CIDP: Analysis of Immunomarkers During COVID-19 mRNA-Vaccination and IVIg-Immunomodulation: An Exploratory Study

Availability of COVID-19 mRNA vaccine for patients with chronic inflammatory demyelinating polyneuropathy (CIDP) treated with intravenous immunoglobulin (IVIg) raises the question of whether COVID-19 mRNA vaccine influences disease activity or IVIg-mediated immunomodulation in CIDP. In this exploratory study, blood samples of CIDP patients on IVIg treatment were longitudinally analyzed before and after vaccination with a COVID-19 mRNA vaccine. A total of 44 samples of eleven patients were characterized at four timepoints by ELISA and flow cytometry in terms of immunomarkers for disease activity and IVIg-immunomodulation. Apart from a significantly lower expression of CD32b on naïve B cells after vaccination, no significant alteration of immunomarkers for CIDP or IVIg-mediated immunomodulation was observed. Our exploratory study suggests that COVID-19 mRNA vaccine does not have a relevant impact on immune activity in CIDP. In addition, immunomodulatory effects of IVIg in CIDP are not altered by COVID-19 mRNA vaccine. This study was registered in the German clinical trial register (DRKS00025759). Graphical Abstract Overview over the study design. Blood samples of CIDP patients on recurrent IVIg treatment and vaccination with a COVID-19 mRNA vaccine were obtained at four timepoints for cytokine ELISA and flow cytometry, to assess key cytokines and cellular immunomarkers for disease activity and IVIg-immunomodulation in CIDP. Supplementary Information The online version contains supplementary material available at 10.1007/s11481-023-10058-x.


Introduction
Chronic inflammatory demyelinating polyneuropathy (CIDP) is a prototypic autoimmune disease of the peripheral nervous system (Lehmann et al. 2019).Pathomechanisms of CIDP include autoreactive T cells (Chi et al. 2010), impaired B cell maturation and reduced expression of regulatory Fc gamma receptor IIb (FcγRIIb, CD32b) on B cells and monocytes, promoting autoantibody production against peripheral nerve myelin and phagocytosis (Press et al. 2003;Tackenberg et al. 2009).Over the last years, several studies tried to establish markers of peripheral blood mononuclear cells (PBMC) and serum cytokines that might reflect disease activity in CIDP.Of those, reduced numbers of CD32b expressing memory B cells and monocytes, as well as increased numbers of CD16 + myeloid dendritic cells, were associated with disease activity and/or poor response to immunoglobulin therapy (Tackenberg et al. 2009;Dyer et al. 2016).Other studies demonstrated that CIDP is associated with a reduction of regulatory T cells, as well as with an increase of monocytes (Matà et al. 2007;Sanvito et al. 2009).

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COVID-19 mRNA vaccine is known to provoke an immune response characterized by a transient increase of pro-inflammatory cytokines (e.g., IL-6), resulting in a SARS-CoV-2 specific proliferation of T cells, B cells, and anti-SARS-CoV-2 antibody-producing plasma cells (Goel et al. 2021;Jordan 2021).In contrast, whole cell counts of naïve, non-naïve, or memory B cells are considered to remain stable across the time course of vaccination (Goel et al. 2021).
Possible adverse effects of COVID-19 mRNA vaccine, like aseptic (peri-)myocarditis, might be caused by a misdirected, possibly stimulus-independent immune response after vaccination, but their exact pathomechanism is currently unknown (Vidula et al. 2021).
It is thus conceivable, yet unknown that COVID-19 mRNA vaccine alters the aberrant immune response in CIDP or impacts the immunomodulatory effects of IVIg.Therefore we here longitudinally examined for the first time the expression of selected cellular and humoral immunomarkers of CIDP and of IVIg-immunomodulation during the vaccination with a COVD-19 mRNA vaccine.

Patient Characteristics and Study Design
Eleven patients (9 male / 2 female, mean age 62 ± 17 years) with CIDP participated in this study.Inclusion criteria were confirmed or probable CIDP (according to the EFNS/PNS criteria (Van Den Bergh et al. 2010)) on recurrent IVIg treatment (every four weeks), the absence of autoantibodies against paranodal junction proteins, vaccination with a COVID-19 mRNA vaccine (Pfizer-BioNTech BNT162b2), and written informed consent for study participation.Exclusion criteria were acute infections or intake of immunosuppressants or systemic corticosteroids within the previous three months.Mean baseline INCAT disability score was 2 ± 2, mean baseline Rasch-built overall disability scale (R-ODS) score was 73 ± 19.
Blood serum samples and peripheral blood mononuclear cell (PBMC) samples were collected at four timepoints: Before vaccination (mean 11 ± 4 weeks before the first vaccine dose), samples were collected before (1 st , on the same day) and one week after IVIg infusion (2 nd ).The mean interval between the first and the second dose of COVID-19 vaccine was 4 ± 1 weeks.Sample collection was repeated two weeks after the second COVID-19 vaccine dose immediately before (3 rd ) and one week after IVIg infusion (4 th ).

Sample Preparation
Blood samples were collected by venipuncture using Sarstedt S-monovettes® (Sarstedt, Nümbrecht, Germany) for serum collection and the BD Vacutainer CPT™ system (BD Biosciences, San Diego, CA, USA) for PBMC isolation.
Serum samples were obtained by centrifugation of whole blood monovettes at 3000 rotations per minute (rpm) for 10 min and then stored at -20 °C for further use.
PBMCs were isolated from the BD Vacutainer CPT™ system according to the manufacturers ' instructions before being stored at -80 °C (15 million PBMCs in 100 µl dimethylsulfoxide [DMSO] and 900 µl fetal bovine serum [FBS]).
All flow cytometry experiments included isotype controls and fluorescence-minus-one controls.Doublet cells were excluded from analyses based on forward scatter-A and forward scatter-H.For each experiment, the lymphocyte and the monocyte population were gated using forward and side scatter.

Statistical Analysis
Statistical analysis was performed using GraphPad PRISM 9.0 software (Graphpad, San Diego, CA, USA).Normality was tested using D'Agostino and Pearson omnibus normality test, before either multiple paired t-tests or Wilcoxon tests were performed.A p-value < 0.05 was considered statistically significant.

Disability before and after COVID-19 vaccination
No changes in disability, measured by the INCAT disability scale (mean INCAT score before and after vaccination: 2 ± 2) and the R-ODS score (mean R-ODS score before and after vaccination: 73 ± 19), occurred after COVID-19 vaccination.Also, no subjective clinical deterioration was reported from CIDP patients after COVID-19 vaccination.

Discussion
To our knowledge, this is the first study that explored a set of presumed markers of disease activity and IVIg-immunomodulation in CIDP patients who underwent vaccination with the novel mRNA vaccine BNT162b2.As such, our study provides important insights into the vaccine response in a chronic autoimmune condition.Our data indicate that in CIDP, immunomarkers for disease activity and IVIgimmunomodulation were not relevantly altered by COVID-19 mRNA vaccine.
The most striking difference in the expression of disease activity markers before and after vaccination was the lower expression of anti-inflammatory CD32b on naïve B cells and an increased expression of pro-inflammatory CD32a.CD32b (FcγRIIb) is known to control antibody generation upon immunization (Takai et al. 1996).As such, it appears attractive to speculate that this decline could be a systemic vaccine effect.However, this hypothesis would need confirmation by similar observations in other IVIg-dependent and -independent conditions.The reduction of CD32b on naïve B cells could be related to CD32a upregulation, as these two immunoglobulin receptors mediate opposing functions on B cells, probably leading to a relative CD32b downregulation when CD32a is upregulated (Nimmerjahn and Ravetch 2007).Regarding its function for the pathogenesis of CIDP, we assume that the decrease of CD32b on naïve B cells is not clinically relevant for the following reasons: 1) CD32b is known to decline subsequent to IVIg treatment on naïve B cells in CIDP within the first week (Dyer et al. 2016).
2) CD32b expression on memory B cells-which are also considered to be critical for CIDP and its treatment with IVIg (Tackenberg et al. 2009)-was not altered.3) None of the patients experienced any clinical deterioration upon the vaccination.Also, the activation of pro-inflammatory CD32a receptors on B cells may reflect a physiological activation of the humoral adaptive immune system for anti-SARS-CoV-2 antibody production, as studies on pneumococcal vaccine showed that CD32a activation plays a crucial role for vaccine response, promoting vaccine-induced antibody generation (Wiertsema et al. 2006).Regarding its role in terms of mediating IVIg efficacy, it has to be noted that although CD32a is known to be suppressed by IVIg, our data and a previous study indicate that this mechanism of action does not seem to play a major role in the pathogenesis in CIDP (Quast et al. 2015).Our observation that none of our patients progressed or relapsed upon the vaccination further supports this conclusion.
Furthermore, other markers for IVIg-immunomodulation in CIDP, like increasing CD32b expression on classical monocytes (Tackenberg et al. 2009) and a decrease of myeloid dendritic cells (Dyer et al. 2016), were not altered after COVID-19 vaccination, further supporting the assumption that COVID-19 mRNA vaccine does not have a relevant impact on immunomarkers for CIDP.
To our knowledge, this study is also the first that explored potential changes in serum cytokine levels subsequent to the vaccination with a COVID-19 mRNA vaccine.Our findings that most of the assessed cytokines showed comparable expression profiles in paired serum samples before and after vaccination further support the notion that the vaccine overall does not impact markers of systemic inflammation.The slightly stronger increase of IL-6 serum levels post IVIg after COVID-19 vaccination could reflect a vaccine-specific immune response (Jordan 2021).This hypothesis is indirectly supported by a recent study that demonstrated that patients under anti-IL6 treatment showed lower antibody responses to COVID-19 mRNA vaccine (Picchianti-Diamanti et al. 2021).Whether our exploratory findings are also transferable to other autoimmune diseases has to be explored by future studies, and recent reviews focusing on COVID-19 vaccination and its potential to influence the course of Guillain-Barré syndrome or systemic lupus erythematosus demonstrate that such studies would raise potential interest (Mason et al. 2021;Lahoz Fernandez et al. 2022).Furthermore, vaccine selection decision-making models might be useful to minimize adverse vaccine effects in patients with autoimmune diseases, as they present with different host factors and a possibly different susceptibility to adverse immunological side effects than the average population (Abdelwahab et al. 2021;Mason et al. 2021).
A recent study demonstrated that anti-SARS-CoV-2 antibody generation is not directly altered by IVIg in CIDP, and that a sufficient vaccine efficacy is assumable in CIDP patients, although CIDP-specific immunological host factors have to be considered (Svačina et al. 2022).
Our study has several limitations, as the small cohort size of eleven patients only provides first exploratory data about the interaction of COVID-19 mRNA vaccine with immunomarkers of CIDP and IVIg-immunomodulation, that will require confirmation by larger follow-up studies also including untreated CIDP patients or unvaccinated IVIg recipients as control cohorts, as well as longitudinal data about the effects of booster vaccinations, that were lacking in our study.Secondly, as COVID-19 mRNA vaccine prevents SARS-CoV-2 from entering human cells via the angiotensin-converting enzyme 2 receptor (ACE2R; Gattinger et al. 2022), and as data about a differential expression of ACE2R in CIDP and healthy subjects, that could generally influence vaccine efficacy in CIDP, are currently lacking, future studies also will have to elucidate ACE2R expression in CIDP.Thirdly, our study did not evaluate the response of CD8-positive cyctotoxic T cells or plasma cells to COVID-19 mRNA vaccine due to its focus on distinct immunomarkers of CIDP.As cyctotoxic T cells and plasma cells play an important role by executing vaccine-induced SARS-CoV-2 immunity (Ssemaganda et al. 2022), future studies should also focus on vaccine-induced alterations of these cell populations in CIDP.On the other hand, our study evaluated CD32a/b expression on B cells, that either enhance or reduce plasma cell-mediated antibody generation (Karnell et al. 2014), therefore a concordance of post-vaccine plasma cell alterations to the CD32 receptor expression patterns observed in our study appears assumable.
In summary, considering the exploratory approach of our study and all its limitations, our study suggests that neither the examined immune mechanisms in CIDP nor key immunomodulatory pathways of IVIg are significantly altered two weeks after full COVID-19 immunization, which is a timepoint assumed to be safe for post-vaccine IVIg administration by expert opinions, but was so far lacking confirmative data (Doneddu et al. 2021).

Fig. 1
Fig. 1 Alterations of immune cell fractions after IVIg administration before and after COVID-19 vaccination.CD32b expression on naïve B cells is significantly reduced after IVIg administration and after COVID-19 vaccination (A).Contrariwise, CD32a expression on naïve and memory B cells increases after COVID-19 vaccination (B,C).IVIg administration significantly decreases CD19 + CD27 + memory B cells in an unvaccinated state, but not after COVID-19 vaccination (D).CD32b expression on classical monocytes is increased after IVIg administration and vaccination (E).IVIg administration tends to reduce CD11c + myeloid dendritic cells, with a significantly reduced cell amount after COVID-19 vaccination (F).Blue arrow = Second dose of COVID-19 mRNA vaccine.* p < 0.05, **p < 0.01

Fig. 2
Fig.2Alterations of serum cytokine concentrations after IVIg administration and COVID-19 vaccination.The relative increase of IL-6 concentrations after IVIg administration is significantly higher after COVID-19 vaccination than in an unvaccinated state, whereas the absolute concentrations remain stable (A).IL-10 concentrations significantly increase after IVIg administration and COVID-19 vaccination, but no significant difference in relative increase is seen after