BORDETELLA PERTUSSIS MPLA AND RIBOFLAVIN AS ADJUVANTS FOR
TRIVALENT INFLUENZA VACCINE
Adjuvanted trivalent influenza vaccines containing 3.75 µg of each antigen strain with alum and riboflavin or Bordetella pertussis MPLA
were tested in mice. Immune response was evaluated by haemagglutination inhibition test, ELISA and antibody affinity test. The highest
increase in antibody titers was obtained with the formulation containing MPLA. Significant increase in HAI titer was observed when
mice received the formulation containing MPLA and alum, with a clear cross reaction between B lineages. Corroborating our previous
results with monovalent vaccine, this study confirmed the adjuvant formulations containing MPLA or Riboflavin as promising adjuvants
to be evaluated in clinical trials.
Influenza vaccine, adjuvants, vitamins, MPLA, TIV, QIV
Seasonal influenza vaccines are usually composed by antigen, i.e.,
split virus, in a physiological buffer containing or not conservatives.
The use of adjuvanted vaccines with reduced antigen amount in
humans have already showed efficacy in a monovalent formulation
(Precioso et al, 2011) and could be an important tool for antigensparing,
for reducing costs or to increase the immune response to
new pandemic strains, taking in account the use of an affordable
At the same time, since 2013, WHO has been recommended the
use of quadrivalent influenza vaccines containing two strains of
type B lineages (Yamagata and Victoria) once both lineages have
been co-circulating (WHO). However, this increase in vaccine
cost might be a trouble in low income and developing countries
either financial and product availability. A formulation that could
elicit cross-reacting antibodies to B lineages would be especially
useful to solve this situation.
In light of these facts and considering previous results for
monovalent A/H1N1 vaccine (Quintilio et al, 2016; Precioso
et al, 2011), in this paper we describe the effect of MPLA and
riboflavin combined to alum as adjuvant as a proof-of-concept
for trivalent influenza vaccine. The effect of the formulations on
eliciting antibody titers and on cross reactivity between B strains
(Yamagata and Victoria) were evaluated in order to elucidate the
choice for better candidate vaccine adjuvant.
Materials and Methods
All animal tests were performed following the guidelines from
Instituto Butantan Animal Care and Use Committee (permit CEUA
Female BALB/c mice (Mus muscullus) aged 6-8 weeks were
provided by Instituto Butantan Animal Facilities. During the
experiments they received balanced food and fresh water ad
Guinea pigs (Cavia porcellus) red blood cells suspension at 1 %
was provided by Influenza Laboratory.
Riboflavin (Vitamin B2) was purchased from Merck. MPLA (Bordetella pertussis Monophosphoryl Lipid A) was produced
at Instituto Butantan as a by-product of pertussis-low vaccine
production [Quintilio et al., 2009]. Alum (Alhydrogel® - Brenntag
Biosector) is a sterilized aluminum hydroxide gel suspension,
which quantity is expressed in mg of Al3+. IgG anti-mousehorsehadish
peroxidaseconjugate for ELISA was purchased from
Sigma-Aldrich. Influenza antigen A/California/7/2009 (H1N1)
pdm09, A/Texas/50/2012, B/Massachusetts/2/2012 (Yamagata
lineage) and B/Brisbane/60/2008 (Victoria lineage) - purified split
influenza vaccine grown in eggs, formaldehyde inactivated - were
produced and kindly provided by Influenza Laboratory at Instituto
Immunization and bleeding
Mice were immunized intraperitoneally (0.5 mL) with trivalent
influenza vaccine (3.75 µg of each antigen strain, considering
2014 Southern hemisphere composition) with or without adjuvant
(32.5 µg of riboflavin with 0.25 mg of alum or 5 µg of MPLA with
0.25 mg of alum/dose). At 21, 60 and 67 days after immunization,
individual serum was obtained by submandibular bleeding and
was kept at 20 °C until antibodies quantification tests. In order
to evaluate immunological memory response, at day 60 the mice
received a booster containing 1/10th of the first dose of trivalent
vaccine without adjuvant.
Haemagglutination Inhibition test (HAI) was performed
individually by using guinea-pigs red blood cells suspension at
1 %. Titers were reported as the reciprocal of the highest sera
dilution that completely inhibits haemagglutination. HAI titer ≥40
is considered a correlate of protection in humans [EMEA, 2015].
ELISAs to determine antibodies titers against each antigen
separately were performed as described previously (Quintilio et
al, 2016). Two fold dilutions of individual sera were incubated in
ELISA 96 wells polystyrene plates (Costar) coated with antigen at
1 µg/mL. HRP anti-mouse IgG conjugate was used to detect IgG
titers with 3,3’,5,5’-tetramethylbenzidine as substrate. Antibody
titers were calculated by sigmoidal curve model (Combistats© -
EDQM) by comparison to the unadjuvanted vaccine at day 21,
which titer was arbitrarily considered 1.
For affinity ELISA measurements, the assays were performed as
described above, but including a potassium thiocyanate (KSCN;
0 to 10 M, dilution factor 1.5) step as described (Guidolin et al,
2013) after the serum incubation (dilution 1/400). The results are
expressed as affinity index (AI): KSCN concentration (mol/L)
needed to displace 50% of the bound antibodies.
Each vaccine strain was tested separately including the B Victoria
strain in order to check cross reactivity. All graphs were prepared
by using GraphPad Prism® 5.01 for MS-Windows™.
Statistical analysis was performed by using GraphPad Prism®
5.01. Kruskal-Wallis and Dunns’ multiple comparison tests were
Based on the previous results, the formulations containing MPLA,
riboflavin and alum were evaluated in order to characterize the
immunological response against the A/H1N1, A/H3N2 and B/
Yamagata influenza antigen in a trivalent vaccine. Besides to the
cross reactivity against B/Victoria lineage individual HAI and
antibody titration by ELISA (IgG) and affinity induced by the
vaccine formulations were evaluated.
Individual HAI results corroborated the previous experiments
(Quintilio et al, 2016). Adjuvanted formulations induced higher
antibody titers than the unadjuvanted vaccine for all strains (Fig.
1). There were no significant differences between formulations
after booster. However, before the booster, for A strains,
especially, the use of adjuvants elicited higher HAI titers. When
the cross-reactivity between B lineages was evaluated, 21 days
after immunization, only MPLA with alum induced mean titer
1/40, the other two groups elicited lower antibodies titer (Fig. 1).
Nevertheless, the HAI titer increased during time and after booster
there was a significant difference between MPLA+alum group and
HAI GMT (the error bars indicate 95% confidence interval): (A) A/H1N1; (B) A/H3N2; (C) B/
Yamagata; (D) B/Victoria. Mice received the trivalent influenza vaccines at day 0 (initial) and a booster
(0.375 µg of each antigen) at day 60. They were bled and the sera were titred with guinea-pig red blood
cells at 21, 60 and 67 days after initial immunization. Dotted line indicates the minimum protective titer. *
indicates statistically significant differences among groups.
Reflecting these HAI results, IgG titer indicated that formulation containing alum+MPLA induced higher titers after booster against
all strains. Despite the positive response against B/Victoria lineage, IgG titers were significantly lower than those observed against B/
Yamagata (Fig. 2).
GMT IgG in mice sera. Mice received non-adjuvanted vaccine (), vaccine containing
riboflavin and alum () and vaccine containing MPLA with alum () at day 0 and were bled at
21, 60 and 67 days after immunization. (A) anti-H1N1, (B) anti-H3N2, (C) anti-B/Yamagata, (D)
anti-B/Victoria. At day 60 all animals received unadjuvanted 0.375 µg of each antigen as booster.
Error bar represents 95% confidence interval.
In order to check if the increased titers were related to higher antibody affinities, an affinity ELISA was performed. After booster, all
groups presented an increased affinity. However, when animals received vaccines with alum and MPLA the affinity index increasing
was remarkable (Table 1). Riboflavin with alum presented an adjuvant effect more evident for A strains, but MPLA with alum was
more effective in all cases.
It is important to emphasize that in this study the influenza vaccine
contained 1/4th of the regular human dose. Meaning that by
following this approach, there could be an important antigen spare,
multiplying by 4 in theory the production plant capacity.
Previous published work (Quintilio et al, 2016) examinated the
use of vitamins and MPLA as adjuvants for vaccines and at that
moment, riboflavin with alum and MPLA with alum were the
most promising adjuvants. Before trying with trivalent vaccine,
one assay with monovalent A/H3N2 and B/Yamagata strains
was performed, confirming the results obtained for A/H1N1
(data not shown). And the same profiles were observed also here
with the trivalent vaccine. Except the affinity ELISA: the results
were different from those obtained previously for adjuvanted
monovalent H1N1 vaccine (Quintilio et al., 2016), likely due to
the presence of other antigens, in our experimental model.
In the studied conditions MPLA with alum induced higher antibody
titers than those obtained with riboflavin and alum. Further studies
must be performed in order to characterize the effect of each
Many studies have been pointed that the use of quadrivalent
vaccines could reduce hospitalizations number and work
absenteeism presenting therefore a good cost-effectiveness ratio
(Gordon et al, 2016; Zerbini et al, 2016). At the same time it is
known that by using adjuvant, cross-reactive antibodies have been
observed in a clinical assay in humans (Camilloni et al, 2009).
results presented here indicate that MPLA with alum may elicit
cross-protection, potentially mimicking a quadrivalent influenza
vaccine containing though 1/4th of the antigen usually present in
In spite of the fact that a human is not a 70 kg mouse, these results
are useful to steer the vaccine development and critical answers
only a clinical trials in humans can bring.
Conflict of interest
WQ collected and analyzed data and wrote the manuscript; FAF
and CM wrote the manuscript; IR idealized the project and wrote
The authors would like to acknowledge Dr Celso Pereira Caricati
for the kind and helpful support, DDTP for the MPLA supply.
Maria Rosa Lange da Silva and Marisa de Souza Silva de Oliveira
for the support in animal housing. Financial support by Fundação
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