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

Inclusion Criteria:

Inclusion criteria are group specific. HBV, HCV and HIV testing are not necessary in the RT
groups as all RT recipients are tested prior to transplant. The investigators will not
restrict volunteers with respect to gender, ethnic or racial group.

Groups 1 (65-75 yrs) and 2 (35-45 yrs) Renal Transplant populations

- End stage renal disease cause either DM2 and/or hypertension (HTN)

- Renal transplant >12 months ago

Group 3: Diabetic/hypertensive 65-75 year old controls

- With DM2 and/or HTN

- Previous immunization with PPV23 >1 year prior

- Willingness to be tested for HIV, HBV and HCV

- "normal kidney function" defined as glomerular filtration rate (GFR) of 60% or above

Group 4: Healthy Control 65-75 yr old

- Without DM2

- May have high blood pressure (systolic>140 and/or diastolic>90) as long as it is well
controlled (systolic<140 and/or diastolic <90) and has not affected kidney function.

- Previous receipt of PPV23 > 1 year prior

- Willingness to be tested for HIV, HBV and HCV

Group 5: Healthy Control 35-45 yr old

- Without DM2.

- May have high blood pressure (systolic>140 and/or diastolic>90) as long as it is well
controlled (systolic<140 and/or diastolic <90) and has not affected kidney function.

- Willingness to be tested for HIV, HBV and HCV and filling out a medical questionnaire
that will include diabetes screening.

Exclusion Criteria:

Exclusion criteria are either applicable to all groups or group specific. Therefore we have
listed the exclusion criteria applicable to ALL groups first. Group specific criteria are
listed under each group.

Exclusion Criteria common to all groups

- Previous immunization with PCV13.

- Pregnancy, no contraceptive practice in women of childbearing age, or breastfeeding

- Known anaphylaxis, hypersensitivity or "bad allergic reaction" to the pneumonia
vaccine. This does not include egg allergy or previous Guillan Barre syndrome.

- Those who received blood products or gamma globulin within 3 months.

- Inability to comprehend or sign the informed consent form

- Previous/present illness that may affect immune response to the vaccine

- previous pneumococcal disease

- disease

- removal of the spleen

- auto-immune disease such as lupus or rheumatoid arthritis

- end-stage liver disease

- cancer

- Significant abnormalities (3xULN and all those considered to be critical values) in
CBC, chemistries including glucose.

- HIV, HBsAg or HCV positivity

- Receipt of PPV23 within 1 year

Groups 1 (65-75 yrs) and 2 (35-45 yrs) Renal Transplant populations

- Medications that are known to affect immune function (chemotherapy, anti-TNF agents)
with the exception of anti-rejection medication.

- Episode of acute rejection within the last 6 month period

Group 3: Diabetic/hypertensive 65-75 year old controls

- Medications that are known to affect immune function (chemotherapy, anti-TNF agents).

- The inclusion/exclusion criteria will be determined by chart review.

Group 4: Healthy Control 65-75 yr old

- Medications that are known to affect immune function (chemotherapy, anti-TNF agents).

- The inclusion/exclusion criteria will be determined by chart review and pregnancy test
for females of child bearing potential.

Group 5: Healthy Control 35-45 yr old

- Medications that are known to affect immune function (chemotherapy, anti-TNF agents).

- The inclusion/exclusion criteria will be determined by chart review and pregnancy test
for females of child bearing potential.