Nanoimmunotherapy: new outlook on allogeneic organ transplantation

One of the main challenges faced in allogeneic organ transplantation is the rejection of the donor tissue by the recipient’s immune system. Using a mixture of immunosuppressive drugs, the acceptance of organ transplantation is highly favorable for a short period of time (Gardiner et al. 2016). However, long term graft survival rates are low due to response from the immune system initiated by innate immune cells which triggers allograft rejection (Liu et al. 2012). While it is known that innate immune cells mediate allograft reject, the specific means by which this is achieve is not fully understood. Learning more about this pathway and the molecular mechanisms involved can lead to the development of specific immunotherapy that can promote long term allograft survival.

A recently published article by Braza et al. in Immunity explored the molecular pathways leading to graft rejection and explored therapeutic approaches that could potentially lead to long term allograft survival. To conduct their experiments, the researchers developed a nanoimmunotherapy strategy which allowed them to deliver proteins to highly targeted regions of the body, in this case, localized to the allograft. Using an experimental transplantation mouse model, they demonstrated that allograft rejection follows a macrophage activation pathway. Additionally, through downregulation of this pathway, by mTOR manipulation and inhibition of co-stimulatory signals, they were able to promote allograft survival indefinitely.

To elucidate the role of macrophage mediated allograft rejection, they performed heart transplant between two genetically different strains of mice. They focused on proteins that might be involved in promoting inflammation and found that vimentin and HMGB1, both agonists to dectin-1 and TLR4, were upregulated in the donor allograft following transplantation. Since macrophages express dectin-1 and TLR4, an increase in TNFα and IL-6 production indicated that vimentin and HMGB1 were able to train macrophages that infiltrated the graft. They then developed a nanoimmunotherapic approach that targets and inhibits the mTOR [referred to as mTORi-HDL in their research] signaling pathway. This pathway is linked to the production of cytokines through trained immunity (Netea et al. 2016). This targeted therapy demonstrated reduced cytokine production during the training period. Interestingly, one area where the mTORi-HDL accumulated was in the bone marrow, where it could potentially facilitates the development of prolonged therapeutic effects by association with myeloid cells and their progenitors.

mTORi-HDL localize to the liver, spleen, kidney, and bone marrow, and
is preferentially taken up by myeloid cells. Uptake of mTORi-HDL
by T and B cells is very limited.

In the allogeneic heart transplant mouse model, mTORi-HDL was preferentially taken up by macrophages compared to other myeloid cells; mTORi-HDL uptake in T cells were poor, suggesting a preference for myeloid cells. Treatment with mTORi-HDL following heart transplantation resulted in lower numbers of macrophages, neutrophils, and DC present in the allograft, blood, and spleen. Additionally, macrophages isolated from heart allografts treated with mTORi-HDL displayed significantly lower production of TNFα and IL-6. So far, Braza et al. have demonstrated that allograft rejection is triggered by the upregulation of proteins such as vimentin and HMGB1 which trains infiltrating macrophages. The attenuation of macrophages can be achieved by using nanoimmunotherapy, mTORi-HDL. This therapy targets myeloid cells, mainly macrophages, and lead to lower number of these cells present in the graft following transplantation. Lastly, to tie it all together, they investigated whether mTORi-HDL nanoimmunotherapy can promote organ transplant acceptance.

To assess the function of infiltrating macrophage in allografts they looked at two different subsets of macrophages. While the mTORi-HDL treatment decreased the number of reactive macrophages, it promoted the number of regulatory macrophages in the allografts. Grafts were rejected when regulatory macrophage population was depleted, suggesting that they play a key functional role in organ transplant acceptance. In fact, the application of mTORi-HDL therapy after heart transplant significantly increased the graft survival after five days. They extended their methods further to create another nanoimmunotherapy that inhibited CD40 – an essential costimulatory molecule required for T cell activation. Remarkably, when the two nanoimmunotherapy were applied together – mTOR inhibition and DC40 inhibition – they led to graft survival past 100 days post heart transplantation without showing any signs of toxicity or off-target side effects. In their model, allograft tolerance is therefore achieved by preventing reactive macrophage production of TNFα and IL-6, and promote regulatory macrophage which leads to CD4⁺ Treg expansion and CD8⁺ T cell inhibition.

Mice with both therapies showed a higher percentage of grafts surviving
past 100 days after transplantation compared to the mice that received one therapy.

This study is important because it demonstrate a significant breakthrough in allograft survival. Braza et al. showed that allogeneic heart transplant in a mouse model can survive, without immune rejection, by using targeted immunotherapies that inhibits mTOR and CD40 signaling. This form of therapy does not rely on global suppression of the immune system, which could lead to infections, cancer and metabolic toxicity (Naesens et al. 2009). Rather, nanoimmunotherapy targets specific cells in a specific region, leading to graft acceptance and survival. Nanoimmunotherapy seem to be a viable option that can be used to promote long term survival of allogeneic organ transplant. While this is promising, further research needs to be conducted to examine how these results translate to humans. Perhaps conducting the same experiments in primates could give us better insights on how this might work in humans. Further research also needs to be conducted on the long term survival of the graft. The research stopped at 100 days post transplantation, however, it would also be interesting to examine the graft at 200 or 500 days after the transplantation. Nonetheless, the results presented in this paper is promising and opens the door to the development of targeted immunotherapy that can be used as a treatment for allogeneic organ transplant.

References:
Braza, Mounia S., et al. "Inhibiting inflammation with Myeloid cell-specific nanobiologics promotes organ transplant acceptance." Immunity 49.5 (2018): 819-828.
Gardiner, Kyle M., et al. "Multinational evaluation of mycophenolic acid, tacrolimus, cyclosporin, and everolimus utilization." Annals of Transplantation 21 (2016): 1-11. 

Liu, Wentao, et al. "Innate NK cells and macrophages recognize and reject allogeneic nonself in vivo via different mechanisms." The Journal of Immunology (2012): 1102997.

Naesens, Maarten, Dirk RJ Kuypers, and Minnie Sarwal. "Calcineurin inhibitor nephrotoxicity." Clinical Journal of the American Society of Nephrology 4.2 (2009): 481-508.
Netea, Mihai G., et al. "Trained immunity: a program of innate immune memory in health and disease." Science 352.6284 (2016): aaf1098.

Lassa Virus Activates Myeloid DCs but Takes Away Their Power

Based on paper published in PLOS Pathogens by a team at the Unit of Biology of Emerging Viral Infections

Lassa virus (LASV) causes lassa fever (LF), which is regularly found in West Africa, with an estimated 300,000 to 500,000 cases and 3,000 to 5,000 deaths every year. The World Health Organization believes that LASV is one of several pathogens likely to cause severe outbreaks in the future since there are no approved vaccines and very limited knowledge on how the disease develops. Recent evidence has shown that dendritic cells (DCs) are an important target of LASV. A key feature of myeloid DCs (a subset of dendritic cells) is that they take in foreign substances and present their proteins on the surface in order to activate T cells (a type of immune cell that determines the specificity of an immune response to foreign substances in the body). Furthermore, mDCs can also produce IFN-1 (an antiviral signaling molecule). Research on primates infected with LASV indicated that an early IFN-1 response along with T cells exposed to the virus contributed to increased survival. This suggested to the authors that mDCs could play a key role during LASV infection.

To further investigate the role of mDCs during LASV infection, the authors began by examining whether LASV and MOPV (a type of virus that is similar to LASV but does not cause disease) activate mDCs. They infected human mDCs with either MOPV or LASV and then used RT-PCR (a technique measures how much a gene is expressed by detecting RNA levels) and flow cytometry (a technique that measures the characteristics of cell components of a solution) to measure the amount of IFN-1 and identify any mDC activation proteins present. The mDCs infected with LASV produced similar levels of IFN-1 to those of MOPV-infected mDCs while similar levels of expression were also seen for several mDCs activation proteins. This result suggested that both MOPV and LASV activate mDCs. The authors also tracked the number of virus particles in mDCs using a Luminex assay (a test that uses color-coded beads with a specific antibody that binds to the molecule of interest followed by the addition of a second antibody that fluoresces). They observed a progressive decrease in the number of virus particles which suggested that the virus was not replicating.

After demonstrating that MOPC and LASV activate mDCs but don’t replicate, the authors sought to investigate the relationship between the IFN-1 response and the lack of viral replication. Schaeffer et al (2018) inhibited the effects of the IFN-1 response by treating mDCs with antibodies (highly specific protein that binds to and is produced in response to another specific protein) that target the receptor protein which IFN-1 binds to and infecting them with LASV or MOPV that expressed mCherry (a fluorescent protein that is inserted into a gene). The authors detected mCherry in the cell after LASV and MOPV infection which indicated that the virus did replicate when IFN-1 was not functional. They further confirmed this by examining the number of virus particles and found that the virus levels increases four days post infection. Collectively, these results suggest that the IFN-1 response inhibits the infection of MOPV or LASV by preventing virus replication.

After demonstrating that both LASV and MOPV activate mDCs, the authors’ next step was to determine whether the mDC response since a major role of mDCs in the body is to activate T cells. To investigate this, T cells were combined with infected or non-infected mDCs and RT-PCR and flow cytometry were used to determine the levels of IFN-1 and activation proteins present on active mDCs. The authors observed that mDCs infected with MOPV produced IFN-1 and expressed the activation proteins in the presence of T cells whereas the LASV-infected did not express the activation proteins or produce IFN-1. These results suggested that there are interactions between T cells and mDCs that can alter the defense response to MOPV and LASV.

After examining the mDCs response, the authors looked at the T cell response to determine if T cells are activated following MOPV or LASV infection. They used flow cytometry to identify the presence of CD69 (an activation marker expressed on activated T cells) on T cells that were exposed to infected or uninfected mDCs. Schaeffer et al (2018) noted that T cells exposed to MOPV-infected DCs expressed the CD69 marker while T cells exposed to LASV-infected mDCs did not. In addition, they found that T cells which were exposed to MOPV-infected DCs had higher levels of perforin and granzyme B (toxic chemicals produced by T cells that kills cells infected by viruses). Together, these results indicate that MOPV-infected DCs activate T cells while LASV-infected mDCs do not.

After demonstrating that LASV-infected mDCs suppress the immune response by not activating T cells, the authors’ next step was to determine what role viral proteins play. They tackled this by exchanging the viral proteins in MOPV and LASV with the corresponding proteins present in the other virus then tested the IFN-1 response. The most striking result was that the MOPV which contained LASV NP (a complex that consists of a nucleic acid bonded to a protein) behaved exactly like normal LASV which suggests that the NP in LASV is responsible for suppressing T cell activation.

The authors showed that both MOPV and LASV can activate mDCs but only MOPV-infected DCs can activate T cells. In addition, they demonstrated that the major immunosuppressive properties of LASV are carried by the NP. Future directions could involve an investigation into the mechanisms behind the crosstalk between T cells and mDCs.

References

Schaeffer, J., Carnec, X., Reynard, S., Mateo, M., Picard, C., Pietrosemoli, N., ... & Baize, S. (2018). Lassa virus activates myeloid dendritic cells but suppresses their ability to stimulate T cells. PLoS pathogens, 14(11), e1007430.

Ogbu, O., Ajuluchukwu, E., & Uneke, C. J. (2007). Lassa fever in West African sub-region: an overview. Journal of vector borne diseases, 44(1), 1.

Collin, M., McGovern, N., & Haniffa, M. (2013). Human dendritic cell subsets. Immunology, 140(1), 22-30.

When "DC" and "Mutations" Don't Connote Superheroes: How Dendritic Cells Lacking B-Arrestin 2 May Be to Blame for Autoimmune Diseases

The immune system deserves credit. For all the times we forget to wash our hands or ignore the five-second rule, our immune system is there to bail us out, never expecting an ounce of appreciation. For all the times bacteria and viruses tried to get ahead in the evolutionary arms race, the immune system continues responds with brilliant advancements in their processes. For all the magic that our immune systems performs on a minute-by-minute basis, however, there is a chink in the proverbial armor of the immune system: autoimmunity. Autoimmune diseases occur  when the body’s natural line of defense identifies a naturally-occurring substance as a threat, whether that be a beneficial bacteria that lives in our intestine or even our own tissues, and proceeds to mount an immune response.

The seriousness of autoimmunity begs the question of how it comes to rise in the first place. Our bodies have evolved to develop an arsenal of lymphocytes, or cells that regulate the immune system, that are specific to specific types of pathogen that we could encounter, whether that be bacteria, virus or fungi (Rosenblum et al., 2015). T-cells are a type of lymphocyte that can either have a killer function against these antigens, or in the case that the object of encounter isn’t dangerous, a tolerance to them. Ultimately, the fundamental basis of autoimmune disease is the presence of T-cells that mistakenly react to our own tissues as they would with pathogens and perform their effector function of killing (Smith et al., 1999).

As one may expect, when autoimmunity arises, the immune system will perform its usual function as if it were fighting a foreign invader. One of the key players in the immune system’s pathogen sensing are DCs. Although they shouldn't be confused with the comic book universe, DCs, or dendritic cells, do play a superhero function in the context of our immune system. They patrol the outskirts of our body (such as the skin, mucous and intestines) on the hunt for anything out-of-the-ordinary and process them in such a way that the immune system will recognize them as something that needs to be taken care of. DCs are truly the bridge between our innate immunity, which regulates the broad, immediate responses we mount against invading bacteria and viruses, and our adaptive immunity, which may take longer to initiate but is more specific to whatever pathogen we are fighting.

Figurably, DC function is also crucial within the context of autoimmunity. In non-autoimmune conditions, DCs can present non-harmful antigen, or processed pieces of a bigger protein, to a T cell to make T cells tolerant to that protein (Ganguly et al., 2013). After DCs process an antigen, they migrate to the nearest T-cell “base” at a nearby lymph node, and present that antigen to them while releasing releasing pro-inflammatory chemicals called cytokines (Ganguly et al., 2013). Ultimately, many of the symptoms of autoimmunity diseases are the same as within normal diseases, such as chronic inflammation to tissues which could prove lethal (Janeway et al., 2001).

As dendritic cells can be powerful positive regulators of autoimmunity, they are also the target of immunotherapies against autoimmune diseases. Therapies often target regulators of DC function to dampen this pro-autoimmune effect. One of these regulators is a group of proteins called arrestins, which regulate survival and motility in pro-inflammatory cells like macrophages. In a recent study published in the Journal of Immunology, researchers identified a specific arrestin, B-arrestin 2, as a regulator of DC migration. Their research aim was to learn about the protein to determine how it affected autoimmunity, and their results were very telling.

First, the researchers found that B-Arrestin 2 is in some way incorporated with limiting the mobility of dendritic cells. They were able to prove this by finding that mouse bone marrow dendritic cells with mutated B-Arrestin proteins showed both more molecular markers signifying DC maturation as well as allowed for more DC migration to draining LNs. They even saw a physiological result in mice as a result of the mutated DCs in the shape of ear swelling!  Keeping in mind that one of the ways to prevent inflammation from autoimmunity is to tamper down the migration of DCs to the lymph nodes, the fact that mutant B-Arrestin allows for increased motility suggests its important regulation.

Not only does nullifying the function of B-Arrestin 2 allow for increased maturation rates and motility of DCs, but it also causes the release of chemicals that encourage T cells to facilitate B-cells to activate against a given antigen. The researchers also went on to identify molecules that interact with B-Arrestin 2 and even chart the pathway that B-Arrestin 2-deficient DCs migrate to the lymph nodes to activate T-cells. Not only does having deficient B-Arrestin 2 prevent its own function, but there are an additional 2500 genes that were expressed differently in the mutated DCs in mice!

Perhaps the most functionally important finding was the fact that mice with DC cells lacking B-Arrestin 2 had worse cases of EAE, or Experimental autoimmune encephalomyelitis. For mice with the mutation, there was a significantly higher clinical score (marking severity of the disease), more infiltration of cells into the spinal cord and demyelination which harms nerve tissue. Another autoimmune disease called SLE (lupus) shows that B-Arrestin 2 deficiency in DCs causes a buildup of different subtypes of DCs in the lymph nodes, a dangerous place for build-up given their ability to prime T-cells against self-tissue for disease developments. Images below show (A) the increased clinical severity, (C/E) increased T-cell accumulation of B-Arrestin 2-deficient mice. The bar chart shows deficient mice in black and the functional mice in white, a clear discrepancy in the amount of cells infiltrating the spinal cord.

The plethora of ways that these authors were able to see the effects of B-arrestin 2-deficiency in DCs in mice open up a world of opportunities in the fight against autoimmune diseases. When the arrestin proteins are knocked out, migration and maturation of DCs allow for T-cells that usually perform inflammatory and killing functions against pathogens actually do so against self-tissue. While it remains to be seen if human dendritic cells behave in similar ways that mouse DCs do, given that mice have proven to be a successful model organisms is promising for the development of immunotherapies. Potential therapy can perhaps restore proper function of arrestin and arrestin-related proteins in DCs, or even introduce DCs with high expression of arrestin into inflammatory sites in autoimmune patients.

Given how impeccable our immune system usually responds, it makes sense that in the rare cases that something goes awry, it has drastic effects. The autoimmunity treatment industry is estimated to be $45.54B by 2022 (PR News Wire) and is one of the top 10 leading causes of death in female children and women due to its sex-hormonal dependence (American Autoimmune Association\). Especially when it comes to key players like dendritic cells, the identification of proteins that could potentially limit symptomology in autoimmunity could have wide-reaching advantages for patients and families affected everywhere.

Works cited:

1. American AutoImmune Association. https://www.aarda.org/news-information/statistics/.
2. Ganguly D, Haak S, Sisirak V, Reizis B. The role of dendritic cells in autoimmunity. Nat Rev Immunol. 2013;13(8):566-77.
3. Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001. Autoimmune responses are directed against self antigens. Available from: https://www.ncbi.nlm.nih.gov/books/NBK27155/
4. Rosenblum MD, Remedios KA, Abbas AK. Mechanisms of human autoimmunity. J Clin Invest. 2015;125(6):2228-33.
5. Yingying Cai, Cuixia Yang, Xiaohan Yu, Jie Qian, Min Dai, Yan Wang, Chaoyan Qin, Weiming Lai, ShuaiChen, Tingting Wang, Jinfeng Zhou, Ningjia Ma, Yue Zhang, Ru Zhang, Nan Shen, Xin Xie, Changsheng Du. Deficiency of β-Arrestin 2 in Dendritic Cells Contributes to Autoimmune Diseases. The Journal of Immunology December 12, 2018, ji1800261; DOI: 10.4049/jimmunol.1800261
6. Smith, D A and D R Germolec. “Introduction to immunology and autoimmunity” Environmental health perspectives vol. 107 Suppl 5,Suppl 5 (1999): 661-5.
7. Wang L, Wang F‐S, Gershwin ME (Research Center for Biological Therapy, the Institute of Translational Hepatology, Beijing 302 Hospital, Beijing, China; and Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, Davis, CA, USA). Human autoimmune diseases: a comprehensive update. (Review).J Intern Med 2015; 278: 369–395.

Could Fetal Immune Reactions Spark Preterm Labor?

Original Paper: Alloreactive fetal T cells promote uterine contractility in preterm labor via TFN-α and IFN-γ

While pregnancy is often characterized as a period of excitement and anticipation, it also marks a time of uncertainty regarding the well-being of the baby. A standard, healthy pregnancy lasts about 40 weeks. However, for some women, labor begins prematurely, before the 37th week of pregnancy. Every year, about 15 million babies are born pre-term, and birth complications are the leading cause of death for children under the age of 5 [1]. Children who survive often face a lifetime of disability and impairment.Though the specific causes of preterm labor are unknown, researchers have found that infection and inflammation are amongst the most common causes of spontaneous preterm labor (PTL) [2]. Considering this, Frascoli et al. seek to understand if an inflammatory environment in mothers with PTL causes an increased activation of fetal immune cells. Typically, research on the maternal-fetal interface has focused on maternal cells tolerating the fetus. By focusing more on fetal cells, Frascoli et al. changed this: what if fetal immune cells are capable of recognizing and targeting maternal cells?

First, we must consider how the mother's womb allows the fetus to develop there for 9 months. Think about it... half of the fetus' genes are from the father, so half of the fetus is essentially foreign to the mother's body. Since this is the case, how does the mother's body know the fetus is not foreign, thereby preventing her immune system from targeting it? Basically, during pregnancy, the fetus is protected from an immune attack through a bunch of complex interactions at the fetal-maternal interface, as shown below: 

Figure 1. (Source: https://www.researchgate.net/figure/Multiple-mechanisms-underlie-maternal-tolerance-of-the-fetus-Mothers-via-changes-that_fig1_7879978)

In the current study conducted by the University of California, San Francisco, Frascoli et al. considered 89 patients with healthy pregnancies and 70 patients with spontaneous PTL. Considering the link between infection/inflammation and PTL, they first examined cytokines in both maternal and fetal cells. Cytokines are important to consider because they are often proinflammatory, which means that when activated, these small signaling molecules promote inflammation. The researchers found that preterm infants exhibit significantly elevated amounts of proinflammatory cytokines as compared to infants born at full-term. Interestingly, no significant differences in cytokine levels between patients with and without PTL. Many of the elevated cytokines are produced by activated dendritic cells- this is important because activated DCs are what interact with T and B cells to mount a sophisticated immune response. Because of this, Farscoli et al. considered whether fetal DCs are activated during PTL. They ultimately found higher levels of activated dendritic cells as well as higher levels of memory T cells in PTL infants as compared to full-term infants. This could indicate that the fetal immune system plays a role in inducing PTL.

Farscoli et al. also found that premature infant cord blood exhibited a higher prevalence of microchimerism. "Microchimerism" basically refers to the phenomena that one can carry a number of cells that originate from another individual (and are therefore genetically distinct from the cells of a host individual) (Figure 2). So in this case, premature infants possessed a higher level of maternal cells than did full-term infants. This led the researchers to hypothesize that higher levels of microchimerism result in the priming of fetal T cells to specifically target maternal antigens. That is, higher levels of microchimerism could prime fetal immune cells to mount an immune response against substances specific to the mother. 

Figure 2. (Source: https://www.omicsonline.org/emerging-questions-in-materno-fetal-microchimerism-2161-038X.S1-002.php?aid=2537) 

To test whether fetal T cells are specifically activated against maternal antigen, Farscoli et al. examined how maternal and fetal T cells interacted with each other as well as with a control. They found significant increases in fetal T cells against maternal antigens in PTL pregnancies as compared to full-term pregnancies. These increases were specific for maternal antigens, as they were not observed when preterm fetal T cells were stimulated with antigens from third-party controls. so, this supports the hypothesis that fetal immune cells could elicit an immune response specifically against maternal antigens. 

Lastly, Farscoli et al. utilized a mouse model to determine the effect of inflammatory cytokines (particularly TFN-α and IFN-γ) on fetal survival. Injection of these activated T cells resulted in increased fetal resorption, whereas transfer of non-activated T cells did not result in increased resorption. Also, increases in resorption appear dependent on TFN-α and IFN-γ, as the transfer of T cells from mice deficient in either cytokine failed to cause resorption. These results are significant in pinpointing TFN-α and IFN-γ as central cytokines in disrupting maternal-fetal tolerance.

Considering these results, Farscoli et al. sought to determine whether the hyperactive immune components of premature infants have a causal relationship with preterm birth. The researchers co-cultured T cells of a premature infant with maternal cells from the smooth muscle of the uterus. The results were striking: T-cells from premature children induced contraction of uterine cells whereas full-term infant T-cells exhibited no effect on uterine contraction. The results are shown in the figure below, where the fetal PTL was the only condition that caused contractions- the full-term fetal and conditions that lacked either TFN-α or IFN-γ did not cause contractions. Thus, TFN-α and IFN-γ must play a key role in inducing uterine contractions during PTL. 

Figure 3

From all of this, researchers concluded that preterm labor could occur when the mother has an infection, which would stimulate inflammatory cytokines like TFN-α and IFN-γ. This maternal immune response could unintentionally induce an immune response in the fetus, causing the fetus to release higher levels of inflammatory cytokines and activatory DCs, eventually leading to uterine contractions. Now that this pathway has been elucidated, the researchers can work towards developing new PTL treatment strategies that manipulate different aspects of this cascade.


References
[1] https://www.who.int/news-room/fact-sheets/detail/preterm-birth
[2] R. Romero, J. Espinoza, L. F. Gonçalves, J. P. Kusanovic, L. Friel, S. Hassan, The role of inflammation and infection in preterm birth. Semin. Reprod. Med. 25, 021–039 (2007). 

Developing a New Brand of Antibodies to Fight a Widespread Fungal Infection

In reference to a new publication in Nature Communications authored by researchers at the University of Aberdeen and the University of Edinburgh.

When you think of the deadliest illnesses in the world, diseases like HIV, malaria, and cancer jump to mind. Strangely, a top contender with all of these conditions is fungal infection. Clocking in at 1.5 million deaths per year, fungi are some of the most dangerous infectious agents on the planet. The most common of these is Candida albicans (C. albicans), the fungus behind common vaginal yeast infections and oral thrush. In people with weakened immune systems (such as HIV patients), these common infections can quickly progress to be life-threatening.

Candidiasis is generally only diagnosed after a blood test, so the infection often isn’t noticed until symptoms have already developed. To prevent infections from progressing that far in the first place, scientists have been working on building vaccines to fungal pathogens. However, these efforts are relatively new and none have been made available to the public as of yet (although one is in Phase II clinical trials).  A new publication in Nature Communications describes a method for generating a fully human-based, highly specific C. albicans antibody which can prepare the immune system to fight off the fungus before an infection ever occurs.

Before we can understand these findings, we need to understand how B cells and antibodies work. B cells are a part of the adaptive immune system which creates long-lasting immune memory against secondary pathogen infection. This system is what stops the same bug from making you sick twice, and also explains why vaccinating with a small piece of a pathogen preps your immune system to face the real thing. When the immune system is seeing a new type of pathogen, T cells (the other major cell in the adaptive immune system) become activated and bind to B cells. This “T cell help” tells the B cells that there is an infection and causes the B cells to multiply. Some of these new B cells will be plasma cells, which quickly secrete lots of antibodies to fight the infection. Other new B cells become memory B cells, which will remain in the body for years in case the same pathogen infects the body again. If so, the memory B cells are quickly activated and the immune response is much faster and more effective the second time around.

The antibodies themselves are receptor proteins made by B cells. They are highly variable in structure and, therefore, in their ability to bind different antigens. When binding an antigen, antibodies can perform a number of different functions to aid the pathogen’s destruction. Their binding can neutralize the pathogen by physical blocking them from breaking into host cells (Lu et al. 2018). They can recruit other immune cell types (such as natural killer cells) to the infected cells to kill them before the pathogen can spread (Lu et al. 2018). As seen in this study, they can also recruit large cells called macrophages to engulf and digest (AKA phagocytose) the antibody-bound infected cells in a process known as opsonization.

Because they are so specific and so good at binding to their antigen of choice, building antibodies to recognize antigens associated with infectious pathogens is a popular field of research at the moment. This Nature Communications article details a new way to make antibodies against a C. albicans antigen (the protein Hyr1, a component of the fungal cell wall).  The process begins by taking memory B cells from the blood of patients who have experienced a Candida infection in the past year. Some of these cells were involved in the response to the Candida infection and produced anti-Candida antibodies. So, the B cells were tested against multiple Candida antigens and the ones whose antibodies bound an antigen were selected. The chosen cells were broken open and had their DNA extracted, specifically the genes which encode for the antibody proteins (called the VH and VL genes). Using a DNA amplification process called RT-PCR, several copies of the VH and VL genes were generated. The genes were then inserted into new cells which multiplied and expressed the VH and VL genes, thus creating the antibodies. Finally, the researchers purified and extracted these antibodies so they could test them in cell cultures and in live mice.

These new antibodies are exceptional because they are monoclonal and fully-human. So rather than transferring whole blood serum (containing lots and lots of different antibodies) from a past patient into a healthy person, the researchers specifically cloned the one antibody that they were looking for. This approach is much more specific than a whole serum transfer, which may cause some sort of graft rejection. This is also an issue if antibodies were originally taken from another animal such as mice, hence why the “fully-human” aspect of the procedure is vitally important.

Just as antibodies tend to do, the anti-Hyr1 antibodies binds its target Hyr1 very specifically. After attaching the antibodies to a fluorescent tag, the researchers could visualize under a microscope the antibodies specifically binding to the cell wall of C. albicans. When tested against mammalian antigens, the anti-Hyr1 antibodies did not show signs of cross reactivity. This is great news for vaccine development, since binding to mammalian antigens could be a sign that the antibody would cause an autoimmune reaction in humans. Even better, the researchers raised other antibodies against a wide host of C. albicans cell wall antigens (anti-whole cell antibodies) which are able to bind other Candida species as well.

Now comfortable that their more expansive anti-whole cell antibodies were specific at targeting only the pathogenic fungus, the researchers added them to cultures of mouse cells and exposed them to  C. albicans infection. Compared to controls, the antibody-injected cultures showed increased phagocytosis of fungal particles by macrophages. Blocking the antibody-binding proteins on macrophages reversed this effect, indicating that it was indeed the antibodies which were stimulating the increased macrophage activity. In live mouse models, the anti-Hyr1 antibodies didn’t increase resistance to fungal infection. However, using the anti-whole cell showed significantly reduced disease symptoms and a smaller presence of fungal cells in the mouse tissues.

As fungal vaccines are in dramatically short supply given the damage they do across the world, this study could provide a strong basis for developments in this emerging field. Not only are the authors addressing a massively understated medical issue, but they are doing it through a widely applicable method for harvesting new recombinant antibodies. This procedure could be valuable in any number of immunological studies, especially those studying vaccine development.

Reference

Lu, L. L., Suscovich, T. J., Fortune, S. M., & Alter, G. (2018). Beyond binding: antibody effector functions in infectious diseases. Nature Reviews Immunology, 18(1), 46.