re demystifying today can be found here, if you want to follow along.
This paper was published in Cell in March 2019 and is an amazing example of existing research being repurposed towards current efforts against COVID-19. This research was conducted by researchers from the University of Washington, including those within the King and Baker groups at the Institute for Protein Design.
In this paper the research team describes how they designed a nanoparticle made of proteins. This nanoparticle can be used to display pieces of a virus that the immune system can recognize and use to learn how to fight off that same virus in the future. The paper specifically focuses on RSV (respiratory syncytial virus), but the techniques and approaches laid down by this research can be used for any manner of viruses- including nCoV-19.
RSV is an enveloped RNA virus- like nCoV-19- that commonly infects humans throughout their lifespan. It is also one of the leading causes of infant mortality wordwide. Developing a safe vaccine for RSV has proved difficult.
A vaccine usually works by showing your body a dead bacteria or virus, or a piece of a dead bacteria or virus. You can think of it as a training video- the army that defends your body (the immune system) is very good at learning the enemy tactics and knowing how to quickly neutralize any attacking pathogens. What a vaccine does is give your immune system a dry run, or a drill. A vaccine works by telling your immune system ‘hey listen up! Anything that looks like this is dangerous- start building your defences now!’ When you’re vaccinated against something, your body already has strong defences built up against it and can eliminate whatever pathogen it is before you get badly sick. One major defence that the immune system uses against viruses is neutralizing antibodies– antibodies that recognize particular parts of the virus and bind to them in a way that stops the virus from proceeding with the process of infection.
For the RSV virus, most of the neutralizing antibodies generated by the body are specific to a particular RSV surface protein that scientists call the ‘F’ protein. Because of this, a lot of vaccine efforts focus on teaching the immune system to recognize this F protein. The RSV F protein is a surface glycoprotein (a protein that also has attached sugar chains) that exists as a trimer (a lot like the S protein of nCoV-19) and is responsible for fusing the virus membrane to the cell membrane during infection (also a lot like the S protein of nCoV-19). It’s known that the F protein often changes its shape significantly over the course of virus + cell membrane fusion. In scientific terms, the F protein goes through big conformational changes while moving form the prefusion to the postfusion states. The prefusion (before fusion) state is relatively unstable and so up until recently most vaccine research focused on the F protein in its postfusion state.
When vaccines against the postfusion state were tested in clinical trials, they found that it was only able to generate a moderate (middle) amount of antibodies in the immune system. Typically very high levels of neutralizing antibodies being generated by a vaccine tend to indicate a lower chance of getting infected again, or immunity.
Recent research has described the structures of both the prefusion and postfusion forms and has also showed that more neutralizing antibodies in the human body tend to target the prefusion state of the F protein. Thanks to this, vaccine development targeting the prefusion form of the F protein has started with a vengeance.
Previous research had shown that when an antigen (the part of a virus or other pathogen that the immune system recognizes; in this case, the F protein) is presented in a dense and repetitive pattern, it can result in a stronger immunity. This is likely because of how the densely packed antigens interact with B cells (a type of cell in the immune system). Self-assembling proteins, proteins that come together in solution to make a particular type of complex, are a very popular way to make a platform for presenting many antigens. The advantage of protein nanoparticle vaccines is that they can be used to form very specific structures that are naturally nontoxic and can be genetically modified to display any antigen that is desired. Also, because nanoparticles are so small they have a very large surface area, which means that they can display a lot of antigens.
Up until recently protein nanoparticle vaccine technology was limited in what it could do because there weren’t that many naturally occurring scaffolds available and the ones that are out there have very clearly defined properties that are difficult to change. Luckily, some of the same scientists that worked on this paper also published another research paper where they described methods to use computer programs to design protein nanoparticles that assemble on their own with exceptional accuracy.
In the paper we’re discussing today the researchers combined their early work into self-assembling protein design with the new breakthroughs in the understanding of the F protein prefusion state to design and build a protein nanoparticle that could be used to induce an immune response and, potentially, immunity against RSV. The researchers found that their nanoparticle induced a 10x stronger immune response than DS-Cav1, which is a leading vaccine candidate already in the clinical-stage of development. That’s a pretty damn big deal.
The research team used their recently designed protein nanomaterials in combination with a technique called computational docking to find which particular nanomaterial building blocks would be the best to fuse with DS-Cav1, a proven RSV antigen.
(Quick recap, an antigen is a part of a virus or bacteria that can be used to train the immune system’s defences)
The goal here was to figure out which building blocks would be able to fuse with the DS-Cav1 antigen and, from there, figure out how to use those building blocks to make a DS-Cav1 displaying nanoparticle. From the computational docking they identified an icosahedral nanoparticle, I53-50, that seemed promising for future work. The researchers call this icosahedral nanoparticle displaying DS-Cav1 “DS-Cav1-I53-50.” When the nanoparticle is fully assembled it looks like the model below:
From the figure you can see that this nanoparticle is a little like a piece of IKEA furniture. It has two parts: I53-50B.4PT1 (let’s just call this part B) and DS-Cav1-153-50A (let’s call this part A). The researchers made part A by putting DNA containing instructions for it in a type of cells called HEK293F cells. They made part B in E.coli bacteria. When they purified both components and mixed them together they found that they assembled (on their own) very efficiently into the target structure- if only IKEA furniture could do the same thing.
They tested the antigenicity (defined in this case as the ability to bind antibodies) of their assembled nanoparticle with a set of antibodies known to bind to the prefusion state of the F protein. They found that the antibodies bound DS-Cav1 (the antigen on its own) in a similar way to how they bound the antigen-displaying nanoparticle. From this they concluded that putting DS-Cav1 onto the surface of a nanoparticle doesn’t make it any less effective at training the immune system (in scientific terms, it doesn’t lower its antigenicity)- which is good news. They then used cryo-electron microscopy (another very cool technique that really deserves a blog of its own) to compare the actual structure of their particle to their computational model and found that the two were highly similar and that adding the DS-Cav1 epitope to the nanoparticle did not change the basic structure of the nanoparticle. As the paper says:
Together, these data establish that two-component protein nanoparticles can display complex glycoprotein antigens and assemble in vitro to generate monodisperse immunogens with high efficiency
Translation- it works so far, and it works well. On with the experiments!
When testing various factors of their new nanoparticle immunogen the researchers found that:
- The prefusion conformation (state) of DS-Cav1 is stabilized when DS-Cav1 is genetically fused to I53-50A
That’s… a lot, I know. Basically put, the researchers tried to stress-test this new molecule. After subjecting it to a range of temperatures and to chemicals known to pull apart proteins they found that the DS-Cav1 was overall more stable, especially at key antigenic sites (parts of the antigen that are recognized by antibodies), when it was mounted on the nanoparticle.
2. DS-Cav1-I53-50 induces a much stronger immune response in mice than DS-Cav1 alone
When mice were immunized with either DS-Cav1 alone OR DS-Cav1-on-the-nanoparticle, researchers found that the induced immune response was 3x higher for DS-Cav1-on-the-nanoparticle and that the levels of neutralizing antibodies generated by the nanoparticle+DS-Cav1 combo was 9x higher. The higher ratio of neutralizing to binding antibodies is an indication of a better quality immune response! The researchers suggest that the nanoparticle helps cause a better immune response because when DS-Cav1 is mounted on the nanoparticle surface the spots on DS-Cav1 can cause the strongest neutralizing immune response are more exposed to B cells, and that the dense grouping of potential epitopes helps with B cell crosslinking (another long story).
To make sure that it was the nanoparticle structure itself that allowed for this better immune response, and not simply the act of fusing DS-Cav1 to the nanoparticle building block, the researchers checked what sort of immune response they’d see if they immunized mice with DS-Cav1 fused to only part A of the nanoparticle (so just the disassembled portions, not an entire nanoparticle). They didn’t see any significant increase in antibody production, which lead them to conclude that the stronger immune response they observed earlier was specifically because of the icosahedral nanoparticle scaffold.
(For reference, an icosahedron is a polyhedron with 20 sides. Yes, D&D nerds, that shape).
3. The nanoparticle scaffold itself causes some immune response when the scaffold without DS-Cav1 is injected into mice, but a much lower immune response when the scaffold WITH Ds-Cav1 is injected into mice.
The nanoparticle scaffold does include proteins that are not typically native to humans (or in this case, mice) and because of this it is completely possible that the immune system will recognize the scaffold itself and launch an immune response against it. After all, the immune system is made to take out any invaders that it sees. The researchers found that while the (disassembled) nanoparticle components cause an immune response the immune response against the fully assembled nanoparticle is much weaker. This is also very good news for the use of this scaffold in vaccine development.
To reiterate that using an amusing analogy- if the immune system is a laser tripwire, the researchers found that throwing the disassembled components of an IKEA table down the hallway ended up triggering the tripwire, whereas putting a fully assembled IKEA table down didn’t.
That analogy works, okay? The table’s legs hold it up from breaking the laser.
4. The strong immune response against DS-Cav1-I53-50 seen in mice was also confirmed in nonhuman primates
That statement is pretty self-explanatory. It means that all this stuff that the researchers saw isn’t just specific to rodent immune systems- it applies to primate immune systems too. It working in primates is a good sign towards eventually developing a human vaccine using the same technology.
This research was groundbreaking when it was published and continues to be groundbreaking today. The scientists involved have designed a system to build self-assembling protein nanoparticles that can display highly complex proteins in a density and conformation that can cause a high quality and very strong immune response. This gives us a shot at making stronger, more versatile and more stable vaccines against a whole bunch of different pathogens.
The Institute for Protein Design announced that this research is being re-purposed for nCov-19 vaccine design. Nanoparticle vaccines displaying the spike protein of SARS-CoV-2 have been made and are currently being tested in mice. I reached out to Dr. Neil King, whose lab is leading many of these efforts, to ask if there was anything else he wanted to add. His response- “we are pushing hard.”
Paper citation: Marcandalli J, Fiala B, Ols S, Perotti M, de van der Schueren W, Snijder J, Hodge E, Benhaim M, Ravichandran R, Carter L, Sheffler W, Brunner L, Lawrenz M, Dubois P, Lanzavecchia A, Sallusto F, Lee KK, Veesler D, Correnti CE, Stewart LJ, Baker D, Loré K, Perez L, King NP. (2019). Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus. Cell. 03 2019. 176(6):1420-1431.e17.