“Subtle Change” in Mothers’ Antibodies During Pregnancy Protects Newborns
“Subtle Change” in Mothers’ Antibodies During Pregnancy Protects Newborns
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Newborn babies make their way into the world and are immediately highly susceptible to infections. Protection is offered by antibodies that are vertically transferred from the babies’ mother, either via the placenta in utero, or via breast milk after birth, until the baby’s immune system develops.
Understanding how maternally transferred antibodies protect newborn babies has been a fundamental research focus in neonatal studies. Previously, antibodies have been considered to only target pathogens that occur outside of cells. However, several pathogens survive by replicating intracellularly, including group B streptococcus (GBS), Escherichia coli, Listeria monocytogenes and Epstein-Barr virus (EBV).
Whether or not maternal vertically transferred antibodies can protect against internally-replicating pathogens has remained a key unanswered question for the field. Now, a new study published in Nature sheds light on this query.
A research team led by Dr. John Erickson, MD and assistant professor at the division of neonatology, Cincinnati Children's Hospital Medical Center, has discovered that a “subtle change” in the structure of sugars that attach to antibodies during pregnancy enables their protection against a wider variety of pathogens – including those that replicate intracellularly.
These findings, the researchers say, could not only pave the way for novel targeted therapies for pregnant mothers and babies, but could also influence the field of antibody-based therapeutics more generally.
Technology Networks had the pleasure of interviewing Erickson to learn more about how the study was conducted, its key findings and the next steps for the research team.
Molly Campbell (MC): Can you discuss the rationale behind this research study, based on previous understandings of antibodies/ maternal antibodies?
John Erickson (JE): It has been long appreciated that mothers pass antibodies to their babies both in utero – across the placenta – and after birth, via breastmilk. This provides a critical layer of immune defence for newborn babies since they are highly susceptible to infections. Indeed, approximately 50% of the worldwide mortality in children < 5 years old occurs in neonates that are < 28 days old, and a large fraction of these ~2.5 million deaths per year are caused by bacteria and viruses that lead to sepsis, pneumonia or diarrheal illness.
A perceived limitation of antibodies is that they are thought to only target pathogens when they are outside of cells (i.e., extracellular). But many pathogens that cause these serious neonatal infections have an intracellular life cycle (i.e., they prefer to replicate inside of cells), where antibodies cannot easily access them. Therefore, a key unanswered question in the field is whether maternally transferred antibodies can protect against the myriad of intracellular infections that afflict neonates.
We utilized Listeria monocytogenes, which possesses an exclusive intracellular lifecycle and causes serious infections in pregnant women and newborn babies, to determine whether maternal vertically transferred antibodies can protect against neonatal Listeria infection. We were fascinated to find that Listeria-specific antibodies from pregnant, but not virgin, mice provide robust protection against Listeria infection in newborns. This told us that antibodies can indeed protect against intracellular infections but was only made evident by studying immunology in the context of pregnancy. Previous attempts to study antibodies against Listeria focused only on adult animals, so moving these studies into the more physiologically relevant context of reproduction enabled this interesting discovery, which has important implications for continued measures to reduce the strikingly high worldwide mortality rate for neonates.
MC: Your study shows that pregnancy changes the structure of sugars attached to antibodies, enabling mothers to protect their babies. Can you explain how this structural change achieves this?
JE: The change that occurs is incredibly subtle. We deciphered the molecular switch that occurs through a long process of elimination. We first ruled out the usual changes that can occur with antibodies, like the different isotypes and their binding specificities. We then reasoned that the change must occur post-translationally, meaning after the antibody is produced, given that pregnant mice were able to convert pre-existing antibodies to this new protective form.
The most common modification to antibodies involves the sugars attached to them, which is called glycosylation. At first glance, the overall sugar pattern on antibodies from virgin and pregnant mice looked identical. But we were struck by an unusually high amount of sialic acid, which is generally thought to be the final step in antibody glycosylation. We decided to remove the sialic acid from the normally protective antibodies from pregnant mice and found their ability to reduce Listeria disease was completely gone. We then utilized a “glycoengineering” approach to remove the sialic acid from Listeria antibodies from virgin mice, and then add back the standard version of sialic acid, which resulted in protection against Listeria when transferred to neonatal mice. We therefore reasoned that a molecular variant of sialic acid must exist in the virgin state that prevents antibodies from being able to protect against Listeria infection in neonates.
The subtle changes ended up being acetylation of the sialic acid on the antibodies. During pregnancy, an enzyme called SIAE is upregulated in both mice and humans, and this enzyme catalyzes the removal of the acetylation from sialic acid. Acetylation is a very small change: it’s the addition of only six atoms! However, these six atoms prevent the acetylated version of sialic acid from being recognized by a specific sialic acid receptor on neonatal B cells – called CD22 – which is essentially an “off” switch on certain subsets of B cells, in particular a subset that secretes a large amount of the inhibitory cytokine IL-10.
Antibodies from virgin mice that possess the acetylated version of sialic acid are unable to bind to CD22 on B cells, and therefore cannot turn off these IL-10 producing B cells. The IL-10 goes on to inhibit other immune cells, which greatly enhances the susceptibility of newborn mice to Listeria infection. For antibodies from pregnant mice that have removed acetylation from the sialic acid, they are then able to bind to CD22 and turn off the IL-10, greatly diminishing the susceptibility of neonates to Listeria infection.
MC: Can you discuss the advanced mass spectrometry techniques (and other methods) that you used to determine the biochemical differences between antibodies in virgin mice, compared to pregnant ones?
JE: We first utilized proteins called “lectins” that bind very specific carbohydrates, which are added in unique ways to other sugars. For example, we utilized lectins from a Pacific crab species, as well as a virus that mainly infects pigs, as both are known to bind very specifically to the acetylated version of sialic acid. These lectins confirmed our suspicion that the Listeria antibodies from virgin mice possessed more of the acetylated version of sialic acid.
We then reached out to our incredible collaborators at the University of Georgia Complex Carbohydrate Research Center, given their extensive experience with advanced mass spectrometry techniques. They utilized a unique approach that correlated a panel of known ions with sialic acid variants and confirmed that Listeria antibodies from virgin mice possessed approximately ten times more acetylated sialic acid compared to antibodies from pregnant mice.
MC: Could you kindly outline the key approaches adopted in this study?
JE: The key approach we utilized was to explore immunology through the lens of pregnancy, where the immune systems of the mother and baby are uniquely intertwined. This enhanced our ability to detect the very subtle, but incredibly important, changes that help to protect newborn babies from serious infections.
Once we showed that mothers can transfer protection to babies against a particularly nasty bacteria that was thought to avoid this type of protection, we then used genetically modified mice to confirm which part of the immune system was responsible.
This revealed to us that antibodies could perform a new job that researchers previously thought was not possible. Time, collaboration and advanced immunological and biochemical techniques then revealed that a tiny molecular change was occurring on the sugars attached to the antibodies. These studies revealed that indeed mothers do know best, even as it relates to producing the very best antibodies to help protect their babies.
MC: This study identified that the “acetylated” form of sialic acid (a sugar attached to antibodies) shifts to the “deacetylated form” in pregnancy. Is sialic acid typically in an acetylated form? Why does this shift occur in pregnancy?
JE: The amount of sialic acid that is acetylated varies between tissues. In general, though, acetylation is usually uncommon and makes up a few percent of the total sialic acid pool. Prior to our study, acetylation was not known to occur on antibody-linked sialic acid, making this one of the striking findings of our paper.
How exactly Listeria infection generates antibodies bearing acetylated sialic acid will be a topic of future study. We think that pregnant women upregulate the enzyme responsible for removing the acetylation in order to enhance the protective ability of antibodies that will be transferred to their babies. This would represent one way of improving “reproductive fitness”.
MC: You say that “The alteration of antibodies that naturally occurs during pregnancy could be replicated to change how antibodies stimulate the immune system, and to “fine tune” their effects”. Please can you expand on how this could be achieved?
JE: The scientific community and biopharmaceutical industry have become adept at producing antibodies for a variety of purposes, but increasingly for treating specific conditions, like cancer, autoimmunity and infections.
However, based on our data, there is incredible untapped potential in the ability to modulate the way these antibodies work by altering their glycosylation – the sugars that are linked to the antibodies. Our findings suggest that even a very small change, like the acetylation of the terminal sialic acid residues on antibodies, can greatly affect their functions.
MC: This research has been conducted mostly in animal models. Can you discuss how you expect it will translate to humans?
JE: You are correct that most of this work was done in animals, which allowed us to specifically manipulate the immune system and glycobiology to determine this fascinating new mechanism of protection for antibodies.
Further animal work is needed to explore the ways in which these unique antibodies are generated and how else they help protect babies. However, we did show in our paper that the key enzyme regulating the acetylation of antibodies, SIAE, is increased in both mouse and human immune cells during pregnancy. This key finding links our results in mice to human pregnancy. It will be important to confirm that antibodies in humans also possess this unique acetylated version of sialic acid and that it decreases during pregnancy.
MC: Are there any limitations to this research that you wish to highlight?
JE: For decades the field of immunology has thought that antibodies play an extremely limited role in protection against intracellular pathogens. The early studies that made this determination utilized Listeria to show that transfer of immune sera, which contains Listeria antibodies, failed to protect recipient mice, whereas the transfer of immune cells was able to protect recipients. We have turned this on its head by looking at a reproductive and developmental context where antibodies can indeed protect against this exclusive intracellular infection. Future work will determine if this approach applies to other intracellular infections, or in other contexts where it might be useful to target something inside of the cell.
Dr. John Erickson, MD, was speaking to Molly Campbell, Senior Science Writer at Technology Networks.