Anand Kumar Andiappan, BTecha,b*
De Yun Wang, MD, PhDc*
Ramani Anantharaman, PhDb
Bani Kaur Suri, MScb
Bernett Teck Kwong Lee, PhDa
Olaf Rotzschke, PhDa
Jianjun Liu, PhDd
Fook Tim Chew, PhDb
Fromathe Singapore Immunology Network (SIgN), Agency for Science, Technology
and Research (A*STAR);bthe Department of Biological Sciences, National Univer-
sity of Singapore;cthe Department of Otolaryngology, National University of Singa-
pore; anddHuman Genetics, Genome Institute of Singapore, Agency for Science,
Technology and Research (A*STAR), Singapore. E-mail: firstname.lastname@example.org.
*These authors contributed equally to this work.
Supported by grants from the Singapore Immunology Network (SIgN-06-006and SIgN-
08-020); the National Medical Research Council (NMRC/1150/2008), Singapore; the
Biomedical Research Council, Singapore; the Agency for Science, Technology and
Research (A*STAR); and the National University of Singapore for the Graduate Re-
search Scholarship for students involved in the study.
Disclosure of potential conflict of interest: A. K. Andiappan, R. Anantharaman, and B.
K. Suri have received research scholarships for PhD studies from the National Univer-
sity of Singapore. F. T. Chew has received grants from the Singapore Immunology
Network, the Singapore Biomedical Research Council, the National Medical Re-
search Council, and the National University of Singapore Graduate Research Schol-
arship; has consultant arrangements with Sime Darby Technology Centre; and is
employed by the National University of Singapore. The rest of the authors declare
that they have no relevant conflicts of interest.
1. Ramasamy A, Curjuric I, Coin LJ, Kumar A, McArdle WL, Imboden M, et al. A
genome-wide meta-analysis of genetic variants associated with allergic rhinitis
and grass sensitization and their interaction with birth order. J Allergy Clin Immunol
2. Gao PS, Rafaels NM, Mu DG, Hand T, Murray T, Boguniewicz M, et al. Genetic
variants in thymic stromal lymphopoietin are associated with atopic dermatitis
and eczema herpeticum. J Allergy Clin Immunol 2010;125:1403-7.
3. Ferreira MAR, Matheson MC, Duffy DL, Marks GB, Hui JN, Le Souef P, et al. Iden-
tification of IL6R and chromosome 11q13.5 as risk loci for asthma. Lancet 2011;
4. Esparza-Gordillo J, Weidinger S, Folster-Holst R, Bauerfeind A, Ruschendorf F, Pa-
tone G, et al. A common variant on chromosome 11q13 is associated with atopic
dermatitis. Nat Genet 2009;41:596-601.
5. Zhou XB, Baron RM, Hardin M, Cho MH, Zielinski J, Hawrylkiewicz I, et al. Iden-
tification of a chronic obstructive pulmonary disease genetic determinant that regu-
lates HHIP. Hum Mol Genet 2012;21:1325-35.
6. Creyghton MP, Cheng AW, Welstead GG, Kooistra T, Carey BW, Steine EJ, et al.
Histone H3K27ac separates active from poised enhancers and predicts developmen-
tal state. Proc Natl Acad Sci U S A 2010;107:21931-6.
7. Karlic R, Chung HR, Lasserre J, Vlahovicek K, Vingron M. Histone modification
levels are predictive for gene expression. Proc Natl Acad Sci U S A 2010;107:
8. Lee GR, Kim ST, Spilianakis CG, Fields PE, Flavell RA. T helper cell
differentiation: Regulation by cis elements and epigenetics. Immunity 2006;24:
9. Brand S, Kesper DA, Teich R, Kilic-Niebergall E, Pinkenburg O, Bothur E,
et al. DNA methylation of TH1/TH2 cytokine genes affects sensitization
and progress of experimental asthma. J Allergy Clin Immunol 2012;129:
Available online December 8, 2012.
Nasopharyngeal carriage with Streptococcus
pneumoniae augments the immunizing effect
of pneumolysin toxoid B
To the Editor:
Streptococcus pneumoniae is a major human pathogen respon-
and sepsis. Morbidity and mortality linked to pneumococcal
disease remain high in sub-Saharan Africa, despite the introduc-
tion of protein-polysaccharide conjugatevaccines (PCVs), which
offer protection against major disease-causing serotypes.1PCVs
are limited in their affordability and their efficacy in high-risk
groups, such as infants.2Furthermore, because the conjugatevac-
cine is limited in its serotype coverage, the major disease-causing
serotypes are being replaced by previously minor serotypes1in
places where vaccination is widespread. Attempts to overcome
the limitations of current vaccination strategies have focused on
developing vaccines around conserved pneumococcal proteins
that vary minimally between serotypes and are indispensible to
bacterial colonization or virulence, such as the cholesterol-
binding, pore-forming toxin pneumolysin and its derivatives.3
infection. At sublytic concentrations, pneumolysin has wide-
ranging effects including activation of the immune system,4in-
Variants of pneumolysin with reduced hemolytic activity have
ing a potent toxin as an immunogen. Pneumolysin toxoid B
(PdB), a pneumolysin derivative with a Trp-433/Phe mutation,
has 0.1% of the hemolytic activity of nativewild-type pneumoly-
sin and has shown promise in immunization studies in animals.7,8
Here we assess the effect of PdB immunization on pneumococ-
cal carriage. It is debatable, in the context of a protein-based
vaccine, whether elimination of carriage would be desirable
because low-level exposure to the pneumococcus might serve to
strengthen and reinforce immune responses induced by vaccina-
tion. Furthermore, in a protein-based approach the problems of
serotype replacement that accompany PCV immunization strate-
gies should not exist, and therefore the issue of the nasopharynx
ameliorated. However, carriage is widely accepted to be a prereq-
uisite for invasive disease, and therefore there is an equally strong
argument to be made for eliminating colonization after vaccina-
tion. We believed this contentious area warranted further investi-
gation in mouse models that have been used previously with great
success to drive forward pneumococcal vaccine development.9
Immunization with PdB in alum adjuvant, administered
a method based on that of Alexander et al,8did not affect mouse
survival in a subsequent invasive pneumonia infection with the
virulent S pneumoniae serotype 2 strain D39 (Table I and see
Fig E1, A, in this article’s Online Repository at www.jacionline.
org), despite inducing a high serum titer of IgG2a anti-
pneumolysin antibodies (Table I and see Fig E1, B) that strongly
inhibited the ability of wild-type pneumolysin to cause red blood
cell lysis in vitro (Table I and see Fig E1, C). Intriguingly,
although PdB immunization did not protect against invasive dis-
I), we did observe a significant decrease in pneumococcal num-
bers colonizing the nasopharynx of PdB-immunized mice after
infection with D39 (Table I and see Fig E1, D). To follow up on
this observation, we performed PdB immunization and chal-
lenged mice 7 days after the final immunization with a dose of
pneumococci that establishes stable long-term nasopharyngeal
colonization but does not cause pneumonia or invasive disease
(Fig 1, A and B). Mice that had received PdB immunization had
significantly reduced pneumococcal numbers in the nasopharynx
at both 3 and 7 days after infection compared with control mice
J ALLERGY CLIN IMMUNOL
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LETTERS TO THE EDITOR 1433
(Fig 1, A), indicating some protection against long-term carriage
in immunized mice. Furthermore, we observed a significant
lymph nodes of PdB-immunized mice at day 7 after infection
compared with that seen in control animals (Fig 1, B). IgA-
producing B-cell numbers increased in all colonized mice over
the course of infection, but no additional increase was seen in
mice that had also received PdB immunization (Fig 1, C).
Because PdB immunization induces strong humoral responses
but does not affect lung bacterial numbers or mouse survival and
because nasopharyngeal carriage is known to induce some anti-
followed by establishment of carriage to affect subsequent
susceptibility to invasive pneumococcal disease (Fig 1, D and E).
Mice were subcutaneously immunized with PdB/alum or PBS/
Twenty-one days after colonization, mice were intravenously
challenged with D39. Strikingly, 80% of mice that received PdB
and were colonized with pneumococci survived subsequent intra-
venous pneumococcal challenge. This compared with an average
of 50% survival of mice that were colonized but did not receive
PdB. Mice with the lowest levels of survival included naive
mice (30%) and mice that received PdB but were not colonized
TABLE I. Immunization with PdB induces antibody and reduces colonization but does not protect against invasive disease
(time of death
(24 h after infection)
lung tissue (48 h
nasopharynx (48 h
Data are presented are means 6 SEMs. Mice were immunized with 2 doses of 20 mg PdB/alum (PdB) or PBS/alum (Control) 14 days apart and 14 days after final immunization
were intranasally infected with 106colony-forming units (CFU) of S pneumoniae.
*P < .001, 1-way ANOVA.
FIG 1. A combination of PdB immunization and colonization with pneumococcus is highly protective
against invasive pneumococcal disease. Mice were immunized with 2 doses of 20 mg PdB/alum (PdB) or
PBS/alum (Control) 14 days apart. Selected groups were then intranasally infected with a colonizing dose
(105colony-forming units [CFU] in 10 mL) of S pneumoniae (colonized). Mice were intravenously challenged
with an acute dose of D39. A, CFU/mg nasopharynx in colonized mice. B and C, ELISpot analysis of cervical
lymph node (CLN) cells. One spot correlates to 1 IgG-producing (Fig 1, B) or IgA-producing (Fig 1, C) B cell.
D, Survival after intravenous challenge. E, Sepsis at 24 hours after challenge. Data are representative of 2
experiments (>10 mice per group). Data in Fig 1, A, B, C, and E, are means 6 SEMs. *P < .05, **P < .01,
and ***P < .001.
J ALLERGY CLIN IMMUNOL
1434 LETTERS TO THE EDITOR
had been colonized or had been PdB immunized and colonized
had significantly fewer bacteria in blood at 24 hours after intrave-
nization provided added protection against sepsis beyond that
seen with colonization alone (Fig 1, E). Because we observed
no increase in the titer of inhibition of sera collected at the time
mice compared with that from mice receiving PdB immunization
alone (data not shown), we believe that the additional protection
afforded by colonization might be mediated through cellular
immunity. This is an area of ongoing investigation.
Collectively, these data suggest that PdB immunization is
highly protective against invasive disease in the context of
additional, low-level exposure to pneumococcus in the nasopha-
rynx. This protection is greater than both that provided by PdB
immunization alone and that provided by previous pneumococcal
carriage. This suggests either that PdB immunization induces
weak immune responses that are then boosted by subsequent
nasopharyngeal exposure to the pneumococcus or more likely
that PdB immunization and pneumococcal exposure induce
differential immune responses that together form highly effective
protection against subsequent invasive disease. The evidence
presented here confirms that PdB immunization induces strong
humoral responses, and therefore perhaps cell-mediated immu-
nity is boosted by carriage. It will be of interest to assess whether
the additional protection afforded by carriage after PdB immu-
nization is serotype specific. Previous work has suggested that
carriage induces complex immune responses directed at a variety
of bacterial protein and polysaccharide antigens,10and therefore
cross-serotype protection seems likely in this model.
The experiments we describe suggest that immunization with a
noncytotoxic pneumolysin derivative is highly protective against
invasive disease in the presence of low-level pneumococcal naso-
pharyngeal carriage. This serves to reinforce immune responses
of carriage might not be a desirable outcome for a pneumococcal
protein vaccine. If the problem of decreasing protection over time
that has been reported for polysaccharide-conjugate vaccines is
also evident in pneumococcal protein vaccines, then maintenance
of low-level carriage in the nasopharynx after vaccination might
serve to reinforce vaccine-induced immunity.
Kingdom) for critical reading of the manuscript.
Daniel R. Neill, PhDa
Sarah Smeaton, PhDb
Mathieu Bangert, BScc
Aras Kadioglu, PhDa,b
FromaClinical Infection, Microbiology and Immunology, Institute of Infection and
Global Health, University of Liverpool, Liverpool, United Kingdom;bthe Department
of Infection, Immunity and Inflammation, University of Leicester, Leicester, United
Kingdom; andcthe Respiratory Infection Group, Liverpool School of Tropical Med-
icine, Liverpool, United Kingdom. E-mail: email@example.com.
Supported by a Medical Research Council PhD studentship and additional funding from
the Institute of Infection and Global Health, University of Liverpool.
Disclosure of potential conflict of interest: S. Smeaton has been supported by a grant
from the Medical Research Council (MRC). A. Kadioglu has been supported by
one or more grants from the MRC, internal University of Liverpool funding, and
the National Institutes of Health. The rest of the authors declare that they have no rel-
evant conflicts of interest.
1. Weinberger DM, Malley R, Lipsitch M. Serotype replacement in disease after
pneumococcal vaccination. Lancet 2011;378:1962-73.
2. ClutterbuckEA, Oh S, Hamaluba M, Westcar S, Beverley PC, Pollard AJ. Serotype-
specific and age-dependent generation of pneumococcal polysaccharide-specific
memory B-cell and antibody responses to immunization with a pneumococcal
conjugate vaccine. Clin Vaccine Immunol 2008;15:182-93.
3. Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneumo-
niae virulence factors in host respiratory colonization and disease. Nat Rev Micro-
4. McNeela EA, Burke A, Neill DR, Baxter C, Fernandes VE, Ferreira D, et al. Pneu-
molysin activates the NLRP3 inflammasome and promotes proinflammatory cyto-
kines independently of TLR4. PLoS Pathog 2010;6:e1001191.
5. Marriott HM, Dockrell DH. Streptococcus pneumoniae: the role of apoptosis in
host defense and pathogenesis. Int J Biochem Cell Biol 2006;38:1848-54.
6. Jounblat R, Kadioglu A, Mitchell TJ, Andrew PW. Pneumococcal behaviorand host
responses during bronchopneumonia are affected differently by the cytolytic and
complement-activating activities of pneumolysin. Infect Immun 2003;71:1813-9.
7. Paton JC, Lock RA, Lee CJ, Li JP, Berry AM, Mitchell TJ, et al. Purification and
immunogenicity of genetically obtained pneumolysin toxoids and their conjuga-
tion to Streptococcus pneumoniae type 19F polysaccharide. Infect Immun 1991;
8. Alexander JE, Lock RA, Peeters CC, Poolman JT, Andrew PW, Mitchell TJ, et al.
Immunization of mice with pneumolysin toxoid confers a significant degree of pro-
tection against at least nine serotypes of Streptococcus pneumoniae. Infect Immun
9. Gonzalez-Fernandez A, Faro J, Fernandez C. Immune responses to polysacchar-
ides: lessons from humans and mice. Vaccine 2008;26:292-300.
10. Richards L, Ferreira DM, Miyaji EN, Andrew PW, Kadioglu A. The immunising
effect of pneumococcal nasopharyngeal colonization; protection against future
colonization and fatal invasive disease. Immunobiology 2010;215:251-63.
Available online December 20, 2012.
French application of the European guidelines
for regulation of allergenic extracts
To the Editor:
As has been implemented in the United States,1,2regulation of
allergen extracts (AEs) used for in vivo diagnosis and immuno-
therapy has also been proposed in Europe.3
In 2001, Directive 2001/83/EC related to medicinal products
(APs) that required marketing authorization (MA).3However, for
Consequently, the European AP market was divided into 2 cate-
gories: (1) industrially produced APs as proprietary medicinal
products that are submitted to MA and (2) ‘‘named patient pro-
ducts’’ (NPPs), which are APs produced for an individual patient
regulated at the national level butare represented by up to 75% to
100% of the AP market in some countries.4-6
In France the NPP status is named allerg? enes pr? epar? es
sp? ecialement pour un individu (APSI [allergens specially pre-
pared for individuals]). In linewith the European guidelines, new
requirements have been introduced by the French authorities.7
(Agence Franc ¸aise de S? ecurit? e Sanitaire des Produits de Sant? e)
with a list of AEs they wish to market. For each AE, a precise
technical file must provide all the pharmacologic, toxicological,
and clinical data supporting its quality and safety but also its
diagnostic and therapeutic interest (see this article’s Online
Repository at www.jacionline.org).
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LETTERS TO THE EDITOR 1435
FIG E1. Immunization with PdB induces antibody levels and reduces colonization but does not protect
against invasive disease. Mice were immunized with PdB/alum (PdB) or PBS/alum (Control) and intranasally
infected with 106colony-forming units (CFU) of S pneumoniae. A, Survival. B, Serum anti-PdB IgG2atiter
after immunizations at day 0 and day 14 and at the study’s end point (time of death after infection).
C, In vitro inhibition of red blood cell lysis by sera. D, CFU/mg in the nasopharynx. Data are representative
of 3 experiments (>10 mice per group) and presented as means 6 SEMs. Significance in 1-way ANOVA with
the Bonferroni posttest. **P < .01 and ***P < .001.
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LETTERS TO THE EDITOR 1435.e1