Peptomer Aluminum Oxide Nanoparticle ... - ACS Publications

Bioconjugate Chem. 1997, 8, 424−433

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Peptomer Aluminum Oxide Nanoparticle Conjugates as Systemic and Mucosal Vaccine Candidates: Synthesis and Characterization of a Conjugate Derived from the C4 Domain of HIV-1MN Gp120 Andreas Frey,†,‡ Marian R. Neutra,† and Frank A. Robey*,§ Department of Pediatrics, Harvard Medical School, and GI Cell Biology Research Laboratory, Children’s Hospital, Boston, Massachusetts 02115, and Oral and Pharyngeal Cancer Branch, The National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892. Received January 30, 1997X

Peptomers are polymers composed of peptides that are specifically cross-linked in a head-to-tail fashion. Recently, a peptomer composed of an amphipathic peptide from the C4 domain of HIV-1MN gp120 was shown to display a prominent R-helical conformation that, as an immunogen, elicited rabbit antibodies recognizing native and recombinant gp120 [Robey et al. (1995) J. Biol. Chem. 270, 23918-23921]. For the present study, we synthesized a conjugate composed of the C4 peptomer covalently linked to calcinated aluminum oxide nanoparticles. The nanoparticles were first reacted with (3-aminopropyl)triethoxysilane to provide an amine load of 15.9 mmol of R-NH2/g of solid. The amine-modified aluminum oxide nanoparticles then were reacted with N-acetylhomocysteine thiolactone at pH 10 to place a reactive thiol on the nanoparticles. A bromoacetylated C4 peptomer, modified at the -amines of lysine residues, then was reacted with the thiolated nanoparticles to give the peptomer covalently linked to aluminum oxide via a thioether bond. The peptomer load was determined to be 16 mg of peptomer/g of particles, a 55% theoretical yield. Particle shape and size of the peptomer-conjugated alumina were analyzed by electron microscopy and displayed a mean maximum diameter of 355 nm and a mean minimum diameter of 113 nm, well within the desired size range of 300 nm believed to be optimal for mucosal immunization purposes. Experimentally determined values of mean particle diameters, specific surface area, and specific peptomer load provided the information necessary to calculate the mean antigen load, which was determined to be 53 000 ( 42 000 peptomer epitopes per particle. Peptomer-alumina conjugates, such as that described here, could form the basis of a new class of biomaterial that combines a chemically defined organic immunogen with a nontoxic chemically defined inorganic adjuvant.

INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) is a pathogen that is transmitted across the mucosal surfaces of the urogenital tract and the rectum (DeSchryver and Meheus, 1990; Amerongen et al., 1991). To intercept the virus on this route of infection, vaccines must be developed that induce a mucosal immune response against HIV-1 (Forrest, 1992; Marx et al., 1993). An important component of mucosal immune protection is antigenspecific secretory immunoglobulin A (sIgA), dimeric or polymeric molecules that are secreted onto mucosal surfaces where they bind pathogens, trap them in mucus, and prevent their further progression (Neutra et al., 1994). Nevertheless, an HIV vaccine should also be able to induce strong systemic humoral and cell-mediated immunity to arm the body against the virus if it breaches the mucosal barrier. A sIgA response is induced only when the vaccine is delivered to the immune system via mucosal surfaces. In the intestine and rectum, antigens, particles, and pathogens are taken up by M cells, a specialized epithelial cell type that occurs exclusively in the epithelium over organized mucosa-associated lymphoid tissue. Selective uptake by M cells is enhanced when the antigen is formulated as a micro- or nanoparticulate material * Address correspondence to this author. Telephone: 301-4964779; fax: 301-402-0823; e-mail: [email protected]. † Harvard Medical School and Children’s Hospital. ‡ Present address: Center for Molecular Biology of Inflammation, University of Muenster, D-48129 Muenster, FRG. § National Institutes of Health. X Abstract published in Advance ACS Abstracts, May 1, 1997.

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ideally of 0.05-1 mm diameter, since only M cells are able to translocate particles of such a size across the tight epithelial barrier (Neutra et al., 1996a,b; Frey et al., 1996). Soluble antigens in the size range of oligopeptides and small proteins are less desireable since they may be taken up by epithelial lining cells and give rise to a state of immunological unresponsiveness that is called oral tolerance (Bland and Warren, 1986). Formulating the antigen in particulate form also is beneficial for systemic vaccinations since mononuclear phagocytes, like macrophages and Kupffer cells, efficiently phagocytose, process, and present antigens that appear in such a particulate form. When antibody-mediated protection against intact pathogens is desired, as for the protection of the mucosal surfaces by sIgA, it is essential that the vaccine be formulated to closely resemble the native structure and conformation of the antigen targets. The native structure and conformation of protein antigens can be altered by aggregation, improper folding, and defects in glycosylation of recombinant proteins or synthetic peptides as well as denaturation and breakdown during the formulation procedure. In addition, antigenic variation in the wildtype pathogen may reduce the efficacy of a vaccine based on protein antigens generated in the laboratory. The development of an effective vaccine against HIV-1 hinges on all of these factors. For effective protection against HIV-1, the antibody response must be directed against the viral envelope glycoproteins gp120 or gp41. Antibodies directed against certain epitopes within the HIV-1 gp120 and gp41, however, were shown to enhance infection of macrophages and monocytes in culture © 1997 American Chemical Society

Peptomer Aluminum Oxide Nanoparticle Conjugates

(Takeda et al., 1988) and to cross-react with immunerelevant host proteins such as HLA-DR (Lasky et al., 1987; Golding et al., 1988) and certain immunoglobulin subclasses (Bjork, 1991). Furthermore, both envelope proteins evade the immune surveillance of the body by continuous variation of their antigenic sites (Starcich et al., 1986; Gurgo et al., 1988). On the virus surface, the envelope proteins gp120/41 are assembled in complex oligomeric structures (Earl et al., 1990) in which the second and third variable regions (V2 and V3) and a segment of the fourth constant region (C4) of gp120 are exposed (Moore et al., 1994). Among those, C4 is of particular importance for the virulence of the virus because it is part of the binding site that interacts with the viral receptor CD4 (Lasky et al., 1987; Cordonnier et al., 1989). As numerous monoclonal antibodies against C4 are neutralizing and broadly cross-reactive between different HIV-1 isolates (Sun et al., 1989; Nakamura et al., 1993), this region appears to be an attractive candidate for an HIV-1 subunit vaccine. However, synthetic C4 peptides do not bind CD4 without being in a solution containing helix-inducing substances such as certain nonionic detergents (Robey et al., 1996), and antibodies raised against monomeric C4 peptides (Robey et al., 1995) do not recognize native or recombinant gp120 glycoprotein. The reason for this is because monomeric C4 peptides display a random coil or β-sheet. Polymerizing the monomer head-to-tail in a coordinate manner renders the product predominantely R-helical and, with the proper adjuvant, enables it to induce antibodies that recognize recombinant as well as native gp120 (Robey et al., 1995). In contrast, polymerizing the monomer randomly (head-to-tail/tail-to-head) and immunizing in Freund’s adjuvant, which could denature secondary structures (Scibienski, 1973; Robey et al., 1995), did not produce antibodies that recognized intact gp120 (Sastry and Arlinghaus, 1991). Thus, if C4 is used in a vaccine formulation, it should not only be polymeric but also be delivered in a nondenaturing environment in order to maintain its R-helical conformation. Aluminum oxohydroxide, phosphate, and hydroxyphosphate compounds are hydrophilic, particulate adjuvants with a long history of safety and efficacy for systemic vaccination (Hem and White, 1995; Gupta et al., 1995). However, the drawbacks of these substances for oral administration are their pH lability and the noncovalent adsorption of the antigen to their surfaces. Thus, the antigen may readily dissociate during gastrointestinal passage, rendering the vaccine ineffective. To circumvent the problems associated with the geltype aluminum compounds, we designed a candidate vaccine in which the antigen is an HIV-1MN gp120 C4 domain peptomer that is covalently conjugated onto the surface of calcinated aluminum oxide nanoparticles. In the present paper, we describe the synthesis and characterization of this conjugate with special emphasis on its immunologically relevant properties, such as particle diameter, antigen load, and degree of polymerization of the individual C4 domain oligopeptide units. EXPERIMENTAL PROCEDURES

Materials. Reagents for Particle, Peptide, and Peptomer Synthesis. R-Aluminum oxide nanoparticles were purchased from Fluka (Ronkonkoma, NY). (3-Aminopropyl)triethoxysilane (98%), nitric acid, anhydrous toluene, toluene, and acetone (all ACS grade) were from Aldrich Chemical (Milwaukee, WI). Deionized ultrapure water was prepared using a Millipore water purification system (Millipore, Bedford, MA). All chemicals used for

Bioconjugate Chem., Vol. 8, No. 3, 1997 425

peptide synthesis were from Applied Biosystems (Foster City, CA). Bromoacetic acid (99+%) and N-acetylhomocysteine thiolactone (99%) were obtained from Aldrich Chemical. N-succinimidyl bromoacetate was synthesized as described previously (Bernatowicz and Matsueda, 1986). Materials and Reagents for Amino Acid Analysis, SDS-PAGE Analysis, and Electron Microscopy. Chemicals for the preparation of aqueous buffers and solutions were obtained from various sources in the highest quality commercially available (Sigma Chemical, St. Louis, MO; Fisher Scientific, Pittsburgh, PA; Aldrich Chemical; Calbiochem-Novabiochem, San Diego, CA). Tris-tricine 10-20% polyacrylamide gels were from Novex (San Diego, CA), and prestained low range protein molecular weight standards were from Gibco-BRL (Gaithersburg, MD). Poly(vinyl formal) (Formvar 15/95) and 150 square mesh copper grids were purchased from Polysciences Inc. (Warrington, PA). Synthetic Procedures. Synthesis of the HIVMN gp120 C4 Domain Peptomer. The general methods for preparing peptomers and their monomeric peptide building blocks have been described in detail previously (Robey and Fields, 1989; Robey, 1994). In brief, cysteinecontaining peptide monomers were synthesized on pmethyl-PAM resin using the standard BOC technology on an Applied Biosystems Model 430 A automated peptide synthesizer on a 0.5 mmol scale. In the last step of the synthesis of the peptide chain, bromoacetic acid anhydride was reacted with the amino terminal amino acid to form the N-R-bromoacetyl-derivatized, fully protected peptide. Deprotection and release of the bromoacteylated peptide from the resin were accomplished by treating the resin with anhydrous hydrogen fluoride containing 10% (v/v) m-cresol. After evaporation of the hydrogen fluoride, the residual resin-peptide mixture was extracted with ethyl acetate followed by extraction of the peptide in 0.1 M acetic acid. The peptide solution was separated from the resin by filtration and dried by lyophilization. Purification of the peptide was accomplished by preparative reversed-phase HPLC on a Vydac C18 column using a 0.1% aqueous trifluoroacetic acid/ acetonitrile gradient. The purified peptide was lyophilized and stored at room temperature in the dark. The N-R-bromoacetyl-derivatized HIVMN gp120 C4 domain peptide was obtained in yields between 50 and 70%. To form the HIVMN gp120 C4 domain peptomer, typically 10 mg of purified N-R-bromoacetyl-derivatized peptide were dissolved in 1 mL of deoxygenated 10 mM TrisHCl and 1 mM EDTA, pH 8.0, and allowed to autopolymerize for 21 h at room temperature under continuous stirring. The reaction was terminated by dialysis against water followed by dialysis against 0.1 M sodium bicarbonate, both at 4 °C using 15 000 MWCO dialysis tubing (Spectrum, Houston, Texas). The peptomer was then end-capped by first reacting it with 10 µL/mL (143 mM) β-mercaptoethanol followed by 32 mg/mL (173 mM) iodoacetamide, each for 1 h at room temperature under continuous stirring. The end-capped peptomer was dialyzed against 0.1 M sodium acetate followed by deionized water, both at 4 °C. At that stage, the peptomer solution was either used directly for bromoacetylation of lysines with N-succinimidyl bromoacetate or lyophilized for long-term storage. When lyophilized, the sodium acetate form of the peptomer was a dry white powder that was stored desiccated at room temperature. Typical yields were 80-90% (referring to the initial amount of N-R-bromoacetyl-derivatized peptide). Bromoacetylation of the HIVMN gp120 C4 Domain Peptomer. A total of 12.4 mg (52.5 mmol) N-succinimidyl bromoacetate was dissolved in 124 µL of DMF, and 22.4

426 Bioconjugate Chem., Vol. 8, No. 3, 1997

µL (∼9 mmol of ester) of this solution was added to a solution of 20 mg of HIVMN gp120 C4 domain peptomer (∼26 mmol of free side chain amine) in 20 mL of water. After 15 min incubation at room temperature, the reaction was terminated by extensively dialyzing against O2free deionized water at room temperature. The resulting 20 mL 1 mg/mL of bromoacetylated peptomer in water was used directly to react with the thiol-derivatized particles: derivatization yield, 28% of the free side chain amine (∼80% of the theoretical value referring to the amount of N-succinimidyl bromoacetate). Surface Activation of the Aluminum Oxide Nanoparticles. The cleaning and surface activation of the alumina was carried out as recommended by Lynn (1975) and Weetall (1976). In a 2-L Erlenmeyer flask, 126.7 g (1.24 mol) of R-aluminum oxide nanoparticles (300 nm nominal diameter, calcinated at 1300 °C, 99.99% pure, >95% R-form) was suspended in 1140 mL of 5% (w/v) nitric acid and heated under swirling for 90 min at 88 °C. The slurry then was allowed to cool to 0 °C in an ice-water bath for 90 min before it was transferred into polyallomer centrifuge bottles, it was centrifuged at 1500g for 10 min at 4 °C, and the supernatant was aspirated. To wash the particles, they were resuspended mechanically in deionized ultrapure water at room temperature and centrifuged at 9500g for 15 min at 4 °C, and the supernatant was removed by aspiration. After a total of 10 washes in 440 mL of deionized ultrapure water each, the particle sediment was transferred into a nitric acidcleaned glass beaker, dried at 250 °C until the weight was constant (21 h), pulverized in a nitric acid-cleaned mortar, and stored desiccated at room temperature in a nitric acid-cleaned glass bottle: yield, 112.2 g (88.6%). Amine Modification of the Activated Aluminum Oxide Nanoparticles. The amine modification of the alumina was adapted from the procedures described by Lynn (1975) and Weetall (1976). In a 2-L round-bottom flask, 50.2 g (0.49 mol) of surface-activated, dry aluminum oxide nanoparticles was suspended in 450 mL of anhydrous toluene; 50 mL (0.21 mol) of (3-aminopropyl)triethoxysilane was added; and the mixture was refluxed under anhydrous conditions for 23 h at 135 °C in an oil bath. Then the suspension was allowed to cool to ambient temperature over 3 h before it was transferred into polyallomer centrifuge bottles and centrifuged at 200g for 5 min; the supernatant was aspirated. To wash the particles, they were resuspended mechanically in 450 mL of fresh toluene at room temperature and centrifuged, and the supernatant was removed by aspiration. After five washes in toluene, 450 mL each (centrifugation conditions: 200g, 5 min, 4 °C), followed by three washes in acetone, 450 mL of each (centrifugation conditions: 5000g, 20 min, 4 °C), the particle sediment was transferred to a nitric acid-cleaned glass beaker and dried for 17 h under vacuum at room temperature followed by 22 h at 115 °C and normal pressure. Then the sediment was pulverized in a nitric acid-cleaned mortar and the amine-modified nanoparticles were stored desiccated at room temperature in an amber bottle: yield, 48.1 g (96% referring to the weight of the underivatized surface activated particles); amine load, 15.9 mmol of R-NH2/g of solid. Thiol Derivatization of the Amine-Modified Aluminum Oxide Nanoparticles. A 250-mg sample (1.57 mmol) of N-acetylhomocysteine thiolactone was added to 1 g of amine-modified aluminum oxide nanoparticles (15.9 mmol R-NH2) in 10 mL of O2-free 0.1 M sodium borate buffer, pH 10, in a 13-mL polypropylene tube. The tube was placed on a rotator, and the reaction was allowed to proceed at room temperature for 45 min under constant

Frey et al.

rotation at 30 rpm. The particles were then washed by centrifuging the buffer-particle mixture at 300g for 5 min at room temperature and resuspending the sediment in O2-free phosphate-buffered saline (PBS). This washing procedure was repeated twice, and the particles were finally suspended in 1 mL O2-free PBS. Coupling of Bromoacetylated HIVMN gp120 C4 Domain Peptomer to Thiol-Derivatized Aluminum Oxide Nanoparticles. A total of 20 mL of a solution of 1 mg/mL bromoacetylated peptomer (∼7.3 mmol bromoacetyl residues) in water was added to 1 mL of thiol-derivatized particles suspended in PBS (700-800 mg of solids) in a 50-mL conical polypropylene tube. The mixture was placed on a rotator and mixed at room temperature for 1 h under constant rotation at 30 rpm. Then, 1 mL of O2-free 0.1 M sodium bicarbonate was added, and the reaction was allowed to proceed for another 65 h. The suspension was then centrifuged at 3000g for 20 min at room temperature and the resulting sediment was washed three times in PBS and five times in deionized water by resuspending and then centrifugating at 4 °C. The final sediment was lyophilized and stored desiccated at room temperature: yield, 684 mg of peptomer nanoparticles containing 16 mg of peptomer/g of particles (55% of the theoretical value). Analytical Procedures. Analysis of the Aluminum Oxide Nanoparticle Derivatives. The amount of free amine that was covalently linked to the aluminum oxide particles was determined with the ninhydrin method of Sarin et al. (1981). The presence of free sulfhydryl groups on the modified aluminum oxide that was formed after the reaction of the free amine with N-acetylhomocysteine thiolactone was determined using Ellman’s reagent (Ellman, 1959), and the amount of peptide conjugated to the aluminum oxide particles was determined by amino acid analysis using the Waters Picotag HPLC system (Waters Corp., Milford, MA). SDS-Polyacrylamide Gel Analysis of Peptomer Preparations. Peptomer in sample buffer [2% (w/v) sodium dodecylsulfate, 10% (v/v) glycerol, 20% (v/v) β-mercaptoethanol, 0.01% (w/v) bromphenol blue] was denatured for 4 min at 100 °C, loaded (1-1.5 mg/lane) onto tris-tricine 10-20% polyacrylamide gradient/SDS gels, and run for 2 h at 40-50 mA in tris-tricine electrophoresis buffer (12.1 g/L tris base, 17.9 g/L tricine, 1 g/L sodium dodecyl sulfate). Gels were fixed for 3.5 h in 10% (v/v) acetic acid and 30% (v/v) methanol, and silver-stained according to the method of Oakley et al. (1980). Circular Dichroism of Peptomer Preparations. CD spectra of the peptides, peptomers, and N--bromoacetylated peptomers were studied using a Jasco Model J-500A/DP-501N CD spectropolarimeter with peptides and peptomers in 10 mM phosphate buffer pH 7.2. Details were described previously (Robey et al., 1995). Densitometry. To determine the relative amounts of individual peptide oligomers in the peptomer preparations, a photographic reproduction of a silver-stained peptomer polyacrylamide gradient gel was scanned with a Microtek Scanmaker III scanner (Microtek Lab Inc., Redondo Beach, CA) at 600 × 600 dpi and analyzed with the NIH Image software package (National Institutes of Health, Bethesda, MD) after one-dimensional vertical background subtraction on an Apple Power PC 7100/66 computer (Apple Inc., Cupertino, CA). Electron Microscopy and Particle Size Determination. A 5-mg sample of surface-activated aluminum oxide nanoparticles, amine-modified nanoparticles, or peptomer-conjugated nanoparticles was suspended in 1 mL of deionized water by agitation and brief sonication (1-2 × 5 s) in a water bath sonicator (Sonorex RK510S,



Bandelin Electronic, Berlin, FRG). The suspensions were serially diluted to concentrations of 500, 50, and 5 mg/ mL particles in water, with sonication between each dilution step. For transmission electron microscopy (TEM), 10 mL of each diluted particle suspension was placed on formvar-coated copper grids, allowed to settle, and dried overnight. Particles were photographed at 14000× and 31000× magnification in a Philips EM 410 transmission electron microscope (Philips Electron Optics, Eindhoven, The Netherlands) using a magnification standard. For scanning electron microscopy (SEM), a drop of each particle suspension was placed on a glass slide precoated with 3 nm of platinum/carbon, allowed to settle, drained, and air-dried overnight before it was coated with platinum/ carbon at an angle of 65° under continuous rotation of the sample. The particles were photographed at 6000× to 60000× magnification in a Hitachi S-5000 field emission scanning electron microscope (Hitachi Instruments Inc., San Jose, CA) using a magnification standard. Particle sizes were determined by measuring the diameters of 125 randomly selected particles of each type on TEM photographs. Determination of Nanoparticle Surface Area and Porosity. The specific surface area of the aluminum oxide nanoparticles was determined by nitrogen adsorption using the multipoint BET method (Brunauer et al., 1938) on a Quantachrome Autosorb 1 automated gas sorption system and by mercury porosimetry on a Quantachrome Autoscan 60 mercury porosimeter. Pore size, pore volume, and pore surface area were determined by mercury porosimetry (mercury intrusion analysis) (Washburn, 1921). Both analyses were performed by Quantachrome Corp. (Boynton Beach, FL).

Scheme 1. Peptomera

Synthesis of HIVMN gp120 C4 Domain

RESULTS

Peptomers are polymers composed of head-to-tail linked synthetic peptides. The peptomer designed for this candidate HIV vaccine is a homopolymer of 18-mer oligopeptides comprised by the amino acid sequence: KIKQIINMWQEVGKAMYAC. The first 17 amino acids of this sequence motif represent amino acids 419-436 of gp120, the HIV-1MN gp120 precursor protein (Gurgo et al., 1988). The sequence is a highly conserved linear epitope in the fourth constant region (C4) of gp120 (between hypervariable regions V4 and V5) (Starcich et al., 1986). It is an essential part of the CD4 receptor binding site of gp120 (Lasky et al., 1987), and it was shown to give rise to virus-neutralizing antibodies (Sun et al., 1989; Nakamura et al., 1993). Synthesis of the HIVMN gp120 C4 Domain Peptomer Aluminum Oxide Nanoparticles. The HIVMN gp120 C4 domain peptomer aluminum oxide nanoparticles were prepared by separately synthesizing the peptomer antigen and the particulate carrier and conjugating both compounds in a terminal step, as outlined in Schemes 1 and 2. First, the peptide monomer for the preparation of the peptomer was synthesized as Cterminal amide on an automated peptide synthesizer. To allow subsequent head-to-tail polymerization via thioether linkages, an additional cysteine, not present in HIV-1MN gp120 at this position, was placed at the carboxy terminal end of the peptide chain. At the amino terminus, a bromoacetyl moiety was introduced by reacting the N-terminal amine of the immobilized, side-chainprotected peptide with bromoacetic acid anhydride (Scheme 1). The entire bifunctional peptide was then deprotected and released from the resin by anhydrous hydrogen fluoride, conditions that had been shown previously not to affect the integrity of the N-R-bromoacetyl

a The C4 peptide used here and before (Robey et al., 1995, 1996) has the amino acid sequence, KIKQIINMWQEVGKAMYAC-NH2.

moiety (Robey and Fields, 1989). To prevent premature polymerization or cyclization after removal of the sulfhydryl protecting group, all subsequent steps involving the monomeric peptide were carried out under acidic conditions. Typical yields of crude N-R-bromoacetylderivatized cysteine-containing peptide were between 50 and 70%. After preparative HPLC, 30% of the expected pure peptide was obtained. Autopolymerization of the N-R-bromoacetyl-derivatized, cysteine-containing peptide was initiated by dissolving the purified peptide in aqueous buffer at slightly alkaline pH (pH 8.0). The reaction was performed at a high monomer concentration (g10 mg/mL) to minimize cyclization reactions (Scheme 1). Under such conditions, the reaction was almost complete after 3 h, at which time most of the detectable free thiols had been consumed. However, as longer polymer chains may be formed preferentially toward the end of the reaction, a prolonged reaction time of 21 h was allowed. As expected, the


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Scheme 2. Synthesis of HIVMN gp120 C4 Domain Peptomer Nanoparticles

Figure 1. Gel analysis of the HIVMN gp120 C4 domain peptomer. Silver stained, reducing 10-20% polyacrylamide/SDS gel. Lane 1: Peptomer of the large-scale preparation used for conjugate preparation. Lane 2: Molecular weight standards. Table 1. Degree of Polymerization of HIVMN gp120 C4 Domain Peptomer

resulting product was not a homogeneous polymer of distinct molecular weight but rather a mixture of peptide oligomers of different chain length (Figure 1; Table 1). This mixture was used for the preparation of the conjugate without further size fractionation or enrichment for a particular oligomer species. Initial attempts to utilize the N-R-bromoacetyl groups that were remaining after termination of the autopolymerzation reaction for conjugating the peptomer onto the thiol-modified aluminum oxide nanoparticles were not successful. To generate reproducible conditions, the peptomer was therefore end-capped by completely removing the reactive groups at the head and tail of the polymer chain before it was prepared for “side on” conjugation by N--bromoacetylation of the lysine side chains (Scheme 2). Bromoacetylation of the lysines was carried out with a 3-fold molar excess of -amino groups to N-succinimidyl bromoacetate in order to guarantee that the labeling occurred statistically in only one out of the three lysines present in a peptide unit. N--Bromoacetylation of the lysines with the activated ester proceeded smoothly, consuming ∼84% of the derivatizable amine (28% of the total -amino groups) within 15 min. The randomly bromoacetylated peptomer was then used without further purification for reaction with the thiol-modified particles. Bromoacetylation of the peptomer did not effect the amount of R-helix that was in the peptomer as compared with the non-bromoacetylated peptomer, and the CD spectrum (Figure 2) of the bromoacetylated peptomer looked virtually identical to the nonbromoacetylated peptomer shown in the earlier publication (Robey et al., 1995). For comparison and as shown before (Robey et al., 1995), the monomeric peptide CD spectrum is given as the broken line in Figure 2 and shows that the peptide itself has very little, if any, helical conformation in the phosphate buffer, pH 7.2.

chain length

% product formeda

monomer dimer trimer tetramer pentamer hexamer heptamer

1.7 22.3 12.9 10.8 8.0 6.2 5.7

chain length

% product formeda

octamer nonamer decamer undecamer dodecamer >dodecamer

4.7 3.5 2.7 2.2 1.6 17.7

a Data are derived from a 600 × 600 dpi scan of a silver-stained SDS-PAA gradient gel. Results are given as means of two measurements. Individual measurements differed less than 5% from the given means.

Figure 2. Conformational study of N--bromoacetylated C4 peptide constructs. CD spectra of N-Ac-peptide-(419-436) (- - -) and N--bromoacetylated peptomer-(419-436) (s).

The thiol-modified particles were prepared from plain R-aluminum oxide nanoparticles as depicted in Scheme 2. First, the surface of the corundum powder was cleaned and activated for subsequent derivatization by treatment with hot dilute nitric acid. To introduce a primary amino function onto the surface of the cleaned aluminum oxide nanoparticles, they were reacted with (3-aminopropyl)-


Peptomer Aluminum Oxide Nanoparticle Conjugates Table 2. Properties of Aluminum Oxide Nanoparticle Conjugates diametera maximum diameter

minimum diameter

surface areab

particle type

mean ( SD (nm)

range (nm)

mean ( SD (nm)

range (nm)

by MP (m2/g)

by BET (m2/g)

conjugate loadc (µmol/g)

surface-activated amino-derivatized peptomer-derivatized

394 ( 140 430 ( 154 355 ( 108

143-871 163-813 158-675

131 ( 52 125 ( 50 113 ( 43

39-358 47-325 42-269

11.9 nd nd

11.9 nd nd

n/a 15.9 7.0

a Particle diameters were determined by transmission electron microscopy. As most particles were of nonspherical shape, the minimum and maximum diameters and the size ranges are given. b Particle surface area was determined after drying the sample at 300 °C either by mercury porosimetry (MP) or by nitrogen adsorption/desorption (multipoint BET method). Due to the high drying temperature for sample preparation, the procedure could not be used for amino- and peptomer-derivatized particles. c Conjugate loads were determined by ninhydrin assay (amino-derivatized particles) or Picotag amino acid analysis (peptomer-derivatized particles). For the peptomerderivatized particles the molar amounts of peptide units on the particles are given. d n/a, not applicable. e nd, not determined.

triethoxysilane (Scheme 2). Assuming that a surface load of 2 mmol/m2 is characteristic for a silane monolayer on a ceramic surface (Larsson, 1983) and the specific surface area of the aluminum oxide nanoparticles is 12 m2/g (Table 2), the silanizing reagent was applied in a 175fold molar excess. The high particle dispersity [2.5% (v/ v) alumina in solvent], in combination with the vast excess of silanizing reagent, effectively prevented crosslinking of the particles as evidenced by the less than 10% increase of the mean particle diameters from before to after the silanization (Table 2). After the surface attached (3-aminopropyl)triethoxysilane was sintered onto the particles, the amount of covalently attached 3-aminopropyl moieties was determined to be 15.9 mmol/g particles, which is equivalent to 1.3 mmol of amine/m2. The modification proved to be largely resistant to mechanical stress because no significant amine loss could be detected after a total of 10 min sonication of a particle suspension in a bath sonicator. To allow conjugation of the N--bromoacetylated peptomer onto the particles via thioether linkages, the amine-modified alumina was reacted at pH 10 with a 100-fold molar excess of N-acetylhomocysteinethiolactone (Scheme 2). The formation of free thiol groups was assayed every 15 min with Ellman’s reagent. After 45 min of reaction, the quantity of free thiol no longer increased and the reaction was terminated. However, though the kinetics of the derivatization could be monitored, it was impossible to determine the absolute amount of free thiol formed because part of the 2-nitro5-thiobenzoic acid that was released through reaction with the free thiols was nonspecifically adsorbed to the particles. The thiol-derivatized aluminum oxide nanoparticles then were reacted with the N--lysyl-bromoacetylated peptomer until no more free sulfhydryl groups were detectable in the reaction mixture. Due to the high particle dispersity of 1% (v/v) solids in the reaction mixture, no cross-linking of the particles was observed. Instead, the mean particle diameters decreased by 1020% when compared to the surface-activated and aminemodified alumina. We attribute this decrease in particle size to abrasion or splitting of the alumina because of mechanical stress during the synthesis. Amino acid analysis of the final conjugate revealed a 55% coupling yield for the peptomer leading to a specific antigen load of 16 mg of peptomer/g of aluminum oxide nanoparticles. Characterization of the HIVMN gp120 C4 Domain Peptomer Aluminum Oxide Nanoparticles. For use of the peptomer alumina as systemic or mucosal vaccines, the most important characteristics are particle size, porosity, and antigen load as well as the chain length of the attached peptomer and its stable covalent coupling to the carrier. Particle shape and size of the surface-activated, aminemodified, and peptomer-conjugated alumina were ana-

Figure 3. HIVMN gp120 C4 domain peptomer-derivatized aluminum oxide nanoparticles. Representative electron micrographs depicting the peptomer-derivatized aluminum oxide nanoparticles. Scanning electron microscopy (A) at high particle density reveals the smooth surface texture that most of the nanoparticles displayed. Transmission electron microscopy (B) at low particle density demonstrates the predominantly elongated shape of the particles and the existence of a “crystalline” subpopulation (arrows) with rugged edges. Scale bar, 250 nm.

lyzed by electron microscopy (Figure 3). There was no evident difference in shape or size between the surfaceactivated starting material and the final peptomer conjugate. However, within each sample, two distinct particle populations were observed. Most of the particles were of ellipsoid or cylindrical shape and displayed a smooth surface texture without sharp corners and edges (Figure 3A). A minor fraction of particle consisted of generally smaller, rugged particles, mostly of non-spherical, irregular shape (Figure 3B, arrows). Because of the elongated shape of all the particles, the particle size is reported as minimum and maximum diameters (Table 2). The final product exhibited a mean maximum diameter of 355 nm and a mean minimum diameter of 113 nm, consistent with the desired size of 300 nm. The porosity and surface area of the particles was determined by mercury intrusion and nitrogen adsorption, respectively. Both techniques require the sample to be completely dry. To meet that requirement, samples have to be heated to 300 °C under high vacuum, conditions under which a peptomer or γ-aminopropyl coating is likely to decompose. As the size, shape, and surface texture of all samples appeared identical when analyzed


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of these data the antigen load could be estimated. The estimation is based on the assumption that the mean particle is of cylindrical shape with hemispherical ends, a model that is the best possible approximation for the heterogeneous population of elongated particles which was observed by EM (Figure 3). For a population of such particles the specific surface area (SSA) is given by

SAA )

4dmax Fdmin(dmax - 1/3dmin)

(1)

where F is the density of the alumina as provided by the manufacturer (3.95 g/cm3) and dmax and dmin are the mean maximum and minimum particle diameters (Table 2), respectively. Using eq 1, the specific surface area of the surface-activated alumina was calculated to be 8.7 ( 3.5 m2/g. This result is in good agreement with the outer surface area of the particles (7.2 m2/g) as determined by mercury intrusion analysis, and the total surface area as determined by nitrogen adsorption or mercury intrusion (Table 2) also lies within the margins of error. We are therefore confident that the assumption of a cylindrical form with hemispheres on each end is an adequate model for the mean particle shape. Assuming such a particle shape, the number of peptide epitopes per mean particle (ne) is given by Figure 4. Pore size analysis of the surface activated aluminum oxide nanoparticles. The differential pore size distribution and the cumulative surface area were obtained from mercury porosimetry. The analysis was carried out with intrusion pressures ranging from 0 to 42 000 N/cm2. The data were calculated assuming a mercury contact angle of 140° and a surface tension of 480 erg/cm2. For greater clarity, only every 20th measured point is shown.

by EM, we considered the underivatized, surface-activated alumina as representative for all particle types in terms of porosity and surface area. The porosity of the surface-activated alumina was determined by mercury intrusion analysis employing the Washburn relationship (Washburn, 1921) (Figure 4). When applying this technique, the interparticle voids as well as every concave surface curvature of the particles, no matter whether it is a shallow indentation, a deep cavity, or a channel, are regarded as pores. In the mercury intrusion analysis of the surface-activated alumina, 40% of the mercuryoccupied interparticle voids (g900 nm) were not included in the pore size analysis. The remainder intruded into pores between 12 and 900 nm without revealing any distinct pore classes. Instead, a nonparametric pore size distribution was observed with the most frequent pore diameter being 115 nm. Seventy-eight percent of the mercury intruded into pores of 76-575 nm and 13% into pores of 12-66 nm diameter. As the 76-575 nm range corresponds to the mean sizes of the particles themselves, these “pores” must be indentations or bulges on the surface of the alumina rather than true holes or channels and, therefore, must represent the outer surface of the particles. They provide 7.2 m2/g or 60% of the total specific surface area. The pores of 12-66 nm diameter can be considered true pores or holes representing the inner surface of the particles. They provide 4.7 m2/g or 40% of the total specific surface area of the particles. This pore diameter also corresponded well with the size of the center holes of some donut-shaped particles that were occasionally observed by EM. The antigen load of individual particles was not directly measurable. However, the mean particle diameters, the specific surface area, and the specific peptomer load were determined experimentally, and with the aid

ne ) 1/4Fπdmin2(dmax - 1/3dmin)SCL NA

(2)

where F is the density of the alumina, dmax and dmin are the mean maximum and minimum diameter, SCL is the specific conjugate load (Table 2), and NA is the Avogadro constant (6.023 × 1023 mol-1). With eq 2, ne was calculated to be 53 000 ( 42 000. Neither the chain length nor the conformation of the peptomer could be obtained experimentally when it was conjugated to the particle surface. These properties can therefore only be estimated on the basis of the chain length distribution in the peptomer solutions and the conformation of the N--bromoacetylated peptomer prior to conjugation. Assuming that no steric constraints or differences in reactivity between peptomer molecules of different chain length exist, the chain length distribution of the immobilized peptomer would be identical to that of the soluble peptomer preparation. The soluble parent peptomer contained polymerization products ranging from the monomeric starting material to dodecamers and higher with a median chain length of 5 peptide units, the monomer made up only 1.7% of that preparation (Table 1). Even if a preference for smaller molecules prevailed in the conjugation reaction, the coupling yield of 55% implies that at least ∼97% of the conjugate consist of dimers and larger molecules. DISCUSSION

Both systemic and mucosal immunization strategies are needed to prevent HIV infection. In this paper, we describe the design, synthesis, and characterization of a nanoparticulate candidate vaccine against HIV-1 that can be adapted for mucosal and systemic administration and that may give rise to broadly cross-reactive, neutralizing antibodies against the viral envelope glycoprotein gp120. To obtain a nanoparticle vaccine of defined particle size and high stability, we chose R-aluminum oxide nanoparticles of 300 nm diameter as antigen carrier. Geltype aluminum oxohydroxide adjuvants have been licensed for decades for use in humans, and R-aluminum oxide has an excellent record of biocompatibility and safety in dentistry and orthopedics (Christel, 1992;


Fairhurst, 1992). Inhaled or ingested corundum powder (R-Al2O3) has been shown to be nontoxic and does not cause granuloma formation, chronic inflammatory reactions, or cancer as is the case for silica dust (Lindenschmidt, 1990). The benefit of a tight, preferably covalent, attachment of the antigen to a particulate carrier was emphasized by the work of Skea and Barber (1993), who bound influenza virus hemagglutinin by a noncovalent antibody bridge to alum gel and achieved a 1500fold increase in immunogenicity. Likewise, Kossovsky et al. (1995) were able to dramatically enhance the immunogenicity of mussel adhesive protein by coupling it onto surface-modified diamond nanoparticles. Derivatization of macroparticulate silica by epoxy- or aminoalkyl silanes for use as high-performance affinity chromatography supports is well documented (Sportsman and Wilson, 1980; Larsson et al., 1983; Anspach et al., 1988; Huisden et al., 1990), but to date there have been no reports of a covalent surface modification and bioconjugation of Al2O3 nanoparticles. As aluminosilicates like garnet or zeolithes are very stable compounds and silica atoms are readily replaced by aluminum in the silicate crystal lattice, we decided to covalently couple our antigen onto the surface of the alumina particles by a (3-aminopropyl)siloxane linker. An amino-derivatization was preferred over a mercaptopropyl or glycidoxypropyl silanization to allow the construction of an N-acetylhomocysteinylamidopropylsiloxane bridge, which should facilitate subsequent release of the antigen in the target cells. The risk of bridging the nanoparticles during silanization to clusters too big to be phagocytosed was anticipated, but it was avoided by using a 175-fold excess of silane and a high particle dispersity throughout the coupling procedure. The increase in particle diameter conferred by silanization, though statistically significant (p < 0.05; one factor ANOVA, Fisher PLSD), was only 10% (data not shown). Thus, the particles still matched the desired size for M cell uptake and phagocytosis by antigen presenting cells. As expected, the silanization product was base-labile but surprisingly sonication/ abrasion-resistant, which facilitated handling and dispersion of the particles by sonication. The silanization yield of 1.3 mmol/m2 was 12 times higher than that which we obtained previously with (3-mercaptopropyl)trimethoxysilane on glass cover slips (Ivanov et al., 1995). Theoretically, a surface load of 2.4 mmol/m2 is achievable for a (3-aminopropyl)siloxane coat when allowing free rotation of the aminopropyl moiety around the Si-C bond (rotation radius: 450 pm). The dramatic increase in derivatization yield as compared to our previous experiments may be explained either by the use of a different silane reagent and a glass support or by the oxidative surface activation and cleaning procedure we applied in this study. Though we did not achieve a complete surface coverage by aminopropylsiloxane, the amine load turned out to be ample for immobilizing the antigen, as only 0.61 mmol of HIV-1MN gp120 C4 domain peptide units/m2 bound to the particle surface. With a median chain length of 5 peptide units/peptomer molecule, the average peptomer surface load was 0.123 mmol of peptomer/m2, which is in good correlation with a surface saturation of 0.121 mmol/m2 reported for oligopeptides coupled onto tresylactivated glass (Massia and Hubbell, 1990). With regard to individual peptide units, we therefore achieved a peptide load five times that of a saturated peptide monolayer. The complete coverage of the particles by antigen also circumvented potential problems associated with the analysis of the thiol-derivatized alumina. When moni-


toring the thiol derivatization of the amine-modified alumina with Ellman’s reagent, part of the reaction product bound to the particles and rendered a quantitative detection of the thiol load impossible. However, in light of the fact that a complete surface coverage of the particles with antigen required only 10% of the amine anchors and that the thiolating N-acetylhomocysteine thiolactone was applied in a 100-fold molar excess, it is unlikely that the thiolation reaction was a yield limiting factor in the conjugation procedure. The particles also interfered with the Picotag amino acid analysis of the immobilized peptomer. While the total antigen load could be determined reliably based on the absolute aliphatic amino acid contents, all dicarboxylic acids like glutamic acid, aspartic acid, S-carboxymethylcysteine, and S-carboxymethylhomocysteine were underrepresented (data not shown). We attribute this phenomenon to the release of aluminum cations from the alumina carrier. Al3+ could have chelated the diacids and thus prevented their labeling by phenylisothiocyanate or changed their motility in the subsequent chromatographic analysis. It is well-known that aluminum ions are able to chelate dicarboxylic acids (Yokel, 1994), but we did not expect the 6 N hydrochloric acid used in the conjugate hydrolysis to liberate Al3+ from the calcinated R-Al2O3, which, in contrast to γ-Al2O3, is considered to be acid resistant. Since S-carboxymethylcysteine is a marker for the polymeric nature of the peptomer and S-carboxymethylhomocysteine is a measure for its covalent coupling to the particle surface, the lack of reliable values for these amino acids forced us to estimate the chain length of the immobilized peptomer on the basis of its soluble unconjugated form. In our view, the problems in obtaining accurate values for S-carboxymethylcysteine and -homocysteine were still outweighed by the biological benefits of the thioether linker technology. First, peptomers are fully biodegradable and their metabolites are predictable, a feature not all synthetic antigens can offer. The biodegradability usually hinges on the nature of the chemical cross-linker. The S-carboxymethylcysteine/homocysteine cross-linkers that are formed in the preparation of a peptomer vaccine are potential substrates for the mercapturic acid pathway of detoxification in which S-substituted cysteines are formed from halogenated xenobiotics and glutathione. The S-substituted cysteine is then N-acetylated to form a mercapturic acid before it is released into the bile and the urine (Jacoby et al., 1984). The S-carboxymethylcysteine and/or S-carboxymethylhomocysteine can join in that pathway before the N-acetylation step and will be readily excreted. Second, we demonstrated previously that HIV-1MN gp120 C4 domain peptomers more closely resemble the native structure of that epitope than individual peptides do (Robey et al., 1995), a finding which should be exploited in designing an HIV-1 gp120 C4 domain vaccine. The polymerization technology also provides additional, immunological benefits for a vaccine. Small oligopeptides are poorly immunogenic because they generally lack repetitive epitopes for T-cell-independent (TI2) immune responses, and they are not large enough to contain additional helper T-cell epitopes that are essential for immunoglobulin class switches and the induction of immunologic memory. To overcome that problem without using a carrier protein or attaching a synthetic T-cell epitope, oligomerization of haptenic peptide anti-


gens was proposed as a means to enhance their immunogenicity. For the study reported previously (Robey et al., 1995), the polymerization of the amphipathic C4 peptide resulted in the peptide assuming an R-helical conformation, and it allowed for the preparation of antibodies that appear to be directed toward the helix. In this case, antibody production may be only B-cell-dependent and T-cell-independent. Therefore, maintaining the helical conformation is most important since this sequence of amino acids is probably in a helical conformation in the parent protein, and it is the parent protein that we are targeting with these antibodies. Although modification of the central W in the C4 peptide (KIKQIINMWQEVGKAMYA) resulted in a loss of helix in the peptomer, it appears that modification of the hydrophilic amino acids in C4 will not influence the ability of C4 to remain helical. The C4 from HIV-1 as compared with the C4 from HIV-2 differs on the hydrophilic face of the helix (Robey et al., 1996), and peptomers from both C4’s display greater than 50% helical content, a property necessary for C4 to bind CD4, the cell receptor for HIV (Robey et al., 1996). It therefore comes as no surprize that the N--bromoacetylation of the peptomer did not change the amount of helix in the peptomer since the bromoacetylation was being performed on the lysine residues, which are assumed to be on the hydrophilic face of the amphipathic helix. Tam (1988) developed the multiple antigenic peptides (MAPs), which consist of a 4- or 8-branch dendritic lysine core onto which four or eight oligopeptide antigens are synthesized colinearly. The MAPs found widespread use and often outperformed the classical peptide carrier protein conjugates (Tam, 1988), but a major drawback of the MAP technology is the limited space on the lysine core. Steric hindrance can occur during the synthesis of long peptides (>10-15 aa) on 8-branch MAP cores, and the tightly packed peptide chains often prevent an antibody response to the more C-terminal amino acids toward the center of the MAP (Francis et al., 1991). As an alternative to the dendritic MAPS, linear head-to-tail peptide polymers have been described. They consisted of 20-30 peptide units prepared either by direct carbodiimide coupling (Borras-Cuesta et al., 1988) or by applying the peptomer technology (Hillman et al., 1990). Both constructs gave rise to antibodies that recognized the native parent protein and did not require cognate T-cell help for induction of the class switch to the IgG isotype. The extent of polymerization that is necessary to achieve such an immunogenicity remains to be determined as both constructs were heterogeneous with respect to their chain length. The studies of Del Giudice et al. (1986) on polymerized NANP peptides from the Plasmodium falciparum CS protein indicate that the immunogenicity of peptide polymers increases with increasing chain length. In light of that observation, we are confident that our HIV-1MN gp120 C4 domain peptomer nanoparticle conjugate is immunogenic even in the absence of T-cell help as it combines the benefits of a linear peptide polymer with the dendritic, collinear peptide organization of a MAP. A summary of the immunogenicity studies will be forthcoming in a future publication. ACKNOWLEDGMENT

We want to thank Dr. R. Reichelt, University of Muenster, Muenster, FRG, for his help with the scanning electron microscopy. We appreciate the professional work of Quantachrome Inc. in analyzing the particle

Frey et al.

surface area and porosity. This work was supported by NIH Research Grants HD17557 and AI34757 to M.R.N. and by NIH Center Grant DK-34854 to the Harvard Digestive Diseases Center. LITERATURE CITED Amerongen, H. M., Weltzin, R. A., Farnet, C. M., Michetti, P., Haseltine, W. A., and Neutra, M. R. (1991) Transepithelial transport of HIV-1 by intestinal M cells: a mechanism for transmission of AIDS. J. Acquired Immune Defic. Syndr. 4, 760-765. Anspach, B., Unger, K. K., Davies, J., and Hearn, M. T. W. (1988) Affinity chromatography with triazine dyes immobilized onto activated non-porous monodisperse silicas. J. Chromatogr. 457, 195-204. Bernatowicz, M. S., and Matsueda, G. R. (1986) Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent. Anal. Biochem. 155, 107-112. Bjork, R. L., Jr. (1991) HIV-1: seven facets of functional molecular mimicry. Immunol. Lett. 28, 91-96. Bland, P. W., and Warren, L. G. (1986) Antigen presentation of epithelial cells of the rat small intestine. II. Selective induction of suppressor T cells. Immunology 58, 9-14. Borras-Cuesta, F., Fedon, Y., and Petit-Camurdan, A. (1988) Enhancement of peptide immunogenicity by linear polymerization. Eur. J. Immunol. 18, 199-202. Brunauer, S., Emmet, P. H., and Teller, E. (1938) Adsorption of gases in multimolecular layers. J. Am. Chem. Soc. 60, 309319. Christel, P. S. (1992) Biocompatibility of surgical-grade dense polycrystalline alumina. Clin. Orthop. 282, 10-18. Cordonnier, A., Montagnier, L., and Emerman, M. (1989) Single amino-acid changes in HIV envelope affect viral tropism and receptor binding. Nature (London) 340, 571-574. Del Giudice, G., Cooper, J. A., Merino, J., Verdini, A. S., Pessi, A., Togna, A. R., Engers, H. D., Corradin, G., and Lambert, P.-H. (1986) The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines. J. Immunol. 137, 2952-2955. DeSchryver, A., and Meheus, A. (1990) Epidemiology of sexually transmitted diseases: the global picture. Bull. W. H. O. 68, 639-654. Earl, P. L., Doms, R. W., and Moss, B. (1990) Oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein. Proc. Natl. Acad. Sci. U.S.A. 87, 648652. Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77. Fairhurst, C. W. (1992) Dental ceramics: the state of science. Adv. Dent. Res. 6, 78-81. Forrest, B. D. (1992) The need for consideration of mucosal immunity in vaccine approaches to AIDS. Vaccine Res. 1, 137-142. Francis, M. J., Hastings, G. Z., Brown, F., McDermed, J., Lu., Y.-A., and Tam, J. P. (1991) Immunological evaluation of the multiple antigen peptide (MAP) system using the major immunogenic site of foot-and-mouth disease virus. Immunology 73, 249-254. Frey, A., Giannasca, K. T., Weltzin, R., Giannasca, P. J., Reggio, H., Lencer, W. I., and Neutra, M. R. (1996) Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting. J. Exp. Med. 184, 1045-1059. Golding, H., Robey, F. A., Gates, F. T., 3rd, Linder, W., Beining, P. R., Hoffman, T., and Golding, B. (1988) Identification of homologous regions in human immunodeficiency virus 1 gp41 and human MHC class II beta 1 domain. J. Exp. Med. 167, 914-923. Gupta, R. K., Rost, B. E., Relyveld, E., and Siber, G. R. (1995) Adjuvant properties of aluminum and calcium compounds. Pharm. Biotechnol. 6, 229-248. Gurgo, C., Guo, H.-G., Franchini, G., Aldovini, A., Collalti, E., Farrell, K., Wong-Staal, F., Gallo, R. C., and Reitz, M. S., Jr.


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