Downloaded from on May 14, 2020 by guest · 2 Authors affiliations Tatiane Queiroz Zorzeto: Center for Investigation in Pediatri cs, Pediatrics Department, State University of Campinas

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Title: Immunogenicity of whole cell low lipopolysaccharide pertussis vaccine in

infants

Running Title: Immunity to whole cell low LPS pertussis vaccine in infants

Authors: Tatiane Queiroz Zorzeto, Hisako Gondo Higashi, Marcos Tadeu Nolasco da

Silva, Emilia de Faria Carniel, Waldely Oliveira Dias, Vanessa Domingues Ramalho,

Taís Nitsch Mazzola, Simone Corte Batista Souza Lima, André Moreno Morcillo,

Marco Antonio Stephano, Maria Ângela Reis de Góes Antonio, Maria de Lurdes

Zanolli, Isaias Raw, Maria Marluce dos Santos Vilela *.

Addresses of the institutions at which the work was performed:

Center for Investigation in Pediatrics, Pediatrics Department, State University of

Campinas Medical School, Campinas, São Paulo, Brazil. Rua Tessália Vieira de

Camargo, 126, Campinas, São Paulo, Brazil. CEP 13083-887.

Tel: 55-19-35218963, Fax: 55-19-35218972.

Butantan Institute. Rua Vital Brasil, 1500, São Paulo, São Paulo, Brazil. CEP 05503-

900. Tel: 55-11-37262139, Fax: 55-11-37261505. E-mail: [email protected].

* Corresponding author: Maria Marluce dos Santos Vilela.

Center for Investigation in Pediatrics, Pediatrics Department, State University of


Camargo, 126, Campinas, São Paulo, Brazil. CEP 13083-887.

Tel: 55-19-35218963, Fax: 55-19-35218972.

E-mail adress: [email protected]

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00339-08 CVI Accepts, published online ahead of print on 4 March 2009

on February 5, 2021 by guest

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Authors affiliations

Tatiane Queiroz Zorzeto: Center for Investigation in Pediatrics, Pediatrics Department,

State University of Campinas Medical School, Campinas, São Paulo, Brazil. Rua

Tessália Vieira de Camargo, 126, Campinas, São Paulo, Brazil. CEP 13083-887. Tel:

55-19-35218989, Fax: 55-19-35218972. E-mail: [email protected].

Hisako Gondo Higashi: Butantan Institute. Rua Vital Brasil, 1500, São Paulo, São

Paulo, Brazil. CEP 05503-900. Tel: 55-11-37262139, Fax: 55-11-37261505. E-mail:

[email protected].

Marcos Tadeu Nolasco Da Silva: Center for Investigation in Pediatrics, Pediatrics

Department, State University of Campinas Medical School, Campinas, São Paulo,

Brazil. Rua Tessália Vieira de Camargo, 126, Campinas, São Paulo, Brazil. CEP 13083-


Emília Faria Carniel: Center for Investigation in Pediatrics, Pediatrics Department, State

University of Campinas Medical School, Campinas, São Paulo, Brazil. Rua Tessália

Vieira de Camargo, 126, Campinas, São Paulo, Brazil. CEP 13083-887. Tel: 55-19-

35218973, Fax: 55-19-35218972. E-mail: [email protected].

Waldely Oliveira Dias: Butantan Institute. Rua Vital Brasil, 1500, São Paulo, São

Paulo, Brazil. CEP 05503-900. Tel: 55-11-37262139, Fax: 55-11-37261505. E-mail:

[email protected].


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Vanessa Domingues Ramalho: Center for Investigation in Pediatrics, State University of


Camargo, 126, Campinas, São Paulo, Brazil. CEP 13083-887. Tel: 55-19-35218989,

Fax: 55-19-35218972. E-mail: [email protected].

Taís Nitsch Mazzola: Center for Investigation in Pediatrics, State University of




Simone Corte Batista Souza Lima: Center for Investigation in Pediatrics, State

University of Campinas Medical School, Campinas, São Paulo, Brazil. Rua Tessália

Vieira de Camargo, 126, Campinas, São Paulo, Brazil. CEP 13083-887. Tel: 55-19-

35218989, Fax: 55-19-35218972. E-mail: [email protected].

André Moreno Morcillo: Center for Investigation in Pediatrics, Pediatrics Department,

State University of Campinas Medical School, Campinas, São Paulo, Brazil. Rua

Tessália Vieira de Camargo, 126, Campinas, São Paulo, Brazil. CEP 13083-887. Tel:

55-19-35218973, Fax: 55-19-35218972. E-mail: [email protected].

Marco Antonio Stephano: Faculdade de Ciências Farmacêuticas. Universidade de São

Paulo. Av. Prof. Lineu Prestes 580. Butantã CEP: 05508-000 - São Paulo, SP – Brasil.

Telefone: 55-11- 30913650, Fax: 55-11- 8156386. E-mail: [email protected].


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Maria Ângela Reis de Góes Monteiro Antonio: Pediatrics Department, State University

of Campinas Medical School, Campinas, São Paulo, Brazil. Rua Tessália Vieira de



Maria de Lurdes Zanolli: Pediatrics Department, State University of Campinas Medical

School, Campinas, São Paulo, Brazil. Rua Tessália Vieira de Camargo, 126, Campinas,

São Paulo, Brazil. CEP 13083-887. Tel: 55-19-35218973, Fax: 55-19-35218972. E-

mail: [email protected].

Isaias Raw: Butantan Institute. Rua Vital Brasil, 1500, São Paulo, São Paulo, Brazil.

CEP 05503-900. Tel: 55-11-37262139, Fax: 55-11-37261505. E-mail:

[email protected].

*Maria Marluce dos Santos Vilela: Center for Investigation in Pediatrics, Pediatrics

Department, State University of Campinas Medical School, Campinas, São Paulo,

Brazil. Rua Tessália Vieira de Camargo, 126, Campinas, São Paulo, Brazil. CEP 13083-



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Abstract

The lack of a clear correlation between the levels of antibody to pertussis

antigens and protection against disease lends credence to the possibility that cell-

mediated immunity (CMI) provides primary protection against disease.

This phase I comparative trial had the aim of comparing the in vitro cellular

immune response and anti-pertussis toxin IgG titers (anti – PT) evaluation of a cellular

pertussis vaccine with low lipopolysaccharide (LPS) content (wPlow) with that of the

conventional whole-cell one (wP). A total of 234 infants were vaccinated at 2, 4, and 6

months with conventional wP or wPlow. Proliferation of CD3+ T cells was evaluated by

flow cytometry after 6 days of peripheral blood mononuclear cells culture, with heat-

killed B. pertussis or phytohemagglutinin (PHA) stimulation. CD3+, CD4+, CD8+ and

TCR γδ+ cells were identified in the gate of blast lymphocytes. IFN-γ, TNF-α, IL-4 and

IL-10 levels in supernatants and serum anti-PT IgG levels were determined using

enzyme–linked immunosorbent assay (ELISA). Net percent CD3+ blasts in cultures

with B. pertussis was higher in the group vaccinated with wP (medians of 6.2% for wP

and 3.9% for wPlow; p=0.029). The frequency of proliferating CD4+, CD8+ and γδ+ cells,

cytokine concentrations in supernatants and the geometric mean titers of anti - PT IgG

were similar between vaccination groups. There was a significant difference between

the T cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of

TCR γδ+ cells in those of B. pertussis (p<0.001). The overall data did suggest that wP

resulted in modestly better specific CD3+ cell proliferation and TCR γδ+ cells

expansion was similar for both vaccines.

Keywords: Pertussis vaccine; Lipopolysaccharide; Cellular immunity, humoral

immunity, infant.


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Introduction

Efforts to immunize against whooping cough began almost as soon as Bordet

discovered the causative bacteria, Bordetella pertussis, in 1912 (8). Whole-cell pertussis

(wP) vaccine has been available since 1940. Yet the road to eliminate circulation of the

pathogen proved arduous. Throughout the world, pertussis remains a major cause of

morbity and mortality among infants (45). Some 20-40 million cases of disease occur

worldwide each year, 90% of which are found in developing countries (44).

Although no causal link has been identified between wP vaccination and

permanent brain damage or death, concerns about systemic reactions after immunization

with wP vaccine have been a major factor in its reduced acceptance in developed

countries (46). This has led to the development of more purified and less locally

reactogenic acellular pertussis (aP) vaccines (21, 22, 43, 47). Similar immunogenicity is

obtained with both types of vaccine, as the most efficacious vaccines of either category

protect >80% of the recipients from clinical disease. Moreover, the considerably higher

development costs of aP result in prices per dose that are unlikely to be currently

affordable for developing countries (46).

Lipopolysaccharide (LPS) possesses endotoxic activity and has also powerful

adjuvant activity. Both these properties are based upon the recognition of the LPS by

the host Toll-like receptor complex TLR4 / MD-2 and the subsequent activation of NF-

κB (35). The relatively high reactogenicity of wP vaccines has been associated with pro-

inflammatory cytokines (2, 28). Hence, a straightforward approach to reduce wP

reactogenicity would be the generation of a pertussis vaccine with a reduced quantity of

LPS. Furthermore, the lack of a clear correlation between the levels of antibody to

pertussis antigens and protection against disease, the persistence of protective immunity

long after the disappearance of pertussis antigen-specific antibody (Ab), and the longer


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duration of T cell responses to pertussis antigens lend credence to the possibility that

cell-mediated immunity (CMI) provides primary protection against disease (16, 34).

This phase I study was then performed to obtain preliminary immunogenicity

data of a new cellular pertussis vaccine with low LPS content (wPlow), in comparison to

the conventional whole-cell pertussis vaccine (wP) used in Brazil, all of which

formulated with diphtheria and tetanus toxoids and given in three primary injections in

infancy.

MATERIALS AND METHODS

Subjects

Infants scheduled to receive the first dose of pertussis vaccine were recruited in

Campinas Public Health Centers, São Paulo, Brazil. A total of 247 infants were

recruited to the study and randomized to receive three doses of wPlow or wP vaccines at

2, 4 and 6 months of age. The preterm (gestational age <37 weeks) and the low birth

weight newborns (weight <2500g), infants whose mothers were younger than 18 years

old or had hepatitis B carrier status, infants with family history of tuberculosis, syphilis

or Human Immunodeficiency Virus (HIV) infection were excluded. Exclusion criteria

also included non compliance, receipt of immunoglobulin therapy or blood products,

any immunodeficiency, malignancy, significant underlying disease or neurological

impairment.


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Vaccines

Vaccines were manufactured by Butantan Institute, São Paulo, Brazil. Bordetella

pertussis cells were treated with an organic solvent and washed in order to perform LPS

extraction. The culture was then detoxified by addition of 0.2 per cent formalin and

bacterial biomass was obtained by tangential flow filtration to formulate wPlow vaccine.

Each 0.5mL dose of both vaccines contained 4 protective units of pertussis, and

2 protective units of diphtheria and tetanus toxoid. The antigens were absorbed onto

1.25 mg of aluminum hydroxide, and 0.2 mg of thimerosal was used as a preservative.

All infants received vaccine from the same batch. The vaccines were visually

indistinguishable and identically packaged and were administered intramuscularly with

the use of standard techniques.

Study design

This prospective, randomized, double-blind comparative trial was conducted

between August 2006 and July 2007, following the principles outlined in the

Declaration of Helsinki. Written informed consent was obtained from all participants’

parent or legal guardian before study procedures were initiated. The study protocol was

approved by the Committee for Ethics in Research from UNICAMP, São Paulo, Brazil.


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Antigens used in cell culture assays

Heat-killed B. pertussis suspension (Butantan Institute, lot IB-CIIn/P14/06),

without thimerosal, was used at 5 x 106 cells/mL. Phytohemagglutinin (PHA, Sigma,

USA) was used as a positive control at 7.5 µg/mL.

T cell proliferation assay

Ten milliliters of heparinized peripheral blood were collected and used to

evaluate immune responses to pertussis vaccination at 7 months of age. The protocol for

proliferation assay was adapted from Gaines & Biberfeld. (20). Peripheral blood

mononuclear cells (PBMC) were isolated by density gradient centrifugation over Ficoll-

Hypaque (Amersham Biosciences, USA), washed, diluted to 1 x 106 cells/mL in RPMI

1640 medium (Sigma, USA) supplemented with 10% human AB serum (Sigma, USA),

1% glutamine (Sigma, USA) and 0.1% gentamycin and stimulated for 6 days with with

heat-killed B. pertussis, phytohemagglutinin (PHA) or medium alone at 37°C with 5%

CO2 in round-bottomed 96-well tissue culture plates (NUNC, Denmark). After

harvesting with 20mM ethylene diamine tetracetic acid (EDTA), samples were

incubated with human immunoglobulin and then stained with anti-human CD3, CD4,

CD8 and T cell receptor (TCR) pan γδ fluorescent antibodies (Beckman Coulter, USA)

before acquisition (Epics XL-MCL flow cytometer, Beckman-Coulter, USA) and

analysis (Expo, Software, Beckman- Coulter, USA). Isotype controls were used to

discriminate positive populations (Beckman Coulter, USA). Only CD3+ T cells were


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used in analysis. Forward and side scatters were used to gate on resting and blast

lymphocytes. Dead cells were excluded from all analyses. CD4+, CD8+ and TCR γδ+

cells were identified in the gate of blast lymphocytes. Proliferation was measured by

CD3+ percent blasts, in which basal proliferation was subtracted from B. pertussis and

PHA-stimulated cultures.

Cytokine quantification in supernatants

For cytokine quantification assay, PBMC were diluted to 2 x 106 cells/mL in

supplemented RPMI and incubated for 48 hours in round-bottomed 96-well tissue

culture plates with B. pertussis, PHA or medium alone at 37°C. Supernatants were

collected and stored at -80°C. Interferon-γ (IFN-γ), Tumor Necrosis Factor-α (TNF-α),

Interleukin 4 (IL-4) and Interleukin 10 (IL-10) levels were determined in duplicate by a

two-Monoclonal Antibody sandwich Enzyme Linked Immunosorbent Assay (two-MAb

ELISA) (R & D Systems) in flat-bottomed MultiSorp ELISA plates (NUNC, Denmark),

according to the manufacturer´s protocols. Recombinant cytokine was used for the

standard curve. The limit of detection was 15.6ρg/mL for IFN-γ and TNF-α, and

31.2ρg/mL for IL-4 and IL-10.

Quantitative determination of anti-PT IgG

Quantitative determination of anti-pertussis toxin IgG (anti-PT ) was performed

blindly on serum samples collected one month after the third dose of vaccine and stored

at -80ºC until tested. Using ELISA (29), anti-PT IgG levels were calculated in IU/mL

by comparison with an U.S. reference human anti-serum (Food and Drug


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Administration, lot 3). To evaluate the titers, the values were transformed into decimal

logarithm and then the mean and the limits of 95% IC were determined. Thereafter, the

antilogarithm and the corresponding 95% IC were calculated.

Quantitative determination of anti-Diphtheria and tetanus IgG

In vitro tests for measuring tetanus and diphtheria antitoxin levels in serum from

both vaccination groups were done by a standardized modified toxin-binding inhibition

(TOBI) assay [41] at the Quality Control Service of Butantan Institute, São Paulo -

Brazil.

Adverse event monitoring

Parents were asked to notify the study staff immediately by phone of any

unexpected or severe reactions. A standardized questionnaire was used to collect

information about any occurrence related to vaccination using scripted questions and

definitions. After the first dose, parents’ compliance to adverse events monitoring was

assessed by phone interview. At the second and third doses and blood sampling,

compliance was assessed by the study nurses during Public Health Center visits.

Statistical considerations

Analyses were carried out with SPSS® for Windows (version 7.5.1, USA).

Mann-Whitney and Friedman nonparametric tests were used in proliferation and

cytokine analyses. Comparisons manifesting a two-tailed p value of <0.05 were


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considered statistically significant. GraphPad Prism (version 4.0, GraphPad Software,

USA) was employed for the figures.

To evaluate the anti-PT titres, calculation of geometric mean titres (GMTs) of

antibodies were performed on log10 transformed data, reporting the antilogarithm. For

each group, GMTs and 95% confidence intervals (CIs) were calculated. For comparison

of the logarithm of the titres, Student’s t test was applied for independent samples.

Comparisons manifesting a two-tailed p value of <0.05 were considered statistically

significant (25).

The differences in the proportions of seroprotection for diphtheria, tetanus and

hepatitis B and 90% confidence intervals (IC 90%) were calculated, as recommended

for non-inferiority studies (10, 36). Differences or ratios equal or lower than 10% were

accepted as the limit for defining non-inferiority of low LPS cellular pertussis vaccine

(wPlow). The null hypothesis of non-inferiority of wPlow vaccine was accepted when the

lower limit of the confidence interval was not lower than -10%.

Results

Study participants

Out of 247 infants initially selected, 234 participated in the entire study

distributed as follows: 115 infants in the low LPS vaccine group (57 male and 58

female) and 119 infants in the whole-cell one (65 male and 54 female). Table 1 shows

the mean and standard deviation values of the infants’ age at the three doses of vaccine

and at blood sampling.


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B. pertussis-specific T cell proliferation

T cell proliferation was measured by flow cytometry (Fig. 1). CD3+, CD4+ and

CD8+ immunophenotyping was carried out in the cultures of 205 infants and TCR γδ+

blast cells were analyzed in 55 children (28 of the low LPS vaccine group).

Median background CD3+ proliferation was 2.3% in the wPlow group and 2.6%

for wP. Net percent CD3+ blasts in cultures with B. pertussis were higher in the group

vaccinated with wP (medians of 6.2% and 3.9% for wP and wPlow, respectively;

p=0.029) but there was no difference in cultures stimulated with PHA (medians of

82.5% and 81.4%; p=0.166) (Fig. 2).

The frequencies of B. pertussis- and PHA-proliferating CD4+, CD8+ and γδ+

cells were similar between vaccination groups (Table 2). On the other hand, there was a

significant difference between the T cell subpopulations for control, B. pertussis- and

PHA-stimulated cultures (Friedman test, p<0.001), with higher percentages of γδ+ cells

in the B. pertussis- ones.

B. pertussis - specific cytokine production

B. pertussis-specific cytokines in cell cultures of infants were determined by

ELISA after 48 hours stimulation. The amount of cytokine secretion in supernatants was

compared among the groups of vaccinees. Undetectable amounts of IL-4 were observed

in stimulated and in unstimulated samples (negative controls). IFN-γ, TNF-α and IL-10

production (Fig. 3) were not different in B. pertussis- and PHA-stimulated cultures of

infants vaccinated with wPlow or wP.


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Anti-PT IgG titers

Pertussis antibody GMTs following three doses of wPlow or wP indicated robust

responses for both vaccines (Table 3) and no statistically significant differences

between the two groups were observed (Student’s t test, p = 0.464).

Diphtheria and tetanus seroprotection

Table 4 shows the percentages and 95% confidence intervals of subjects

protected to diphtheria and tetanus. As the lower limit of the confidence interval was not

lower than -10% for the three studied parameters, the null hypothesis of non-inferiority

of wPlow vaccine was accepted.

Adverse events

Within the first 2-3 days of each injection, the parents and legal guardians of the

vaccinees reported local reactions, as erythema ≥20mm (wPlow, 3.6% and wP 1.4%)

and pain (wPlow, 5.3% and wP 2.3%). Likewise, systemic reactions as body temperature

≥ 39ºC were observed in both groups (wPlow, 3.8% and wP, 2.0%). Irritability was

reported after 4.4% of wPlow and 2.9% of wP injections, respectively. Severe systemic

adverse effects after each DTP vaccine dose were not reported. None of the participants

withdrew from the study because of vaccine-related adverse events. There were no

significant differences between wPlow and wP groups.


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Discussion

Studies of immune responses to pertussis vaccines suggest that both B- and T-

cell responses are elicited in humans (5, 7, 14) and mice (4, 33). Using a murine model

of infection, it has been demonstrated that adoptive transfer of CD4+ T cells from

immune mice confers protection from B. pertussis challenge in the absence of

detectable Ab response but also passive Ab transfer protected mice from B. pertussis

infection (27). In an attempt to better understand the CMI responses to pertussis

vaccines in infants, we evaluated the T cell in vitro proliferation specific to B. pertussis

at one month after the third dose of vaccine.

We used a flow cytometry-based lymphocyte proliferation assay that allowed the

characterization of specific subpopulation expansion in response to B. pertussis. We

found that infants vaccinated with both wP and wPlow exhibited similar frequencies of B.

pertussis-proliferating CD4+, CD8+ and γδ+ cells, although total CD3+ proliferation in

wP group was significantly higher.

We report here, for the first time that, in humans, high percentages of γδ+ cells

are found in B. pertussis stimulated cultures (medians of 20.3% for wP and 16.8% for

wPlow), suggesting that these cells may play an important role in pertussis-specific

response. T cells expressing the γδ receptor were identified in the mid 1980s (11, 12,15,

39) and are disproportionately abundant within epithelial surfaces, including those in the

lung (26). Their intraepithelial distribution and capacity to recognize nonprotein

antigens, sometimes in a non-major histocompatibility complex-restricted fashion, has

led to their consideration as part of the first line defense against pathogens (23). In

humans, large expansion of γδ+ T cells after Bacillus Calmette Guérin vaccination and

during infection with Mycobacterium tuberculosis, Listeria monocytogenes, Brucella


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melitensis, and Ehrlichia chaffeensis suggests their importance in host response (32,

42).

The activation of γδ+ T lymphocytes can lead to IFN-γ production that is

instrumental in the upregulation of both macrophage and natural killer (NK) cell

functions central to early antibacterial protection prior to the αβ+ T cell response (23).

IFN-γ also influences the downstream acquisition of a Th1 phenotype by αβ T cells

(42). γδ+ T cells thus bridge the innate and acquired immune response by providing

initial protection of epithelia from invasion and injury in instances where αβ T cells are

not yet operational and then down-regulating the antigen-specific adaptive immune

response after the danger has passed to minimize potential immune-mediated injury

(40).

Although little is known about the importance of γδ+ T lymphocytes during B.

pertussis infection, a role for these cells in the response of infected children has

previously been suggested by the migration of circulating γδ+ T cells to the airways (9).

Moreover, using a murine aerosol challenge model, Zachariadis and co-workers

demonstrated that the absence of γδ+ T cells could influence the subsequent adaptive

immune response to B. pertussis antigens, as evidenced by a shift from a Th1 to a Th2

type response against filamentous hemagglutinin (FHA) in γδ TCR -/- mice (48).

Th1 cytokines are associated with protection in various B. pertussis infection

models, and in particular, in humans, protection from pertussis after infection or

vaccination is determined by the presence of Th1 cytokines such as interleukin-12 (IL-

12) and IFN-γ (37). Also, the clearance of B. pertussis or protection induced by wP

vaccines is dependent on the production of Th1 cytokines, since IFN-γ or IFN-γ

receptor-defective mice and mice depleted of NK cells, which infiltrate the lung and

secrete IFN-γ early in infection, develop disseminating lethal infections (13).


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In this study, ELISA measurements of cytokine levels in culture supernatants

demonstrated significant increases in IFN-γ and TNF-α secretion by B. pertussis

stimulated PBMC. Since there was no increase in IL-4 secretion, we speculate that the

cytokine production in response to B. pertussis was more Th1-like for both vaccines.

However our assay did not permit the identification of the cells which are secreting

these cytokines. Similar Th1 cytokine secretion profiles for adults (3) and children (6,

31, 37) have been reported for cellular pertussis vaccines. In contrast, T cells from

children immunized with acellular pertussis vaccines can also secrete IL-5 following

stimulation with B. pertussis antigens and generate a type 2 effector response (38).

Because of the bias against Th1-cell-polarizing cytokines, it was initially thought

that the neonatal immune system was generally impaired or depressed. However,

mounting evidence suggests that, under some circumstances, human neonates seem able

to develop mature Th-cell responses, ranging from deficient or deviant to fully mature,

depending on the conditions of antigen exposure (reviewed in ref. 1). Our results

demonstrate that infants are able to mount Th1 responses to B. pertussis after wP or

wPlow administration.

The induction of protective Th1 responses by immunization with wP or by

previous infection with B. pertussis has been associated with IL-12 production by

macrophages or dendritic cells (DC), and this has been linked with LPS present in the

wP preparations and in the live bacteria (30). LPS signaling through TLR4 in innate

immune cells play a critical role in the generation of inflammatory cytokines, IL-12, IL-

23, and IL-1, which direct the induction of Th-1 and Th-17 cells in mice immunized

with wP. Furthermore, the cytokines secreted by these T cell subtypes promote bacterial

killing by macrophages, a response that is further enhanced at the effector level by

TLR4-mediated activation of macrophages. Thus, TLR4 plays a critical role in the


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induction and in the effector phase of the protective cellular immune response to B.

pertussis induced by vaccination (24), which could explain the lower proliferation of

CD3+ T cells observed in wPlow group.

Additionally, the null hypothesis of non-inferiority of wPlow vaccine was

accepted because wPlow and wP elicited a similar anti-PT IgG geometric mean titer and

did not interfere in tetanus or diphtheria seroconversion. Theoretically, wPlow with 95%

less LPS would have weaker adjuvant activity and therefore lower antibody response. In

this sense we can understand the significantly higher total CD3+ proliferation in wP

group as a result of the LPS linked to the host Toll-like receptor complex TLR4 / MD-2

and the subsequent activation of NF-κB (35). Because there is no defined parameter for

pertussis seroprotection (18), we could not estimate differences in the proportions of

protected infants.

Our surveillance system for severe adverse events following immunization was

able to detect an excellent primer safety profile of wPlow and wP vaccines. DTP

vaccine changes from manufacturer to manufacturer, because of the different Bordetella

pertussis strains used for production. Some manufacturers even use two different

strains. There is a rare, although serious risk of a severe adverse event related to the

pertussis component of DTP. The risk of encephalopathy and convulsions varies

according to the origin of the vaccine and the strain used for production. These risks

have been estimated in 1/19,496 for febrile convulsions, 1/76,133 for convulsion

without fever and 3/106 for encephalopathy. (17, 19). Whole cell pertussis vaccine

available in Brazil (licensed by Butantan Institute) has been administered free of charge

to almost 100% of infants in the past 15 years. More than 50 million doses have been

used, without major adverse effects. According to the Brazilian Ministry of Health

pertussis incidence has declined from 10.8 in 1990 to 0.55 per 100,000 in 2005. In the


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same period, diphtheria incidence decreased from 0.45 to 0.01 per 100,000, and tetanus

incidence from 1.0 to 0.25 per 100,000.

Since there was a similar pattern of response in both groups of vaccination, it is

reasonable to assume that the low LPS cellular pertussis vaccine is capable to induce B.

pertussis specific response and, consequently, is immunogenic. Therefore, our data

endorse further investigations with wPlow vaccine. On the other hand, it must be

recognized that, although low LPS cellular pertussis vaccine was similar to the

conventional whole-cell one, based on Th1-polarized effector response and specific γδ+

T cell expansion the overall data did suggest that wP performed modestly better

specific CD3+ cell proliferation.

Acknowledgements

We are indebted to the participating parents and study nurses, whose intimate

collaboration formed the backbone of the trial. This study was supported by Fundação

de Amparo à Pesquisa do Estado de São Paulo (FAPESP), São Paulo, Brazil, grant

number 2005/03539-1, and by Financiadora de Estudos e Projetos (FINEP), Brazil,

grant number 01040957/0.


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Table 1. Distribution of infants vaccinated with low LPS cellular pertussis (wPlow) or

whole-cell pertussis (wP) vaccines by age (in months) at the three doses of vaccine and

at blood sampling.

Time Group n a

Age (months)

Mean ± SD b

wPlow 124 2.1 ± 0.5 1st dose

wP 123 2.1 ± 0.3

wPlow 121 4.2 ± 0.5 2nd dose

wP 121 4.2 ± 0.4

wPlow 120 6.3 ± 0.6 3rd dose

wP 119 6.3 ± 0.4

wPlow 117 7.7 ± 0.7 Blood sampling

wP 119 7.7 ± 0.6

(a): number of individuals; (b): standard deviation.


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Table 2: Percentages of CD4+, CD8+ and γδ+ blast cells determined by flow cytometry in PBMCa cultures incubated with medium

alone (control), B. pertussis or phytohemagglutinin (PHA) from infants vaccinated with low LPS cellular pertussis (wPlow) or whole-

cell pertussis (wP) vaccines.

Median % of T cell (95% CI)b

wPlow wP

nc Control B. pertussis PHAd nc Control B. pertussis PHAd

CD4+ 102 53.2

(49.6 – 58.3)

49.8

(45.9 - 52.7)

78.8

(74.7 - 80.5) 103

51.0

(47,4 – 55.8)

47.7

(45.0 - 51.6)

73.6

(69.6 - 76.2)

CD8+ 102 29.6

(25.6 – 33.1)

23.5

(22.3 - 26.5)

18.0

(15.4 - 20.7) 103

32.3

(28.7 – 34.8)

27.0

(25.4 - 29.7)

19.6

(17.1 - 24.8)

γδ+ 28

5.7

(4.0 – 7.0)

16.8

(13.0 - 24.4)

1.9

(1.4 - 3.4) 27

5.9

(4.1 – 8.1)

20.3

(15.0 - 24.8)

1.9

(1.3 - 2.7)

(a): Peripheral blood mononuclear cell; (b): 95% confidence interval for median; (c): number of individuals; (d): phytohemagglutinin


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Table 3. Geometric mean titer (GMT) and 95% confidence interval (CI) for anti-PT IgG

serum levels of infants vaccinated with three doses of low LPS cellular pertussis (wPlow)

or whole cell pertussis (wP).

Group na GMT (95% CI)b

wPlow 114 12.65 IU/mL [10.46 – 15.30]

wP 116 14.07 IU/mL [11.36 – 17.42]

(a): number of individuals; (b): geometric mean titer and 95% confidence interval


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Table 4. Geometric mean titer (GMT) and 95% confidence intervals (CI) for IgG Anti-

Tetanus and Anti-Difteria serum levels of seven-month-old infants vaccinated with three

doses of low LPS (wPlow) or whole-cell pertussis (wP) vaccines.

wPlow wP

Anti-Tetanus ≥ 0,10 IU/mL

n/N

% [95% CI]

107/112

95.5 [89.9– 98.5]

111/113

98.2 [93.8 – 99.8]

Anti-Diphtheria ≥ 0,10 IU/mL

n/N

% [95% CI]

104/113

92.0 [85.4 – 96.3]

107/112

95.5 [89.9 – 98.5]

n: number of subjects considered protected; N: total of subjects in evaluation


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Vaccine

T.Q. Zorzeto, M.T.N. Da Silva, T.N. Mazzola, V.D. Ramalho, S.C.B.S. Lima, A.M.

Morcillo, E.F. Carniel, M.A.R.G.M. Antonio, M.L. Zanolli, H.G. Higashi, I. Raw,

M.M.S. Vilela.

Figure 1:

Resting

89,3%

Blasts

3,3%

Resting

58,8%

Blasts

15,1%

Resting

3,7%

Blasts

31,8%

Forward light scatter

Sidelightscatter

CD3+

38,2% 42,2% 46,7%

(A)

(B)

Control B. pertussis PHA

Resting

89,3%

Blasts

3,3%

Resting

58,8%

Blasts

15,1%

Resting

3,7%

Blasts

31,8%

Forward light scatter

Sidelightscatter

CD3+

38,2% 42,2% 46,7%

(A)

(B)

Control B. pertussis PHA


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M.M.S. Vilela.

Figure 2:

Bp PHA Bp PHA0

25

50

75

100

Low LPS Whole-cell

n = 102 n = 103

CD

3+

perc

en

t b

lasts

p = 0.029

wPlow wP


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M.M.S. Vilela.

Figure 3A:

Control Bp PHA Control Bp PHA

0

250

500

1000200030004000

n = 32 n = 34

IFN

- γγ γγ c

on

ce

ntr

ati

on

(pg

/mL

)

wPlow wP


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M.M.S. Vilela.

Figure 3B:


0

500

1000

1000

2000

3000

n = 32 n = 34

wPlow wP

TN

F- αα αα

co

nc

en

tra

tio

n (

pg

/mL

)


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M.M.S. Vilela.

Figure 3C:


0

100

200

300

400

IL-1

0 c

on

ce

ntr

ati

on

(p

g/m

L)

n = 32 n = 34

wPlow wP


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Fig. 1: Flow cytometry of unstimulated (control), B. pertussis (Bp) and phytohemagglutinin

(PHA) stimulated peripheral blood mononuclear cells of a 7-month-old infant vaccinated

with low lipopolysaccharide cellular pertussis. After gating on CD3+ cells (A), events were

analyzed for size and complexity (FS and SS gates), from which resting and blast

lymphocytes were separated (B). T lymphocyte subsets (CD4+, CD8+ e TCR δγ+) were then

verified in blast lymphocytes.

Fig. 2: Distribution of CD3+ percent blasts determined by flow cytometry of B. pertussis

(Bp: �) and phytohemagglutinin (PHA: �) stimulated peripheral blood mononuclear cells

from low lipopolysaccharide cellular pertussis (wPlow) or whole-cell pertussis (wP)

vaccinated infants. Medians (indicated by bars) of CD3+ blasts in Bp-stimulated cultures

were 3.9% and 6.2% for wPlow and wP vaccines, respectively. Medians of CD3+ blasts in

PHA-stimulated cultures were 81.4% and 82.5%, respectively; n: number of individuals.

Fig.3: IFN-γ (A), TNF-α (B) and IL-10 (C) concentration determined by ELISA (values are

given in picograms per milliliter) by peripheral blood mononuclear cells in cultures without

stimuli (control: �), with B. pertussis (Bp: �) or phytohemagglutinin (PHA: •) stimulation

in infants immunized with low lipopolysaccharide cellular pertussis (wPlow) or whole-cell

pertussis (wP) vaccines. Bars indicate medians and Mann-Whitney test was used to verify

significances; n: number of individuals.


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Downloaded from on May 14, 2020 by guest · 2 Authors affiliations Tatiane Queiroz Zorzeto: Center for Investigation in Pediatri cs, Pediatrics Department, State University of Campinas