Vaccine xxx (2017) xxx–xxx

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Short communication

A cross-reacting material CRM197 conjugate vaccine induces diphtheria

toxin neutralizing antibody response in children and adolescents
infected or not with HIV
Giselle P. Silva a, Rafaela S. Santos a, Wânia F. Pereira-Manfro a, Bianca Ferreira b, Daniella M. Barreto b,
Ana Cristina C. Frota b, Cristina B. Hofer b,c, Lucimar G. Milagres a,⇑
a
State University of Rio de Janeiro, Department of Microbiology, Immunology and Parasitology, Rio de Janeiro, Brazil
b
Instituto de Puericultura e Pediatria Martagão Gesteira, Rio de Janeiro, Brazil
c
Preventive Medicine Department, School of Medicine, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

a r t i c l e i n f o a b s t r a c t

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.

1. Introduction require detoxification with formaldehyde, causing much less

Active and efficient immunization programs against diphtheria
are based on the use of a formaldehyde-detoxified preparation of
diphtheria toxoid to induce protective neutralizing antibody
responses [1]. However, anti-diphtheria antibody levels decrease
with increasing age, and frequent booster vaccinations are
required to maintain herd immunity in the adult population
[2–4]. The cross-reacting material (CRM197) of diphtheria toxin is
a genetically detoxified preparation of the toxin that does not
Abbreviations: DT-Nab, diphtheria toxin neutralizing antibody; HI, HIV-infected
children/adolescents; HC, healthy controls; MenC, serogroup C meningococcus;
CRM197, cross-reacting material; DTP, diphtheria-tetanus-pertussis; HAART, highly
active antiretroviral therapy; SBA, serum bactericidal antibody; V1, before immu-
nization; V2, one-two months after first dose; V3, one year after first dose; V4, one-
two months after booster; MTT, brometo de [3-(4,5-dimetiltiazol-2yl)-2,5-difenil
tetrazolium].
⇑ Corresponding author at: Av. Prof Manoel de Abreu, 444, terceiro andar,
Disciplina de Microbiologia, CEP: 20550–170, Rio de Janeiro, Brazil.
E-mail addresses: milagreslucimar@gmail.com, lucimar@uerj.br (L.G. Milagres).
adverse effects in humans [5].
The recommended vaccination schedule against diphtheria in
Brazil consists of three doses of diphtheria-tetanus-pertussis
(DTP) vaccine in the first year of life, followed by booster doses
at 15 months and 4 years of age. After that, a booster dose should
be given at every 10 years. Currently, meningococcal serogroup C
conjugate vaccines is recommended in a regimen of two doses
8 weeks apart for previously unvaccinated HIV-infected toddlers,
children and adolescents, with a booster 5 years later [6].
We have previously reported the bactericidal antibody response
to Neisseria meningitidis C (MenC) after vaccination (1 injection) of
children/adolescents, HIV-vertically infected (HI) and healthy con-
trols (HC), with a conjugate vaccine using CRM197 as the carrier
protein [7]. The objective of the present study was to analyze the
diphtheria toxin neutralizing antibody (DT-Nab) response induced
by the conjugate vaccine (C polysaccharide-CRM197) in a sampling
of the previously reported study [7]. For the present study, HI vac-
cinees received a booster dose of vaccine about one year after the
first vaccination. We also report the association of DT-Nab

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
cell levels and viral load.
2. Patients and methods
We conducted a prospective cohort study at the Instituto de
Puericultura e Pediatria Martagão Gesteira, Universidade Federal do
Rio de Janeiro (IPPMG/UFRJ), Rio de Janeiro, Brazil, to investigate
the seroconversion rate after MenC vaccination in HIV-vertically
infected individuals. The study was approved by the IPPMG Institu-
tional Review Board (IRB, number 24/09) and Brazilian Ministry of
Health Ethics Commission (Comissão Nacional de Ética em Pesquisa,
CONEP, number 15578). Details about the inclusion and exclusion
criteria, IRB and participant’s consent were previously published
[7]. Due to a weak antibody response after the first dose of vaccine,
HI patients received a booster dose of the vaccine about one year
later. HC children with matched age were also included in this
study. However, they received only one injection of the vaccine,
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
given [8]. The conjugate vaccine (Novartis; C Polysaccharide/
CRM197) was administered at the recommended dose
(10 lg/0.5 ml). Most (93%) of the HI patients of the present study
was receiving HAART for about 6.5 years (median).
2.1. Serum samples
For HI, blood samples were collected before (Visit 1) vaccina-
tion, 1–2 months after one dose (Visit 2), before booster
(10–12 months after first dose, Visit 3) and 1–2 months after
boosting (Visit 4) using tubes in the absence of anticoagulant.
Serum samples were stored at 20°C.
For HC group, that received only one vaccine injection, we col-
lected blood samples before (visit 1), 1–2 months after vaccination
(visit 2) and one year later (visist 3).
In total, we analyzed 168 blood samples from 42 HI patients,
with median age of 12 years, and 150 blood samples from 50 HC
individuals with median age of 10 years.
2.2. Bactericidal assay
Serum bactericidal antibody (SBA) titers were measured as pre-
viously described [7,9], using human complement source.
2.3. Diphtheria toxin (DT) neutralization test
The DT neutralization test in Vero cells (green monkey renal
episerum) was used. The concentration of serum diphtheria anti-
toxin in cell culture was estimated according to the procedure
described by Miyamura et al. [10], with some modifications. Serial
2-fold dilutions (25 mL) of serum were mixed with 25 mL DT equiv-
alent to four times the minimal cytotoxic dose (0.26 ng/mL) (Insti-
tuto Nacional de Controle de Qualidade em Saúde, Fundação
Oswaldo Cruz, Rio de Janeiro, Brazil) and incubated for 1 h at
37°C. Then, 50 mL suspension containing 2.5  105 Vero cells/mL
in modified Eagle’s medium supplemented with 10% fetal calf
serum was added to each well. The plates were gently shaken, cov-
ered and incubated at 37°C/5%CO2 for 6 days. On the basis of con-
current testing with a reference serum (equine antiserum from
Instituto Nacional de Controle de Qualidade em Saúde, Fundação
Oswaldo Cruz, Rio de Janeiro Brazil; 10 IU/ml antibody to DT),
the antibody titer is reported as IU/mL. An antitoxin-positive con-
trol serum, a toxin control, a test serum control, and a cell control
were run for every plate. Neutralizing antibody titer was defined as
the highest dilution of serum neutralizing toxin that killed 100%
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
Vero cells. Cell viability was determined by the MTT assay [11].
Neutralizing antibody levels were categorized according to inter-
nationally accepted ranges: <0.01 IU/mL (non-protective), 0.01–
0.09 IU/mL (basic protection), and 0.1 IU/mL (full protection)
[12–14].
2.4. Statistical analysis
The levels of significance of the differences between groups
were examined by, either the Mann-Whitney test (unpaired sam-
ples) or the Wilcoxon matched-pair test (paired samples), as non-
parametric data were obtained. The correlation between different
measurements of immune response was analyzed using Spearman
rank test, after graph analyses. These analyses were performed
with the GraphPad-Prism software, version 7 (GraphPad Software,
Inc., USA). The differences between the percentage of seroconver-
sion and seroprotection were analyzed by software Epi InfoTM
6.04d, CDC.
3. Results and discussion
3.1. CRM197 activates antibody response against DT
Fig. 1A shows the DT-Nab response in HI individuals. We
observed a significant (P < 0.0001) increase of specific antibodies
after the first immunization (median of 0.31 IU/ml) compared with
pre-immunization levels (median of 0.01 IU/ml). One year after
immunization, there was a significant decrease of DT-Nab levels
(median of 0.03 IU/ml; P < 0.0001) but it was still higher than
pre-immunization levels (0.01 IU/ml).
Vaccine boosting induced a marked increase of DT-Nab levels
(median of 0.32 IU/ml; P < 0.0001), reaching similar levels as
detected after the first dose.
Fig. 1B shows the DT-Nab response for HC group. There was a
huge (P < 0.0001) increase of antibody levels against DT at visit 2
(median of 5) when compared to pre-vaccination levels (median
of 0.16). One year after vaccination (V3), it was detected a signifi-
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
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-
nificantly (P < 0.0001) higher than the response of HI group at all
time points analyzed. All together, these data indicate that
CRM197 protein was able to activate the memory response induced
by the diphtheria toxoid administered during childhood especially
in HC group and to a less extension in HI group. CRM197 also
primed the immune system of HI patients since we detected a
booster response after the second conjugate vaccine
administration.
3.2. Protection status to diphtheria differs between HI and HC groups
Table 1 shows the protection status to diphtheria based on DT-
Nab levels for HI and HC groups. Before vaccination, 21 HI patients
(50%) had no protection against diphtheria (0.01 IU/ml of anti-
body), 13 (31%) had basic protection (above 0.01–0.09 IU/ml) and
only 8 (19%) showed complete protection (0.1 IU/ml) against
the disease. Therefore, based on DT-Nab levels, most of the individ-
uals infected with HIV (81%) were not fully protected against diph-
theria before vaccination with the conjugate vaccine.
In contrast, for HC group, about half of the individuals (56%) had
complete protection (0.1 IU/ml) before immunization, 16 (32%)
had basic protection (up 0.01–0.09 IU/ml) and 6 subjects (12%)
had no protection against diphtheria (0.01 IU/ml). The frequency
of HC patients with protective DT-Nab levels was significantly
 G.P. Silva et al. / Vaccine xxx (2017) xxx–xxx
Fig. 1. Neutralizing antibody levels to diphtheria toxin (DT-Nab) before immu-
nization (V1) with the MenC vaccine (MenC-CRM197), one-two months after the
first dose (V2), one year after first dose (V3) and one-two months after boosting
(V4) for HIV-infected individuals (HI) (A) and V1, V2 and V3 for healthy controls
(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.
(P < 0.0003) higher compared with HI group. Besides, HI group had
a higher (P < 0.00001) proportion of unprotected individuals.
The median age of HI and HC groups did not differ significantly;
median of 12 years (IQR; 9.7–15 years) for HI group, and median of
10 years (IQR; 8–13 years) for HC group. Concerning to the number
of previous immunization against diphtheria, about 81% of HI
patients with recorded vaccinations against diphtheria (n = 37)
received four to six doses of diphtheria vaccine before the conju-
gate vaccine and seven (19%) patients received 2 or 3 injections
of diphtheria vaccine. The median of time elapsed between the last
diphtheria vaccine and the first dose of conjugate vaccine was of
10 years for HI group and 4.9 years for HC group. For the latter
group, 94% of the 35 individuals, with vaccination records, received
4 or 5 diphtheria vaccine injections before the conjugate vaccine
and 6% were vaccinated twice before the conjugate vaccine.
Altogether, these data suggest that the lower DT-Nab preva-
lence in HI vaccinees compared to HC group was independent of
age range and number of previous vaccination against diphtheria,
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
3
Table 1
Protective status to diphtheria based on antibody levels to DT before and after each C
polysaccharide-CRM197 vaccine dose.
Non-protection Basic protection Full protection
HI group
V1 21 (50%) 13 (31%) 8 (19%)
V2 12 (28.6%) 6 (14.4%) 24 (57%)
V3 19 (45.2%) 7 (16.7%) 16 (38.1%)
V4 10 (23.8%) 5 (11.9%) 27 (64.3)
HC group
V1 6 (12%) 16 (32%) 28 (56%)
V2 0 (0%) 2 (4%) 48 (96%)
V3 2 (4%) 2 (4%) 46 (92%)
Non-protection 0.01 UI/ml, basic protection 0.01–0.09 UI/ml, full protection
0.1 UI/ml. V1: before immunization, V2: one-two months after first dose, V3: one
year after first dose, V4: one-two months after booster.
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
DTaP) vaccine in HI or HC children/adolescents. A study in infants
aged 6–12 weeks in South Africa measured antibodies to DT by
Luminex multiplex-immunoassay and reported a similar frequency
(77–100%) of individuals with protective antibody levels to DT in
Hib vaccine [16].
The duration of the DT-Nab response was evaluated one year
after the first dose of the conjugate vaccine. At this time (V3), there
was an important decrease of DT-Nab levels but the frequency of
protected HI individuals was still higher (38.1%) than the one
detected before vaccination (19%) (Table 1). HC group had a longer
lasting DT-Nab response compared with HI group. More than 90%
of HC individuals had full protection one year after vaccination.
After the second dose of vaccine in HI group (V4, Table 1) 64% of
vaccinees showed full protection against diphtheria. Compared
with the HC group that reached 96% of protection against DT after
one dose of vaccine, this data indicate that the high frequency
(24%) of unprotected HI patients after two vaccinations may be
related to the secondary immune system deficiency of those indi-
viduals and/or characteristics of the immunogen, since the second
vaccination did not induce a significant increase in DT-Nab anti-
body response compared with the response detected after the first
vaccination (V2). Of note, we detected a significant booster
response against MenC in HI group, which showed a frequency of
55% and 83% of responders (based on bactericidal antibody levels)
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.

2.5 Conflict of interest

0.0
None.
0 2 4 6 8 10
Neutralizing antibodies to DT (IU/ml)-V4
Author’s checklist

B) 50 GPS an RSS performed the neutralization assay and supported

r = 0.33 the data analysis.
P = 0.033 GPS and WFPM performed the bactericidal assays.
40
BF, DMB, ACCF and CBH recruited the patients, collected biolog-
% of CD4 T cells - V2

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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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