1. Microreviewcmi_1609 934..942
Optimizing vaccine development
Daniel F. Hoft,1
* Vladimir Brusic2
and
Isaac G. Sakala1
1
Division of Infectious Diseases, Allergy & Immunology,
Saint Louis University School of Medicine, St. Louis,
MO, USA.
2
Cancer Vaccine Center, Dana-Farber Cancer Institute,
Boston, MA, USA.
Summary
Optimizing the development of modern molecular
vaccines requires a complex series of interdiscipli-
nary efforts involving basic scientists, immunolo-
gists, molecular biologists, clinical vaccinologists,
bioinformaticians and epidemiologists. This re-
view summarizes some of the major issues that
must be carefully considered. The intent of the
authors is to briefly describe key components of
the development process to give the reader an
overview of the challenges faced from vaccine
concept to vaccine delivery. Every vaccine re-
quires unique features based on the biology of the
pathogen, the nature of the disease and the target
population for vaccination. This review presents
general concepts relevant for the design and
development of ideal vaccines protective against
diverse pathogens.
Topic 1: Antigen discovery
There are many important considerations in the develop-
ment of an effective vaccine (see Table 1), but the first
issue in vaccine development is antigen choice. A vaccine
must induce memory immune responses capable of rec-
ognizing the intended vaccine target. These immune
responses should be directed against highly conserved
structures expressed by the pathogen. Use of highly con-
served antigens in vaccines can minimize the chances of
the pathogen achieving the immunological escape that
can occur when hypervariable regions are used as
vaccine antigens. These hypervariable regions rapidly
accumulate mutational changes and provide wider diver-
sity of immunological epitopes at the pathogen population
level. Targeting required virulence factors can further
focus vaccine responses on functional protection. Immu-
nological destruction or neutralization of a key virulence
factor in theory could render a pathogen innocuous even
without preventing pathogen infection/replication.
In addition to choosing antigens that are highly con-
served and crucial for pathogen virulence, antigen choice
requires considerations of the biology of the specific patho-
gen to better predict which antigens could be ideal targets.
For example, extracellular and intracellular pathogens are
best targeted by antibody responses and T-cell responses
respectively. For immunological protection against an
extracellular pathogen, linear as well as conformational
peptide, polysaccharide, glycopeptide and glycolipid
epitopes expressed on the surface of the pathogen can
provide targets for neutralization by high-affinity antibody
responses. These high-affinity antibody responses can
prevent pathogen infection and/or opsonize the pathogen
for uptake and killing by professional phagocytes. The
induction of optimal antibody responses normally requires
help from CD4+ helper T cells. Therefore, vaccines
designed to induce antibody responses protective against
an extracellular pathogen should include both antibody
epitopes and helper T-cell epitopes.
Conversely, for an intracellular pathogen, the conven-
tional ab T cells reactive with short peptide epitopes pre-
sented by major histocompatibility complex (MHC) class I
and II proteins on the surface of an infected cell are critical
for protective immunity. T cells unlike antibodies can rec-
ognize infected cells expressing short pathogen-derived
peptides presented by MHC surface molecules. The acti-
vated T cells can inhibit intracellular pathogen growth by: (i)
production of cytokines capable of activating intracellular
microbicidal activities, (ii) direct induction of infected cell
apoptosis, or (iii) the release of cytolytic granules contain-
ing perforin, granzymes and other components that can
lead to cytolysis of the infected cells. The diversity of
allotypic MHC alleles expressed by human populations,
and the consequent variations in the relevant epitope
specificity of protective T cells in different individuals, result
in additional complexities regarding vaccine antigen
choice for the development of T-cell vaccines designed to
protect against intracellular pathogens. Combinations of
Received 24 February, 2011; revised 4 April, 2011; accepted 7 April,
2011. *For correspondence. E-mail: hoftdf@slu.edu; Tel. (+1)
314 977 5500; Fax (+1) 314 771 3816.
Cellular Microbiology (2011) 13(7), 934–942 doi:10.1111/j.1462-5822.2011.01609.x
First published online 2 June 2011
© 2011 Blackwell Publishing Ltd
cellular microbiology

2. epitope targets that at least the majority of vaccine recipi-
ents would be able to mount effective responses against
must be selected. Recent advances in bioinformatics have
led to the ability to predict T-cell epitopes with the capacity
for widely promiscuous binding to multiple different
common HLA types (‘superepitopes’ or ‘epibars’), which
theoretically should be immunogenic in more than 90% of
diverse populations (Meister et al., 1995; Sette and Sidney,
1998; Southwood et al., 1998; Wang et al., 2008; Gregory
et al., 2009; Moise et al., 2011).
Finally, antigen choice should take into consideration the
principle of immunodominance.Avaccine needs to be able
to induce potent immune responses that can easily recog-
nize and act early during pathogen invasion. Naturally
immunodominant T- and B-cell epitopes can provide effec-
tive targets for protective immunity. In contrast, strongly
immunodominant epitopes can prevent the development
of more broadly protective immune responses, particularly
important for immunity against a highly mutable pathogen.
Furthermore, recent reports have suggested that some
pathogens may have evolved to utilize immunodominance
for their own advantage, in some cases to enhance the
long-term survival of certain chronic pathogens in their
hosts (Martin et al., 2006; Tzelepis et al., 2008). Thus,
depending on the specific pathogen and how the pathogen
has evolved with the host’s immune system, the inclusion
of immunodominant epitopes could be beneficial or detri-
mental for optimal vaccine efficacy.
All of these critical issues for vaccine antigen selection
discussed above must be carefully considered in the
context of the specific pathogen being targeted.
Topic 2: T helper subset differentiation and
protective immunity
The next issue for vaccine development is the need to
determine the type of T helper (Th) subset needed to
induce the relevant protective immune responses. CD4+
Th cells can differentiate into different effector subsets
that produce distinct cytokine profiles characterized as
Th1, Th2, Th3/regulatory T cells (Treg), Th17 and T folli-
cular helper (Tfh) cells (Zhou et al., 2009; O’Shea and
Paul, 2010). Th1 cells produce IFN-g, TNF-a and IL-2
important for activating intracellular microbicidal activities
and for the generation of CD8+ cytolytic T lymphocytes
(CTL), all of which are required for control of intracellular
pathogens. Th2 cells produce IL-4, IL-5 and IL-13 which
enhance antibody responses and antibody-dependent
cellular responses required for control of extracellular
pathogens. Th3/Treg cells produce TGF-b, IL-10 and/or
IL-35, and are associated with potent secretory IgA
responses protective against mucosally invasive patho-
gens and minimization of immunopathology during
chronic infections. Th17 cells produce IL-17, IL-21 and
other cytokines/chemokines that enhance inflammatory
recruitment of neutrophils and additional T cells, macroph-
ages and dendritic cells. Th17 cells may be important for
both direct control of extracellular pathogens along epi-
thelial surfaces and indirect enhancement of protection
against intracellular pathogens by recruitment of Th1 cells
(Khader et al., 2007). Tfh produce IL-21 in germinal
centres important for B-cell activation, differentiation and
affinity maturation.
Depending on the nature of the target pathogen, a
vaccine may need to induce Th1 responses to protect
against an intracellular pathogen, Th2 responses to
protect against an extracellular pathogen, or Th17 and
Th3 responses for protection against epithelial infection/
invasion. Specific differentiation factors have been identi-
fied as critical for the generation of each Th subset. IL-12,
IL-4, TGF-b, TGF-b plus IL-6, and IL-21 induce Th1, Th2,
Th3, Th17 and Tfh respectively (Spolski and Leonard,
2010; Zhu et al., 2010). Each of these driving cytokines
trigger distinct transcriptional activation programmes
Table 1. Key considerations for development of a vaccine.
Consideration Comments
1. Antigen discovery Conserved virulence factors, B/T epitopes depending on pathogen biology,
immunodominance
2. Relevant CD4+ Th subset target Th1 versus Th2 versus Th17 depending on pathogen biology
3. Need for CD8+ T cells For intracellular pathogens, especially if replicates in cytoplasm and/or infects
non-haematopoietic cells
4. Specific memory subset needed Central memory (Tcm) versus peripheral effector memory (Tpem)
5. Avoidance of excessive Treg Treg inhibit effector T-cell development but may be necessary for the generation of
long-term memory
6. Adjuvant selection Alum salts enhance Ab responses; Toll-like receptor agonists, oil/water emulsions enhance
T cells
7. Vectors/delivery format B- versus T-cell responses, ideally mimic natural pathogen invasion strategy
8. Schedules and routes of vaccination Mucosal versus systemic vaccinations depending on pathogen biology, heterologous
boosters
9. Immunological networks/biomarkers Identify molecular signatures associated with optimal immune response, mucosal versus
cutaneous trafficking
10. Phase I through III clinical trials Optimization of dosing, safety first, then immunogenicity and then protective efficacy
Optimizing vaccine development 935
© 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942

3. associated with differential master switches (e.g. Tbet,
Gata3 and RORgt for Th1, Th2 and Th17 respectively).
The Th1 and Th2 immune profiles may represent the most
stable end-point phenotypes among these subsets, and
have been shown to be programmable in long-term
memory cells (Swain, 1994). In contrast, Th17, Th3/Treg
and Tfh subsets may represent more transient, less dif-
ferentiated states with more plasticity/reversion capacity
(Zhou et al., 2009; O’Shea and Paul, 2010). The above
mentioned differential driving cytokines, and other factors
affecting the distinct transcriptional activation associated
with these subsets, can be used to selectively induce the
most relevant immune responses for the target pathogen
(Hoft and Eickhoff, 2002).
Additional subsets of T cells may be important targets
for new vaccines, broadening the recognition potential of
the immune response and/or facilitating the rapid recall of
immunity. Certain T cells can recognize ceramides and
other lipids presented by non-polymorphic CD1 mol-
ecules, and can rapidly produce cytokine responses upon
restimulation (Hiromatsu et al., 2002; Vincent et al.,
2003). Similarly, the g9d2 TCR+ human T-cell subset can
recognize phospholipids in an MHC-unrestricted fashion,
can rapidly produce cytokines capable of enhancing Th1
immunity and can directly inhibit intracellular pathogen
replication (Hoft et al., 1998; Shen et al., 2002; Morita
et al., 2007; Spencer et al., 2008). Inclusion of key cera-
mide and/or lipid antigens for specific activation of NKT
and/or gd T cells may be important for the future develop-
ment of some types of optimal vaccines.
Topic 3: CD8+ T cells
Conventional CD4+ T cells are stimulated by peptide
epitopes presented by MHC class II molecules which
generally sample extracellular antigens taken up by
pinocytosis/phagocytosis, or antigens synthesized by
pathogens that persist and/or replicate within the endoso-
mal compartments of infected cells. CD8+ T cells are
stimulated by foreign peptides presented by MHC class I
molecules which sample cytoplasmic contents of infected
cells. These CD8+ T cells contain cytolytic granules with
perforin and granzymes responsible for membrane
damage and apoptosis induction in infected target cells.
Another molecular component of human cytolytic granules
is granulysin which can mediate direct microbicidal activity
against bacterial pathogens (Stenger et al., 1998). In addi-
tion to this classical effector degranulation response with
subsequent lysis and death of the recognized target cell,
CD8+ T cells can trigger the inflammasome and/or lead to
non-lytic effects that enhance intracellular suppression of
certain pathogens like HSV (Knickelbein et al., 2008;
Metkar et al., 2008). Because CD8+ T cells are generally
stimulated by cytoplasmic peptides, vaccines that result in
synthesis of antigens within the antigen presenting cell
(e.g. live attenuated vaccine vectors and DNA expression
vectors) are needed to induce optimal vaccine-specific
responses. However, cross-presentation of soluble extra-
cellular antigens by dendritic cells and B cells to CD8+ T
cells can occur with the right adjuvant conditions (e.g. TLR
signals that induce a ‘danger’ response including produc-
tion of IL-12) (Bevan, 2006; Hoft et al., 2007).
Topic 4: Memory T-cell subsets
Different subsets of memory immune cells have been
identified with complementary roles (Sallusto et al., 1999;
2004; Willinger et al., 2005). CCR7-expressing central
memory T cells (Tcm) with high proliferative potential
re-express lymph node homing receptors which allow for
accumulation in lymph nodes. CCR7 negative, peripheral
effector memory T cells (Tpem) are distributed throughout
peripheral tissues, have little proliferative potential, but
are able to rapidly provide protective effector functions at
the initial peripheral sites of pathogen rechallenge. Tcm
serve as efficient immune memory storage facilities
waiting for dendritic cells from the periphery to bring early
antigens upon remote pathogen rechallenge to the drain-
ing lymph nodes where Tcm undergo restimulation, pro-
liferative expansion and effector differentiation to dampen
later waves of pathogen replication and spread. Tpem
provide a first line of adaptive immune defence and gen-
erally require persistence of antigen for their prolonged
presence. Although still controversial, in general for long-
term immune protection, the Tcm memory subset is prob-
ably the best response to focus on inducing to provide
optimal vaccine efficacy. However, experimental vaccines
designed to maintain long-term induction of Tpem with
chronically persisting vaccine vectors, with the goal of
inducing optimal protection against initial epithelial inva-
sion immediately upon pathogen exposure are being
investigated (Hansen et al., 2009).
In addition to proliferative expansion and effector T-cell
function, the polyfunctionality of vaccine-induced memory
immune responses is another important feature for con-
sideration in vaccine development. For example, several
studies have found that antigen-specific IFN-g production
is not the only effector function important for optimal pro-
tective immunity. Seder et al. demonstrated that the
capacity of CD4+ T cells to produce all three Th1 cytok-
ines, IFN-g, TNF-a and IL-2, was a much better predictor
of the relative protective capacity provided by various
leishmania vaccines in murine models than IFN-g produc-
tion alone (Darrah et al., 2007). Similarly, CD8+ T cells
capable of further proliferative expansion and production
of both IFN-g and perforin were associated with delayed
HIV disease progression while CD8+ T cells capable of
producing only IFN-g were not (Migueles et al., 2002).
936 D. F. Hoft, V. Brusic and I. G. Sakala
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4. Consistent with these other results, we have shown that
IFN-g production alone does not correlate with the ability
of human T cells to inhibit intracellular mycobacteria (Hoft
et al., 2002). Therefore, polyfunctional assessments of
the memory T cells induced by vaccination are important
for clinical development of the most effective vaccines.
Topic 5: Treg
Regulatory T cells are important for negative regulation of
immunity, and the prevention of autoimmune diseases
(Bluestone and Abbas, 2003). Natural and induced Treg
develop in the thymus versus periphery, respectively, and
can limit over-exuberant immune-mediated inflammation.
Natural Treg express Foxp3, a master transcription factor
that induces a detailed genome-wide molecule pro-
gramme that maintains the Treg phenotype, and suppress
effector T-cell responses through contact-dependent and
-independent mechanisms. Induced Treg may or not
express Foxp3, but still can inhibit effector T-cell
responses through the production of IL-10 and/or TGF-b.
Treg in general are critical for maintaining homeostasis of
the immune system and minimizing the pathological
effects of immune activation. However, many infectious
pathogens induce Treg which can interfere with the devel-
opment of optimal protective immunity. In fact, Belkaid
et al. demonstrated that Treg can be responsible for the
persistence of a chronic infection (Belkaid et al., 2002),
indicating that some pathogens have learned to deliber-
ately utilize the Treg response for their own advantage.
Therefore, the efficacies of some vaccines and/or immu-
notherapies directed against certain pathogens might be
enhanced by limiting or actively inhibiting Treg responses.
Furthermore, there is some evidence that certain T-cell
epitopes can at least preferentially induce Treg rather
than effector T-cell responses (Massa et al., 2007). Avoid-
ing Treg biasing epitopes obviously could be important for
the development of some types of vaccines.
In addition to the natural and induced Treg discussed
above, natural ligands produced in vivo during comple-
ment activation can trigger signalling via CD46 on human
T cells leading to negative feedback induction of IL-10
production in Th1 cells after peak immune responses are
finished and no longer needed (Kemper et al., 2003;
Cardone et al., 2010). Several pathogens have evolved to
utilize CD46 as a specific target receptor for infection of
host cells, and engagement between these pathogens
and CD46 can trigger negative feedback inhibition of cell-
mediated immune responses (Kemper and Atkinson,
2007). These negative regulatory effects triggered during
initial infection can prevent the induction of optimal
vaccine-induced immunity. We have shown that BCG
immunity in humans is reduced by the effects of CD46
cross-linking on T cells, and natural ligands produced
during BCG infection/replication can specifically enhance
CD46 signalling (Truscott et al., 2010).
Learning how to prevent these downregulatory signal-
ling events may be important for learning how to induce
more protective vaccine-induced immune memory. On the
other hand, IL-10 produced by Treg has been implicated
as a requirement for the induction of long-term memory,
perhaps by inhibiting apoptosis in early effector T cells
(Belkaid et al., 2002; Foulds et al., 2006). Therefore,
further research efforts exploring the effects of Treg/
CD46-mediated regulation are necessary to optimize
future vaccine strategies.
Topic 6: Adjuvants
Specific adjuvants can enhance immune memory and
shape the phenotype of the recall response. Until very
recently, the only adjuvants approved for human use were
aluminium salts, which can increase the half-life of an
antigen, improve uptake by professional phagocytes and
trigger the inflammasome via NALP3 sensing (Eisenbarth
et al., 2008). Alum adjuvants clearly increase the titres of
specific antibodies generated by vaccination, but are not
optimal adjuvants for the induction of CD4+ Th1 cell and
CTL responses important for the control of intracellular
pathogens. Newer adjuvants including oil/water mixtures
with toll-like receptor triggering ligands (e.g. lipid A for
TLR4, unmethylated CpG dinucleotide motifs for TLR9,
etc.) are becoming available which can greatly enhance
Th1 and CTL responses (Coffman et al., 2010).
Classic immunological work has confirmed that Th1 and
Th2 priming can induce stable differentiated phenotypes
that maintain their original bias after remote recall stimu-
lations (Swain, 1994). Systemic IL-12 administration (or
administration of a TLR ligand such as a CpG oligonucle-
otide that stimulates IL-12 production by dendritic cells
and macrophages) during vaccination in animals can
clearly bias for Th1/CTL response generation. Systemic
IL-4 given during vaccination can skew for long-term Th2
memory immune cells. It is not clear yet whether Th3,
Treg, Th17, NKT and/or gd T cells can develop similar
stable long-term differentiated effector memory cells.
However, driving cytokines have been identified for at
least short-term induction of these latter immune subsets
(e.g. TGF-b/IL-10 for Th3/iTreg generation; TGF-b plus
IL-6, IL-21 and IL-23 for Th17 generation) (Zhu et al.,
2010) and future research will focus on the importance of
these additional subsets and their potential for attaining
long-term stable immune memory phenotypes. Additional
research will need to explore the relative values of using
different membrane and cytoplasmic foreign sensors
(TLR, RLR and NLR) individually and in combinations to
induce the best vaccine-specific responses appropriate
for each unique pathogen.
Optimizing vaccine development 937
© 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942

5. Topic 7: Vectors/delivery format
Next for consideration are a diverse group of delivery
systems all designed to result in antigens being synthe-
sized within or translocated into the cytoplasm of antigen
presenting cells in order to enhance T-cell stimulation and
facilitate class I presentation to CD8+ CTL. Many viral and
bacterial live attenuated vectors have been developed
and shown to induce potent cell mediated immunity in
animal models. These live attenuated vectors are highly
immunogenic usually producing multiple pathogen-
associated molecular patterns that signal through TLR/
RLR/NLR, providing the inflammatory signals and
co-stimulation required to optimally induce protective
immune responses. In addition, these live attenuated
vectors can be delivered through more natural routes of
pathogen invasion (e.g. through mucosal surfaces) poten-
tially inducing more relevant regional immune responses
for protection against the initial infection. However, by
inducing potent inflammation these highly immunogenic
live vectors can be associated with undesired reactoge-
nicity, or may induce vector-specific immunity that can
reduce the efficacy of booster vaccinations with the same
vaccine.
DNA vaccines, consisting of recombinant plasmids
encoding vaccine antigens under the control of a eukary-
otic promoter and containing TLR9 stimulatory CpG
motifs, also deliver genes for expression inside the cyto-
plasm of antigen presenting cells. These plasmid vaccine
vectors have induced very potent Th1 and CTL responses
in animal models but have not been as successful in
humans to date. However, methods involving electropo-
ration of plasmid into the superficial epidermis with an
applied electrical current recently have been developed
which result in enhanced targeting and uptake by resident
Langerhan and other dendritic cells, and are likely to
improve human DNA vaccinations (Hirao et al., 2011; Lin
et al., 2011).
Liposomal and cationic structures have been devel-
oped that can allow translocation of soluble protein mol-
ecules into the cytoplasm of antigen presenting cells.
These additional delivery ‘vectors’ are potentially advan-
tageous for mucosal vaccinations as these materials tend
to be taken up by mucosal surfaces and concentrate
within highly phagocytic cells (Heurtault et al., 2010;
Mishra et al., 2010). This is a very active area of research
currently, although so far there are no licensed vaccines
consisting of liposomal/cationic formulated antigens.
Topic 8: Schedules and routes of vaccination
In addition to determining the right B- and T-cell epitopes,
optimizing the capacity for selective induction of the
appropriate immune subsets required, as well as selec-
tion of the ideal adjuvant and delivery system, the sched-
ules and routes of administration of each new vaccine
must be optimized. For pathogens that invade through or
are shed from mucosal tissues, mucosal vaccinations
might induce optimal protection against initial infection
and/or secondary transmission. Activation of T and B cells
within immune inductive sites lining mucosal tissues leads
to the upregulation of surface molecules on these cells
important for allowing access to peripheral mucosal sites
after circulation in blood. In fact, a network of mucosal
immunity known as the common mucosal immune system
results from the expression of mucosal homing molecules
on immune cells activated in one mucosal tissue that can
lead to enhanced distribution of these immune cells along
most if not all mucosal tissues. Specific integrin com-
plexes, additional adhesion molecules and chemokine
receptors are involved in the specific dissemination of
memory immune T and B cells to mucosa (Kunkel and
Butcher, 2002). For example, the a4b7 integrin complex is
upregulated on the surface of lymphocytes activated in
the Peyer’s patches. This integrin specifically binds to
MadCAM1 on endothelial cells and triggers transendothe-
lial migration from the vasculature into the peripheral
mucosal tissues. In contrast, intradermal or subcutaneous
vaccinations induce cutaneous lymphocyte antigen (CLA)
expression on T and B cells activated in the skin or lymph
nodes receiving lymphatic drainage from cutaneous sites.
CLA is important for recognition of molecular structures
lining endothelial cells in cutaneous microcapillaries
required for transpedesis into the peripheral cutaneous
tissues. Therefore, by changing the route of a vaccination,
regional immune responses relevant for protection
against specific pathogens can be selectively enhanced.
The optimal number of booster vaccinations must be
determined for each new vaccine. Generally, more than 1
dose of a vaccine is required to induce optimal immune
responses. Booster vaccinations lead to higher titre anti-
body responses with increased affinity resulting from
somatic hypermutation induced by recurrent stimulation
within the hypervariable regions critical for antigen recog-
nition. Two- to four-week intervals between booster vac-
cinations historically have worked well for vaccines
designed to induce protective antibody responses. Much
less is known regarding the optimal boosting intervals for
induction of Th1, CTL and other specific T-cell subsets.
Although somatic hypermutation is thought not to occur in
antigen-specific T cells, the most relevant clones are
selectively amplified in response to booster vaccinations
based on affinity reactions between MHC/peptide and
TCR. These higher avidity T cells can lead to enhanced
numbers of antigen-specific T cells, potency of effector
responses and longevity of antigen-specific memory.
Whether boosting T-cell responses after completion of
their differentiation into maximal numbers of resting Tcm
938 D. F. Hoft, V. Brusic and I. G. Sakala
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6. could improve long-term T-cell immune memory is
another important hypothesis to test.
As mentioned above, vectors can induce vector-
specific immunity that can limit the effectiveness of
booster vaccinations with the same vaccine vector.
Another important concept to investigate during vaccine
schedule optimization is whether distinct molecular
vaccine formats presenting the same vaccine antigens in
heterologous prime/boosting combinations can enhance
vaccine-induced protective memory immunity (Schneider
et al., 1998; Wei et al., 2010). Priming with a DNA vaccine
followed by boosting with a viral vectored vaccine, when
both vaccines encode the same pathogen-specific anti-
gens, can greatly enhance at least the total numbers of
antigen-specific T cells that develop and persist long term.
This technique of using heterologous prime/boosting
schedules can overcome the problems encountered due
to vector-specific immunity that develops during homolo-
gous prime/boosting vaccinations, and may maximize the
breadth and phenotype of immune subsets induced by
providing T-cell stimulations in the context of wider com-
binations of TLR/RLR/NLR.
Topic 9: Immunological memory networks
The ability to study whole genome-wide expression pat-
terns in cells after different states of activation/
differentiation promises to revolutionize the way we
approach vaccinology (Pulendran et al., 2010). Genome-
wide expression comparisons of effector T cells and
resting Tcm have demonstrated that the most potent
effector cells represent the most terminally differentiated
cells with the least capacity for self-renewal (Willinger
et al., 2005). In contrast, Tcm are less terminally differen-
tiated, have the best self-renewal/expansion capacity and
because of increased expression of anti-apoptotic genes
provide more long-term populations of antigen-specific
memory T cells.
Genome-wide expression pattern analyses have impli-
cated basic metabolic pathways in effector versus
memory T-cell generation. The mTOR pathway, activated
by the presence of numerous substrates and important for
growth and activation of primarily activated T cells, can
bias for development of mostly short-lived effector T cells
(Araki et al., 2009). Specific inhibitors of the mTOR
pathway (e.g. rapamycin and metformin) can lead to
increases in long-term memory T cells present after vac-
cination. Further network analyses of immune cells have
identified vitamin D metabolism as important for maximiz-
ing the cutaneous trafficking potential of memory T and B
cells, while vitamin A metabolism is involved in the induc-
tion of mucosal trafficking potential (Sigmundsdottir and
Butcher, 2008). The near future should bring additional
genome-wide expression studies that identify gene net-
works induced early on after vaccination that can predict
optimal long-term protective immunity and characterize
the more detailed factors involved in differential develop-
ment of Th1/Th2/Th3/Treg/Th17 immune phenotypes and
distinct lymphocyte homing programmes.
To demonstrate the power of molecular transcriptomal
analyses, we include preliminary data that we have
recently generated studying human volunteers with previ-
ous exposure to mycobacterial antigens. We have con-
ducted a series of tuberculosis (TB) vaccine trials with the
overall goal of learning how to improve the protective
capacity of new TB vaccines. To our knowledge, there are
no published reports of human T-cell antigen-specific
molecular transcriptomes. Transcriptomes expressed in
subsets of unactivated, total polyclonal human memory
T-cell subsets have been studied, but not antigen-specific
populations or the differences between rested and acti-
vated memory T-cell responses. In mice, TCR transgenic
models have facilitated studies of antigen-specific tran-
scriptional profiles by providing highly purified populations
of T cells that can be obtained with relative ease in naïve,
activated effector and memory states. These pure popu-
lations ensure all gene expressions being measured are
related to the antigen-specific populations of interest. In
humans, the frequencies of T cells specific for a given
vaccine or pathogen usually are < 1–10% of the total
T-cell population. Thus, it has been assumed that antigen-
specific T cells must be purified in order to study complex
gene expression pathways in a minority subset of antigen-
specific T cells relevant for a given vaccine or infection.
Practical issues with this approach include the small
number of T cells recovered, the consequent need for
amplification techniques to study the mRNA expressed,
and potential alteration in gene expression profiles due to
labour intensive in vitro manipulations. To address these
feasibility concerns, we completed a pilot experiment
which clearly demonstrates that TB-specific transcrip-
tional profiles can be studied with a relatively simple
approach not requiring purification of antigen-specific T
cells. We detected BCG-induced changes in gene expres-
sion among total memory CD4+ T cells (CD4+CD45RO+)
purified from three PPD+ persons (VTR #1–3 in Fig. 1A).
As shown in Fig. 1A, we detected greater than 50-fold
increases in IL-2 mRNA at 24 h in T cells stimulated with
BCG-infected compared with uninfected DC. These
results demonstrate that we can detect marked increases
in expression of the IL-2 target gene despite the fact that
BCG-specific T cells likely represent only a minor fraction
of all polyclonal CD4+ memory T cells, and indicate that
stimulation of T cells for 24 h with BCG-infected DC
(moi = 20) represent reasonable conditions for genome
expression studies of BCG-specific T-cell responses. We
prepared cDNA and cRNA from RNA samples harvested
from CD4+ memory T cells co-cultured with uninfected
Optimizing vaccine development 939
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7. and BCG-infected DC for 24 and 48 h from these three
volunteers and performed Affymetrix hybridizations with
HG-U133 Plus 2 Affymetrix chips. The data were normal-
ized and ANOVA used to identify genes with significant
changes in expression comparing BCG-infected and unin-
fected stimulation conditions. We found 3098 genes
significantly altered by stimulation with BCG-infected
DC and 608 upregulated at both the 24 and 48 h time
points. Gene set enrichment analysis (GSEA) looking for
networks of genes altered by BCG antigen-specific stimu-
lation indicates that many gene sets classically associ-
ated with immune response pathways (T-cell receptor
signalling, Jak-Stat signalling, apoptosis, cytotoxicity,
cytokine–receptor interactions and integrin-medicated cell
adhesion) are highly represented among the significantly
altered gene expression patterns (http://cvc.dfci.harvard.
edu/share_folder/IMN). Therefore, we can use a relatively
simple strategy to purify total memory CD4+ T cells and
study the expression of a highly diverse set of human
genes induced by antigen-specific stimulation. We are
currently using this strategy to identify specific alterations
in the antigen-specific gene set associated with mucosal
versus cutaneous BCG vaccination, and which genes
predict the best functional long-term memory responses
after BCG vaccination. The overall goal is to determine
new biomarkers that can be used to more rapidly and
accurately assess mucosal and systemic immunogenicity
of iterative TB vaccination approaches.
Topic 10: Clinical development/safety and
surrogate markers
Once a vaccine is ready for clinical testing, the focus first
becomes safety. Phase I dose escalation trials are
designed to identify the safest and most immunogenic
vaccine doses. Phase II trials expand safety analyses into
larger numbers of volunteers and begin to address the
efficacy of vaccination. Phase III trials are designed to
provide sufficient statistical power to definitively address
vaccine efficacy and include detailed reactogenicity
assessments. Throughout the clinical development
pathway from phase I to phase III, it is important to have
surrogate markers of protective immunity that can be
assessed in vaccinated volunteers. Ideally, correlates of
protection should be known to help direct vaccine optimi-
zation. Unfortunately, correlates of protection are not
Fig. 1. Human molecular transcriptomal
analyses can help identify important biological
responses required for successful vaccines. In
(A), memory CD4+ T cells were purified from
three PPD+ persons with Miltenyi negative
selection kits resulting in > 97% pure memory
CD4+ T cells. Memory CD4+ T cells were
stimulated with autologous DC (20:1 T : DC
ratio) that were uninfected or infected with a
BCG moi of 4, 20 or 100. Total RNA was
harvested at 24, 48 and 72 h. We completed
qRT-PCR for IL-2 mRNA to determine what
conditions gave us the best ability to see
BCG-induced changes. RNA harvested from
the optimal conditions was used to identify the
Affymetrix molecular signatures associated
with BCG-specific stimulation demonstrating
that the expression of 3098 genes was
significantly involved (data not shown). (B)
depicts the major phases of T-cell activation
and the serial points of transcriptomal
analyses being used to identify: (i) gene
expression patterns involved in programming
long-term protective immune memory, and (ii)
the differential gene expression patterns that
predict T-cell responses capable of providing
optimal mucosal versus systemic immunity.
940 D. F. Hoft, V. Brusic and I. G. Sakala
© 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 934–942

8. known for many of the human pathogens that remain key
challenges for vaccine development. For HIV, TB and
many other major world pathogens, only partial informa-
tion regarding surrogates/correlates of protective immu-
nity is available. This shortcoming makes iterative
research critical involving the empirical development of
vaccine candidates, clinical development of experimental
vaccines and refinement of second-generation vaccines
based on enhanced targeting of new surrogates/
correlates identified in vaccine trials with prototype vac-
cines. Finally, once a vaccine is shown to be safe and
efficacious in humans, additional research is necessary to
assess ongoing effectiveness of the vaccine for preven-
tion of infection and disease due to the target pathogen
under real-world conditions, and to provide quality control
for continual production of effective vaccines.
Concluding remarks
Vaccine development is a complex process involving mul-
tiple different specialists, careful thought into the specific
vaccine design, as well as laborious testing and evalua-
tion. We have discussed some of the key issues important
for the generation of a successful vaccine. Because of
space limitations we have left out many additional steps
that are necessary for this process including detailed
animal testing of immunogenicity, protective capacity and
toxicity. We hope that the reader now has a more com-
plete appreciation of the detailed requirements for devel-
opment of a successful vaccine.
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