The “Dirty Little Secret”* About Vaccines

Note: This originally appeared on the SciBox section of the Oxford SciBar. Check out the site, there’s a host of podcasts of talks and other articles there.

 

In some ways, the immune system’s like a brain: it can learn, it can adapt and, so it’s ready for the next time, it can remember. This is, perhaps, its single most important feature.

The immune system has the chance to learn a unique signature on the surface of an invader every time we’re infected by something, be it virus, bacteria or even a parasite. Once it learns these signatures the body produces antibodies or ’killer cells’ to fight it. When the infection has been defeated, special cells – B-cells – remember it, for as long as a life time. If the invader returns it won’t have such an easy time: the immune system will be ready.

Like most things that learn, the immune system can also be taught – in this case, by using vaccines. Depending on what the target disease is, a vaccine might be a weakened, or dead, version of a virus or bacteria or some specific component of them. Vaccines too can provide a life time of immunity.

For vaccines it’s not just quantity that counts – it’s also quality. And the quality of a vaccine, in terms of its effectiveness, can be improved by the addition of an adjuvant. Adjuvants act like boosters for vaccines, amplifying the immune system’s response. But not all adjuvants are created equal: some work better with one vaccine, but not others. So the search to find new, improved ones is still on.

Now, a team led by Professor Quentin Sattentau, at the Sir William Dunn School of Pathology at Oxford University, has shown that by using a polymer, called PEI, as an adjuvant, mice were completely protected from an otherwise lethal dose of flu virus. With just a single dose of vaccine and PEI adjuvant.

PEI is more commonly known in the life sciences as a ‘transfection reagent’, something which can safely shuttle things like DNA into the interior of cells, and its discovery as an adjuvant was somewhat serendipitous.

Quentin’s team started looking for new adjuvants about 10 years ago. Around that time there was a hypothesis that molecules which stimulate certain immune cell receptors, called TLRs, should act as good adjuvants.

“We’d been messing about with nucleic acid based TLR agonists, things called CpG and RNA mimics and stuff…”

“We’d been messing about with nucleic acid-based TLR agonists, things called CpG and RNA mimics and stuff, and we’d been injecting them and finding that they weren’t actually very good,” says Quentin, laughing.

But his team did realise that some of these molecules also had receptors inside the cell. So they turned to transfection reagents, to shepherd these agonists into cells. He asked one of his students to try this out and see if they got an increased immune response.

“And he said ‘yeah, we do. And it works, but, when I leave out the TLR agonists, we still get a really good immune response’,” Quentin says, laughing again. “Fortunately, we’d done the negative control, and the negative control was positive. So that kind of was the lucky break, because we then realised that transfection reagents could be adjuvants in their own right.” Of all the transfection reagents they tested, one was a clear winner: PEI.

The most commonly used adjuvant, in humans, is a substance called alum – an aluminium salt – and has been used since about the 1920s. In the US and Canada, alum is the only adjuvant approved for human use. When Quentin’s team compared alum and PEI in mice they were shocked by the results: “…on an injectable basis we found that you could use about 10,000 fold less PEI and get the same potency as alum.” If PEI proved safe for use in humans, that could represent a significant cost saving.

But Quentin still had a nagging feeling. The data suggested that alum and PEI worked in similar ways. But there was one more thing he wanted to try: to give the vaccine and PEI as a nasal droplet, to deliver it mucosally, rather than injecting. And it worked. Well.

“I thought ‘Aha!’”, says Quentin

“I thought ‘Aha!’,” said Quentin, “this is something special because alum doesn’t work mucosally and the other mucosal adjuvants that are out there, experimental mucosal adjuvants, have problems.”

If PEI was up to the job, you could give the vaccines needle free, as a nasal spray, or preferably under the tongue. “If it works under the tongue, you could just give someone a pastel and say ‘stick that under your tongue until it’s dissolved.’ That might be a better way,” says Quentin. It certainly would for the needle-phobic.

The team then quickly moved on to testing PEI as a mucosal adjuvant from there. They turned to an experimental HIV vaccine they’d been working on. The vaccine uses a fragment of the HIV virus called gp140. And their initial results in mice are compelling.

Cut away model of HIV

gp140 closely mimics a molecule HIV uses to stick to, and invade, its target: T-cells. As such it’s one of the least variable target signatures on HIV and probably one of the best for a vaccine. The team compared two different adjuvants – CpG and CTB – to PEI in mice. They all generated about the same amount of antibodies to gp140, but when they looked at how good these antibodies were – how strongly they bound to gp140 – the winner was clear: PEI.

Perhaps more importantly, antibodies from PEI-treated mice recognised gp140 in its ‘native conformation’ – it maintained the shape of the signature as it exists ‘in the wild’. “That was an unexpected, but pleasing result. The same would probably be true for other glycoproteins from other viruses,” said Quentin, “you know, we didn’t test it, but I suspect it’s probably true for HA from influenza and GD from herpes which are glyco-proteins too.”

“That was an unexpected, but pleasing result.”

It’s a ‘pleasing result’ because when a vaccine is made you have to process, store and deliver it under the right conditions to maintain its ‘native conformation’ when it reaches the immune system. But it’s not always possible; even subtle changes to a vaccine’s composition can cause different creases, nooks and crannies to appear in the signature compared to the wild version. A vaccine that makes the immune system recognise the ‘native conformation’ should give better protection from the real-world virus.

Next on the list was Flu. A fatal dose of it.

Quentin’s team showed that mice given a single nasal dose of vaccine with PEI as an adjuvant then exposed to an otherwise lethal dose of flu were completely protected from the virus. They showed no signs of weight loss – a kind of proxy measure of disease severity. In the other groups given one of two other adjuvants with vaccine, vaccine alone or no vaccine, mice lost between 10–25% of their starting body weight. With the different adjuvants the mice weren’t completely protected.

PEI also gave much higher avidity – the tightness with which an antibody binds to its target – than any of the other adjuvants. As with gp140 the team suspected this improved immunity was due to much better recognition of the ‘native conformation’ of the vaccine and how well antibodies bound to the flu vaccine.

Such complete protection from a single dose is almost unheard of. Think back to vaccines you may have had; normally you’ll need a ‘booster’ shot a few months later.

They did one more test with PEI, this time using an HSV-2 (herpes) vaccine followed by a potentially fatal dose of the HSV-2 virus. This time the mice were primed with the vaccines and adjuvant combinations as before, but were also ‘boosted’ twice before being given the HSV-2 virus. The results were much the same as with Flu. Only the adjuvanted vaccines gave the mice protection from HSV-2 and PEI gave much better protection from disease after just a single dose than other vaccine/adjuvant combinations.

But how?

Historically, it was a mystery how adjuvants worked. But at least we did know they worked. Even alum has only recently had its mechanism of action – mostly – worked out. Even so, there are still questions.

Quentin’s group acknowledged this. “The biggest problem, I think, that the adjuvant field has had is in understanding how adjuvants work,” he says, “and so I think the biggest puzzle we had to solve was understanding the mechanism of how PEI worked.”

They painstakingly teased apart how PEI works. They found that it releases double stranded DNA (dsDNA) from cells. Normally dsDNA shouldn’t be found outside of the cell. The immune system recognises this and uses it as a signal that there’s damage. That essentially puts the immune system on high alert, locally, to seek out what the danger was, and deal with it; priming it to respond more. “It’s a pretty clear result, and we’re very pleased with that,” says Quentin.

The inner workings of a Killer T-cell (blue) attacking its target.

What the group also showed breaks a dogma in immunology: that you need a ‘Th1 response’ if you’re going to combat viruses. The Th1 response uses a specific type of killer cell called CTLs. Their job is to sweep in when a virally infected cell is recognised and to execute them, stopping the virus from spreading. “…what we’ve shown here,” says Quentin, “is that actually you don’t, at least in mice, you can protect them really well against flu and herpes, without any CTLs that are detectable.”

Since it’s not a Th1 response, Quentin feels this may mean that PEI could have applications beyond viruses. “…perhaps, this adjuvant will be even more useful in vaccines against bacterial infections. So that’s another thing that we are intending to look at very soon,” he says, “and possibly also parasitic infection, […] it may turn out that PEI would be very good for adjuvanting parasitic vaccines, like malaria.”

While PEI is very promising as an adjuvant, it’s still very early days. There’s a number of scientific and safety testing hurdles to get over before we are likely to see it in human vaccines. Quentin estimates that, all being well, we may see the first in man trials using PEI within four years. This may be optimistic but, if PEI is as effective as it promises to be, we may be seeing it. And we may well need to.

The 2009 swine flu outbreak is a prime example of why we need safe, approved, adjuvants.

During the height of the outbreak, as manufacturers were racing to make a reliable vaccine in sufficient quantities, the American government ordered 251 million doses (they later halved this order). Without an adjuvant. Now, suppose that an adjuvant could double the effectiveness of a vaccine, instead of a 30 µg dose, you might need 15 µg. With the relatively small extra cost of adding a reliable adjuvant, you could double that coverage to 502 million people.

With an eye on the future, in the early days of a new pandemic, with vaccine production levels low, and there’s not enough to go around yet, we can’t afford not to look into new and safe adjuvants.

Reference:  Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigen, Wegmann, F., Gartlan, K. H., Harandi, A. M., Brinckmann, S. A., Coccia, M., Hillson, W., Kok, W. L., et al. (2012). Nat Biotech, advance on.

http://dx.doi.org/10.1038/nbt.2344

Top image by Image (CC) by Markus Schöpke

*for why adjuvants were called the dirty little secret of immunology/vaccines, have a look at the wikipedia entry here.