Keystone meeting in Rio: Breakthroughs and surprises

“Breakthrough” isn’t a term scientists use often when they talk about a finding. But according to co-organizer Rino Rappuoli of Novartis, attendees of the Keystone meeting on Advancing Vaccines in the Genomics Era, which took place from Oct. 31st until Nov. 4 in Rio de Janeiro, heard talks on not just one, but two breakthroughs, both published in Science on the first day of the meeting.

The first is the structure of a near-native version of the HIV Envelope (Env) trimer, the protein spike on the HIV surface that the virus uses to enter its target cells. Determining its structure has been a major goal of the HIV field for a long time. The second is the proof that in principle, it is possible to use a potent neutralizing antibody as a starting point to design a vaccine immunogen, at least for respiratory syncytial virus (RSV), the leading cause of hospitalization in the US for children under five. 

Attendees also heard a lot about advances in systems biology, an emerging branch of biology where researchers try to measure the parameters of biological “systems” in their entirety, such as changes in the expression levels of most or all genes in the genome. Researchers hope that such a comprehensive approach will provide new insights into how vaccines affect the immune system, and one day help predict whether a vaccine candidate is likely to work.

The fine structure of the HIV trimer

The instability of the HIV trimer has long hindered efforts to obtain its molecular structure at a truly useful resolution. But as mentioned in our previous report from AIDS Vaccine 2013 in Barcelona, an effort led by John Moore at Weill Cornell Medical College in New York has over the years identified apparently effective ways to stabilize the protein without disrupting it too much.

Researchers also compared trimers from many different HIV variants modified this way for stability, and eventually selected an Env trimer called BG505, isolated from the derivative of an HIV clade A founder virus from an infected child in Kenya. They then analyzed its structure by X-ray crystallography and cryo EM. 

At the meeting, Ian Wilson and Andrew Ward from The Scripps Research Institute presented the details of these studies. Wilson, who presented the X-ray crystallography work (Science 2013, doi: 10.1126/science.1245625), said that he and his colleagues tried to grow crystals of BG505 bound to many different broadly neutralizing antibodies (bNAbs), and found that the best crystals formed when BG505 was bound to the bNAb PGT122. Ward and colleagues used cryo EM to determine the structure of the same BG505 trimer bound to a different bNAb, PGV04 (Science 2013, doi: 1126/science.1245627). 

The structures obtained through X-ray and cryo-EM confirm and complement each other, said Ward, who presented the cryo EM work: For the X-ray structure analysis, researchers had to shave most sugars off of the trimer to be able to crystallize it, while cryo EM allowed them to leave on all of the sugars. 

And even though the X-ray structure doesn’t quite have atomic level resolution at 4.7 Ångstroms, Ward said he is confident that it’s accurate because it agrees with the 5.8 Ångstrom cryo EM structure. “This is really it,” he said, adding that one challenge now is to further improve the structures to get closer to atomic-level resolution. Ward also wants to bring the cryo EM structure even closer to the native trimer than it already is, by adding back the so-called membrane proximal external region, which the researchers removed to make the trimers more soluble and prevent protein aggregation. 

For the first time, the BG505 structures show the Env trimer in its entirety, including the relative position of the variable loops 1, 2 and 3. “The biggest thing we learned was that the epitopes are a lot more complicated than previously thought,” Ward said, referring to the target sites of bNAbs. This restricts the angle an antibody can come in to bind. For example, he said, “you have to come in and navigate this very straight path in order to get to the CD4 binding site.” 

As a result of the studies, researchers now also know how to make trimers stable enough to use them as immunogens in a candidate vaccine. “[BG505] is a stable trimer and we know that it doesn’t fall apart, so it can be used as an immunogen,” Wilson said, adding that the next goal is to make similar trimers from other HIV clades. “It’s really the start of a new generation of immunogens and vaccines that weren’t previously accessible,” Ward added. 

While the BG505 structures are consistent with each other and with a cryo EM structure published on Oct. 23rd by Sriram Subramaniam and colleagues (Nat. Struct. Mol. Biol. 2013, doi: 10.1038/nsmb.2711), they differ from a previous structure published by Joseph Sodroski and colleagues (Proc. Natl. Acad. Sci. 110, 12438, 2013). This suggests that Sodroski’s structure, which has recently been the focus of controversy fueled by critical comments published by leading structural biologists, might not be accurate, Ward said.

On the way to an RSV vaccine? 

The design of a candidate vaccine against RSV, reported by Peter Kwong from the Vaccine Research Center at the National Institute of Allergy and Infectious Diseases, may also be important for HIV vaccine development, as it is shows that, in principle, the structures targeted by potent neutralizing antibodies can be used as a starting point for the design of immunogens. 

Earlier this year, Kwong and colleagues determined the crystal structure of a potent neutralizing antibody called D25 to the prefusion form of RSV F, the protein RSV uses to enter its target cells. In the new study, they used their knowledge of this structure to find ways to stabilize the prefusion RSV F protein without the D25 antibody bound to it (Science 2013, doi: 10.1126/science.1243283). 

When they used this stabilized prefusion protein as an immunogen in mice and monkeys, they found that it induces neutralizing antibody levels that are many times higher than the titers needed for protection, and about 10-fold higher than the titers induced by a post-fusion form of the RSV F protein, which is currently being developed as a vaccine candidate. Next, Kwong and colleagues plan clinical trials with the new RSV immunogen.

Conference co-organizer Bali Pulendran from Emory University was clearly impressed with the RSV results. “This is one of the first examples of an approach where you go from a structure through rational design to construct an immunogen that’s highly immunogenic,” he said.

Given the recent descriptions of the HIV Env structure, Kwong also plans to try a similar approach to design immunogens for an HIV vaccine. D25-like antibody responses to RSV seem very common in people, so one important message of the RSV study for HIV, Kwong said, is that it’s important to choose an antibody as a starting point for such efforts that’s not just broadly neutralizing and potent, but also commonly made in people. “It’s very, very important to look at what humans make naturally at high titer. If you want a vaccine that everyone could make, see what people make,” he said, adding that he is currently performing detailed analyses to find the most common bNAbs in HIV-infected people. 

An additional challenge is that HIV-specific bNAbs take 1-2 years to fully develop. That’s why Kwong and others are also studying in detail how bNAbs develop in infected individuals, so they can design immunogens that can accelerate the maturation of bNAbs. 

Systems biology: From validation to surprise 

One approach to understanding how vaccines work is to measure gene expression changes of most, if not all, of the genes in immune cells following immunization. If the systems biology part of the meeting was any indication, the strategy is notably effective. 

Rafick Sékaly from the Vaccine and Gene Therapy Institute of Florida, for example, reported that induction of inflammation-related genes in elderly people before vaccination corresponds with lower responses to flu vaccination. This suggests that age-related systemic inflammation is one reason why vaccines have less of an effect in elderly people. 

But inflammation is only bad for vaccine responses if it gets out of control, Sékaly said. Normally, the body tries to keep inflammation in check by boosting expression of certain genes that turn it down. But in the elderly people who showed lower vaccine responses, Sékaly and colleagues didn’t find such genes upregulated, suggesting that only inflammation that’s left unchecked lowers vaccine responses. Next, Sékaly wants to test if reducing inflammation before vaccination can improve responses to yellow fever and hepatitis B vaccines. 

One advantage of the “systems” approach is that it involves measuring everything. This means that researchers are not constrained by their preconceptions of what to expect. That’s why systems biology can also lead to unexpected insights, something that became especially clear in a talk by Pulendran.

When Pulendran and his colleagues measured global gene expression changes in response to flu vaccination, they found that the upregulation of a gene called TLR5 one week after vaccination correlated with the level of the later antibody response to the vaccine. That surprised Pulendran, because TLR5 is a receptor that senses bacterial flagellin, which is not present in viruses. So, at first, Pulendran and colleagues thought the flu vaccine they were studying might be contaminated with bacteria. 

Turned out it wasn’t. But further investigation revealed that mice without TLR5, or without bacteria in their gut, showed less differentiation of plasma cells, the cells that produce antibodies. This suggests that the sensing of our own bacteria might help induce the antibody response to vaccines, and that things that disturb them, like antibiotics, might be harmful to some vaccine responses.

Pulendran and colleagues have also been trying to find common signatures of gene expression changes that might make it possible to predict later antibody responses to vaccines. Interestingly, they have found that gene expression changes in response to certain classes of vaccines share common signatures that differ from other classes of vaccines: For example, vaccines based on live viruses like flu mist or yellow fever vaccine have similar signatures, which differ from the signature of carbohydrate vaccines.

In his final remarks to the attendees, Rappuoli noted that systems biology has until recently been in a validation phase: Researchers used approaches like measuring changes of global gene expression largely to confirm things that were already known. That’s a good thing, he said, because it confirmed that the approach works. But now, he said, findings like Pulendran’s TLR5 results show that the field is ready to discover new things.

Rappuoli ended the meeting with a glimpse of the future. Systems biology, he said, might eventually help accelerate the development of candidate vaccines which, he noted, can take about 15 years to move from bench-top to clinic. “That’s a long time,” he said, adding that while clinical trials today test, say, 10 parameters in 10,000 people, systems biologists might be able to find ways to do the same thing with much less effort. “I want to leave you with [a] dream for vaccine development,” he said. “If you could use [this] technology [to test] ten people [with] 40,000 data points per person to predict what’s going to happen, probably vaccine development would be much faster.”