Tadashi Tomo,Mitsuo Takei,Makoto Matsuyama,Iehisa Matsuyama,Kazuhiro Matsuyama
Examination of the Effect of Switching Dialysis Membranes on the Immune Response to Influenza Virus Vaccination in Hemodialysis Patients
Polymethyl methacrylate membrane,Influenza vaccination,Anti-Influenza antibody,Immune response,Beta-2-microglobulin
Introduction In this study, we examined the effect of switching dialysis membranes on the response to influenza virus vaccination in HD patients. Methods This study consisted of two phases. In phase 1, antibody titers were measured and compared between HD patients and healthy volunteers (HVs) before and after vaccination against influenza virus. Using antibody titers 4 weeks after vaccination, HD patients and HVs were classified according to seroconversion (i.e., antibody titers against all four strains were >20-fold) or non-seroconversion (i.e., antibody titer against at least one strain was <20-fold). In the phase 2, we examined whether the change in the dialysis membrane from a polysulfone (PS) to a polymethyl methacrylate (PMMA) membrane affected the response to vaccination in HD patients without seroconversion in response to the vaccine the previous year. Patients with seroconversion and non-seroconversion were classified as responders and nonresponders, respectively. Additionally, we compared clinical data. Results In the phase 1, 110 HD patients and 80 HVs were enrolled, and their seroconversion rates were 58.6% and 72.5%, respectively. In the phase 2, 20 HD patients without seroconversion in response to the vaccine the previous year were enrolled, and the dialyzer membrane was changed to PMMA 5 months before annual vaccination. After annual vaccination, 5 and 15 HD patients were categorized as responders and nonresponders, respectively. In the responders, β2-microglobulin, white blood cell counts, platelet counts, and serum albumin levels (Alb) were all higher than in the nonresponders. Conclusion The responsiveness to vaccination against influenza virus was lower in HD patients compared with HVs. Changing the dialysis membrane from PS to PMMA appeared to affect the response to vaccination in HD patients.
Examination of the Effect of Switching Dialysis Membranes on the Immune Response to Influenza Virus Vaccination in Hemodialysis Patients

In this study, we examined the effect of switching dialysis membranes on the response to influenza virus vaccination in HD patients.

This study consisted of two phases. In phase 1, antibody titers were measured and compared between HD patients and healthy volunteers (HVs) before and after vaccination against influenza virus. Using antibody titers 4 weeks after vaccination, HD patients and HVs were classified according to seroconversion (i.e., antibody titers against all four strains were >20-fold) or non-seroconversion (i.e., antibody titer against at least one strain was <20-fold). In the phase 2, we examined whether the change in the dialysis membrane from a polysulfone (PS) to a polymethyl methacrylate (PMMA) membrane affected the response to vaccination in HD patients without seroconversion in response to the vaccine the previous year. Patients with seroconversion and non-seroconversion were classified as responders and nonresponders, respectively. Additionally, we compared clinical data.

In the phase 1, 110 HD patients and 80 HVs were enrolled, and their seroconversion rates were 58.6% and 72.5%, respectively. In the phase 2, 20 HD patients without seroconversion in response to the vaccine the previous year were enrolled, and the dialyzer membrane was changed to PMMA 5 months before annual vaccination. After annual vaccination, 5 and 15 HD patients were categorized as responders and nonresponders, respectively. In the responders, β2-microglobulin, white blood cell counts, platelet counts, and serum albumin levels (Alb) were all higher than in the nonresponders.

The responsiveness to vaccination against influenza virus was lower in HD patients compared with HVs. Changing the dialysis membrane from PS to PMMA appeared to affect the response to vaccination in HD patients.

In hemodialysis (HD) patients, the innate and adaptive immune systems are both impaired [1]. Although the underlying mechanisms are not fully understood, the accumulation of certain uremic toxins, chronic low-grade inflammation, and oxidative stress has been implicated [2]. As the result of immune dysfunction, it is reported that HD patients have a lower response to vaccination against viruses such as hepatitis B virus [3] and influenza virus [4]. Influenza viruses frequently cause seasonal epidemic infections, so the guidelines for HD patients recommend annual vaccination before the epidemic season to protect against influenza infection. In recent years, coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has spread worldwide. HD patients are considered to be at higher risk of developing severe symptoms and have a higher mortality rate associated with COVID-19 infection compared with healthy persons [5]. It is reported that vaccination against SARS-CoV-2 would be effective in protecting against the aggravation of COVID-19 symptoms in HD patients [6]. Therefore, improvement of the poor vaccine response in HD patients would be beneficial. Polymethyl methacrylate (PMMA) membrane has unique properties, such as its homogenous structure and adsorption removal ability. Unlike polysulfone (PS) membranes that remove uremic toxins mainly by diffusion and filtration, PMMA membranes can more effectively remove middle- and high-molecular-weight substances as a result of their adsorption removal properties [7]. Many studies have reported the clinical benefits of dialyzers using PMMA membrane, including the improvement of dialysis itchiness [8, 9], the nutritional status of the patient [10], their immune response [11, 12, 13], and their prognosis [14, 15]. Regarding the improvement in the immune response, it was reported that the response to vaccine against hepatitis B virus could be improved in HD patients who were classed as nonresponders previously, when the dialyzer was changed to BK-F, a dialyzer that uses PMMA membrane [11, 12]. Furthermore, it was shown that removal of soluble CD40 (sCD40), which plays a role in antibody production and was not removed by the dialyzer using PS membrane, may be related to this improvement [11, 12, 16]. In this study, we investigated the response to influenza vaccine in dialysis patients compared with healthy persons. In addition, we examined whether changing the dialysis membrane affects the response to influenza vaccine in HD patients who were previous nonresponders and investigated the characteristics of patients who responded after the change in dialysis membrane.

To investigate the response to the influenza virus vaccine, HD patients were enrolled. The exclusion criteria were as follows: (1) patients who had a history of adverse reactions to inactivated vaccines, (2) patients who had received influenza vaccines within 27 days from receiving other live vaccines or 6 days from receiving other inactivated vaccines, (3) patients who showed a fever or cold symptoms at the time of influenza vaccination, (4) patients who had taken immunosuppressants, such as steroids, or those with immune diseases, (5) patients who received hemodiafiltration (HDF), and (6) patients who were determined to be ineligible for this study by the attending physician. For the purposes of comparison, healthy volunteers (HVs), who had not received dialysis therapy, were enrolled as a control.

Antibody titers against four strains of influenza virus including influenza A (H1N1), influenza A (H3N2), and two types of influenza B before and after vaccination were measured by a hemagglutination inhibition test by a commercial company (SRL Inc., Tokyo, Japan). The starting dilution was 10 times and the dilution factor was 2. The antibody titers of samples with dilution rates less than 10 times were set to 0. The antibody titers against four strains of influenza virus in HD patients and HVs were measured before and 4 weeks after vaccination. The threshold of antibody titers shown by SRL Inc. was less than 10-fold. Regarding the antibody titers before vaccination, HD patients and HVs whose antibody titers against all four influenza strains were 20-fold or higher were regarded as “positive,” and those whose antibody titer against at least one strain was less than 20-fold were regarded as “negative.” Regarding the antibody titers 4 weeks after vaccination, HD patients and HVs whose antibody titers against all four influenza strains were 20-fold or higher were regarded as demonstrating “seroconversion,” and those whose antibody titer against at least one strain was less than 20-fold were regarded as demonstrating “non-seroconversion.” In addition, patients who demonstrated seroconversion and non-seroconversion were classified as “responders” and “nonresponders,” respectively.

Our study comprised two phases (Fig. 1). In the “phase 1 study,” the response to the vaccine for influenza virus in HD patients was investigated and compared with that of HVs. Antibody titers against influenza virus were measured before and after vaccination. Blood samples for biological tests were collected before vaccination in the 2016/2017 season. In the “phase 2 study,” we examined whether switching of the dialysis membrane affected the response to vaccine in HD patients who had been classified as negative and did not undergo seroconversion in the previous year (phase 1 study). HD patients who had been dialyzed with a PS-type dialyzer and did not undergo seroconversion in the phase 1 study were selected, and their dialyzers were switched to PMMA dialyzers (NF-H; Toray Inc., Tokyo, Japan). After 6 months, patients were vaccinated against influenza virus. The antibody titers against influenza virus were measured before and after vaccination. Blood samples for biological tests were collected before vaccination in the 2017/2018 season.

The vaccine types and strains used in the three seasons, from 2015 to 2018, are shown in Table 1. With the exception of influenza A (H3N2) virus A/Swiss/9715293/2013 in the 2017/2018 season, the vaccine types and strains in the three seasons were the same.

This study was reviewed and approved by the Second Institutional Review Board of Adachi Kyosai Hospital, Shinwakai Healthcare Corporation, in accordance with the Declaration of Helsinki. It was initiated after registration in the University Hospital Medical Information Network Center (UMIN ID: UMIN000019455). Informed consent was obtained from all patients before their participation in the study.

Results are presented as the mean ± standard deviation or the median (minimum − maximum value). For statistical analyses, the t test, Fisher's exact test, and the Brunner-Munzel test were performed using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan). The level of significance was set at p < 0.05.

In total, 110 HD patients and 80 HVs were enrolled in this study in the 2016/1017 season. Compared with the HVs, the percentage of males and the age of HD patients were significantly higher (Table 2). When comparing the antibody titers against influenza virus, the percentage of patients who had undergone seroconversion in response to the vaccine was significantly higher among the HVs (72.5%) than among the HD patients (58.2%) (Fig. 2a, p < 0.05). HD patients and HVs were classified as positive or negative based on their antibody titers against four strains of influenza virus, which were measured before vaccination. Among HD patients, 15 were positive and 95 were negative (Table 2). Among the HVs, 22 were positive and 58 were negative (Table 2). The percentage of individuals with negative antibody titers was significantly higher for HD patients than for HVs (p < 0.05). All of the HD patients and HVs that were classified as having positive antibody titers underwent seroconversion after vaccination. Among HD patients and HVs classified as having negative antibody titers, the percentages of seroconversion were 51.6% and 62.1%, respectively, which showed no significant difference (Table 2).

We selected 20 HD patients who had not undergone seroconversion during the phase 1 study. These patients were treated with PS-type dialyzers, and their dialyzers were switched to PMMA dialyzers, NF-H, 6 months before vaccination. These 20 patients were also classified as negative according to their antibody titers before vaccination in the phase 2 study. After vaccination in the 2017/2018 season, 5 and 15 HD patients underwent seroconversion (25.0%) and non-seroconversion (75.0%), respectively (Fig. 2b), and these patients were considered to be responders and nonresponders, respectively. When comparing several factors, such as dialysis conditions and nutritional status, before and after switching of dialyzers among the 20 HD patients, a remarkable decrease in serum albumin levels was observed, but no other significant differences were detected (Table 3). When comparing responders and nonresponders, white blood cell (WBC) counts and platelet counts were significantly higher and β2-microglobulin (β2-MG) levels were significantly lower in responders than in nonresponders. These phenomena were observed before and after switching to NF-H. In nonresponders only, there was significant reduction in serum albumin levels after switching to NF-H (Table 3).

In our phase 1 study comparing the responses to vaccination against influenza virus, seroconversion rates in HD patients and HVs were 58.2% and 72.5%, respectively. This result was consistent with several reports showing that the seroconversion rates in HD patients were lower than those in HVs [4, 17]. To counteract the low response to vaccine, an increase in the dose or the number of vaccinations has been trialed [4, 18, 19]; however, the resulting efficacies are controversial, particularly in HD patients [18, 19, 20]. The occurrence of adverse events and the increased costs must also be considered when such countermeasures are applied. One way to avoid these limitations would be to improve the response to vaccine by changing the dialysis membrane and/or the dialysis modality. Regarding changing the modality, Nongnuch and coworkers reported that seroconversion rates for influenza vaccine were not significantly different between HD and HDF therapies, although seroprotection in patients treated with HDF was sustained for longer compared with those treated by HD [21]. Regarding the change in dialysis membranes, some studies have reported that the use of PMMA dialyzers has the potential to improve the response to vaccine against HBV in HD patients who were treated with PS-type dialyzers and were classified as nonresponders to the vaccine [11, 12, 16]. These reports showed the removal of soluble CD40 sCD40, which can suppress the interaction between T and B cells, as the possible mechanism involved. Additionally, it has been reported that a PMMA dialyzer, BK-F, can influence the modulation of both the percentage of natural killer (NK) cells expressing NKG2D and the overexpression of MICA, a marker of inflammation induced by cellular stress [13]. This modulation of immune cells might contribute to the improvement in the response to vaccine. Recently, de Précigou et al. [22] reported no improvement in the vaccine response with a PMMA dialyzer compared with high-flux PS. The abovementioned improvement in the response to vaccine by PMMA dialyzers, BK-F, is also controversial. However, we investigated whether the change in dialysis membrane affected vaccination and the differences between responders and nonresponders to vaccination in HD patients who had not responded to influenza vaccine in the previous year. In our second phase study, we selected 20 HD patients, who had been classified as negative and had not undergone seroconversion during the previous year and were treated with PS-type dialyzers, and examined whether the change in dialysis membranes would influence their responsiveness to vaccine. After the change of PMMA dialyzer, vaccination induced seroconversion in 5 patients but seroconversion was not detected in the other 15 patients, which were classified as responders and nonresponders, respectively. According to comparisons of the clinical data between responders and nonresponders, there were significant differences in β2-MG levels, WBC counts, platelet counts, and serum albumin levels before dialysis. After the change of dialysis membranes, a significant difference was only detected in serum albumin levels between the responders and nonresponders. Because it is reported that serum albumin levels correlate positively with the responsiveness to vaccine [23], we propose that the response to vaccine observed in patients without decreased serum albumin levels was a result of the change in dialysis membrane in our study. Regarding the other three indexes measured (i.e., β2-MG levels, WBC counts, and platelet counts), there were significant differences between responders and nonresponders before and after the change of dialysis membrane but the membrane change was not related to these significant differences. Since platelets function as immune cells [24, 25, 26], it would be expected that patients with higher platelet and WBC counts had better responses to vaccine. β2-MG has been reported to be a negative regulator of the immune system [27], so it would be assumed that the immune response to vaccine would be improved in patients with lower β2-MG levels than those with higher β2-MG levels. However, these patients did not actually respond to vaccine in the phase 1 study. Furthermore, these patients responded to vaccine after the change in dialysis membrane without any remarkable change in the platelet and WBC counts or the β2-MG levels. These findings suggest that immune suppressors, such as sCD40, may be removed by the change in dialysis membrane. In this pilot study, sCD40 levels were not measured; however, this will be the subject of a future study.

This study has several limitations. First, the design of this pilot study was a single-arm trial to obtain preliminary results. Second, the sample size was small. Third, the serum levels of substances, such as sCD40, presumed to be related to immune dysfunction in HD patients were not measured before and after the change in dialysis membrane. Furthermore, the profiles or functions of immune cells, including NK cells, were not compared before and after the change of membrane. Based on the results of our pilot study, we plan to conduct a double-arm trial with a large sample size in the future to clarify whether the change of dialysis membrane would be beneficial in the response to vaccine.

The responsiveness to vaccination against influenza virus was substantially lower in HD patients compared with HVs. Additionally, the change of dialysis membrane from PS to PMMA appeared to affect the response to vaccine in HD patients who had not previously responded. According to the comparison of clinical data between the seroconversion and non-seroconversion groups, there were significant differences in four indexes: β2-MG levels, WBC counts, platelet counts, and serum albumin levels.

This study was reviewed and approved by the Second Institutional Review Board of Adachi Kyosai Hospital, Shinwakai Healthcare Corporation, in accordance with the Declaration of Helsinki (Approval date: October 22, 2015). Written informed consent was obtained from all the patients before they were enrolled into this study. It was initiated after registration in the University Hospital Medical Information Network Center (UMIN ID: UMIN000019455).

The authors have no conflict of interest to disclose.

This study was funded by Toray Industry Inc.

Tadashi Tomo planned the study, collected data, analyzed the data, and wrote the paper. Mitsuo Takei, Makoto Matsuyama, Iehisa Matsuyama, and Kazuhiro Matsuyama assisted in data collection and data analysis.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.