Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving standard of care therapy

Vaccine xxx (2017) xxx–xxx

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Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving standard of care therapy Per Nived a,b,⇑, Johanna Nagel a, Tore Saxne a, Pierre Geborek a, Göran Jönsson c, Lillemor Skattum d, Meliha C. Kapetanovic a a

Department of Clinical Sciences Lund, Section of Rheumatology, Lund University, Skåne University Hospital, SE-221 85 Lund, Sweden Department of Infectious Diseases, Central Hospital Kristianstad, J A Hedlunds väg 5, SE-291 85 Kristianstad, Sweden Department of Clinical Sciences Lund, Section of Infectious Diseases, Lund University, Skåne University Hospital, SE-221 85 Lund, Sweden d Department of Laboratory Medicine, Section of Microbiology, Immunology and Glycobiology, Lund University, Lund, and Clinical Immunology and Transfusion Medicine, Region Skåne, Sweden b c

a r t i c l e

i n f o

Article history: Received 8 January 2017 Received in revised form 10 May 2017 Accepted 16 May 2017 Available online xxxx Keywords: Systemic vasculitis Pneumococcal vaccination Pneumococcal conjugate vaccine

a b s t r a c t Aim: To study the effect of standard of care therapy on antibody response and functionality following immunization with 13-valent pneumococcal conjugate vaccine (PCV13) in patients with primary systemic vasculitis compared to healthy controls. Methods: 49 patients with vasculitis and 49 controls received a single dose (0.5 ml) PCV13 intramuscularly. Ongoing treatments: azathioprine (AZA; n = 11), cyclophosphamide (CYC; n = 6), methotrexate (MTX; n = 9), rituximab (n = 3); anti-TNF (n = 2), mycophenolate mofetil (n = 2), prednisolone alone (n = 15) and no active treatment (n = 2). Specific antibody concentrations for serotypes 6B and 23F were determined using ELISA and opsonophagocytic activity (OPA) assay (23F) was performed, on serum samples taken immediately before and 4–6 weeks after vaccination. Proportion of individuals with putative protective antibody concentration (1.0 µg/mL) and positive antibody response (2-fold increase from prevaccination concentration) for both serotypes were calculated and groups were compared. Results: At baseline, 6 patients (12%) and 12 controls (24%) had protective antibody levels for both serotypes. After vaccination, antibodies increased for both serotypes in patients and controls (p < 0.001), 32 patients (65%) and 35 controls (71%) reached protective level for 6B, and 32 patients (65%) and 37 controls (76%) for 23 F. Compared to controls, patients had lower prevaccination geometric mean concentration (23F, p = 0.01) and a numerical trend towards lower prevaccination level (6B) and postvaccination levels (both serotypes). Patients with prednisolone alone had lower prevaccination OPA (p < 0.01) compared to controls. OPA increased after vaccination in both patients and controls (p < 0.001), but improvement was better in controls (p = 0.001). AZA, CYC or MTX, but not prednisolone alone, tended towards a lower proportion of patients reaching protective antibody levels (p = 0.06), compared to controls. Conclusions: Pneumococcal conjugate vaccine was safe and immunogenic in patients with established vasculitis. Treatment with DMARDs, mostly AZA, CYC and MTX but not systemic prednisolone may impair antibody response. Trial registration. ClinicalTrials.gov Identifier: NCT02240888. Registered 4 September, 2014 Ó 2017 Published by Elsevier Ltd.

Abbreviations: PPV23, pneumococcal polysaccharide vaccine; PCV7, pneumococcal conjugate vaccine; IPD, invasive pneumococcal disease; ANCA, antineutrophil cytoplasmic antibody; PR3-ANCA, ANCA directed against proteinase 3; MPO-ANCA, ANCA directed against myeloperoxidase; P-ANCA, perinuclear/nuclear staining; GMC, geometric mean Ab concentrations; ARR, antibody response ratio; ELISA, enzyme-linked immunosorbent assay; OPA, opsonophagocytic activity; DMARDs, disease modifying anti-rheumatic drugs; MTX, methotrexate; AZA, azathioprine; CYC, cyclophosphamide; anti-TNF, tumour necrosis factor inhibitors. ⇑ Corresponding author at: Department of Infectious Diseases, Central Hospital Kristianstad, J A Hedlunds väg 5, SE-291 85 Kristianstad, Sweden. E-mail address: [email protected] (P. Nived).

1. Introduction Modern immunosuppressive therapy greatly improves the survival of patients with systemic vasculitis, but comes at the expense of potentially serious infectious complications [1]. Infection is the most common cause of death within the first year of diagnosis in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, and remains a major cause of both mortality and morbidity several years after disease onset [2–4]. Vaccines play an important role in

http://dx.doi.org/10.1016/j.vaccine.2017.05.044 0264-410X/Ó 2017 Published by Elsevier Ltd.

Please cite this article in press as: Nived P et al. Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving standard of care therapy. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.044

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the prevention of such infections, but there is concern that immunosuppressive therapy may lead to impaired immune responses. Streptococcus pneumoniae (pneumococcus) is one of the leading vaccine-preventable causes of serious infections, e.g. pneumonia, sepsis and meningitis. Invasive pneumococcal disease (IPD) is defined as an infection confirmed by the isolation of pneumococci from a normally sterile site (e.g. the bloodstream). Immunocompromised patients have both increased incidence and mortality of IPD, compared to healthy controls [5,6], and in a recent study from Canada, persons immunocompromised by underlying disease or treatment comprised 28% of all IPD cases [7]. Elevated risks of IPD have been demonstrated in patients previously hospitalized with immune-mediated diseases, for example a 5 times greater risk in patients with polyarteritis nodosa than an age-matched healthy population [8]. In patients with giant cell arteritis, the rate of severe infections during the first year after diagnosis was doubled, compared to an age and sex-matched general population, and a prednisolone dose of >10 mg/day after 1 year of treatment was associated with increased infectionrelated mortality (hazard ratio 4.6) [9]. Twenty-three-valent pneumococcal polysaccharide vaccine (PPV23) consists of capsular polysaccharides of 23 common IPDcausing serotypes, and in pneumococcal conjugate vaccines (PCVs) a subset of these (e.g. 13 serotypes in PCV13) have been covalently linked to a carrier protein, consisting of a non-toxic mutant of the diphtheria toxin. In contrast to the T cell-independent reactions caused by pneumococcal polysaccharide vaccine (PPV), PCV has been demonstrated to elicit T cell-dependent immune reactions stimulating the proliferation of memory B cells, thus improving immunological memory [10]. In a large trial from the Netherlands, immunocompetent adults (65 years of age) received either PCV13 or placebo and a 75% efficacy of PCV13 in preventing IPD was shown [11]. Little is known about the immunogenicity and efficacy of pneumococcal vaccination using pneumococcal conjugate vaccine in immunocompromised persons. Randomized trials of HIV-infected patients in Malawi and Uganda, have indicated that PCV7, but not PPV23, was effective in preventing IPD [12,13]. In the United States, the Centers for Disease Control and Prevention (CDC) recommends that adults with immunocompromising conditions, asplenia, cochlear implants and cerebrospinal fluid leaks, receive immunization with a dose of PCV13, followed in at least 8 weeks by a dose of PPV23 [14]. Our group have previously reported that antibody responses to PPV23 [15] and 7-valent pneumococcal conjugate vaccine (PCV7) [16] are impaired in chronic arthritis patients during treatment with methotrexate (MTX), but not with tumour necrosis factor (TNF) inhibitor monotherapy. We did not observe any difference in the immunogenicity of PCV7 compared to PPV23, in patients with rheumatoid arthritis treated with immunomodulating drugs [17]. Morgan et al. recently reported that patients with systemic vasculitis were safely vaccinated with PCV7, but vaccine responses were highly variable with a median antibody response rate of 46% of patients responding to each antigen, using the threshold 0.35 µg/ mL [18]. To date there is little knowledge regarding the immunogenicity of PCV13 in individuals with systemic vasculitis. The aim of this study was to investigate the effect of standard of care therapy on antibody (Ab) response following immunization using 13valent pneumococcal conjugate vaccine (PCV13) in patients with systemic vasculitis compared to healthy controls.

2. Material and methods 2.1. Patient inclusion Adult patients who had established systemic vasculitis diagnoses and were regularly monitored at the Department of

Rheumatology in Lund and Malmö at Skåne University Hospital were eligible for this study. Patients had to fulfil the American College of Rheumatology criteria for different systemic vasculitides [19]. Ongoing treatment at the time of vaccination was noted as a basis for later patient stratification. Patients were excluded from the study if anti-rheumatic treatment had been changed within 4 weeks before and 6 weeks after vaccination, if they had been previously vaccinated with PPV23 within 1 year, had a history of allergic reaction at previous vaccinations, were pregnant, or had an ongoing infection. Healthy control subjects were recruited from the staff and relatives at the department of Rheumatology in Lund. 2.2. Vaccination protocol All participants received a single 0.5 mL dose of PCV13 (Prevenar 13Ò, Pfizer) administered as an intramuscular injection in the deltoid muscle by a rheumatology nurse. At the time of vaccination, a clinical examination was performed by a rheumatologist and data were collected on disease and treatment characteristics and previous vaccinations using a structured protocol. All patients were encouraged to monitor and report possible adverse or unexpected effects of the vaccination, as well as changes in rheumatic disease. Adverse events (AEs) and adverse drug reactions (ADRs) were recorded according to the Guideline for Good Clinical Practice and Clinical Safety Data Management [20]. 2.3. Pneumococcal serotype-specific IgG measurement Serum samples were collected immediately before and 4– 6 weeks after vaccination. Serotype-specific IgG antibody concentrations for pneumococcal serotypes 6B and 23F, both included in PCV13, were quantified using enzyme-linked immunosorbent assay (ELISA) meeting World Health Organization (WHO) standard, described previously [21]. The method was executed with two minor modifications. In short, ELISA plates were coated with 1 µg pneumococcal capsular polysaccharides (PS) 6B or 23F. In order to diminish nonspecific binding to capsular PS, dilutions of human sera were absorbed with pneumococcal cell wall PS, and then added to the ELISA plates. In contrast to the WHO protocol, 22F PS was not used for absorption. Goat anti-human IgG antibodies, conjugated with alkaline phosphatase, followed by addition of the substrate, nitrophenyl phosphate, were used for the detection of serotype-specific antibodies (anti-6B and anti-23F IgG). The optical density, proportional to the amount of anti-6B and anti23F IgG present in the serum, was measured with an ELISA plate reader at 405 nm. The assay was calibrated with international reference serum 89SF, that was kindly provided by Dr. C. Frasch, Bethesda, MD, USA [22]. This is also a modification to the latest WHO protocol which utilizes reference serum 007sp [21]. The lower limit of detection was 0.01 mg/L. 2.4. Opsonophagocytic activity (OPA) assay OPA assay was performed for pneumococcal serotype 23F. The method has been described by Martinez et al. [23] and was executed with some modifications. The analysis was conducted with samples from 48 systemic vasculitis patients, and 36 healthy controls included in this study. Pneumococci of serotype 23F obtained from Statens Serum Institut in Copenhagen, were cultured, killed by addition of glutaraldehyde and subsequently frozen in aliquots as described previously [24]. Killed bacteria were thawed and incubated for 20– 30 min in the dark with FITC (fluorescein isothiocyanate; F7250, Sigma-Aldrich, St. Louis, MO, USA) in sodium carbonate buffer, subsequently washed 3 times in VBS-CaMg (veronal-buffered saline with 0.15 mM Ca2+ and 0.5 mM Mg2+).

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FITC-labeled bacteria (5  107/mL), 20 µL suspended in VBSCaMg were incubated with 10 µL of heat-inactivated patient or control serum (prediluted 1/16 in VBS-CaMg) for 30 min at 37 °C. Subsequently 20 µL of baby rabbit serum (CL3441, Cedarlane, USA) was added and incubation was continued for 30 min at 37 °C. Polymorphonuclear neutrophils from healthy donors, obtained as previously described [24] were preincubated with PE (Phycoerythrin)-labeled anti-CD66 (551480, BD Biosciences, Franklin Lakes, NJ, USA) and subsequently added to the opsonized bacteria at a final concentration of 800 cells/mL. After incubation for 30 min at 37 °C, cells were analysed with BD Accuri C6 flow cytometry (BD Biosciences). Results were expressed as percentage of cells (PE positive events) with significant uptake of bacteria (events positive for both PE and FITC). Inter-assay variation was compensated for by adjusting values to the mean value of a serum with high opsonizing ability, included in each analysis. A negative control consisting of bacteria preincubated only with BSA and no serum was also included in each analysis.

2.5. Statistical analysis Differences between groups were analysed using the Chi-square test and the Mann-Whitney U test when appropriate. Geometric mean Ab concentrations (GMCs) and 95% confidence intervals were calculated from log-transformed values. Pre- and post-PCV13 GMCs for all patients and controls were compared using paired samples t-test. Pre- to postvaccine OPA and antibody changes in subgroups were compared using Wilcoxon signed rank test. Differences in GMCs between all patients and controls were analysed using independent samples t-test. To study the impact of different anti-inflammatory treatments, we used the antibody response ratio (ARR, i.e., the ratio of post- to prevaccination antibody levels). A positive antibody response was defined as an ARR of 2. Proportions of individuals with positive antibody responses and postvaccination Ab concentrations 1.0 µg/mL (putative protective level)

with 95% confidence intervals (95% CI) were calculated for both serotypes. Proportions of patients with putative protective levels pre- and postvaccination (matched pairs) were compared using McNemars test. Correlation between OPA and ELISA titers were calculated using Spearman’s rank correlation test. Statistical calculations were performed using IBMÒ SPSSÒ version 23 and diagrams were drawn using Graphpad PrismÒ version 6.

3. Results Demographics, diagnoses, disease characteristics, treatment and prior pneumococcal vaccinations are shown in Table 1. Forty-nine patients and 49 controls entered the study, and none were lost to follow-up. Patients were slightly older than controls; median age 57 years vs 65 years (p = 0.001). Patients were diagnosed with Granulomatosis with polyangiitis (GPA) (n = 21), Eosinophilic granulomatosis with polyangiitis (EGPA) (n = 8), Giant cell vasculitis (n = 13), Takayasu’s disease (n = 4) or other vasculitis (n = 3). Ongoing treatment (unchanged since at least 4 weeks) was azathioprine (AZA with/without prednisolone, n = 11), MTX (with/ without prednisolone, n = 9), cyclophosphamide (CYC with prednisolone, n = 6), mycophenolate mofetil (with prednisolone, n = 2), prednisolone (without disease-modifying anti-rheumatic drugs [DMARDs], n = 15), rituximab (with DMARDs and prednisolone, n = 3), TNF inhibitors (with/without DMARD and prednisolone, n = 2). One patient had no active treatment. Forty-three patients (88%) were treated with glucocorticoids, i.e. oral prednisolone with median dose 10 (range 2.5–65) mg per day. Patients receiving biologic treatments were excluded from subgroup analyses because of small numbers. The remaining patients were stratified into two groups based on their treatment, as follows: Patients receiving MTX, AZA or c CYC, with or without prednisolone, Group 1, n = 26); Patients receiving prednisolone (without DMARDs, n = 15, Group 2). Median age did not differ between group 1 and controls, but group 2 were older than con-

Table 1 Demographic, diagnoses, disease characteristics, treatments and previous pneumococcal vaccination at the time of vaccination.a All patients (n = 49)

Group 1 (n = 26)

Group 2 (n = 15)

Controls (n = 49)

Age, median (range) years Sex, % females

65 (22–85) 51

61 (26–85) 50

70 (56–85) 40

57 (17–85) 63

Diagnoses, n (%): Granulomatosis with polyangiitis (GPA) Eosinophilic granulomatosis with polyangiitis (EGPA) Giant cell vasculitis Takayasu arteritis Other vasculitis

21 (43) 8 (16) 13 (27) 4 (8) 3 (6)

16 (62) 5 (19) 0 2 (8) 2 (8)

1 (7) 1 (7) 13 (87) 0 0

– – – – –

Disease characteristics: Disease duration, mean (range) years C-reactive protein, median (range) mg/L Erythrocyte sedimentation rate, median (range) mm/h

5.1 (0–42) 2.3 (0–141) 10 (2–80)

5.8 (0–42) 2.2 (0–84) 10 (2–44)

2.5 (0–18) 5.4 (0–141) 14.5 (6–80)

– 0.8 (0–12) –

Ongoing treatments, n (%): Azathioprine (AZA) Cyclophosphamide (CYC) Methotrexate (MTX) Mycophenolate mofetil Prednisolone Rituximab TNF inhibitor No active treatment Prednisolone dose, median (range) mg/day

11 (22) 6 (12) 9 (18) 2 (4) 42 (86) 3 (6) 2 (4) 2 (4) 7.5 (0–65)

11 (42) 6 (23) 9 (35) 0 22 (85) 0 0 0 5 (0–60)

0 0 0 0 15 (100) 0 0 0 12.5 (2.5–65)

– – – – – – – – –

Previous vaccinations: PPV23, n (%)

4 (8)

2 (8)

1 (7)

3 (6)

a

Group 1 = Vasculitis patients receiving methotrexate (MTX), azathioprine (AZA) or cyclophosphamide (CYC) (with or without prednisolone and without biologics). Group 2 = Vasculitis patients receiving prednisolone (without DMARDs).

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3.1. Vaccine safety

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Pre- to postvaccination serotype-specific antibody geometric mean concentrations (GMCs) increased for 6B and 23F, in both patient and control groups (p < 0.001, Fig. 1). Postvaccination GMC (95% C.I.) for 6B and 23F were respectively, in patients 1.7 (0.9–2.9) µg/mL and 1.8 (1.2–2.8) µg/mL and in controls 3.1 (1.9– 5.0) µg/mL and 3.3 (2.0–5.5) µg/mL. In patient subgroups 1 and 2, pre- to postvaccine antibody increases were found for both serotypes. Patients had lower antibody levels, i.e. GMCs, for 23F prevaccination (p = 0.01) and numerically but not statistically significant lower prevaccination level for 6B and postvaccination levels for both 6B and 23F, compared to controls. 3.3. Putative protective levels

(n =4 9)

Geometric mean concentration ( g/mL) (95% CI)

B

A

The vaccine was safe and generally well tolerated. In total, 13 patients and 16 controls experienced some pain or tenderness around the injections site, redness, or headache lasting for a few days. A few patients described slightly increased body temperature and one patient reported intensive general muscle pain and difficulties to move the upper arm where the vaccine was injected but these symptoms resolved completely. An additional patient reported a transitional reduction of symptoms of his vasculitis lasting for 1.5 days.

0

9)

Geometric mean concentration ( g/mL) (95% CI)

A

Fig. 1. Pre- and postvaccination antibody levels (geometric mean concentrations; 95% CI) for serotype 6B (A) and 23F (B) (* = p  0.05, ** = p  0.01, *** = p  0.001).

Prior to vaccination, the proportions of patients with antibody concentrations 1.0 µg/mL (95% C.I.) were: 31% (17–44) for 6B and 22% (10–35) for 23F. The proportions of patients with putative protective levels, for 6B and 23F, increased pre- to postvaccination for all vasculitis patients, subgroups and controls (Table 2). After vaccination, the proportions reaching protective level for 6B and 23F (95% C.I.), respectively, were 65% (51–79) for both serotypes in the patient group, compared to 71% (58–85) and 76% (63–88) in the control group. Two of the 3 patients treated with rituximab did not respond to vaccination, i.e. postvaccination levels for both serotypes were below 1.0 µg/mL. For both serotypes combined, group 1 compared to controls, tended towards a lower proportion of individuals reaching antibody concentrations 1.0 µg/mL (p = 0.06, Fig. 2). In contrast, there was no difference between group 2 and controls. 3.4. Antibody response ratios

trols (p = 0.003). In group 1, 81% of patients were diagnosed with either GPA or EGPA, in contrast to the predominant diagnosis of group 2, i.e. giant cell vasculitis in 87% of patients.

Although numerically lower, no significant differences were found in the proportions of individuals with a positive antibody response (i.e. 2-fold increase from prevaccination levels),

Table 2 Proportions of individuals with putative protective antibody levels (1.0 µg/mL) pre- and postvaccination. All patients (n = 49)

Group 1 (n = 26)

Group 2 (n = 15)

Controls (n = 49)

Pneumococcal serotype 6B: Pre-PCV13, mean % (95% CI) Post-PCV13, mean % (95% CI) P of difference

31 (17–44) 65 (51–79) < 0.001

31 (12–50) 62 (42–82) 0.039

33 (6–60) 73 (48–99) 0.031

37 (23–51) 71 (58–85) < 0.001

Pneumococcal serotype 23F: Pre-PCV13, mean % (95% CI) Post-PCV13, mean % (95% CI) P of difference

22 (10–35) 65 (51–79) < 0.001

23 (6–40) 65 (46–85) 0.001

27 (1–52) 73 (48–99) 0.039

37 (23–51) 76 (63–88) < 0.001

Both serotypes: Post-PCV13, mean % (95% CI)

49 (34–63)

42 (22–63)

60 (32–88)

61 (47–75)

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postvaccination OPA were lower in patients (6.7%) compared with controls (31.9%, p = 0.001). Patients on DMARDs (group 1) had lower postvaccination OPA compared with controls (p = 0.043). Patients treated with prednisolone alone (group 2) had lower both pre- and postvaccination OPA compared with controls (p = 0.007 and p = 0.002). 3.6. Correlations between ELISA IgG and OPA After vaccination, there were significant correlations between serotype 23F antibody levels measured by ELISA and OPA percent uptake, in both patients (correlation coefficient = 0.33, p = 0.02) and controls (correlation coefficient = 0.54, p = 0.001). Correlation between pre- to postvaccination difference in ELISA and OPA for all patients and controls is shown in Fig. 4B. 4. Discussion

Fig. 2. Proportion of individuals with putative protective antibody response (i.e. 1.0 µg/mL) for both serotypes (6B and 23F) after vaccination. (a: p = 0.06).

between all patients, group 1, group 2 and controls, for 6B, 23F or both serotypes combined (Fig. 3).

3.5. Opsonophagocytic activity (OPA) Both controls (n = 36) and patients (n = 48) showed a significant increase in OPA with serotype 23F after vaccination (both p < 0.001, Fig. 4A). Median prevaccination OPA were numerically lower in patients (0.0%) compared to controls (6.2%, ns). Median

Fig. 3. Proportion of individuals with positive antibody response (i.e. 2-fold increase in pre- to postvaccination antibodies) for both serotypes.

In this study, we found that pneumococcal conjugate vaccine was safe and immunogenic in patients with systemic vasculitis receiving standard of care therapy. In other words, significant pre- to postvaccination increases were shown in both serotypespecific antibody titers and in the proportions of patients reaching putative protective antibody levels (i.e. 1.0 µg/mL), for pneumococcal serotypes 6B and 23F. Our results are in agreement with the previous study by Morgan et al., which showed significant improvement of antibodies above threshold 0.35 µg/mL after PCV7 vaccination in patients with systemic vasculitis in remission [18]. Patients receiving MTX, AZA or CYC may have impaired antibody responses to PCV, indicated by the lower proportion of this group reaching putative protective levels after vaccination compared to controls. Most patients (85%) in this treatment group were diagnosed with GPA, EGPA or MPO-vasculitis. In contrast, for patients with glucocorticoid treatment (without DMARDs), most of them diagnosed with giant cell arteritis (87%), the proportion of patients reaching Ab levels 1.0 µg/mL after vaccination, was not reduced compared to controls. We found no correlation between antibody response ratios and prednisolone dose. This is in line with results from our previous study reporting that ongoing prednisolone treatment did not impair the immune response to pneumococcal polysaccharide vaccine in patients with rheumatoid arthritis [15]. Although we found significant increases in OPA and correlations between ELISA and OPA, the correlation was weaker and postvaccination improvement of OPA was lower in patients compared to controls. Notably, monotherapy with prednisolone resulted in lower pre- and postvaccination OPA, probably a reflection of both higher age and higher dosage of prednisolone in this patient group. To our knowledge, this is the first study of both antibody response and functionality of antibodies to pneumococcal conjugate vaccination in patients with systemic vasculitis. Since this patient group is particularly vulnerable to severe infections, e.g. invasive pneumococcal disease, the prevention of these infections should be a priority. Generally, the optimal approach is to vaccinate well before the initiation of immunosuppressive treatment, but for patients with rapidly progressive necrotizing vasculitis the timing is often an issue. The clinical implication of this study is that PCV can elicit adequate immune responses in patients with systemic vasculitis. Despite the lower antibody levels in patients, all should be encouraged to have pneumococcal vaccination, since significant improvements were seen in all groups. Further strengths of this study were the use of different approaches to evaluate antibody responses, i.e. increases in antibody titres, proportions of patients with 2-fold increase, those reaching above a putative protective threshold and analysis of

Please cite this article in press as: Nived P et al. Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving standard of care therapy. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.044

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A 100 p<0.001

p<0.001

80

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ns

60 p=0.001

40

20

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ro ls

nt Po

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ac

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Spearman r r0,4697 95% confidence interval 0,2863 to 0,6199

60

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40

20

-50

50

100

150

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Fig. 4. (A) Proportion of phagocytes with uptake of pneumococcal serotype 23F. (B) Correlation between serotype 23F post-prevaccination ELISA (µg/mL) and phagocytosis (percentage change).

functionality of these antibodies with OPA. The fact that only a few patients and controls, 4 and 3 individuals respectively, had received PPV23 (all at least a year prior to this study), minimized the previously described risk of hyporesponse when PCV13 is administered within a year of PPV23 vaccination [25]. Furthermore, no patients or controls were lost to follow-up. An obvious limitation to this study is that serotype-specific antibodies were only determined for two of the PCV13 serotypes, i.e. 6B and 23F. Compared to the other vaccine serotypes, the antigen amount of 6B is double in PCV13, and a lower immunogenicity of this serotype has been described in several studies [26]. However, pneumococcal serotypes 6B and 23F were among the seven most common IPD-causing serotypes in the population of Scania, Sweden, found in 436 pneumococcal blood isolates during 2009– 10 (Personal communication with the Department of Microbiology, Lund). In this study, the use of a flow cytometric-type OPA assay, although a validated method, may be considered a limitation

compared to the killing-type assay which has become the standard method for vaccine evaluations [27]. Because OPA was used to compare functionality of antibodies in different groups, differences between killing-type OPA and the flow-cytometric OPA used by us may be considered a minor issue. Also, OPA was only determined for one serotype (i.e. 23F). Although the use of a single serotype is suboptimal, the authors are not aware of any evidence to contradict the assumption that the response to serotype 23F is representative of the response to the different PCV13 serotypes. In the clinical trials preceding the licensure of PCV13, similar OPA titres (all non-inferior to PPV23) were found for the 12 serotypes in common with PPV23 [28]. The age difference between patients and controls could possibly lead to an underestimation of vaccine responses in the patient group. The age difference was most pronounced in the prednisolone only group, but in this group vaccine responses were comparable to controls. Further the study was limited by heterogeneity of diagnoses and treatments, and it was not

Please cite this article in press as: Nived P et al. Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving standard of care therapy. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.044

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possible to draw reliable conclusions on small treatment subgroups. The use of a putative protective threshold to evaluate pneumococcal immune responses is a controversial subject. The World Health Organization (WHO) has recommended a protective threshold 0.35 µg/mL of serotype-specific IgG following conjugate immunization in children [21], but the relevance of this threshold for protection against invasive pneumococcal disease (IPD) in adults is unclear. Pneumococcal antibody levels have been shown to increase with age and inversely the incidence of IPD decreases, except for the elderly; although the proportion of older people with antibody levels 0.35 µg/mL remains high, the incidence of IPD is elevated compared to young adults [29]. Using the putative protective threshold 1.0 µg/mL may be more appropriate in adults [30]. We have previously seen that patients with more robust antibody responses (i.e. >1.0 µg/mL) after vaccination with pneumococcal conjugate vaccine were less likely to suffer from serious infections [31]. In addition, measuring vaccine serotypespecific IgG concentrations do not necessarily represent the immunity in vivo, and should be considered surrogate markers in the absence of real efficacy data, i.e. the ability of a vaccine to prevent disease. Romero-Steiner et al. have shown that opsonophagocytic activity, but not IgG concentrations, was reduced in elderly individuals (median age 85.5 years) [30]. A recent study found correlation between OPA and ELISA in healthy controls (median age 68 years) but not in patients with multiple myeloma (median age 80 years) [32]. Although we found correlation between OPA and ELISA, it was weaker and postvaccine OPA were lower in patients compared to controls, in contrast to comparable antibody levels. 5. Conclusion Pneumococcal conjugate vaccine is safe and immunogenic in patients with systemic vasculitis receiving standard of care therapy but immune response may be impaired in patients treated with AZA, CYC or MTX. However, immunosuppressed vasculitis patients previously not immunised against pneumococci should be encouraged to be vaccinated. Further studies are needed to evaluate the effect of immunosuppressive therapy and the possible benefit of booster doses. Declarations Ethics approval and consent to participate The study was approved by the regional ethics committee at Lund University, Sweden (Dnr 2011/341). Consecutive patients fulfilling inclusion criteria were invited to participate in the study. Oral and written information was provided to all subjects who were invited to participate, and written consent was obtained from each participant before enrolment. Consent for publication Not applicable. Availability of data and material The datasets for the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests.

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Funding The study was supported by grants from the Swedish Rheumatism Association, the Medical Faculty of Lund University, Alfred Österlund´s Foundation, The Crafoord Foundation, Greta and Johan Kock´s foundation, The King Gustaf V Foundation, Lund University Hospital, Inger Bendix Foundation, Apotekare Hedbergs Foundation and Anna-Greta Crafoord Foundation. Authors’ contributions The study was conceived by MCK, TS, PG, GJ, LS, JN and PN. PN wrote the manuscript and all authors have revised it critically for important intellectual content. All authors have approved the final version of the manuscript for submission. Acknowledgments This study could not have been conducted without the excellent contributions of the nurses in the clinic of Rheumatology at Skåne University Hospital. References [1] Smith RM, Jones RB, Jayne DRW. Progress in treatment of ANCA-associated vasculitis. Arthritis Res Ther 2012;14:210. http://dx.doi.org/10.1186/ar3797. [2] Flossmann O, Berden A, de Groot K, Hagen C, Harper L, Heijl C, et al. Long-term patient survival in ANCA-associated vasculitis. Ann Rheum Dis 2011;70:488–94. http://dx.doi.org/10.1136/ard.2010.137778. [3] McGregor JG, Negrete-Lopez R, Poulton CJ, Kidd JM, Katsanos SL, Goetz L, et al. Adverse events and infectious burden, microbes and temporal outline from immunosuppressive therapy in antineutrophil cytoplasmic antibodyassociated vasculitis with native renal function. Nephrol Dial Transplant 2015;30(Suppl 1):i171–81. http://dx.doi.org/10.1093/ndt/gfv045. [4] Goupil R, Brachemi S, Nadeau-Fredette A-C, Déziel C, Troyanov Y, Lavergne V, et al. Lymphopenia and treatment-related infectious complications in ANCAassociated vasculitis. Clin J Am Soc Nephrol 2013;8:416–23. http://dx.doi.org/ 10.2215/CJN.07300712. [5] Klemets P, Lyytikäinen O, Ruutu P, Ollgren J, Nuorti JP. Invasive pneumococcal infections among persons with and without underlying medical conditions: implications for prevention strategies. BMC Infect Dis 2008;8:96. http://dx.doi. org/10.1186/1471-2334-8-96. [6] van Hoek AJ, Andrews N, Waight PA, Stowe J, Gates P, George R, et al. The effect of underlying clinical conditions on the risk of developing invasive pneumococcal disease in England. J Infect 2012;65:17–24. http://dx.doi.org/ 10.1016/j.jinf.2012.02.017. [7] Shigayeva A, Rudnick W, Green K, Chen DK, Demczuk W, Gold WL, et al. Invasive pneumococcal disease among immunocompromised persons: implications for vaccination programs. Clin Infect Dis 2016;62:139–47. http://dx.doi.org/10.1093/cid/civ803. [8] Wotton CJ, Goldacre MJ. Risk of invasive pneumococcal disease in people admitted to hospital with selected immune-mediated diseases: record linkage cohort analyses. J Epidemiol Commun Health 2012;66:1177–81. http://dx.doi. org/10.1136/jech-2011-200168. [9] Schmidt J, Smail A, Roche B, Gay P, Salle V, Pellet H, et al. Incidence of severe infections and infection-related mortality during the course of giant cell arteritis: a multicenter, prospective, double-cohort study. Arthritis Rheumatol (Hoboken, NJ) 2016;68:1477–82. http://dx.doi.org/10.1002/art.39596. [10] Clutterbuck EA, Lazarus R, Yu L-M, Bowman J, Bateman EAL, Diggle L, et al. Pneumococcal conjugate and plain polysaccharide vaccines have divergent effects on antigen-specific B cells. J Infect Dis 2012;205:1408–16. http://dx. doi.org/10.1093/infdis/jis212. [11] Bonten MJM, Huijts SM, Bolkenbaas M, Webber C, Patterson S, Gault S, et al. Polysaccharide conjugate vaccine against pneumococcal pneumonia in adults. N Engl J Med 2015;372:1114–25. http://dx.doi.org/10.1056/NEJMoa1408544. [12] French N, Gordon SB, Mwalukomo T, White SA, Mwafulirwa G, Longwe H, et al. A trial of a 7-valent pneumococcal conjugate vaccine in HIV-infected adults. N Engl J Med 2010;362:812–22. http://dx.doi.org/10.1056/NEJMoa0903029. [13] French N, Nakiyingi J, Carpenter LM, Lugada E, Watera C, Moi K, et al. 23-valent pneumococcal polysaccharide vaccine in HIV-1-infected Ugandan adults: double-blind, randomised and placebo controlled trial. Lancet 2000;355:2106–11. [14] Centers for Disease Control and Prevention (CDC). Use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine for adults with immunocompromising conditions: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Morb Mortal Wkly Rep 2012;61:816–9. [15] Kapetanovic MC, Saxne T, Sjöholm A, Truedsson L, Jönsson G, Geborek P. Influence of methotrexate, TNF blockers and prednisolone on antibody

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Please cite this article in press as: Nived P et al. Immune response to pneumococcal conjugate vaccine in patients with systemic vasculitis receiving standard of care therapy. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.044