Gene Elements That Regulate Streptococcus Pneumoniae Virulence and Immunity Evasion

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Current Gene Therapy, 2013, 13, 51-64
51
Gene Elements that Regulate Streptococcus pneumoniae Virulence and

Immunity Evasion
Pamela A. Nieto1,2, Sebastin A. Riquelme1,2, Claudia A. Riedel1,5, Alexis M. Kalergis1,2,3,4,
Susan M. Bueno1,2,4,*
1
Millennium Institute on Immunology and Immunotherapy 2Departamento de Gentica Molecular y Microbiologa,

Facultad de Ciencias Biolgicas and 3Departamento de Reumatologa, Facultad de Medicina, Pontificia Universidad
Catlica de Chile; 4INSERM, UMR 1064, Nantes, France; 5 Departamento de Ciencias Biolgicas, Facultad de Ciencias
Biolgicas and Facultad de Medicina, Universidad Andrs Bello
Abstract: Streptococcus pneumoniae is one of the most important aetiological agents of bacterial pneumonia and meningitis in the world. This bacterium can cause severe inflammation of lung tissue and disseminate to the central nervous system. Although B cell activation and antibody secretion is considered one of the most important events in the prevention or
clearance of bacterial infection by the host, dendritic cells (DCs) and T cells play a fundamental role in the generation of
the protective immunity required to prevent the pathogenesis caused by S. pneumoniae infection. Here we review recent
studies that have evaluated the impact of DCs and T cells on S. pneumoniae infection and the gene elements encoding
virulence factors used by this bacterium to interfere with the appropriate function of these immune cells. This knowledge
could be relevant for generating new prophylactic and therapeutic tools and to prevent the severe infection caused by this
pathogen.
Keywords: Streptococcus pneumoniae, virulence factors, dendritic cells, T cells, adaptive immunity.
INTRODUCTION
Streptococcus pneumoniae (or pneumococcus) is a microbial pathogen that causes over 44% of total hospitalizations due to bacterial respiratory tract infections in children
under 2 years old [1]. The infection caused by this bacterium
has several manifestations, including otitis media, sinusitis,
upper respiratory tract infections, and other serious lifethreatening symptoms, such as septicemia and meningitis
[2]. Pneumococcus is a common colonizing bacterium of the
respiratory tract in humans, especially in young children [3].
In fact, close to 60% of children attending daycare carry this
pathogen in the nasopharynx [4]. This bacterium can be
horizontally transmitted through contaminated secretions,
and individuals with poor mucosal immunity can suffer persistent colonization or infection of the lower respiratory tract.
In children, the most common manifestation of pneumonia is
bronchial pneumonia, which compromises bronchioles and
alveoli [5]. Pneumococcal pneumonia is characterized by
inflammation of lung tissue due to bacterial colonization of
the lower respiratory tract, which can cause fever, malaise,
dyspnea, and productive cough [3]. This infection can progress to a severe disease with acute respiratory failure, septic
shock, and death.
*Address correspondence to this author at the Departamento de Gentica

Molecular y Microbiologa, Facultad de Ciencias Biolgicas, Pontificia
Universidad Catlica de Chile. Avenida Libertador Bernardo OHiggins
340, Santiago 8331010, Chile; Tel: 56-2-686-2842; Fax: 56-2-686-2185;
E-mail: sbueno@bio.puc.cl, susan.bueno@gmail.com
1-53/13 $58.00+.00
Another life-threatening disease caused by S. pneumoniae is pneumococcal meningitis, which mainly affects newborns and people living in low-income countries [6]. This
type of disease is caused when pneumococci, which colonize
the upper respiratory tract mucosa, cross the brain blood barrier (BBB) and reach the Central Nervous System (CNS) [7].
Several studies have suggested that pneumococcus binds to
the platelet-activating factor receptor (PAFR) [8, 9] and to
laminin receptors [10], which are expressed on the surface of
the brain microvascular endothelial cells. As a result, the
bacterium crosses the BBB and penetrates into the brain. At
this location, several cell wall components, such as lipoteichoic acid (LTA), peptidoglycan, and also DNA are released from the bacterium by means of an autolysis mechanism [7]. These molecules are then recognized by host cells
and trigger an inflammatory response [7]. Upon recognition
of inflammatory cytokines, innate immune cells such as neutrophils are recruited from the vasculature to the brain and
cause a pyogenic inflammation [7]. Brain infection by
pneumococcus can lead to several secondary neurological
alterations, such as cerebral ischemia, brain edema, and increased intracranial pressure [11]. If the infection is not
opportunely treated, mortality can reach up to 100%. Due to
the morbidity related to pneumonia and meningitis caused by
this bacterial pathogen, it is highly relevant to understand the
role of immune cells in the initiation of an efficient immune
response and also in the capacity of pneumococcus to cause
infection in the host.
Importantly, a worldwide concern of pneumococcus infection is the arising of antibiotic resistant strains. For in 2013 Bentham Science Publishers
52 Current Gene Therapy, 2013, Vol. 13, No. 1
stance, in the United States it has been reported that 40% of

clinical isolates of pneumococcus are resistant to penicillin
and 28% are resistant to macrolides [12], whereas in Latin
America it has been reported that 38% of the S. pneumoniae
strains isolated have decreased susceptibility to beta-lactams
[13]. The increased number of pneumococcus strains resistant to antibiotics also highlights the importance of studying
the immune response against this pathogen and the bacterial
factors responsible for preventing the appropriate initiation
and activation of protective immunity in the host.
Recent reports have shown that the pneumococcus genome encodes several virulence factors that interfere with
the proper function of dendritic cells (DCs) and with the activation of T cells. Such virulence factors contribute significantly to pneumococcus pathogenicity, because DCs and T
cells are thought to play an important role in controlling S.
pneumoniae infection and dissemination. In this review we
discuss recent studies about the role of DCs and T cells in S.
pneumoniae infection and immunity, as well as the virulence
factors encoded by the pneumococcus genome that interfere
with the proper function of T cell immunity.
EARLY IMMUNE RESPONSE TO S. PNEUMONIAE
INFECTION
The respiratory tract (RT) is constantly exposed to environmental antigens, which could be innocuous or harmful for
the host. The bronchial and tracheal epithelia constitute a
strong mechanical barrier against incoming pathogens and,
together with alveolar macrophages (AMs) and sentinel DCs,
work as the first line of defense to prevent infection Fig.
(1A) [14, 15]. S. pneumoniae is detected by patternrecognition receptors (PRRs), such as Toll-like receptors
(TLRs) expressed by immune and epithelial cells [16].
Among these, TLR2 recognizes pneumococcal cell wall
components, such as lipoproteins and LTA [17, 18], while
TLR4 recognizes the pneumococcus toxin pneumolysin (Ply)
[19]. TLR9, which is an endosomal receptor, recognizes unmethylated pneumococcal CpG DNA [20]. Mice lacking
either one of these receptors show only a minor increase in
susceptibility when infected with pneumococcus, which indicates some redundancy in their functions [19-22]. Lung
epithelial cells, through TLRs, respond to recognition of the
pathogen-associated molecular patterns (PAMPs) of S.
pneumoniae by secreting inflammatory mediators, such as
IL-6, IL-8, IL1-, IL1- and TNF-, chemotactic mediators
(i.e. chemokines), and antimicrobial substances (i.e. - and
-defensins) Fig. (1A) [3, 23, 24]. Among other cytokines,
AMs secrete IL1-, IL1-, and TNF-, contributing to the
strong chemoattractant action that induces the migration of
neutrophils and other mononuclear cells to the infected tissue
Fig. (1A) [3, 21].
Other receptors that are relevant in the early response
against pneumococcus infection are the NLRP3 inflammasome, which is activated by Ply [25], and scavenger receptors, such as SR-A and MARCO [26]. The absence of these
proteins in mice significantly increases the susceptibility to
the infection caused by S. pneumoniae [27-29]. Another important component of the innate immune response in S.
pneumoniae infection is the complement. Along these lines,
the C-type lectin SIGN-related 1 (SIGN-R1), which is a re-
Nieto et al.
ceptor expressed by macrophages that recognizes and binds

to capsular polysaccharides of pneumococci [30-32], activates the classical complement pathway through C1q binding
on SIGN-R1 coated pneumococci [33]. Studies with mice
lacking specific components from the classical or alternative
pathway suggest that the classical pathway is more relevant
for protection against pneumococcus [34].
Several virulence factors of S. pneumoniae that alter the
early innate immune response and the initiation of an adaptive immune response have been described and will be discussed below.
GENES ENCODING VIRULENCE FACTORS CONTRIBUTING TO S. PNEUMONIAE INFECTION
Several S. pneumoniae molecules, such as proteins and
carbohydrates, have been linked to the capacity of this pathogen to colonize the respiratory tract epithelium and to cause
inflammation [2, 35]. The most important virulence factor of
this pathogen is the capsule (Table 1, Fig. 2), a thick layer of
polysaccharides that extends from the cell wall. A capsular
polysaccharide (CPS) is formed mainly by monosaccharides
of D-glucose, D-galactose, L-rhamnose, N-acetyl D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine,
N-acetyl-D-fucosamine or D-glucoronate, connected by
glycosidic linkages [36]. In turn, these polysaccharides are
connected to the bacterial cell wall through a covalent
linkage to the peptidoglycan [37]. Ninety-three different
capsular serotypes have been reported for the pneumococcus,
but despite this diversity, the majority of pneumococcal CPS
are synthetized by the same metabolic pathway [38]. The
genes needed for the synthesis of the capsule are all located
in the cps chromosomal locus, between genes dexB and aliA
[39]. Apart from polysaccharides, the capsule also contains
some non-sugar components, such as cell wall proteins that
extend and protrude outside the capsule Fig. (2). The
pneumococcal capsule acts as a hydrated shield that protects
the bacterial surface from recognition and interaction with
components of the host immune system. Further, an antiphagocytic activity has been also shown for the capsule,
because it prevents opsonophagocytosis and impairs the
recognition of cell wall-deposited C3b/C3bi. In addition, this
virulence factor also promotes the adhesion of bacteria to the
respiratory epithelium [35, 40].
This bacterium also possesses an arsenal of surface lytic
enzymes (Table 1, Fig. 2), such as hyaluronidases, neuraminidases, and serine proteases, which degrade components of
the extracellular matrix, such as glycolipids and oligosaccharides found in host body fluids and cellular surfaces [41, 42].
The hyaluronidase is encoded by the gene hylA [42, 43]. It
breaks down the hyaluronic acid from the mammalian connective tissue and extracellular matrix, helping bacterial
spreading and colonization. Neuraminidases (also known as
sialidases) are present in all strains of pneumococcus and
exist in three isoforms, encoded by the genes nanA, nanB,
and nanC [44]. They cleave N-acetylneuraminic acid from
glycolipids, oligosaccharides, and lipoproteins of host cell
surfaces and body fluids [45], a process required to unmask
potential binding sites for the pathogen and also to cause
damage to the host. Finally, the serine protease PrtA [46]
(Table 1, Fig. 2) plays an unknown role in virulence, given
Gene Elements That Regulate Streptococcus pneumoniae Virulence and Immunity Evasion
Current Gene Therapy, 2013, Vol. 13, No. 1
53
Alveolus
TNF
IL-1b IL-1a
CD4 T cell
Monocytes
Macrophage
activation
A
FIRST INNATE RESPONSE
Streptococcus pneumoniae
Neutrophils
SECOND BURST
OF INNATE CELLS
Alveolar
macrophage
Blood torrent
Respiratory
epithelium
ENHANCEMENT
OF INNATE
IMMUNITY
IL-17
Plasmatic cell
Dendritic cell
IL-8
Antigen sampling
IL-1a
IL-6
IL-1b defensins
TNF
T cell
chemokines
IFN
Presentation of antigen
loaded on MHC-II
Phagocytosis of
pneumococcus
Maturation
of DC
Apoptosis
of DC
Supression
of CD4+ T cells
CD4+ T cell
IL-17
IL-22
GM-CSF
Activation of CD4+ T cell

and differentiation to TH1
IL-12
IL-17F
IL-6
IL-2
B
Prevention of phagocytosis
by the capsule, PavA
TNF-
CD4+ Th17 cell

IL-10
TGF-
ACTIVATION OF
ADAPTIVE
IMMUNITY
B cell
Pneumolysin
Plasmatic cell
Contribution to
humoral immunity
against
pneumococcal
polysaccharides
Regulatory CD8+ T cell

Serotype 1
pneumococcus
Fig. (1). Model of adaptive immune response generated against Streptococcus pneumoniae. A. The response against the bacterium begins
when pneumococcus is detected by epithelial cells of the respiratory tract, alveolar macrophages (AMs), and sentinel dendritic cells (DCs).
This recognition leads to the activation of AMs, which secrete cytokines that will recruit monocytes and neutrophils to the site of infection.
Epithelial cells also secrete cytokines, defensins, and chemokines that will induce an inflammatory state. B. The pneumococcus prevents DC
phagocytosis by masking its antigens with the capsule and also by the indirect action of the protein PavA. In some cases the pneumococcus
will be successfully phagocytized, inducing the maturation of the DC, which will change its morphology and expression of surface molecules.
Some DCs will undergo apoptosis due to the phagocytized bacteria or due to the effects of pneumolysin. C. The mature DC will present processed pneumococcus antigens bound to MHC class II molecules to CD4+ T cells (the Zwitterionic polysaccharide from ST1 strains can also
be presented this way), secrete IL-12, and aid in the production of antibodies by B cells, thus activating an adaptive response. D. Activated
CD4+ T cells will differentiate into a TH1 phenotype and will secrete the corresponding cytokines. E. CD4+ TH1 T cells will contribute to
humoral immunity by enhancing the production of antibodies by B cells. F. Regulatory CD8+ T cells will secrete cytokines to suppress the
action of CD4+ T cells, thus regulating inflammation, as has been seen in a model with serotype 3 pneumococcus. In a model with serotype 1,
the activation of regulatory CD8+ T cells would proceed by enhancement of TCR crosslinking. G. T cells would secrete IL-17 at the early
stages of pneumonia, contributing to the recruitment of more monocytes and neutrophils and to the enhancement of their functions. At a later
stage T cells would prevent the action of macrophages and DCs by directly killing these cells. H. CD4+ TH17 T cells secrete IL-17 that will
promote an inflammatory state by recruiting more monocytes and neutrophils to the site of infection. The concerted participation of DCs, B
cells, and T cells, together with the non-specific actions of macrophages and neutrophils, ultimately lead to clearance of the infection.
that a PrtA-negative mutant strain of pneumococcus is attenuated in a murine model of disease [46].
Another important virulence determinant found in pneumococcus is Ply [2, 47] (Table 1, Fig. 2), which is a cytoplasmic protein found in several Gram-positive bacteria and
in most of the strains of pneumococcus, although different
alleles of the gene ply have been found [48, 49]. This molecule is released upon autolysis of pneumococcus in a manner
independent of autolysin activity [50, 51]. The toxic effects
of Ply are due to the initial binding of cholesterol in the host

cell membranes and the subsequent polymerization with
other 50 sub-units, to form large pores that produce hemolysis of mammalian cells [47, 52-54]. This molecule also inhibits complement deposition, mainly affecting the classical
pathway [55]. S. pneumoniae strains lacking this protein due
to the deletion or mutations of the ply gene are attenuated in
animal models of pneumococcal pneumonia, cause reduced
inflammation in lungs, and delayed recruitment of neutrophils to the airways [56-58].
Table 1.
Nieto et al.
Major Virulence Factors of Streptococcus pneumoniae
Virulence Factor
Role in Virulence
Location
Gene Coordinates*
NCBI Gene ID*
Gene(s) Name
References
Capsule
Masks bacterial antigens, prevents

phagocytosis. Zwitterionic polysaccharides promote CD4+ T cell
activation in a DC-contact dependent manner but activates CD8+ T
cells only by TCR cross-linking in
the absence of APCs.
Over the cell

wall
312482-332438
(locus)
Chromosomal
locus cps
[38, 39, 142]
Pneumolysin (Ply)
Binds cholesterol, polymerizes, and

forms membrane pores that produce hemolysis. Inhibits complement deposition. It is proposed to
positively influence phagocytosis
in DCs and it is associated to decrease DCs maturation. Induce DC
apoptosis. Promotes T cell migration and IL-17A secretion.
Cytoplasm
1721457-1722872
4441611
ply
[29, 47-58,
113, 118,
122]
Hyaluronidase
(HylA)
Breaks hyaluronic acid. Helps

bacterial spread and colonization.
Cell wall
Not reported in
strain D39
Not reported in
D39
hylA
[42, 43]
Neuraminidase A
(NanA)
Cleaves N-acetylneuraminic acid.

Causes damage to the host and unmask binding sites for the pathogen.
Cell wall
1522475-1525468
4442379
nanA
[44, 45]
Serin protease
(PrtA)
Unknown
Cell wall
569948-576382
4442925
prtA
[46]
Autolysin (LytA)
Bacterial autolysis, release of

degradation products that induce
inflammation.
Cell wall
1729601-1730557
4442230
lytA
[58, 67-70]
Pneumococcal
surface protein A
(PspA)
Interferes with the complement

system. Binds lactoferrin and
apolactoferrin. Also used as a good
immunogenic antigen that mediates
humoral response and complement
activation.
Cell wall
128356-130215
4441373
pspA
[62, 72-83]
Pneumococcal
surface protein C
(PspC)
Binds to complement regulatory

protein factor H, and to the human
secretory IgA.
Cell wall
1995044-1997149
4441159
pspC
[84-88]
Pneumococcal
surface adhesin A
(PsaA)
Adheres to E-cadherin.
Cell membrane
1478217-1479146
4442424
psaA
[59, 61-64]
Pneumococcal
adherence and
virulence factor A
(PavA)
Prevents phagocytosis by DCs.
Cell wall
868727-870382
4442613
pavA
[110, 115,
142]
*From Streptococcus pneumoniae strain D39, NC_008533.1 [174].
The pneumococcal surface adhesin A (PsaA) [59] (Table

1, Fig. 2) is a substrate-binding lipoprotein, part of an ABC
transporter of manganese ions composed also by the ATPbinding protein PsaB and the permease PsaC [60]. The receptor of PsaA is E-cadherin [61] and mutations in the psaA
gene decrease bacterial adhesion to cells, increase sensitivity
to oxidative stress, and reduce bacterial virulence [62-64].
Other important proteins associated to virulence are cholin-binding proteins (CBPs), which are surface exposed proteins that non-covalently bind to phosphorylcholine by repeated choline-binding domains [65, 66]. The major cell wall
hydrolase LytA (the autolysin) was the first reported CBP
from pneumococcus [67, 68] (Table 1, Fig. 2). LytA is required for cell division, cell lysis in stationary phase, or after
treatment with penicillin [69]. As a virulence factor it promotes inflammation, allowing the release of cell wall degradation products and Ply from the cytoplasm [58, 70]. Apart
from LytA, there are other three hydrolytic enzymes: LytB,
LytC, and CbpE, which are involved in nasopharyngeal
colonization [65].
The pneumococcal surface proteins A (PspA) and C
(PspC) are two other virulence factors belonging to the
family of CBPs [71] (Table 1, Fig. 2). PspA is present in
virtually all strains of S. pneumoniae, although it is highly
variable serologically because of the high heterogeneity in
the N-terminal region of the pspA gene [72-74]. The variants
of PspA have been classified into three families (1, 2 and 3),
each composed of different clades [75]. PspA acts by interfering with complement activation [76] and also by binding
lactoferrin and apolactoferrin, protecting bacteria against
these bactericidal molecules [77, 78]. Among the known
pneumococcal antigens, PspA is perhaps the best studied and
considered a good candidate for a protein-based vaccine due
to its immunogenicity and because it elicits an antibody response that enhances complement deposition and protects
against nasal colonization, pneumonia and sepsis in mice
[62, 79, 80]. Moreover, the PspA families are able to generate antibodies that show cross-protection among different
serotypes of the same family [75, 81, 82]. The observation
that PspA-deficient strains of pneumococcus showed
reduced virulence [83] supports the importance of this protein in bacterial fitness in the host. Same as PspA, PspC (also
called CbpA) presents high variability and has been classified into 11 groups [84]. It binds to the complement regulatory protein factor H [85] and to the human secretory IgA
[86, 87]. It is considered a good candidate for a proteinbased vaccine as it shows cross-reactivity with different
strains [88].
All these virulence determinants allow pneumococcus to
cause severe infection in the lungs, which results in inflammation and compromised normal oxygen supply. As will be
described below, these virulence factors also affect DC and
T cell function.
ROLE OF DCs IN THE INITIATION AND ACTIVATION OF ADAPTIVE IMMUNITY AGAINST S.
PNEUMONIAE
The initiation of adaptive immunity against S. pneumoniae requires professional antigen presenting cells (APCs),
such as DCs. DCs recognize and degrade bacterial antigens,
then go through a process of maturation that allows them to
present antigens as peptide-MHC complexes to nave T cells
[89-93]. DCs link innate and adaptive immunity by directly
recognizing pathogens at the site of infection, in order to
activate bacteria-specific T cells [92-96]. After detection of
PAMPs, immature DCs become activated, undergoing
several functional and phenotypic changes collectively
known as maturation. These changes are triggered by the
engagement of TLRs, NOD proteins and NOD-like receptors
(NLRs), which are expressed by immature DCs and translate
PAMP recognition into cellular responses [97-99]. As a result of maturation costimulatory proteins, anti-bacterial
molecules, and proinflammatory cytokines are actively produced by DCs [92, 100-102]. Additionally, a mature DC
55
becomes capable of translocating MHC class II molecules

bound to the captured and processed antigen to the cell surface. Antigen capture by activating Fc and Fc receptors
promotes the generation of pMHC-I and -II complexes and
the expression of costimulatory molecules (such as CD80
and CD86), and T cell adhesion molecules (such as CD48
and CD58), all of which become upregulated in response to
the maturation signals [91]. The morphology of a mature DC
changes; it expresses long membrane extensions that will
increase the chance of interaction with T cells [91]. Mature
DCs also secrete cytokines that define the nature of T cell
effector function, which is activated during antigen recognition [103-105]. Due to maturation, DCs also modify their
profile of chemokine receptors and acquire the capacity to
migrate from peripheral sites of infection to regional lymph
nodes, where nave T cells normally reside [106-108].
At the RT, DCs act as sentinel cells forming a network
all over the conducting airways and in the lung parenchyma
Fig. (1A) [15]. They are strategically situated to sample the
inhaled antigens and rapidly deliver them to the local lymph
nodes, where the activation of antigen-specific nave T cells
occurs [109]. Upon infection of the respiratory tract by bacterial pathogens, DCs can readily detect and capture them.
This process activates maturation and the concomitant expression of several molecules on the DC surface Fig. (1B)
[110]. Phagocytized bacteria are processed by proteases that
partially digest antigens, and bacterial derived peptides are
loaded onto MHC-II molecules and translocated to the DC
surface, to be presented to CD4+ T cells Fig. (1C) [111].
Although there are no studies demonstrating that DCs can
cross-present S. pneumoniae antigens to CD8 + T cells, DCs
are relevant to the proliferation and migration of CD8 + T
cells in response to S. pneumoniae infection, independently
of antigen presentation [112].
Due to the relevant role of DCs in the protection of the
respiratory tract, as several other bacterial pathogens, S.
pneumoniae has virulence factors that prevent the proper
function of DCs during pneumococcal infection. For instance, S. pneumoniae capsule severely impairs phagocytosis
by DCs Fig. (1B) [113]. In agreement with this notion is the
observation that non-encapsulated mutant strains of S. pneumoniae or strains that produce lower amounts of capsular
polysaccharide are rapidly captured and degraded by DCs
[110]. Of interest, intracellular antigen availability is required during antigen degradation and presentation by DCs
to T cells [114]. Pathogens that evade phagocytosis block the
possibilities for intracellular antigens loads, rendering DCs
unable to activate T cells [114]. Thus, S. pneumoniae may
exploit phagocytosis evasion as a mechanism to disrupt the
communication between DCs and T cells.
Another virulence factor that contributes to the prevention of DC phagocytosis is the pneumococcal adherence and
virulence factor A (PavA) (Table 1). This is an outer cell
surface protein first identified as an adhesin for immobilized
fibronectin, whose inactivation attenuated the virulence of
the bacterium in a mouse sepsis model [115]. Recently, it has
been described that this protein can directly prevent phagocytosis by DCs Fig. (1B) [110]. Later, it was shown that
pavA mutants were attenuated in a mouse meningitis model,
and new evidence suggested that PavA modulates other viru-
Nieto et al.
Fig. (2). Schematic representation of the major virulence factors of S. pneumoniae. Different virulence factors from Streptococcus
pneumoniae are shown with their respective localization in the bacterium (capsule, cell wall, cell membrane and cytoplasm). The capsule is
shown as a brown layer, where polysaccharides are represented as branched structures. From left to right: Serin Protease (PrtA), Pneumococcal Surface Adhesin A (PsaA), Pneumococcal Adherence and Virulence factor A (PavA), Pneumococcal Surface Protein A (PspA),
Hyaluronate Lyase (HylA), Pneumococcal Surface Protein C (PspC), Neuraminidase A (NanA), Pneumolysin (Ply), and Autolysin (LytA).
The components of the cell wall lipoteichoic acid (LTA) and teichoic acid (TA) are also shown. Disruption of the cell membrane and cell
wall mediated by LytA allows the liberation of Ply to the extracellular medium.
lence determinants associated to adherence [116]. Furthermore, pavA mutants are attenuated in mouse long-term nasopharyngeal carriage and pneumonia models. Mice infected
with these mutant strains show reduced mortality and absence of bacteremia, although the bacteria cause persistent
infection and chronic pneumonia [117]. Studies performed
with human monocyte-derived DCs and a non-encapsulated
strain of plypavA S. pneumoniae showed that the absence
of PavA enhanced pneumococcal internalization into DCs,
and once internalized, it negatively affected the release of
several cytokines that promote T cell proliferation [110].
It has also been observed that Ply alters DC function
(Table 1). Although PavA was found to protect against
phagocytosis, Ply was found not to influence the pneumococcus uptake by human DCs [110]. Contrarily to these findings, another study found that Ply does contribute to the bacterial uptake by human DCs, since a strain lacking the protein is phagocytized 50% less than the wild type (WT) strain
[113]. In addition, another report described that the intracellular expression of Ply downregulates the production of IL12 p70 and IL-8 by human DCs and inhibits their maturation.
In fact, DCs infected with a ply mutant significantly upregulated the expression of activation markers CD80 and CD86,
in comparison to DCs infected with the WT strain [113].

These data suggest that Ply could be acting as a reducer of
DCs maturation, impairing the costimulatory signals proportioned to T cells during antigen presentation. This could prevent the trigger of adaptive immune responses.
Besides preventing phagocytosis by DCs, S. pneumoniae
has also been shown to induce apoptosis in these cells Fig.
(1B). In murine DCs, apoptosis is induced by two mechanisms: a caspase-independent mechanism, which relies on
Ply expression, and through a caspase-dependent mechanism, produced by the interaction of bacterial components
located below the capsule with TLRs on the DC surface
[118]. In human DCs it has been described that Ply induces
apoptosis through the activation of caspases, showing a more
pronounced effect when a nonencapsulated strain of S.
pneumoniae is used, probably due to the higher phagocytosis
of these bacteria [113]. However, both in murine and human
DCs, it has been described that the internalization of S.
pneumoniae is not a prerequisite for Ply to induce apoptosis
Fig. (1B) [113, 118].
The protective role of DCs against pneumococcal infection is supported by studies showing that mice lacking the
CC chemokine ligand 2 (CCL2) show more severe pneumonia after S. pneumoniae infection, as a result of impaired
recruitment of lung mononuclear phagocytes, including conventional DCs [119]. However, these observations are opposed by in vivo experiments showing that DCs might play a
negative role in the outcome of pneumococcal infection. A
study performed by Winter et al. [120] showed that the use
of FMS-like tyrosine kinase 3 ligand (Flt3L) to increase the
frequency of myeloid DCs in the lungs of mice negatively
impacts the clearance of pneumococci. Mice pretreated with
Flt3L and infected with S. pneumoniae showed increased
vasculitis, microthrombus formation, lung permeability, elevated bacterial loads in lungs, and increased mortality [120].
This is probably due to increased amounts of proinflammatory cytokines, such as IL-6, KC, and MIP-1, detected in the lungs of Flt3L-treated mice after 4 days of infection. Accordingly, treatment of these mice with an antiCCL2 blocks monocytes and reduces lung permeability after
pneumococcus infection [120]. These data suggest that DCs
drastically contribute to S. pneumoniae colonization, but still
remains non addressed whether live virulent S. pneumoniae
could be exploiting DCs as a carrier to promote its own
transport, either as extracellular-attached bacteria or as intracellular pathogen, thus reaching more easily lung tissues or
systemic circulation and causing bacterial colonization and
spreading. This mechanism has been previously described
for other pathogens such as Salmonella typhimurium [102,
114] and HIV-1 [121]. However, further research is needed
to explore this hypothesis for S. pneumoniae.
ROLE OF T CELLS IN S. PNEUMONIAE INFECTION
T cells have been shown to arrive soon after the infection
takes place with S. pneumoniae, appearing near inflamed
areas of the lungs 24-48 hours post-infection, which suggest
that these cells play a fundamental role in early stages of the
infection [122]. However, instead of an increase in the total
T cell number after S. pneumoniae infection, which would
indicate an active infiltration of peripheral T cells, what apparently happens is a rapid relocation of the lung residing T
cell population. Thus, these cells become more localized
around the inflamed bronchioles 24 hours post-infection, and
relocate around perivascular tissue after 48 hours [123]. It
has also been suggested that T cells are able to migrate
quickly upon detection of S. pneumoniae derived molecules,
such as Ply [122].
In terms of adaptive immunity, it has been described that
T cells are fundamental for protection after immunization
with either vaccines or previous infection with different serotypes of this bacterium. Below we will refer to the role of
different T cell populations in pneumococcus infection and
protective immunity.
-CD4+ T CELLS
CD4+ T cells, also denominated helper T cells (TH),
contribute to the adaptive immune response mainly through
the secretion of specific cytokines [124]. At date, the most
described TH phenotypes are TH1, TH17, TH2, and Tregs [125].
Combinations of these CD4+ T subtypes promote the
clearance of pathogenic bacteria.
57
CD4+ T cells have been extensively studied in models of

S. pneumoniae nasopharyngeal colonization. Kadioglu et al.
(2004) found that when nave MHC class II-deficient mice
(hence lacking CD4+ T cells and potential TH subsets) were
intranasally inoculated with pneumococcus, the infection
persisted longer in lungs and blood in comparison to WT
mice, suggesting that CD4+ T cells contribute in early host
defense [122]. It seems that CD4+ T cells are necessary for
the clearance of pneumococcal colonization through a process that is proposed to be mediated by TH1 cells and their
secretion of IFN- [21]. However, other studies have suggested that the presence of CD4+ T cells is detrimental in the
early stages of murine pneumonia, because of their contribution to inflammation and progression of the disease [126].
Nave MHC class II-deficient mice and CD4+ T cell-depleted
mice showed reduced mortality after intranasal infection
with S. pneumoniae, although no significant differences were
observed in the bacterial load in airways, blood, and spleen
[126]. The presence of CD4+ T cells early during infection
with pneumococcus in WT mice was related to increased
amounts of cytokines in serum, such as IL-1, IL-10, MIP1, and MIP-2 [126]. It is possible that these cytokines promote immunopathology upon pneumococcus infection and
increase the severity of the disease.
CD4+ TH17 cells appear to have great significance in the
response against pneumonia, as several studies have shown
that IL-17 secretion by these cells contributes to the clearance of the infection [122, 127, 128]. RAG1-/- mice receiving CD4+ T cells from whole cell vaccine (WCV)immunized mice show reduced colonization with pneumococcus, as compared to mice transferred with CD4+ T cells
from mice immunized only with a control immunogen
(Cholera Toxin) [127]. However, T cells from WCVimmunized mice whose IL-17A contained a targeted deletion
are unable to protect against colonization. In this study it was
described that IL-17A was enhancing the phagocytic activity
of neutrophils against pneumococcus Fig. (1H) [127].
Using a proteomic screening approach, pneumococcal T
cell antigens capable of generating a TH17 response have
been identified [129]. Stimulation of mice with these antigens protects against colonization and elicits TH17 responses
[129]. This protection is abrogated when mice are depleted
of CD4+ T cells or treated with anti IL-17A [129]. In another
study, which used a model of murine protection against
pneumonia, it was shown that the pretreatment of mice with
a sublethal dose of a clinical strain of pneumococcus serotype 1 generates protection against a lethal infection with the
same strain [130], which correlates with increased expression of IL-17-related genes and with an increment of the IL17A+ population, 40% of which correspond to CD4+ T cells
[130]. Protected mice also show a more pronounced recruitment of PMNs to the lungs.
In humans, TH17 cells also appear to play a fundamental
defense role against pneumococcus [131]. Cocultures of human monocytes and CD4+ T cells have shown that monocytes produce IL12p40 in the presence of live pneumococci,
which triggers a TH1 response. Instead, the use of heat-killed
pneumococci triggers a TH17 response, through TLR2 signaling [131]. Moreover, T cell responses against some
pneumococcal antigens have been evaluated in sera of
young, adult, and elderly individuals, finding a predominant

TH17 response combined with a TH1 or TH2 response [132].
Other studies that have evaluated several immunization
strategies to prevent pneumococcus infections have shown
the fundamental role of CD4+ T cells in the specific protection against S. pneumoniae. For instance, intranasal immunization using live pneumococcus or a killed nonencapsulated
WCV protected antibody-deficient mice against colonization, indicating that antibodies were not necessary for this
protection [133]. T-cell deficient mice and CD4+ T cell depleted mice were not protected by intranasal immunization
with WCV, which suggested a role for CD4+ T cells in the
protection against recolonization [133]. Intranasal immunization of mice with pneumococcal proteins also elicits a
mechanism of protection dependent on CD4+ T cells and
independent of antibodies [134]. In agreement with this notion, BALB/c DO11.10 Rag-/- mice that lack mature B and T
cells, except for CD4+ T cells specific for the OVA323-339
peptide, are protected against subsequent colonization with
an unrelated strain of S. pneumoniae expressing PspA-OVA,
when they are previously immunized with WCV-OVA
[135].
As described before, pneumococcus impairs its uptake in
DCs and this virulence mechanism can further impact the
activation of CD4+ T cells by these cells. New studies are
required to describe how the clearance of pathogenic S.
pneumoniae is enhanced by the synergistic work between
CD4+ T cells and DCs.
-CD8+ T CELLS
CD8+ T cells are effector cells that contribute to the host
defense against microbes, secreting IFN- and recognizing
infected cells expressing foreign peptide antigens associated
to MHC-I molecules, which are eliminated through the secretion of perforin and granzyme [136]. Therefore, CD8 + T
cells are important in the defense against intracellular pathogens, as it has been observed in different models of infection
[137, 138]. Despite this, increasing evidence demonstrates
that CD8+ T cells participate in the response against infections caused by extracellular pathogens and their role is apparently to modulate the CD4+/TH17 response [139, 140]. In
the S. pneumoniae infection model the same contribution of
CD8+ T cells has been observed, but only to a few specific
serotypes Fig. (1F) [141, 142].
In a study using a conjugate vaccine of pneumococcal
CPS3 and tetanus toxoid (TT), immunized CD8-/- mice
showed higher titers of Ig antibodies compared to WT mice
[143]. Even though they were protected against a lethal systemic challenge with pneumococcus, the capacity to survive
did not improve when these animals were intranasally challenged with bacteria [143]. However, sera from immunized
CD8-/- mice did protect nave WT mice, but when CD8-/mice were immunized with immune sera from WT or CD8-/mice, there was no improvement in their survival after a lethal intranasal challenge [143].
Later it was seen that CD8+ T cells contribute to the resistance against a lethal infection caused by a serotype 3 strain
in mice [141]. In this model, CD8+ T cells were shown to
contribute to survival, while CD4+ T cells were found to be
Nieto et al.
dispensable. CD8-/- mice showed increased IL-17 production

by CD4+ T cells and a decrease in the Tregs (CD4+ CD25 +
Foxp3+) population. This suggests that a restriction for IL-17
secretion by a regulatory T CD8+ subset could exist in vivo,
which might be serotype-specific.
It has been shown that glycosylated zwitterionic components of the bacterial surface can promote T CD8 + cell activation [142]. Zwitterionic polysaccharides (ZPs), expressed
in the surface of S. pneumoniae trigger regulatory roles for
CD8+ T cells. These CD8+ T cells do not express the CD28
molecule in their surface, making up a CD8+CD28- population unable to receive costimulation from CD80 and CD86
expressed in the surface of mature APCs. On the contrary,
they are able to mediate the production of IL-10 and TGF-
(two important immunosuppressive molecules) Fig. (1F)
[142]. For CD8+ cells, this new concept of regulatory cells
(Tregs) activation by means of ZPs arises as a mechanism to
control TH-dependent exacerbated inflammation during the
immune response, because CD8+CD28- cells suppress CD4 +
T cell activity either in vitro as well as in vivo [142]. Interestingly, activation of these regulatory cells is APC-T cell contact-independent, but requires TCR engagement/ crosslinking, suggesting that soluble ZPs derived from S. pneumoniae
can, by themselves, mediate a suppressor adaptive immune
response as an evolved mechanism displayed by the host to
counteract exacerbated inflammation during bacterial clearance.
Interestingly, in other model it has been shown that T
CD8+CD28- cells can block the CD4+ T cells activation mediated by antigen-loaded APCs by recognition of class I
pMHC [144]. These CD4+ T cells fall into an anergic state,
then secrete decreased levels of the auto-stimulatory/clonal
expanding IL-2 cytokine [144]. This is very similar to what
was observed for reduced priming of CD4+ T cells in cocultures with APCs in the presence of CD8+CD28- T cells
(generated by pneumococcal ZP stimulation) [142]. However, reduction in CD4+ T cell activation by ZP-generated
CD8+CD28- T cells was reported as T cell-APC contactindependent, suggesting an important role for soluble suppressive factors such as IL-10 and TGF- that are secreted
by these regulatory cells [142]. These observations suggest
that Tregs can exert their regulatory/suppressive role either by
contact with class I pMHC or stimulation by soluble factors
[145].
Recently, it has been shown that under S. pneumoniae
infection, IL-10 and TGF- expression is necessary to promote bacterial clearance in the Balb/c mice model [146].
Mice that received blocking peptides for TGF- developed
acute inflammation with detrimental consequences for the
animals. In these TGF--suppressed mice, blood bacterial
levels were drastically increased, and a dramatic decrease in
Foxp3+ regulatory T cell recruitment to the lungs was observed [145]. In addition, it was shown that recruitment of
Foxp3+ IL-10-producing regulatory cells to the lungs was
also necessary for host protection [145]. These observations
suggest that both TGF-- and IL-10-secreting CD8+CD28- T
cells, produced by S. pneumoniae-derived ZPs, could be mediating the anti-exacerbated TH response that will favors host
recovery. Finally, CD8+ T cells have been shown to contribute to the generation of Ig responses against pneumococcal
antigens, but their contribution is relatively small in comparison to CD4+ T cells [147].
- T CELLS
T cells are a rare population of immunomodulatory T
cells present in many tissues [148]. The exact ligands recognized by the TCR are not known, but there is evidence
that T cells respond to microbial products and to stressed
epithelial cells [149, 150]. T cells have been implicated in
the resolution phase of a sub-lethal infection with S. pneumoniae in mice. Upon resolution, their numbers increase
over 30-fold, and they are proposed to regulate the influx of
AMs and DCs into the infected lungs through the regulation
of inflammatory mediators, but also by directly killing both
nave and infected AMs and pulmonary DCs Fig. (1G) [151].
In TCR-/- mice, an increment in the number of AMs and
pulmonary DCs is observed but eventually their levels normalize, which suggests that T cells contribute to this
process but they are not essential [151].
In other models of infection, T cells have been implicated in the early production of IL-17A. In a murine pneumococcal infection model, T cells have also been pointed
to be the main producers of IL-17A Fig. (1G), a process dependent on the presence of Ply, given that mice infected with
a Ply-deficient strain showed significantly lower concentrations of IL-17A [29]. This finding supports the notion that
T cells would be the main cells producing IL-17A, but as
previously mentioned, it has been described that CD4+ T
cells also have an important role in this process.
S. pneumoniae infections enhance neutrophil recruitment
to the lungs [152]. Interestingly, this recruitment was associated with T cells presence. Thus, these T cells participate
in the lung bacterial clearance mechanisms, mainly by TNF production [152]. However, not all T cells are recruited
to the lungs during S. pneumoniae infection. It has been recently described that this infection increases the activation
and proliferation of resident V1, V4 and V6 T cells in the
lungs, but not in associated lymphoid tissue [153]. Nevertheless, other consequences for S. pneumoniae infection, such as
meningitis, display increased T cells numbers in other
tissues different from the lungs [154]. Patients suffering S.
pneumoniae-mediated meningitis displayed over 30% increased amounts of T cells in blood, which could be mediating the inflammation [154]. Thus, T cells proliferation
and tissue homing could be associated with the kind of disease caused by S. pneumoniae. Still remains unknown
whether T cells could be involved during acute otitis media, sepsis, sinusitis, and keratitis, the other common manifestations for pneumococcal infections.
THERAPEUTIC
STRATEGIES
PNEUMOCOCCAL DISEASES
TO
PREVENT
To date, polysaccharide conjugate vaccines (PCVs)

against S. pneumoniae are available and used worldwide
[155-157]. These vaccines consist on purified capsule polysaccharides conjugated to carrier proteins, which can be protective and prevent further pneumococcal pneumonia and
meningitis caused by certain serotypes [155, 158]. A vaccine
containing 7 capsular serotypes has been shown to reduce the
incidence of pneumonia and meningitis caused by S. pneu-
59
moniae by up to 65% in children under 5 years old in the

United States [156]. These PCVs promote T cell responses
and induce B cell memory, providing a protective response
in small children [159, 160]. However, despite their protective capacity, the manufacture of PCVs is of high-cost and
the use of these vaccines has led to serotype-replacement
where non-vaccine serotypes start to predominate after massive vaccination [157, 161]. Therefore, the design of a new
type of vaccine, more accessible and with broad protection is
considered a priority.
In the last decade new strategies have emerged for the
design of vaccines against this pathogen. The term Reverse
Vaccinology refers to the scanning of the bacterial genome
to identify for genes that encode proteins suitable for use as
antigens in a vaccine, which are then synthetized in vitro and
assayed in vivo [162, 163]. Good candidates are proteins
predicted to be expressed on the bacterial surface and conserved among different strains. This strategy was applied to a
clinical isolate of S. pneumoniae, in which the search for
motifs that predicted a surface localization of the protein led
to a selection of 130 proteins. When mice were immunized
with these proteins, 6 of them conferred immune protection
against a pneumococcal infection: pneumococcal histidine
triad A protein (PhtA), endo-beta-N-acetylglucosaminidase
(LytB), 1,4-beta-N-acetylmuramidase (LytC), pneumococcal
vaccine antigen A (PvaA) serine protease (PrtA, two different antigens). In addition, these proteins were broadly distributed among various strains and showed immunogenicity
during human infection [163].
Several pneumococcal proteins are currently being studied for their potential use as vaccines in clinical trials.
Along these lines, PspA is one of the promising candidates
because despite existing in several isoforms, antibody crossreactivity has been observed between various S. pneumoniae
strains [164, 165]. Protein- and DNA-based vaccines have
been evaluated in mice, both inducing protection but differing in the type of TH call and IgG antibody responses generated [166, 167]. Phase I studies in humans using recombinant PspA have shown that this protein is immunogenic and
that sera from immunized individuals can protect mice
against a fatal pneumococcal infection [165]. Furthermore,
PspA is able to elicit strong T cell mediated immune
responses in adults with pneumonia [168]. Also, a recombinant attenuated Salmonella vaccine (RASV) strain has been
used as a vehicle for delivery of PspA [169]. Other pneumococcal proteins under study as potential antigens for vaccination are the histidine triad protein D (PhtD) and the monovalent choline-binding protein A (PcpA). Either individually or
as a bivalent vaccine, both have shown immunogenicity in
phase I trial in humans [170, 171]. Further studies are required to define whether these proteins are able to elicit similar responses in the pediatric population.
Given that nasopharyngeal colonization precedes bacterial invasion to the lower airways, several studies have aimed
to designing a vaccine that would prevent this step of S.
pneumoniae infective cycle. Because immunity to nasal
colonization was demonstrated to be CD4+ T cell dependent
[133], a new strategy was used to identify potential vaccine
candidates. As mentioned above, a proteomic screening approach allowed the identification of pneumococcal T cell
antigens capable of activating murine TH17 cells [129]. Interestingly, among the protective TH17 antigens identified,
traditional antibody targets, such as PsaA and PspA, were
not included. Therefore, antigens recognized by TH17 cells
appear to differ significantly from antigens that are traditionally targeted by antibodies [129]. Phage display library is
another useful tool for the identification of novel bacterial
antigens. After construction, the library is screened against
sera from infected patients or immunized mice. This approach has allowed the identification of pneumococcal proteins containing B cell epitopes, such as the histidine triad
protein E (PhtE), PspA, and the IgG1 protease [172, 173].
In summary, the generation of new and universal pneumococcal vaccines, that will help fighting the diseases
caused by this pathogen and also will be an alternative to the
current PCVs, is a process in progress that is leading to
promising candidates currently under clinical evaluation.
CONCLUDING REMARKS
S. pneumoniae is still one of the most important causes of
respiratory-associated diseases worldwide. Therefore, understanding the interaction of this pathogen with cellular components of the immune system, such as DCs and T cells is
highly relevant. DCs are important components of the adaptive immune response, classified as the best APCs. Given the
low efficiency in phagocytizing encapsulated strains of S.
pneumoniae, and the virulence genes used by the pneumococcus to overcome this process, the activation of pneumococcus-specific T cells by DCs might be dampen by this
bacterium, which might prevent its clearance form infected
tissues. Some studies point to a negative contribution of DCs
in the course of pneumococcal pneumonia, suggesting that
they lead to an exacerbated inflammatory response. Further
studies are needed to clarify the role of DCs in the response
against pneumococcus.
Nieto et al.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflicts of interest.
ACKNOWLEDGEMENTS
The authors are supported by the following grants:
FONDECYT N. 1070352, FONDECYT N. 1050979, FONDECYT N. 1040349, FONDECYT N. 1100926, FONDECYT N. 1110397, FONDECYT N. 1100971, FONDECYT
N. 1110604, and Millennium Institute on Immunology and
Immunotherapy (P09-016-F). PAN and SAR are CONICYT
fellows, AMK is a Chaire de la Rgion Pays de la Loire de
Chercheur tranger d'Excellence.
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Gene Elements that Regulate Streptococcus pneumoniae Virulence and

Millennium Institute on Immunology and Immunotherapy 2Departamento de Gentica Molecular y Microbiologa,

*Address correspondence to this author at the Departamento de Gentica

52 Current Gene Therapy, 2013, Vol. 13, No. 1

stance, in the United States it has been reported that 40% of

ceptor expressed by macrophages that recognizes and binds

Current Gene Therapy, 2013, Vol. 13, No. 1

Activation of CD4+ T cell

CD4+ Th17 cell

Regulatory CD8+ T cell

of Ply are due to the initial binding of cholesterol in the host

54 Current Gene Therapy, 2013, Vol. 13, No. 1

Major Virulence Factors of Streptococcus pneumoniae

NCBI Gene ID*

Masks bacterial antigens, prevents

Over the cell

[38, 39, 142]

Binds cholesterol, polymerizes, and

Breaks hyaluronic acid. Helps

Cleaves N-acetylneuraminic acid.

Bacterial autolysis, release of

Interferes with the complement

Binds to complement regulatory

Prevents phagocytosis by DCs.

*From Streptococcus pneumoniae strain D39, NC_008533.1 [174].

The pneumococcal surface adhesin A (PsaA) [59] (Table

Current Gene Therapy, 2013, Vol. 13, No. 1

becomes capable of translocating MHC class II molecules

56 Current Gene Therapy, 2013, Vol. 13, No. 1

in comparison to DCs infected with the WT strain [113].

Current Gene Therapy, 2013, Vol. 13, No. 1

CD4+ T cells have been extensively studied in models of

58 Current Gene Therapy, 2013, Vol. 13, No. 1

young, adult, and elderly individuals, finding a predominant

dispensable. CD8-/- mice showed increased IL-17 production

To date, polysaccharide conjugate vaccines (PCVs)

Current Gene Therapy, 2013, Vol. 13, No. 1

moniae by up to 65% in children under 5 years old in the

60 Current Gene Therapy, 2013, Vol. 13, No. 1

There is also need to dissect the exact role of CD4+,

Drummond P, Clark J, Wheeler J, Galloway A, Freeman R, Cant

Malley R, Henneke P, Morse SC, Cieslewicz MJ, Lipsitch M,

Current Gene Therapy, 2013, Vol. 13, No. 1

62 Current Gene Therapy, 2013, Vol. 13, No. 1

exhibit a requirement for Zn and Mn resulting from inactivation of

pathogen immunity and prevent autoimmunity. Immunotherapy

Current Gene Therapy, 2013, Vol. 13, No. 1

pneumococci suggest a crucial protective role in the host response

64 Current Gene Therapy, 2013, Vol. 13, No. 1

Received: August 30, 2012

Revised: November 21, 2012

Accepted: November 21, 2012

2005 in a population of children who received 7-valent

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