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Article history: Anti-diphtheria antibody levels decrease with aging, and frequent booster vaccinations are required to
Received 21 March 2017 maintain herd immunity. We analyzed the diphtheria toxin neutralizing antibody (DT-Nab) response
Received in revised form 24 May 2017 induced by a conjugate vaccine (meningococcal C polysaccharide-CRM197) in HIV-vertically infected
Accepted 26 May 2017
(HI) children and adolescents and healthy controls (HC) with matched age. We report the association
Available online xxxx
of DT-Nab with the bactericidal antibodies to serogroup C meningococcus (MenC). Before vaccination,
21 HI patients (50%) had no protection against diphtheria (0.01 IU/ml of antibody) and only 8 (19%)
Keywords:
showed complete protection (0.1 IU/ml). About half of the HC (56%) had complete protection before
CRM197
Conjugate vaccine
immunization and 6 subjects (12%) had no protection against diphtheria. After one and two vaccine injec-
Diphtheria neutralizing antibody tions, 96% of HC and 64% of HI vaccinees, respectively, showed full protection against diphtheria. These
Bactericidal antibody data indicate that CRM197 was able to induce primary and/or booster response in both groups of
individuals.
Ó 2017 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2017.05.080
0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Silva GP et al. A cross-reacting material CRM197 conjugate vaccine induces diphtheria toxin neutralizing antibody
response in children and adolescents infected or not with HIV. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.080
2 G.P. Silva et al. / Vaccine xxx (2017) xxx–xxx
response with the bactericidal antibody response to MenC, CD4 T Vero cells. Cell viability was determined by the MTT assay [11].
cell levels and viral load. Neutralizing antibody levels were categorized according to inter-
nationally accepted ranges: <0.01 IU/mL (non-protective), 0.01–
2. Patients and methods 0.09 IU/mL (basic protection), and 0.1 IU/mL (full protection)
[12–14].
We conducted a prospective cohort study at the Instituto de
Puericultura e Pediatria Martagão Gesteira, Universidade Federal do 2.4. Statistical analysis
Rio de Janeiro (IPPMG/UFRJ), Rio de Janeiro, Brazil, to investigate
the seroconversion rate after MenC vaccination in HIV-vertically The levels of significance of the differences between groups
infected individuals. The study was approved by the IPPMG Institu- were examined by, either the Mann-Whitney test (unpaired sam-
tional Review Board (IRB, number 24/09) and Brazilian Ministry of ples) or the Wilcoxon matched-pair test (paired samples), as non-
Health Ethics Commission (Comissão Nacional de Ética em Pesquisa, parametric data were obtained. The correlation between different
CONEP, number 15578). Details about the inclusion and exclusion measurements of immune response was analyzed using Spearman
criteria, IRB and participant’s consent were previously published rank test, after graph analyses. These analyses were performed
[7]. Due to a weak antibody response after the first dose of vaccine, with the GraphPad-Prism software, version 7 (GraphPad Software,
HI patients received a booster dose of the vaccine about one year Inc., USA). The differences between the percentage of seroconver-
later. HC children with matched age were also included in this sion and seroprotection were analyzed by software Epi InfoTM
study. However, they received only one injection of the vaccine, 6.04d, CDC.
as per recommendation at the time of this study, in healthy chil-
dren and youth, aged 1–25 years, a single MenC dose should be 3. Results and discussion
given [8]. The conjugate vaccine (Novartis; C Polysaccharide/
CRM197) was administered at the recommended dose 3.1. CRM197 activates antibody response against DT
(10 lg/0.5 ml). Most (93%) of the HI patients of the present study
was receiving HAART for about 6.5 years (median). Fig. 1A shows the DT-Nab response in HI individuals. We
observed a significant (P < 0.0001) increase of specific antibodies
2.1. Serum samples after the first immunization (median of 0.31 IU/ml) compared with
pre-immunization levels (median of 0.01 IU/ml). One year after
For HI, blood samples were collected before (Visit 1) vaccina- immunization, there was a significant decrease of DT-Nab levels
tion, 1–2 months after one dose (Visit 2), before booster (median of 0.03 IU/ml; P < 0.0001) but it was still higher than
(10–12 months after first dose, Visit 3) and 1–2 months after pre-immunization levels (0.01 IU/ml).
boosting (Visit 4) using tubes in the absence of anticoagulant. Vaccine boosting induced a marked increase of DT-Nab levels
Serum samples were stored at 20°C. (median of 0.32 IU/ml; P < 0.0001), reaching similar levels as
For HC group, that received only one vaccine injection, we col- detected after the first dose.
lected blood samples before (visit 1), 1–2 months after vaccination Fig. 1B shows the DT-Nab response for HC group. There was a
(visit 2) and one year later (visist 3). huge (P < 0.0001) increase of antibody levels against DT at visit 2
In total, we analyzed 168 blood samples from 42 HI patients, (median of 5) when compared to pre-vaccination levels (median
with median age of 12 years, and 150 blood samples from 50 HC of 0.16). One year after vaccination (V3), it was detected a signifi-
individuals with median age of 10 years. cant drop in the level of DT-Nab (median of 0.62, P < 0.0001).
Notice that, although the median levels of V1 and V3 antibodies
2.2. Bactericidal assay were low, the median of V3 (0.62) was higher (P < 0.0001) than
V1 (median = 0.16). The DT-Nab response of HC vaccinees was sig-
Serum bactericidal antibody (SBA) titers were measured as pre- nificantly (P < 0.0001) higher than the response of HI group at all
viously described [7,9], using human complement source. time points analyzed. All together, these data indicate that
CRM197 protein was able to activate the memory response induced
2.3. Diphtheria toxin (DT) neutralization test by the diphtheria toxoid administered during childhood especially
in HC group and to a less extension in HI group. CRM197 also
The DT neutralization test in Vero cells (green monkey renal primed the immune system of HI patients since we detected a
episerum) was used. The concentration of serum diphtheria anti- booster response after the second conjugate vaccine
toxin in cell culture was estimated according to the procedure administration.
described by Miyamura et al. [10], with some modifications. Serial
2-fold dilutions (25 mL) of serum were mixed with 25 mL DT equiv- 3.2. Protection status to diphtheria differs between HI and HC groups
alent to four times the minimal cytotoxic dose (0.26 ng/mL) (Insti-
tuto Nacional de Controle de Qualidade em Saúde, Fundação Table 1 shows the protection status to diphtheria based on DT-
Oswaldo Cruz, Rio de Janeiro, Brazil) and incubated for 1 h at Nab levels for HI and HC groups. Before vaccination, 21 HI patients
37°C. Then, 50 mL suspension containing 2.5 105 Vero cells/mL (50%) had no protection against diphtheria (0.01 IU/ml of anti-
in modified Eagle’s medium supplemented with 10% fetal calf body), 13 (31%) had basic protection (above 0.01–0.09 IU/ml) and
serum was added to each well. The plates were gently shaken, cov- only 8 (19%) showed complete protection (0.1 IU/ml) against
ered and incubated at 37°C/5%CO2 for 6 days. On the basis of con- the disease. Therefore, based on DT-Nab levels, most of the individ-
current testing with a reference serum (equine antiserum from uals infected with HIV (81%) were not fully protected against diph-
Instituto Nacional de Controle de Qualidade em Saúde, Fundação theria before vaccination with the conjugate vaccine.
Oswaldo Cruz, Rio de Janeiro Brazil; 10 IU/ml antibody to DT), In contrast, for HC group, about half of the individuals (56%) had
the antibody titer is reported as IU/mL. An antitoxin-positive con- complete protection (0.1 IU/ml) before immunization, 16 (32%)
trol serum, a toxin control, a test serum control, and a cell control had basic protection (up 0.01–0.09 IU/ml) and 6 subjects (12%)
were run for every plate. Neutralizing antibody titer was defined as had no protection against diphtheria (0.01 IU/ml). The frequency
the highest dilution of serum neutralizing toxin that killed 100% of HC patients with protective DT-Nab levels was significantly
Please cite this article in press as: Silva GP et al. A cross-reacting material CRM197 conjugate vaccine induces diphtheria toxin neutralizing antibody
response in children and adolescents infected or not with HIV. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.080
G.P. Silva et al. / Vaccine xxx (2017) xxx–xxx 3
Table 1
Protective status to diphtheria based on antibody levels to DT before and after each C
polysaccharide-CRM197 vaccine dose.
but may have been influenced by the time of the last immunization
against diphtheria, which was lower for HC individuals compared
with HI group.
The median age of initiation of HAART therapy was 4.9 years
(IQR 3.4–9.8 years), a relatively later time, that mighty had influ-
enced the low DT-Nab response to diphtheria of HI group. Initia-
tion of HAART in HIV-1 vertically-infected children within the
first year of life permits the normal development and maintenance
of the memory B cell compartment. On the contrary, memory B
cells from patients treated later in time are remarkably reduced
and their function is compromised regardless of viral control [15].
After the first dose of the conjugate vaccine (V2, Table 1), it was
observed that the frequency of non-protected HI individuals
decreased from 50% to 28.6% (n = 12, P = 0.04). Individuals with
basic protection dropped from 31% to 14.4% (n = 6, P = 0.07) and
the frequency of individuals with complete protection tripled from
19% to 57% (n = 24, P = 0.0003). For HC group, only two individuals
(4%, P < 0.00001) remained partially protected after vaccination
and 96% (P < 0.000001) of the patients were fully protected. These
data indicate that CRM197 was able to induce primary and/or boos-
ter response in both group of individuals. We have no knowledge
of DT-Nab studies in humans vaccinated with CRM197. We are also
not aware of any report about DT-Nab response induced by DTP (or
Fig. 1. Neutralizing antibody levels to diphtheria toxin (DT-Nab) before immu-
DTaP) vaccine in HI or HC children/adolescents. A study in infants
nization (V1) with the MenC vaccine (MenC-CRM197), one-two months after the aged 6–12 weeks in South Africa measured antibodies to DT by
first dose (V2), one year after first dose (V3) and one-two months after boosting Luminex multiplex-immunoassay and reported a similar frequency
(V4) for HIV-infected individuals (HI) (A) and V1, V2 and V3 for healthy controls (77–100%) of individuals with protective antibody levels to DT in
(HC) (B). Each symbol represents one volunteer. Lines are median of antibody levels.
* HC and HI children one month after 3 doses of combined DTP-
P < 0.05, **P < 0.01, ***P < 0.0001.
Hib vaccine [16].
The duration of the DT-Nab response was evaluated one year
(P < 0.0003) higher compared with HI group. Besides, HI group had after the first dose of the conjugate vaccine. At this time (V3), there
a higher (P < 0.00001) proportion of unprotected individuals. was an important decrease of DT-Nab levels but the frequency of
The median age of HI and HC groups did not differ significantly; protected HI individuals was still higher (38.1%) than the one
median of 12 years (IQR; 9.7–15 years) for HI group, and median of detected before vaccination (19%) (Table 1). HC group had a longer
10 years (IQR; 8–13 years) for HC group. Concerning to the number lasting DT-Nab response compared with HI group. More than 90%
of previous immunization against diphtheria, about 81% of HI of HC individuals had full protection one year after vaccination.
patients with recorded vaccinations against diphtheria (n = 37) After the second dose of vaccine in HI group (V4, Table 1) 64% of
received four to six doses of diphtheria vaccine before the conju- vaccinees showed full protection against diphtheria. Compared
gate vaccine and seven (19%) patients received 2 or 3 injections with the HC group that reached 96% of protection against DT after
of diphtheria vaccine. The median of time elapsed between the last one dose of vaccine, this data indicate that the high frequency
diphtheria vaccine and the first dose of conjugate vaccine was of (24%) of unprotected HI patients after two vaccinations may be
10 years for HI group and 4.9 years for HC group. For the latter related to the secondary immune system deficiency of those indi-
group, 94% of the 35 individuals, with vaccination records, received viduals and/or characteristics of the immunogen, since the second
4 or 5 diphtheria vaccine injections before the conjugate vaccine vaccination did not induce a significant increase in DT-Nab anti-
and 6% were vaccinated twice before the conjugate vaccine. body response compared with the response detected after the first
Altogether, these data suggest that the lower DT-Nab preva- vaccination (V2). Of note, we detected a significant booster
lence in HI vaccinees compared to HC group was independent of response against MenC in HI group, which showed a frequency of
age range and number of previous vaccination against diphtheria, 55% and 83% of responders (based on bactericidal antibody levels)
Please cite this article in press as: Silva GP et al. A cross-reacting material CRM197 conjugate vaccine induces diphtheria toxin neutralizing antibody
response in children and adolescents infected or not with HIV. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.080
4 G.P. Silva et al. / Vaccine xxx (2017) xxx–xxx
after one and two doses of the vaccine, respectively (data not (r = 0.27, P = 0.055) between the two kind of antibody responses
shown). (data not shown).
Fig. 2B illustrates the positive (r = 0.33, P = 0.033) association
3.3. DT-Nab levels association with SBA against MenC, CD4 count and between DT-Nab levels and CD4 T cells percentage measured
viral load 1 month after the first vaccine injection (V2). We did not observe
any significant association between DT-Nab levels and nadir CD4
Fig. 2A shows the correlation analysis between DT-Nab levels T cells or age of the patients.
and MenC bactericidal antibody response after two doses of vac- A significant negative correlation was observed between viral
cine in HI group. The results showed an important positive associ- load detected at immunization (V1) and DT-Nab levels in V1
ation (r = 0.65, P < 0.0001) between DT-Nab response and SBA (r = 0.326, P = 0.035), V2 (r = 0.31, P = 0.048) and V4
response to MenC. For the HC group, that received only one dose (r = 0.531, P = 0.0003, Fig. 2C). These data agree with our previous
of the conjugate vaccine we could also see a positive correlation report [7] that undetectable viral load at immunization was asso-
ciated with immune response to MenC.
In conclusion, this study suggests that HI group should receive a
Bactericidal antibody titers (log2 ) - V4
A) 15.0
r = 0.65
differentiated immunization schedule against diphtheria, includ-
ing booster injections within shorter period of time compared with
12.5
P < 0.0001
n = 42 the current immunization schedule. The data also reinforce the
necessity of keeping HC children/adolescents up to date with their
10.0 vaccinations against diphtheria since only about half of them
showed complete protection against diphtheria before vaccination.
7.5
CRM197, the carrier of the conjugate vaccine, was useful for boost-
5.0
ing and keeping up date the immunity of children and adolescents.
0.0
None.
0 2 4 6 8 10
Neutralizing antibodies to DT (IU/ml)-V4
Author’s checklist
n = 41
ical specimens, immunized patients and did the patient follow-up.
30 LGM designed the experiments, did the data analysis, assisted
the statistical analysis, wrote the paper and obtained funds.
20
Acknowledgments
10
We thank to the legal guardians of children and adolescents
involved in this study. This work was supported by Fundação de
0
0 2 4 6 8 10
Amparo a Pesquisa do Estado do Rio de Janeiro - FAPERJ (grant #
E-26/112.645/2012), Brazil e CNPq (Grant # 449775/2014-3),
Neutralizing antibodies to DT (IU/ml) - V2 Brazil.
C) 5
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response in children and adolescents infected or not with HIV. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.080
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Please cite this article in press as: Silva GP et al. A cross-reacting material CRM197 conjugate vaccine induces diphtheria toxin neutralizing antibody
response in children and adolescents infected or not with HIV. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.080