VACCINE NEXT GEN
Molecular clamp technology may prove to be the future of vaccines, but in the fight against COVID-19 it was famously abandoned at the 11th hour. MANUELA CALLARI reports.
Public confidence put paid to Australia’s Covid-busting molecular clamp technology. Or did it? MANUELA CALLARI picks apart the approach that may save us from the next pandemic, and the one after that...
Keith Chappell is a laconic man with sideburns that wouldn’t be out of place in a hipster brewery. His tone is quiet and calm, even as he discusses the news that derailed 11 months of intense research to try and solve the global problem that has changed our lives.
“You have your heart and mind set on, ‘We’re going to save some lives – we’re going to help the world get back to normal through this devastating pandemic.’ And to have that hope taken away from us was incredibly hard to deal with,” he says.
An associate professor at the University of Queensland, Chappell has spent much of his career trying to find ways to stabilise viral surface proteins. He began studying flaviviruses – viruses transmitted to humans by mosquitoes and ticks that cause the most prevalent viral infections worldwide – during his PHD at UQ. Then he spent three years at the Instituto Salud Carlos III in Madrid working on stabilising the Respiratory Syncytial Virus (RSV) surface fusion protein, the target for a potential RSV vaccine.
When he returned to Australia in 2011, he began to search for a stabilising method that is both versatile and quick. Together with Paul Young and Daniel Watterson, he created the molecular clamp technology.
In January 2020, the research team received funding from the Coalition for Epidemic Preparedness to boost the technology and develop new vaccines to help stop the world’s next epidemic.
Only days later, we woke up to find an unknown virus – then named SARS-COV-2 – quickly spreading
across the globe, killing thousands. The UQ team partnered with global biotech company CSL and rapidly got to work trying to apply their patented clamp technology to the new virus. The vaccine was one of Australia’s most promising, but after strenuous work and encouraging early results, its trial was abandoned when numerous participants returned false-positive HIV tests.
It appeared that the team’s innovative molecular clamp technology was to blame. But could this technology still be the future of vaccines?
Vaccines the Australian way
Bacteria can replicate on its own, but a virus must use the mechanisms of the human cells to proliferate. In the case of SARS-COV-2, protein stalks called spike proteins, which stick out of the virus surface, attach to the ACE2 receptors in human cells. These receptors are attached to our cells’ membrane and play a role in regulating blood pressure. When the key-like spike protein unlocks the cell, the virus can then insert its genetic material into the cell to replicate, making us unwell.
Molecular clamp technology works as a scaffold that holds the spike protein in the right shape for the immune system to make antibodies.
Traditional vaccines contain either inactivated or live but attenuated viruses. Inactivated vaccines – such as some types of flu vaccines – contain viruses treated with heat, radiations or chemicals, such as formalin or ß-propiolactone, so they cannot replicate, but can still trigger an immune response. Similarly, live, attenuated vaccines, like measles and polio vaccines, contain viruses weakened in the lab. The virus is still viable and stimulates an immune response, but it cannot cause disease.
Both come with risks. “You have to make sure that your pathogen is genuinely inactivated so that you don’t unintentionally infect people, but it still maintains the immunogenicity that will elicit a neutralising response,” says Daniel Wrapp, a postdoctoral fellow at the University of Texas in the US.
“One issue scientists have run into when trying to inactivate or attenuate viruses to use them in vaccines is that they can unintentionally cause greater infection upon exposure to the genuine pathogen,” he explains.
That happened in 1966, when a vaccine against RSV, which infects children before they turn two, was tested in the US. The trial had dreadful consequences. Many children still caught the virus, some suffered worse symptoms than usual, and two toddlers died due to enhanced symptoms. A safe vaccine for RSV has still not been found.
It appears that the formalin scientists used to inactivate the virus changed the shape of the antigen, suggesting that the failure was primarily a result of the immunisation triggering the creation of poorly designed antibodies.
Using a subunit of the virus, such as the SARSCOV-2 spike protein, avoids the problems of a pathogen replicating in the human body, which is why many of the research teams working on COVID-19 vaccines target the spike rather than dealing with the whole virus.
But to do this, scientists have to figure out how to preserve the shape of the spike protein as it shows on the surface of SARS-COV-2.
The spike protein is anchored to the virus surface through a region that traverses the viral membrane. When the spike fuses with the ACE2 receptor, it undergoes a significant change, refolding into a highly stable post-fusion form. Similarly, the isolated spikes
– in pre-fusion form – are naturally unstable and rearrange into the stable post-fusion state, which is a very different shape.
Targeting the post-fusion form could cause the immune system to produce antibodies designed for the wrong enemy. They would not protect us from the virus and could enhance the disease, as happened with the RSV vaccine.
“It’s really important to be able to maintain pre-fusion [form],” says Fasséli Coulibaly from the Department of Biochemistry and Molecular Biology at Monash Biomedicine Discovery Institute. “And that’s exactly what the clamp is able to do.”
To make the synthetic version of the spike protein, the UQ team used genetic technology to create a gene that encodes the spike protein and a fragment of an HIV protein known as gp141, replacing the transmembrane domain to hold the spike in shape and stop the transformation to its post-fusion form.
The stitched-together gene sequence is delivered into Chinese hamster ovary cells. These are placed into bioreactors for about 10 days, at the end of which the code is translated into the polypeptide. The HIV fragment naturally self-assembles, clamping the spike into its native structure and giving the same shape the spike would typically have when attached to the surface of the virus.
“At the time, I knew the clamp was a very solid technology,” says Damian Purcell, Professor of Virology at the University of Melbourne and head of the Department of Microbiology and Immunology at the Peter Doherty Institute. Purcell, who studies HIV, has helped the UQ team develop methods to measure the quality and the breadth of the immune response elicited by the vaccine against SARS-COV-2.
He explains that 50 years of HIV research has led scientists to discover that the HIV envelope spike’s soluble form – the HIV antigen – can be manufactured by introducing mutations around the gp141 region to stabilise the structure. Purcell has encoded these mutations in the HIV vaccine that his team is currently trialling, which shows promising results in animals.
The UQ group found that they could further modify HIV gp141 and stitch it onto the base of many different virus surface proteins that are similar to the coronavirus spikes.
Over the past decade, Chappell and his collaborators have tested the clamp on several viruses, including RSV, influenza, Ebola, Nipah, Lassa fever and MERS coronavirus, so were confident that it would work well for this SARS coronavirus. The approach was ready, it was fully funded and was “the best lead candidate we had in Australia,” says Purcell.
The team started work on SARS-COV-2 at the end of January 2020. Within just three weeks, the first
The vaccine does not infect people because it does not contain any genetic material of HIV. “The worst thing that could happen is that it might actually protect you from HIV infection.”
vaccine was ready to test. After preclinical studies testing safety and dosing in animals, phase one human clinical trials were launched in July. The vaccine was safe and elicited a high immune response.
What was unclear then was whether the small HIV fragment would also elicit an immune response. The UQ team knew that was a possibility and informed all participants of the risk. But because HIV tests are built to detect human antibodies, there was no way to test cross-reactivity in animal studies before clinical trials.
“The human immune system is extremely good at recognising material that it deems ‘foreign’,” says Wrapp. “If you inject a non-human protein, then it is pretty much inevitable that your immune system will eventually raise antibodies against that protein.”
But because the clamp weighs only about 10% of the whole polypeptide, the widespread assumption among scientists was that it wouldn’t register. “I think if you talked to me – and probably others – we’d say the chances were very low,” says Purcell.
At the 57-day mark, study participants were tested for a number of effects, including HIV reactivity. Gradually, the results started returning, and evidence mounted: patients were testing positive to HIV. The immune system had made antibodies against SARS-COV-2, but it had also made antibodies against the clamp. When an HIV diagnostic test finds those antibodies, it registers the patient positive for HIV as a correlation.
“When SARS-COV-2 hit, there were just so many issues, problems and unknowns that we had to get around. We were really run off our feet, and this was really down low on our priority list,” says Chappell.
“Everything was done right, from my point of view, and I think that’s the opinion of the [scientific] community, as far as I could tell from my colleagues,” says Coulibaly. “If anything, it’s actually a good demonstration that the system is robust and detects things that weren’t likely, and we stopped the problem before vaccines.”
The vaccine does not infect people with HIV because it does not contain any genetic material of HIV, and thus the virus cannot replicate. “The worst thing that could happen is that it might actually protect you from HIV infection as well,” adds Purcell.
But the widespread use of this vaccine would interfere with HIV testing and demand new HIV diagnostics. To add to this, the risk to public confidence in vaccines was high.
“It would have been incredibly difficult to explain to 100% of the population that no, this is not HIV, it’s a small fragment of one particular protein,” says Chappell. “There is still a huge amount of stigma attached to those three letters, and it’s disappointing.”
On 11 December, 2020 the trial was abandoned.
Being prepared
The idea of sticking spike proteins together to lock them into the pre-fusion state isn’t new. “That’s something people have been working on for quite some time,” says Chappell.
“It sort of came out of necessity,” says Wrapp. “When working with these proteins in the lab, a lot of them are so unstable that when we try to express them, we are getting no pre-fusion whatsoever.”
Wrapp is a postdoc fellow in Mclellan Lab, a research group at the University of Texas that works on protein stabilisation. The group has engineered two mutations called proline mutations that, when added to the spike, inhibit the transition from pre- to post-fusion form.
Almost 10 years ago, they started studying a virus called HKU1, which causes mild respiratory symptoms similar to the common cold. Using a technique called cryo-electron Microscopy (crem) – which can observe molecular structure at an atomic level, using extremely cold temperatures to stop molecules vibrating – they solved the HKU1 spike protein’s atomic structure and could engineer the two stabilising proline mutations. They then transferred the mutations into the structure of the MERS coronavirus spike.
Studying the atomic structure of a spike protein from a new virus can take months, but thanks to the previous painstaking work done on closely related viruses, the Mclellan Lab team were able to quickly engineer those stabilising mutations into SARSCOV-2, a new pathogen that no one had heard of before.
“You need to have a lot of basic discovery science on an ongoing basis – to have things in the bag of
The clamp technology is easily scalable, allowing it to rapidly produce large amounts of the antigen protein needed for the vaccine.
information that you can deploy when the chips are down,” says Purcell.
In the past months, the Mclellan team has worked closely with the National Institutes of Health – the primary agency of the US government responsible for biomedical and public health research. Their proline mutations are now incorporated into the Pfizer and Moderna vaccines, which have been approved in some parts of the world, and into the Novavax and Johnson & Johnson vaccines, both currently under clinical trials.
While the proline mutations worked well for COVID-19 vaccines, the need for a technology readily applicable to a broad range of pathogens is notably urgent. Human activity and environmentally destructive practices are creating the perfect conditions for zoonotic pathogens like SARS-COV-2 to emerge. The risk of a global pandemic occurring in the future will continue.
“The big benefit of the molecular clamp is that we don’t need to know the structure ahead of time,” Chappell says. “We’ve shown that the technology works for a whole range of virus families. It’s broadly applicable.”
The technology is also easily scalable, allowing it to rapidly produce large amounts of the antigen protein needed for the vaccine, and it enables the production
of proteins otherwise difficult to express and reduces the amount of protein needed for each dose of vaccine. The UQ team has also developed a purification mechanism so that any vaccine candidate is purified using the same methodology. “That’s a huge advantage,” says Chappell.
“It’s an exciting technology because it gives you a generic platform for something you can adapt quite easily to different antigens and different diseases,” adds Coulibaly.
Never before have so many different new technologies been developed in such a short time. Innovative vaccine technologies, such as the molecular clamp and the MRNA approach used in other vaccines being rolled out, are going to be crucial in dealing with future pandemics.
“It’s incredibly disappointing that this vaccine candidate is not being progressed,” says Chappell, but he is confident there are ways to solve the HIV test cross-reactivity.
The UQ researchers are back in the lab to restart from where they began in January 2020. They had been tweaking the clamp to remove parts of HIV gp141 known to elicit significant antibody responses. Now, they are trying to find ways to reduce the clamp antigenicity even more, to hide it from the immune system completely.
The team is also running through a broad panel of similar bundled proteins to find candidates that can replace the HIV clamp while achieving the same stability levels.
“I wholeheartedly believe that we’ve shown that the technology works and that it’s safe,” says Chappell. “We do need to go back to square one, but we’ve learnt a lot over the past months. We’ll be back, I guess.”
MANUELA CALLARI is a Sydney-based freelance science writer specialising in health. This is her first feature for Cosmos.
“I wholeheartedly believe that we’ve shown that the technology works and that it’s safe ... We’ve learnt a lot over the past few months. We’ll be back, I guess.”