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

Table 3 Promising vaccine formulations for the treatment of viral infections
Cell cultures and no vaccination in animal models were employed
N/A not applicable
Lipid(s) and sterol used
Virus type
Cell line/animal model 
used
Administration route Promising liposome 
formulation
References
DOTMA
Hepatitis C
HepG2 cells and BALB/c 
mice
Transcutaneous
DOTMA
[
85
]
DC-Chol
Hepatitis B
Lymph node cells, BALB/c, 
OF1 and B10.M mice
Subcutaneous
DC-Chol
[
84
]
DMPC, DMPC/DMPG, 
DC-Chol/DOPE, DSTAP/
Chol, DDA/Chol, DOTAP/
Chol, DMTAP/Chol and 
CCS/Chol
Influenza H3N2
Splenocytes from BALB/c 
and C57BL/6 mice
Intranasal
DMTAP/Chol and DOTAP/
Chol
[
45
]
DDA
Respiratory Syncytial Virus BALB/c mice
Intranasal
DDA
[
88
]
DOPC/OPPE (Influenza 
virosome) and DOPC/
OPPE (liposomes)
Influenza
MDMs, MDDCs, 16HBE14o 
cells, PHNECs and 
EPCam + cells
N/A
DOPC/OPPE (Influenza 
virosome)
[
86
]

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De Serrano and Burkhart  J Nanobiotechnol (2017) 15:83 
(−) bacteria also contain an important immunogenic 
molecule and pathogenicity factor, the lipopolysaccha-
ride (LPS) [
90
, 
91
]. LPS has been used to increase the 
fusogenicity of cationic liposomes [
92
]. In that study, 
researchers developed a carrier system to incorporate 
LPS into mammalian cell membranes via DOPE (1,2-dio-
leoyl-sn-glycero-3-phosphoethanolamine):DOTAP (1:1 
wt. ratio) liposomes. The team of researchers demon-
strated that high LPS concentrations on immortalized 
fibroblasts generated the activation of macrophages, 
starting the elimination of LPS-bearing cells.
Additionally, bacteria will have unique cell compo-
nents, like genetic material, lipids or proteins, that could 
serve as adjuvants or antigen markers for subunit vaccine 
development, depending on the molecule chemical char-
acteristics. Li et al. investigated the potential use of cati-
onic (DDA-based) mannosylated liposomes to deliver the 
model DNA plasmid pGL4.10 (encoding luc2) [
19
]. The 
plasmid was protected from nuclease degradation by the 
liposomes. The cationic mannosylated liposomes showed 
high uptake and transfection, activating bone marrow 
DCs (BMDCs). BMDCs activation was characterized 
by the upregulation of CD80, CD86 and CD40. Another 
study by Nakanishi et al. studied the effects of positively, 
negatively and neutrally charged liposomes administered 
subcutaneously in the immune responses of the fragment 
A of diphtheria toxin (DTA) and ovalbumin (OVA) [
93
]. 
Cationic liposomes were composed of phosphatidylcho
line:cholesterol:stearylamine (PC:Chol:SA, 4:5:1 molar 
ratio), anionic liposomes were composed of PC:Chol:l-
α-dimirystoylphosphatidic acid (PC:Chol:DMPA, 4:5:1) 
and neutral liposomes were composed of PC:Chol 
(1:1). Positively charged liposomes could induce potent 
antigen-specific cytotoxic T cell responses. However, 
DTA-containing cationic liposomes were cytotoxic to 
macrophages. In contrast, empty cationic liposomes or 
DTA-loaded anionic and neutral liposomes were not 
cytotoxic. CD8
+
OVA responses were highly induced 
by positively charged liposomal vaccines, potentially 
presenting the processed antigen through MHC I. Both 
research articles presented us the utilization of liposomes 
to investigate and test the effects on immune responses 
during vaccination. The immune responses meas-
ured varied, but making it clear that cationic liposomes 
induced the required cells (macrophages and DCs) to 
obtain the appropriate responses.
The liposomal vaccine studies mentioned above serve 
as the basis for the following articles which incorporate 
bacterial antigens for the development of prophylactic 
vaccines for certain infections. Puangpetch et al. devel-
oped a cationic-based liposomal formulations incor-
porating CpG ODN and to determine the prolongation 
and mechanisms of the immune responses [
2
]. The 
researchers employed the etiologic agent of melioido-
sis, Burkholderia pseudomalei, as the infection model in 
BALB/c mice. Cationic and not neutral liposomes admin-
istered intramuscularly granted protection against the 
bacterial challenge study. Prominent levels of IFN-γ were 
observed 2 days postinfection, but lowered by a CpG 
ODN-loaded cationic liposome pre-treatment. Neutro-
phils were not activated by the cationic liposomes with 
CpG ODN, but macrophages were stimulated by the 
formulation due to nitric acid production and low intra-
cellular bacterial burden (30 days post vaccination). An 
additional study involving mannosylated liposomes con-
taining the meningococcal PorA (from Neisseria men-
ingitidis) focused its attention on cell interaction [
94
]. 
Anionic (PG- (phosphatidylglycerol) and PS-based) and 
cationic (DMTAP-based) liposomes were formulated, 
and one of the anionic formulations was mannosylated 
(PC:PG:Chol + Man-PE (mannosyl phosphatidylethan-
olammine). When exposing the formulations to human 
and murine DCs, researchers observed an increase of 
liposome-cell interaction in the anionic mannosylated 
liposomes and cationic liposomes when compared to ani-
onic formulations alone. The result indicated that adding 
mannosyl moieties to liposomes generated a mannose 
receptor (MR)-mediated cell interaction. The murine 
DCs were confirmed to present the markers MHC II
+
, 
CD11c
+
and CD11b
+
, meanwhile human DCs presented 
CD40
+
, CD1a
+
and both MHC I and II. Researchers in 
the field should address studies that investigate route of 
administration effects on immune responses. With the 
studies presented here, it is difficult to determine what 
best route of administration we should follow for future 
prophylactic vaccine development. We recommend 
investigating the optimized formulations in different 
administration route studies.
The use of cationic liposomes seems of importance for 
the development of adequate vaccine formulations. The 
studies presented above demonstrate that by employ-
ing cationic phospholipids like DOTAP, DMTAP and 
DDA, would improve cell interaction levels, allow ade-
quate antigen presentation and induce strong immune 
responses. These effects will insure that the vaccine 
will work properly and optimally. For the benefit of the 
reader, Table 
4
presents a summary of the most relevant 
literature that optimized vaccine formulations for the 
treatment of bacterial infections. Additionally, previous 
research on cationic liposomes have discussed the cyto-
toxicity potential of such formulations. DDA has been 
determined to be safe as no relevant cytotoxic effects 
were determined in studies by Hilgers and Snippe and 
Gall [
47
, 
95
]. Only local inflammatory reactions mani-
fested as swelling were observed in mice (when admin-
istered alone). Contrasting results are found for DOTAP 

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De Serrano and Burkhart  J Nanobiotechnol (2017) 15:83 
and DOTMA cationic lipids. DOTAP has been found to 
be not cytotoxic to macrophages in a study by Jin et al. 
[
96
], but Romøren et al. determined that macrophage-
derived cell lines were found to be affected by the cati-
onic lipid [
97
]. The cytotoxic response differences might 
be due to structural and morphological characteris-
tics in the formulations. Jin et al. employed Tween 20 
and tricaprin (part of the solid core) to form solid lipid 
nanoparticles, meanwhile Romøren et al. just prepared 
liposomes. Further contradictory information is available 
for DOTMA, which can be cytotoxic for RAW 264.7 cells 
at all lipid ratios (DOTMA + DOPE), but not to human 
umbilical endothelial cells or mouse fibroblasts cells [
98
]. 
Kurosaki et al. determined that erythrocytes undergo 
agglutination and hemolysis when exposed to DOTMA-
based liposomes [
99
]. However, an earlier study by Kuro-
saki et al. determined lower cytotoxicity for erythrocytes, 
showing no agglutination and hemolysis, when DOTMA 
was formulated with N-laurylsarcosine, and Chol, vita-
min E and Chol or egg PC and Chol [
100
]. Future work 
should include the analysis of lipids alone and formulated 
with other lipids to elucidate the cytotoxic effects. Addi-
tional work should be done for these and other bacterial 
infections lacking proper prophylactic vaccines, leading 
to outcome improvement from the infection.