Because vaccination against Streptococcus pneumoniae is recommended in patients with immunodeficiency or chronic diseases (1, 2), we recently addressed the question of how effective vaccination was in kidney transplant recipients with clinically stable condition (3). This question is worth considering because most mortality associated with the severe influenza pandemics was attributable to bacterial coinfection, especially with pneumococci (4). Worldwide, 1.6 million people die each year after pneumococcal infection, especially young children, seniors, and patients with immunodeficiency (5).
Capsular polysaccharides are the main virulence factors of S. pneumoniae, a gram-positive bacterium. Antibodies against these polysaccharides protect against invasive infection, the most severe form of the disease (6). To date, more than 90 polysaccharides (serotypes) have been characterized (7). In adults, the protection against invasive pneumococcal infection after vaccination was estimated as 50% to 95% (8–10). Of note, antibody response after vaccination is described as dependent on age and pneumococcal serotype (10–13).
Our previous study has shown that, at month 1 after pneumococcal vaccination, 43 kidney transplant recipients with clinically stable condition display almost normal concentrations of pneumococcal antibodies (3). In that study, we have used the first commercially available assay for the measurement of 14 different serotype-specific polysaccharide antibodies against pneumococci (xMAP pneumococcal immunity panel; Luminex, Oosterhout, the Netherlands).
It was the aim of the present study to (1) define in detail the long-term efficacy of pneumococcal vaccination in 49 kidney transplant recipients with clinically stable condition and (2) determine clinical parameters influencing the decrease of antibody concentrations after vaccination. We are aware of only one study (14) analyzing serotype-specific long-term responses toward a 23-valent pneumococcal vaccine in kidney transplant recipients. Our study extends this previous study by including twofold more patients and considering responses toward 14 serotypes by Luminex instead of 7 serotypes by enzyme-linked immunosorbent assay (ELISA) method. Apart from the strength of the initial antibody response (14), parameters defining the decrease of antibody concentrations have not been reported so far in this patient group.
Pneumococcal vaccination was performed in 49 kidney transplant recipients with clinically stable condition, of whom 44 achieved immunosuppression by calcineurin inhibitors (26 received cyclosporine A and 18 received tacrolimus). Five patients were treated by azathioprine and mycophenolate mofetil (MMF), and all but three patients received prednisone.
One month after vaccination, antibody concentrations against 14 of 14 capsular polysaccharides—included in the pneumococcal immunity panel—were significantly (P<0.0001) elevated as compared with the values before vaccination (Fig. 1A). At month 15 after vaccination, 11 of 14 antibody concentrations were still significantly (P<0.001) higher than at baseline. For a clearer quantitative assessment, antibody concentrations were summed up in each patient and added (sum of antibody concentrations; Fig. 1B). The sum of antibody concentrations was 2.9-fold higher (P<0.0001) at month 1 than before vaccination (median [range], 53.6 mg/L [4.5–132.4 mg/L] vs. 18.2 mg/L [2.9–55.5 mg/L]). Antibody concentrations were 2.3-fold higher (P<0.0001) at month 15 than at baseline (41.3 mg/L [4.9–105.0 mg/L]). The antibody concentration at month 15 was 77% of the initial antibody response (at month 1).
The percentage of patients with protective antibody responses (9) was higher at month 15 versus before vaccination (Fig. 1C,D). According to a recent study by Borgers et al. (9) using the same method for antibody measurement, responses were defined as protective if they were greater than the fifth percentile of healthy controls. Depending on the serotype, 20% to 80% (median, 55%) of kidney transplant patients showed protective antibodies before vaccination, 65% to 90% (median, 82%) at month 1, and 47% to 88% (median, 68%) at month 15.
The number of pneumococcal serotypes recognized was 8 (0–13) before vaccination and significantly (P<0.0001 each) increased at month 1 (13 [0–14]) and month 15 (11 [0–14]) after vaccination (Fig. 2). However, at month 15, the number of serotypes with protective antibodies already displayed a significant decrease (P<0.0001) as compared with month 1. In comparison with 75 healthy controls tested at month 1 after vaccination (9), the number of serotypes with protective antibodies was lower in kidney transplant recipients at months 1 and 15. However, the total amount of antibodies was higher in 47 (96%) of 49 patients at month 1 and in 45 (92%) of 49 patients at month 15 after vaccination as compared with before vaccination.
Moreover, Spearman rank correlation analysis showed that older patients displayed a more pronounced decrease in antibodies (months 1–15). Significant correlations were observed for polysaccharides 19A and 23F (r=0.30 and P=0.04 each).
Women versus men showed a lower concentration of pneumococcal antibodies before vaccination (sum of antibodies, 14.0 mg/L [2.9–50.4 mg/L] vs. 20.1 mg/L [6.8–55.5 mg/L]; P=0.05) and displayed protective antibodies toward a lower number of serotypes (7 [0–12] vs. 10 [2–13]; P=0.006). The increase in antibody concentrations (month 1 vs. before vaccination) was less pronounced in women (antibodies against polysaccharide 14, P=0.03). The sum of antibodies in women versus men increased by 30.8 mg/L (0.7–95.6 mg/L) versus 37.5 mg/L (0.0–110.8 mg/L) at month 1 after vaccination and decreased by 10.1 mg/L (0–39.1 mg/L) versus 19.0 mg/L (0.0–62.8 mg/L) (months 1–15). In women, pneumococcal antibody concentrations and the number of serotypes with protection were lower at all time points, but interestingly, antibodies diminished to a lesser extent after vaccination.
The immunosuppressive drug regimen (cyclosporine A, tacrolimus, or non–calcineurin inhibitor group) showed a significant impact on antibody concentrations. Patients treated with tacrolimus versus cyclosporine A showed lower pneumococcal antibody concentrations before vaccination and at month 15 after vaccination. They also displayed a greater decrease of antibodies (months 1–15; antibodies against polysaccharide 8, P=0.006).
Serum creatinine at month 15 after vaccination was significantly correlated with the decrease (months 1–15) in antibody concentrations (antibodies against polysaccharide 18C, r=0.26 and P=0.04). Patients with better kidney function lost fewer antibodies in the long term.
As expected, for all pneumococcal serotypes antibody responses at months 1 and 15 were significantly correlated (P<0.005 each), indicating that those patients with high antibody concentrations at month 1 still showed high antibody concentrations at month 15 (Table 1A). Interestingly, there was a significant correlation between increase and decrease of antibody concentrations (months 1–0 vs. 1–15) (Table 1B). Patients displaying a greater increase in antibodies initially after vaccination also showed a greater decrease in the long term. Spearman rank correlation analysis indicated a statistically significant correlation of changes in antibody concentrations (increase and decrease) for 11 of 14 serotypes tested.
Finally, multinomial logistic regression analysis showed that antibody concentrations at month 15 and the decrease in antibody concentrations (months 1–15) correlated significantly with patient age, gender, immunosuppressive drug regimen, and kidney function as detailed in Table 2.
In summary, our main findings are that (1) 77% of the initial antibody response (at month 1) remained detectable 15 months after vaccination in kidney transplant recipients with clinically stable condition and (2) the long-term decrease in antibodies was minor in younger kidney transplant recipients, in women, in patients receiving cyclosporine A versus tacrolimus, and in patients with better kidney function.
Our results show that, at month 15 after pneumococcal vaccination, kidney transplant recipients with clinically stable condition displayed 77% of initial antibody concentrations (determined at month 1). As compared with healthy controls, this decline in antibody concentrations may be more rapid. Mufson et al. (15) reported that, in healthy controls 4 years after vaccination, 90% of initial antibodies against pneumococci were detectable and that, 5 to 6 years thereafter, 76% of antibodies were still detectable. These findings imply that the decline in antibodies in kidney transplant recipients at month 15 would be comparable with the decline 5 to 6 years after vaccination in healthy controls.
There are several differences between the study by Kumar et al. (14) cited previously—also analyzing long-term responses toward a 23-valent pneumococcal vaccine—and our own study. Their study contained 24 kidney transplant recipients, tested immunity toward 7 serotypes (instead of 14), used the ELISA format, determined antibodies at week 8 and 3 years after vaccination, and assessed antibody concentrations in a different way. Interestingly, the decline of antibodies from week 8 to year 3 after vaccination (14) was similar to the decline from months 1 to 15 in our study (76% and 77%, respectively). For these calculations, we summed up antibody concentrations for 7 (14) and 14 serotypes.
Another follow-up study by Rytel et al. (16) on healthy controls and kidney transplant recipients at years 1, 2, and 3½ after vaccination indicates that, although differences were nonsignificant, kidney transplant recipients tended to have lower pneumococcal antibody levels. Furthermore, Marrie et al. (17) reported that, at 1 year after immunization of renal allograft recipients, antibody concentrations declined by 14% to 37% from the month 1 levels. Thus, our data are consistent with the previous reports, although, in the two mentioned studies on kidney transplant recipients (16, 17), another vaccine (14-valent vaccine) and another detection system for antibodies (radioimmunoassay) were used and although, in the study by Rytel et al. (16), most kidney transplant recipients were splenectomized. The long-term comparative data in healthy controls and kidney transplant recipients (16) can only be roughly estimated because they were not displayed in numbers but as a graph using a logarithmic scale. In our own cohort, none of the patients underwent splenectomy. It is very likely that the decline in antibodies may be less pronounced in our own cohort as compared with the group of mostly splenectomized patients (16) because splenectomy had been reported to result in a faster fall of antibodies (18), which is attributable to the dependency of antibody formation against capsular polysaccharides on the marginal zone of the spleen.
It may be even more important to consider the number of serotypes with protective antibodies. Such a calculation relies on a proper definition of protective antibodies and should take into account that protective antibody concentrations seem to be serotype specific (9, 19, 20). Such an analysis had not yet been presented in kidney transplant recipients. Our own definition of serotype-specific levels of protective antibodies is based on a recent publication by Borgers et al. (9) who applied the Luminex method for the first time to detect serotype-specific pneumococcal antibodies in healthy controls and patients with immunodeficiency experiencing recurrent infections. We could demonstrate that the number of serotypes with protective antibodies increased from a median value of 8 before vaccination to 13 at month 1 and declined to 11 at month 15 (maximum value of serotypes with protection as determined by Luminex assay, 14). An average protection against 11 of 14 serotypes should persist until month 15. We could show that, at month 15 after vaccination, the rate of protection was 47% to 88% (median, 68%), depending on the serotype. In comparison, we observed a protection rate of 20% to 80% (median, 55%) before vaccination and 65% to 90% (median, 82%) at month 1. There were two serotypes where approximately half of the patients had already lost protective antibodies at month 15 (polysaccharides 4 and 12F). Of note, the initial antibody responses against both serotypes were the lowest. Luckily, invasive pneumococcal infection in German adults is caused, according to the most recent data available (21), by serotype 4 only in 5.2% and by serotype 12F in 1.7%. Protection against the most abundant serotypes 3 and 14 causing 12.5% and 9.4% of invasive pneumococcal infection, respectively, was detectable in 61.2% and 85.7% of patients, respectively, at month 15. There is a small study on seven kidney transplant children and young adults (aged 9–27 years) indicating that, 1 year after pneumococcal vaccination, three (43%) displayed protective antibodies (22). Depending on the serotype, the protection rate observed by Kumar et al. (14) 3 years after vaccination was 0% to 90% (median, 43%). In contrast to our definition of protection, an antibody titer greater than 200 (22) or a twofold increase in titer from baseline and an absolute titer of 0.35 μg/mL or greater (14)—irrespective of the serotype—were considered as protective in these studies. Other definitions of protective antibodies and other time points could explain the differences from our study. We have to clarify that we did not determine antibodies by a functional assay such as an opsonophagocytic assay (23).
Another approach to quantify vaccination responses would be to calculate the proportion of patients with an increase in antibody concentrations. In our own cohort, the sum of antibodies increased in 47 (96%) of 49 patients at month 1 and in 45 (92%) of 49 patients at month 15 after vaccination. A recent study by Pourfarziani et al. (24) also using the vaccine Pneumovax 23 (Sanofi Pasteur MSD, Leimen, Germany) resulted in very similar data in 37 kidney transplant recipients at month 1 (increase in 49 [96%] of 51 patients) and a slightly lower vaccination response 1 year after vaccination as compared with our own data at month 15 (increase in 38 [85%] of 45 patients). In that study, total IgG antibodies were determined by ELISA. The minor differences as compared with our data are either just observed by chance or the serotype-specific measurement of IgG by Luminex method could be more sensitive in detecting changes of antibody concentrations. In line with the assumption of a slightly more sensitive Luminex assay, the study by Pourfarziani et al. (24) observed a 2.6-fold increase of antibodies at month 1 and a 2.1-fold increase at year 1 as compared with a 2.9-fold increase of antibodies at month 1 and a 2.3-fold increase at month 15 in our own cohort.
The clinical parameters influencing the decrease of antibody concentrations in the long term after vaccination are partly comparable with those determining the strength of antibody responses in the short term. Previously, age has been identified as an influencing factor of pneumococcal antibody responses at weeks 2 to 3 after vaccination in 58 healthy individuals (11); and kidney function, as an influencing factor of antibodies at month 1 in 43 kidney transplant recipients (3). Other studies (14, 24–26) observed no correlation of antibody concentrations with age, gender, immunosuppressive drugs, or kidney function at weeks 2 to 8 after pneumococcal vaccination. To the best of our knowledge, the initial antibody response after vaccination was the only factor defined to affect long-term antibody responses in kidney transplant recipients (14). Our own analysis in a larger patient cohort could confirm this finding (Table 1A) and define further influencing factors. In our view, the most interesting finding was the influence of gender on antibody responses. Women showed lower pneumococcal antibody concentrations and protection toward a lower number of pneumococcal serotypes before vaccination. Multivariate analysis indicated a significant influence of gender on the decline of antibodies. Further studies are necessary to clarify if female kidney transplant recipients display an impaired immunity toward pneumococci.
Differences in antibody responses in patients taking tacrolimus versus patients taking cyclosporine A cannot be caused by direct effects on T cells as responses to polysaccharide antigens such as pneumococcal polysaccharides are known as T cell independent. Presumably, the effect is indirect and mediated by cytokines produced by T cells (27). It could be shown previously (28) that tacrolimus and cyclosporine A both affected IgG production after anti-CD40 monoclonal antibody and CpG stimulation. As compared with in vivo drug concentrations in our patient cohort, the in vitro concentrations used in that study (28), however, were approximately 3% to 25% (tacrolimus) and 33% to 100% (cyclosporine A). Thereby, it is difficult to quantitatively translate the in vitro into in vivo effects.
What is the clinical implication of our findings? Currently, in adults with immunosuppression or chronic disease, revaccination against pneumococci is recommended after 5 years (1, 2). In kidney transplant recipients, a shorter interval, for example, of 3 years as suggested by Rytel et al. (16) and our own data may be superior for protective antibodies. Of note, the policy of revaccination has recently changed in individuals 65 years or older; only single revaccination is recommended (1, 2). According to the expert information about Pneumovax 23, two vaccinations with an interval of less than 5 years could lead in seniors to a higher rate of local and systemic adverse reactions after vaccination. Because of the knowledge about a possible increase in adverse reactions after repetitive vaccinations and our own findings, we suggest a compromise between the avoidance of adverse reactions and an adequate protection against invasive pneumococcal infection in immunocompromised patients. Our results support the need for revaccination in kidney transplant recipients. To define the optimal time point for revaccination, additional testing (e.g., at 2, 3, 4, and 5 years after vaccination) is necessary. At the current stage, we would like to encourage physicians taking care of kidney transplant recipients to follow the recommendations (1, 2) and perform revaccinations against pneumococci after 5 years. Assays that detect functional antibodies and a continuation of the follow-up after vaccination should facilitate the decision to shorten the interval between pneumococcal vaccinations despite safety concerns.
MATERIALS AND METHODS
This study was performed in 49 kidney transplant recipients with clinically stable condition (21 women and 28 men; median age, 53 years [range, 29–74 years]). Inclusion criteria were absence of acute infection, no rejection episode within the last 3 months, changes in serum creatinine level of less than 15% within 3 months, interval to kidney transplantation at least 4 months, and no pregnancy. The median (range) serum creatinine concentrations were 1.3 mg/dL (0.7–4.9 mg/dL) at the time of vaccination, 1.3 mg/dL (0.6–4.9 mg/dL) at month 1 after vaccination, and 1.4 mg/dL (0.8–6.2 mg/dL) at month 15 after vaccination. Details on kidney function are presented in Table 3. The median interval between transplantation and vaccination was 6.5 years (range, 5 months to 12 years). In none of the patients, rejection or considerable adverse effects occurred after vaccination. Twenty-six patients were treated with cyclosporine A (whole-blood trough level, 100–150 ng/mL; tested by TDx [monoclonal] fluorescence polarization immunoassay; Abbott Laboratories Limited, Wiesbaden, Germany); and 18 patients, with tacrolimus (whole-blood trough level, 4–10 ng/mL; tested by IMx tacrolimus assay; Abbott Laboratories Limited). In detail, the immunosuppressive regimen consisted of cyclosporine A alone (n=1), cyclosporine A/prednisone (n=17), cyclosporine A/azathioprine (n=1), cyclosporine A/azathioprine/prednisone (n=3), cyclosporine A/MMF/prednisone (n=3), tacrolimus/prednisone (n=11), tacrolimus/azathioprine/prednisone (n=6), tacrolimus/MMF/prednisone (n=2), azathioprine/MMF (n=1), and azathioprine/MMF/prednisone (n=4). Altogether, 12 patients were on triple therapy. This study does not include a control group such as patients on dialysis in whom the effect of renal impairment irrespective of immunosuppressive drug therapy could be assessed.
After informed consent was obtained, each subject received one dose of a standard, 23-valent pneumococcal vaccine (0.5 mL of Pneumovax 23) by intradeltoid administration. This unconjugated vaccine contains 25 μg each of 23 pneumococcal serotypes: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.
Determination of Antibody Concentrations
Antibodies against 14 pneumococcal capsular polysaccharide antigens (serotypes) were determined before vaccination and at months 1 and 15 after vaccination by multiplexed bead assay (xMAP pneumococcal immunity panel). Here, reactions take place on the surface of polystyrol microbeads that are color-coded using different fluorescent intensities of two dyes. The 14 serotypes contained in the xMAP assay (1, 3, 4, 6B, 7F, 8, 9N, 9V, 12F, 14, 18C, 19A, 19F, and 23F) are all components of Pneumovax 23. The protocol for antibody determination exactly followed the manufacturer’s instructions. Plates were read on the Luminex 100 IS device, and Luminex IS 2.3 software was used for analysis.
The definition of antibody titers considered as protective relied on data published by Borgers et al. (9). Here, the cutoff value for protection was defined as the fifth percentile of antibody concentrations determined in 75 vaccinated, healthy controls. This means that the protection rate in healthy controls was assumed as 95%. Depending on the serotype, cutoff concentrations varied between 0.24 and 1.45 mg/L. This estimation of protective antibody concentrations takes into account that protective antibodies are serotype dependent; cutoff values are in the range of previous publications (19, 29, 30).
Reactions before and after vaccination were compared by Wilcoxon matched pairs test; and those in patients and healthy controls, by Mann-Whitney U test. Correlation analyses of antibody results at different time points and of antibody results and clinical parameters (patient age and serum creatinine concentration) were performed by Spearman rank correlation test. The influence of gender and immunosuppressive therapy (cyclosporine A, tacrolimus, or non–calcineurin inhibitor group) on antibody results and the influence of gender, age, and immunosuppressive therapy on kidney function were analyzed by Mann-Whitney U test. These analyses were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). Furthermore, factors influencing antibody concentrations were defined by multinomial logistic regression analysis (SPSS 12.0G for Windows, version 12.0.1; SPSS Inc., Chicago, IL). P values less than 0.05 were considered as significant.
The authors thank Monika Huben and Sabine Wortmann for their excellent technical assistance.
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