Thesis
Reference
Immune responses to wild-type or vaccine influenzavirus in solid organ transplant recipients
L'HUILLIER, Arnaud
Abstract
Solid organ transplantation (SOT) has become a commonly performed procedure for patients with end-organ disease. Many SOT patients' way of life is associated to the same community exposure to influenza as the general population. However, mostly because of the lifelong immunosuppressive regimen, SOT patients are at increased risk for influenza-associated morbidity and mortality. This is further complicated by the reduced vaccine immunogenicity among SOT patients, making the only efficient preventive strategy suboptimal. In this context, there is a need to thoroughly evaluate immune responses to natural influenza infection and influenza immunization in SOT patients. This will improve clinical management by detecting patients most at-risk for severe influenza, but also optimize prevention by improving vaccine immunogenicity. The line of research described in this work reflects an ongoing attempt to better understand the humoral and cellular immune responses against influenza natural infection and immunization in SOT patients, which immunological and clinical factors may be associated with protection against (re)infection and [...]
L'HUILLIER, Arnaud. Immune responses to wild-type or vaccine influenzavirus in solid organ transplant recipients. Thèse de privat-docent : Univ. Genève, 2020
DOI : 10.13097/archive-ouverte/unige:146780
Available at:
http://archive-ouverte.unige.ch/unige:146780
Disclaimer: layout of this document may differ from the published version.
Clinical Medicine Section Department of Pediatrics, Gynecology and Obstetrics
IMMUNE RESPONSES TO
WILD-TYPE OR VACCINE INFLUENZAVIRUS IN SOLID ORGAN TRANSPLANT RECIPIENTS
Thesis submitted to the Faculty of Medicine of the University of Geneva
for the degree of Privat-Docent by
Arnaud G. L’HUILLIER
Geneva 2020
TABLE OF CONTENTS
A. SUMMARY 3
B. INTRODUCTION 4
1) Influenza virus 4
Classification 4
Structure 4
Viral cycle 5
Antigenic drift and shift 5
Epidemiology 6
Influenza vaccine 6
2) Transplantation 7
Introduction 7
Transplantation and infectious diseases burden 7 Transplantation and immune responses to infections and vaccines 9
3) The interplay between influenza and immune responses in SOT 9 Epidemiology and clinical presentation of influenza in SOT 9
Influenza immunization after SOT 10
Adaptive immune responses to influenza infection and immunization 10 Vaccine immunogenicity in the SOT population 13
C. Study 1 : Summary 15
D. Study 2 : Summary 16
E. Study 3 : Summary 17
F. Study 4 : Summary 18
G. CONCLUSION 19
H. REFERENCES 26
A. SUMMARY
Over the last decades, solid organ transplantation (SOT) has become a commonly performed procedure for patients – when possible - with end-organ disease. Because of improving quality of life and survival, many SOT patients’ way of life is associated to the same community exposure to microorganisms as the general population, such as exposure to influenza. However, mostly because of the lifelong immunosuppressive regimen required to avoid graft rejection, sometimes associated with other chronic comorbidities, SOT patients are at increased risk for influenza-associated morbidity and mortality. This is further complicated by the reduced vaccine immunogenicity among SOT patients, mostly related to their immunosuppressive regimens, making the only efficient preventive strategy suboptimal. In this context, there is a need to thoroughly evaluate immune response to natural influenza infection and influenza immunization in SOT patients, and to better understand the interactions between the virus and the host immune system. This will improve clinical management by detecting patients most at-risk for severe influenza, but also optimize prevention by improving vaccine immunogenicity. The line of research described in this work reflects an ongoing attempt to better understand the humoral and cellular immune responses against influenza natural infection and immunization in SOT patients, which immunological and clinical factors may be associated with protection against (re)infection and whether some biomarkers of disease severity can be identified.
B. INTRODUCTION
Solid organ transplantation (SOT) is an increasingly performed procedure for patients with end-organ failure. Mostly because of the lifelong immunosuppressive regimen required to avoid graft rejection, SOT patients are at increased risk for severe infections, including common infections acquired in the community, such as influenza, one of the most frequent cause of respiratory infections worldwide1. Influenza is responsible for significant morbidity and mortality2, especially among high-risk groups such as SOT patients3,4. Therefore, a better understanding of the interactions between the virus and the immune system of the SOT patient is necessary to improve influenza prevention and management strategies, which will be covered in this work.
1) Influenza virus Classification
Influenzaviruses are negative-sense single-stranded RNA viruses that belong to the Orthomyxoviridae family5. Two different genus (type) of influenza epidemiologically relevant in the human population: influenza A and influenza B6. Influenza A subtypes are designated based on their 17 different hemagglutinin (H1 to H17) and ten different neuraminidase (N1 to 10) proteins, with influenza A/H1N1 and influenza A/H3N2 being the most common circulating subtypes since the 1970s7. On the other hand, influenza B viruses are classified into two lineages (Yamagata-like and Victoria-like) based on the hemagglutinin (HA) glycoprotein. Both lineages have been co-circulating during most influenza seasons for the last four decades8.
Structure
Influenzaviruses have an envelope derived from infected human cells with spikes consisting of HA and neuraminidase (NA) proteins. Influenza A and B viruses both possess eight RNA segments: PB1, PB2 and PA (all coding for the RNA-dependent RNA polymerase), HA, NP (coding for the nucleoprotein), NA, M (coding for the matrix protein and the ion channel) and NS1 (coding for the non-structural proteins)9.
Viral cycle
Influenzaviruses infect human cells through binding of their HA to the sialic acid molecules present of the surface of host cells10. Upon attachment, the virus is then endocytosed and the fusion of the viral envelope with the endosomal membrane releases the nucleocapsid in the cytoplasm. The nucleocapsid is then transported to the nucleus where the negative- stranded viral RNA (vRNA) is copied into both positive-stranded messenger RNA (mRNA) and complementary RNA (cRNA). mRNAs return to the cytoplasm for translation into proteins whereas the cRNA serves as a template to generate more copies of the vRNA in the form a nucleocapsid. Nucleocapsids are then exported to the cytoplasms where the budding of the membrane containing HA and NA proteins allows for the generation of complete virions1. At this point, the NA protein is responsible for the cleavage of the sialic acid molecules, allowing for the release of complete virions.
Antigenic drift and shift
Continuous genetic changes in the viral genome have a major impact on influenza immunity and the design of seasonal influenza vaccine. Antigenic drifts are changes related to point mutations and recombinations, which lead to the emergence of new variants7 responsible for seasonal epidemics and the need for yearly modifications in the influenza vaccine. Drifts happen in both influenza types, even though less rapidly in influenza B11. Antigenic shifts are major genetic changes happening only in case of reassortment between two different influenza A subtypes. Although rare, antigenic shifts can be the cause of pandemic if there if there is no pre-existing immunity in the population and efficient human-to-human transmission6. The latest influenza pandemic happened in 2009 when a novel influenza A(H1N1) that originated from pigs and was antigenically distinct from previously circulating H1N1 viruses started circulating worldwide12. Since 2009, the so-called pandemic H1N1 strain has remained dominant over other influenza A/H1N1 strains2.
Epidemiology
Influenza has a typical seasonal circulation, with epidemics happening yearly during the winter in the Northern hemisphere13. Outbreaks typically begin abruptly and peak after a few weeks, with a total duration of two to three months13. Every year, influenza A/H1/N1, A/H3/N2, B/Yamagata and B/Victoria lineages co-circulate6,7. Just in the U.S., the burden of influenza for the 2018-2019 season has been estimated to be more than 531’000 admissions and more than 36’400 deaths2. In terms of costs, it has been estimated that each year, the influenza-associated healthcare costs vary between two to six billions US$
just in the U.S.14. Influenza-related morbidity and mortality is overrepresented among patients with chronic comorbidties3 and immunosuppression is a well-defined risk factor for influenza-related morbidity and mortality15.
Influenza vaccine
There are two types of influenza vaccines. The intranasal live attenuated influenza vaccine (LAIV) and the inactivated injectable vaccine (IIV). Influenza vaccines are either trivalent (containing one H1N1, one H3N2 and one B strain) or quadrivalent (containing one H1N1, one H3N2 and two B strains). Twice yearly, the World Health Organization (WHO) reviews surveillance and clinical data to provides recommendations about the strains that should be included in the vaccine for the upcoming season16. Because the strains included in the vaccine are selected months before the onset of influenza season, mismatches between vaccine and circulating strains are a major determinant of vaccine effectiveness (VE). As a consequence, VE against influenza types and subtypes vary significantly, but overall VE ranges around 50%17.
In the U.S., yearly influenza immunization is recommended for all persons ≥ 6 months as long as there is no contraindication18. In Switzerland, yearly influenza immunization is recommended for persons with increased risk of complications, such as persons ≥ 65 years old, pregnant women, premature infants, patients with chronic comorbidities (including end-organ disease and organ transplantation) as well as those in regular contact with persons at increased risk of complications19. It can also be proposed to all persons ≥ 6 months who wish to receive it.
2) Transplantation Introduction
The first successful human-to-human SOT was described in 1955 and consisted of a kidney transplantation between two identical twins20. For genetically different persons, irradiation was initially used to reduce the recipient’s immune response against the graft without frank success, until the use of oral steroids and azathioprine in the mid-sixties revolutionized the field by drastically improving patient survival21. Since then, kidney transplantation became a routine procedure for patients with end-organ failure. Another major breakthrough in transplantation happened with the use of calcineurin inhibitors such as ciclosporin in the eighties, allowing for a focused immunosuppression targeting T- cells21. Consequently, other transplantation programs such as heart and liver transplant – which were initially limited to a very few centers - also started to become routine procedures21. Currently, SOT is the preferred choice in many end-organ failure situations.
For example, kidney transplantation has been associated with better long term outcomes when compared to dialysis22. Consequently, SOT is increasingly proposed to improve patients’ quality of life and survival, with more than 36’000 transplantations performed in 2018 in the U.S.23. Among organ transplant, kidney are by far the most frequently SOT performed, followed by liver23. Survival rates at one year are excellent, reaching 90% or more for most organs24. On the other hand, organ shortages and death while on the transplant list are significant, with currently more than 100’000 SOT candidates on the waitlist in the U.S.24.
Transplantation and infectious diseases burden
A well-balanced immunosuppressive regimen is the key factor for the overall success of SOT. The main causes of post-transplant morbidity and mortality are directly related to the amount of immunosuppression: too little immunosuppression increases the risk of graft rejection whereas too much immunosuppression increases the risk of infections25,26. The infectious risk is mainly determined by two factors: epidemiologic exposures and the net state of immunosuppression26. Epidemiologic exposures encompasses exposure to persistent infections (such as herpesviruses and hepatitis viruses), travel-related exposures (such as S. stercoralis or T. Cruzi), community exposures (such as respiratory
viral infections, including influenza) and nosocomial exposures27. The net state of immunosuppression is primarily determined by the choice, dosage and duration of immunosuppressive therapy26. The standard immunosuppressive regimen is a two- or three-drug regimen based of oral glucocorticoids, antinucleotides (such as mycophenolate or azathioprine) and immunophilin binding agents (such as cyclosporin or tacrolimus)28,29. Glucocorticoids are broad immunosuppressants inhibiting mostly innate and cell-mediated immunity whereas antinucleotides interfere with DNA synthesis, reducing lymphocyte replication. On the other hand, immunophilin binding agents inhibit the production of cytokines by CD4+ T-cells, especially IL-2.28,29. Alongside the immunosuppressive regiment, other factors contribute to the net state of immunosuppression, such as underlying immunodeficiencies, previous chemotherapies or antimicrobial therapies, integrity of mucocutaneous barriers, post-surgical complications, cytopenia, metabolic comorbidities and infections with immunomodulatory viruses such as cytomegalovirus (CMV) also play a role26.
Clinical manifestations and sites of infection following SOT depend on several factors such as the type of transplant, the time since SOT, the net state of immunosuppression and the infecting microorganism26,27. The transplanted organ is an important factor in the type of infection patients can encounter. Indeed, the site of transplant is one of the most common sites of infection30. In terms of risk of infection, the first six months following SOT is widely accepted as the most at-risk period, which is mainly related to a higher net state of immunosuppression but also to frequent post-surgical complications26,27. Several challenges face clinicians following SOT. First, besides usual pathogens encountered in the general population, SOT patients are also at increased risk for infection by microorganisms that are usually not pathogenic in healthy individuals (known as opportunistic pathogens) because of the immunosuppressive therapies. Then, SOT patients may also lack typical symptoms or biomarkers of infection such as fever, erythema and leukocytosis27 . Then, viral infections are associated with allograft dysfunction through direct tissue damage and immunologically-mediated injury31. This has been shown for community-acquired respiratory viral infections, especially among lung transplant recipients32.
Transplantation and immune responses to infection and vaccines
Mainly because of the iatrogenic immunosuppression, immune responses to natural infections following transplantation are weaker when compared to the general population33-37. The same is true for immunization where vaccine immunogenicity in SOT patients is lower than in the general population38,39. Second, even if SOT patients mount an immune response after immunization, vaccine-induced immunity wanes faster than the general population39-41 and SOT patients require more frequent booster doses41. Another challenge in vaccines and transplantation is the fact that even though SOT patients are more at risk for vaccine-preventable diseases, live vaccines are generally contraindicated after SOT because their immunosuppression increases the risk of vaccine-induced infection42,43.
3) The interplay between influenza and immune responses in SOT Epidemiology and clinical presentation of influenza in SOT
The incidence and seasonality of influenza infection is similar in SOT patients when compared to the general population44,45. Incidence of influenza ranges from 3 to 42 cases per 1000 person-years after liver and lung transplantation, respectively46.
Clinical presentation of influenza infection among SOT patient do not significantly differ from symptoms in the general population, with fever and respiratory symptoms being most frequently reported4. However, influenza infection is more severe in SOT patients than in the general population, causing a significant burden in terms of morbidity and mortality4. If most SOT patients with natural infection will completely recover4, up to 30%
develop complications such as pneumonia, intensive care unit (ICU) admission and death4,47. Some risk factors for severe influenza infection in the post-transplant setting are absence of seasonal immunization, delayed antiviral treatment, pneumonia, diabetes,
< 3 months post-SOT and anti-lymphocyte globulin use4,47,48. It is still debated whether influenza infection is associated with organ rejection or not45.
Influenza immunization after SOT
The only effective preventive strategy against influenza is yearly immunization, which is strongly recommended in SOT patients19,42,43,49. As live vaccines such as LAIV are contraindicated in SOT patients because of the risk of vaccine-induced infection42,43, only the IIV should be used after SOT42,43,45. Compared to the LAIV which mimics more accurately natural influenza infection by triggering a broad humoral and cellular response, the IIV elicits strong humoral responses but weak mucosal IgA and cell-mediated responses50,51. Despite yearly vaccination with the IIV, a significant proportion of SOT patients develop influenza4, which is likely because of suboptimal immunogenicity of vaccines in the SOT setting, and the fact that vaccine strains differ from those circulating52-54. Nevertheless, SOT patients with natural influenza despite previous immunization have been shown to have lower viral loads and reduced severity of disease compared to those who did not receive the vaccine4,47.
Adaptive immune responses to influenza infection and immunization
The adaptive immune system is divided in two arms, both of which are important in the immune response against influenza55: cell-mediated immunity and humoral immunity.
The cell-mediated immunity – also known at T-cell immunity - allows for the direct killing of pathogens and helps in the production of neutralizing antibodies. On the other hand, the humoral immunity – also known as B-cell immunity - is mainly directed towards the production of neutralizing antibodies, but also supports opsonization and complement fixation as well as antibody-dependent cellular cytotoxicity55. Despite many decades of research, it is yet unclear which arm is better correlated with protection against re- infection. So far, the vast majority of the available data regarding the development of immunity against natural influenza infection is about humoral immunity, which is at least partially related to the fact that the assessment of antigen-specific cell-mediated immunity is significantly more expensive and labor-intensive when compared to the assessment of humoral immunity. Furthermore, there is no consensus on protective thresholds and no assay standardization in the evaluation of cell-mediated immunity.
Humoral immunity. Natural influenza infection and immunization elicit the production of antibodies against HA and NA, which have been shown to reduce the risk of
(re)infection56. Hemagglutinin-inhibiting antibodies (HIA) who prevent the binding of the virus to the host cell are most widely accepted surrogate of protection against influenza infection57, with titers of 1:40 or above considered as seroprotective in the immunocompetent population56. However, antibodies generated following infection with an influenza (sub)type confer only limited or absent protection against infection against other (sub)types58. Besides protection against (re)infection, antibodies also play a role in reducing the viral load and improving the clinical outcome59. In immunocompetent patients with influenza natural infection, seroconversion rates vary between 82% and 95%
and more than 90% of patients reach seroprotective HIA titers following infection33-36. Among immunocompromised cohorts, data are lacking and cohorts are usually limited in size but seroprotection rates after natural influenza infection are generally lower than the general population. For example, only 60% of hematopoietic stem cell transplant recipients reached seroprotective titers after influenza infection60. Similarly, among a cohort of 19 SOT patients, less than 40% reached seroprotective titers after infection37. In the transplant setting, there is a significant amount of data about humoral responses following influenza immunization but there is a lack of data about humoral responses to natural infection. Studies involving a large cohort of SOT patients with natural influenza infection are needed to better evaluate seroconversion and seroprotection after natural infection. Also, there is a need to evaluate if factors such as prior immunization, viral kinetics or clinical endpoints can influence HIA responses.
Cellular immunity. Studies in elderly immunocompetent persons, which are considered as poor vaccine responders, have suggested that cell-mediated responses could be a better correlate of protection against influenza than humoral immunity61,62. Data have also shown that even though humoral immunity may protect against infection, cell-mediated immunity contributes to reduce disease severity63. The cellular immunity is commonly divided into CD8+ and CD4+ T-cell immunity. During natural influenza infection, the virus encounters the components of the innate immune system, which eventually triggers the generation of influenza-specific CD8+ and CD4+ T-cells, both of which are important to control the infection. CD8+ T-cells differentiate into cytotoxic T cells which kill infected cells through production of effector molecules and cytokines, eventually controlling viral replication64. At the same time, CD4+ T-cells activate B-cells which will eventually
produce antibodies, but also facilitates CD8+ and macrophage responses64. In the evaluation of cellular responses to infection, antigen-specific cells producing more than one cytokine are of particular interest. There is no widely accepted definition for these so- called polyfunctional cells but available literature usually considers cells producing at least two cytokines among TNF-α, IFN-γ, and IL-2 as polyfunctional65,66. Polyfunctional cells are widely accepted as good correlates of the quality of the T-cell response67 because they have been shown to be producing higher amount of cytokine and CD40L per cell and to have increased degranulation capacities during influenza natural infection68. In terms of clinical outcome, an increased percentage of polyfunctional T-cells was associated with reduced disease severity among influenza-infected pregnant women69, confirming previously published animal data70,71. The superior functionality of polyfunctional cells has also been documented in other infections than influenza and in vaccine studies65,66,72. In the SOT setting, it has been shown that antigen-specific CD8+
and CD4+ T-cells responses were present in only 36% and 50% of patients at 7 months following influenza infection37. However, there is a lack of data about the breadth of cellular responses against influenza in the immediate post-infectious period, as well as which factors influence the development of cellular immunity after infection. Moreover, data are lacking about whether SOT patients infected with one influenza subtype develop cellular responses against other influenza subtypes.
The CD4+ T-cell immunity is further subdivided into Th1 and Th2 responses. The Th1 response is mainly directed at pathogen clearance through phagocytic activity and opsonizing antibodies, but also facilitates cell-mediated immunity against intracellular pathogens through CD8+ T-cell and macrophage stimulation73,74. The levels of the prototypical Th1 cytokine, IFN-γ, have been shown to increase during influenza infection75-77 but not to be associated with disease severity among immunocompetent patients77-79. IFN-γ’s typical proinflammatory role74 which enhances pathogen eradication but also induces inflammatory tissue damage, might explain why IFN-γ levels might seem protective in some studies76,80,81 and deleterious in others82. These differences could also be due to the fact that only some of these studies included H3N2- or influenza B-infected individuals, the latter being associated with higher IFN-γ levels78. On the other hand, Th2 immunity contributes to the development of humoral immunity against extracellular
pathogens and the reduction of inflammation73. During an infection, Th2 cytokines such as IL-13 and IL-4 stimulate the humoral responses73. In immunocompetent patients with influenza natural infection, it is still debated whether the level of Th2 cytokines is associated with increased or decreased disease severity75,79-82. Another cytokine of interest is IL-10, which is the most potent anti-inflammatory cytokine83. IL-10 inhibits Th1, natural killer cells and macrophages and consequently reduces tissue damage83. Consequently, IL-10 also impairs pathogen clearance which is why many consider this cytokine as a double-edged sword83. Like IFN-γ, IL-10 levels are increased during natural influenza infection75-77. Among immunocompetent patients with influenza infection, IL-10 levels have been associated with increased disease severity75,76,78,84,85, such as pneumonia and ICU admission78,79,82, even though this has not been confirmed in other datasets77,80. As a consequence, the delicate balance between the two arms of the T-helper immunity play an important role in the pathogenesis and recovery from various to infections. The lack of data in SOT patients associated with the increased severity of natural infection after SOT highlights the importance to better understand T- helper responses and cytokine levels during influenza infection in order to evaluate whether the level of some cytokines may be useful to predict disease severity.
Vaccine immunogenicity in the SOT population
It is well admitted that natural infection elicits stronger immune responses than immunization. For example, the humoral and cellular responses elicited by primary varicella-zoster virus (VZV) infection is stronger than the response following VZV vaccination86,87. Regarding influenza, natural infection induces a stronger humoral response with higher antibody levels and a more sustained response than immunization in healthy adults9,88,89. Similarly, cellular responses following natural infection are stronger than those following vaccination90,91. Given the particular immune setting following transplantation, it is not clear whether cellular responses following influenza vaccination are also weaker than those following natural infection in SOT patients.
As mentioned above, the IIV elicits strong humoral responses but weak mucosal IgA and cell-mediated responses in the general population50,51. As for natural influenza infection, immunization also elicits polyfunctional T-cells in immunocompetent persons92. In
immunocompetent adults 65 years old or above, the IIV elicits stronger CD4+ than CD8+
responses61,93. Given the immunosuppressive therapy and then increased susceptibility to severe influenza infection, there is a need to better understand cellular responses following IIV in the SOT population.
Some inherent factors have been shown to influence the response to influenza vaccine among SOT patients. For example, higher IL-6/IL-2 ratios or polymorphisms with reduced expression of IL-28B gene have been associated with better seroconversion rates following immunization94,95. Similarly, clinical factors such as lung transplantation, high doses of mycophenolate and age 65 or above have been associated with decreased vaccine responses52,96-99. The lower vaccine responses in lung transplant recipients when compared to other organ transplant recipients is likely related to the net state of immunosuppression53,99,100.
Several approaches have been evaluated to improve immunogenicity in the post- transplant setting. The use of booster doses or adjuvants, as well as changing the mode of delivery of the vaccine have not shown to significantly improve vaccine immunogenicity52,54,101,102. Recently, the use of a high-dose (HD) influenza vaccine, containing fourfold more HA per strain than the standard-dose (SD) vaccine, has been shown in a randomized setting to induce significantly greater geometric mean titers (GMT) and to increase seroconversion (SC) rates among transplant recipients96. In persons 65 years or older, another group of poor vaccine responders, the HD vaccine has also shown greater immunogenicity and efficacy103-106, and is now recommended in this age group107. Studies have shown the importance of cell-mediated immunity to protect against influenza infection or to reduce influenza disease severity61-63. Surprisingly, there was no difference in cellular immunity between the SD and the HD vaccine among non- immunocompromised adults108,109. Given the stronger humoral immunogenicity of the HD vaccine among SOT recipients, and the importance of cellular responses against influenza, there is a need to evaluate whether the HD vaccine also elicits stronger cell- mediated responses among SOT patients when compared to the SD vaccine.
C. Study 1: Summary
Humoral response to natural influenza infection in solid organ transplant recipients
Am J Transplant. 2019;19:2318–2328
If humoral responses to influenza immunization have been widely evaluated in the transplant setting, studies involving a large cohort of transplant patients with natural influenza infection are needed to better evaluate seroconversion and seroprotection after natural infection. In this prospective multicentre cohort study, humoral responses were measured in 196 transplant patients with influenza infection at presentation and 28 days later. Association between seroresponses and clinical or virological factors were also evaluated. Among influenza A-infected patients (n=116), strain-specific seropositivity was 44% at diagnosis and 65% at day 28. Thirty-three percent of patients seroconverted.
Vaccine recipients were less likely to seroconvert whereas lung transplant recipients were more likely to seroconvert (p=0.024 and 0.002, respectively). A subset of 30 patients was responsive to neither immunization nor to natural infection. No correlation between viral kinetics and humoral responses was seen. Overall, this study provides novel data on seroresponses to influenza natural infection in a large cohort of transplant recipient, as well as on the role of previous immunization, organ transplant and viral kinetics in antibody responses.
D. Study 2: Summary
T-cell responses following Natural Influenza Infection or Vaccination in Solid Organ Transplant Recipients
SCIENTIFIC REPORTS 2020 (in press) https://doi.org/10.1038/s41598-020-67172-6
Little is known about the breadth of cellular responses against influenza among SOT recipients. The aim of this work was to evaluate CD4+ and CD8+ responses against influenza in a cohort of SOT patients with natural influenza A and B infection. During the 2017-2018 influenza season, peripheral blood mononuclear cells (PBMCs) were collected at influenza diagnosis and 28 days later in 31 SOT patients. Flow cytometry and intracellular cytokine staining was used to measure infection-elicited influenza-specific CD4+ and CD8+ T-cell responses and to compare it against CD4+ and CD8+ T-cell responses following IIV in SOT patients. Natural infection elicited significant CD4+ T-cell responses, with a 37-fold and 7-fold increase in polyfunctional cells among influenza A/H3N2 and B-infected patients, respectively (p=0.006 and 0.004, respectively).
Moreover, CD4+ T-cell responses were superior following natural infection compared to those following IIV for influenza A/H1N1 (p=0.026), A/H3N2 (p=0.041) and B (p=0.004).
T-cell responses against influenza A/H1N1 could not be evaluated because of the almost absence of circulation of influenza A/H1N1 during this season. These data show that natural influenza infection triggers a significant increase in CD4+ T-cell responses in SOT patients and that those responses are stronger than those elicited by IIV, suggesting that prior infection likely elicits better protection against reinfection than immunization.
E. Study 3: Summary
Cytokine Profiles and Severity of Influenza Infection in Transplant Recipients
The Journal of Infectious Diseases 2019;219:535–9
The T-helper immunity following natural influenza infection has not been well studied in the SOT setting. This, associated with the increased severity of natural infection after SOT, highlights the importance to better understand T-helper responses and cytokine levels during influenza infection in order to evaluate whether the level of some cytokines may be useful to predict disease severity. In this study, we evaluated serum Th1 and Th2 cytokines as well as IL-10 levels during at influenza infection and 28 days later in a large cohort of 277 transplant patients with influenza infection. The levels of the Th2 cytokine IL-13 were associated with protection against pneumonia and ICU admission, whereas the IFN-γ/IL-13 ratio, as a corollary of the Th1/Th2 ratio, was associated with an increased risk of pneumonia and ICU admission, suggesting a protective role of the Th2 response.
Moreover, levels of the anti-inflammatory IL-10 cytokine was associated with an increased risk of pneumonia and ICU admission, possibly through a delayed pathogen clearance secondary to the anti-inflammatory properties of this cytokine. These findings were independent of the influenza viral load.
F. Study 4: Summary
Cell-Mediated Immune Responses After Influenza Vaccination of Solid Organ Transplant Recipients: Secondary Outcomes Analyses of a Randomized Controlled Trial
The Journal of Infectious Diseases 2020;221:53–62
Despite yearly vaccination, SOT patients are still at increased risk for severe influenza infection. If humoral responses following IIV have been widely studied in SOT patients, there is a need to better understand cellular responses against the vaccine in this population. Moreover, there is a need to evaluate whether the HD vaccine also elicits stronger cellular responses than the SD vaccine in SOT patients. The aim of this study was to evaluate and compare CD4+ and CD8+ T-cell responses against the SD and the HD vaccine. PBMCs were collected before and 28 days after vaccination in 60 SOT patients randomized to receive the SD or the HD vaccine during the 2016-2017 influenza season. Flow cytometry and intracellular cytokine staining was used to measure vaccine- elicited influenza-specific CD4+ and CD8+ T-cell responses. The HD vaccine elicited significantly greater monofunctional CD4+ and CD8+ T-cell responses against all vaccine strains (H1N1, H3N2 and B). This was also the case for polyfunctional responses, with higher median vaccine-elicited antigen-specific polyfunctional CD4+ T-cells in recipients of the HD vaccine after stimulation with influenza A/H1N1 (p=0.003), A/H3N2 (p=0.008), and B (p=0.001). Also, vaccine-elicited antigen-specific polyfunctional CD8+ T-cells were higher following the HD vaccine after stimulation with influenza B (p=0.002). These data provide novel evidence that the HD vaccine elicits greater cell-mediated responses than the SD vaccine in SOT patients, confirming data about stronger humoral responses following the HD vaccine in SOT patients. This provides additional support to preferentially use the HD vaccine after transplantation.
G. CONCLUSION
The complex interplay between the pathogen and the host immune response is evident following natural influenza infection. Despite decades of research, it is yet unclear how exactly does the virus shape the immune system and which host factors are the best correlates against (re)infection. This is further complicated in the transplant setting because of the dysregulated immune system secondary to the iatrogenic immunosuppression.
The first manuscript of this thesis evaluated humoral responses against influenza in a large cohort of SOT patients. Seroprotective rates at baseline were 39% and 70% for influenza A and influenza B, respectively. At day 28, seroprotective titers were seen in 64% of patients after influenza A and 81% of the patients after influenza B infection, with overall, seroconversion rates of only 33% of patients against influenza A and 30% against influenza B infection. Antibody responses in our cohort were significantly lower than in the general population, where seroconversion rates are reported in 82-95% and seroprotective HIA titers in 93-98% of patients following natural influenza infection33-36. Seroprotection rates measured at day 28 in our study were however higher than those observed by Baluch et al, where only 37% of SOT patients were seroprotected at 7 months after natural influenza infection37. This is most likely related to waning of antibodies following natural influenza infection, which is well described after influenza infection in immunocompetent populations110, and probably further worsened in the transplant setting where waning of antibodies is even faster39-41. The fact that many SOT patients remained seronegative at day 28 after infection is concerning because it suggests that these patients could be at risk for another infection in case of re-exposition with the same strain. This could also suggest that HIA antibodies are not a reliable surrogate of protection in the SOT setting, which is also suggested by the high proportion of SOT patients with influenza B infection despite seroprotective titers at baseline.
Interestingly, lung transplant recipients were more likely to seroconvert when compared to other transplant recipients. This is surprising because of the higher net state of immunosuppression among lung transplant recipients. The fact that lung and non-lung transplant recipients had similar HIA titers at baseline can reasonably rule out the hypothesis that seroconversion rates were higher among lung transplant recipients simply
because they were more likely to be seronegative at baseline. One can hypothesize that the pro-inflammatory local environment present in a significant proportion of lung transplant recipients contributes to stronger antibody responses despite a higher net state of immunosuppression111,112. The fact that previously immunized SOT patients were less likely to seroconvert likely reflects a study bias where patients who did mount a strong humoral response to the vaccine were protected against influenza infection and therefore not enrolled in the current study. Finally, this study showed that viral load was not an important factor in the seroresponse. This is surprising given the fact that the use of convalescent serum or immunoglobulins has been shown to reduce duration of viral shedding59,113,114. Altogether, these data suggest that even though antibodies contribute to viral clearance, they might not be crucial to clear the virus if appropriate and early antiviral therapy is given. More studies are needed to determine whether the accepted seroprotective cut-off of 1:40 is applicable in the SOT setting. Further studies are also needed to evaluate the role in viral clearance of alternate neutralizing antibodies or non- neutralizing antibodies directed against neuraminidase or conserved influenza antigens115.
The second manuscript of this work evaluated influenza-elicited cell-mediated responses in SOT patients and highlighted that influenza infection elicited primarily CD4+ T-cell responses. The predominant CD4+ T-cell response following influenza immunization is in line with previous data looking at long-term responses to influenza in SOT patients37. Several factors might explain the virtually absent CD8+ T-cell response following natural influenza infection in our cohort. First, the antigen cocktail used for cell stimulation could potentially have underestimated CD8+ responses because it the inactivated virus was calibrated on HA concentration, which predominantly stimulates CD4+ responses whereas CD8+ responses are mostly stimulated by internal proteins such as NP and M1116. However, it is not known whether the other antigens included in the cocktail were in lower, similar or higher respective concentration than HA. Second, the immunosuppressive treatments taken by the patients and to which PBMCs were exposed could preferentially affect CD8+ responses, which might explain why other data in SOT patients also show a predominant CD4+ response against influenza despite the use of live virus for cell stimulation37. Our data showed that infection-elicited responses were not
only monofunctional but also polyfunctional, the latter being considered as a marker of the quality of the T-cell response65-72. When compared to T-cell responses following IIV, responses following infection were stronger, confirming previous data in the general population showing that natural infection elicits stronger cell-mediated responses than immunization, not only for influenza90,91 but also for other viruses87. This was true despite a possibly higher net state of immunosuppression in the infection cohort, with a greater proportion of patients on prednisone, with higher daily doses, and an overrepresentation of lung transplant recipients. Several findings of this study were surprising. First, transplant patients infected with influenza A/H3N2 appeared mount an immune response against influenza A/H1N1 as well. This heterosubtypic immunity may be mediated by a T-cell response directed towards conserved influenza antigens117. Unfortunately, the lack of influenza A/H1N1 circulation during the study period did not allow to evaluate whether the infection with influenza A/H1N1 also elicited significant H3N2 responses118. Second, there was no difference in T-cell responses at baseline between patients who did receive the IIV at the beginning of the season and those who did not, suggesting a lack of sustained cell-mediated responses following the vaccine, as previously shown for humoral responses against vaccine-preventable diseases in the SOT setting39-41. In conclusion, our data show that natural influenza infection elicited a predominant CD4+ T- cell response after SOT, at levels significantly higher than following the IIV, and that among influenza A-infected patients, these responses appeared to be cross-reactive with other influenza subtypes. Further studies are needed to evaluate whether our findings are applicable to influenza A/H1N1 infection, as our study was performed during a season where influenza A/H1N1 was virtually absent118. More studies are also needed to define which influenza proteins and epitopes are responsible for the effector response and to better define cell-mediated thresholds for protection against (re)infection. Finally, additional data is needed to evaluate whether the predominant CD4+ response seen in our cohort is confirmed with the use of antigens that predominantly stimulate the CD8+
response, not only in transplant patients but also in the general population who doesn’t have immunosuppression as a potential confounding factor.
In the third manuscript of this thesis, we evaluated the balance of Th1 to Th2 responses, as well as levels of IL-10 at influenza diagnosis and 28 days later in a large cohort of SOT patients. Our data showed that an immune responses skewed towards Th1 was associated with a higher incidence of pneumonia or ICU admission, as reflected by an increased IFN-γ/IL-13 ratio. Available data suggest that crude values of Th1 or Th2 cytokines might not be reliable predictors of disease severity75-82. However, the ratio of Th1/Th2 cytokines might be better at assessing the delicate balance between the Th1 and the Th2 response in response to viral infections. The second important finding of this work was the association between IL-10 levels and influenza disease severity, confirming available data in the immunocompetent setting75,76,78,84,85. More specifically, our data are in line with previous studies showing and association between IL-10 levels, pneumonia and ICU admission78,79,82, which is likely related to IL-10’s potent anti-inflammatory effects impairing pathogen clearance83. Our findings were independent on viral load, highlighting that the immune response is an independent contributor to influenza disease severity.
Unfortunately, our findings were mostly significant at day 28, meaning that the evaluation of those cytokines might be of limited clinical relevance as a predictor of influenza severity following transplantation. In the future, more studies are needed to evaluate whether these cytokines or others might help guide reduction of immunosuppression in case of severe influenza infection among SOT patients. Furthermore, more studies are needed in immunocompromised and immunocompetent patients to evaluate whether other cytokines could be clinically relevant predictors of influenza disease severity.
The last study of this thesis compared T-cell responses among SOT patients following the HD and the SD vaccine and showed that the recipients of the HD vaccine mounted significantly stronger cell-mediated responses against influenza A/H1N1, influenza A/H3N2 and influenza B, which was consistent with published data also showing better humoral responses among SOT recipients receiving of the HD vaccine96. However, our data are not in line with data in immunocompetent adults were no difference in cellular responses could be demonstrated between SD and HD recipients108,109, despite the better humoral immunogenicity in the HD group103-106. The similar cellular immunogenicity of both vaccines in immunocompetent populations may be related to the fact that the SD vaccine already elicits a reasonable degree of cellular responses in these patients and
therefore the incremental benefit of the HD vaccine may not be visible61,62,109. Following transplantation, the SD vaccine elicits poor cellular responses, allowing us to demonstrate a significant difference. The non-significant overrepresentation of kidney transplant recipients in the HD group despite randomization could have partially contributed to the better responses among recipients of the HD vaccine. Surprisingly, despite better humoral and cellular responses among recipients of the HD vaccine, we were unable to find any correlation between cellular and humoral responses. In the general population, the correlation between cellular and humoral responses following the SD vaccine is debated51,100,109,119; for the HD vaccine, the only published study showed a moderate correlation between CD4+ and antibody responses109. Most vaccine studies in transplant have not been able to find a correlation between the two arms of the adaptive response53,100,120, with the exception of one study that found that seroconversion was correlated with the proportion IL4+CD4+ T-cells following stimulation with influenza A/H1N1121. Even though we were not able to find a strong correlation between antibody and cellular responses, patients who seroconverted were more likely to have higher cellular responses, especially after stimulation with influenza B. Our findings showing better cellular responses among recipients of the HD vaccine are of particular relevance providing that cell-mediated responses might be a better correlate of protection against (re)infection in poor vaccine responders61,62, but also reduce disease severity63. The better cell-mediated immunogenicity of the HD vaccine was more evident for CD4+ than CD8+ T-cell responses, which could be have several explanations. First, some data have shown that the IIV elicits a predominant CD4+ response51,61,93. Second, as mentioned above, the antigen cocktail used for cell stimulation was calibrated on HA concentration and could potentially potentially have underestimated CD8+ responses116. The better cellular immunogenicity of the HD vaccine was evident not only for monofunctional but also for polyfunctional cells, which are correlated with the quality of the T-cell response65-
72. Despite lower than CD4+ responses, CD8+ responses following the HD vaccine were nevertheless detectable, unlike CD8+ responses following natural infection shown in our previous study. The difference between CD8+ responses seen in those two different settings could be explained by a possibly skewed immune response secondary to the intramuscular use of highly antigenic peptides which fails to mimic the mucosal immune
response seen during natural infection and possibly directed towards different epitopes.
It could also be related to the emergence of cytotoxic T lymphocytes escape quasispecies in the absence of immune pressure during natural infection, which does not happen following vaccination122. Lung transplant recipients were more at risk to have an absent CD4+ T-cell response to the vaccine when compared to non-lung transplant recipients, which is consistent with previous data and most likely related to the higher net state of immunosuppression53,99,100. Our data provide additional evidence about the better immunogenicity of the HD vaccine following SOT96. These data, associated with a similar safety profile to the SD vaccine96, strengthens the fact that the HD vaccine should be preferred after SOT. Studies following the sustainability of both cellular and humoral responses are needed to evaluate whether the stronger immunogenicity of the HD vaccine persists at six or twelve months post immunization.
Besides the future perspectives introduced earlier, other areas of research need to be explored when evaluating the interactions between influenza and the immune system in transplant patients. For example, there is a need to perform an in-depth evaluation of the humoral response against the influenza epitopes other than the widely studied HA in transplant patients with natural infection and those who received the IIV. Indeed, little is known about humoral responses against conserved epitopes such as NP, M, NS1 nor against other variable epitopes such as NA123-127. Some data have shown that anti-M2 antibodies reduced viral load and mortality whereas anti-NA antibodies correlated with milder influenza infection126,128. Protein microarrays allow for the simultaneous profiling of the humoral response to a wide range of antigens129-134 and will be used to investigate the breadth of the humoral response against natural influenza infection and immunization in SOT patients. Another field which would benefit from further research is the study of some specific immune cell populations, such as T follicular helper (Tfh), T-regulatory (Treg) cells or innate lymphoid cells (ILC). Tfh cells are a subset of CD4+ T-cells that mainly reside within the germinal center where they interact with B cells to generate antibody responses against influenza135. Dysfunctional Tfh responses have been associated with poor influenza vaccine responses in healthy and transplant populations94,136,137. On the other hand, T-reg cells are a CD4+ T-cell population with a strong immunosuppressive effects that are induced upon influenza infection138. They
have an important role in suppressing the inflammatory responses and stimulating tissue repair, notably through the attenuation of effector responses139. Then, ILC are considered as the innate equivalent of T-cells140 and have been shown to accumulate in the lungs during influenza infection and to play a role in restoring airway integrity141,142. Despite their importance, neither of those subpopulations have been studied following natural influenza infection in the transplant setting. Finally, another area of research could be the use of single-cell RNA sequencing to evaluate how influenza infection impairs the host cellular transcriptional profile when compared to the absence of infection. This technique allows for the evaluation of host transcriptome at the single cell level143,144. Although already performed in healthy patients with influenza infection145, performing single-cell RNA sequencing on influenza-infected SOT patients would contribute to better understand how the immunosuppression affects transcriptional profiles of the immune cells at the mucosal level.
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