glycoprotein that is widely used for examining antigen-specific
cellular and humoral responses in mice. It is an inherently weak
immunogen when administered alone, but can induce strong antigen-specific responses when paired with the proper adjuvant.
Archaeosomes are liposomes that are traditionally comprised of
total polar lipids or semisynthetic glycerolipids derived from
Archaebacteria. They have been shown to exhibit strong adjuvant
activity in multiple preclinical models [1]. We recently developed an
SLA archaeosome formulation composed of a single sulfated glycolipid named sulfated lactosylarchaeol (SLA; 6
0 -sulfate-β-D-Galp(1,4)-β-D-Glcp-(1,1)-archaeol) that when admixed or used to
encapsulate antigen results in the induction of strong, long-lasting
cellular and humoral immune responses in mice with a favorable
safety profile [2, 3].
In our vaccination model, we inject OVA alone or in combination with SLA to the tibialis anterior (TA) muscles (Fig. 1). At days
1 and 3 postvaccination, TA muscles and draining LNs (inguinal
and popliteal) are harvested and processed for flow cytometric
analysis of immune cell types. Antigen uptake is determined by
calculating the number of cells positive for OVA conjugated with
Alexa Fluor 647 fluorochrome (OVA-AF647) per immune cell
type. The impact of immunization on immune cell recruitment
can be assessed by quantifying cell number regardless of positivity
for antigen. Using this method, we have demonstrated the impact
of SLA-based adjuvants on the magnitude and kinetics of immune
cell recruitment to the injection site and draining LNs and how this
modulates cellular uptake and distribution of antigen [2–5].
2 Materials
2.1 Vaccine
Formulation
1. Adjuvant: sulfated lactosylarchaeol (SLA; 6
0 -sulfate-β-D-Galp(1,4)-β-D-Glcp-(1,1)-archaeol) (National Research Council
Canada) (see Note 1).
2. Antigen: Ovalbumin (Hyglos GmbH, Bernried am Starnberger
See, Germany) labeled with Alexa Fluor
® 647-NHS Ester dye
(Thermo Fisher Scientific, Pittsburgh, PA, USA)
(OVA-AF647) (see Note 2).
3. Negative control: 1Â phosphate-buffered saline (PBS)
(pH 7.4) (see Note 3).
2.2 Intramuscular
Injection
1. Insulin syringes, 0.5 cc.
2. Polyethylene (PE)-20 tubing.
2.3 Muscle
Collection
and Processing
1. R10 complete medium: RPMI containing 10% heat-inactivated
fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% glutamine, and 55 μM 2-mercaptoethanol.
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Gerard Agbayani et al.
cellular and humoral responses in mice. It is an inherently weak
immunogen when administered alone, but can induce strong antigen-specific responses when paired with the proper adjuvant.
Archaeosomes are liposomes that are traditionally comprised of
total polar lipids or semisynthetic glycerolipids derived from
Archaebacteria. They have been shown to exhibit strong adjuvant
activity in multiple preclinical models [1]. We recently developed an
SLA archaeosome formulation composed of a single sulfated glycolipid named sulfated lactosylarchaeol (SLA; 6
0 -sulfate-β-D-Galp(1,4)-β-D-Glcp-(1,1)-archaeol) that when admixed or used to
encapsulate antigen results in the induction of strong, long-lasting
cellular and humoral immune responses in mice with a favorable
safety profile [2, 3].
In our vaccination model, we inject OVA alone or in combination with SLA to the tibialis anterior (TA) muscles (Fig. 1). At days
1 and 3 postvaccination, TA muscles and draining LNs (inguinal
and popliteal) are harvested and processed for flow cytometric
analysis of immune cell types. Antigen uptake is determined by
calculating the number of cells positive for OVA conjugated with
Alexa Fluor 647 fluorochrome (OVA-AF647) per immune cell
type. The impact of immunization on immune cell recruitment
can be assessed by quantifying cell number regardless of positivity
for antigen. Using this method, we have demonstrated the impact
of SLA-based adjuvants on the magnitude and kinetics of immune
cell recruitment to the injection site and draining LNs and how this
modulates cellular uptake and distribution of antigen [2–5].
2 Materials
2.1 Vaccine
Formulation
1. Adjuvant: sulfated lactosylarchaeol (SLA; 6
0 -sulfate-β-D-Galp(1,4)-β-D-Glcp-(1,1)-archaeol) (National Research Council
Canada) (see Note 1).
2. Antigen: Ovalbumin (Hyglos GmbH, Bernried am Starnberger
See, Germany) labeled with Alexa Fluor
® 647-NHS Ester dye
(Thermo Fisher Scientific, Pittsburgh, PA, USA)
(OVA-AF647) (see Note 2).
3. Negative control: 1Â phosphate-buffered saline (PBS)
(pH 7.4) (see Note 3).
2.2 Intramuscular
Injection
1. Insulin syringes, 0.5 cc.
2. Polyethylene (PE)-20 tubing.
2.3 Muscle
Collection
and Processing
1. R10 complete medium: RPMI containing 10% heat-inactivated
fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% glutamine, and 55 μM 2-mercaptoethanol.
514
Gerard Agbayani et al.