REVIEW OF LITERATURE
3. REVIEW OF LITERATURE
3. 1. Vaccine delivery using biodegradable polymer particles
Biodegradable polymer particles have the potential to be a successful vaccine
delivery system as they can promote efficient antigen presentation (Men et a/.,
1999, O'Hagan and Singh, 2003, Carcaboso eta/., 2004). Particles particularly
made from poly lactide-co-glycolide (PLGA) or poly lactide (PLA), not only work
as a delivery system but also provide adjuvant activity (O'Hagan and Singh,
2003, Jiang eta/., 2005). These polymeric particulate delivery systems have the
capacity to present the antigen both by MHC class I (MHC I) and MHC class II
(MHC II) pathway and thus can activate both humoral and cellular response (Men
et a/., 1999, O'Hagan and Singh, 2003, Carcaboso et a/., 2004). Immune
responses from many polymer entrapped antigens reveal that micron sized range
particles promote humoral response where· as nanoparticles (<1 ~m) promote
cellular response (Harding and Song, 1994, Gutierro eta/., 2002). Biodegradable
polymer particle based delivery systems are currently being much sought for
single dose vaccines and for different therapeutic applications (Hanes et a/.,
1997, Gupta eta/., 1998, Lima and Rodrigues Junior, 1999, Cleland, 1999).
Polymers like PLA or PLGA have a long history of human use in surgery as
suture materials (Lima and Rodrigues Junior, 1999) and in other controlled
release formulations (Okada eta/., 1991, Plosker and Brogden, 1994).
In polymeric microspheres based vaccine delivery systems, antigens are
physically entrapped in a solid sphere. Among various methods reported in the
literature (Wand et a/., 1990), water-in-oil-in-water (W1/0N'h) double emulsion
solvent evaporation method has been most widely used for the preparation of
polymer particles (Hanes eta/., 1997, Tamber eta/., 2005). Various process and
formulation variables related to this process have been identified which can be
modified and controlled so as to obtain the particles of desirable characteristics
such as size, encapsulation efficiency, protein loading, porosity etc. (Cleland,
1998). Stability of encapsulated protein is an essential part of vaccine
development when a biodegradable polymer matrix is used for controlled release
13
applications. Early attempts to develop controlled release vaccine formulations
resulted in a poor in vitro - in vivo correlation (Gupta et a/., 1997) and failed to
offer any specific advantage over the equal dose of conventional vaccine (Men et
a/., 1995). This was attributed to the instability of the antigens during preparation
and biodegradation of particles (Hanes et a/., 1997). During encapsulation the
protein is exposed to many potentially damaging conditions such as
aqueous/organic interfaces, elevated temperatures, sonication, vigorous
agitation, hydrophobic surfaces, detergents etc. (Brunner eta/., 1999, Fu eta/.,
2000, Schwendeman, 2002). Many excipients such as trehalose, BSA, poly vinyl
alcohol (PVA), gelatin, methyl cellulose, mannitol, hydrophobic dextrans, urea,
sucrose, Tween 80, y-hydroxypropylcyclodextrins, alginate, heparin, poloxamer
etc. have been shown to improve the stability of proteins during encapsulation
(Cleland and Jones, 1996, Crotts and Park, 1997, Blanco and Alonso, 1998,
Johansen eta/., 1998a, Sanchez eta/., 1999, van de Weert eta/., 2000, Rouzes
et a/., 2000, Krishnamurthy et a/., 2000). Choice of excipients in the external
aqueous phase has also been reported to exert significant effect on the
encapsulation efficiency and release profiles of entrapped antigen from polymer
particles (Coombes eta/., 1998, Pean eta/., 1998). It is essential to add exipients
which will take care of the stability problem during the primary emulsion step;
lyophilization; and during polymer degradation (Zhu et a/., 2000). In our
laboratory, serum albumin (e.g. RSA) (2.5% w/v), sodium bicarbonate (NaHC03)
(2% w/v) and sucrose (10% w/v) were used during particle formulation to take
care of antigen stability during different steps of particle formulation which proved
to be highly effective in protecting the antigen from denaturation (Raghuvanshi et
a/., 1998, Srinivasan eta/., 2005, Katare and Panda, 2006b).
Both PLA and PLGA polymer degrade through bulk erosion and erode mostly
through the hydrolysis of the ester bonds (Shah eta/., 1992, Vert eta/., 1995, I
Grizzi et a/., 1995, Gopferich, 1996). These polymers undergo homogeneous
'bulk erosion' i.e. the rate of water penetration into the matrix is faster than the
rate of polymer degradation and degradation occurs through random hydrolytic
chain scission of the swollen polymer (Wu, 1995a). The observed erosion rate of
14
a polymeric matrix system is thus strongly dependent on the ability of water
molecules to penetrate into the polymeric matrix. Various factors have been
reported to affect the hydrolytic degradation behaviour of biodegradable
polyesters like PLA and PLGA (Shive and Anderson, 1997). These are:
-Water permeability and solubility (hydrophilicity I hydrophobicity)
- Chemical composition
-Mechanism of hydrolysis (noncatalytic, autocatalytic, enzymatic)
-Additives (acidic, basic, monomers, solvents, drugs)
-Morphology (crystalline, amorphous)
-Device dimensions (size, shape, surface to volume ratio)
-Porosity
- Glass transition temperature (glassy, rubbery)
- Molecular weight and molecular weight distribution
- Physico-chemical factors (ion exchange, ionic strength, and pH)
-Sterilization
- Site of implantation
Poly D, L-lactide (PLA) is better suited for use as vaccine delivery system than
PLGA as it is more hydrophobic. The rate of degradation of PLA is slower than
the hydrophilic PLGA and it elicits better immune response (Cleland eta/., 1998,
Rahguvanshi et a/., 2001b). Therefore, PLA (45 KDa) was used in the
preparation of all the formulations in this study. Nevertheless, both PLGA and
PLA have been used as vaccine delivery systems worldwide for various vaccine
applications. Many PLGA and PLA microencapsulated vaccine antigens have
been evaluated in a variety of animal models for protection against challenge,
antibody responses or cell-mediated immune responses (O'Hagan and Singh,
2003, Jiang et a/., 2005). In a study by Shi et a/., hepatitis 8 surface antigen
(HBsAg) was encapsulated in PLGA microspheres by water-in-oil-in
water/solvent evaporation method (Shi eta/., 2002). Although single injections of
stable and unstable microsphere preparations (12 IJg antigen dose) elicited low
antibody responses to HBsAg in C3H mice, admixture of stable microspheres
15
and small amount of alum-adsorbed HBsAg (3 j.Jg antigen in alum; 9 j.Jg antigen
in microspheres) increased the antibody levels but not so in case of the unstable
preparation. Audran eta/ .. reported enhancement of antibody response in BALB/c
mice to single injections of PLGA microspheres encapsulating TT upon
incorporation of a number of stabilizers (e.g., BSA, trehalose, calcium carbonate
or phosphate, g-hydroxypropylcylcodextrin) with the microencapsulated antigen
(Audran et a/., 1998). There are several reports of mixing TT containing
microspheres with aluminum adjuvants resulting in a high antibody response in
animals after a single injection (Raghuvanshi eta/., 2002, Katare et a/., 2003).
Potent and long lasting ~ystemic antibody responses and mixed T helper (Th)
1/Th2 immune response after nasal immunization with malaria antigen loaded
PLGA microparticles have also been reported (Carcaboso et a/., 2004).
Preparation of DNA-loaded microparticles with PLAIPLGA polymers using
different methods has also been investigated (Jilek eta/., 2005). The effects of
DNA-loaded microparticles on transfection and stimulation of dendritic cells
(DCs) in vitro have also been extensively examined using a mouse DC line and a
reporter plasmid DNA encoding the green fluorescent protein (GFP) (Jilek eta/.,
2005).
3. 2. lmmunogenicity of PLA particle based vaccines: Interactions with
APCs- Effect of size
Particulate antigens are known to be more immunogenic that soluble antigens in
vivo. Efficient targeting of antigen to the professional antigen presenting cells
(APCs) has been reported as the major factor contributing to the generation of an
immune response. In this regard, size of the antigen-entrapped particle is a major
parameter affecting the presentation of antigen to the APCs. Uptake of a
pathogen or antigen by these APCs normally takes either of three pathways
(Conner and Schmid, 2003). Firstly, APCs are capable of engulfing particles or
microorganisms non-specifically. Secondly, phagocytes are equipped with
several cell-surface receptors that recognize pathogen surfaces for receptor
mediated endocytosis. Thirdly, phagocytic cells can take up soluble substances
16
in pinocytic vacuoles by a process called macropinocytosis (Trombetta and
Mellman, 2005). There has been much ambiguity and debate over whether the
immunogenicity of particulate antigens can be explained solely by the enhanced
uptake of the particles by the professional APCs and more so over the size of
particles that can be engulfed by the APCs for antigen presentation to initiate an
effective immune response. It has been reported that while non-phagocytic
eukaryotic cells can internalize particles < 1 1-1m in size (Rejman eta/., 2004), the
professional phagocytic APCs- dendritic cells (DCs) and macrophages - can
internalize and present particulate antigens of size >1 1-Jm (Shen eta/., 1997). It
has been suggested that for efficient antigen presentation particle size should be
between 1-10 1-1m (Oh and Swanson, 1996, Shen eta/., 1997, Tomazic-Jezic et
,a/., 2001). Nevertheless, efficient primary immune responses have been
observed with particles of size greater than that of APCs (~1 0 1-Jm) (Tabata eta/.,
1996, Shi eta/., 2002, Gutierro eta/., 2002).
Generation of different magnitudes and kinetics of immune response from
microparticles, nanoparticles and their physical mixture with and without
adjuvants like alum suggests that size of particulate antigen (and use of
adjuvants) is an important parameter affecting immune response (Katare et a/.,
2003). Very few studies have been carried out to evaluate the effect of polymer
particle size on immune response. In one such study, Nakaoka et a/. observed
an inverse relationship between the size of particles and antibody response in
mice following intraperitoneal administration (Nakaoka et a/., 1996). In our
laboratory we have conclusively shown that for humoral response the optimum
particle size range should be between 2-8 1-Jm (Katare eta/., 2005). PLGA or PLA
based nanoparticles are extensively taken up by non-phagocytic eukaryotic cells,
macrophages and DCs (Lutsiak eta/., 2002, Panyam and Labhasetwar, 2002,
Kanchan and Panda, 2007); however such clear uptake studies with large sized
polymer particles have not been shown conclusively (Walter eta/., 2001, Jones
eta/., 2002, Peyre eta/., 2004). As the size of PLAIPLGA particles changes from
nanometer range to > 1 1-Jm, drastic reduction in uptake of polymer particles by
cells is observed (Desai eta/., 1996, Horisawa eta/., 2002) and 0.5 1-1m has been
17
suggested as the cut-off size for efficient phagocytosis (Foster eta/., 2001, Lai et
a/., 2007, Hirota eta/., 2007). It is quite possible that bigger sized particles are
localized in the cell membrane (Lacasse eta/., 1998) and deliver antigen into
phagosomes which have the capacity to present the antigen via MHC II pathway
(Ramachandra eta/., 1999). Most of these studies used polystyrene particles and
there are enough evidences which suggest that surface chemistry of particles
does influence the uptake rate (Oh and Swanson, 1996, Tomazic-Jezic et a/.,
2001, Foged eta/., 2005), thus a clear picture on size based cellular uptake is
never observed. But rarely has anyone suggested the role of surface chemistry
of particulate delivery systems in terms of immunogenicity and correlated the
uptake of different sized particles with antibody response. In our laboratory, we
have conclusively shown that for antibody response, hydrophobic PLA particles
are better than hydrophilic polymer particles (Raghuvanshi et a/., 2002) and
microparticles (2-8 !Jm size) entrapping tetanus toxoid elicit optimal antibody
titers (Katare eta/., 2005). Immunization with higher sized polymeric particles (>
10 IJm and < 50 IJm) as well as nanoparticles also elicited comparable antibody
titers but lower than that observed for microparticles of 2-8 !Jm size (Katare eta/.,
2003, Katare et a/., 2005). High antibody titers have also been reported from
immunization with bigger sized particles (20 IJm) entrapping HBsAg (Shi eta/.,
2002). As bigger particles can never be internalized by APCs, whether these
particles promote humoral response by virtue of their phagocytic potential or not
is still ambiguous. The mechanism of antigen delivery and its presentation to
APCs from polymeric particles which is considered to be the major factor
contributing to generation of sustained antibody response from single point
immunization is still elusive.
Over the last decade or so, much emphasis has been given to phagocytic uptake
and trafficking studies of model antigens using biodegradable polymer particles
to provide more detailed view of the molecular mechanism of antigen
presentation from such particulate delivery systems. Various fluorescent markers
have been used for visualizing the cellular uptake and distribution by
fluorescence microscopy and confocal laser scanning microscopy (CLSM)
18
(Tabata and lkada, 1988, Torche eta/., 1999, Newman eta/., 2000). In order to
have a better understanding of the principle mechanism involved in generation of
immune responses from particle based vaccine delivery it is appropriate to study
the intracellular distribution as well as tissue uptake of such particles primarily in
professional APCs - macrophages, dendritic cells (DCs) and 8 cells.
Macrophages and dendritic cells are active in eliminating the intruders by
phagocytosis (mainly by macrophages), a mechanism that is followed by antigen
presentation to generate adaptive immunity. While phagocytosis is a common
feature of macrophages and various monocytes, macro-pinocytosis seems to be
a unique property of dendritic cells. Quantitative analysis of the particle uptake by
professional APCs as also by non-professional APCs like endothelial cells has
also been performed previously by analyzing the extracted fluorescent dye from
micro- and/or nano-particles after their phagocytic uptake (Davda and
Labhasetwar, 2002). It has been shown that nanoparticles are efficiently taken up
by human vascular smooth muscle cells (VSMCs) (Panyam eta/., 2003) and also
microparticles (_:: 2 ~m) in mouse and pig peritoneal macrophages (Tabata and
lkada, 1988, Torche eta/., 1999, Newman eta/., 2000). Most of the fluorescent
dyes including Texas red dextran, Fluorescein isothiocyanate (FITC), Rhodamine
6 GX, and Rhodamine 8 used in fluorescent particle preparation undergo
leaching during uptake and therefore hinder in the correct visualization of the
uptake profile. Recently a fluorescent dye, 6-coumarin, has been reported to be
an efficient probe for visualizing the particle uptake at the cellular level as it
hardly leaches (Panyam eta/., 2003).
3. 3. Modulation of Immune Response
3. 3. 1. Humoral response from antigen entrapped in polymer particles
The early protection conferred by most existing vaccines is primarily based on
antibody-dependent mechanisms, and the quality of these responses, including
antibody avidity, is a determining factor of its efficacy (Lambert eta/., 2005). The
duration of protection relies largely on 8-cell memory responses, although
persistence of antibody production is often crucial for combating rapidly invasive
19
infections. T-cell responses to existing vaccines are undoubtedly of importance .
but they have not been extensively analyzed. Antigenic· variation and strain
diversity are complicating factors that have so far been addressed in a case-by
case manner. Vaccine-induced T -cell responses have been demonstrated for
most protein or live vaccines that are routinely used. T helper (Th) cell and
occasionally cytotoxic T lymphocyte (CTL) responses can be measured after
immunization with inactivated vaccines, such as hepatitis 8, pertussis, diphtheria,
tetanus and influenza vaccines, as well as with a number of live vaccines,
including measles, mumps, rubella, varicella, vaccinia and bacille Calmette
Guerin (BCG). However, the relative importance of T cell-mediated effector
mechanisms in the protection achieved with these vaccines is often unknown
(Lambert eta/., 2005).
Both PLGA (Esparza and Kissel, 1992, Alonso et a/., 1994, Men et a/., 1995,
Audran eta/., 1998, Gupta eta/., 1998, Tobio eta/., 1999, Sasiak eta/., 2000,
Raghuvanshi et a/., 2001b) and PLA (Raghuvanshi et a/., 2002, Katare eta/.,
2003, Katare eta/., 2005) have been extensively used towards the development
of single dose vaccine against tetanus toxoid (TT). These studies have
demonstrated protection after challenge or high levels of neutralizing antibodies
that persisted for a year or longer. Polymeric particles have the capacity to be
tailored into different sizes ranging from nanoparticles (< 1 J.Jm) to macroparticles
(> 50 J.Jm) and therefore are widely used as injectables for achieving immune
response. Most of the time these particles have been used either by
intramuscular, intradermal or intraperitoneal injections for achieving long lasting
immune response from single dose systemic immunization. Apart from TT, other
antigens such as diphtheria toxoid (DT) and hepatitis B surface antigen (HBsAg)
have been most extensively used as model systems for the development of
single dose vaccine (Johansen eta/., 1998b, Singh eta/., 1998, Johansen eta/.,
1999, Shi eta/., 2002, Peyre eta/., 2004).
20
3. 3. 2. Cellular immune response from polymer particle entrapped antigen
CD8+ T cells play a vital role in protective immunity against many intracellular
pathogens and cancer, but are notoriously difficult to activate with vaccination
and immunotherapy (Raychaudhuri and Rock, 1998, Ada, 2001). As smaller
sized polymer particles (nanoparticles, <1 !Jm) have the ability to enter the cells,
it is expected that they will deliver the antigen intracellularly resulting in MHC I
presentation. A hypothetical mechanism has been previously proposed for the.
endosomal escape of biodegradable nanoparticles following cellular
internalization (via endocytosis) according to which nanoparticles undergo
surface charge reversal (anionic to cationic) in the acidic pH of endo-lysosomes.
This facilitates an interaction of nanoparticles with the vesicular membranes,
leading to transient and localized destabilization of the membrane, thereby
resulting in the escape of nanoparticles into the cytosol (Panyam et a/., 2002,
Vasir and Labhasetwar, 2007). It was reported that a significant fraction of
nanoparticles undergoes exocytosis and only 15% of the internalized
~ nanoparticles escape into the cytosolic compartment. However, the fraction of
ln nanoparticles that escapes the endosomal compartment seems to remain in the
- cytoplasmic compartment and release the encapsulated vaccine or therapeutic in l
l :r::_ a sustained manner as the polymer degrades slowly. Fig. 3. 1 depicts a typical
f""'> intracellular trafficking pathway for nanoparticles (NPs) as vaccine or drug-carrier
systems.
It has been shown that particulate antigens (known to be more immunogenic)
favor T helper (Th) 1 response than soluble antigen in vivo (Yang eta/., 1993,
Newman eta/., 1998, Brewer eta/., 1998). The mechanisms by which exogenous
particulate antigens elicit cytotoxic T cell (CTL) response via major
· histocompatibility complex (MHC) I presentation are based on two models: (a)
cross presentation i.e. fusion of phagocytic cup (phagosome) with endoplasmic
reticulum membrane, resulting in MHC I presentation of the antigen and (b) cross
priming i.e. apoptotic antigen presenting cells (APCs)- mainly macrophages that
engulf the pathogen are taken up by dendritic cells (DCs) to cause MHC I
presentation (Sigal et a/., 1999, Heath and Carbone, 2001, Brewer et a/., 2004,
571,1€4'5 l~\~\2-b\}=-
571.9645 K1312 Ev
1111111111111111111111111111111 TH15147
21
Fifis eta/. , 2004, Lehner and Cresswell. 2004, Kaufmann and Schaible, 2005) .
But still ambiguities exist related to the various explainations for the mechanism
of uptake by APCs. Before substantial experimental evidence for cross
presentation had become available, it was assumed that different mechanisms of
endocytosis resulted in only MHC 11-restricted antigen presentation of
extracellular antigen exclusively to CD4+ T cells. The mechanisms that allow
APCs to selectively present extracellular antigen to CDS+ effector T cells (cross
presentation) or to CD4+ T helper cells are not yet fully resolved (Zinkernagel ,
2002b, Guermonprez et a/., 2003, Houde et a/., 2003, Yewdell and Haeryfar,
2005, Touret eta/., 2005).
Endosomal escape
•• ~ [!]
PE ~.____.. • RE ./ ,~ ~ - - ~ -,, \!) ,' , \
\ / f5l : Nucleus :
r:l / L!J' , L!J ... / .... -~ ' ... .. __ _ ~ .... Endo-lys ~............_ 0 .· .. ···
......... . 4 ,-' •' ~ , .. ' !.:\ ~ . ...
Lys ~ : • • Sustalned release
~-- ( of therapeytle
Sustained effect
Cytoplasmic transport
Figure 3. 1. Schematic drawing of steps involved in cytosolic delivery of
therapeutics using polymeric nanoparticles (NPs). (1) Cellular association
of NPs, (2) Internalization of NPs into the cells by endocytosis, (3)
Endosomal escape of NPs, (4) Release of therapeutic in cytoplasm, (5)
Cytosolic transport of therapeutic agent, (6) Degradation of protein/drug
either in lysosomes or in cytoplasm, (7) Exocytosis of NPs. [PE: Primary
endosomes, RE: Recycling endosomes, Endo-lys: Endo-lysosomes, Lys:
Lysosomes, Solid circles represent polymeric NPs].
22
It has been recently reported that APCs use distinct endocytic mechanisms to
simultaneously introduce soluble antigen into separate intracellular
compartments, which were exclusively presented to cos+ or CD4+ T cells.
Specifically, the mannose receptor (MR) supplied an early endosomal
compartment distinct from lysosomes, which was committed to cross
presentation (Burgdorf eta/., 2007). These findings implied that antigen does not
require intracellular diversion to access the cross-presentation pathway, because
it can enter the pathway already during endocytosis. Although this applies only to
soluble antigens (considered to be weak immunogens) that have strong affinity
for MR this gives a possible direction for encapsulated antigens that can be
released from the particles near the APCs. A role of uptake mechanisms in
classical cross-priming (Bevan, 1976) still remains to be shown.
There has been a major issue and debate over the size of particles that can be
engulfed by the APCs for efficient antigen presentation to initiate an effective
immune response. A recent report suggests that antigen formulated in relatively
large particles (560 nm) results in more efficient antigen presentation by
macrophages as they are targeted in early phagosomes than those entrapped in
relatively smaller sized particles (155 nm) which rapidly move to lysosomal
compartments (Brewer et a/., 2004). Another report suggested release of high
levels of IFN-y from DCs in draining lymph nodes and high antibody titers in mice
when they were immunized with nano-beads (40-50 nm) covalently conjugated
with the antigen (Fifis et a/., 2004). Differences in size and release pattern of
polymer entrapped antigen(s) are known to influence the way the antigen(s)
is/are presented by APCs to activate na·ive CD4+ T cells. These then differentiate
upon activation into either T helper (Th) 1 or Th2 cells resulting in either cell
mediated or humoral immune responses respectively (Storni eta/., 2005). Th1
cells are associated with IFN-y, IL-12 and TNF-a whereas Th2 cells typically
produce IL-4, IL-5, IL-10 and IL-13. CD4+ T cell polarization depends both on the
duration of antigenic stimulation and the cytokine environment to which the cells
are exposed (Bird eta/., 1998, Langenkamp eta/., 2000). The two CD4+ T cell
subsets regulate each other. Once one subset becomes dominant, it is hard to
23
shift the response to the other subset. IL-10 can inhibit the development of Th1
cells by acting on the APCs, whereas IFN-y can prevent the activation of Th2
cells (Storni eta/., 2005). The amount and sequence of the antigen that initiates
the response also influence the differentiation of CD4+ T cells into distinct effector
subsets, with high and low density of peptide on the surface of antigen
presenting cells stimulating Th1 or Th2 cell responses, respectively (Rogers and
Croft, 1999, Ruedl et a/., 2000). Hence, when stability of the antigen is
compromised, as may occur in poly (lactide-co-glycolide) microspheres
(Johansen et a/., 1998b, Johansen et a/., 2000a), this might also have ~-
consequences for the Th1ffh2 skewing of the immune response. This 'means '"':"
\
that particulate antigens can be effective immunogens in therapeutic,
prophylactic and vaccination scenarios with possibility of modulation of immune
response to either humoral or cell mediated responses.
3. 4. Candidate antigens
3. 4. 1. Tetanus toxoid and Diphtheria toxoid
Tetanus is a vaccine-preventable disease with neonatal tetanus alone accounting
for an estimated 200,000 deaths in 2000 (Vandelaer et a/., 2003). The WHO
recommends a TT vaccination regime consisting of two equal doses of alum
adsorbed TT (containing 0.85 mg aluminium/dose) given intramuscularly at least
four weeks apart followed by the third dose 6-12 months later for the prevention
of tetanus and neonatal tetanus. Subsequently, a booster dose every 10 years is
recommended. According to WHO, anti-TT antibody titers are considered
protective if circulating antibody levels are >0.5 IU/ml. Since compliance with this
multi-dose vaccine regimen of conventional tetanus toxoid (TT) vaccine is difficult
to achieve, it has been targeted as one of the priority projects as the first single
dose vaccine using polymer microspheres by the World Health Organization
(WHO) (Dietz et a/., 1996). Most investigations on single-dose vaccines have
been performed with model antigens, birth control vaccines (Singh eta/., 1995)
and TT (Johansen et al., 1998a, Audran eta/., 1998). Many other antigens such
Z4
as diphtheria toxoid (DT) (Singh et a/., 1991) considered being weak
immunogens should be equally good candidates for microparticle based
vaccines. The conventional adult dose regime for DT vaccination involves two
equal doses of alum adsorbed DT given intramuscularly at least four weeks apart
followed by the third dose 6-12 months later. According to WHO, anti-DT
antibody titers are considered protective if circulating antibody levels are >0.5
IU/ml. Circumstantially, the diphtheria incidence had also increased (Johansen
et a/., 1999). Combined single-dose vaccines containing typically diphtheria,
tetanus and pertussis toxoids would be a future goal in the developmeht of a new
generation of antigen delivery systems.
Microencapsulated TT has been reported to be better immunogen than soluble
TT. Hazrati et a/. reported that 20 Lf of TT when given in microsphere form was
more immunogenic than the same amount of soluble TT (Hazrati eta/., 1992). It
was also observed that even if the free antigen is given in three divided doses (at
0, 4 and 12 weeks), TT in microparticles was better than TT in solution, up to 15
weeks. Alonso et a/., 1993 reported that when 5 Lf of soluble TT and
encapsulated TT in PLA (molecular weight 3000) and PLGA (molecular weight
1 ,00,000) of 9 IJm and 80 IJm size range respectively, were injected to mice,
encapsulated TT gave higher antibody titres throughout 24 weeks study period.
Johansen eta/., 200Gb described the pre- clinical optimizations of final candidate .. TT vaccine formulations in guinea pigs and showed that the most efficacious
vaccines were small-sized particles{< 5 IJm), immunized with admixed alum and
fabricated from fast-degrading polymers. Previous studies in our laboratory have
showed the effect of excipients, load and dose of entrapped antigen, and more
importantly the size of PLA particles in generation of long term antibody response
against TT from single point immunization (Raghuvanshi eta/., 2001 b, Katare et
a/., 2005, Katare and Panda, 2006a, Katare and Panda, 2006b). lmmunogenicity
and single dose application of DT microencapsulated in different types of PLA
and PLGA particles (1-5 !Jm and 15-60 IJm) prepared by the methods of spray
drying and coacervation have been studied in guinea pigs (Johansen et a/.,
1999).
25
3. 4. 2. Hepatitis B surface antigen
Hepatitis 8 vaccines are produced either by rigorous purification of hepatitis 8
surface antigen (H8sAg) particles from the plasma of persistently infected
individuals or as a result of DNA cloning and expression of the H8V surface (S)
gene, the major constituent of H8sAg protein (Moynihan et a/., 2002). H8sAg
exists as a 25 KDa protein self assembled into 18-22 nm subviral particles. The
widely accepted schedule of administering 10 IJg of alum adsorbed hepatitis 8
vaccine at 0, 1 and 6 months has been used in humans since (Snyder and
Pickering, 2000). According to WHO, anti-H8sAg antibody titers are considered
protective if circulating antibody levels are >1 0 miU/mL (Lambert et a/., 2005).
However, the need for multiple injections combined with the social and cultural
resistance to the use of syringes has meant that universal coverage has not
reached those levels to contain the spread of H8V in communities where the
virus is highly endemic. A microencapsulation system that can be exploited to
produce H8sAg in powder form for delivery by alternative routes would represent
an important breakthrough. In this regard, a successful development of room
temperature stable, controlled release formulation using oligosaccharide ester
derivatives (OEDs) of trehalose and a synthetic peptide analogue of H8sAg has
been reported (Moynihan et a/., 2002). High antibody titers have also been
reported from immunization with bigger sized particles (20 IJm) entrapping
H8sAg (Shi et a/., 2002). Enhancement of T helper (Th) type 1 immune
responses against hepatitis 8 virus core antigen by PLGA nanoparticle vaccine
delivery have also been reported recently (Chong eta/., 2005). Strong systemic
and mucosal immune responses to surface-modified PLGA microspheres
containing recombinant H8sAg have been reported after intranasal
administration (Jaganathan and Vyas, 2006). A very recent report suggests
enhancement of immune response of H8sAg loaded PLA microspheres against
hepatitis 8 through incorporation of alum and chitosan (Pandit et al., 2007). A
number of approaches are currently being tested particularly for the delivery of
subunit vaccines against H8sAg, and in recent years, a number of groups have
devoted their efforts to develop nano/microparticles prepared from PLAIPLGA
polymers as vaccine delivery systems with the goal of inducing both humoral and
cellular immune responses (Bharali eta/., 2007).
3. 5. Alum as additional adjuvant
The aluminium compounds, originally identified as adjuvants over 70 years ago,
remain unique in their widespread application to human vaccines. Given this
history, it is surprising that the physicochemical interactions between aluminium
compounds and antigens are relatively poorly understood. Recent developments
in our understanding of the physicochemical and biological aspects of research
into aluminium adjuvants have offered many insights (Brewer, 2006).
The application of alum in clinical vaccines has largely been limited to situations
where protection is afforded by T helper {Th) 2 cell -related phenomenon; in
particular neutralizing antibody production (Brewer et a/., 1996, Rimaniol et a/.,
2004). The underlying mechanisms by which alum induces Th2 responses are
still not completely understood. Though alum (-aluminium hydroxide and
aluminium phosphate) has been used for many years as a vaccine adjuvant, little
is known about its mechanism of action (Guy, 2007). Two most commonly cited
mechanisms are: (a) formation of an antigen depot, and (b) immunostimulation
(HogenEsch, 2002). A recent report suggests promotion of antigen independent
B cell immune responses induced by alum (Jordan eta/., 2004). In mice these
responses have been characterized by the appearance of antigen specific lgG1
antibody in the absence of lgG2a, as well as production of IL-4 and IL-5 following . in vitro restimulation of in vivo primed lymphocytes (Rimaniol eta/., 2004). Alum
?tlso initiates strong antigen specific Th2 responses in the absence of IL-4 or IL-
13 mediated signaling in which presence of either may compensate the absence
of the other (Brewer eta/., 1999).
Since most vaccines are usually administered systemically by intramuscular
injections, histological examination of injection sites have been performed
showing a clear predominance of macrophages following the injection of
aluminum hydroxide-containing vaccines in animals, and the data obtained
27
illustrate the key role of this cell type in the physiological reaction to aluminum
hydroxide containing vaccines (Gherardi et a/., 2001). Previous reports have
shown aluminium hydroxide (AIOOH)-gel (alum) to increase vascular
permeability and toxic effects in macrophages with weak hemolytic effect (Goto
eta/., 1993). In vitro effects of aluminum hydroxide adjuvants (alum) on isolated
macrophages have been studied (Rimaniol eta/., 2004). Many reports suggest a
muscle reaction to alum containing vaccine injection resulting in a granuloma
with striking muscle fascia infiltration by aluminum-loaded macrophage, a
histological entity called macrophagic myofasciitis (MMF) that is also found in
some human vaccines. Altogether, these studies strongly suggested that
macrophages play a critical role in vaccine-induced immune responses and alum
induces macrophage differentiation towards a specialized antigen-presenting cell
type (Rimaniol eta/., 2004). The role of such cells in aluminium adjuvanticity has
been highlighted- macrophages were shown to be activated by aluminium to
present antigen and a previously unknown population of IL-4-producing cells was
shown to be required for alum-induced in vivo priming and expansion of antigen
specific B cells (Jordan et a/., 2004). Recently it has been shown that antigen
(alpha casein) internalization by dendritic cells was enhanced when the antigen
remained adsorbed to aluminium-containing adjuvant (phagocytosis) following
administration than when administered in soluble form (macropinocytosis)
(Morefield eta/., 2005).
For the induction of antibody responses it was initially thought that the antigen
needs to be adsorbed to the adjuvant surface. In such a scenario, the adjuvant
forms a depot at the vaccination site from which antigen is released, transforming
a soluble antigen into a particulate one to favour APC uptake (Guy, 2007). It has
been shown previuosly that the ability of aluminium adjuvants to increase
internalisation of antigen for generation of antibody response is dependent on
association of antigen· with aluminium adjuvants-particles and presumably
phagocytic uptake (Brewer, 2006). Significantly, the ability of aluminium
adjuvants to adsorb antigens is critically dependent on the environmental milieu
that the vaccine is delivered into with protein rich solutions, such as tissue culture
28
....,
medium or interstitial fluid being capable of displacing antigens previously
adsorbed to aluminium adjuvants. This is particularly true of antigens that bind to
aluminium adjuvants through electrostatic interactions rather than ligand
exchange, for example, ovalbumin (Brewer, 2006). Therefore, a reason that
aluminium adjuvants may fail on their own is through lack of antigen association
with adjuvant not only during formulation, but also following administration, and
consequently a loss of antigen targeting to phagocytes or uptake via
phagocytosis. It is now been realized that the degree of adsorption may change
following intramuscular or subcutaneous administration of the vaccine (Jiang et
a/., 2006). This is because the composition of interstitial fluid is different than the
composition of the vaccine formulation. Components of interstitial fluid such as
phosphate anion, citrate anion, or fibrinogen have been found to cause elution of
the antigen (Jiang eta/., 2006) thereby citing the limitations of antigen adsorbtion
on alum.
On the other hand, biodegradable polymer particles prepared using PLA and
PLGA offer much advantage as potent adjuvants and vaccine delivery systems
and have been extensively pursued for development of single dose vaccines
(Hanes eta/., 1997, Sasiak eta/., 2000, Lofthouse, 2002, Katare eta/., 2003,
O'Hagan and Singh, 2003, Katare eta/., 2005, Katare and Panda, 2006b). But
immune response is still lower than the response achieved from conventional
multiple doses of alum adsorbed antigen. It has therefore been suggested to
incorporate alum as an additional adjuvant along with polymer encapsulated
antigen. Previous studies in our laboratory have suggested that co-administration
of alum and PLA microparticle entrapped antigen (single dose) enhances
antibody titers better than alum adsorbed antigen (two doses) or microparticle
entrapped antigen alone (single dose) lasting for many months post
immunization (Katare et a/., 2003). Proof of principle studies have been
performed where improved immune response comparable and even better than
that from alum adsorbed antigen dose has been reported by use of additional
adjuvants in conjunction with polymer entrapped antigen (Singh et a/., 1998,
Gupta eta/., 1998, Shi eta/., 2002). It has also been reported that microparticles
29
alone generate low levels of lgG1 but presence of alum along with microparticles
improves lgG1 levels, a known Th2 response (antibody response) indicator, '
considerably (Katare and Panda, 2006a).
The main objective is to elicit long lasting immune responses and thus minimize
immunizations. Enhanced immune response have also been reported with a
combination of alum and biodegradable nanoparticles containing tetanus toxoid
indicating synergistic adjuvant effect of biodegradable nanoparticles in
combination with alum (Raghuvanshi et a/., 2001a). Overall, microparticle
entrapped antigen along with alum elicit better antibody response in comparison
to either alum adsorbed antigen or nanoparticle entrapped antigen in presence of
alum (Katare eta/., 2005, Katare and Panda, 2006a, Kanchan and Panda, 2007) . . But there exist no reports that explain the mechanism of action of alum in
conjunction with particle based immunization as adjuvant for generation of long
lasting antibody response or their interaction with macrophages that form the
predominant antigen presenting cell (APC) type for such immunization modalities
(Rimaniol et a/., 2004). There has also been much emphasis on continuous
presence versus single contact of antigen with the host immune system for
generation of antibody response (Ochsenbein eta/., 2000a, Gourley eta/., 2004).
Mode of delivery of polymer entrapped antigen to APCs and duration of its
presence near the APCs is very critical for the type and duration of immune
response and needs to be elucidated clearly. The role of alum can be critical .
here in prolonging the stay of the released antigen and its presentation to the
APCs that may lead to improved antibody response.
3. 6. Immunological memory and its maintenance
Vaccines represent one of the greatest successes of medicine over the last
century. Interestingly, all working vaccines protect hosts via neutralizing
antibodies. This includes the Classical childhood vaccines against bacterial
toxins, measles, poliomyelitis, and smallpox (Zinkernagel, 2003). The greatest
attribute of a successful vaccine has been considered to be its ability to confer
protection in terms of both strength and duration upon infection and reinfection.
30
This signifies that memory is a hallmark of immunity and a requirement for a
successful vaccine. With the exception of T cell-independent polysaccharide
based vaccines, all existing successful vaccines induce immunological memory.
It is believed that there are two arms of immunological memory, humoral
immunity that includes preexisting antibody, memory B cells and plasma cells;
and cellular immunity that includes memory cos+ and CD4+ T cells. The relative
importance of humoral and cellular immunity in protection against reinfection has
been of high interest. It is widely believed that both humoral and cellular immunity
have evolved to provide distinct effector functions. Preexisting specific antibodies
can directly bind virus particles, extracellular bacteria, and parasites. Their role is
to said to provide the first line of defense by neutralizing or opsonizing invading
pathogens. T cells on the other hand cannot recognize free pathogens but
instead recognize infected cells by interacting with microbial antigens (peptides)
bound on major histocompatibility complex (MHC) class I (CDS T cells) or class II
(CD4 T cells) molecules (Gourley eta/., 2004). Yet the magnitude and duration of
memory differs considerably between vaccine types and it is essential to
understand determinants that enhance or limit the duration of vaccine efficacy.
The ability of a vaccine antigen to generate protection is a complex challenge
that goes beyond the antibody response itself. Protection persists as long as
actively induced or passively acquired antibodies are present in blood and is
mainly due to opsono-phagocytic mechanisms (antibodies binding to bacteria
and thus facilitating their phagocytosis and elimination), with some contribution of
direct bactericidal effects.
Memory antibody response is largely responsible for protection against
reinfection with most known acutely lethal infectious agents and thus is the basis
for most clinically successful vaccines. But there is a lot of confusion about the
concept of immunological memory and immunity (Zinkernagel, 2003).
Immunological memory has been described well in certain textbooks (Janeway et
a/., 2001, Goldsby et a/., 2000). According to Janeway et a/., memory is "the
ability of the immune system to respond more rapidly and effectively to
pathogens that have been encountered previously" and reflects the pre-existence
31
of clonally expanded populations of antigen-specific lymphocytes (Janeway eta/.,
2001 ). Memory responses which are called secondary depending on the number
of exposures to antigen also differ qualitatively from primary responses. This is
particularly clear in the case of the antibody response where the characteristics
of antibodies produced in secondary and subsequent responses are distinct with
respect to affinity-avidity from those produced in the primary response to the j
same antigen. But it is argued that since such studies have been performed
using model antigens like ovalbumin, sheep red blood cells or phenolic haptens,
the cited textbook definitions are not sufficient to explain immunity - that is,
improved survival after exposure to infections causing acute disease.
A school of thought exists that believes that there probably is no special
immunological memory as defined by textbooks that is biologically important
(Zinkernagel, 2003). Instead, protective immunity is considered to be simply
maintained in an antigen-driven manner to guarantee host and species survival.
In particular, increased protective neutralizing-antibody titers that are antigen
dependent guarantee survival of offspring and of the species, whereas antigen
activated T cells are important to protect individual hosts against their persistent
low-level infections (Zinkernagel, 2002a).
Herein lies the more pertinent issue - the reasons behind the generation and
maintenance of memory response. This attains more significance when we look
from the perspective of vaccine strategies. Maintenance of long-term antibody
responses is critical for protective immunity against many pathogens. However,
the duration of humoral immunity and the role played by memory 8 cells remain
poorly defined (Amanna et a/., 2007). A long standing debate about whether
specific memory is maintained by distinct populations of long lived plasma cells
(antibody secreting cells) that can persist without antigen or by long lived
memory cells that are under continuous stimulation by residual antigen/antigen
re-exposure still exists (Zinkernagel, 2002a, Gourley eta/., 2004).
32
3. 6. 1. Role of antigen in generation of immunological memory
3. 6. 1. 1. Evidences against antigen persistence
An important question that still needs to be addressed is: How are memory 8
cells maintained in the absence of re-exposure to the pathogen, and whether
they actually require antigen for their survival? Adoptive transfer experiments
suggested that antigen was important for the long term maintenance of memory
· 8 cells (Askonas eta/., 1970, Gray and Skarvall, 1988). It has been proposed
that the source of antigen for this maintenance was retained on follicular dendritic
cells trapped in immune complexes (Mandel eta/., 1980, Tew eta/., 1990, Gray,
1993, Zinkernagel eta/., 1996). However, recent studies have demonstrated that
memory 8 cells can persist in mice in the absence of detectable immune
complexes (Hannum et a/., 2000). Wherein mice were engineered so that the
only form of immunoglobin M (lgM) was membrane bound and thus lacked the
ability to form immune complexes. Memory 8 cells although at lower numbers
than control mice could be maintained in these mice in the absence of immune
complexes. Elegant experiments by Rajewsky's group using a novel transgenic
approach largely confirmed that antigen was not required for the maintenance of
memory 8 cells (Maruyama eta/., 2000). It was suggested that the presence of a
8-cell receptor (8CR) is required for survival of all peripheral 8 cells (Lam eta/.,
1997). The importance of 8CR and 8 cell activation factor (8AFF) (Gourley eta/.,
2004) as also a role for CD40L (Liu eta/., 1989, Gray eta/., 1994) for the survival
of memory 8 cells has been highlighted. It is possible that low affinity interactions
with self-antigen may promote the survival of both memory and naive 8 cells.
Natural re-exposure to antigen is the most important mechanism for maintaining
antibody levels against commonly reoccurring or endemic pathogens. Low-grade
chronic or latent infections that provide a continuous or sporadic antigenic
stimulation are also believed to drive 8CR-dependent differentiation of both
memory 8 cells and naive 8 cells into antibody-secreting plasma cells. In the
absence of re-exposure to the pathogen, however, antibody levels can still be
maintained for many years. Classical examples of long-term protective immune
memory in the absence of re-exposure to antigen include: (a) measles immunity
33
on the Faroe Islands (65 years), (b) yellow fever immunity in Virginia (75 years),
and (c) polio immunity in remote Eskimo villages in Alaska (40 years) (Gourley et
a/., 2004). There are two current hypotheses to explain the longevity of the
antibody response in the absence of re-exposure to antigen. The first proposed
independently by Slifka et a/. and Manz et a/. was that antibody levels are
maintained by the presence of long-lived plasma cells with very long half-life, on
the order of 150 to 300 days in the bone marrow that secrete specific antibody for
extended periods, potentially for the life of an individual (Manz et a/., 1997, Slifka
eta/., 1998). The second proposed by Bernasconi eta/. states that memory B
cells are continually differentiating into plasma cells in an antigen independent
manner due to bystander or polyclonal activation (Bernasconi eta/., 2002).
Given the experimental evidence available to date it is argued that multiple
mechanisms come into play to maintain serum antibody levels in an antigen
independent fashion (Fig. 3. 2, adapted from Gourley eta/., 2004). There are no
available data directly examining the longevity of plasma cells in humans but
studies using murine model have shown that long-lived plasma cells (-1 0%) can
survive for the entire lifespan of a mouse (Slifka et a/., 1998). Based on these
evidences one can argue for the possibility that plasma cells could survive and
secrete antibody for extended periods in humans as well in an antigen
independent manner and hence explain the reasons behind long lasting memory
antibody response.
3. 6. 1. 2. Evidences in favor of antigen persistence
It is argued that B cells cannot differentiate and mature to become antibody
producing plasma cells in the absence of antigen (Gray and Skarvall, 1988,
Ochsenbein eta/., 2000a, Zinkernagel, 2002). B cells process antigen bound to
surface immunoglobulin in order to present the relevant peptides on major
histocompatibility complex (MHC) class II molecules on their surface and to
receive signals from specific T helper cells. This process is necessary for B cells
to mature to plasma cells, but it is not sufficient to prime naive T cells.
34
I)
2)
3)
Long-lived p lasn1a ce 11
(bone n1arrow)
Bystander ac~h·ation . Y y lA\ (T cell mffi1ated) ..,. Wi)y y · Y. ~ Innate signals y . y Y
l\.1 emory B cell ( pG L rs etc) . • .. plasma ceU
Short or long-lived?
Short or long-lived?
Figure 3. 2. Multiple mechanisms to generate long-term persisting
antibody. (1) The presence of long-lived plasma cells in the bone
marrowgenerated during the primary infection or vaccination produce
antibody for extended periods, potentially lifelong. (2) The turnover of
memory B cells into short-lived or long-lived plasma cells due antigen
independent polyclonal activation. Polyclonal activation could include non
specific CD4 T cell help or activation by innate signals such as CpG or LPS.
(3) The differentiation of memory B cells into short-lived plasma cells due
to the presence of persistent antigen, for example, during a chronic
infection or due tore-exposure to antigen.
35
Naive T helper cells are efficiently induced only by antigen presenting cells
(APCs); including dendritic cells (DCs) presenting helper peptides via MHC class
II. After priming, increased precursor frequencies of specific T and B cells have
been readily demonstrated in humans or mice (Beverley, 1990, Zinkernagel et .
a/., 1996, Ahmed and Gray, 1996, Slifka eta/., 1998). But primed T and B cells
without specific antigen have been demonstrated not to be protective by
themselves,· as demonstrated using adoptive transfer experiments (Steinhoff et
a/., 1995, Ochsenbein eta/., 2000b).
It is also advocated that protection requires pre-existent neutralizing antibody
titers, which are produced only by antigen-triggered B cells maturing to plasma
cells. Some experiments have suggested that plasma cells may have a very long
half-life, on the order of 150 to 300 days (Manz eta/., 1997, Slifka eta/., 1998).
However, that experimental evidence was considered to be flawed by the
argument that such studies relied on antibody responses against nonprotective
antigens composed of multiple undefined determinants (Zinkernagel, 2002a) ..
Evidence most prominently used to highlight immunological memory being very
long-lasting in the absence of antigen re-exposure is the historical
epidemiological measles viral infection in South Pacific Islands or the Faroe
Islands (Ahmed, 1992, Gourley et a/., 2004). This conclusion was based on
evidence that survivors of measles epidemics were still immune 60-70 years
later when ship crews brought a new epidemic to these islands. It was, however,
shown that crippled measles virus may persist in many more hosts than had
previously been suspected, if not in all of them (Zinkernagel, 2002a).
There is evidence to suggest that protective antibody titers usually decrease over
time e.g. against diphtheria, tetanus toxins, or measles vaccines (Guris et a/.,
1996, Ochsenbein eta/., 1999). All these observations showed that protective
neutralizing or opsonizing antibody responses are antigen dependent
(Zinkernagel, 2003).
Studies on B cell memory have also been carried out in mice using rabies-like
cytopathic vesicular stomatitis virus, the noncytopathic lymphocytic
36
choriomeningitis virus (Armstrong and WE), and after immunization with various
inert viral antigens (Ochsenbein eta/., 2000a). It was reported that memory B
cells were long-lived in the absence of antigen, nondividing, and relatively
resistant to irradiation; and must be stimulated by antigen to differentiate to short
lived antibody-secreting plasma cells, a process that is also efficient in the bone
marrow and always depends on radiosensitive, specific T help. For vaccines to
induce long-term protective antibody titers, they need to repeatedly provide, or
continuously maintain antigen in minimal quantities over a prolonged time period
in secondary lymphoid organs or the bone marrow for sufficient numbers of long
lived memory B cells to mature to short:-lived plasma cells. Because the half-life
of serum lg is considered to be less than 3 weeks (Talbot and Buchmeier, 1987,
Vieira and Rajewsky, 1988), continuous antibody production is believed to be
necessary to maintain lgG antibody titers over a prolonged period. Therefore, to
maintain long-term antibody titers, a continuous differentiation of B cells to
plasma cells apparently must take place (Ochsenbein eta/., 2000a). This study
suggested that B cell memory is characterized by a pool of antigen-independent
long-lived B cells with higher frequencies than found in unprimed mice (Schittek
and Rajewsky, 1990, Gray eta/., 1996). It was also indicated that memory B cells
require additional encounter with specific antigen to differentiate to antibody
secreting plasma cells but do so only if appropriate T help is available. It is widely
accepted that antigen may persist on follicular dendritic cells as antigen-antibody
complexes that restimulate B cells (Szakal et a/., 1989, Tew et a/., 1990,
Maclennan et a/., 1992, Gray et a/., 1996). It is possible that dependent on the
definition of memory and the assay method used, immunological memory may
not necessarily correlate with protective immunity (Zinkernagel et a/., 1996,
Ahmed and Gray, 1996). Thus, the term 'immunological memory' (quicker and
better) can be considered to be mostly antigen independent and produced by
increased precursor frequencies, whereas protection is determined by antigen
dependent preexisting antibody titers or activated T cells (Zinkernagel, et a/.,
2007). This understanding needs to be kept in mind while deciding vaccine
strategies. ·
37
The importance and relevance of plasma cells can not be undermined here. It
has been emphasized that although highly frequent memory B cells are reported
to be long-lived (Schittek and Rajewsky, 1990, Ochsenbein et a/., 2000a) and
independent of antigen, they do not produce antibodies and the~efore cannot
mediate the type of immediate protection necessary against acutely cytopathic
infectious agents (Steinhoff et a/., 1995). Maintenance of protective antibody
titers is considered to be dependent on a continuous or repetitive stimulation by
antigen from within or outside of the host. This suggests that rather than memory
B cells, effector B cells, i.e. plasma cells secreting neutralizing antibodies, are the
bearer of protective immunity. Therefore, for improved antibody-protection by B
cell vaccination, antigen persistence in some form over a prolonged period of
time in the host; or persistence of antigen or repetitive exposure from the outside
(e.g., polio virus) has been stated to be essential (Ochsenbein eta/., 2000a).
3. 6. 1. 3. Neutral views on immunological memory
A recent finding suggests that peripheral memory B cells and antibody-secreting
plasma cells may represent independently regulated cell populations and may
play different roles in the maintenance of protective immunity (Amanna et a/.,
2007). The study involved analysis of duration of humoral immunity to common
vaccinia virus, measles, mumps, rubella, varicella-zoster virus, and Epstein-Barr
virus and nonreplicating vaccine antigens (tetanus and diphtheria) in 45 subjects
for a period of up to 26 years.
Very recent works have thrown some light on the duration of antibody responses
as a function of the life span of plasma cells where the role of persisting antigen
in maintaining B cell memory has been tested (Gatto eta/., 2007). It was reported
that antibody titers were long-lived, but declined continuously with a t112 of -80
days, which corresponded to the life span of plasma cells. The germinal center
(GC) reaction, which lasted for up to 100 days, was shown to be dependent on
antigen associated with follicular dendritic cells; and early GCs produced
massive numbers of plasma and memory B cell precursors, whereas the late
antigen-dependent GCs are dispensable for the maintenance of antibody levels
38
and B cell memory. The findings again suggested an independent existence and
function of the two important cell populations in maintaining protection- Memory
B cells and Plasma cells.
It has been reported that analogous to maintenance of memory B cells, memory
CDS+ T cells do not require antigen for survival or homeostasis (Lau eta/., 1994,
Maruyama et a/., 2000). Instead recent studies have identified a role for
cytokines IL-7 (for survival) and IL-15 for maintenance of memory CDS T+ cells.
Another study evaluated whether T cell memory reflects increased precursor
frequencies of specific long-lived T cells and/or a low-level immune response
against some form of persistent antigen (Kundig et a/., 1996). Antivirally
protective CDS+ T cell memory was analyzed mostly in the original vaccinated
host to assess the role of antigen in its maintenance. It was concluded that T cell
mediated protective immunity against the usual peripheral routes of reinfection is
antigen dependent.
To summarize, the success of current vaccines is based on the induction of long
lived antibody responses. Despite this, relatively little is known about how the
duration of antibody responses is regulated. It seems plausible that long-lived
plasma cells may maintain antibody titers for long periods of time in an antigen
independent fashion, as has been observed in individuals exposed to vaccinia
virus (Crotty eta/., 2003). In contrast, antigen can persist on follicular dendritic
cells (FDCs) for long periods of time, and potentially contribute to the
maintenance of antibody responses in an antigen-dependent fashion (Mandel et
a/., 1980, Tew eta/., 1990, Zinkernagel eta/., 1996). Antigen may also persist in
the absence of FDCs, for example during latent viral infections or after
vaccination, in which the use of adjuvants results in local antigen depots
(Zinkernagel, 2003). Hence, the relative contribution of long-lived bone marrow
plasma cells and continually differentiating memory B cells to the maintenance of
humoral memory remains controversial (McHeyzer-Williams and Ahmed, 1999,
Manz eta/., 2002, Zinkernagel, 2003) . •
39
3. 6. 2. Antigen entrapped polymer particles as model system to evaluate
memory antibody response
For evaluating memory antibody response, it is difficult to create experimental . conditions that exclude antigen persistence (Gatto et a/., 2007). However,
biodegradable polymer particles with different porosity and release patterns of
the entrapped antigen can be used to mimic conditions of antigen persistence
versus initial bolus of antigen to address this issue from vaccination point of view.
The role of alum can be critical here in prolonging the stay of the released
antigen and its presentation to the antigen presenting cells (APCs). These
controlled antigen release systems can be designed to release entrapped
antigen over a long period of time (weeks to months) or quick early burst release
of antigen in one go following a single immunization (Morris et a/., 1994, Hanes
et a/., 1997, Cleland, 1999). The release of encapsulated proteins from the
polymer particles occurs by the combination of diffusion through pores and
subsequent polymer biodegradation (Jalil and Nixon, 1990). Release rates of the
antigens can be further modified by varying polymer composition, antigen
content, size and porosity of particles, physicochemical properties of antigens
and by inclusion of additives (Wu, 1995a).
To put things into proper perspective, it is safe to believe that memory is indeed a
hallmark of immunity and a prerequisite for a successful vaccine. Still an
important topic of concern is efficacy of vaccine in terms of quality of such a
recall response. Although it may be reassuring to rely on memory responses for
long-term protection, it is advised to be aware of the limitations of recall
responses when microbial invasion can be faster than 8-cell reactivation as
reported in case of toxoid-based vaccines. Protection by toxoid-based vaccines
is known to require persistence of antitoxin antibodies at the time of toxin
exposure. A large diphtheria outbreak in the former Soviet Union indicated that in
the context of a short incubation period (1-5 days), pathogen-induced
reactivation of immune memory was not sufficient to protect against diphtheria
even when most adults were immunized in childhood. This indicated that they
had lost immunity over time (Golaz eta/., 2000). Conversely, memory induced by
40
multiple doses of vaccine is reported to be quite efficient for ensuring long-lived
protection against hepatitis B (Banatvala eta/., 2000). The sustained efficacy of
hepatitis B immunization is believed to rely on a rather long viral incubation
period (4-12 weeks between exposure and hepatitis) that exceeds the few days
required for the reactivation of HBsAg-specific memory cells. After acute
infection, unless immediately neutralized by a sufficient level (>10 miU/mL) of
circulating antibodies, hepatitis B virus reaches the liver and initiates its
replication within hepatocytes. In vaccinated subjects, viral replication is believed
to rapidly drive the activation of vaccine-induced memory cells into effector cells
capable of interrupting viral replication before the onset of chronic liver disease
thereby confering protection to preimmunized individuals.
Although T-cell effector responses are not considered involved in the short-term
protection induced by most existing vaccines, they are likely to have a role in
long-term effects and it would be unreasonable to ignore this arm of vaccine
responses in the development of new vaccines. Vaccine-induced T-cell
responses have been demonstrated for most protein or live vaccines that are
routinely used e.g. inactivated vaccines, such as hepatitis B, pertussis,
diphtheria, tetanus and influenza vaccines, as well as with a number of live
vaccines, including measles, mumps, rubella, varicella, vaccinia and bacille
Calmette-Guerin (BCG) (Lambert eta/., 2005). However, the relative importance
of T cell-mediated effector mechanisms in the protection achieved with these
vaccines is often unknown. BCG is the only licensed tuberculosis vaccine known,
which relies on T cell-dependent mechanisms to confer protection.
Keeping the above mentioned concerns in mind, the use of biodegradable
polymer particles offers a viable alternative and a possible model system to
analyze the role of antigen in generation of memory antibody response. It offers
the choice of a wide window for strategic designing of vaccine formulations
keeping in mind the immunological profile of the target disease and the target
population at the initial steps of vaccine design.
41