CVI Accepted Manuscript Posted Online 21 January 2015 Clin. Vaccine Immunol. doi:10.1128/CVI.00714-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Vaccine Self-assembling Immune Matrix is a new delivery platform that enhances immune
2
responses to rHBsAg in mice
3 4
Rafaella F. Q. Grenfell*a,b, Lisa M. Shollenberger*a, E. Farah Samlia, Donald A. Harna#
5 6
Department of Infectious Diseases, College of Veterinary Medicine and The Center for Tropical
7
and Emerging Global Diseases, University of Georgia, Athens, GA, USAa. Schistosomiasis
8
Laboratory, Rene Rachou Research Center, Oswaldo Cruz Foundation, Belo Horizonte, Minas
9
Gerais, Brazilb.
10 11
Running Title: Adjuvanticity of Vaccine Self-assembling Immune Matrix
12 13
#Address correspondence to Donald A. Harn, [email protected].
14
*R.F.Q.G. and L.M.S. contributed equally to this work.
1 of 23
15
ABSTRACT
16 17
Vaccination remains the most effective public health tool to prevent infectious diseases. Many
18
vaccines are marginally effective and need enhancement for immune compromised, elderly and
19
very young populations. To enhance immunogenicity, we exploited the biphasic property of the
20
(RADA)4 synthetic oligopeptide to create VacSIM™ (Vaccine Self-assembling Immune
21
Matrix), a new delivery method. VacSIM™ solution can easily be mixed with antigens,
22
organisms and adjuvants for injection. Post-injection, the peptides self-assemble into hydrated
23
nanofiber gel-matrices, forming a depot with antigens and adjuvants in the aqueous phase. We
24
believe the depot provides slow release of immunogens, leading to increased activation of
25
antigen presenting cells that then drive enhanced immunogenicity. Using recombinant hepatitis B
26
surface antigen (rHBsAg) as a model immunogen, we compared VacSIM™ delivery to delivery
27
in alum or complete Freund’s adjuvant (CFA). Delivery of the rHBsAg antigen to mice via
28
VacSIM™ without adjuvant elicited higher specific IgG responses than when rHBsAg was
29
delivered in alum or CFA. Evaluating IgG subtypes shows a mixed Th1/Th2 type response
30
following immunization with VacSIM™, which was driven further toward Th1 with addition of
31
CpG as adjuvant. Increased specific IgG endpoint titers were observed in both C57BL/6 and
32
BALB/c mice, representative of Th1 and Th2 environments, respectively. Restimulation of
33
splenocytes suggests VacSIM™ does not cause an immediate pro-inflammatory response in the 2 of 23
34
host. Overall, these results suggest that VacSIM™, as a new delivery method, has potential to
35
enhance immunogenicity and efficacy of numerous vaccines.
36 37
INTRODUCTION
38 39
Vaccines remain the single greatest public health tool to combat infectious diseases. Vaccine
40
formulation and delivery are key to the ability of vaccines to induce the desired immune
41
responses. One goal of vaccine delivery is to present vaccine antigens in a manner that enhances
42
antigen presenting cell (APC) activation, leading to antigen/organism uptake and processing of
43
vaccine antigen(s). Delivery methods or adjuvants that safely enhance vaccine
44
immunogenicity/efficacy are desirable for vaccines that are marginally effective, or for vaccines
45
administered to low-responders, or immune compromised populations. Additional goals are to
46
reduce the number of doses required to induce effective, vaccine responses and/or to reduce the
47
amount of vaccine/dose, especially when a single dose of vaccine is administered, as with annual
48
influenza vaccine. Lastly, in pandemics, a vaccine that produces high titers after a single
49
administration would be beneficial. Recent advances in the understanding of how innate
50
mechanisms influence adaptive immunity have led to more rational design in the development of
51
new vaccine adjuvants and delivery systems.
52 3 of 23
53
Aluminum salts were the first adjuvants licensed for human vaccines in the 1920s. The licensure
54
of non-aluminum salt adjuvants took an additional 80 years (1). One reason for this long gap is
55
that the principles of adjuvant activity were largely unknown, thus the development of adjuvants
56
was empirical, Moreover, many adjuvants, including Freund’s adjuvant, were reactogenic and
57
not acceptable for licensure (2). Recent methods to improve vaccine delivery have taken several
58
approaches, including, virosomes (3-5), vector-based (6-8), liposome based (9-11) and more
59
traditional formulation with adjuvants (12-17). Each of these methods has some drawbacks,
60
either in terms of reactogenicity, regulatory issues, product stability or time required for
61
formulation, however each of these methods focuses on presenting the vaccine as a particulate.
62 63
To enhance vaccine immunogenicity over that seen when conventional alum-based delivery
64
methods are used, we focused on identifying ways to deliver vaccines such that vaccine antigens
65
are released over time. This led to our development of a new vaccine delivery method we call
66
VacSIM™ (Vaccine Self-Assembling Immune Matrix). VacSIM™ is based on the unique
67
properties of the (RADA)4 synthetic oligopeptide and other biopolymers (18-22). The (RADA)4
68
synthetic oligopeptide was created by Zhang (22), and commercialized by 3-D Matrix Inc. for
69
cell scaffolding, and is currently in clinical trials for wound healing (PuraStat®), tissue repair
70
(23), and dental implant scaffolding (PuraMatrix) (24), and as such, has already undergone third
71
party reactogenicity and toxicity testing (25). VacSIM™ is composed solely of the (RADA)4 4 of 23
72
synthetic oligopeptide. Thus, it is biocompatible and biodegradable. Ex vivo, a 1.0% VacSIM™
73
solution is in liquid phase, allowing the flexibility to mix virtually any antigen, organism and
74
adjuvant. Upon injection into tissue, VacSIM™ self-assembles into hydrated nanofibers (26),
75
forming a gel matrix depot, which entraps and concentrates vaccine components in the aqueous
76
phase at the injection site (27). We hypothesize that the vaccine depot allows slow egress of
77
antigen out of the gel pores, leading to persistent release of antigen, which is considered to be
78
important in the development of robust adaptive immunity and memory responses (28, 29). We
79
theorize that slow release of antigen and possible cellular infiltration of the VacSIM™ gel-depot,
80
increases activation and maturation of antigen presenting cells that then drive more robust
81
adaptive responses. Further, it is possible that the gel depot protects vaccine components from
82
degradation.
83 84
VacSIM™ is different from first and second generation hydrogels, such as alginates and
85
methacrylates, as well as microneedle and layer-by-layer technology, as all of these require
86
polymerization ex vivo (30-35). Further, it is different from other self-assembling peptide (SAP)
87
technologies, such as the Q11 peptide, where the antigenic motif must be directly conjugated to
88
the SAP (36-38). In contrast, VacSIM™ is prepared by simply mixing SAP and antigens prior to
89
administration.
90 5 of 23
91
The biphasic property of VacSIM™, coupled with the inert nature of the resultant vaccine gel
92
depot, provides novel technology that can be translated for use in a multitude of vaccines. In this
93
study, we tested the ability of VacSIM™ to enhance specific immune responses to the
94
recombinant hepatitis B surface antigen.
95 96
MATERIALS & METHODS
97 98
Immunizations. Five to seven week old female BALB/c or C57BL/6 mice were purchased from
99
Harlan Laboratories, housed in specific pathogen-free conditions and allowed to acclimate for
100
one week prior to manipulation. All animal work was performed in accordance with all
101
applicable policies and approved by the institutional animal care and use committee. Mice were
102
immunized subcutaneously with recombinant Hepatitis B surface antigen (rHBsAg) adw subtype
103
(5 g, Fitzgerald Industries, Inc. Massachusetts, USA) ± CpG (50 g ODN 1826, InvivoGen,
104
Inc. California, USA), via alhydrogel (250 g, Inject Alum, Thermo Fisher Scientific, Inc.,
105
Pittsburgh, PA, USA) ± CpG, by VacSIM™ ± CpG, in Freund's (13 l, Sigma-Aldrich Co.
106
Missouri, USA) or sham-immunized with VacSIM™ or phosphate buffered saline (PBS), at a
107
maximum volume of 200 l per injection site. Three or four weeks later, as indicated, mice were
108
administered a second, identical immunization.
109 6 of 23
110
Antibody Responses. For the kinetic experiments, blood samples were collected from all
111
immunized and control mice weekly, beginning week -1 prior to primary immunization.
112
rHBsAg-specific antibodies in sera were analyzed by ELISA. Briefly, plates were coated with 4
113
g/ml rHBsAg before singly diluted sera (1:800 for IgA, IgG2a and 1:1200 for IgM, IgG, IgG1)
114
was added. Antibodies specific for rHBsAg were detected by HRP-conjugated secondary
115
antibodies (1:2000 α-IgA, 1:2500 α-IgM, and 1:2500 α-IgG from Santa Cruz; 1:1000 α-IgG1 and
116
1:1000 IgG2a from Invitrogen). Plates were developed with SureBlue TMB 1-component
117
substrate (KPL) and stopped by addition of 2N sulfuric acid. To evaluate the IgG1/IgG2a ratio,
118
we multiplied the absorbance values by the dilution factors of the sera to normalize prior to
119
dividing. For non-kinetic studies, all methods remain the same except that mice were bled prior
120
to immunization, and post each subsequent immunization. Each sample of serum was diluted
121
serially for endpoint titer antibody analysis and samples with undetectable antibody were
122
assigned a value below the detection limit.
123 124
ELISpots. Splenocytes were obtained 3weeks post-prime or 2 weeks post-boost, then stimulated
125
for 20 hours to evaluate HBsAg-specific cell-mediated responses using IFN ELISpot, according
126
to the manufacturer’s instructions (BD Biosciences, San Francisco, CA, USA). Briefly, single
127
cell suspensions (3 and 1.5 × 105 cells per well) were cultured at 37°C with 5% CO2 for 20 hours
128
in complete medium [RPMI-1640 (Hyclone, Thermo Scientific, Utah, USA) supplemented with 7 of 23
129
10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, antimycotic, non-essential amino acids
130
and β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Cells were stimulated with 1
131
μg/ml Concanavalin A (ConA), 20 µM HBs specific peptide [S28-39, IPQSLDSWWTSL
132
(synthesized at greater than 95% purity [Biosynthesis Inc., Lewisville, TX, USA and dissolved in
133
DMSO prior to dilution in culture media]) or left unstimulated. ELISpot plates were developed
134
with AEC substrate and spot forming units (SFU) were counted using an Immunospot Analyzer
135
(C.T.L.). The SFU value was expressed as mean of triplicate cultures per mouse.
136 137
Flow cytometry. Splenocytes collected at 3 weeks post prime and 2 weeks post boost were
138
stimulated for 3 days with 5 µM HBs S28-39 peptide and 40 U/ml rIL-2 (Peprotech) for
139
assessment of molecular specificity. Briefly, HBs peptide was bound with H2-Ld DimerX
140
reagent (BD) at 37oC overnight and incubated with secondary antibody (A85-1-PE, BD) and
141
isotype control (mIgG1λ, BD). Two million re-stimulated splenocytes were stained with
142
DimerX-HB reagent, α-CD8 Pacific Blue antibody (BD) and viability dye (Live/Dead Near
143
Infrared fixable dye, Invitrogen). Live cells were acquired using a BD LSRII flow cytometer and
144
analyzed with FACS Diva software (BD).
145 146
Cytokine responses. Splenocytes were isolated two weeks post boost and tested for rHBsAg-
147
specific cytokine production. Single cell suspensions (1.5 × 106/ml) were cultured at 37°C with 8 of 23
148
5% CO2 in complete medium and stimulated with 1 μg/ml ConA, 5 μg/ml rHBsAg or left
149
unstimulated. Levels of TNF-α and IFN were quantified after 24 and 48 hours culture, IL-5
150
after 48 hours, IL-4 at 48 and 72 hrs and IL-10 after 72 hours, each in triplicate, by ELISA,
151
according to manufacturer’s instructions (BD).
152 153
Statistical analyses. Except for the kinetic studies in Figure 1A, all other data are represented by
154
box and whisker plots, ranging from the minimum value to the maximum, with the mean
155
displayed as a plus (+) sign. Statistical analyses (one- or two-way ANOVAs with Bonferroni
156
post tests) were performed using Prism 5 (GraphPad, California, USA). Differences were
157
considered statistically significant when p values were ≤ 0.05 and are denoted by asterisks.
158 159
RESULTS
160 161
Using recombinant hepatitis B surface antigen (rHBsAg) as a model immunogen, we compared
162
the VacSIM™ delivery to rHBsAg in alum or Freund’s, +/- CPG as a model adjuvant for use
163
with VacSIM™ and alum.
164 165
VacSIM™ enhances and sustains specific humoral immunity
166 9 of 23
167
To assess humoral immunity, total rHBsAg-specific IgA, IgM and IgG titers were quantified at
168
various times post immunization and are presented in Figure 1A. Mice immunized with
169
VacSIM™-delivered rHBsAg had higher specific IgM levels than CFA-delivered antigen
170
(Figure 1B, upper panel, p
1
Vaccine Self-assembling Immune Matrix is a new delivery platform that enhances immune
2
responses to rHBsAg in mice
3 4
Rafaella F. Q. Grenfell*a,b, Lisa M. Shollenberger*a, E. Farah Samlia, Donald A. Harna#
5 6
Department of Infectious Diseases, College of Veterinary Medicine and The Center for Tropical
7
and Emerging Global Diseases, University of Georgia, Athens, GA, USAa. Schistosomiasis
8
Laboratory, Rene Rachou Research Center, Oswaldo Cruz Foundation, Belo Horizonte, Minas
9
Gerais, Brazilb.
10 11
Running Title: Adjuvanticity of Vaccine Self-assembling Immune Matrix
12 13
#Address correspondence to Donald A. Harn, [email protected].
14
*R.F.Q.G. and L.M.S. contributed equally to this work.
1 of 23
15
ABSTRACT
16 17
Vaccination remains the most effective public health tool to prevent infectious diseases. Many
18
vaccines are marginally effective and need enhancement for immune compromised, elderly and
19
very young populations. To enhance immunogenicity, we exploited the biphasic property of the
20
(RADA)4 synthetic oligopeptide to create VacSIM™ (Vaccine Self-assembling Immune
21
Matrix), a new delivery method. VacSIM™ solution can easily be mixed with antigens,
22
organisms and adjuvants for injection. Post-injection, the peptides self-assemble into hydrated
23
nanofiber gel-matrices, forming a depot with antigens and adjuvants in the aqueous phase. We
24
believe the depot provides slow release of immunogens, leading to increased activation of
25
antigen presenting cells that then drive enhanced immunogenicity. Using recombinant hepatitis B
26
surface antigen (rHBsAg) as a model immunogen, we compared VacSIM™ delivery to delivery
27
in alum or complete Freund’s adjuvant (CFA). Delivery of the rHBsAg antigen to mice via
28
VacSIM™ without adjuvant elicited higher specific IgG responses than when rHBsAg was
29
delivered in alum or CFA. Evaluating IgG subtypes shows a mixed Th1/Th2 type response
30
following immunization with VacSIM™, which was driven further toward Th1 with addition of
31
CpG as adjuvant. Increased specific IgG endpoint titers were observed in both C57BL/6 and
32
BALB/c mice, representative of Th1 and Th2 environments, respectively. Restimulation of
33
splenocytes suggests VacSIM™ does not cause an immediate pro-inflammatory response in the 2 of 23
34
host. Overall, these results suggest that VacSIM™, as a new delivery method, has potential to
35
enhance immunogenicity and efficacy of numerous vaccines.
36 37
INTRODUCTION
38 39
Vaccines remain the single greatest public health tool to combat infectious diseases. Vaccine
40
formulation and delivery are key to the ability of vaccines to induce the desired immune
41
responses. One goal of vaccine delivery is to present vaccine antigens in a manner that enhances
42
antigen presenting cell (APC) activation, leading to antigen/organism uptake and processing of
43
vaccine antigen(s). Delivery methods or adjuvants that safely enhance vaccine
44
immunogenicity/efficacy are desirable for vaccines that are marginally effective, or for vaccines
45
administered to low-responders, or immune compromised populations. Additional goals are to
46
reduce the number of doses required to induce effective, vaccine responses and/or to reduce the
47
amount of vaccine/dose, especially when a single dose of vaccine is administered, as with annual
48
influenza vaccine. Lastly, in pandemics, a vaccine that produces high titers after a single
49
administration would be beneficial. Recent advances in the understanding of how innate
50
mechanisms influence adaptive immunity have led to more rational design in the development of
51
new vaccine adjuvants and delivery systems.
52 3 of 23
53
Aluminum salts were the first adjuvants licensed for human vaccines in the 1920s. The licensure
54
of non-aluminum salt adjuvants took an additional 80 years (1). One reason for this long gap is
55
that the principles of adjuvant activity were largely unknown, thus the development of adjuvants
56
was empirical, Moreover, many adjuvants, including Freund’s adjuvant, were reactogenic and
57
not acceptable for licensure (2). Recent methods to improve vaccine delivery have taken several
58
approaches, including, virosomes (3-5), vector-based (6-8), liposome based (9-11) and more
59
traditional formulation with adjuvants (12-17). Each of these methods has some drawbacks,
60
either in terms of reactogenicity, regulatory issues, product stability or time required for
61
formulation, however each of these methods focuses on presenting the vaccine as a particulate.
62 63
To enhance vaccine immunogenicity over that seen when conventional alum-based delivery
64
methods are used, we focused on identifying ways to deliver vaccines such that vaccine antigens
65
are released over time. This led to our development of a new vaccine delivery method we call
66
VacSIM™ (Vaccine Self-Assembling Immune Matrix). VacSIM™ is based on the unique
67
properties of the (RADA)4 synthetic oligopeptide and other biopolymers (18-22). The (RADA)4
68
synthetic oligopeptide was created by Zhang (22), and commercialized by 3-D Matrix Inc. for
69
cell scaffolding, and is currently in clinical trials for wound healing (PuraStat®), tissue repair
70
(23), and dental implant scaffolding (PuraMatrix) (24), and as such, has already undergone third
71
party reactogenicity and toxicity testing (25). VacSIM™ is composed solely of the (RADA)4 4 of 23
72
synthetic oligopeptide. Thus, it is biocompatible and biodegradable. Ex vivo, a 1.0% VacSIM™
73
solution is in liquid phase, allowing the flexibility to mix virtually any antigen, organism and
74
adjuvant. Upon injection into tissue, VacSIM™ self-assembles into hydrated nanofibers (26),
75
forming a gel matrix depot, which entraps and concentrates vaccine components in the aqueous
76
phase at the injection site (27). We hypothesize that the vaccine depot allows slow egress of
77
antigen out of the gel pores, leading to persistent release of antigen, which is considered to be
78
important in the development of robust adaptive immunity and memory responses (28, 29). We
79
theorize that slow release of antigen and possible cellular infiltration of the VacSIM™ gel-depot,
80
increases activation and maturation of antigen presenting cells that then drive more robust
81
adaptive responses. Further, it is possible that the gel depot protects vaccine components from
82
degradation.
83 84
VacSIM™ is different from first and second generation hydrogels, such as alginates and
85
methacrylates, as well as microneedle and layer-by-layer technology, as all of these require
86
polymerization ex vivo (30-35). Further, it is different from other self-assembling peptide (SAP)
87
technologies, such as the Q11 peptide, where the antigenic motif must be directly conjugated to
88
the SAP (36-38). In contrast, VacSIM™ is prepared by simply mixing SAP and antigens prior to
89
administration.
90 5 of 23
91
The biphasic property of VacSIM™, coupled with the inert nature of the resultant vaccine gel
92
depot, provides novel technology that can be translated for use in a multitude of vaccines. In this
93
study, we tested the ability of VacSIM™ to enhance specific immune responses to the
94
recombinant hepatitis B surface antigen.
95 96
MATERIALS & METHODS
97 98
Immunizations. Five to seven week old female BALB/c or C57BL/6 mice were purchased from
99
Harlan Laboratories, housed in specific pathogen-free conditions and allowed to acclimate for
100
one week prior to manipulation. All animal work was performed in accordance with all
101
applicable policies and approved by the institutional animal care and use committee. Mice were
102
immunized subcutaneously with recombinant Hepatitis B surface antigen (rHBsAg) adw subtype
103
(5 g, Fitzgerald Industries, Inc. Massachusetts, USA) ± CpG (50 g ODN 1826, InvivoGen,
104
Inc. California, USA), via alhydrogel (250 g, Inject Alum, Thermo Fisher Scientific, Inc.,
105
Pittsburgh, PA, USA) ± CpG, by VacSIM™ ± CpG, in Freund's (13 l, Sigma-Aldrich Co.
106
Missouri, USA) or sham-immunized with VacSIM™ or phosphate buffered saline (PBS), at a
107
maximum volume of 200 l per injection site. Three or four weeks later, as indicated, mice were
108
administered a second, identical immunization.
109 6 of 23
110
Antibody Responses. For the kinetic experiments, blood samples were collected from all
111
immunized and control mice weekly, beginning week -1 prior to primary immunization.
112
rHBsAg-specific antibodies in sera were analyzed by ELISA. Briefly, plates were coated with 4
113
g/ml rHBsAg before singly diluted sera (1:800 for IgA, IgG2a and 1:1200 for IgM, IgG, IgG1)
114
was added. Antibodies specific for rHBsAg were detected by HRP-conjugated secondary
115
antibodies (1:2000 α-IgA, 1:2500 α-IgM, and 1:2500 α-IgG from Santa Cruz; 1:1000 α-IgG1 and
116
1:1000 IgG2a from Invitrogen). Plates were developed with SureBlue TMB 1-component
117
substrate (KPL) and stopped by addition of 2N sulfuric acid. To evaluate the IgG1/IgG2a ratio,
118
we multiplied the absorbance values by the dilution factors of the sera to normalize prior to
119
dividing. For non-kinetic studies, all methods remain the same except that mice were bled prior
120
to immunization, and post each subsequent immunization. Each sample of serum was diluted
121
serially for endpoint titer antibody analysis and samples with undetectable antibody were
122
assigned a value below the detection limit.
123 124
ELISpots. Splenocytes were obtained 3weeks post-prime or 2 weeks post-boost, then stimulated
125
for 20 hours to evaluate HBsAg-specific cell-mediated responses using IFN ELISpot, according
126
to the manufacturer’s instructions (BD Biosciences, San Francisco, CA, USA). Briefly, single
127
cell suspensions (3 and 1.5 × 105 cells per well) were cultured at 37°C with 5% CO2 for 20 hours
128
in complete medium [RPMI-1640 (Hyclone, Thermo Scientific, Utah, USA) supplemented with 7 of 23
129
10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, antimycotic, non-essential amino acids
130
and β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Cells were stimulated with 1
131
μg/ml Concanavalin A (ConA), 20 µM HBs specific peptide [S28-39, IPQSLDSWWTSL
132
(synthesized at greater than 95% purity [Biosynthesis Inc., Lewisville, TX, USA and dissolved in
133
DMSO prior to dilution in culture media]) or left unstimulated. ELISpot plates were developed
134
with AEC substrate and spot forming units (SFU) were counted using an Immunospot Analyzer
135
(C.T.L.). The SFU value was expressed as mean of triplicate cultures per mouse.
136 137
Flow cytometry. Splenocytes collected at 3 weeks post prime and 2 weeks post boost were
138
stimulated for 3 days with 5 µM HBs S28-39 peptide and 40 U/ml rIL-2 (Peprotech) for
139
assessment of molecular specificity. Briefly, HBs peptide was bound with H2-Ld DimerX
140
reagent (BD) at 37oC overnight and incubated with secondary antibody (A85-1-PE, BD) and
141
isotype control (mIgG1λ, BD). Two million re-stimulated splenocytes were stained with
142
DimerX-HB reagent, α-CD8 Pacific Blue antibody (BD) and viability dye (Live/Dead Near
143
Infrared fixable dye, Invitrogen). Live cells were acquired using a BD LSRII flow cytometer and
144
analyzed with FACS Diva software (BD).
145 146
Cytokine responses. Splenocytes were isolated two weeks post boost and tested for rHBsAg-
147
specific cytokine production. Single cell suspensions (1.5 × 106/ml) were cultured at 37°C with 8 of 23
148
5% CO2 in complete medium and stimulated with 1 μg/ml ConA, 5 μg/ml rHBsAg or left
149
unstimulated. Levels of TNF-α and IFN were quantified after 24 and 48 hours culture, IL-5
150
after 48 hours, IL-4 at 48 and 72 hrs and IL-10 after 72 hours, each in triplicate, by ELISA,
151
according to manufacturer’s instructions (BD).
152 153
Statistical analyses. Except for the kinetic studies in Figure 1A, all other data are represented by
154
box and whisker plots, ranging from the minimum value to the maximum, with the mean
155
displayed as a plus (+) sign. Statistical analyses (one- or two-way ANOVAs with Bonferroni
156
post tests) were performed using Prism 5 (GraphPad, California, USA). Differences were
157
considered statistically significant when p values were ≤ 0.05 and are denoted by asterisks.
158 159
RESULTS
160 161
Using recombinant hepatitis B surface antigen (rHBsAg) as a model immunogen, we compared
162
the VacSIM™ delivery to rHBsAg in alum or Freund’s, +/- CPG as a model adjuvant for use
163
with VacSIM™ and alum.
164 165
VacSIM™ enhances and sustains specific humoral immunity
166 9 of 23
167
To assess humoral immunity, total rHBsAg-specific IgA, IgM and IgG titers were quantified at
168
various times post immunization and are presented in Figure 1A. Mice immunized with
169
VacSIM™-delivered rHBsAg had higher specific IgM levels than CFA-delivered antigen
170
(Figure 1B, upper panel, p
