Immunization strategies to augment oral vaccination with DNA and viral vectors expressing HIV envelope glycoprotein

Vaccine 20 (2002) 1295–1307

Immunization strategies to augment oral vaccination with DNA and viral vectors expressing HIV envelope glycoprotein Andrzej Wierzbicki a,1 , Irena Kiszka b,2 , Hiroshi Kaneko a,3 , Dariusz Kmieciak c,1 , Thomas J. Wasik a,4 , Jaroslaw Gzyl c , Yutaro Kaneko d , Danuta Kozbor a,∗ b

a Center for Neurovirology, MCP Hahnemann University, Philadelphia, PA 19102, USA Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107, USA c Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, PA 19122, USA d Pharmaceutical Division, Ajinomoto Co. Inc., Tokyo 104, Japan

Received 12 February 2001; received in revised form 8 October 2001; accepted 6 November 2001

Abstract Induction of mucosal immunity to the human immunodeficiency virus (HIV) envelope (env; gp160) glycoprotein has been demonstrated with orally administered recombinant vaccinia virus (rVV) vectors and poly(dl-lactide-co-glycolide) (PLG)-encapsulated plasmid DNA expressing gp160. In this study, we investigated the effect of an oral DNA-prime/rVV-boost vaccine regimen in conjunction with adjuvants on the level of gp160-specific cellular and humoral responses in BALB/c mice. We demonstrated that DNA priming followed by a booster with rVV expressing gp160 (vPE16) significantly augmented env-specific immunity in systemic and mucosal tissues of the immunized mice. Association of vPE16 with liposomes and coadministration of liposome-associated ␤-glucan lentinan or IL-2/Ig-encoded plasmid DNA-encapsulated in PLG microparticles triggered the optimal cell-mediated immune (CMI) responses. Lentinan was found to increase env-specific type 1 cytokine production and cytotoxic T-lymphocyte (CTL) activities but had no effect on humoral responses. On the other hand, IL-2/Ig-mediated increases in both type 1 and 2 activities were associated with higher levels of env-specific CTL and antibody responses. Results of these studies demonstrated the effectiveness of oral vaccines with DNA and rVV vectors in conjunction with immunomodulators in inducing specific immune responses in systemic and mucosal tissues. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: HIV; Vaccine; Oral immunization

1. Introduction Induction of human immunodeficiency virus (HIV)specific immune responses in mucosal as well as systemic compartments of the immune system is a critical feature of an effective AIDS vaccine. Because over 90% of HIV-seropositive individuals acquire the infection via ∗ Corresponding author. Present address: Center for Neurovirology and Cancer Biology, Temple University, 1900 North 12th Street, Philadelphia, PA 19122, USA. Tel.: +1-215-204-6860; fax: +1-215-204-6528. E-mail address: [email protected] (D. Kozbor). 1 Present address: Department of Biochemistry and Molecular Biology, University of Medical Sciences, Poznan, Poland. 2 Present address: Institute of Molecular Biology, Jagiellonian University, Krakow, Poland. 3 Present address: Department of Rheumatology, Juntendo University, Tokyo, Japan. 4 Present address: Department of Molecular Biology, Biochemistry and Biopharmacy, Medical University of Silesia, Katowice, Poland.

mucosal surfaces [1], this strongly implies that HIV-specific immunity at mucosal sites is critical for the control of infection in many individuals exposed to the virus. The presence of HIV and simian immunodeficiency virus (SIV)-specific cytotoxic T-lymphocytes (CTLs) has been detected in genital and gastrointestinal mucosal tissues following infection with the pathogenic viruses [2–4]. Additionally, protection against mucosal challenge in monkeys vaccinated with attenuated strains of SIV has been easier to achieve than protection against intravenous challenge [5]. However, despite a compelling need to provide better understanding of the ability of virus-specific immune responses to prevent or contain HIV or SIV replication in mucosal tissues, the ability of different AIDS vaccine strategies to induce mucosal immune responses has not been systematically explored. Accumulating evidence suggests that an oral route of immunization or delivery of adjuvants might represent an effective approach for providing protection against infections acquired via mucosal surfaces [6–11]. Induction of mucosal

0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 1 ) 0 0 4 8 0 - 7

1296

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

immunity in the gut has been demonstrated with orally delivered DNA vaccines encapsulated in microparticles made of biologically erodable polymer [8,12,13]. The encapsulation has not only protected the plasmid DNA from gastric environment, but due to the propensity of polymeric spheres toward antigen presenting cell (APC) uptake, it has also increased intracellular delivery of DNA designed to activate immune responses [12,13]. Separate studies performed with orally administered recombinant viral vaccines demonstrated that rVV administered by the oral route generated mucosal and systemic immune responses to the recombinant products as well as antigens of the viral vector [14,15]. Results of these experiments prompted us to investigate whether the efficacy of orally delivered env-specific DNA vaccine could be enhanced by a boost with rVV expressing gp160. This prime/boost immunization strategy was further utilized to analyze the effect of orally administered adjuvants including the ␤-glucan lentinan and IL-2/Ig on the level of env-specific systemic and mucosal immune responses. Immunologically active glucans, i.e. (1–3)-␤-linked glucose polymers with antitumor and antimicrobial activities, occur as a primary component in the cell wall of bacteria and fungi or are secreted extracellularly by various microorganisms [16–19]. Recently, it has been shown that ␤-glucans activate macrophages, neutrophils, and NK cells to kill sensitive tumor cells through binding to the (CD11b/CD18) CR3 receptor [16,17,20]. They are also capable of stimulating secretion of IL-1, TNF-␣, GM-CSF, and IL-6 from macrophages and enhancing T cell responses to cellular antigens [21,22]. Because of the intrinsic ability of ␤-glucans to stimulate innate immunity, some of them including lentinan and schizophyllan have been applied clinically for tumor immunotherapy [23–26]. Among cytokines, IL-2 has been used clinically in humans to increase CD4 counts in HIV-infected individuals and as adjunctive therapy for metastatic renal cell carcinoma and melanoma [27,28]. Administration of plasmid IL-2 has also been reported to augment cellular and antibody responses elicited by a hepatitis B virus DNA vaccine [29]. Subsequent studies have demonstrated that the effect of IL-2 on the DNA vaccine-elicited immune response was influenced by the temporal relationship between antigen and cytokine delivery; injection of protein or plasmid IL-2 or IL-2/Ig either before or coincident with gp120-encoded plasmid DNA suppressed the gp120-specific immune response, whereas, injection of plasmid IL-2/Ig after DNA vaccine amplified this immune response [30]. Furthermore, IL-2/Ig was found to be more effective than IL-2 as a DNA vaccine adjuvant because of a longer in vivo half-life of the cytokine/Ig fusion protein compared with the native cytokine [31]. Recently, IL-2/Ig-augmented DNA vaccination has been shown to control viremia and prevent clinical AIDS after a homologous pathogenic SHIV-89.6P challenge in rhesus monkeys [32]. In this study, we investigated the effect of an oral DNA-prime/rVV-boost vaccine strategy in conjunction with lentinan and plasmid IL-2/Ig on env-specific cellular

and humoral responses. To facilitate uptake of lentinan through the intestinal epithelium, lentinan was associated with liposomes and administered daily during the entire immunization period. This regimen exhibited the most effective antitumor activities of lentinan in the murine system [33]. The IL-2/Ig-encoded plasmid DNA was encapsulated in PLG microparticles and delivered 2 days after the env-specific vaccine. The relative efficacy of lentinan and plasmid IL-2/Ig as oral adjuvants was examined by measuring the levels of env-specific cellular and humoral responses in systemic and mucosal tissues of the immunized mice.

2. Materials and methods 2.1. Reagents Lentinan (∼500 kDa) was obtained from Ajinomoto Central Laboratories (Kawasaki, Japan). For the in vivo studies, lentinan was resuspended in PBS at a final concentration of 2 mg/ml and sonicated for 2.5 min (Branson Sonifier 450, Danbury, CT) with the following setting: duty cycle, 50; output control, 7. After incubation on ice, the sonication was repeated for additional 60 s. This treatment resulted in a homogenous preparation of lentinan with no visible particles. The env gene segment was cloned in the SmaI and NotI restriction sites of the pCI plasmid (Promega, Madison, WI) as described [8]. The IL-2/Ig plasmid consisting of murine IL-2 and a noncytolytic murine Fc␥2a mutated to eliminate its antibody-dependent cell-mediated cytotoxicity and complement binding properties [30] was a generous gift from Dr. N.L. Letvin (Beth Israel Deaconess Medical Center, Boston, MA). 2.2. Viruses The rVV vectors expressing the full-length HIV-1IIIB gp160 (vPE16; [34]), and the WR strain of nonrecombinant vaccinia virus (vac; [35,36]) were provided by Dr. B. Moss (Laboratories of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD). 2.3. Encapsulation of plasmid DNA in PLG microparticles Controlled-release microparticles with entrapped plasmid DNA encoding gp160 or IL-2/Ig fusion protein were prepared with a poly(dl-lactide-co-glycolide) polymer (Sigma, St. Louis, MO) using the water-in-oil-in water solvent evaporation method [8,13]. Briefly, 200 mg of PLG dissolved in 6 ml of dichloromethane (Sigma) was mixed by vortexing with 0.3 ml of TE buffer (pH 8.0) containing 3 mg of plasmid DNA and sonicated for 1 min. The resulting solution was emulsified in 8% polyvinyl alcohol solution (PVA) (Sigma) using a PowerGen 125 homogenizer (Fisher Scientific,

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

Pittsburgh, PA). The emulsion was then poured into 100 ml of 8% PVA and stirred magnetically for 4 h at room temperature to allow solvent evaporation and microparticle formation. The microparticles were isolated by centrifugation, washed 4 times in water, and freeze dried. The final product was stored in a desiccator at −20 ◦ C. The microsphere size profile was analyzed on a Coulter Counter (Miami, FL). DNA integrity was determined by dissolving 25 mg of the PLG microparticles in 500 ␮l of chloroform. After adding 500 ␮l of water, DNA was extracted by ethanol precipitation and analyzed on an agarose gel. To measure incorporation of DNA, the PLG microparticles were dissolved in 0.1 M NaOH at 100 ◦ C for 10 min, and DNA content was determined by the A260 measurement. Incorporation of DNA into microparticles ranged from 1.3 to 2.1 ␮g of DNA per mg of PLG. 2.4. Liposome preparation To prepare vPE16- or lentinan-associated cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1, 2-dilauroyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Alabaster, AL) were dissolved in chloroform at a molar ratio of 1:1:0.5. A lipid monolayer was formed in glass pear bottom vial connected to a rotary evaporator (Labconco, Kansas City, MO), and the lipid solution was thoroughly ventilated using a nitrogen stream [37–39]. The lipid film was wetted with PBS containing either vPE16 vaccinia virus or lentinan, and liposomes were then prepared by intense vortex dispersion and 4 min sonication. The liposome preparation was purified on a BIO-GEL A-50m column (Bio-Rad Laboratories, Hercules, CA). The titer of liposome-associated vPE16 was determined by plating serial 10-fold dilutions on a human HuTK− 143B indicator cells, staining with crystal violet and counting plaques at each dilution. The concentration of lentinan encapsulated in liposomes was determined using the Fungitec G test (Seikagaku Co. Inc., Japan) according to the manufacturer’s protocol. 2.5. Oral vaccination Six-week-old BALB/c (H-2d ) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free microisolator environment. Mice were immunized 3 times (on days 0, 7, and 14) with PLG-encapsulated plasmid DNA (10 ␮g) delivered orally using a 24-gauge feeding needle (Popper & Sons Inc., New Hyde Park, NY). For a booster immunization with the live vector, mice primed with the DNA vaccine received orally 107 PFU of vPE16 or liposome-associated vPE16 1 week after the DNA vaccine. Control mice received orally PLG-encapsulated plasmid pCI followed by vac-associated liposomes. Lentinan-encapsulated in liposomes was delivered on a daily basis during the entire immunization period. The PLG-encapsulated plasmid DNA encoding IL-2/Ig

1297

(10 ␮g) was administered orally 2 days after immunization with the env-specific vaccine. 2.6. Cell purification Induction of env-specific T helper (Th) cell and CTL responses in Peyer’s patches (PP), lamina propria (LP), and spleen was analyzed 3 weeks after specific immunization. Lymphocytes from LP were dissociated into single cells by enzymatic digestion as described [40]. Briefly, the large and small intestines were dissected from individual mice and fecal material was flushed from the lumen with medium. After PP were identified and removed from the intestinal wall, the intestines were opened longitudinally, cut into short segments, and washed extensively in complete medium. To remove the epithelial cell layer, tissues were placed into 20 ml of 1 mM EDTA and incubated twice (for 40 min and then for 20 min) at 37 ◦ C with stirring. After the EDTA treatment, tissues were washed in complete medium for 10 min, placed into 50 ml of RPMI 1640 medium containing 10% FCS and incubated with stirring for 15 min at 37 ◦ C. To isolate LP lymphocytes, tissues were cut into small pieces and incubated in medium containing collagenase type VIII, 300 units/ml (Sigma) for 50 min at 37 ◦ C with stirring. The collagenase dissociation procedure was repeated twice and the isolated cells were pooled and washed again. To remove dead cells and tissue debris, cells were passed through a cotton–glass wool column (Fisher Scientific) and then layered onto a discontinuous gradient containing 75 and 40% Percoll (Sigma). After 20 min centrifugation (600 × g) at 4 ◦ C, the interface layer between the 75 and 40% Percoll was carefully removed and washed with medium. This procedure provided over 95% viable lymphocytes with a cell yield of 1.5 × 106 to 2 × 106 lymphocytes per mouse. Spleen cells were aseptically removed and a single cell suspension was prepared by gently teasing the cells through sterile screens. 2.7. In vitro stimulation assay To measure env-specific Th cell responses, lymphocytes from spleen (3 × 106 cells/ml), PP and LP (2 × 106 cells/ml) were incubated in 96-well plate (Linbro, ICN Biomedicals Inc., Aurora, OH) with 3 ␮g/ml of recombinant gp160 (rgp160; Immunodiagnostics Inc., Bedford, MA) or medium only. On the third day of stimulation, culture supernatants were collected and analyzed for IFN-␥, IL-2, and IL-4 production by the ELISA assay (QuantikineTM , R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. The level of cytokine production from triplicate or duplicate wells was calculated after subtracting the background values from unstimulated cultures. 2.8. Anti-gp160 antibody ELISA A direct ELISA assay was used to determine the presence of env-specific antibodies in sera, fecal samples,

1298

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

vaginal washes, and saliva. Ninety-six-well Maxisorp ELISA plates (Nunc, Naperville, IL) were coated overnight at 4 ◦ C with 100 ␮l of 3 ␮g/ml rgp160 (Immunodiagnostics Inc.) as described [8]. The rest of the assay was conducted at room temperature. Following a wash with PBS containing 0.05% Tween-20 (PBS/Tween-20), the wells were blocked for 2 h with a solution containing 2% BSA (Sigma) and 0.05% Tween-20 in PBS. Sera were prepared from murine blood samples, serially diluted in PBS/Tween-20 and added to the ELISA wells. For each dilution, duplicate wells were used. After incubation at room temperature for 1 h, the plates were washed 3 times and then incubated with a 1/10,000 dilution of a peroxidase-conjugated goat anti-mouse Ig (IgG, IgM, and IgA; Sigma) in PBS/Tween. The plates were washed, developed with O-phenylenediamine (0.4 mg/ml; Sigma) in 0.05 M phosphate–citrate buffer containing 0.03% sodium perborate (Sigma), stopped with 0.4N sulfuric acid, and analyzed at 450 nm with an ELISA plate reader (Dynatech MRX, Chantilly, VA). Sample dilutions were considered positive if the optical density recorded for that dilution was at least 2-fold higher than the optical density recorded for a naive sample at the same dilution [41]. The level of env-specific IgA in fecal and vaginal washes as well as saliva was measured by ELISA using 1/1,000 dilution of peroxidase conjugated-goat anti-mouse IgA (Sigma). Fecal samples (100 mg) were mixed with 1 ml of PBS, incubated at room temperature for 15 min, vortexed, and centrifuged in a microcentrifuge for 10 min. Supernatants were collected and stored at −20 ◦ C until assayed for anti-gp160 antibodies. Saliva was obtained after stimulation with pilocarpine (5 mg/kg of body weight injected i.p.), and vaginal washes were collected in 50 ␮l of PBS as described [41]. 2.9. CTL assay Lymphocytes from spleen were cultured at 2×106 cells/ml in 24-well culture plates with medium containing 1 ␮M of the env peptide I10 (RGPGRAFVTI, amino acids 318-327) [42] and 10% T cell stimulatory factor (T-STIMTM Culture Supplement, Collaborative Biomedical Products, Bedford, MA) as a source of exogenous IL-2. After 3 days of stimulation, cells were split and cultured in medium supplemented with 0.3 ng/ml of recombinant mouse IL-2 (Pharmingen, San Diego, CA). Cytolytic activity of CTL lines was analyzed after 6 days of cultures by a standard 4 h 51 Cr release assay against 17Cu cells (a clone derived from the H-2d -positive 3T3 fibroblasts, provided by Dr. M. Wysocka, The Wistar Institute, Philadelphia, PA) infected with vPE16 or vac. Specific lysis was calculated with the formula: 100 × ([cpm experimental release − cpm spontaneous release]/[cpm maximum release − cpm spontaneous release]), where spontaneous release was cpm from target cells incubated with medium alone and maximum release was cpm from target cells incubated with 1% Triton X-100.

2.10. Neutralization assay The neutralization assay of the HIV recombinant clone pIIIB was performed using as target cells PHA-stimulated peripheral blood lymphocytes (PBL) as described [43,44]. The pIIIB clone [43] was provided to us by Dr. B.R. Cullen (Duke University Medical Institute, Durham, NC). It contains a modified form of the replication-competent HxB3 provirus derived from the HTLV-IIIB isolate in which the single base pair frame-shift mutation present in the vpr gene of HxB3 has been repaired [43]. The pIIIB virus stock was prepared from COS-1 cells transfected with the pIIIB plasmid as described [43]. Briefly, at ∼72 h after transfection, the COS-1 cultures were fed with fresh medium containing PHA-stimulated PBL at a density of 106 cells/ml. Cells were cultured with the transfected COS-1 cells for 3 days, aspirated, washed, and maintained in the expanded culture for 4 more days. On day 7 after infection, supernatants were removed and filtered through a 0.45-␮m filter. The virus was titrated in replicate cultures of PBL derived from a single donor in flat-bottom wells of 96-well tissue culture plates in complete RPMI 1640 medium with rIL-2 by the end-point-dilution method [45], aliquoted, and kept frozen at −80 ◦ C until further use. For the antibody-mediated neutralization, the virus stock diluted to 25 50% tissue culture infectious doses (TCID50 ) in 50 ␮l of complete RPMI medium was preincubated with an equal volume of serially diluted heat-inactivated (35 min at 56 ◦ C) sera for 1 h at 37 ◦ C in 96-well tissue culture plates (Corning). For each serum dilution, triplicate wells were used. Sera from mice immunized with PLG-encapsulated control plasmid and liposome-associated vac served as controls for nonspecific neutralization. To each well, 0.1 ml of complete medium with rIL-2 containing 105 PHA-induced blasts was added. Following 4 h incubation at 37 ◦ C, half of the volume of each well was replaced with fresh, complete RPMI medium. Following centrifugation of the plate (5 min at 1500 rpm), half of the volume of each well was again replaced with fresh medium. This procedure was repeated twice. The p24-antigen concentration in each well was evaluated 7 days after infection by a p24-antigen-capture assay (NEN Life Science Products, Inc., Boston, MA) according to the manufacturer’s procedures. Each assay plate contained uninfected and infected cell controls and a serum from an individual with AIDS as a positive control. Neutralization titers were reported as the reciprocals of the serum dilutions at which the production of the p24-antigen was reduced by 90%. 2.11. Statistical analysis The significance of differences in the means of IFN-␥, IL-2, and IL-4 production and env-specific antibody titers between groups of mice was determined by the unpaired Student’s t-test [46] using JMP software (SAS Institute Inc., Cary, N.C.). Results were considered statistically significant for P < 0.05.

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

3. Results 3.1. Systemic cellular and humoral responses to the env glycoprotein induced by oral vaccination with DNA and rVV expressing gp160 In the initial experiments, we explored approaches for optimizing the efficacy of orally administered DNA- and viral vector-based vaccines. For oral immunization of BALB/c mice (four to five mice per group), the DNA and viral vectors were administered separately or sequentially with the env-specific DNA vaccine used for priming and vPE16 delivered as a booster. The level of env-specific IFN-␥ responses in cell-free culture

1299

supernatants derived from rgp160-stimulated splenocytes was analyzed 3 weeks after specific immunization. Fig. 1A shows that the DNA-prime/rVV-boost vaccination regimen with PLG-encapsulated plasmid DNA encoding gp160 and vPE16 elicited 2- to 3-fold higher IFN-␥ responses to rgp160 than immunization with the single vector alone (P < 0.005). Furthermore, the association of vPE16 with cationic liposomes elicited approximately 40% increases in env-specific IFN-␥ production compared with responses induced by immunization with the free virus (P < 0.006). Responses of a significantly higher level were also induced when liposome-associated vPE16 was applied in the DNA-prime/rVV-boost regimen (P < 0.001), suggesting that an oral vaccine with liposome-associated rVV might

Fig. 1. HIV env-specific IFN-␥ and CTL responses in spleen induced by oral vaccines with DNA and rVV vectors expressing gp160. (A) Splenocytes from mice immunized with env-specific DNA vaccine or control vectors were stimulated for 3 days with 3 ␮g/ml of rgp160. The level of IFN-␥ in cell-free culture supernatants was determined by the ELISA assay. Background values from unstimulated cultures were subtracted from all values given. The data represent the mean ± S.D. of four to five separate experiments. (B) Splenocytes were stimulated with the env-specific H-2d -restricted peptide I10 for 6 days and the level of env-specific CTL responses was examined against vPE16-infected 17Cu target cells using a standard 51 Cr-release assay. In parallel, each culture was analyzed for CTL responses against 17Cu cells infected with the nonrecombinant vaccinia virus (vac) as a negative control. The latter values were subtracted from those obtained with vPE16-infected target cells for analysis of env-specific CTL responses. All CTL experiments were run in triplicate samples with S.D. < 10%. Results are presented as the mean ± S.D. of at least three independent experiments.

1300

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

be more effective for induction of specific IFN-␥ responses than immunization with the free virus. To determine whether the effect achieved with the oral DNA-prime/vPE16-boost immunization strategy on env-specific IFN-␥ responses could also be obtained in cytotoxic activities, we analyzed the level of env-specific CTL responses in spleen of the immunized mice by a standard 51 Cr-release assay. Splenocytes were stimulated with the env-specific, H-2d -restricted peptide I10 [42] for 6 days prior to the killing assay. The CTL activities in I10-stimulated cultures were analyzed against 17Cu cells infected with vPE16. In parallel, each culture was analyzed for CTL responses against 17Cu cells infected with the nonrecombinant vaccinia virus (vac) as a negative control. The latter values were subtracted from those obtained with vPE16-infected target cells for analysis of env-specific CTL activities. As shown in Fig. 1B, immunization with vPE16 or gp160-encoded plasmid DNA elicited a similar level of env-specific CTL activity. The association of the viral vector with cationic liposomes had a small enhancing effect on the development of CTL responses. Consistent with the profile of IFN-␥ production, the highest env-specific cytotoxic responses were detected in cultures derived from mice immunized with PLG-encapsulated plasmid DNA encoding gp160 followed by free or liposome-associated vPE16. The ELISA assay used to analyze humoral responses to gp160 revealed detectable levels of env-specific antibodies in sera of mice immunized orally with PLG-encapsulated plasmid DNA encoding gp160 (titer: 2500 ± 720) or vPE16 (titer: 3400 ± 650) (Fig. 2). The DNA-prime/rVV-boost immunization strategy generated significant increases in env-specific antibody responses compared with the antibody levels induced with vPE16 (P =0.013) or gp160-encoded plasmid DNA-encapsulated in PLG

microparticles (P = 0.005). In contrast to cellular responses, the association of vPE16 with cationic liposomes had no significant effect on the titer of env-specific antibodies. 3.2. Mucosal IFN-γ and antibody responses to the env glycoprotein To determine whether a delivery system based on the prime/boost immunization strategy with PLG-encapsulated plasmid DNA and rVV expressing gp160 would enhance the level of specific responses in mucosal tissues, cultures were established from PP and LP of the immunized mice and examined for production of IFN-␥ after stimulation with rgp160. As shown in Fig. 3, IFN-␥ responses to rgp160 in gut-associated lymphoid tissues were detectable in all animals immunized with the env-specific vaccines. The association of vPE16 with cationic liposomes induced higher levels of env-specific IFN-␥ responses in PP and LP than immunization with the free virus (P = 0.008 and 0.04, respectively). Additional increases in the level of env-specific responses in PP were elicited by priming with gp160-encoded plasmid DNA-encapsulated in PLG microparticles and a booster with free (P = 0.002) or liposome-associated vPE16 (P < 0.0001). Similarly, the DNA-prime/rVV-boost vaccination regimen with free and liposome-associated vPE16 increased env-specific IFN-␥ production in LP compared with responses induced by the DNA vaccine (P = 0.0009 and 0.0001, respectively). The differences in immunization strategies were also reflected in the level of env-specific IgA responses in fecal samples, vaginal washes and saliva. As shown in Fig. 4A, gp160-specific mucosal IgA responses were present in fecal samples derived from mice immunized orally with the

Fig. 2. Induction of env-specific antibody responses by oral vaccines with DNA and rVV vectors expressing gp160. Sera prepared from murine blood samples were serially diluted and analyzed for gp160-specific antibody responses by the ELISA assay. Bars represent the mean ± S.D. of four independent experiments.

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

1301

Fig. 3. Induction of env-specific IFN-␥ responses in mucosal tissues by oral vaccination with DNA and viral vectors expressing gp160. Cells isolated from PP and LP were stimulated for 3 days with 3 ␮g/ml of rgp160. The level of IFN-␥ in cell-free supernatants from rgp160-stimulated and unstimulated cultures was determined by the ELISA assay. Background values from unstimulated cultures were subtracted from all values given. Results are presented as the mean ± S.D. of four separate experiments.

env-specific DNA vaccine, and were further enhanced by a booster with vPE16 (P < 0.01). In contrast to cellular responses, no differences in the level of gp160-specific IgA responses in gut-associated lymphoid tissues were measured in mice boosted with free or liposome-associated vPE16. The env-specific IgA responses were also detectable in vaginal washes (Fig. 4B) and saliva (Fig. 4C) in mice immunized with the DNA-prime/vPE16-boost regimen. However, they were at background levels after immunization with the single vector alone. 3.3. Effect of lentinan and plasmid IL-2/Ig on systemic responses to the env glycoprotein Experiments were performed to analyze the stimulatory effect of orally delivered lentinan and plasmid IL-2/Ig on vaccine-elicited immune response to gp160 in systemic and mucosal tissues. For the oral delivery, 10 ␮g of lentinan

was encapsulated in liposomes to facilitate uptake of this ␤-glucan through the epithelial tissue of the gut. The liposomal preparation of lentinan was administered together with the env-specific vaccine and continued on a daily basis during the entire immunization period. The effect of lentinan on env-specific cellular and humoral responses was compared with that of IL-2/Ig-encoded plasmid DNA. The latter was encapsulated in PLG microparticles and administered orally 2 days after specific immunization. The effect of lentinan and IL-2/Ig on env-specific IFN-␥ responses was first analyzed in rgp160-stimulated splenocytes derived from BALB/c mice immunized orally by the prime/boost strategy with gp160-encoded plasmid DNA-encapsulated in PLG microparticles and liposome-associated vPE16. As shown in Table 1, the highest increases in env-specific IFN-␥ production were measured in cultures established from mice fed with the liposomal formulation of lentinan. This group of mice

Table 1 Effect of liposome-associated lentinan and PLG-encapsulated plasmid IL-2/Ig on env-specific cytokine responses in spleen Inoculuma

PLG PLG PLG PLG PLG PLG

pCI/liposome-vac pCI/liposome-vac + liposome-lentinan pCI/liposome-vac + PLG pIL-2/Ig env-pCI/liposome-vPE16 env-pCI/liposome-vPE16 + liposome-lentinan env-pCI/liposome-vPE16 + PLG pIL-2/Ig

Cytokinesb (pg/ml) IFN-␥

IL-2

310 ± 260 430 ± 273 360 ± 190 21700 ± 4200 42200 ± 6000 38000 ± 5320

91 120 143 6300 10400 9200

IL-4 ± ± ± ± ± ±

66 92 77 790 1300 1100

34 23 28 69 76 214

± ± ± ± ± ±

25 16 19 16 18 25

a Mice were immunized orally with gp160-encoded plasmid DNA-encapsulated in PLG microparticles and liposome-associated vPE16. Lentinan was associated with liposomes and administered daily during the entire immunization period. PLG-encapsulated plasmid DNA encoding IL-2/Ig was delivered 2 days after the specific vaccine. b Splenocytes from mice immunized with the specific vaccine or control vectors were incubated for 3 days with 3 ␮g/ml of rgp160. The level of IFN-␥, IL-2, and IL-4 in cell-free supernatants was determined by ELISA. Background values from unstimulated cultures were subtracted from all values given. Results represent the mean ± S.D. of at least four independent experiments.

1302

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

Fig. 4. Env-specific mucosal IgA responses in mice immunized orally with DNA and viral vectors expressing gp160. The level of env-specific IgA in stool (A), vaginal washes (B) and saliva (C) was determined by ELISA. Results are presented as the mean ± S.D. of at least four independent experiments.

maintained approximately 2-fold higher level of IFN-␥ in rgp160-stimulated cultures compared with animals immunized in the absence of any adjuvant treatment (P = 0.006). The changes in the level of IFN-␥ in rgp160-stimulated splenocytes derived from mice immunized in the presence of liposome-associated lentinan were also associated with increases in IL-2 production (P = 0.002). On the other hand, lentinan had no effect on env-specific IL-4 responses. Administration of PLG-encapsulated plasmid DNA encoding IL-2/Ig during specific vaccination also augmented env-specific IFN-␥ and IL-2 production in

rgp160-stimulated splenocytes (P = 0.008 and 0.009, respectively). However, in contrast to lentinan, delivery of plasmid IL-2/Ig resulted in approximately 3-fold increases in IL-4 production compared with responses in mice immunized in the absence of any adjuvant treatment (P < 0.001). Additional experiments were conducted to determine whether the adjuvant effect of orally delivered lentinan or IL-2/Ig-encoded plasmid DNA on env-specific type 1 responses could also be reflected in cytotoxic activities. The level of CTL responses in the immunized animals was assessed in splenocytes stimulated with the H-2d -restricted

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

immunodominant V3 loop peptide for 6 days prior to the 51 Cr-release assay. The target cells consisted of 17Cu cells infected either with vPE16 or vac. Consistent with the profile of rgp160-induced IFN-␥ responses, oral delivery of 10 ␮g of liposome-associated lentinan or PLG-encapsulated plasmid DNA encoding IL-2/Ig elicited the highest increases in the level of env-specific CTL activities (Fig. 5). Lower killing of vPE16-infected target cells was detected in mice immunized in the absence of any adjuvant treatment. The effect of lentinan and IL-2/Ig plasmid on the level of env-specific serum antibody responses differed. As shown in Table 2, liposome-associated lentinan had no stimulatory effect on the level of env-specific humoral responses.

1303

In contrast, oral administration of PLG-encapsulated plasmid DNA encoding IL-2/Ig induced significant increases in anti-gp160 serum antibody titer (P = 0.004). 3.4. Neutralizing antibody responses induced by vaccination with DNA and viral vectors expressing the env glycoprotein Next, we analyzed neutralizing activities of env-specific antibodies in sera of mice immunized orally with the env-specific vaccines. Analysis of p24-antigen levels in culture supernatants of PHA-stimulated PBL infected with the pIIIB virus in the presence or absence of the immune sera revealed that immunization with PLG-encapsulated plasmid DNA expressing gp160 or vPE16 was not effective in inducing neutralizing antibodies (Table 2). The titer of env-specific antibodies capable of neutralizing pIIIB infection of PHA-stimulated cells showed small increases after a booster with vPE16. Among four mice immunized by the prime/boost strategy with PLG-encapsulated plasmid DNA encoding gp160 and vPE16, one animal elicited env-specific antibodies able to neutralize the infection at a serum dilution of 1:32. At this dilution, sera from the remaining three animals inhibited the infection by 30–70%. A similar profile of neutralizing activities was detected in sera from mice primed with env-specific DNA vaccine and boosted with liposome-associated vPE16. Additional experiments with sera derived from mice immunized in the presence of adjuvants revealed that administration of liposome-associated lentinan had no effect on the level of neutralizing antibody response (Table 2). On the other hand, three out of four animals immunized in the presence of PLG-encapsulated plasmid DNA encoding IL-2/Ig generated antibodies that were able to inhibit pIIIB infection at dilutions 1:32 or 1:64, suggesting that increases in the env-specific antibody titer in sera of IL-2/Ig-treated mice were associated with higher neutralizing activity. 3.5. Effect of liposome-associated lentinan and PLG-encapsulated plasmid IL-2/Ig on env-specific mucosal responses

Fig. 5. Effect of orally administered liposome-associated lentinan and PLG-encapsulated plasmid IL-2/Ig on env-specific CTL responses. Mice were immunized orally with gp160-encoded plasmid DNA-encapsulated in PLG microparticles and liposome-associated vPE16. Lentinan was associated with liposomes and administered daily during the entire immunization period. PLG-encapsulated plasmid DNA encoding IL-2/Ig was delivered 2 days after the specific vaccine. Mice immunized with PLG-encapsulated control plasmid and liposome-associated vac in the presence or absence of adjuvant treatment mice served as controls. The env-specific CTL responses were analyzed by 51 Cr-release assay in I10 peptide-stimulated cultures against vPE16-infected 17Cu cells. The percent of lysis with vac-infected cells used as a negative control was subtracted from the presented values. All CTL experiments were run in triplicate samples with S.D. < 10%. Results are presented as the mean ± S.D. of at least three independent experiments.

Further studies were carried out to investigate the effect of lentinan and plasmid IL-2/Ig on env-specific mucosal immunity elicited by the prime-boost immunization strategy with PLG-encapsulated plasmid DNA encoding gp160 and liposome-associated vPE16. We first analyzed the level of env-specific IFN-␥, IL-2, and IL-4 production in rgp160-stimulated lymphocytes isolated from LP of the adjuvant-treated mice. As shown in Table 3, IFN-␥ and IL-2 responses to rgp160 in mucosal tissues of the gut induced by immunization with the env-specific vaccine were increased by administration of liposome-associated lentinan (P < 0.001 and 0.005, respectively). Likewise, oral administration of PLG-encapsulated plasmid IL-2/Ig increased the levels of env-specific IFN-␥ and IL-2 responses (P = 0.003

1304

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

Table 2 HIV env-specific antibody responses and neutralizing titers in mice immunized orally with env-specific vaccines in the presence or absence of liposome-associated lentinan or PLG-encapsulated plasmid IL-2/Ig Immunization regimena Prime

Boost

vPE16 Liposome-vPE16 PLG env-pCI PLG-pCI PLG env-pCI PLG env-pCI PLG env-pCI PLG env-pCI

– – – Liposome-vac vPE16 Liposome-vPE16 Liposome-vPE16 Liposome-vPE16

Adjuvantb

Anti-gp160 antibody titerc

– – – – – – Lentinan pIL-2/Ig

3400 3250 2500 83 5600 7200 7000 19800

± ± ± ± ± ± ± ±

Neutralization titerd

<10 <10 <10 <10 18 ± 20 ± 22 ± 44 ±

650 900 720 75 900 2100 2422 4900

10 8 12 24

a Mice were immunized orally with gp160-encoded plasmid DNA-encapsulated in PLG microparticles and vPE16 delivered separately or sequentially with env-specific DNA vaccine used for priming and vPE16 delivered as a booster. In some experiments, mice were immunized with liposome-associated vPE16. Control mice were immunized with PLG-encapsulated control plasmid and liposome-associated vac. b Lentinan was associated with liposomes and administered daily during the entire immunization period. PLG encapsulated plasmid DNA encoding IL-2/Ig was delivered 2 days after specific vaccine. c Sera prepared from murine blood samples were serially diluted and analyzed for env-specific antibody titers by ELISA. For each serum dilution, triplicate wells were used. Results represent mean ± S.D. of four independent experiments. d The neutralizing antibody activity in sera was measured against the pIIIB virus and PHA-stimulated PBL used as target cells. Values shown represent the reciprocals of serum dilutions at which a 90% inhibition of virus infection was observed. The data are presented as the mean ± S.D. of at least three independent experiments.

Table 3 Effect of liposome-associated lentinan and PLG-encapsulated plasmid IL-2/Ig on env-specific cytokine responses in mucosal tissues Inoculuma

Cytokinesb (pg/ml) IFN-␥

PLG PLG PLG PLG PLG PLG

pCI/liposome-vac pCI/liposome-vac + liposome-lentinan env-pCI/liposome-vac + PLG pIL-2/Ig env-pCI/liposome-vPE16 env-pCI/liposome-vPE16 + liposome-lentinan env-pCI/liposome-vPE16 + PLG pIL-2/Ig

110 146 122 4200 8100 6700

± ± ± ± ± ±

IL-2 80 65 74 520 850 865

30 52 44 1100 2700 2300

IL-4 ± ± ± ± ± ±

21 29 27 340 590 610

11 13 17 31 51 97

± ± ± ± ± ±

9 10 10 12 9 18

a Mice were immunized orally with gp160-encoded plasmid DNA-encapsulated in PLG microparticles and liposome-associated vPE16. Lentinan was associated with liposomes and administered daily during the entire immunization period. PLG-encapsulated plasmid DNA encoding IL-2/Ig was delivered 2 days after env-specific vaccine. b Lymphocytes isolated from LP of the immunized and control mice were stimulated for 3 days with 3 mg/ml of rgp160. Production of IFN-␥, IL-2, and IL-4 in cell-free supernatants was determined by ELISA. Background values from unstimulated cultures were subtracted from all values given. Results represent the mean ± S.D. of four independent experiments.

Fig. 6. Effect of orally delivered liposome-associated lentinan and PLG-encapsulated plasmid DNA encoding IL-2/Ig on mucosal IgA responses to the env glycoprotein. The level of env-specific IgA in stool, vaginal washes, and saliva was determined by ELISA. Results are presented as the mean ± S.D. of four to five independent experiments.

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

and 0.01, respectively). Furthermore, delivery of plasmid IL-2/Ig, but not liposome-associated lentinan, augmented env-specific IL-4 production in rgp160-stimulated cultures (P < 0.001). Additional experiments were carried out to examine the effect of liposome-associated lentinan and PLG-encapsulated plasmid IL-2/Ig on mucosal IgA responses to the env glycoprotein in mice immunized with the DNA-prime/liposomevPE16-boost regimen (Fig. 6). Consistent with the profile of serum antibody responses, the env-specific IgA level in fecal and vaginal washes or saliva was not affected by the lentinan treatment. In contrast, intragastric administration of IL-2/Ig-encoded plasmid DNA-encapsulated in PLG microparticles significantly augmented vaccine-induced anti-gp160 IgA responses in fecal samples (P = 0.007), vaginal washes (P = 0.02), and saliva (P = 0.04). 4. Discussion The primary goal of HIV vaccine development is to identify an immunogen that can be formulated in a way such that the vaccine is effective and affordable for worldwide use. In this study, we demonstrated that the prime-boost immunization strategy based on orally delivered PLG-encapsulated plasmid DNA encoding gp160 and liposome-associated vPE16 vaccinia virus was capable of inducing potent cellular and humoral immune responses to the env glycoprotein in mucosal and systemic tissues. This is also the first study that used orally delivered liposome-associated lentinan and PLG-encapsulated plasmid encoding IL-2/Ig for significant increases in gp160-specific immune responses. Because mucosal immunity constitutes an impressive first-line defense system, the described oral route of immunization and the vaccine regimen might represent an effective approach for providing protection against infections acquired via mucosal surfaces. Additionally, oral vaccines have the advantage in that they obviate the need for needles, which is a major cause of iatrogenic infections in developing countries. The ability of intragastric vaccination alone or in combination with orally delivered adjuvants to elicit immune responses to HIV antigens in mucosal tissues has not been systematically explored. Analysis of polio-specific mucosal responses in humans vaccinated with live oral polio vaccine, inactivated polio vaccine, or combinantions of these vaccines, showed that induction of specific mucosal immune responses required priming with the live oral polio vaccine [47]. Evidence for the importance of mucosal homing can also be found in pulmonary influenza virus infection, where the ability of adoptively transferred CD8+ T cells to migrate to the site of infection is critical for protection [48]. While the DNA prime/viral vector-boost vaccination regimen is particularly effective at inducing virus-specific CD8+ T cells in systemic tissues [49–51], more information is needed regarding the mucosal homing ability of cells induced by these vaccines. Previously, we have demon-

1305

strated that oral immunization with PLG-encapsulated plasmid DNA encoding gp160 protected mice against mucosal challenge with rVV expressing the homologous env glycoprotein [8]. In this report, we showed that the efficacy of orally delivered env-specific DNA vaccine could be significantly augmented by a booster with vPE16. Unfortunately, we were not able to study the protective immunity after mucosal immunization with vPE16 because the cross-reactive vaccinia virus-specific responses induced during a booster would recognize any other vaccinia viruses that could be used to challenge. Although, liposomes have been extensively used as a delivery system for variety of antigens including proteins and peptides, their use in association with viral vectors for oral vaccination remains to be investigated. The increases in immune responses to gp160 after association of vPE16 with liposomes may be due to the liposome-mediated protection of the virus against the gastric environment as well as an adjuvant effect of cationic liposomes on immune responses in vivo [52]. Furthermore, this technique might be particularly useful for delivery of replication-attenuated recombinant viruses because the association with liposomes may improve viral dissemination in vivo. Finally, liposome-associated ␤-glucans delivered during immunization may augment vaccine-induced immune responses due to a high binding affinity between ␤-glucans and the CR3 (CD11b/CD18) receptor expressed on macrophages and dendritic cells [16,17]. Our results of an increased level of env-specific Th1 and CTL activities in mice fed with the liposomal formulation of lentinan are consistent with the reported ability of ␤-glucans to activate APC through their interaction with the CD11b subunit of CR3 [16,17]. This interaction results in induction of a variety of cytokines including IL-1, TNF-␣, GM-CSF, and IL-6 [21], suggesting that lentinan-mediated upregulation of expression of immunoregulatory cytokines and costimulatory molecules may promote efficient antigen priming in vivo. Although, the complete range of activation events induced by ␤-glucans has not been fully defined, accumulating evidence suggests that ␤-glucans with the highest affinity for the receptor are most effective in leukocyte activation [53]. Because the cytotoxic host defense function of ␤-glucans against tumors or microbial infections is specific for C3-opsonized target cells [54], the mechanism of action of ␤-glucans in promoting host defense relies on the specificity of the antibody. This distinguishes ␤-glucans from other “nonspecific” biological response modifiers, including cytokines. The differences in the mechanism of action between ␤-glucans and cytokines may also be useful for vaccine development because these immunomodulators may have a synergistic effect on vaccine-elicited immune responses. In our studies, lentinan was found to increase env-specific type 1 cytokine production and CTL activities but had no effect on humoral responses. On the other hand, IL-2/Ig-mediated increases in both type 1 and 2 activities were associated

1306

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307

with higher levels of env-specific CTL and antibody responses. Additionally, treatment of the immunized mice with IL-2/Ig-encoded plasmid DNA increased neutralizing antibody responses induced by the env-specific vaccine. Because lentinan and IL-2/Ig exhibit their immunostimulatory activities through a different mechanism of action, it is possible that immune responses to the env glycoprotein can be further optimized by administration of lentinan at the time of immunization followed by delivery of plasmid IL-2/Ig after the specific vaccine. In summary, results of our experiments demonstrated that oral administration of liposome-associated lentinan or PLG-encapsulated plasmid DNA encoding IL-2/Ig augmented vaccine-elicited immune responses to the env glycoprotein. The findings that association of adjuvants with liposomes or PLG microparticles facilitated their uptake in the gastrointestinal tract may also be applicable to other vaccines and immunotherapeutic approaches. Furthermore, the reported ability of ␤-glucans to enhance innate immunity and the lentinan-mediated increases in cellular responses elicited by the env vaccine, suggest that lentinan may be effective in enhancing the efficacy of DNA- and viral vector-based vaccines in humans. This, together with the use of IL-2/Ig-encoded plasmid DNA that augmented immune responses elicited by the specific DNA vaccine in SHIV-89.6P challenged monkeys with uniformly good outcomes [32], makes the strategy of orally delivered adjuvants particularly attractive for enhancing the therapeutic effect of vaccines in HIV-infected patients.

Acknowledgements We are grateful to Drs. N.L. Letvin (Beth Israel Deaconess Medical Center, Boston, MA), Brian R. Cullen (Duke University Medical Institute, Durham, NC), B. Moss (Laboratories of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD), and M. Wysocka (The Wistar Institute, Philadelphia, PA) for reagents. This work was supported by research grants from the National Institute of Allergy & Infectious Diseases (AI/HD39148 and AIDE48370), the Japan Health Sciences Foundation, and Ajinomoto Co. Inc., Japan.

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

References [1] Kresina TF, Mathieson B. Human immunodeficiency virus type 1 infection, mucosal immunity, and pathogenesis and extramural research programs at the National Institutes of Health. J Infect Dis 1999;179(3):S392–6. [2] Musey L, Hu Y, Eckert L, Christensen M, Karchmer T, McElrath J. HIV-1 induces cytotoxic T-lymphocytes in the cervix of infected women. J Exp Med 1997;185:293–303. [3] Couedel-Courteille A, Le Grand R, Tulliez M, Guillet JG, Venet A. Direct ex vivo simian immunodeficiency virus (SIV)-specific cytotoxic activity detected from small intestine intraepithelial

[19]

[20]

[21]

[22]

lymphocytes of SIV-infected macaques at an advanced stage of infection. J Virol 1997;71:1052–7. Murphey-Corb M, Wilson LA, Trichel AM, et al. Selective induction of protective MHC class I-restricted CTL in the intestinal lamina propria of rhesus monkeys by transient SIV infection of the colonic mucosa. J Immunol 1999;162:540–9. Johnson RP, Lifson JD, Czajak SC, et al. Highly attenuated vaccine strains of simian immunodeficiency virus protect against vaginal challenge: inverse relationship of degree of protection with level of attenuation. J Virol 1999;73:4952–61. Childers NK, Zhang SS, Michalek SM. Oral immunization of humans with dehydrated liposomes containing Streptococcus mutans glucosyltransferase induces salivary immunoglobulin A2 antibody responses. Oral Microbiol Immunol 1994;9:146–53. Czerkinsky C, Anjuere F, McGhee JR, et al. Mucosal immunity and tolerance: relevance to vaccine development. Immunol Rev 1999;170:197–222. Kaneko H, Bednarek I, Wierzbicki A, et al. Oral DNA vaccination promotes mucosal and systemic immune responses to HIV envelope glycoprotein. Virology 2000;267:8–16. McGhee JR, Mestecky J. In defense of mucosal surfaces: development of novel vaccines for IgA responses protective at the portals of entry of microbial pathogens. Infect Dis Clin N Am 1990;4:315–41. Mestecky J, Eldridge JH. Targeting and controlled release of antigens for the effective induction of secretory antibody response. Curr Opin Immunol 1991;3:492–5. Chen SC, Jones DH, Fynan EF, Farrar GH, Clegg JCS, Greenberg HB, et al. Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles. J Virol 1998;72:5757–61. Hedley ML, Curley J, Urban R. Microspheres containing plasmid-encoded antigens elicit cytotoxic T-cell responses. Nat Med 1998;4:365–8. Jones DH, Corris S, McDonald S, Clegg JC, Farrar GH. Poly(dl-lactide-co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration. Vaccine 1997;15:814–7. Bender BS, Rowe CA, Taylor SF, Wyatt LS, Moss B, Small AP. Oral immunization with a replication-deficient recombinant vaccinia virus protects mice against influenza. J Virol 1996;70:6418–24. Gherardi MM, Esteban M. Mucosal and systemic immune responses after oral delivery of vaccinia virus recombinants. Vaccine 1999;17:1074–83. Vetvicka V, Thornton BP, Wieman TJ, Ross GD. Targeting of natural killer cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and ␤-glucan-primed CR3 (CD11b/CD18). J Immunol 1997;159:599–605. Vetvicka V, Thornton BP, Ross G. Soluble ␤-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J Clin Invest 1996;98:50–61. Diller IC, Mankowski ZT, Fisher ME. The effect of yeast polysaccharides on mouse tumors. Cancer Res 1963;23:201–10. Di Luzio NR, Williams DL, McNamee RB, Edwards BF, Kitahama A. Comparative tumor-inhibitory and anti-bacterial activity of soluble and particulate glucan. Int J Cancer 1979;24:773–9. Thornton BP, Vetvicka V, Pitman M, Goldman RC, Ross GD. Analysis of the sugar specificity and molecular location of the ␤-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J Immunol 1996;156:1235–46. Borchers AT, Stern JS, Hackman RM, Keen CL, Gershwin ME. Mushrooms, tumors, and immunity. Proc Soc Exp Biol Med 1999;221:281–93. Hamuro J, Rollinghoff M, Wagner H. ␤(1–3)-glucan-mediated augmentation of alloreactive murine cytotoxic T-lymphocytes in vivo. Cancer Res 1978;38:3080–5.

A. Wierzbicki et al. / Vaccine 20 (2002) 1295–1307 [23] Chihara G. Lentinan and its related polysaccharides as host defense potentiators: their application to infectious diseases and cancer. In: Masihi KN, Lange W, editors. Immunotherapeutic prospects of infectious diseases. Berlin: Springer-Verlag, 1990. p. 9–18. [24] Kaneko Y, Chihara G. Potentiation of host resistance against microbial infections by lentinan and its related polysaccharides. In: Friedman H, Klein TW, Yamaguchi H, editors. Microbial infections. New York: Plenum Press, 1992. p. 201–15. [25] Matsuoka H, Seo Y, Wakasugi H, Saito T, Tomada H. Lentinan potentiates immunity and prolongs the survival time of some patients. Anticancer Res 1997;17:2751–5. [26] Taguchi T, Furue H, Kimura T, Kondo T, Hattori T, Ogawa N. Clinical efficacy of lentinan on neoplastic diseases. Adv Exp Med Biol 1983;166:181–7. [27] Kovacs JA, Vogel S, Albert JM, et al. Controlled trial of interleukin-2 infusions in patients with the human immunodeficiency virus. N Engl J Med 1996;335:1350–6. [28] Raz E, Watanabe A, Baird SM, et al. Systemic immunological effects of cytokine genes injected into skeletal muscle. Proc Natl Acad Sci USA 1993;90:4523–7. [29] Chow YH, Huang WL, Chi WK, Chu YD, Tao MH. Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2. J Virol 1997;71:169–78. [30] Barouch DH, Santra S, Steenbeke TD, et al. Augmentation and suppression of immune responses to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration. J Immunol 1998;161:1875– 82. [31] Landolfi NF. A chimeric IL-2/Ig molecule possesses the functional activity of both proteins. J Immunol 1991;146:915–9. [32] Barouch DH, Santra S, Schmitz JE, et al. Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Science 2000;290:486–92. [33] Suga T, Shiio T, Maeda YY, Chihara G. Antitumor activity of lentinan in murine syngeneic and autochthonous hosts and its suppressive effect on 3-methylcholanthrene-induced carcinogenesis. Cancer Res 1984;44:5132–7. [34] Earl PL, Koenig S, Moss B. Biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: analysis of proteins with truncations and deletions expressed by recombinant vaccinia viruses. J Virol 1991;65:331–41. [35] Mackett M, Smith GL, Moss B. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci USA 1982;79:7415–9. [36] Moss B. Genetically engineered poxviruses for recombinant gene expression, vaccination and safety. Proc Natl Acad Sci USA 1996;93:11341–8. [37] Felgner PL, Tsai YJ, Sukhu L, et al. Improved cationic lipid formulations for in vivo gene therapy. Annu NY Acad Sci 1995;772:126–39. [38] Gregoriadis G, Ryman BE. Fate of protein-containing liposomes injected into rats: an approach to the treatment of storage diseases. Eur J Biochem 1972;24:485–91. [39] Harokopakis E, Hajishengallis G, Michalek SM. Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system. Infect Immun 1998;66:4299–304.

1307

[40] Belyakov IM, Derby MA, Ahlers JD, et al. Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T-lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge. Proc Natl Acad Sci USA 1998;95:1709–14. [41] Staats HF, Nichols WG, Palker TJ. Mucosal immunity to HIV-1: systemic and vaginal antibody responses after intranasal immunization with the HIV-1 C4/V3 peptide T1SP10 MN(A). J Immunol 1996;157:462–72. [42] Takahashi H, Nakagawa Y, Leggatt GR, et al. Inactivation of human immunodeficiency virus (HIV)-1 envelope-specific CD8+ cytotoxic T-lymphocytes by free antigenic peptide: a self-veto mechanism? J Exp Med 1996;183:879–89. [43] Hwang SS, Boyle TJ, Lyerly HK. Cullen BR. Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science 1991;253:71–4. [44] Jagodzinski PP, Wustner J, Kmieciak D, et al. Role of the V2, V3, and CD4-binding domains of gp120 in curdlan sulfate neutralization sensitivity of HIV-1 during infection of T-lymphocytes. Virology 1996;226:217–27. [45] Dulbecco R. The nature of viruses. In: Dulbecco R, Ginsberg HS, editors. Virology. 2nd ed. London: Lippincott. 1988. p. 1–26. [46] Winer BJ. Design and analysis of factorial experiments. In: Maytham W, Shapiro A, Stern J, editors. Statistical principals in experimental design. New York: McGraw Hill. 1971. p. 224–51. [47] Herremans TM, Reimerink JH, Buisman AM, Kimman TG, Koopmans MP. Induction of mucosal immunity by inactivated poliovirus vaccine is dependent on previous mucosal contact with live virus. J Immunol 1999;162(8):5011–8. [48] Cerwenka A, Morgan TM, Harmsen AG, Dutton RW. Migration kinetics and final destination of type 1 and type 2 CD8 effector cells predict protection against pulmonary virus infection. J Exp Med 1999;189(2):423–34. [49] Hanke T, Samuel RV, Blanchard TJ, et al. Effective induction of simian immunodeficiency virus-specific cytotoxic T-lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen. J Virol 1999;73(9):7524–32. [50] Amara RR, Villinger F, Altman JD, et al. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 2001;292:69–74. [51] Xiang ZQ, Pasquini S, Ertl HCJ. Induction of genital immunity by DNA priming and intranasal booster immunization with a replication-defective adenoviral recombinant. J Immunol 1999;162:6716–23. [52] Ishii N, Fukushima J, Kaneko T, et al. Cationic liposomes are a strong adjuvant for a DNA vaccine of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 1997;13:1421–8. [53] Ross GD. Membrane complement receptors. In: Lachmann PJ, Peters DK, Rosen FS, Walport MJ, editors. Clinical Aspects of immunology. Oxford: Blackwell. 1993. p. 241–64. [54] Xia Y, Vetvicka V, Yan J, Hanikyrova M, Mayadas T, Ross GD. The ␤-glucan-binding lectin site of mouse CR3 (CD11/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells. J Immunol 1999;162:2281–90.