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′ ′
Tanimura et al. [23b]
Fujimoto et al. [23c]
Figure4.5 Chemical structures of various lipid As and lipid A analogs.
Previous structure–activity relationship studies[4a, 24] have revealed that agonistic and antagonistic effects can be controlled by the number of acyl chains and phosphate groups and the chain length (Figures4.4 and4.5). That is, hexa-acylated E. coli
lipid A (1) is an agonist, whereas tetra-acylated lipid IVa (3) is an antagonist. MPL504
(6) (Figure4.5), which is E. coli lipid A (1) without 1-phosphate, shows weaker IL-6-
inducing activity than E. coli lipid A (1)[23]. MPL504 (6) is less dependent on CD14,
a glycosylphosphatidylinositol-anchored receptor known to serve as a co-receptor
for TLR4. Additionally, TLR4/MD2 dimerization in response to MPL is much lower
than the response to E. coli LPS. MPL504 (6) has shown CD14-independent but
MyD88-dependent TNFα-producing ability and TRIF-dependent CD86 upregulation and IFNβ-inducing ability [23b]. Similar to MPL504 (6), MPL505 (7), which
lacks a 4′-phosphate, also exhibits mild immunomodulatory effects. However, while
the ability of MPL504 (6) to induce IL-18 production is lower than that of E. coli
LPS, MPL505 (7) exhibits the same IL-18 induction level as E. coli LPS [23c].
Therefore, these MPLs are expected to be developed as future adjuvants with different adjuvant effects. As described below, such structural modifications help regulate
the effects of lipid A as a potential adjuvant.
LPS consists of an O-antigen polysaccharide part that is characteristic of each
bacterial species, a core oligosaccharide part that has a high degree of commonality
in chemical structure across bacterial species, and a lipid A component (Figure4.1).
We also investigated the structure–activity relationship of lipid A connected with
the partial structure of the core oligosaccharide. There are R-mutant bacteria consisting of LPS lacking the O-antigen polysaccharide moiety, and the E. coli Re-mutant
has a Re-LPS (8) consisting of lipid A linked to Kdo disaccharide. To elucidate the
effect of Kdo on lipid A activity, we synthesized Re-LPS (8), Kdo-506 (9), and KdoMPL504 (10) (Figure 4.5), showing for the first time that Kdo enhances lipid A
activity[25].
4.3 Vaccines Containing Natural LPS as Adjuvants
Vaccines are designed to attenuate or inactivate pathogens and their toxins. Most
vaccines for bacteria contain natural bacterial components, and some of which
would act as natural adjuvants. LPS is considered the main natural adjuvant in vaccines derived from Gram-negative bacteria. Here are some examples of vaccines
that contain LPS.
4.3.1 Cholera Vaccines
Live-attenuated and inactivated whole-cell vaccines have been developed for cholera[26]. These vaccines include V. cholerae LPS and act as natural adjuvants.
An injectable whole-cell cholera vaccine was used in the 1960s, mainly in the
USA and Japan, and was administered subcutaneously twice every five to seven
days. The immune response rate was 50%, and protection continued for only six
months. Side effects have been reported, and the World Health Organization recommended its discontinuation.
®
Dukoral
nit, is an oral vaccine that was licensed in Sweden in 1991. This vaccine has an efficacy rate of 85–97% with few side effects. The duration of protection is approximately
two to three years. It is licensed mainly in Europe, Canada, South Asia, and Latin
America.
Live-attenuated oral cholera vaccines, Orochol
using the V. cholerae Inaba strain, whose cholera toxin A subunit (toxic) was deleted.
This vaccine is approved in the same country as Dukoral and has a similar effectivity
rate and effectivity period. However, the production and sale of these vaccines have
been discontinued.
, comprising killed whole bacteria and recombinant cholera toxin B subu-
®
and Mutacol®, were developed
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4.3.2 Salmonella enterica Serovar Typhi Vaccines
Live-attenuated, inactivated whole-cell, and subunit vaccines have been developed
for Salmonella enterica serovar Typhi[27].
®
Vivotif Berna
, derived from the attenuated typhoid Ty21a strain, is an oral liveattenuated vaccine developed in Switzerland. This vaccine is effective for more than
two years and has few side effects. However, the administration of this vaccine to
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118
children under five years of age is not recommended. It is licensed in Africa, Europe,
Asia, the USA, and South America.
Inactivated whole-cell vaccines (heat–phenol-inactivated or acetone-inactivated)
were first developed in 1896. However, these have reported side effects, and most
countries have withdrawn the use of these vaccines.
®
Typhim Vi
and Typherix®, using the virulence (Vi) capsular polysaccharide antigen purified from Salmonella Typhi, are subunit vaccines. Because the Vi capsular
polysaccharide antigen was checked by the endotoxin test, LPS was omitted. This
vaccine is effective for several years with a single injection. Its side effects are similar
to those of whole-cell-inactivated vaccines but are relatively mild. It is licensed in
Europe, Africa, Asia, Australia, and the USA.
4.3.3 Other Vaccines
Bexsero®, a vaccine against meningococcal group B (MenB), was developed by
Novartis. It contains a meningococcal outer membrane vesicle, and LPS derived
from meningococcal outer membrane vesicles can function as adjuvants in these
®
vaccines[28]. In contrast, the MenB vaccine Trumenba
uses a recombinant lipo-
protein containing the TLR2ligand bound to the protein antigen[29].
For Bordetella pertussis, subunit and inactivated vaccines have been developed
and are used as diphtheria, tetanus, and pertussis combination vaccine or diphtheria, pertussis, tetanus, and inactivated poliovirus combination vaccine. In most
combination vaccines, the safer acellular pertussis vaccine is commonly used.
However, a more effective whole-cell vaccine is still being used.
Similarly, for other Gram-negative bacteria, such as Haemophilus influenzae
type b and Neisseria meningitidis (serogroups A, B, C, Y, and W-135), vaccines have
also been developed, but all of them are capsular polysaccharide based vaccines;
hence LPS was not included.
Therefore, vaccines that might contain LPS are widely used, and, in some cases,
LPS may act as an adjuvant. In addition, it has been reported that some LPS could
retain their immunostimulatory effects even when administered orally[30], in which
case side effects would decrease compared to injected whole-cell vaccines with LPS.
4.4 LPS and Lipid A in the Environment or Fermented
Foods as Adjuvants
Immunomodulatory functions of LPS and lipid A in the environment or fermented
foods have been reported; thus, LPS and lipid A are attracting attention as safe adjuvant candidates.
The Gram-negative bacterium Pantoea agglomerans, which is widely present in
the soil and plants such as wheat, rice, sweet potato, apple, and pear, has been
Heine and coworkers [34]
detected during the fermentation process of rye bread [31]. P. agglomerans LPS
exhibits immunostimulatory effects via oral administration [30]. P. agglomerans
lipid A is a mixture of E. coli lipid A (1) and Salmonella minnesota lipid A (11)[32]
(Figure4.6), which are both agonists.
Kurozu (fermented black vinegar), an Asian fermented food, contains LPS derived
from Acetobacter spp. This genus of Gram-negative bacteria is used in acetic acid
fermentation. The chemical structure of Acetobacter pasteurianus LPS[35] and its
lipid A 12[33] (Figure4.6) have been reported recently. Although A. pasteurianus
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Hashimoto et al. [33]
Figure4.6 Chemical structures of various lipid As.
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LPS had weaker immunostimulating effects than E. coli LPS, A. pasteurianus LPS
and lipid A are still expected to be novel adjuvants because of their safety as foodderived compounds.
The hygiene hypothesis states that exposure to environmental microbes during
early childhood reduces the risk of developing allergic diseases. In the search for
bacteria with an allergy-suppressing ability, Acinetobacter lwoffii F78[34] was found
in livestock feed. A. lwoffii LPS selectively induces T helper 1 (Th1) cell-derived
cytokines, such as IL-12 and IFN-γ, which exhibit anti-allergic effects. A. lwoffii
F78LPS and its lipid A 13 (Figure4.6) have potential as novel adjuvants.
4.5 Synthetic and Semisynthetic Lipid As as Adjuvants
3D-MPL (2) [5] (Figure4.2), which has a 4′-monophosphate structure similar to
MPL504 (6), was developed by GSK. By optimizing the lipid A structure, especially
the acyl and phosphate groups, 3D-MPL (2) has been successfully attenuated and is
currently being derivatized and produced from S. minnesota R595LPS. 3D-MPL (2)
selectively activates the TRIF-dependent pathway of the two signaling cascades
downstream of TLR4/MD2 (Figure4.3).
GSK has developed the liposome adjuvant AS01, a mixture of 3D-MPL (2), cholesterol, and QS21 (a saponin derived from the South American native tree Quillaja
saponaria). AS01was applied to the herpes zoster vaccine, composed of recombinant glycoprotein E of varicella-zoster virus and AS01.
Infectious sporozoites are injected into human blood via salivary glands during
blood collection by Anopheles vector mosquitoes during infection with Plasmodium
falciparum, the causative agent of malaria. Therefore, the development of vaccines
targeting sporozoite surface proteins has been pursued. The recombinant protein
RTS,S consists of a segment of a sporozoite protein and the hepatitis B virus (HBV)
surface antigen, and the malaria vaccine candidate RTS,S/AS01has been developed
by GSK and is currently in phase III clinical trials. Adjuvant AS02, which consists of
3D-MPL (2), oil emulsion, squalene, and QS21, has also been developed by GSK,
and the malaria vaccine RTS,S/AS02 is currently in phase III clinical trials.
GSK has also developed the adjuvant AS04, a mixture of 3D-MPL (2) and aluminum salts. Cell-mediated immune responses are induced by AS04, which exhibits
antiviral effects. AS04 is practically used as an adjuvant for the human papillomavi-
®
rus (HPV) vaccine Cervarix
Furthermore, the MPL mimic RC-529 (14) (Figure4.7) was approved as an adjuvant for the HBV vaccine in Argentina in 2003.
Lipid A adjuvants such as MPL can induce anti-inflammatory cytokines, including IL-10, while modulating the induction of inflammatory cytokines such as IL-6
by their chemical structural modifications[36]; therefore, lipid A adjuvants have a
low risk of developing adjuvant-induced autoimmune diseases.
and the HBV vaccine Fendrix®.
Figure4.7 RC-529.
4.6 Developing Novel Lipid A Adjuvants
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4.6.1 Parasitic Bacterial Lipid As
Recently, we undertook a lipid A-mediated host–bacterial chemical ecology study.
Considering human symbiotic bacterial components as a pool of safe immunomodulators, we elucidated the immunomodulatory function of symbiotic bacterial lipid
As, thereby investigating the lipid A-mediated chemical communication between
host and symbiotic bacteria and developing their lipid As into safe and effective
adjuvants[23c, 37].
Helicobacter pylori, which lives in the stomach, causes gastric ulcers, and
Porphyromonas gingivalis, an oral bacterium, is a causative agent of periodontal dis-
ease. Extracted LPS from these parasitic bacteria has weak immunostimulatory
effects and is associated with chronic inflammation and atherosclerosis[38]. H. pylori
and P. gingivalis lipid As 15–21 are heterogeneous and have several different structures (Figure4.8). The ability of these parasitic bacterial lipid As to regulate TLR4/
MD2was suggested to be a factor in the specific biological activity of the aforementioned parasitic bacterial LPS[39]. E. coli lipid A (1) (Figure4.1) consists of six fatty
chains (C12–C14), whereas H. pylori lipid As 15, 16 have fewer (three to four) but
longer (C16–C18) fatty chains. Regarding phosphate groups, E. coli lipid A (1) has
two phosphate groups at the 1 and 4′-positions, whereas H. pylori lipid A has an
MPL structure. That is, 15a, 16a have a phosphate group only at the 1-position, and
15b, 16b have an ethanolamine phosphate group only at the 1-position. P. gingivalis
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Figure4.8 Chemical structures of parasitic bacterial LPS partial structures.
lipid As 18–21 have three to five fatty chains (C15–C17), including chains with terminal branches, and only the 1-position is phosphorylated. Parasitic bacterial lipid
As 15–21 have the following common structural features: Compared to canonical
E. coli lipid A (1), the fatty chains are longer and more diverse, and only 1-position
is phosphorylated.
We have chemically synthesized these parasitic bacterial partial structures 15–21
comprehensively and evaluated their immunostimulatory functions (cytokineinducing activities) in human peripheral whole blood. Their antagonistic effects on
TLR4/MD-2were evaluated by competition assays using E. coli LPS. Parasitic bacte-
rial lipid As 15a, 16a, 18, 19, which have three to four fatty acid chains and one
normal phosphate group, showed antagonistic activity in the induction of proinflammatory cytokines, such as IL-6 and TNF-α. In contrast, H. pylori lipid A 15b,
16b with three to four fatty acid chains and one ethanolamine phosphate group and
P. gingivalis lipid A 20 with five fatty acid chains and one normal phosphate group
showed IL-6- and TNF-α-inducing activity; however, the degree of activity was
markedly lower than that of E. coli LPS. As shown in Figure4.1, canonical E. coli
lipid A (1) is linked to the polysaccharide part via Kdo and the immunostimulatory
effect of lipid A is enhanced by the introduction of Kdo for E. coli lipid A[25]. In
contrast, for H. pylori lipid A, 17a with Kdo added to antagonist 15a showed stronger
antagonistic effects than 15a, and 17b with Kdo added to weak agonist 15b switched
to an antagonist [37a]. For H. pylori LPS, Kdo-lipid A, but not lipid A itself, was
found to be the active principle. All parasitic bacterial lipid As 15-21 induced IL-12
and -18, which are involved in chronic inflammation, and 15a, 16a, 17-19 were
found to selectively induce IL-12 and IL-18. Because the combination of IL-12 and
IL-18induces IFN-γ, which is involved in antitumor and anti-allergic responses,
H. pylori lipid As, which selectively induce IL-12 and IL-18, are promising adjuvant
candidates. LPS-mediated IL-18induction was reported to be dependent on the
4.7 Symbiotic Bacterial Lipid As
TRIF pathway [40]; however, a TRIF-independent pathway has also been
reported[41]. The molecular mechanism of selective cytokine induction triggered
by parasitic bacterial lipid As remains unclear. In 2014, it was reported that caspases
4, 5, and 11 are cytosolic LPS receptors [42]. Therefore, there may be a TLR4independent pathway in humans via caspase 4 or 5 for caspase-1 activation, which
is upstream of IL-18induction.
4.7 Symbiotic Bacterial Lipid As
Parasitic bacterial lipid A research has suggested that parasitic bacteria evolve to
escape the innate immune responses of the host, and their LPS/lipid A show antagonistic or extremely weak agonistic effects that favor infection of the host.
Furthermore, these results indicate that parasitic bacteria might induce chronic
inflammatory diseases while avoiding the bactericidal effects derived from acute
inflammation (host immune response), suggesting that the activity of lipid A profoundly reflects the characteristics of the bacteria, i.e. that there is the presence of a
lipid A-mediated bacterial–host chemical ecology (chemical communication
between bacteria and host via lipid A). Thus, we hypothesized that symbiotic bacteria would have extremely low toxic immunomodulators and their components
would be associated with maintaining homeostasis, and we chose symbiotic bacterial lipid A as a pool of safer immunomodulators.
Kiyono and Kunisawa revealed that the Gram-negative bacteria Alcaligenes faeca-
lis inhabits gut-associated lymphoid tissues (GALT), Peyer’s patches, which play an
important role in the maintenance of homeostasis[43]. We hypothesized that A. fae-
calis lipid A has a homeostatic function, which is a key factor in establishing symbiotic relationships with the host, and could be applied as a safe, low-toxicity
immunomodulator. Therefore, we performed purification, structural determination, and functional analysis of A. faecalis LPS. Canonical E. coli produce LPS consisting of tens to hundreds of sugar residues; however, some species produce
lipooligosaccharides (LOS) with short sugar chains. We have revealed that A. faeca-
lis produces a LOS consisting of a nonasaccharide (Figure4.9)[37c].
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Figure4.9 Chemical structures of A. faecalis LOS structures.
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The extracted LOS fraction from A. faecalis significantly promoted IgA antibody
production without toxicity and was comparable to that of toxic E. coli LPS, suggesting that the A. faecalis component is a promising safe adjuvant. Furthermore, the
antibody production enhancement of the extracted A. faecalis LOS was TLR4dependent, and it was suggested that lipid A is an adjuvant function core [44].
Structural analysis showed that A. faecalis lipid A was a mixture of 22–24 with different acyl chain patterns (Figure4.10). Thus, we chemically synthesized 22–24 and
Figure4.10 Chemical structures of synthesized A. faecalis lipid As.
evaluated their functions using human monocytic cells and found that only
hexa-acylated A. faecalis lipid A 22 (Hexa-AfLA) showed immunostimulatory activity, which was almost identical to that of the extracted A. faecalis LOS, indicating that
Hexa-AfLA is the active component of A. faecalis LOS[37c]. In vivo studies in mice
confirmed that Hexa-AfLA has the same useful adjuvant effect (enhancement of
antigen-specific IgA and IgG production and Th17-mediated protective immunity)
as A. faecalis LOS without toxicity[45]. The efficacy of Hexa-AfLA as a safe nasal
vaccine adjuvant has been demonstrated in S. pneumoniae infection models [45],
making it an extremely promising adjuvant for vaccines against infectious diseases.
Hexa-AfLA from GALT resident A. faecalis can regulate the induction of IgA,
which is responsible for maintaining the homeostasis of mucosal immunity, suggesting that Hexa-AfLA is a regulator of intestinal mucosal immunity. Based on
bacterial–host chemical ecology research, we have succeeded in identifying key
compounds for intestinal mucosal immunity by focusing on symbiotic bacteria
inhabiting the GALT and immunoregulatory tissues in the gut and have found
promising adjuvants that can safely regulate mucosal immunity.
4.8 Lipid A-Based Self-Adjuvanting Vaccines
The self-adjuvanting strategy promotes more efficient antibody production by complexing the antigen with the adjuvant. Recently, many studies on this strategy, especially those using lipopeptide adjuvants (Pam
published[6, 46]. The antigen–adjuvant complex is actively taken up by dendritic
cells via an innate immune ligand (adjuvant), which activates the immune system
and induces cytokine production, resulting in efficient antibody production
(Figure4.11). The advantage of this strategy is that the antigen and adjuvant are
taken up by the same dendritic cells and can trigger a specific immune response. It
is also excellent in terms of quality retention and safety control because it is easy to
obtain high-purity products. As for complexing methods between antigens and
adjuvants, one is based on covalent bond formation[46], and the other is based on
liposomes or self-aggregate formation[47]. In this section, covalent bond formationtype self-adjuvanting vaccines using lipid A are described.
Guo and coworkers synthesized an MPL-based self-adjuvanting vaccine, which is
a covalently bound MPL adjuvant and antigen. They reported a complex MPL with
GM3[48] or α-2,9-oligosialic acid (meningococcal antigen)[49] (Figure4.12a) and
confirmed enhanced antibody production. Lewicky and Jiang synthesized an MPL
mimic, RC-529 (14), conjugated with a Thomsen–Friedenreich antigen (a tumorassociated carbohydrate antigen) (Figure4.12b)[50]. Codée and coworkers synthesized a lipid A mimic, CRX-527, conjugated with peptide antigen (Figure4.12c), and
T-cell immune responses against the antigen and specific killing of target cells
expressing the antigen were observed[51]. Trumenba, the aforementioned vaccine
against N. meningitidis group B, is a recombinant lipoprotein with TLR2-stimulating
activity and is also a type of self-adjuvanting vaccine.
CSK4 and TLR2ligands), have been
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