Pneumococcal Conjugate Vaccine in Aging Renal Transplant

Objectives / Specific Aims

Normal aging is characterized by changes in the immune system that result in increased susceptibility to infections, poor response to new vaccine antigens, loss of protection offered by previous vaccinations and decreased immune surveillance. Despite the chronic inflammation associated with immunosenescence, the aging population is living longer. Consequently, individuals >65 years of age, are the most rapidly growing population amongst those with end stage renal disease (ESRD) and account for more than 18% of renal transplant (RT) recipients.

The incidence of pneumococcal disease is significantly higher in both elderly and those with RT and the combination of these factors is likely additive, if not synergistic, for invasive pneumococcal disease (IPD). It is recommended that both elderly>65 and RT recipients be vaccinated with a regimen that includes both the 13-valent pneumococcal conjugate vaccine (PCV13) and the 23-valent pneumococcal polysaccharide vaccine (PPV23). However, small immunogenicity studies performed in the transplant populations have not shown superiority of a PCV containing regimen. Moreover, the addition of PCV to the pneumococcal vaccine regimen does not improve protective immunity in this population. Studies to date fail to elucidate the possible foundation of the disappointing immune responses to the PCV regimens, data essential for the development of a rational and effective next generation pneumococcal vaccine.

Specific Aim 1. The investigators will define immune responses by measuring serum antibody and functional antibody responses to PPS 14, 19A and 23F following PCV13 vaccination in RT recipients 65-75 years of age and compare these to: RT recipients 35-45 years of age and persons with DM/HTN but normal function 65-75 years of age to dissect out the age and RT components respectively. Healthy persons 35-45 and 65-75 years of age will be studied as age appropriate reference.

Specific Aim 2. The investigators will measure and characterize the antigen-specific B cell subset response following immunization with PCV13 in the RT recipients 65-75 years of age and compare them to each of the groups described in Specific Aim 1 using flow cytometry and fluorescently labeled PPS and monoclonal antibodies. These measures will be correlated with post-immunization functional antibody activity, a surrogate of protection.

Specific Aim 3. The investigators will measure TNFR expression by B cells following immunization with PCV13 in 65-75 year old RT and compare them to each of the groups described in Specific Aim 1. Gene expression, with focus on the B cell activating factor (BAFF) system, will be measured in PPS-specific and non-PPS specific B cells using single cell genomics and flow cytometry. These measures will be correlated with post-immunization functional antibody activity, a surrogate of protection.

The central hypothesis is that the aging RT population responds poorly to PCV13 vaccination reflecting the combined effects of aging and RT. The investigators postulate that both the number of memory B cells and expression of tumor necrosis factor (TNF) superfamily receptors, which play crucial roles in the response to pneumococcal polysaccharides (PPS), are deficient in the elderly RT population and contribute to poor pneumococcal vaccine responses.

The investigators have developed fluorescently labeled PPS allowing us to study the nature and surface receptors of PPS-specific B cells. The preliminary data demonstrate that RT recipients 1. Respond poorly to pneumococcal immunization as measured by antibody titer and functional antibody activity. 2. RT recipients and healthy elderly have lower absolute number of both IgM and switched memory B cells. 3. The number of PPS-specific IgM and switched memory B cells are significantly lower in RT recipients and 4. TACI and BAFF-R, members of the TNF superfamily receptors, expression is significantly lower in the PPS-specific memory B cells in the RT population versus healthy controls. The overall objective of this proposal is to characterize the immune response and explore possible mechanisms of poor vaccine responsiveness following immunization with PCV in the rapidly growing group of elderly with RT. As the RT population is a heterogeneous group the investigators will study only those in whom the underlying cause of renal failure is diabetes mellitus type 2 (DM2) and/or hypertension (HTN).

Background

Streptococcus pneumoniae is the most common bacteria isolated from the elderly with community acquired pneumonia. The elderly are at high risk of pneumococcal infection, have an increased incidence of blood stream infection and a higher mortality rate compared to younger adults. It is projected that between 2000 and 2040, the number of Americans 65 years of age will more than double. The rapid growth of the aging population has resulted in a significant increase in elderly individuals with end-stage renal disease (ESRD). In 2014, there were >300,000 older adults with ESRD, a seven-fold increase compared to the 1990's. In addition, individuals with ESRD are living longer, disproportionally increasing the number of elderly with ESRD. Moreover, older (>65 yrs) individuals now account for 18.4% of the 17,000 renal transplants performed in the US annually and are by far the fastest growing population requiring renal replacement therapy, i.e. dialysis or renal transplantation. Although the renal transplant (RT) population is susceptible to a host of infections, S. pneumoniae is the most common bacterial respiratory pathogen and the incidence of invasive pneumococcal disease (IPD) is estimated to be 12.8 fold higher than in the general population. Moreover, aging individuals with RT have two distinct risk factors for pneumococcal disease, namely age and RT accounting for the high incidence of disease in this population.

Accordingly, pneumococcal vaccination is recommended for all adults 65 years of age and all RT recipients. In 2012 the vaccination recommendations by the Advisory Committee on Immunization Practices (ACIP) for these immune compromised adults, were changed to include vaccination with the 13-valent pneumococcal conjugate vaccine (PCV13). Although highly successful in infants, immunogenicity studies have overall failed to demonstrate superiority of PCV based regimens in older adults. Similarly, a prime-boost regimen based on sequential immunization with PCV7 and the 23-valent pneumococcal polysaccharide vaccine, Pneumovax (PPV23), has not demonstrated improved responses in RT and liver transplant populations. The RT population 65 years of age is rapidly growing and represents a unique subpopulation with combined immunodeficiencies related to aging and immunosuppressive drugs related to transplant. Both aging and RT result in a marked increased risk of pneumococcal disease with decreased immune response to pneumococcal vaccination. The immune response to vaccination with PCV is poorly characterized in the aging and transplant populations, and may not be more effective than PPV23 alone, implying the need for alternate vaccine/adjuvant strategies. Moreover, studies characterizing and comparing antigen-specific B cell responses in well-defined RT populations are lacking.

The investigators have developed directly labeled fluorescent PPS to enumerate and characterize PPS-specific B cells. This allowed the analysis of antigen-specific B cell responses to pneumococcal vaccination. The results of these studies demonstrated that the immune response to PPV in young adults consists predominantly of IgM memory B cells. In contrast, in the elderly who are deficient in memory B cells, particularly IgM memory B cells, the predominant B cell phenotype in response to PPS consists of switched memory B cells (IgG/A+IgM-CD27+). HIV+ individuals, are also deficient in CD27+IgM+ B cells yet the predominant response consists of PPS-specific CD27+IgM+ B cells, albeit at significantly reduced numbers. Thus, the cause of poor pneumococcal vaccine responses varies according to study population.

Several investigators and the preliminary data show that like the elderly and HIV-positive, absolute number and percentage of memory B cells are significantly lower in stable RT recipients, at 6 months to 5 years post-RT, compared to healthy subjects. Moreover, low number and percentage of IgM memory B cells correlate with poor antibody responses to PPV23.

This may however not be the only cause for poor vaccine responses. The ability to isolate antigen-specific B cells allows us to explore other deficits and/or changes in surface markers and gene expression potentially implicated in poor vaccine responses. It has been well described that members of the TNFR family and certain Toll like receptors (TLR) play a crucial role in the immune response to pneumococcal vaccination. As shown in preliminary data, the investigators have found decreased TACI and BAFFR expression in PPS-specific B cells derived from RT recipients. Although the number of RT recipients studied is very small, it was a consistent finding and merits further study. Adjuvants that specifically upregulate TACI expression, such as CpG-ODN, could therefore effectively increase immune responsiveness to pneumococcal vaccines. This strategy has shown to increase immune and memory responses to the conjugate pneumococcal vaccine in HIV-positive persons. The central hypothesis is that the elderly RT population responds poorly to PCV13 reflecting combined effects of aging and RT that result in abnormalities in both number of memory B cells and TNF superfamily expression, which play crucial roles in the response to polysaccharide antigens. The investigators will test this hypothesis by measuring PPS-specific antibody and opsonophagocytic antibody responses, PPS-specific B cell phenotypes and surface and gene expression of the TNFR with known role in response to pneumococcal vaccination and correlate these individually with antibody and functional antibody responses. This has important clinical implications as deficient TNFR expression can be modulated by the addition of adjuvants such as CpG-ODN.

Intervention to be studied The intervention to be studied is the immune response to immunization with PCV13 or Prevnar13. This FDA approved vaccine is given as part of the standard of care in Groups 1-4. The experimental part of the protocol in these groups will be blood draws at days 0, 7, 30 and years 1 and 2 in these groups.

In Group 5 two interventions will occur: 1. Immunization with the FDA approved PPV23 or Pneumovax 23 and 2. PCV13 a FDA approved vaccine. In addition, blood samples will be obtained at days 0, 7, 30 year 1 and year2.

Both PPV23 and PCV13 are FDA approved vaccines for use in humans. PCV13 is recommended for all individuals enrolled in Groups 1-4. It is not recommended as routine part of care in Group 5 enrolled individuals however it has never shown to be harmful in this group of individuals.

Study Endpoints The primary endpoint of this study is: quantify the antibody titers in mg/mL and opsonophagocytic antibody titer calculated as serum dilution, number of polysaccharide specific B cells, and absolute number of cells/mL induced by vaccination with PCV13.

Secondary endpoint of the study is to describe differences in gene expression of 56 genes to be determined by single cell PCR and comparing these between groups.

The primary safety endpoint of the study is measured as minimal versus moderate local side effect. Minimal side effect measured as no impairment in activity. Moderate local side effect is a side effect affecting use of the extremity for less than 24 hours.

Inclusion and Exclusion Criteria/ Study Population

Age ranges were chosen based on previous published and unpublished studies performed in the investigators' laboratory indicating that a significant decrease in pneumococcal vaccine response starts to appear at age 65 and that prior to age 50 the response is similar to individuals 20-30 age of years, i.e. optimal range. The investigators chose the 35-45 age group specifically, and not 20-30, as renal transplants recipients with cause of renal failure is type II diabetes or hypertension are well represented in the MUSC transplant population.

Individuals will be screened for eligibility depending on the group they are recruited for:

Group 1 Older RT group (Age 65-75) Group 2 Younger RT group (Age 35-45) The elderly (65-75 years) and young (35-45 years) renal transplant recipient groups 1 and 2 seen at the MUSC and VA transplant clinics whose end-stage renal disease was caused by hypertension (HTN) and/or DM II who are at least 1 year post-transplant and due for their PCV 13 vaccination will be asked to participate in the study. Those that do not wish to participate in the study will receive the clinical standard of care.

Group 3 Individuals with DM II and/or HTN age 65-75. Individuals with the diagnosis DM II and/or HTN due for routine visits at the general internal medicine or endocrinology clinic at the Ralph H Johnson VA Medical Center will be screened for normal (Glomerular filtration rate 60) versus abnormal (GFR<60) who are due for their standard of care PCV13 vaccine will be asked to participate.

Group 4: 65-75 years old without DM2 or renal impairment due for routine visits at the general internal medicine or endocrinology clinic at the RHJ VAMC who are due for their standard of care vaccination with PCV13.

Group 5: Healthy persons 35-45 years of age without DM or renal impairment willing to participate in a study to receive PPV23, if they have not received this previously or/and PCV13, at least 1 year after PPV23 vaccination.

Diversity: the investigators will attempt to mimic the local renal transplant recipient population consisting of a disproportionately high number of males (>60%) of African-American decent, 50%. This gender and race pattern of distribution is consistent with the patient population of the Ralph H. Johnson VA Medical Center outpatient clinics, where the investigators will be recruiting most of the control populations (Groups 3-5). The investigators therefore anticipate a diverse population as a result of eligibility at the recruitment sites.

Children will NOT be included in this study as this study is uniquely aimed at defining the immune response to PCV13 in ADULTS. The immune response to PCV13 is age-dependent and therefore completely different in children, as is the recommended vaccine frequency.

Number of Subjects 275

Setting The MUSC Renal Transplant clinic is located on the 9th floor of the Rutledge Tower. The RT recipients at the Ralph H. Johnson VA Medical Center are followed at the Nephrology outpatient clinics on the first floor. Nephrologists, physician assistants, pharmacists and nursing personnel are present at both facilities. These individuals are well trained in the administration of vaccines.

The study and consent and HIPAA forms will be explained to the potential participant by the study staff in a private room Blood draws will be performed by staff trained in phlebotomy or phlebotomists at the facility laboratories.

The group 5, healthy volunteers, 35-45 years of age will be asked to come to the P.I.'s laboratory at the Strom Thurmond Building Room 411 and study, consent and HIPAA explained in private by the study personnel. Either PPV23 or PCV13 will be administered by Dr. Westerink. Blood samples will be obtained at the MUSC laboratory.

Study Sites

1. The MUSC Renal Transplant clinic is located on the 9th floor of the Rutledge Tower

2. The nephrology clinic at the Ralph H. Johnson VA Medical Center

3. the P.I.'s laboratory at the Strom Thurmond Building Room 411

Recruitment Methods

- Potential study participants will be recruited from Ralph H. Johnson Veterans Affair Medical Center and Medical University of South Carolina during the appointment with their treating physician. Flyers will be posted in the waiting room areas of the clinics and attending physicians and clinic nurses will be asked to keep this study in mind and mention availability of these studies to their patients when ordering PCV13 for them.

- Potential study subjects will be explained the purpose of the study and how their information was obtained (from a treating physician or a chart review); further the eligibility criteria will be explained and what involves in participating in the study: procedures, estimated time of commitment, any reasonably foreseeable risks, benefits, and compensation. In a similar manner, healthy volunteers will be recruited through flyer advertisement on MUSC campus as well as in VA.

- Potential study subjects will be identified by reviewing medical records and by physician's recommendations.

- Flyers will be used to recruit study subjects as well as broadcast emails for MUSC and VA employees and volunteers.

Consent Process

Informed Consent will be obtained in a private room from all participants at either:

1. The MUSC Renal Transplant clinic is located on the 9th floor of the Rutledge Tower Or 2.The nephrology clinic at the Ralph H. Johnson VA Medical Center Or 3. the P.I.'s laboratory at the Strom Thurmond Building Room 411

Consent will be sought of competent adults who express interest in the study. The purpose of the study, the study details regarding gathering health information and obtaining blood samples, the potential benefit and risks involved, including risk of blood draw and vaccination, will be explained in detail and in layman terms, as well as the non-standard of care explained to the potential subject (i.e. blood draws for groups 1-4, vaccination protocol and blood draws for group 5). The subject will be encouraged to ask questions and they will be asked to answer questions regarding essential parts of the study to ensure proper understanding of the study requirements. The investigators will explicitly explain that the study is completely voluntary, and they can withdraw from the study at any time. Additionally the investigators will explain that participation is independent of the medical care the subject receives at the Ralph H Johnson VA medical center or MUSC.

Consent will be obtained as described above in the presence of the P.I. or study coordinator. The potential volunteer will be given written information, and opportunity for questions will be provided. Volunteers will be questioned concerning the study to assure complete understanding.

The consent will only be obtained from the participant him or herself and not from a surrogate.

There will be no waiting period between consent and beginning of the study The study and consent form will be reviewed in depth and after each separate section the participant will be asked to summarize the section in his or her own words to ensure understanding.

Vulnerable populations, i.e. employees at the P.I.'s institution will be recruited, however they will ONLY be recruited if they have

1. no personal relationship with the PI 2 are not directly employees of the PI or a division where they could be vulnerable to coercion or undue influence.

3. it will be made clear to all these participants that participation is strictly voluntary 4.recruitment will only be performed in an in personal mass email

Study Design / Methods The overall objective of this proposal is to characterize the immune response and explore possible mechanisms of poor vaccine responsiveness following immunization with PCV in the rapidly growing group of elderly with RT. As the RT population is a heterogeneous group the investigators will study only those in whom the underlying cause of renal failure is diabetes mellitus type 2 (DM2) and/or hypertension (HTN). There will be 5 study groups as outlined above.

Volunteers from all 5 study groups will be recruited as above. All individuals from study groups 1-4 who meet the study criteria will receive Prevnar13 on day 0. Group 5 volunteers will be recruited during the first year of the study and those who have not previously received PPV23 will receive PPV23 on day 0 and PCV13 on day 366. Blood samples will be obtained as outlined below and results obtained in various groups will be compared.

Timing of blood samples Peripheral blood samples will be collected at day 0 (pre-immune), day 7, and day 30 (4 weeks post-PPV23) for groups 1-4 and at days 366, 373 and 396 for group 5. In addition, day 5, and 10 blood samples will be obtained from a limited (n=10) number of elderly RT participants to determine the optimal time point for circulation of PPS-specific B cells. Yearly blood samples will be obtained thereafter for serum antibody and OPA analyses to test longevity of antibody and OPA responses. Samples from days 0, 30 and yearly samples will be used for antibody titers and opsonophagocytic assays. Samples from day 0 (day of vaccination with PCV13) and day 7 will be used for flow cytometric analysis. The day 7 time point for isolation of antigen-specific B cells was specifically chosen as the investigators have extensively studied temporal dynamics of antigen-specific B cell in peripheral blood samples (unpublished studies) and have found that the highest number of antigen-specific B cells are present in the peripheral circulation at day 6 and 7 with rapid (70+%) decline at day 8 in healthy young, elderly including those with DM2 and HTN, and HIV-positive. At day 30, a minimal number of PPS-specific B cells were present. Others have described similar findings.

However, it is possible that day 7 may not be the optimal time point for the isolation of antigen-specific B cells in the RT populations. Therefore, the investigators will obtain blood samples on days 5, 7, and 10 from the first 10 participants in the aging RT group to redefine optimal time point for antigen-specific B cell isolation in this population. The first 10 participants in Group 1 will be recruited from both MUSC and the VAMC. The optimal time point will be applied to all subsequent RT participants.

BAFF concentration will be measured at day 0.

All groups: All recruited subjects will agree to be tested for HIV by rapid HIV test, HBsAg and HCV if their status is not available in their health records. In addition they will agree to have CBC and Complete metabolic profile (CMP) and hemoglobin A 1c (HbA1c) should a recent (<6 months old) result not be available.

Groups 1-4

All individuals in groups 1-4 agree to

1. Receive PCV13 (Prevnar13): this is STANDARD of care in all group 3 recruited and enrolled subjects

2. Experimental part of the protocol they will agree to:

In the first 10 vaccine recipients in groups 1 : donate blood specimens: at 7 different times of up to 60 mL per visit: day 0, day of vaccination: at day 5, day 7, day 10 and at day 30 and yearly thereafter up to 2 years. Samples drawn at days 5, 7 and 10 are used to determine the optimal time point of isolation of PPS-specific B cells.

Participants 11-40 in group 1 and all Group 2: will donate blood samples at 5 different time points of up to 60 mL per visit: day 0, day of vaccination: at optimal time point being either day 5 or day 7 or day 10 and at day 30 and yearly thereafter up to 2 years.

The optimal time point of isolation of PPS-specific B cells post-vaccination in both young and elderly healthy and diabetic populations had been determined by us in previous studies, however this may vary in the transplant population. The investigators will therefore test this in the first 10 RT subjects enrolled in both groups 1 and 2. The first 10 participants in Group 1 will be recruited from both MUSC and the VAMC.

Groups 3 and 4: There will be 5 visits: blood draws of up to 60 mL per visit: day 0, day of vaccination: at day 7 and at day 30 and yearly thereafter up to 2 years.

Group 5

1. Receive PPV23 (Pneumovax) on day 0 of enrollment if they have never received PPV23. This is to assure that all participants in the study have been immunized with PPV23 at least 1 year prior to receiving PCV13.

2. Receive PCV13 (Prevnar13) on day 366

2. Experimental part of the protocol they will agree to: All vaccine recipients donate blood samples up to 60mL per visit: at 5 different times: day 366 day 373, 396 and yearly thereafter up to 3 years, i.e. referred to years 1, 2 and 3.

In group 5, healthy 35-45 year olds, PPV23 and PCV 13 may not be routine part of care (in those without any underlying disease or tobacco use) and vaccination with PCV13/PPV23 is not part of routine care in any of these individuals, however neither vaccination regimen is contra-indicated in these age groups and is NOT associated with a higher rate of adverse events exceeding vaccination in other age groups. In fact, significant benefit may be derived from either vaccine regimen.

The vaccine(s) administered to the participants are FDA approved vaccinations and vaccination protocols. The standard and recommended dose of vaccine will be administered either by a qualified nurse or physician and both vaccines are considered low risk. In groups 1-4 PCV13 will be administered per standard of care and is not part of the experimental protocol. In group 5, both PPV23 and PCV 13 are not routine part of care and are part of the experimental protocol. These vaccines have however been extensively studied in this and other populations and are considered low risk and 70-80% protective against pneumococcal disease.

The risk associated with blood draws is minimal.

The data to be collected will be stored using an excel spread sheet. The data include:

- Cause of end stage kidney disease if applicable

- Date of kidney transplant

- Complete blood count (CBC)

- comprehensive chemistry profile including renal and liver functions

- HbA1c

- serum albumin as measure of nutrition

- HbsAg

- HCV and HIV test

- Pre- and post-immunization serum antibody titers and opsonophagocytic antibody titers to PPS14, 19A and 23F in ug/mL determined by ELISA IgM and switched memory B cell number and percentage determined by flow cytometry

- PPS+ B cell number and percentage determined by flow cytometry

- Serum BAFF concentration by ELISA

- Gene expression of 56 genes will be studies using single cell PCR analysis