Liposomal vaccine formulations as prophylactic agents: design considerations for modern vaccines

Table 5 Candidiasis treatment with potential liposomal vaccines
Lipid(s) and sterol used Fungi or disease
Cell line/animal model 
used
Administration route Promising liposome 
formulation
References
PC/Chol
C. albicans and C. tropi-
calis
BALB/cByJ mice
Intravenous
PC/Chol
[
106
]
PC/Chol
BALB/c mice
Intravenous
PC/Chol
[
107
]
DMPC/DMPG
C. albicans
ICR mice
Subcutaneous
DMPC/DMPG
[
108
]
EPD/DOGS-NTA-Ni
BALB/c mice
Intradermal
EPD/DOGS-NTA-Ni
[
109
]
ICR mice
Intradermal
[
110
]
DDA:MO
Macrophages and BALB/c 
mice
Subcutaneous
DDA:MO
[
111
]
BALB/c mice
Subcutaneous
[
112
]

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De Serrano and Burkhart  J Nanobiotechnol (2017) 15:83 
Parasitic infections and liposomal vaccines
Parasitism and malaria
Parasitism is a non-mutual, biological interaction in 
which the parasite lives inside the host and derives its 
own nutrients at the host’s expense. Parasites can be 
classified as macroparasites (visible with the naked eye) 
like helminths, or microparasites (which are smaller) 
like viruses, bacteria or protozoa. A textbook exam-
ple of a parasite is Plasmodium vinckei, causative agent 
of malaria. In malaria, the parasite (Plasmodium sp.) is 
transmitted by mosquito bites. The parasite’s sporozoites 
reside in the liver (in humans), developing into merozo-
ites that infect human red blood cells, initiating the red 
blood cell cycle. When appropriate, the merozoites will 
developed into gametocytes that infect more red blood 
cells that are taken up by mosquitoes during the bites. 
This initiates the mosquito stages (gametes, ookinetes 
and oocysts). Oocysts are transmitted to the host, initiat-
ing the liver stage.
Postma et al. developed a novel desferrioxamine B 
(DFO) delivery system based on liposomes to treat 
malaria [
113
]. DFO is a siderophore that chelates ferric 
iron (iron is an vital component of red blood cells). Iron is 
an important nutrient for P. vinckei as it infects red blood 
cells. Contrasting to previous liposomal vaccine articles, 
the researchers investigated the lipid to drug composi-
tion and the bilayer fluidity effects on DFO delivery and 
protection from infection. The lipids were all anionic in 
charge due to the presence of egg PG. Three different 
treatments of DFO were analyzed in the study: (1) mul-
tiple free DFO subcutaneous injections, (2) intraperito-
neal infusion of free DFO and (3) multiple subcutaneous 
DFO-loaded liposomes injections in C57B1/6J female 
mice. Parasitemia was suppressed by multiple subcutane-
ous injections of free DFO before and during infection, 
but injections prior to infection did not. Suppression of 
parasitemia and long-term survival was observed for 
intraperitoneal infusion of free DFO 1 day before infec-
tion or by subcutaneous injections of liposomal DFO 
prior to infection (day-1). Bilayer rigidity was studied 
by incorporating Chol (intermediate rigidity) and DSPC 
(high rigidity) in the liposomes, demonstrating that no 
relationship exists that affect the liposome antimalarial 
function. However, drug-to-lipid ratio affected the anti-
malarial activity of the liposomal-based vaccine, suggest-
ing that low drug-to-lipid ratios are the best formulation 
parameter at combating the infection. When liposomal 
DFO was administered to mice, a long-term protection 
against malaria was observed (days 7 and 8, 400 mg/kg/
day). This article proves that utilizing a common sidero-
phore (DFO) as an iron chelator in combination with 
liposomes, improve the therapeutic and prophylactic 
effects of the vaccine. However, no immune response 
studies were performed, lacking essential information 
about the mechanisms of the vaccine when immuniza-
tion occurs.
A pre-clinical report of the malaria vaccine RTS,S was 
published by Stewart et al. [
114
]. The RTS,S/AS02A vac-
cine utilizes the circumsporozoite protein as antigen. 
Investigators in this report evaluated the effects of cer-
tain adjuvants (AS01B, AS02A, AS05 and AS06) which 
vary in the concentration of MPL, QS21 or CpG and 
their formulation delivery system (emulsion vs. formula-
tion). AS01B was the only liposomal formulation in the 
study. Rhesus macaques were immunized by intramuscu-
lar injection with the different RTS,S/adjuvant combina-
tions and specific antibodies, IFN-γ and IL-5 levels were 
determined after weeks 14 and 34. All regimes were safe 
and presented elevated antibody titers (except for AS06-
containing vaccine formulation). RTS,S/AS01B presented 
higher levels of IFN-γ at weeks 14 and 34, and the high-
est IFN-γ to IL-5 ratio when compared to RTS.S/AS02A. 
Leishmaniasis.
Another parasitic disease is leishmaniasis caused by 
the parasite Leishmania sp. It is transmitted by sand-
flies to mammals, transferring metacyclic promastigotes 
via feeding. The metacyclic promastigotes invade mac-
rophages and granulocytes, developing into amastigotes 
which multiply by simple division, eventually causing 
macrophage lysis. Further, amastigotes infect new mac-
rophages or are transferred to the sandflies during feed-
ing. Amastigotes transform into procyclic promastigotes 
in the gut, maturing into metacyclic promastigotes by 
simple division. The disease is common in certain regions 
of Asia, Africa, South and Central America and even 
southern Europe. The disease is divided into three major 
syndromes, cutaneous, mucosal or visceral leishmaniasis. 
Visceral leishmaniasis poses a major risk of death inci-
dence [
115
].
Three seminal articles provide valuable information 
regarding the studies and development efforts towards 
a prophylactic vaccine for leishmanial infections [
116
–
118
]. First, Bhowmick et al. presented the immunother-
apy effects of leishmanial antigens in liposomes [
116
]. 
The researchers correlated the efficacy of soluble leishma-
nial antigens (SLAs) from Leishmania donovani promas-
tigote membrane incorporated in neutral (lecithin:Chol), 
negative (lecithin:Chol:PA (phosphatidic acid)) and posi-
tively (lecithin:Chol:SA) charged liposomes intraperi-
toneally administered. L. donovani was eliminated from 
the liver and spleen when SLAs were present in cationic 
lipids. IL-4 and IL-10 were downregulated when SLA-
cationic liposomes were administered to the mice and 
the immunomodulatory response presented the T
H
1 
cytokines IFN-γ and IL-12. Subsequently, Banerjee et al. 
presented two articles which cover the study of cationic 

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Page 15 of 23
De Serrano and Burkhart  J Nanobiotechnol (2017) 15:83 
stearylamine liposomes for the development of a visceral 
leishmaniasis vaccine. In the first published article by 
the team of researchers, amphotericin B (AmB) is used 
in association with stearylamine (cationic) liposomes as 
a novel therapeutic approach [
117
]. When administered 
to BALBc mice, the leishmanial parasite was eliminated 
from the liver and spleen. Moreover, when comparing to 
the conventional liposomal formulation AmBisome, the 
intravenous administration of AmB-SA-PC liposomes 
induced the production of IFN-γ from CD8
+
and CD4
+
T cells. At the same time, the formulation reduced the 
toxicity effects of the drug by reducing TNF-α levels. In 
the splenic supernatant culture, IL-10 was downregu-
lated, causing the production of IL-12 and nitric oxide 
during the AmB-SA-PC liposomes treatment. Also, 
Banerjee et al. investigated the effects of liposome charge 
in an antileishmanial assay [
118
]. Researchers provided 
evidence of membrane disruption caused by the cati-
onic stearylamine liposomes in promastigotes and amas-
tigotes. No toxicity in murine peritoneal macrophages 
and human erythrocytes was detected. These studies 
confirmed the prophylactic effect of SA-PC liposomes 
against leishmanial infections.