Humoral immune responses play a major role in naturally acquired immunity to malaria, but are slow to develop and ineffectively maintained. Although this may be partially due to the complex nature of ...Plasmodium parasites and the high degree of antigenic variation, there is evidence that the parasite also actively alters B cell function. We integrate recent findings on the effect of Plasmodium falciparum (Pf) on B cells and the association of parasite exposure with altered B cell proportions, such as the expansion of atypical memory B cells. We propose a model of how the parasite may mediate these effects by direct interaction with B cells via the cysteine-rich interdomain region 1 α (CIDRα) of the erythrocyte membrane protein 1 (PfEMP1) and modulation of the host B cell activating factor (BAFF) immune pathway, and how this may compromise protective immune memory by interfering with B cell differentiation.
Mass cytometry enables highly multiplexed profiling of cellular immune responses in limited‐volume samples, advancing prospects of a new era of systems immunology. The capabilities of mass cytometry ...offer expanded potential for deciphering immune responses to infectious diseases and to vaccines. Several studies have used mass cytometry to profile protective immune responses, both postinfection and postvaccination, although no vaccine‐development program has yet systematically employed the technology from the outset to inform both candidate design and clinical evaluation. In this article, we review published mass cytometry studies relevant to vaccine development, briefly compare immune profiling by mass cytometry to other systems‐level technologies, and discuss some general considerations for deploying mass cytometry in the context of vaccine development.— Reeves, P. M., Sluder, A. E., Raju Paul, S., Scholzen, A., Kashiwagi, S., Poznansky, M. C. Application and utility of mass cytometry in vaccine development. FASEB J. 32,5‐15 (2018). www.fasebj.org
Introduction
Q fever, caused by the intracellular bacterium
Coxiella burnetii
, is considered an occupational and biodefense hazard and can result in debilitating long-term complications. While ...natural infection and vaccination induce humoral and cellular immune responses, the exact nature of cellular immune responses to
C. burnetii
is incompletely understood. The current study seeks to investigate more deeply the nature of long-term cellular recall responses in naturally exposed individuals by both cytokine release assessment and cytometry profiling.
Methods
Individuals exposed during the 2007-2010 Dutch Q fever outbreak were grouped in 2015, based on a
C. burnetii
-specific IFNγ release assay (IGRA), serological status, and self-reported clinical symptoms during initial infection, into asymptomatic IGRA-negative/seronegative controls, and three IGRA-positive groups (seronegative/asymptomatic; seropositive/asymptomatic and seropositive/symptomatic). Recall responses following
in vitro
re-stimulation with heat-inactivated
C. burnetii
in whole blood, were assessed in 2016/2017 by cytokine release assays (n=55) and flow cytometry (n=36), and in blood mononuclear cells by mass cytometry (n=36).
Results
Cytokine release analysis showed significantly elevated IL-2 responses in all seropositive individuals and elevated IL-1β responses in those recovered from symptomatic infection. Comparative flow cytometry analysis revealed significantly increased IFNγ, TNFα and IL-2 recall responses by CD4 T cells and higher IL-6 production by monocytes from symptomatic, IGRA-positive/seropositive individuals compared to controls. Mass cytometry profiling and unsupervised clustering analysis confirmed recall responses in seropositive individuals by two activated CD4 T cell subsets, one characterized by a strong Th1 cytokine profile (IFNγ
+
IL-2
+
TNFα
+
), and identified
C. burnetii
-specific activation of CD8 T cells in all IGRA-positive groups. Remarkably, increased
C. burnetii
-specific responses in IGRA-positive individuals were also observed in three innate cell subpopulations: one characterized by an IFNγ
+
IL-2
+
TNFα
+
Th1 cytokine profile and lack of canonical marker expression, and two IL-1β-, IL-6- and IL-8-producing CD14
+
monocyte subsets that could be the drivers of elevated secretion of innate cytokines in pre-exposed individuals.
Discussion
These data highlight that there are long-term increased responses to
C. burnetii
in both adaptive and innate cellular compartments, the latter being indicative of trained immunity. These findings warrant future studies into the protective role of these innate responses and may inform future Q fever vaccine design.
Controlled human malaria infection with sporozoites is a standardized and powerful tool for evaluation of malaria vaccine and drug efficacy but so far only applied by exposure to bites of Plasmodium ...falciparum (Pf)-infected mosquitoes. We assessed in an open label Phase 1 trial, infection after intradermal injection of respectively 2,500, 10,000, or 25,000 aseptic, purified, vialed, cryopreserved Pf sporozoites (PfSPZ) in three groups (N = 6/group) of healthy Dutch volunteers. Infection was safe and parasitemia developed in 15 of 18 volunteers (84%), 5 of 6 volunteers in each group. There were no differences between groups in time until parasitemia by microscopy or quantitative polymerase chain reaction, parasite kinetics, clinical symptoms, or laboratory values. This is the first successful infection by needle and syringe with PfSPZ manufactured in compliance with regulatory standards. After further optimization, the use of such PfSPZ may facilitate and accelerate clinical development of novel malaria drugs and vaccines.
Cellular responses to Plasmodium falciparum parasites, in particular interferon-gamma (IFNγ) production, play an important role in anti-malarial immunity. However, clinical immunity to malaria ...develops slowly amongst naturally exposed populations, the dynamics of cellular responses in relation to exposure are difficult to study and data about the persistence of such responses are controversial. Here we assess the longevity and composition of cellular immune responses following experimental malaria infection in human volunteers. We conducted a longitudinal study of cellular immunological responses to sporozoites (PfSpz) and asexual blood-stage (PfRBC) malaria parasites in naïve human volunteers undergoing single (n = 5) or multiple (n = 10) experimental P. falciparum infections under highly controlled conditions. IFNγ and interleukin-2 (IL-2) responses following in vitro re-stimulation were measured by flow-cytometry prior to, during and more than one year post infection. We show that cellular responses to both PfSpz and PfRBC are induced and remain almost undiminished up to 14 months after even a single malaria episode. Remarkably, not only 'adaptive' but also 'innate' lymphocyte subsets contribute to the increased IFNγ response, including αβT cells, γδT cells and NK cells. Furthermore, results from depletion and autologous recombination experiments of lymphocyte subsets suggest that immunological memory for PfRBC is carried within both the αβT cells and γδT compartments. Indeed, the majority of cytokine producing T lymphocytes express an CD45RO(+) CD62L(-) effector memory (EM) phenotype both early and late post infection. Finally, we demonstrate that malaria infection induces and maintains polyfunctional (IFNγ(+)IL-2(+)) EM responses against both PfRBC and PfSpz, previously found to be associated with protection. These data demonstrate that cellular responses can be readily induced and are long-lived following infection with P. falciparum, with a persisting contribution by not only adaptive but also (semi-)innate lymphocyte subsets. The implications hereof are positive for malaria vaccine development, but focus attention on those factors potentially inhibiting such responses in the field.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
, the causative agent of Q fever, is a Gram-negative intracellular bacterium transmitted via aerosol. Regulatory approval of the Australian whole-cell vaccine Q-VAX® in the US and Europe is hindered ...by reactogenicity in previously exposed individuals. The aim of this study was to identify and rationally select
epitopes for design of a safe, effective, and less reactogenic T-cell targeted human Q fever vaccine. Immunoinformatic methods were used to predict 65 HLA class I epitopes and 50 promiscuous HLA class II
epitope clusters, which are conserved across strains of
. HLA binding assays confirmed 89% of class I and 75% of class II predictions, and 11 HLA class II epitopes elicited IFNγ responses following heterologous DNA/DNA/peptide/peptide prime-boost immunizations of HLA-DR3 transgenic mice. Human immune responses to the predicted epitopes were characterized in individuals naturally exposed to
during the 2007-2010 Dutch Q fever outbreak. Subjects were divided into three groups: controls with no immunological evidence of previous infection and individuals with responses to heat-killed
in a whole blood IFNγ release assay (IGRA) who remained asymptomatic or who experienced clinical Q fever during the outbreak. Recall responses to
epitopes were assessed by cultured IFNγ ELISpot. While HLA class I epitope responses were sparse in this cohort, we identified 21 HLA class II epitopes that recalled T-cell IFNγ responses in 10-28% of IGRA+ subjects. IGRA+ individuals with past asymptomatic and symptomatic
infection showed a comparable response pattern and cumulative peptide response which correlated with IGRA responses. None of the peptides elicited reactogenicity in a
exposure-primed guinea pig model. These data demonstrate that a substantial proportion of immunoinformatically identified HLA class II epitopes show long-lived immunoreactivity in naturally infected individuals, making them desirable candidates for a novel human multi-epitope Q fever vaccine.
Q fever is a zoonotic disease caused by the highly infectious Gram-negative coccobacillus,
Coxiella burnetii
(
C. burnetii
). The Q fever vaccine Q-VAX
®
is characterised by high reactogenicity, ...requiring individuals to be pre-screened for prior exposure before vaccination. To date it remains unclear whether vaccine side effects in pre-exposed individuals are associated with pre-existing adaptive immune responses to
C. burnetii
or are also a function of innate responses to Q-VAX
®
. In the current study, we measured innate and adaptive cytokine responses to
C. burnetii
and compared these among individuals with different pre-exposure status. Three groups were included: n=98 Dutch blood bank donors with unknown exposure status, n=95 Dutch village inhabitants with known natural exposure status to
C. burnetii
during the Dutch Q fever outbreak of 2007-2010, and n=96 Australian students receiving Q-VAX
®
vaccination in 2021. Whole blood cytokine responses following
ex vivo
stimulation with heat-killed
C. burnetii
were assessed for IFNγ, IL-2, IL-6, IL-10, TNFα, IL-1β, IP-10, MIP-1α and IL-8. Serological data were collected for all three cohorts, as well as data on skin test and self-reported vaccine side effects and clinical symptoms during past infection. IFNγ, IP-10 and IL-2 responses were strongly elevated in individuals with prior
C. burnetii
antigen exposure, whether through infection or vaccination, while IL-1β, IL-6 and TNFα responses were slightly increased in naturally exposed individuals only. High dimensional analysis of the cytokine data identified four clusters of individuals with distinct cytokine response signatures. The cluster with the highest levels of adaptive cytokines and antibodies comprised solely individuals with prior exposure to
C. burnetii
, while another cluster was characterized by high innate cytokine production and an absence of
C. burnetii
-induced IP-10 production paired with high baseline IP-10 levels. Prior exposure status was partially associated with these signatures, but could not be clearly assigned to a single cytokine response signature. Overall, Q-VAX
®
vaccination and natural
C. burnetii
infection were associated with comparable cytokine response signatures, largely driven by adaptive cytokine responses. Neither individual innate and adaptive cytokine responses nor response signatures were associated retrospectively with clinical symptoms during infection or prospectively with side effects post-vaccination.
Long-lasting and sterile protective immunity against Plasmodium falciparum can be achieved by immunization of malaria-naive human volunteers under chloroquine prophylaxis with sporozoites delivered ...by mosquito bites (CPS-immunization). Protection is mediated by sporozoite/liver-stage immunity. In this study, the capacity of CPS-induced antibodies to interfere with sporozoite functionality and development was explored.
IgG was purified from plasma samples obtained before and after CPS-immunization from two separate clinical trials. The functionality of these antibodies was assessed in vitro in gliding and human hepatocyte traversal assays, and in vivo in a human liver-chimeric mouse model.
Whereas pre-treatment of sporozoites with 2 mg/ml IgG in the majority of the volunteers did not have an effect on in vitro sporozoite gliding motility, CPS-induced IgG showed a distinct inhibitory effect in the sporozoite in vitro traversal assay. Pre-treatment of P. falciparum sporozoites with post-immunization IgG significantly inhibited sporozoite traversal through hepatocytes in 9/9 samples when using 10 and 1 mg/ml IgG, and was dose-dependent, resulting in an average 16% and 37% reduction with 1 mg/ml IgG (p = 0.003) and 10 mg/ml IgG (p = 0.002), respectively. In vivo, CPS-induced IgG reduced liver-stage infection and/or development after a mosquito infection in the human liver-chimeric mouse model by 91.05% when comparing 11 mice receiving post-immunization IgG to 11 mice receiving pre-immunization IgG (p = 0.0008).
It is demonstrated for the first time that CPS-immunization induces functional antibodies against P. falciparum sporozoites, which are able to reduce parasite-host cell interaction by inhibiting parasite traversal and liver-stage infection. These data highlight the functional contribution of antibody responses to pre-erythrocytic immunity after whole-parasite immunization against P. falciparum malaria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
T cell-mediated immunity plays a central role in the control and clearance of intracellular
infection, which can cause Q fever. Therefore, we aimed to develop a novel T cell-targeted vaccine that ...induces pathogen-specific cell-mediated immunity to protect against Q fever in humans while avoiding the reactogenicity of the current inactivated whole cell vaccine. Human HLA class II T cell epitopes from
were previously identified and selected by immunoinformatic predictions of HLA binding, conservation in multiple
isolates, and low potential for cross-reactivity with the human proteome or microbiome. Epitopes were selected for vaccine inclusion based on long-lived human T cell recall responses to corresponding peptides in individuals that had been naturally exposed to the bacterium during a 2007-2010 Q fever outbreak in the Netherlands. Multiple viral vector-based candidate vaccines were generated that express concatemers of selected epitope sequences arranged to minimize potential junctional neo-epitopes. The vaccine candidates caused no antigen-specific reactogenicity in a sensitized guinea pig model. A subset of the vaccine epitope peptides elicited antigenic recall responses in splenocytes from C57BL/6 mice previously infected with
However, immunogenicity of the vaccine candidates in C57BL/6 mice was dominated by a single epitope and this was insufficient to confer protection against an infection challenge, highlighting the limitations of assessing human-targeted vaccine candidates in murine models. The viral vector-based vaccine candidates induced antigen-specific T cell responses to a broader array of epitopes in cynomolgus macaques, establishing a foundation for future vaccine efficacy studies in this large animal model of
infection.
Removal of dead cells in the absence of concomitant immune stimulation is essential for tissue homeostasis. We recently identified an injury-induced protein misfolding event that orchestrates the ...plasmin-dependent proteolytic degradation of necrotic cells. As impaired clearance of dead cells by the innate immune system predisposes to autoimmunity, we determined whether plasmin could influence endocytosis and immune cell stimulation by dendritic cells - a critical cell that links the innate and adaptive immune systems. We find that plasmin generated on the surface of necrotic cells enhances their phagocytic removal by human monocyte-derived dendritic cells. Plasmin also promoted phagocytosis of protease-resistant microparticles by diverse mouse dendritic cell sub-types both in vitro and in vivo. Together with an increased phagocytic capacity, plasmin-treated dendritic cells maintain an immature phenotype, exhibit reduced migration to lymph nodes, increase their expression/release of the immunosuppressive cytokine TGF-β, and lose their capacity to mount an allogeneic response. Collectively, our findings support a novel role for plasmin formed on dead cells and other phagocytic targets in maintaining tissue homeostasis by increasing the phagocytic function of dendritic cells while simultaneously decreasing their immunostimulatory capacity consistent with producing an immunosuppressive state.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
