1
CHAPTER 1 INTRODUCTION
1.1 Currently Licensed Vaccines
Since Edward Jenner’s discovery that cowpox could prevent human from the threat
of smallpox infection, vaccines have become the most important and successful strategy
to protect against infectious diseases.1-3 Currently, there are several types of licensed
vaccines available, including attenuated or killed pathogens, toxoids, proteins or
polysaccharides from those pathogens, and so on (Table 1.1).4
Table 1.1 Examples of licensed vaccines1,4
Vaccine type
Vaccine examples
Live mycob
Tuberculosis
Live attenuated
Influenza (intranasal), Measles, Mumps, Polio (Sabin), Rotavirus, Rubella, Varicella,
Yellow Fever
Killed
Hepatitis A, Pertussis (whole cell), Polio (Salk), Rabies,
Killed, subunit
Influenza
Toxoid
Diphtheria toxoid, Tetanus toxoid,
Protein
Hepatitis B, Pertussis (acellular),
PS
Hib PS, Meningococcal PS, Pneumococcal PS, Typhoid PS
PS-protein
Hib glycoconjugates, Meningococcal conjugates, Pneumococcal conjugates
Virus-like-
particle
Papillomavirus
Hib: Haemophilus influenzae type B; PS: polysaccharide
Vaccines derived from bacterial and viral particles or subunits, such as influenza
vaccine5,6, Bacillus Calmette-Guerin (BCG)7 and measles, and from attenuated or killed
2
pathogens are relatively easily accessed and developed.8 Hence, once an epidemic
surges and the pathogen of the disease is found, it is theoretically quick to design a
vaccine based on the pathogen itself. Currently, most of the successful vaccines in clinical
applications are developed empirically. For example, the seasonal influenza vaccines are
always developed based on the specific influenza virus components which vary year to
year.5,6
Despite the great success of whole cell and subunit/component-based vaccines,
there are still concerns or issues about them. First, the number of vaccines is still limited,
and there are still many diseases that have not been covered.1,9 Second, many of the
vaccines have limited immunological efficacy or are only effective in certain populations
not including young children who need more protection against pathogens.10-12 For
example, to control the spread of typhoid fever by routine vaccination, three licensed
vaccines, parenteral inactivated whole-cell vaccine, oral attenuated S. typhi Ty21a
vaccine, and parenteral Vi polysaccharide vaccine, confer only about 70% protection in
older children and adults and do not protect young children.11,13 safety issues and the
difficulties in quality control are other important issues associated with these vaccines.
Potential incomplete attenuation and contamination caused by pathogenic residuals are
big impediments to vaccine safety.14,15
In recent decades, vaccines derived from some specific antigenic components of
pathogens, including both proteins or polysaccharide (PS), have been developed, such
as vaccines against Hib12,16-19, N. meningitidis10,20 and S. pneumoniae21-24. Regarding
these vaccines, the antigenic components are structurally defined and can be easily
accessible from cultured bacteria in purified form. Therefore, these vaccines play an
3
important role in the study of vaccine immunology and the interactions between antigens
and the immune systems. Understanding the mechanism of vaccines and immunological
interactions may help us design and optimize vaccines that can elicit more effective and
long-term immunity.25
Proteins and carbohydrates of pathogens are two major components that can be
attractive targets for vaccine development. Pathogen proteins can often elicit strong and
T cell-dependent immune responses, so they have been useful and popular targets for
vaccine design. On the other hand, carbohydrates alone typically only introduce short-
term and T cell-independent immunity,26-29 especially in infants and children. Therefore,
pathogen polysaccharides have been mainly used as vaccines for adults. To overcome
this problem, carbohydrates have been coupled with carrier proteins to form conjugate
vaccines.22 In this way, the immune response induced against carbohydrate antigens can
be switched to T cell-dependent immunity which is more potent and functional. In addition,
the immune responses induced by conjugate vaccine have long-term effects and better
immunological activities.23,24 In the meantime, carbohydrates are exposed on the cell
surface, making them easily recognized by and interacting with the immune system.30
Consequently, the unique carbohydrates as antigens of both pathogen and cancer cells
have become particularly important targets for various immune studies and for vaccine
design.1,30 As the main topic of the present dissertation, carbohydrate-based vaccines will
be the focus of the introduction.
4
1.2 Carbohydrate-Based Vaccines
The unique glycans expressed by pathogens, including bacteria, parasites, fungi
and viruses, as well as the abnormal glycans on tumor cells, are very attractive markers
for the design and development of novel carbohydrate-based vaccines.1,31-33 First, the
glycans expressed by pathogens or the abnormal glycans expressed by tumors often
have distinctive chemical structures and are exposed on the cell surface, thus they are
easy targets as antigens for the immune system.1,10,34-37 Second, carbohydrates are
typically highly conserved and are often the most abundant antigens on the cell surface.
Consequently, development of carbohydrate–based vaccines against various diseases
(Figure 1.1) has been a hot area in recent decades, and many new and highly effective
vaccines have been developed.1,31,38 For example, as mentioned above, current licensed
vaccines against typhoid fever confer only about 70% protection in older children and
adults and have no protection to young children.11,13 To deal with the issue, Lin et al
developed a new conjugate vaccine based on the capsular polysaccharide of Salmonella
typhi, which has been demonstrated to be safe and strongly immunogenic and have
shown more that 90% efficacy in children two to five years old.11
Despite that some specific carbohydrates on the cell surface can be potentially
ideal antigens for the development of vaccines, they still have problems and limitations.
Most importantly, carbohydrates alone usually have low immunogenicity and are T cell-
independent antigens,30,31 thus even if they are immunogenic, they typically elicit T cell-
independent immune responses and fail to activate T cell immunity.26-29 As a result, the
inducing immune responses are often weaker and shorter antibody responses lacking
necessary immune memory.30,31 Moreover, carbohydrates alone usually cannot induce
5
robust immune responses in children under 5 years old. Therefore, although a number of
antibacterial vaccines made of pure polysaccharides have been developed, they are only
used in adults in clinic.
Figure 1.1 Carbohydrate antigens have been employed for various vaccine designs
A widely adopted strategy that has been employed to overcome the problem of
low immunogenicity of carbohydrate antigens is to link them to a large, immunologically
active protein carrier to form glycoprotein conjugates. This would not only improve the
immunogenicity of carbohydrates but also switch them to T cell-dependent antigens and
formulate functional vaccines. In this way, many effective carbohydrate-based vaccines
have been developed, which will be discussed in detail in the following sections.
However, polysaccharide-protein conjugates as vaccines have complex structure
and are difficult to analyze and control in terms of quality and purity.39-42 To overcome this
problem, in recent years, new vaccine designs and strategies have been explored, such
as conjugate vaccines derived from chemical synthetic carbohydrate antigens that are
6
homogeneous and fully synthetic conjugate vaccines with well-defined structure. These
vaccines may provide consistent quality as well as improved safety.1,38 To meet this
demand, new carrier molecules, in addition to proteins, have been exploited for the
development of synthetic glycoconjugate vaccines.42-45
1.3 Carbohydrate-Based Cancer Vaccines
1.3.1 Tumor-associated carbohydrate antigens
The association of abnormal glycosylations with tumor progression was first
discovered by Meezan et al. in 1969 with the demonstration that many glycans on cancer
cells differ from that on normal cells.36,46 The abnormal glycosylations include loss or
overexpression of certain glycans, presence of truncated glycans, and insertion of new
glycans.47,48 A variety of abnormal glycans on tumor cells have been identified by
immunohistochemical staining with lectin or monoclonal antibody (mAb) or by MS
analysis.49,50 Abnormal glycans expressed on cancer cells are termed tumor-associated
carbohydrate antigens (TACAs),72,92-95 some of which are depicted in Figure 1.2.
TACAs are widely detected in the majority of common human cancers, including
lung, breast, colorectal, ovarian, prostate, and pancreatic cancers. TACAs have been
correlated to specific types of cancers, and each type of malignant tissue is often
characterized by a distinct set of TACA expression as shown in Table 1.2.49,50 TACAs are
thus employed as important biomakers for clinical diagnosis of cancer. On the other hand,
TACAs are also attractive targets for anti-cancer vaccine and immunotherapy
8
Table 1.2 Expression profiles of TACAs on malignant tissues49,50
Tumor
Tumor-associated carbohydrate antigens
sLex
Lex
sLea
Lea
sTn
Tn
TF
Ley
Globo H
PSA
CD2
CD3
FucosylGM1
GM2
B cell lymphoma
✓
✓
Breast
✓
✓
✓
✓
✓
✓
✓
Colon
✓
✓
✓
✓
✓
Lung
✓
✓
✓
✓
✓
Melanoma
✓
✓
✓
Neuroblastoma
✓
✓
✓
✓
Ovary
✓
✓
✓
✓
✓
Prostate
✓
✓
✓
✓
✓
Sarcoma
✓
✓
✓
Small cell lung
✓
✓
✓
✓
✓
Stomach
✓
✓
✓
✓
✓
✓
✓
✓
✓
1.3.2 TACA-based cancer vaccines
TACA-based cancer vaccines have been widely explored despite of the potential
problems associated with TACAs as described previously. In general, the production of
immunity or antibodies induced by antigens depends on the cooperative function of two
types of lymphocytes, B cells and helper T cells. However, the carbohydrate antigens are
T cell-independent antigens and can hardly stimulate helper T lymphocytes. As a result,
they only elicit B cell immunity which lacks long-term immunological memory and induce
IgM antibodies that have low relatively affinity to antigens. On the other hand, the
involvement of helper T cells in the immune reactions (T cell immunity) and the induction
9
of IgG antibodies, which correlate to long-term immunological memory, higher antibody
affinity and improved antibody-dependent cell-mediated cytotoxicity,31,44,48,52 are
important for effective cancer therapy.
To formulate effective therapeutic cancer vaccines, the common strategy is to
covalently link TACAs to a carrier protein to form conjugate vaccines that can induce T
cell immunity. Keyhole limpet hemocyanin (KLH) is one of the most widely used carrier
proteins for the design and development of glycoconjugate cancer vaccines because of
its outstanding immunostimulatory ability. By coupling TACAs to proteins, the immune
responses to TACAs can switch from T cell-independent to T cell-dependent and lead to
the production of IgG antibodies with higher affinity.22,27 The approach has been widely
and successfully utilized to develop a number of TACAs-based vaccines. Some of these
vaccines, such as Globo H-KLH and sTn-KLH conjugates as well as others, have been
tested in clinical trials.53,54 Some of the vaccines studied and the carrier proteins used are
listed in Table 1.3.
Table 1.3 Examples of carbohydrate-based anticancer vaccines
Target antigen
Spacer/other epitope
Carrier
Cancer type
Monomeric vaccine
Globo H
CH2CH2
KLH,
BSA
Breast55
MMCCH
KLH
Prostate, breast56,57
Fuc-GM1
Ceramide-reductive amination
KLH
Small-cell lung58
GD2
Ceramide-lactone
KLH
melanoma59
10
GD3
Ceramide-reductive amination
KLH
Melanoma60,61
GM2
Ceramide-reductive amination
KLH
Melanoma62,63
GM3
Proteoliposome
OMPC
Melanoma64
Ley
CH2CH2
KLH
Ovarian39
sTn
Crotyl linker
KLH
Breast, covarian and
colorectal65-68
PSA, NP-PSA
Reductive amination
KLH
Small-cell lung69
Tn
MBS
KLH,
PAM
Prostate70
TF
MBS
KLH
Prostate71
sTn
sTn(c) crotyl linker-MMCCH
KLH
Breast72
Gb3-MUC5Ac
Gb2-norleucine-MUC5Ac-MBS
KLH
Ovarian73
GM3NPhAc
Immunotherapy based on
glycoengineering
KLH
Melanoma74,75
sTnNPhAc
Immunotherapy based on
glycoengineering
KLH
Epithelial cells related
cancer76,77
Polyvalent vaccine (pooled monomeric vaccines)
GD3, Ley, MUC1 and
MUC2
(GD2)-reductive amination
(Ley)-CH2 CH2
(MUC1, MUC2)-MBS
KLH
Melanoma
Ovarian
Breast78
GM2, Globo H, Ley, Tn
and TF MUC1 (32mer)
(GM2)-reductive amination
(Ley, Globo H)-MMCCH
(MUC1, Tn, TF)-MBS
KLH
Prostate79
GM2, Globo H, Ley, Tn,
TF, sTn and MUC1
(GM2)-reductive amination
(Ley, Globo H)-MMCCH
KLH
Epithelial ovarian,
11
(MUC1, Tn, TF, sTn)-MBS
Fallopian tube or
peritoneal80
Unimolecular polyvalent vaccine (consists of multi-antigens on unimolecule)
Globo H, Tn, sTn, TF,
Ley and GM2
Diaminopropyl-MBS
KLH
Breast and prostate81-
83
Globo H, GM2, Tn, sTn
and TF
Diaminopropyl-MUC1-alanine-MBS
KLH
Breast84
Multicomponent vaccine
Fucosyl GM1
FucGM1-norleucine-MHC II binding
peptide-MBS
KLH
Small-cell lung85
Tn (Two components)
Pam3Cys-aminobutyl-di-Tn86
Ley (two components)
Pam3Cys-peptide-(Ley)3
Ovarian87
Tn, TF or STF
Pam3CysSK4-ethylene glycol-MUC1
Breast88
Tn and TF (three-
component branched)
Pan-DR epitope-Lys-MUC1-Tn-Ala-
MUC1-TF
Breast89
Tn (three component)
TLR-2 ligand –Th epitope-MUC1-Tn
Breast42,43
Tn (four component)
TLR-2 ligand –CD8+ epitope
(OVA257-264)-CD4+ epitope (Pan-
DR)- Tn
RAFT
Breast90,91
sTnNPhAc
sTnNPhAc-linker-MPLA;
immunotherapy based on
glycoengineering
Epithelial cells related
cancer92,93
GM3NPhAc
GM3NPhAc-linker-MPLA;
immunotherapy based on
glycoengineering
Melanoma45
12
(c): cluster; Ala: alanine; BSA: Bovine serum albumin; CRM: Diphtheria toxin mutant; Fuc: Fucosyl; Gb3:
Globosyltriaoside; GD: Ganglioside antige; Globo H: Globohexaosylceramide; GM: Gangliosidoses; KLH:
keyhole limpet hemocyanin; Ley: Lewisy antigen; MBS: M-maleimidobenzoyl-N-hydroxysuccinamine ester;
MMCCH: 4-(4-N-maleimidomethyl) cyclohexane-1-carbonylhydrazide; MUC: Mucin; MUC5Ac: Mucin 5,
subtypes A and C; NP: N-propionylated; NPhAc: N-phenyl acetyl; OMPC: Neisseria meningitidis serogroup
B outer membrane protein complex; OVA: Ovalbumin; PAM: Palmitic acid; Pam3Cys: Tripalmitoyl-S-
glyceryl-cysteinylserine; Pan-DR: CD4+ T-helper epitope peptide; PSA: polysialic acid; RAFT:
Regioselectively addressable functionalized template; SK4: Serine-lysine-lysine-lysine-lysine; STF: sialic
Thomsen-Friedenreich; sTn: Sialyl 2-6-α-N-acetylgalactosamine; TF: Thomsen-Friedenreich; Th: T helper;
Tn: 2-6-α-N-acetylgalactosamine; TT: Tetanus toxoid.
As tumor cells have multiple TACAs expressed on their surface, and in different
stages of development, they have different kinds of TACAs. Therefore, polyvalent cancer
vaccines containing multiple antigens is desirable for targeting transformed tumor cells.
These polyvalent vaccines can be achieved by using a mixture of several monomeric
vaccines individually containing one antigen78-80 or a unimolecular polyvalent vaccine
consisting multi-antigens81-83 (Table 1.3). The pooled polyvalent vaccine of GD3-KLH, Ley-
KLH, MUC1-KLH and MUC2-KLH with QS021 adjuvant was found to elicit strong antigen-
specific IgG and IgM antibodies.78 Another vaccine pooled from GM2-KLH, Globo H-KLH,
Ley-KLH, TF-KLH, Tn-KLH, sTn-KLH and MUC1-KLH had similar results.80 However, this
mixture vaccine required more amount of carrier proteins and pre-validated vaccine
component. To promote the polyvalent vaccine, the unimolecular vaccines consisting
multi-antigens were developed. The KLH conjugates with Globo H, Tn, sTn, TF, Ley and
GM2 was demonstrated to generate antigen-specific IgM and IgG antibodies which was
similar as the other unimolecular polyvalent vaccines.81-83
13
Although these semi-synthetic vaccines have potential to be further used in clinic,
they have some limitations. The reproducibility of protein conjugates is not consistent from
batch-to-batch which induces the difficulty in safety control and quality control.9,42,43,94 To
further overcome these problems, some new construct of vaccines with fully-synthetic
chemical structures are in development. The examples of full-synthetic carbohydrate
vaccine having robust immunogenicity without the use of a protein carrier or adjuvant
were composed of TACAs and an immunologically active lipopeptide, tripalmitoyl-s-
glyceryl-cysteinylserine (Pam3Cyc).42,43,94 Our group developed novel full-synthetic anti-
cancer vaccine using monophosphoryl lipid A (MPLA) as a immunostimulatory carrier and
build-in adjuvant and the animal studies demonstrated these vaccines induced excellent
immune response, especially T cell immune response.45,92
Another strategy for new construct of vaccines was developed by Huang’s group
that they used tobacco mosaic virus and bacteriophage Qbeta virus-like particles as
carrier to stimulate immune response.95,96 The key advantage of virus-like particles carrier
is the ability to present antigens in an organized and high density manner. The antigen-
specific antibodies generated by these vaccines included IgG isotypes which reacted
strongly with the native Tn antigens on human leukemia cells.95,96
1.3.3 A novel cancer immunotherapeutic strategy
Although promising, most vaccines made of natural TACAs failed in clinical trials,
because of the lack of sufficient capability to generate effective immune responses.31 This
problem is mainly caused by the immunotolerence of TACAs resulting from the similar
14
structure to self-antigens that induces the absence of immunological supervision.31,36,37
Therefore, an approach to break immunotolerance to TACAs is employing unnatural
TACA analogs instead of natural TACAs to induce strong immune response, especially T
cell-mediated immunity which is required for successful cancer therapy. This hypothesis
was proved by some earlier studies that unnatural TACA analogs really provoked strong
immune response including both IgM and IgG antibodies.97,98 However, the antibodies
elicited by the unnatural TACA analogs may have limited cross-reactivity to the natural
TACAs on cancer cells which compromised the efficacy of immunotherapy. To overcome
this problem, a novel immunotherapy strategy based on artificial TACA vaccine and cell
glycoengineering was developed in our group.31,74,75,93 This novel strategy comprised of
two steps. First, a vaccine with artificial glycan resident, such as artificial sialic acid
residues, will be used to stimulate the immune response including a strong T cell immunity.
Immediately after immune response being elicited, a precursor (mannosamine
corresponding to sialic acids) with the same artificial modification will be employed to
glycoengineer the target cells and enforce the cells specifically expressing the
corresponding glycan antigens on cell surface.74 Subsequently, the pre-stimulated
antibodies can recognized the target cells and kill them with the help of other stimulated
components in immune system. (Figure 1.3) To carry out this novel immunotherapy
strategy, our group have demonstrated that an array of cancer cells were efficiently
engineered to express unnatural GM3 and sTn analogs, that is N-phenylacetyl GM3
(GM3NPhAc) and sTn (sTnNPhAc) by the treatment of precursor, N-phenylacetyl-D-
mannosamine (ManNPhAc). The previous studies also revealed that vaccines based on
GM3NPhAc and sTnNPhAc can provoked strong T cell-mediated immune responses
15
which exhibited strong and specific complements-dependent cytotoxicity (CDC) to the
pre-glycoengineered melanoma cell lines.31,45,74-77,93 This strategy was verified as a novel
and selective cancer immunotherapy although the selectivity of this strategy for tumor in
vivo need to be further investigated.31
Figure 1.3 A novel immunotherapy strategy based on glycoengineering
1.4 Carbohydrate-Based Antibacterial Vaccines
The polysaccharides (PS), either a capsule or lipopolysaccharide, uniquely
expressed on the surface of bacterial pathogens are attractive targets for anti-bacterial
vaccine designs and immunotherapies.1 Recently, since the antibiotic-resistant problems
in traditional clinical treatment of bacterial pathogens, the development of capsular
polysaccharides-based vaccines have engendered increasing excitement.20,99-101 For
many bacterial infections, some vaccines have been made from the fragment of bacterial
capsular polysaccharide, such as Haemophilus influenza type b (Hib), Neisseria
meningitides A, C, Y and W-135, and Salmonella typhi, and have been approved for
clinical using.1 (Table 1.4)
16
Table 1.4 Examples of licensed carbohydrate-based vaccines1
Target pathogen
Vaccine
Manufacture (Trade name)
Haemophilus influenzae
type b (Hib)
Glycoconjugate, polysaccharide with
tetanus toxoid (TT)
Sanofi Pasteur (ActHIB,1993);
GlaxoSmithKline Biologicals
(Hiberix, 1998, EU)
Diphtheria toxoid (DT), TT and acellular
pertussis adsorbed, inactivated poliovirus
and Hib-TT conjugate vaccine
Sanofi Pasteur (Pentacel)
Hib conjugate (meningococcal protein
conjugate)
Merck & Co (PedvaxHIB, 1990)
Hib conjugate (meningococcal protein
conjugate) and hepatitis B (recombinant)
vaccine
Merck & Co (Comvax)
Synthetic oligosaccharides with TT
conjugate
Licensed in Cuba (Quimihib
2004)33
Neisseria meningitides
A, C, Y and W-135 102
Glycoconjugate, meningococcal
polysaccharide with DT
Sanofi Pasteur (Menactra,
2005)
Meningococcal polysaccharide without
carrier
GlaxoSmithKline (ACWY Vax,
1981)
Meningitidis A, C, Y and W-135
polysaccharide with CRM197
Novartis (Menveo, 2010)
Salmonella typhi
Vi capsular polysaccharide
Sanofi Pasteur (Typhim Vi, )
Streptococcus
pneumoniae
Capsular polysaccharide with CRM197
Wyeth (Prevnar 13, 2000)
Capsular polysaccharide
Merck & Co (Pneumovax 23,
1983)
(From the US Food and Drug Administration website and www.cdc.gov/vaccine )
17
However, the heterogeneity and complexity of capsular polysaccharides cause the
difficulties and complicacy in the development of efficient carbohydrate-specific
vaccines.39-42 Moreover, using naturally derived polysaccharides to produce protein
conjugate vaccines presents the challenges in quality control and safety standards as
described above. Therefore, to overcome these problems, synthetic glycans with well-
characterized carrier proteins or full-synthetic vaccines with a well-defined synthetic
carrier can be the future directions.33,103
The first approved full-synthetic glycan-based vaccine, Quimi-Hib, was developed
in Cuba to prevent from Hib infection.33 The synthetic ribosylribitol-phosphate (RRP)
repeat units have been proven efficient in provoking immunity and can be accessed by
large-scale good manufacturing practice production. This promising production strategy
of vaccine incorporating a synthetic bacterial carbohydrate antigen reduced the safety
problems which existed in the purification of saccharide fragments from bacteria culture
and provided effective vaccine as already-licensed vaccine for human use. This strategy
was therefore feasible and can be further applied in the development of vaccines against
other pathogens.33 However, the reassembling Hib polysaccharide fragments were the
mixture of several RRP repeat units but not single structure.
To further study the structure-immunogenicity relationships of saccharide moieties
in a conjugate vaccine and optimize a well-defined glycoconjugate vaccine, in our group,
two series of oligosaccharide fragments, mimicking the antigens from Hib or N.
meningitidis, respectively, were synthesized and were conjugated with KLH or MPLA
carriers individually. The chapter 4 of this dissertation will focus on the immunological
18
studies of these semi-synthetic and full-synthetic anti-bacterial vaccines in animal, and
the investigation of structure-immunogenicity relationships. (Figure 1.4)
Figure 1.4 Anti-bacterial vaccines based on capsular polysaccharides
1.5 Conclusion
Carbohydrate antigens are attractive targets for immunotherapy and vaccine
development against bacteria, fungi, virus and cancers. However, there are a lot of
problems existing in the development of efficient vaccines. To overcome these problems,
many approaches have been explored. In our lab, a novel immunotherapy strategy based
on glycoengineering was developed to overcome the immunotolerance of TACAs.
Another novel construct of vaccine based on monophosphoryl lipid A (MPLA) was
explored. The achievements based on these two pioneering ideas will be exhibited in this
dissertation including the investigation of glycoengineering efficiency, a series of anti-
cancer vaccines based on TACAs with MPLA as carrier, and two series of anti-bacterial
vaccines.
19
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28
CHAPTER 2 Quantifying the Efficiency of Sialic Acids Metabolic Engineering
2.1 Introduction of Sialic Acid Metabolic Engineering
Sialic acids are a family of nine-carbon keto carbohydrates, and the most common
member is N-acetyl neuraminic acid (Neu5Ac, Figure 2.1). Neu5Ac is usually present at
the non-reducing end of glycans and plays important roles in various biological processes,
such as specific recognition, binding and regulatory events.1,2 It is also well known that
Neu5Ac is typically overexpressed on the surface of cancer cells3 and many of the
uniquely or excessively expressed glycans on cancer cells, known as tumor-associated
carbohydrate antigens (TACAs),4 contain the Neu5Ac residue. Some sialo-TACAs, such
as α-2,8-polysialic acid, sTn antigen, sialy Lewis antigens and the GM and GD
gangliosides, are abundantly expressed by a number of tumors. Therefore, sialo-TACAs
are useful targets for the design and development of new strategies for cancer diagnosis
and immunotherapy.5-7 However, most native TACAs are poorly immunogenic and
tolerated by the patients’ immune system; therefore, they may not be able to induce robust
antitumor immune responses, especially T cell-dependent immune responses, in cancer
patients.
Figure 2.1. Structure of Neu5Ac
To deal with the immunotolerance problem of TACAs, we have explored a novel
strategy for cancer immunotherapy based on metabolic engineering of cancer cell surface
sialo-TACAs.8,9 A key step of this strategy is to give cancer cells an unnatural analog of
29
N-acetyl-D-mannosamine (ManNAc), the native biosynthetic precursor of Neu5Ac, to
bioengineer cancer cells to express an unnatural sialo-TACA analog10,11 (Figure 2.2) that
is more distinguishable and immunogenic than the natural TACA to enable effective cancer
immunotherapy.8 Using antigen-specific antibodies combined with bioassays such as
ELISA and flow cytometry,12,13 we have demonstrated that N-phenylacetyl-D-
mannosamine (ManNPhAc) could metabolically engineer cancer cells to express sialo-
TACA analogs, such as analogs of sTn and GM3, carrying unnatural N-phenylacetylsialic
acid (Neu5PhAc) residues. However, these were only qualitative and semiquantitative
analyses. To ultimately determine the efficiency of ManNPhAc to metabolically engineer
cancer cells, a reliable method to quantify the ratio of Neu5Ac and Neu5PhAc expressed
by cancer cells is required, which is the aim of this research.
Figure 2.2 Metabolic engineering of sialic acids on the cancer surface
2.2 Results and Discussion
In the literature several methods have been described for the analysis of sialic
acids, such as colorimetric and fluorometric assays after derivatization with thiobarbituric
acid14,15 and liquid chromatography (LC) with UV,16 fluorescence17-27 and mass
spectrometry (MS) 28-32 detection. Except for LC-MS, sialic acids must be derivatized to
be photometrically detectable. In addition to thiobarbituric acid,14,15 a number of reagents
30
including 3,4-diaminotolenene,32 1,2-diamino-4,5-dimethoxybenzene,17 O-
phenylenediamine,20 and 1,2-diamino-4,5-methylenedioxybenzene (DMB),21,22,24-29,33-35
have been used for sialic acid derivatization, taking advantage of the special α-keto acid
functionality. Among these methods, DMB derivatization for HPLC-fluorescence and LC-
MS analyses was the most popular due to the high sensitivity and selectivity of the
reaction involved. In this research, we aimed at a potentially broadly applicable method
that uses simple instrument. Therefore, we became interested in a method that was
based on HPLC separation and UV detection. To make sialic acids UV-detectable, we
planned to employ DMB for sialic acids derivatization.29,35,36 As shown in Figure 2.3, the
resulting DMB derivatives had strong maximum absorbance at 370 nm, which was utilized
for quantitative analysis. Consequently, our design for the sialic acid analysis was to
release the total sialic acids from cell surfaces under mild acidic conditions, derivatize
them with DMB and finally subject the mixture to HPLC analysis with UV detection at 370
nm.
Figure 2.3 Reactions between sialic acids and DMB and structures of the reaction
products
To verify that DMB derivatization of sialic acids can be used for quantitative
analysis and that the HPLC signals of corresponding sialic acid derivatives have a linear
relationship with the concentrations of Neu5NAc and Neu5PhAc and to establish proper
calibration curves, different concentrations of standard Neu5NAc and Neu5PhAc samples
31
were treated with DMB and then applied to HPLC-UV analysis. It was revealed that the
reaction was clean and complete and the HPLC retention times (C18 column, 0.5 μm,
250 × 4.6 mm; eluted with water and acetonitrile containing 0.1% trifluoroacetic acid (TFA):
acetonitrile concentration changed from 5% to 25% in 10 min, kept at 25% for 10 min,
then from 25% to 70% in 15 min; flow rate: 1mL/min) for the Neu5Ac and Neu5PhAc
derivatives 2.3 and 2.4, which were confirmed with MS, were 9.87 min and 15.45 min,
respectively. As shown in Figure 2.4, under the tested conditions (i.e., Neu5Ac and
Neu5PhAc concentrations in the range of 15-130 µM), the observed HPLC peak areas of
2.3 and 2.4 were in good linear relationship (coefficient: 0.9814 and 0.9763, respectively)
with the Neu5Ac and Neu5PhAc concentrations in the samples. These results have
demonstrated that the method could be used for the quantification of Neu5Ac and
Neu5PhAc. Equations, HPLC peak area = 2067 [Neu5Ac] + 18,835 and HPLC peak area
= 1832 [Neu5PhAc] – 1,141, were used to calculate the concentrations or quantities of
Neu5Ac and Neu5PhAc in samples, respectively.
N e u 5 A c C o n c e n t r a tio n ( M )
P e a k a r e a
0 50 100 150
0
100000
200000
300000
400000
R2= 0 .9 8 1 4
N e u 5 P h A c c o n c e n t ra t io n (M )
peak area
0 50 100 150
0
50000
100000
150000
200000
R2= 0 .9 7 6 3
Figure 2.4 A linear correlation between the peak areas of Neu5Ac or Neu5PhAc
derivative and the concentrations of Neu5Ac or Neu5PhAc was demonstrated. The
coefficient of determination (R2) is displayed for each graph.
32
For the analysis of biological samples derived from cells, there are several issues
that may be of concern. First, the detachment of sialic acids from the cell surface may not
be complete, which can affect the observed amount of sialic acids. However, this is a
systematic error that may affect the sample recovery but not the ratio of Neu5Ac and
Neu5PhAc, the latter of which is actually our focus. Second, the collected sialic acids
need to be derivatized before analysis, while under the condition that the samples contain
numerous impurities, whether the derivatization reaction will be affected is another
question, despite that the above experiments have proved the reaction efficiency in the
absence of impurities. Third, samples derived from metabolically engineered cells will
contain variable ratios of Neu5Ac and Neu5PhAc, which can be immensely different.
Whether this can be accurately reflected is another question.
To address these issues, we then probed the reliability and accuracy of the
proposed method under more complex situations, such as for the analysis of biologic
samples or samples with big differences in the Neu5Ac and Neu5PhAc concentrations.
To probe the reliability of the method, we added different amounts of Neu5PhAc in the
culture of SKMEL-28, a melanoma cell line, and then examined its recovery upon a series
of treatments involved in the real sample analysis, including incubation with acetic acid
employed to detach sialic acids from cells, lyophilization, reaction with DMB, and
eventually HPLC analysis. As shown in Table 2.1, the recovery rates of the externally
added Neu5PhAc in various samples were excellent (97-99%, average 98±1%). In this
study, the recovery of Neu5Ac from cells was not examined, as the added Neu5Ac would
interfere with naturally exiting Neu5Ac. However, we anticipated that its recovery rate
should not be lower than that of Neu5PhAc. To probe the accuracy of the method, we
33
prepared a series of Neu5Ac and Neu5PhAc mixtures in ratios ranging from 100:0 to
100:8 and applied them to analysis by the proposed method. As shown in Table 2.2, the
experimental results were in good agreement with the expected ratios, and in average
the accuracy was above 93%. These results have clearly demonstrated the reliability and
accuracy of the proposed method for the analysis of Neu5Ac and Neu5PhAc expressed
by metabolically engineered cells.
Table 2.1 Recovery analysis of Neu5PhAc from the SKMEL-28 cell culture
Added Neu5PhAc (nmol)
65
130
130
130
Detected Neu5PhAc (nmol)
63
128
127
129
Recovery rate (%)
97
98
98
99
Table 2.2 Analysis of Neu5Ac and Neu5PhAc mixtures prepared in different ratios
Sample number
1
2
3
4
5
Standard samples
Neu5Ac:Neu5PhAc
100:0
100:2
100:4
100:6
100:8
Experimental results
Neu5Ac:Neu5PhAc
100:0
100:1.9(±0.1)
100:3.7(±0.1)
100:5.9(±0.3)
100:7.1(±0.4)
After the proposed method was verified with standard samples under different
conditions, it was applied to the analysis of metabolically engineered cancer cells. In
these studies, SKMEL-28 cells were incubated with various concentrations of ManNPhAc
(0, 5, 10, 20, and 30 mM) for 2-4 days. Then, ca. 1.0 × 106 cells were harvested from
each culture, washed, and treated with 2 M acetic acid at 80 ºC for 2 h to release sialic
acids from cells. This was followed by lyophilization and reaction with excessive DMB at
50 ºC for 3 h.29 After the reaction mixtures were filtered off through a 0.22 μm film, they
34
were subjected to analysis by RP-HPLC using gradient eluents of water and acetonitrile
containing 0.1% TFA. Figure 2.5 shows the typical HPLC diagram of a sample. As labeled
in the figure, the DMB derivatives of Neu5Ac and Neu5PhAc 2.3 and 2.4 had the same
retention times as that of the standard samples. To further prove their identity, 2.3 and
2.4 were collected and subjected to MALDI-MS analysis, which gave the correct masses
[m/z: calculated, 425.2 and 501.2; observed, 426.3 and 502.5 (M+H+), respectively].
Evidently, the samples also contained a small number and amount of other impurities,
which appeared at different positions and did not affect the analysis.
Figure 2.5 HPLC diagram of a sample derived from 106 SKMEL-28 cells treated with 30
mM of ManNPhAc for 3 days. The retention times for the DMB derivatives of Neu5Ac and
Neu5PhAc 2.3 and 2.4 were about 9.90 and 15.50 min, respectively.
The amounts of Neu5Ac and Neu5PhAc in samples obtained from 106 SKMEL-28
cells were calculated based on the HPLC peaks of 2.3 and 2.4 according to the standard
curves shown in Figure 2.4, and the results are presented in Figure 2.6. As anticipated,
significant Neu5PhAc expression was observed for cells incubated with 5 mM of
0
50000
100000
150000
200000
250000
0 5 10 15 20
Retention Time (min)
Peak Intensity
3
4
35
ManNPhAc (Figure 2.6B), and the amount of expressed Neu5PhAc increased
proportionally with the increase of ManNPhAc concentration in the cell culture. However,
Neu5PhAc expression was still much lower than that of Neu5Ac even at a quite high
concentration (30 mM) of ManNPhAc. Furthermore, it was found that natural Neu5Ac
expression was also elevated in the presence of high concentrations of ManNPhAc.
Typically, one should expect that natural product biosynthesis would be depressed on the
addition of an unnatural biosynthetic precursor due to either competition or inhibition. In
contrast, it was very interesting to notice that the presence of ManNPhAc seemed to
promote not only Neu5PhAc expression but also Neu5Ac biosynthesis, which is unique
and can be of biological significance, so it is worthy further investigation. Conclusively,
the results suggested that ManNPhAc could effectively glycoengineer SKMEL-28 cells to
express unnatural Neu5PhAc in a concentration-dependent manner in the range of 5-30
mM, whereas Neu5Ac expression was only slightly affected under the same condition. It
was also evident that only a small fraction of Neu5Ac on the cell surface was substituted
with Neu5PhAc upon metabolic engineering using ManNPhAc, a process that had to
compete with the endogenous biosynthetic pathway.
36
(A) (B)
N e u 5 A c E x p r e s s io n
0 m M
5 m M
10 m M
20 m M
30 m M
0
1 0
2 0
3 0
4 0
P r e c u r s o r C o n c e n t r a t io n
N e u 5 A c n m o l/1 0 E 6 c e lls
N e u 5 P h A c E x p r e s s i o n
0 m M
5 m M
1 0 m M
20 m M
3 0 m M
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
P r e c u r s o r C o n c e n t r a t io n
N e u 5 P h A c n m o l/1 0 E 6 c e lls
Figure 2.6 The amounts of Neu5Ac (A) and Neu5PhAc (B) expressed by SKMEL-28 cells
(106) that were incubated with 0, 5, 10, 20 and 30 mM of ManNPhAc for 3 days,
determined by HPLC. Error bar represents the standard deviation of three parallel
experiments.
To further define the efficiency of cell metabolic engineering using ManNPhAc, the
percentage of Neu5PhAc expressed on the cell surface in terms of total sialic acids was
calculated according to the following equation, Neu5PhAc% = Neu5PhAc /(Neu5Ac +
Neu5PhAc) x 100%, and the results are shown in Figure 2.7(A). The percentage was
about 2.2% and 5.8% for cells incubated with 5 mM and 30 mM of ManNPhAc for 3 days,
respectively.
37
Figure 2.7 The percentages of Neu5PhAc expressed on SKMEL-28 cells incubated with
0, 5, 10, 20 and 30 mM of ManNPhAc for 3 days (A), and the percentages of Neu5PhAc
on SKMEL-28 cells incubated with 5 and 20 mM of ManNPhAc for 2, 3, 4 and 9 days (B),
respectively. The error bar represents the standard deviation of three parallel experiments.
Finally, we examined the influence of incubation time on the cell engineering
efficiency. In these studies, we incubated cancer cells with 5 mM and 20 mM of
ManNPhAc for 2, 3, 4 and 9 days, and then examined their Neu5PhAc expression. The
results (Figure 2.7B) indicated that the percentage of Neu5PhAc increased during the
first 3 days to reach peak values at day 3 and then decreased slightly to retain a consistent
level of Neu5PhAc after day 4. The reason for decreased Neu5PhAc expression after day
3 was not clear, but it might be due to the change of metabolic activities of the cell, as we
found that after 3 days of incubation cell proliferation and growth became slower.
Nevertheless, it was ultimately disclosed that cancer cells could be effectively engineered
(A) (B)
Neu5PhAc Expression
0 mM
5 mM
10 mM
20 mM
30 mM
0
2
4
6
8
Precursor Concentration
Neu5PhAc Percentage (%)
38
to express a significant level of unnatural Neu5PhAc to make the cells distinct and that
this could persist for a long period.
2.3 Conclusion
We have developed a convenient and reliable method for quantitative analysis of
sialic acids expressed by cells. This method was based on the specific reaction between
DMB and the α-keto acid functionality of sialic acids to generate products that had strong
UV absorption, enabling easy HPLC analysis with UV detection. After the consistency
and accuracy of this method were verified with standard samples under various conditions,
it was used to analyze metabolically engineered cancer cells, which expressed both
natural Neu5Ac and unnatural Neu5PhAc.
The protocols for this analysis were rather straightforward. After incubation with
ManNPhAc, cells were subjected to acetic acid-promoted hydrolysis to release sialic
acids on the cell surface. Then, the sample was lyophilized and treated with DMB. The
reaction mixture was directly applied to HPLC analysis to determine the amounts and
ratio of Neu5Ac and Neu5PhAc. The method is not only applicable to the analysis of
Neu5Ac and Neu5PhAc but also generally useful for the analysis of other sialic acids.
While this is a simple, reliable and quantitative method, it does have limitations.
For example, it is not easily applicable to the analysis of cells incubated with µM
concentration of ManNPhAc, probably because the Neu5PhAc expression level was too
low. However, Neu5PhAc expression on these cells was detected by flow cytometry with
the help of antibody-labeling.13 Evidently, flow cytometry is more sensitive, albeit not
precisely quantitative, than HPLC analysis. Another problem for the flow cytometry
method is that it relies on antigen-specific antibodies that are not always readily available.
39
Therefore, a combination of these two analytical methods can portrait the whole picture
concerning the metabolic engineering of cell surface sialic acids with unnatural ManNAc
derivatives as biosynthetic precursors.
This research has revealed for the first time that more than 2% of Neu5Ac was
substituted for Neu5PhAc upon metabolic engineering with ManPhNAc. We have
previously shown by means of flow cytometry13 and antibody-mediated cytotoxicity37 that
cancer cells could be engineered to express Neu5PhAc at low µM concentrations (20-50
µM) of ManNPhAc. Under these conditions, the expression level of Neu5PhAc should be
much lower than 2%, but the engineered cells could be recognized by Neu5PhAc-specific
antibodies and undergo functional immune reactions. This information is useful not only
for our immunotherapy but also for other studies that are based on metabolic engineering
of cell surface sialic acids or other sugars. A particularly interesting but unexpected
discovery was that natural sialic acid expression also increased in the presence of
ManNPhAc, which could be only disclosed from quantitative analysis. This may be of
biological significance, as sialic acid expression is related to various biological activities,
and it is worthy further investigation.
2.4 Experimental Section
Materials, reagents and animals
SKMEL-28 cell line, Dulbecco’s Modified Eagle’s Medium (DMEM) for cell culture,
and fetal bovine serum (FBS) were purchased from American Type Culture Collection
(ATCC). Penicillin-streptomycin and trypsin-EDTA were purchased from Invitrogen.
40
ManNPhAc was synthesized by a procedure previously reported by our laboratory.13 DMB,
sodium hydrosulfite, β-mercaptoethnal and other chemicals were purchased from Sigma.
Metabolic engineering of tumor cell
SKMEL-28 cells were cultured in DMEM containing 10% of FBS, 1% of penicillin-
streptomycin and various concentrations (0 to 30 mM) of ManNPhAc at 37 oC. After a
certain number of days of incubation, cells were harvested upon treatment with trypsin-
EDTA solution, and finally washed twice with PBS to be readied for further analysis.
Hydrolysis of sialic acids
A cell sample (containing about 1.0×106 cells) was dissolved in a 2 M solution of
acetic acid (2 mL). The mixture was stirred at 80 ºC for 2 h, and then lyophilized to get a
powder product that contained sialic acids released from cells.
DMB derivatization of sialic acids and HPLC analysis
The powder product obtained above was mixed with DMB (0.5 mg, excessive) in
1.4 M acetic acid containing 18 mM of sodium hydrosulfite and 0.75 M of β-
mercaptoethnal.29 After the reaction mixture was stirred at 50 ºC for 3 h, it was filtered off
through a 0.22 μm film, and the filtrate was analyzed by RP-HPLC (C18 column, 250 ×
4.6 mm, 0.5 μm) using gradient eluents of water (A) and acetonitrile (B) containing 0.1%
TFA at flow rate of 1mL/min. Elution started with 5% B, which was increased gradually to
25% B in 10 min. Elution of the column with 25% B was kept for 10 min, followed by
gradual increase of B composition to 70% within 15 min, and the elution was kept under
this condition to the end of an experiment. Finally, the column was washed and balanced
41
with 5% B for 40 min before the next experiment. The eluents were monitored with a UV
detector at 370 nm, at which wavelength DMB derivatives of sialic acids had the maximum
absorption. All of the HPLC peaks were separately collected, and the fractions containing
the anticipated DMB derivatives of sialic acids were further analyzed and verified by
MALDI-MS.
Establishment of standard calibration curves and equations used to quantify
Neu5Ac and Neu5PhAc
A Neu5Ac or Neu5PhAc standard sample (1, 2, 4 or 6 µg) was mixed with a DMB
reaction solution (150 µL containing 0.5 mg DMB, excessive) in 1.4 M acetic acid, 18 mM
of sodium hydrosulfite and 0.75 M of β-mercaptoethnal. The reaction mixture was stirred
at 50 °C for 3 h, and then filtered off through a 0.22 µm film. The filtrate (10 µL) was
analyzed by RP-HPLC (C18 column, 250 × 4.6 mm, 0.5 µm) using gradient eluents of
water and acetonitrile containing 0.1% TFA at flow rate of 1 mL/min as described above.
The eluents were monitored with a UV detector at 370 nm. The peak areas were plotted
against the sample concentration to obtain the calibration curves and equations.
Determining the recovery rate of Neu5PhAc from cell culture
SKMEL-28 cell was cultured, harvested, and washed twice with PBS. To the cell
sample (about 1.0 × 106 cells, in 1 mL PBS solution) was added 65 or 130 nmol of a
Neu5PhAc standard sample. The cell sample was added to an acetic acid solution (final
volume 2 mL and final concentration 2 M). The mixture was stirred at 80 °C for 2 h. The
reaction mixture was lyophilized to obtain a powder product that was reacted with a DMB
solution (150 µL, containing 0.5 mg DMB) in 1.4 M acetic acid containing 18 mM of sodium
42
hydrosulfite and 0.75 M of β-mercaptoethnal. The reaction mixture was finally subjected
to HPLC analysis as described above.
Determining the Neu5Ac and Neu5PhAc ratios in standard samples
A series of standard samples containing different ratios of Neu5Ac and Neu5PhAc
(100:0, 100:2, 100:4, 100:6, 100:8 mol/mol) were prepared. About 20 nmol of each
sample was added to a DMB solution (150 µL, containing 0.5 mg DMB) in 1.4 M acetic
acid containing 18 mM of sodium hydrosulfite and 0.75 M of β-mercaptoethnal. After the
reaction mixture was stirred at 50 °C for 3 h, it was filtered off through a 0.22 µm film, and
10 µL of the filtrate was finally subjected to RP-HPLC analysis as described above.
2.5 Appendices
Mass Spectrum Confirmation
The fraction of 15.45 min of HPLC was collected to be further confirmed by MALDI-
TOF MS (Figure 2.8). In the standard Neu5PhAc-DMB mass spectrum, the MALDI peaks,
501.800 (M+H+) and 523.676 (M+Na+) confirmed that the standard Neu5PhAc was
successfully labeled with DMB. In the HPLC fraction of 15.4 min collected from cell
samples treated with precursor, the peaks, 502.536 (M+H+) and 524.258 (M+Na+),
confirmed that the cells expressed the modified sialic acid as expected . The mass error
was due to the system shift of m/z value in MALDI machine.
43
Figure 2.8 MALDI MS of Neu5PhAc-DMB: (A) Sample was from reaction solution of
standard Neu5PhAc after DMB derivatization; (B) HPLC fraction sample of 15.4 min in
the cell samples which was treated with 20 mM precursor ManNPhAc for 2 days.
523.676
501.800
0
1000
2000
3000
4000
5000
Intens. [a.u.]
200 400 600 800 1000
m/z
502.536
524.258
0
10
20
30
40
Intens. [a.u.]
100 200 300 400 500 600 700 800 900 1000 1100
m/z
(A) Standard Neu5PhAc-DMB
(B) HPLC fraction of 15.4 min
in treated cell samples
[M+H]+
[M+Na]+
[M+H]+
[M+Na]+
44
2.6 Reference
(1) Troy, F. A., 2nd Glycobiology 1992, 2, 5.
(2) Schauer, R. Advances in experimental medicine and biology 1988, 228, 47.
(3) Takano, R.; Muchmore, E.; Dennis, J. W. Glycobiology 1994, 4, 665.
(4) Hakomori, S. Advances in cancer research 1989, 52, 257.
(5) Hakomori, S. Advances in experimental medicine and biology 2001, 491, 369.
(6) Ragupathi, G. Cancer Immunol. Immunother. 1998, 46, 82.
(7) Yin, Z.; Huang, X. Journal of carbohydrate chemistry 2012, 31, 143.
(8) Guo, Z.; Wang, Q. Current opinion in chemical biology 2009, 13, 608.
(9) Pan, Y.; Chefalo, P.; Nagy, N.; Harding, C.; Guo, Z. Journal of medicinal chemistry 2005, 48,
875.
(10) Mahal, L. K.; Bertozzi, C. R. Chem. Biol. 1997, 4, 415.
(11) Keppler, O. T.; Horstkorte, R.; Pawlita, M.; Schmidt, C.; Reutter, W. Glycobiology 2001, 11,
11R.
(12) Wang, Q.; Zhang, J.; Guo, Z. Bioorg. Med. Chem. 2007, 15, 7561.
(13) Chefalo, P.; Pan, Y.; Nagy, N.; Guo, Z.; Harding, C. V. Biochemistry 2006, 45, 3733.
(14) Hammond, K. S.; Papermaster, D. S. Analytical biochemistry 1976, 74, 292.
(15) Skoza, L.; Mohos, S. The Biochemical journal 1976, 159, 457.
(16) Tzanakakis, G. N.; Syrokou, A.; Kanakis, I.; Karamanos, N. K. Biomedical chromatography :
BMC 2006, 20, 434.
(17) Hara, S.; Yamaguchi, M.; Takemori, Y.; Nakamura, M.; Ohkura, Y. Journal of
chromatography 1986, 377, 111.
(18) Hara, S.; Takemori, Y.; Yamaguchi, M.; Nakamura, M.; Ohkura, Y. Analytical biochemistry
1987, 164, 138.
45
(19) Hara, S.; Yamaguchi, M.; Takemori, Y.; Furuhata, K.; Ogura, H.; Nakamura, M. Analytical
biochemistry 1989, 179, 162.
(20) Anumula, K. R. Analytical biochemistry 1995, 230, 24.
(21) Kawabata, A.; Morimoto, N.; Oda, Y.; Kinoshita, M.; Kuroda, R.; Kakehi, K. Analytical
biochemistry 2000, 283, 119.
(22) Luchansky, S. J.; Argade, S.; Hayes, B. K.; Bertozzi, C. R. Biochemistry 2004, 43, 12358.
(23) Luchansky, S. J.; Argade, S.; Hayes, B. K.; Bertozzi, C. R. Biochem. 2004, 43, 12358.
(24) Dehnert, K. W.; Baskin, J. M.; Laughlin, S. T.; Beahm, B. J.; Naidu, N. N.; Amacher, S. L.;
Bertozzi, C. R. Chembiochem : a European journal of chemical biology 2012, 13, 353.
(25) Goon, S.; Schilling, B.; Tullius, M. V.; Gibson, B. W.; Bertozzi, C. R. Proceedings of the
National Academy of Sciences of the United States of America 2003, 100, 3089.
(26) Lemieux, G. A.; Bertozzi, C. R. Chemistry & biology 2001, 8, 265.
(27) Saxon, E.; Luchansky, S. J.; Hang, H. C.; Yu, C.; Lee, S. C.; Bertozzi, C. R. Journal of the
American Chemical Society 2002, 124, 14893.
(28) Galuska, S. P.; Geyer, H.; Weinhold, B.; Kontou, M.; Rohrich, R. C.; Bernard, U.; Gerardy-
Schahn, R.; Reutter, W.; Munster-Kuhnel, A.; Geyer, R. Analytical chemistry 2010, 82, 4591.
(29) Morimoto, N.; Nakano, M.; Kinoshita, M.; Kawabata, A.; Morita, M.; Oda, Y.; Kuroda, R.;
Kakehi, K. Analytical chemistry 2001, 73, 5422.
(30) van der Ham, M.; de Koning, T. J.; Lefeber, D.; Fleer, A.; Prinsen, B. H.; de Sain-van der
Velden, M. G. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
2010, 878, 1098.
(31) van der Ham, M.; Prinsen, B. H.; Huijmans, J. G.; Abeling, N. G.; Dorland, B.; Berger, R.; de
Koning, T. J.; de Sain-van der Velden, M. G. Journal of chromatography. B, Analytical technologies in the
biomedical and life sciences 2007, 848, 251.
46
(32) Wang, D.; Zhou, X.; Wang, L.; Wang, S.; Sun, X. L. Journal of chromatography. B, Analytical
technologies in the biomedical and life sciences 2014, 944, 75.
(33) S.J., L.; Bertozzi, C. R. ChemBioChem. 2004, 5, 1706.
(34) Lin, S. L.; Inoue, S.; Inoue, Y. Carbohydr. Res. 2000, 329, 447.
(35) Nakamura, M.; Hara, S.; Yamaguchi, M.; Takemori, Y.; Ohkura, Y. Chem. Pharm. Bull. 1987,
35, 687.
(36) Liu, T.; Guo, Z.; Yang, Q.; Sad, S.; Jennings, H. J. The Journal of biological chemistry 2000,
275, 32832.
(37) Wang, Q.; Zhang, J.; Guo, Z. Bioorganic & medicinal chemistry 2007, 15, 7561.
47
CHAPTER 3 Development of Anti-Cancer Vaccines Using Monophosphoryl Lipid
A as a Carrier Molecule and Built-in Adjuvant
3.1 Introduction of Monophosphoryl lipid A (MPLA)
3.1.1 Lipopolysaccharide
The endotoxins were first found in heat-killed bacteria Vibrio cholera by Richard
Pfeiffer and Robert Koch in the nineteenth century and were associated primarily with
Gram-negative bacteria.1 They are distinguished from exotoxin because the former are
not secreted to the culture media by bacteria but only released upon lysis of bacteria cells.
The principal endotoxin, lipopolysacchride (LPS) which is a family of complex glycolipid,
is the major component of the outer monolayer of Gram-negative bacteria cell outer
membrane.2 LPS was discovered as a heat-stable and amphiphilic components
containing a hydrophobic lipid A as a membrane anchor domain, an oligosaccharide ‘core’,
and a distal polysaccharide (Figure 3.1).3
Figure 3.1 The structure of outer layer of outer membrane of Gram-negative bacteria.
48
The LPS layer of the outer membrane plays important roles in bacteria activities,
such as reacting to the changes of environment and inhibiting toxic compounds (e.g.,
antibiotics) to protect the bacteria cell, working for nutrient transport, and mediating
interaction with the host.4 The outer membrane with LPS can associate with divalent
cations to provide an effective permeability barrier for the bacteria cell against external
stress factors.5-7 Moreover, LPS has been demonstrated to be an extremely powerful
immunostimulators and elicit toxic effects by inhibiting cellular functions or killing host
cells. According to present knowledge about LPS, it interacts with various types of host
cells including mononuclear cells, endothelial cells, smooth muscle cells,
polymorphonuclear granulocytes, thrombocytes and so on through its lipid A component.
Consequently, LPS has engendered considerable interests for antibiotics development
because of its important role in antibiotics resistant and for immunotherapy development
because of its strong immunostimulatory ability.
3.1.2 Lipid A structure-activity relationships
In general, the lipid A domain of LPS determines its principal immunostimulatory
ability because lipid A itself is a potent toxin that can be specifically and sensitively
recognized by immune system.8 Lipid A binds specifically to the toll or interleukin-1
receptor domain-containing adaptor-inducing interferon-β (TRIF) of toll-like receptors
(TLRs) in association with MyD88 to induce a downstream signaling cascade, stimulate
the release of cytokines and chemokines, such as tumor necrosis factor-α (TNF-α),
interleukin-1β (IL-1β), IL-6 and interferon-β (IFN-β), and upgrade immune cell
49
expression.9 Although the immunostimulatory activity of lipid A can be extremely useful,
its pro-inflammatory and septic properties are a serious problem.10
Regardless of the source, all lipid As contain the highly conserved construct as
illustrated in Figure 3.2. They have several lipids, varying considerably in chain length,
saturation, number and distribution, linked to the N-2-, N-2’-, O-3- and O-3’- positions and
two phosphate groups linked to the O-1 and O-4’- positions of the β-1,6-linked
disaccharide of D-glucosamine (GlcNH2).
Figure 3.2 An example of lipid A structure.
Biological assays demonstrated that the number, structure and position of lipids
and the degree of phosphorylation are important factors determining the bioactivity of lipid
A.2,3 To learn the structure-activity relationships of lipid A, studies were performed using
50
a murine macrophage cell line and human peripheral monocytes. The mediator-inducing
capacity of various lipid A derivatives carrying synthetic partial structures were
quantitatively analyzed. The results are schematically summarized in Figure 3.3.2-4
Figure 3.3 Schematic representation of lipid A structure-activity relationships.
As showed in Figure 3.3, once one of the phosphoryl groups in lipid A was
removed, the bioactivity was reduced significantly (by a factor of 102 as compared to
parent lipid A). The resulting monophosphoryl lipid A (MPLA) derivatives still had strong
immunostimulatory ability but much reduced toxicity, thus they can be very useful. The
monosaccharide derivatives had the least activity (reduction of activity by a factor of more
than 107). In addition, substituting the α-anomeric phosphate group with the β-anomer
significantly reduced the bioactivity, and modifications in the hydrophobic region of lipid
chains had dramatic effects as well.2-4
The number of lipid chains also affected the bioactivity significantly.2-4,11 A typical
lipid A that exhibits the full spectrum of immunological and endotoxic activities consists of
51
six lipids that can be distributed symmetrically (3+3), such as in the lipid A of Neisseria
meningitides, or asymmetrically (4+2), such as in the lipid A of Escherichia coli, on the
two GlcNH2 units. Abstraction of one acyl group (resulting in pentaacyl lipid A) or addition
of a fatty acid (resulting in heptaacyl lipid A) reduced the bioactivity by a factor of 102.
However, abstraction of two acyl groups (resulting in tetraacyl lipid A) caused complete
loss of the mediator-inducing capacity (a factor of > 107). Therefore, tetraacyl lipid A was
an antagonist for the signaling activation rather than an agonist. The hydroxide group in
the lipid chains also affect the immunostimulatory activity of lipid A by about a factor of
101.2,4
To further investigate the action mechanisms and the structure-activity
relationships of lipid A, its interaction with the toll-like receptor 4 (TLR4)-MD2 signaling
pathway that is activated by LPS or lipid A binding was analyzed.12 Most lipid A molecules
can be detected at picomolar levels by TLR4, a receptor of the innate immune system,
present on macrophages and endothelial cells. TLR4 is a class 1 transmembrane
receptor with an extracellular domain, a single membrane spanning helix and a globular
cytoplasmic domain, the TIR (Toll interleukin-1 receptor) domain. Another important lipid-
binding protein is MD2 which can form a TLR4-MD2 heterodimer.13 Binding of LPS to
TLR4-MD2 is mediated by the intercalation of the acyl chains of lipid A into the
hydrophobic core of the MD2 β-sandwich pocket that is similar to that formed by the
immunoglobulin domains of antibody molecules.11-19
Generally, hexa-acyl lipid A is a TLR4 agonist, whereas tetra-acyl lipid A that has
different binding conformation with MD2 is an antagonist.18,19 For the tetra-acyl lipid A,
two of the acyl chains, but four for hexa-acyl lipid A, can fully extended into the binding
52
pocket of MD2, while two are bent in the center. The tetra-acyl lipid A-MD2 ligand does
not induce a conformational change in the receptor, which makes it an antagonist. The
rest of lipid A, including the diglucosamine backbones, is fully exposed.17 The MD2-LPS
complex binds TLR4 to form a higher order structure, creating an activation cluster that
stimulates the following MyD88-dependent signal pathway. This signal pathway finally
leads to production of inflammatory cytokines such as IL-12, IL-1, IL-6 and TNFα.20
In conclusion, LPS, especially its lipid A domain, has potent immunostimulatory
ability that can be very useful for various immunological studies and immunotherapeutic
applications. However, it is worth mentioning that the structure-activity relationship
studies of lipid A were performed in vitro instead of in vivo.
3.1.3 Monophosphoryl lipid A
From the structure-activity relationship investigations described above, it is clear
that the two phosphate groups in lipid A are essential for its endotoxic activity. However,
modification of its O-1-position, for example, removal of the phosphate group at this
position to generate monophosphoryl lipid A (MPLA), was shown to greatly reduce its
endotoxicity (at most only 0.08% of toxicity remains)21 with limited impact (by a factor of
102)4 on its immunostimulatory activity. As a result, MPLA has emerged as a potential
adjuvant used in human vaccine formulation.22 The clinical grade of MPLA, called MPL
adjuvantTM manufactured by GlaxoSmithKline showed approximately 0.1% the toxicity of
LPS in pre-clinical rabbit pyrogenicity assays.23 MPL adjuvant is usually added to an
adjuvant system, such as alum and water-oil form adjuvant, instead of replacing the other
adjuvant. For example, MPL adjuvant has been widely used in adjuvant systems, such
as AS01, AS02, and AS04. AS04 is a licensed adjuvant system containing MPLA and
53
alum, which elicits a major immune reaction including antibodies and Th1 immune
responses (Table 3.1).
Table 3.1 Adjuvant types and their mechanisms of action
Adjuvant
type
Examples
Mechanisms of action
Type A
MPLA
Agonists for TLRs, indirectly, activating APCs and triggering the
secretion of cytokines22
Type B
Alum, MF59, liposomal
adjuvants
Non-specific adjuvant, enhancing antigen presentation by building
a depot at the injection site, leading to a high local antigen
concentration and improving uptake by APCs24,25
Type C
Freud’s adjuvant,
nanoparticles, toxin-derived
adjuvant, flagellin
Enhancing signal, co-stimulatory molecular, a novel super agonist,
directly stimulating T cells, inducing cytokine storm26
3.1.4 Introduction to adjuvants
Adjuvants are materials that can improve immune responses. This adjuvant idea
was first recognized by William Coley who employed bacterial products to treat cancer
patients.3 Typically, there are three types of adjuvants (Table 3.1). MPLA (formulated in
AS04) is a type A adjuvant that stimulates TLR4 signal pathway and triggers a polarized
Th1 response and has been approved for clinical application in Europe.27,28
Currently, there are four licensed adjuvants in human clinical application: Alum,
MF59, AS03, and AS04.29 Despite that these adjuvants have been widely utilized in clinic
(Table 3.2), there is still an urgent need of new adjuvants for usage with relatively
immunologically challenging vaccine, such as cancer vaccines. More importantly, new
54
adjuvants are needed for the design and development of novel constructs of vaccines.
There are also commercial adjuvants, such as Freund’s complete/incomplete adjuvant
(CFA/IFA), Titermax Gold adjuvant, QS21 and so on, which are widely used in animals
and in research laboratories but not in human.27
Table 3.2 Licensed adjuvants28,30
Adjuvant
Immune active
component
Pattern recognition
receptors (PRRs)
Major immune
response
Vaccine
Alum
Aluminum salts
NLRPs inflammasome
Ab. Th2(mice) Ab.
Th1+Th2 (humans)
Various
MF59
Squalene in emulsion of
oilwater
Pro-inflammatory
responses
Ab. Th1+Th2
Influenza/
pandemic flu
AS03
Squalene in emulsion of
oil-water + a tocopherol
Pro-inflammatory
responses
Ab. Th1+Th2
Pandemic
influenza
AS04
MPL +Alum
TLR4+NLRP3
inflammasome
Ab. Th1
HBV (Fendrix),
HPV (Cervarix)
Liposomes
Oil-water emulsion
HAV, Flu
Ab: antibodies; Alum: aluminum hydroxide; AS03: Adjuvant System 03; AS04: Adjuvant System 04; HBV:
hepatitis B virus; HPV: human papillomavirus; MPL: monophosphoryl lipid A; NLRP3: nucleotide-binding
oligomerization domain, leucine-rich repeat and pyrin domain containing 3; Th: helper cells; TLR4: Toll-like
receptor 4.
3.1.5 MPLA-based full-synthetic vaccine
In addition to the advantages of semi-synthetic glycoconjugate vaccine described
in chapter 1, full-synthetic vaccines with a well-defined structure have more improvements.
55
Firstly, glycan-protein conjugates usually have non-consistent loading of saccharides
which induces the difficulties in quality control. But the full-synthetic vaccines will have
consistent composition. In addition, the protein conjugates always need adjuvant for
stimulating the immune response, while the full-synthetic vaccines could have a built-in
adjuvant which will be more convenient to preparation and be further improved in vaccine
design. With the help from the technology of organic synthesis, the full-synthetic vaccines
have much stronger potential to provide immunological prevention against diseases.
Therefore, based on the direction of full-synthetic vaccine, one of our research
projects has focused on the development of fully synthetic TACA-based cancer vaccines
using MPLA as a carrier. We have been interested in MPLA as a carrier because of its
attractive immunostimulatory properties. We anticipated that MPLA would be able to not
only improve the immunogenicity of tumor-associated carbohydrate antigens used for
vaccine development but also act as a built-in adjuvant to formulate self-adjuvanting
vaccines. The new vaccine design was to have the carbohydrate antigen linked to the
non-reducing end of MPLA through a spacer as shown in Figure 3.4.
Figure 3.4 A new construct of full-synthetic vaccine based on MPLA
56
In our previous investigation, a MPLA analog of the lipid A structure derived from
Neisseria meningitides, was coupled to a GM3 antigen, which is overexpressed on
melanoma cancer cell surface, and its synthetic analog N-phenylacetyl GM3
(GM3NPhAc).31,32 The resulting conjugates, such as the MPLA-GM3NPhAc conjugate
illustrated in Figure 3.5, were evaluated as vaccines that can be used for cancer
immunotherapy based on cell surface sialic acid metabolic engineering.31,33-37 It was
revealed that these conjugates alone could induce robust immune responses in animals
in the absence of an external adjuvant (Figure 3.5), suggesting that, as an
immunostimulant, MPLA was functional not only as a vaccine carrier but also as a built-
in adjuvant.31
Figure 3.5 The structure of MPLA-GM3NPhAc and immune results.
Encouraged by the above interesting discoveries, the present work aimed at: (1)
further optimizing the lipid A structure as a vaccine carrier and built-in adjuvant, thus a
series of MPLA derivatives were designed and synthesized for systematic structure-
activity relationship analysis in the context of conjugate vaccines, and (2) developing new
57
fully synthetic carbohydrate-based anticancer vaccines with the optimized lipid A as the
carrier molecule.
58
3.2 Evaluation of MPLA-sTnNPhAc Conjugates as Cancer Vaccines and
Optimization of the MPLA Structure as a Vaccine Carrier
3.2.1 Introduction
Sialyl-Tn (sTn, Figure 3.6) is a mucin-associated carbohydrate antigen that is
overexpressed by a variety of tumors, such as breast, prostate, colorectal, ovarian, gastric
and pancreatic carcinoma rather than normal cells,38-54 and it is relatively cancer-specific
as it is rarely expressed on normal cells. In addition, the expression of sTn antigen is an
independent indicator for cancer diagnosis.39,41,43 Moreover, sTn antigen is a predictor of
metastatic potential which associates with more aggressive cancer status in breast
cancer.38,39 Therefore sTn is an attractive target for anti-cancer vaccine and
immunotherapy development. In 2003, a semi-synthetic cancer vaccine, Theratope®,
was developed by Biomira Inc., which had the sTn antigen linked to keyhole limpet
hemocyanin (sTn-KLH conjugate). This vaccine was successful in phase I and II clinical
trials with excellent results in stimulating antibody- and cell-mediated immune responses
against the tumor-associated antigen, but eventually failed in phase III clinical trial,
because it eventually failed to induce robust immune responses in cancer patient.55-61
Figure 3.6 The structure of sTn antigen
59
Inspired by the results of MPLA-GM3 conjugates31, here we have designed and
synthesized four monophosphoryl analogs (including one from previous exploration) of
the lipid A of N. meningitides, containing lipids of different chain lengths and linkages as
well as different patterns of lipidation (Figure 3.7). To investigate the immunological
properties of these MPLA derivatives as vaccine carriers and adjuvants, we conjugated
them with N-phenyl acetyl sTn (sTnNPhAc), a modified TACA that can be further used in
our immunotherapy strategy based on glycoengineering, and examined in mice the
specific immune responses induced by the resulting MPLA-sTnNPhAc conjugats 3.1-3.4
(Figure 3.7). Through these studies, we anticipated to identify novel vaccine carriers and
adjuvants with improved immunological properties useful for the development of fully
synthetic glycoconjugate cancer vaccines.
Figure 3.7 The structures of designed MPLA derivatives and their sTn conjugates.
3.2.2 Results and Discussion
Immunological investigation of the MPLA-sTnNPhAc conjugates 3.1-3.4 was
carried out with female C57BL/6J mouse, a well-characterized inbred strain for vaccine
60
evaluation and comparable to the human in immune reactions.62 To improve the solubility
of these conjugates in buffer, they were incorporated in liposomes formed by using 1, 2-
distearoyl-sn-glycero-3-phosphocholine and cholesterol. Delivering glycoconjugate
vaccines in liposomal forms can further improve their immunogenicity.63,64 For
immunization, the liposomal preparations of 3.1-3.4 were each injected subcutaneously
(s.c.) to a group of six mice. In the meantime, groups of mice were simultaneously
inoculated with emulsions of 3.1-3.4 liposomes and Titermax Gold adjuvant to evaluate
the potential influence of an external adjuvant on the activities of these glycoconjugate
vaccines. The adopted vaccination schedule was to inject into each mouse 0.1 mL of a
vaccine preparation containing ca. 3 µg of sTnNPhAc on day 1, 14, 21 and 28,
respectively. Each mouse was subjected to bleeding on day 0 before the first injection
(used as blank controls) and on day 27 and 38 after immunization. The blood samples
were treated according to standard protocols to prepare antisera for the analysis of
sTnNPhAc-specific antibodies by enzyme-linked immunosorbent assay (ELISA) with the
human serum albumin (HSA) conjugate of sTnNPhAc (sTnNPhAc-HSA)35 as a capture
antigen. In addition to total antibodies, antibody isotypes such as IgG1, IgG2a, IgG3 and
IgM were also individually assessed. Antibody titers were calculated from linear
regression analysis of the curves of the optical density (OD) value against serum dilution
number, and were defined as the dilution number yielding an OD value of 0.2.31
The ELISA results of antisera obtained with 3.1 without the use of an external
adjuvant (Figure 3.8A) revealed that the conjugate itself provoked a strong sTnNPhAc-
specific immune response in mice. More importantly, in addition to IgM antibody, a high
titer of IgG3 and some IgG1 antibodies were also observed, suggesting a T cell-
61
dependent immune response.65,66 Interestingly, 3.1 plus Titermax Gold failed to stimulate
a significant antibody response (Figure 3.8B), although IgM antibodies were observed.
These results suggest that the external adjuvant had an inhibitory effect on the T cell-
mediated immune response against 3.1 but a relatively small influence on the B cell
response. The results agreed well with our previous report about other MPLA-TACA
conjugates.31
The ELISA results of 3.2 (Figure 3.8C and 3.8D) were similar to that of 3.1 in terms
of the type of immune responses provoked and the influence of Titermax Gold. However,
the IgM and IgG antibody titers for 3.2 were much higher than that for 3.1. Moreover, 3.2
stimulated a strong immune response in all of the mice. Another interesting finding was
that 3.2 also induced a significant level of IgG1 antibody which is usually observed with
neoglycoprotein vaccines. The ELISA results of 3.3 (Figure 3.8E and 3.8F) and 3.4
(Figure 3.8G and 3.8H) were similar to that of 3.1 and 3.2, but their antibody titers were
lower than that of 3.2 and higer than that of 3.1. In all of these cases, Titermax Gold
showed inhibition of immune responses against the conjugate vaccines.
62
Figure 3.8 ELISA results of day 38 antisera of mice immunized with 3.1 alone (A), 3.1
plus Titermax Gold (B), 3.2 alone (C), 3.2 plus Titermax Gold (D), 3.3 alone (E), 3.3 plus
63
Titermax Gold (F), 3.4 alone (G) and 3.4 plus Titermax Gold (H), respectively. The titers
of various sTnNPhAc-specific antibodies are displayed. Each dot represents the antibody
titer of an individual mouse, and the black bar shows the average antibody titer of a group
of six mice.
To further verify that the detected antibodies were indeed sTnNPhAc-specific, a
competitive ELISA experiment for the pooled mouse antisera obtained with 3.2, 3.3, 3.4
and 3.2 plus adjuvant was performed. In this study, various concentrations (0, 0.00152,
0.0152, 0.152, 1.52, 15.2 and 152 µM) of free sTnNPhAc were added to compete with
sTnNPhAc-HSA attached to the ELISA plate for antibody binding. Again, alkaline
phosphatase-linked goat anti-mouse kappa antibody was employed as the secondary
antibody. The OD values at 405 nm reflected the levels of total antibodies bound to the
sTnNPhAc-HSA-coated plates. These studies (Figure 3.9) have revealed that sTnNPhAc
had concentration-dependent inhibition of antibody binding to the plates and that, at
concentrations higher than 152 µM, sTnNPhAc could essentially completely inhibit the
binding, proving that the elicited antibodies were indeed specific to the sTnNPhAc antigen.
64
Figure 3.9 Competitive ELISA results of the pooled mouse antisera obtained with
conjugates 3.2, 3.3, 3.4 and 3.2 plus adjuvant. The OD values at 405 nm reflected the
levels of total antibodies bound to the sTnNPhac-HSA-coated ELISA plates in the
presence of specified concentrations of sTnNPhAc.
The above ELISA results disclosed that the MPLA conjugates 3.1-3.4 alone, that
is, in the absence of any external adjuvant, provoked a robust antigen-specific immune
response in mice. More importantly, they stimulated high titers of IgG3 and IgG1
antibodies, indicating a T cell-mediated immune response desirable for cancer
immunotherapy.65,66 Therefore, all of the synthetic MPLA derivatives could act both as a
vaccine carrier and as an adjuvant to effectively enhance the immunogenicity of
sTnNPhAc and to efficiently promote a T cell-dependent immune response toward
TACAs.20,31,67 These properties of MPLA should be particularly useful for the design and
development of new fully synthetic carbohydrate-based conjugate vaccines.
0
0.5
1
1.5
2
2.5
3
0.00152 0.0152 0.152 1.52 15.2 152
3.2
3.3
3.4
3.2 + adjuvant
sTnPhAc concentration (μM)
OD (405 nm) valune
65
Titermax Gold is a commercial adjuvant that is commonly used with vaccines such
as carbohydrate-protein conjugates37 to improve their immunogenicity. However, we
showed in this work that it had an inhibitory impact on the immunological activities of 3.1-
3.4. As Titermax Gold mainly affect the T cell-mediated immune response. We proposed
that Titermax Gold might interact with MPLA in the conjugate vaccine to prevent MPLA
from binding to cell surface TLRs and affect its engagement in T cell-mediated
immunological pathways. Another hypothesis is that Titermax Gold might interact with
MPLA to affect vaccine delivery to the lymph system or antigen presenting cells (APCs)
required for effective T cell-mediated immunity.68,69 This discovery and further detailed
studies may help understand vaccine adjuvants, such as their binding sites, functional
mechanisms and so on.
Despite the fact that 3.1-3.4 stimulated similar patterns of immune responses in
mice, they did show differences in immunological activity. For example, conjugate 3.2
elicited much higher and more consistent titers of both total antibodies and IgG2 antibody
than 3.1, 3.3 and 3.4 (Figure 3.9). Moreover, all of the mice showed strong immune
responses to 3.2 but not to other conjugates. Clearly, 3.2 was the best vaccine among
the MPLA conjugates investigated.
Structurally, 3.1-3.4 were different only in their MPLA moiety, and their antisera
were obtained by means of the same immunization protocol and schedule. Consequently,
any difference in their immunological activity should reflect the impact of the MPLA
structure. Conjugate 3.2 contained the monophosphoryl form of natural lipid A of N.
meningitides (H44/76 strain).70 In 3.1, the two free hydroxyl groups on the lipid chains of
66
the MPLA moiety were removed; 3.3 and 3.4 were different in that their MPLA contained
different lengths and different numbers of lipid chains than that of 3.2.
As all these conjugates had essentially the same immunological profile, it seems
that the free hydroxyl groups on the lipid chains and the length and number of lipid chains
of MPLA had a quantitative, rather than qualitative, impact on its biological activities. As
discussed in 3.1.2 with Figure 3.3, it is clear that the hydroxyl groups on the lipid chains
play an important role in the lipid A interaction with its receptors so that the endotoxin of
lipid a has a reduce factor about 10 comparing with natural form. Although the level of
antibody titers is not exactly the same as the endotoxin activities of MPLA, the decreasing
trend of immunostimulatory activity of MPLA in conjugate 3.1 compared with that in 3.2
agrees with the trend of lipid A’s endotoxin activities. Additionally, elongating the length
of lipids at the 3-O- and 3’-O- positions (conjugate 3.3) seem to have a relatively small
impact, although such changes did result in reduced immunological activity and some
inconsistence of immune responses in individual mice. Usually, as described in 3.1.2,
lessened or additional one lipid chains linked to these lipids have a reduced impact of
endotoxin activities about 102.2 What’s more, lipid A totally loses its endotoxin activity to
be a LPS antagonist if two lipid chains are removed.4 In our case, the additional two lipid
chains linked to these lipids as an octa-lipid chains MPLA is not like tetra-lipid chains one
that loses immunological activity, but helps to elicit a considerable high level of antibody
titers (conjugate 3.4) which only a little lower than that of 3.2.
67
3.2.3 Conclusion
In summary, four MPLA derivatives were coupled with sTnNPhAc to form fully
synthetic glycoconjugate cancer vaccines. Studies on the resulting MPLA-sTnNPhAc
conjugates revealed that they elicited strong and T cell-dependent immune responses
without the use of any external adjuvant. Our previous work revealed that antisera derived
from mice immunized with MPLA conjugates could effectively bind to and kill cancer cells
metabolically engineered to express the corresponding antigen.31 MPLA has thus been
demonstrated to be a useful platform for the development of new vaccine carriers and
adjuvants and for the development of novel types of fully synthetic carbohydrate-based
cancer vaccines with self-adjuvanting properties. Our results have also revealed that
MPLA derivatives containing six lipid chains exhibited more potent immunostimulatory
activities than those with eight lipid chains (conjugate 3.4) and that the lipid structure and
length had a significant impact on the immunology of MPLA. The monophosphoryl form
of natural N. meningitides lipid A was found to have the most promising immunological
properties and its sTnNPhAc conjugate elicited the most potent and the most consistent
T cell-dependent anti-sTnNPhAc immunity. As a result, the MPLA moiety in 3.2 was
identified as the first generation of optimized vaccine carriers and adjuvants that is under
further optimization and additional investigation.
On the other hand, Titermax Gold was found to inhibit the immunological activity
of MPLA-sTnPhAc conjugates, whereas it has the opposite influence on the activity of
protein-sTnNPhAc conjugates.33,35 It is proposed that Titermax Gold may interact with
MPLA to affect its binding to cell surface receptors and/or its delivery to the lymph system
or antigen presenting cells. It is anticipated that these issues may be clarified by studies
68
utilizing labeled MPLA derivatives and conjugates, the results of which should be useful
for understanding the immunostimulatory and adjuvant activities and the functional
mechanisms of MPLA and Titermax Gold, which is a very important topic in cancer
vaccine immunology.
3.2.4 Experimental Section
Materials, Reagents, and Animals
Glycoconjugates 3.1, 3.2, 3.3 and 3.4 conjugates were synthesized by Mohabul
Mondal. STnNPhAc-HSA conjugate were synthesized according to the procedures
previously reported by our laboratory. Titermax Gold adjuvant, 1, 2-distearoyl-sn-glycero-
3-phosphocholine, and cholesterol were purchased from Sigma-Aldrich. The SKMEL-28
and B16F0 cancer cell line, Dulbecco’s Modified Eagle’s Medium (DMEM) for cell culture,
and fetal bovine serum (FBS) were purchased from American Type Culture Collection.
Penicillin-streptomycin and trypsin-EDTA were purchased from Invitrogen.
Alkaline phosphatase linked goat anti-mouse kappa, IgM, IgG1, IgG2a, and IgG3
antibodies and FITC-labeled goat anti-mouse kappa antibody were purchased from
Southern Biotechnology. Female C57BL/6 mice of 6-8 weeks age used for immunological
studies were purchased from the Jackson Laboratory.
General Procedure for the Preparation of Liposomes of Glycoconjugates 3.1-3.4
The mixture of a specific MPLA conjugate 3.1-3.4 (60 μg of sTnNPhAc moiety,
0.102 μmol), 1, 2-distearoyl-sn-glycero-3-phosphocholine (M.W. 790.15, 0.52 μg, 0.664
69
μmol), and cholesterol (M.W. 386.66, 0.197 μg, 0.51 μmol) (a 10:65:50 molar ratio) was
dissolved in CH2Cl2 and MeOH (1:1, v/v, 2 mL) in a 10 mL vial. Then, the solvents were
removed in vacuum to form a thin lipid film on the vial wall, which was hydrated by adding
2.0 mL of HEPES buffer (20 mM, pH 7.5) containing NaCl (150 mM) and shaking the
mixture in 40 ⁰C water bath, and then shaking it by vortex mixer for several times to form
a milky suspension. The milky suspension was finally sonicated for 1 min to obtain the
desired liposomes.
General Procedure for the Preparation of Emulsion of liposomes and Adjuvant
The prepared liposomes with double concentration were mixed with Titermax Gold
adjuvant (1:1, v/v) and to form an emulsion according to the manufacturer’s protocol.
Immunization of Mouse
Each group of six female C57BL/6 mice were immunized on day 1 by
subcutaneous (sc) injection of 0.1 mL of the liposomal solution of a specific
glycoconjugate 3.1-3.4 containing 3 μg of the carbohydrate antigen (5.1 nmol) or by
injection of an emulsion of the liposomal solution of a specific glycoconjugate vaccine and
Titermax Gold adjuvant prepared according to the manufacturer’s protocol (each 1 mL of
the liposomal solution mixed with 1 mL of Titermax Gold adjuvant). Following the initial
immunization, mice were boosted 3 times on day 14, day 21 and day 28 by sc injection
of the same conjugate and by the same immunization protocol. Blood samples of each
mouse were collected through the leg veins prior to the initial immunization on day 0 and
after immunization on day 27 and day 38 and were clotted to obtain antisera that were
stored at -80 ⁰C before use.
70
Protocols for ELISA
ELISA plates were treated with 100 μL of a solution of sTnNPhAc-HAS conjugate
(2 ug/ml) dissolved in coating buffer (0.1 M bicarbonate, pH 9.6) at 4 ⁰C overnight, and
then at 37 ⁰C for 1 h, which was followed by treatment with blocking buffer (10% BSA in
PBS solution with NaN3) and washing 3 times with phosphate-buffered saline (PBS)
containing 0.05% Tween-20 (PBST). Thereafter, a pooled or an individual mouse
antiserum with serial half-log dilutions from 1:300 to 1:656100 in PBS was added to the
coated ELISA plates (100 μL/well), which was followed by incubation at 37 ⁰C for 2 h. The
plates were then washed with PBS and incubated at rt for another 1 h with a 1:1000
diluted solution of alkaline phosphatase linked goat anti-mouse kappa, IgM or IgG2a
antibody or with a 1:2000 diluted solution of alkaline phosphatase linked goat anti-mouse
IgG1 and IgG3 antibody (100 μL/well), respectively. Finally, these plates were washed
with PBS and developed with 100 μL of p-nitrophenylphosphate (PNPP) solution (1.67
mg/mL in buffer) for 30 min at rt, followed by colorimetric readout using a BioRad 550
plate reader at 405 nm wavelength. The optical density (OD) values were plotted against
antiserum dilution values, and a best-fit line was obtained. The equation of the line was
employed to calculate the dilution value at which an OD of 0.2 was achieved, and the
antibody titer was calculated at the inverse of the dilution value.
Protocols for Competitive ELISA
The protocols for competitive experiment had the same process as ELISA of
antibody investigation described above. The only difference was that free sTnNPhAc
saccharide was added to compete with antibody binding of plate-bound sTnNPhAc-HSA.
Hence, the ELISA plate was coated with sTnNPhAc-HSA in 4 ⁰C overnight and then
71
incubated in 37 ⁰C for 1 h. After washed with PBS buffer, the plate was treated with
blocking buffer in rt (room temperature) for 1 h. After that, the plate was washed with PBS
and then, added 50 μL PBS solution containing 0, 0.003, 0.03, 0.30, 3.0, 30.4 and 304
μM of free sTnNPhAc and 50 μL of pooled antisera (1:150 dilutions) in PBS to each well
(the final concentrations of free sTnNPhAc were 0, 0.0015, 0.015, 0.15, 1.5, 15.2 and 152
μM in 1:300 diluted pooled antisera solution) at the same time, and incubated in 37 ⁰C for
2 h. Then, the plate was washed and incubated with alkaline phosphatase linked goat
anti-mouse kappa antibody (1:1000 dilution), washed, reacted with PNPP solution and
finally collected the reading at 405 nm as described above.31
72
3.3 MPLA Carrier Applied in Globo H-Based Vaccine
3.3.1 Introduction
Globo H (GH) is a hexasaccharide specifically overexpressed on a number of
cancer cells, such as breast, lung, ovary, stomach and small-cell lung cancer cells but
weakly expressed on normal cells.71-76 It was first discovered from human breast cancer
cell line MCF-7 in 1983 by Hakomori.77-79 The structure of Globo H is showed in Figure
3.10 which was first assigned by Hakomori80 and colleagues and was fully synthesized
by several groups.79,81-84 The immunocharacterization of Globo H attracted interests in
this antigen which was studied via the monoclonal antibodies (mAb) MBr1 and VK9.78,85
As a TACA described before, Globo H is an attractive target for the development
of an immunotherapy and anti-cancer vaccines against a variety of cancer such as breast
carcinoma, small-cell lung carcinoma, and several malignant cancers. Base on the
structure of Globo H, a therapeutic cancer vaccine, full-synthetic Globo H conjugated to
keyhole limpet hemocyanin (KLH) with adjuvant QS-21, against breast and prostate
cancer, showed hopeful results in clinical trials.79,83,86 This vaccine was now in Phase III
clinical trial with Globo H synthesized by the one-pot method.84
73
Figure 3.10 Structure of Globo H
To further improve the efficiency of Globo H conjugated vaccine, in Chi-Huey
Wong’s group, Globo H was conjugated to different carrier proteins including KLH,
diphtheria toxoid cross-reactive material (CRM) 197 (DT), tetanus toxoid (TT), and BSA
with the combination of various adjuvants such as QS-21 and α-galactosylceramide
C34.81 The results showed that, compared with the phase III clinical trial vaccine, Globo
H-KLH, the Globo H-DT with C34 adjuvant vaccine elicited higher antibody titer of IgG
isotype which has high selectivity for not only Globo H but also the Globo H related
epitopes, including stage-specific embryonic antigen 3 (SSEA3) and SSEA4. In additional,
they developed a novel vaccine candidate, SSEA4-DT with C34 adjuvant, which can
stimulate strong immune response with high IgG antibodies which are highly specific for
SSEA4 antigen.81
In our group, Globo H was successfully synthesized which can be further linked
with carriers as anti-cancer vaccine. Based on the previous investigation, MPLA showed
excellent characteristics as a build-in carrier and adjuvant. Therefore, the MPLA was
applied in Globo H-based anti-cancer vaccine design. The structure of Globo H-KLH (3.6)
74
and Globo H-MPLA (3.7) were showed in Figure 3.11.This Globo H-MPLA conjugate
contains the MPLA moiety which is the structure used in conjugate 3.2 which has the best
result in our above evaluation.87
Figure 3.11 The structure of Globo H-KLH (3.6) and Globo H-MPLA (3.7)
3.3.2 Results and Discussion
Immunological investigation of Globo H-KLH (3.6) and Globo H-MPLA (3.7)
conjugates was carried out with female C57BL/6J mouse. The KLH conjugate was
dissolved in PBS solution and then mixed with Freund’s complete adjuvant (CFA) as an
emulsion to immunize mice. To improve the solubility of the MPLA conjugate and also
improve the immunogenicity of vaccine, the MPLA conjugate was incorporated in
liposomes formed by using 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol
the same as described in previous MPLA-sTnNPhAc conjugate project.63,64 For
immunization, the adjuvant preparation of 3.6 and the liposomal preparation of 3.7 was
injected subcutaneously (s.c.) to a group of six mice. The inoculation schedule was to
inject into each mouse 0.1 mL of a vaccine preparation containing ca. 3 µg of Globo H
75
moiety on day 1, 14, 21 and 28, respectively. The blood of each mouse was collected on
day 0 before the first inoculation (used as blank controls) and on day 27 and 38 after
immunization. The blood samples were used to prepare antisera according to the protocol.
Then the antisera were further analyzed by ELISA the same as previous described in
MPLA-sTnNPhAc project. Globo H-HSA was used as capture reagent, and goat anti-
mouse antibodies with AP enzyme were used as second antibodies. The total antibody
titer and the antibody titers of each isotype, IgG1, IgG2a, IgG2b, IgG3 and IgM were
calculated from the curves of the optical density (OD) value against serum dilution number,
and the titers were defined as the dilution number yielding an OD value of 0.1.31,87
The ELISA results of antisera induced by 3.6 with CFA as an external adjuvant
(Figure 3.12) indicated that Globo H-KLH (3.6) conjugate provoked a strong Globo H-
specific antibody response (kappa: about 38,000) in mice. Moreover, the high antibody
titer (average about 35,000) of IgG1 and some positive antibody titer of IgG2b and IgG3
suggested that T cell-dependent immunity was involved. Generally, six out of six mice
had strong Globo H specific antibody titers which means every mouse had been
immunized successfully. In addition, five out of six mice had high IgG1 antibody titer.
The ELISA results of antisera elicited by Globo H-MPLA (3.7) had similar antibody
response (the average total antibody titer was about 60,000) including high antibody titer
of IgG1 isotype ( about 64,000) which indicated that Globo H-MPLA (3.7) had excellent
immunogenicity to stimulate immune response especially T cell immunity. Excitingly, the
total antibody and IgG1 isotype of Globo H-MPLA (3.7) had even higher antibody titer
than that elicited by Globo H-KLH (3.6) conjugate. Additionally, five out of six mice (one
had 8741 much lower than others) in this group had been successfully immunized by
76
conjugate 3.7 indicated by the high kappa antibody titer. And, five out of six mice (one
had low antibody titer 3853) in this group had high antibody titer of IgG1 isotype.
Figure 3.12 ELISA results of day 38 antisera of mice immunized with 3.6 plus CFA
adjuvant (A) and 3.7 in liposome form (B), respectively. The titers of Globo H specific
antibodies are displayed. Each dot represents the antibody titer of an individual mouse,
and the black bar shows the average antibody titer of a group of six mice.
These results suggested that the MPLA conjugate had an excellent potential for
design as a build-in adjuvant and carrier. This phenomenon was discovered here that a
small molecule vaccine can work as well as large molecule vaccine with protein carrier.
What’s more, the protein carrier usually with T cell epitopes can help provoked T cell
immunity like the results here provoked by Globo H-KLH (3.6) (Figure 3.12). However, in
our new discovery, MPLA, usually considered as a TLR-based adjuvant,18,19 can serve as
a stimulator to elicit T cell-mediate immunity as well.
kapp a
Ig G 1
Ig G 2 a
Ig G 2 b
Ig G 3
Ig M
0
50000
100000
150000
Is o t y p e
A n t ib o d y T ite r
k a p p a
Ig G 1
Ig G 2 a
Ig G 2 b
Ig G3
Ig M
0
20000
40000
60000
80000
Is o t y p e
A n t ib o d y T it e r
(A)
(B)
3.6 + CFA
3.7 liposome
77
Figure 3.13 The kinetic ELISA results of antisera induced by conjugates 3.6 and 3.7.
ELISA results of Globo H-specific antibody in the antisera pooled from mice immunized
with (A) conjugate 3.6 plus CFA adjuvant and (B) 3.7 in liposome form, respectively, on
day 21 (blue line), day 27 (red line) and day 38 (green line). X-axis is the diluted factor
from 300 to 8100 in logarithmic scale, and y-axis is the OD value at 405 nm reading each
well after 30 min PNPP reaction. (C) Calculated antibody titer of pooled antisera obtained
from mice immunized with 3.7 in liposome form on day 0, day 14, day 21, day 27 and day
38.
The time courses of the induction of total antibody pooled from six mice immunized
by 3.6 or 3.7 were shown in Figure 3.13. The antibody titers generally increased with the
more immunization boost. Day 27 antisera were obtained after three boosts and day 38
were obtained after four boosts. Three boosts of conjugates both 3.6 and 3.7 can already
0
10000
20000
30000
40000
50000
60000
d0 d14 d21 d27 d38
Antibody Titer
3.7 liposome
(C)
78
elicit significant immune response. The fourth boost could improve the immune response
significantly, nearly twice higher than day 27 result. Therefore, both the vaccines required
four times immunization injection.
Cell surface reactivity of these antisera obtained from mice immunized with 3.6
plus CFA adjuvant or 3.7 in liposome form was evaluated by fluorescence-activated cell
sorting (FACS) technology on Globo H positive MCF-7 cell line (Figure 3.14) and Globo
H negative SKMEL-28 cell line as a negative control (Figure 3.15). In these studies, both
kinds of cells were incubated with normal mouse sera or pooled antisera obtained from
mice immunized with 3.6 or 3.7, respectively. Thereafter, the cells were cultured with
fluorescein isothiocyanate (FITC)-labeled goat anti-mouse kappa antibody, followed by
FACS analysis.
79
Figure 3.14 FACS results of MCF-7 cells. (A) MCF-7 cell without any treatment; (B) MCF-
7 cells stained with normal sera and FITC labeled 2nd antibody; (C) MCF-7 cells stained
with antisera obtained from mice immunized with 3.6 plus CFA adjuvant; (D) MCF-7 cells
stained with antisera obtained from mice immunized with 3.7 in liposome form; (E) Gated
cells percentage of positive cells and (F) Mean fluorescent intensity of cells stained with
normal sera and the other two antisera.
80
Figure 3.15 FACS results of SKMEL-28 cells. (A) SKMEL-28 cell without any treatment;
(B) SKMEL-28 cells stained with normal sera and FITC labeled 2nd antibody; (C) SKMEL-
28 cells stained with antisera obtained from mice immunized with 3.6 plus CFA adjuvant;
(D) SKMEL-28 cells stained with antisera obtained from mice immunized with 3.7 in
liposome form.
Figure 3.16 Overlay plots of FACS results of MCF-7 (A) and SKMEL-28 (B) cell lines
stained with normal sera (green), pooled antisera from mice immunized with 3.6 plus CFA
adjuvant (blue) and 3.7 liposome (red), respectively.
From the FACS results (Figure 3.16) of MCF-7 cell line and SKMEL-28 cell line
which were stained with normal sera and antisera obtained from mice immunized with
conjugate 3.6 and 3.7, the conclusion that both conjugate 3.6 and 3.7 can elicit strong
immune response and Globo H-specific antibodies was further confirmed. The MCF-7
cells with Globo H expressed on cell surface can be significantly stained with the antisera
81
containing Globo H-specific antibodies, while the SKMEL-28 cell had negative results.
What’s more, the gated cells percentage of positive cells of antisera induced by 3.7 was
significantly higher than that of antisera induced by 3.6 plus CFA adjuvant. This
phenomenon suggested that 3.7 conjugates may have better immunological
characteristics than that of conjugate 3.6 which was consistent with the results of ELISA.
In addition, the MFI of cells stained with antisera induced by 3.7 are significantly higher
than that of 3.6 which further confirmed the results of ELISA and gated cells percentage.
Therefore, both ELISA and FACS results confirmed that both conjugates can elicit strong
immune response and MPLA conjugate 3.7 may have even better results than KLH
conjugate 3.6.
3.3.3 Conclusion
In summary, both Globo H-KLH and –MPLA conjugates can provoke strong Globo
H-specific immune response, especially the T cell immunity. Moreover, Globo H-MPLA
was proved that have better capability to generate antibodies than that of KLH conjugate.
It’s the first time we discovered that small molecular glycoconjugate with MPLA carrier
had better immunological capability than that of glycoprotein. Although the mechanism of
how MPLA conjugate elicited stronger immune response was not clear, the MPLA-based
new construct of vaccine really had charming immunological properties, such as external
adjuvant-free and the ability of stimulate T cell-mediated immunity. Some of the Globo H-
KLH conjugate was already in phase III clinical trial for the therapy of breast and prostate
cancers. Therefore, our new construct vaccine, Globo H-MPLA which showed better
ELISA and FACS results than KLH conjugate, should have magnificent hope to be further
82
applied in clinical trial. For future directions, it is feasible and interesting to prepare Globo
H-specific monoclonal antibodies (mAbs) using in the further immunotherapeutic studies.
The improvement of the novel construct using MPLA as carrier is also another interesting
direction. The construct may be promoted by coupling with other components, such as T
cell epitopes, multivalent antigens and so on. What’s more, the successful strategy of
vaccine designs with a TLR ligands also can be moved forward to other TLR ligands,
lectin ligands and so on.
3.3.4 Experimental Section
Materials, Reagents, and Animals
Glycoconjugates 3.6, 3.7 and Globo H-HSA were synthesized by Guochao Liao.
Freund’s complete adjuvant (CFA), 1, 2-distearoyl-sn-glycero-3-phosphocholine, and
cholesterol were purchased from Sigma-Aldrich. The MCF-7 and SKMEL-28 cancer cell
line, Dulbecco’s Modified Eagle’s Medium (DMEM) for cell culture, and fetal bovine serum
(FBS) were purchased from American Type Culture Collection (ATCC). Penicillin-
streptomycin and trypsin-EDTA were purchased from Invitrogen.
Alkaline phosphatase (AP) linked goat anti-mouse kappa, IgM, IgG1, IgG2a, IgG2b
and IgG3 antibodies and FITC-labeled goat anti-mouse kappa antibody were purchased
from Southern Biotechnology. Female C57BL/6 mice of 6-8 weeks age used for
immunological studies were purchased from the Jackson Laboratory.
Protocol of vaccine preparation
83
The loading of Globo H-KLH is about 8%. According to KLH m.w. 350~400 kDa,
each KLH molecule has 29~33 Globo H residents.
Table 3.3 Dose of KLH conjugate 3.6 and MPLA conjugate 3.7
Conjugate
Globo H amount/ ug
Globo H amount/nmol
1 Dose amout/µg
30 Dose/µg
3.6
3
2.83
37.5
1125
3.7
6
5.7
16
500
Preparation of liposomes of glycoconjugates 3.7. The protocol was similar to that
reported in the literature.31,87 Briefly, the mixture of a specific conjugate 3.7 (0.5 mg, 0.176
μmol), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (0.872 mg, 1.1 μmol), and
cholesterol (0.33 mg, 0.85 μmol) (in a 10:65:50 molar ratio) was dissolved in CH2Cl2,
MeOH and H2O (about 3:3:1, v/v, 2 mL) in a vial. The solvents were then removed in
vacuum to form a thin lipid film on the vial wall, which was hydrated by adding 3.0 mL of
HEPES buffer (20 mM, pH 7.5) containing NaCl (150 mM) and shaking the mixture by
vortex.
Preparation of emulsion of KLH conjugate 3.6. The protocol was similar to that
reported in the literature. Generally, conjugate 2 (1.125 mg) was dissolved in 1.5 mL 1X
PBS solution and mixed with 1.5 mL Freund’s Complete Adjuvant (CFA) following the
manufacturer’s protocol to form an emulsion.
Immunization of mouse
Each group of six female C57BL/6J mice were inoculated on day 1 by
subcutaneous (s.c.) injection of 0.1 mL of the liposomal solution or CFA emulsion of a
84
specific glycoconjugate. Following the initial immunization, mice were boosted 3 times on
day 14, day 21, and day 28 by s.c. injection of the same conjugate and by means of the
same immunization protocol. Mouse blood samples were collected prior to the initial
immunization on day 0 and after immunization on day 21, day 27 and day 38, and were
clotted to obtain antisera that were stored at -80 °C before use. The animal protocol (#A
02-10-14) for this investigation was approved by the Institutional animal Care and Use
Committee (IACUC) of Wayne State University, and all animal experiments were
performed in compliance with the relevant laws and institutional guidelines.
ELISA protocol
ELISA plates were treated with 100 μL of a solution of Globo H-HSA conjugate (2
μg/mL) dissolved in coating buffer (0.1 M bicarbonate, pH 9.6) at 37 °C for 1 h, which was
followed by treatment with a blocking buffer and washing 3 times with phosphate-buffered
saline (PBS) containing 0.05% Tween-20 (PBST). Then, the pooled or an individual
mouse antiserum with serial half-log dilutions from 1:300 to 1:656100 in PBS was added
to the coated plates (100 μL/well) and incubated at 37 °C for 2 h. The plates were wased
with PBST and incubated at rt for another hour with a 1:1000 diluted solution of alkaline
phosphate (AP) linked goat anti-mouse kappa, IgG1, IgG2a, IgG2b, IgG3 and IgM
antibody (100 μL/well), respectively. Finally, the plates were washed with PBST and
developed with 100 μL of a p-nitrophenylphosphate (PNPP) solution (1.67 mg/mL in buffer)
for 30 min, 1 h or 2 h (Due to the low reading, the longer reaction time made the reading
higher; all groups will be compared in the same reaction condition) at rt for colorimetric
readout using a microplate reader at 405 nm wavelength. For titer analysis, OD values
were plotted against antiserum dilution values, and a best-fit logarithm line was obtains.
85
The equation of this line was used to calculate the dilution value at which an OD of 0.1
(this value will be further modified according the data to get better plots) was achieved,
and the antibody titer was calculated at the inverse of the dilution value.
Protocols for FACS Assay
MCF-7 cell line (which can expressed Globo H antigen) was chose to be performed
this experiment, and SKMEL-28 cell line (which was confirmed that Globo H antigen
negative) was chose to be as negative group. MCF-7 cell line was incubated in ATCC-
formulated Eagle's Minimum Essential Medium (EMEM) containing 10% FBS and 1%
antibiotics. SKMEL-28 cell line was incubated in ATCC-formulated Dulbecco's modified
Eagle's medium (DMEM) containing 10% FBS and 1% antibiotics.
Then these cells were harvested by being treated with trypsin-EDTA solution. Cells
(about 1.0 ×106) were washed twice with FACS buffer (PBS containing 5% FBS) and
incubated with 50 µL of normal mouse sera (1:10 dilution) at 4 °C for 15 min. Then the
cell were washed with FACS buffer and incubated with 50 µL certain pooled antisera of
day 38 (1:10 dilution) at 4 °C for 30 min. After that, cells were washed again with FACS
buffer and incubated with FITC-linked goat anti-mouse kappa antibody (2 µL in 50 µL
FACS buffer) at 4 °C for 30 min. Finally, cells were washed and suspended in 0.8 mL of
FACS buffer, and then sent to FACS analysis on a Becton Dickinson LSR II Analyzer.
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93
CHAPTER 4 Development of Novel Carbohydrate-Based Vaccines against
Haemophilus Influenzae Type B and Neisseria Meningitidis Serotype C
4.1 Vaccines against Haemophilus Influenzae Type B
4.1.1 Introduction
Haemophilus influenzae type b (Hib) is an important human pathogen that can
cause various diseases, such as bacterial meningitis and pneumonia, and it usually
strikes children under the age of 5 years old. Before Hib vaccine available, bacterial
meningitis caused by Hib was a life-threatening disease among young children.
(www.cdc.gov/vaccines) Hib-caused disease have been under control in developed
countries by the introduction of successful conjugate vaccines beginning from 1990s.1
However, according to the estimation of the World Health Organization (WHO), Hib still
remains a life-threatening disease in developing country that causes approximately three
million serious illnesses and an estimation of 386,000 deaths per year among children
under the age of 5 years old.2,3 (World Health Organization website (2005):
http://www.who.int/mediacentre/factsheets/fs294/en/)
It has been well known for many years that the capsular polysaccharide (CPS) of
Hib, a polymer of repeating ribosyl ribitol phosphate (RRP) units4 (Figure 4.1), is an
attractive target for developing vaccines against Hib. Currently, the commercial available
Hib vaccines were made from the CPS extracted from bacteria by conjugation with carrier
proteins such as tetanus toxoid (TT) (ActHib, 1993, Sanofi Pasteur; Hiberix, 1998, EU)
and diphtheria toxoid (DT) (Pentacel, Sanofi Pasteur), which are protective against the
diseases caused by Hib.
94
Figure 4.1 The structure of polyribosylribitol phosphate (RRP) on Hib surface.
However, the Hib RRP polysaccharides used for vaccine production, which were
isolated from the supernatants of bacterial cell cultures, are often heterogeneous and
contaminated with other antigenic components that may cause safety problems and
quality control problems. To deal with the issue, conjugate vaccines based on well-defined
synthetic carbohydrate antigens have attracted great interest. After decades of
development and optimization, the first semi-synthetic glycoconjugate vaccine against
Hib, Quimi-Hib™ (Heber Biotech), which was derived from the conjugation of synthetic
polyribosylribitol phosphate and tetanus toxoid (TT), was approved and commercialized
in Cube and became a part of Cuba’s national vaccination program in 2004.5-7 This
vaccine was also confirmed to stimulate higher long-term protective antibody titers than
that of licensed products composed with polysaccharide extracted from Hib bacteria.5,6
This research has provided a proof of principle for the development of effective bacterial
vaccines based on oligosaccharides in addition to CPSs. The great success of Quimi-
Hib™ has encouraged the exploration of a series of vaccines against Neisseria
meningitidis serogroup C8 and W1359 and Streptococcus pneumoniae10.
Actually, as early as in 1990s, Peeters’ group already synthesized a series of Hib
CPS derivatives, including trimeric and tetrameric RRP, and conjugated them to carrier
95
proteins such as DT and TT via a thioether linkage.11 Immunological studies indicated that
all of these conjugates elicited RRP-specific antibody responses with an increasing
IgG/IgM antibody ratio in both mice and monkeys. Moreover, the tetramer conjugates
elicited higher RRP-specific antibody titers than the trimer conjugates, suggesting that the
tetrameric RRP was a better immunogen. In addition, the results of adult monkeys
immunized with tetrameric RRP-TT conjugate were as good as that of oligosaccharide-
CRM197 conjugate, which elicited protective levels of serum antibodies in human infants
after two or three boosts.
Additional investigation about synthetic RRP conjugate vaccine against Hib was
carried out by Chong’s group.12 They coupled synthetic oligosaccharides carrying RRP
repeating units to synthetic peptides, which contained potent T-helper cell determinants
and B-cell epitopes of Hib outer membrane proteins (OMPs), P1, P2 and P6. Animal
studies of these conjugates revealed that some of them could elicit high titers of both
RRP-specific and OMP-specific IgG antibodies. Moreover, they investigated the factors
that might affect the conjugate immunogenicity. They found that the magnitude of RRP-
specific antibody responses significantly depended on the relative spatial orientations of
RRP and T-cell determinants and that the antibody response increased when a multiple
antigenic peptide carrier was used. It was found that three RRP repeating units were
optimal to elicit RRP-specific antibodies. Lipidation of the peptide-RRP conjugates was
found to have a minimal effect on the immune response.
In conclusion, the above studies have clearly demonstrated that a carbohydrate
hapten conjugated with a peptide can provoke strong immune response, and especially
help elicit T cell-dependent immune response. Furthermore, the antisera raised by these
96
conjugates were confirmed to be protective against Hib infection in an infant rat model.
4.1.2 Our Vaccine Designs
The main aims of this project were to systematically explore the structure-activity
relationships of oligo-RRP as antigens and develop effective RRP-based Hib vaccines.
According to the literature, Hib-derived capsular polysaccharides used in some licensed
vaccines can contain oligosaccharides as short as five RRP repeating units.13 Moreover,
trimeric and tetrameric RRPs were demonstrated to have excellent immunostimulatory
ability.11,12 For example, in Quimi-Hib™, the saccharide moiety has an average of eight
RRP repeating units.5
Based on the above discoveries, our group designed tri-, tetra- and pentameric
RRPs, which were successfully synthesized by Dr. Guochao Liao, and linked them to KLH
and MPLA to form conjugates 4.1-4.3 and 4.7-4.9 (Figure 4.2). In the meantime, these
oligosaccharides were also coupled with HSA to get conjugates 4.4-4.6 that were utilized
as the capture reagents in ELISA experiment. The new RRP-KLH conjugates 4.1-4.3 and
RRP-MPLA conjugates 4.7-4.9 were evaluated as vaccines in mice.
97
Figure 4.2 The structure of designed olio-RRPs and their KLH conjugates (4.1-4.3), HSA
conjugates (4.4-4.6), and MPLA conjugates (4.7-4.9)
4.1.3 Results and Discussion
4.1.3.1 KLH Conjugates 4.1-4.3 as Vaccines
After the KLH conjugates 4.1, 4.2 and 4.3 and HSA conjugates 4.4, 4.5, and 4.6
were synthesized, their weight% loadings of the saccharide moiety were examined, and
the results were showed in Table 4.1. The saccharide loadings of HSA conjugates were
estimated by means of MS. In this case, the average molecular weights of HSA and HSA
conjugates were obtained by MALDI TOF MS (Figure 4.3). Then, the saccharide loadings
of were calculated according to the following equation:
% saccharide loading = [conjugate MW - HSA MW]/conjugate MW
where the HSA MW was 67 kDa. The calculated saccharide loadings of 4.4, 4.5 and 4.6
were about 13.7%, 8.4% and 7.4%, respectively. Their saccharide loadings were also
examined by a chemical method,14,15 which gave slightly different results, namely that the
loadings of 4.4, 4.5 and 4.6 were about 8%, 8.6% and 9.6%, respectively. The differences
may be caused by the systematic errors of the chemical method, in which the presence
98
of protein and the existence of saccharides in the form of conjugate might have some
impacts on the carbohydrate analysis. Nevertheless, the results suggested that resultant
glycoconjugates had the proper saccharide loadings as vaccines, as it has been observed
that glycoconjugates with sugar loading in the range of 5-20% produce the optimal
immunological results. For the KLH conjugates, as KLH is huge and exists in difficult
forms, which makes its measurement by MS very difficult, their loadings were only
examined by the chemical method.14,15 Their loadings were also in the acceptable range
of 8~9%.
Table 4.1 The loadings of saccharide moiety in protein conjugates*
Conjugates
Loading (wt %)
Average saccharide
residues per protein
4.1 (Hepta-Sacc-KLH)
8.4
27
4.2 (Nona-Sacc-KLH)
8.4
21.5
4.3 (Undecyl-Sacc-KLH)
9.0
19.2
4.4 (Hepta-Sacc-HSA)
7.88 (13.7)**
8
4.5 (Nona-Sacc-HSA)
8.60 (8.4)**
3.7
4.6 (Undecyl-Sacc-HSA)
9.57 (7.4)**
2.7
* Data provided by Dr. Guochao Liao.
**The values in () were calculated according to the MALDI TOF MS data (Figure 4.4).
99
Figure 4.3 MALDI TOF MS results of HSA conjugates 4.4, 4.5 and 4.6.
Immunological evaluations of RRP-KLH conjugates 4.1-4.3 were carried out with
female C57BL/6J mouse. For initial immunization, each KLH conjugate containing ca. 3
µg of the carbohydrate antigen mixed with Titermax Gold adjuvant as an emulsion (0.1
mL) was injected subcutaneously (s.c.) to each mouse in a group of six. Four more boost
immunizations were carried out in these mice on day 14, day 21, day 28 and day 38,
respectively, using the same vaccines.
The immunological responses to conjugates 4.1-4.3 were evaluated by analyzing
the antigen-specific antibody titers in the mouse sera by ELISA. For this purpose, each
mouse was subjected to bleeding on day 0 before the initial injection (used as the blank
control) and on day 27, day 38 and day 48 after immunization. The blood samples were
treated to prepare sera according to standard protocols and the antisera were stored at -
80 °C before further analysis by ELISA with the corresponding HSA conjugates 4.4-4.6
as capture antigens. Antibody titers were calculated from the logarithm fit of the curves of
100
the OD value against serum dilution number, and were defined as the dilution number
yielding an OD value of 0.5.
Kinetic results of the immune responses
To investigate the changes in immune responses upon each boost immunization,
we analyzed the total antibody (kappa) titers in the sera obtained at different date points.
Figure 4.4 shows the antigen-specific total antibody titers of pooled sera. Clearly, all of
the three conjugates elicited strong immune response after 4 boost immunizations, and
the antibody titers of the day 48 antisera were significantly higher than that of day 38
antisera. Most significantly, some of the conjugates, e.g. 4.2, did not stimulate strong
responses until the 4th boost immunization.
Figure 4.4 ELISA results of total antibodies (kappa) of the pooled antisera collected on
day 0, day 38 and day 48 from mice immunized with: (A) 4.1 plus Titermax Gold adjuvant;
(B) 4.2 plus Titermax Gold adjuvant; (C) 4.3 plus Titermax Gold adjuvant. (D) Calculated
antigen-specific total antibody titers of the conjugates.
It is worth pointing out that conjugate 4.2 was somehow not well soluble in 1X PBS.
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The low antibody titer of 4.2 after first 3 boost immunizations might be due to this problem.
Thereafter, the method to prepare vaccines for immunization was improved. The
conjugates were first dissolved in 10X PBS (high ionic concentration can help the
conjugates to dissolve) and then diluted to 2X PBS buffer, which were finally mixed with
adjuvant. Furthermore, for the initial and first 3 boost immunization, the route of vaccine
administration was subcutaneous, but the 5th boost immunization was intramuscular. All
these changes might have helped conjugate 4.2.
In conclusion, conjugates 4.1-4.3 could elicit robust immune responses, and the
induced immune responses were dependent on the frequencies of boost immunization.
With the increase in the number of boost immunization, the immune responses became
stronger. The results also suggested that immune responses might be dependent on the
antigen structure, as the 3 conjugates were different. In addition, it seems that the
preparation and administration methods of the vaccines also had some impact on the
immune response, which is an interesting question worth further investigation.
Isotypes of antibodies
In addition to the total antibody titer which shows the overall immune response to
a specific vaccine, we have also examined the titers of various antibody isotypes of the
antiserum obtained with each vaccine to show the quality of the immune response. The
ELISA results of day 48 antisera are depicted in Figure 4.5. Each dot represents the
antibody titer of an individual mouse, and the black bar represents the average antibody
titer of a group of six mice. It is evident that, in addition of IgM antibodies, all of the three
conjugates 4.1-4.3 could elicit high levels of IgG1 antibodies, indicating the involvement
of T cell-mediated immunity. In the case of 4.1 and 4.2, significant levels of IgG2a and
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IgG3 antibodies were also elicited. More detailed analyses revealed that the two mice
with lower IgG1 antibody titers had higher IgG2a antibody titers and that the mice with
higher IgG1antibody titers always had lower IgG2a antibody titers. This phenomenon was
consistent with the influence of cytokines on antibody isotype switching (Table 4.2).
Nevertheless, each mouse showed high titer of at least one kind of IgG antibody. On the
other hand, the elicitation of high titers of IgG1 antibody indicated the production of IL-4
cytokine.
Figure 4.5 ELISA results of antigen-specific antibody isotypes in day 48 antisera of mice
immunized with: (A) 4.1 plus Titermax Gold adjuvant, (B) 4.2 plus Titermax Gold adjuvant,
(C) 4.3 plus Titermax Gold adjuvant, respectively. Each dot represents the antibody titer
of an individual mouse, and the black bar represents the average antibody titer of a group
of six mice.
Table 4.2 Influence of cytokines on antibody isotype switching16
Cytokine
IgG1
IgG2a
IgG2b
IgG3
IgM
IL-4
Induces
Inhibits
Inhibits
Inhibits
IFN-γ
Inhibits
Induces
Induces
Inhibits
TGF-β
Induces
Inhibits
Inhibits
Previously, we found that KLH-carbohydrate conjugates usually elicited IgG1
antibodies whereas lipid A-carbohydrate conjugates typically elicited IgG1 and IgG3
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antibodies.14,17-19 However, the KLH conjugates here elicited various isotypes of IgG
antibodies. We proposed that different administration routes of vaccines might be one of
the factors that influence the immune responses. Depending on whether the vaccine is
administered by s.c. or i.m. injection, the antigen will meet different sets of antigen
presenting cells (APCs) in the dermal area and in the muscle. As a result, the APCs will
present the antigenic determinant in MHC-II molecule complex to the T helper cells in the
nearest local draining lymph nodes which are different as well.20
Although 4.1-4.3 elicited similar immune responses, the IgG1, IgG2a and IgG3
antibody titers induced by the higher oligomer 4.3 (pentamer of the RRP repeating unit)
were significantly lower than that induced by the other two conjugates. Literature results
also indicated that the trimer and tetramer of the RRP repeating unit showed good
immunogenicity.11,12 These results combined suggest that the immunogenicity of a poly-
saccharide is not necessarily increased by simply increasing its length. This information
should be especially useful and helpful for the design and development of fully synthetic
conjugate vaccines based on oligosaccharide antigens.
Cross reactions
To further investigate the properties of antisera induced by different conjugates,
cross reactions between different antisera and antigens were carried out. The results are
shown in Figure 4.6.
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Figure 4.6 ELISA results of the cross reactions between antigens displayed in 4.4-4.6
and pooled antisera of mice immunized with 4.1, 4.2 and 4.3.
In this study, the antisera were pooled for each group of 6 mice immunized with
conjugates 4.1, 4.2 and 4.3, respectively. Conjugates 4.4, 4.5 and 4.6 were used to coat
plates for ELISA analysis. It is clear that the antisera induced by the three conjugates had
significant cross reactions, meaning the antibodies induced by each vaccine can at
different levels recognize and bind the other two oligosaccharide antigens as well. Thus,
they share some common structural motifs. On the other hand, the reactivities did show
some differences. In general, antisera showed higher titers to the carbohydrate antigens
same to the ones in the vaccines used to induce antisera. In addition to the structural
factor, the loading percent of saccharides in the HSA conjugates and the concentrations
of antibodies in the antisera may also affect the results shown in Figure 4.6. This makes
it complex to derive any firm conclusion.
Nevertheless, this high level of cross reactions indicated that the structural motif
of the polysaccharide repeat unit was probably the antigen determinant, and at least a
part of the antibodies in the antisera were against this common motif. Alternatively, the
results may be interpreted that the oligosaccharide antigens might have been degraded
105
in vivo, so that antibodies against different lengths of saccharides have been elicited. In
the latter case, the antisera induced by 4.3 should show higher cross reactions to 4.4 and
4.5 than that of 4.1 to 4.5 and 4.6, which is not conclusive. However, degradability of
vaccines is indeed an important factor influencing vaccine immunity. Usually proteins
vaccines are digested into pieces by APCs to become suitable for loading onto the MHC
molecules for presentation to the T cells. However, how carbohydrate antigens are
processed in the immune system is largely unclear presently.
4.1.3.2 MPLA Conjugates 4.7-4.9 as Vaccines
Structurally well-defined and characterized conjugates 4.7-4.9 were synthesized
by Dr. Guochao Liao in the Guo laboratory. Immunological studies of these conjugates
were performed using female C57BL/6 mice, following the protocols described for
conjugates 4.1-4.3, except that different vaccine preparations and adjuvant schedules
were involved. Because MPLA conjugates had low solubility in aqueous buffer, they were
administered in the form of liposomes, prepared with 1,2-distearoyl-sn-glycero-3-
phosphocholine and cholesterol. According to literature,17,21 this not only can improve the
solubility of vaccines but also can increase their immunogenicity, as the liposomes may
help vaccine delivery in vivo. Again, antibody titers were calculated from the logarithm fit
of the curves of OD value against serum dilution number, and were defined as the dilution
number yielding an OD value of 0.1.
Kinetic results of the immune responses
Experiment to examine the antibody response kinetics against MPLA conjugates
was performed with 4.7. Thus, 1 day to 9 days after the 5th boost immunization, mouse
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antisera were obtained and antigen-specific total antibodies were evaluated by ELISA to
give normalized antibody titers. The results are shown in Figure 4.7. Clearly, 1 day after
the boot immunization, high titers of antibodies started to appear, and it reached the
maximum on day 2. Thereafter, the antibody titers started to decline, and at about day 9,
the antibody titer became relatively small, even though significant immune response
remained. These results demonstrated that the immune responses induced by previous
immunizations were memorized and immediate and strong immune responses could be
generated in the mice upon contact of the same antigen.
Figure 4.7. ELISA results of the anti-kappa (total) antibody titers (OD 405 = 0.1) in the
pooled antisera of mice immunized with conjugate 4.7 obtained on day 1, 2, 4, 5, 7 and 9
after the 5th boost immunization.
Isotypes of antibodies
The titers of antibody isotypes in the antisera obtained from mice two days after
the 5th immunization with 4.7-4.9 were analyzed according to protocols described
previously. As shown in Figure 4.8, all conjugates induced high titers of IgG3 antibodies,
which is consistent with the pattern of immune responses to glycolipid conjugates.17,18 In
the meantime, significant levels of IgG1 and IgG2a antibodies were also observed in the
0
20000
40000
60000
80000
100000
120000
d1 d2 d4 d5 d7 d9
Kappa
Antibody Titer
107
antisera. These results indicated T cell-mediated immune responses, which is important
for the vaccines. There were some differences in terms of antibody titers among these
conjugates, but the differences were much less obvious than that observed with KLH
conjugates 4.1-4.3. Overall, conjugate 4.7 showed the highest IgG antibody, once again
suggesting that longer saccharides are not necessarily better antigens/immunogens or
induce stronger immune responses.
Figure 4.8. ELISA results of different isotypes of antigen-specific antibodies in day 2
antisera of mice after final boost immunization with 4.7, 4.8 and 4.9. Each dot represents
the antibody titer of an individual mouse, and the black bar represents the average
antibody titer of a group of six mice.
Cross reactions
Figure 4.9 shows the cross reactions of HSA conjugates 4.4-4.6 with all three
groups of pooled antisera derived from mice immunized with MPLA conjugates 4.7-4.9.
The results were very similar to that of the KLH conjugates (Figure 4.6). Once again, all
antisera had significant cross reactions with all three different oligosaccharide antigens,
suggesting that the three vaccines 4.7-4.9 might share some common antigenic motifs in
terms of the RRP structure.
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Figure 4.9 ELISA results of the cross reactions between 4.4-4.6 and the antisera derived
from mice immunized with 4.7-4.9.
4.1.3.3 Evaluation of the Hib-binding Property of Antisera
Although we have demonstrated that the synthetic vaccines could induce strong
antigen-specific immunities, the coating antigens utilized in the ELISA experiments were
respective oligosaccharides. The key question is whether the antibodies can recognize
the carbohydrate antigens on the bacterial cell surface and interact with bacterial cells.
To answer this question, the cell binding properties of these antisera were evaluated by
two different experiments. One is the direct binding experiment, which was very similar to
the regular ELISA except that Hib cells were used as the capture reagents. The other
experiment was competitive ELSA. For the former experiment, Hib (ATCC 10211) cells
instead of HSA conjugates were utilized to coat 96-well plates and then pooled mouse
antisera obtained from mice immunized with conjugate 4.1-4.3 and 4.7-4.9 were added
to the plates. After washing, AP-linked goat anti-mouse kappa antibody was added for
109
detecting antibodies bound to Hib cell-coated plates, reflected by the OD values at 405
nm. The ELISA results are showed on Figure 4.10.
Figure 4.10 ELISA results of antibody-Hib binding experiments. (A) and (B): results of
the 1:100 diluted pooled antisera of conjugates 4.1-4.3 and 4.7-4.9; and (C) and (D):
results of the 1:300 diluted pooled antisera of 4.1-4.3 and 4.7-4.9 (d0: the pooled sera
before immunization used as negative controls). The error bars represent standard
deviations of three parallel experiments.
The pooled antisera before immunization (d0) were used as blank controls which
had an OD value below 0.2. For the pooled antisera derived from all KLH conjugates, the
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OD values were similar, ca. 0.85-0.95 (1:100 diluted sera) and 0.6-0.8 (1:300 diluted sera).
The result confirmed that the antisera against KLH conjugates could successfully
recognize and bind to Hib cells. For the antisera of MPLA conjugates, at 1:100 dilution,
significantly higher OD values (0.3-0.5) than that of the d0 antisera were observed, but
the difference was not significant with 1:300 diluted sera. These results correspond well
with the observed antibody titers induced by various conjugate vaccines (Figures 4.5 and
4.8). In conclusion, these results have demonstrated that antisera elicited by KLH and
MPLA conjugates could bind to Hib cells.
Competitive ELISA experiment was performed to confirm above antibody binding
to Hib cells and verify its specificity. The experimental protocols were similar to that for
regular ELISA except that, in the step of adding diluted antisera solution to coated plates,
Hib cells were mixed to bind with the antibodies in the antisera solution to compete with
the HSA-antigen conjugates. The results (Figure 4.11) showed that with the increase of
Hib cells added to the plate well to compete with HSA conjugates coated in the plates,
the plate OD values (representing antibodies bound to plates) decreased significantly in
a concentration dependent manner. The antisera of two KLH conjugates 4.1-4.3 and 4.7
had similar results, though it seems that the competition for 4.1 is more effective than the
others. Therefore, the competitive ELISA results have verified the specific binding of
antisera obtained from mice immunized with our synthetic vaccines to Hib cells.
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Figure 4.11. Hib competitive ELISA results using: (A) 1:2700 diluted antisera of mice
immunized with KLH conjugate 4.1; (B) 1:900 diluted antisera of mice immunized with
MPLA conjugate 4.7; (C) 1:300 diluted antisera of mice immunized with 4.2; (D) 1:900
diluted antisera of mice immunized with 4.3. The amounts of Hib cells used in the
experiments are represented by the OD value (X-axis) of cells at 600 nm, and higher OD
means higher cell concentrations. Error bars are the standard deviation of three parallel
experiments.
4.1.4 Conclusions
In summary, KLH conjugates 4.1-4.3, and MPLA conjugates 4.7-4.9, can elicit
strong immune response, especially the T cell-mediated immune response. The capbility
of conjugates to generate antibodies followed the order: 4.1>4.3≈4.2, and 4.7≥4.9>4.8,
112
respectively. Therefore, the Hepta-saccharide had the best results both in KLH conjugate
and MPLA conjugate. This capability order also suggested that longer saccharides are
not necessarily better antigens/immunogens or induce stronger immune responses. The
cross reaction between the three structures was significant which revealed that vaccines
might share some common antigenic motifs in terms of the RRP structure. The future
works about this project can focused on the investigation about minimum antigen
determinant which can provide a principle to direct the design of more effective vaccines.
To achieve this purpose, the technology of monoclonal antibodies (mAbs) can be
employed to produce RRP-specific mAb for the study of binding properties with different
oligomer antigens. This kind of mAb binding studies was well-established in the
exploration of fungal vaccines by Bundle’s group.22
4.1.4 Experiment Section
KLH conjugates
Table 4.3 Doses of KLH conjugates used in animal studies
Conjugates
Loading (%)
Each mouse (µg)
6 mice (µg)
6mice 4 times (µg)
Hepta-KLH
8.4
35.7
214.2
856.8
Nona-KLH
8.4
35.7
214.2
856.8
Undecyl-KLH
9.0
33.3
199.8
799.2
All glycoconjugates were synthesized by Dr. Guochao Liao. Titermax Gold
adjuvant, 1, 2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol were purchased
from Sigma-Aldrich. Alkaline phosphatase linked goat anti-mouse kappa, IgM, IgG1,
IgG2a, and IgG3 antibodies and FITC-labeled goat anti-mouse kappa antibody were
113
purchased from Southern Biotechnology. Female C57BL/6J mice of 6-8 weeks age used
for immunological studies were purchased from the Jackson Laboratory. The doses of
KLH conjugates were showed in Table 4.3.
General Procedure for the Preparation of Emulsion of Conjugates
Each conjugate (30 doses) was dissolved in small 0.3 ml 10× PBS buffer and then
was diluted to 1.5 ml 2× PBS solution. The solution was mix with 1.5 ml Titermax Gold
adjuvant (1:1, v/v) and to form an emulsion according to the manufacturer’s protocol.
(Usually, 1× PBS buffer works well to dissolve the KLH conjugates. However, these
conjugated are difficultly dissolved in 1× PBS buffer. The high ionic concentration of 10×
PBS buffer may help the conjugates to dissolve in water-based buffer.)
Immunization of Mouse
Each group of six female C57BL/6 mice were immunized on day 1 by
subcutaneous (sc) injection of 0.1 mL of the emulsion of the conjugate vaccine and
Titermax Gold adjuvant prepared according to the manufacturer’s protocol. Following the
initial immunization, mice were boosted 4 times on day 14, day 21, day 28 and day 38 by
sc injection of the same conjugate and by the same immunization protocol. Blood samples
of each mouse were collected through the leg veins prior to the initial immunization on
day 0 and after immunization on day 27, day 38 and d 48 were clotted to obtain antisera
that were stored at -80 ⁰C before use.
Protocols for ELISA
ELISA plates were treated with 100 ul of a solution of individual conjugates (4.4 -
4.6) (2 µg/ml) dissolved in coating buffer (0.1 M bicarbonate, pH 9.6) at 4 ⁰C overnight,
and then at 37 ⁰C for 1 h, which was followed by treatment with blocking buffer (10% BSA
114
in PBS solution with NaN3) and washing 3 times with phosphate-buffered saline (PBS)
containing 0.05% Tween-20 (PBST). Thereafter, a pooled or an individual mouse
antiserum with serial half-log dilutions from 1:300 to 1:656100 in PBS was added to the
coated ELISA plates (100 µL/well), which was followed by incubation at 37 ⁰C for 2 h. The
plates were then washed with PBS and incubated at rt for another 1 h with a 1:1000
diluted solution of alkaline phosphatase linked goat anti-mouse kappa, IgM or IgG2a
antibody or with a 1:2000 diluted solution of alkaline phosphatase linked goat anti-mouse
IgG1 and IgG3 antibody (100 µL/well), respectively. Finally, these plates were washed
with PBS and developed with 100 µL of p-nitrophenylphosphate (PNPP) solution (1.67
mg/mL in buffer) for 30 min at rt, followed by colorimetric readout using a BioRad 550
plate reader at 405 nm wavelength. The optical density (OD) values were plotted against
antiserum dilution values, and a best-fit line was obtained. The equation of the line was
employed to calculate the dilution value at which an OD of 0.5 was achieved, and the
antibody titer was calculated at the inverse of the dilution value.
MPLA conjugates
4.7 (Hepta-Sacc-MPLA): MW=2959.22 14.8 μg/mouse,
4.8 (Nona-Sacc-MPLA): MW=3305.45 16.53 μg/mouse,
4.9 (Undecyl-Sacc-MPLA) MW=3651.68 18.26 μg/mouse,
Dose: 5.0 ×10-9 mol/mouse, about 9.4 μg saccaride per mouse.
General Procedure for the Preparation of Liposomes of Glycoconjugates 4.7-4.9.
The mixture of each specific MPLA conjugate (4.7-4.9) (75.2 μg of saccharide
moiety, 0.04 μmol), 1, 2-distearoyl-sn-glycero-3-phosphocholine (M.W. 790.15, 200 μg,
115
0.26 μmol), and cholesterol (M.W. 386.66, 77 μg, 0.20 μmol) (a 10:65:50 molar ratio) was
dissolved in CH2Cl2 and MeOH (1:1, v/v, 2 mL) in a 10 mL vial. Then, the solvents were
removed in vacuum to form a thin lipid film on the vial wall, which was hydrated by adding
0.80 mL of HEPES buffer (20 mM, pH 7.5) containing NaCl (150 mM) and was finally
sonicated for 1 h to obtain the desired liposomes. (This kind of conjugates were difficultly
dissolved in water-based buffer. So, a long time sonication was need for making milk-like
liposome form.)
Immunization of Mouse
Each group of six female C57BL/6 mice were immunized on day 1 by
subcutaneous (sc) injection of 0.1 mL of the liposomal solution of a specific
glycoconjugate 1-3 containing 9.4 μg of the carbohydrate antigen (5.0 nmol) or by
injection of an emulsion of the liposomal solution of a specific glycoconjugate vaccine.
Following the initial immunization, mice were boosted 3 times on day 14, day 21 and day
28 by sc injection of the same conjugate and by the same immunization protocol. Blood
samples of each mouse were collected through the leg veins prior to the initial
immunization on day 0 and after immunization on day 27 and day 38 and were clotted to
obtain antisera that were stored at -80 ⁰C before use.
The ELISA results of day 27 and day 38 antisera showed that the antibody titer
was very low which may be caused by that antibody may disappear on day 7 and day 10
after the boost. The maximum antibody titer may appear on the earlier days after the
boost. Therefore, the blood was collected again on day 1, 2, 4, 5, 7, and day 9 after
another boost and the antisera were analyzed.
116
Protocols for ELISA
ELISA plates were treated with 100 ul of a solution of HSA conjugates 4.4-4.6 (2
ug/ml) dissolved in coating buffer (0.1 M bicarbonate, pH 9.6) at 4 ⁰C overnight, and then
at 37 ⁰C for 1 h, which was followed by treatment with blocking buffer (10% BSA in PBS
solution with NaN3) and washing 3 times with phosphate-buffered saline (PBS) containing
0.05% Tween-20 (PBST). Thereafter, a pooled or an individual mouse antiserum with
serial half-log dilutions from 1:300 to 1:656100 in PBS was added to the coated ELISA
plates (100 uL/well), which was followed by incubation at 37 ⁰C for 2 h. The plates were
then washed with PBS and incubated at rt for another 1 h with a 1:1000 diluted solution
of alkaline phosphatase linked goat anti-mouse kappa, IgM or IgG2a antibody or with a
1:2000 diluted solution of alkaline phosphatase linked goat anti-mouse IgG1 and IgG3
antibody (100 uL/well), respectively. Finally, these plates were washed with PBS and
developed with 100 uL of p-nitrophenylphosphate (PNPP) solution (1.67 mg/mL in buffer)
for 30 min at rt, followed by colorimetric readout using a BioRad 550 plate reader at 405
nm wavelength. The optical density (OD) values were plotted against antiserum dilution
values, and a best-fit line was obtained. The equation of the line was employed to
calculate the dilution value at which an OD of 0.1 was achieved, and the antibody titer
was calculated at the inverse of the dilution value.
Protocols for Hib binding ELISA experiments
Haemophilus influenzae type b (Hib, ATCC 10211) was chose to be performed this
experiment. Hib was incubated in ATCC #814 agar medium in 37 °C with 5% CO2
atmosphere for 24 h.
117
Bacterial cells were collected and resuspended in 10% buffered formalin
phosphate (4% w/w formaldehyde, 0.4% w/w sodium phosphate monobasic monohydrate,
0.65% w/w sodium phosphate dibasic anhydrous, 1.5% w/w methanol) for 15 min. Then
the fixed bacteria solution was centrifuged in 1000 rpm for 5 min. The chocolate agar
medium percipitated in the bottom was removed, and the supernate containing bacterial
cells was used to coat the 96-well plates. Each well of 96-well plate was coated with 100
µL bacteria solution which had an OD value of 1 at 600 nm. The 96-well plate was dried
off under biological cabinet in room temperature and formed a thin bacterial film on the
bottom of the well.
The bacteria-coated plate was then treated with BSA blocking buffer (200 µL/well)
in r.t. for 1 h and then washed three times with PBS containing 0.05% Tween-20 (PBST).
Then, the pooled antisera from mice immunized with 4.1-4.3 or 4.7-4.9 with dilutions
1:100 or 1:300 in PBS were added to the coated plate (100 µL/well), respectively, and
incubated at 37 °C for 2 h. The plates were washed with PBST and incubated at r.t. for
another one hour with a 1:1000 diluted solution of alkaline phosphate (AP) linked goat
anti-mouse kappa antibody (100 µL/well). Finally, the plates were washed with PBST and
developed with 200 µL of a p-nitrophenylphosphate (PNPP) solution (1.67 mg/mL in
PNPP buffer). After reacting for 30 min at r.t., 100 µL solution obtained from each well
was used for colorimetric readout using a microplate reader at 405 nm wavelength. OD
values represented the binding ability of antisera to bacteria.
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Protocol for Hib Competitive ELISA
The protocol for this experiment was the same as for regular ELISA described in above
except that Hib solution (in PBS) was utilized to compete with plate-bound antigen-HSA
(4.4-4.5) for antibody binding. Thus, after an ELISA plate was treated with different
antigen-HSA and the blocking solution sequentially and then washed with PBS, 100 µL
of the pooled antisera obtained from mice immunized with conjugates (1:300, 1:900 or
1:2700 dilution) in PBS containing different concentration Hib (OD 600 nm value at 0.001,
0.01, 0.1, and 1) was added to each well of the ELISA plate, respectively. Then, the plate
was incubated in 37 °C for 2 h and washed. Finally, the plate was treated with alkaline
phosphatase linked goat anti-mouse kappa antibody (1:1000 dilution), washed,
developed with PNPP, and finally subjected to colorimetric readout at 405 nm wavelength,
as described above.
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4.2 Vaccines against Neisseria Meningitides Serogroup C
4.2.1 Introduction
Neisseria meningitides remains a leading cause of bacterial meningitis and
septicaemia in the United States and other countries.23-26 According their capsular
polysaccharides, they are classified into 13 serogroups.27,28 In worldwide, most cases of
meningococcal diseases were caused by serogroup A, B, C, Y and W-135.27 Right now,
some multivalent polysaccharide vaccines, such as MPSV4 (Menomune; Sanofi-Pasteur,
1981), ACWY Vax (GlaxoSmithKline) and MCV4 (Menactra; Sanofi Pasteur, 2005), are
available to provide protection against serogroup A, C, Y and W-135.28
In 1981, the first meningococcal vaccine, MPSV4 (Menomune; Sanofi Pasteur),
based on polysaccharides from serogroup A, C, Y and W-135, was licensed in US.26
However, generally, the polysaccharide vaccines have some marked limitations, such as
lacking of induction of immunological memory, relatively short duration of protection and
low immunogenicity in infants.26,29
To overcome the limitation of low immunological efficiency of vaccines, conjugate
vaccines in which polysaccharides extracted from Neisseria meningitidis are covalently
linked to a carrier protein have been developed. Therefore, in 2005, the first such product,
the quadrivalent meningococcal vaccine MCV4 (Menactra; Sanofi Pasteur) containing
polysaccharides from N. meningitidis serogroup A, C, Y and W-135 conjugated
individually to diphtheria toxoid (DT), has been approved in United States.26,29
The second such product, MenACWY-CRM (Menveo; Novartis Vaccines and
Diagnostics), another quadrivalent conjugate vaccine, containing N. meningitidis
serogroup A, C, Y and W-135 oligosaccharides conjugated individually to
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Corynebacterium diphtheria cross-reactive material (CRM197) was approved by the US
FDA in 2010, to prevent invasive meningococcal disease in people aged 11-55 years.29,30
For serogroup B, the polysaccharide of capsular is α[2,8]-linked polysialic acid that
is identical structure to polysialic acid in fetal neural tissue and therefore may be
immunotolerated in human immune system.27 One of the general strategy to overcome
this immunotolerance problem is to modify the carbohydrate antigen to be a foreign
structure to the host as a vaccine using for immunization. Then the antibody elicited by
the modified carbohydrate-based vaccine may further cross-react with the natural glycan
on the pathogen surface. Bruge’s group developed a group B meningococcal vaccine,
consisting of N-propionylated (NPr) B capsular polysaccharide conjugated to tetanus
toxoid (TT) carrier protein resulting in moderately higher NPr B polysaccharide-specific
antibody titers with a certain cross reaction with B polysaccharide antigen.31 However,
the induced antibodies lack functional activity in vitro. Thus, further investigation about
this strategy are still needed.
The other method to develop the vaccine against serogroup B Neisseria
meningitidis is based on the surface-exposed proteins expressed by the genome
sequence. Rappuoli’s group and their collaborators developed a total of 350 candidate
antigens expressed in Escherichia coli, purified and used to immunize mice.32 The surface
exposed proteins conserved in sequence across a range of strains can induce a
bactericidal antibody response which may be not only against group B N. meningitidis but
also against other serogroups and species of pathogenic Neisseria.32
In conclusion, the above studies have clearly demonstrated that a carbohydrate
hapten conjugates with a peptide are effective vaccine design against meningitidis. To
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overcome the immunotolerance of serogroup B N. meningitidis antigen, α[2-8]-linked
polysialic acids, the vaccines based on modified carbohydrate antigens, or the proteins
expressed on pathogen surface were explored.
4.2.2 Our Vaccine Designs
The previous development of full-synthetic carbohydrate-based vaccines with
protein carrier or MPLA carrier to against cancer and Hib, encouraged us to develop the
vaccines against meningitidis based on the capsular polysaccharide antigens of
serogroup C, α[2-9]-linked polysialic acids. Therefore, our group designed dimer, trimer,
tetramer and pentamer sialic acids, which were successfully synthesized by Guochao
Liao, and linked them to KLH and MPLA to form conjugates 4.10-4.13 and 4.18-4.22.
(Figure 4.12). In the meantime, there oligosaccharides were also coupled with HSA to
get conjugates 4.14-4.17 that were utilized as the capture reagents in ELISA experiment.
These new glycoconjugates, 4.10-4.13 and 4.18-4.22 were evaluated as vaccines in mice.
122
Figure 4.12 The structures of designed [α2-9] linked oligo sailic acids KLH conjugates
(4.10-4.13), HSA conjugates (4.14-4.17), and MPLA conjugates (4.18-4.22)
4.2.3 Results and Discussion
4.2.3.1 KLH Conjugates 4.10-4.13 as Vaccines
After the KLH conjugates 4.10-4.13 and HSA conjugates 4.14-4.17 were
synthesized, their weight% loading of the saccharide moiety were examined by the same
method described in Hib vaccines, and the results were showed in Table 4.4.
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Table 4.4 The loading of saccharide moiety in protein conjugates 4.10-4.17
Conjugates
Loading (wt %)
4.10 (Dimer-KLH)
7.52
4.11 (Trimer-KLH)
11.49
4.12 (Tetramer-KLH)
7.89
4.13 (Pentamer-KLH)
6.83
4.14 (Dimer-HSA)
8.58
4.15 (Trimer-HSA)
11.48
4.16 (Tetramer-HSA)
10.92
4.17 (Pentamer-HSA)
7.79
Immunological studies of oligo-sialic acid-KLH conjugates (4.10-4.13) were carried
out with female C57BL/6J mouse. For initial immunizations, each glycolconjugate
containing 4.7×10-9 mol saccharide residues mixed with Titermax Gold adjuvant as an
emulsion (0.1 mL) was injected intramuscular (i.m.) in mice (3 µg Dimer-sialic acid moiety
per mouser, see experiment section). More boost immunization were carried out in these
mice by subcutaneous (s.c.) injection on day 14, day 21 and day 28, using the same
vaccines. Each mouse was subjected to bleeding on day 0 before the first injection (used
as blank controls) and on day 27 and day 38 after immunization. The blood samples were
treated according to standard protocols to prepare antisera for the analysis of antigen-
specific antibodies by ELISA with the HSA conjugates 4.14-4.17 as capture reagents. In
addition to total antibodies, antibody isotypes such as IgG1, IgG2a, IgG2b, IgG3 and IgM
were also individually assessed. Antibody titers were calculated from linear regression
124
analysis of the curves of the optical density (OD) value against the logarithmic scale of
serum dilution number, and were defined as the dilution number yielding an OD value of
0.2.
Figure 4.13 showed the antibody titers of antigen-specific antibodies in d38
antisera obtained from mice immunized with 4.10-4.13. Each dot represents the antibody
titer of an individual mouse, and the black bar represents the average antibody titer of a
group of six mice. The results revealed that all the conjugate 4.10-4.13 can elicit strong
immune responses, especially the high level of IgG1, IgG2a and IgG2b antibodies
indicating the involvement of T cell-mediated immunity. In additionally, all the mice had
similar antibody titer and antibody isotype. The ratio of successful immunization could be
considered as 100% that means all individual can be successfully provoked to produce
immune response by the vaccine.
Figure 4.13 ELISA results of antigen-specific antibody isotypes in day 38 antisera of mice
immunized with (A) 4.10 plus Titermax Gold adjuvant, (B) 4.11 plus Titermax Gold
125
adjuvant, (C) 4.12 plus Titermax Gold adjuvant, (D) 4.13 plus Titermax Gold adjuvant,
respectively. Each dot represents the antibody titer of an individual mouse, and the black
bar shows the average antibody titer of a group of five or six mice.
In all the isotypes, IgG2b was extremely higher than the other isotype for all the six
mice. According Table 4.2, it indicated that TGF-β cytokine was involved in this immune
response. The IgG1 was also showed good antibody titers which may indicated that IL-4
cytokines was involved. The corresponding T cell immune pathway can be figure out
according this relationship between cytokines and antibody isotype switching (Table
4.2).16 Falk Nimmerjahn and Jeffrey V. Ravetch33 found out that different isotype of IgG
can bind to different Fcγ receptors which included activation and inhibitory receptors.
Based on the activating-to-inhibitory (A/I) ratio of the receptors IgG binding to, the
hierarchy of activity for the IgG subclasses was thus IgG2a≥IgG2b>IgG1>>IgG3.33
Therefore, in this result, the high antibody titer of IgG2b elicited by the vaccines could be
considered that antibody IgG2b has very strong immune activities.
Furthermore, among IgG subclasses, IgG 2a and 2b are generally considered to
be the most potent for activating effector responses and dominate antiviral immunity,
which also supported the application potential of this anti-bacteria vaccine.34-38
In another aspect, carrier protein KLH should make an important role in IgG isotype
switch because that protein antigens usually elicit a thymus-dependent response
generally represented by the IgG1, 2a and 2b, whereas carbohydrate antigens can elicit
thymus-independent responses that induce IgG3 antibody secretion.39 Hence, in the
carbohydrate antigen vaccine, carrier protein was very important to improve the
immunogenicity. Moreover, the adjuvant, Titermax Golden adjuvant, worked well with
poly-sialic acids KLH conjugates.
126
Kappa antibody isotype standing for the total antibodies, and IgG2b isotype
representing T cell-mediated immunity, were selected to compare the immunogenicity of
conjugates (Figure 4.14). There was no statistical difference between group Dimer 4.10
and Trimer 4.11. Interestingly, the kappa antibody titers induced by the higher oligomer
(4.12 and 4.13) were significantly lower than that induced by 4.11, but the IgG2b antibody
titers were almost the same for all the conjugates. The saccharide loading of HSA
conjugates was closed to each other which may hardly affect the antibody titers. Hence,
the trend of kappa antibody titer may due to the immunogenicity of oligo-sialic acids
antigen only. Therefore, the capability of conjugates to generate the kappa antibodies
followed the order 4.11≈4.10>4.12≈4.13, while the capability to generate IgG2b was
similar.
Figure 4.14 The antigen-specific antibody titers of kappa (A and B) and IgG2b (C and D)
127
isotypes for all the four glycoconjugates 4.10-4.13. In (A) and (C), each dot represents
the antibody titer of an individual mouse, and the black bar shows the average antibody
titer of a group of five or six mice. In (B) and (D), the error bar represented the standard
deviation of six or five individual mice.
Kinetic plots
Kinetic
Liao-Dimer-Sial-KLH
Liao-Trimer-Sial-KLH
Liao-Tetramer-Sial-KLH
Liao-Pentamer-Sial-KLH
0
50000
100000
150000 d1
d28
d38
Group of mice
Antibody Titer
Figure 4.15 The kinetic plot of glycoconjugates (4.10-4.13). The pooled antisera of day
28 was obtained from mice immunized three times with glycoconjugates, respectively.
The pooled antisera of day 38 was obtained ten days later after the fourth boost from the
mice immunized with glycoconjugates, respectively.
For all the four groups, antibody titer of kappa on day 38 was slightly higher than
day 28. After third or fourth administration, the antibody titer had already reached a high
level which indicated that three or four times injection was sufficient for the immunization.
Cross reaction
To further investigate the properties of antisera induced by different conjugates,
cross reactions between different antisera and antigens were carried out. The protocol of
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cross reaction was similar as that in anti-Hib vaccines. The results are shown in Figure
4.16.
Cross Reaction
Dimer
Trimer
Tetramer
Pentamer
0
50000
100000
150000 Dimer-HSA
Trimer-HSA
Tetramer-HSA
Pentamer-HSA
Antisera
Antibody Titer
Figure 4.16 The ELISA results of cross reactions between the pooled antisera obtained
from mice immunized with conjugates (4.10-4.13) dimer-, trimer-, tetramer- and
pentamer-sialic acid-KLH and the capture reagents (4.14-4.17) dimer-, trimer-, tetramer-
and pentamer-sialic acid-HSA.
It is clear that the antisera induced by the four conjugates had significant cross
reactions, meaning the antibodies induced by each vaccine can recognize and bind the
other oligosaccharide antigens as well. Thus, they share some common structural motifs
that should be dimer-sialic acid. This high level of cross reactions indicated that the
structural motif of dimer-sialic acid was probably the minimum antigen determinant, and
at least a part of the antibodies in the antisera were against this common motif.
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4.2.3.2 MPLA Conjugates 4.18-4.22 as Vaccines
The MPLA conjugates 4.18-4.22, as structural well-defined and characterized
constructs, were synthesized by Dr. Guochao Liao in our laboratory. Immunological
studies of these conjugates were performed using female C57BL/6J mice, following the
similar protocols described for anti-Hib MPLA conjugates 4.7-4.9.
All the conjugates 4.18-4.22 were incorporated in liposomes formed by using 1,2-
distearoyl-sn-glycero-3-phosphocholine and cholesterol to improve the solubility in buffer
and further improve the immunogenicity. For immunization, the liposomal preparations of
conjugate 4.18-4.22 were each injected subcutaneously (s.c.) to a group of five or six
mice. Each female C57BL/6J mouse was injected with 0.1 mL of a vaccine preparation
containing ca. 14.0 nmol of saccharide moiety on day 1, 14, 21 and 28, respectively. Each
mouse was subjected to bleeding on day 0 before the first injection (used as blank
controls) and on day 27 and 35 after immunization. The blood samples were treated
according to standard protocols to prepare antisera for the analysis of antigen-specific
antibodies by ELISA with the HSA conjugates (4.14-4.17) as capture antigens. In addition
to total antibodies, antibody isotypes including IgG1, IgG2a, IgG2b, IgG3 and IgM were
also individually analyzed. Antibody titers were calculated from linear regression analysis
of the curves of the optical density (OD) value against logarithmic scale of serum dilution
number, and were defined as the dilution number yielding an OD value of 0.2.
Isotypes of antibodies
Figure 4.17 shows the isotypes of antibodies in the antisera obtained from mice
immunized with conjugate 4.18-4.22.
130
Figure 4.17 The ELISA results of antigen-specific antibody titers in day 35 antisera of
mice immunized with (A) 4.18 alone, (B) 4.18 plus Freund’s Completed Adjuvant (CFA),
(C) 4.19 alone, (D) 4.20 alone, (E) 4.21 alone and (F) 4.22 alone, respectively. Each dot
represents the antibody titer of an individual mouse, and the black bar shows the average
antibody titer of a group of five or six mice.
The ELISA results of antibody titers in antisera obtained with 4.18-4.22 without the
use of an external adjuvant (Figure 4.17 except (B)) suggested that all the conjugates
provoked a strong antigen-specific immune response in mice. Moreover, the high
antibody titer of IgG2b isotype and some IgG1 and IgG3 antibody isotypes revealed that
a T cell-dependent immune response was involved.17,18 According to previous discussion,
IgG2b has an extremely high immunological activity which further confirmed that this
conjugate had strong immunogenicity and could be further developed to be a
vaccine.33,37,38 In addition, no significant difference between the antisera obtained from
mice immunized with conjugate 4.18 alone and plus CFA adjuvant either in kappa or in
IgG isotypes. This result confirmed the great adjuvant function of MPLA as a build-in
131
carrier and the possibility of adjuvant-free vaccine preparations.17,18
Tetramer-sialic acid saccharide was conjugated with two kinds of MPLA including
the best one we optimized in chapter 3 and another one without two hydroxyl groups in
the acetyl-chains (Figure 4.12).18 The ELISA results of antisera obtained from mice
immunized with 4.20 and 4.22 indicated that both the conjugates can provoke strong
immune response in mice. However, the antibody titers of antisera obtained with 4.20
were significantly higher than that of 4.22 which revealed that the MPLA structure without
two hydroxyl groups had much lower immunostimulatory ability.17,40 In another aspect,
this result confirmed that MPLA as a build-in adjuvant and as a carrier played an
extremely important role in the immunostimulatory pathway that even a small change in
the MPLA structure can induced a significant decrease in the final antibody production.
To enhance the immunogenicity of MPLA conjugates, the optimized structure was the
best choice for vaccine design.
The ELISA results of antisera obtained from mice immunized with conjugate 4.21
were interesting that the total antibody titer was significantly decreased but the antibody
titer of IgG2b isotype was almost the same as the other conjugates. The phenomenon
was similar as that in KLH conjugates. This means that the pentamer-sialic acid-MPLA
conjugate may have a comparable potential to be designed as a vaccine.
In conclusion, all these conjugates without external adjuvant can elicit strong
immune response, especially the T cell-dependent immunity. The MPLA as a build-in
adjuvant and a carrier plays an extremely important role in the immunostimulatory
pathway although the details of antibody production and antibody isotype switching were
not clear. Moreover, with the length of saccharide moiety increasing, the total antibody
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titer was decreased slightly but the antibody titer of IgG2b isotype was consistent.
Cross Reaction
To investigate the recognition pattern of provoked antibodies, the cross reactions
between different length saccharides and the antisera obtained from mice immunized with
different saccharides conjugates were carried out (Figure 4.18). Antisera of 4.18 had the
highest antibody titer with Dimer-sialic acid-HSA, but had a significant lower cross
reactivity with longer sialic acid-HSA. This phenomenon revealed that, although dimer-
sialic acid did induce anti-dimer antibodies, these antibodies may be not protective due
to the low reactivity with other oligomer antigens which should be more popular on
bacteria surface. However, the cross reactivity of other conjugates (4.19-4.22) were
strong and similar as that in KLH conjugates (4.10-4.13).
C r o s s r e a c ti o n
Dim e r
Tr i m e r
Te t r a m e r 1
Te tr a m e r 2
Pe n t a m e r
0
50000
100000
150000
D im e r- H S A
T r im e r- H S A
T e t ra m e r- H S A
P e n ta m e r-H S A
A n t is e ra
A n t ib o d y T it e r
Figure 4.18 The ELISA results of cross reactivity of the antisera induced by conjugates
(4.18-4.22) dimer-, trimer-, tetramer- and pentamer-sialic acid-MPLA and the different
capture reagents (4.14-4.17) from dimer- to pentamer-sialic acid-HSA.
The antibodies elicited by vaccines should be more effective if they can cross react
with wild-spectrum oligosaccharide antigens. Thus the proper number of oligomer in this
kind vaccine construct should be higher than 2. Combining with the conclusion in above
133
isotype discussion, trimer-sialic acid conjugates was the best design with best capability
to generate effective antibodies. The cross reactivity was an important topic and worth to
be further investigated.
4.2.4 Conclusion
In summary, all the oligo-sialic acid-KLH and -MPLA conjugates can stimulate
extremely strong immune response, especially high level of IgG2b antibody titer which
indicated T cell-dependent immunity successfully involving. The IgG2b considered as one
of the most powerful antibody isotypes was particularly concentrated. In each group, all
five or six mice can be successfully immunized. Among these conjugates, trimer-sialic
acid was the best candidate for both KLH and MPLA constructs. Dimer-sialic acid may be
too short for a protective vaccine because the low cross reactivity of antibodies. The
results of MPLA conjugates further confirmed the potential immunostimulatory ability of
MPLA which can be developed to be an efficient carrier and build-in adjuvant. In future
works, the deeply investigation of structure-activity relationships of oligo-sialic acid
antigens should be an interesting and meaningful direction. For this purpose, the
monoclonal antibodies (mAb) can be prepared and the interactions of mAb and oligomer
antigens can be further explored by NMR binding studies.22
4.2.5 Experiment Section
Materials, reagents and animals
All glycoconjugates were synthesized by Dr. Guochao Liao. Titermax Gold
adjuvant, 1, 2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol were purchased
134
from Sigma-Aldrich. Alkaline phosphatase linked goat anti-mouse kappa, IgM, IgG1,
IgG2a, and IgG3 antibodies and FITC-labeled goat anti-mouse kappa antibody were
purchased from Southern Biotechnology. Female C57BL/6J mice of 6-8 weeks age used
for immunological studies were purchased from the Jackson Laboratory.
N.M group C vaccine poly-sialic acid-KLH conjugate:
Saccharide about 4.66×10-9 mol per mouse (3 µg Dimer-sialic acid moiety per
mouse per time to scale up). The loading percentages are shown in Table 4.4 above.
Table 4.5 Dose of each KLH conjugate (4.10-4.13) used for immunizing mice
Dose
Saccharide/µg
Conjugates/µg
24 doses/µg
30 doses/mg
Dimer (4.10)
3.00
39.89
957.36
1.1967
Trimer (4.11)
4.35
37.89
909.36
1.1367
Tetramer (4.12)
5.71
72.376
1737
2.171
Pentamer (4.13)
7.07
103.47
2483
3.104
N.M group C vaccine poly-sialic acids-MPLA conjugates:
13.98×10-9 mol (9 µg dimer sialic acid moiety, 33.59 µg conjugate) per mouse
Table 4.6 Dose of each MPLA conjugate (4.18-4.22) used for immunizing mice
Dose
nmol
M.W.
1 dose/ug
30 doses/ug
Dimer (4.18)
13.98
2402.9432
33.59
1.007
Trimer (4.19)
13.98
2694.1978
37.66
1.1298
Tetramer 1 (4.20)
13.98
2985.4523
41.736
1.2521
Tetramer 2(4.22)
13.98
2953.4535
45.81
1.239
Pentamer (4.21)
13.98
3276.7069
41.29
1.3743
135
General Procedure for the Preparation of Emulsion of KLH Conjugates
Each conjugate (30 doses, 1.19 mg of 4.10, 1.14 mg of 4.11, and 2.17 mg of 4.12,
and 3.10 mg of 4.13, respectively) was dissolved in 1.5 ml 1× phosphate-buffered saline
(PBS) buffer. The solution was mix with 1.5 ml Titermax Gold adjuvant (1:1, v/v) and to
form an emulsion according to the manufacturer’s protocol.
General Procedure for the Preparation of Liposomes of MPLA Glycoconjugates
The mixture of a specific MPLA conjugate (4.18-4.22) (13.98 nmol for each dose),
1, 2-distearoyl-sn-glycero-3-phosphocholine (M.W. 790.15), and cholesterol (M.W.
386.66) (a 10:65:50 molar ratio) was dissolved in CH2Cl2 and MeOH (1:1, v/v, 2 mL) in a
10 mL vial. Then, the solvents were removed in vacuum to form a thin lipid film on the vial
wall, which was hydrated by adding 2.0 mL of HEPES buffer (20 mM, pH 7.5) containing
NaCl (150 mM) and shaking the mixture in 40 ⁰C water bath, and then shaking it by vortex
mixer for several times to form a milky suspension. The milky suspension was finally
sonicated for 1 min to obtain the desired liposomes.
The liposome of MPLA conjugates with double concentration of the front one, was
mixed with the same volume of Completed Freud’s adjuvant (CFA) to form a uniform
formula according to the manufacturer’s protocol.
Immunization of Mouse
For KLH conjugates: Each female C57BL/6J mice in a group of six was immunized on
day 1 by intramuscular (i.m.) injection of 0.1 mL of the emulsion of the conjugate vaccine
and adjuvant prepared according to the manufacturer’s protocol. Following the initial
immunization, mice were boosted 3 times on day 14, day 21, and day 28 by subcutaneous
(sc.) injection of the same conjugate prepared by the same protocol. Blood samples of
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each mouse were collected through the leg veins prior to the initial immunization on day
0 and after immunization on day 28 and d 38 were clotted to obtain antisera that were
stored at -80 ⁰C before use.
For lipid A conjugates: Each female C57BL/6J mice in a group of six was immunized
on day 1 by subcutaneous (sc.) injection of 0.1 mL of the liposome of the conjugate
vaccine with/without adjuvant prepared according to the manufacturer’s protocol.
Following the initial immunization, mice were boosted 3 times on day 14, day 21, and day
28 by subcutaneous (sc.) injection of the same conjugate prepared by the same protocol.
Blood samples of each mouse were collected through the leg veins prior to the initial
immunization on day 0 and after immunization on day 18, day 28 and d 35 were clotted
to obtain antisera that were stored at -80 ⁰C before use.
Protocols for ELISA
ELISA plates were treated with 100 ul of a solution of HAS conjugate (2 ug/ml)
dissolved in coating buffer (0.1 M bicarbonate, pH 9.6) at 4 ⁰C overnight, and then at 37
⁰C for 1 h, which was followed by treatment with blocking buffer (10% BSA in PBS solution
with NaN3) and washing 3 times with phosphate-buffered saline (PBS) containing 0.05%
Tween-20 (PBST). Thereafter, a pooled or an individual mouse antiserum with serial half-
log dilutions from 1:300 to 1:656100 in PBS was added to the coated ELISA plates (100
uL/well), which was followed by incubation at 37 ⁰C for 2 h. The plates were then washed
with PBS and incubated at rt for another 1 h with a 1:1000 diluted solution of alkaline
phosphatase linked goat anti-mouse kappa, IgM, IgG2a and IgG2b antibody or with a
1:2000 diluted solution of alkaline phosphatase linked goat anti-mouse IgG1 and IgG3
antibody (100 uL/well), respectively. Finally, these plates were washed with PBS and
