Content uploaded by Samuel M Behar
Author content
All content in this area was uploaded by Samuel M Behar on Nov 10, 2014
Content may be subject to copyright.
The next generation: tuberculosis vaccines that elicit protective
CD8+ T cells
Samuel M. Behar, M.D., Ph.D.,
Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital and
Harvard Medical School, Smith Building Room 516C, One Jimmy Fund Way, Boston, MA 02115.
Phone: (617)-525-1033, Fax: (617)-525-1010
Joshua S.M. Woodworth, B.S., and
Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital and
Harvard Medical School, Smith Building Room 516C, One Jimmy Fund Way, Boston, MA 02115.
Phone: (617)-525-1065, Fax: (617)-525-1010
Ying Wu, M.D.
Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital and
Harvard Medical School, Smith Building Room 516C, One Jimmy Fund Way, Boston, MA 02115.
Phone: (617)-525-1042, Fax: (617)-525-1010
Samuel M. Behar: sbehar@rics.bwh.harvard.edu; Joshua S.M. Woodworth:
joshua_woodworth@student.hms.harvard.edu; Ying Wu: ywu0@partners.org
Summary
Tuberculosis continues to cause considerable human morbidity and mortality across the world,
particularly in people coinfected with HIV. The emergence of multidrug resistance makes the
medical treatment of tuberculosis even more difficult. Thus, the development of a tuberculosis
vaccine is a global health priority. Here we review the data concerning the role of CD8
+
T cells in
immunity to tuberculosis and consider how CD8
+
T cells can be elicited by vaccination. Many
immunization strategies have the potential to elicit CD8
+
T cells, and we critically review the data
supporting a role for vaccine-induced CD8
+
T cells in protective immunity. The synergy between
CD4
+
and CD8
+
T cells suggests that a vaccine which elicits both T cell subsets has the best
chance at preventing tuberculosis.
Keywords
Mycobacterium tuberculosis; BCG; vaccine; bacterial infection; CD4; CD8; T cell; animal
models; microbial immunity
1. Introduction
In the 21
st
century, tuberculosis remains a plague in many parts of globe. In addition to the
8–10 million cases of tuberculosis that occur each year, nearly a third of the world’s
population is latently infected, which represents an enormous potential reservoir of disease.
The development of multidrug resistant (MDR)
1
and extensive drug resistant (XDR) strains
Correspondence to: Samuel M. Behar, sbehar@rics.bwh.harvard.edu.
1
Abbreviations: Ad, adenovirus; APC, antigen presenting cells; β2m, β2 microglobulin; BCG, Bacille Calmette Guérin; DC, dendritic
cell; ER, endoplasmic reticulum; HIV, human immunodeficiency virus; IFNγ, interferon-γ; IV, intravenous; mAb, monoclonal
antibody; MDR, multidrug resistant; MHC, major histocompatibility; TAP, transporter associated with antigen processing; TLR, Toll-
like receptor; VV, vaccinia virus; XDR, extensive drug resistant;
NIH Public Access
Author Manuscript
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
Published in final edited form as:
Expert Rev Vaccines
. 2007 June ; 6(3): 441–456. doi:10.1586/14760584.6.3.441.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
of Mycobacterium tuberculosis make the task of treating tuberculosis even more daunting
and highlight the need for strategies to prevent transmission, such as prophylactic
vaccination of susceptible individuals. The only vaccine available against M. tuberculosis is
the attenuated strain of M. bovis known as Bacille Calmette Guérin (BCG), which is used in
many parts of the world. Unfortunately, data suggest that while BCG vaccination can reduce
the incidence of severe infantile forms of tuberculosis such as meningoencephalitis and
disseminated tuberculosis, BCG appears ineffective at preventing the most common adult
form of tuberculosis, pulmonary tuberculosis. Therefore, there is wide spread consensus that
a new vaccine against tuberculosis is needed – a vaccine that would be effective against
pulmonary tuberculosis – the form of disease that is most contagious and represents the
greatest threat to public health.
Abundant experimental and clinical data supports a critical role for T cells in immunity to
M. tuberculosis. Consequently, eliciting immune T cells is proposed to be an essential
property of any vaccine that is capable of providing protection against tuberculosis.
Designing the best vaccine for clinical trials requires an understanding of the T cell subsets
that contribute to protective immunity against tuberculosis. T cells come in a variety of
flavors, each with unique properties and specialized functions. For example, CD4
+
T cells
recognize peptide antigens derived from proteins that enter the cell’s endocytic system and
are processed and presented by the class II MHC pathway. In contrast, CD8
+
T cells
recognize peptide antigens, derived from cytosolic proteins, such as those generated in
virally infected cell, which are presented by the class I MHC pathway. In addition to MHC-
restricted CD4
+
and CD8
+
T cells, other T cell subsets exist that are restricted by
nonclassical MHC molecules. CD1-restricted T cells are one such well-characterized subset
of T cells. CD1 is a family of non-polymorphic antigen presenting molecules that present
lipid and glycolipid antigens to T cells. Group 1 CD1 (CD1a, CD1b, and CD1c), can present
mycobacterial lipid antigens to human T cells, which are often CD4
−
and CD8
−
(so called
“double negative” T cells). In contrast, group 2 CD1 (CD1d) is the restricting element for
NKT cells, which are almost always CD4
+
or CD4
−
8
−
. In designing a vaccine to prevent
tuberculosis, it is important to consider which T cell subsets are desirable to activate. In this
review, we will discuss the evidence that class I MHC-restricted CD8
+
T cells are important
in immunity to M. tuberculosis. An overview of vaccine strategies designed to elicit M.
tuberculosis-specific CD8
+
T cells will be presented. Finally, we will critically examine the
how these strategies perform with respect to eliciting CD8
+
T cells that recognize
mycobacterial antigens and mediate protective immunity.
2. CD8
+
T cells mediate host defense against M. tuberculosis
Although extensive experimental data supports the contention that T cells play a critical role
in mediating immunity to M. tuberculosis, the data that support an essential role for T cells
in people is not as abundant. One exception concerns the role of CD4
+
T cells. Here,
abundant clinical data supports the observation that people infected with human
immunodeficiency virus 1 (HIV-1) have a greater risk of developing both primary and
reactivation tuberculosis. Since one of the central immunological defects following HIV
infection is a loss of CD4
+
T cells and their function, the association is commonly
interpreted as evidence that CD4
+
T cells mediate immunity to tuberculosis in people. In
contrast, clinical data supporting a role for CD8
+
T cells is not available; however, it is clear
from a variety of experimental systems that CD8
+
T cells are an important component of
protective immunity to M. tuberculosis.
The transfer of cells or serum from immune donors into naïve recipients can passively
transfer immunity. When done experimentally, such adoptive transfer experiments were
classically used to distinguish between cell-mediated versus humoral immunity. For
Behar et al. Page 2
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
immunity to tuberculosis, Lefford found that splenocytes but not serum from BCG
vaccinated mice could protect irradiated recipient mice against BCG or M. tuberculosis [1].
The capacity of splenocytes from BCG vaccinated mice to transfer protection was
significantly impaired if T cells were depleted from the splenocytes prior to transfer. As
better T cell purification techniques became available, Orme and his colleagues confirmed
that T cells from BCG vaccinated mice could provide protection to T cell deficient recipient
mice challenged with virulent M. tuberculosis [2]. Orme also determined the differential
ability of CD4
+
and CD8
+
T cells to provide protection to mice challenged with M.
tuberculosis [3]. Splenic CD4
+
and CD8
+
T cells from mice infected intravenously (IV) with
M. tuberculosis were transferred into uninfected irradiated mice, which were then
challenged with 10
5
CFU of M. tuberculosis by the IV route and protection was assessed 10
days later by determining the bacterial burden in the lungs. Both CD4
+
and CD8
+
splenic T
cells mediated protection; however, CD4
+
T cells provided greater protection and acquired
the capacity to transfer protection earlier than CD8
+
T cells. These kinetics may reflect a
dependence of CD8
+
T cell function on CD4
+
T cell helper function and reflect a
requirement for CD4
+
T cell licensing of CD8
+
T cell effector function. Using the same
model, Orme showed that memory T cells also provide protection to naïve irradiated mice
challenged with M. tuberculosis; however, in the case of memory T cells, only CD4
+
and
not CD8
+
T cells were effective [4]. Similarly designed studies have shown that T cells
mediate protection against challenge with M. bovis BCG [5,6]. Britton’s lab established an
adoptive transfer model using RAG-1 −/−mice, which lack B and T cells, as
immunodeficient hosts to study T cell mediated protection against mycobacterial infection.
They find that protection against aerosolized M. bovis BCG can be mediated by the adoptive
transfer of both CD4
+
and CD8
+
T cells. In all of these studies, CD8
+
T cells provide less
protection than an equivalent number of CD4
+
T cells [6]. Nevertheless, these adoptive
transfer experiments show that CD8
+
T cells, even in the absence of CD4
+
T cells, can
mediate protection against tuberculosis.
Administration of monoclonal antibodies (mAb) to CD4 and CD8 is used to deplete T cells
subsets in mice, and this experimental approach has been used to determine whether CD4
+
and/or CD8
+
T cells are required for immunity to tuberculosis. Antibody mediated depletion
has a more robust effect in thymectomized mice, since they lack the capacity to regenerate
new CD4
+
and CD8
+
T cells. Muller et al treated thymectomized C57BL/6 mice with
monoclonal antibodies (mAb) specific for CD4 or CD8 and then infected the mice with M.
tuberculosis by the IV route. Depletion of either CD4
+
or CD8
+
T cells impaired control of
bacterial replication in the spleen when measured 3 wks after infection [7]. Pedrazzini et al
examined the effect of depleting CD4
+
or CD8
+
T cells on the growth of M. bovis BCG in
mice. The growth of BCG in the spleen and lung was enhanced in anti-CD4 mAb treated
mice, reaching a maximum by day 45 (30-fold increase), but subsequently returned to
normal levels by day 100 post-infection. In contrast, treatment with anti-CD8 had no effect
on bacterial levels [5]. Similar experiments were done by Cox et al using BCG to infect both
resistant (C57BL/6) and susceptible (CBA/Ca) mouse strains. Anti-CD4 treatment resulted
in a 0.5–0.6 log increase in splenic CFU in both mouse strains. The effect of anti-CD8 mAb
treatment was strain dependent: CD8
+
T cell depletion had no effect in CBA/Ca mice, while
there was a modest exacerbation (0.3–0.4 log) in C57BL/6 mice [8]. Overall, these results
suggest that CD8
+
T cells have a role in mediating immunity to mycobacterial infection, and
this effect is more important in resistance to virulent bacterial strains.
The most convincing data that CD8
+
T cells are required for host immunity after M.
tuberculosis infection comes from experiments using “knockout” mouse strains. Knockout
mouse strains that lack protein expression of β2 microglobulin (β2m), transporter associated
with antigen presentation-1 (TAP-1), and the class I MHC heavy chains (K
b
D
b
), all have
impaired class I MHC antigen presentation, either because they lack class I MHC structural
Behar et al. Page 3
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
proteins (β2m and K
b
D
b
) or because they lack proteins (TAP-1) that transport peptides from
the cytosol into the ER. Since expression of class I MHC/peptide complexes in the thymus is
required for positive T cell selection during T cell ontogeny, all three of these knockout
mouse strains lack conventional MHC-restricted CD8
+
T cells. Following infection with
virulent M. tuberculosis by the IV route, all three of these knockout mouse strains are more
susceptible than appropriate genetically intact control mice [9–12]. Although caveats exist
concerning the interpretation of these experiments (discussed in reference [13]), these data
support the hypothesis that CD8
+
T cells are required for optimum immunity to tuberculosis.
Many of the studies described above that demonstrate a role for CD8
+
T cells in the control
of M. tuberculosis infection were performed in mice infected with M. tuberculosis by the IV
route. Robert North addressed the relative importance of CD4
+
and CD8
+
T cells following
infection by the aerosol route [14]. After aerosol infection with virulent M. tuberculosis,
class II MHC knockout mice (which lack conventional CD4
+
T cells) are much more
susceptible than β2m knockout mice (which lack conventional CD8
+
T cells), as measured
by survival. Similarly, C57BL/6 mice treated with anti-CD4 mAb succumbed earlier than
mice treated with anti-CD8 mAb. Nevertheless, mice without CD8
+
T cells were always
more susceptible than immunocompetent “wild-type” control mice. Mice treated with the
combination of anti-CD4 plus anti-CD8 mAb were more susceptible than mice treated with
anti-CD4 alone, suggesting that CD8
+
T cells have a unique non-redundant role.
Most studies that have compared the contribution of CD8
+
and CD4
+
T cells to anti-
mycobacterial immunity have found that the protective effect of CD8
+
T cells is less than
that described for CD4
+
T cells. This comparison, however, is an oversimplification. First,
the mouse may not accurately model all stages of the natural history of M. tuberculosis
infection in people. For example, latency, which is arguably one of the most important but
least understood phases of infection, is not easily studied in animal models. Using a
modified version of the Cornell latency model, in which small numbers of bacteria persist
despite antibiotic therapy, van Pinxteren et al showed that although CD4
+
T cells were
critical during acute infection, CD8
+
T cells were of greater importance during latency [15].
Thus, the effector functions of different T cell subsets may be particularly well suited to
controlling bacterial replication at different phases of disease.
A second caveat is that certain features of an optimum CD8
+
T cell response are dependent
on CD4
+
T cells for licensing or “help”, and mice lacking CD4
+
T cells have suboptimal
CD8
+
T cell effector function and memory responses [16]. These principles are derived from
a variety of model systems but are likely to be important for adaptive immunity following
M. tuberculosis infection. For example, the cytotoxic activity of CTL elicited following M.
tuberculosis infection is defective in CD4 knockout mice, perhaps because of a need for
IL-2 and IL-15 [17]. Such findings are taking on even more importance since we now
appreciate that antigen-specific CD8
+
T cells elicited following infection have cytotoxic
activity in vivo [18,19]. Through the use of vaccination studies, Hill’s group has shown that
an protection correlates with an optimum CD8
+
T cell response to Ag85A, which is
critically dependant upon CD4
+
T cell help [20]. Thus, hosts lacking class II MHC-restricted
CD4
+
T cells may not be able to mount an optimal CD8
+
T cell response to M. tuberculosis,
and consequently studies that deplete CD4
+
T cells, whether genetically or by treatment with
mAb, are likely to overestimate the contribution of CD4
+
T cells relative to CD8
+
T cells.
After reviewing the literature, we believe that immunological synergy exists between the
CD4
+
and CD8
+
T cell response to M. tuberculosis and that the best chance for inducing
protective immunity is to elicit antigen-specific T cells from both T cell subsets.
Behar et al. Page 4
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
3. Eliciting CD8
+
T cells and measuring protection
Bacteria and extracellular particulate antigens are internalized by antigen presenting cells
(APC) via phagocytosis, receptor-mediated endocytosis or macropinocytosis. An array of
diverse hydrolytic enzymes resides in the endocytic compartment and can catabolize even
intact bacteria. Peptides generated by proteolysis can bind class II MHC and the resulting
complex is transported to the cell surface for recognition by CD4
+
T cells. The preferential
entry of exogenous antigens into the class II MHC pathway makes the job of eliciting CD4
+
T cells by vaccination relatively straightforward. Both inactivated pathogens (e.g., influenza
A) and purified components (e.g., tetanus toxoid), can stimulate antigen-specific CD4
+
T
cells. The development of newer adjuvants approved for human use promises to make this
process even more effective. In contrast, inducing antigen-specific CD8
+
T cells is more
complicated. Antigens that are recognized by CD8
+
T cells are usually derived from
cytosolic proteins, such as viral proteins that are found in the cytosol during virus assembly.
Cytosolic antigens are cleaved by the proteasome, a large enzymatic structure which
normally functions to catabolize proteins that the cell has targeted for degradation [21]. The
resulting peptides are transported into the endoplasmic reticulum (ER) by transport proteins
including TAP-1 and tapasin. Once in the ER, peptides compete for binding to “empty”
class I MHC and β2m heterodimer [22]. After a trimeric complex is formed, it is transported
to the cell surface where it can be recognized by CD8
+
T cells. Therefore, the trick to
inducing antigen-specific CD8
+
T cells is to target the antigen of interest to the cytosol of an
APC.
Several innovative methods have been developed to target proteins to the cytosol for the
purpose of eliciting CD8
+
T cell responses. These approaches fall into three main categories.
One strategy is to alter physicochemical properties of the APC using techniques such as
osmotic shock, which can be used in vitro to deliver extracellular proteins to the cytosol. A
second approach takes advantage of defined physiological properties of APC. For example,
if the minimal epitope has been defined for a particular antigen, the corresponding synthetic
peptide can be cultured with the APC in vitro. If the peptide binds to class I MHC with high
affinity, it can bind directly to cell surface class I MHC proteins, bypassing the requirement
for cytosolic processing. DC are the APC of choice, since they uniquely prime naïve cells
when injected back into the host. Cells also have a propensity to take up DNA under certain
conditions, and this has led to the concept of DNA vaccination. DNA encoding the antigen
of interest is administered by the parenteral route and several delivery methods have been
developed including intramuscular or intradermal injection or epidermal delivery via a gene
gun. The DNA is taken up by local cells and gene expression leads to antigen production,
some of which is acquired by local DC, which can migrate to the regional LN and induce an
immune response. DNA vaccination is tremendously versatile and the gene encoding the
antigen can be engineered to target the protein to either the class I or class II MHC pathway
by changing the signal peptide. In addition, adjuvant-like signals can be provided by co-
vaccination with plasmids encoding cytokines or using immunostimulatory DNA sequences
that activate toll-like receptors (TLR). The third, and perhaps most successful vaccine
approach, is to highjack the properties of pathogens that naturally target or enter the cell’s
cytosol. The CD8
+
T cell response is an important component of adaptive immunity to most
viruses and many intracellular bacteria. Attenuated versions of several viruses and bacteria
have been developed as successful vaccines. For example, M. bovis BCG, an attenuated
form of M. bovis that is related to M. tuberculosis, is the current vaccine for tuberculosis and
elicits antigen-specific CD8
+
T cells in both experimentally infected animals [23] and in
BCG vaccinated people [24]. Similarly, vaccinia is a pox virus that successfully protects
people against smallpox infection. Using recombinant DNA methodology, both of these
vectors can be engineered to express antigens of interest for the purpose of eliciting a strong
immune response.
Behar et al. Page 5
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
The “prime-boost” strategy is increasingly being considered in the design of vaccines. An
attenuated pathogen, such as BCG, can be used to “prime” an immune response, which is
then boosted by a second immunization using a distinct vaccine that shares only a subset of
antigens with the first vaccine, such as adenovirus or vaccinia virus expressing recombinant
mycobacterial proteins. Boosting repeatedly with the same vaccine can lead to a suboptimal
T cell response because neutralizing antibodies ultimately limit the functional availability of
the administered antigens to restimulate T cells. In contrast, by using a different vector for
the boost, a primary immune response is generated to the “vector”, but a secondary (and
hence more effective) response to the recombinantly expressed protein antigens. Although
this approach shares some of the disadvantages of subunit vaccines such as the problems
inherent in vaccinating an immunogenetically heterogeneous population with a single
antigen, it is nevertheless a powerful way to elicit a robust adaptive immune response to
defined antigens.
One strategy would be to prime the immune system with one vaccine, such as a DNA
vaccine, and boost with a second vaccine, such as a recombinant virus. In this case both the
DNA vaccine and the recombinant virus express the same mycobacterial antigen. In
practice, this strategy would be most important for eliciting a potent immune to M.
tuberculosis antigens not present in BCG. However, most of the world’s population,
particularly in tuberculosis endemic regions, has been vaccinated with BCG. The protective
effect of BCG against childhood disseminated TB ensures its continued use over the
foreseeable future. In the BCG vaccinated population, the role of any novel vaccine would
be to boost an immune system previously primed with BCG. Thus, many of the new
vaccines against tuberculosis are designed around such a BCG prime-boost strategy.
Ultimately, vaccination is designed to protect the host against infectious disease. Because
tuberculosis is a chronic infection, it is not trivial to determine the protective efficacy of
vaccines. For animal studies, vaccinated subjects are challenged with M. tuberculosis and
then analyzed to determine whether or not protective immunity has been induced. Although
there is considerable variability in how protection is determined, there is an ongoing effort to
standardize these studies [25–27]. First, challenge with M. tuberculosis is generally not
performed until at least 4 weeks after the last immunization because of concern that non-
specific activation by the vaccine, particularly of the innate immune system, may lead to an
appearance of protection. Although many different routes of infection are used for animal
models of tuberculosis, the IV and aerosol routes are the most commonly used. The aerosol
route is increasingly preferred because of the use of low numbers of bacteria (e.g., 100–300
CFU/mouse) and similarity to the route of infection in people. It is imperative that virulent
bacteria are used in all of these studies. Growth of less virulent bacteria is more easily
inhibited by the innate immune system, whereas greater synergy between innate and
adaptive immune pathways is required for the control of more virulent bacteria. How one
determines whether vaccinated subjects are protected by the vaccine can be divided into
“microbial” and “host” endpoints. Most commonly, the bacterial burden is measured,
generally 4 weeks after infection which is near the peak of the adaptive immune response, as
well as a later time point, usually 2–4 months after infection, which is during the plateau or
chronic phase of infection. When possible, the bacterial burden in both the lung and the
spleen should be measured. This is particularly important following aerosol infection
because the immune mechanisms that limit bacterial growth in the lung may be different
from how the immune system prevents systemic dissemination and controls the infection in
other tissues. Many vaccines appear to be more effective at controlling systemic infection
than pulmonary infection. Unfortunately, short-term control of the bacterial burden does not
always correlate with long-term survival [28]. In part, this is because the pulmonary
pathology caused by M. tuberculosis arises not only from direct toxic effects of the bacteria,
but also as a consequence of immune-mediated tissue injury. Survival studies are an
Behar et al. Page 6
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
important way to measure the long-term benefit of vaccination [29,30], and integrate both
the beneficial effect of improved bacterial control and whether improved microbial control
is at the cost of greater immune-mediated inflammation that paradoxically results in more
tissue damage. In practice BCG has been the standard for comparison, regardless of the
measure of protection used. This both reflects the desire to match, if not improve upon, the
current M. tuberculosis vaccine, as well as allow for the comparison of different vaccine
strategies.
4. Do CD8
+
T cells mediate vaccine-induced immunity?
To address whether eliciting mycobacterial antigen-specific CD8
+
T cells should be a goal
of vaccine development for tuberculosis, we will review studies that use different
vaccination strategies to induce M. tuberculosis-specific CD8
+
T cell responses. One
difficulty in interpreting the literature is that many of these vaccines elicit both CD4
+
and
CD8
+
T cell responses, and it is difficult to distinguish between the effect of CD4
+
and
CD8
+
T cells in the standard M. tuberculosis challenge models. Conversely, because few
mycobacterial epitopes that are recognized by CD8
+
T cells have been defined, it is difficult
to know whether the failure of vaccines designed to elicit CD8
+
T cells to induce protective
immunity is due to a failure to elicit antigen-specific CD8
+
T cells, or because the CD8
+
T
cells elicited by vaccination do not recognize M. tuberculosis infected cells, or whether the
M. tuberculosis-specific CD8
+
T cells lack effector functions required to protect mice from
tuberculosis.
What is acceptable evidence that CD8
+
T cells elicited by vaccination protect mice against
tuberculosis? Just as a variety of techniques have been used to show the role of CD8
+
T cells
in immunity to tuberculosis following infection, similar approaches have been used to prove
that CD8
+
T cells elicited by vaccination protect mice against tuberculosis in a challenge
model. One way is to transfer CD8
+
T cells from vaccinated mice to naïve recipients to
determine whether the elicited CD8
+
T cells provide passive immunity against challenge
with virulent M. tuberculosis. Investigators have depleted CD4
+
and CD8
+
T cells in vivo
after vaccination to show the role of different T cell subsets in resistance to tuberculosis.
Sophisticated strategies include the use of vaccines that preferentially elicit CD8
+
T cell
responses in mice that lack CD4
+
T cells, with the assumption that any protective effect can
be ascribed to CD8
+
T cells. As we will discuss, although these studies have a great deal of
promise, there exist many caveats and the results need to be carefully interpreted.
DC vaccination
Dendritic cells (DC) are the ultimate APC [31]. More than any other APC, DC efficiently
prime naïve T cells and this is their most important in vivo function. Their capacity to
activate antigen-specific T cells and initiate the adaptive immune response is based on four
important properties. First, they constitutively sample the extracellular milieu by
macropinocytosis [32]. Second, they efficiently present antigen, in part because they do not
rapidly degrade foreign proteins, which prolongs the half life of antigens and increases the
probability that antigenic peptides will be presented to T cells [33]. Third, an array of
costimulatory molecules is expressed by DC, particularly following their activation and
maturation, which enhances their ability to activate T cells [34]. Finally, unlike
macrophages, DC have the capacity to recirculate. Immature DC are found in all major
tissue beds, and when they encounter pathogens or other danger signals, they undergo
activation and maturation, which allows them to migrate to the regional LN. In the LN, DC
can interact with naïve T cells until ones are encountered that are specific for the antigens
presented by the DC. Instead of delivering protein and adjuvant into the skin and expecting
that the vaccine is picked up by DC, a more efficient T cell vaccination strategy is to add
specific antigens to DC in vitro, and then inject the DC into the host. The expectation is that
Behar et al. Page 7
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
the DC will traffic back to the LN and trigger an adaptive immune response to the specific
antigens it carries.
In principle, DC vaccination can protect mice from virulent tuberculosis. Administration of
BCG-infected DC induces anti-mycobacterial immunity and provides some protection to
mice challenged with aerosolized M. tuberculosis [35]. The power of DC vaccination is
based on its ability to focus the immune response on a select group of protective antigens.
Conversely, one can screen candidate proteins using DC vaccination in order to identify
ones that are protective. Loading the DC with antigen can be as simple as culturing the cells
with the intact protein antigen. For the purposes of generating CD8
+
T cell responses, this
strategy has the added benefit since the cells can be subjected to osmotic shock to load the
protein into the cytosol, or if the minimal epitopes have been defined, synthetic peptides can
be used to exogenously load the cell surface class I MHC. Another way to load antigens is to
infect DC with a virus expressing a mycobacterial protein. This approach has been used to
identify mycobacterial antigens recognized by human CD8
+
T cells. Lewinsohn et al
infected human DC with recombinant adenovirus expressing the protein Mtb39 [36]. CD8
+
T cells from individuals with latent tuberculosis recognized autologous DC infected with
rAd.Mtb39, demonstrating that Mtb39-specific CD8
+
T cells are elicited in people after M.
tuberculosis infection. Vaccinating with DC infected with recombinant adenovirus could be
even more efficient than vaccinating with the virus alone, since in vivo, many non-APC
would be infected, which would be unable to directly prime T cells. This strategy has been
tested by Malowany et al who infected DC with recombinant adenovirus expressing the
mycobacterial protein Antigen 85A (AdAg85A) [37]. Vaccination of mice with AdAg85A-
infected DC induced a persistent CD4
+
and CD8
+
T cell response to Ag85A, which was
clearly superior to the T cell response induced by DC pulsed either with Ag85A protein or
with the synthetic peptides corresponding to the class I and class II MHC-restricted Ag85A
epitopes. The Ag85A-specific T cells produced interferon-γ (IFNγ) and had cytotoxic
activity in vivo. Interestingly, the T cell response following vaccination was dominated by
CD8
+
T cells. This type of vaccination induced protection that was comparable to BCG and
clearly better than DC pulsed with Ag85A peptides or protein. In a similar study, DC that
were retrovirally-transduced with Ag85A were used to vaccinate mice [38]. Vaccination of
mice with transduced DC induced Ag85A-specific CD4
+
and CD8
+
T cells. The frequency
of antigen-specific T cells was not determined so the relative efficiency of retrovirally-
transduced DC can not be compared to the use of adenovirus-infected DC. Of note,
protection induced by Ag85A transduced DC under these conditions was modest, with ~2-
fold reduction in the pulmonary CFU 4 weeks after IV infection with H37Rv. The approach
of immunizing with DC infected with replicative-defective recombinant viruses appears to
be a promising way to elicit antigen-specific CD4
+
and CD8
+
T cells.
While the use of DC infected with recombinant viruses is efficient at activating CD4
+
and
CD8
+
T cells, these studies have not addressed whether the CD8
+
T cells generated by these
methods are protective. Adenovirus-infected DC could be used to answer this question,
since antigen-specific CD8
+
T cells are efficiently stimulated. Another way to address the
relative importance of CD4
+
and CD8
+
T cells following DC vaccination is to use synthetic
peptides corresponding to well-characterized epitopes from Ag85A recognized by CD4
+
or
CD8
+
T cells. McShane et al used Ag85A peptide-pulsed DC to vaccinate mice and found
that DC pulsed with synthetic peptides recognized by both CD4
+
and CD8
+
T cells induce
protective immunity against M. tuberculosis comparable to BCG [20]. In contrast, DC
pulsed separately with either the class I or class II MHC-restricted peptide could not induce
protection. Apparently, both peptides need to be presented by the same DC to induce
optimum immunity since mixing DC that had been separately pulsed with each peptide did
not protect mice from challenge with M. tuberculosis. Only DC pulsed with both peptides
simultaneously were capable of inducing protection. Under these conditions, the frequency
Behar et al. Page 8
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
of Ag85A-specific CD8
+
T cells was higher in mice vaccinated with DC pulsed with both
peptides compared to mice vaccinated with a mixture of DC pulsed with each peptide
separately. This may reflect a dependence of the CD8
+
T cell response on CD4
+
T helper
cells and provides evidence for the critical role played by conventional class I MHC-
restricted CD8
+
T cells.
Lastly, it is important to remember that tuberculosis is a disease that in large part is a
consequence of immunopathology. Enhancement of the immune response always has the
potential to worsen the outcome of infection. Intranasal vaccination of mice with DC pulsed
with Ag85A protein led to priming of antigen-specific CD4
+
T cells but was not associated
with a reduction in CFU upon M. tuberculosis challenge [39]. However, mice that received
Ag85A/DC, had a dramatic increase in the recruitment of CD4
+
and CD8
+
T cell
recruitment into the lung and greater tissue pathology compared to control mice that were
vaccinated with ovalbumin pulsed DC. The increase in tissue pathology without any
evidence of improved bacterial control, suggests that this strategy led to an over-exuberant
immune response that may have been detrimental for the host. Thus, an ideal vaccine would
reduce the bacterial burden and diminish the tissue inflammation.
Intracellular bacteria
Bacteria, especially those that are intracellular pathogens, elicit potent CD8
+
T cell
responses and can be used in the development of vaccines that generate both CD4
+
and
CD8
+
T cell responses. As detailed above, both M. tuberculosis and M. bovis BCG elicit
CD4
+
and CD8
+
T cell responses [40]. In fact, given the extensive clinical experience with
BCG, it is being used as a antigen-delivery system for the development of other vaccines.
For example, HIV and hepatitis C proteins are being expressed in BCG and other
mycobacterial strains [41]. Vaccination of experimental animals with these recombinant
BCG strains elicits antigen-specific CD8
+
T cell responses [42].
To improve the ability of BCG vaccination to prevent tuberculosis, recombinant BCG is
being developed that over-expresses immunodominant antigens that have the potential to
elicit both CD4
+
and CD8
+
T cell responses. Thus, BCG over-expressing Ag85B has been
shown to be superior to conventional BCG at protecting guinea pigs from virulent
tuberculosis [43,44]. In addition to over-expressing selected mycobacterial antigens, BCG
vaccine strains have been engineered to co-express certain host genes, primarily cytokines
such as GM-CSF, which may provide an adjuvant or immunostimulatory effect [45]. One of
the principal advantages of vaccines based on mycobacteria is that they are more
antigenically “diverse” compared to subunit vaccines. From an immunological perspective,
a M. tuberculosis based vaccine should have a greater chance of eliciting a protective
immune response in a genetically diverse population than a subunit vaccine, which may
have only a limited number of epitopes, presented only by a restricted number of HLA
alleles. One hypothesis to explain why BCG vaccination fails to prevent pulmonary
tuberculosis is that its loss of certain genes during attenuation included key targets of
protective immunity. Consequently, recombinant BCG strains have been developed that
overexpress the immunodominant antigen ESAT-6, which is expressed by M. tuberculosis
but not by BCG [46]. Pym et al have shown that ESAT6-expressing BCG is superior to
BCG alone at inducing protection [46]. BCG has been engineered in other ways to improve
its ability to elicit protective immunity. For example, Stefan Kaufmann’s group has created a
recombinant BCG strain that should be better able to prime CD8
+
T cells. Listeriolysin is a
protein normally expressed by Listeria monocytogenes, which functions to help the bacteria
translocate from the phagosome into the cytosol. Grode et al engineered urease C
−
BCG to
express listeriolysin. Deleting urease C from BCG prevents alkalinization of the phagosome,
leading to a more optimum pH for listeriolysin activity [47]. Vaccination with this
engineered recombinant BCG strain led to improved protection against virulent M.
Behar et al. Page 9
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
tuberculosis as measured by lung CFU and survival. Macrophages infected with the
listeriolysin-expressing BCG also underwent more apoptosis compared to macrophages
infected with conventional BCG, which could also lead to greater cross-priming of CD8
+
T
cells [48,49]. These findings highlight the possibility that pro-apoptotic vaccines may
generate a better CD8
+
T cell response. It should be noted, however, that it has not yet been
addressed whether the increased protection afforded by the engineered BCG is due to an
increased CD8
+
T cell response, it’s proposed mechanism of action [50,51].
The desire to have a vaccine strain that closely resembles the pathogen M. tuberculosis has
led to the development of attenuated vaccine strains of M. tuberculosis that have well-
defined mutations. Laboratory selected mutants are generally the consequence of single gene
defects that are amenable to precise characterization. A important class of these mutants are
auxotrophs of M. tuberculosis. Auxotrophic strains are unable to grow under conventional
conditions unless media is supplemented with certain nutrients. Thus, a leucine auxotroph
requires growth media that is supplemented with leucine. Auxotrophs of M. tuberculosis and
other well-defined mutants such as the phoP deletion, potentially have even a better safety
profile than BCG, which may be advantageous for use in HIV-infected individuals; yet,
except for the defined defects, they express a full complement of antigens comparable to
pathogenic M. tuberculosis [52–55]. Nevertheless, safety concerns over using attenuated M.
tuberculosis in people, particularly in those that may be immunocompromised because of
HIV infection, has led investigators to develop strains with a least two independent
mutations. One potential problem with attenuated M. tuberculosis is their susceptibility to
growth restriction by the innate immune mechanisms may lead to an impaired induction of
adaptive immunity. Although attenuated M. tuberculosis strains exist that are capable of
inducing protection, the relationship between bacterial persistence, induction of memory
immunity, and protection, have not been clearly delineated in these examples [52,53,56].
Furthermore, pre-existing immunity to environmental mycobacteria may limit the
effectiveness of attenuated M. tuberculosis strains.
Other non-pathogenic mycobacterial strains have also been considered as vaccines such as
the nontuberculous bacteria M. vaccae and M. smegmatis [41,57]. All of these various
strains have the potential to elicit both CD4
+
and CD8
+
T cell responses. However, whether
bacterial strains that are unable to persist long-term are capable of eliciting long term
protective immunity remains to be determined [58]. If this is true, the less virulent, and
hence safer, vaccine strains, may not elicit truly long-lived immunity. Other bacteria,
including Listeria monocytogenes and Salmonella species, have also been developed as
delivery systems that elicit CD8
+
T cell responses. Although these bacteria elicit potent
CD8
+
T cell responses, they are also human pathogens and thus they require engineering so
they are safe for human administration.
Viral Vectors
Viruses are intracellular pathogens that assemble new daughter virions in the cytoplasm of
infected cells. Viral proteins enter the class I MHC pathway leading to the presentation of
viral antigens to CD8
+
T cells, which are an important component of immunity to most
viruses. Certain attenuated viruses can be engineered to express mycobacterial proteins and
vaccination with these recombinant viruses stimulates CD8
+
T cells. Two viruses with these
properties that are being developed as vaccines are adenovirus and vaccinia.
Recombinant Adenovirus
Many microbial pathogens invade the host at mucosal sites and mucosal vaccination may be
more effective at defending against these infectious diseases. Although it is debatable
whether M. tuberculosis is a true mucosal pathogen, much of the respiratory system,
Behar et al. Page 10
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
particularly the upper airway, is a mucosal site. This has led to interest in whether mucosal
based vaccines would provide superior protection against tuberculosis. One encouraging
approach has been the use of recombinant adenovirus. Adenovirus is a human pathogen that
causes mild upper respiratory infections and infects respiratory epithelial cells. Replication
defective adenovirus has been engineered as a vehicle for gene transfer and has also been
investigated as a vector for vaccination against a variety of infectious diseases including
AIDS and malaria. Zhou Xing has tested the feasibility of developing an adenovirus-based
vaccine for tuberculosis. A secreted form of Antigen 85A was cloned into a replication-
deficient serotype 5 human adenovirus (AdAg85A) [59]. Intranasal vaccination of mice with
AdAg85A activated both Ag85A-specific CD4
+
and CD8
+
T cells, which accumulated in
the lung, and protected mice in a M. tuberculosis respiratory challenge model. Treatment of
vaccinated mice with anti-CD4 or anti-CD8 impaired protection only slightly, whereas
treatment with both antibodies significantly impaired vaccine-induced protection. The
performance of AdAg85 is extremely encouraging, both alone and following priming by a
DNA vaccine. Follow-up studies have investigated the mechanism of protection in more
detail. Santosuosso et al showed that compared to IM administration, AdAg85A specifically
induced Ag85A-specific CD4
+
and CD8
+
T cells in the airway, and these T cells were
cytolytic and produced IFNγ upon activation [60]. Furthermore, these airway lumen derived
T cells were capable of protecting naïve recipient mice against respiratory tuberculosis.
Based on the potent antigen-specific CD8
+
T cell response elicited by this vaccination
strategy, this approach may provide further insight into the relative contribution of CD4
+
vs.
CD8
+
T cells in vaccine-induced protection against tuberculosis.
Pre-existing immunity to all vaccine vectors is a real consideration that may lead to the
blunting of effective vaccine-induced protection via premature clearance of the vaccine or a
skewed memory response towards vector antigens only. Indeed pre-existing immunity to
environmental mycobacteria is a prevailing hypothesis to explain the failure of BCG in
certain geographical regions [61]. Since adenovirus serotype 5 is a common viral infection
among people, recombinant adenovirus serotype 5-based vaccines are particularly
vulnerable to failure because of pre-existing immunity. The development of rAd systems
that use relatively rare adenovirus serotypes [62], or replace rAd5 coat proteins with those
from rare Ad serotypes [63] are strategies currently being pursued to circumvent anti-vector
immunity. Additionally, phase II trials using rAd5-based HIV vaccines will provide
additional information about the extent to which pre-existing immunity negatively affects
the efficacy of these vaccines.
Recombinant Vaccinia Virus
Vaccinia virus is an enveloped virus of the poxvirus family. It has a linear double-stranded
DNA and is unique in its cytoplasmic site of replication. Through its successful use in the
worldwide eradication of smallpox, vaccinia virus has proven itself as an important and
effective vaccine. Although protective immunity to vaccinia virus is incompletely
understood, infection with vaccinia induces virus-specific CD4
+
and CD8
+
T cells [64,65],
which have been shown to mediate protective effects against lethal infection, in the absence
of protective antibodies [66,67]. Recombinant vaccinia virus (rVV) has been appreciated as
a highly valuable expression and cloning vector for mammalian cells for nearly twenty-five
years. Almost immediately following the advent of rVV, development of rVV-based
vaccines began. The ability of rVV to generate effective heterologous antigen-specific CD8
+
T cell responses following immunization focused much of the early work on the
development of anti-viral vaccines for influenza, hepatitis B, and herpes simplex 1 and 2
viruses [68–70]. Indeed, in several cases the protective effect of rVV has been specifically
associated with pathogen-specific CD4
+
or CD8
+
T cells [71–73].
Behar et al. Page 11
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Vaccinia virus has many advantageous characteristics as a vaccine. It is highly immunogenic
with a broad host cell tropism. The vaccinia genome does not integrate itself into the host
genome. Recombinant technologies allow precise insertion, via homologous recombination,
into its large (~190 kbp) viral genome large that can accommodate large inserts. In contrast
to other large genome viruses, vaccinia does not appear to disrupt class I or class II MHC
processing and presentation.
Although, vaccinia virus has a strong efficacious history, safety concerns of this live vaccine
have lead to the development of attenuated vaccinia strains. Modified vaccinia virus Ankara
(MVA) was developed by greater than 570 passages in chicken embryo fibroblasts as a safer
smallpox vaccine. MVA has lost the promiscuous tropism of wild-type vaccinia virus, and is
unable to grow in human cells, as well as most other mammalian cells, in culture (Reviewed
in [74,75]). A safer vaccinia virus vector is of special importance considering the high
incidence of M. tuberculosis in HIV-infected individuals, and an early study of MVA safety
in HIV-patients is encouraging [76].
Although much of the initial vaccine development using rVV was focused on viral
pathogens, rVV expressing M. tuberculosis antigens have been studied since 1990 [77].
Administration of rVV expressing secreted Mtb 19kD or 38kD glycoproteins protected mice
against M. tuberculosis challenge although the immunological basis for protection was not
elucidated [78]. In 2000, Malin et al characterized the M. tuberculosis-specific cellular
immune response following vaccination with rVV expressing an M. tuberculosis antigen
[79]. Splenocytes isolated from mice immunized with rVV expressing Ag85A or Ag85B
secreted IL-2 and IFNγ in recall responses to the Ag85 complex, suggesting the stimulation
of M. tuberculosis-specific T cells. The first evidence that M. tuberculosis-specific CD8
+
T
cells could be elicited by vaccinia immunization was shown with rVV expressing the M.
tuberculosis early-secreted antigen MPT64 (rVV-MPT64) [80]. Feng et al reported the
generation of CTL specific for a MPT64-derived peptide vaccination of mice with rVV-
MPT64 [80]. Interestingly, the CTL generation was similar between rVV-MPT64 (the
Western Reserve vaccinia strain) and attenuated MVA-MPT64 (MVA expressing MPT64).
Furthermore, a prime-boost strategy in which mice were first immunized with a DNA
vaccine encoding MPT64 and then boosted with rVV-MPT64 or MVA-MPT64 further
increased the frequency of MPT64-specific CD8
+
T cells. Although it was never determined
whether vaccination with rVV-MPT64 or MVA-MPT64 could provide protection in an M.
tuberculosis challenge model, a similar DNA-rVV prime-boost strategy enhanced the
protective efficacy of MVA expressing ESAT-6 [71]. However, in this study, M.
tuberculosis-specific CD8
+
T cells could not be detected and the vaccine-induced protection
was presumably mediated by the measurable M. tuberculosis-specific CD4
+
T cell response.
The use of prime-boost strategies to enhance the efficacy of rVV may be of potential value
in improving M. tuberculosis vaccine design. Indeed, priming animals with BCG and then
boosting with MVA expressing Ag85A (MVA-Ag85A) enhances survival and decreases
bacterial burden after aerosol M. tuberculosis challenge compared to BCG alone [81,82].
MVA-Ag85A boosting of mice previously vaccinated with high doses of BCG enhanced
CD4
+
and CD8
+
T cells responses to Ag85A peptides. However, when a sub-optimal BCG
dose was used, MVA-Ag85A administration led to a boost in M. tuberculosis-specific CD4
+
T cells but not of the CD8
+
T cells. Because the boost did lead to an increase in protection,
this data argues that Ag85A-specific CD4
+
T cells were responsible for protective effect.
Regardless of the cellular basis for the protection mediated by MVA expressing M.
tuberculosis antigens, its success, particularly in cooperation with BCG, has propelled MVA
into clinical trials. Phase I trials show a good safety profile. People vaccinated with MVA-
Ag85A generated IFNγ-secreting CD4
+
T cells specific for Ag85; however, no Ag85A-
Behar et al. Page 12
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
specific CD8
+
T cells responses were detected. Ag85-specific T cells were increased in
subjects pre-exposed to BCG or environmental mycobacterium [83,84].
DNA vaccination
Since DNA vaccination can effectively induce cellular immunity, especially CD8
+
CTL and
Th1 response, genetic immunization is a promising approach for developing vaccines
against intracellular pathogens including M. tuberculosis. In the past few years, several
plasmid DNAs encoding an immunodominant antigens from M. tuberculosis have been
reported to induce protective immunity in animal models. Huygen et al showed that DNA
vaccines based on Ag85A reduced bacterial burdens in organs after aerosol or IV M.
tuberculosis challenge by establishing a cellular immune response characterized by antigen-
specific IFNγ production and CD8
+
CTL activity [85]. Vaccination with plasmid DNA
encoding the 38 kDa protein of M. tuberculosis also elicited antigen-specific CD8
+
T cell
responses [86]. Similarly, immunization of mice with DNA encoding Mtb72 fusion protein
elicits a strong CD4+ and CD8+ T cell response and this vaccine prolongs the survival of
guinea pigs following aerosol challenge with M. tuberculosis [87]. Additionally, DNA
vaccines have a therapeutic effect in mice infected with M. tuberculosis. When M.
tuberculosis infected BALB/c mice were given four doses of plasmid DNA encoding HSP65
of M. leprae, the number of viable bacteria in the spleen and lungs was dramatically reduced
up to 5 months later [88]. The therapeutic effect was associated with CD8
+
lung cell
activation and restored production of IFNγ induced by HSP65 DNA [89]. However, it has
been noted that HSP60/lep DNA vaccines induce severe pulmonary necrosis in an
immunotherapeutic model [90,91]. These findings raise a safety issue with regard to DNA
vaccines. Furthermore, in contrast to the encouraging results using mice, the clinical
experience with DNA vaccines has been disappointing [92,93] and new methods are needed
to enhance the immunogenicity of DNA vaccines.
DNA vaccines have a number of potential advantages compared to more conventional
vaccines such as peptide or attenuated virus. DNA vaccines are easily prepared and stable.
Plasmid DNA does not induce vector immunity and therefore can be repeatedly
administrated without side effects. Furthermore, DNA is able to be maintained in cells for
long-term expression of the encoded antigen. Thus, maintenance of immunologic memory is
possible. However, the level of protection induced by DNA vaccines has varied for different
antigens and generally is less than that induced by BCG immunization. Therefore, strategies
to increasing the effectiveness of DNA vaccines are needed. Prime-boost immunization
consisting of plasmid DNA and BCG have been developed in order to increase the immune
responses, particularly the CD8
+
T-cell responses. Priming with Ag85B-expressing DNA
and boosting with BCG was more effective than BCG immunization in protecting B6 mice
against aerosol M. tuberculosis challenge [94]. The enhanced protection observed following
prime-boost was diminished after CD8
+
T cell depletion, suggesting that the augmentation
of host resistance was partially mediated by CD8
+
T cells [94]. Several factors may
contribute to the improved protection. First, priming with DNA vaccines can focus the
immune response toward the dominant mycobacterial antigen, and away from potentially
harmful or non-protective antigens. Second, DNA vaccine priming is capable of increasing
antigen-specific CD4
+
and CD8
+
T cell responses and may be more potent than
mycobacteria at priming naïve CD8
+
T cells. Third, BCG immunization may effectively
amplify mycobacterium-specific Th1 and CD8
+
T responses primed by DNA immunization.
Prime-boost strategies can increase protection against M. tuberculosis either by priming with
DNA or by boosting with DNA vaccines. This may have practical implications since
Derrick et al reported that waning BCG-induced anti-tuberculosis protective immunity could
be boosted in aging mice by vaccinating with a DNA vaccine expressing an ESAT6-Antigen
85B fusion protein [95]. Interestingly, the number of CD8
+
cells secreting IFNγ post
Behar et al. Page 13
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
challenge with M. tuberculosis, was substantially elevated indicating that BCG vaccination
induced memory CD8
+
T cells that could be expanded by the DNA boost and ultimately
activated by the infection [96].
Other approaches have been investigated to increase the effectiveness of DNA vaccination
against tuberculosis including the codelivery of plasmid expressing cytokines, such IL-12
[97] or IL-2 [98], or coimmunization with DNA vectors encoding multiple antigens [99].
Alternatively, multiple CpG motifs can be added to the DNA backbone to increase its
adjuvant effects. Furthermore, the development of new DNA vaccines that incorporate the
Sindbis virus RNA replicase increase the immunogenicity of conventional DNA vaccines
and allow lower doses of DNA to be administered [100]. For example, Derrick et al found
that a Sindbis virus-based DNA vaccine encoding the M. tuberculosis Ag85B was able to
provide protection comparable to that of BCG based both on a reduction in CFU and a
prolongation in survival [29]. Based on data showing that apoptotic vesicles prime CD8
+
T
cells, there is interest in developing pro-apoptotic vaccines. DNA vaccines encoding fusion
proteins in which the antigen is fused to a pro-apoptotic protein such as CD95 may increase
their effectiveness [101]. Finally, coadminstration of DNA and proteins or live vectors in
prime-boost strategies have been used to augment the protective response to M. tuberculosis
infection [102]. A combination of these approaches may be necessary to obtain clinically
significant long-lived protective efficacy for DNA vaccines in humans.
HIV
+
individuals co-infected with M. tuberculosis are very frequently associated with a drop
in CD4
+
T cell counts. The development of a new vaccine against M. tuberculosis for use in
HIV
+
persons has been considered unlikely because of the presumed essential roles that
CD4
+
T cells play in controlling M. tuberculosis infections. Derrick et al demonstrated that
immunization with a DNA vaccine cocktail protected CD4
−/−
mice against an aerosol
infection with M. tuberculosis [103], suggesting that it may be possible to develop effective
M. tuberculosis vaccines in HIV-infected populations. By contrast, DNA vaccines encoding
mycobacterial antigens Ag85A, Ag85B and PstS3 from M. tuberculosis were ineffective in
mice lacking CD4
+
T cells [104]. Although the specific reasons for these disparate results
are unclear, the use of different mouse strains, M. tuberculosis antigens, the mode and
timing of infectious challenge may have contributed to the differing outcomes. In the latter
study, reconstitution of CD4
+
T cell compartment in CD4
−/−
mice restored DNA vaccine-
mediated anti-mycobacterial immune responses and protection [104]. CD4
+
T cell counts
are known to increase under effective and highly active anti-retroviral therapy in HIV-
infected patients. Taken together, the findings provide evidence to support that DNA
vaccination is a promising direction for the treatment of HIV and M. tuberculosis co-
infected patients.
Finally, an interesting hybrid approach uses attenuated intracellular bacteria as a delivery
system for DNA vaccines. Dietrich et al cloned Ag85A, Ag85B or MPB51 into a plasmid
that was stably carried by the L. monocytogenes Δ2 mutant [105]. Following infection of
APC, this mutant escapes from the phagosome into the cytosol, and in the cytosol it
undergoes self-destruction but releases its plasmids. This approach to DNA vaccination
appears to be able to induce protective immunity against M. tuberculosis [106]. Additional
work including a detailed immunological analysis will be required to determine whether this
ability to target plasmid delivery makes immunization more efficient.
Subunit vaccines
The most effective and practical way to elicit a CD8
+
T cell response, whether
experimentally or clinically, is to vaccinate with a viral vector or with plasmid DNA (for
example see [107]). However, Skeiky et al have evaluated the vaccine 72F. The 72F vaccine
consists of the Mtb39 protein inserted into the middle of the Mtb32 protein. Vaccination
Behar et al. Page 14
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
with plasmid DNA encoding 72F elicits a robust CTL response against Mtb32
93–102
and
generates protective immunity in both mice and guinea pigs [87]. Interestingly, when the
vaccine is formulated as a polyprotein vaccine, it elicits a strong CTL response against
Mtb32
93–102
, an epitope that elicits CD8
+
T cells after DNA vaccination. A critical variable
appears to be the adjuvant – and the adjuvant AS01B seems to be particularly effective at
stimulating a CD8
+
T cell response. Although it is not yet clear whether the elicited CD8
+
T
cells contribute to the protective effect induced by the DNA vaccine or the adjuvanated
polyprotein, it raises the possibility that protein subunit vaccines coupled with appropriate
adjuvants can stimulate CD8
+
T cells in addition to CD4
+
T cells.
Expert Commentary
An important issue in designing tuberculosis vaccines is to effectively incorporate advances
in immunology. This is particularly relevant for vaccines designed to elicit CD8
+
T cells
because the target antigens are still being defined. Furthermore, the way CD8
+
T cells are
elicited may affect both their antigen specificity and function. During the past five years, the
progress in defining mycobacterial antigens recognized by human and murine CD8
+
T cells
has led to the identification of several vaccine candidates that can now be tested for their
ability to protect experimental animals against virulent M. tuberculosis (reviewed in [13]).
Despite the availability of other animal models, the mouse is still the most facile model to
determine induction of adaptive immunity and protection as measures of vaccine efficacy.
Establishing whether a particular antigen elicits protective immunity is a task that is subject
to various pitfalls. A critical issue is whether vaccination elicits an adaptive immune
response. For antigens recognized by CD4
+
T cells, this can be measured post-vaccination
by stimulating the subject’s leukocytes with the immunizing antigen and assaying the T cell
recall response either by a cytokine assay (e.g., IFNγ production) or by measuring T cell
proliferation. If pure recombinant protein is not available, crude mixtures of antigen such as
M. tuberculosis sonicate or PPD are often sufficient, as long as the vaccinating antigen is
represented in the crude antigen preparation. In contrast, this strategy will not work for
CD8
+
T cells as it is necessary to get the antigen into the cytosol of the APC. For
investigators committed to the study of a single antigen, the cDNA encoding the antigen can
be used to transfect tumor cell lines to create artificial APC expressing the antigen.
However, this limits the choice of APC. A more versatile approach uses defined peptides.
As more mycobacterial epitopes recognized by CD8
+
T cells are defined, investigators will
no longer be forced to empirically determine whether vaccine-induced CD8
+
T cells are
protective.
Although defining the precise epitopes recognized by CD8
+
T cells is important, particularly
during the early stages of vaccine development, it is important to recognize that most T cell
epitopes specifically bind to a single MHC molecule. One of the strengths of the mouse
model, the availability of genetically defined inbred strains, is also an important limitation
for vaccine studies. Most tuberculosis vaccines are tested in one or two commonly used
mouse strains. The majority of studies use C57BL/6 mice and this practice significantly
reduces the potential immunogenetic diversity of the host. This is because C57BL/6 mice
express a single class II MHC molecule (I-A
b
) and two class I MHC molecules (K
b
and D
b
).
Humans have three distinct class I MHC molecules (HLA-A, -B, -C) and three distinct class
II MHC molecules (HLA-DP, -DQ, -DR), and since most people have two different alleles
that encode these antigen-presenting molecules, each individual potentially expresses 12
distinct MHC proteins. Thus, the probability that a peptide derived from a mycobacterial
protein will bind to a MHC molecule is greater for human APC than for murine APC. This
effect in demonstrated by CD8
+
T cell recognition of CFP10. Neither C57BL/6 nor BALB/c
mice generate a CD8
+
T cell response to CFP10; in contrast, nearly 30% of the CD8
+
T cells
in the lungs of infected C3H (H-2
k
) mice are specific for CFP10 [18]. After infection with
Behar et al. Page 15
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
M. tuberculosis, many people with diverse MHC haplotypes, mount a CD8
+
T cell response
to CFP10 [108,109]. Thus, the CD8
+
T cell response of C57BL/6 mice is not predictive of
the human CD8
+
T cell response, probably because of its limited MHC diversity. In vivo
models with greater MHC diversity may better predict true “antigenicity” albeit with the risk
of significantly complicating the immunological analysis.
As more effort is applied to developing tuberculosis vaccines that elicit CD8
+
T cells, new
challenges are encountered. CD8
+
T cells induced by vaccination may recognize different
mycobacterial epitopes than CD8
+
T cells elicited following natural infection. After
administration of an Ag85A DNA vaccine, the array of epitopes recognized by both CD4
+
and CD8
+
T cells is broader compared to the number of epitopes recognized by T cells from
M. tuberculosis infected mice [110]. In fact, CD8
+
T cells from DNA vaccinated mice
recognize three peptide epitopes from Ag85A that were not recognized by CD8
+
T cells
from M. tuberculosis infected mice. One explanation is that the frequency of Ag85A-
specific CD8
+
T cells significantly increases following vaccination, whereas it remains very
low after infection. If this were true, these vaccine-induced CD8
+
T cells may enhance
protective immunity, but only if the relevant Ag85A peptide epitopes are processed and
presented by M. tuberculosis infected cells.
In another dramatic example of how the way a vaccine is delivered can alter which epitopes
elicit immune T cells, Peter Andersen developed a fusion protein consisting of ESAT6
covalently linked to Ag85B as a subunit vaccine [107]. This vaccine elicits a CD4
+
T cell
dominated response to ESAT6
1–15
and Ag85B
241–255
, and induces protective immunity in
C57BL/6 mice. To potentially enhance vaccine efficacy, the gene construct encoding the
ESAT6/Ag85B fusion protein was inserted into adenovirus. Interestingly, rAd
ESAT6/Ag85B
elicits T cells that recognized a significantly different repertoire of epitopes, including a
CD8
+
T cell dominated response to ESAT6
15–29
. In contrast, the frequency of CD4
+
T cells
recognizing ESAT6
1–15
and Ag85B
241–255
were markedly reduced. Interestingly, CD8
+
T
cells specific for ESAT6
15–29
have never been detected following M. tuberculosis infection.
Despite eliciting antigen-specific T cells capable of producing IFNγ, the rAd
ESAT6/Ag85B
form of the vaccine failed to protect mice. Perhaps this is due to the failure of ESAT6
15–29
presentation by M. tuberculosis infected cells, so that even though abundant, these CD8
+
T
cells are unable to recognize infected macrophages and therefore can not contribute to host
immunity. The rules governing peptide selection for presentation by class I MHC have not
been completely defined. Thus, even with the precise definition of epitopes recognized by
CD8
+
T cells, the task of developing a vaccine that elicits the desired CD8
+
T cells is still
formidable. With our increasing appreciation that CD8
+
T cells serve to protect the host
against tuberculosis, there is renewed interest in developing immunization strategies that can
induce protective CD8
+
T cells. Testing this hypothesis, i.e., that immunization strategies
designed to elicit CD8
+
T cells, such as rVV and listeriolysin-expressing BCG, actually
induce protective CD8
+
T cells, is required as these vaccines move forward in clinical
development.
Five-year View
The appreciation that CD8
+
T cells play an important non-redundant role in host immunity
against M. tuberculosis infection has led to a greater interest in their stimulation by
vaccination and their role in vaccine-induced immunity. During the past few years, several
mycobacterial antigens that are recognized by human and murine CD8
+
T cells have been
defined, some of which are immunodominant antigens after experimental infection ([18,111]
and reviewed in [13]), and large-scale antigen discovery efforts are underway, particularly to
define the mycobacterial epitopes recognized by human CD8
+
T cells. These efforts will
Behar et al. Page 16
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
define the prospective vaccine candidates but also will provide reagents that can be used to
monitor the immunological success of eliciting antigen-specific CD8
+
T cells.
In parallel, because of the complexity of the immune response elicited by many vaccines,
more in vivo models are beginning to be applied in a more sophisticated way in order to
definitively demonstrate the protective potential of vaccine elicited CD8
+
T cells. Because
CD4 −/− mice have an unusual population of CD4
−
CD8
−
T cells that are class II MHC-
restricted and can provide Th1-like function, their usefulness in assessing protection
mediated solely by CD8
+
T cells is more limited than previously thought. Instead, traditional
adoptive transfer models using flow cytometry to sort pure populations are being introduced
into the BL3 setting, and the function of CD8
+
T cells can be better evaluated in class II
MHC or RAG-1 knockout mice.
We have recently learned that the development of both CD8
+
T cell memory and certain
CD8
+
T cell effector functions are dependent upon CD4
+
T cells. We expect that during the
next few years, a greater emphasis will be placed on understanding the synergy between
CD4
+
and CD8
+
T cell responses and elucidating how to optimally stimulate these T cells
simultaneously. This is an important goal since many of the vaccines that are in clinical
development, including recombinant BCG, M. tuberculosis auxotrophs, DNA vaccines, and
recombinant viruses, have the potential to elicit both antigen-specific CD4
+
and CD8
+
T
cells.
Perhaps most important, as tuberculosis vaccines start clinical phase I/II trials, we will have
a unique opportunity to learn about both the CD4
+
and CD8
+
T cell response, not in animal
models, but in people. This data will provide invaluable insight about how host
immunogenetics, antigen diversity, and specific vaccine strategies all interact to establish a
protective T cell response.
Acknowledgments
This work was supported by National Institutes of Health grant R01 AI47171 and American Lung Association
Career Investigator Award to SMB.
Reference List
1. Lefford MJ. Transfer of adoptive immunity to tuberculosis in mice. Infect Immun. 1975; 11:1174–
1181. [PubMed: 806520]
2. Orme IM, Collins FM. Adoptive protection of the Mycobacterium tuberculosis-infected lung.
Dissociation between cells that passively transfer protective immunity and those that transfer
delayed-type hypersensitivity to tuberculin. Cell Immunol. 1984; 84:113–120. [PubMed: 6421492]
3*. Orme IM. The kinetics of emergence and loss of mediator T lymphocytes acquired in response to
infection with Mycobacterium tuberculosis. J Immunol. 1987; 138:293–298. Classic study
showing that following M. tuberculosis infection, CD4
+
and CD8
+
T cells acquire the capacity to
transfer resistance against tuberculosis to naïve mice. [PubMed: 3097148]
4. Orme IM. Characteristics and specificity of acquired immunologic memory to Mycobacterium
tuberculosis infection. J Immunol. 1988; 140:3589–3593. [PubMed: 3129497]
5. Pedrazzini T, Hug K, Louis JA. Importance of L3T4+ and Lyt-2+ cells in the immunologic control
of infection with Mycobacterium bovis strain bacillus Calmette-Guerin in mice. Assessment by
elimination of T cell subsets in vivo. J Immunol. 1987; 139:2032–2037. [PubMed: 3114383]
6. Feng CG, Britton WJ. CD4+ and CD8+ T cells mediate adoptive immunity to aerosol infection of
Mycobacterium bovis bacillus Calmette-Guerin. J Infect Dis. 2000; 181:1846–1849. [PubMed:
10823799]
Behar et al. Page 17
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
7. Muller I, Cobbold SP, Waldmann H, Kaufmann SH. Impaired resistance to Mycobacterium
tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. Infect Immun.
1987; 55:2037–2041. [PubMed: 3114143]
8. Cox JH, Knight BC, Ivanyi J. Mechanisms of recrudescence of Mycobacterium bovis BCG infection
in mice. Infect Immun. 1989; 57:1719–1724. [PubMed: 2656521]
9*. Sousa AO, Mazzaccaro RJ, Russell RG, Lee FK, Turner OC, Hong S, Van Kaer L, Bloom BR.
Relative contributions of distinct MHC class I-dependent cell populations in protection to
tuberculosis infection in mice. Proc Natl Acad Sci U S A. 2000; 97:4204–4208. Comprehensive
study ranking the contribution of β2m, TAP1, CD1, CD8 and perforin to resistance against
tuberculosis. [PubMed: 10760288]
10. Behar SM, Dascher CC, Grusby MJ, Wang CR, Brenner MB. Susceptibility of mice deficient in
CD1D or TAP1 to infection with Mycobacterium tuberculosis. J Exp Med. 1999; 189:1973–1980.
[PubMed: 10377193]
11. Rolph MS, Raupach B, Kobernick HH, Collins HL, Perarnau B, Lemonnier FA, Kaufmann SH.
MHC class Ia-restricted T cells partially account for beta2-microglobulin-dependent resistance to
Mycobacterium tuberculosis. Eur J Immunol. 2001; 31:1944–1949. [PubMed: 11433392]
12. Urdahl KB, Liggitt D, Bevan MJ. CD8+ T cells accumulate in the lungs of Mycobacterium
tuberculosis-infected Kb−/−Db−/− mice, but provide minimal protection. J Immunol. 2003;
170:1987–1994. [PubMed: 12574368]
13*. Woodworth JS, Behar SM. Mycobacterium tuberculosis-specific CD8+ T cells and their role in
immunity. Crit Rev Immunol. 2006; 26:317–352. A critical review examining the role of CD8
+
T
cells in immunity to tuberculosis. Includes a comprehensive discussion of which mycobacterial
antigens are recognized by CD8
+
T cells following infection. [PubMed: 17073557]
14**. Mogues T, Goodrich M, Ryan L, LaCourse R, North R. The Relative Importance of T Cell
Subsets in Immunity and Immunopathology of Airborne Mycobacterium tuberculosis Infection in
Mice. J Exp Med. 2001; 193:271–280. An elegant study that defines the relative contribution of
different T cell subsets and various effector molecules to host resistance against pulmonary
tuberculosis. [PubMed: 11157048]
15. van Pinxteren LA, Cassidy JP, Smedegaard BH, Agger EM, Andersen P. Control of latent
Mycobacterium tuberculosis infection is dependent on CD8 T cells. Eur J Immunol. 2000;
30:3689–3698. [PubMed: 11169412]
16. Smith CM, Wilson NS, Waithman J, Villadangos JA, Carbone FR, Heath WR, Belz GT. Cognate
CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity. Nat Immunol. 2004;
5:1143–1148. [PubMed: 15475958]
17. Serbina NV, Lazarevic V, Flynn JL. CD4(+) T cells are required for the development of cytotoxic
CD8(+) T cells during Mycobacterium tuberculosis infection. J Immunol. 2001; 167:6991–7000.
[PubMed: 11739519]
18*. Kamath AB, Woodworth J, Xiong X, Taylor C, Weng Y, Behar SM. Cytolytic CD8+ T Cells
Recognizing CFP10 Are Recruited to the Lung after Mycobacterium tuberculosis Infection. J
Exp Med. 2004; 200:1479–1489. First identification of an immunodominant antigen that elicits a
CD8
+
T cell response that is cytolytic in vivo. [PubMed: 15557351]
19. Romano M, Denis O, D’Souza S, Wang XM, Ottenhoff TH, Brulet JM, Huygen K. Induction of in
vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport
receptor of Mycobacterium tuberculosis. J Immunol. 2004; 172:6913–6921. [PubMed: 15153510]
20. McShane H, Behboudi S, Goonetilleke N, Brookes R, Hill AV. Protective immunity against
Mycobacterium tuberculosis induced by dendritic cells pulsed with both CD8(+)- and CD4(+)-T-
cell epitopes from antigen 85A. Infect Immun. 2002; 70:1623–1626. [PubMed: 11854254]
21. Yewdell JW, Reits E, Neefjes J. Making sense of mass destruction: quantitating MHC class I
antigen presentation. Nat Rev Immunol. 2003; 3:952–961. [PubMed: 14647477]
22. Cresswell P, Ackerman AL, Giodini A, Peaper DR, Wearsch PA. Mechanisms of MHC class I-
restricted antigen processing and cross-presentation. Immunol Rev. 2005; 207:145–157. [PubMed:
16181333]
Behar et al. Page 18
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
23. Majlessi L, Rojas MJ, Brodin P, Leclerc C. CD8+-T-cell responses of Mycobacterium-infected
mice to a newly identified major histocompatibility complex class I-restricted epitope shared by
proteins of the ESAT-6 family. Infect Immun. 2003; 71:7173–7177. [PubMed: 14638811]
24. Smith SM, Malin AS, Pauline T, Lukey Atkinson SE, Content J, Huygen K, Dockrell HM.
Characterization of Human Mycobacterium bovis Bacille Calmette-Guerin-Reactive CD8+ T
Cells. Infect Immun. 1999; 67:5223–5230. [PubMed: 10496899]
25. McMurray DN. A coordinated strategy for evaluating new vaccines for human and animal
tuberculosis. Tuberculosis. 2001; 81:141–146. [PubMed: 11463235]
26. Kamath AT, Fruth U, Brennan MJ, Dobbelaer R, Hubrechts P, Ho MM, Mayner RE, Thole J,
Walker KB, Liu M, Lambert PH. New live mycobacterial vaccines: the Geneva consensus on
essential steps towards clinical development. Vaccine. 2005; 23:3753–3761. [PubMed: 15893612]
27. Orme IM, McMurray DN, Belisle JT. Tuberculosis vaccine development: recent progress. Trends
Microbiol. 2001; 9:115–118. [PubMed: 11239788]
28. Kamath AB, Behar SM. Anamnestic Responses of Mice following Mycobacterium tuberculosis
Infection. Infect Immun. 2005; 73:6110–6118. [PubMed: 16113332]
29. Derrick SC, Yang AL, Morris SL. Vaccination with a Sindbis Virus-Based DNA Vaccine
Expressing Antigen 85B Induces Protective Immunity against Mycobacterium tuberculosis. Infect
Immun. 2005; 73:7727–7735. [PubMed: 16239577]
30. Romano M, D’Souza S, Adnet PY, Laali R, Jurion F, Palfliet K, Huygen K. Priming but not
boosting with plasmid DNA encoding mycolyl-transferase Ag85A from Mycobacterium
tuberculosis increases the survival time of Mycobacterium bovis BCG vaccinated mice against low
dose intravenous challenge with M. tuberculosis H37Rv. Vaccine. 2006; 24:3353–3364. [PubMed:
16488518]
31. Mellman I, Steinman RM. Dendritic cells: specialized and regulated antigen processing machines.
Cell. 2001; 106:255–258. [PubMed: 11509172]
32. Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the
mannose receptor to concentrate macromolecules in the major histocompatibility complex class II
compartment: downregulation by cytokines and bacterial products. J Exp Med. 1995; 182:389–
400. [PubMed: 7629501]
33. Delamarre L, Pack M, Chang H, Mellman I, Trombetta ES. Differential lysosomal proteolysis in
antigen-presenting cells determines antigen fate. Science. 2005; 307:1630–1634. [PubMed:
15761154]
34. Fujii S, Liu K, Smith C, Bonito AJ, Steinman RM. The linkage of innate to adaptive immunity via
maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and
CD80/86 costimulation. J Exp Med. 2004; 199:1607–1618. [PubMed: 15197224]
35. Demangel C, Bean AG, Martin E, Feng CG, Kamath AT, Britton WJ. Protection against aerosol
Mycobacterium tuberculosis infection using Mycobacterium bovis Bacillus Calmette Guerin-
infected dendritic cells. Eur J Immunol. 1999; 29:1972–1979. [PubMed: 10382760]
36. Lewinsohn DA, Lines RA, Lewinsohn DM. Human dendritic cells presenting adenovirally
expressed antigen elicit Mycobacterium tuberculosis--specific CD8+ T cells. Am J Respir Crit
Care Med. 2002; 166:843–848. [PubMed: 12231495]
37. Malowany JI, McCormick S, Santosuosso M, Zhang X, Aoki N, Ngai P, Wang J, Leitch J,
Bramson J, Wan Y, Xing Z. Development of cell-based tuberculosis vaccines: genetically
modified dendritic cell vaccine is a much more potent activator of CD4 and CD8 T cells than
peptide- or protein-loaded counterparts. Mol Ther. 2006; 13:766–775. [PubMed: 16343993]
38. Nakano H, Nagata T, Suda T, Tanaka T, Aoshi T, Uchijima M, Kuwayama S, Kanamaru N, Chida
K, Nakamura H, Okada M, Koide Y. Immunization with dendritic cells retrovirally transduced
with mycobacterial antigen 85A gene elicits the specific cellular immunity including cytotoxic T-
lymphocyte activity specific to an epitope on antigen 85A. Vaccine. 2006; 24:2110–2119.
[PubMed: 16352377]
39. Gonzalez-Juarrero M, Turner J, Basaraba RJ, Belisle JT, Orme IM. Florid pulmonary
inflammatory responses in mice vaccinated with Antigen-85 pulsed dendritic cells and challenged
by aerosol with Mycobacterium tuberculosis. Cell Immunol. 2002; 220:13–19. [PubMed:
12718935]
Behar et al. Page 19
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
40. Pithie AD, Rahelu M, Kumararatne DS, Drysdale P, Gaston JS, Iles PB, Innes JA, Ellis CJ.
Generation of cytolytic T cells in individuals infected by Mycobacterium tuberculosis and
vaccinated with BCG. Thorax. 1992; 47:695–701. [PubMed: 1440463]
41. Cayabyab MJ, Hovav AH, Hsu T, Krivulka GR, Lifton MA, Gorgone DA, Fennelly GJ, Haynes
BF, Jacobs WR Jr, Letvin NL. Generation of CD8+ T-cell responses by a recombinant
nonpathogenic Mycobacterium smegmatis vaccine vector expressing human immunodeficiency
virus type 1 Env. J Virol. 2006; 80:1645–1652. [PubMed: 16439521]
42. Uno-Furuta S, Matsuo K, Tamaki S, Takamura S, Kamei A, Kuromatsu I, Kaito M, Matsuura Y,
Miyamura T, Adachi Y, Yasutomi Y. Immunization with recombinant Calmette-Guerin bacillus
(BCG)-hepatitis C virus (HCV) elicits HCV-specific cytotoxic T lymphocytes in mice. Vaccine.
2003; 21:3149–3156. [PubMed: 12804842]
43. Horwitz MA, Harth G, Dillon BJ, Maslesa-Galic’ S. Recombinant bacillus Calmette-Guerin (BCG)
vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce
greater protective immunity against tuberculosis than conventional BCG vaccines in a highly
susceptible animal model. PNAS. 2000; 97:13853–13858. [PubMed: 11095745]
44. Horwitz MA, Harth G. A New Vaccine against Tuberculosis Affords Greater Survival after
Challenge than the Current Vaccine in the Guinea Pig Model of Pulmonary Tuberculosis. Infect
Immun. 2003; 71:1672–1679. [PubMed: 12654780]
45. Murray PJ, Aldovini A, Young RA. Manipulation and potentiation of antimycobacterial immunity
using recombinant bacille Calmette-Guerin strains that secrete cytokines. Proc Natl Acad Sci U S
A. 1996; 93:934–939. [PubMed: 8570663]
46. Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths KE, Marchal G,
Leclerc C, Cole ST. Recombinant BCG exporting ESAT-6 confers enhanced protection against
tuberculosis. Nat Med. 2003; 9:533–539. [PubMed: 12692540]
47**. Grode L, Seiler P, Baumann S, Hess J, Brinkmann V, Nasser EA, Mann P, Goosmann C,
Bandermann S, Smith D, Bancroft GJ, Reyrat JM, van SD, Raupach B, Kaufmann SH. Increased
vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-
Guerin mutants that secrete listeriolysin. J Clin Invest. 2005; 115:2472–2479. A mutant of BCG
that has been engineered to express listeriolysin and this recombinant BCG is highly effective as
a vaccine in a mouse model. [PubMed: 16110326]
48**. Winau F, Weber S, Sad S, de Diego J, Hoops SL, Breiden B, Sandhoff K, Brinkmann V,
Kaufmann SH, Schaible UE. Apoptotic vesicles crossprime CD8 T cells and protect against
tuberculosis. Immunity. 2006; 24:105–117. Demonstrates that not only do apoptotic vesicles
prime CD8+ T cells in vivo, but in addition, these vesicles elicit a protective adaptive immune
response. [PubMed: 16413927]
49**. Schaible UE, Winau F, Sieling PA, Fischer K, Collins HL, Hagens K, Modlin RL, Brinkmann
V, Kaufmann SH. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I
and CD1 in tuberculosis. Nat Med. 2003; 9:1039–1046. Shows that apoptotic vesicles from
infected cells are taken up by DC and presented to class I MHC and CD1-restricted human T
cells. [PubMed: 12872166]
50. Hess J, Miko D, Catic A, Lehmensiek V, Russell DG, Kaufmann SH. Mycobacterium bovis
Bacille Calmette-Guerin strains secreting listeriolysin of Listeria monocytogenes. Proc Natl Acad
Sci U S A. 1998; 95:5299–5304. [PubMed: 9560270]
51. Conradt P, Hess J, Kaufmann SH. Cytolytic T-cell responses to human dendritic cells and
macrophages infected with Mycobacterium bovis BCG and recombinant BCG secreting
listeriolysin. Microbes Infect. 1999; 1:753–764. [PubMed: 10816080]
52. Martin C, Williams A, Hernandez-Pando R, Cardona PJ, Gormley E, Bordat Y, Soto CY, Clark
SO, Hatch GJ, Aguilar D, Ausina V, Gicquel B. The live Mycobacterium tuberculosis phoP
mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in
mice and guinea pigs. Vaccine. 2006; 24:3408–3419. [PubMed: 16564606]
53. Sambandamurthy VK, Derrick SC, Jalapathy KV, Chen B, Russell RG, Morris SL, Jacobs WR Jr.
Long-term protection against tuberculosis following vaccination with a severely attenuated double
lysine and pantothenate auxotroph of Mycobacterium tuberculosis. Infect Immun. 2005; 73:1196–
1203. [PubMed: 15664964]
Behar et al. Page 20
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
54. Sampson SL, Dascher CC, Sambandamurthy VK, Russell RG, Jacobs WR Jr, Bloom BR,
Hondalus MK. Protection elicited by a double leucine and pantothenate auxotroph of
Mycobacterium tuberculosis in guinea pigs. Infect Immun. 2004; 72:3031–3037. [PubMed:
15102816]
55. Sambandamurthy VK, Wang X, Chen B, Russell RG, Derrick S, Collins FM, Morris SL, Jacobs
WR Jr. A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated and protects
mice against tuberculosis. Nat Med. 2002; 8:1171–1174. [PubMed: 12219086]
56. Sambandamurthy VK, Derrick SC, Hsu T, Chen B, Larsen MH, Jalapathy KV, Chen M, Kim J,
Porcelli SA, Chan J, Morris SL, Jacobs WR Jr. Mycobacterium tuberculosis DeltaRD1
DeltapanCD: a safe and limited replicating mutant strain that protects immunocompetent and
immunocompromised mice against experimental tuberculosis. Vaccine. 2006; 24:6309–6320.
[PubMed: 16860907]
57. Skinner MA, Yuan S, Prestidge R, Chuk D, Watson JD, Tan PL. Immunization with heat-killed
Mycobacterium vaccae stimulates CD8+ cytotoxic T cells specific for macrophages infected with
Mycobacterium tuberculosis. Infect Immun. 1997; 65:4525–4530. [PubMed: 9353029]
58. Lagranderie MR, Balazuc AM, Deriaud E, Leclerc CD, Gheorghiu M. Comparison of immune
responses of mice immunized with five different Mycobacterium bovis BCG vaccine strains.
Infect Immun. 1996; 64:1–9. [PubMed: 8557324]
59*. Wang J, Thorson L, Stokes RW, Santosuosso M, Huygen K, Zganiacz A, Hitt M, Xing Z. Single
mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides
potent protection from pulmonary tuberculosis. J Immunol. 2004; 173:6357–6365. Nice
demonstration of the use of recombinant adenovirus to elicit protective immunity against
tuberculosis in the murine model. [PubMed: 15528375]
60. Santosuosso M, Zhang X, McCormick S, Wang J, Hitt M, Xing Z. Mechanisms of mucosal and
parenteral tuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits
sustained accumulation of immune protective CD4 and CD8 T cells within the airway lumen. J
Immunol. 2005; 174:7986–7994. [PubMed: 15944305]
61. Andersen P, Doherty TM. The success and failure of BCG - implications for a novel tuberculosis
vaccine. Nat Rev Microbiol. 2005; 3:656–662. [PubMed: 16012514]
62. Havenga M, Vogels R, Zuijdgeest D, Radosevic K, Mueller S, Sieuwerts M, Weichold F, Damen I,
Kaspers J, Lemckert A, van MM, van d V, Holterman L, Hone D, Skeiky Y, Mintardjo R,
Gillissen G, Barouch D, Sadoff J, Goudsmit J. Novel replication-incompetent adenoviral B-group
vectors: high vector stability and yield in PER.C6 cells. J Gen Virol. 2006; 87:2135–2143.
[PubMed: 16847108]
63. Roberts DM, Nanda A, Havenga MJ, Abbink P, Lynch DM, Ewald BA, Liu J, Thorner AR,
Swanson PE, Gorgone DA, Lifton MA, Lemckert AA, Holterman L, Chen B, Dilraj A, Carville A,
Mansfield KG, Goudsmit J, Barouch DH. Hexon-chimaeric adenovirus serotype 5 vectors
circumvent pre-existing anti-vector immunity. Nature. 2006; 441:239–243. [PubMed: 16625206]
64. Harrington LE, Most RR, Whitton JL, Ahmed R. Recombinant vaccinia virus-induced T-cell
immunity: quantitation of the response to the virus vector and the foreign epitope. J Virol. 2002;
76:3329–3337. [PubMed: 11884558]
65. Xu R, Johnson AJ, Liggitt D, Bevan MJ. Cellular and humoral immunity against vaccinia virus
infection of mice. J Immunol. 2004; 172:6265–6271. [PubMed: 15128815]
66. Belyakov IM, Earl P, Dzutsev A, Kuznetsov VA, Lemon M, Wyatt LS, Snyder JT, Ahlers JD,
Franchini G, Moss B, Berzofsky JA. Shared modes of protection against poxvirus infection by
attenuated and conventional smallpox vaccine viruses. Proc Natl Acad Sci U S A. 2003;
100:9458–9463. [PubMed: 12869693]
67. Snyder JT, Belyakov IM, Dzutsev A, Lemonnier F, Berzofsky JA. Protection against lethal
vaccinia virus challenge in HLA-A2 transgenic mice by immunization with a single CD8+ T-cell
peptide epitope of vaccinia and variola viruses. J Virol. 2004; 78:7052–7060. [PubMed:
15194781]
68. Smith GL, Murphy BR, Moss B. Construction and characterization of an infectious vaccinia virus
recombinant that expresses the influenza hemagglutinin gene and induces resistance to influenza
virus infection in hamsters. Proc Natl Acad Sci U S A. 1983; 80:7155–7159. [PubMed: 6580632]
Behar et al. Page 21
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
69. Moss B, Smith GL, Gerin JL, Purcell RH. Live recombinant vaccinia virus protects chimpanzees
against hepatitis B. Nature. 1984; 311:67–69. [PubMed: 6472464]
70. Cremer KJ, Mackett M, Wohlenberg C, Notkins AL, Moss B. Vaccinia virus recombinant
expressing herpes simplex virus type 1 glycoprotein D prevents latent herpes in mice. Science.
1985; 228:737–740. [PubMed: 2986288]
71. McShane H, Brookes R, Gilbert SC, Hill AV. Enhanced immunogenicity of CD4(+) t-cell
responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost
vaccination regimen for murine tuberculosis. Infect Immun. 2001; 69:681–686. [PubMed:
11159955]
72. Moss B. Genetically engineered poxviruses for recombinant gene expression, vaccination, and
safety. Proc Natl Acad Sci U S A. 1996; 93:11341–11348. [PubMed: 8876137]
73. Gonzalez-Aseguinolaza G, Nakaya Y, Molano A, Dy E, Esteban M, Rodriguez D, Rodriguez JR,
Palese P, Garcia-Sastre A, Nussenzweig RS. Induction of protective immunity against malaria by
priming-boosting immunization with recombinant cold-adapted influenza and modified vaccinia
Ankara viruses expressing a CD8+-T-cell epitope derived from the circumsporozoite protein of
Plasmodium yoelii. J Virol. 2003; 77:11859–11866. [PubMed: 14557672]
74. Sutter G, Staib C. Vaccinia vectors as candidate vaccines: the development of modified vaccinia
virus Ankara for antigen delivery. Curr Drug Targets Infect Disord. 2003; 3:263–271. [PubMed:
14529359]
75. Drexler I, Staib C, Sutter G. Modified vaccinia virus Ankara as antigen delivery system: how can
we best use its potential? Curr Opin Biotechnol. 2004; 15:506–512. [PubMed: 15560976]
76. Cosma A, Nagaraj R, Buhler S, Hinkula J, Busch DH, Sutter G, Goebel FD, Erfle V. Therapeutic
vaccination with MVA-HIV-1 nef elicits Nef-specific T-helper cell responses in chronically
HIV-1 infected individuals. Vaccine. 2003; 22:21–29. [PubMed: 14604567]
77. Lyons J, Sinos C, Destree A, Caiazzo T, Havican K, McKenzie S, Panicali D, Mahr A. Expression
of Mycobacterium tuberculosis and Mycobacterium leprae proteins by vaccinia virus. Infect
Immun. 1990; 58:4089–4098. [PubMed: 2123833]
78. Zhu X, Venkataprasad N, Ivanyi J, Vordermeier HM. Vaccination with recombinant vaccinia
viruses protects mice against Mycobacterium tuberculosis infection. Immunology. 1997; 92:6–9.
[PubMed: 9370917]
79. Malin AS, Huygen K, Content J, Mackett M, Brandt L, Andersen P, Smith SM, Dockrell HM.
Vaccinia expression of Mycobacterium tuberculosis-secreted proteins: tissue plasminogen
activator signal sequence enhances expression and immunogenicity of M. tuberculosis Ag85.
Microbes Infect. 2000; 2:1677–1685. [PubMed: 11137041]
80. Feng CG, Blanchard TJ, Smith GL, Hill AV, Britton WJ. Induction of CD8+ T-lymphocyte
responses to a secreted antigen of Mycobacterium tuberculosis by an attenuated vaccinia virus.
Immunol Cell Biol. 2001; 79:569–575. [PubMed: 11903615]
81. Goonetilleke NP, McShane H, Hannan CM, Anderson RJ, Brookes RH, Hill AV. Enhanced
immunogenicity and protective efficacy against Mycobacterium tuberculosis of bacille Calmette-
Guerin vaccine using mucosal administration and boosting with a recombinant modified vaccinia
virus Ankara. J Immunol. 2003; 171:1602–1609. [PubMed: 12874255]
82. Williams A, Goonetilleke NP, McShane H, Clark SO, Hatch G, Gilbert SC, Hill AV. Boosting
with poxviruses enhances Mycobacterium bovis BCG efficacy against tuberculosis in guinea pigs.
Infect Immun. 2005; 73:3814–3816. [PubMed: 15908420]
83**. McShane H, Pathan AA, Sander CR, Keating SM, Gilbert SC, Huygen K, Fletcher HA, Hill AV.
Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and
naturally acquired antimycobacterial immunity in humans. Nat Med. 2004; 10:1240–1244. First
use of recombinant vaccinia virus expressing M. tuberculosis antigen 85A in human. [PubMed:
15502839]
84. McShane H, Pathan AA, Sander CR, Goonetilleke NP, Fletcher HA, Hill AV. Boosting BCG with
MVA85A: the first candidate subunit vaccine for tuberculosis in clinical trials. Tuberculosis
(Edinb). 2005; 85:47–52. [PubMed: 15687027]
85. Huygen K, Content J, Denis O, Montgomery DL, Yawman AM, Deck RR, DeWitt CM, Orme IM,
Baldwin S, D’Souza C, Drowart A, Lozes E, Vandenbussche P, Van Vooren JP, Liu MA, Ulmer
Behar et al. Page 22
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
JB. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med. 1996;
2:893–898. [PubMed: 8705859]
86. Fonseca DPAJ, aissa-Trouw B, van Engelen M, Kraaijeveld CA, Snippe H, Verheul AFM.
Induction of Cell-Mediated Immunity against Mycobacterium tuberculosis Using DNA Vaccines
Encoding Cytotoxic and Helper T-Cell Epitopes of the 38-Kilodalton Protein. Infect Immun. 2001;
69:4839–4845. [PubMed: 11447158]
87. Skeiky YA, Alderson MR, Ovendale PJ, Guderian JA, Brandt L, Dillon DC, Campos-Neto A,
Lobet Y, Dalemans W, Orme IM, Reed SG. Differential immune responses and protective efficacy
induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA
or recombinant protein. J Immunol. 2004; 172:7618–7628. [PubMed: 15187142]
88. Lowrie DB, Tascon RE, Bonato VLD, Lima VMF, Faccioli LH, Stavropoulos E, Colston MJ,
Hewinson RG, Moelling K, Silva CL. Therapy of tuberculosis in mice by DNA vaccination.
Nature. 1999; 400:269–271. [PubMed: 10421369]
89. Bonato VL, Goncalves ED, Soares EG, Santos Junior RR, Sartori A, Coelho-Castelo AA, Silva
CL. Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of
CD8+ cells, interferon-gamma recovery and reduction of lung injury. Immunology. 2004;
113:130–138. [PubMed: 15312144]
90. Taylor JL, Turner OC, Basaraba RJ, Belisle JT, Huygen K, Orme IM. Pulmonary necrosis resulting
from DNA vaccination against tuberculosis. Infect Immun. 2003; 71:2192–2198. [PubMed:
12654841]
91. Turner J, Rhoades ER, Keen M, Belisle JT, Frank AA, Orme IM. Effective Preexposure
Tuberculosis Vaccines Fail To Protect When They Are Given in an Immunotherapeutic Mode.
Infect Immun. 2000; 68:1706–1709. [PubMed: 10678993]
92. Rosenberg SA, Yang JC, Sherry RM, Hwu P, Topalian SL, Schwartzentruber DJ, Restifo NP,
Haworth LR, Seipp CA, Freezer LJ, Morton KE, Mavroukakis SA, White DE. Inability to
immunize patients with metastatic melanoma using plasmid DNA encoding the gp100 melanoma-
melanocyte antigen. Hum Gene Ther. 2003; 14:709–714. [PubMed: 12804135]
93. Triozzi PL, Aldrich W, Allen KO, Carlisle RR, LoBuglio AF, Conry RM. Phase I study of a
plasmid DNA vaccine encoding MART-1 in patients with resected melanoma at risk for relapse. J
Immunother. 2005; 28:382–388. [PubMed: 16000957]
94. Feng CG, Palendira U, Demangel C, Spratt JM, Malin AS, Britton WJ. Priming by DNA
immunization augments protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin
against tuberculosis. Infect Immun. 2001; 69:4174–4176. [PubMed: 11349095]
95. Derrick SC, Yang AL, Morris SL. A polyvalent DNA vaccine expressing an ESAT6-Ag85B fusion
protein protects mice against a primary infection with Mycobacterium tuberculosis and boosts
BCG-induced protective immunity. Vaccine. 2004; 23:780–788. [PubMed: 15542202]
96. Goter-Robinson C, Derrick SC, Yang AL, Jeon BY, Morris SL. Protection against an aerogenic
Mycobacterium tuberculosis infection in BCG-immunized and DNA-vaccinated mice is associated
with early type I cytokine responses. Vaccine. 2006; 24:3522–3529. [PubMed: 16519971]
97. Triccas JA, Sun L, Palendira U, Britton WJ. Comparative affects of plasmid-encoded interleukin
12 and interleukin 18 on the protective efficacy of DNA vaccination against Mycobacterium
tuberculosis. Immunol Cell Biol. 2002; 80:346–350. [PubMed: 12121223]
98. Cai H, Yu DH, Tian X, Zhu YX. Coadministration of interleukin 2 plasmid DNA with combined
DNA vaccines significantly enhances the protective efficacy against Mycobacterium tuberculosis.
DNA Cell Biol. 2005; 24:605–613. [PubMed: 16225391]
99. Kamath AT, Feng CG, Macdonald M, Briscoe H, Britton WJ. Differential Protective Efficacy of
DNA Vaccines Expressing Secreted Proteins of Mycobacterium tuberculosis. Infect Immun. 1999;
67:1702–1707. [PubMed: 10085007]
100. Kirman JR, Turon T, Su H, Li A, Kraus C, Polo JM, Belisle J, Morris S, Seder RA. Enhanced
immunogenicity to Mycobacterium tuberculosis by vaccination with an alphavirus plasmid
replicon expressing antigen 85A. Infect Immun. 2003; 71:575–579. [PubMed: 12496215]
101. Nimal S, Thomas MS, Heath AW. Fusion of antigen to Fas-ligand in a DNA vaccine enhances
immunogenicity. Vaccine. 2007; 25:2306–2315. [PubMed: 17239500]
102. Huygen K. Plasmid DNA vaccination. Microbes Infect. 2005; 7:932–938. [PubMed: 15878683]
Behar et al. Page 23
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
103. Derrick SC, Repique C, Snoy P, Yang AL, Morris S. Immunization with a DNA vaccine cocktail
protects mice lacking CD4 cells against an aerogenic infection with Mycobacterium tuberculosis.
Infect Immun. 2004; 72:1685–1692. [PubMed: 14977976]
104. D’Souza S, Romano M, Korf J, Wang XM, Adnet PY, Huygen K. Partial reconstitution of the
CD4+-T-cell compartment in CD4 gene knockout mice restores responses to tuberculosis DNA
vaccines. Infect Immun. 2006; 74:2751–2759. [PubMed: 16622212]
105. Dietrich G, Bubert A, Gentschev I, Sokolovic Z, Simm A, Catic A, Kaufmann SH, Hess J, Szalay
AA, Goebel W. Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by
attenuated suicide Listeria monocytogenes. Nat Biotechnol. 1998; 16:181–185. [PubMed:
9487527]
106. Miki K, Nagata T, Tanaka T, Kim YH, Uchijima M, Ohara N, Nakamura S, Okada M, Koide Y.
Induction of Protective Cellular Immunity against Mycobacterium tuberculosis by Recombinant
Attenuated Self-Destructing Listeria monocytogenes Strains Harboring Eukaryotic Expression
Plasmids for Antigen 85 Complex and MPB/MPT51. Infect Immun. 2004; 72:2014–2021.
[PubMed: 15039321]
107*. Bennekov T, Dietrich J, Rosenkrands I, Stryhn A, Doherty TM, Andersen P. Alteration of
epitope recognition pattern in Ag85B and ESAT-6 has a profound influence on vaccine-induced
protection against Mycobacterium tuberculosis. Eur J Immunol. 2006; 36:3346–3355. [PubMed:
17109467] Interesting study on how the delivery of antigens alters the immundominance of the
epitopes and can have a profound influence on vaccine-induced protection
108*. Shams H, Klucar P, Weis SE, Lalvani A, Moonan PK, Safi H, Wizel B, Ewer K, Nepom GT,
Lewinsohn DM, Andersen P, Barnes PF. Characterization of a Mycobacterium tuberculosis
peptide that is recognized by human CD4+ and CD8+ T cells in the context of multiple HLA
alleles. J Immunol. 2004; 173:1966–1977. The promiscuity of CFP10-derived peptides for
multiple HLA alleles provides an explanation for why it is such an immunodominant antigen
following M. tuberculosis infection of people. [PubMed: 15265931]
109. Lewinsohn DM, Zhu L, Madison VJ, Dillon DC, Fling SP, Reed SG, Grabstein KH, Alderson
MR. Classically restricted human CD8+ T lymphocytes derived from Mycobacterium
tuberculosis-infected cells: definition of antigenic specificity. J Immunol. 2001; 166:439–446.
[PubMed: 11123322]
110. Denis O, Tanghe A, Palfliet K, Jurion F, van den Berg TP, Vanonckelen A, Ooms J, Saman E,
Ulmer JB, Content J, Huygen K. Vaccination with plasmid DNA encoding mycobacterial antigen
85A stimulates a CD4+ and CD8+ T-cell epitopic repertoire broader than that stimulated by
Mycobacterium tuberculosis H37Rv infection. Infect Immun. 1998; 66:1527–1533. [PubMed:
9529077]
111. Kamath A, Woodworth JS, Behar SM. Antigen-specific CD8+ T cells and the development of
central memory during Mycobacterium tuberculosis infection. J Immunol. 2006; 177:6361–6369.
[PubMed: 17056567]
Behar et al. Page 24
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Key issues
• Antigen-specific CD8
+
T cells are elicited as part of the adaptive immune
response following infection with Mycobacterium tuberculosis and contribute to
host defense.
• Optimum expression of CD8
+
T cell effector functions require help or licensing
that is dependent upon CD4
+
T cells.
• CD8
+
T cells are elicited by several of the vaccine strategies that are being
considered for use in people at risk for tuberculosis. These include recombinant
BCG and M. tuberculosis auxotrophs, viral vectors such as recombinant
adenovirus and vaccinia, and DNA vaccines.
• Data exists to indicate that CD8
+
T cells contribute to the protective efficacy of
vaccines used against tuberculosis, and argues for the development of vaccines
that will stimulate both M. tuberculosis-specific CD4
+
and CD8
+
T cells.
• Before the role of vaccine-induced CD8
+
T cells in protection can be assessed,
the mycobacterial antigens they recognize need to be defined so the
immunological efficacy of vaccination can be determined.
Behar et al. Page 25
Expert Rev Vaccines. Author manuscript; available in PMC 2011 July 12.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
