Vaccine 39 (2021) 4723–4732

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

Antibodies response induced by recombinant virus-like particles from

Triatoma virus and chimeric antigens from Trypanosoma cruzi
Aline Maria Vasconcelos Queiroz a,b, Yulia Aleksandrovna Yanshina c, Emily Thays da Silva Rodrigues b,
Fred Luciano Neves Santos d, Paola Alejandra Fiorani Celedon e, Sweta Maheshwari f, Sandra Beatriz Gabelli f,
Carla Stephanie Peucelle Rubio g,h, Aritz Durana i, Diego M.A. Guérin g,h,⇑, Marcelo Sousa Silva a,b,c,⇑
a
Postgraduate Programme in Pharmaceutical Sciences, Federal University of Rio Grande do Norte, Rua Gen, Gustavo Cordeiro de Farias, 384, 59012-570 Natal, Brazil
b
Immunoparasitology Laboratory, Department of Clinical and Toxicological Analysis, Federal University of Rio Grande do Norte, Rua Gen, Gustavo Cordeiro de Farias, 384,
59012-570 Natal, Brazil
c
Global Health and Tropical Medicine, Institute of Hygiene and Tropical Medicine, Universidade Nova de Lisboa, Rua da Juqueira, 100, 1800-166 Lisbon, Portugal
d
Advanced Public Health Laboratory, Gonçalo Moniz Institute, Fiocruz, Rua Waldemar Falcão, 121, 40296-710 Salvador, Brazil
e
Molecular Biology Institute of Paraná, Rua Professor Algacyr Munhoz Mader, 3775, 81350-010 Curitiba, Brazil
f
School of Medicine, Johns Hopkins University, 725 N Wolfe St, Baltimore, MD 21205, USA
g
Departamento de Bioquímica y Biología Molecular, Instituto Biofisika, Universidad del País Vasco (UBF, CSIC, UPV-EHU), B° Sarriena S/N, 48940 Leioa, Bizkaia, Spain
h
Ikosaedrika Biologicals S.L. ZITEK Edificio Rectorado UPV/EHU, B° Sarriena S/N, 48940 Leioa, Vizcaya, Spain
i
Instituto Biofisika (CSIC, UPV/EHU), Fundación Biofísica Bizkaia, B° Sarriena S/N, 48940 Leioa, Vizcaya, Spain

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

Article history: Background: The infection caused by the protozoan Trypanosoma cruzi affects humans and is called as
Received 29 November 2020 Chagas disease. Currently, the main measures available to reduce the incidence of this disease are drug
Received in revised form 4 May 2021 treatment and vector control. Traditionally, the development of vaccines occurs mainly through the
Accepted 14 May 2021
use of antigenic candidates of the etiologic agent in the form of a vaccine preparation. Virus-like particles
Available online 27 May 2021
(VLPs) are structures analogous to viral capsids composed essentially of structural proteins and are
widely used in vaccination protocols because of their immunostimulatory properties. In this context,
Keywords:
the objective of this study was to use strategies in a murine immunization model to characterize the
Virus-like particles (VLPs)
Chagas disease
immunostimulatory capacity of VLPs from Triatoma virus (TrV-VLPs), analysed in the presence or absence
Trypanosoma cruzi of the aluminium vaccine adjuvant. In parallel, to characterize the immunogenic behaviour of four T. cruzi
Adjuvant chimeric recombinant proteins (mix-IBMP) associated with TrV-VLPs or aluminium vaccine adjuvant.
Humoral immune response Method: We immunized BALB/c mice once or twice, depending on the strategy, and collected serum sam-
Triatoma virus ples at 15, 30 and 45 days after the immunization. Subsequently, serum samples from animals immu-
nized with TrV-VLPs were used to determine total IgG, IgG1, IgG2a, IgG2b and IgG3 anti-TrV-VLPs by
enzyme-linked immunosorbent assay (ELISA).
Results: Data obtained demonstrate the ability of TrV-VLPs to preferably induce IgG2b and IgG3 type
antibodies in the absence of aluminium adjuvant. In fact, the use of aluminium did not interfere with
the total IgG profile of anti-TrV-VLPs. Interestingly, mix-IBMP had a better profile of total IgG, IgG1
and IgG3 subclasses when mixed with TrV-VLPs.
Conclusion: In conclusion, these results suggest the potential of TrV-VLPs as a vaccine adjuvant and the
use of T. cruzi chimeric antigens as a rational strategy for the development of vaccines against the exper-
imental model of Chagas disease.
Ó 2021 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors at: Departamento de Bioquímica y Biología Molecular,

Instituto Biofisika, Universidad del País Vasco (UBF, CSIC, UPV-EHU), B° Sarriena S/
N, 48940 Leioa, Bizkaia, Spain (D. Guerin). Postgraduate Programme in Pharma- 1. Introduction
ceutical Sciences, Federal University of Rio Grande do Norte, Rua Gen, Gustavo
Cordeiro de Farias, 384, 59012-570 Natal, Brazil (M. Silva).
Vaccines represent an effective and successful intervention in
E-mail addresses: fred.santos@fiocruz.br (F. Luciano Neves Santos), paolaf-
c@ibmp.org.br (P. Alejandra Fiorani Celedon), gabelli@jhmi.edu (S. Beatriz Gabelli), the control and prevention of several infectious diseases, such as
diego.guerin@ehu.es (D.M.A. Guérin), mssilva@ihmt.unl.pt (M. Sousa Silva). yellow fever, influenza, human papillomavirus, hepatitis B, tuber-

https://doi.org/10.1016/j.vaccine.2021.05.039
0264-410X/Ó 2021 Elsevier Ltd. All rights reserved.
A. Maria Vasconcelos Queiroz, Y. Aleksandrovna Yanshina, E. Thays da Silva Rodrigues et al.
culosis, measles, mumps, etc. Vaccines may be prepared in differ-
ent ways, mainly depending on the nature of the antigen used in
the vaccine preparation. Thus, most vaccines available on the mar-
ket consist of live or attenuated microorganisms, protein subunits,
and toxoids. Lately and since the 80 s, virus-like particles (VLPs)-
based vaccines have been widely used in new immunization
regimens.
VLPs are made up of multiple proteins that form the cap-
somer of a viral particle and have a structural and conforma-
tional virus organization that can properly activate the immune
system of numerous hosts during an immunization process [1].
VLPs have been widely used in the production of new vaccines
such as respiratory syncytial virus vaccines [2], vaccines against
different dengue virus serotypes [3], malaria protozoan [4], most
recently influenza A virus [5], and currently as vaccine candidate
platform against SARS-CoV-2 [6], among other infectious disease
agents. In this context, VLPs are a versatile component in the
development of new vaccines, mainly due to their conforma-
tional and immunological characteristics, enabling the activation
of the immune system and the development of immune memory
[7,8].
The search for antigenic candidates for the development of
vaccines against protozoa still represents a major challenge for
modern vaccinology. Important infections caused by protozoa
remain without vaccines available for control, such as Chagas
disease, leishmaniasis, sleeping sickness, malaria, and toxoplas-
mosis, among others. Since the protozoans responsible for these
infectious diseases have a complexity of evasion mechanisms
and the ability to manipulate their hosts’ immune systems.
The most rational strategy for vaccine development is certainly
the use of multiple antigenic candidates in a single vaccine
preparation [9]. In this context, chimeric recombinant antigens
could be an interesting strategy to increase the antigenic reper-
toire of a vaccine preparation against these diseases.
Recently, Santos and colleagues produced four chimeric
recombinant proteins from Trypanosoma cruzi, the etiological
agent of Chagas disease, called as Institute of Molecular Biology
of Paraná (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) [10].
The immunodominant regions of these four chimeric T. cruzi
recombinant proteins have been used in the serological diagnosis
of Chagas disease [10–12]. In parallel, our research group had
produced and characterized recombinant VLPs from Triatoma
virus (TrV) [13], a viral agent capable of infecting various tri-
atomine species [14]. TrV-VLPs had been studied by our lab as
a potential adjuvant for the production of new vaccines. TrV-
VLPs are derived from an insect virus [15], they are recombi-
nantly produced in the baculovirus expression system [13] and
they can be modified to insert new antigens on their capsid sur-
face [16].
In this context, this work aims to characterize the immunostim-
ulatory property of TrV-VLPs and T. cruzi chimeric recombinant
proteins (IBMPs) through their ability to induce polarized humoral
immune response during an immunization process in BALB/c mice.
To this end, specific anti-TrV-VLPs antibodies were evaluated in
mice immunized with TrV-VLPs alone, and TrV-VLPs mixed with
aluminium, a vaccine adjuvant popularly used in vaccination
experimental models. Also, specific anti-IBMPs antibodies were
evaluated in mice immunized with mixed IBMP proteins, in the
presence of TrV-VLPs or aluminium. The data presented in this
work show the ability of TrV-VLPs to induce specific antibodies
during the immunization process in mice, and in the presence or
absence of aluminium adjuvant. Interestingly, the TrV-VLPs immu-
nization process preferentially induced a polarized profile of IgG2b
and IgG3 anti-TrV-VLPs antibodies in both groups of immunized
animals.
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Vaccine 39 (2021) 4723–4732
2. Material and Methods
2.1. Production and purification of recombinant virus-like particles
from Triatoma virus
Viruses and cells: Two baculovirus vectors (BVs), BV_P1 and
BV_NS, were employed to produce TrV-VLPs by co-infection in
H5 insect cells (Invitrogen). BVs were prepared as described previ-
ously [14] and used to infect cells grown in suspension (under
magnetic stirring) in Express Five Media serum-free (Gibco).
Recombinant BVs were grown and titrated as previously described
[17].
Cell infection: Bottles (2L, Celstir Thermofisher) containing H5
cells at a concentration of about 106 cells/mL were infected with
BV_P1 or coinfected with BV_P1 and BV_NS at an MOI of 2p.f.u.
of each virus per cell. During cell growth, prior to, and during infec-
tion, bottles were pumped up with filtered air (using a syringe fil-
ter of 0.22 um cut-off, and a regular fish tank air pump) by
introducing a stain steel tube from one of the top nozzles. At
48 h post-infection, cells were harvested by centrifugation, obtain-
ing several pellets per bottle, each of about 10 mL volume. Cells
were resuspended in lysis buffer [50 nM Tris/HCl pH73; 500 nM
NaCl; 1 nM MgCl2; 0.25% (v/v) Igepal CA-63 and Protease inhibitor
cocktail (1 tablet per 10 mL)], followed by lysis using freeze–thaw
cycles and sonication. TrV-VLPs were purified using sucrose cush-
ion and 10–30% sucrose gradient, fractionated, dialyzed to remove
the sucrose, and the resulting solution (rich in TrV-VLPs as mea-
sured by UV absorption) was loaded onto a Sephacryl S-500 HR
(GE Healthcare) column. After concentration with Amicon tubes
to reach a protein concentration of about 0.2 mg/mL, TrV-VLPs
were characterized using Sodium dodecyl sulfate-Polyacryamide
gel electrophoresis (SDS-PAGE) (silver stained) and by anti-VP1
Western blot analysis. This procedure was performed as described
previously [14].
We used a 10% SDS-PAGE gel to estimate the size of TrV-VLPs
according to the molecular weight (kDa) and the bacterial endo-
toxin content for recombinant TrV-VLPs was detected using
ToxinSensorTM Gel Clot Endotoxin Assay Kit (Genscript). This test
detects endotoxins from Gram-negative bacteria using a Gel-Clot
technique in a semi-quantitative way. Following the instructions
of the kit, different dilutions of the sample (from 1:1 to
1:100,000) were tested.
2.2. Expression and purification of Trypanosoma cruzi chimeric
antigens
Synthetic genes encoding T. cruzi proteins (IBMP-8.1, IBMP-8.2,
IBMP-8.3 and IBMP-8.4) were obtained from a commercial supplier
(GenScript, Piscataway, NJ, USA) and were subcloned into the
pET28a vector. The antigens were expressed in Escherichia coli
BL21 Star (DE3) cells that had been grown in Luria-Bertani medium
and induced with 0.5 mM isopropyl-b-D-1-thiogalactopyranoside
(IPTG), as described in Santos et al. (2016). The proteins were puri-
fied from the soluble fraction of the total extract of bacterial lysate
using affinity chromatography followed by ion-exchange chro-
matography, and were then quantified by means of the fluoromet-
ric method (Qubit 2.0, Invitrogen Technologies, Carlsbad, CA, USA),
following the manufacturer’s protocol. All recombinant proteins
were stored at 20 °C until used in immunization protocols.
2.3. Immunization in a mouse model
Mice used in this study were obtained from and kept at the
vivarium of the Health Sciences Centre at the Federal University
of Rio Grande do Norte (UFRN – Brazil). All procedures of immu-
A. Maria Vasconcelos Queiroz, Y. Aleksandrovna Yanshina, E. Thays da Silva Rodrigues et al.
nization and blood collection protocol were approved and per-
formed according to the indications of the Ethics Committee on
Animal Use (CEUA – UFRN, Protocol Number 068.080/2017).
Briefly, 8-week-old BALB/c female Mus musculus mice were used
in the immunization protocols. Animals were immunized subcuta-
neously using syringe and needle (OmnicanÒ 100, B. Braun, Ger-
many). In the first immunization strategy, the first group of
animals was administered with TrV-VLPs (100 mg) in sterile phos-
phate saline (PBS), and the second group was administered with
TrV-VLPs (100 mg) mixed with the 10% aluminium hydroxide adju-
vant (Vetec Quimica Fina, Rio de Janeiro, Brazil) in PBS and the
third group is non-immunized animals.
In the second immunization protocol, 50 mg of TrV-VLPs mixed
with 50 mg of mix-IBMP (12,5 mg of each IBMP protein) in PBS were
administered in the first group, the second group was administered
with mix-IBMP (50 mg) mixed with the 10% aluminum hydroxide
adjuvant in PBS, the third group was administered with mix-
IBMP (50 mg) in PBS, and the fourth group was administered only
with PBS. For both strategies, on different days (15, 30 and 45 days)
of the single-dose immunization, blood samples from immunized
animals were obtained and processed to obtain sera. Serum sam-
ples from the immunized animals were used for screening of
anti-TrV-VLPs antibodies by enzyme-linked immunosorbent
immunoassay (ELISA).
2.4. Obtaining sera from mice immunized with TrV-VLPs and IBMPs
antigens
In order to determine anti-TrV-VLPs and anti-IBMPs antibodies
from the immunized BALB/c mice, serum samples were extracted
from the coagulation of whole blood, extracted by terminal tail
excision of the immunized animals. Briefly, whole blood samples
were obtained and maintained at 37 °C for 30 min and then at
5 °C for one hour. Subsequently, the samples were centrifuged at
5,000 rpm for 10 min. Serum samples were fractionated and stored
at 20 °C until use.
2.5. Determination of anti-TrV-VLPs and anti-IBMPs antibodies in
immunized mice
For the evaluation of the humoral immune response in the
immunized animals, serum samples were used to investigate the
different subtypes of specific anti-TrV-VLPs and anti-IBMPs anti-
bodies (total IgG, IgG1, IgG2a, IgG2b and IgG3). For this, an indirect
enzyme-linked immunosorbent assay (ELISA) was used, according
to the methodology previously developed in our laboratory [16].
Briefly, 96-well microplates (Nunc-Immuno MicroWell, USA) were
coated with 50 ng/well of purified recombinant TrV-VLPs or IBMP-
8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4 in 0.1 M carbonate buffer pH
8.5 (overnight at 4 °C). The microplates were washed three times
with PBS-0.05% Tween 20 (Promega, USA) and blocked for one hour
at artificial air temperature in the laboratory (25 °C) with 1% (w/v)
skim milk powder in PBS. After three washes, a serial dilution (IgG
total: 1/10 – 1/180; IgG subclass: 1/20 – 1/160) of immunized mice
sera was added for one hour at artificial air temperature. Then,
anti-mouse horseradish peroxidase (HRP)-conjugated: anti-
mouse IgG (diluted 1/40,000; Sigma-Aldrich, USA), anti-mouse
IgG1 (diluted 1:2,000; Caltag Laboratories. Burlingame, CA), anti-
mouse IgG2a (diluted 1/2,000; Caltag Laboratories. Burlingame,
CA), anti-mouse IgG2b (diluted 1/2,000; Thermo Fisher: Invitrogen,
USA) and anti-mouse IgG3 (diluted 1/2,000; Sigma-Aldrich, USA)
were added. Next, all HRP-conjugated antibodies were incubated
for 30 min with a substrate solution, composed of 10 mL of citrate
buffer pH 5.0 with 10 mg of OPD (o-Phenylenediamine dihy-
drochloride; Sigma-Aldrich, USA) and 10 ml of hydrogen peroxide
30% (Sigma-Aldrich, USA). Then, sulphuric acid 4 N was used to
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Vaccine 39 (2021) 4723–4732
stop the solution. All sera from immunized mice were assayed in
triplicate and final reaction measured at 490 nm by spectropho-
tometer (Epoch, BioTek Instruments, Winooski, VT, USA). Serum
samples obtained from unimmunized mice (pre-immune sera)
were used as the control group in the determination of anti-TrV-
VLPs and anti-IBMPs antibodies subtypes. GraphPad Prism 6 soft-
ware for Windows (GraphPad Software, La Jolla, CA) was used for
the statistical analysis.
2.6. Temperature treatment and TEM observation of recombinant TrV-
VLPs
In order to obtain the protein of interest to study their particu-
lar characteristic, TrV-VLPs purification was performed. Particles
were purified in NMT buffer (500 Nm Tris/HCL pH 7.3; 100 nM
NaCl and 10 nM MgCl2) in a 0.1 mg mL 1 (protein) concentration
and stored for one and a half year at 4 °C. Sample aliquots of about
50 ml each were stored in 0.5 mL capped plastic tubes for one week
at 80 °C, 20 °C, room temperature, and 37 °C. All samples were
mounted on carbon-coated copper grids, previously polarized by
the glow discharge method, stained (3% w/v uranyl acetate), and
then analysed under TEM. Images were acquired using a Phillips
EM208S transmission electron microscope (nominal resolution of
0.34 nm) combined with a Morada camera.
3. Results
Using the baculovirus-insect cell expression system, recombi-
nant TrV-VLPs were successfully produced for studies in the mur-
ine immunization model. Subsequently, purified recombinant TrV-
VLPs were further visualized by electron microscopy and SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1), and
immunocharacterized by Western Blotting (results not show),
according to our previous study targeting particles of 37.3 kDa
[14]. In addition, the bacterial endotoxin test was performed on
the TrV-VLPs to detect endotoxins from Gram-negative bacteria
using a gel-clot technique in a semi-quantitative way. This proce-
dure used to detect the presence of bacterial’ origin toxins in
human biological and drugs was approved by the FDA. It was found
that the concentration range of the endotoxins present in the TrV-
VLPs was 25–250 EU/mL, a range that according to the kit instruc-
tions corresponds to a negative value (results not shown). Based on
that, the particles were considered free of toxins that could come
from the expression procedure, and therefore we can be sure that
the immune response to TrV-VLPs was not enhanced due to bacte-
rial detritus contamination.
Next, in order to evaluate the immunostimulatory ability of
recombinant TrV-VLPs to induce humoral immune response, an
immunization process was established in a murine model. Then,
the production of anti-TrV-VLPs IgG antibodies was analysed in
serum samples from BALB/c immunized animals. Mice were
immunized with a single TrV-VLPs dose (100 mg/ animal), adminis-
tered subcutaneously in the presence or absence of aluminium
adjuvant. Anti-TrV-VLPs antibodies profile was analysed at 15, 30
and 45 days after primary immunization. It was observed that ani-
mals immunized with a single dose of TrV-VLPs exhibit the ability
to induce total anti-TrV-VLPs IgG antibodies similar to animals
immunized with the TrV-VLPs mixed with aluminium adjuvant
(Fig. 2). These data were compared with unimmunized animal sera
(pre-immune sera).
Subsequently to the characterization of total anti-TrV-VLPs IgG
antibodies in animals immunized with a single dose (primary
immunization 100 mg/animal) of TrV-VLPs (Fig. 2), the animals
were subjected to the second dose of TrV-VLPs (50 mg/animal).
After seventy days of immunization, the profile of the different
A. Maria Vasconcelos Queiroz, Y. Aleksandrovna Yanshina, E. Thays da Silva Rodrigues et al. Vaccine 39 (2021) 4723–4732

Fig. 1. Recombinant TrV-VLPs produced in the baculovirus expression system. Left panel: Electron micrograph of negative staining samples (3% w/v uranyl acetate) of
purified recombinant TrV-VLPs. Bar 200 nm. Right panel: SDS-polyacrylamide gel electrophoresis. In lanes labeled as TrV-VLPs1 and TrV-VLPs2, 2.5 mg and 5 mg of TrV-VLPs
were added, respectively. Lane labeled as M represent molecular weight markers (BlueRay Prestained Protein Marker - Jena Bioscience) and the corresponding molecular
weights of the markers are shown in the lane labelled as kDa.

Fig. 2. Determination of total anti-TrV-VLPs IgG antibodies in animals immunized with a single dose of TrV-VLPs subcutaneously. The bars represent the antibody profile of
animals immunized with TrV-VLPs in the absence (VLP) or presence of aluminium (VLP-A) and anti-TrV-VLPs IgG antibody profile of unimmunized (pre-immune) animals is
represented by the black bar. Each bar represents the mean absorbances obtained in each group of immunized animals and their respective standard deviations, after 15
(15D), 30 (30D) and 45 (45D) days post-immunization. Serum samples were analyzed at 1/100 dilution (v/v). Absorbance at 490 nm is shown on Y axis. Differences between
mean values were considered significant when p < 0.05. Signs ‘‘*” and ‘‘#” represent differences observed in the immunized groups compared to the negative control and
difference between the immunized groups, respectively.

anti-TrV-VLPs IgG antibody subtypes (total IgG, IgG1, IgG2a, IgG2b,
and IgG3) was determined in the serum samples of all immunized
animals. The data presented in Fig. 3 show that both groups of
immunized animals (TrV-VLPs in the presence or absence of alu-
minium) had predominantly TrV-VLPs-specific IgG2b and IgG3
antibodies when analysed by ELISA. Regarding immunoglobulin
of the IgG3 subtype, animals immunized with TrV -VLPs have titers
44 times higher when compared to the control group and 1.5 times
higher when compared to animals immunized with VLP-A. Addi-
tionally, immunized animals show little or no production of anti-
TrV-VLPs IgG1 and IgG2a antibodies compared to unimmunized
(pre-immune) animals.
The results of the characterization of the immunostimulatory
activity of T. cruzi antigenic candidates in mix form (mix-IBMP)
with the concomitant TrV-VLPs or aluminium for total IgG anti-
IBMP-8.1 are shown in Fig. 4A, for anti-IBMP-8.2 in Fig. 4B, for
anti-IBMP-8.3 in Fig. 4C, and for anti-IBMP-8.4 in Fig. 4D.
The data show the presence of higher total IgG anti-IBMPs in
the groups immunized with mix-IBMP in association with TrV-
VLPs or aluminium, when compared to the negative control. How-
ever, for all proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4.)
and at all times, the group immunized with the traditional adju-
vant has a superior profile of total IgG against each protein. For
groups immunized with the aluminium, it was observed that for

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A. Maria Vasconcelos Queiroz, Y. Aleksandrovna Yanshina, E. Thays da Silva Rodrigues et al. Vaccine 39 (2021) 4723–4732

Fig. 3. Determination of different subtypes anti-TrV-VLPs IgG antibodies in animals immunized with TrV-VLPs after 70 days post-immunization. Each bar represents the
mean absorbance obtained in each group of immunized animals and their respective standard deviation, after 70 days post-immunization. Each serum sample was analyzed
at 1/20 dilution (v/v) by ELISA. Absorbance at 490 nm is shown on Y axis. Differences between means were considered significant when p < 0.05. The graphical signs ‘‘*” and
‘‘#” mean differences observed in the immunized groups compared to the negative control and difference between the immunized groups, respectively.

Fig. 4. Determination of anti-IBMP-8.1 (A), anti-IBMP-8.2 (B), anti-IBMP-8.3 (C) and anti-IBMP-8.4 (D) IgG antibodies profile in animals immunized with mix-IBMP mixed
TrV-VLPs (mix-IBMP + VLP) or in presence of aluminum (mix-IBMP + Alum), respectively. Negative control is represented by the black bar. Bars represent mean absorbances
obtained in each group of immunized animals and their respective standard deviations, after 15 (15D), 30 (30D) and 45 (45D) days post-immunization. Each serum sample
was analyzed at 1/100 dilution (v/v) by ELISA. Absorbance at 490 nm is shown on Y axis. Differences between means were considered significant when p < 0.05. The graphical
signs ‘‘*” and ‘‘#” mean differences observed in the immunized groups compared to the negative control and difference between the immunized groups, respectively. Above
D30 bar (D) graphic sign ‘‘



” means that 30 days after immunization at 1/100 dilution absorbance exceeded the adopted wavelength of 490 nm.

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A. Maria Vasconcelos Queiroz, Y. Aleksandrovna Yanshina, E. Thays da Silva Rodrigues et al. Vaccine 39 (2021) 4723–4732

proteins IBMP-8.2, IBMP-8.3 and IBMP-8.4 at the last point (D45,
the level of total IgG starts to decline  1.1  (4B),  1.5  (4C)
and  1.3  (4D) to respect point (D30).
The profile of IgG anti-TrV-VLPs antibodies was different when
TrV-VLPs were administered with the mix-IBMP compared to TrV-
VLPs administered alone after 15, 30 and 45 days after the first
immunization (results not shown). At the same time, the adminis-
tration of TrV-VLPs mixed with the mix-IBMP makes the formula-
tion more immunogenic, mainly for the IBMP-8.2 and IBMP-8.4
antigens, in relation to the production of IgG antibodies (results
not shown). Another interesting observation is the fact that there
seems to be an immunogenic competition between the different
antigens of T. cruzi and the TrV-VLPs, since different levels of IgG
antibodies have been observed for the different antigens (results
not shown).
The mix-IBMP mixed with TrV-VLPs, mix-IBMP plus aluminium
and mix-IBMP groups were evaluated at 30 days (D30) after the
first immunization for the production of IgG1 anti-IBMPs (Fig. 5)
and IgG3 (Fig. 6) antibodies anti-IBMP-8.1, anti-IBMP-8.2, anti-
IBMP-8.3 and anti-IBMP-8.4.
It was observed that in the group associated with aluminium
there was a production of the IgG1 type antibody, mainly against
IBMP-8.4 (5D). Possibly, this is inherent in aluminium, because
when the mix-IBMP is administered in isolation there is no produc-
tion of IgG1 against the proteins. The production of IgG1 in the
mix-IBMP group associated plus TrV-VLPs against IBMP-8.2 is
inherent in protein, because when the particles are administered
alone there is no production of this type of antibody, not even
when associated with particles of aluminium (Fig. 3).
Data shown in Fig. 6 referring to the IgG3 type antibodies, exhi-
bit a significant difference against IBMP-8.1 (6A) and IBMP-8.2 (6B)
proteins when mix-IBMP is mixed with TrV-VLPs in relation to the
group with only the administration of the mix-IBMP or of the mix-
IBMP mixed with the traditional aluminium adjuvant. A different
antibody profile was found against IBMP-8.3 (6C), in which there
is a significantly greater difference in the mix-IBMP group com-
pared to the others, and against IBMP-8–4 (6D), in which the lar-
gest difference is found when the association is with aluminium.
The association with aluminium, in addition to induce the pro-
duction of the significant IgG3 antibodies (Fig. 6), also induces the
production of IgG1 antibodies against all proteins, mainly against
IBMP-8.4 (Fig. 5). Unlike when the mix-IBMP is in association with
TrV-VLPs, there is only IgG3 antibodies induction. Besides that, the
stability of these recombinant TrV-VLPs was evaluated in relation
to temperature, to understand whether this variable interferes
with the assembly of the empty particle. The analysis of TrV-
VLPs stability under different temperature storage can be observed
in Fig. 7. All temperatures treated particles look spherical and no
small or ‘‘open” particles were observed in the grids that could
be attributed to particles disassembly.
4. Discussion
VLPs are considered safe because they do not have the viral gen-
ome [18] and also mimic the immunogenic potential of many
viruses due to structural epitopes present in capsid conformation
[19]. The particulate structure of VLPs can be efficiently recognized
by antigen-presenting cells [19] and is therefore considered impor-
tant for exogenous antigen display. Therefore, VLPs have been a
reliable and safe tool for the development of new vaccines. In the
context of vaccine adjuvants, the use of aluminium salts as an

Fig. 5. Determination of subtype anti-IBMPs IgG1 antibodies profile in animals immunized (after 30 days) with mix-IBMP mixed TrV-VLPs (mix-IBMP + VLP) or in presence of
aluminum (mix-IBMP + Alum), respectively. The negative control (PBS) is represented by the black bar. Each bar represents the mean absorbance obtained in each group of
immunized animals and their respective standard deviation, after 30 days post-immunization. Each serum sample was analysed at 1/40 dilution (v/v) by ELISA. Absorbance at
490 nm is shown on Y-axis. Differences between means were considered significant when p < 0.05. The graphical signs ‘‘*” and ‘‘#” mean differences observed in the
immunized groups compared to the negative control and difference between the immunized groups, respectively.

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A. Maria Vasconcelos Queiroz, Y. Aleksandrovna Yanshina, E. Thays da Silva Rodrigues et al. Vaccine 39 (2021) 4723–4732

Fig. 6. Determination of subtype anti-IBMPs IgG3 antibodies profile in animals immunized (after 30 days) with mix-IBMP mixed TrV-VLPs (mix-IBMP + VLP) or in presence of
aluminium (mix-IBMP + Alum), respectively. The negative control (PBS) is represented by the black bar. Each bar represents the mean absorbance obtained in each group of
immunized animals and their respective standard deviation, after 30 days post-immunization. Each serum sample was analyzed at 1/40 dilution (v/v) by ELISA. Adopted
absorbance of 490 nm. Differences between means were considered significant when p < 0.05. The graphical signs ‘‘*” and ‘‘#” mean differences observed in the immunized
groups compared to the negative control and difference between the immunized groups, respectively.

Fig. 7. TrV-VLPs stability. TEM images of TrV-VLPs stained particles obtained after storage at different temperatures (see Materials and Methods). (A) Purified particles stored
at 4 °C for 1.5 years (18 months); (B) Particles from A stored for one week at room temperature; (C) Particles from A stored one week at 20 °C; (D) Particles from A stored for
one week at 37 °C. Black bars correspond to 100 nm. Antibody response induced by recombinant VLPs from Triatoma virus and chimeric antigens from Trypanosoma cruzi.

adjuvant is currently licensed in most vaccine preparations [20],
however, its use is related to episodes of severe local adverse reac-
tions, such as pruritic subcutaneous nodules and hypersensitivity
[21] and motor neuron degeneration [22]. Given these limitations,
this study contributes to the characterization and evaluation of the
immunostimulatory action of TrV-VLPs for their ability to induce
antibodies, in the absence of aluminium adjuvant.
The recombinant VLPs-TrV used in the murine immunization
model was produced using the Bac-to-Bac TM system (Invitrogen,
USA) of baculovirus gene expression [13]. This system is widely
used in the production of VLPs for vaccine development [23]. The
VLPs used in this study are a versatile tool for the development
of vaccines due to the conformational and immunological charac-
teristics that confer their immunostimulatory property [7]. Data
presented in this study show that TrV-VLPs have the ability to
induce different specific subtypes antibodies and that this ability
is independent of the use of aluminium as an adjuvant. Other stud-
ies have previously shown that the use of aluminium salts can
increase HPV16L1-VLPs antigen-specific antibody titters [24]. This
was not observed in our study (Fig. 2).
A study published by Ko and collaborators shows the ability of a
VLP to increase serum levels of anti-flagellin antibodies when this
antigen is formulated with the adjuvant VLP [25]. Our data show that
animals immunized with TrV-VLPs preferentially produce IgG2b
and IgG3-specific antibodies (Fig. 3). In addition, there is no produc-
tion of IgG1 (Figs. 3 and 5), as in the work of Ko et al. (2019), where
the production of anti-flagellin IgG1 antibodies with Th2 polariza-
tion was observed. This finding is especially important for the

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A. Maria Vasconcelos Queiroz, Y. Aleksandrovna Yanshina, E. Thays da Silva Rodrigues et al.
rational design of vaccines, since the induction of polarized immu-
nity of the Th2 type can hinder the protection mechanisms, when
the infectious agents develop an intracellular phase of infection
[26]. In the context of Chagas disease, the induction of a polarized
immunity of the Th2 type may not have an impact on the protection
mechanisms against the infection, since the Th1 versus Th2 polar-
ization mechanisms are involved in the resistance and susceptibility
mechanisms, respectively in the course of T. cruzi infection [27].
Induction in the production of IgG2b and IgG3 antibodies
(Fig. 3) suggests the immunomodulatory potential of TrV-VLPs as
an immunization strategy against T. cruzi, since the presence of
anti-T. cruzi antibodies of the IgG2b type is associated with protec-
tive mechanisms during infection [28]. Regarding antibodies of the
IgG3 type, they have the ability to opsonize and destroy infected
cells [29]. Thus, it was observed in this study that the isolated
TrV-VLPs achieve a greater profile of IgG3 production, compared
with their use in combination with the conventional adjuvant
(Fig. 3). Animals immunized only with TrV-VLPs after 15 days, after
the second immunization, showed 81 times more IgG3 antibodies
when compared to the negative control group (results not shown).
The use of structural protein as an adjuvant can induce Th1-
type cytokines. This result was seen in a study using flagellin as
an adjuvant. The authors observed that its isolated use, in low con-
centrations, induced specific antibodies [30]. Likewise, TrV-VLPs
are particles made up of structural proteins organized in the form
of a viral capsid. Here we demonstrate that TrV-VLPs are capable of
inducing specific antibodies after 70 days of immunization. Thus,
TrV-VLPs, based on viral structural proteins, naturally have the
ability to induce specific T-cell-dependent antibodies [31].
In vaccine formulations, the use of chimeric recombinant pro-
teins is interesting and viable. Protein expression systems, such
as baculoviruses, are processes in modern biotechnology that are
effective for the production of proteins [32], and in the case of chi-
meric proteins are composed of distinct gene sequences of antigens
in a single molecule, enabling an increase in immunogenicity by
offering to the immune system a variety of epitopes [9]. Strategies
using chimeric molecules have also shown efficacy for serological
diagnosis of diseases, for example, human and canine Chagas dis-
ease [10,12,33]. It was shown in a study for malaria the importance
of recombinant antigens in inducing a strong antibody response in
a murine model [34].
In our study, we showed that T. cruzi recombinant chimeric
antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) are efficient
in inducing antibodies in mice, especially when administered with
aluminium and VLPs, showing a higher total IgG antibodies
response with the use of aluminium (Fig. 4). Although aluminium
induces a greater humoral response similar to VLPs, the toxicity
of this vaccine adjuvant is often reported as a disadvantage
[27,28]. Even the IBMP-8.4 protein presenting in its antigenic
repertoire more T. cruzi epitopes [35], in an immunization protocol
in a murine model, the IBMP-8.2 (Fig. 4B) protein induces greater
production of total IgG antibodies when associated with TrV-
VLPs. It is possible that the presentation of the IBPM-8.2 protein
to the immune system is more effective due to existence of more
immunogenic antigens available for recognition [36].
A study published by Thema and colleagues observed the ability
of VLPs to induce IFN-c production and a Th1-type polarized
immune response [37]. The IFN-c cytokine produced by Th1 lym-
phocytes inhibits the proliferation of Th2 cells and this phe-
nomenon could explain the down regulation of the production of
IgG1 antibodies for the IBMP mix-associated with VLPs-TrV
(Fig. 5). This non-induction in production has already been seen
to immunize TrV-VLPs in the absence or presence of aluminium
(Fig. 3). There is the secretion of B cell-activating cytokines through
the Th1 type response and consequently, there is the production of
related antibodies [38].
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Vaccine 39 (2021) 4723–4732
Regarding the IgG3 antibody subtype (Fig. 6), the results pre-
sented here are interesting in the context of inducing immunity
in the Chagas disease, since the ability to produce IgG3 antibodies
is conditioned by the production of cytokines of the type Th1 that
also favours the induction of cytotoxic CD8+ T lymphocytes [29].
The induction of IgG3 production when using TrV-VLPs, instead
of IgG1 and IgG3 as in the aluminium group, shows the advantage
of TrV-VLPs in inducing the production of Th1-type cytokine-
dependent antibodies.
The VLP technology has interesting characteristics for the pro-
duction of new, safe, stable, and efficient vaccines [39,40]. Modified
or natural VLPs can increase the immunogenicity of a given vaccine
formulation [41,42]. The data presented in this study corroborate
the literature regarding the use of non-infectious VLPs, which
increases the immunogenicity of vaccine antigens, thus being an
interesting strategy for the development of new vaccines.
There are data in the literature reporting the use of VLPs as vac-
cine adjuvants for the induction of Th1-type polarized immune
response [30]. In these scenarios, we believe that during the TrV-
VLPs immunization process the immune response is of the Th1 type,
regarding antibody-induced immunity induction, we observed that
TrV-VLPs have the same ability to stimulate antibodies when used
alone or when mixed with aluminium. In addition, T. cruzi chimeric
recombinant proteins were also able to stimulate antibodies when
used alone, or when used in vaccine formulations with aluminium
or TrV-VLPs. Thus, we have shown the intrinsic immunostimulatory
ability of TrV-VLPs and T. cruzi chimeric recombinant proteins to
produce antibodies. These data open the possibility of using TrV-
VLPs as a vaccine adjuvant. However, further studies need to be per-
formed to characterize other immunity-inducing mechanisms, such
as T-cell mediated immunity, cytokine production, and characteri-
zation of immune memory.
Finally, thermal study of TrV-VLPs shows that these particles are
very robust and do not break apart when frozen. Thinking about the
potential use of them as adjuvant, this feature is in contrast with
adjuvant-based aluminium-salts because vaccines formulated with
these adjuvants have the drawback that they cannot be frozen [43].
Moreover, TrV-VLPs particles can be stored for long periods at 4 °C,
or even remain unaltered for one week at room temperature and
37 °C. Given this is the temperature of humans’ body, it can be
assured that, the intrinsic feature of TrV-VLPs particulate form to
stimulate PAMPs (Pathogen-associated molecular pattern) of the
immune system, will not be compromised.
5. Conclusions
In conclusion, the data obtained in this study demonstrate the
particular ability of TrV-VLPs to preferentially induce specific
anti-TrV-VLPs IgG2b and IgG3 antibodies in the murine model of
immunization. These data contribute to the potentiation of the
use of TrV-VLPs as vaccine adjuvant in vaccine formulations. TrV-
VLPs can be used to increase the immunogenicity of a given vac-
cine formulation, mainly in the production of IgG3 type antibodies,
as demonstrated here for recombinant antigens of T. cruzi, namely
IBMP-8.1, 8.2 and 8.4 proteins. However, the immune mechanisms
induced by TrV-VLPs and T. cruzi chimeric recombinant proteins
need to be more clearly understood, especially the induction of
the polarized humoral and cellular immune response.
Funding
This research was funded by Consejo Superior de Investigaciones
Científicas (grant number COOPB20503 - CSIC, Spain) and Global
Health and Tropical Medicine [grant number GHTM-UID/
multi/04413/2013] and partially supported by grants to DMAG,
A. Maria Vasconcelos Queiroz, Y. Aleksandrovna Yanshina, E. Thays da Silva Rodrigues et al.
from the Ministerio de Ciencia e Innovación [BFU2012-36241],
Grupos Investigación UPV/EHU 2018 [GIU18/172], and Gobierno
Vasco [Elkartek KK-2017/00008], Programa de empresas Innovado-
ras A1 2020 EKINZAILE, The Basque Country, Spain. This study also
was supported by Coordenação de Aperfeiçaomento de Pessoal de
Nível Superior (CAPES, Brazil).
Declaration of Competing Interest
D.M.A.G. is inventor of the pending patent PCT/EP2015/050054.
Both C.S.P.R. and D.M.A.G., are co-founders and shareholders of
Ikosaedrika Biologicals S.L., the enterprise licensee of the named
patent. The other authors declare no conflict of interest.
Acknowledgments
A.M.V.Q thanks the financial support provided by CAPES/Brazil
through postgraduate. MSS thanks to CNPq/Brazil for the Research
Grant (Bolsa de Produtividade). DMAG thanks a traveling grant to
visit JHU from Fundación Biofísica Bizkaia (Spain). C.S.P.R. thanks
a summer fellowship from the Basque Country government ‘‘Becas
Ikasiker de Colaboración 2019”. We thank Dr. Yana Li (JHU) for tech-
nical assistance. TEM images were collected at the Servicio General
de Microscopía Analítica y de Alta Resolución en Biomedicina (SGI-
KER, UPV/EHU). We are grateful to Paulo Fanado for editing this
manuscript.
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