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Author: Donna Huber

Macrocyclic lactone anthelmintic-induced leukocyte binding to Dirofilaria immitis microfilariae: Influence of the drug resistance status of the parasite

The macrocyclic lactone anthelmintics are the only class of drug currently used to prevent heartworm disease. Their extremely high potency in vivo is not mirrored by their activity against Dirofilaria immitis larvae in vitro, leading to suggestions that they may require host immune functions to kill the parasites. We have previously shown that ivermectin stimulates the binding of canine peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) to D. immitis microfilariae (Mf). We have now extended these studies to moxidectin and examined the ability of both drugs to stimulate canine PBMC and PMN attachment to Mf from multiple strains of D. immitis, including two that are proven to be resistant to ivermectin in vivo. Both ivermectin and moxidectin significantly increased the percentage of drug-susceptible parasites with cells attached at very low concentrations (<10 nM), but much higher concentrations of ivermectin (>100 nM) were required to increase the percentage of the two resistant strains, Yazoo-2013 and Metairie-2014, with cells attached. Moxidectin increased the percentage of the two resistant strains with cells attached at lower concentrations (<10 nM) than did ivermectin. The attachment of the PBMCs and PMNs did not result in any parasite killing in vitro. These data support the biological relevance of the drug-stimulated attachment of canine leukocytes to D. immitis Mf and suggest that this phenomenon is related to the drug resistance status of the parasites.

Tessa Berrafato, Ruby Coates, Barbara J. Reaves, Daniel Kulke, Adrian J. Wolstenholme. 2019. Int J Parasitol Drugs Drug Resist.; 10:45-50. doi: 10.1016/j.ijpddr.2019.04.004.

Invited Speaker Spotlight: Marc-Jan Gubbels

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About the Speaker

Marc-Jan GubbelsMarc-Jan Gubbels received his Ph.D. at the University of Utrecht, the Netherlands, on diagnostics and vaccines for tick-transmitted Theileria and Babesia parasites of cattle. He then came to the CTEDG for a post-doc with Dr. Boris Striepen on Toxoplasma gondii. In 2005, he transitioned into an independent faculty position at Boston College and continued working on Toxoplasma. His lab is using and developing forward, reverse and functional genetic tools using enzymatic as well as fluorescent protein reporter assays in combination with cell sorting and fluorescence microscopy to learn more about the parasite’s cell biology.

Marc-Jan Gubbels’s Talk

Dr. Gubbels will give the following talk at 4:25 pm.

Of the Toxoplasma gondii Basal Complex Proteome: Cell Division, Apical Annuli and Beyond

Klemens Engelberg1, Suyog Chavan1, Tyler Bechtel2, Victoria Sánchez-Guzmán1, Allison Drozda1, Eranthie Weerapana2 and Marc-Jan Gubbels1
1Department of Biology, Boston College, Chestnut Hill, MA, 2Department of Chemistry, Boston College, Chestnut Hill, MA

Toxoplasma gondii replicates by an internal budding mechanism producing two daughter parasites per division round. Budding is driven by cortical cytoskeleton assembly and concludes with the actions of the basal complex (BC). Although the BC is reminiscent of the contractile ring in higher eukaryotes, its composition, mechanism and controls differ substantially. To deepen our insights in this unusual cytokinesis apparatus, we dissected its proteomic composition by reciprocal proximity-dependent biotinylation experiments (BioID). This identified numerous undefined proteins, several of which with critical roles in cell division, next to hints at multiple phosphorylation-based controllers. Next, we assembled a protein-protein interaction network using interaction probability predictions, which defined several sub-complexes as well as protein hubs connecting the complexes. Furthermore, temporal resolution across the budding process revealed components uniquely associated with BC initiation, its expansion and, surprisingly, its mature phase, hinting at functions beyond cell division. Serendipitously, some of the BC proteins were also present in the enigmatic apical annuli, which comprise 5-6 donut shaped structures toward the basal end of the cytoskeleton. Assessment of the annuli resolved their architecture and provided hints toward a function in internal budding, thereby highlighting an underappreciated aspect of cell division.

 

More information about the Molecular Parasitology & Vector Biology Symposium and the schedule of presentions are available on our website. The deadline to register for the symposium is April 24.

Invited Speaker Spotlight: Tiffany Weinkoff

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About the Speaker

Tiffany WeinkoffMacrophages are the major cell type infected by Leishmania parasites and the goal of my research is to define the relationship between Leishmania parasites, macrophages and the vasculature. Throughout my graduate studies and postdoctoral positions, I have dedicated my efforts toward understanding parasite-host interactions. As a graduate student at the University of Georgia carrying out research at the Centers for Disease Control and Prevention under Dr. Patrick Lammie, I was exposed to high caliber basic research in the CTEGD while simultaneously confronted with important public health issues in field settings related to neglected tropical diseases. My predoctoral research focused on the ability of monocytes to modulate lymphatic endothelial cell function and how this interaction contributed to the pathogenesis of human filarial disease. For my first postdoctoral position, I moved to the laboratory of Dr. Fabienne Tacchini-Cottier at the University of Lausanne in Switzerland. In Switzerland I gained experience using the mouse model to study the immunologic mechanisms involved in susceptibility to Leishmania infection. For my second postdoctoral position, I went to the laboratory of Phillip Scott at the University of Pennsylvania, a world-renowned professor and institution in the field of immunoparasitology. My initial project in the Scott lab, addressed the role of M2 macrophages as safe havens during Leishmania infection. For my second project in the Scott lab, I merged the knowledge and experience gained from my predoctoral research studying myeloid cells and vessels in filariasis with the technical experience of my first postdoctoral position to address the roles of innate cells in vivo in leishmaniasis. In the past months, I have transitioned to an Assistant Professor at the University of Arkansas for Medical Sciences (UAMS) where I have joined an elite group of vibrant and enthusiastic scientists with expertise in host-pathogen interactions. At UAMS, I am expanding upon previous studies examining vascular remodeling and the role of the VEGF-A/VEGFR-2 signaling pathway in lymphangiogenesis during Leishmania infection. In the future, I will work in collaboration with Dr. Camila Oliveira in Bahia, Brazil in a new project focusing on vascular remodeling during murine and human Leishmania braziliensis infection. These studies will provide important insights into our current understanding of the role of the vasculature during human disease.

Tiffany Weinkoff’s Talk

Dr. Weinkoff will give the following talk at 2:45 pm

The Role of Myeloid Cells in Vascular Remodeling during Leishmania major Infection

Tiffany Weinkopff
Department of Microbiology and Immunology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA

Our lab investigates the mechanistic basis for the pathogenesis of disease driven by Leishmania, a protozoal parasite that causes cutaneous lesions. Following infection, lesion severity is often increased by an exaggerated inflammatory response. As a result, the inflammatory response can maintain disease even after the parasite infection has been controlled. Given vascular remodeling contributes to the magnitude of many inflammatory conditions, it is hypothesized that the manipulation of factors promoting angiogenesis or lymphangiogenesis would alter lesion severity in leishmaniasis. Our findings demonstrate that murine Leishmania major infection leads to dramatic changes in vessel morphology, number and permeability. At the peak of infection, VEGF-A and VEGFR-2 expression are upregulated and VEGFR-2 blockade led to a reduction in lymphatic endothelial cell proliferation and simultaneously increased lesion size without altering the parasite burden. We showed VEGF-A/VEGFR-2 signaling promotes lymphangiogenesis to restrict tissue inflammation in leishmaniasis. Given VEGF-A/VEGFR-2 signaling contributes to lesion resolution, we are currently investigating the cellular and molecular mediators driving VEGF-A production. We found that macrophages are the predominant cell type expressing VEGF-A during L. major infection and that parasites can directly induce VEGF-A production by macrophages in vitro. Given Leishmania parasites activate HIF-1α and this transcription factor induces VEGF-A expression, we analyzed the expression of HIF-1α during infection. We showed that macrophages are the major cell type expressing HIF-1α during infection and that parasite-induced VEGF-A production is mediated by HIF activation. We are presently examining VEGF-A expression in mice deficient in HIF signaling specifically in myeloid cells. To date, we have shown that LysMCre ARNTf/f mice express less VEGF-A than LysMCre ARNTf/+ control mice following infection, and we are exploring how decreased myeloid VEGF-A production influences vascular remodeling and lesion resolution in these animals. Altogether, these studies suggest macrophage HIF-dependent VEGF-A production contributes to lymphatic remodeling during L. major infection.

 

More information about the Molecular Parasitology & Vector Biology Symposium and the schedule of presentions are available on our website. The deadline to register for the symposium is April 24.

Invited Speaker Spotlight: James Morris

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About the Speaker

James MorrisJames Morris is currently a Professor of Genetics and Biochemistry at Clemson University.  He earned his BS at The College of William and Mary in Williamsburg VA in 1990 and an M.S. in Entomology at the University of Georgia in Athens GA in 1992.  He completed his Ph.D. in Cellular Biology at the University of Georgia in 1997, characterizing the enzymology of a glycosylphosphatidylinositol phospholipase C from the African trypanosome, Trypanosoma brucei, under the supervision of Dr. Kojo Mensa-Wilmot.  Following his Ph.D., Jim moved to Baltimore MD to work in the laboratory of Dr. Paul T. Englund, where he was part of the team that developed the first RNAi-based library for forward genetics in any organism – in this case, the African trypanosome.  As part of this work, the team developed the vector pZJM (Jim is the “J”) that is still widely used for silencing genes in the parasite.

In 2003, Jim moved to Clemson University as an Assistant Professor in the Department of Genetics and Biochemistry, where he has remained to date.  His team has focused on resolving the mechanisms that protozoan parasites use to sense and metabolize the important sugar glucose during infection of their human host.  Through these studies, parasite-specific components of the sugar sensing and uptake pathway have been identified and, in an on-going collaborative effort, small molecule inhibitors of the pathways with anti-parasitic activity have been developed.  While his team has historically focused on the African trypanosome, more recent work on the malaria parasite Plasmodium falciparum and the brain-eating amoeba Naegleria fowleri suggests that exploiting the sugar metabolism pathways of these single-celled invaders may also prove useful in the development of new therapeutics.

James Morris’s Talk

Dr. Morris will give the following talk at 12:05 pm.

Pour Some Sugar on Me: Glucose, Development, Drug Discovery, and the African Trypanosome

James C. Morris
Eukaryotic Pathogens Innovation Center, Clemson University

Glucose is critical for the infectious blood stages of the African trypanosome, Trypanosoma brucei, serving as both a key metabolic agent and an important signaling molecule. While lack of the hexose is toxic to the proliferative long slender life stage of the parasite, the absence of glucose initiates differentiation in the non-dividing short stumpy (SS) form. These parasites demonstrate hallmarks of development into the next lifecycle stage, the procyclic form (PF) parasite, that include resumption of growth and expression of PF-specific antigens. Both SS differentiation and the growth of the resulting PF parasites is inhibited by glucose and non-metabolizable glucose analogs, with the latter observation suggesting a potential receptor-mediated mechanism for perception of the sugar. The importance of the hexose to the parasite for both metabolic a developmental needs suggests that glucose uptake or distribution inhibitors would be potentially useful anti-parasitic compounds. To identify small molecule inhibitors of glucose acquisition, we developed parasites that endogenously express FRET-based protein glucose sensors in the cytosol or glycosomes. Using these cells, we have completed a 25,000-compound pilot screen and have identified inhibitors with useful medicinal chemistry properties that have potential as a new line of lead compounds against the parasite.

 

More information about the Molecular Parasitology & Vector Biology Symposium and the schedule of presentions are available on our website. The deadline to register for the symposium is April 24.

Invited Speaker Spotlight: Matthew Collins

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About the Speaker

Matt CollinsMatthew Collins obtained his Ph.D. studying under mentor Dr. Rick Tarleton at the University of Georgia; his thesis project focused on the role of CD8+ T cells in controlling chronic T. cruzi in nonlymphoid tissue. During Infectious Diseases fellowship at the University of North Carolina, he worked with Dr. Aravinda de Silva to study specificity and cross-reactivity of human antibody responses to flaviviruses such as dengue and Zika virus. He is now an Assistant Professor at Emory University where he leads a translational arbovirology research program. Goals of the group are to understand basic aspects of human adaptive immune responses to sequential related viral infection and to develop and implement serologic tools to support epidemiologic studies and the development of vaccines and diagnostic tests.

Matt Collins’s Talk

Dr. Collins will present the following talk at 9:55 am.

Epitope Targets of the Human Antibody Response to Zika Virus Infection

Matthew H. Collins1,2, Huy A. Tu3,4, Ciara Gimblet-Ochieng5, Guei-Jiun Alice Liou5, Ramesh S. Jadi5, Stefan W. Metz5, Ashlie Thomas5, Benjamin D. McElvany4, Edgar Davidson6, Benjamin J. Doranz6, Yaoska Reyes7, Natalie M. Bowman2, Sylvia Becker-Dreps8, Filemón Bucardo7, Helen M. Lazear5, Sean A. Diehl3,4, Aravinda M. de Silva5
1Department of Medicine, Emory University 2Department of Medicine, University of North Carolina-Chapel Hill 3Cellular, Molecular, and Biomedical Sciences Program, University of Vermont 4Department of Microbiology and Molecular Genetics, University of Vermont 5Department of Microbiology and Immunology, University of North Carolina 6Integral Molecular, Inc. 7Department of Microbiology, National Autonomous University of Nicaragua 8Departments of Family Medicine and Epidemiology, University of North Carolina-Chapel Hill

Zika virus (ZIKV) transmission became a global public health emergency after the recent epidemic in Latin America and beyond revealed rare but dire manifestations of infection such as severe birth defects and Guillain-Barré syndrome. The emergence of ZIKV in areas where other related flaviviruses such as dengue are endemic creates challenges in accurately diagnosing infections, conducting reliable surveillance, as well as in understanding the distinguishing aspects of the host immune response to ZIKV due to antibody (Ab) cross-reactivity. Because vaccines represent a key strategy for prevention of infectious diseases and typically rely on robust antibody responses, we sought to analyze the durable antibody responses in individuals infected by ZIKV as a first flavivirus infection. We observed complex populations of antibodies that bind to epitopes on intact virions, simpler epitopes on envelope protein monomers as well as envelope subdomains. Moreover, strong neutralizing antibody responses that minimally cross-react with dengue viruses were consistently detected. To better understand the molecular determinants of the neutralizing antibody response to ZIKV and to develop tools that could aid vaccine development, we isolated two potently neutralizing monoclonal antibodies (mAbs) from one primary ZIKV case and mapped key amino acid residues involved in mAb binding and neutralization by multiple complimentary methods including generation of neutralization escape mutants and alanine scanning mutagenesis. The mAbs recognize different epitopes centered on domain I and domain II of the viral envelope protein. Functionally, both mAbs were protective in a lethal mouse model of ZIKV infection. Ongoing work is examining the prevalence of these specific Ab responses at the population level. This work provides new knowledge and tools that may be useful as diagnostic reagents or as therapeutics and will advance vaccine development.

 

More information about the Molecular Parasitology & Vector Biology Symposium and the schedule of presentions are available on our website. The deadline to register for the symposium is April 24.

Database offers tool for global health collaborations

 

As the big data revolution continues to evolve, access to data that cut across many disciplines becomes increasingly valuable. In the field of public health, one barrier to sharing data is the need for users to fully comprehend complex methodological details and data variables in order to properly conduct analyses.

The Clinical Epidemiology Database, ClinEpiDB.org, aims to address these barriers by not only providing access to huge volumes of data, but also providing tools to help interpret complex global epidemiologic research studies. The development of ClinEpiDB has been led by the University of Georgia’s Institute of Bioinformatics, University of Pennsylvania’s School of Arts and Sciences and its Perelman School of Medicine, and the University of Liverpool’s Institute of Integrative Biology.

On March 7, ClinEpiDB released data, methodology and documentation from “The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development” (MAL-ED) study. The MAL-ED study represents a nearly decade-long research collaboration between the Foundation for the National Institutes of Health (FNIH), Fogarty International Center, and an international network of investigators.

The MAL-ED study was designed to help identify environmental exposures early in a child’s life that are associated with shortfalls in physical growth, cognitive development, and immunity. The study characterizes gut function biomarkers on the causal pathway from environmental exposure to growth and development deficits and assesses diversity across geographic locations with respect to exposures and child health and development. The MAL-ED consortium has published a significant library of peer-reviewed publications and ClinEpiDB now makes the MAL-ED data highly visible and accessible in new and exciting ways.

“It is great to see how investments and effort directed at data being Findable, Accessible, Interoperable and Reusable—i.e., F.A.I.R—are beginning to bear fruit,” said Jessica Kissinger, UGA Distinguished Research Professor of Genetics and co-principal investigator on the Bill & Melinda Gates Foundation award that funded the ClinEPi Development. “Too many important studies are buried in the scientific or medical literature and not easily accessible or reusable in moving the frontier in the important battles related to infectious disease and human health. This multi-institutional, multiple-funder, interdisciplinary approach is working.”

ClinEpiDB is also home to the Global Enteric Multicenter Study (GEMS) which contains data from more than 22,000 children from seven sites in South Asia and Africa and was the largest-ever study to investigate the causes to moderate-to-severe diarrheal illness in children in lower- to middle-income countries. The most recent ClinEpiDB release also contains data from GEMS1A, a continuation of the GEMS study that broadened its scope to include less-severe diarrheal episodes. The addition of MAL-ED adds to the growing resource of high-quality maternal and child global health data.

“Over 10 years, our international network of investigators collaborated through MAL-ED to better understand the complicated relationships among intestinal infections, nutrition and other environmental exposures on child development,” said Michael Gottlieb, FNIH deputy director of science (retired) and lead PI for the MAL-ED study. “The MAL-ED Network generated a high-quality data set, possibly the largest of its kind, on various research areas from cognitive abilities to gut function to immunological response. We are pleased to make this dataset available through ClinEpiDB so it can be used by researchers far into the future to increase scientific understanding, test new research hypotheses and design and implement better intervention strategies to reduce childhood morbidity and mortality.”

MAL-ED sites (located in Iquitos, Peru; Fortaleza, Brazil; Haydom, Tanzania; Limpopo, South Africa; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Vellore, India; Dhaka, Bangladesh) allowed for comparisons to be made among and between children living in geographically and culturally diverse urban and rural environments and in countries at different levels of economic development.

MAL-ED data in ClinEpiDB account for over 1.3 million observations covering anthropometrics, nutrition, vaccination status, diarrheal and respiratory disease episodes and countless other details collected by community field workers in 2009-2014. The current release includes longitudinal data from children followed two times a week for the first 24 months of life.

Future data releases will contain data for some children up to 5 years of age. ClinEpiDB allows users to walk through these data easily via an intuitive interface, enabling point-and-click filtering, simple queries and more complex “search strategies.”

See https://youtu.be/535PcFrBH8M for a video introduction to this resource. ClinEpiDB will continue to grow and provide increased access to malaria and maternal and child health global datasets thus facilitating epidemiologic research in an open data environment while protecting patient identity.

Multiple Praziquantel Treatments of Schistosoma mansoni Egg-Negative, CCA-Positive Schoolchildren in a Very Low Endemic Setting in Egypt Do Not Consistently Alter CCA Results

Forty-four Schistosoma mansoni egg-negative/circulating cathodic antigen (CCA) low-positive (trace or 1+) children in three districts of very low prevalence in Egypt were given three sequential praziquantel (PZQ) treatments. Stool and urine specimens were collected 3 months following the initial treatment, and 3 weeks following the second and following the third PZQ treatments, which were conducted 5 weeks apart. Stool specimens were examined by Kato-Katz (four slides/stool sample) and all S. mansoni egg-negative stools were further tested by the “miracidia hatching test” (MHT). Urine samples were examined by the point-of-care CCA assay (POC-CCA). Over the study period, all stool samples from study subjects remained S. mansoni egg negative and MHT negative. Of the POC-CCA test results, in the first day of the study 3 months following the initial treatment, 29.5% were negative, 61.4% CCA trace positives, and 9.1% CCA 1+ positives. Following each PZQ treatment, the test results fluctuated between 1+, trace, and negative, but did not consistently decrease. The proportions of POC-CCA-positive results obtained in the first day (70.5%) as compared with the last day of the study (72.7%) in all of the three districts were very similar. We conclude that CCA trace and 1+ readings, in Kato-Katz S. mansoni egg-negative children in this area with very low levels of intestinal schistosomiasis, are not consistently altered or rendered consistently negative following repeated PZQ treatments and are therefore likely to represent false-positive readings. This finding is of critical importance for countries such as Egypt as they approach elimination.

Ayat A. Haggag, Miriam Casacuberta Partal, Amal Rabiee, Khaled M. Abd Elaziz, Carl H. Campbell Jr., Daniel G. Colley and Reda M. R. Ramzy. 2019. Am J Trop Med Hyg. doi: 10.4269/ajtmh.18-0961

Order Your CTEGD Shirts by April 23

shirts

Now is your chance to get a short sleeve shirt, long sleeve shirt, and/or a sweatshirt with CTEGD’s logo. Orders must be placed by April 23. If you are attending the symposium or would like to pick up your shirt at UGA, then choose “Pickup at Shirtworks” at checkout. We will pick up the shirts for distribution at the Molecular Parasitology & Vector Biology Symposium on May 1. Any shirts not picked up at the symposium will be available in the CTEGD main office in Coverdell Center.

If you are outside of UGA and not attending the symposium, you can have your order shipped directly to you.

Your purchase of a shirt supports the activities of CTEGD. Place your order TODAY: http://bit.ly/2V9wqNn

Field Testing Integrated Interventions for Schistosomiasis Elimination in the People’s Republic of China: Outcomes of a Multifactorial Cluster-Randomized Controlled Trial

Despite significant progress, China faces the challenge of re-emerging schistosomiasis transmission in currently controlled areas due, in part, to the presence of a range of animal reservoirs, notably water buffalo and cattle, which can harbor Schistosoma japonicuminfections. Environmental, ecological and social-demographic changes in China, shown to affect the distribution of oncomelanid snails, can also impact future schistosomiasis transmission. In light of their importance in the S. japonicum, lifecycle, vaccination has been proposed as a means to reduce the excretion of egg from cattle and buffalo, thereby interrupting transmission from these reservoir hosts to snails. A DNA-based vaccine (SjCTPI) our team developed showed encouraging efficacy against S. japonicum in Chinese water buffaloes. Here we report the results of a double-blind cluster randomized trial aimed at determining the impact of a combination of the SjCTPI bovine vaccine (given as a prime-boost regimen), human mass chemotherapy and snail control on the transmission of S. japonicum in 12 selected administrative villages around the Dongting Lake in Hunan province. The trial confirmed human praziquantel treatment is an effective intervention at the population level. Further, mollusciciding had an indirect ~50% efficacy in reducing human infection rates. Serology showed that the SjCTPI vaccine produced an effective antibody response in vaccinated bovines, resulting in a negative correlation with bovine egg counts observed at all post-vaccination time points. Despite these encouraging outcomes, the effect of the vaccine in preventing human infection was inconclusive. This was likely due to activities undertaken by the China National Schistosomiasis Control Program, notably the treatment, sacrifice or removal of bovines from trial villages, over which we had no control; as a result, the trial design was compromised, reducing power and contaminating outcome measures. This highlights the difficulties in undertaking field trials of this nature and magnitude, particularly over a long period, and emphasizes the importance of mathematical modeling in predicting the potential impact of control intervention measures. A transmission blocking vaccine targeting bovines for the prevention of S. japonicum with the required protective efficacy would be invaluable in tandem with other preventive intervention measures if the goal of eliminating schistosomiasis from China is to become a reality.

Gail M. Williams, Yue-Sheng Li, Darren J. Gray, Zheng-Yuan Zhao, Donald A. Harn, Lisa M. Shollenberger, Sheng-Ming Li, Xinglin Yu, Zeng Feng, Jia-Gang Guo, Jie Zhou, Yu-Lan Dong, Yuan Li, Biao Guo, Patrick Driguez, Marina Harvie, Hong You, Allen G. Ross and Donald P. McManus. Front Immunol. 2019 Apr 3;10:645. doi: 10.3389/fimmu.2019.006452019.