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Keywords:

  • Influenza;
  • liver transplantation;
  • pneumococcus;
  • renal transplantation;
  • vaccine

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Different immunosuppressant regimens vary in their effects on antibody responses to vaccination. The combination of prednisolone and azathioprine has only a minor effect, whereas the addition of ciclosporin attenuates protective antibody responses to influenza vaccination. The effect of sirolimus, a new immunosuppressant, on vaccine responses has been little studied.

Thirty-two hepatic or renal transplant patients randomized to calcineurin inhibitor-based or sirolimus-based immunosuppression were vaccinated against influenza and pneumococcus. Following tri-valent influenza vaccination, a similar rise in antibody titer occurred in sirolimus and calcineurin inhibitor (CNI) treated patients, though sirolimus treated patients developed a ‘protective’ titer to more influenza antigens. The pneumococcal polysaccharide vaccine was equally effective in both groups. Hence, vaccination guidelines in place for CNI treated patients are likely to be appropriate for transplant recipients maintained on sirolimus.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Following solid organ transplantation, patients are both more susceptible to contracting microbial infection and at greater risk of serious complications. Pneumococcal infections cause disease including pneumonia, meningitis and septicaemia, with pneumococcal pneumonia estimated to affect approximately 1 in a 1000 adults every year in the United States and Europe (1). In interpandemic periods, the influenza viruses A and B generally cause self limiting disease in healthy adults. Immunosuppressed patients, however, are at particular risk of major complications including pneumonitis and secondary bacterial pneumonia. An association between influenza infection and both acute rejection (in renal transplant recipients) and obliterative bronchiolitis (in lung transplant recipients) has been suggested in small series (2,3).

Vaccination is effective in preventing both influenza (in 70–80% of people (4)) and pneumococcal infection in 60–70% of people (1). A number of studies have measured antibody responses to vaccination in solid organ transplant recipients. Early studies of recipients maintained on prednisolone and azathioprine found no difference in antibody titers compared with healthy controls (5,6). Responses to influenza vaccination are reduced by ciclosporin (a calcineurin inhibitor [CNI]) and further dampened by subsequent addition of mycophenolate mofetil (MMF) (7,8).

The use of sirolimus (rapamycin) is increasing in solid organ transplantation. Recent trials in transplant recipients have demonstrated that there is no increase in acute rejection rates in transplant recipients maintained on a sirolimus-, compared with a CNI-based regimen (9), and as a maintenance agent it is associated with a significantly better glomerular filtration rate (10). One non-randomized study has examined influenza vaccine responses in patients treated with sirolimus and CNI compared with CNI-based therapy alone (11). More patients in the sirolimus treated group had seroprotective titers postvaccination (though CNI dose, which was presumably lower in this subgroup, was not given). In the absence of previous direct comparisons, we have compared the immune response to influenza (T-dependent) and pneumococcal (T-independent) vaccinations in patients randomized to receive either CNI or sirolimus.

Subjects and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Study population

A total of 32 patients were recruited from existing studies underway in Cambridge comparing renal function in transplant recipients randomized to either CNI-based immunosuppression or a sirolimus-based regimen (10, Watson et al., submitted). Patients were randomized to either continuing treatment with CNI or switching to sirolimus, and glomerular filtration rates were compared at 12 months. A total of 23 patients were enrolled from a study which had recruited patients at least 3 months post renal transplant (10) and nine from one that enrolled individuals at least 6 months post hepatic transplantation (Watson et al.). Patients on stable immunosuppressive therapy for at least 2 months, with no previous adverse reaction to vaccination or to the vaccine components, not pregnant and not receiving dialysis treatment, were deemed eligible and approached for this study.

Influenza vaccine

Each subject was vaccinated in a 5- week period in autumn 2004 with a 0.5 mL intramuscular injection of influenza vaccine from a single batch. The split virion influenza vaccine complied with the WHO recommendation for the northern hemisphere winter 2004–2005 to contain A/New Caledonian/20/99(H1N1)-like virus, A/Fujian/411/2002(H3N2)-like virus and B/Shanghai/361/2002-like virus.

Pneumococcal vaccine

Four weeks after vaccination with the influenza vaccine, each subject received 0.5 mL intramuscular injection of pneumovax II from a single batch. The 0.5 mL vaccine contained 25 μg of each of the following 23 pneumococcal serotypes: 1, 2, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F.

Immunization regimen

Serum was collected on the day of influenza vaccination from all subjects and again 4-weeks later. On that day subjects were given the pneumococcal vaccine and further serum collected 8 weeks later.

Serology

IgG recognizing pneumococcal polysaccharide of 7 serotypes was measured by the Bio-Plex Protein Array System. Pneumococcal Reference Standard Serum 89SF-3 was provided by Dr. Carl Frasch, Center for Biological Evaluation and Review, Food and Drug Administration, Rockville, USA. Pneumococcal polysaccharides were coupled to microspheres as previously described (12). Fifty microliters of microsphere mixture was added to each well of a pre-wetted 96-well filter plate and liquid aspirated using a vacuum manifold filtration system. The 50 μL of standard and (diluted) test sera were added and left for 30 min at 37°C on a plate shaker. After washing three times with 200 μL PBS-Tween, 50 μL of anti-human IgG-R-PE (1 μg/mL in blocking buffer) was added and incubated for 30 min at 37°C on a plate shaker. After a further wash with 200 μL PBS-T, the beads were re-suspended in 100 μL PBS-T on plate shaker. The plate was then run on the Bio-Plex system (5 μL injection volume/100 microspheres per set). Data were analyzed using Biorad Bio-Plex Manager V3.0® (Biorad Laboratories, Hercules, CA). A serotype specific antibody value ≥ 0.35 μg/mL was considered protective, in accordance with WHO recommendations (13).

Influenza antibody titers were analyzed in the Enteric, Respiratory, and Neurological Virus Laboratory, Central Public Health Laboratory, Colindale, London. Antibodies to A/NCAL/20/99 (H1N1), A/FUJIAN/411/02 (H3N2) and B/JIANGSU/10/03 were tested by hemagglutination inhibition (HI). Antibody levels were expressed as serial dilutions with a level of 1:40 or more considered as protective (14).

Statistical analyses

Nonparametric group data was initially rank transformed and then analyzed by a repeated measure ANOVA, whilst a chi-squared test was used to compare group proportions. Multivariate analysis was performed (multiple regression for pneumococcal data—outcome measured as number of serotypes showing a twofold rise in antibody level post vaccination compared with pre-vaccination levels; multiple logistic regression for influenza data—outcome measured as presence or absence of protection post vaccination against either one or more of, or three of the influenza strains) using the following parameters as independent factors: serum creatinine, number of immunosuppressive agents, type of transplant, previous immunization against the relevant infective agent, and use of CNI/sirolimus. Where the introduction of an independent factor into the multiple regression analysis led to either complete or quasi-complete separation of the matrix then that factor was removed and subsequent statistical analysis of the role of that factor in determining the outcome was performed using a Chi-squared test.

Ethics

Ethical approval for the study was obtained (Local Research Ethics Committee reference number 03/292). All patients gave written, informed consent before enrolment in the study.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

All eligible patients who had been enrolled in previous studies (10, Watson et al.) were invited to participate. Twenty three of 30 eligible renal, and 9 of 19 liver, transplant patients agreed to take part. The patient groups had similar demographics and clinical characteristics (Table 1). There were nonsignificant trends towards older age and poorer renal function in the CNI group (age, p = 0.21: creatinine p = 0.20). None of the study participants had received depleting antibody treatment (e.g. anti-thymocyte globulin, anti-lymphocyte globulin, rituximab or alemtuzumab). No episodes of rejection occurred during the period of the study, and no adverse reactions to vaccination were reported. All 32 patients recruited into the study received the influenza vaccine. One patient withdrew from the study before measurement of antibody response; hence 31 patient results were available for analysis of influenza antibody titers. Two of the patients being treated with tacrolimus at the time of the influenza vaccine switched to sirolimus based treatment at the time of the pneumovax vaccination. One patient discontinued treatment with sirolimus after receiving pneumovax, but prior to antibody measurement. Pneumococcal antibody levels for these three patients were not included in the analysis, thus 28 patients were analyzed.

Table 1.  Characteristics of subject groups
TacrolimusCiclosporinSirolimus 
  1. 1Values given as mean (standard deviation) followed by range.

  2. 2At time of first vaccination.

n12515
Male:Female6:65:09:6
Age (years)1,258.2 (9.9) 45.8 (6.4) 49.7 (10.4)
41–7441–5539–72
Serum level1,28.38 (2.6) 108.2 (35.0) 8.45 (3.3)
2.9–14.169–1513.1–15.5
Creatinine (μmol/dL)1,2200 (51.9)157.1 (53.2) 146.7 (42.3)
99–270143–26388–262
Months from transplant to61.8 (21.8)51.5 (17.8) 51.6 (19.5)
First vaccination133–9542–9617–90
Liver allograft504
Renal allograft7511 
Monotherapy303
With prednisolone alone012
With azathioprine alone113
With prednisolone + azathioprine736
With prednisolone + MMF101

Following pneumococcal vaccination, mean antibody levels rose significantly in both patient groups for all 7 serotypes of pneumococcus (p < 0.05) except 6B (p = 0.07). There was no significant difference in absolute antibody levels, or in the change in antibody levels, between patients taking CNI and those randomized to sirolimus (Figure 1A–G).

image

Figure 1. Pneumococcal responses in immunosuppressed transplant patients. (A–G) show pneumococcal IgG values for each serotype, comparing individuals treated with CNI (•) and sirolimus (▪) before and 8 weeks after vaccination. The geometric mean is shown by a bar, and levels considered not protective by grey shading. (H) shows the percentages of patients with antibody levels that more than doubled post vaccination to each serotype.

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Eight weeks after vaccination, patients maintained on CNI had antibody levels greater than ≥0.35 μg/mL (the level determined by WHO to confer protection against disease (13)) to a mean of 6.4 of the 7 antigens against which they had been vaccinated. This compares to a mean of 6.3 antigens in patients maintained on sirolimus. It should be noted, however, that prior to vaccination, patients in the CNI group had antibody levels ≥0.35 μg/mL to a mean of 5 of 7 antigens, and those in the sirolimus group to 4.9 antigens, and that 12 of the 27 patients in the trial had received pneumovax previously, 10 of these in the last 10 years (in accordance with UK Department of Health guidelines). Thus we also looked for a response to vaccination as defined by a two fold rise in antibody concentration. The percentages of patients in each group whose antibody levels doubled are shown in Figure 1H. In the CNI group, antibody levels doubled to a mean of 4.5 of 7 antigens following vaccination, compared with 4.1 in the sirolimus group. Of the titers that were ≤0.35 μg/mL prior to vaccination, 81% (25 of 31) in the CNI group doubled following vaccination, compared with 67% (22 of 33) in the sirolimus group (p = 0.21). There was no significant difference between the groups in the number of serotypes to which antibody levels doubled, and multiple regression analysis showed that creatinine, number of immunosuppressant agents and type of transplant did not have a significant effect on responses (sirolimus compared with CNI p = 0.369). Previous vaccination was, however, associated with a reduction in the number of antigens against which antibody levels doubled (p = 0.0019).

Three different outcome measures were used to assess the immune response to influenza vaccination. Firstly, we compared pre and post vaccination antibody titers and found that after influenza vaccination, mean antibody titers rose significantly in both groups for all three influenza antigens (p < 0.0005). There was no significant difference in the rise in antibody titers between patients taking CNI and those taking sirolimus (Figure 2A–C). Secondly, we assessed the percentage of individuals with a protective antibody titer after vaccination. We found that all 15 patients in the sirolimus group had a protective antibody titer (≥1:40)(14) to at least one of the influenza antigens against which they had been vaccinated, compared with 12 of 16 patients in the CNI group, p = 0.038 (chi-squared test, see methods) (Figure 2D). The percentages of individuals with protective titers post vaccination in the sirolimus group were 73% (11 of 15), 100% (15 of 15) and 67% (10 of 15) for H1N1, H3N2 and B influenza subtypes respectively, and in the CNI group were 44% (7 of 16), 81% (13 of 16) and 44% (7 of 16).

image

Figure 2. Influenza vaccine responses in immunosuppressed transplant patients. (A–C) show antibody titers (serial dilution) measured by HI assay, comparing individuals treated with CNI (•) and sirolimus (▪) before and 4 weeks after vaccination. The geometric mean titer is shown by a bar, and titers considered not protective by grey shading. A dilution of <1:10 is assigned a value of 1. Numbers in the shaded area refer to the number of individuals in each group with a value of 1, i.e. a dilution <1:10. (D) shows the percentage of individuals in each treatment group who had protective titer to 0, 1 or more and all three influenza antigens post vaccination. The p values refer to analysis by the chi-squared test.

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Lastly, we determined the number of patients in each group who had protective antibody titers pre-vaccination compared with the number of patients protected after vaccination. Pre-vaccination, no patients in either group had protective antibody titers to all three influenza antigens. Following vaccination, 5 of the 16 patients in the CNI group had protective titers to all three antigens, compared with 9 of 15 in the sirolimus group (multiple logistic regression, sirolimus compared with CNI use p = 0.221). Of 46 titers obtained post vaccination from the CNI group, 27 were protective. This is significantly fewer than the 36 of 43 titers that were protective in the sirolimus group (p = 0.005). Creatinine, number of immunosuppressant agents, type of transplant and previous influenza immunization had no significant effect on vaccine responses (multiple logistic regression).

None of the study participants suffered an influenza infection during the subsequent influenza season. The majority of the study participants undergo yearly influenza vaccination as recommended by the UK Department of Health. A total of 14 of 16 patients in the CNI group had been vaccinated against influenza in the autumn of 2003 (and the remaining two had been vaccinated the previous year). Fourteen of 15 patients in the sirolimus group had been vaccinated in 2003, and the remaining patient had never received an influenza vaccination. For the winters of both 2002/2003 and 2003/2004, the WHO recommended vaccine contained the H1N1 strain A/New Caledonian/20/99-like virus, the H3N2 strain A/Moscow/10/99-like virus and B strain B/Hongkong/330/2001-like virus. The H3N2 and B virus strains therefore differed from those in the 2004/2005 vaccination. At the start of the study, 15 of the participants had protective antibody titers to H1N1, 12 to H3N2 and none to B.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

After pneumococcal vaccination, almost all anti-pneumococcal IgG titers from study participants were in excess of the protective titer recommended by WHO. As was expected from previous studies (e.g. 15), our patients had ‘protective’ titers to 4.9 of the 7 antigens before vaccination. These initial levels reflect previous exposure either to S. pneumoniae or to the vaccine—12 of the 27 patients in the trial had received pneumovax previously. Nonetheless, if an immune response to vaccination is indicated by a two-fold rise in antibody concentration, the majority did respond despite their initial level, and there was no difference between the groups. Previous studies have also demonstrated the efficacy of pneumococcal vaccination in transplant recipients at eliciting protective antibody levels (16), although small sample sizes and differences in methodology make comparisons between studies difficult.

Most measures of responses to influenza vaccination were similar between the two groups. There was a trend to increased vaccine responses in the sirolimus group for some of the parameters measured. As patient numbers were small, we cannot be confident this reflects a real increase in vaccine responses in sirolimus treated patients compared with CNI, but the data suggests there is likely to be at least equivalence between the two groups. The study by Hayney et al. (11) in 68 lung transplant recipients maintained on CNI found that the 24 patients also treated with sirolimus had increased rates of seroprotection post influenza vaccination compared with those on other CNI based regimens. However, it is not clear if the CNI dose was lower in the sirolimus group, nor how many patients in each group were also treated with MMF, which was found to significantly reduce seroprotective titers. Thus these studies hint that vaccine responses may be increased in sirolimus treated patients, but larger studies are needed to see if this effect is real. Although we cannot prove equivalence in a study this size, it nonetheless seems likely that responses in sirolimus treated patients are at least as good as those in CNI treated patients.

This study focused on a comparison between the effects of sirolimus and CNI on vaccine responses and did not contain a healthy control group. A number of studies have compared pneumococcal or influenza vaccine responses in transplant recipients to healthy controls. Following influenza vaccination, titers are consistently reduced in those individuals on CNI, particularly if also on MMF, compared with healthy controls (7,8,11). However, with the exception of patients on MMF, the majority of transplant recipients in these studies do mount antibody responses to levels deemed protective against infection. Similarly, responses to pneumococcal vaccination are lower in transplant recipients than in healthy controls (16,17). A study in heart transplant recipients (maintained on ciclosporin, azathioprine and corticosteroids) compared responses to both influenza and pneumococcal vaccinations with those of normal controls (18). Post vaccination, all study participants (controls and transplant recipients) attained protective pneumococcal antibody titers. Fewer of the transplant patients had protective influenza antibody titers post vaccination, although 69% mounted a protective response to one or more influenza antigens. Our study suggests that most transplant recipients treated with sirolimus also mount protective responses to influenza and pneumococcal vaccinations, with response rates similar to those seen in CNI treated patients. All of our patients were at least 1 year post transplantation and we cannot extrapolate our data to predict vaccine responses in the early posttransplant period. It seems likely that at this time, when immunosuppressant levels are generally higher, vaccine responses will be less robust, although the majority of studies have, like ours, excluded patients within 6 months of transplantation. Further studies would be needed to determine the efficacy of vaccination in this period.

Our study included both renal and liver transplant recipients maintained on 1, 2 or 3 immunosuppressant therapies. Multivariate analyses did not show an effect of type of organ allograft or number of immunosuppressant drugs on vaccine responses. Although the majority of transplant recipients responded well to vaccination, some patients did not mount protective titers. There is some debate about the role of booster immunization in such non-responding immunosuppressed patients. Repeat influenza vaccination for initial non-responders was advocated by Soesman et al., after finding that a repeat vaccination in liver transplant recipients increased the frequency of protective response to all three strains from 68–80% (19). Other studies, however, did not demonstrate a significant improvement in vaccine response with a second dose (8,20). Similarly controversial is the most appropriate measure of response to T-dependent vaccination. Antibody titers have been shown to correlate with clinical protection (13,14). Measurement of IFNγ ELISpots as a read-out of T-cell immunity has shown variable results, with a reduction in CNI-treated patients compared with healthy individuals in one study (21) not confirmed in a second smaller study (22), and this is an area which requires further investigation.

A number of conclusions can be drawn from the data obtained in our study, despite a relatively small sample size. A number of transplant recipients do not mount a protective response to influenza vaccination and it would seem prudent to maximize vaccination of healthy family members and health care workers to reduce exposure to influenza. Overall, both T-dependent and T-independent vaccines are effective in the majority of transplant recipients (though care should be taken in extrapolating these results to patients soon after transplantation on higher immunosuppressant doses). The efficacy of pneumococcal polysaccharide vaccine questions whether there would be any advantage of conjugate pneumococcal vaccine in transplant recipients. Finally, we propose that vaccination guidelines in place for CNI treated patients can be applied to transplant recipients maintained on sirolimus, as our results suggest that vaccines are at least as effective in these patients.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Funding sources: L.C.W is supported by an MRC Clinical Research Training Fellowship and K.G.C.S. by a Wellcome Research Leave Award for Clinical Academics.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
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