Genomics and Epigenomics of the Elderly Response to Pneumococcal Vaccines
Phase: PHASE4
Conditions: Pneumonia, Aging
Sponsor: The Jackson Laboratory

Protocol 
Title:  Genomics and Epigenomics of the Elderly Response to Pneumococcal Vaccines 
NCT number: NCT03104075 
Document Date: July 09, 2019

Protocol Version 5.1,   9 July 2019
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Genomics and Epigenomics of the Elderly Response
to Pneumococcal Vaccines
Clinical Protocol
Principal Investigator 
George Kuchel, MD
Clinical Protocol:
Director, UConn Center on Aging 
UConn Health, Farmington, CT
Co-Investigator
Jacques Banchereau, Ph.D. 
Clinical Protocol,
Director, Immunological Sciences
Principal Investigator
The Jackson Laboratory for Genomic Medicine
LaboratoryAnalysis
Farmington, CT
Co-Investigator
Duygu Ucar, Ph.D. 
Clinical Protocol,
Assistant Professor
LaboratoryAnalysis
The Jackson Laboratory for Genomic Medicine
Farmington, CT
Sponsor:
The Jackson Laboratory
Funding agency:
National Institute On Aging of the National Institutes of 
Health under Award Number R01AG052608.
Key Personnel:
Lisa Kenyon-Pesce, MPH
Study & Recruitment Coordinator
 
       UConn Center on Aging
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TABLE OF CONTENTS
   Page
TABLE OF CONTENTS................................................................................................................2
PRÉCIS.........................................................................................................................................4
Specific Aims.............................................................................................................................4
Design and Outcomes...............................................................................................................4
Sample Size, Population, Interventions and Duration...............................................................4
1 
OBJECTIVES........................................................................................................................4
1.1 
Specific Aim 1................................................................................................................5
1.2 
Specific Aim 2................................................................................................................7
1.3 
Specific Aim 3................................................................................................................9
2 
BACKGROUND, Rationale AND SIGNIFICANCE............................................................11
2.1 
Background and rationale............................................................................................11
2.2 
Significance.................................................................................................................16
3 
STUDY DESIGN..................................................................................................................16
3.1 
Characteristics of the Study Population.......................................................................17
3.2 
Sampling Plan..............................................................................................................17
4 
SELECTION AND ENROLLMENT OF PARTICIPANTS....................................................17
4.1 
Inclusion Criteria..........................................................................................................18
4.2 
Exclusion Criteria.........................................................................................................18
4.3 
Study Enrollment Procedures......................................................................................19
5 
STUDY INTERVENTIONS..................................................................................................19
5.1 
Interventions................................................................................................................19
5.2 
Handling of Study Interventions...................................................................................21
6 
STUDY PROCEDURES......................................................................................................23
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6.1 
Schedule of Events......................................................................................................23
6.2 
Description of Study Visits...........................................................................................24
7
RISKS and PROTECTIONS...............................................................................................28
7.1 
Potential Risks to Subjects..........................................................................................28
7.2 
Adequacy of Protection Against Risks.........................................................................29
7.3 
Adverse Events and Serious Adverse Events.............................................................31
7.4 
Reporting Procedures..................................................................................................32
7.5 
Treatment for Adverse Events.....................................................................................32
7.6 
Data and Safety Monitoring.........................................................................................32
8 INTEGRATIVE DATA ANALYSIS......................................................................................33
8.1 
Statistical Methods.......................................................................................................33
8.2 
Power Calculations......................................................................................................33
9 
DATA COLLECTION AND Management..........................................................................34
9.1 
Data Collection Forms (CRF)......................................................................................34
9.2 
Data Management.......................................................................................................35
9.3 
Quality Assurance........................................................................................................36
9.4 
Protocol Deviations......................................................................................................36
9.5 
Unanticipated Problems...............................................................................................36
10 PARTICIPANT RIGHTS AND CONFIDENTIALITY............................................................37
10.1 Institutional Review Board (IRB) Review.....................................................................37
10.2 Informed Consent........................................................................................................37
10.3 Genomic Data and Sample Sharing............................................................................37
10.4 HIPAA Authorization....................................................................................................37
11 REFERENCES....................................................................................................................38
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PRÉCIS
Study Title:  Genomics and Epigenomics of the Elderly Responseto
 Pneumococcal Vaccines
Specific Aims
To complete a 40 subject clinical study with the following aims:
1. To vaccinate healthy elderly with two distinct pneumococcal vaccines, collect longitudinal 
blood samples and assess pneumococcal-specific antibody responses.
2. To establish the transcriptional and epigenetic profiles of elderly blood 
immune cells 
linked 
with antibody responses to pneumococcal vaccination.
3. To examine the functional status of immune cells in the elderly stratified according to their 
pneumococcal vaccine responder status.
 
Design and Outcomes  
This is a prospective, single-site, randomized, then open-label study designed to develop a 
detailed transcriptional and epigenetic profile of the immune response to pneumococcal 
vaccination with conjugated and non-conjugated polysaccharide vaccines in the senescent 
immune system of older adults. 
Sample Size, Population, Interventions and Duration 
In this study, 40 healthy adults ages 60 and older that have never received pneumococcal 
vaccination, will be randomized in a 1:1 ratio to receive Prevnar-13 (Pfizer), a conjugated 13-
valent vaccine or Pneumovax 23 (Merck), a non-conjugated 23-valent vaccine.  Following 
randomized assignment of vaccine, the study will be open-label.
Six (6) study visits will occur over about 70 days, with an optional 7
th visit for participants to 
receive a second vaccination with the other pneumococcal vaccine one to two years after 
randomization.  Participants will provide blood samples for transcriptional, epigenetic and 
biological analyses pre- and post-vaccination.  
1 OBJECTIVES
The goal of our project is to assess the alterations to immune blood cells associated with the 
response of healthy elderly subjects to two pneumococcal vaccines, unconjugated (PPSV23) and 
conjugated (PCV13) polysaccharide vaccines. APCs, Tfh and B-cell subsets will be analyzed at 
different time points after vaccination. Phenotype, function, transcriptome and epigenetic 
analyses will be carried out on selected high responders and non-responders. These studies 
would provide us with the unique opportunity to 
investigate the human 
immune response of elderly 
individuals—a primary target for these vaccines—to a TI and TD antigen. Furthermore, a detailed 
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assessment of circulating immune cells from healthy elderly could enable us to identify the 
immune deficits that underpin non-responsiveness; such insights could help to improve vaccine 
efficacy.
     Our detailed analyses will include: i) RNAseq of whole blood and cell subsets, to generate 
profiles of both coding RNAs and ncRNAs [1]; ii) ATACseq, to assess chromatin state and 
determine whether the genome is poised towards specific immune responses; and iii) in vitro 
cellular immunology studies to determine the function of APCs, T cells and B cells in vaccine-
induced immune responses. We will keep all samples to further assess genotype using high-
density SNP chips. Note that this study is not designed to establish baseline phenotypes 
predictive of vaccine responsiveness.
     We expect to find that PPSV23 and PCV13 will activate different pathways leading to 
pneumococcal-specific antibody responses. Our hypothesis is that the PCV13 response pattern 
will resemble the response pattern we observed with Fluzone® inasmuch as both vaccines are 
considered to be TD. Significant differences in responsiveness can, however, be brought by 
molecular components specific to each vaccine, i.e., the polysaccharide component of PCV13 or 
the viral nucleic acids that might be included in Fluzone®. Our preliminary data in vivo in young 
adult volunteers and in vitro with APC subsets suggest that PPSV23 might trigger different 
immune pathways to elicit specific antibody responses. While the consensus 
is that 
its effects are 
TI, our observed induction of activated T cells in vivo and the presence of mutated IgG and IgA 
hints to more complex mechanisms, which might include both TD and TI mechanisms of B-cell 
activation [2]. 
1.1 Specific Aim 1
To vaccinate healthy elderly with two distinct pneumococcal vaccines, collect longitudinal 
blood samples and assess pneumococcal-specific antibody responses.
Hypothesis: We hypothesize that PPSV23 and PCV13 differentially activate the circulating 
immune cells and the blood transcriptome of healthy elderly individuals.
Rationale: Our studies in healthy young adults showed that Fluzone® and pneumococcal 
vaccines activate different immune cell subsets in vivo (data not shown). In addition, we showed 
that individuals vaccinated with PPSV23 and Fluzone® display a different blood transcriptome at 
day 1 (Fig. 1). Thus, different vaccines 
might induce the generation of specific 
antibodies through different mechanisms. 
Here, our goal is to generate profiles of 
responses  
to  
conjugated  
and 
unconjugated  
polysaccharide-based 
pneumococcal vaccines and to correlate 
the elicited immune profiles with antibody 
responses.
Approach: The overall flow of the work is 
illustrated in Fig. 1. We will examine 
whether the induction of pneumococcus 
Figure 1. Overview of work.
PatientVaccination(Pneumovax / Prevnar) 
Day 0 Day 1 
Day 10 
Day 28 
Whole blood 
Phenotyping
ATAC-Seq
PBMC
Thawandsorting
Cryopreservation
RNA-Seq
Day 60 
Serum
*
*
*
*
5highresponders vs.5lowresponders toPPV23andPCV13(RNASeq,ATAC-Seq) 
FunctionalStudies
Day-7
Abtiter
Serum
Abtiter
Tempus
RNA-Seq
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                       Table 1. Sample processing details
-7
0
1
10
28
60
50 ml 10 ml 50 ml 50 ml 50 ml 50 ml
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
PBMC
X
X
X
X
X
X
Monocytes
X
X
CD4 T cells
X
X
X
B cells
X
X
X
CD1c DCs
X
X
Whole blood
X
X
X
X
X
X
PBMC
X
X
X
X
X
X
Monocytes
X
X
CD4 T cells
X
X
X
B cells
X
X
X
CD1c DCs
X
X
Tfh
X
B cells
X
ATAC-Seq
RNA-Seq
In vitro studies
Day
Blood Volume
Ab titers
Opsonophagocytic Assay
Flow cytometry
CBC
Cell sorting
serotype–specific antibodies at day 28 correlates with the activation of T cells (as measured by 
flow cytometry) at day 10 and the activation of APCs at day 1 (measured by flow cytometry and 
transcriptomics). In a cohort of 40 healthy elderly naïve to pneumococcal vaccination we will: 1) 
vaccinate with either PPSV23 or PCV13; 2) longitudinally collect blood, serum and blood cell 
samples from each group; 3) perform the experiments that require immediate processing, such 
as flow cytometry analysis; and 4) cryopreserve all the other samples for downstream genomics 
and cellular immunology studies. Our earlier studies permitted us to draw robust conclusions on 
the transcriptome profiles in blood cells and on the role of Tfh activation in the generation of 
Fluzone®-specific antibody responses. 
Elderly Populations: We will focus on the healthy adults >60 years of age that meet eligibility 
criteria (Section 4). 40 healthy community–dwelling elderly from areas surrounding the University 
of Connecticut Health Center will be accrued. Volunteers will be randomized to receive either the 
PCV13 or PPSV23 vaccine. CDC currently recommends that healthy older adults with no history 
of previous pneumococcal vaccination receive PCV13 followed by PPSV23 at least 12 months 
later. All participants will be offered a boost dose with the other vaccine at one to two years later, 
at no cost to them.
Rationale for selected time points: We will collect blood (50 ml) at days –7, 1, 10, 28 and 60 
and (10 mL) on day 0 prior to 
vaccination with PPSV23 or 
PCV13 (Table 1). 
Baseline (Day -7 and 0): 
Blood will be collected on 
these dates to assess baseline 
immune status. We will only 
collect 10ml of blood on day 0 
so as to avoid collecting nearly 
100ml on two consecutive 
days. Day 1 coincides with the 
peak of the innate response to 
vaccines in blood. We found 
that PPSV23 induces a 
transcriptional  
signature 
reflecting the activation of 
myeloid compartment. We will establish whether this transcriptomic signature is observed in 
elderly cohorts. Day 10 
is when T-cell responses (including Tfh cells) peak. This time point allows 
us to determine whether PPSV23/PCV13 also activates a Tfh subset in elderly and whether a 
lack of Tfh activation correlates with a lack of anti-pneumococcal antibody responsiveness as we 
showed with Fluzone®. The activation of CD4+ Tfh subsets will be measured as a function of 
ICOS expression levels in conjunction with CXCR5, CCR6 and CXCR3. In addition, a portion of 
the blood samples collected at day 10 will be used for transcriptome analysis. Day 28 (± 3 days) 
reflects the peak of the circulating antibody response. We will assess the induction of serotype-
specific anti-pneumococcal antibody titers at days 28. Day 60 (± 5 days) will provide us with 
additional blood samples containing memory cells that can be used to eventually confirm the day 
28 in vitro data. Details of sample processing are given in Table 1. We will cryopreserve PBMCs 
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and sorted cell populations for genetic and cellular immunology experiments at all time points. If 
cell yield prevents cryopreservation of PBMCs, we will recruit additional subjects to reach our 
proposed number of 40.
Antibody responses: We will analyze the breadth and magnitude of antibody responses by 
assessing PPSV23/PCV13 serotype-specific IgG titers at day 0 and 28, as recommended by the 
World Health Organization, and using the Opsonophagocytic assay. This assay (serotypes 4, 6B, 
14 and 23F) will be performed using serotype–specific S.pneumoniae incubated with serial-diluted 
heat-inactivated sera [3]. Newborn rabbit serum is added as a source of complement and 
differentiated HL-60 cells are added. Sera are tested in duplicate and results obtained using the 
Opsotiter 1 software. We will use U-scores to rank antibody response by titer, affinity and number 
of serotypes (as done for antigen-specific T cells in response to DC vaccines [4-6]. Our 
collaborator, Dr Nahm, will assess serotype-specific anti-polysaccharide responses. His lab will 
also measure antibody avidity and opsonizing capacity as described [7]. 
CMV status. Influenza vaccine studies in the elderly have revealed a critical role for CMV status 
[8]. In a single study, CMV status did not appear to influence antibody responses to PPSV23 
vaccine in older adults, yet its role in other aspects of immune responses or in baseline pre-
vaccination status cannot be ruled out[9]. As a result, we will obtain CMV status using antibody 
titers and PCR-based technologies [10], and will include this variable in our analytic plan.
Analytical Approaches: 
   1. Multicolor flow cytometry: 0.5 ml of blood will be used to determine the profile of circulating 
immune cells. We designed five multi-color monoclonal antibody cocktails that will permit us to 
establish the detailed phenotype of T-cell subsets, monocytes, DCs and B-cell subsets. We 
included a set of antibodies to identify NKT cells, as these cells have been reported to contribute 
to responses to PPSV23 vaccination [11].
   2. Cryopreservation of isolated PBMCs and sorting of purified populations: We have validated 
monoclonal antibody cocktails that permit us to sort T-cell subsets, monocytes, DCs and B cells 
for cryopreservation (Table 1). PBMCs will be frozen in 4–5 aliquots (recovering on average 
20x106 cells); 1–2 will be used for ATAC-seq and 2–3 for functional studies. After thawing we will 
sort monocytes, CD1cDCs, CD45RO+CD4+ T cells, and B cells for ATAC-seq and RNA-seq. We 
aim to collect 15,000–25,000 of each type of cells. 
   3. Cryopreservation of whole blood RNA: Two Tempus tubes (1 ml each) will be stored for 
further analysis of coding RNA and ncRNAs.
   4. Sample prioritization: Our preliminary data show that we can perform ATAC-seq on 
cryopreserved cells. Previous studies [12] show that T/B-cell cultures can be done with 
cryopreserved and thawed cell samples. If we encounter low cell yields, which might limit the cell 
culture experiments, we can draw from the blood samples acquired at day 60. 
1.2 Specific Aim 2
To establish the transcriptional and epigenetic profiles of elderly blood immune cells 
linked with antibody responses to pneumococcal vaccination.
Hypothesis:  We hypothesize that transcriptome and epigenome profiling of blood and sorted 
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immune cells will reveal the coding and non-coding regulatory sites associated with different 
immune responses following vaccination with PPSV23 and PCV13 in healthy elderly.
Rationale: The non-coding regions of the genome contain a variety of RNA species, such as 
microRNAs (miRNAs) and long non–coding RNAs (lncRNAs), which play fundamental roles in 
regulating gene expression [1, 13, 14]. The study of these regulatory RNAs is becoming 
increasingly relevant to the fields of immunology and aging. Recent studies in aging mice and 
humans reported the up- and downregulation of miRNA expression in PBMCs among other 
tissues [15, 16], yet little is known about the status of ncRNAs within elderly immune cells, 
particularly in response to vaccination. In addition to transcriptome differences, it is conceivable 
that epigenetic remodeling also contribute to decline 
in the 
immunity of elderly. Current epigenetic 
approaches require large numbers of cells, making them impractical for detailed analysis of 
primary blood–derived human immune cells. In a recent major advance, a transposase-based 
method to sequence genomes, ATAC-seq, was adapted to interrogate chromatin accessibility. 
ATAC-seq generates representative chromatin accessibility maps from as few as 500–5,000 cells, 
making it an ideal approach to investigate genome-wide epigenetic patterns at coding and 
noncoding regulatory sites in primary human immune cell samples. 
To investigate our hypothesis we will apply RNA-seq, rather than DNA microarrays, to generate 
transcriptional profiles, as this will enable quantitative assessment of both coding RNAs and 
ncRNAs. We will also use ATAC-seq to resolve the epigenetic landscape of immune cells in 
healthy elderly in the context of vaccine responses.
Approach:
1. Blood transcriptomics by RNA-seq: We will carry out RNA-seq of whole blood samples at 
baseline, day 1 and day 10. Samples collected from days 28 and 60 will be kept frozen for future 
studies. RNA-seq on purified cell subsets will be performed using purified cells from the five 
lowest 
and five highest responders for each vaccine. Sequencing will be performed at JAX-GM with an 
Illumina HiSeq2500.
2. Blood epigenomics analysis by ATAC-seq: We will analyze PBMCs, CD4+ T cells, monocytes, 
CD1cDCs and B cells (25x103 cells per assay). We are also developing assays with lower cell 
numbers to address the scarcity of some populations, and are encouraged by reports of single 
cell ATAC-seq [17]. We will generate a database describing the DNA accessibility landscape of 
major immune–cell subsets from elderly individuals at steady state. The data will also include the 
DNA accessibility landscape of monocytes, T and B cells analyzed at different time points 
following vaccination with the two pneumococcal vaccines. For these in-depth studies, we will 
select the five highest and five lowest responders for each vaccine. 
Correlative analysis: We will correlate the increased levels of day 28 anti-pneumococcal 
antibodies as defined in Aim 1 with all the parameters collected in this aim. Parameters include: 
1) cellular composition at baseline, day 1 and day 10 post-vaccination; 2) transcriptome analysis 
on blood and purified immune cell subsets at baseline, day 1 and day 10; 3) ATAC-seq data on 
PBMCs and purified cell subsets at the different time points and 4) pneumococcal colonization at 
baseline and at day 28. A detailed description of the statistical approaches is given below (see 
Integrative Data Analysis section).
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1.3  Specific Aim 3
To examine the functional status of immune cells in the elderly stratified according to 
their pneumococcal vaccine responder status.
Hypothesis: We hypothesize that lack of in vivo response to PPSV23 and PCV13 depends on 
alterations to APCs, Tfh cells or B cells.
Rationale: Aging impacts the immune system at steady state as well as its capacity to respond 
to challenges such as infection or vaccination. In advanced age, there are i) reduced numbers of 
B cells responding to influenza as well as lower antibody avidity to carbohydrate antigens [18-22] 
and ii) increased numbers of memory cells occupying tissue niches that is linked with lower 
migration and maintenance of naïve B and T cells into tissues. These changes, associated with 
the biased differentiation of CD4+ T-cells into Th17 cells, diminish the overall capacity of CD8+ 
and CD4+ cells to mount diverse and robust responses to antigens [23, 24]. Nonetheless, major 
gaps remain in our knowledge of elderly T/B-cell responses. There is also a need to better 
understand the alterations in function of innate immune cells including DCs and macrophages in 
advanced age. We propose two sub-aims to further characterize the functions of key 
immune cell 
types 
in the healthy elderly, thereby complementing the genomics and epigenomics data gathered 
in Aim 2.
Aim 3a: Determine whether a specific immune–cell subset can be linked with the lack of 
response to pneumococcal vaccines. Here, we will seek to determine whether ex vivo 
responses to vaccines can be related to in vivo–derived transcriptional profiles of the blood at 
steady state and after vaccination as well as to antibody responses. We will analyze PBMCs and 
sorted monocytes, CD1c+DCs, CD4+ T cells and B cells.
PBMC function: We will activate PBMCs from five of each of PPSV23 and PCV13 responders 
and non-responders with medium alone, PPSV23 or PCV13. PMA plus ionomycin and 
lipopolysaccharide (LPS) will be used as positive controls. We will measure cell activation by flow 
cytometry at day 1 and 2. We will measure cytokine production at day 2 (IL-1, IL-6, IL-10, IL-21, 
TNF and IFNg) post-stimulation. Specific antibody 
levels will be measured at day 12 as described 
[3, 20, 25]. Some experiments will be carried out by culturing PBMCs with a few selected 
polysaccharides from S. pneumoniae, such as PP13 and PP23F [18, 20, 21].
Monocyte and CD1c+DC function: we will select baseline monocytes/CD1c+DCs from five 
elderly subjects not responsive to PPSV23 and five not responsive to PCV13 and compare them 
to monocytes/CD1c+DCs from responders. Monocytes/CD1c+DCs will be isolated using CD14-
beads and cultured in medium, with either of the two pneumococcal vaccines or LPS as an 
activation control. Before and after stimulation we will: 
1) Determine the phenotype of cultured cells by flow cytometry at day 1 post-activation using 
various markers, including MHC class-II, CD40, 80, 83 and 86.
2) Measure the secretion of cytokines (IL-1, IL-6, TNF and IL-10) by ELISA. 
3) Assess the transcriptome of monocytes after six hours of activation using RNA-seq. 
4) Analyze monocyte differentiation into either DCs or macrophages following culture with GM-
CSF with or without IL-4 [26]. We will measure the differentiation potential of the elderly 
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monocytes by assessing the phenotype of cultured cells and analyzing their ability to induce a 
mixed lymphocyte reaction with allogeneic CD4+T cells. We will also measure, after six days of 
co-culture, the production of cytokines by flow cytometry after activation with PMA and ionomycin 
[27-29]. Cytokines to be measured include IL-21 and CXCL-13, which are specifically secreted 
by Tfh cells; IFN-g (Th1); IL-4 (Th2) and IL-17 (Th17). CD1c+DC function will be measured as 
described for monocytes [30, 31].
B-cell function: B cells will be 
isolated from PBMCs by magnetic sorting and will then be cultured 
with the vaccines alone or together with SAC (i.e., fixed Staphylococcus aureus strain Cowan) 
and IL-2 [32, 33]. Alternatively we will use a combination of CD40-L and either IL-10 or IL-21, 
which results in potent cell proliferation and differentiation into plasma cells [34]. Ig secretion will 
be measured at day 12 by ELISA. Differentiation into plasma cells will be assessed by loss of 
CD20 and acquisition of CD138 [35].
Aim 3B: Determine whether the CD4+T cells can induce B cells to differentiate into plasma 
cells secreting anti-pneumococcal antibodies in vitro. 
For these studies, we will use day 28 and day 60 cells from vaccine responders versus non-
responders. 
Assess antigen specificity of T-cell subsets: We will use cultures of purified CD4+ T-cells and 
B cells as we described. Briefly, purified CD4+T cells will be cultured with either of the two 
vaccines or a mixture of the polysaccharides (ATCC) for which the elderly showed an antibody 
response. For the PCV13 cohort we will also test the reactivity to protein carrier alone (CRM 197 
from LBL). T-cell activation with selected antigens can be measured by induction of CD154 after 
six hours of culture in the presence of monensin and brefeldin[12]. Cytokine expression will be 
assessed by flow cytometry after intracytoplasmic staining with anti-IFNg, IL-2, IL-10, IL-17 and 
IL-21 as described. A second set of experiments will be carried out with separated 
CXCR5+CD4+CD45RO+ T cells (Tfh cell) or CXCR5-CD4+CD45RO+ T cells (non-Tfh cells). 
Assess T-cell subset helper activity in B cells: To analyze the ability of T cells to activate B 
cells, we will use a dual approach as designed for Fluzone®-vaccinated individuals. Purified T–
cell subsets (5X103 per well) will be cultured with sorted naïve (IgD+CD27-CD3-) or memory 
(CD27+CD3-CD19+) B cells (5X10
3 per well) in the presence of 1ug/ml staphylococcal 
enterotoxin B to test polyclonal B-cell activation and differentiation. Experiments will also be 
carried out with purified IgM+ memory B cells. B-cell activation will be monitored by induced 
expression of CD138 and CD38 after 14 days of culture. Culture supernatants will be harvested 
at day 14 for measurement of IgM, IgG and IgA by ELISA. For antigen-specific responses, 
memory B cells will be loaded with the relevant antigen and cultured for six days with the Tfh and 
non-Tfh CD4+ T-cell subsets as shown earlier. Supernatants will be harvested at day 14 and 
assessed for anti-pneumococcal polysaccharide antibodies by ELISA. 
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2 
BACKGROUND, RATIONALE AND SIGNIFICANCE
2.1 Background and rationale
Aging, pneumococcal vaccines and S. pneumoniae infection: The declining ability of the 
aging immune system to combat infection is a major threat to the health, independence and 
survival of older adults [36-39]. Cellular immunosenescence associated with a hyper-
inflammatory state has been linked to many diseases common in the elderly, including infectious 
diseases [36, 40, 41]. Beyond evidence of poor immune-cell responses with declines in naïve T 
cells, we know little about the mechanisms of immunosenescence [41-43].
    Pneumococcus infection is one common condition in the elderly for which a better 
understanding of immunosenescence and immune responses to vaccines is urgently needed [8, 
44-50]. The polysaccharide pneumococcal vaccine PPSV23 has long remained the only vaccine 
recommended for prevention of pneumococcal infection in healthy adults >65 and children and 
adults 2 to 64 years of age with certain health conditions. However, 
its efficacy was felt to be poor, 
especially against non-bacteremic pneumonia and in advanced age. Although young and old 
subjects might demonstrate similar increases in antibody levels post-PPSV23 immunization, the 
functional activity of these antibodies is lower in the elderly. In contrast, the 13-valent 
pneumococcal conjugate PCV13 has superior immunogenicity against some serotypes. The 
CAPiTA trial [51], a randomized placebo-controlled trial of PCV13 involving nearly 85,000 Dutch 
individuals ≥65 years, indicates that PCV13 has 75% efficacy against invasive pneumococcal 
infections caused by PCV13 serotypes and 45% efficacy against non-bacteremic variants. In 
contrast, evaluation of a national program of PPSV23 vaccination in Taiwan [52] in individuals 75 
years and older revealed that vaccination in the previous year was associated with a 60% 
reduction in pneumonia hospitalization, a 76% reduction in invasive pneumococcal disease and 
>90% reduction in related death in this vulnerable population. In spite of these striking findings, 
no definitive conclusions can be drawn with regards to the comparative efficacy of the two 
vaccines, since in both studies PCV13 or PPSV23 were each compared to placebo and not each 
other. The 2015 CDC Guidelines recommend, for healthy older adults (≥ 65 years) who have not 
received a pneumococcal vaccine, that PCV13 be administered first followed by PPSV23 ≥ 1 year 
later, and for those that have received PPSV23, a dose of PCV13 ≥ 1 year later [53]. Thus, the 
two vaccines may remain complementary to each other, with benefits dependent on which 
serotypes are most responsible for community-acquired pneumonia in a given population, but 
many questions as to the nature of (and variability in) vaccine responsiveness remain 
unanswered. 
     Even within the 
landscape of broad 
late-life vulnerability to 
infectious diseases, pneumococcal 
infection assumes an overwhelmingly important role [54]. Thus, preventive strategies such as 
vaccination remain crucial. It is known that T- and B-cell receptor repertoires become more 
skewed during aging, and likely contribute to the limited specific immune responses to vaccines 
or new infections [44]. However, we lack fundamental insight into the transcriptional and 
epigenetic signatures that could be predictive of immunosenescence and reduced vaccine 
responsiveness at the personalized level—and could enable us to identify and target individuals 
most at risk of complications from poor responsiveness to pneumococcal and potentially other 
vaccines.
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Immune responses to vaccination are complex and depend on the coordinated actions of 
specific B- and T-cell subpopulations. B-cell responses are classified as T-cell-dependent (TD) 
or T-cell-independent (TI) based on the requirement for T-cell help in antibody production [55]. 
TD antigens are processed then presented by MHC class-II molecules for recognition by cognate 
helper T cells. Mouse studies show that B1 cells and splenic marginal zone B cells play major 
roles in the TI response [56]. In contrast, the human counterpart of mouse B1 cells, the IgM+ 
memory B cells, remains the object of some controversy [57-61]. 
     Several studies have identified the importance of pneumococcal polysaccharide (PS)-IgM 
antibodies 
in generating protective 
immunity against S. pneumoniae [62]. These antibodies are 
in 
part produced by human IgM memory B cells [7, 32, 33]. This population of IgM+ memory B cells 
is reduced in the elderly, splenectomized persons, infants less than two years of age and a 
subgroup of Common variable–immunodeficiency patients—all of whom respond poorly to PS 
vaccines and are susceptible to infections with encapsulated microbes [63]. These findings 
support the concept that IgM+ memory B cells are important in generating responses to TI 
antigens. It is however unlikely that IgM+ memory B cells are solely responsible for anti-PS 
antibody production. Indeed, switched memory B cells (CD27+IgM-) secrete higher levels of anti-
PS antibody than CD27+IgM+ memory cells following in vitro stimulation. This might be due to 
additional PS-responsive B-cell subsets and/or the versatile role that IgM memory cells play in 
pneumococcal antibody responses. 
     Follicular helper T cells (Tfh) play a critical role in the generation of high-affinity memory B 
cells [12, 64-69]. The memory Tfh cell compartment in human blood is composed of subsets that 
differentially express the chemokine receptors CXCR3 and CCR6 and display different functions 
[67]. Recently, we demonstrated a direct correlation between activation of Flu antigen–specific 
Tfh1 responses and the generation of antibody responses upon vaccination [12](see Preliminary 
Studies). However, how Tfh cells regulate antibody responses to pneumococcal vaccines in 
elderly is unknown. 
Systems biology of immunity and vaccine responsiveness in humans: We [70] and others 
[71-76] used systems biology approaches to investigate immune responses to vaccines. We 
found that young adults mount distinct responses to Flu (Fluzone®) and pneumococcal (PPSV23) 
vaccines (see Preliminary Studies). Our results provide proof-of-principle that we can detect 
global 
immune responses elicited by different vaccines and suggest that comparative analyses of 
these differences will be critical for understanding the immune mechanisms underpinning 
successful vaccination. Here, we propose to apply these and other novel immunogenomic 
approaches to further understand how protective immunity against pneumococcal antigens is 
established in healthy elderly using two pneumococcal vaccines.
PRELIMINARY STUDIES
I) IMMUNOGENOMICS OF PNEUMOCOCCAL VACCINATION.
Different vaccines elicit distinct transcriptional profiles in blood cells. To investigate the 
innate and adaptive immune responses to Flu (Fluzone®) and pneumococcal (PPSV23) 
vaccines, we performed transcriptional profiling of whole blood using DNA microarrays. These 
studies revealed significant differences in the quality and magnitude of transcriptional responses 
at different time points after each vaccination. Specifically, we found that influenza vaccination 
elicited type-I interferon signaling signatures, while PPSV23 vaccination was associated with an 
acute 
inflammatory response signature 
linked with myeloid cells. The day 7 plasmablast response 
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induced by both vaccines was more pronounced after PPSV23 vaccination. These findings 
provide proof-of-principle that a transcriptomics approach can be used to identify distinct global 
immune responses elicited by different vaccines.
Decoding innate responses to PPSV23 vaccine in vitro. In an effort to decipher the 
mechanisms leading to blood signatures in vivo, we assessed the transcriptional profiles of 
various antigen-presenting cells (APCs) exposed to vaccines in vitro [77]. Monocytes, in vitro–
derived IL-4 dendritic cells (DCs), blood CD1c+ DCs and CD141+ DCs from 4–5 donors were 
stimulated with vaccines for six hours. Their transcriptional fingerprints yielded 23,060 transcripts. 
A modular analysis, initially developed to characterize the blood transcriptome [78-81], identified 
42 differentially expressed modules, forming four groups of transcriptionally active conditions. We 
then analyzed 22 modules induced by Fluzone®, PPSV23 or human papilloma virus (HPV) 
vaccine in IL-4 DCs, monocytes or CD1c+ DCs. Importantly for the proposed studies, PPSV23 
induced unique modular signatures in monocytes, including IL-1, NFkB and type II IFN–related 
modules. We will further develop these analyses and determine the mechanisms underlying 
differential responses of APCs to PPSV23 and PCV13. APCs from elderly 
individuals, responding 
or not to PPSV23 or PCV13, will be compared to determine whether non-responsiveness can be 
linked with specific APC alterations.
II) ADAPTIVE IMMUNE RESPONSES TO VACCINATION. To begin to define the cell types that 
comprise adaptive 
immune responses to vaccination, we performed detailed phenotypic analyses 
of T-cell subsets taken from healthy young adults vaccinated or not with Flu or PPSV23 vaccine. 
We found that vaccination induced ICOS expression almost exclusively on blood CXCR3+ Tfh 
cells and that the induced ICOS+CXCR3+ Tfh cell population was enriched with cells specific for 
Flu antigens. The increase in ICOS+CXCR3+ Tfh cells in blood at day seven post-vaccination 
correlated with an increase in antibody titers at Day 28. Isolated ICOS+CXCR3+ Tfh cells induced 
antigen-loaded memory B cells to produce Flu-specific Abs in vitro through secretion of IL-10 and 
IL-21. Thus, the analysis of blood Tfh subsets led us to discover biomarkers reflecting vaccine 
efficacy and provided 
insights 
into vaccine mode of action. Further studies 
in healthy young adults 
vaccinated with PPSV23 indicated 1) a plasmablast response on days 7 and 10 and 2) the 
emergence of ICOS+ Tfh cells in blood peaking at day 10. However, the status of Tfh cells in 
elderly has not been well characterized. We will address this gap by profiling the Tfh cells of 
healthy elderly vaccinated with either PPSV23 or PCV13.
III) IMMUNOGENOMICS OF BLOOD CELLS FROM OLDER ADULTS. We recently launched a 
systematic analysis of the status of immune cells in the blood of elderly subjects (age 65 and 
older; mean=75.3 yrs). Our approach included 1) assessment of cell composition by 
polychromatic flow cytometry; 2) transcriptional profiling by RNA-seq and 3) assessment of 
epigenetic landscape of PBMCs as well as sorted subsets by ATAC-seq (Assay for Transposase 
Accessible Chromatin or ATAC-Seq)[82]. ATAC-seq captures open (i.e., transcriptionally active) 
chromatin sites and can be used to reveal various categories of epigenetic information, including 
the genomic 
locations of open and closed chromatin regions and their interplay with DNA-binding 
proteins, as well as of individual nucleosomes. Importantly, ATAC-seq generates information via 
a simple two-step protocol that requires only a few thousand cells, making it uniquely suited to 
study epigenomic profiles of human clinical samples with a systems biology approach, where 
small cell numbers are a limiting factor.
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Reproducibility of ATAC-seq datasets: To determine the feasibility of ATAC-seq for studying 
the epigenome of immune cells in the elderly, we conducted a pilot study in PBMCs (50,000 cells 
per experiment) of ten healthy young (20–30 yrs) and ten healthy old (>65 yrs) individuals. Each 
sample was run in triplicate to confirm reproducibility. ATAC-seq datasets were also generated in 
FACS-sorted B cells and in naïve and memory CD4+ and CD8+ T cells. We identified ATAC-seq 
peaks, i.e., open chromatin sites, in each of these PBMC samples. First, we confirmed that our 
identified ATAC-seq peaks are consistent with PBMC histone modification mark profiles 
generated by the Roadmap consortium, where ATAC-seq peaks are enriched in active marks 
(H3K9ac, H3K36me3, H3K4me1) and depleted in inactive marks (H3K9me3 and H3K27me3) 
(data not shown). Next, we calculated pairwise correlations between ATAC-seq datasets based 
on read count distribution across the whole genome and clustered the resulting correlation matrix 
(using hierarchical clustering algorithm) to identify similarities between ATAC-seq datasets 
globally. 
     Our analyses showed that ATAC-seq datasets are reproducible and capture cell-type-specific 
epigenetic 
landscapes. We found a good match between observed and expected open chromatin 
sites around cell-type-specific genes, e.g., the CD14 gene promoter had an open chromatin site 
in CD14+ monocytes (data not shown). We also identified cell-type-specific open chromatin sites 
in CD4+ and CD8+ cells by comparing memory and naïve ATAC-seq samples. We found that 
cell-type-specific open sites lose their specificity with aging both in CD4 and CD8 cells, with the 
change being more dramatic in CD8+ T cells. This preliminary study confirms that our ATAC-seq 
data generation and analysis workflow yields highly reproducible and high-quality ATAC-seq 
datasets from PBMCs and sorted cell populations. 
ATAC-seq identifies age-associated epigenetic changes in open chromatin sites: In our 
next set of ATAC-seq analyses, we sought to identify open chromatin sites remodeled with aging 
in PBMCs, i.e., open chromatin sites closing with age (“young specific”) or relatively closed 
chromatin sites opening with age (“old specific”). To do this we used an algorithm designed to 
capture differential sites from read count data based on negative binomial distributions; this 
allowed us to capture a total of ~20,000 differentially open chromatin sites (at 0.05 FDR cut-off, 
out of 100,000 consensus ATAC-seq peaks) between age groups, where ~11,000 were young 
specific and ~9000 old specific.
      Among these differential sites, we found a remarkable genomic distribution bias between old-
specific and young-specific open chromatin sites, whereby young-specific sites were mostly at 
promoters, whereas old-specific sites were enriched in intergenic and intronic regions. We 
confirmed that these differentially open chromatin sites separate age groups (i.e., elderly and 
young PBMCs) using the first two components of a principal component analysis (PCA) (data not 
shown). Among these differential open chromatin sites, we were further intrigued to find age-
dependent closure of chromatin sites at the IL-7R promoter. This decline 
in chromatin accessibility 
correlated with a significant decrease in IL7R expression, as measured by RNA-seq. We also 
confirmed by flow cytometry that IL7R expression is decreased in elderly CD8+ T cells (data not 
shown). Moreover, among our “young-specific” differentially open chromatin sites, we identified 
~60 promoter and 
intergenic ATAC-seq peaks targeting other genes 
in the IL7 signaling pathway, 
which is in agreement with the literature, as alterations in lymphocyte homeostasis related to the 
IL7/IL7R pathway have been reported in old mice and humans [83-87]. These analyses reveal 
that the epigenome of the IL7R signaling pathway significantly closes with aging, thereby 
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uncovering a putative mechanism to explain the decreased ability of elderly individuals to deal 
with new antigenic challenges. They also further validate our approach using ATAC-seq to 
identify 
age-dependent differences in the epigenomes of healthy individuals.  Here, we will extend these 
findings to determine whether vaccine responders and non-responders show differential 
expression and differential epigenetic profiles of immune-related molecular pathways. We will 
also analyze whether responders, 
in contrast to non-responders, reactivate their IL-7R pathways. 
Modular interpretation of age-associated epigenetic changes. To systematically interpret the 
biological meaning of the remodeled open chromatin sites identified via ATAC-seq, we employed 
the transcriptional immune module analysis described in Fig. 1B, composed of gene sets that are 
coordinately expressed in PBMCs in a wide range of diseases (28 modules in total from 239 
microarray profiles) [79-81, 88]. We developed and used this stable modular framework 
extensively to interpret immune signatures associated with diverse diseases and immune 
responses. We first identified putative (i.e., nearest) gene targets for ATAC-seq open chromatin 
sites. For each module, we then calculated the average log-fold change of read counts per gene 
between old and young samples for genes imputed to differential open chromatin sites. We next 
isolated modules whose chromatin landscape significantly changes with age, identifying 12 
modules that are differentially open in samples from young individuals, including a module of T-
cell regulators, and five modules that are differentially open in samples from elderly, including 
modules associated with neutrophil and platelet function and inflammation. We also investigated 
whether we can capture differences in module profiles of individual epigenomes when compared 
to population averages (i.e., all samples). 
     We observed systematic and distinct differences in the relative openness of certain modules 
in samples from elderly versus young 
individuals, e.g., neutrophils vs. T-cell modules, which helps 
us to interpret the regulatory implications of epigenetic changes. Module-based functional 
analysis revealed that i) young-specific open chromatin sites mostly occur around promoters and 
are associated with modules related to T cell functions and ribosomal proteins; and 
ii) old-specific 
open chromatin sites mostly occur at intronic/intergenic sites and are associated with modules 
related to inflammation and neutrophil and platelet activity. Our modular analyses also helped us 
to identify outlier samples, including an old individual with a chromatin profile similar to samples 
from young individuals and vice versa. Modular analysis of immune epigenomes thus reinforced 
and enhanced our ATAC-seq analyses to provide greater insights into the immunogenomic 
profiles of elderly individuals. Here, it will be used to interpret epigenetic remodeling changes 
induced by vaccination in the elderly and to study whether responders and non-responders have 
different modular profiles before and after vaccination.
ATAC-seq datasets can be obtained from frozen samples. We next performed ATAC-seq on 
fresh and cryopreserved PBMCs from two different individuals. Our analyses revealed that fresh 
and frozen samples yield very similar genome-wide read-count profiles (data not shown), with 
only 44 peaks out of 75,365 total consensus open chromatin sites being different. These results 
confirm that ATAC-seq profiles can be obtained from frozen samples. This will enable us to 
retrospectively generate ATAC-seq datasets after vaccination responses are quantified, thereby 
significantly reducing experimental costs. 
Differentially expressed non-coding RNAs in aging. Aging is associated with a complex 
transcriptional signature that includes changes to ncRNA profiles in addition to coding genes [89-
96]. ncRNAs include small transcripts such as microRNAs and lncRNAs [1]. Although many 
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ncRNAs have been 
identified, only a few have been fully characterized 
in terms of their regulatory 
roles in important cellular processes, including aging. To determine whether ncRNAs are 
remodeled in elderly human blood, we used limma software to identify differentially expressed 
ncRNAs between old (n=8) and young (n=10) RNA-seq PBMC samples (p <0.05). This analysis 
revealed 24 ncRNAs differentially expressed in aging. We will expand our preliminary datasets 
and analyses to analyze the expression of ncRNAs in aging and determine their relationship to 
the vaccine response status. 
2.2 Significance
Pneumonia due to Streptococcus pneumoniae infection is a serious public health challenge 
among elderly populations. S. pneumoniae is the leading cause of community-acquired and in-
hospital pneumonia in the United States (US) and globally, and a major cause of morbidity and 
mortality in the elderly. According to the CDC, an estimated 400,000 hospitalizations from 
pneumococcal pneumonia occur annually in the US; the case-fatality rate is 5–7% and may be 
>50% among elderly persons. Although pneumococcus-specific vaccines exist, elderly 
individuals display reduced responses for reasons that are as yet unclear, leaving many in this 
group highly vulnerable to infection and the consequences thereof. This project focuses on 
understanding the immune alterations associated with aging that affect responses to the two 
available S. pneumoniae vaccines, which differ respectively in composition and elicited host 
immune response. A better understanding of how elderly populations respond to these vaccines 
could be leveraged to improve overall efficacy and protection in elderly individuals—an outcome 
of significant public health relevance. Specifically, the proposed study would enable the 
identification of targets that could be modified to reactivate specific 
immune cells and/or pathways 
to improve vaccine response rates. Furthermore, the data generated would represent an 
important resource for future studies of elderly individuals with chronic diseases. 
The declining ability of the aging immune system to combat infection is a major threat to the 
health, independence and survival of older adults (1-4). Cellular immunosenescence associated 
with a hyper-inflammatory state has been linked to many diseases common in the elderly, 
including infectious diseases (1, 5, 6). Beyond evidence of poor immune-cell responses with 
declines in naïve T cells, we know little about the mechanisms of immunosenescence (6-8).
3 
STUDY DESIGN
This prospective, single-site, randomized, then open-label study 
is designed to develop a detailed 
transcriptional and epigenetic profile of the immune response to pneumococcal vaccination with 
conjugated and non-conjugated polysaccharide vaccines in the senescent immune system of 
older adults. This knowledge may lead to development of more effective vaccines through 
increased understanding of the effects of immunosenescence on mechanisms of immune 
response to pneumococcal vaccination in the elderly.
Forty (40) healthy adults ages 60 and older that have never received pneumococcal vaccination, 
will be randomized 
in a 1:1 ratio to receive Prevnar-13 (Pfizer), a conjugated 13-valent vaccine or 
Pneumovax 23 (Merck), a non-conjugated 23-valent vaccine. Following randomized assignment 
of vaccine, the study will be open-label.
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The first six (6) study visits are planned to occur over 67 days at Days -7, 0, 1, 10, 28 (±3 d)  and 
60 (± 5d). Participants will provide blood samples for transcriptional, epigenetic and biological 
analyses pre- and post-vaccination.  
One to two years after receiving the randomly-assigned vaccination, participants may opt to 
receive administration of a second pneumococcal vaccine with the vaccine that they did not 
receive by random assignment at Visit 2 (Day 0).  This second vaccine will be provided at no 
charge to the participant. Administration of this vaccine will occur at an optional Visit 7 for 
participants who choose to receive the second vaccine and will be scheduled at the participant’s 
convenience one-two years after receiving the first pneumococcal vaccine.
If the participant opts to receive the second vaccine within the study and attends optional Visit 7, 
blood samples for genomic and biologic analysis will be collected at the visit. 
3.1 Characteristics of the Study Population
Number:    
40
Age Range:  
60 and older
Health Status:   
Healthy, as defined by eligibility criteria
 
Have never received pneumococcal vaccination
Duration of Participation: 6 visits* over approximately 70 days 
*1 optional additional visit at 1-2 years for vaccination with the other vaccine not provided at Visit 
2 (Pneumovax 23 / Prevnar 13, Prevnar 13/ Pneumovax 23).   Optional Visit 7 to be scheduled at 
the convenience of the participant within 1-2 years for second vaccination with the other vaccine 
at no cost to the participant and for blood sample collection.
3.2 Sampling Plan
The study sample will be drawn from the population of healthy elderly in the catchment area of 
UConn Health in Farmington, CT.  
4 
SELECTION AND ENROLLMENT OF PARTICIPANTS 
Forty (40) healthy men and women, ages 60 and older, from all ethnic backgrounds, that have 
never received pneumococcal vaccination will be recruited to this study over a two-year period. 
The National Health Interview Survey 2013 found that nationally, 40% of adults ages 65 and 
older have not received pneumococcal vaccination (1), which supports successful recruitment 
in this population. Recruiting participants aged 60 and up should enhance our recruitment efforts 
to ensure recruitment goals are met. 
Dr. George Kuchel, the Principal Investigator of the clinical protocol, will utilize UConn Center 
on Aging recruitment resources for this project. The UConn Center on Aging Geriatric 
Recruitment & Community Outreach Core provides a centralized infrastructure for the 
recruitment of older research volunteers from the community.  Over more than a decade Lisa 
Kenyon-Pesce, MPH, has managed this core. Such recruitment expertise, together with a 
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26,000-name database has permitted the successful completion of nearly 100 clinical studies 
involving older adults. Furthermore, under Dr. Kuchel’s leadership, the UConn Center on Aging 
has an established track record of recruiting more than 145 young and older adults each year 
for ongoing NIH-funded studies (PO1AG021600, R01AG048023) of the immune response to 
influenza vaccination in the elderly. Other recruitment methods will include recruitment flyers 
placed in local clinics, letters mailed to the Center on Aging database, use of broadcast email 
in the UConn Health system, and newspaper advertisements. Retention will be encouraged by 
planning visits at convenient times for the participants and by providing financial compensation 
for study participation.
Vulnerable Populations
Vulnerable populations will not be enrolled in this research study.
Collaborating Sites
All recruitment, enrollment, clinical data and sample collection will occur at the UConn Center 
on Aging 
located on the UConn Health campus 
in Farmington, CT under the direction of Clinical 
PI Dr. George Kuchel. 
All transcriptional, epigenetic and biological analysis of samples will occur at the Jackson 
Laboratory for Genomic Medicine under the direction of Laboratory Analysis PI, Jacques 
Banchereau, Ph.D.
4.1 Inclusion Criteria 
Participants must meet all of the following inclusion criteria to participate in this study:
• Able and willing to provide written informed consent
• Male or Female, 60 years of age or older
• Willing to receive pneumococcal vaccination with Prevnar 13 (Wyeth/ Pfizer) or 
Pneumovax 23 (Merck), as randomly assigned.
• Available to attend 6 study visits over 67 days (Visit 7 
is optional at Day 365-720).
4.2 Exclusion Criteria 
Candidates meeting any of the following criteria will be excluded from study participation: 
• Previous pneumococcal vaccination with Prevnar 13 or Pneumovax 23.
• History of anaphylactic/anaphylactoid or severe allergic reaction to any 
component of Pneumovax 23, Prevnar 13 or any diphtheria toxoid-containing 
vaccine. 
• Established diagnosis of diabetes 
• History of receiving Zostavax (shingles vaccine) within previous 4 weeks. (Study 
entry may be delayed to satisfy a 28-day interval between vaccinations)
•  Known history of any of the following co-morbid conditions:
Malignancy (participants without a recurrence in the last 5 years will be 
allowed)
Congestive Heart Failure 
Cardiovascular Disease (unstable ≤ 6 months*)
Kidney disease
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Renal failure 
Impaired hepatic function 
Autoimmune disease such as: Rheumatoid Arthritis, systemic lupus 
erythematosus (SLE), Inflammatory Bowel Disease, etc.
Use of medicines during past 6 months known to alter immune response 
such as high-dose corticosteroids
HIV, AIDS or other Immunodeficiency
Recent (≤ 3 months) trauma or surgery
Current substance and/or alcohol abuse
* Unstable disease is defined as a change in therapy or hospitalization for worsening disease.   
4.3 Study Enrollment Procedures 
Forty (40) healthy men and women, ages 60 and older, from all ethnic backgrounds, that have 
never received pneumococcal vaccination will be recruited to this study over a two-year period.
Recruitment 
letters will be prepared, approved by IRB and mailed to potentially eligible 
individuals 
from the UConn Center on Aging recruitment database that describe the study and provide 
contact information to obtain more information from a member of the study team. All persons 
listed in the recruitment database agreed to be contacted about future research projects during 
prior 
interactions with the Center on Aging clinical research team. Other recruitment methods may 
include recruitment flyers placed in local clinics, use of broadcast email in the UConn Health 
system, newpaper/print advertisements, outreach to collaborating research clinics in urban areas 
or through physician referrals to the study.
Interested persons will phone the study team in response to the letter, broadcast email, flyer, 
referral or other advertisement to complete an IRB-approved preliminary screening questionnaire 
that asks general and aggregated questions to determine eligibility while minimizing collection of 
Protected Health Information. Progression through the screening questionnaire will stop when a 
response is given that renders the caller ineligible. Demographic information (age, gender, race, 
and ethnicity) will be collected for all callers for transparent reporting of participant selection, but 
individual identifiers and contact information will only be collected after the preliminary screening 
questionnaire has been successfully completed, indicating preliminary eligibility.  A copy of the 
IRB approved Informed Consent Form (ICF) will be provided to potential subjects for review prior 
to the informed consent/screening visit, whenever possible.  The ICF may be provided by fax, 
email, 
interoffice mail, hand delivery or by US Mail. The participant will then be scheduled for Visit 
1, when the informed consent process will be initiated and the ICF signed before full screening or 
any other study procedures are performed. 
5 
STUDY INTERVENTIONS 
5.1 Interventions
For 2015, the CDC revised recommendations for pneumococcal vaccination of adults age 65 and 
older that have never received pneumococcal vaccination, to include one dose of PPSV23 
(Pneumovax 23) and one dose of PCV13 (Prevnar 13).  Their recommendation is to administer 
PCV13 first followed by PPSV23 ≥ 1 year later[53].  Although pneumococcal vaccine is 
recommended for healthy adults to receive at age 65 or older, it is routinely given to children and 
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adults 2 to 64 years of age, with various chronic disease.  Subjects who elect to participate in this 
study would be receiving the vaccination as a healthy adult, at an earlier age.
While PCV13 demonstrated efficacy in reducing incidence of community acquired pneumonia 
(CAP) due to serotypes of S. pneumoniae included in the vaccine, it did not demonstrate efficacy 
in community acquired pneumonia from any cause with reported vaccine efficacy of 5.1%, 95% 
CI, (−5.1 to 14.2) (2). PPSV23 includes 11 serotypes that are not included in PCV13 and elicits 
immune response by a different mechanism than the conjugated vaccine.  Since reduced 
response to pneumococcal vaccination in patients age 65 and older limits the benefits of 
vaccination, it is critical to develop additional understanding of aging-related immunosenescence 
to find ways to increase response to vaccination in this population.  Each vaccine has potential 
benefits and limitations.  PCV13 and PPSV23 have not been compared directly for effectiveness 
in reducing 
incidence or severity of CAP through a randomized, controlled clinical trial (3).  Studies 
to date have used antibody response as outcome measures rather than clinical efficacy and have 
focused on the 12 serotypes of S. pneumoniae that PPSV23 and PCV13 share.  This study looks 
to develop a deeper understanding of the mechanisms of diminished response to both vaccines 
in the elderly.
To elucidate transcriptional and epigenetic profiles of the mechanisms of response to each 
vaccine in the elderly, enrolled participants will be randomly assigned to receive pneumococcal 
vaccination with Prevnar-13 (PCV13) or Pneumovax-23 (PPSV23). These vaccines are FDA 
approved for administration in this population and will be administered by a Registered Nurse at 
the dose and by route (intramuscular injection), as approved.
Randomized assignment to group will be in a 1:1 ratio utilizing block randomization in blocks of 
10.  A Randomization Plan will be prepared by the Data Manager at the UConn Center for Aging 
using Randomization software. Randomization assignments will be revealed to the research 
nurse when the subject meets eligibility for randomization and is assigned the next available 
sequential randomization number.  Participants will receive the vaccine that was randomly 
assigned to their sequential randomization number in the study and will be offered a boost dose 
with the non-assigned vaccine one to two years later.
Participants will be informed of the vaccine that was administered and will be provided with 
information about the vaccine as provided by the manufacturer.  Pneumovax 23 has an FDA- 
approved Patient Product Information Sheet that will be provided to participants receiving 
Pneumovax 23.  Participants that receive Prevnar 13 will receive a Fact Sheet about the vaccine 
prepared by Pfizer.  Each participant will also be given a pneumococcal vaccine fact sheet from 
CDC for the vaccine administered.
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Summary of Prescribing Information for Prevnar 13 and Pneumovax 23 (4, 5) :
Prevnar-13
Pneumovax-23
Dose 
Adults 18 years and older: a 
single dose 0.5 mL dose.
Adults 50 Single 0.5-mL dose
Route of 
Administration
Intramuscular injection
Intramuscular injection
Indications
In adults 18 years of age and 
older, Prevnar 13 is indicated 
for active immunization for the 
prevention of pneumonia and 
invasive disease caused by S. 
pneumoniae serotypes 1, 3, 4, 
5, 6A, 6B, 7F, 9V, 14, 18C, 
19A, 19F and 23F. 
Pneumovax 23 
is approved for 
use 
in persons 50 years of age 
or older. Pneumovax 23 is 
indicated  
for  
active 
immunization  
for  
the 
prevention of pneumococcal 
disease caused by the 23 S. 
pneumoniae 
serotypes 
contained in the vaccine (1, 2, 
3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 
11A, 12F, 14, 15B, 17F, 18C, 
19F, 19A, 20, 22F, 23F, and 
33F)
Contraindications
Severe allergic reaction (e.g., 
anaphylaxis)  
to  
any 
component of Prevnar 13 or 
any  
diphtheria  
toxoid-
containing vaccine.
Severe allergic reaction (e.g., 
anaphylaxis)  
to  
any 
component of Pneumovax 23.
Limitations of 
Use and 
Effectiveness
Prevnar 13 does not protect 
against disease caused by S. 
pneumoniae serotypes that 
are not in the vaccine.
Pneumovax 23 will not 
prevent disease caused by 
capsular types of S. 
pneumoniaeother than those 
contained in the vaccine.
Source
FDA- approved Prescribing 
Information, Prevnar 13
FDA- approved Prescribing 
Information, Pneumovax 23
Vaccine Administration
Both Prevnar-13 and Pneumovax 23 will be administered at the dose, by the route and in the 
population currently approved for marketing by FDA, therefore administered consistent with 
routine clinical care.  Vaccine will be administered by a Registered Nurse using best practices 
and will be administered by intramuscular injection in the deltoid.  Participants will be required 
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to remain in the clinic for 30 minutes after vaccine administration to be monitored for life-
threatening allergic reactions.  The nurse will discharge the participant after 30 minutes has 
elapsed. 
5.2 Handling of Study Interventions 
The FDA-approved vaccines to be administered in this study, Pneumovax-23 and Prevnar-13, 
will be ordered and received by the Research Pharmacist at UConn Health, per University 
policy.  Once the study has IRB approval and is ready to initiate, the research pharmacist will 
dispense all vaccine doses to the UConn Center for Aging research team where they will be 
stored during the active study period. The UConn Center on Aging Clinical Research unit has 
a medication refrigerator in which vaccines will be stored until use.  Temperature logs will be 
kept for the refrigerator 24 hours per day, 7 days per week, using an electronic temperature 
monitoring device that records temperature every 15 minutes.  Logs will be downloaded 
weekly during the study period with a copy of the log added to the study regulatory binder. 
Vaccinations will be given by an RN at Visit 2.  Randomization and Vaccination Forms will be 
used by the study team to record the sequential randomization number assigned to each 
participant when eligibility was confirmed, the participant ID (PID) for the study, the vaccine 
that was administered, expiration date of the dose, date of administration and the signature of 
the nurse that administered the vaccine.  The sticker from the dose packaging will be added to 
the form.
Participants will be informed of the vaccine that was administered to them and will be provided 
with information about the vaccine as provided by the manufacturer.  Pneumovax 23 has an 
FDA- approved Patient Product Information Sheet that will be provided to participants 
receiving Pneumovax 23.  Participants that receive Prevnar 13 will receive a Fact Sheet about 
the vaccine prepared by Pfizer (Appendix 1).  Participants will also receive a handout from 
CDC about the vaccine that was administered.
Each participant will be given a Documentation of Pneumococcal Vaccine Administration
as a record of their vaccination to share with their physician.
-to next page for study schema-
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6 
STUDY PROCEDURES
6.1 Schedule of Events
Intervention
Day -7
Visit 1
Day 0 
Visit 2
Day 1 
Visit 3
Day 10 
(± 1 d)
Visit 4
Day 28 
(± 3 d)
Visit 5
Day 60
(± 5 d)
Visit 6 
Day 
365-730
Optional
Visit 7
Informed Consent
Prior to study 
activities
x
Screening Form
x
x
Vital Signs (blood 
pressure, heart 
rate, temperature)                       
x
x
x
x
x
x
x
Body weight
x
x
x
x
Height
x
Medical History
x
Medical History 
update
x
x
x
Concomitant 
Medications
x
x
x
x
Adverse Events
x
x
x
x
x
x
x
Peripheral blood 
specimen (50 mL)       x
x
x
x
x
x
Peripheral blood 
specimen (10 mL)
(pre-vaccination)
x
Vaccination with 
Prevnar 13 or 
Pneumovax 23 by 
randomized 
assignment
x
Vaccination with 
Prevnar 13 or 
Pneumovax 23,
Other than 
received at V1
x
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(optional)
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6.2 Description of Study Visits
Visit 1:  Day -7.  Approximate visit time = 1 hour.
Consent Procedure
At the conclusion of the preliminary phone screening, study personnel briefly explain the study 
and provide potential subjects a copy of the approved ICF for review, in advance, whenever 
possible. Subjects will be encouraged to discuss study participation with family members 
and/or trusted advisors. At Visit 1, the ICF will be reviewed with the subject section by section 
by a qualified member of the study team in a private area.  Subjects will be given the 
opportunity to ask questions and to have them fully answered. Subjects who elect to enroll 
will sign and date the consent form. The member of the study team conducting the informed 
consent discussion will also sign the ICF.  A copy of the ICF signed by the consenter and the 
subject will be provided to the subject.  This process will be documented by the Documentation 
of Consent Form that will be stored 
in the research record under a unique participant 
identifier 
(PID).  The original signed ICF will be stored separately from the research record and with 
other study documents that contain personal identifiers (HIPAA Authorization, W-9, or other).
The HIPAA Authorization form will be provided to the participant to review and sign to 
authorize the use and disclosure of their Protected Health Information collected for use in this 
study.  
Enrollment
The participant is considered enrolled and receives a Participant ID (PID) number in 
the study once the ICF has been signed. 
Screening  
After informed consent, the following study activities will be performed:
Medical History Form 
Recording of Concomitant Medications 
Taking and recording of body temperature (°F) oral or ear
Screening Form completed with subject by interview 
Once all inclusion and exclusion criteria have been reviewed and the subject has been 
determined to have met all eligibility criteria, the subject proceeds to randomization.
Randomization

If the subject does not meet all eligibility criteria during the screening process, the 
subject is not randomized to vaccine and is not assigned a randomization number.  

If study entry is deferred to meet a 28 day interval from receipt of Zostavax, blood will 
not be drawn at this time. Visit 1 procedures (other than Consent) will be rescheduled 
and performed when the 28 day interval between vaccinations has been met.

Once the subject has met all eligibility criteria, they are assigned the next available 
sequential randomization number on the Randomization Log.  The PID is entered in 
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the space next to the number on the Randomization Log and the Randomization 
number is entered on the Randomization and Vaccination Form for this PID to be used 
at Visit 2. 

Visit 2 is scheduled 7 days from Visit 1. There is no window for sample collections in 
this study.  
Baseline measures

Vital signs (blood pressure, heart rate, and respiration rate), weight and height are 
recorded.

Blood draw of 50 mL:  Blood will be collected peripherally by phlebotomy by qualified 
research staff using best practices.  The tubes will be labeled with PID, Visit number 
and date of collection. Fifty (50) mL will be drawn and securely transferred to JAX-GM 
for processing and analysis. Three (3) mL will be sent by JAX-GM to Quest Diagnostics 
Laboratory or to another JAX campus for a complete blood count (CBC) with differential 
testing for research purposes only. The results of the CBC blood test will be coded with 
the PID number only and will not be placed in medical records. Results will be sent 
back to Jackson Laboratory for use in research testing. Results will not be shared with 
research participants, however, if the study doctor feels that blood sample shows 
abnormalities during research testing, he may contact participant and advise them to 
follow-up with their personal physician.
 Visit 2:  Day 0:  Vaccination Visit.  Approximate visit time = 15-30 minutes.
At Visit 2, the following procedures will be performed:
Body Temperature and Blood Draw
Taking and recording of body temperature °F (oral or ear)
If the participant has a body temperature of 100.5°F (38° C) or greater, Visit 2 will be 
deferred and rescheduled for a minimum of 3 days in the future. 
A deferral of vaccination due to fever will not require an additional Visit 1 blood draw 7 
days in advance of vaccination as long as vaccination occurs within 14 days of the 
original Visit 1 blood draw.  
Participants with a temperature below 100.5°F will proceed to blood draw.
Blood draw of 10 mL:  Blood will be collected peripherally by phlebotomy by qualified 
research staff prior to vaccination.  The tube will be labeled with PID, Visit number and 
date of collection and will be securely transferred to JAX-GM for processing and 
analysis.
The research nurse reviews the Randomization and Vaccination form and identifies 
the assigned vaccine from the Randomization Plan for the randomization number.
The subject is informed of the randomized vaccine assignment and given information 
about the vaccine.
Data Collection
Adverse events
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Concomitant medication use
Vaccination
The research nurse obtains one dose of the assigned vaccine from the refrigerator and 
prepares the dose per the package insert.
The vaccine dose label (sticker) is placed in the designated area of the Visit 2 Source 
Document Form.
The area of the deltoid where the vaccine will be injected is wiped with alcohol
The vaccine is injected intramuscularly in the deltoid by the RN.
The participant must remain at the clinic for 30 minutes after vaccine administration to be 
monitored for life-threatening allergic reactions.  Visits are scheduled for study visits 3-6.  
There is no visit window for follow-up visits.  Visits must be performed on the day indicated. 
The nurse will discharge the participant after 30 minutes has elapsed.
 Visit 3:  Day 1:  Follow-up visit.  Approximate visit time = 15 minutes.
  Weight, Blood pressure, heart rate, temperature: taken and recorded on source document.
Data Collection
Adverse events
Concomitant medication use
Blood Draw
Blood draw of 50 mL:  Blood will be collected peripherally by phlebotomy by 
qualified research staff prior to vaccination.  The tube will be labeled with PID, Visit 
number and date of collection. The sample will be securely transferred to JAX-GM 
for processing and analysis following the study visit.
Visit 4:  Day 10 (± 1 day) :  Follow-up visit.  Approximate visit time = 15 minutes.
Weight, Blood pressure, heart rate, temperature: taken and recorded on source document.
Data Collection
Adverse events
Concomitant medication use
Blood Draw
Blood draw of 50 mL:  Blood will be collected peripherally by phlebotomy by 
qualified research staff prior to vaccination.  The tube will be labeled with PID, Visit 
number and date of collection. The sample will be securely transferred to JAX-GM 
for processing and analysis following the study visit.
 Visit 5:  Day 28 (± 3 days):  Follow-up visit.  Approximate visit time = 15 minutes.
Weight, Blood pressure, heart rate, temperature: taken and recorded on source document.
Data Collection
Adverse events
Concomitant medication use
Medical History Review and update
Blood Draw
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
Blood draw of 50 mL:  Blood will be collected peripherally by phlebotomy by qualified 
research staff using best practices.  The tubes will be labeled with PID, Visit number 
and date of collection. Fifty (50) mL will be drawn and securely transferred to JAX-GM 
for processing and analysis. Three (3) mL will be sent by JAX-GM to Quest Diagnostics 
Laboratory or to another JAX campus for a complete blood count (CBC) with differential 
testing for research purposes only. The results of the CBC blood test will be coded with 
the PID number only and will not be placed in medical records. Results will be sent 
back to Jackson Laboratory for use 
in research. Results will not be shared with research 
participants, however, if the study doctor feels that blood sample shows abnormalities 
during research testing, he may contact participant and advise them to follow-up with 
their personal physician.
Planning for Optional Visit 7 at Visit 6 or earlier:   
To receive vaccination with the other pneumococcal vaccine (Prevnar or Pneumovax) that was 
not randomly assigned and administered at Visit 2, 1-2 years after administration of randomly 
assigned vaccine at Visit 2.
Discuss the option to schedule vaccination with the other vaccine one-two years 
from when the randomly assigned vaccine was administered.  
Ask the participant if they are interested in receiving the second vaccine at one-
two years free of charge and document their choice on the Visit 7 opt-in form.
Participants interested in second vaccination:
Document participant choice on Visit 7 opt-in form.
Schedule the visit 365-730 days from date of Visit 2 vaccination.  
Confirm with the participant that they agree to be contacted to be reminded of this 
visit two weeks prior and again the day before planned Visit 7.
Participant may change their mind about receiving second vaccine at any time.
Participants not interested in second vaccination:
Document participant choice on the Visit 7 opt-in form.
Study participation ends at Visit 6.  Complete End of Study form.
Participants can change their mind before the two-year anniversary of Visit 2 and decide 
that they would 
like to receive the other pneumococcal vaccine free of charge.  Participants 
will be 
instructed to contact the study team 
if they would 
like to receive the second vaccine.
 Visit 6:  Day 60 (± 5 days) :  Follow-up visit.  Approximate visit time = 15 minutes
Weight, Blood pressure, heart rate, temperature: taken and recorded on source document.
Data Collection
Adverse events
Concomitant medication use
Medical History Review and update
Blood Draw
Blood draw of 50 mL:  Blood will be collected peripherally by phlebotomy by qualified 
research staff prior to vaccination.  The tube will be 
labeled with PID, Visit number and 
date of collection. The sample will be securely transferred to JAX for processing and 
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analysis following the study visit.
Visit 7:  Day 365-730 (optional) Second vaccine visit Approximate visit time = 30 minutes.
Data Collection
Adverse events
Concomitant medication use
Medical History Review and update
Screening Form for Visit 7 Vaccination
Body Temperature.  Temperature is taken and recorded on the Visit 7 CRF.
If the participant has a body temperature of 100.5°F (38° C) or greater, Visit 7 will 
be deferred and rescheduled for a minimum of 3 days in the future. 
Body Temperature and Blood Draw
Body Temperature.  Temperature is taken and recorded on the Visit 7 CRF.
If the participant has a body temperature of 100.5°F (38° C) or greater, Visit 7 will 
be deferred and rescheduled for a minimum of 3 days in the future. 
Participants with a temperature below 100.5°F (38° C) will proceed to blood draw.
Blood draw of 50 mL:  Blood will be collected peripherally by phlebotomy by 
qualified research staff prior to vaccination.  The tube will be labeled with PID, Visit 
number and date of collection. The sample will be securely transferred to JAX-GM 
for processing and analysis following the study visit.
Vaccination
The research nurse reviews the Randomization and Vaccination form and identifies 
the vaccine administered at Visit 2.  The other vaccine will be administered at Visit 
7. (Prevnar at V2 receives Pneumovax at V7, Pneumovax at V2 receives Prevnar at 
V7.  
The research nurse obtains one dose of the Visit 7 vaccine from the refrigerator and 
prepares the dose per the package insert.
The vaccine dose label (sticker) is placed in the designated area of the Vaccination 
Form for Visit 7.
The area of the deltoid where the vaccine will be injected is wiped with alcohol
The vaccine is injected intramuscularly in the deltoid by the RN.
The participant is given the patient information handout for the vaccine received.
The RN completes and signs the Vaccination Form for Visit 7.
The participant must remain at the clinic for 30 minutes after vaccine administration to be 
monitored for life-threatening allergic reactions.  The nurse will discharge the participant after 30 
minutes has elapsed and note the time on the Vaccination Form for Visit 7.
7
RISKS AND PROTECTIONS 
7.1 Potential Risks to Subjects
Risk to Confidentiality:  There is a potential risk to confidentiality due to the protected health 
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information collected and stored in the subject’s research record.  
Risk from Blood draw:   There may be a minor amount of discomfort due to the phlebotomy.  
There is a minor risk of bruising (< 1%), infection at the phlebotomy site (< 1%) or dizziness 
following the blood draw (<1%).
Risk from Information on Cytomegalovirus (CMV) Serology and Infection:  Given the 
frequency of CMV positive titers 
in the aged population and the potential 
influence on 
immune 
responses to pneumococcal vaccines (6), individual samples will be evaluated in the Jackson 
Laboratory for Genomic Medicine as to CMV serology and infection.  
Risk from Vaccination with Prevnar-13 
As stated in Prevnar 13 Prescribing Information the following risks are known to occur after 
vaccination with Prevnar 13: 
In adults aged 50 years and older the commonly reported solicited adverse reactions were 
pain at the injection site (>50%), fatigue (>30%), headache (>20%), muscle pain (>20%), 
joint pain (>10%), decreased appetite (>10%), 
injection site redness (>10%), 
injection site 
swelling (>10%), limitation of arm movement (>10%), chills (>5%) or rash (>5%). 
In adults, antibody responses to Prevnar 13 were diminished when given with inactivated 
trivalent influenza vaccine (TIV).
Prevnar 13 does not protect against disease caused by S. pneumoniae serotypes that are 
not in the vaccine.
Risk from  Vaccination with Pneumovax-23 
As stated in Pneumovax-23 Prescribing Information, risks known to occur from vaccination 
with Pneumovax-23 are as follows:
The most common adverse reactions, reported in >10% of subjects vaccinated with 
Pneumovax 23 in clinical trials, were: injection-site pain/soreness/tenderness (60.0%), 
injection-site swelling/induration (20.3%), headache (17.6%), injection-site erythema 
(16.4%), asthenia and fatigue (13.2%), and myalgia (11.9%).  
Pneumovax 23 will not prevent disease caused by capsular types of S. pneumoniae other 
than those contained in the vaccine.
In a randomized clinical study, a reduced immune response to Zostavax® as measured 
by ELISA was observed in individuals who received concurrent administration of 
Pneumovax 23 and Zostavax compared with individuals who received these vaccines 4 
weeks apart. Consider administration of the two vaccines separated by at least 4 weeks. 
Risks from OPTIONAL second vaccination one year after initial vaccine (Visit 7)
Risk to participants randomized to Prevnar 13 who opt to receive Pneumovax 23 a year 
after initial vaccination. 
At one year, this sequence is consistent with CDC recommendations for 2015, therefore 
risk is consistent with that of routine medical care. Data is available only for antibody 
response to the 12 serotypes that the two vaccines have in common, and for these, the 
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second vaccination at one year results in similar antibody levels as an initial Prevnar 13 
vaccination. 
Risk to participants randomized to Pneumovax 23 who opt to receive Prevnar 13 one year 
after initial vaccination.
In patients 60-64 years old,  antibody response to the 13 serotypes in Prevnar 13 were 
found to be lower when given a year after Pneumovax 23 than when Prevnar is given as 
an initial vaccination. There is no available data for antibody response to all 23 serotypes 
of S. pneumoniae in Pneumovax 23 when followed by Prevnar 13. 
7.2 Adequacy of Protection Against Risks
This study will be conducted under the supervision of the Principal Investigator, Dr. Kuchel. 
Best medical practices will be followed during all procedures. UConn Health emergency care 
procedures will be followed 
if an adverse event or medical emergency occurs.  The study site 
is located on the campus of a tertiary care hospital that is available for treatment of medical 
emergencies.
Protection against Risk to Confidentiality:  
All study visits will occur in a private room at the UConn Center for Aging at UConn Health in 
Farmington, CT.
Research records will be labeled with a participant ID number (PID), an assigned unique 
identifier that is not derived from any patient identifiers.  All contents of the research record 
will be labeled with the assigned PID.  Research records will be stored in a secure area. A 
complete record of the subject
's pertinent history and documentation of the clinical visits will 
be kept on case report forms and will be stored in a secure area. Research records will be 
accessible only to the study team directly involved in conduct of the clinical protocol. 
Any study documents (Informed Consent Form, HIPAA Authorization, Visit 7 Opt-in form) that 
contain the participant’s name will be kept in a separate file apart from the research record 
and will be stored in a secure location. A master key that links participant names and PIDs 
will be maintained in a separate and secure location.
The database manager follows the Data Safety and Security Policy and Confidentiality 
agreement. These Policies provide procedures to assure that volunteer information remains 
confidential. All persons in the Recruitment Database have been asked to give permission to 
leave their name in the database. This policy will be included in this study. 
The study database is password protected and secure, thus allowing only certain individuals 
access to the information. In addition, the database can be monitored in order to track the 
date, time, and individual who entered the database.
Samples/specimens will be labeled with PID, date, and visit number when they are delivered 
to the Jackson Laboratory for Genomic Medicine (JAX) for processing, storage and analysis.  
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A coded clinical data set 
linked to samples by PID will be provided to JAX for use 
in analysis.  
Results of this study when published will not identify subjects by name.
Certificate of Confidentiality:
This research is covered by a Certificate of Confidentiality from the National Institutes of 
Health. The researchers with this Certificate may not disclose or use 
information, documents, 
or biospecimens that may identify you in any federal, state, or local civil, criminal, 
administrative, legislative, or other action, suit, or proceeding, or be used as evidence, for 
example, if there is a court subpoena, unless you have consented for this use. Information, 
documents, or biospecimens protected by this Certificate cannot be disclosed to anyone else 
who is not connected with the research except, if there is a federal, state, or local law that 
requires disclosure (such as to report child abuse or communicable diseases but not for 
federal, state, or local civil, criminal, administrative, legislative, or other proceedings, see 
below); if you have consented to the disclosure, including for your medical treatment; or if it 
is used for other scientific research, as allowed by federal regulations protecting research 
subjects. 
The Certificate cannot be used to refuse a request for information from personnel of the 
United States federal or state government agency sponsoring the project that is needed for 
auditing or program evaluation by NIH National Institute of Allergy and Infectious Diseases 
(NIAID) which is funding this project. You should understand that a Certificate of 
Confidentiality does not prevent you from voluntarily releasing information about yourself or 
your involvement in this research. If you want your research information released to an 
insurer, medical care provider, or any other person not connected with the research, you 
must provide consent to allow the researchers to release it.
The Certificate of Confidentiality will not be used to prevent disclosure as required by federal, 
state, or local law of information about elder, spousal abuse, reportable communicable 
diseases. The investigators on this study will report this information to State officials if it 
becomes known to them. The Certificate of Confidentiality will not be used to prevent 
disclosure for any purpose you have consented to in this informed consent document.
A description of this clinical study will be available on http://www.ClinicalTrials.gov. This 
Website will not include information that can identify participants. At most, the Web site will 
include a summary of the results. Participants will be informed in the consent that they can 
search this Website at any time.
Protection against Risk from Blood Draw:
Blood will be drawn peripherally via venipuncture at all visits.  The volume drawn will be 50 
mL at Visits 1, 3, 4, 5, 6 & (optional visit) 7 and 10 mL at Visit 2.  Blood will be drawn by 
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experienced, trained research staff in a clinic setting on a hospital campus.  The area where 
the needle will be inserted will be wiped with alcohol before and after the draw.  A band-aid 
will be placed over the site.  Emergency treatment is accessible on campus for any severe 
complications from blood draw. 
Protection from Risk from Information on CMV Serology and Infection:  
Information about CMV infection will not be shared with individual subjects or their 
physicians. In addition to the 
importance of maintaining the “firewall” between our clinical and 
genomic studies, there is at this time no evidence that a positive CMV serology should result 
in any specific course of action on the part of the patient or his/her physician. As a result, a 
decision to share such information would only result in unnecessary anxiety and confusion.
Protection against Risk of Vaccination with Prevnar 13:
A detailed medical history questionnaire will be completed by the participant and reviewed 
by a qualified member of the study team to identify contraindications to vaccination and to 
determine study eligibility. For eligible participants, assigned vaccine will be administered by 
a Registered Nurse as a single dose, given intramuscularly in the deltoid.  Participants will 
be required to remain on site for 30 minutes after vaccine administration so that they can be 
monitored for adverse events.  
Protection against Risk of Vaccination with Pneumovax 23: 
In addition to protections listed under vaccination with Prevnar 13, participants will be asked 
about recent vaccination with Zostavax for shingles within the prior 28 days from date of 
planned vaccination or if they are planning to receive Zostavax in the 28 days following.  In 
order to optimize response to all vaccines, study entry may be delayed to allow 28 days 
between vaccine administrations.  If study entry is delayed, eligibility criteria will be reviewed 
and confirmed prior to randomization.
Protection against Risk from optional second vaccination one to two years after 
initial vaccine 
(Visit 7):
Participants that opt into a second vaccination with the vaccine not assigned at baseline will 
receive the benefits of two vaccinations for pneumococcal infection at no cost to them. Both 
vaccines are FDA approved for healthy adults 65 and older and children and adults 2 to 64 
years of age with certain chronic health conditions.  Vaccinations are given at the dose and 
via the route (intramuscular injection) as approved.  Medical history will be reviewed and 
eligibility re-confirmed before the second vaccine is administered by an RN at optional Visit 
7.
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7.3 Adverse Events and Serious Adverse Events 
An adverse event (AE) is defined as any unfavorable and unintended diagnosis, symptom, 
sign, syndrome or disease which either occurs during the study, having been absent at 
baseline, or if present at baseline, appears to worsen. Adverse events will be collected and 
recorded at each visit regardless of their relationship to the study intervention or to study 
participation.
A serious adverse event (SAE) is defined as any untoward medical occurrence that results 
in death, is life threatening, requires inpatient hospitalization or prolongation of existing 
hospitalization, results in persistent or significant disability/incapacity, or is a congenital 
anomaly.  
7.4 Reporting Procedures
The Clinical Principal Investigator will review all AEs and SAEs to assess causality.  The IRB 
requires reporting of unexpected adverse events that may represent an unanticipated 
problem involving risks to subjects or others (UPIRSO). Such events are to be reported to 
UConn Health IRB using the Problem Report Form within the IRIS electronic IRB submission 
system.
7.5 Treatment for Adverse Events
UConn Health does not provide insurance coverage to compensate for injuries incurred 
during research.  UConn Health does not offer free care.  However, treatment for a research 
related injury can be obtained at UConn Health for the usual fee.   Medical care at UConn 
Health will be available to study participants for treatment of an adverse event at normal cost.  
7.6 Data and Safety Monitoring
Data and Safety Monitoring Plan
The Data & Safety Monitoring Plan for this study describes the components of the study that 
will be monitored by the PI, study coordinator and data manager once annually.   
Recruitment, drop outs, adverse events, unanticipated problems, data integrity and 
confidentiality, participant privacy and the general conduct of the study will be reviewed.  
Minutes of the annual DSMP review will be kept in the regulatory binder and provided to IRB 
at study continuation.  The vaccines given in this study are given at the dose, via the route 
and in the population for which they are approved by FDA, and study samples are limited to 
peripheral blood draws 
in healthy older adults, thus this study poses minimal risk to research 
subjects. As a result, a Data Safety Monitoring Board (DSMB) 
is not 
indicated.  Nevertheless, 
robust procedures are in place to ensure confidentiality of research data.
Procedures in place to ensure confidentiality of research data are as follows:
1. Only authorized individuals have access to any data, used or stored (via electronic format 
or as hard copy records).  Only designated research staff and 
investigators will be granted 
access.  
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2. Logon IDs and passwords for access to the UConn Center on Aging shared network drive 
and the University’s online resources are initially assigned by the Information Technology 
Department (IT).
3. All data (clinical, recruitment and schedule-based) are stored in password-protected 
databases on the secured network drive.  Only approved personnel have access to these 
databases and passwords.
4. All data collected on data forms are stored in locked drawers located in the Center on 
Aging research facility for scanning and verification purposes.  Files are stored in locked 
cabinets in rooms that are locked when not in use. All data are backed up on the shared 
network drive for UCHC.
8  INTEGRATIVE DATA ANALYSIS 
8.1 Statistical Methods 
The breadth and magnitude of IgG titers will be analyzed individually and together using best-
response and multivariate U-statistic approaches [4, 97]. Differences among groups (e.g., 
responder status) and/or time of immunological variables (e.g., flow, RNAseq) will be assessed 
using generalized linear mixed-model analyses with appropriate distributional assumptions (e.g., 
normal, negative binomial) and link functions (e.g., identity, log). Propensity scores [98] will be 
adjusted for possible confounds such as gender, age, race and ethnicity, while the Bonferroni 
correction or Benjamini and Hochberg false discovery rate (FDR) will account for multiple testing 
[99]. Correlation of clinical and immunological variables will be conducted using the Pearson or 
Spearman Correlation Coefficient depending on whether or not the assumptions of linearity and 
homoscedasticity are met. For class prediction, a variety of methods including k-nearest 
neighbors, radial basis machine, random forests, boosted trees and support vector machines will 
be employed and validated using leave-one-out cross-validation. Unsupervised analysis of high 
dimensional data will be conducted using heatmaps, hierarchical cluster analysis and principal 
component analysis. Lastly, attrition and missing data are issues in longitudinal [100] and elderly 
studies [101]. “Missingness” will be characterized and multiple imputation applied when 
appropriate [102-104]. Drs. Churchill (JAX) and Chaussabel (SIDRA) will assist with statistics and 
bioinformatics as needed.
8.2 Power Calculations  
Power calculations are based on the ability to identify an adequate number of differentially 
expressed genes from RNAseq data to conduct downstream analyses and gain biological 
insights. Twenty patients per cohort adequately powers (>90%) this study for the detection of at 
least 6,000 differentially expressed genes. This calculation is established on conservative 
estimates obtained from RNAseq data of the flu pilot study. This study contained eight high 
responders and six low responders from the 2011 flu season measured at baseline, day 1, and 7 
post vaccination Out of 37,649 genes, 6,412 to 10,915 genes displayed a >2 fold difference 
between responder status and/or time points Other specific parameters used for the power 
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calculation include a two-sided two-sample t-test, standard deviation of 0.7 (base 2 log scale), 
and false discovery rate of 1%. The PASS 2008 statistical package was used for power analysis.
9 DATA COLLECTION AND MANAGEMENT
Data Collected from Human Subjects Specifically for Research Purposes
Medical and medication history provided by the participant: Prior to the first collection of blood at 
Visit 1, 
individuals who have consented to participate 
in the study will be asked to fill out a detailed 
health history questionnaire with past and current medical conditions with date of diagnosis, 
including current and prior medication use, and vaccine history.
At all visits, participants will be asked about recent health history (e.g., colds, flu, pneumonia-like 
symptoms, hospitalizations, infections) and concomitant medication use since the previous 
review.
Vital signs (blood pressure, heart rate, temperature) and adverse events will be collected by a 
qualified member of the study team at each visit.
Height at Visit 1 only, weight at all visits.
Participant Identification (PID) number:  
A unique participant identifier (PID) not derived from any patient identifiers will be assigned to 
each participant once written informed consent for study participation has been obtained.  All 
clinical data will be linked to the PID in the database.  Participant names or other personal 
identifiers will not be included in the study data set; all data will be identified only by PID.
Access to individually identifiable private information about human subjects
Access to clinical research records and identifiable study data will be restricted to the study team 
involved 
in the conduct of the clinical protocol. Coded clinical data will be provided to the JAX-GM 
laboratory staff for analysis linked with study samples by the PID.  HIPAA personal identifiers will 
not be included in the clinical dataset provided to JAX-GM for use in analysis. The key linking the 
PID to participant identifiers will be stored separately and securely from the research records at 
the UConn Center on Aging.  The code key will not be provided to the JAX investigator or staff at 
any time.  JAX Lab staff will have access to coded samples and data only.  Dr. Banchereau, as a 
co-investigator on the clinical protocol may be present at clinical team meetings when identifiable 
information is present. He will not record or disclose participant identifiers and he will not receive 
the code key at any time. 
9.1 Data Collection Forms (CRF)
Forms to be completed for this study include:
Phone Pre-screening Form:  
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A partial waiver of consent and HIPAA Authorization will be requested for preliminary phone 
screening of study participants.  No personal identifiers will be added to phone pre-screening 
forms until the form has been successfully completed. Pre-screening forms for participants that 
provide informed consent and enter the study will be stored with the identifiable forms in the 
Informed Consent/HIPAA Authorization Form binder for the study that 
is kept separate from coded 
CRF.
The Visit 1 Source Documentation Form 
The Visit 1 Source Documentation Form is labeled with the PID and includes the following data:
Documentation of Informed Consent: 
To confirm content of the informed consent discussion, that signatures/dates were 
collected and that the participant was given a copy of the signed ICF.  
Screening Form:  
This form lists all eligibility criteria.  Participants must meet all inclusion criteria and not 
have any of the exclusion criteria to be randomized to vaccine.
Medical History Form including medication use
Visit 1 Procedures Documentation 
The sequential randomization number assigned per the Randomization Log
Visit 2 Source Documentation Form:  
Checklist for Visit 2 procedures labeled with the PID and date records the members of 
the study team that contributed to visit procedures.  
Randomization and Vaccination Data:  
The sequential randomization number assigned at Visit 1 the vaccine assigned per the 
Randomization Plan for that number, the sticker from the vaccine dose administered, date 
of administration, confirmation of 30 minute waiting period at the clinic after vaccination 
and the signature of the RN that administered the vaccine.
Visits 3-5:  Source Documentation Forms: 
These visit forms labeled with the PID and date are identical other than visit number. Members 
of the study team that collected data at this visit, vital signs and blood draw documentation are 
recorded on these visit forms. 
Visit 7 Opt-in Form:  
This form is completed at any time during study participation but must be completed before the 
end of Visit 6.  The participant will indicate their intention to receive the second vaccine (not 
administered at Visit 2) at the optional Visit 7 as described in study procedures section or will 
decline the optional visit and second vaccine. The participant will sign and date the form and will 
be given a copy of the signed form for their records.  This form will be stored with the ICF and 
HIPAA since it has a signature.  Visit 7 will be scheduled for participants that choose to attend 
the optional Visit 7.
Visit 6 Source Documentation Form
This form captures members of the study team that collected data at this visit, vital signs and 
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blood draw documentation.  Completion of Visit 7 Opt-in form is recorded and the selection of 
the participant made on the form.
Visit 7 (optional visit) Procedures Documentation Form: 
This form is a checklist for Visit 7 procedures for participants that opt for a second 
vaccination.
Vaccination administration detail  for Visit 7:  
The research nurse will identify the original assigned vaccine and the other vaccine to be 
given at Visit 7 on this form.  The sticker from the vaccine dose will be placed on the form.  
The time and date of vaccine administration and confirmation of the 30 minute post-
vaccine observation period with time of dismissal will be recorded by the research RN.
9.2 Data Management 
Data Collection Forms (CRF) will be 
labeled with the Participant ID code, Subject 
initials and Visit 
Date and will be stored in the Research Record.  Subject initials will be included on forms to aid 
the research staff in confirming that data is being collected for the correct participant.  Participant 
initials will not be included in the coded dataset that is provided for use at JAX-GM.  Clinical data 
provided to JAX-GM will be labeled only with the PID. 
9.3 Quality Assurance 
Training 
 A study initiation meeting will be attended by study staff in which the protocol, visit procedures, 
secure storage of research records, secure management of electronic databases and sharing of 
coded data with JAX, sample labeling protocol, and the process for sample transfer to the 
Jackson Laboratory for Genomic Medicine will be reviewed.
Delegation of Responsibilities
A Delegation of Responsibilities log will be completed at the Initiation Visit for members of the 
study team that will be entering data on CRF, determining eligibility, labeling samples or 
administering vaccines.  This log will be kept in the study regulatory binder.
9.4 Protocol Deviations
 A cumulative Protocol Deviation Log will be kept electronically by the study coordinator with a 
copy added to the regulatory binder.  Deviations from the protocol will be entered on this log with 
a Note to File labeled with PID and no personal identifiers added to the regulatory binder with a 
copy to the research record that describes the deviation, date when 
it was 
identified, the corrective 
action taken to prevent recurrence, whether or not the deviation met criteria of an Unanticipated 
Problem, and the date of IRB notification.  
Incidents of non-compliance, defined as any action that 
is taken or occurs that 
is not 
in accordance 
with an IRB approved study, IRB policies or regulations or failure to follow the requirements and 
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determinations of the IRB, that is within the control of the study team must be reported to the IRB 
within 5 days of becoming aware of the occurrence.
9.5 Unanticipated Problems
Per UConn Health policy for the purpose of reporting Unanticipated Problems to IRB, internal 
adverse events that may also represent an unanticipated problem are defined as those events, 
experiences or outcomes that are:
1. Unexpected (in terms of nature, severity or frequency) given (a) the research procedures that 
are described in the protocol-related documents, such as the IRB approved research protocol 
and informed consent document; and (b) the characteristics of the subject population being 
studied; and
2. Related or possibly related to participation in the research (i.e., there is a reasonable
possibility that the incident, experience, or outcome may have been caused by the procedures 
involved in the research).  Any internal event meeting these criteria must be reported to the IRB, 
which will then make the final determination as to whether the research places subjects or others 
at a greater risk of harm (including physical, psychological, economic or social) than was 
previously recognized.
10 PARTICIPANT RIGHTS AND CONFIDENTIALITY 
10.1Institutional Review Board (IRB) Review
This protocol and the informed consent form and any subsequent modifications will be reviewed 
and approved by the UConn Health IRB before use.
10.2Informed Consent
The informed consent process will begin after a participant successfully completes phone 
screening and plans to enter the study.  A copy of the IRB approved Informed Consent Form 
(ICF) will be provided to the participant in advance for review, whenever possible.  
At Visit 1, a qualified member of the study team will review the ICF, section by section, with the 
volunteer in a private room prior to any study procedures. Questions from the participant will be 
encouraged, and will be fully answered.   Once the form has been fully reviewed and all questions 
answered, the volunteer will be asked if they would like to participate in the study.  If they agree 
to participate, they will be asked to sign and date the ICF indicating their consent.  The member 
of the study team obtaining consent will sign the ICF as well and a copy of the form signed by 
both the subject and the consenter will be provided to the subject.  The participant will be asked 
to verbally confirm continued Informed Consent at each study visit before procedures are 
performed.
The Informed Consent process will be documented by the consenter on the Documentation of 
Informed Consent Form 
labeled with the PID and will be stored 
in the Research Record.  Original 
signed and dated ICFs will be stored separately and securely away from the research record 
with other identifiable documents that contain participant identifiers.
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10.3 Genomic Data and Sample Sharing
Participants will provide or decline consent within the ICF for sharing of their randomly recoded 
(new code that is different than the study code) genomic data in public and/or controlled access 
scientific databases.  The study database will include a field for whether consent for genomic 
data sharing was provided or declined by the participant and if consent provided was for public 
and/or limited access databases.  This information will be included in the dataset when provided 
to JAX-GM so that it can be provided to dbGAP (NIH database of Genotypes and Phenotypes) 
at the conclusion of the study to ensure that the wishes of the participant regarding use of their 
data and samples are respected.
Participants will provide or decline consent for sharing of coded samples that remain after study 
analysis is completed with other researchers and to be used in other studies. This information 
will be included as a variable in the coded dataset provided for analysis to JAX-GM.
10.4 HIPAA Authorization
Study participants must provide written authorization at study entry for use and disclosure of their 
Protected Health Information that is recorded and used in this study in order to participate.  
Participants may withdraw their Authorization at any time, but will then be withdrawn from the 
study. Study data will be stored 
in a password-protected database coded by Participant ID (PID).  
The key linking PID and participant name will not be shared with the JAX staff performing 
laboratory analysis. As a co-investigator, Dr. Banchereau will be actively engaged 
in the conduct 
of this research and although he will not be provided with the key linking PID and identity of 
participants, he may have identifiable information disclosed to him during meetings with the 
clinical study team. Dr. Banchereau will be 
listed 
in the HIPAA Authorization form for this reason.
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