Vaccine 24 (2006) 1680–1686

A preliminary evaluation of alternative adjuvants to alum using arange of established and new generation vaccine antigens

Manmohan Singha,∗, Mildred Ugozzolia, Jina Kazzaza, James Cheskoa,Elawati Soenawana, Donatella Mannuccib, Francesca Tittab, Mario Contornib,

Gianfranco Volpinib, Giuseppe Del Guidiceb, Derek T. O’Hagana

a Vaccines Research, Chiron Vaccines, 4560 Horton St., M/S 4.3, Emeryville, CA 94608, USAb Chiron S.p.A, 1 Fiorentina Road, 5300 Siena, Italy

Received 6 July 2005; received in revised form 22 August 2005; accepted 26 September 2005Available online 6 October 2005

Abstract

Although alum is the most commonly used vaccine adjuvant, it has some limitations for use with the next generation recombinant antigens.W P andt Ag, MenC potent forM ines. Ont ticles werec with othera©

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entlyines.om-s tonju-re isr thetivesalum

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e explored the use of alternative adjuvant formulations (poly lactide co-glycolide (PLG) microparticles, MF59 emulsion, CAl-yrosine suspension) in comparison with five different vaccine antigens—namely, diphtheria toxoid (DT), tetanus toxoid (TT), HBs

conjugate and MB1. The results indicated that although alum was optimal for bacterial toxoid based vaccines, it was not highlyB1, Men C or HBsAg antigens. MF59 emulsion stood out as a good alternative to alum for TT, HBsAg, MB1 and Men C vacc

he other handl-tyrosine suspension and CAP did not enhance immune responses over alum with most antigens. PLG microparomparable or better than alum with both MB1 and Men C conjugate vaccine. The study indicates that it is possible to replace alumdjuvant formulations like MF59 and PLG and maintain and/or improve immune responses with some vaccine antigens.2005 Elsevier Ltd. All rights reserved.

eywords: PLG microparticles; Alum; CAP;l-Tyrosine; MF59 emulsion

. Introduction

The use of aluminium hydroxide (generically calledlum), as an adjuvant was first described by Glenny et al.[1] in926. Since then, both aluminum hydroxide and aluminiumhosphate have been selected as the preferred adjuvants forost human vaccines. In most cases the selection is basedrimarily on alum’s long history of safe use in humans.owever, although alum performs as a good adjuvant foracterial toxoids, including diphtheria and tetanus toxoidsDT and TT), it has significant limitations for some newereneration vaccine antigens. Alum is a poor inducer of Th1ellular immune responses and stimulates the production ofgE antibodies, which is consistent with Th2 cellular immuneesponse[2–4]. Unfortunately, a Th2 based immune response

∗ Corresponding author. Tel.: +1 510 923 7877; fax: +1 510 923 2586.E-mail address: manmohansingh@chiron.com (M. Singh).

is not likely to offer optimal protection against several imptant infectious diseases, including TB, HIV and HCV.addition, in recent years there have been some concerelation to safety issues with alum, including some destions of nodules and erythema[5,6]. However, in a recencomprehensive evaluation of the safety of alum adsovaccines, it was definitively concluded that these arefor widespread use[7]. Nevertheless, the challenge remathat alum-based adjuvants may not be suitable or sufficipotent to allow the development of new generation vaccSome next generation vaccines will comprise complex cbination products, with several different kinds of antigenbe included, which could be protein polysaccharide cogates, recombinant proteins and traditional toxoids. Thea real concern that alum may not prove to be effective fodevelopment of new generation vaccines and that alternaare needed. Furthermore, the total amount of acceptabledose for human vaccines (1.0–1.5 mg/vaccination) limit

264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2005.09.046

M. Singh et al. / Vaccine 24 (2006) 1680–1686 1681

potential to carry several antigens and adjuvants within thesame formulation. For example, the delivery of multiple anti-gens on alum like the Pnc-9-Men C (Wyeth Vaccines, UK) hasa limitation on total alum that can be used. This prompted usto evaluate alternative adjuvants and delivery systems, whichmay have the potential to be used with a variety of vaccineantigens.

The adjuvant approaches that were chosen to be evalu-ated were based on a number of factors, including previousexperience in humans with similar vaccines (calcium phos-phate, CAP), previous experience in humans with differentkinds of vaccines (tyrosine), our own experience both pre-clinically and clinically with a range of vaccines (MF59) andencouraging pre-clinical experience to date (PLG). MF59emulsion is an oil-in-water emulsion that is part of a fluvaccine (FLUAD)TM which is already used in more than 20countries[8]. MF59 has also been evaluated in the clinic withseveral different antigens (HIV gp120/gp140, HSV gB/gD,HBV) in thousands of human subjects and has demonstratedexcellent safety and tolerability[8]. Earlier studies with HBV,Hib and Men C in non-human primates established the poten-tial of MF59 adjuvant to be used with a range of vaccines,for the induction of more potent responses than alum[9,10].Hence, MF59 appears to be a good alternative to alum, butits relative potency needs to be established with a more broadrange of vaccines.

bio-c s),rp s asd cord[ d theu vac-c b-l clesi tigen[ sedt ingh ventsaa ilityi PLGm cci-n le ofeW gensf edu nses[ nanta faceo thea eyc lud-iM i-g the

use of alum as a broadly applicable delivery system forvaccines.

Another formulation that was selected for comparisonwith alum is l-tyrosine suspension, sincel-tyrosine hasbeen shown to be a safe adjuvant for human vaccine and ispart of an allergy vaccine licensed in Europe (Tyrosin TUTM

by Allergy Therapeutics)[26]. Earlier work with long-chainstearyl esters of tyrosine (stearyl tyrosine) however did notshow great enhancement in comparison to alum with TTas an antigen[33]. l-Tyrosine being an amino acid hasan excellent safety profile and it is also widely used as ahealth supplement. We used a micro-fluidized suspensionof l-tyrosine as an alternative adjuvant for comparisonwith alum. We do not believe thatl-tyrosine amino acid byitself has previously been evaluated with vaccines againstinfectious diseases, but its established safety profile made itan attractive approach to evaluate.

Calcium phosphate has previously been shown to be aneffective adjuvant for a number of vaccines, including diph-theria and tetanus toxoids, and therefore was selected forcomparison with alum[27]. CAP has been included in a suc-cessfully marketed vaccine for humans, but was graduallyreplaced with aluminum hydroxide in the 1960s. However,its established safety profile made it interesting for evaluationwith new generation vaccines.

In this paper, we compared the immunogenicity of fivea a-t ect-i howb r dif-f ectedb relatew bac-t ort ore,s relyq -v ucee func-t

2

2

erc ec-i ers-b dem ical( s-p ark.l em-i nust ccal

Microparticles prepared from the biodegradable andompatible polymers, the poly lactide co-glycolides (PLGepresent a new approach for vaccine delivery[11]. Theseolymers have already been widely used in humanrug delivery systems and have an excellent safety re

11]. Several groups over the last decade have explorese of PLG microparticles with entrapped antigens asine delivery systems[12–15]. However, a common proem with the encapsulation of antigens into micropartis denaturation or degradation of the entrapped an16–19]. During microencapsulation, antigens are expoo a range of potentially damaging conditions, includigh shear, aqueous/organic interfaces, organic solnd low pH within the degrading polymer[18,19]. In anttempt to overcome the problems of antigen instab

n microparticles, we have developed novel chargedicroparticles which were prepared with dioctylsulfosuate (DSS) as the particle stabilizer, and were capabfficient adsorption of an antigen onto their surface[20,21].e have recently reported that novel recombinant anti

rom Neisseria meningitidis serotype B can be deliversing this approach and induce potent immune respo

22]. We also demonstrated that a complex recombintigen from HIV-1 (gp120) was adsorbed on the surf microparticles, without changing the structure ofntigen[23]. An advantage of microparticles is that than also be used to deliver additional adjuvants, incng CpG, which can be adsorbed on the surface[24], or

PL, which can be entrapped[25]. The adsorption of antens onto PLG microparticles provides an alternative to

ntigens (TT, DT, Men C, Men B, HBsAg) in combinion with the different alum alternative adjuvants. By selng different types of antigens, we wanted to evaluateroadly applicable and robust these adjuvants were fo

erent kinds of vaccines. The five antigens were also selecause the antibody response to them is known to corith protection, either using functional assays (serum

ericidal activity for MenB and MenC, diphtheria toxin)hrough quantitation of antibodies (e.g. HBsAg). Therefide-by-side comparison of different adjuvants is not meuantitative, but also qualitative[28]. The different adjuant formulations were evaluated on their ability to indnhanced antibody responses and wherever possible,

ional antibody responses in mice.

. Materials and methods

.1. Chemicals reagents

RG503, poly (d,l-lactide-co-glycolide) 50:50 co-polymomposition (intrinsic viscosity 0.4 from manufacturers spfications) was obtained from Boehringer Ingelheim (Peturg, VA, USA). Dioctylsulfosuccinate (DSS), USP graannitol and sucrose came from Sigma-Aldrich Chem

St. Louis, MO, USA). Alum hydroxide and calcium phohate were obtained from Brenntag Biosector, Denm-Tyrosine powder was obtained from Sigma-Aldrich Chcals, St. Louis, in a powder form. Diphtheria toxoid, tetaoxoid, hepatitis B surface antigen, group C Meningoco

1682 M. Singh et al. / Vaccine 24 (2006) 1680–1686

conjugate vaccine (Men C conjugate) and recombinant MB1from N. meningitidis serotype B (Men B) were all procuredfrom Chiron Vaccines, Siena, Italy and respective proteinconcentration confirmed for each antigen in Emeryville,USA. MF59 emulsion was obtained from Chiron Vac-cines, Marburg. U96-Nunc Maxisorp plates (Nalgene NuncInternational, Rochester, NY and Costar, Cambridge, MA),goat anti-mouse IgG-HRP conjugate (Caltag Laboratories,Burlingame, CA), and TMB Microwell Peroxidase SubstrateSystem (Kirkegaard & Perry Laboratories, Gaithersburg,MD) were used for the ELISA.

2.2. Methods

2.2.1. Preparation of anionic PLG microparticles forprotein adsorption

Anionic microparticles were prepared by a solvent evap-oration technique[23]. Briefly, microparticles were preparedby homogenizing 10 ml of 6% (w/v) polymer solution inmethylene chloride, with 2.5 ml PBS using a 10-mm probe(Ultra-Turrax T25 IKA-Labortechnik, Germany) thus form-ing a water in oil emulsion which was then added to 50 mlof distilled water containing 6 ug/ml DSS and homogenizedat very high speed using a homogenizer with a 20-mm probe(ES-15 Omni International, GA, USA) for 25 min in an icebath. This resulted in a water in oil in water emulsion whichw d them ltingm

inedu stru-m lvernZ f thes nsioni zeda ed asP

2sus-

p mgp Mh leftoo ose)w s thel teinl asa

2

p nsionw -115h e at1 d ten

times through a microfluidizer from Microfluidics at 90 psi.2.5 ml of 10 mg/ml tyrosine solution in water was incubatedovernight with protein at a target load of 0.5% (w/w) in10 mM phosphate buffer pH 7. The suspension was thencentrifuged and the supernatant was analyzed for unboundprotein by size exclusion HPLC. The amount of bound pro-tein was calculated against a standard curve. All antigenswere adsorbed at 0.5% (w/w) loading levels.

2.2.4. Preparation of alum and CAP adsorbedformulations

Adsorption to both alum and calcium phosphate suspen-sions was carried out 1 mg/ml concentration and optimizedfor each antigen. The adsorption was subsequently confirmedby both SDS–PAGE and western blots (data not shown).

2.2.5. MF59 emulsion formulationPre-formed MF59 emulsion was obtained from Chi-

ron Vaccines in Marburg and tested for size distributionusing a Zetasier (Malvern Instruments). The emulsion wasdiluted 1:1 with the respective antigen solution prior toimmunization.

2.2.6. Formulation characterizationThe size of the microparticle and thel-tyrosine formu-

lation was measured by laser light scattering (Mastersizer,M ul-s lverni romt mgo de er 1 hb

ona ysesf ga-t wasr ibed.T for5 ntw thes tigena ons aredw n ofM t fort ntrola

2

fivea . Alla e for-m oupw own

as stirred at 1000 rpm for 12 h at room temperature, anethylene chloride was allowed to evaporate. The resuicroparticles had 0.05% DSS (w/w).The size distribution of the microparticles was determ

sing a particle size analyzer (Master Sizer, Malvern Inents, UK). The zeta potential was measured using a Maeta analyzer (Malvern Instruments, UK). PLG content ouspension was measured by aliquoting a 1 ml suspento each of three pre-weighed vials, which were lyophilind weighed again and the average net weight was usLG content/1 ml suspension.

.2.2. Adsorption of antigen to microparticlesTo prepare microparticles with adsorbed vaccines, a

ension containing 100 mg of PLG was incubated with 1rotein in 10 ml total volume of 10 mM of PBS or 10 mistidine pH 5.5 buffer (optimized for each antigen) andn a lab rocker (aliquot mixer, Miles Laboratories) at 4◦Cvernight. Formulation stabilizers (mannitol and sucrere then added to the suspension. The suspension wa

yophilized. Formulations were prepared with a 1% prooad for TT, MB1, HBsAg and Men C. DT antigen wdsorbed at a 0.5% (w/w) protein loading.

.2.3. Preparation of l-tyrosine suspensionTen milligrams per milliliter suspension ofl-tyrosine

owder in purified DI water was prepared and the suspeas then homogenized for 10 min using an Omni ESomogenizer (GA, USA) with a 20 mm generator prob5,000 rpm. The homogenized suspension was passe

n

alvern, UK). The size distribution of the MF59 emion was measured on a Zetasizer 2000 instrument (Manstruments, UK). The 1 h release for all five antigens fhe PLG formulation was measured by reconstituting 10f lyophilized formulation with 1 ml of purified water anstimating the amount released in the supernatant afty Bicinchoinic protein assay (BCA).

The evaluation of integrity and identity of antigenslum and CAP was carried out by Western blot anal

or all antigens on alum and CAP. Briefly, after centrifuion of the formulation aliquot of 1.2 ml, the supernatantemoved for analysis by SDS–PAGE as previously descrhe pellet was resuspended in extraction buffer, boiledmin and centrifuged 10 min at 13,000× g, the supernataas removed to a fresh tube. A volume equivalent totandards loaded was loaded on SDS–PAGE. The andsorption was controlled by Sialic Acid determinationupernatants. A standard curve of sialic acid was prepith six points (0, 5, 10, 15, 20 and 25 ug) and a solutioen C-CRM with the components of the vaccine, excep

he adjuvant, was added to the samples as positive cond all the calculations are relative to this value.

.3. Mouse studies

A total of five mouse studies were carried out with thentigens and five adjuvant formulations (including alum)ntigens were tested at two dose levels with each vaccinulation, except for Men C. Groups of 10 animals per grere used. CD1 were used for MB1 antigen as it was sh

M. Singh et al. / Vaccine 24 (2006) 1680–1686 1683

to give consistent response with MB1 antigen in earlier stud-ies. Balb/C were used for all other studies. All mice studieshad two immunizations at days 0 and 28 except Men B whichwas immunized at days 0, 21 and 35 (as previously described)[22]. All mice were bled at 2 weeks after the last immuniza-tion and sera analyzed by ELISA for circulating antibodytiters.

2.4. Determination of antibody responses to variousantigens

Enzyme-linked immunosorbent assay (ELISA) designedto measure antigen specific antibodies were performed onmice sera at week 6 for DT, TT, HBsAg and Men C. ForMB1 the ELISA was carried out at week 7 after three immu-nizations. The immunoassay for each antigen was optimizedusing standard sera as a control. Briefly, purified antigenswere coated onto Nunc Maxisorp U bottom plates at 1 ug/ml.Sera were tested at 1:100 and 1:400 dilution followed by serialthree fold dilutions. Horseradish peroxidase-conjugated goatanti mouse IgG (CALTAG diluted 1:40,000) was used as sec-ond antibody. After the 1-h incubation at 37◦C, plates werewashed to remove unbound antibody. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was used to develop the platesand the color reaction was blocked after 15 min by additiono rumd ISAa eachg .

2

bac-t ofpS ento ysis.T pre-v ons ively.S

2

tesa edi ea-

surement of diphtheria toxin neutralizing antibodies is anacceptable correlate for protection with diphtheria vaccines.Assays were performed on pools of each group.

2.7. Statistical analysis

Serum antibody titers are reported as geometric mean titer.Significant differences among groups were ascertained usingthe ANOVA factorial test at the 95% confidence interval(StatView 4.4 software; Abacus Concepts, Inc.).

3. Results and discussion

3.1. Size distribution

The particle size as measured by laser light scattering forPLG was determined to be 1.5± 0.6�m and 2.0± 2.4�mafter antigen adsorption. The size distribution of the MF59emulsion was found to be 185± 10 nm. Thel-tyrosine for-mulation had a size distribution of 2.5± 0.8�m prior toantigen adsorption and 3.0± 1.6�m after antigen adsorption.The zeta potential measurement of all the formulations exceptPLG was very comparable to each other.Table 1details thephysical characterization. The adsorption of antigens on var-i thati anyc ly ton

3

u-l totall nti-g tala datan pre-d

3

3

f 5 and5 lumw

TP

F V) (%)

PlCM

N

f 2N HCL. The titers reported are the reciprocal of the seilutions that gave an optical density at 450 nm of 0.5 ELbsorbency units. The geometric mean titers (GMT) forroup were calculated and compared with other groups

.5. Complement-mediated serum bactericidal activity

The measurement of complement-mediated serumericidal activity (SBA) in serum is a accepted correlaterotective efficacy for Men B and Men C vaccines[28]. TheBA assay measures the ability of antibody to fix complemn the surface of the bacterium and trigger bacterial lhe extent of serum bactericidal activity was assayed asiously described[28,29]using baby rabbit complementera collected at weeks 6 and 7 for Men C and B, respectBA assay was performed on pools of each group.

.6. Diphtheria toxin neutralization assay

This assay was performed in 96-well microtiter placcording to Miyamura et al.[30]. Titers are represent

n EU/ml for each group based on pooled sera. The m

able 1hysical characterization of various vaccine formulations

ormulation Mean particle size (�m) Zeta potential (m

LG 1.5–2.5 −45 ± 8-Tyrosine 2.5 −8 ± 3AP 6–7.5 −12± 3F59 0.20 −10 ± 3

.A.: Not Applicable.

ous formulations is a consequence of several factorsnclude electrostatic and hydrophobic interactions. In mases negatively charged proteins adsorb very effectiveegatively charged PLG surface.

.2. Antigen release

The 1 h release for all five antigens from the PLG formation was measured to be between 5 and 20% of theoad for all formulations. The amount of unadsorbed aen from thel-tyrosine, CAP and alum was <5% of the tontigen load and was confirmed by SDS–PAGE gels (ot shown). The antigen in the MF59 emulsion remainsominantly unassociated in the aqueous phase.

.3. Mouse studies

.3.1. Tetanus toxoidIn the mouse study carried out with TT (Fig. 1a), all

ormulations induced responses at both the doses (0..0 Lf). But the responses with MF59 emulsion and aere higher than those with CAP, PLG andl-tyrosine. At

Antigen loading level (%, w/w) Adsorption efficiency

1.0 >901.0 >951.0 >951:1 (Dilution) N.A.

1684 M. Singh et al. / Vaccine 24 (2006) 1680–1686

Fig. 1. (a) Serum anti-TT antibody titers in Balb/C mice after two immunizations at weeks 0 and 28 and bleedout at 2 weeks post second immunization.Geometric mean titer (GMT± S.E.) presented for each group. (b) Serum anti-DT antibody titers in Balb/C mice after two immunizations at weeks 0 and 28and bleedout at 2 weeks post second immunization. Geometric mean titer (GMT± S.E.) is represented for each group along with toxin neutralization titers in(EU/ml). (c) Serum anti-Men C polysaccharide antibody titers in Balb/C mice after two immunizations at weeks 0 and 28 and bleedout at 2 weeks post secondimmunization. Geometric mean titer (GMT± S.E.) is represented for each group along with serum bactericidal titers (SBA). (d) Serum anti-HBsAg antibodytiters in Balb/C mice after two immunizations at weeks 0 and 28 and bleedout at 2 weeks post second immunization. Geometric mean titer (GMT± S.E.) isrepresented for each group. (e) Serum anti-MB1 from Men B antibody titers in CD1 mice after three immunizations at weeks 0, 21 and 35 and bleedout at 2weeks post third immunization. Geometric mean titer (GMT± S.E.) is represented for each group along with serum bactericidal titers (SBA).

the low dose (0.5 Lf) MF59 emulsion induced significantlyhigher responses than all other formulations (p < 0.05).

3.3.2. Diphtheria toxoidIn the study carried out with DT (Fig. 1b), all formulations

exceptl-tyrosine induced responses at both the doses (0.5 and5.0 Lf). The responses with alum were higher than those withMF59, CAP, PLG andl-tyrosine. At the low dose (0.5 Lf)l-tyrosine induced significantly lower responses to all other

formulation (p < 0.05). In the toxin neutralization assay bothalum and PLG induced higher titers (>9.0 EU/ml) at the 5.0 Lfdose than all other formulations.

Responses with both the toxoids indicated that alum wasan effective adjuvant for these vaccines. This finding con-firms the utility of alum as a successful adjuvant for bacterialtoxoids (TT, DT, DPT, etc.) and in combinations using thesetoxoids (DPT-Hib, DtaP-Hib). MF59 emulsion was also anequally effective adjuvant for TT vaccine and therefore has

M. Singh et al. / Vaccine 24 (2006) 1680–1686 1685

the potential to be used as a part of a vaccine formulationsneeding TT along with other antigens.

3.3.3. Men C conjugate vaccineIn the study with Men C conjugate vaccine (Fig. 1c), all

formulations induced serum responses to the polysaccharideantigen. Both MF59 emulsion and PLG induced higher serumand bactericidal titers than alum and higher bactericidal anti-bodies thanl-tyrosine and CAP. CAP was good in inducingantibody titers but did not result in high bactericidal titersunlike PLG and MF59.

MF59 has also been shown earlier to induce enhancedbactericidal titers in infant baboons with Men C/Hib vacci-nation[10]. It may therefore offer an alternative to alum fordelivering Men C and Hib conjugates. This makes it a highlysuitable candidate along with alum for delivery of conjugatevaccines. MF59 may also have the advantage of deliveringseveral conjugates within the same formulation unlike alumwhere dose restrictions may not allow higher amounts of alumto be used.

PLG microparticles were also equally effective in inducingstrong responses with the conjugate adsorbed on the surface.PLG microparticles may help in stabilizing the conjugatesupon lyophilization if required and may offer a stable andeffective way of delivering conjugates in comparison to alumo lifeo

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dju-v ane ithh ns inM 100-f ineo forem poorr f thep redo

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sa nif-ih hert alt andP

Our earlier work with MB1 [23] also demonstratedthat PLG microparticles were very effective in generatingenhanced bactericidal responses with MB1. This has nowbeen reproduced with several other MB antigens (unpub-lished data). This general observation, applies to otherpathogens too which had antigens identified by reverse vacci-nology, adsorbed on PLG microparticles (unpublished data).PLG microparticles offer a suitable way to stabilize the anti-gens through adsorption followed by lyophilization.

The data shows that for all the five antigens tested, at leastone of the formulations either was comparable to or supe-rior to alum. MF59 emulsion overall outperformed all otherformulations and induced robust responses with most anti-gens. PLG formulation was the next most effective for MenB and Men C conjugate vaccine. CAP only induced betterresponse than alum for Men C conjugate whilel-tyrosinewas the least potent of all the formulations in the currentevaluation.

The main difference in presentation of the antigen in MF59versus alum, CAP, PLG andl-tyrosine was that in MF59 theantigen remains free in the aqueous phase of the emulsion.This enables better antigen integrity and stability, resulting inpotent responses. This lack of association of the antigen withthe emulsion droplets does not in any way inhibit the responseto that antigen as was seen with several antigens evaluated.It has been proposed that lymph node-resident dendritic cellsc iza-t

po-n dt Thisw od-e ent.O pe-c thes se-q

tingt ionsf (TTa nanta l andp ringa auseo iousa tests ator.F o bep le.

A

ar-i ilsonf

r MF59. PLG microparticles may also improve the shelff the vaccine formulation as it is freeze dried.

.3.4. Hepatitis B surface antigenIn the study with HBsAg (Fig. 1d), serum titers at th

ower 0.5 ug dose with MF59 were significantly higherll formulations. At the higher 5.0 ug dose, MF59 and aere significantly higher than all other formulations (PL-tyrosine and CAP) (p < 0.05).

MF59 was previously also shown to be an effective aant for HBsAg in primates[9] and in humans. Heinemt al.[31] summarized the clinical evaluation of MF59 wepatitis B vaccine containing the PreS and S antigeF59 and showed that the vaccine in MF59 induced >

old GMT titers than that seen with the licensed vaccn alum. So this result was unsurprising. MF59 thereight be helpful in generating protective responses in

esponders to HBsAg. Currently a small percentage oopulation responds very poorly to HBsAg antigen deliven alum[9].

.4. MB1 from Men B

In this study with recombinant MB1 (Fig. 1e), serum titert the lower dose (2 ug) with PLG and MF59 were sig

cantly higher than those with alum andl-tyrosine. At theigher dose (20 ug) PLG and MF59 were significantly hig

han all other formulations (p < 0.05). The serum bactericiditers at 10ug dose were significantly higher for MF59LG in comparison to alum, CAP andl-tyrosine.

an acquire MF59 emulsion after intramuscular immunion by uptake of the apoptotic macrophages[32].

Some of these adjuvants, including MF59, are coments of approved vaccines (FLUAD)TM and could be use

o replace vaccines currently administered on alum.ould need extensive further evaluation in larger animal mls and formulation stability and compatibility assessmther formulations like PLG microparticles may have s

ific advantages over MF59 with some antigens, wheretability at 4◦C could be improved by adsorption and subuent lyophilization.

Overall the study shows initial encouraging data indicahat it is possible to replace alum with alternative formulator a variety of antigens ranging from bacterial toxoidsnd DT), polysaccharide conjugate (Men C), recombintigens (HbsAg and Men B) and to generate functionarotective titers with these vaccines. The need for explolternative formulations for each antigen may arise becf its physio-chemical properties and interaction with vardjuvants. The next step in this evaluation will be toome of these formulations along with an immunopotentiurther immunization and challenge studies will need terformed to establish clinically-relevant proof of princip

cknowledgements

We would like to thank Nelle Cronen for her help in prepng the manuscript. Thanks are also due to Jacqueline Wor carrying out the vivarium work.

1686 M. Singh et al. / Vaccine 24 (2006) 1680–1686

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