University of Groningen Ageing and vaccination in transplant patients Wang, Lei

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University of Groningen

Ageing and vaccination in transplant patients

Wang, Lei

DOI:

10.33612/diss.160954435

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Wang, L. (2021). Ageing and vaccination in transplant patients. University of Groningen. https://doi.org/10.33612/diss.160954435

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Chapter 8

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1 Summary of findings in this thesis

Along with the increasing numbers of organ transplantations performed in this era, the age of the transplant recipients also goes up (1). Despite the improvement of graft operation and medication, patients still face serious mortality which is caused by graft failure, cardiovascular disease, malignancies and infectious complications (2). Advanced age is considered to significantly contribute to these complications. Varicella zoster virus (VZV) is a human α-herpes virus which can establish a lifelong latency in the dorsal root ganglia after primary infection. Reactivation of VZV leads to herpes zoster (HZ). The risk factors for developing HZ and its related complications include advancing age and weakening of immune system (3). Transplant patients as well as the patients on the waiting list for transplantation are facing higher risk of HZ due to their immunocompromised status (4). Currently, there are two HZ vaccines available on the market: a live-attenuated vaccine (Zostavax®_{, Merck Sharp &}

Dohme) and a recombinant subunit vaccine (Shingrix®_{, GlaxoSmithKline).}

Due to the impaired immune response to the vaccination by administration of immunosuppressants after transplantation and the safety concerns of live-attenuated vaccine usage in immunocompromised patients, immunization before transplantation is highly recommended (5). In this thesis, we focused on two aspects: one is investigating ageing of the immune system in elderly and transplanted patients; the other one is studying the safety and immunogenicity of HZ vaccine in transplant patients, and the influence of ageing on the immune response to this vaccine.

In Chapter 2, we reviewed the literature in recent years about the diagnosis of HZ, treatments for HZ and its complication postherpetic neuralgia (PHN), and furthermore the prevention of HZ, especially in transplant patients. Generally, the lesions of HZ present in unilateral dermatomal distribution with localized neurological pain (6). However, it’s easy to confuse HZ with other cutaneous diseases in clinical diagnosis since HZ in transplant patients is often presented with atypical mucocutaneous forms (7). As a supplementary diagnostic tool, the polymerase chain reaction (PCR) is a highly sensitive test which can detect VZV virus in vesicle fluid swab, biopsies or blood samples (8). VZV-specific antibody levels of transplant patients should be measured by serologic testing before transplantation to evaluate the risk of developing serious varicellosis (chickenpox) in seronegative patients and HZ in seropositive patients after transplantation (6). Once diagnosis is confirmed, antiviral agents are prescribed 149

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to control the replication of VZV in infected cells. The complicated condition and fragile organ function of transplant patients may influence the drug metabolism and tolerability (9). Therefore, the type, dosage and side effects of treatments for HZ and its complications should be prescribed on the basis of patients’ personalized condition. As for the prophylaxis for HZ, both live-attenuated vaccine and recombinant subunit vaccine were found to be well-tolerated in selected transplant patient groups, and the latter vaccine showed better efficacy although more studies are still needed. Other than vaccination, long-term antiviral prophylaxis is also effective for preventing HZ. In transplant patients, cytomegalovirus (CMV) infection is one of the most prevalent infectious complications (10). The antiviral agents (ganciclovir and its valyl-ester valganciclovir) used to prevent CMV infections also show excellent inhibition on VZV activity (11, 12). However, during antiviral prophylaxis the risk for developing HZ is still higher in transplant patients than in the general population and discontinuation of prophylaxis could not prevent HZ later on (13). Vaccination before transplantation is still a promising strategy for prevention of HZ in transplant patients.

To assess the safety and immunogenicity of HZ vaccination in transplant patients, as described in Chapter 3, we vaccinated 68 end-stage pulmonary disease (ESPD) patients with one dose of Zostavax® _{while they were newly}

screened for lung transplantation. We also enrolled 37 patients on the waiting-list as un-vaccinated controls. Both VZV-specific humoral and cellular immunity were evaluated by an in-house glycoprotein VZV enzyme-linked immunosorbent assay and interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assay, respectively. During around 2 years (median) follow-up, 29 vaccinated patients underwent lung transplantation and no vaccine-related serious adverse events were found in this study. Besides, there was no sign of vaccination affecting the transplant outcome. A robust humoral and cellular response to the vaccine was observed, especially in younger patients, which was highest within 3 months after vaccination and was not influenced by gender or type of ESPD. Multilevel analysis showed that age, CMV serostatus and immunity to VZV before vaccination impacted the subsequent immune response to the vaccine. VZV-specific cell-mediated immunity (CMI) was significantly reduced by the short-term immunosuppressant after transplantation and a better recovery of VZV-CMI was seen 6 months after transplantation in vaccinated patients. We concluded that patients awaiting lung

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transplantation could benefit from HZ vaccine, also in patients who are younger than the normally recommended age at which people get this vaccination.

Impairment of the immune system caused by immunosenescence could affect the efficacy of HZ vaccination. Less expression of co-stimulatory surface receptor CD28 on T cells and accumulation of age associated B cells (ABCs) were found to be strongly related to ageing. In Chapter 4, we measured the frequencies of T and B cell subsets, especially aged cell subsets (CD28- T cells and ABCs), before and after vaccination in ESPD patients who received one dose Zostavax®_{. Humoral and cellular immune response to HZ vaccine were}

also evaluated. Although CD4+CD28- and CD8+CD28- cells were both positively correlated with age, we didn’t find a relationship between the frequencies of CD28- T cells pre-vaccination and the immune response to the HZ vaccine. Higher frequencies of ABCs before vaccination correlated with a low immune response to the HZ vaccine, especially the cellular immune response to HZ vaccine. This might be due to the inflammatory microenvironment caused by cumulative ABCs, which in turn disturbed the T cell response to the HZ vaccine (14).

In Chapter 5 we described the effect of transplantation on ageing of T and B cells in end-stage renal disease (ESRD) patients before and after (median 2.7 years) kidney transplantation, in comparison with age- and sex-matched healthy controls (HC). Before transplantation, there was no difference in frequencies of T and B cell compartments compared with HC. Three years after transplantation, a significant shift in proportions of naive to memory T cells happened, especially in older patients. Percentages CD28- T cells in CMV seropositive HC and ESRD patients were significantly higher compared to that in CMV seronegative participants. There was no difference in CD28- T cell frequencies in patients before and after transplantation, and also not between young and old patients. Our data indicated that CMV latency drove the lower expression of CD28 in transplant patients more than the age at transplantation. Just like T cells, higher percentage of memory B cells and lower percentages of naïve B cells were found after transplantation. Patients with viral or bacterial infections after transplantation had higher percentage of ABCs.

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Studying the VZV-specific immunity before and after kidney transplantation could give a better understanding on strategies for prevention of HZ in this patient group. In Chapter 6, we described the effect of kidney transplantation on humoral and cellular immunity to VZV. Transplant patients showed significantly lower VZV-specific immunity than matched HC. After transplantation, VZV IgG levels significantly decreased while the number of VZV-stimulated IFN-γ-producing cells did not change when comparing it with their numbers before transplantation. Patients who experienced rejection or any acute infection other than HZ after transplantation had a lower number of IFN-γ-producing cells, which could reflect the suboptimal immune system of these patients. In response to VZV and polyclonal stimulation, frequencies of cytokine production CD4+ T cells were shown to be higher before transplantation, but did not differ between transplant patients and HC. No difference in expression of inhibitory receptor programmed cell death protein 1 (PD-1), but a higher expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) was observed after transplantation both after VZV and polyclonal stimulation.

As described in Chapter 7, we successfully established an in vitro model to study the cellular senescence effect on VZV infection. The senescent status of human dermal fibroblasts (HDFs) was achieved by continuously passaging until more than 20 passages and this was confirmed by the expression of senescence-associated β-galactosidase. VZV-infected human lung fibroblasts effectively replicated in both young (passage 3-10) and senescent (passage20-25) HDFs and a typical cytopathic effect was observed. VZV infection induced increased phosphorylation level of c-Jun N-terminal kinase (JNK)/p38 and their downstream substrates 48 hours after infection but no significant difference was found in these two pathways in response to VZV infection during replication-induced cellular senescence.

2 General discussions and future perspectives

In the studies described in this thesis, we reported that the ageing of immune system was affected by transplantation as well as its further impact on HZ vaccine response. This part discusses key findings of this thesis and future directions on ageing and HZ vaccine in transplant patients.

2.1 Ageing in transplant patients 152

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Ageing-associated immunosenescence can affect all immune compartments both in function and regulation. In elderly transplant patients, elevated mortality is observed with increasing age of the recipients, and more than half of all mortalities are caused by complications such as cardiovascular disease, infections, or malignancies and they are aggravated by age and immunosuppressive therapy (15). Although the rate of acute rejection in older recipients is lower, acute rejections occurring in older recipients are more harmful on graft survival than in younger recipients (16). Studying immunosenescence and immune remodeling in transplant patients could show vital clinical values for individualizing transplant protocols to improve graft outcomes. T cells play a key role in tolerance and rejection, and most age-related immune remodeling are considering T cells. In older people, decrease in the frequency of naïve T cells and increase in the frequency of differentiated memory T cells are demonstrated. This shift, from naïve to memory phenotypes, leads to the significant shrinkage of T cell repertoire diversity (17). The thymus involution already starts at age 1, but the contribution of the thymus to T cell generation is really limited in elderly with a decline from ~16% to <1% throughout lifetime, and the output of naïve T cells is more and more relying on homeostatic self-renewal at higher ages (18). Therefore, the decreased number of naïve T cells with age is a combination of thymic involution and peripheral expansion to antigen-experienced memory or effector cells (15, 18). Premature ageing of the immune system, possibly caused by oxidative stress and inflammatory microenvironments in ESRD patients was reported (19). In contrast, we showed in Chapter 5 similar frequencies of T cell subsets in patients before kidney transplantation compared with age-matched HC. Major differences in T cell subsets frequencies were seen after transplantation and they were probably caused by immunosuppressive treatment.

CD28 plays an important role in T cell activation, proliferation, and survival, and the accumulation of CD28- T cells is one of the most remarkable changes in age-associated changes of T cells (20). Previous data showed up-regulated CD28 expression on CD4+ T cells during acute rejection (AR) episodes and CD28 may have the potential to become a predictive biomarker of AR (21). CD28- T cells are considered to be terminally differentiated senescent cells, with shorter telomeres than the CD28+ T cells (20). CD28- T cells have a great ability of cytotoxicity and express large amount of effector molecules 153

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(granzymes and perforins) and pro-inflammatory cytokines such as INF-γ (22). The loss of CD28 expression with age is considered to be mainly the result of repeated antigenic stimulation but could also be driven by proinflammatory environments during new or persistent infections. Previous studies have shown that the accumulation of CD28- T cells mainly occur in the CD8 compartment (22), which is in agreement with our observation in Chapter 5 that the median frequency of CD4+CD28- T cells was 2.6%, while the median frequency of CD8+CD28- T cells was 39.3% in patients before kidney transplantation. Although it is often said that CD28- T cells accumulate with chronological age, CMV infection plays an important contributory role in this process as the number of persons with CMV infection is also increasing with age. In Chapter 4 and Chapter 5, significantly higher frequencies of CD28- T cells were observed in CMV seropositive HC and in ESRD or ESPD patients. Our data suggested that CD28- T cell expansion in transplant patients is more driven by CMV latency than ageing or transplantation. Similar results were found in other studies (23-26). This is supported by the fact that about 10% of the total circulating T cells are CMV-specific and a large portion of CD28- T cells is CMV-specific (19, 22) but it is unknown whether these cells also recognize other antigens. It is therefore crucial that all studies related to ageing of the immune system and immunity in transplant patients take the CMV status into account.

B cells are known to be mainly responsible for humoral immunity, but they are also important to boost cellular immunity. The age-related remodeling on B cells is still controversial due to different cellular markers to identify functional B cell subsets as well as the variations between enrolled individuals (27). Immunosuppressive drugs after transplantation could also affect the number and phenotype of B cells. In one study, kidney transplant recipients under combined treatment with tacrolimus, mycophenolate mofetil, and steroids had more B cells with a memory-like B-cell phenotype when comparing it with before transplantation (28). We described consistent results in Chapter 5 and we also found significantly lower frequency of switched memory B cells after transplantation, compared with pre-transplantation patients and HC. Switched memory B cells can generate rapid secondary antibody responses to the re-exposed antigens (29). The decreased frequency of this B cell subset could account for the high rate of infections after transplantation.

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Accumulation of a unique B cell subpopulation, named age associated B cells (ABCs) during ageing, was identified by characteristic profile of CD11c+CD11b+CD21-CD23-Tbet+ expression both in aged mice and in humans (14). There is ample evidence that antigens associated with toll-like receptors (TLR) 7 or TLR9 ligands and inflammatory microenvironment (especially IFN-γ) are necessary to drive ABC differentiation (29). ABCs can impair the generation of young pro-B cells by secreting high amounts of tumor necrosis factor (TNF)-α and then hinder B cell genesis. ABCs are elevated in autoimmune diseases but how ABCs are developing in transplant patients is still not fully known. In Chapter 5 we showed a higher frequency of ABCs after kidney transplantation but we could not prove that this was mainly driven by the transplantation itself. We did find that patients with viral or bacterial infections after transplantation during follow-up had significant increase in ABCs frequencies, which is in line with the notion that acute and chronic infections are associated with emerging ABCs (14).

2.2 VZV immunity in transplant patients

Most of the transplant patients are seropositive for VZV and are facing a high risk for developing HZ caused by VZV reactivation. Advanced age and an immunocompromised status are risk factors for HZ (6). In a population-based retrospective study using data from more than 4 million inhabitants in Valencia Region of Spain (2009–2014), overall incidence of HZ in subjects with no immunocompromised condition was 4.7 per 1,000 person-years, which was increased significantly with age, reaching 9.5 per 1,000 person-years in ≥80 age group. Immunocompromised subjects had higher incidence of HZ in all age groups than immunocompromised-free subjects (30). Although the molecular mechanisms underlying the establishment of VZV latency and prevention of reactivation remain largely unknown as will be discussed further in section 2.5, the waning of VZV-specific CMI, especially the level of VZV-specific CD4 T cells is found to be correlated to the occurrence of HZ (3). This is supported by the evidences that humoral immunity against VZV is relatively stable while VZV-CMI declines in elderly and immunocompromised patients (31). A large-scale prospective cohort study showed that the severity of HZ skin lesions and the incidence of HZ and its complications was inversely associated with VZV-specific CMI, and not with humoral immunity (32). In Chapter 6, we show that kidney transplant patients had lower levels of VZV-specific CMI at 3 years after transplantation compared with HC at that moment but the levels before 155

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and after transplantation were not significantly different. In Chapter 3, we showed that VZV-specific CMI returned to its pre-transplant level at a median of 7.6 months post-transplantation in unvaccinated lung transplant patients and a better recovery of VZV-CMI was seen in vaccinated lung transplant patients. In patients who do get HZ, the VZV-specific CMI level was shown to be associated with the onset of HZ and the severity of skin lesions (32). Our observation that VZV-CMI did not markedly change following kidney/lung transplantation reinforces that vaccination before transplantation could be beneficial.

2.3 Immunosenescence and immune response to HZ vaccination in transplant patients

Zostavax®_{is the first licensed HZ vaccine which contains at least a 14 times}

higher titer of the same live attenuated Oka/Merck strain that is present in the primary varicella vaccine (Varivax®_{, Merck Sharp & Dohme) (33). Currently}

Zostavax®_{has been administered and studied in different immunocompromised}

patients with hematologic malignancies, hematopoietic stem cell transplantation (HSCT) and ESRD. Although overall Zostavax®_was

well-tolerated and no significant adverse events were reported in either cohort, Zostavax®_{is still not recommended and especially contraindicated in highly}

immunocompromised patients because of safety concerns and small numbers of participants enrolled in these studies (34). Lung transplant patients receive more intensive immunosuppression thus they have an even higher risk of HZ among all types of solid organ transplantation (4). The study described in Chapter 3 is the first study about the safety and efficiency of Zostavax®_in

lung transplant candidates. We showed that the ESPD patients had a good humoral response to the HZ vaccine that persisted even more than 1 year after vaccination. In the 29 patients who underwent lung transplantation after vaccination, with a median 16.6 months follow-up post-transplantation, we did not observe impact of HZ vaccine on transplant outcomes and we detected a rebound of VZV-CMI level after a short-term drop post-transplantation, which was higher than in unvaccinated transplant patients.

The efficacy of Zostavax®_{significantly decreases with the age of the vaccinees,}

from 70% in 50-59 age group down to 18% in >80 age group (35). Immunosenescence has been suggested as a key factor of poor vaccine responses in older adults. During immunosenescence, a lower frequency of

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naïve cells signifies limited activation and differentiation of naïve cells in response to new pathogens and vaccines and meanwhile more terminally differentiated and senescent cells could impair the ability of an active response to previously experienced antigens, including Zostavax®_{. In this way}

immunosenescense could limit the ability to induce a VZV-specific adaptive immune response (36). Weinberg et al. compared the VZV-specific CMI to Zostavax®_{in young and older adults, and they found that older vaccinees}

displayed higher VZV-specific CD8+CD57+ senescent T cells both before and after vaccination than young vaccinees. They also showed that older adults had decreased numbers of dual-functional VZV-specific CD4+ and CD8+ Th1 responses (defined by co-expression of IFN-γ, IL-2, and CD107) after vaccination compared with young vaccinees (35, 37). Since only exceedingly small percentage of T cells is specific for VZV, it is difficult to accurately measure VZV-specific senescent T cells and investigate its relation with vaccine response. There is some evidence that shows that CD28- T cells as well as ABCs are associated with a poor response to influenza vaccine in elderly (36, 38). We investigated if these senescent phenotypes also affected the response to Zostavax®_{. In}_{Chapter 4, we found that a higher frequency of ABCs rather}

than the level of CD28- T cells before vaccination in ESPD patients correlated with a lower cellular immune response to Zostavax®_{. Due to the small number}

of patients and variance of age, we could not draw firm conclusions, but a better understanding of the role of ABCs during immunosenesence is warranted. We did see by multilevel analysis in Chapter 3 that CMV seropositive vaccinees had lower VZV-CMI response to HZ vaccine. This suggest that CMV latency could accelerate ageing of the immune system and thus can contribute significantly to the overall impairment of the immune system and the waning of a good immune response to HZ vaccination.

In the studies of this thesis, all the participants were younger than 70 years and immunosenescence might even be more severe in septuagenarians and octogenarians. It is expected that we will enroll even older patients in future transplant studies and in this way we will enlarge the age variance. Besides, as we have shown that CMV latency has a significant effect on the ageing process and the complexity of donor/recipient CMV serostatus, it is difficult to investigate other factors because of the large effect of CMV on immunosenescence. It could be interesting to focus more on donor CMV-/recipient CMV- transplant patients and explore in this particular subgroup the

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effect of transplantation itself on the impairment of the adaptive immune system. In addition, it is necessary to follow up the patients with HZ vaccination in our studies to see the efficacy on preventing HZ occurrence. Although only one vaccinated individual developed HZ 3 years later and we think that it was not vaccine related, it will be useful to detect the VZV virus in the HZ rash by PCR and sequencing analysis to identify whether it was wild type or vaccine strain. This will provide even more evidence on the safety of this live-attenuated vaccine.

2.4 Shingrix®_{-as alternative HZ vaccine in transplant patients}

In 2017, a recombinant subunit HZ vaccine (Shingrix®_{) containing VZV}

glycoprotein E (gE) and the AS01B adjuvant system was approved by the Food

and Drug Administration. According to the Advisory Committee on Immunization Practices (ACIP) recommendations, Shingrix® _{is preferred over}

Zostavax®_{for the prevention of HZ in adults aged≥50 years. Shingrix}®_should

be administered in 2 doses (0.5 mL each) intramuscularly with 2-6 months interval (39). Unlike Zostavax®_{, the overall efficacy against HZ of Shingrix}®

was 97.2% and was not affected by age, with an efficacy of 97.9% in ≥70 year group (40). In an immunogenicity study of Shingrix®_{, 97.8% of the recipients}

developed robust humoral (anti–gE antibody) responses and CMI (gE-specific CD4 T-cell) that persisting for 3 years after vaccination (41). The adjuvant system probably brings a higher protection of HZ in older individuals but it also raises reactogenicity and results in a relative higher frequency of systemic reactions. Solicited injection-site reactions occurred in 81.5% of Shingrix®

recipients, which was about 7 times higher than placebo recipients (40). In a post-hoc analysis based on the population in two large placebo-controlled efficacy studies (ZOE-50/70), efficacy of Shingrix® _{remained high in recipients}

with selected medical conditions present at enrollment such as hypertension, diabetes and asthma. Persons with immunocompromised conditions or on immunosuppressive therapy were excluded from this study (42). Currently, one clinical trial (NCT03993717) is determining the effect of Shingrix®_vaccination

at different time points after vaccination and a new clinical trial is being setup (NCT04128189) that will focus on a 3rd boost vaccination after kidney transplantation.

HSCT patients have the highest incidence of HZ and HZ related complications 158

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among all kinds of immunocompromised patients (30, 34). In spite of a good preventive effect on HZ by given at least 1 year antiviral prophylaxis following HSCT, incidence of HZ was high and a concern about the tolerability of long-term use of antiviral agents is raised in HSCT patients (43).In a phase III, randomized, observer-blinded study, 1846 adult patients who underwent autologous HSCT were enrolled and received first doses Shingrix® _{(or placebo)}

50 to 70 days after transplantation and the second dose 1 to 2 months thereafter. During the 21 months (median) follow-up, a calculative 68.2% vaccine efficacy was observed. One month after the second dose, the highest humoral and cellular immune responses to Shingrix®_{were detected, followed}

by a decline subsequently but levels remained higher than baseline 24 months after the second dose (44). Besides, Shingrix®_{was also reported to be}

well-tolerated post-transplantation in small numbers of kidney or heart transplant patients (45, 46). Currently the experiences of Shingrix® _{were all with}

post-transplant administration. The efficacy of this vaccine could be interfered by the immunosuppressive therapy and the patients’ vulnerable immune status after transplantation. In the future, it will be interesting to investigate the possibility of giving Shingrix®_{before transplantation or before initiation of}

immunosuppressive therapy in HSCT, lung and kidney transplant patients, especially in elderly patients. For allogenic HSCT it could even be interesting to immunize the donor before harvesting the stem cells. At the same time, it is recommended to investigate the optimal time interval between 2 doses of Shingrix®_{in these populations.}

2.5 Molecular mechanism of VZV reactivation

The molecular mechanism of how VZV reactivates from latent status in the neuron is still largely unclear although the waning of VZV-CMI is believed to be related to this process (3). Currently, small animals such as guinea pigs and mice were used to study VZV latency. These animals with experimental VZV infection do not show typical varicella but ganglionic neurons can be infected later on (47). However, these animal models do not fully replicate VZV reactivation in human as seen due to VZV human-restricted nature of latency in dorsal ganglia. So as alternative also human neurons obtained post-mortem are widely studied (47, 48). By analysis of human neurons from autopsy, VZV gene expression was found to be highly restricted in human ganglia. The proportion of infected neurons might increase due to the loop that VZV travels anterograde to causes skin lesions and then it travels back to the ganglia to 159

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infect more neurons (49). Of course, the virus genome transcription might not fully reflect the real replication pattern in vivo because of possible changes of ganglia after death. Recently, there are some advances in development of neuronal in vitro models by inducing differentiation of the stem cells into human neurons (48). By investigating human neurons derived from embryonic stem cells, the JNK pathway was found to be activated during VZV infection and inhibition of JNK limited VZV reactivation in vitro (50). More studies on additional neuronal signaling pathways during VZV reactivation are still needed. In Chapter 7, we established a replicative senescence in vitro model. We realized that this model did not simulate the reactivation process in neurons but it could mimic the VZV development in the skin epidermal layers of elderly after release from the neurons as second step in the reactivation. We found no difference of VZV replication between young and senescent HDFs and also activation of the JNK/p38 signal pathway was similar in young and old HDFs. In this study we have established an in vitro model that could form the basis of a more complex in vitro model where we can investigate how VZV specific T cells can prevent the further spreading of the virus within the skin during reactivation. It is still necessary to further confirm virus loads and transcriptional levels of JNK/p38 downstream signaling components in senescent HDFs. As next step, VZV specific T cells derived from T cell cloning could be introduced into this in vitro system. This would allow us to gain better understand the molecular mechanisms how VZV-specific adaptive cellular immune responses can prevent the reactivation of the latent VZV virus. Insight into such molecular mechanisms can lead to possible new targets of therapy in the increasing populations of elderly transplant patients.

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