picked up by DCs at the injection site, small (20–200 nm) nanoparticles and virus-like particles
(~30 nm) are also found in lymph node (LN)-resident DCs and macrophages, suggesting free drainage
of these particles to the LNs, where most DCs are concentrated. DCs appear to be strictly required for
the transport of large particles from the injection site to LNs (Manolova et al. 2008), with smaller
(25 nm) particles being more efficiently accumulated by DCs than larger ones (100 nm) (Reddy et al.
2007). Particles that are too small, however (less than ~10 nm, which is the case of most MP/APol
complexes), are taken up by antigen-presenting cells less efficiently than those that are close in size to a
virus (typically a few tens of nm) or a bacterium (typically in the μm range) (see e.g. Bachmann and
Jennings 2010, Kushnir et al. 2012, Yue and Ma 2015, and references therein; note, however, that the
amphiphilicity of MP/APol complexes may perhaps compensate for their small size; see Seong and
Matzinger 2004). This dependence has led to the development of vaccines in which the immunogen is
either aggregated into or carried by particles of the appropriate size, for instance, gold nanoparticles
(reviewed in Vartak and Sucheck 2016). Some of the tagged APols would be well adapted to this use.
For instance, the thiomorpholine-carrying version of A8-35 (Chap 4., Table 4.5; F. Giusti and
M. Zoonens, unpublished data) could be used to adsorb MP/APol complexes onto gold beads of
optimal size (Fig. 15.6, ③; cf. Suárez-Suárez et al. 2013).
Finally, the charge carried by endocytosed particles is known to play an important role in
determining their fate. A positive charge, such as that conferred by cationic lipids to DNAdelivering liposomes, improves the cytosolic release of antigens by affecting the integrity of the
endosomal membrane (see e.g. Li and Szoka 2007; Conwell and Huang 2005; Vartak and Sucheck
2016). The pH-sensitive character of the amphipathic polymer PP-50 – poly (L-lysine iso-phthalamide)
grafted with phenylalanine – appears to favor its internalization (Mercado et al. 2016). The currently
available panel of APols offers a wide diversity of behaviors, whose impact on vaccine efficiency
deserves to be explored:
(i) A8-35, A8-75, and styrene-maleic acid co-polymers (SMA) are negatively charged at the
neutral pH of the serum and will protonate and become insoluble at the lower pH (5.0–5.5)
of the lumen of endosomes, which, in the case of methacrylate-based particles, has been
shown to enhance CD8
+ T cell responses (see Keller et al. 2014, Trimaille and Verrier 2015,
and references therein).
(ii) Sulfonated APols (SAPols; Dahmane et al. 2011) will remain negatively charged following
internalization.
(iii) Non-ionic APols (NAPols; Bazzacco et al. 2012; Sharma et al. 2012) will remain
uncharged.
(iv) Phosphorylcholine-based APols (PC-APols; Diab et al. 2007a, b), due to the presence of a
secondary amine, carry a positive charge at neutral and acidic pH.
(v) Perhaps most interestingly, imidazole-carrying derivatives of A8-35 (ImidAPol; F. Giusti
and M. Zoonens, unpublished data discussed in Chap. 4, § 4.4.2) will turn from anionic to
zwitterionic and then cationic as the pH drops, a process that can be modulated by the
density of imidazole moieties (Fig. 15.6, ④).
This range of physical-chemical properties provides the vaccinologist with a broad array of new
tools to modulate the interactions of MP/APol particles with cells and the release of the immunogens
into the cytosol (it is worth noting, in this respect, that delivery to anionic liposomes of siRNA
associated with the zwitterionic APol PMAL-B-100 has been modeled by molecular dynamics and
experimentally tested; see Li et al. 2015). It would perhaps also be conceivable to trap, along with the
MP used as an immunogen, a pH-sensitive fusogenic lipid, whose integration into liposomes has been
shown to strengthen the immune response (Sato et al. 2012; Miyabe et al. 2014).
Note that all of the approaches shown in Fig. 15.6 could easily be combined.
15.2 Using Amphipols to Formulate a Subunit Vaccine Against Chlamydia trachomatis
671
(~30 nm) are also found in lymph node (LN)-resident DCs and macrophages, suggesting free drainage
of these particles to the LNs, where most DCs are concentrated. DCs appear to be strictly required for
the transport of large particles from the injection site to LNs (Manolova et al. 2008), with smaller
(25 nm) particles being more efficiently accumulated by DCs than larger ones (100 nm) (Reddy et al.
2007). Particles that are too small, however (less than ~10 nm, which is the case of most MP/APol
complexes), are taken up by antigen-presenting cells less efficiently than those that are close in size to a
virus (typically a few tens of nm) or a bacterium (typically in the μm range) (see e.g. Bachmann and
Jennings 2010, Kushnir et al. 2012, Yue and Ma 2015, and references therein; note, however, that the
amphiphilicity of MP/APol complexes may perhaps compensate for their small size; see Seong and
Matzinger 2004). This dependence has led to the development of vaccines in which the immunogen is
either aggregated into or carried by particles of the appropriate size, for instance, gold nanoparticles
(reviewed in Vartak and Sucheck 2016). Some of the tagged APols would be well adapted to this use.
For instance, the thiomorpholine-carrying version of A8-35 (Chap 4., Table 4.5; F. Giusti and
M. Zoonens, unpublished data) could be used to adsorb MP/APol complexes onto gold beads of
optimal size (Fig. 15.6, ③; cf. Suárez-Suárez et al. 2013).
Finally, the charge carried by endocytosed particles is known to play an important role in
determining their fate. A positive charge, such as that conferred by cationic lipids to DNAdelivering liposomes, improves the cytosolic release of antigens by affecting the integrity of the
endosomal membrane (see e.g. Li and Szoka 2007; Conwell and Huang 2005; Vartak and Sucheck
2016). The pH-sensitive character of the amphipathic polymer PP-50 – poly (L-lysine iso-phthalamide)
grafted with phenylalanine – appears to favor its internalization (Mercado et al. 2016). The currently
available panel of APols offers a wide diversity of behaviors, whose impact on vaccine efficiency
deserves to be explored:
(i) A8-35, A8-75, and styrene-maleic acid co-polymers (SMA) are negatively charged at the
neutral pH of the serum and will protonate and become insoluble at the lower pH (5.0–5.5)
of the lumen of endosomes, which, in the case of methacrylate-based particles, has been
shown to enhance CD8
+ T cell responses (see Keller et al. 2014, Trimaille and Verrier 2015,
and references therein).
(ii) Sulfonated APols (SAPols; Dahmane et al. 2011) will remain negatively charged following
internalization.
(iii) Non-ionic APols (NAPols; Bazzacco et al. 2012; Sharma et al. 2012) will remain
uncharged.
(iv) Phosphorylcholine-based APols (PC-APols; Diab et al. 2007a, b), due to the presence of a
secondary amine, carry a positive charge at neutral and acidic pH.
(v) Perhaps most interestingly, imidazole-carrying derivatives of A8-35 (ImidAPol; F. Giusti
and M. Zoonens, unpublished data discussed in Chap. 4, § 4.4.2) will turn from anionic to
zwitterionic and then cationic as the pH drops, a process that can be modulated by the
density of imidazole moieties (Fig. 15.6, ④).
This range of physical-chemical properties provides the vaccinologist with a broad array of new
tools to modulate the interactions of MP/APol particles with cells and the release of the immunogens
into the cytosol (it is worth noting, in this respect, that delivery to anionic liposomes of siRNA
associated with the zwitterionic APol PMAL-B-100 has been modeled by molecular dynamics and
experimentally tested; see Li et al. 2015). It would perhaps also be conceivable to trap, along with the
MP used as an immunogen, a pH-sensitive fusogenic lipid, whose integration into liposomes has been
shown to strengthen the immune response (Sato et al. 2012; Miyabe et al. 2014).
Note that all of the approaches shown in Fig. 15.6 could easily be combined.
15.2 Using Amphipols to Formulate a Subunit Vaccine Against Chlamydia trachomatis
671