How Antibodies and Complement Orchestrate Protective Immune Responses Against Bacteria
Suzan Rooijakkers, PhD, Professor, UMC Utrecht
Antibodies: A Potential Alternative to Antibiotics
Immunotherapies could be an alternative to antibiotics, according to Suzan Rooijakkers, PhD, a Professor at UMC Utrecht, who uses therapeutic antibodies to fight hospital-acquired infections.
Rooijakkers told delegates at the Antibody Engineering and Therapeutics Europe conference about the approach, explaining the idea is to use antibodies that trigger the body’s complement system to fight infection.
“There are several ways in which an antibody could help cure a bacterial infection. For instance, antibodies are now being developed that block the adhesion of bacteria to host cells. There are other developmental antibodies that block or neutralize important adherence factors for bacteria,” she said.
“In my lab we are mostly interested in whether we can use antibodies to actually engage our own immune system to better kill bacteria. And for that, an important prerequisite is that the antibodies can bind to the bacterial surface via the fab domains and then subsequently activate the immune system by the Fc domain.”
The specific focus is on antibodies that trigger the complement cascade, a part of the immune system that induces inflammatory responses that fight infection.
Gram-Positive Infections
Most often, the complement cascade is associated with fighting infection caused by gram-negative organisms via the formation of membrane attack complex (MAC) pores that damage their membranes.
However, the complement mechanism can also be harnessed to combat gram-positive bacteria, according to Rooijakkers.
“Gram-positive bacteria are actually resistant to MAC formation, but still, complement activation is really important in immune defence against these bugs,” she said.
Rooijakkers explained that complement labels gram-positive bacteria with proteins called opsonins, which help phagocytes kill them. Also, during complement activation, chemo-attractants are formed that allow neutrophils to find the infection site and kill the organism responsible.
Rooijakkers and her team developed therapeutic antibodies that trigger complement activation.
“So we know that complement can be activated on bacteria spontaneously or via lectin pathway or the alternative pathway. It’s also known that specific antibodies can potently enhance this. However, the question is what is a good antibody in driving this?” she said.
Initial efforts have sought to identify “good antigens” that complement activating antibodies bind. However, this has been proven challenging.
Other research has concentrated on understanding how antibody Fc regions impact complement activation, according to Rooijakkers, who cited a 2014 as the basis for the idea.
“Basically the paper showed that complement activation by antibodies was different than we previously thought. Initially, we thought that the antibodies could just randomly bind to a target cell surface, but in their paper, they showed that antibodies actually need to come together to form an organized cluster of hexamers held together by Fc-Fc interactions.
“And this hexamer of IDGs is the perfect platform for C1q molecules to dock on and activate complement,” she said. Subsequent research showed introducing point mutations to antibody Fc regions enhances hexamer formation and elevates complement activation levels.
Antibacterial Antibodies
This work was the prompt for Rooijakkers and her team.
“We started collaborating to study whether this also holds through for antibacterial antibodies” she said, noting that the initial studies focused on antibodies against the bacteria streptococcus pneumoniae.
“Strep pneumonia is a common cause of community-acquired infections but also hospital-acquired infections. This bacterium has a very thick capsule, and there is also a lot of variation in this capsule, which makes it difficult to develop effective vaccines.
“Another problem with strep pneumonia is that it is becoming increasingly resistant to antibiotics. So we thought potentially there’s room for antibodies here.”
The team started generating mAbs against strep pneumonia bacteria, introducing some mutations in the Fc region that have been shown to enhance clustering.
The mutations on their own made minor impact; however, when the researchers added complement, the effect was dramatic, according to Rooijakkers
“So to our surprise, we saw that the wild type antibodies against the capsule had absolutely no capacity to activate complement on these bacteria. In contrast, when we introduced these mutations, then we saw a very potent complement activation.”
Protein A
The researchers also study staphylococcus aureus, the leading cause of hospital-acquired infections.
“Staph aureus is really good at evading the human immune system. It has evolved a lot of molecules by which it can use to, for instance, block complement or neutrophils. Also, it has a protein called staphylococcal protein A – aka protein A - it uses to block IgG [antibodies].”
Protein A is anchored to the cell wall of staph aureus and has five repeated IgG binding molecules. It is used to isolate antibodies.
However, protein A also blocks the activity of antibodies through a hitherto unknown mechanism, which is what Rooijakkers has set out to research.
“It became evident that protein A actually binds to the same interface where antibodies need to form IgG hexamers. So we started to investigate our hypothesis that maybe protein A blocks antibodies by blocking hexamer formation,” she said.
“So long story short, we showed that protein A blocks the formation of these hexamers in solution. We also used high speed AFM [atomic force microscopy] to demonstrate that protein A can prevent the formation of higher order IgG oligomers.”
The finding has obvious therapeutic application, according to Rooijakkers, explaining that by modifying an IgG1-type antibody into a separate subclass of antibody – IgG3 –not bound by protein A, the team found a way of preventing staph aureus from evading the immune response.
“We showed that if we engineer them into an IgG3, then protein A can no longer block these antibodies. So this is actually a way in which we can evade the immunization mechanism from the bacteria,” Rooijakkers said.
