Calcium phosphate: a substitute for aluminum adjuvants?

Abstract

Introduction: Calcium phosphate was used as an adjuvant in France in diphtheria, tetanus, pertussis and poliomyelitis vaccines. It was later completely substituted by alum salts in the late 80’s, but it still remains as an approved adjuvant for the World Health Organization for human vaccination.
Area covered: Thus, calcium phosphate is now considered as one of the substances that could replace alum salts in vaccines. The aim of this paper is to draw a review of existing data on calcium phosphate as an adjuvant in order to bring out the strengths and weaknesses for its use on a large scale.
Expert commentary: Calcium phosphate is a compound naturally present in the organism, safe and already used in human vaccination. Beyond comparisons with the other adjuvants, calcium phosphate represents a good candidate to replace or to complete alum salts as a vaccine adjuvant.

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Calcium phosphate: a substitute for aluminum
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Jean-Daniel Masson, Michel Thibaudon, Laurent Bélec & Guillemette
Crépeaux
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Crépeaux (2016): Calcium phosphate: a substitute for aluminum adjuvants?, Expert Review of
Vaccines, DOI: 10.1080/14760584.2017.1244484
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REVIEW
Calcium phosphate: a substitute for aluminum adjuvants?
Jean-Daniel Masson
a
, Michel Thibaudon
b
, Laurent Bélec
c
and Guillemette Crépeaux
d,e
a
Association E3M (Entraide aux Malades de Myofasciite à Macrophages), Monprimblanc, France;
b
Pharmacien « Service des Allergènes », de
l’Institut Pasteur, Paris, France;
c
Laboratoire de Microbiologie, hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, &
Faculté de Médecine Paris Descartes, Université Paris Descartes, Sorbonne Paris Cité, Paris, France;
d
École nationale vétérinaire d’Alfort,
Maisons-Alfort, France;
e
Inserm U955 E10, Université Paris Est Créteil, Créteil, France
ABSTRACT
Introduction: Calcium phosphate was used as an adjuvant in France in diphtheria, tetanus, pertussis
and poliomyelitis vaccines. It was later completely substituted by alum salts in the late 80’s, but it still
remains as an approved adjuvant for the World Health Organization for human vaccination.
Area covered: Thus, calcium phosphate is now considered as one of the substances that could replace
alum salts in vaccines. The aim of this paper is to draw a review of existing data on calcium phosphate
as an adjuvant in order to bring out the strengths and weaknesses for its use on a large scale.
Expert commentary: Calcium phosphate is a compound naturally present in the organism, safe and
already used in human vaccination. Beyond comparisons with the other adjuvants, calcium phosphate
represents a good candidate to replace or to complete alum salts as a vaccine adjuvant.
ARTICLE HISTORY
Received 12 May 2016
Accepted 30 September
2016
Published online 25 October
2016
KEYWORDS
Vaccine; adjuvant; calcium
phosphate; safety; immune
response; aluminum salts
1. Introduction
Adjuvants used in vaccines can be defined as substances
added to antigens to enhance immunity response to these
antigens [1,2]. First pastorian vaccines, such as rabies vaccine,
were developed at the end of the nineteenth century without
adjuvants because they were designed from whole viruses or
bacteria. The notion of adjuvant was developed at the begin-
ning of the twentieth century when traditional technique
failed to produce an efficient immunity response in horses
with diphtheria and tetanus purified toxoids [3,4].
(Toxoid = toxin with intact immunogenic properties, but
whose toxicity has been destroyed by heat or formaldehyde for
example.)
Concomitantly, alum salts (aluminum hydroxide Al(OH)
3
and aluminum phosphate AlPO
4
) have shown their effective-
ness to produce a quality immune response with the same
toxoids as in horses. Indeed, the injection of alum potassium
(KAl(SO
4
)
2
) with diphtheria toxoid produced higher plasma
concentration of antitoxin than the same vaccine without
alum [5].
The substantially equivalent result in humans explains that
aluminum salts are now the most frequently used adjuvants
although other substances such as oil in water emulsion,
saponin, or polysaccharides have been developed too [4].
Adjuvants such as aluminum salts aim to improve the
vaccine antibody response in order to make them more effec-
tive [3]. However, one should recall controversies on these
adjuvants, notably the one dealing with hepatitis B and
human papillomavirus vaccine, causing autoimmune or neu-
rological diseases [6]. Among unusual reactions to vaccines
containing aluminum hydroxide, macrophagic myofasciitis is
an inflammatory lesion described in 1998 [7] and recognized
as a ‘distinctive histopathological entity that may be caused by
intramuscular injection of Al-containing vaccines’[8,9].
Diseases attributed to vaccines, quoted in this article, have in
common the presence of neurological disorders. The neuro-
toxicity of aluminum is now well recognized, and the possible
relationship between aluminum and chronic neurological dis-
eases such as Alzheimer’s disease has been the key issue of
studies for many years [4,10,11].
The total safety of aluminum adjuvant for human use is still
debated [12]. Many studies demonstrate the link between
vaccinal aluminum and different diseases, but the French
National Academy of Medicine has not admitted this relation-
ship yet [4,13].
Nevertheless, the question of vaccine safety is of significant
concern and associations of patients advocate, as a precaution
measure, the substitution of Al-adjuvant by calcium phos-
phate, which is another adjuvant already used in human
vaccination.
Calcium phosphate was initially developed by the Pasteur
Institute in hydroxyapatite form (Ca
10
(PO
4
)
6
(OH)
2
) although
the patent describes it as the tricalcium phosphate form (Ca
3
(PO
4
)
2
) (see Section 2.1.)[14]. This adjuvant was used in
diphtheria, tetanus, pertussis, and poliomyelitis vaccines
[15–17]. The preparation method of this compound is
detailed in Section 2.1. Although calcium phosphate was
later substituted by alum salts in the late 1980s, when the
industrial sectors of Pasteur Institute and Merieux Institute
merged, it still remains as an approved adjuvant for the
World Health Organization (WHO) for human vaccination.
CONTACT Guillemette Crépeaux guillemette.crepeaux@gmail.com Inserm U955 E10, Université Paris Est Créteil (UPEC), 8 rue du Général Sarrail, Créteil
94010, France
EXPERT REVIEW OF VACCINES, 2016
http://dx.doi.org/10.1080/14760584.2017.1244484
© 2016 Informa UK Limited, trading as Taylor & Francis Group

Thus, calcium phosphate is now considered as one of the
substances that can replace the alum salts in vaccines
[18,19]. The nonspecific use of calcium phosphate with any
antigen has already been proposed but was never fully
researched due to the success of the aluminum adju-
vants [20].
However, since 1984, Pasteur Institute has marketed
delayed-effect allergens adsorbed on calcium phosphate in
order to highlight the delay effect of calcium phosphate and
its immunogenic effect. As the use of allergens in specific
desensitization required an increasing number of injections
and since the only delayed-effect allergens were allergens on
aluminum hydroxides (alum-pyridine or aluminum hydroxide
in situ), Pasteur Institute then moved towards the only alter-
native to aluminum hydroxide: calcium phosphate [21].
Recent demands of associations of patients to use again
calcium phosphate instead of vaccinal aluminum have revived
studies on this molecule, but very few data are available in the
scientific literature. Opinions differ about efficiency and inno-
cuity of calcium phosphate adjuvants. In this context, we
intend to draw a review of existing data on calcium phosphate
as an adjuvant in order to bring out the strengths and weak-
nesses for its use on a large scale.
2. Adsorption capacity and adjuvanticity
In the lights of the currently available scientific literature, we
should clearly define the concepts of adsorption capacity and
antigenicity, which sometimes seem confused. We will there-
fore consider that adsorption capacity is the adjuvant capacity
to adsorb a certain amount of antigen per unit of volume.
Antigenicity is the capacity of an adjuvant to induce an ade-
quate immune response to vaccination in terms of antibody
blood concentration or title. In other words, its capacity to
present an antigen in an efficient manner to induce the opti-
mal production of antibodies by the organism according to
the initial antigen dose injected. This point was also funda-
mental in the choice of calcium phosphate as adjuvant
adsorbed with allergens for specific desensitization [21].
2.1. Preparation and quality of calcium phosphate
Two preparation methods are possible to obtain vaccinal cal-
cium phosphate. The first is the in situ co-precipitation with
disodium hydrogen phosphate, calcium chloride, and antigen
(gel form). The second is the direct adsorption onto a preformed
gel (suspension form) [15,20,22–24]. The calcium phosphate gel
form is prepared by contacting an antigen with an aqueous gel
obtained through a reaction between an aqueous solution of
dibasic sodium phosphate (Na
2
HPO
4
) and an aqueous solution
of calcium chloride (CaCl
2
). The calcium chloride solution is
poured into the phosphate solution as rapidly as possible (within
3 min), in a proportion substantially equal to 1 mol of Na
2
HPO
4
for 1 mol of CaCl
2
, stirring constantly. The mixture thus obtained
is stirred on and on while its pH is adjusted to a value very near 7
with sodium hydroxide, and the gel formed is subsequently
decanted and washed. The gel is composed of calcium and
phosphate ions in proportions such that the ratio Ca/P is 1.62–
1.85 and exhibits a settling rate of 1–20 mm in 10 min at 20°C
when containing 0.07 atoms Ca per liter [14,17,22–24].
Calcium phosphate can be found in many forms presenting
different Ca/P ratios (Table 1). The theoretical molecular composi-
tion of calcium phosphate used in vaccination is Ca
3
(PO
4
)
2
[25],
but in the filed patent, the ratio Ca/P is from 1.62 to 1.85, which
approximates the ratio of hydroxyapatite (Ca
10
(PO
4
)
6
(OH)
2
)[14].
Although the ratio Ca/P is not sufficient to define a compound, it
seems that the calcium phosphate adjuvant is not chemical trical-
cium phosphate (Ca
3
(PO
4
)
2
) as the name implies but hydroxyapa-
tite (hereafter referred to as calcium phosphate) under different
forms with different Ca/P ratios including the calcium-deficient
(non-stochiometric) hydroxyapatite (Ca
9
(HPO
4
)(PO
4
)
5
(OH)) that
have the same ratio Ca/P as tricalcium phosphate [26]. The pre-
sence of a hydroxyl group is important as compounds containing
metallic hydroxyls exhibit a pH-dependent surface charge. In
addition, adsorption of phosphorylated antigens by ligand
exchange with surface hydroxyls may also occur, thus impacting
on the adsorption capacity of the adjuvant [26–28].
The quality of the calcium phosphate gel depends on the
concentration of the reactants and the rate at which the
reactants are mixed. The best adjuvant quality was obtained
in equimolar solution with a quick mixing (shorter than 3 min)
[17]. Indeed, in the patent, the author observed that the very
rapid addition of the calcium salt leads to a calcium phosphate
compound that differs from brushite (CaHPO
4●
2H
2
O) and
other forms [14]. Slow mixing of the reactants (longer than
3 min) results in a gel with a calcium-to-phosphorus ratio of
1.35–1.55, whereas quick mixing of the reactants (10 s) gives a
calcium-to-phosphorus ratio of 1.83 (Table 2)[14,17].
The mixing rate of reactants also affects the physical char-
acteristics and adsorption of antigens onto the gel. Calcium
phosphate gel prepared by rapid pouring of calcium chloride
into disodium hydrogen phosphate and mixing within 10 s
results in a gel with a lower pH and that sediments more
slowly than the calcium phosphate gel made by slow mixing
Table 1. Presentation of calcium phosphate compositions (formula, name, and
Ca/P ratio).
Ca/P ratio Formula Name
0.5 Ca(H
2
PO
4
)
2●
H
2
O Monocalcium phosphate monohydrate
1.0 CaHPO
4●
2H
2
O Hydrated calcium phosphate/brushite
1.0 CaHPO
4
Anhydrous calcium phosphate/monetite
1.33 Ca
8
H
2
(PO
4
)
6●
5H
2
O Octacalcium phosphate
1.5 Ca
3
(PO
4
)
2
Tricalcium phosphate/whitlockite
1.5 Ca
9
(HPO
4
)(PO
4
)
5
(OH) Calcium-deficient hydroxyapatite
1.67 Ca
10
(PO
4
)
6
(OH)
2
Hydroxyapatite
2.0 CaO
●
Ca
3
(PO
4
)
2
Tetracalcium phosphate/hilgenstockite
Table 2. Effect of reactant mixing time (equal volumes of 0.07 M disodium
hydrogen phosphate and 0.07 M calcium chloride) on the chemical composition
of calcium phosphate gel.
Concentration (mg/mL) in
Reaction time
a
Reactants Gel
Ca P Ca P Ca/P ratio
10 s 1.41 1.07 1.38 0.76 1.83
10 min 1.47 1.15 1.40 1.03 1.35
20 min 1.43 1.12 1.39 0.92 1.51
30 min 1.44 1.07 1.39 0.90 1.55
a
Time taken to pour calcium chloride into disodium hydrogen phosphate.
2J.-D. MASSON ET AL.

of reactants (≥10 min) (Table 3). Calcium phosphate adjuvant
precipitated by rapid mixing adsorbed 100% of diphtheria
toxoid, while the adjuvant produced by slow mixing only
adsorbed 58% of the same dose of diphtheria toxoid
[14,17,26]. Some authors suggested a method of preparation
with established conditions to produce calcium phosphate gel
with constant properties [20].
2.2. Adsorption capacity
The calcium phosphate adsorption capacity is dependent on
many factors and involves several phenomena such as hydro-
gen bonds or electrostatic attractions [26,29]. pH plays a major
role in the determination of this capacity although it should
be close to a physiological value for human vaccination [17].
The point of zero charge (pH at which the surface charge of a
molecule is nil) is 5.5 for calcium phosphate, 5 for aluminum
phosphate, and 11.1 for aluminum hydroxide. The net surface
charge or zeta potential is positive when the pH is below the
point of zero charge or isoelectric point. Consequently, at
physiological pH, commercial calcium phosphate and alumi-
num phosphate adjuvants exhibit a negative zeta potential,
but aluminum hydroxide exhibits a positive zeta potential
[26,30,31]. An isoelectric point also characterizes antigens. As
a general guideline, for many antigens, adsorption is best
accomplished in the pH interval between the isoelectric
point of the antigen and the point of zero charge of the
adjuvant [32]. The pH-dependent zeta potential suggests
that calcium phosphate adjuvant can adsorb positively
charged antigens at physiological pH by electrostatic attrac-
tion. The negative zeta potential of calcium phosphate
explains the effective electrostatic adsorption of lysozyme.
Indeed, lysozyme has an isoelectric point of 11 and has a
positive surface charge at physiological pH [26]. In the same
way, this explains why aluminum hydroxide is more efficient
to adsorb diphtheria and tetanus toxoid having a respective
isoelectric point at 4.1 and 5 [33,34].
However, zeta potential of calcium phosphate cannot
explain the good adsorption of casein, which is also negatively
charged at pH 7.4. Another bonding form between calcium
phosphate and the antigen is possible in addition to electro-
static attractions. The hydroxyl group in commercial calcium
phosphate adjuvant makes it a candidate to strongly adsorb
phosphorylated antigens as the adsorption of phosphate
anion by hydroxylated mineral surfaces occurs by ligand
exchange or hydrophobic attractive forces [28]. Previous stu-
dies on aluminum hydroxide adjuvant have demonstrated that
phosphorylated proteins and antigens are adsorbed by ligand
exchange too [35,36]. Adsorption capacity is greatly impacted
by the presence of hydroxyl group on adjuvants. Indeed, it
enables the adsorption of antigen with the same surface
charge as the adjuvant. Moreover, this adsorption is strong,
and it is not affected by pH variation [26].
The last factor influencing the adsorption capacity is the
specific surface area. This parameter takes into account the
shape and the size of adjuvant particles in order to determine
the real surface in contrast with the apparent surface. Calcium
phosphate, as aluminum hydroxide, can be found in particle
form. But contrary to aluminum hydroxide, calcium phos-
phate, which is a main component of bone or teeth, is a
biodegradable and biocompatible material. In fact, calcium
phosphate has been shown to be degraded by macrophages
[37]. As the particle size decreases, the specific surface area
increases. For example, two tricalcium phosphate powders
used as cement have been characterized. The first one has a
median particle size of 10.88 µm and a specific surface area of
0.54 m
2
/g, while the second one has a median particle size of
2.22 µm and a specific surface area of 2.73 m
2
/g [38].
Nanoparticles are therefore the form that presents the highest
specific surface area due to its very small size. Since the
adsorption capacity increases like the specific surface area,
hydroxyapatite nanoparticles are the calcium phosphate
form with the best adsorption capacity [39].
Due to its zeta potential, the presence of a hydroxyl group,
and its specific surface area, calcium phosphate presents an
adsorption capacity equal or higher than aluminum hydroxide
for ovalbumin and various fractions of snake venom [40,41]. To
conclude, calcium phosphate can have a good adsorption
capacity of positively charged and phosphorylated antigens,
and it is a biodegradable and biocompatible adjuvant even at
a nanoscale.
2.3. Antigenicity
The calcium phosphates antigenicity is a topic raising many
questions, and it must be highlighted that properties of cal-
cium phosphate required for the antigenicity have not been
fully characterized yet. Previous studies showed diverging and
random results depending on the biological model, the used
antigen, and the form (suspension or gel; see Section 2.1.)of
the preparation. It seems that the adjuvant adsorption capa-
city does not necessarily determine its antigenicity. Indeed, for
a comparable adsorption rate in antigen of snake venom,
calcium phosphate gel presents a greater antigenicity than
aluminum hydroxide in mice, so antigenicity does not corre-
late only with adsorption capacity [41]. Suspension or gel form
of calcium phosphate also would have a different antigenicity,
and gel form seems to have a higher antigenicity [41,42]. This
antigenicity difference could come from the different proper-
ties of the compounds. Indeed, it seems likely that gel type
and suspension type are not caught in the same way by
phagocytes which are the source of immunogenicity [42].
Moreover, this different way taken by the suspension form
could cause troubles because if calcium phosphate suspension
Table 3. Effect of reactant mixing time (equal volumes of 0.07 M disodium
hydrogen phosphate and 0.07 M calcium chloride) on the physical character-
istics of calcium phosphate gel.
Height
b
(mm) of clear solution in
Reaction
time
a
pH of
gel
mL/L of NaOH
to bring the
pH to 6.85 5 min 10 min 20 min 75 min 17 h
10 s 5.7 14.8 2.5 3.8 6.3 21.2 85.0
10 min 6.5 1.2 48.0 82.0 92.0 100.0 105.0
20 min 6.0 7.0 48.0 83.0 91.0 95.0 105.0
30 min 6.1 5.2 57.0 89.0 95.0 105.0 109.0
a
Time taken to pour calcium chloride into disodium hydrogen phosphate.
b
Height of clear liquid at top of gel in 125-mm-long tube containing 50 mL of
gel, held at 20°C.
EXPERT REVIEW OF VACCINES 3

replaces aluminum hydroxide in vaccine formulations at the
same concentration, then the potency test might show that
some lots of vaccine would fail to pass the test because of
small lot-to-lot variations [33].
Recent studies have shown that the physicochemical prop-
erties of particles such as the size, shape, surface charge, and
surface area are important parameters that influence the
adsorption capacity, antigenicity, and innocuity (for advanced
information, see [43]) [26,38]. Generally, particulates with a
comparable size to pathogens such as virus are easily recog-
nized and destroyed by dendritic cells or macrophages [44].
Small particles (20–200 nm) are preferentially caught via endo-
cytosis by dendritic cells, and larger particles (<5 µm) are
phagocytosed by macrophages, leading to efficient coadmi-
nistered antigen presentation and subsequent adaptive
immune response [45]. Comparison of six sizes of calcium
phosphate particles from 40 nm to 5 µm in mice highlighted
that particles of size from 100 to 400 nm present the greater
antigenicity. However, we should be careful with the trials of
size because it seems that an optimal size range exists for each
particulate adjuvant [37]. Commercial calcium phosphate pre-
sents a mean particle size distribution of 9.2 µm to 905 nm
[46]. Antigenicity is also impacted by the vaccination schedule
and booster injection (an extra amount of vaccine that is later
injected to maintain an adequate level of antibody). Indeed, in
humans, if antibody production to diphtheria and tetanus
toxoids measured after the first injection is more important
with aluminum hydroxide than with tricalcium phosphate, the
final antibody production measured after the booster is more
important with tricalcium phosphate [24,47].
As suggested by their successful uses in the past, the
antigenicity of calcium phosphate-based vaccines may be
compatible with human vaccination. The use of allergens
adsorbed on calcium phosphate during desensitization treat-
ment showed excellent immunogenicity [48]. In humans, the
calcium phosphate antigenicity calculated by serum antibody
concentration is important for diphtheria, tetanus, poliomyeli-
tis, pertussis vaccines, and five associations of these various
vaccines. Moreover, post-vaccine reaction rates are low and
often benign including allergic subjects to the vaccine anti-
gens [49–51].
Without attempting to compare the antigenicity of calcium
phosphate with alum salts, these studies demonstrate the
effectiveness of calcium phosphate as adjuvant for mentioned
vaccines. Calcium phosphate is therefore likely to be reused in
human vaccination without discrimination of individuals aller-
gic to toxoids.
Indeed, anti-polio vaccine based on calcium phosphate
allows sustainable vaccination of total polio-unimmunized
persons [50]. Furthermore, associations of antigens diphtheria,
tetanus, pertussis, and polio, possible with calcium phosphate,
have allowed the simplification of the vaccination schedule
[49]. Rare and mild local reactions make that kind of vaccines
well tolerated, including for persons previously allergic to
antigens, for which vaccination was until contraindicated
[50,51]. The generally lower antigenicity of calcium phosphate
could be actually a reflection of its lower capacity adsorption
than aluminum adjuvants with certain antigens and in certain
preparation conditions [52]. Despite its lower antigenicity than
alum salts, calcium phosphate has demonstrated its effective-
ness making it usable in vaccination [31,53].
On the other hand, it seems obvious for some authors that
the improved vaccine safety comes at the expense of immu-
nogenicity resulting from a compromise between antigenicity
and safety [54,55]. The inflammatory or danger-signal model
of adjuvant action implies that increased vaccine reactogeni-
city is the inevitable price for improved antigenicity. Hence,
adjuvant reactogenicity may be avoidable only if it is possible
to separate inflammation from adjuvant action [56]. This is an
encouraging observation for the nanoparticular calcium phos-
phate because inflammasome activation could not be
required for its antigenicity [37]. Other factors such as the
age of the vaccinated organism could play a role in vaccine
and adjuvant effectiveness [57]. Similarly, data obtained using
animal model may not always be reported to humans, as
reflected in the saying that ‘mice lie’[56]. Human trials
would be more appropriate to provide information specifically
about the ability of calcium phosphate to induce an appro-
priate immune response.
Under well-defined conditions, highly purified toxoids
adsorbed on calcium phosphate gel produce a highly immu-
nogenic vaccine to induce great immune response [1,20]. A
study onto various allergens adsorbed on calcium phosphate
(dust mites, pollens, and cat hairs) in both adults and children
allowed a very good response at the level of desensitization
and especially a lack of secondary reactions. Indeed, allergens
adsorbed on calcium phosphate were considered safe and
effective [21].
To conclude, antigenicity of calcium phosphate is size
dependent, and the optimal size range would be 100–
400 nm. The preparation form affects antigenicity, and the
gel form should be preferentially used. Finally, beyond the
comparison with alum salts, calcium phosphate has demon-
strated its effectiveness for use in human vaccination although
some additional analyses are needed.
3. Antigenicity, immunoglobulins, and antibody
3.1. Immunoglobulins and antibody
Immunoglobulin (Ig) and antitoxin production as antigenicity
depends on many factors such as antigen, way of preparation,
and particle size. Thus, tetanus toxoid adsorbed on calcium
phosphate or aluminum hydroxide used in Guinea pigs
showed that calcium phosphate is less efficient.
Indeed, the production of IgG 1a and 1b is lower with
calcium phosphate compared to aluminum hydroxide [58].
However, the same tetanus toxoids or diphtheria toxoids
adsorbed on tricalcium phosphate at higher concentration
induce in both animals and humans similar or greater IgG
production than what is induced by aluminum adjuvants,
especially after the booster [29,47,59]. The use of different
forms of calcium phosphate (hydroxyapatite, moneite, and
tricalcium phosphate) at different doses makes complex the
comparison of the results of these studies. It is difficult to say if
the concentration or the type of compound is the most
impacting on the antibody response, but both parameters
must have an impact on this factor. As a natural constituent
4J.-D. MASSON ET AL.

of the human organism [15], calcium phosphate provides a
slow release of adsorbed antigens at the injection site. This
slow release induced high antibody production with calcium
phosphate gel [41]. In humans, the concentration of anti-
tetanus antibodies is equal after calcium phosphate or alumi-
num vaccine injection. On the other hand, 3.5 months after
the booster, antibody concentration is much greater in people
who have been vaccinated with calcium phosphate vaccines.
Furthermore, the number of not reacting subjects after two
injections is lower with calcium phosphate-based vaccines
[24]. It was also possible to make successful multi-antigen
vaccines on calcium phosphate (diphtheria, tetanus, polio,
Bacille Calmette Guérin (BCG), yellow fever, and measles) as
well as simultaneous injections, including a hepatitis B vaccine
[17,60].
In addition, with the adjuvant concentration, animal model,
antigen used, and others, an important factor can also operate
to explain the difference of antibody production between
studies, the particle size. As already mentioned in Section
2.3, small particles (20–200 nm) are preferentially caught via
endocytosis by dendritic cells, and larger particles (<5 µm) are
phagocytosed by macrophages, leading to efficient coadmi-
nistered antigen presentation and subsequent adaptive
immune response [45]. This size-dependent antigenicity has
been shown by various particulates. Some adjuvants have a
nanometric optimal size, and others have a micrometric opti-
mal size, so it seems that an optimal size range exists for each
particulate adjuvant. Calcium phosphate particles of 100–
400 nm present the greater antibody production and the
most virus neutralization for influenza in mice [37].
Although animal model results are divergent depending on
antigen and adjuvant concentrations, human data suggest
that calcium phosphate is an effective adjuvant and poten-
tially more effective than alum after the booster. It has a
reasonable capacity to adsorb antigens, induces high levels
of IgG antibodies, and does not increase IgE production
[15,17,61].
3.2. Immunoglobulin E
Vaccine adjuvants generally cause the preferential synthesis of
one or more antigen group [62]. Calcium phosphate gel has a
major advantage because it does not induce an increase of the
rate of IgE during its use in booster in both human and
animals, contrary to aluminum hydroxide [58,63,64].
The extended production of IgE could cause allergy in case
of regular injections [58]. Calcium phosphate induces a very
low IgE production probably because it is a natural constituent
of the body [15]. The use of a higher concentration of calcium
phosphate may also lead to the IgE production, but only at
concentrations higher than those used for human vaccina-
tion [65].
Calcium phosphate formulation also plays a role in the IgE
response. Used in humans or in mice, monetite (CaHPO
4
)or
tricalcium phosphate may induce low IgE levels like aluminum
hydroxide in function of adsorbed antigen, while this effect is
not found in Guinea pigs [47,59]. Tricalcium phosphate is
defined by the authors as more effective but does not
represent a reliable alternative to aluminum hydroxide in
terms of their comparable IgE production in study conditions
[47,58].
In any event, the presence of specific IgE antibodies in
human serum must be regarded as a potential negative effect
which can appear also after booster immunization, especially
for hyperimmunized and allergic persons [58]. Human IgE
production is conditioned by a hereditary factor; however,
many authors associate the increasing frequency of allergic
diseases with the intensive mass immunization programs with-
out regard for the possible mechanisms [66,67].
Indeed, the IgE presence in the body could stimulate an IgE
response to other immunogens which come into contact with
the immune system of the body at the same time. This might
result in the development of an IgE response to different
environmental antigens and in some cases lead to clinically
manifested reactions of hypersensitivity [58], while the IgE
production by aluminum vaccine has been known for a long
time [68–71].
4. Innocuity
Like most adjuvants, calcium phosphate may cause small
under-skin nodules, which can be palpable for 30 days after
injections. These nodules then tend to be resorbed leaving a
final scarry lesion with clumps of calcium deposit surrounded
by a thin and mostly acellular fibrous layer [72–74]. However,
the under-skin nodules may be avoided with deeper intramus-
cular injection [50].
It should be noted that calcium phosphate is a compound
classified as safe and biocompatible by the FDA [39,75], due to
its natural occurrence in the organism. While having similar
properties with alum salts, calcium phosphate has the advan-
tage that it is a natural compound to the human organism and
may therefore be exceptionally well tolerated. Calcium phos-
phate particles are biodegradable material caught via endocy-
tosis by macrophage or dendritic cells and degraded in
lysosome [37,76]. Products of biodegradation (Ca
2+
and
PO
43−
) are also physiologically present in blood in relatively
high concentrations [39,77,78]. Nonbiodegradable particles
such as aluminum hydroxide, widely used for human vaccines,
can be retained for a long time, raising the question of bio-
persistence and biotranslocation, thus inducing potential toxi-
city (for review, see Gherardi et al. [79]). Thus, biodegradable
particles are supposed to be less toxic than nonbiodegradable
particles [80]. The excellent biocompatibility of calcium phos-
phate makes it a biomaterial used in orthopedics and dental
prostheses [75].
4.1. Inflammatory reaction
The route of administration of vaccines, particularly those
adsorbed onto mineral adjuvants such as calcium phosphates
or alum salts, plays an important part to avoid adverse local
reactions [81]. In subcutaneous injections, calcium phosphate
induced active inflammation involving neutrophils and macro-
phages [42]. It should be noted that the subcutaneous way is
now replaced by the intramuscular way because
EXPERT REVIEW OF VACCINES 5

subcutaneous injections induce nodules which are not
observed when the vaccine is administered intramuscularly
[50,81]. Used in Guinea pigs, intramuscular injections of cal-
cium phosphate suspension cause histopathological effects
similar to those of aluminum hydroxide [42]. However, the
gel form causes more moderate reactions compared to the
suspension form or to aluminum adjuvants [42,58]. It seems
likely that gel form is not easily caught by phagocytes. This
may explain the fact that the inflammation due to the suspen-
sion form is severer than that induced by the gel form.
The calcium phosphate gel inflammatory reaction does not
last more than 4 weeks, contrary to the one induced by
aluminum adjuvants that can last twice as long [42]. Some
local reactions following vaccination, such as severe pain,
while not directly damaging to physical health, may still
have a strong negative impact on the public’s perception of
the risk–benefits of immunization and thus should be lim-
ited [56].
The shorter inflammatory reaction observed with calcium
phosphate gel may be explained by an easier dissemination of
calcium phosphate that is a natural constituent of the human
body [15]. Unlike aluminum adjuvants, calcium phosphate is
not toxic in vitro for macrophages at the concentration range
used in vaccination [42,82].
This is an important point to consider calcium phosphate as
an adjuvant for human vaccination and for specific immunity
because the role of macrophages is recognized in the induc-
tion of immune response [62]. Moreover, the inflammatory
reaction remains short even at high concentration of calcium
phosphate [42]. This makes the use of calcium phosphate
vaccine possible, even in case of overconcentration, a condi-
tion potentially required depending on the antigen used.
4.2. Antigen–adjuvant interaction and innocuity
Histological reactions are not equal in the presence of single
adjuvant, single antigen, or combinations of both. Predicting
the vaccine local reaction seems complicated as antigen
adsorbed on calcium phosphate influences the overall safety
of the vaccine.
For instance, in rabbits, when considered individually, cal-
cium phosphate as in tetanus toxoid injection cause a moder-
ate reaction with respective presence of a granuloma or
vascular inflammation, which can cause small bruises.
However, the combination of two molecules may induce an
intense granulocytic reaction from the sixth hour accompa-
nied by cytolysis of polynuclear leukocytes in the twenty-
fourth hour.
The association of three antigens on calcium phosphate
caused a relatively similar reaction to tetanus toxoid adsorbed
alone on calcium phosphate in rabbits, but the addition of
pertussis antigen caused a granulocytes influx to the third day,
which may lead to a lesion similar to acute fibrinous inflam-
mation [73].
Moreover, the vaccine preparation quality is crucial to
ensure safety. Indeed, some of the local vaccine effects may
directly reflect chemical irritation due to a non-physiological
pH or osmolarity and salt concentrations toxic for cells [56].
4.3. Hemolytic activity
Calcium phosphate gel shows high hemolytic activity in
human, mouse, sheep, and rabbit blood samples taken after
vaccination. Indeed, analyses show a high rate of free hemo-
globin, indicating the erythrocytes lysis in vitro. This activity is
reduced by 50% when the adjuvant is used in suspension
form [82].
The hemolytic effect is dose dependent and remains equal
to the one induced by aluminum compounds at concentration
less than 1.3 mg/mL of calcium, leading to the establishment
of a superior limit of 1.3 mg of calcium phosphate per vaccine
dose by the WHO and the European Pharmacopoeia
[26,39,40,59].
If calcium phosphate concentration is higher than 1.3 mg,
the hemolytic effect of calcium phosphate becomes higher
than that produced by aluminum hydroxide probably because
this concentration might disrupt the delicate chemical balance
of the organism. However, it is possible to greatly reduce the
calcium phosphate hemolytic effect by the addition of phos-
phate ions to the solution. This addition also decreases the
adsorption capacity of the adjuvants [40,83]. However, adjust-
ments of adjuvant and phosphate ions concentrations allow to
obtain aluminum hydroxide and calcium phosphate solutions
with the same adsorption capabilities and hemolytic
effect [40].
It seems important to point out that these results come of
in vitro experiments and that they are not unanimously
accepted. Indeed, calcium phosphate, while exhibiting a
proved hemolytic activity, is not designed, as an adjuvant, to
be injected into vascular vessels but intramuscularly or sub-
cutaneously injected, thus leading to consider that kind of
data as invalid from a clinical point of view [84]. It therefore
seems important to respect strictly intramuscular injection
mode to avoid hemolytic reaction at the injection site [84].
This example perfectly illustrates the fact that available safety
data on a vaccine are strictly specific to the vaccine and its
way of administration [19].
4.4. Nano-particular form and innocuity
Recent studies present data on the safety of calcium phos-
phate nanoparticles. Keeping in mind that this specific form
does not probably have the same characteristics as micropar-
ticles, we wanted to add recent data to the present review.
Among adjuvant developments, many researches now focus
on nanoparticle compounds. Indeed, their small size provides a
highsurfaceareatovolumeratios[39]. Apart from its excellent
storage stability, cold preparation, and relatively inexpensive
development, calcium phosphate nanoparticles have positive
characteristics in all areas studied [85,86].
In mice, calcium phosphate nanoparticles with herpes or
Epstein–Barr viruses induce a sustainable total IgG, IgG2a, and
IgA production without producing IgE. No inflammation evi-
dence at the injection sites and no sign of toxicity have been
shown in this study [85–87]. In studies on cultured fish, bird,
and human cells, this nanoparticle adjuvant was readily
absorbed and dissolved in lysosomes prior to being evacuated
out of the cells [86,88–90].
6J.-D. MASSON ET AL.

It was suggested to develop a single-dose vaccine with
sustained-release capabilities [85]. This form is effective for
vaccination through mucosal way (intranasaly and intravagin-
ally), allowing systemic vaccination [85,87].
Alum salts selectively stimulate humoral immune
responses, especially type-2 helper (Th2) immune responses,
which are characterized by the production of interleukin (IL)-4
and IL-5 and the induction of IgE and IgG1 [91–93]. Calcium
phosphate nanoparticles exist as rod shaped and sphere
shaped. Rod-shaped one can induce both cellular immune
response (development of T lymphocytes) by type-1 helper
(Th1) way and humoral immune response by Th2 way
[37,94,95]. Microparticle-sized adjuvants may not be able to
induce the same response due to a greater size [87,96–99].
Nanoparticle adjuvants also allow the optimization of a selec-
tive cell response for the antigen release [39,100].
However, in mice, a comparison of two tetanus vaccines
based on calcium or aluminum phosphate nanoparticle allowed
to highlight the presence of histological lesions in brain, kidney,
liver, and injected muscle. These lesions included hepatocellular
necrosis infiltrated by mononuclear cells, hydropic degenera-
tion, and appearance of Küppfer cells. They are generally less
acute with the calcium phosphate except for muscle damage
showing signs of hemorrhage [101].
The nanoparticle compounds are currently too new to
ensure a sufficient view on their safety. These compounds,
unlike the microparticle form, have higher permeability
through cell membranes and thus influence the physiology
of most cells and translocation to other distant organs
[101,102]. This high membrane permeability requires further
studies to bring out the advantages and disadvantages for
vaccination [101,103,104].
4.5. Old studies, in situ data
The interest for calcium phosphate greatly decreased after
discontinuation of its use in the 1980s, and thus, research on
this molecule was progressively reduced. At present, there are
only a few studies on the safety of calcium phosphate micro-
particles used as adjuvant, and the most recent was published
at the end of the 1990s [42]. To the best of our knowledge,
calcium phosphate does not seem to show evidence of any
activity likely to cause allergic problems or reactions, making it
incompatible with its use for human or animal vaccination. On
the contrary, calcium phosphate seems to be very well toler-
ated [17,25,42,51,58,105,106].
Furthermore, calcium phosphate was used in France until
the mid-1980s, mainly for the diphtheria–pertussis–tetanus
vaccine group, without any mention of adverse reactions by
physicians. It has been successfully used in the pentavalent
human vaccination (smallpox, yellow fever, measles, BCG, and
tetanus), without any reported adverse reaction [60].
According to several authors, calcium phosphate is a nat-
ural constituent of the human organism, thus providing a
greater guarantee for the absence of adverse effects, and
may be considered as safer than aluminum hydroxide [107].
Other authors recognize that an ascertained safety profile
makes it interesting for the evaluation of new vaccines, but
keeping in mind that as with adjuvanticity, studies conducted
on animals to test the safety of vaccines and of their adjuvants
do not guarantee that they are a perfect reflection of the
human situation and would thus require new studies [31,56].
5. Perspectives
Within the scientific literature on the calcium phosphate as a
vaccine adjuvant, some publications highlight several perspec-
tives for its use, potentially deserving further investigations.
In the 1990s, calcium phosphate was studied for the devel-
opment of an anti-HIV vaccine in rabbits [108]. Thanks to the
adsorption of the HIV-1 gp160 antigen on calcium phosphate
gel [25], the final vaccine allowed a good immune response in
rabbits. Although not leading to the production of anti-HIV
vaccine in humans, these studies highlighted the calcium
phosphate capabilities as adjuvant for the development of
future promising vaccines [108,109].
Calcium phosphate has been the subject of recent research
for the development of new vaccines. Another study shows the
possible use of calcium phosphate in the hepatitis B DNA vac-
cine development on murine model [83]. This vaccine induces
protective humoral- (through antibodies) and cell-mediated
immune responses (through CD8 T lymphocytes) [83].
Calcium phosphate nanoparticles remain a development
requiring further research. Indeed, this new formulation may
present several advantages such as the adsorption capacity,
duration of the immune response, and induction of cellular
response or mucosal immunity, but their different adverse
effects are not fully characterized or understood [39,85,87,89].
Finally, in association with mesoporous silica, calcium phos-
phate has been studied to develop a cancer vaccine on mur-
ine model [110]. Cancer vaccines allow the activation of
immune cells, which specifically recognize and destroy tumor
cells without harming normal cells. The use of cancer vaccines
attempts to harness the specific power of the immune system
for cancer treatment. The crucial point for cancer vaccines is
the inclusion of an appropriate adjuvant [111]. In this role, the
combination of mesoporous silica with calcium phosphate or
Zn- and Mg-tricalcium phosphates allows a higher potent
systemic antitumor immunity, which inhibits the growth of
tumor both in vitro and in vivo [110,111].
In addition, since the end of the 1990s, the allergens
adsorbed on the Pasteur Institute calcium phosphate were
taken over by the Stallergènes laboratory and are still very
widely disseminated at the international level, such as in some
specific desensitization treatments by Stallergènes-Greer.
6. Expert commentary
Before considering a compound for use as an adjuvant in
human vaccines, it is necessary to consider several factors,
mainly to determine experimentally if the preparation is sui-
table and safe. These factors include safety by tests of acute
and chronic toxicity, local and general tolerance teratogeni-
city, biodegradability, carcinogenicity or co-carcinogenicity,
the degree of immunopotentiation, unacceptable or pharma-
cologic adverse effects, and the definition of action [62].
Calcium phosphate has successfully passed these various
tests including high efficiency in terms of concentration of
EXPERT REVIEW OF VACCINES 7

circulating antibody, which allowed its use in several human
vaccines for 20 years [1,4].
In the development of novel adjuvants for vaccination, several
tracks are promising but represent expensive and long-lasting
research. The immediate use of calcium phosphate is therefore
desirable because the information collected for human vaccina-
tion has demonstrated that its use is possible. The lack of data on
this molecule does not allow a strict conclusion about its safety.
However, the available human data suggest an adequate ability of
calciumphosphatetoproduceaneffectiveimmuneresponse
with diphtheria, tetanus, polio, and pertussis antigens in certain
defined conditions [25]. Calcium phosphate is a compound natu-
rally present in the organism, characterized as safe by the FDA. To
the best of our knowledge, no study has revealed adverse effects
when used under clinical conditions; thus, no reason seems to
justify the sidelining of this adjuvant in human vaccination for
cited antigens. This is confirmed by its use in specific desensitiza-
tion for more than 30 years.
To conclude, beyond the knowledge of differences between
calcium phosphate and aluminum salts, and comparisons to
determine if calcium phosphate is more or less efficient than
aluminum salts, we showed in this review that calcium phosphate
and specially the hydroxyapatite form represents a good candi-
date to replace or to complete alum salts as a vaccine adjuvant
(Table 4). The calcium phosphate use as nonspecific adjuvant for
other antigens should be subjected to further research, focusing
particularly on the response of the compound gel form that has
already been used in humans. This compound is present in the
International Pharmacopoeia and is part of the additives
approved by the WHO. It is also mentioned asa possible adjuvant
in the European Pharmacopoeia. In addition, the president of
French Technical Committee of Vaccinations admitted that cal-
cium phosphate is as effective as aluminum. Finally, the patent of
calcium phosphate is now in the public domain, making it acces-
sible for further utilization in different future vaccines.
7. Five-year view
Research on calcium phosphate should be resumed. In the
next 5 years, studies and clinical trials necessary for the
calcium phosphate reuse in human vaccination could be com-
pleted. This would offer a greater range of vaccines. This wider
range would enable patients to choose an adjuvant. Patients
who do not want to be vaccinated with aluminum adjuvants
would thus have access to immunization with calcium phos-
phate, thus ensuring a great vaccinal coverage.
Key issues
●Calcium phosphate was used as a vaccine adjuvant.
●It was replaced by aluminum salts at the end of 80’s.
●Aluminum salts raise safety questions.
●Calcium phosphate can be as effective as aluminum salts.
●It is a natural compound of the human body.
●Human data highlight the good tolerance of calcium
phosphate.
●The widespread use of calcium phosphate requires further
studies.
Acknowledgments
We are grateful to E & R Jandot for their careful revision and critical
comments on this paper.
Funding
This work has been carried out with E3M association with the financial
support of Mutuelle Familiale.
Declaration of interest
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
Documentary research methodology
The aim of this study has been to make an international state of the art
about the use of calcium phosphate as an adjuvant in vaccine. Exhaustive
documentary research has been conducted in order to establish a
Table 4. Summary of advantages and disadvantages of the use of calcium phosphate (CaP) as an adjuvant in human vaccines.
Advantages Disadvantages
●CaP was used as an adjuvant up to 1985; data are available to use it again in a
fast but not expensive way compared to the development of new adjuvants
●CaP is present in the general monographic on human vaccines of the
European Pharmacopoeia 8.0
●CaP is classified as safe and biocompatible by the US FDA
●CaP is a natural compound of the human body, thus suggesting its good
tolerance for individuals
●Adsorption capability is equivalent to aluminum adjuvants, depending on
preparation mode, considered antigens, and particle size
●Booster antigenicity is potentially better than with aluminum adjuvants
●CaP is used in allergic desensitization
●CaP does not induce immunoglobulin E production that may cause an allergic
reaction
●Inflammatory reaction is moderate and shorter with CaP than with aluminum
adjuvants
●CaP does not lead to macrophagic toxicity
●Good tolerance of CaP is highlighted by the lack of negative return during its
commercial use
●Several forms of preparations exist: suspension and gel form (the suspension
form does not appear suited for vaccinal use), micro- and nano-metric size,
some formulation (see Table 1), and sphere shape or rod shape
●Sensitivity of the quality of the gel form depends on the conditions of
preparation
●Global primo-injection antigenicity is weaker than with aluminum adjuvants
●Due to severe hemolytic effect with intravascular injection, intramuscular
injection is imperative
8J.-D. MASSON ET AL.

quantitative and qualitative usable data assessment. All published data
concerning advantages and disadvantages of the use of calcium phos-
phate have been sought. Points of View or recommendations on the
inclusion of the calcium phosphate in vaccines or more broadly on the
adjuvant safety have been identified. Documentary sources database, grey
literature, relevant internet sites as well as (French) regulatory information
have been sorted, assessed and interviewed in a systematic way. The
recommendations, meta-analysis and systematic reviews have been
sought in Medline from key words: (‘Adjuvants, Immunologic’[Mesh] OR
Adjuvant*) And (vaccine* Or immunol*)[title] then scientific publications
on the use of calcium phosphate as adjuvant from key words: ‘Calcium
phosphate’[Supplementary Concept] OR ‘Calcium Compounds’[Mesh] OR
‘Calcium Phosphates’[Mesh] OR calcium phosphate Or CaP Field: Title AND
(‘Adjuvants, Immunologic’[Mesh] or adjuvant* Field: Title). 160 references,
with various quality and relevance, have been obtained on the basis of
this research and a hundred of them have proved workable.
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