Single Cell Molecular Analysis of Influenza Vaccine Induced T Cell Responses in Adults 65 Years of Age or Older
Phase: PHASE4
Conditions: Flu Vaccine
Sponsor: Vanderbilt University Medical Center

Single Cell Molecular Analysis of Influenza Vaccine Induced T
Cell Responses in Adults 65 Years of Age or Older
NCT04077424
November 11, 2019

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Single cell molecular analysis of influenza vaccine induced T cell responses in adults 65 
years of age or older  
 
 
 
PRINCIPAL INVESTIGATOR: 
Spyros Kalams, MD 
Associate Professor of Medicine 
Vanderbilt University Medical Center 
 
s.kalams@vumc.org 
 
 
 
Co-Investigators: 
H. Keipp Talbot, MD MPH 
Jessica L. Castilho, MD MPH 
Natasha Halasa, MD MPH 
 
 
 
 
 
 
 
 
 
 

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1.0 
BACKGROUND AND RATIONAL FOR THE STUDY 
Each year influenza virus is associated with approximately 5,546 deaths in adults ≥65 years in 
the United States.1 The reasons for the disproportionate morbidity and mortality of influenza in 
older individuals are not well defined, and they increase with age.2 In fact, persons aged 85 
years and older are 32 times more likely to die of influenza and associated pneumonia than 
those only 65-69 years of age.2 Annual influenza vaccination has been recommended for adults 
≥65 years3 of age since 1960 to reduce excess morbidity and mortality in older adults. However, 
a steady and systemic declined in the immune system with increasing age, termed 
Immunosenescence,4 appears to reduce vaccine effectiveness (VE) in older adults.5 
The reasons for decreasing response to vaccination with age are multifactorial and may be related 
to changes in adaptive immune responses, including decreased T cell 6-8 and B cell 9,10 function. 
T cell phenotypic changes associated with aging include a gradual loss of CD4+ and CD8+ naïve 
cells 11, and an accumulation of late-stage differentiated CD4+ and CD8+ memory cells with loss 
of CD28 expression6,12-14. CD28 is a costimulatory molecule required for activation, proliferation, 
and differentiation of T cells 15.  
Immune senescence is defined as phenotypic and functional changes in T cell populations 
associated with aging. These include skewing of the phenotype of CD4+ and CD8+ T cells toward 
a “late stage differentiated” phenotype, characterized by lack of expression of the costimulatory 
molecules CD27 and CD28 16. Expansion of this T cell phenotype has been associated with 
cytomegalovirus (CMV) seropositivity and CMV antigen recognition 17, suggesting an antigen 
driven expansion of these cell populations. Despite their decreased proliferative ability, these cells 
are functional, as they are still able to produce cytokines and maintain lytic capability (expressing 
granzyme and perforin) 17-22. Poor vaccine responses in older persons have been linked to 
expansion of memory T cell populations with decreased CD28 expression, elevated levels of 
exhaustion markers (increased PD-1, CTLA-4 and LAG-3), and an accumulation of T cells with 
decreased proliferative potential (increased CD57 and KLRG1) 23-26. 
Herpesvirus infection is ubiquitous in humans, and is relevant for immune senescence and aging. 
It is estimated that >90% of the adult population globally is infected with at least one HHV.27 These 
include herpes simplex virus (HSV), CMV, varicella zoster virus, and other HHV. In 
immunocompetent persons, most of these are latent infections that do not contribute to overt 
clinical disease over the course of a lifetime. There is mounting evidence, however, that 
maintaining HHV latency comes at a cost to the immune system, even in the immunocompetent 
host 27. CMV is a beta herpesvirus, and like the other viruses in this family, causes chronic latent 
infection 28,29. The incidence of CMV seropositivity increases with age 28,30-32, and has been 
associated with poor responses to influenza vaccination in elderly individuals 12,33, and with 
reports of negative 34 and beneficial 35 effects in young individuals. The links between CMV 
infection and influenza vaccine response therefore are not straightforward. A recent study 
evaluated antibody responses to a new antigen strain (H1N1pdm in 2009) in adults 18-52 years 
and found diminished antibody responses associated with age, but not with CMV infection 36. Poor 
immune responses in the setting of chronic CMV infection have been linked to accumulation of 
late-stage differentiated senescent T cells with diminished helper function12,22,33,37 38.  
The mechanism by which CMV infection may alter the host’s response to new infections or 
vaccination is not clear. However, the chronic nature of the infection leads to a feature of the 
immune response termed “memory inflation”. Classical CD4+ and CD8+ memory T cell responses 
against viruses expand during primary infection and contract to low magnitudes during post 
infection resolution 18. However, CD8+ T cell responses to select epitopes of human CMV (HCMV) 
39,40, rhesus CMV 41, and murine CMV (MCMV) 42,43 persist for decades at very high magnitudes 
after primary infection or during latency. This phenomenon is termed “memory inflation” and has 

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been best characterized among CMV- specific CD8+ T cells that consist primarily of CD45RO+ 
CCR7- CD27- T cells (effector memory T cells [TEM]) and their CD45RA+ revertants, CD45RO- 
CCR7- CD27- T cells (effector memory RA+ [TEMRA]) 21,44-47. We have recently identified this same 
feature of CMV-specific CD4+ T cells 19. We will evaluate CD4+ CMV-specific T cell memory 
inflation in this cohort of elderly influenza participants to determine whether it contributes to poor 
vaccine responses.   
The term “older adults” represents a diverse population of individuals over 65 years of age 
ranging from physically and mentally fit to physically and/or cognitively-impaired with very 
different requirements for daily care ranging from those who are entirely independent to those 
who must rely on others for their complete care. Hence when performing studies in older adults, 
it is essential to describe the population being studied in explicit detail to interpret study results 
and for generalizability of findings. One key characteristic of older adults that may differ greatly 
is the degree of frailty. Frailty is the conceptualization of a phenotype of poor physiologic 
reserve and poor resistance to stressors and hence is associated with a high risk of morbidity 
and death from diseases.48 One of the most accepted methods to measure frailty is one 
described by Fried et al,49 that is operationalized by declines in lean body mass, strength, 
endurance, balance, walking performance, and low activity. Frailty as a syndrome in the 
geriatric population encompasses a person’s chronic medical conditions, functional status, and 
risk of mortality.50 Frailty may be a better predictor of the immune response in older adults than 
chronologic age.51 In previous studies, frailty scales have predicted vaccine response to 
polysaccharide pneumococcal vaccine better than age 52 and to be correlated with poor 
antibody response to influenza vaccination.53  
Immune responses to both influenza and influenza vaccines are moderated by prior exposure to 
influenza and influenza vaccines. The original concept was called antigenic sin54 but recently 
has been termed imprinting.55 Influenza A viruses can be divided into two phylogenetic groups.  
The first influenza A group that a person is exposed to will provide better protection for the 
remaining influenza viruses within the group but not in the other group.55  The birth cohort 
ranging from 1918 to 1957 is the majority of the population ≥65 years of age was first exposed 
to an H1N1 virus leaving this birth cohort at increased morbidity and mortality to H3N2 group 
viruses.    
The current circulating H3N2 has adapted well to humans and evades antibody binding by 
glycosylating common antigenic sites. These same glycosylations make growth for vaccine 
production in eggs difficult.  Egg adaption can change the vaccine strain less well-matched to 
the circulating strain.  
As a strategy to increase immunogenicity of influenza vaccines, FDA approved high-dose (HD) 
influenza vaccine (Fluzone® High-Dose, IIV-HD) containing 4-times more hemagglutinin (HA) 
(60µg HA/strain) than the standard-dose (SD) vaccine (15µg HA/strain) for people aged ≥ 65 
years in 2009.56 In clinical trials, the high dose vaccine demonstrated significantly higher HAI 
antibody (Ab) responses (seroconversion and seroprotection rates) without an increase in 
clinically relevant systemic adverse reactions, but a slight increase in local reactions.57 The 
large phase IIIb/IV study of HD, led by Dr. Talbot, showed improved clinical efficacy with a 
relative VE of 24%. In 2015, the FDA approved an MF-59 adjuvanted inactivated influenza 
vaccine (FLUAD™, aIIV) for the same population of older adults. The adjuvanted vaccine, 
FLUAD, has been in use in Europe for over 25 years with an excellent safety and tolerability 
experience.58 The effectiveness of the MF-59 adjuvanted vaccine was compared with standard 
influenza vaccine and was found to be 63% more effective.59 A recombinant inactivated 
influenza vaccine (rIIV)comprised of 45mcg hemagglutinin per strain (total of 180mcg) was 
licensed for use in the United States in 2013. Likely due to the higher dose of antigen, the rIIV 
was shown to have higher efficacy in adults ≥50 years of age in a trial during the 2014-2015 

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season.60  At this time, the, the Advisory Committee on Immunization Practices recommends all 
adults ≥65 years of age receive an influenza vaccine but does not preferentially recommend any 
vaccine for this population. 
2.0 
Specific Aims: 
Studies of immune function in elderly populations have been limited. We propose to advance 
multiple high-priority fields simultaneously: T cell immunology, immunologic aging and immune 
senescence, precision medicine and vaccine responses, and the relationship between cellular 
immune responses to chronic CMV infection and response to influenza vaccination in 
immunocompetent older populations.  
Specific Aim 1. To perform HAI and microneutralization assay individuals who receiving influenza 
vaccine. 
Specific aim 2. Determine whether profile of immune senescence is  predictive of vaccine 
responses in elderly individuals receiving IIV-HD, aIIV3 or rIIV. 
Specific aim 3. Determine whether inflated CMV-specific immune responses in elderly 
populations are responsible for the immune senescence profile of elderly individuals and limits 
their ability to mount immune responses to vaccines 
Specific aim 4. Determine whether measures of frailty are associated with immune senescence 
and decreased ability to respond to vaccines. 
STUDY PROCEDURES 
3.0 
Eligibility for enrollment.  
Community dwelling adults ≥65 years of age without contraindication for influenza vaccination 
will be eligible. Participants will not be excluded based on comorbid conditions in order to 
comprehensively capture the spectrum of the aging population. 
 
Accural Goal 
Our goal is to enroll 120 people each year. 
4.0 
Inclusion/Exclusion Criteria  
Inclusion Criteria  
1. Age ≥65 years 
2. Independent Living (including Assisted Living) 
Exclusion Criteria  
1. Unable to understand the consent or the study.  
2. Allergic to any vaccine components, excluding eggs. 
3. History of Guillain-Barre. 
4. Anaphylaxis egg allergy 
5. Residing in a long-term care facility such as a nursing home. 
 
 

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5.0 
Study procedures: 
Data Collection: During the first year of the study, the following forms will be designed to 
complete the objectives of this study. 
1. REDCap Case Report Form: Administered by the enrollers, and includes: demographics; 
height and weight; past medical history; history of recent falls and hospitalizations; active 
medications; alcohol use; use of home oxygen; questions regarding ability to perform 
activities of daily activity; frailty measures (see section on Frailty Metrics); sarcopenia 
assessment; depression symptom screen; cognitive assessment; and contact information for 
the participant.  
2. Frailty Metrics: As mentioned earlier, the evaluation of frailty is very important in the 
assessment of vaccine responses. Frailty can be measured by multiple methods. One of the 
most accepted tools is the Fried Frailty Evaluation.49 This tool evaluates shrinkage (loss of 
mean body mass), weakness, endurance, slowness, and physical activity on a scale of 0-5. 
This tool is predictive of falls, worsening mobility or disability in the activities of daily living, 
and in predicting hospitalization and death over a three-year period. This measure of frailty 
is not synonymous with comorbidities or functional status. A composite frailty measure will 
be calculated using each of the evaluation scales and utilized to compare the safety profile 
and ultimately the immune responses.61 This measure will be based on the five parameters 
of weight loss, exhaustion, low activity level, slowness, and weak strength, described below.   
1. Weight Loss 
Meets criteria for frailty if answers yes to: 
Self-report of loss of >10 pounds unintentionally over the past year 
2. Exhaustion 
Meets criteria for frailty if answers: 
Self-report of “moderate” or “most of the time” for either: 
1. I felt that everything I did was an effort in the last week: 
a. Rarely or none of the time (<1 day) 
b. Some or little of the time (1-2 days) 
c. Moderate amount of the time (3-4 days) 
d. Most of the time 
 
2. I could not get going in the last week: 
a. Rarely or none of the time (<1 day) 
b. Some or little of the time (1-2 days) 
c. Moderate amount of the time (3-4 days) 
d. Most of the time 
3. Low Activity Level 
Meets criteria for frailty if answers: 
Self-reported “limited a lot” for: 
Does your health limit vigorous activities such as running, lifting heavy objects, 
or participating in strenuous sports? 
a. No, not at all 
b. Yes, limited a little 
c. Yes, limited a lot 
4. Slowness 
Meets criteria for frailty if: 
Time to walk 15 feet (4.57 meters) at usual pace (averaged over 2 trials) 
Men                                                 Women                
> 7sec for height < 173cm               > 7sec for height < 159cm 
> 6sec for height > 173cm               > 6sec for height > 159cm 
 
 
 
 
 

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5. Weakness 
Meets criteria for frailty if: 
Grip strength measured by hand dynamometer (averaged over 3 trials of 
dominant hand): 
Men                                                 Women                
< 29kg for BMI < 24                        < 17kg for BMI < 23                
< 30kg for BMI 24.1-26                   < 17.3kg for BMI 23.1-26                
< 30kg for BMI 26.1-28                   < 18kg for BMI 26.1-29               
< 32kg for BMI >28                         < 21kg for BMI >29                
(BMI: body mass index) 
Participants with total scores of 3 or greater meet criteria for frailty. Those participants with a 
higher composite score (and therefore a greater degree of frailty) will be expected to have lower 
reactogenicity to vaccine and likely a lower serologic response, but this hypothesis will be 
tested. 
3. Five-times Sit-to-Stand (5STS) Test for Sarcopenia. All participants will be undergo two 
trials of timed duration to go from sitting position to standing position and back to sitting five 
times. The average of the 2 trials will be used for the result. The 5STS is a routinely-used 
geriatric assessment for sarcopenia and fall risk. An average 5STS time of >12 seconds is 
indicative of fall risk 62.  
4. The Montreal Cognitive Assessment (MoCA): The MoCA is a validated, performance-
based tool used to detect cognitive impairment with score ranges from 0 to 30 points. It will 
be administered at enrollment. The following ranges may be used to grade severity: 18-26 = 
mild cognitive impairment, 10-17 = moderate cognitive impairment and < 10 = severe 
cognitive impairment. This tool will be included as many geriatricians feel cognitive 
impairment is often associated with frailty. As presence of depressive symptoms may affect 
cognition and memory, we will also include a depression symptom screen as part of the 
assessment. All participants will complete the Patient Health Questionnaire as part of 
routine care.  The results of the Patient Health Questionnaire performed during routine 
medical care will be included in the chart review. 
5. Baseline clinical laboratory assessment 
At the first visit, blood specimen collection will also include evalution of two clinical tests to 
be conducted by the Vanderbilt Laboratory: CMV serology and CD4/CD8 lymphocyte ratio 
calculation. Detection of  serum CMV IgM and IgG antibodies will be conducted on all 
participants. Calculation of absolute CD4 and absolute CD8 lymphocytes will also be 
performed using the clinical lab for calculation of CD4/CD8 ratio. Inversion of the CD4/CD8 
in older adults has been found to correlate with measures of adaptive immune senescence, 
CMV seropositive, as well as frailty and falls 63-65. 
 
          
 
Visit 1  
(D0) 
Visit 2 
(D7[+/2days]) 
Visit 3 (D28 
[-4/±14 
days]) 
Visit 4 
(Month 6/ 
D180[+/-14 
days]) 
Optional 
Baseline Data Collection: 
- 
Case report form 
- 
Frailty Metrics 
- 
5STS 
- 
MoCA 
X 
 
 
 

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- 
Clinical laboratory 
tests 
Blood Draw 
X 
X 
X 
X 
Vaccination 
X 
 
 
 
 
Recruitment and Retention: Although most industry-sponsored vaccine studies in older adults 
seek to recruit a very healthy, mobile population, in this study we will pay special attention to the 
enrollment of older adults ≥80 years of age and those who are frail so we can get a more 
comprehensive assessment of the safety and immunogenicity of these two vaccines in a larger 
spectrum of older adults. The population in Davidson County is 61% white and 27% black with 
37,787 woman and 25,574 men ≥65 years of age who reside in the County.  
Informed Consent in Older adults: Previous research conducted by our group to identify viral 
respiratory disease in hospitalized patients has led our team to work closely with our Institutional 
Review Board to improve the consenting process for older adults. Consents have been 
simplified to include only necessary information and they are printed in larger fonts. Each 
enroller also utilizes magnifiers and hearing amplifiers to assist the participants. In addition, we 
also have utilized a standardized assessment to determine a person’s ability to self-consent 
wherein they are asked specific questions to determine their understanding of the consent form. 
These practices will be used for the proposed study.  
Table : Enrollment Plan for 2019-2020 
Samples (100 subjects) Tests 
Amount of Blood/Participant 
Prevaccination (Day 0) PBMC/HLA + Ab + CBC w/diff 
45 ml +10 ml + 10ml = 65 ml 
Day 7 (+/- 2 days) 
PBMC + Ab + CBC w/diff  
45 ml +10 ml + 10 ml= 65 ml 
Day 28 (-4/+14 days) 
PBMC + Ab + CBC w/diff  
45 ml +10 ml + 10 ml= 65 ml 
Month 6/Day 180  
(+/- 14 days) 
PBMC + Ab + CBC w/diff 
45 ml +10 ml + 10 ml= 65 ml 
Total 
 
270 ml 
PBMC = peripheral blood mononuclear cells 
Ab= antibody 
Approximately 100 adults ≥65 years of age will be enrolled. After consent, blood and serum will 
be collected and then subjects will be randomized to either high dose influenza vaccine or 
adjuvanted influenza vaccine (both FDA approved vaccines). Vaccination will be done by an 
unblinded nurse. All further evaluations and analysis will be done by a blinded staff member. 
Medical history, relevant medications, and frailty status will be assessed at the first visit. 
Subjects will return (Table 3) for blood and serum draws on days 7 (+/- 2 days), 28 (-4/+14 
days) and for an optional visit at 6 months/180 days (+/- 14 days) (after influenza season).  
Influenza vaccination 
The ACIP recommends influenza vaccine for all adults ≥65 years of age. This includes the egg-
based vaccines, the cell-based vaccines and the recombinant vaccines. This study will use the 
egg-based high-dose inactivated influenza (IIV-HD), the egg-based adjuvanted influenza 
vaccine (aIIV), and the recombinant influenza vaccine (rIIV). Each of these vaccines are 
recommended and licensed in adults ≥65 years of age and will be used in this study according 
to licensure.   

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Subjects Randomized: Subjects will be randomized 1:1:1 using the REDCap System, hosted 
at Vanderbilt. Since this is a web-based system, Randomization in REDCap allows users to 
create their custom allocation list, which will serve as a lookup table for deciding how to 
randomize subjects. First, users will set up their randomization model and all its parameters, 
after which REDCap will provide some examples of how to set up the allocation table. The 
randomization table must be generated outside of REDCap using other software (e.g., SAS, 
Stata, R) before users will finally upload it. By letting users create their own allocation table 
outside of REDCap, they can structure their allocations and assignments (e.g., block sizes, 
permutations, stratification balancing). The figure below demonstrates the options to set up the 
randomization model. 
 
 
6.0 
Laboratory Methods  
Specimen processing for peripheral blood mononuclear cells (PBMCs): 
Blood will be collected from a vein. Study personnel will collect the blood samples, label without 
personal identifiers, and deliver to the VUMC Cell Processing Core (Directed by Dr. Kalams) for 
preparation of serum and and isolation of peripheral blood mononuclear cells (PBMCs). Serum 
will be frozen until further testing at Vanderbilt University Medical Center for HAI, MN testing, B 
and T cell influenza-specific responses. Processing, cryopreservation, storage, and 
maintenance of sample logs will be done according to standard operating procedures that have 
been in place and maintained for over 15 years All clinical labs will be sent to the local lab. 
Specimen processing for serum: 
Whole blood drawn in a tube without anticoagulant will be left at room temperature for a 
minimum of 30 minutes and maximum of 2 hours. The samples will then be placed at 4ºC. 

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Samples will be processed within 24 hours of collection. Tubes will be placed in a table top 
centrifuge and serum will be clarified by centrifugation at 3000 rpm for 10 minutes at 4ºC. 
Serum will be aliquotted into labeled cryovials with an adjustable pipette. Aliquot size will be 
determined based on the testing to be performed. Serum will then be stored at –80ºC in labeled 
fiberboard boxes until shipping or testing is performed. 
HAI Assay:  
The hemagglutination inhibition assay (HAI) assay is adapted from the CDC laboratory-based 
influenza surveillance manual and we have extensive experience in its use. Briefly, three parts 
of receptor destroying enzyme (RDE) will be added to one part sera and incubated overnight in 
a 37°C water bath. Then the RDE will be inactivated by incubation in a 56°C water bath for 30 
minutes. Following inactivation of RDE, PBS will be added to the sample for a final serum 
concentration of 1:10. Turkey red blood cells (RBC) will be washed twice and diluted in PBS to a 
final concentration of 5% RBC. The cells are kept at 4°C and expire in one week. Twenty-five 
microliters of RDE-treated sera are then serially diluted across the plate (2-fold). Twenty-five 
microliters of prepared flu antigen tittered to 4HAU is added to the diluted sera. The plates are 
then incubated at room temperature for 30 minutes followed by the addition of 50 μl of 5% 
turkey RBC. Then, the plates are mixed and the RBCS are allowed to settle for 1 hour at room 
temperature. The test will be read by observing a lack of agglutination in the button RBC that is 
settled in the bottom of the well. The HAI titer is determined by the reciprocal dilution of the last 
well with lack of agglutination. Every plate will have a virus and a serum control. The virus 
control will consist of PBS + 4HAU of antigen + turkey RBC. The cell control will consist of PBS 
+ RBC. A negative titer is reported as a <10. All samples are tested in duplicate and results are 
expected to be +/- one well. If duplicate results are more than one well different the sample will 
be repeated. The lower of the two results within one well will be recorded as the titer. 
7.0 
Statistical Analysis: 
This is an exploratory study. Antibody titers will be compared pre- and post-vaccination on the 
samples collected. Paired measures of cell mediated immunity will be compared before and 
after vaccination. In addition, post-vaccination measures of cell mediated immunity will be 
compared across age strata and correlated with serologic response to vaccination. Analyses will 
be done accounting for age, frailty, and underlying medical conditions.  
Data Management: All data will be collected and stored under the REDCap database system. 
This system is a secure web application for building and managing online surveys and 
databases. While REDCap can be used to collect virtually any type of data, it is specifically 
geared to support data capture for research studies. The REDCap Consortium is composed of 
1,920 active institutional partners in 103 countries who utilize and support REDCap in various 
ways including the CDC. The REDCap application allows users to build and manage online 
surveys and databases quickly and securely, and is currently in production use or development 
build-status for more than 273,000 projects with over 372,000 users spanning numerous 
research focus areas across the consortium. This system will allow each site to access the 
database and will provide a secure means to send a study dataset without personal identifiers 
upon request to the CDC.  
8.0 
REFERENCES 
1. 
Centers for Disease C, Prevention. Estimates of deaths associated with seasonal 
influenza --- United States, 1976-2007. MMWR Morb Mortal Wkly Rep 2010;59:1057-62. 
2. 
Thompson WW, Shay DK, Weintraub E, et al. Mortality associated with influenza and 
respiratory syncytial virus in the United States. Jama 2003;289:179-86. 
3. 
Eickhoff TC, Sherman IL, Serfling RE. Observations on excess mortality associated with 
epidemic influenza. Jama 1961;176:776-82. 

VERSION: 4.0 
DATE: November 11, 2019 
 
 
 
 
10 
 
4. 
Reber AJ, Chirkova T, Kim JH, et al. Immunosenescence and Challenges of Vaccination 
against Influenza in the Aging Population. Aging and disease 2012;3:68-90. 
5. 
Weiskopf D, Weinberger B, Grubeck-Loebenstein B. The aging of the immune system. 
Transplant international : official journal of the European Society for Organ Transplantation 
2009;22:1041-50. 
6. 
Goronzy JJ, Fulbright JW, Crowson CS, Poland GA, O'Fallon WM, Weyand CM. Value 
of immunological markers in predicting responsiveness to influenza vaccination in elderly 
individuals. Journal of virology 2001;75:12182-7. 
7. 
Gupta S, Bi R, Su K, Yel L, Chiplunkar S, Gollapudi S. Characterization of naive, 
memory and effector CD8+ T cells: effect of age. Exp Gerontol 2004;39:545-50. 
8. 
Pawelec G, Barnett Y, Forsey R, et al. T cells and aging, January 2002 update. Front 
Biosci 2002;7:d1056-183. 
9. 
Frasca D, Diaz A, Romero M, Blomberg BB. The generation of memory B cells is 
maintained, but the antibody response is not, in the elderly after repeated influenza 
immunizations. Vaccine 2016;34:2834-40. 
10. 
Frasca D, Diaz A, Romero M, et al. Intrinsic defects in B cell response to seasonal 
influenza vaccination in elderly humans. Vaccine 2010;28:8077-84. 
11. 
Vallejo AN. CD28 extinction in human T cells: altered functions and the program of T-cell 
senescence. Immunological reviews 2005;205:158-69. 
12. 
Derhovanessian E, Theeten H, Hahnel K, Van Damme P, Cools N, Pawelec G. 
Cytomegalovirus-associated accumulation of late-differentiated CD4 T-cells correlates with poor 
humoral response to influenza vaccination. Vaccine 2013;31:685-90. 
13. 
Saurwein-Teissl M, Lung TL, Marx F, et al. Lack of antibody production following 
immunization in old age: association with CD8(+)CD28(-) T cell clonal expansions and an 
imbalance in the production of Th1 and Th2 cytokines. J Immunol 2002;168:5893-9. 
14. 
Vallejo AN, Weyand CM, Goronzy JJ. T-cell senescence: a culprit of immune 
abnormalities in chronic inflammation and persistent infection. Trends Mol Med 2004;10:119-24. 
15. 
Acuto O, Michel F. CD28-mediated co-stimulation: a quantitative support for TCR 
signalling. Nature reviews Immunology 2003;3:939-51. 
16. 
Soderberg-Naucler C, Fornara O, Rahbar A. Cytomegalovirus driven 
immunosenescence-An immune phenotype with or without clinical impact? Mechanisms of 
ageing and development 2016. 
17. 
Pera A, Vasudev A, Tan C, Kared H, Solana R, Larbi A. CMV induces expansion of 
highly polyfunctional CD4+ T cell subset coexpressing CD57 and CD154. Journal of leukocyte 
biology 2016. 
18. 
Klenerman P, Oxenius A. T cell responses to cytomegalovirus. Nature reviews 
Immunology 2016;16:367-77. 
19. 
Abana CO, Pilkinton MA, Gaudieri S, et al. Cytomegalovirus (CMV) Epitope-Specific 
CD4(+) T Cells Are Inflated in HIV(+) CMV(+) Subjects. J Immunol 2017;199:3187-201. 
20. 
Jackson SE, Sedikides GX, Mason GM, Okecha G, Wills MR. Human Cytomegalovirus 
(HCMV)-Specific CD4(+) T Cells Are Polyfunctional and Can Respond to HCMV-Infected 
Dendritic Cells In Vitro. Journal of virology 2017;91. 
21. 
Pachnio A, Ciaurriz M, Begum J, et al. Cytomegalovirus Infection Leads to Development 
of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular 
Endothelium. PLoS pathogens 2016;12:e1005832. 
22. 
Turner JE, Campbell JP, Edwards KM, et al. Rudimentary signs of immunosenescence 
in Cytomegalovirus-seropositive healthy young adults. Age (Dordr) 2014;36:287-97. 
23. 
Henson SM, Akbar AN. KLRG1--more than a marker for T cell senescence. Age (Dordr) 
2009;31:285-91. 

VERSION: 4.0 
DATE: November 11, 2019 
 
 
 
 
11 
 
24. 
Messaoudi I, Slifka MK. Editorial: Profiling senescent influenza-specific T cells in the 
elderly. Journal of leukocyte biology 2013;93:819-21. 
25. 
Dolfi DV, Mansfield KD, Polley AM, et al. Increased T-bet is associated with senescence 
of influenza virus-specific CD8 T cells in aged humans. Journal of leukocyte biology 
2013;93:825-36. 
26. 
Leng Q, Bentwich Z, Borkow G. CTLA-4 upregulation during aging. Mechanisms of 
ageing and development 2002;123:1419-21. 
27. 
Virgin HW, Wherry EJ, Ahmed R. Redefining chronic viral infection. Cell 2009;138:30-50. 
28. 
Pawelec G, McElhaney JE, Aiello AE, Derhovanessian E. The impact of CMV infection 
on survival in older humans. Current opinion in immunology 2012;24:507-11. 
29. 
Pawelec G, Derhovanessian E. Role of CMV in immune senescence. Virus research 
2011;157:175-9. 
30. 
Ahlfors K. IgG antibodies to cytomegalovirus in a normal urban Swedish population. 
Scand J Infect Dis 1984;16:335-7. 
31. 
Hecker M, Qiu D, Marquardt K, Bein G, Hackstein H. Continuous cytomegalovirus 
seroconversion in a large group of healthy blood donors. Vox Sang 2004;86:41-4. 
32. 
Staras SA, Dollard SC, Radford KW, Flanders WD, Pass RF, Cannon MJ. 
Seroprevalence of cytomegalovirus infection in the United States, 1988-1994. Clinical infectious 
diseases : an official publication of the Infectious Diseases Society of America 2006;43:1143-51. 
33. 
Trzonkowski P, Mysliwska J, Szmit E, et al. Association between cytomegalovirus 
infection, enhanced proinflammatory response and low level of anti-hemagglutinins during the 
anti-influenza vaccination--an impact of immunosenescence. Vaccine 2003;21:3826-36. 
34. 
Frasca D, Diaz A, Romero M, Landin AM, Blomberg BB. Cytomegalovirus (CMV) 
seropositivity decreases B cell responses to the influenza vaccine. Vaccine 2015;33:1433-9. 
35. 
Furman D, Jojic V, Sharma S, et al. Cytomegalovirus infection enhances the immune 
response to influenza. Science translational medicine 2015;7:281ra43. 
36. 
van den Berg SPH, Wong A, Hendriks M, Jacobi RHJ, van Baarle D, van Beek J. 
Negative Effect of Age, but Not of Latent Cytomegalovirus Infection on the Antibody Response 
to a Novel Influenza Vaccine Strain in Healthy Adults. Frontiers in immunology 2018;9:82. 
37. 
Derhovanessian E, Maier AB, Hahnel K, McElhaney JE, Slagboom EP, Pawelec G. 
Latent infection with cytomegalovirus is associated with poor memory CD4 responses to 
influenza A core proteins in the elderly. J Immunol 2014;193:3624-31. 
38. 
van Leeuwen EM, Remmerswaal EB, Vossen MT, et al. Emergence of a CD4+CD28- 
granzyme B+, cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus 
infection. J Immunol 2004;173:1834-41. 
39. 
Waller EC, Day E, Sissons JG, Wills MR. Dynamics of T cell memory in human 
cytomegalovirus infection. Med Microbiol Immunol 2008;197:83-96. 
40. 
Klarenbeek PL, Remmerswaal EB, ten Berge IJ, et al. Deep sequencing of antiviral T-
cell responses to HCMV and EBV in humans reveals a stable repertoire that is maintained for 
many years. PLoS pathogens 2012;8:e1002889. 
41. 
Cicin-Sain L, Sylwester AW, Hagen SI, et al. Cytomegalovirus-specific T cell immunity is 
maintained in immunosenescent rhesus macaques. J Immunol 2011;187:1722-32. 
42. 
Bolinger B, Sims S, Swadling L, et al. Adenoviral Vector Vaccination Induces a 
Conserved Program of CD8(+) T Cell Memory Differentiation in Mouse and Man. Cell reports 
2015;13:1578-88. 
43. 
Dekhtiarenko I, Ratts RB, Blatnik R, et al. Peptide Processing Is Critical for T-Cell 
Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine 
Vectors. PLoS pathogens 2016;12:e1006072. 

VERSION: 4.0 
DATE: November 11, 2019 
 
 
 
 
12 
 
44. 
Munks MW, Cho KS, Pinto AK, Sierro S, Klenerman P, Hill AB. Four distinct patterns of 
memory CD8 T cell responses to chronic murine cytomegalovirus infection. J Immunol 
2006;177:450-8. 
45. 
Sierro S, Rothkopf R, Klenerman P. Evolution of diverse antiviral CD8+ T cell 
populations after murine cytomegalovirus infection. European journal of immunology 
2005;35:1113-23. 
46. 
Burgers WA, Riou C, Mlotshwa M, et al. Association of HIV-specific and total CD8+ T 
memory phenotypes in subtype C HIV-1 infection with viral set point. J Immunol 2009;182:4751-
61. 
47. 
Snyder CM, Cho KS, Bonnett EL, van Dommelen S, Shellam GR, Hill AB. Memory 
inflation during chronic viral infection is maintained by continuous production of short-lived, 
functional T cells. Immunity 2008;29:650-9. 
48. 
Barker WH, Mullooly JP. Impact of epidemic type A influenza in a defined adult 
population. Am J Epidemiol 1980;112:798-811. 
49. 
Fried LP, Tangen CM, Walston J, et al. Frailty in older adults: evidence for a phenotype. 
J Gerontol A Biol Sci Med Sci 2001;56:M146-56. 
50. 
Fried LP, Ferrucci L, Darer J, Williamson JD, Anderson G. Untangling the concepts of 
disability, frailty, and comorbidity: implications for improved targeting and care. J Gerontol A Biol 
Sci Med Sci 2004;59:255-63. 
51. 
Rockwood K, Song X, MacKnight C, et al. A global clinical measure of fitness and frailty 
in elderly people. Cmaj 2005;173:489-95. 
52. 
Barker WH, Mullooly JP. Pneumonia and influenza deaths during epidemics: 
implications for prevention. Archives of internal medicine 1982;142:85-9. 
53. 
Yao X, Hamilton RG, Weng N-p, et al. Frailty is associated with impairment of vaccine-
induced antibody response and increase in post-vaccination influenza infection in community-
dwelling older adults. Vaccine 2011;29:5015-21. 
54. 
Francis T. On the Doctrine of Original Antigenic Sin. Proceedings of the American 
Philosophical Society 1960 104:572-8. 
55. 
Gostic KM, Ambrose M, Worobey M, Lloyd-Smith JO. Potent protection against H5N1 
and H7N9 influenza via childhood hemagglutinin imprinting. Science 2016;354:722-6. 
56. 
www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2009/ucm195483.htm. FDA 
approves a high dose seasonal influenza vaccine specifically intended for people ages 65 and 
older. . 2009. 
57. 
Falsey AR, Treanor JJ, Tornieporth N, Capellan J, Gorse GJ. Randomized, double-blind 
controlled phase 3 trial comparing the immunogenicity of high-dose and standard-dose 
influenza vaccine in adults 65 years of age and older. J Infect Dis 2009;200:172-80. 
58. 
Podda A. The adjuvanted influenza vaccines with novel adjuvants: experience with the 
MF59-adjuvanted vaccine. Vaccine 2001;19:2673-80. 
59. 
Van Buynder PG, Konrad S, Van Buynder JL, et al. The comparative effectiveness of 
adjuvanted and unadjuvanted trivalent inactivated influenza vaccine (TIV) in the elderly. Vaccine 
2013;31:6122-8. 
60. 
Dunkle LM, Izikson R, Patriarca P, et al. Efficacy of Recombinant Influenza Vaccine in 
Adults 50 Years of Age or Older. N Engl J Med 2017;376:2427-36. 
61. 
Buchman AS, Wilson RS, Bienias JL, Bennett DA. Change in frailty and risk of death in 
older persons. Experimental aging research 2009;35:61-82. 
62. 
Tiedemann A, Shimada H, Sherrington C, Murray S, Lord S. The comparative ability of 
eight functional mobility tests for predicting falls in community-dwelling older people. Age Ageing 
2008;37:430-5. 

VERSION: 4.0 
DATE: November 11, 2019 
 
 
 
 
13 
 
63. 
Wikby A, Johansson B, Olsson J, Lofgren S, Nilsson BO, Ferguson F. Expansions of 
peripheral blood CD8 T-lymphocyte subpopulations and an association with cytomegalovirus 
seropositivity in the elderly: the Swedish NONA immune study. Exp Gerontol 2002;37:445-53. 
64. 
Johnstone J, Parsons R, Botelho F, et al. T-Cell Phenotypes Predictive of Frailty and 
Mortality in Elderly Nursing Home Residents. J Am Geriatr Soc 2017;65:153-9. 
65. 
Luz Correa B, Ornaghi AP, Cerutti Muller G, et al. The inverted CD4:CD8 ratio is 
associated with cytomegalovirus, poor cognitive and functional states in older adults. 
Neuroimmunomodulation 2014;21:206-12.