Understanding the immune response to seasonal influenza vaccination in older adults: A systems biology approach

Abstract

Annual vaccination against seasonal influenza is recommended to decrease disease-related mortality and morbidity. However, one population that responds suboptimally to influenza vaccine is adults over the age of 65 years. The natural aging process is associated with a complex deterioration of multiple components of the host immune system. Research into this phenomenon, known as immunosenescence, has shown that aging alters both the innate and adaptive branches of the immune system. The intricate mechanisms involved in immune response to influenza vaccine, and how these responses are altered with age, have led us to adopt a more encompassing systems biology approach to understand exactly why the response to vaccination diminishes with age. Here, the authors review what changes occur with immunosenescence, and some immunogenetic factors that influence response, and outline the systems biology approach to understand the immune response to seasonal influenza vaccination in older adults.

Full text

10.1586/ERV.12.61
985
ISSN 1476-0584
© 2012 Expert Reviews Ltd
www.expert-reviews.com
Review
Influenza vaccinology is rapidly changing. From
the point of view of vaccine recommendations,
we have moved from a ‘one size fits all’, risk-based
approach to a population approach that now calls
for all Americans aged 6 months and older to be
immunized annually. However, as far as which
vaccine formulation to use, we have moved to a
more individualized, directed approach [1]. This
is evident in the recent licensure and availability
of both high-dose trivalent influenza vaccines
(HD-TIVs) and intradermal trivalent influenza
vaccines, respectively, in the USA [201]. In Europe,
an MF59-adjuvanted vaccine is available, and the
pipeline of influenza vaccine development con-
tinues to grow.
Within this field is an area of special concern:
immunosenescence and the resulting decreased
immunogenicity and efficacy of influenza vaccines
in older persons. This concern results from the
reality that the older individual is more suscepti-
ble to morbid infection, may be unable to mount
an effective vaccine-induced protective response,
and is likely to have concomitant comorbidi-
ties that either contribute to the higher rates of
morbidity and mortality if influenza infection
results, and/or further impairs the development of
an effective immune response to vaccine. While
these are issues of considerable inquiry, to date,
the understanding of immunosenescence remains
limited. In turn, this impairs our ability to devise
vaccines or adjuvants that can overcome such
barriers. For this reason, an expanded research
agenda and approaches to understand immuno-
senescence and its relationship to vaccine-induced
immunity is essential.
In this review, the authors summarize data on
the epidemiology of influenza in older persons,
the current understanding of immunosenescence,
the role of immunosenescence in reduced vaccine
immunogenicity and finally, discuss a systems
biology and vaccinomics approach to unraveling
the impact of immunosenescence on decreased
vaccine immunogenicity and the application of
such knowledge to the development of improved
influenza vaccines for older persons [2].
Epidemiology of influenza in older
adults
While older adults suffer the highest rates of hos-
pitalization and mortality, they neither have the
Nathaniel D Lambert
1
,
Inna G Ovsyannikova
1
,
V Shane Pankratz
2
,
Robert M Jacobson
1,3
,
Gregory A Poland*
1
1
Mayo Clinic Vaccine Research
Group, Mayo Clinic, Guggenheim
611C, 200 1st Street SW, Rochester,
MI 55905, USA
2
Department of Health Sciences
Research, Mayo Clinic, Guggenheim
611C, 200 1st Street SW, Rochester,
MI 55905, USA
3
Department of Pediatric and
Adolescent Medicine, Mayo Clinic,
Guggenheim 611C, 200 1st Street SW,
Rochester, MI 55905, USA
*Author for correspondence:
Tel.: +1 507 284 4456
Fax: +1 507 266 4716
poland.gregory@mayo.edu
Annual vaccination against seasonal influenza is recommended to decrease disease-related
mortality and morbidity. However, one population that responds suboptimally to influenza
vaccine is adults over the age of 65 years. The natural aging process is associated with a
complex deterioration of multiple components of the host immune system. Research into this
phenomenon, known as immunosenescence, has shown that aging alters both the innate and
adaptive branches of the immune system. The intricate mechanisms involved in immune response
to influenza vaccine, and how these responses are altered with age, have led us to adopt a more
encompassing systems biology approach to understand exactly why the response to vaccination
diminishes with age. Here, the authors review what changes occur with immunosenescence,
and some immunogenetic factors that influence response, and outline the systems biology
approach to understand the immune response to seasonal influenza vaccination in older adults.
Understanding the immune
response to seasonal influenza
vaccination in older adults:
a systems biology approach
Expert Rev. Vaccines 11(8), 985–994 (2012)
Keywords: bioinformatics • immunogenetics • immunosenescence • inuenza • seasonal inuenza vaccine
• systems biology • vaccinomics • vaccine-induced immunity
Expert Review Vaccines
2012
11
8
985
994
© 2012 Expert Reviews Ltd
10.1586/ERV.12.61
1476-0584
1744-8395
Understanding the immune response to seasonal influenza vaccination in older adults
Lambert, Ovsyannikova, Pankratz, Jacobson & Poland
Expert Rev. Vaccines
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highest rates of infection nor represent major contributors to local
outbreaks. Local outbreaks begin suddenly and unaccountably,
peak over a 2–3-week period and then persist for 2–3 months.
The timing and nature of these outbreaks remain unpredictable,
unexplained and a target for scientific speculation [3–6] . In an
outbreak, the first cases of influenza appear in school-aged chil-
dren and then spread to adults, including older adults, infants
and younger children. Attack rates vary from 10 to 20% in the
general population, reaching attack rates in the general popula-
tion of more than 50% during a pandemic; attack rates can be
extraordinarily high in institutional settings.
Despite having no higher attack rate than in younger adults,
influenza’s effects are more significant in older adults. Barker’s
study focusing on the impact of influenza infection in the
frail elderly showed a decline in functional status measurable
3–4 months after infection on at least one major function (e.g.,
bathing, dressing or mobility) for 25% of older patients residing
in nursing homes as compared with 15.7% of controls – ran-
domly selected residents living in the same facility not contracting
influenza or influenza-like illness during the same outbreak [7].
As mentioned earlier, older adults also have higher rates of
hospitalization and mortality. Thompson et al. used the CDC’s
influenza-infection surveillance data and the National Hospital
Discharge Survey data to estimate the annual influenza-related
hospitalization rates in the USA [8]. The results showed that hos-
pitalization greatly increased with age in those aged 65 years and
older; specifically, the rates increased with each 5-year block of age
from 65 to 69, to 85 years and older. Where pneumonia or influ-
enza was listed as the primary diagnosis, the average hospitalization
rate was 36.8 per 100,000 person-years, but this increased in older
persons from 37.9 for those 50–64 years old, to 71.1 for those
65–69 years old, to 127.8 for those 70–74 years of age, to 302.2
for those 80–84 years old, and 628.6 for those aged 85 years and
older. Furthermore, the length of hospital stay also increased with
age from a median of 3 days for those less than 5 years of age, to 4
days for those 5–49 years of age, to 6 days for those 50–74 years
of age, to 7 days for those 75 years and older.
Death rates from pneumonia and influenza in the USA have
ranged from 5000 to 50,000 a year as a result of cardiovascular
and respiratory pathology and depending upon the circulating
influenza strain. While hospital rates in older adults approximate
the hospital rates in infants and children younger than 2 years of
age, the fatality rate associated with the elderly is much higher.
Thompson et al. found in their study that mortality rates due to
influenza have increased from the years 1976 to 1999, which they
explained in part due to the aging of the US population [9]. While
the mortality rates from underlying pneumonia or influenza for
those younger than 50 years of age ranged from 0.3 per 100,000
person-lives, the rates increased at 50 years of age and above [9].
The rates were 1.3 per 100,000 person-lives for those 50–64 years
of age and 22.1 person-lives for those 65 years and older [9]. The
increased death rate found in this study matched findings of other
investigations [9,10]. Increasing the risk of mortality are the pres-
ence of high-risk medical conditions; Nordin et al. found the
lowest risk of mortality among those with no high-risk medical
conditions who were 65–74 years of age, and the highest risk of
mortality among those with high-risk medical conditions who
were 75 years and older [10] .
Using 2003 data, Molinari et al. estimate that the total financial
burden of seasonal influenza infection in the USA amounts to
$10.4 billion a year and that the older population bear 64% of
the total economic burden [11] . Efforts to target the reduction of
the disease burden in the older population therefore would have
a substantial impact on the expense of seasonal influenza [11] .
Vaccine efficacy & induced immune response in older
adults
Vaccine efficacy against influenza illness in older adults is difficult
to measure and reliable data are scarce. To date, there has been only
one placebo-controlled trial of influenza vaccine efficacy against
laboratory-confirmed illness in older adults [12]. The study esti-
mated protection from influenza illness at approximately 50%. An
alternative and widely accepted approach is the measurement of
influenza-specific antibody titers as a correlate of protection. Titers
are traditionally measured using a hemagluttination inhibition
(HAI) assay, which quantifies the ability of hemagglutinin (HA)-
specific antibodies to block N-acetylneuraminic acid-mediated
viral agglutination of red blood cells [13,14]. Using the set guidelines
of this assay, vaccine protection can be assessed based on patient
seroconversion (fourfold increase in antibody titers postvaccina-
tion) and seroprotection (HAI antibody titers ≥1:40 postvaccina-
tion). Although some discrepancies exist in studies focusing on
antibody response to influenza vaccine in older adults, a quantita-
tive review concluded that HA-neutralizing antibodies are consid-
erably lower in vaccinated older adults than in younger adults [15].
There is also a correlation between health status in older adults and
HAI titers, with healthy older adults having statistically significant
higher levels of HAI titers than those with chronic diseases [16].
A strain-specific robust humoral response to influenza is nec-
essary to prevent primary infection, but eventual viral clearance
is dependent on the presence of CD8
+
T cells directed toward
conserved regions of the virus [17] . Influenza-specific CD8
+
T cells
produce antiviral mediators and directly kill infected cells [18].
Another approach used to measure cellular-mediated efficacy
of influenza vaccines against laboratory-confirmed disease is to
quantify the ratio of IFN-γ:IL-10 and the cytolytic enzyme gran-
zyme B from T cells postvaccination [19] . Specifically, granzyme
B production has been reported as a direct method of assess-
ing vaccine failure and subsequent illness in older adults [20,21] .
Furthermore, several studies have demonstrated a defect in the
production of IFN-γ and granzyme B in CD8
+
T-cell subsets
obtained from vaccinated older adults [22–24].
To overcome the diminished immune response observed
in older adults, an ‘increase the firepower’ approach has been
adopted. HA concentrations for each strain of 60 μg or more, as
compared with 15 μg of HA in the standard trivalent inactivated
vaccine (SD-TIV), result in increased immunogenicity for
influenza A strains and noninferiority for influenza B in older
adults [25,26]. This led to the formulation of an US FDA-licensed
high-dose vaccine for adults 65 years or older [202]. Each HD-TIV
Lambert, Ovsyannikova, Pankratz, Jacobson & Poland

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contains 60 µg of HA antigen for each H1N1, H3N2 and B strain
contained in the SD-TIV. The HD-TIV was more immunogenic
for both influenza A virus strains in older adults than the SD-TIV
in a Phase III trial [27] . However, both antibody and cell-mediated
immune responses in older adults vaccinated with the HD-TIV
never achieve the same levels observed in young adults vaccinated
with a standard-dose vaccination [28]. Although the antibody titers
achieved with the high-dose influenza vaccine in older adults may
be effective against circulating influenza strains, the increasing
emergence of deadlier strains demands the development of
vaccines that focus on more than just increasing antigen dose.
An aging immune system may not be able to mount a sufficiently
protective response to current or novel strains regardless of the
amount of antigen present without the addition of adjuvants or
newer methods of antigen delivery.
Immunosenescence
A key factor driving vaccine failure in older adults is immuno-
senescence. Immunosenescence is a broad term used to describe
complex alterations in the immune response attributed to aging.
As the immune system ages, there is a significant increase in sus-
ceptibility to infection, autoimmunity and cancers, and a decrease
in vaccine-induced immunity [29]. At a cellular level, immuno-
senescence is a combination of diminished immune cell num-
bers and function, coupled with an inappropriate/unregulated
inflammatory response that results in less than ideal immunity.
The following sections summarize published work that addresses
the influence of immunosenescence on the innate and adap-
tive immune systems and how these properties may diminish
vaccination response in older adults.
Immunosenescence & the innate response
The innate branch of the immune system affords the host ability
to respond rapidly and nonspecifically to an invading pathogen by
host pattern recognition receptors (PRRs) [30]. Specifically, influ-
enza virus has been shown to interact with innate signaling media-
tors, Toll-like receptors (TLRs; e.g., TLR7), Nod-like receptors
(e.g., NLRP3, NOD2) and RIG-I-like receptors [31–34] . Along
with initial pathogen clearance, innate immunity is also responsi-
ble for the genesis of the adaptive response by recruiting immune
effector cells [35]. An age-related deficiency in innate immunity
can negatively influence any subsequent adaptive response. As
described below, there is mounting evidence that the phenotypic
responses of many components of innate immunity are influenced
by immunosenescence.
Monocytes, dendritic cells, NK cells and other innate immu-
nity cells express TLRs [36]. The interactions between conserved
molecular patterns present on microbial pathogens and TLRs lead
to a MyD88 or TRIF-dependent induction of proinflammatory
cytokines and the upregulation of type I interferons [37] . There is
increasing evidence that a combination of inappropriate activation
of TLRs and diminished function in response to many ligands is
present in an aged population. Peripheral blood mononuclear cells
from older adults produce decreased levels of IL-6 and TNF-α
and TLR1 surface expression levels are reduced after stimulation
with a TLR1/2 ligand [38]. In the context of viral infection, pro-
inflammatory cytokine production and TLR3 expression levels
are increased on West Nile virus-infected macrophages from older
human donors, which may result in an inappropriate inflamma-
tory response [39]. Plasmacytoid dendritic cells from aged donors
secrete decreased amounts of both IFN-1 and IFN-III after stimu-
lation with both the TLR7 ligand CpG and live influenza virus,
which is due to impairment in IRF-7 phosphorylation [40]. These
plasmacytoid dendritic cells also exhibit diminished induction
and priming of CD4/CD8 T-cell immunity. Panda et al. dem-
onstrated a correlation between defects in cytokine response from
aged human dendritic cells stimulated with TLR ligands and
diminished influenza vaccine-induced antibody production [41] .
Taken together, impaired TLR response in immune cells from
older adults directly affects both cellular and humoral immunity
to influenza.
CD80 and CD86 are costimulatory molecules expressed on
antigen-presenting cells and help activate T cells after interac-
tion with CD28 [42,43] . Costimulatory molecule expression on
TLR-activated monocytes can predict influenza vaccine immune
response in both young and older adults; in one study, TLR-
induced CD80 levels were approximately 68% less in older adults
(p = 0.0002) compared with young adults [44]. A decreased ability
to interact with and activate effector T cells would ultimately
result in both a deficient cellular and humoral response to vaccine.
NK cells are vital to the clearance of viral infection by the
production of IFN-γ and lysis of infected cells [45] . Multiple stud-
ies have highlighted the importance of NK cells during influ-
enza infection in both humans and mice [46–49]. NK cell activity
in human subjects is augmented by influenza vaccination [50].
Interestingly, the overall numbers of NK cells are increased in
healthy older adults [51] . However, the function and number of
NK cells decrease with diminished health status, and NK activity
correlates with health status and HAI titers in vaccinated older
adults [16 ,52]. Any perturbation in NK cell function would be
detrimental to the development of a protective immune response
to infection.
In contrast to the many diminished responses associated with
immunosenescence is the subclinical hyperinflammatory state
known as ‘inflamm-aging’ [53]. Immune cells isolated from older
adults produce higher concentrations of inflammatory cytokines,
such as IL-1β, IL-6 and TNF-α after stimulation. Serum IL-6
levels increase with age in humans and are associated with
disability and geriatric frailty [54–56]. Constant inflammation
could leave a host susceptible to infection by not having the
ability to recognize a true inflammatory response to a pathogen.
This is true in a mouse model of systemic herpes viral infection,
where an elevated state of inflammation increases susceptibility
[57] . Vaccination failure and susceptibility to influenza illness may
be a result of too much inflammation and not enough regulation.
Immunosenescence & the adaptive response
Bone marrow-derived T-cell progenitors undergo development
and selection in the thymus and emerge as mature naive T cells
[58]. One of the more dramatic observations associated with aging
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Expert Rev. Vaccines 11(8), (2012)
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is thymic involution, which results in a measurable decrease in
circulating levels of new naive T cells [59] . Surprisingly, there is
no change in overall circulating T-cell numbers with age [60].
Research postulates that T-cell homeostasis and the production of
new T cells is maintained through clonal expansion of peripheral,
antigen-specific T cells [61] .
An adverse effect of new T cells produced from existing T cells
is a decrease in the diversity of T-cell receptors (TCRs) [62]. A
robust immune response to influenza infection is dependent on
TCR diversity and there is evidence of a decrease in influenza-
specific CD8
+
T-cell repertoire in older adults [22,63]. T-cell popu-
lation diversity is also diminished in older adults after lifelong
exposure to certain antigens and the accumulation of memory
T cells [64].
A decline in T-cell diversity and massive expansion of memory
T-cell clones has also been linked to persistent viral infections.
For example, chronic infection with CMV in the older popula-
tion results in extensive accumulation of exhaustive, high-affinity,
CMV-specific memory T cells [65] . CMV-specific CD8
+
T cells
also produce higher levels of IFN-γ, which could partly explain
age-associated ‘inflamm-aging’ [66]. The abundant numbers of
CMV-specific memory T cells alone can alter homeostasis and
decrease the amount of circulating naive T cells.
The expression of the costimulatory molecule CD28, which is
needed for differentiation of naive T cells after initial antigen expo-
sure, on CD8
+
T cells decreases with age [67]. There is also a direct
link between a decrease in CD28 expression (CD8
+
CD28
-
T cells)
and a poor immune response to influenza vaccine. In one study, a
10% proportional increase in CD8
+
CD28
-
cells correlated with a
24% decrease in humoral response to influenza [68]. The presence
of other late effector T-cell subsets (CD8
+
KLRG1
hi
CD57
hi
) is also
inversely correlated with influenza vaccine immunogenicity [69].
The identification of specific cellular subsets in older adults that
successfully predict immune outcome could be a powerful tool in
developing the next generation of vaccines.
A portion of decreased humoral response in older adults can
also be attributed to a deficiency in extrinsic cellular signaling
between CD4
+
T cells and B cells [70] . Senescent CD4
+
T cells
express lower levels of CD154 (CD40L) and this molecule is cru-
cial for stimulation of B cells. Antibody response in older adults is
also altered by a shift in B-cell homeostasis from naive to effector
cells similar to that observed in T cells [71] . B-cell class switching,
recombination and somatic hypermutation are also defective in
older populations [72] . This defect would result in an inability to
produce high-affinity antibodies against influenza.
In summary, immunosenescence and its contribution to sub-
optimal vaccine response in older adults is a complex and multi-
faceted process. The majority of research has focused on pinpoint-
ing singular components of the immune system responsible for
a diminished response. In reality, many key systems contribute
to immunosenescence. A successful model to predict and define
vaccine outcome in older adults must therefore take into account
not only individual aspects of the aging immune system, but
also other systems, such as epigenomic, genomic, proteomic and
transcriptomic factors.
Immunogenetic factors associated with host responses
to seasonal influenza vaccine
Relationships between genetic polymorphisms (and nongenetic
factors) and immune response to influenza vaccine in the human
population have been reported [73–76]. With regard to human influ-
enza infection, evidence was found for a heritable predisposition
to the development of severe influenza virus infection and death,
strongly suggesting genetic associations with the immune response
to influenza infection [77,78]. It is also thought that the predisposi-
tion to a fatal outcome of influenza illness also depends on envi-
ronmental, nutritional, demographic and virologic factors [79]. The
authors of these reports comment that “… it is important to iden-
tify those genes associated with the ability to respond (to influenza)
with protective immunity after natural or vaccine challenge’’ [77].
One specific gene responsible for the anti-inflammatory response
to severe influenza infection is the inducible heat shock protein
gene, heme oxygenase-1 (HO-1) [80]. Recent studies have demon-
strated the lungs of mice that were infected with highly pathogenic
strains of influenza virus exhibited increased levels of HO-1 gene
expression and a decrease in the expression levels of antioxidants
Gpx3 and Prdx5 [81]. Furthermore, impaired antibody produc-
tion in response to influenza vaccination was observed in aged
HO-1-deficient mice [82]. Importantly, a recent study suggested
that decreased influenza vaccine response in humans is associated
with polymorphisms in the HO-1 gene [82]. Also, in a genome-
wide association study of 147 influenza-vaccinated individuals,
promoter SNP rs743811 and intronic SNP rs2160567 in the
HO-1 and constitutively expressed isoform HO-2 genes, respec-
tively, were found to be associated with decreased H1-specific HAI
titers following influenza vaccine [82]. Thus, the HO-1 and other
gene polymorphisms should be investigated to better understand
possible genetic determinants for influenza disease and vaccine
effectiveness.
Host genetic polymorphisms probably play a significant role in
immunity against influenza vaccine. There is limited immuno-
genetic information available to explain significant interindividual
variations observed in immune response to influenza vaccines.
Population-based association studies revealed the importance of
HLA and other immunity-related gene polymorphisms in influ-
enza vaccine-induced humoral immunity [73,76]. HLA class I and
class II molecules present antigenic epitopes to CD8
+
and CD4
+
T cells, respectively, and initiate adaptive immune responses.
Influenza-derived peptide presentation by HLA class I and class
II molecules induces T-cell populations with diverse specificities
and functions [83,84]. Various HLA class I (A*2, A*11, B*27 and
B*35) and class II (DRB1*07, DRB1*13 and DQB1*06) alleles
have been reported to correlate with the serologic response to
influenza vaccination [73,76,85] . These differences in HLA class I
and class II pathway presentations of immunodominant epitopes
are likely the source for some proportion of the interindividual
variation in influenza vaccine-induced immune responses.
Preliminary data from the candidate gene studies demon-
strate significant correlations between influenza H1-specific HAI
antibody levels and single nucleotide polymorphisms (SNPs)
in cytokine (IFNG, IL6, IL12A, IL12B and IL18), and
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cytokine receptor (IFNAR2, TNFRSF1A, IL1R, IL2RG, IL4R,
IL10RB and IL12RB) genes (range of p values 0.005–0.045) [76].
Associations were also discovered between polymorphisms in genes
regulating vitamin A receptor retinoic acid receptor γ and innate
immunity (TLR4) and variations in influenza H1-specific antibody
levels [Poland GA, Ovsyannikova IG, Jacobson RM, Unpublished Data]. For
example, in the pilot studies, an increased frequency of the minor
allele of the 5’UTR SNP (rs7398676; p = 0.08) in the retinoic acid
receptor γ gene was associated with protective serum H1 antibody
titers (median HAI titer of 1:320) after influenza vaccine. Similarly,
an intronic SNP (rs1927907; p = 0.1) in the TLR4 gene was margin-
ally correlated with higher H1 antibody levels (median HAI titer of
1:320); however, a larger sample size is needed in order to improve
statistical power and confidence. These preliminary data provide
evidence that the immune-related gene polymorphism is associated
with influenza H1-specific antibody titers after vaccination.
In addition to the findings associated with TLR4, it has been
demonstrated that gene polymorphisms in TLR4 (that recognizes
lipopolysaccharide) may influence innate immune responses to
respiratory syncytia virus and influence the predisposition to severe
respiratory syncytia virus disease [86,87]. Another study of the tran-
scriptional targets of immune responses to influenza virus in human
peripheral blood mononuclear cells following influenza vaccination
demonstrated a high expression of interferon-induced and -regu-
lated genes, including IFN-γ-induced protein precursor 10 (IP-10)
gene, suggesting their function in immune response to influenza
antigens [78]. In addition, the RIG-I gene is involved in the influ-
enza virus-specific production of IFN-β. Also, influenza virus non-
structural protein-1 has been demonstrated to interact with RIG-I
and inhibit the RIG-I pathway, thereby inhibiting the generation
of IFN-β [88]. By understanding genetic influences on the genera-
tion of immunity due to vaccination, it is feasible to develop new
vaccines against influenza [1,89–92]. By applying knowledge on the
interactions of various pathways of key gene families critical to
developing protective immune responses, it is feasible to gain an
understanding of the host response to influenza vaccine antigens.
Systems biology approach
Each of the aforementioned components is an important individual
contributor to the ability of an older adult, or indeed any individ-
ual, to mount an effective immune response following vaccination
against influenza; evidence supporting their roles has been well
established. While this understanding has come through extensive
studies, these investigations have primarily focused on relatively
small components of the immune system. In order to fully under-
stand the way in which vaccines induce protection against foreign
antigens, it is important to take a broader view of the components
of the system that together give rise to immunity. In the study of
biological processes, approaches are being developed that address
this more expansive view and use more comprehensive modeling
techniques that are integrated with existing biologic knowledge
bases [93–96]. These approaches comprehensively integrate data gath-
ered from a variety of often high-throughput, high-dimensional
assays with human-collated models of biological function. These
approaches, which have come to be known as systems biology,
have not coalesced into a single defined entity, but rather encom-
pass a broad class of methods that all seek to arrive at a deeper
and global understanding of biological processes and the complex
inter-relationships of systems that compose an organism [97–100].
The general idea guiding the study of systems biology is that to
understand the full process by which specified biologic systems
function, one needs both empirical data and structured models of
existing knowledge. In the current era, the availability of technol-
ogy to extract data measuring a wide variety of both inputs and
outputs of molecular systems is greater than ever. Thus, it is rela-
tively simple to obtain simultaneous detailed information about
genomic, transcriptomic, proteomic and other measures. There
is also an ever-increasing knowledge base consisting of models of
genetic and protein networks, as well as other models of immune
function. The key to effective systems biology research is to effec-
tively apply robust multivariate statistical analyses of these data
in the context of the existing biological knowledge base. These
analyses make it possible to either modify known models or to
confirm and refine already-described models. Such approaches
carry the promise of making it possible to more deeply understand
the initiation and maintenance of immune responses [2,96,100].
The current research group has initiated a series of studies within
the context of a systems biology approach in order to more deeply
understand the mechanistic underpinnings and complexities of
diminished vaccine response in older adults. The authors organize
information from a wide variety of sources, including genetic poly-
morphisms, gene transcripts, epigenetics, genetic pathways and
protein–protein interactions. Using these data sources as inputs,
the authors employ a variety of state-of-the-art statistical models
and approaches to determine the extent to which the interplay of
these data clarify existing models or bring new understandings that
may lead to the development of novel biological models.
Specifically, a systems biology generated immune profile will be
constructed around time points associated with distinct temporal
stages of influenza vaccine response. The baseline data, or day 0,
correspond with prevaccination immunity. Days 3, 28 and 75
will be associated with innate, adaptive and the immune sys-
tem’s return to homeostastis, respectively. The authors will then
have a distinct set of data points for each individual over a broad
duration of immune response to seasonal influenza vaccination.
The innovation of this study lies in pairing traditional influenza
vaccination outcomes, such as cellular and humoral measures,
with flow cytometric markers for adaptive and innate immunity,
proteomics and cutting-edge technologies such as next-generation
mRNA sequencing (Figure 1). The authors also incorporate assays
to quantify and compare the contribution of immunosenescence
markers to vaccine response by measuring TCR diversity, CD28
expression and TCR excision circles analysis.
Once all data have been generated and analyzed in the con-
text of the current biological knowledge base, the authors will
utilize the findings to examine the entire spectrum of biological
responses and compare and contrast them across a range of ages
and immunization strategies. This will enable the authors to com-
prehensively understand interactions among the components of
the aging immune system and their impact on the development
Understanding the immune response to seasonal influenza vaccination in older adults

Expert Rev. Vaccines 11(8), (2012)
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and maintenance of immunity to influenza vaccine. Ultimately,
this will lead to a method of more directed development of vac-
cines against influenza, perhaps by ‘reverse engineering’ around
identified genetic or cellular elements.
Similar approaches have already been applied and these have led
to novel information relative to processes by which immunogenic-
ity might be induced. The earliest example of this is the case of yel-
low fever vaccine, where a large collection of data gathered across
multiple time points were analyzed with multivariate statistical
techniques to identify a collection of gene signatures that predicted
the immunogenicity of the YF-17D vaccine [96]. Importantly, these
gene signatures were validated in an independent sample set; an
important step in the research process when complex statistical
approaches are applied with the goal of integrating information
across a number of high-throughput technologies and existing
knowledge bases. The advantage offered by this approach to study-
ing immune responses is in its focus on simultaneously studying
a large number of input and output data; something that more
closely approximates the reality of the complex interactions that
take place within living organisms mounting an immune response
against an antigen. Classical approaches that are typically used to
study correlates of immunity tend to focus on simple associations
between a single input and a single output, perhaps while adjust-
ing for a small number of potentially contributing factors, and
are therefore not able to provide insight into the full cellular and
immunologic milieu. Because of this, it is essential that research
be extended into the realm of systems biology, where information
across a wide range of data sources can be integrated to provide
insight into the immunologic processes.
Moving forward
This review has briefly outlined the epidemiology of influenza
in older persons, acknowledging the high rates of morbidity and
mortality that older adults experience as a result of influenza
infection. In addition, the huge economic costs associated with
influenza resulting in increased medical care, lost work and lost
time in school, in tandem with annual epidemics of influenza
(and periodic pandemics), combine to make prevention of influ-
enza a major public health concern. An additional and pertinent
temporal trend must also be recognized, and that is the rapid
increase in the aging of populations throughout the world. For
example, in the USA, the fastest growing segment of the popu-
lation is individuals over the age of 85 years. The implications
are considerable. Older persons are increasing in number, have
increased rates of illness, hospitalization, medical care use and
death from increasing virulent strains of influenza, in the context
of yearly epidemics, and respond poorly to current influenza vac-
cines. It therefore becomes imperative that the research agenda be
expanded to both understand the mechanisms that result in poor
immunity in older persons, and use such information to devise
more immunogenic influenza vaccine candidates.
Critical to our work, and to progress in the field, is to ‘unravel’
the complexity of the immune response in older persons, and to
understand how it differs from younger persons. The task is daunt-
ing, although made easier by the plethora of high-throughput,
high-dimensional technologies rapidly becoming available at an
affordable price. A more serious obstacle, however, are the bioinfor-
matics personnel and processes needed to analyze and make sense
of such data. Consider that the combination of transcriptomic,
other immunophenotyping and sequencing data can result in a
terabyte of data in just one experiment involving a single subject.
Analyzing such data in the context of models built on the current
understanding of the immune response network theory and a vac-
cinomics approach requires a significant investment in devising
and testing bioinformatics models [1,89–92,101]. In many cases, the
current models are simply insufficient and reflect the difficulty
in reducing extremely complex systems to more simple models.
As the authors have reviewed, immunosenescence has far-
reaching implications in terms of generating immune responses
on innate, adaptive, T-cell and B-cell function. Further research
is needed on the critical changes and impairments that together
result in immunosenescence, and possibilities for reversing adverse
changes associated with the aging immune system. Important
findings have been published, and progress made – but there is a
long way to go to meet the challenge of protecting an aging popu-
lation against infectious diseases for which they are particularly
susceptible.
Seasonal inactivated influenza A/H1N1 vaccine
Humoral response Innate response
Genomics
Proteomics
Epigenomics
Cellular response
Transcriptomics
Immune profile over time
(day 0, 3, 28, 75)
Expert Rev. Vaccines © Future Science Group (2012)
Figure 1. Systems biology approach to developing an
influenza A/H1N1 vaccine-induced immune profile.
Multifunction immune and systems analysis over the duration of
vaccine response will be used to determine individual immune
outcomes, functional pathways and longitudinal immune profiles
that will lead to the explanation and prediction of immune
response to influenza A/H1N1 vaccine. This will be accomplished
using a fusion of traditional measures of humoral, cellular and
innate immunity, paired with measures of gene regulation and
large-scale analysis of protein response. Immune response to
seasonal influenza vaccination will be measured after in vitro
stimulation of subject peripheral blood mononuclear cells with
live influenza A/California/H1N1 virus. Assays specific to markers
of immunosenescence will also be used to measure the influence
of age on immune response to vaccine.
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Expert commentary
With the approval of an HD-TIV for older adults, novel vaccines
are being developed to address the issue of immunosenescence.
However, as stated previously, both the humoral and cellular
responses in HD-TIV-vaccinated older adults do not reach the same
level as those in SD-TIV-vaccinated younger adults [28]. With the
emergence of highly pathogenic influenza strains and a decrease in
vaccine response in older adults, we have to consider a different and
more directed approach to vaccine research and development. To
truly understand why vaccine efficacy decreases with age, we will
have to decrease our dependence on reductionist-based science [102].
Although the immune system can be thought of as the summation
of multiple smaller parts (innate and adaptive), aging causes too
many complex alterations to these systems to attempt to understand
the whole of immunosenscence by focusing on a single component.
Our own work, and that of others, is directed at just such issues.
Importantly, the NIH has developed and funded a research pro-
gram that seeks to uncover drivers of immune response to viral and
other vaccines. The Human Immunology Project Consortium is
currently funding seven centers throughout the USA to perform
exactly the systems-level research work described above [203]. In
addition, through this program funds are available to finance
preliminary human-based studies that are consistent with the pri-
orities of the consortium, and that are performed in collaboration
with one of the funded primary centers.
Five-year view
Recent innovative work demonstrates that a systems biology
approach can be successful in elucidating predicative markers of
immune response [96]. We believe that our approach, and others
like it, will be adopted to not only gain a thorough understanding
of host interactions with vaccines, but will also be applied to the
interactions between host and specific pathogens.
Our model is unique in that we focus on the influence of immuno-
senescence on seasonal influenza vaccination, but immunosenescence
is a contributing factor in host response to other viral vaccines as
well. Interestingly, the elderly exhibit a delayed antibody response
to yellow fever vaccination (YF-17D) and an increase in adverse
events [103]. In addition, both cell-mediated immunity and antibody
response against herpes zoster vaccine declines with age [104]. If we
are successful in developing a holistic predictive immune profile to
seasonal influenza vaccination, this model can be applied to research
focusing on other vaccine systems and the contribution of immu-
nosenescence. Such work will be accelerated by increasing complex
bioinformatc models that will allow us to understand the simulta-
neous contributions of genetic, proteomic, epigenetic and cellular
systems; and ever-expanding, high-dimensional, high-throughput,
whole systems-level assays becoming available.
Financial & competing interests disclosure
GA Poland is the chair of a Safety Evaluation Committee for investigational
vaccine trials being conducted by Merck Research Laboratories. GA Poland
offers consultative advice on new vaccine development to Merck & Co., Inc.,
Avianax, Theraclone Sciences (formally Spaltudaq Corporation), MedImmune
LLC, Liquidia Technologies, Inc., Emergent BioSolutions, Novavax, Dynavax,
EMD Serono, Inc., Novartis Vaccines and Therapeutics and PAXVAX, Inc.
He is also the co-inventor of intellectual property licensed to TapImmune Inc.
IG Ovsyannikova is a co-inventor of intellectual property licensed to TapImmune
Inc. R Jacobson is a member of a safety review committee for a postlicensure
study funded by Merck & Co. concerning the safety of a human papillomavirus
vaccine. He is also a member of a data monitoring committee for an inves-
tigational vaccine trial funded by Merck & Co. He also serves as a principal
investigator for two studies, including one funded by Novartis International
for its licensed meningococcal conjugate vaccine and one funded by Pfizer,
Inc. for its licensed pneumococcal conjugate vaccine. The authors acknowledge
support from NIH grant U01AI089859 for this work. The authors have no
other relevant affiliations or financial involvement with any organization or
entity with a financial interest in or financial conflict with the subject matter
or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Key issues
• Older adults have a significantly higher rate of influenza-related morbidity and mortality.
• Vaccine efficacy is decreased in older adults, and compromises efforts to protect the elderly.
• Immunosenescence is associated with complex and multifaceted changes in both the innate and adaptive response to influenza.
• Our previous work has demonstrated that immunogenetic factors contribute to immune response variations to seasonal influenza
vaccination.
• A systems biology approach incorporates assays aimed at measuring complex interactions between the aging host and immune
responses to seasonal influenza vaccine, and complex statistical models that aid in understanding these interactions.
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Lambert, Ovsyannikova, Pankratz, Jacobson & Poland