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