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Citation: Mbhele, Z.; Thwala, L.;
Khoza, T.; Ramagoma, F. Evaluation
of Aluminium Hydroxide
Nanoparticles as an Efficient
Adjuvant to Potentiate the Immune
Response against Clostridium
botulinum Serotypes C and D Toxoid
Vaccines. Vaccines 2023,11, 1473.
https://doi.org/10.3390/
vaccines11091473
Academic Editors: Mariusz
Skwarczynski and Alan Cross
Received: 25 June 2023
Revised: 26 August 2023
Accepted: 8 September 2023
Published: 10 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Article
Evaluation of Aluminium Hydroxide Nanoparticles as an
Efficient Adjuvant to Potentiate the Immune Response against
Clostridium botulinum Serotypes C and D Toxoid Vaccines
Ziphezinhle Mbhele 1,2, Lungile Thwala 1,3 , Thandeka Khoza 2and Faranani Ramagoma 1, *
1Onderstepoort Biological Products, 100 Old Soutpan Road, Onderstepoort, Pretoria 0110, South Africa;
ziphezinhle@obpvaccines.co.za (Z.M.); lthwala@csir.co.za (L.T.)
2
Discipline of Biochemistry, School of Life Sciences, University of KwaZulu Natal, Pietermaritzburg Campus,
Private Bag X01, Scottsville 3209, South Africa; khozat1@ukzn.ac.za
3Council for Scientific and Industrial Research, National Laser Centre, Pretoria 0001, South Africa
*Correspondence: faranani@obpvaccines.co.za; Tel.: +27-12-522-1561; Fax: +27-12-522-1591
Abstract:
Clostridium botulinum serotypes C and D cause botulism in livestock, a neuroparalytic
disease that results in substantial economic losses. Vaccination with aluminium-based toxoid vaccines
is widely used to control the spread of botulism. Aluminium-based adjuvants are preferred owing
to their apparent stimulation of the immune responses to toxoid vaccines when compared to other
adjuvants. The aim of our study was to evaluate aluminium hydroxide nanoparticles as a potential
substitute for alhydrogel in the botulism bivalent vaccine. Botulism vaccines were formulated
with either alhydrogel or nanoalum and comparative efficacy between the two formulations was
conducted by evaluating the immune response in vaccinated guinea pigs. A significant increase in
immunological parameters was observed, with the antibody titres higher in the serum of guinea pigs
(20 IU/mL of anti-BoNT C/D) injected with nanoalum-containing vaccine than guinea pigs inoculated
with the standard alhydrogel-containing vaccine (8.7 IU/mL and 10 IU/mL of anti-BoNT C and
anti-BoNT D, respectively). Additionally, the nanoalum-containing vaccine demonstrated potency
in a multivalent vaccine (20 IU/mL of anti-BoNT C/D), while the standard alhydrogel-containing
vaccine showed a decline in anti-BoNT C (5 IU/mL) antibody titres.
Keywords: botulism; toxoids; vaccines; alhydrogel; nanoalum
1. Introduction
Botulism is a severe neuroparalytic disease caused by Clostridium botulinum toxins [
1
].
C. botulinum bacteria are divided into six serotypes (A–F) according to their genetic, phys-
iological, and metabolic characteristics. Serotypes A, B, E, and, to a lesser extent, F, are
responsible for human botulism, while animal botulism arise from infection with serotypes
C and D, whereas C. botulinum C/D and D/C neurotoxin mosaics are mainly associated
with avian botulism [
2
]. In livestock, botulism is mainly acquired through the ingestion of
the toxins from contaminated water or silage. The disease may also be contracted through
wound and intestinal toxic infections [
3
]. Upon infection, the botulism neurotoxins are ab-
sorbed into the bloodstream and carried to the peripheral cholinergic nerve terminal where
they block the release of acetylcholine, resulting in the flaccid paralysis of the muscles [
4
].
The disease is characterised by progressive paralysis or onset death in severe cases.
Protection against botulinum intoxication relies on the presence of specific neutralizing
antibodies during exposure [
5
]. In acute cases, antibody therapy is effective, but not always
accessible due to high costs [
6
]. Consequently, vaccination is the most effective control
measure for botulism. The most widely used vaccines are bivalent (C. botulinum C and
D) vaccines that are formulated with formaldehyde-inactivated toxins derived from the
fermentation of C. botulinum [
7
]. Outbreaks in vaccinated cattle have been a source of
Vaccines 2023,11, 1473. https://doi.org/10.3390/vaccines11091473 https://www.mdpi.com/journal/vaccines
Vaccines 2023,11, 1473 2 of 12
concern about the quality and efficacy of the existing vaccines [
8
] Recently, recombinant
protein-based Botulinum vaccines have been evaluated as a potential alternative to the
chemically treated toxoids. Recombinant vaccines have been shown to present low toxicity
and good immunogenicity among other advantages [
9
,
10
]; however, these have not reached
the market. The downstream processes used in the purification of recombinant antigens
require expensive purification procedures, making them unattractive from an industrial
perspective [
7
]. Therefore, innovative methods are still being explored to improve the
efficacy of native Botulinum toxoids for vaccine production.
Current vaccines against botulism are formulated with aluminium hydroxide as
an adjuvant [
1
]. Despite the controversy around its mechanism of action, aluminium
hydroxide is the most well-established adjuvant and is often the first choice for vaccine
development [
11
]. Recently, researchers have discovered a direct link between the size of
the adjuvant material and the immune response. Adjuvants at the nanoscale have been
shown to induce a robust and long-lasting immunity when compared to their microparticle
counterparts [
11
,
12
]. In that context, prospects of aluminium hydroxide nanoparticles are
emerging as potential vaccine adjuvants. Conventional aluminium hydroxide particles
form aggregates of 1 to 10
µ
m size. These particles predominantly recruit antigen presenting
cells (APCs) to the site of injection for the uptake of antigens and delivers them to the
lymph node to generate an immune response. Aluminium hydroxide nanoparticles, on the
other hand, range from 80 to 600 nm, and are therefore able to traffic to the lymph nodes
where there is a dense population of APCs, improving the delivery of antigens to the APCs
and the overall immune response [
12
,
13
]. This makes nanoalum an attractive alternative
to conventional aluminium hydroxide, since antigen specific T cell activation is restricted
within the lymph nodes [14].
The aim of this study was to evaluate the potency of aluminium hydroxide nanoparti-
cles (nanoalum) as a potential substitute for conventional alhydrogel in a multicomponent
vaccine. Alhydrogel and nanoalum were each formulated with BoNT C and BoNT D
toxoids to form a bivalent botulism vaccine and tested in guinea pigs. The two adju-
vants were further tested in a multivalent vaccine, containing the Botulinum toxoids and
two additional bacterial antigens.
2. Materials and Methods
2.1. Preparation and Characterization of Nanoalum
Nanoalum was prepared from alhydrogel (Onderstepoort Biological Products (OBP),
Pretoria, South Africa) used in commercial vaccines. The alhydrogel was mixed with 50%
(w/v) of 5 kDa polyacrylic acid (PAA) (Sigma, Ronkonkoma, NY, USA) to a final concentra-
tion of 2.7% (v/v) [
15
]. The mixture was homogenised at 10,000 rpm for 10 min at room
temperature (RT). The average size and polydispersity index (PDI) of the nanoalum and
alhydrogel were determined via dynamic light scattering (DLS) and the zeta (
ζ
) potential
was calculated from the electrophoretic mobility values determined using Laser Doppler
Anemometry (LDA), BoNT, measured using a Zetasizer
®
Nano-ZS, ZEN 3600, Malvern
instruments, (Worcestershire, UK) equipped with a red laser light beam (
λ
= 632.8 nm).
For the particle size and PDI measurements, the nanoalum and alhydrogel samples were
diluted 50
×
in milliQ water (18.2 M
Ω
-cm), and for the
ζ
-potential they were diluted 50
×
in 1 mM of KCl. The shape and properties of the alhydrogel and nanoalum were analysed
using a transmission electron microscope (TEM, Joel 2010, 80 kV, Philips, Amsterdam,
The Netherlands). The samples were deposited on a copper grid and allowed to dry before
viewing under the TEM. The viscosity and conductivity of the adjuvants were measured
at RT using a viscometer (AMTEK Brookfield, Middleborough, MA, USA) at 5 rpm and
100 rpm for the alhydrogel and nanoalum, respectively.
2.2. Adsorption Capacity of Alhydrogel and Nanoalum
The antigen adsorption capacity of alhydrogel and nanoalum was analysed via SDS-
PAGE using formaldehyde-inactivated C. botulinum Type D toxoid (OBP, SA) as a model
Vaccines 2023,11, 1473 3 of 12
antigen. The BoNT D toxoid vaccine was formulated with (10% v/v) either alhydrogel or
nanoalum and stirred for 24 h at RT. The formulation was sampled at 0 h and 24 h; the
samples were centrifuged at 13,000
×
g, 10 min, 4
◦
C. The supernatant was analysed on a
7.5% non-reducing SDS-PAGE according to the method described by Laemmli, 1970 [
16
].
The inactivated BoNT D toxoid was included as a reference (initial sample), and the same
result would be expected for BoNT C.
2.3. Adjuvant Safety and Vaccine Formulation
Nanoalum and alhydrogel were mixed with saline (OBP) to a final concentration of
20% (v/v). The adjuvant mixtures were administered SC in guinea pigs at a 2 mL dose on
day 0 and day 28 to mimic the vaccinations. The animals were monitored for up to 14 days
after the second dose for any local reactions [10].
Bivalent vaccines (C. botulinum type C and D toxoids) and multivalent vaccines (con-
taining BoNT C and D toxoids, and two additional antigens derived from C. perfringens
and B. anthracis) were formulated to the minimum lethal dose (MLD
50
) of
×
10
4
and
×
10
5
,
respectively, as described by Schantz and Kautter [
17
], with 10% (v/v) of either the alhy-
drogel or nanoalum in a final volume of 200 mL. The vaccines were stirred at 100 rpm for
24 h at 4
◦
C. The formulated vaccines were tested for sterility by inoculating 20 mL of the
thioglycolate broth medium and soya broth with 0.5 mL of the vaccine sample, and they
were incubated for 14 days at 37 ◦C under aerobic and anaerobic conditions [10].
2.4. Immunisation of Guinea Pigs
Immunizations were carried out as described by Gil et al. (2013) [
18
] with minor modi-
fications. Duncan Hartley female guinea pigs aged 3–4 months, weighing 200 to 310 g, were
acclimatized for three days prior to immunization. The vaccine groups were divided as fol-
lows: Group A—bivalent alhydrogel, Group B—bivalent nanoalum, Group C—multivalent
alhydrogel, Group D—multivalent nanoalum, and Group E—placebo, and each group was
assigned eight guinea pigs. A volume of 2 mL of the vaccine, including the control, was
administered subcutaneously (SC) on day 0 and a booster was administered SC on day 28.
On day 35, blood samples were collected from the guinea pigs via cardiac puncture and
centrifuged, 3000
×
g, 10 min, to obtain sera [
18
]. The sera from each group were pooled
and stored at −20 ◦C until further use in toxin neutralization studies.
2.5. Evaluation of Neutralising Antibodies
Toxin neutralising antibodies were evaluated in a toxin neutralization assay in mice as
described by Rosen et. al., 2016 [
19
]. Briefly, the sera obtained in Section 2.4 was used to
prepare serial 1.2-fold dilutions of each antitoxin preparation. Standard antitoxin prepara-
tions were concurrently diluted to concentrations of 0.08, 0.10, 0.12, and 0.14 International
Units per mL (IU/mL). All antitoxin dilutions were then incubated for 30 min at 37
◦
C
with a toxin test dose of 10
6
MLD
50
BoNT/C and 10
5
MLD
50
BoNT/D toxins. The abil-
ity of the anti-Botulinum antitoxins to neutralise the toxins was studied using a mouse
assay to evaluate their potency via intravenous challenge. Each mixture was injected into
8-week-old CD-1
®
IGS female mice weighing between 18 and 22 g at 0.2 mL per mouse with
4 mice assigned to each group. Survival was monitored for three days. Antitoxin potency
was calculated based on the lowest dilution of antitoxin that failed to protect the animals,
compared to that of the standard antitoxin. The animals were monitored for three days,
and the number of live and dead mice were recorded for each serum dilution (Figure 1) [
18
].
Titrations were also performed with standard anti-toxins C and D as controls (1st British
standard 01/508 Botulinum type C equine antitoxin, 01/510 Botulinum D equine antitoxin,
NIBSC, Potters Bar, UK).
Vaccines 2023,11, 1473 4 of 12
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Figure 1. Schematic representation of immunisation of guinea pigs and toxin neutralization assay
in mice. BoNT C and BoNT D were formulated as a bivalent vaccine as well as a multivalent vaccine
with two additional antigens. The vaccine was administered subcutaneously on day 0 (1st injection)
and day 28 (booster injection). On day 35, the guinea pigs were bled, and the blood samples were
centrifuged to obtain sera, which was evaluated in a toxin neutralisation assay. Live mice indicate
neutralisation of the toxin and death indicates the absence of toxin neutralisation.
2.6. Statistical Analysis
R Console version 3.2.1 (analytical software) was used to determine the significant
differences among the vaccine groups and the adjuvant groups, using a t-test on the mean
values [20].
2.7. Ethics Statement
Ethical clearance for the animal procedures was obtained from the Onderstepoort
Biological Products (OBP) Animal Ethics Commiee (South African Veterinary Council
Facility Registration Number: FR1514054) and the Department of Agriculture, Land Re-
form and Rural Development under Section 20 of the Animal Diseases Act (Act 35 of 1984),
Protocol reference number:12/11/11(b)/1914(HP).
3. Results
3.1. Preparation and Characterisation of Nanoalum
The morphology of the adjuvants was analysed under a transmission electron micro-
scope (Figure 2). The alhydrogel displayed filamentous particles which formed aggregates
that were dispersed via homogenization (Figure 2A, B). The addition of PAA prior to ho-
mogenisation resulted in the formation of nanoalum, which displayed plate-like particles
(Figure 2C). The properties and particle sizes of the adjuvants were analysed using dy-
namic light scattering (DLS). The untreated alhydrogel was 3400 nm with a PDI of 1.0 (Figure
3A) and a ζ potential of +10 mV (Figure 3B). Following homogenisation, the alhydrogel
particle size was reduced to an average of 620 nm (Figure 3A) and the nanoalum had an
average diameter of 265 nm, and the addition of PAA shifted the ζ-potential to −53 mV
(Figure 3B). The viscosity of the alhydrogel was 471 cP with a conductivity of 164.1 µS/cm,
and these were reduced to 2.94 cP (water-like consistency) and 5.60 µS, respectively, in the
nanoalum samples (Figure 3C). Furthermore, the nanoalum particles maintained a stable
size conformation over six months of storage at 4 °C, whereas the alhydrogel reaggregated
to 1555 nm (Figure 3A). The untreated alhydrogel particles’ sizes could not be determined
via DLS after 6 months.
Figure 1.
Schematic representation of immunisation of guinea pigs and toxin neutralization assay in
mice. BoNT C and BoNT D were formulated as a bivalent vaccine as well as a multivalent vaccine
with two additional antigens. The vaccine was administered subcutaneously on day 0 (1st injection)
and day 28 (booster injection). On day 35, the guinea pigs were bled, and the blood samples were
centrifuged to obtain sera, which was evaluated in a toxin neutralisation assay. Live mice indicate
neutralisation of the toxin and death indicates the absence of toxin neutralisation.
2.6. Statistical Analysis
R Console version 3.2.1 (analytical software) was used to determine the significant
differences among the vaccine groups and the adjuvant groups, using a t-test on the mean
values [20].
2.7. Ethics Statement
Ethical clearance for the animal procedures was obtained from the Onderstepoort
Biological Products (OBP) Animal Ethics Committee (South African Veterinary Council
Facility Registration Number: FR1514054) and the Department of Agriculture, Land Reform
and Rural Development under Section 20 of the Animal Diseases Act (Act 35 of 1984),
Protocol reference number:12/11/11(b)/1914(HP).
3. Results
3.1. Preparation and Characterisation of Nanoalum
The morphology of the adjuvants was analysed under a transmission electron micro-
scope (Figure 2). The alhydrogel displayed filamentous particles which formed aggregates
that were dispersed via homogenization (Figure 2A,B). The addition of PAA prior to ho-
mogenisation resulted in the formation of nanoalum, which displayed plate-like particles
(Figure 2C). The properties and particle sizes of the adjuvants were analysed using dynamic
light scattering (DLS). The untreated alhydrogel was 3400 nm with a PDI of 1.0 (Figure 3A)
and a
ζ
potential of +10 mV (Figure 3B). Following homogenisation, the alhydrogel particle
size was reduced to an average of 620 nm (Figure 3A) and the nanoalum had an average
diameter of 265 nm, and the addition of PAA shifted the
ζ
-potential to
−
53 mV (Figure 3B).
The viscosity of the alhydrogel was 471 cP with a conductivity of 164.1
µ
S/cm, and these
were reduced to 2.94 cP (water-like consistency) and 5.60
µ
S, respectively, in the nanoalum
samples (Figure 3C). Furthermore, the nanoalum particles maintained a stable size confor-
mation over six months of storage at 4
◦
C, whereas the alhydrogel reaggregated to 1555 nm
(Figure 3A). The untreated alhydrogel particles’ sizes could not be determined via DLS
after 6 months.
Vaccines 2023,11, 1473 5 of 12
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Figure 2. Characterization of alhydrogel and nanoalum particle morphology via transmission elec-
tron microscopy. (A) Untreated alhydrogel particles. (B) Alhydrogel after homogenization at 10,000
rpm. (C) PAA-stabilized nanoalum. The scale bar was 100 nm for alhydrogel and 50 nm for
nanoalum.
Figure 2.
Characterization of alhydrogel and nanoalum particle morphology via transmission electron
microscopy. (
A
) Untreated alhydrogel particles. (
B
) Alhydrogel after homogenization at 10,000 rpm.
(C) PAA-stabilized nanoalum. The scale bar was 100 nm for alhydrogel and 50 nm for nanoalum.
Vaccines 2023, 11, x FOR PEER REVIEW 5 of 12
Figure 2. Characterization of alhydrogel and nanoalum particle morphology via transmission elec-
tron microscopy. (A) Untreated alhydrogel particles. (B) Alhydrogel after homogenization at 10,000
rpm. (C) PAA-stabilized nanoalum. The scale bar was 100 nm for alhydrogel and 50 nm for
nanoalum.
Figure 3. Cont.
Vaccines 2023,11, 1473 6 of 12
Vaccines 2023, 11, x FOR PEER REVIEW 6 of 12
Figure 3. Physiochemical characterization of alhydrogel and nanoalum. (A) Particle sizes and poly-
dispersity index (PDI), and (B) zeta potential analysis via dynamic light scaering on a zetasizer.
(C) Viscosity and conductivity measured using a viscometer. ND: not determined.
3.2. Adsorption Capacity of Alhydrogel and Nanoalum
The adsorption capacity of alhydrogel and nanoalum was studied using the BoNT D
toxoid, which appears as a wide band which is >250 kDa (after inactivation with formal-
dehyde), with a pI of 5.4. The toxoid was formulated in a total volume of 2 mL with 10%
(w/v) of either alhydrogel or nanoalum, and allowed to incubate for 24 h. The formulation
was sampled at 0 h and 24 h and clarified via centrifugation to obtain a supernatant. The
resultant supernatant was analysed on a 7.5% non-reducing SDS-PAGE gel (Figure 4) for
the presence of any unbound antigens. Due to the difference in charges, the toxoid was
adsorbed to the alhydrogel immediately following formulation, while the nanoalum
showed a low adsorption over the 24 h incubation period as indicated by the presence of
the toxoid in the supernatant. This low adsorption was expected as the addition of PAA
shifted the ζ-potential of the adjuvant to a negative charge.
Figure 4. Adsorption capacity of alhydrogel and nanoalum analysed via SDS-PAGE. Alhydrogel
and nanoalum were formulated with C. botulinum type D toxoid and incubated for 24 h. Samples
were taken at 0 h and 24 h, centrifuged, and the resulting supernatants were analysed on a 7.5%
non-reducing SDS-PAGE. M: Molecular weight marker. Initial sample: inactivated toxoid sample
before formulation.
Figure 3.
Physiochemical characterization of alhydrogel and nanoalum. (
A
) Particle sizes and
polydispersity index (PDI), and (
B
) zeta potential analysis via dynamic light scattering on a zetasizer.
(C) Viscosity and conductivity measured using a viscometer. ND: not determined.
3.2. Adsorption Capacity of Alhydrogel and Nanoalum
The adsorption capacity of alhydrogel and nanoalum was studied using the BoNT D
toxoid, which appears as a wide band which is >250 kDa (after inactivation with formalde-
hyde), with a pI of 5.4. The toxoid was formulated in a total volume of 2 mL with 10%
(w/v) of either alhydrogel or nanoalum, and allowed to incubate for 24 h. The formulation
was sampled at 0 h and 24 h and clarified via centrifugation to obtain a supernatant. The
resultant supernatant was analysed on a 7.5% non-reducing SDS-PAGE gel (Figure 4) for
the presence of any unbound antigens. Due to the difference in charges, the toxoid was ad-
sorbed to the alhydrogel immediately following formulation, while the nanoalum showed
a low adsorption over the 24 h incubation period as indicated by the presence of the toxoid
in the supernatant. This low adsorption was expected as the addition of PAA shifted the
ζ-potential of the adjuvant to a negative charge.
Vaccines 2023, 11, x FOR PEER REVIEW 6 of 12
Figure 3. Physiochemical characterization of alhydrogel and nanoalum. (A) Particle sizes and poly-
dispersity index (PDI), and (B) zeta potential analysis via dynamic light scaering on a zetasizer.
(C) Viscosity and conductivity measured using a viscometer. ND: not determined.
3.2. Adsorption Capacity of Alhydrogel and Nanoalum
The adsorption capacity of alhydrogel and nanoalum was studied using the BoNT D
toxoid, which appears as a wide band which is >250 kDa (after inactivation with formal-
dehyde), with a pI of 5.4. The toxoid was formulated in a total volume of 2 mL with 10%
(w/v) of either alhydrogel or nanoalum, and allowed to incubate for 24 h. The formulation
was sampled at 0 h and 24 h and clarified via centrifugation to obtain a supernatant. The
resultant supernatant was analysed on a 7.5% non-reducing SDS-PAGE gel (Figure 4) for
the presence of any unbound antigens. Due to the difference in charges, the toxoid was
adsorbed to the alhydrogel immediately following formulation, while the nanoalum
showed a low adsorption over the 24 h incubation period as indicated by the presence of
the toxoid in the supernatant. This low adsorption was expected as the addition of PAA
shifted the ζ-potential of the adjuvant to a negative charge.
Figure 4. Adsorption capacity of alhydrogel and nanoalum analysed via SDS-PAGE. Alhydrogel
and nanoalum were formulated with C. botulinum type D toxoid and incubated for 24 h. Samples
were taken at 0 h and 24 h, centrifuged, and the resulting supernatants were analysed on a 7.5%
non-reducing SDS-PAGE. M: Molecular weight marker. Initial sample: inactivated toxoid sample
before formulation.
Figure 4.
Adsorption capacity of alhydrogel and nanoalum analysed via SDS-PAGE. Alhydrogel
and nanoalum were formulated with C. botulinum type D toxoid and incubated for 24 h. Samples
were taken at 0 h and 24 h, centrifuged, and the resulting supernatants were analysed on a 7.5%
non-reducing SDS-PAGE. M: Molecular weight marker. Initial sample: inactivated toxoid sample
before formulation.
Vaccines 2023,11, 1473 7 of 12
3.3. Evaluation of the Potency of Alhydrogel and Nanoalum Vaccine Formulation
3.3.1. Safety and Protective Efficacy of Alhydrogel- and Nanoalum-Containing Vaccines
The animals were inoculated 28 days apart with 20% (v/v) of nanoalum and alhy-
drogel and monitored over 42 days. No reactions were observed in any of the animals;
thus, nanoalum was reported as safe for use in guinea pigs. Neither fungal nor bacterial
contaminates were detected in any of the vaccine and control samples.
The potency of the alhydrogel formulation was compared with that of the nanoalum
formulation. The botulism bivalent vaccine contained BoNT C and D antigens. The
multivalent vaccine contained BoNT C and BoNT D antigens, and two other bacterial
antigens. The overall protective efficacy of the alhydrogel- and nanoalum-containing
vaccines was informed by the total number of mice that survived in the toxin neutralization
assay. In the bivalent formulations, the nanoalum vaccine demonstrated 100% protection
against a challenge with BoNT toxins, while the maximum protection achieved with the
alhydrogel vaccine was only 60% against the BoNT C and BoNT D challenge (Figure 5). In
the multivalent vaccine study, the alhydrogel-containing vaccine achieved 40% and 55%
protection against the BoNT C and BoNT D challenge, respectively, while 100% of the mice
survived in the nanoalum vaccine group.
Vaccines 2023, 11, x FOR PEER REVIEW 7 of 12
3.3. Evaluation of the Potency of Alhydrogel and Nanoalum Vaccine Formulation
3.3.1. Safety and Protective Efficacy of Alhydrogel- and Nanoalum-Containing Vaccines
The animals were inoculated 28 days apart with 20% (v/v) of nanoalum and alhydro-
gel and monitored over 42 days. No reactions were observed in any of the animals; thus,
nanoalum was reported as safe for use in guinea pigs. Neither fungal nor bacterial con-
taminates were detected in any of the vaccine and control samples.
The potency of the alhydrogel formulation was compared with that of the nanoalum
formulation. The botulism bivalent vaccine contained BoNT C and D antigens. The mul-
tivalent vaccine contained BoNT C and BoNT D antigens, and two other bacterial antigens.
The overall protective efficacy of the alhydrogel- and nanoalum-containing vaccines was
informed by the total number of mice that survived in the toxin neutralization assay. In
the bivalent formulations, the nanoalum vaccine demonstrated 100% protection against a
challenge with BoNT toxins, while the maximum protection achieved with the alhydrogel
vaccine was only 60% against the BoNT C and BoNT D challenge (Figure 5). In the multi-
valent vaccine study, the alhydrogel-containing vaccine achieved 40% and 55% protection
against the BoNT C and BoNT D challenge, respectively, while 100% of the mice survived
in the nanoalum vaccine group.
Figure 5. Protective efficacy of alhydrogel- and nanoalum-adjuvanted botulism vaccines in a toxin
neutralization assay. The antisera from guinea pigs vaccinated with the different vaccine groups
(alhydrogel- and nanoalum-adjuvanted vaccines) were evaluated in a mice challenge against MLD
50
of BoNT C and D toxins. Bivalent vaccine: containing BoNT C and BoNT D, Multivalent vaccine:
BoNT C, BoNT C, and two other bacterial antigens.
3.3.2. Quantification of Toxin-Neutralizing Antibodies
The neutralizing antibody titres were quantified using toxin neutralization assay
mice. The bivalent alhydrogel formulation induced 8.7 IU/mL of neutralizing antibodies
against the BoNT C toxin, and 10 IU/mL against the BoNT D toxin. The bivalent nanoalum
formulation was able to induce 20 IU/mL of neutralizing antibodies against the two toxins
(Figure 6A, B). In the multivalent alhydrogel vaccine, anti-BoNT C showed a significant
decrease (p < 0.0001) in antibody titres, from 8.7 IU/mL to 5 IU/mL, while anti-BoNT D
titres were maintained at 10 IU/mL. Interestingly, the nanoalum formulation maintained
20 IU/mL antibody titres against the BoNT toxoids (Figure 6A, B). The BoNT alhydrogel
and nanoalum vaccines were able to surpass the minimum required antibody levels, i.e.,
5 IU/mL of anti-BoNT C and 2 IU/mL of anti-BoNT D [19]. There was a significant differ-
ence (p < 0.0001) between the antibody titres induced by the alhydrogel vaccines and
nanoalum vaccines.
Figure 5.
Protective efficacy of alhydrogel- and nanoalum-adjuvanted botulism vaccines in a toxin
neutralization assay. The antisera from guinea pigs vaccinated with the different vaccine groups
(alhydrogel- and nanoalum-adjuvanted vaccines) were evaluated in a mice challenge against MLD
50
of BoNT C and D toxins. Bivalent vaccine: containing BoNT C and BoNT D, Multivalent vaccine:
BoNT C, BoNT C, and two other bacterial antigens.
3.3.2. Quantification of Toxin-Neutralizing Antibodies
The neutralizing antibody titres were quantified using toxin neutralization assay mice.
The bivalent alhydrogel formulation induced 8.7 IU/mL of neutralizing antibodies against
the BoNT C toxin, and 10 IU/mL against the BoNT D toxin. The bivalent nanoalum
formulation was able to induce 20 IU/mL of neutralizing antibodies against the two toxins
(Figure 6A,B). In the multivalent alhydrogel vaccine, anti-BoNT C showed a significant
decrease (p< 0.0001) in antibody titres, from 8.7 IU/mL to 5 IU/mL, while anti-BoNT D
titres were maintained at 10 IU/mL. Interestingly, the nanoalum formulation maintained
20 IU/mL antibody titres against the BoNT toxoids (Figure 6A,B). The BoNT alhydrogel
and nanoalum vaccines were able to surpass the minimum required antibody levels,
i.e., 5 IU/mL of anti-BoNT C and 2 IU/mL of anti-BoNT D [
19
]. There was a significant
difference (p< 0.0001) between the antibody titres induced by the alhydrogel vaccines and
nanoalum vaccines.
Vaccines 2023,11, 1473 8 of 12
Vaccines 2023, 11, x FOR PEER REVIEW 8 of 12
Figure 6 Neutralizing antibody titres against BoNT C and D toxins induced by alhydrogel- and
nanoalum-adjuvanted vaccines. The guinea pigs were immunized with a Botulinum type C and D
bivalent vaccine; seven days after booster immunization, the guinea pigs were bled, and the neu-
tralizing antibodies obtained from the sera were quantified in a toxin neutralization assay. (A) Anti-
BoNT C titres. (B) Anti-BoNT D titres. The doed lines represent the minimum required titres. Bi-
valent vaccine: containing BoNT C and BoNT D; Multivalent vaccine: BoNT C, BoNT C, and two
other bacterial antigens ND: not detected. * p < 0.0001.
4. Discussion
Botulism continues to threaten livestock production; thus, routine immunizations are
performed to control outbreaks. Recent reports of botulism outbreaks in vaccinated ani-
mals have raised concerns about the efficacy of vaccines against botulism [3, 8, 21]. There
are several reports where commercial livestock Botulinum vaccines failed to elicit the re-
quired minimum antibody titres following their approval [22, 23]. These observations
highlight the need for alternative vaccine strategies to induce robust and durable protec-
tive immunity against botulism. Therefore, this study was aimed at evaluating nanoalum
as a potential substitute for alhydrogel to improve the efficacy of Botulinum toxoid vac-
cines when administered as a bivalent vaccine or in combination with other antigens.
To date, Botulinum toxoid vaccines are predominantly adjuvanted with aluminium
salts to aid the induction of a protective immune response [1]. The aluminium hydroxide
particles disaggregate during mixing, thus allowing a uniform distribution of the ad-
sorbed antigen in the vaccine [24]. This is a valuable characteristic, especially for vaccines
which contain multiple antigens that are likely to compete for adsorption [24].
Resting alhydrogel readily reaggregates after mixing [25]; therefore, the size reported
for alhydrogel after homogenisation may indicate a disaggregated system which contin-
ues to form aggregates over time. The size of the aggregates may vary, depending on the
conditions of homogenisation; as a result, aluminium hydroxide particles are highly het-
erogenous and sometimes too big to characterise using dynamic light scaering [25]. Sev-
eral studies have reported on the application of aluminium hydroxide nanoparticles to
optimize the adjuvant activity in vaccines [15, 26, 27]. Unlike the conventional aluminium
adjuvants, nanoalum particles are structurally well-defined and easier to characterise for
quality control purposes. In the present study, the addition of PAA contributed to the
uniformity and maintained the stability of the particles at 4 °C over a 6-month period.
Similar results have been reported, where PAA contributed to the formation and stabili-
zation of the nanoparticles at different storage conditions [15]. PAA-stabilized nanoalum
has also demonstrated the ability to withstand multiple freeze–thaw cycles, making it an
ideal adjuvant for a cold-chain environment. However, the stability of nanoalum vaccines
Figure 6.
Neutralizing antibody titres against BoNT C and D toxins induced by alhydrogel- and
nanoalum-adjuvanted vaccines. The guinea pigs were immunized with a Botulinum type C and D
bivalent vaccine; seven days after booster immunization, the guinea pigs were bled, and the neutraliz-
ing antibodies obtained from the sera were quantified in a toxin neutralization assay. (
A
) Anti-BoNT
C titres. (
B
) Anti-BoNT D titres. The dotted lines represent the minimum required titres. Bivalent
vaccine: containing BoNT C and BoNT D; Multivalent vaccine: BoNT C, BoNT C, and two other
bacterial antigens ND: not detected. * p< 0.0001.
4. Discussion
Botulism continues to threaten livestock production; thus, routine immunizations
are performed to control outbreaks. Recent reports of botulism outbreaks in vaccinated
animals have raised concerns about the efficacy of vaccines against botulism [
3
,
8
,
21
]. There
are several reports where commercial livestock Botulinum vaccines failed to elicit the
required minimum antibody titres following their approval [
22
,
23
]. These observations
highlight the need for alternative vaccine strategies to induce robust and durable protective
immunity against botulism. Therefore, this study was aimed at evaluating nanoalum as a
potential substitute for alhydrogel to improve the efficacy of Botulinum toxoid vaccines
when administered as a bivalent vaccine or in combination with other antigens.
To date, Botulinum toxoid vaccines are predominantly adjuvanted with aluminium
salts to aid the induction of a protective immune response [
1
]. The aluminium hydroxide
particles disaggregate during mixing, thus allowing a uniform distribution of the adsorbed
antigen in the vaccine [
24
]. This is a valuable characteristic, especially for vaccines which
contain multiple antigens that are likely to compete for adsorption [24].
Resting alhydrogel readily reaggregates after mixing [
25
]; therefore, the size reported
for alhydrogel after homogenisation may indicate a disaggregated system which contin-
ues to form aggregates over time. The size of the aggregates may vary, depending on
the conditions of homogenisation; as a result, aluminium hydroxide particles are highly
heterogenous and sometimes too big to characterise using dynamic light scattering [
25
].
Several studies have reported on the application of aluminium hydroxide nanoparticles to
optimize the adjuvant activity in vaccines [
15
,
26
,
27
]. Unlike the conventional aluminium
adjuvants, nanoalum particles are structurally well-defined and easier to characterise for
quality control purposes. In the present study, the addition of PAA contributed to the
uniformity and maintained the stability of the particles at 4
◦
C over a 6-month period.
Similar results have been reported, where PAA contributed to the formation and stabiliza-
tion of the nanoparticles at different storage conditions [
15
]. PAA-stabilized nanoalum
has also demonstrated the ability to withstand multiple freeze–thaw cycles, making it an
ideal adjuvant for a cold-chain environment. However, the stability of nanoalum vaccines
Vaccines 2023,11, 1473 9 of 12
at different temperatures must be carefully investigated as alum-containing vaccines are
known to lose potency upon freeze–thawing [25].
The potency of alhydrogel is generally attributed to its interaction with antigens [
28
].
Acidic antigens adsorb onto alhydrogel based on electrostatic charge. Adsorption may
result in partial unfolding, exposing additional hydrophobic residues, which further
strengthen the adsorption [
24
]. The antigens are then slowly released in the interstitial
fluid for a prolonged exposure to the immune system and induce type 2 (Th2)-biased
immune responses [
29
,
30
]. Nanoparticles, on the other hand, allow for a greater adsorption
capacity due to the larger surface area-to-volume ratio [
13
]. In this study, PAA-stabilised
nanoalum showed a weaker adsorption capacity due to the shift in electrostatic charge.
However, the was a stronger immune response when compared to alhydrogel. These
results agree with a series of studies that reported an inverse relationship between the
strength of antigen adsorption and the resultant immune response [
13
,
15
,
31
,
32
]. It is the
effective adsorption of the antigen in the interstitial fluid that improves the immunological
response [
32
]. The small size of nanoparticles facilitates a more efficient accumulation
of the vaccine in the lymph nodes and induces a more balanced (Th1/Th2), robust, and
durable immune response [
30
]. The eluted antigens are taken up by the antigen-presenting
cells via micropinocytosis while the bound antigen is internalised through phagocytosis.
Phagocytosis was shown to be more efficient compared to micropinocytosis in antigen
uptake and was enhanced when the adjuvant aggregates were smaller [
33
]. Furthermore,
it is important to highlight the relationship between the low viscosity of the nanoalum
and the induced immune response observed in this study. This is because high viscosity
has been a major constraint in the widespread application of some potent vaccines due to
the reactogenicity at the site of injection [
34
]. Even though alum-based adjuvants are the
key benchmark for the evaluation of new adjuvants, they have also been associated with
some adverse side effects [
27
]. A good adjuvant should demonstrate a balance between
safety and efficacy; therefore, the water-like consistency of nanoalum can be expected to
minimise local reactions at the injection site. Additionally, the nanoalum suspension also
maintained homogeneity during incubation, while alhydrogel particles readily separated
from the liquid phase, forming a heterogenous mixture (results not shown).
Toxoid vaccines must induce specific levels of antibody titres in immunized animals
to certify their efficacy [
35
]. In the case of botulism, 5 IU/mL and 2 IU/mL are the
minimum required neutralising antibody levels for BoNT C and BoNT D, respectively.
The formulations evaluated in this study exceeded these requirements, with the exception
of BoNT C in the alhydrogel-adjuvanted multivalent vaccine that induced the minimum
required antitoxin level. Moreover, there was a significant decrease (p< 0.0001) in the
anti-BoNT C titres from the bivalent vaccine when compared to the multivalent vaccine.
This observation is common with vaccines containing two or more antigens due to the
antigenic competition between the antigens [
24
]. This phenomenon has been reported for
BoNT C and D toxoids vaccines; thus, these antigens must be carefully proportioned to
ensure a sufficient immune response to each component [
22
]. Additionally, BoNT D is more
immunogenic when compared to BoNT C, thus eliciting higher antibody titres, as reported
in several studies [
22
,
23
,
36
]. Interestingly, this variation was not observed with the vaccines
containing nanoalum, as the BoNT antigens achieved the same levels of toxin-neutralising
antibody titres. Comparable anti-BoNT D titres have been achieved with a recombinant
bivalent botulism vaccine, while the immunogenicity of BoNT C remained inferior [
9
].
Moreover, the potency demonstrated by nanoalum in the bivalent vaccine was maintained
in the multivalent vaccine. Although experimental models do not always accurately mimic
the immune responses in target animals [
37
], the nanoalum vaccines used in this study
have the potential to meet or exceed the minimum required protective immunity in target
animals. Nevertheless, further experiments in field animals are required to evaluate the
extent to which the nanoalum-containing vaccines induce an immunological response.
In addition to a stronger IgG immune response, nanoalum has also demonstrated
the ability to augment a Th1 immune response in ways that micron-sized aluminium
Vaccines 2023,11, 1473 10 of 12
hydroxide could not [
15
,
38
]. The findings reported here also highlight the improved
antigen delivery offered by nanoalum. This study further reports that nanoalum could be
an ideal alternative, where the antigen concentrations exceed the absorption capacity of
the standard alum-based adjuvants, such as in multicomponent vaccines [
24
]. However,
the potency of nanoalum still requires critical evaluation. A study conducted previously
reported that the enhanced immune response in a nanoalum-adjuvanted vaccine was
associated with overactivation of the innate immune response as well as local and systemic
reactogenicity [
39
]. Nevertheless, the reactogenicity may be addressed via dose reductions.
The success of nanoalum in the potentiation of an immune response offers an added
advantage of dose reduction.
5. Conclusions
In this study, we converted conventional alhydrogel to nanoalum and evaluated it
as an efficient adjuvant to improve the immune response to botulism bivalent vaccines
as well as in a multicomponent vaccine. Nanoalum demonstrated superior adjuvating
ability when compared to alhydrogel and can be used to improve the immune response to
botulism vaccines. Additionally, the subjugation of antigenic competition demonstrated
by nanoalum in a multivalent vaccine could open a new avenue for vaccine development.
While experimental trials conducted in experimental animals provide some information
on the nanoalum, these same results cannot be guaranteed in target animals. Therefore,
further studies are required to evaluate the safety, potency, and duration of the immune
response conferred by nanoalum vaccines in livestock.
Author Contributions:
Conceptualization, L.T. and F.R.; methodology, L.T. and Z.M.; software,
Z.M.; formal analysis, Z.M. and L.T.; investigation, Z.M.; resources, L.T. and F.R.; data curation,
Z.M.; writing—original draft preparation, Z.M.; writing—review and editing, L.T., T.K. and F.R.;
supervision, and L.T., T.K. and F.R.; project administration, Z.M.; funding acquisition, F.R. All authors
have read and agreed to the published version of the manuscript.
Funding:
Ziphezinhle Mbhele would like to thank the Department of Science and Technology—National
Research Foundation (DST-NRF), South Africa, for the grant holder-linked Professional Development
Programme (PDP) with Grant UID 127779. Onderstepoort Biological Products funded the study.
Institutional Review Board Statement:
All animal experiments were carried out according to the
guidelines of the Onderstepoort Biological Products (OBP) Animal Ethics Committee (South African
Veterinary Council Facility Registration Number: FR1514054) and the Department of Agriculture,
Land Reform and Rural Development under Section 20 of the Animal Diseases Act (Act 35 of 1984),
Protocol reference number: 12/11/11(b)/1914(HP).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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