1556-6811/09/$08.00⫹0 doi:10.1128/CVI.00339-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Immunogenicity of a Whole-Cell Pertussis Vaccine with Low
Lipopolysaccharide Content in Infants
䌤
Tatiane Queiroz Zorzeto,
1,4Hisako Gondo Higashi,
2Marcos Tadeu Nolasco da Silva,
1,4Emilia de Faria Carniel,
1,4Waldely Oliveira Dias,
2Vanessa Domingues Ramalho,
1Taís Nitsch Mazzola,
1Simone Corte Batista Souza Lima,
1Andre´ Moreno Morcillo,
1,4Marco Antonio Stephano,
3Maria A
ˆ ngela Reis de Go´es Antonio,
4Maria de Lurdes Zanolli,
4Isaias Raw,
2and Maria Marluce dos Santos Vilela
1,4*
Center for Investigation in Pediatrics1and Pediatrics Department,4State University of Campinas Medical School,
Rua Tessa´lia Vieira de Camargo 126, Campinas, Sa˜o Paulo, Brazil CEP 13083-887; Butantan Institute, Rua Vital Brasil, 1500, Sa˜o Paulo, Sa˜o Paulo, Brazil CEP 05503-9002; and Faculdade de
Cieˆncias Farmaceˆuticas, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes 580, Butanta˜, Sa˜o Paulo, Brazil CEP 05508-0003
Received 17 September 2008/Returned for modification 3 November 2008/Accepted 10 February 2009
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 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 (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wPlowvaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wPlowvaccine. Proliferation of CD3ⴙT cells was evaluated by flow cytometry after 6 days of peripheral
blood mononuclear cell culture with stimulation with heat-killedBordetella pertussisor phytohemagglutinin (PHA). CD3ⴙ, CD4ⴙ, CD8ⴙ, and T-cell receptor ␥␦-positive (␥␦ⴙ) cells were identified in the gate of blast
lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in super-natants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3ⴙblasts in cultures withB. pertussisin the group vaccinated with wP was higher than
that in the group vaccinated with the wPlowvaccine (medians of 6.2% for the wP vaccine and 3.9% for the wPlow vaccine;Pⴝ0.029). The frequencies of proliferating CD4ⴙ, CD8ⴙ, and␥␦ⴙcells, cytokine concentrations in
supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations forB. pertussisand PHA cultures, with a higher percentage of␥␦ⴙcells in theB. pertussiscultures (P< 0.001). The overall data did suggest that wP vaccination
resulted in modestly better specific CD3ⴙcell proliferation, and␥␦ⴙcell expansions were similar with the two
vaccines.
Efforts to immunize against whooping cough began almost as soon as Bordet discovered the causative bacterium, Borde-tella pertussis, in 1912 (8). A whole-cell pertussis (wP) vaccine has been available since 1940. However, the road to eliminate circulation of the pathogen has proven arduous. Throughout the world, pertussis remains a major cause of morbidity and mortality among infants (45). Some 20 million to 40 million cases of disease occur worldwide each year, 90% of which are found in developing countries (44).
Although no causal link between wP vaccination and per-manent brain damage or death has been identified, concerns about systemic reactions after immunization with a wP vac-cine 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 per-tussis (aP) vaccines (21, 22, 43, 47). Similar immunogenici-ties are 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 coun-tries (46).
Lipopolysaccharide (LPS) possesses endotoxic activity and also has powerful adjuvant activity. Both of these properties are based upon the recognition of the LPS by the host Toll-like receptor (TLR) complex TLR4/MD-2 and the subsequent ac-tivation of NF-B (35). The relatively high reactogenicity of wP vaccines has been associated with proinflammatory cytokines (2, 28). Hence, a straightforward approach to reducing 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 (Ab) to per-tussis antigens and protection against disease, the persistence of protective immunity long after the disappearance of
pertus-* Corresponding author. Mailing address: Center for Investigation in Pediatrics and Pediatrics Department, State University of Campinas Med-ical School, Rua Tessa´lia Vieira de Camargo 126, Campinas, Sa˜o Paulo, Brazil CEP 13083-887. Phone: 55-19-35218963. Fax: 55-19-35218972. E-mail: marluce@fcm.unicamp.br.
䌤Published ahead of print on 4 March 2009.
sis antigen-specific Ab, and the longer duration of T-cell re-sponses to pertussis antigens lend credence to the possibility that cell-mediated immunity provides primary protection against disease (16, 34).
This phase I study was thus performed to obtain preliminary immunogenicity data on a new cellular pertussis vaccine with low LPS content (wPlowvaccine), in comparison to the
con-ventional wP vaccine used in Brazil, both of which are formu-lated with diphtheria and tetanus toxoids and given in three primary injections during infancy.
MATERIALS AND METHODS
Subjects.Infants scheduled to receive the first dose of pertussis vaccine were recruited in Campinas Public Health Centers, Sa˜o Paulo, Brazil. A total of 247 infants were recruited to the study and randomized to receive three doses of wPlowor wP vaccine at 2, 4, and 6 months of age. The preterm (gestational age,⬍37 weeks) and low-birth-weight (weight,⬍2,500 g) newborns, infants whose mothers were younger than 18 years old or had hepatitis B carrier status, and infants with a family history of tuberculosis, syphilis, or human immunode-ficiency virus infection were excluded. Exclusion criteria also included noncom-pliance, receipt of immunoglobulin (Ig) therapy or blood products, and any immunodeficiency, malignancy, or significant underlying disease or neurological impairment.
Vaccines.Vaccines were manufactured by the Butantan Institute, Sa˜o Paulo, Brazil.Bordetella pertussiscells were treated with an organic solvent and washed in order to perform LPS extraction. The culture was then detoxified by the addition of 0.2% formalin, and the bacterial biomass was obtained by tangential flow filtration to formulate the wPlowvaccine.
Each 0.5-ml dose of either vaccine contained four protective units of pertussis toxin and two protective units of diphtheria and tetanus toxoids. The antigens were adsorbed 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 ad-ministered intramuscularly with the use of standard techniques.
Study design.This prospective, randomized, double-blind comparative trial was conducted between August 2006 and July 2007 and followed the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all participants’ parents or legal guardians before study procedures were initiated. The study protocol was approved by the Committee for Ethics in Research of the State University of Campinas, Sa˜o Paulo, Brazil.
Antigens used in cell culture assays.A suspension of heat-killedB. pertussis (lot IB-CIIn/P14/06; Butantan Institute), without thimerosal, was used at a con-centration of 5⫻106cells/ml. Phytohemagglutinin (PHA; Sigma) was used as a positive control at 7.5g/ml.
T-cell proliferation assay.Ten milliliters of heparinized peripheral blood was collected and used for the evaluation of immune responses to pertussis vaccina-tion at 7 months of age. The protocol for the proliferavaccina-tion assay was adapted from the method of Gaines and Biberfeld (20). Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Ficoll-Hypaque (Amersham Biosciences), washed, diluted to 1⫻106cells/ml in RPMI 1640 medium (Sigma) supplemented with 10% human AB serum (Sigma), 1% glutamine (Sigma), and 0.1% gentamicin, and stimulated for 6 days with heat-killedB. pertussis, PHA, or medium alone at 37°C with 5% CO2in round-bottom 96-well tissue culture plates (Nunc, Denmark). After being harvested with 20 mM EDTA, samples were incubated with human Ig and then stained with anti-human CD3, CD4, CD8, and T-cell receptor (TCR) pan-␥␦fluorescent Abs (Beckman Coulter) before acquisition (Epics XL-MCL flow cytometer; Beck-man-Coulter) and analysis (Expo software; Beckman Coulter). Isotype controls were used to discriminate positive populations (Beckman Coulter). Only CD3⫹ T cells were used in analysis. Forward and side scatters were used to gate resting and blast lymphocytes. Dead cells were excluded from all analyses. CD4⫹, CD8⫹, and TCR␥␦-positive (␥␦⫹) cells were identified in the gating of blast lympho-cytes. Proliferation was measured by percentages of CD3⫹blasts, in which basal proliferation was subtracted fromB. pertussis- and PHA-stimulated cultures.
Cytokine quantification in supernatants.For the cytokine quantification as-say, PBMC were diluted to 2⫻106cells/ml in supplemented RPMI medium and incubated for 48 h in round-bottom 96-well tissue culture plates withB. pertussis, PHA, or medium alone at 37°C. Supernatants were collected and stored at ⫺80°C. Gamma interferon (IFN-␥), tumor necrosis factor alpha (TNF-␣), inter-leukin-4 (IL-4), and IL-10 levels were determined in duplicate by a
two-mono-clonal-Ab sandwich enzyme-linked immunosorbent assay (ELISA) (R & D Sys-tems) in flat-bottom MultiSorp ELISA plates (Nunc, Denmark), according to the manufacturer’s protocols. Recombinant cytokine was used for the standard curve. The limits of detection were 15.6 pg/ml for IFN-␥and TNF-␣and 31.2 pg/ml for IL-4 and IL-10.
Quantitative determination of anti-PT IgG.Quantitative determination of anti-pertussis toxin (anti-PT) IgG was performed in a blind manner on serum samples collected one month after the administration of the third dose of vaccine and stored at⫺80°C until tested. Using ELISA (32), anti-PT IgG levels were calculated in IU/ml by comparison with a U.S. reference human antiserum (lot 3; Food and Drug Administration). To evaluate the titers, the values were transformed into decimal logarithms, and then the means and the limits of the 95% confidence interval (CI) were determined. Thereafter, the antilogarithm and the corresponding 95% CI were calculated.
Quantitative determination of anti-diphtheria toxin and anti-tetanus toxin IgG.In vitro tests for measuring tetanus and diphtheria antitoxin levels in sera from both vaccination groups were done by a standardized modified toxin-binding inhibition assay (41) at the Quality Control Service of the Butantan Institute, Sa˜o Paulo, Brazil.
Adverse event monitoring.Parents were asked to notify the study staff imme-diately by phone of any unexpected or severe reactions. A standardized ques-tionnaire using scripted questions and definitions was used to collect information about any occurrence related to vaccination. After the first dose, parents’ com-pliance to monitoring of adverse events was assessed by phone interview. At the administrations of the second and third doses and blood sampling, compliance was assessed by the study nurses during visits to the public health center.
Statistical considerations.Analyses were carried out with SPSS for Windows (version 7.5.1). Mann-Whitney and Friedman nonparametric tests were used in proliferation and cytokine analyses. Comparisons manifesting a two-tailedP value of⬍0.05 were considered statistically significant. Prism software (version 4.0; GraphPad Software) was employed for the figures.
To evaluate the anti-PT titers, calculation of the geometric mean titers (GMTs) of Abs was performed on log10-transformed data, and we report the antilogarithms. For each group, GMTs and 95% CIs were calculated. For com-parison of the logarithm of the titers, Student’sttest was applied for independent samples. Comparisons manifesting a two-tailedPvalue of⬍0.05 were considered statistically significant (25).
The differences in the proportions of subjects with seroprotection against diphtheria and tetanus and 90% CI were calculated as recommended for non-inferiority studies (10, 36). Differences or ratios equal to or lower than 10% were accepted as the limit for defining noninferiority of the wPlowvaccine. The null hypothesis of noninferiority of the wPlowvaccine was accepted when the lower limit of the CI was not lower than⫺10%.
RESULTS
Study participants.Out of 247 infants initially selected, 234 participated in the entire study and were distributed as follows: 115 infants in the low-LPS-content vaccine group (57 males and 58 females) and 119 infants in the whole-cell vaccine group (65 males and 54 females). Table 1 shows the mean ages (with standard deviations) of the infants at the administrations of the three doses of vaccine and at blood sampling.
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␥␦⫹
blast cells in 55 children (28 in the low-LPS-content vaccine group) were analyzed.
Median background CD3⫹
proliferation levels were 2.3% in the wPlowvaccine group and 2.6% in the wP vaccine group.
The net percentage of CD3⫹
blasts in cultures withB. pertussis
in the group vaccinated with the wP vaccine was higher than that in the group vaccinated with the wPlowvaccine (medians
of 6.2% and 3.9% for those immunized with wP and wPlow
The frequencies of proliferating CD4⫹ , CD8⫹
, and␥␦⫹ cells inB. pertussis- and PHA-stimulated cultures were similar for the vaccination groups (Table 2). On the other hand, there was a significant difference between the T-cell subpopulations for control andB. pertussis- and PHA-stimulated cultures (Fried-man test,P⬍0.001), with higher percentages of␥␦⫹
cells in theB. pertussis-stimulated cultures.
B. pertussis-specific cytokine production.B. pertussis-specific cytokines in cell cultures of infants were determined by ELISA after 48 h of stimulation. For the groups of vaccinees, the amounts of cytokine secretion in supernatants were compared. IL-4 was undetectable in stimulated and unstimulated samples (negative controls). Levels of IFN-␥, TNF-␣, and IL-10 pro-duction (Fig. 3) were not different betweenB. pertussis- and PHA-stimulated cultures of infants vaccinated with the wPlow
or wP vaccine.
Anti-PT IgG titers.Anti-PT Ab GMTs following three doses of wPlow or wP vaccine indicated robust responses to both
vaccines (Table 3), and no statistically significant differences between the two groups were observed (Student’sttest,P⫽
0.464).
Diphtheria and tetanus seroprotection.Table 4 shows the percentages (with 95% CIs) of subjects protected against
diph-FIG. 1. Flow cytometry of unstimulated (control) andB. pertussis- and PHA-stimulated PBMC of a 7-month-old infant vaccinated with a cellular pertussis vaccine with low LPS content. After gating of CD3⫹cells (A), events were analyzed for size and complexity (forward-scatter and
side-scatter gates), from which resting and blast lymphocytes were separated (B). T-lymphocyte subsets (CD4⫹, CD8⫹, and␦␥⫹) were then verified
in blast lymphocytes.
FIG. 2. Distribution of CD3⫹blasts (percentages) determined by
flow cytometry ofB. pertussis(Bp;f)- and PHA (Œ)-stimulated PBMC from wPlowvaccine- or wP vaccine-immunized infants. Medians (indi-cated by bars) of CD3⫹blasts inB. pertussis-stimulated cultures were
3.9% and 6.2% for wPlowand wP vaccines, respectively. Median per-centages of CD3⫹blasts in PHA-stimulated cultures were 81.4% and
82.5%, respectively. n, number of individuals. TABLE 1. Distributions of infants vaccinated with the wPlowor wP
vaccine by age at the administrations of the three doses of vaccine and at blood sampling
Time Vaccine na Age (mo)b
1st dose wPlow 124 2.1⫾0.5
wP 123 2.1⫾0.3
2nd dose wPlow 121 4.2⫾0.5
wP 121 4.2⫾0.4
3rd dose wPlow 120 6.3⫾0.6
wP 119 6.3⫾0.4
Blood sampling wPlow 117 7.7⫾0.7
wP 119 7.7⫾0.6
aNumber of individuals. bMean
theria and tetanus. As the lower limits of the CIs were not lower than⫺10% for the three studied parameters, the null hypothesis of noninferiority of the wPlowvaccine was accepted.
Adverse events.Within the first 2 to 3 days after each injec-tion, the parents and legal guardians of the vaccinees reported local reactions, such as erythema (ⱖ20 mm; 3.6% for the wPlow
vaccine group and 1.4% for the wP vaccine group) and pain (5.3% for the wPlowvaccine group and 2.3% for the wP vaccine
group). Likewise, systemic reactions (body temperature,
ⱖ39°C) were observed in both groups (3.8% for the wPlow
vaccine group and 2.0% for the wP vaccine group). Irritability was reported after 4.4% of wPlowvaccine injections and 2.9%
of wP vaccine injections, respectively. Severe systemic adverse effects after each diphtheria-tetanus-pertussis (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 wPlowand wP
vaccine groups.
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). By use of a murine model of infection, it has been demonstrated that adoptive transfer of CD4⫹
T cells from immune mice confers protection againstB. pertussis dur-ing challenge in the absence of a detectable Ab response, but also, passive Ab transfer protected mice fromB. pertussis in-fection (27). In an attempt to better understand the cell-me-diated immune responses to pertussis vaccines in infants, we
evaluated the in vitro proliferation of T cells specific to B. pertussisat one month after the third dose of vaccine was given. We used a flow cytometry-based lymphocyte proliferation assay that allowed the characterization of specific subpopula-tion expansion in response toB. pertussis. We found that in-fants vaccinated with the wP vaccine and those vaccinated with the wPlowvaccine exhibited similar frequencies ofB. pertussis
-specific proliferating CD4⫹ , CD8⫹
, and ␥␦⫹
cells, although total CD3⫹
cell proliferation in the wP vaccine group was significantly higher.
We report here for the first time that, in humans, high percentages of␥␦⫹
cells are found inB. pertussis-stimulated cultures (medians of 20.3% for wP and 16.8% for wPlow),
suggesting that these cells may play an important role in the pertussis-specific response. T cells expressing the␥␦receptor were identified in the mid-1980s (11, 12, 15, 39) and are dis-proportionately abundant within epithelial surfaces, including those in the lung (26). Their intraepithelial distribution and capacity for recognizing nonprotein antigens, sometimes in a non-major histocompatibility complex-restricted fashion, have led to their consideration as part of the first-line defense against pathogens (23). In humans, large expansion of␥␦⫹
T cells afterMycobacterium bovisbacillus Calmette-Gue´rin vac-cination and during infection withMycobacterium tuberculosis,
Listeria monocytogenes, Brucella melitensis, and Ehrlichia chaffeensis suggests their importance in the host response (31, 42).
The activation of ␥␦⫹
T lymphocytes can lead to IFN-␥
production, which is instrumental in the upregulation of both
FIG. 3. IFN-␥(A), TNF-␣(B), and IL-10 (C) concentrations determined by ELISA (values are given in picograms per milliliter) by PBMC in cultures without stimuli (control), withB. pertussis(Bp), or with PHA (F) stimulation in infants immunized with wPlowor wP vaccine. Bars indicate medians, and the Mann-Whitney test was used to verify significances. n, number of individuals.
TABLE 2. Percentages of blast cells determined by flow cytometry in cultures of PBMC that were obtained from vaccinated infants and incubated with medium alone,B. pertussis, or PHA
T-cell type
wPlow wP
na Median % of specific T cells (95% CI) na Median % of specific T cells (95% CI)
Controlb B. pertussis PHA Controlb B. pertussis PHA
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)
␥␦⫹ 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)
macrophage and natural killer (NK) cell functions central to early antibacterial protection prior to the␣⫹
T-cell response (23). IFN-␥also influences the downstream acquisition of a Th1 phenotype by␣⫹
T cells (42).␥␦⫹
T cells thus bridge the innate and acquired immune response by providing initial pro-tection of epithelia from invasion and injury in instances where
␣⫹
T cells are not yet operational and then downregulating the antigen-specific adaptive immune response after the dan-ger has passed to minimize potential immune-mediated injury (40).
Although little is known about the importance of ␥␦⫹ T lymphocytes duringB. pertussisinfection, a role for these cells in the response of infected children has previously been sug-gested via the migration of circulating␥␦⫹
T cells to the air-ways (9). Moreover, using a murine aerosol challenge model, Zachariadis and coworkers demonstrated that the absence of
␥␦⫹
T cells could influence the subsequent adaptive immune response toB. pertussisantigens, as evidenced by a shift from a Th1-type response to a Th2-type response against filamen-tous hemagglutinin in TCR␥␦⫺/⫺
mice (48).
Th1 cytokines are associated with protection in various B. pertussisinfection models, and in particular, in humans, pro-tection against pertussis after infection or vaccination is deter-mined by the presence of Th1 cytokines such as IL-12 and IFN-␥(37). Also, the clearance of B. pertussisor protection induced by wP vaccines is dependent on the production of Th1 cytokines, since IFN-␥- or IFN-␥receptor-defective mice and mice depleted of NK cells, which infiltrate the lung and secrete IFN-␥ early in infection, develop disseminating lethal infec-tions (13).
In this study, ELISA measurements of cytokine levels in culture supernatants demonstrated significant increases in IFN-␥and TNF-␣secretion byB. pertussis-stimulated PBMC. Since there was no increase in IL-4 secretion, we speculate that the cytokine production in response toB. pertussis was more Th1-like for both vaccines. However, our assay did not permit the identification of the cells which were secreting these cyto-kines. Similar Th1 cytokine secretion profiles for adults (3) and children (6, 30, 37) in response to cellular pertussis vaccines have been reported. In contrast, T cells from children immu-nized with aP vaccines can also secrete IL-5 following stimu-lation withB. pertussisantigens 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 condi-tions of antigen exposure (reviewed in reference 1). Our results
demonstrate that infants are able to mount Th1 responses toB. pertussisafter wP or wPlowvaccine administration.
The induction of protective Th1 responses by immunization with the wP vaccine or by previous infection withB. pertussis
has been associated with IL-12 production by macrophages or dendritic cells, and this has been linked with LPS present in the wP preparations and in the live bacteria (29). LPS signaling through TLR4 in innate immune cells plays a critical role in the generation of inflammatory cytokines IL-12, IL-23, and IL-1, which direct the induction of Th1 and Th17 cells in mice immunized with wP vaccine. Furthermore, the cytokines se-creted by these T-cell subtypes promote bacterial killing by macrophages, a response that is further enhanced at the effec-tor level by TLR4-mediated activation of macrophages. Thus, TLR4 plays a critical role in the induction and in the effector phase of the protective cellular immune response toB. pertussis
induced by vaccination (24), which could explain the lower level of proliferation of CD3⫹
T cells observed in wPlow
vac-cine group.
Additionally, the null hypothesis of noninferiority of wPlow
vaccine was accepted because the wPlowand wP vaccines
elic-ited similar anti-PT IgG GMTs and did not interfere in tetanus or diphtheria seroconversion. Theoretically, the wPlowvaccine,
having 95% less LPS than the conventional vaccine, would have weaker adjuvant activity and therefore produce a lower-level Ab response. In this sense, we can understand the signif-icantly higher level of total CD3⫹
proliferation in the wP vaccine group as a result of the LPS being linked to the host TLR complex TLR4/MD-2 and the subsequent activation of NF-B (35). Because there is no defined parameter for per-tussis 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 wPlowand wP vaccines. DTP vaccines differ from
manufacturer to manufacturer because of the different Borde-tella pertussisstrains 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 compo-nent of the DTP vaccine. The risks of encephalopathy and convulsions vary according to the origin of the vaccine and the strain used for production. These risks have been estimated as 1/19,496 for febrile convulsions, 1/76,133 for convulsion with-out fever, and 3/1,000,000 for encephalopathy (17, 19). The wP vaccine available in Brazil (licensed by the Butantan Institute) has been administered free of charge to almost 100% of infants
TABLE 3. GMTs and 95% CI for serum anti-PT IgG levels of infants vaccinated with three doses of wPlowor wP vaccine
Vaccine group na Titer (IU/ml)
Geometric mean 95% CI
wPlow 114 12.65 10.46–15.30
wP 116 14.07 11.36–17.42
aNumber of individuals.
TABLE 4. Protection against tetanus toxin and diphtheria toxin in 7-month-old infants vaccinated with three doses of the
wPlowor wP vaccine
Ab (ⱖ0.10 IU/ml) and vaccine
No. of subjects considered protected/total
% of subjects protected
Mean 95% CI
Anti-tetanus toxin IgG
wPlow 107/112 95.5 89.9–98.5
wP 111/113 98.2 93.8–99.8
Anti-diphtheria toxin IgG
wPlow 104/113 92.0 85.4–96.3
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 per 100,000 in 1990 to 0.55 per 100,000 in 2005. In the same period, diphtheria incidence decreased from 0.45 per 100,000 to 0.01 per 100,000 and tetanus incidence decreased from 1.0 per 100,000 to 0.25 per 100,000.
Since there were similar patterns of responses in the two vaccinated groups, it is reasonable to assume that the low-LPS-content cellular pertussis vaccine is capable of inducing aB. pertussis-specific response and, consequently, is immunogenic. Therefore, our data endorse further investigations with the wPlowvaccine. On the other hand, it must be recognized that,
although the low-LPS-content cellular pertussis vaccine was similar to the conventional whole-cell one, based on Th1-polarized effector response and specific␥␦⫹
T-cell expansion, the overall data did suggest that the wP vaccine performed modestly better with regard to specific CD3⫹
T-cell prolifera-tion.
ACKNOWLEDGMENTS
We are indebted to the participating parents and study nurses, whose intimate collaboration formed the backbone of the trial.
This study was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, Sa˜o Paulo, Brazil, grant number 2005/03539-1, and by Financiadora de Estudos e Projetos, Brazil, grant number 01040957/0.
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