Researchers at La Jolla Institute for Immunology (LJI) have discovered that time may be a key factor in allowing the immune system to generate antibodies capable of neutralizing HIV, a virus that to date has proven to be one of the most elusive using existing vaccination strategies. Scientists have traditionally thought that in the body’s germinal centers (GCs) antibody-producing B cells spent only weeks perfecting their weaponry against viral threats. The newly reported LJI research shows that a “slow delivery, escalating dose” vaccination strategy can prompt B cells to spend months mutating and evolving their pathogen-fighting antibodies. This finding could represent an important step toward developing effective, long-lasting vaccines against pathogens such as HIV, influenza, malaria, and SARS-CoV-2.

“This shows the immune system can do really extraordinary things if you give it the opportunity—and that in some vaccine contexts, patience really is a virtue,” said LJI professor Shane Crotty, PhD, who is senior author of the team’s study, published in Nature, and titled, “Long-primed germinal centers with enduring affinity maturation and clonal migration.” In their paper, the team noted, “These findings indicate that patience can have value in allowing antibody diversification and evolution in GCs over extended periods of time, and this long-prime, slow-delivery immunization approach holds promise for difficult vaccine targets.”

Most pathogens look alien to the immune system, and are covered in unfamiliar proteins. When the body’s dendritic cells see these strange proteins, they signal to helper T cells to start training an army. The immune system’s B cells get the signal that there is an invading pathogen, and they are presented with an antigen—a molecular marker.

Germinal centers are microscopic structures that form in special lymphoid tissues throughout the body. The germinal centers are critical in the fight against pathogens, as they give B cells a place to mutate and test out their antibodies. “Germinal centers are the engines of antibody evolution,” the team noted. “To accomplish this evolution, GC B cells (BGC cells) proliferate rapidly—every 4–6 h.” So while B cells that don’t mutate and improve their antibodies over time are eliminated, germinal center B cells with useful mutations get sent out into the body to tackle the pathogen. “It really is evolution,” said Crotty. “This process can work incredibly well and lead to antibodies that become a thousand times better at binding the virus.”

Germinal centers can also be thought of as a kind of pop-up shop. Once the threat has passed, the germinal centers collapse. No one knows yet exactly why this happens, although there must be some kind of underlying molecular signal.

For Crotty’s lab, a big question has been how to get germinal centers to stay open for longer. Timing is important because some pathogens can only be neutralized by highly specialized antibodies. HIV is one of the toughest pathogens against which to develop effective antibodies. HIV is covered with an invisibility cloak of sugar molecules, and the virus can change its shape as it enters cells. This stealth and shape-shifting power makes it really hard for immune cells to spot useful antigen targets on HIV.

The germinal centers instead start pushing out B cells that make “low affinity” antibodies. These antibodies cannot bind and neutralize HIV in a very effective way. HIV’s shifting structure can also lead to high-affinity antibodies that can bind really tightly, but onto the wrong targets. So for HIV, the authors wrote, “ … an ideal vaccine should generate cross-reactive or broadly neutralizing antibodies that can protect against variants; however, so far, no broadly neutralizing antibodies against HIV have been elicited in the serum of either humans or nonhuman primates (NHPs) by vaccination.”

Left to right: LJI professor Shane Crotty, PhD, and LJI postdoctoral fellow Harry Sutton, PhD. [Matt Ellenbogen, La Jolla Institute for Immunology]

Crotty thinks that B cells just need more time to mutate. “It takes a long time, and many cell divisions, before you just get lucky and one of the right mutations finally happens,” he said. The idea is that the longer B cells can mutate and perfect themselves in germinal centers, the more likely that the B cells will luck into producing broadly neutralizing antibodies against HIV.

For years, Crotty and his collaborators in the LJI Center for Infectious Disease and Vaccine Research, and the Scripps Research-led Consortium for HIV/AIDS Vaccine Development, have worked to solve pieces of the HIV vaccine puzzle. The Crotty Lab co-led pivotal studies into the use of promising new vaccine ingredients and how to best activate B cells against HIV. The newly reported study highlights the importance of stretching out the period where B cells can evolve in the germinal centers.

For this study, collaborators at the Tulane National Primate Research Center gave rhesus monkeys immunizations every two days for 12 days. The series of seven shots contained an escalating dose of the HIV Env protein antigen, the HIV protein that the researchers wanted the immune system to learn to target.

“That pattern mimics a natural infection more so than just a single immunization,” explained LJI postdoctoral fellow Harry Sutton, PhD, who served as co-first author of the new study with former LJI instructor Jeong Hyun Lee, PhD, now a senior scientist with the IAVI Neutralizing Antibody Center at Scripps Research.

Two groups of monkeys then received a booster vaccine after 10 weeks, while a third group did not. The researchers tracked immune responses by examining monkey lymph nodes. The team also monitored B cell development in individual germinal centers. Their work revealed that the germinal centers stayed active, and B cells continued to evolve for six months after the initial series of seven shots. As Sutton pointed out, the study was set to end after six months, but the germinal centers may have lasted even longer if the research had continued.

The scientists also performed genetic sequencing to analyze immune cell memory and antibody binding abilities. They found that monkeys given the seven-shot series but no booster had a stable and durable population of anti-HIV antibodies after six months. “… using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal center B (BGC) cells that last for at least six months,” the team noted. “A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization.” These non-boosted animals also had greater numbers of T follicular helper cells that were ready to recognize the HIV antigen and launch B cells into battle. The boosted animals did have a second “peak” in antibody numbers after their booster shot, but they didn’t end up with the same high-quality antibodies.

The slow delivery, escalating dose strategy appeared to result in a better payoff. The large dose of antigen had likely given the immune system enough of a taste of the virus that the germinal centers were ready to stay open, and the B cells were prompted to evolve for as long as possible to address the perceived threat. “Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure,” the investigators wrote. “Numerous BGC cell lineage phylogenies spanning more than the six-month germinal center period were identified, demonstrating continuous germinal center activity and selection for at least 191 days with no further antigen exposure.”