B CELL RESPONSES TO HUMAN FLAVIVIRUS VACCINATION AND SARS-COV-2 INFECTION

B CELL RESPONSES TO HUMAN FLAVIVIRUS VACCINATION AND
SARS-COV-2 INFECTION
Tyler Sandberg
Stockholm 2022
B cell responses to human flavivirus vaccination and SARS-CoV-2 infection
THESIS FOR DOCTORAL DEGREE (Ph.D.)
By
Tyler Sandberg
The thesis will be defended in public at Karolinska Institutet ANA-Futura, Alfred Nobels Allè 8, 18th of March, 2022 at 9:30.
Principal Supervisor: Professor Hans-Gustaf Ljunggren Karolinska Institutet Department of Medicine, Huddinge Center for Infectious Medicine Co-supervisor(s): Kim Blom, PhD Karolinska Institutet Department of Medicine, Huddinge Center for Infectious Medicine Professor Karin Loré Karolinska Institutet Department of Medicine, Solna Division of Immunology & Allergy
Opponent: Professor Tomas Bergström, MD-PhD University of Gothenburg Institute of Biomedicine Department of Infectious Diseases Examination Board: Associate Professor Guro Gafvelin Karolinska Institutet Department of Clinical Neuroscience Therapeutic Immune Design Unit Associate Professor Anna Lundgren University of Gothenburg Institute of Biomedicine Department of Microbiology & Immunology Associate Professor Anna Överby Umeå University Department of Clinical Microbiology Section of Virology
This thesis is dedicated to my family. I wouldn’t have made it here without you.
POPULAR SCIENCE SUMMARY OF THE THESIS No other medical intervention has had such a significant impact on human health as that of vaccines. Vaccination has become a topic of interest since the debut of the COVID-19 pandemic. Vaccines are designed to teach the immune system to recognize critical parts of a pathogen so that if you were ever exposed to it in real life, your immune cells and antibodies could prevent infection or severe disease. There are several different vaccine platforms used today, the most common using weakened live viruses or inactivated viruses in vaccine formulations. During the COVID-19 pandemic, mRNA vaccine technology was approved for the first time in humans, saving an estimated 500,000 lives already.
One of the most effective and successful vaccines in history is the yellow fever virus (YFV) vaccine. First developed by Max Theiler, who later won the Nobel Prize in Medicine for his discovery, the vaccine uses a weakened strain of the virus that, upon administration, leads to a mild infection. The immune events following vaccination lead to sustained antibody titers, memory T cells that can kill virus-infected cells and memory B cells that can quickly produce antibodies if infected with the virus. The YFV vaccine has long been considered a model vaccine as it provides lifelong immunity from a single dose. Scientists have long used the vaccine to learn more about viral infections and the immune responses following vaccination.
In this thesis, we used the YFV vaccine to understand how human B cells respond to vaccination. In Paper I, we found that the YFV vaccine elicits a strong B cell response during the first two weeks after vaccination. The vaccine also leads to the development of protective levels of antibodies, and we were able to identify virus-specific memory B cells circulating in the donor samples by the end of the study. Similarly in Paper II, B cell responses to other vaccines including the Tick-borne encephalitis (TBEV) vaccine and Japanese encephalitis (JEV) vaccine were also studied. An important question we wanted to investigate in Paper II was how human B cell responses would be affected if the YFV vaccine was given at the same time as either TBEV or JEV vaccines. We found that most healthy donors in the studies developed antibodies against the respective viruses after vaccination with no differences in donors receiving two vaccines compared with donors receiving only one of the vaccines.
At the beginning of the COVID-19 pandemic, our focus shifted towards studying human immune responses to SARS-CoV-2 infection in hospitalized patients. In Papers III and IV, we found that COVID-19 patients had strong early B cell and T cell responses with high levels of antibody responses. These early immune events led to detectable virus-specific memory B cells and memory T cells in addition to antibodies at 5- and 9-months after infection, likely contributing to protection from reinfection or severe disease.
The findings in this thesis contribute to our understanding of how, specifically, B cells respond to vaccination and infection. This knowledge can be used to help design new vaccines and vaccination strategies or even improve upon current vaccines to prevent severe human infection and disease.
ABSTRACT Viral infections pose a major threat to global heath. As specific antiviral treatments are lacking against many human viruses, vaccination is the most effective medical intervention to prevent severe disease and death. Delineating the immune events following viral vaccination and infection can help in the design of new vaccines and therapeutics. The aims of this thesis were to characterize human B cell responses following yellow fever virus (YFV) vaccination (Paper I), following concomitant vaccination with YFV and the Tick-borne encephalitis virus (TBEV) vaccine or Japanese encephalitis virus (JEV) vaccine (Paper II), and during acute and convalescent SARS-CoV-2 infection (Papers III and IV).
In Paper I, healthy volunteers were vaccinated with the YFV vaccine and blood samples were taken at up to five time points afterwards to characterize the magnitude, kinetics, and specificity of the humoral immune response. Activation in the Th1-polarized circulating T follicular helper cell population was observed 7 days following vaccination, coinciding with increased germinal center activity as measured by serum CXCL13 levels. Peak YFV-E specific plasmablast expansion was observed at day 14 following vaccination. Additionally, the frequencies of IgG+ plasmablasts at day 14 correlated with day 90 neutralizing antibody (nAb) titer magnitude, suggesting that plasmablasts may be used as an early marker indicating later protective immunity. YFV-E specific memory B cells were also detectable at day 28 and 90 as well as protective titers of nAbs. These findings provide insights into immune events that lead to the development of B cell immunity following YFV vaccination.
In Paper II, we investigated the feasibility and effectiveness of concomitant vaccination with YFV vaccines and either TBEV or JEV vaccines. 145 healthy volunteers were recruited into a prospective open label, non-randomized clinical trial and received either YFV, TBEV, or JEV vaccines only or YFV vaccine with either TBEV or JEV vaccines. Blood and serum samples were taken at baseline and up to ten timepoint following vaccination. The development of virus-specific nAbs was not affected by concomitant vaccination when comparing the vaccine cohorts. Importantly adverse events were mild and not affected by concomitant vaccination suggesting that the vaccination strategy should be considered effective and safe.
In Papers III and IV, early immune events and ultimately immune memory was investigated in COVID-19 patients during and after hospitalization. Increased germinal center activity with a Th1-polarized circulating T follicular helper cell activation was observed that coincided with SARS-CoV-2-specific expanded antibody secreting cells during acute COVID-19. The majority of patients also had detectable levels of nAbs during acute disease. In Paper IV, SARS-CoV-2-specific nAb titers persisted in patients at 5- and 9-months post infection as well as virus-specific polyfunctional memory T cells and memory B cells regardless of COVID-19 severity during hospitalization.
Together, the findings in this thesis contribute to our understanding of humoral responses to different types of flavivirus vaccines as well as infection with SARS-CoV-2.
LIST OF SCIENTIFIC PAPERS
I. Sandberg JT, Ols S, Löfling M, Varnait R, Lindgren G, Nilsson O, Rombo L, Kalén M, Loré K, Blom K, and Ljunggren HG. Activation and Kinetics of Circulating T Follicular Helper Cells, Specific Plasmablast Response and Development of Neutralizing Antibodies Following Yellow Fever Virus Vaccination. Journal of Immunology, 2021, 207 (4), pp. 1033-1043.
II. Sandberg JT, Löfling M, Varnait R, Emgård J, Al-Tawil N, Lindquist L, Gredmark-Russ S, Klingström J, Loré K, Blom K, and Ljunggren HG. Safety and immunological responses of concomitant vaccination with flavivirus vaccines: results from an open label non-randomized clinical trial. Manuscript.
III. Varnait R, Garcia M, Glans H*, Maleki KT*, Sandberg JT*, Tynell J, Christ W, Lagerqvist N, Asgeirsson H, Ljunggren HG, Ahlén G, Frelin L, Sällberg M, Blom K, Klingström J, and Gredmark-Russ S. Expansion of SARS-CoV- 2–Specific Antibody-Secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients. Journal of Immunology, 2020, 205 (9), pp. 2437-2446. *Authors contributed equally.
IV. Sandberg JT*, Varnait R*, Christ W, Chen P, Muvva JR, Maleki KT, Garcia M, Dzidic M, Folkesson E, Skagerberg M, Ahlén G, Frelin L, Sällberg M, Eriksson LI, Rooyackers O, Sönnerborg A, Buggert M, Björkström NK, Aleman S, Strålin K, Klingström J, Ljunggren HG, Blom K, Gredmark-Russ S, and The Karolinska COVID-19 Study Group. SARS-CoV-2-specific humoral and cellular immunity persists through 9 months irrespective of COVID-19 severity at hospitalization. Clinical & Translational Immunology, 2021, 10: e1306. *Joint first authors.
SCIENTIFIC PAPERS NOT INCLUDED IN THE THESIS
SI. Blom K, Sandberg JT, Loré K, and Ljunggren HG. Prospects for induction of CD8 T cell-mediated immunity to Zika virus infection by yellow fever virus vaccination. Journal of Internal Medicine. 2017, 282 (3) pp. 206-208.
SII. Blom K, Cuapio A, Sandberg JT, Varnaite R, Michaëlsson J, Björkström NK, Sandberg JK, Klingström J, Lindquist L, Gredmark Russ S, Ljunggren HG. Cell- Mediated Immune Responses and Immunopathogenesis of Human Tick-Borne Encephalitis Virus-Infection. Frontiers in Immunology. 2018, 9:2174.
SIII. Cornillet M, Strunz B, Rooyackers O, Ponzetta A, Chen P, Muvva JR, Akber M,
Buggert M, Chambers BJ, Dzidic M, Filipovic I, Gorin J-B, Gredmark-Russ S, Hertwig L, Klingström J, Kokkinou E, Kvedaraite E, Lourda M, Mjösberg J, Maucourant C, Norrby-Teglund A, Parrot T, Perez-Potti A, Rivera-Ballesteros O, Sandberg JK, Sandberg JT, Sekine T, Svensson M, Varnaite R, , Eriksson LI, Aleman S, Strålin K, Ljunggren H-G and Björkström NK (2021), COVID-19 specific metabolic imprint yields insights into multi organ-system perturbations. European Journal of Immunology. https://doi.org/10.1002/eji.202149626
CONTENTS 1 INTRODUCTION ......................................................................................................... 3
1.1 Adaptive immunity ............................................................................................... 3 1.1.1 B cells ....................................................................................................... 3 1.1.2 CD4+ T cells ............................................................................................. 4
1.2 Flaviviruses & flavivirus vaccines ....................................................................... 5 1.2.1 YFV .......................................................................................................... 6 1.2.2 TBEV ....................................................................................................... 6 1.2.3 JEV ........................................................................................................... 6 1.2.4 Flavivirus vaccines ................................................................................... 6
1.3 SARS-CoV-2 ...................................................................................................... 10 1.3.1 Clinical manifestation ............................................................................ 10
2 RESEARCH AIMS ..................................................................................................... 11 3 MATERIALS AND METHODS ................................................................................ 13
3.1 Ethical considerations ........................................................................................ 13 3.2 Sample collection and processing ...................................................................... 14 3.3 Flow cytometry .................................................................................................. 14 3.4 Serological analyses ........................................................................................... 14
3.4.1 ELISA .................................................................................................... 15 3.4.2 Neutralization tests ................................................................................. 15
3.5 FluoroSpot Assays ............................................................................................. 16 3.5.1 B cell FluoroSpot ................................................................................... 16 3.5.2 T cell FluoroSpot ................................................................................... 17
3.6 Analysis of soluble markers ............................................................................... 18 3.6.1 ELISA .................................................................................................... 18 3.6.2 Multiplex immunoassay ......................................................................... 18 3.6.3 Proteomic analysis – Olink immune response panel ............................. 19 3.6.4 Viral load – real-time PCR .................................................................... 19
3.7 Statistics .............................................................................................................. 19 4 RESULTS & DISCUSSION ....................................................................................... 21
4.1 B cell immune responses following flavivirus vaccination ............................... 21 4.1.1 B cell and T cell activation .................................................................... 21 4.1.2 YFV replication and soluble factors ...................................................... 22 4.1.3 Germinal center assessment ................................................................... 23 4.1.4 Plasmablast expansion ........................................................................... 24 4.1.5 Serological responses ............................................................................. 26 4.1.6 Safety assessment ................................................................................... 27
4.2 B cell immune responses to SARS-CoV-2 infection ........................................ 28 4.2.1 B cell and T cell activation during acute COVID-19 ............................ 28 4.2.2 Germinal center assessment during acute COVID-19 .......................... 29 4.2.3 Plasmablast expansion during acute COVID-19 ................................... 30 4.2.4 Serological outcomes during and following SARS-CoV-2
infection .................................................................................................. 32 4.2.5 Persistence of immunological memory ................................................. 33
5 CONCLUSIONS ......................................................................................................... 37 6 FUTURE PERSPECTIVEs ......................................................................................... 39 7 ACKNOWLEDGEMENTS ........................................................................................ 41 8 REFERENCES ............................................................................................................ 45
LIST OF ABBREVIATIONS
C-X-C chemokine receptor
enzyme-linked immunoassay
envelope protein
major histocompatibility complex class II
neutralizing antibodies
nucleocapsid protein
severe acute respiratory syndrome coronavirus 2
spike subunit 1
somatic hyper mutation
3
1.1 ADAPTIVE IMMUNITY
As a result of the COVID-19 pandemic, vaccines, the immune system, antibodies, B cells, and T cells have become common topics of discussion in our everyday lives. The mechanisms behind how we develop immune memory following vaccination or infection are complex and still being delineated today. The human immune system can be broadly divided into two parts, the innate and the adaptive immune systems. The innate immune system acts as the first responder following infection or vaccination. Innate immune cells start inflammatory responses, take up antigen, and bring them to the draining lymph nodes thereby acting as a bridge to adaptive immune responses. The adaptive immune system consists of antigen-specific responses from B cells and T cells that arise in the days or weeks following antigen exposure1. A hallmark feature of the adaptive immune system is the development of immunological memory so that future exposure to the pathogen leads to a more robust and rapid response, contributing towards preventing severe disease.
1.1.1 B cells
Following infection or vaccination, antigen-specific B cells will, ideally, become activated and differentiate into antibody secreting cells (ASCs) producing neutralizing antibodies (nAbs) or memory B cells. The process starts when antigen makes its way to the draining lymph nodes where B cells capable of binding the specific antigen via their surface B cell receptor (surface IgD/IgM) undergo a quick activation (Figure 1). In an extrafollicular reaction, B cells rapidly differentiate into ASCs such as plasmablasts that produce large amounts of antibodies within the first weeks following vaccination or infection. Plasmablasts, which are short lived ASCs, have been shown to play a role in clearing active infections and typically show up in circulation one to two weeks following infection or vaccination secreting large amounts of pathogen- specific antibodies2–5. A follicular reaction is often initiated when activated cognate CD4+ helper T cells provide costimulatory signals to B cells to enter or initiate the germinal center reaction6,7. B cells upregulate CXCR5 to migrate towards higher concentrations of CXCL13 produced by follicular dendritic cells (FDC) and T follicular helper (Tfh) cells in the B cell follicle8,9. During the germinal center reaction, activated B cells undergo rapid proliferation and somatic hypermutations (SHM) in the dark zone to generate higher affinity B cell receptors. With new mutations in the BCR, B cells exit to the light zone to test the BCR against antigen carried by FDCs. Tfh cells provide signaling to B cells with advantages mutations to return to the dark zone for more SHM or further differentiation. High affinity B cells are ultimately chosen for further differentiation into memory B cells or ASCs like plasmablasts or long-lived plasma cells6. Plasma cells migrate to the bone marrow where they produce antibodies and memory B cells patrol secondary lymphoid organs for their specific antigen10. Memory B cells that meet their antigen quickly differentiate into ASCs or even better memory B cells with
4
higher affinity BCRs3,11. Together long-lived plasma cells and memory B cells are key players in providing protective immunity from pathogens.
1.1.2 CD4+ T cells
The two main subsets of T cells carry out very different functions during and after vaccination or infection. CD4+ T cells are commonly referred to as helper T cells and play a critical role in coordinating adaptive immune responses. Their main function is to help other immune cells carry out their effector functions through cytokine signaling12,13. Their distinct helper functions are used in their classification as well including different T helper cells (Th1, Th2, Th17, and Th22 cells), T regulator cells and Tfh cells14. As their name suggests, Tfh cells reside in follicles of secondary lymphoid organs. Here they play an essential role in establishing germinal center reactions. Vaccine- or infection-activated Tfh cells express the chemokine receptor CXCR5 and downregulate the lymph node homing receptor CCR7, directing them into B cell follicles with higher concentrations of CXCL13 produced by FDCs15,16. There, Tfh cells also secrete high levels of CXCL13 aiding in the organization of germinal centers17. Activated B cells that have undergone SMH in the germinal center migrate to these CXCL13 rich areas. Here, Tfh cells provide co-stimulation to activated cognate B cells via CD40-CD40-L interaction and the production of IL-21. These signals drive B cell proliferation and is critical both in the selection of high-affinity B cells and support memory B cell development6,18,19. Increases in peripheral
Figure 1 | Germinal center reaction. In response to antigen reaching secondary lymph organs, antigen-specific B cells become activated. Dendritic cells (DCs) process and present antigen on MHC-II molecules to activate the cells that in turn signal cognate B cells to enter germinal center (GC) reactions or differentiae into plasmablasts. B cells entering the dark zone of the GC undergo mass proliferation and SHM honing the B cell receptor. B cells migrate towards the light zone to test the new B cell receptor and present to cognate germinal center Tfh cells. Advantageous mutations are selected and signals from Tfh cells direct the B cell to differentiate into memory B cells, plasmablasts, or long-lived plasma cells.
5
CXCL13 levels have been shown to coincide with lymph node germinal center activity16. This has suggested that serum or plasma CXCL13 levels can act as a biomarker indicating the activation of germinal centers and thereby, adaptive immune system activation following vaccination or infection16. Deeper insights into human germinal center reactions and Tfh cell responses has been hampered due to the invasiveness of extracting human lymph nodes. More recently, however, a subset of CD4+ T cells in the periphery have been identified that share phenotypic and functional characteristics of germinal center Tfh cells that are believed to correspond with their germinal center counterparts20. These circulating Tfh (cTfh) cells have been shown to correlate with later neutralizing antibody titers and play an important role in developing immune memory21,22. The expression of CXCR3 on cTfh cells has also revealed that they can polarize towards Th1 functional cells following vaccination with HPV and influenza vaccines making them a cell of interest when delineating immune responses following infection or vaccination23,24.
1.2 FLAVIVIRUSES & FLAVIVIRUS VACCINES
Arboviral infections continue to pose a global health threat affecting nearly every continent. The virus family Flaviviridae is a large contributor to this threat with human pathogens such as yellow fever virus (YFV), Tick-borne encephalitis virus (TBEV), Zika virus, Japanese encephalitis virus (JEV), Dengue virus, and West Nile virus. The reach of flaviviruses are also increasing with recent outbreaks of Zika and YFV in South America, TBEV across Europe and northern Asia and JEV spreading in SE Asia and even reaching Australia25–28 (Figure 2). No specific antiviral treatment exists against these viruses, so vaccination is the main strategy used to control the spread and preventing severe infection. Fortunately, we do have vaccines against several of these viruses, three of which are used in the current thesis; YFV, TBEV, and JEV vaccines. The YFV vaccine uses a live attenuated strain of YFV that, after a single dose, provides lifelong immunity. TBEV and JEV vaccines, on the other hand are whole, inactivated
Figure 2 | Global distribution of YFV, TBEV and JEV. Map adapted from the Center for Disease Control and the World Health Organization epidemiological maps. Yellow denotes YFV endemic areas, blue denotes TBEV endemic areas, and red denotes JEV endemic areas.
6
virus vaccines using Alum as adjuvant and require multiple booster doses to provide and maintain immunity. The differences in immune responses and effectiveness from vaccination have inspired scientists to further unravel the complex mechanisms contributing to the development of immunity against these flavivirus infections. The following sections aim to provide an overview of the current understanding of humoral and B cell responses induced by flavivirus vaccines included in the thesis.
1.2.1 YFV
YFV infection causes over 200,000 infections and 30,000 deaths annually. The YFV is transmitted to human through bites from infected Aedes aegypti mosquitos leading to varying symptoms depending on the severity of the infection29,30. Following a 3-6-day incubation period, flu-like symptoms arise including fever, headache, and joint pain typically lasting between three and four days before subsiding. In roughly 15% of cases, symptoms can worsen and include nausea, vomiting, renal failure, jaundice, and hemorrhaging leading to a 50% mortality rate within 10-14 days.31
1.2.2 TBEV
An estimated 13,000 TBEV infections occur annually transmitted by bites from infected Ixodes ticks and even through ingestion…