Induction of Systemic and Mucosal Cross-Clade Neutralizing Antibodies in BALB/c Mice Immunized with Human Immunodeficiency Virus Type 1 Clade A Virus-Like Particles Administered by Different Routes of Inoculation

We have recently developed a candidate human immunodeficiencyvirus type 1 (HIV-1) vaccine model, based on virus-like particles(VLPs) expressing gp120 from a Ugandan HIV-1 isolate of cladeA (HIV-VLP_As), which shows the induction of neutralizing antibodiesas well as cytotoxic T lymphocytes (CTL) in BALB/c mice by intraperitoneal(i.p.) administration. In the present study, immunization experimentsbased on a multiple-dose regimen have been performed with BALB/cmice to compare different routes of administration. i.p. andintranasal (i.n.), but not oral, administration induce systemicas well as mucosal (vaginal and intestinal) immunoglobulin G(IgG) and IgA responses. These immune sera exhibit >50% exvivo neutralizing activity against both autologous and heterologousprimary isolates. Furthermore, the administration of HIV-VLP_Asby the i.n. immunization route induces a specific CTL activity,although at lower efficiency than the i.p. route. The HIV-VLP_Asrepresent an efficient strategy to stimulate both arms of immunity;furthermore, the induction of specific humoral immunity at mucosalsites, which nowadays represent the main port of entry for HIV-1infection, is of great interest. All these properties, and thepossible cross-clade in vivo protection, could make these HIV-VLP_Asa good candidate for a mono- and multicomponent worldwide preventivevaccine approach not restricted to high-priority regions, suchas sub-Saharan countries.

INTRODUCTION

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Introduction

The development of an effective, safe, and affordable vaccinestrategy represents a crucial goal for both industrialized anddeveloping countries. In fact, although highly active antiretroviraltherapy induces dramatic reductions in both human immunodeficiencyvirus (HIV)-related morbidity and mortality, it fails to eradicatethe viral infection and selects resistant viral populationswhich cannot be controlled with alternative "salvage" strategies(21, 39).

Considering the discordant reports on the role of the humoraland cellular immune responses in the containment of HIV type1 (HIV-1) disease progression (5, 9, 34, 41, 45, 47), a preventiveHIV-1 vaccine should elicit both virus-specific neutralizingantibodies and cytotoxic T lymphocytes (CTLs). In this perspective,we have chosen a vaccine approach based on virus-like particles(VLPs), a model currently under investigation as a potentialvaccine for different human viruses, such as hepatitis viruses,papillomavirus, rotavirus, parvovirus, and Norwalk virus (25,30, 33, 36, 46, 53). The HIV-targeted VLPs (HIV-VLPs) are basedon the fact that the HIV-1 Pr55^gag precursor protein assemblesas immature, nonreplicating, noninfectious VLPs with effectiveinduction of both arms of the immune response (10, 16, 27, 29,51). Furthermore, HIV-VLPs can be employed to deliver additionalantigenic structures, such as whole proteins or specific individualepitopes; in particular, specific HIV-VLPs have been developedto present an entire gp120 molecule, anchored through the transmembraneportion of the Epstein-Barr virus gp220/350 (Env-Gag-hybridVLPs), and to increase the expression and stability of the glycoproteinon the surfaces of VLPs without affecting its oligomerization(10). The gp120 glycoprotein selected for these HIV-VLPs derivesfrom a Ugandan HIV-1 clade A isolate (11, 12), which representsthe second most prevalent HIV-1 subtype worldwide (approximately25%) and is predominant in many developing countries. TheseHIV-VLPs (HIV-VLP_As) show strong in vivo immunogenicity in BALB/cmice in the absence of adjuvants, and HIV-1-specific CTLs aswell as cross-clade neutralizing antibodies have been detectedin immunized animals (13).

The transmission of HIV-1 infection during heterosexual or homosexualintercourse accounts for as much as 80% of AIDS globally (59).Infection can involve both free and cell-associated HIV-1, which,for successful sexual transmission, engages in different strategiesto cross the mucosal barriers of both the intestinal and genitaltracts in order to infect CD4⁺ T cells (7, 31, 35, 49, 56).Mucosal secretory immunoglobulin A (sIgA) specific for HIV-1envelope glycoproteins is consistently detected in seropositivesubjects (3, 18) and has been strongly associated with protectionfrom HIV-1 infection in uninfected individuals having unprotectedsexual intercourse with HIV-1-seropositive partners (19, 32,38, 42).

Considering this epidemiological and experimental evidence,it seems reasonable to believe that specific mucosal immunityis extremely relevant for controlling the primary HIV-1 infection.In this perspective, the nasal route of immunization, betterthan the oral route, appears to elicit both mucosal and systemicimmunity with a limited induction of mucosal tolerance, whichmay compromise local vaccination with soluble antigens (8).These mucosal vaccination strategies, furthermore, would overcomethe major problems faced in developing countries related tothe necessity of disposable sterile syringes and needles, whichnot only result in higher costs but also, if not properly handled,may facilitate the spread of HIV-1 infection.

Several mucosal vaccination approaches have been developed inrecent years for human pathogens transmitted mainly throughthe mucosal system, including influenza A virus (57), Vibriocholerae (62), human papillomavirus (6), rotavirus (15), andherpes simplex virus type 2 (23). For HIV-1, in particular,a number of different mucosal vaccine strategies have been studied,including bacterial vectors (17, 28, 37, 50), proteins (2, 52),live-attenuated vectors (4, 26), inactivated HIV-1 (1, 20),VLPs (61), and naked DNA (40, 55).

We report here the induction of systemic and mucosal humoralresponses as well as specific CTL activity in mice after intraperitoneal(i.p.) as well as intranasal (i.n.) immunization with HIV-VLP_As,without any adjuvant. In contrast, in our model, neither a humoralnor a cellular immune response was obtained following oral administration,as previously shown for rotavirus and parvovirus VLPs (46, 54).Both systemic and mucosal antibody responses exhibit >50%ex vivo neutralization activity against autologous as well asheterologous primary isolates, indicating that the HIV-VLP representsan efficient approach to be employed alone or as part of a multicomponentvaccine strategy.

MATERIALS AND METHODS

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Materials and Methods

Production of recombinant baculovirus and HIV-VLP_As. Recombinant baculoviruses expressing both the subtype A gp120_UG-Epstein-Barrvirus transmembrane fusion gene and the Pr55^gag cDNA were producedas previously described (10). Briefly, the HIV-1 coding sequenceswere transferred in a single step, by site-specific transposition,from the pFastBac dual-transfer vector (Gibco-BRL) to the baculovirusDNA. The baculovirus DNA was obtained in a DH10Bac Escherichiacoli bacterial strain (Gibco-BRL) modified to propagate botha baculovirus shuttle vector (bacmid), which contains mini-attTn7attachment sites, and the pMON7124 helper plasmid, which providesthe Tn7 transposition functions in trans. The recombinant baculovirusDNA was used to transfect SF9 insect cells, and supernatantswere collected at 48 to 72 h posttransfection; different multiplicitiesof infection were then used to infect High Five cells and scaleup the production of VLPs. Particles were purified through acontinuous 10-to-60% sucrose gradient, and their productionwas confirmed by standard electron microscopy analysis (10).

Immunization experiments. Female BALB/c mice, each group consisting of three animals 6to 8 weeks of age, were administered 20 or 100 µg purifiedVLPs by the i.p., i.n., or oral route. Control mice were treatedwith endotoxin-free phosphate-buffered saline (PBS). The immunizationschedule was based on a 4-dose regimen, where the booster inoculationswere administered at weeks 3, 7, and 9 after the primary vaccinedose. All the immunization protocols were performed in two independentexperiments without addition of adjuvants.

(i) i.n. vaccination. Prior to inoculation, mice were anesthetized by halothane. Animalswere treated by placing 200 µl of inoculum, 50 µlat time, into alternating nostrils and allowing mice to inhale.Animals were monitored until consciousness was regained.

(ii) Oral vaccination. Mice were gavaged with 40 µl of 5% bicarbonate bufferimmediately prior to oral inoculation with VLPs, in order toneutralize stomach acidity. Each mouse was then gavaged withVLPs in a final volume of 200 µl. No adverse signs werenoted in any mice receiving i.n. or oral inoculation.

Sample collection and processing. (i) Serum samples. Blood samples were collected from each animal 1 week after eachvaccination dose by puncture of the retro-orbital vein. Afterthe last dose, the entire blood volume was collected by tailbleeding; serum was obtained by standard methods and storedat –80°C until use.

(ii) Vaginal secretions. Vaginal secretions were collected from each animal 1 week aftereach vaccination dose by pipetting 50 µl of PBS in andout of the vagina gently until a discrete clump of mucus wasrecovered. This usually took four to eight cycles of pipettingand required cutting the pipette tip back to a diameter of 1to 2 mm. A second vaginal wash with 50 µl of PBS was thendone to ensure more complete recovery of the vaginal secretions,and this material was combined with the first wash. Vaginalwashes were centrifuged at 12,000 x g for 10 min shortly aftercollection to separate the mucus from the PBS wash solution.The mucus and supernatant were then frozen separately at –80°C.The PBS wash solution contained a cocktail of proteinase inhibitors(153.8 nM aprotinin, 3.2 µM bestatin, and 10 µMleupeptin). In order to obtain complete recovery of sIgA andpresumably also IgG from the vaginal mucus, samples were thawed,weighed, and extracted twice for 2 h each time in 100 µlof PBS per sample, with rotation at 20 rpm in a 12-ml polystyrenetube at 4°C. The two extracts and the original wash supernatantwere pooled, made up to 300 µl per sample, and frozenat –80°C until needed (48).

(iii) Fecal pellets. Fecal antibody samples were collected from each mouse 1 weekafter each vaccination dose. Fecal pellets were resuspendedat a 10% (wt/vol) concentration in a stool diluent (10 mM Tris,pH 7.5, 100 mM NaCl, 1 mM CaCl₂, 0.05% Tween 20, 5 mM sodiumazide, supplemented with 1 mg of aprotinin/ml and 10 mg of leupeptin/ml).After incubation on ice for 20 min, suspensions were centrifugedat 18,000 x g for 15 min in order to remove fecal solids. Processedfecal antibody samples were stored at –80°C.

Serological assays. The presence and the titers of IgG and IgA antibodies, specificfor the native conformational HIV-VLP_As used in the immunizationprotocols as well as for the Env and Gag epitopes, were evaluatedin samples obtained from immunized mice by an enzyme-linkedimmunosorbent assay (ELISA). Ninety-six-well MICROTEST assayplates (Becton Dickinson) were coated with 2.5 µg of HIV-VLP_As;alternatively, plates were coated either with 0.5 µg ofthe gp120-Env V3 loop peptide (CTRPYNNTRQSTHIGPGQALYTTNIIGDIRQAHC)(catalog no. EVA7017.1), representing the consensus sequenceof Ugandan clade A HIV-1 strains, or with 0.5 µg of 20-merpeptides with a 10-amino-acid overlap (derived from the HIV-SF2isolate) spanning the entire p24^gag protein (catalog no. ARP788.1-22).Both the V3 loop and p24 peptides were obtained through theNIBSC Centralised Facility for AIDS Reagents (http://www.nibsc.ac.uk/catalog/aids-reagent/catalog_2002).

Antigens were allowed to adhere to the plates by incubationat +4°C overnight, and nonspecific protein binding to theplates was blocked by incubation with 5% (wt/vol) dry milk inPBS for 1 h at room temperature (RT). Diluted mouse sera ormucosal fluids were added to the wells and incubated for 1 hat RT. After three washes, wells were treated for 1 h at RTwith peroxidase-conjugated goat anti-mouse IgG or IgA; afterthree additional washes, the reaction was visualized with 0.075%4-chloro-1-naphthol in 0.056% hydrogen peroxide and stoppedwith 2 N sulfuric acid. Absorbance was determined at 492 nm,and reactions were considered positive when the mean absorbancefor immunized animals exceeded the mean absorbance of equaldilutions of sera collected from control animals by a factorof 3. Serum ELISA results are expressed as the log₁₀ geometricmean titer of the last positive dilution point from the animalsof each set in the two independent experiments. Results forantibodies in vaginal or fecal samples are expressed as theoptical density (OD) obtained with 1:5-diluted fluids (54).

HIV-1 neutralization assay. The neutralization assay was performed using as a target theU87 human glioma cell line transfected with the CCR5 chemokinereceptors (14). The autologous clade A Ugandan and heterologousclade B Italian field isolates, used for the assays, are HIV-1primary isolates identified in asymptomatic, recently seroconvertedindividuals by coculture with fresh peripheral blood mononuclearcells and frozen down with no additional passages on lymphocytecell lines (11). Primary isolates replicate on CCR5-expressingU87 cells, and they were titrated in these cells for the exvivo neutralization assays. No signs of replication are detectableon CD4- or CD4/CXCR4-expressing U87 cells.

U87 target cells were seeded in 96-well plates at a densityof 1 x 10⁴ cells/well. On the following day, 10 50% tissue cultureinfective doses (TCID₅₀) of both viruses were mixed with serialdilutions of serum (1:20 to 1:80) or with mucosal fluids (1:5)and incubated for 1 h at 37°C. As controls, the viruseswere either not treated or mixed with matched serum dilutionsfrom a preimmunized animal. The mixture was added to well-adherentCCR5-expressing U87 cells in triplicate and incubated overnight.After three washes with phosphate-buffered saline, 200 µlof complete medium was added per well and cultures were incubatedfor 7 days. The levels of HIV-1 p24 antigen in the supernatantswere determined at days 4 and 7 postinfection (p.i.) with acommercially available kit (NEN DuPont); the percentage of neutralizationwas calculated relative to a dilution-matched control. Cellviability was microscopically verified during incubation andat the end of the incubation period in all wells. The test wasscored positive when, on day 7 p.i., the p24 concentration was $≥$ 130 pg/ml in control wells and the tested serum dilution exhibiteda >50% neutralization effect.

CTL assay. Single-cell suspensions of spleens were obtained from immunizedand control mice and cocultured in the presence of interleukin-2(5 IU/ml) for 7 to 14 days with irradiated Env- or Gag-expressingsyngeneic splenocytes (stimulators) in order to propagate specificeffector T cells. In particular, stimulator cells were inducedfor 24 h with 5 µg/ml of phytohemagglutinin (PHA; Sigma),infected with 1 PFU/10³ cells of Env- or Gag-recombinant vacciniavirus, and, after an additional 16 to 24 h, ${gamma}$ -irradiated with2,000 rads. For cytolytic assays, target syngeneic H-2^d P815cells were infected with 1 PFU/10³ cells of either Gag- or Env-vacciniavirus 16 to 24 h prior to the start of the assay. Both Gag-and Env-vaccinia viruses were obtained from the NIBSC CentralisedFacility for AIDS Reagents; the gag sequence derives from theclade B HIV-1_BH10 isolate (14), and the env sequence derivesfrom a Ugandan clade A HIV-1 isolate (24). Infected target cellswere pulsed for 1 h at 37°C with ⁵¹Cr (Amersham PharmaciaBiotech) (30 µCi/10⁶ cells) and washed three times withculture medium before the addition of effector cells. Specificlysis was measured in a triplicate assay performed with 5 x10³ target cells mixed with different ratios of effector cells(effector to target cell [E:T] ratio, 25:1 to 3.125:1) in atotal volume of 200 µl. After a 5-h incubation at 37°C,100 µl culture supernatant was collected for countingthe amount of ⁵¹Cr release. Positive and negative controls,in the absence of effector cells, were treated with or without1 N HCl, respectively. The percentage of specific lysis wascalculated as follows: 100 x [(specific release – spontaneousrelease)/(total release – spontaneous release)].

Statistical analyses. Intergroup comparisons were performed with the Mann-WhitneyU test (for univariate nonparametric group analysis). All Pvalues were two tailed and considered significant if less than0.05.

RESULTS

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Results

Induction of serum antibody response by HIV-VLP_As injected by different routes. The humoral immune efficacy of HIV-VLP_As, produced in insectcells and purified on a 10-to-60% continuous sucrose gradient(10), was evaluated for 18 BALB/c mice, divided into six groups,by following a 4-dose immunization schedule and comparing differentroutes of administration. In particular, one animal group wastreated with an i.p. injection of 20 µg purified VLPs,which has been shown previously to induce specific antibodyas well as CTL responses (13); two animal groups received i.n.administration of 20 or 100 µg purified VLPs; two animalgroups received oral administration of 20 or 100 µg purifiedVLPs; and the control group was i.p. injected with PBS alone.At weeks 3, 7, and 9, each group was treated with the same amountof antigen received in the primary vaccination dose, and a weeklater, blood samples were drawn from the retro-orbital vein.A second experiment with the same group distribution and modalitywas later performed independently, starting 2 weeks after theend of the first experiment. No animals showed any signs oftoxicity, and all remained healthy up to the end of the vaccinationprotocol.

In order to evaluate specific HIV-VLP_A-induced serum antibodytiters, ELISAs were performed on microwell plates coated withHIV-VLP_As. The data showed that serum IgG titers increased aftereach vaccination dose administered by the i.p. or i.n. route(up to 1:81,920 for the i.p. and 1:10,240 for the i.n. route),while the oral route did not induce a detectable antibody titer(Fig. 1A). The i.p. immunization protocol induced statisticallysignificantly higher systemic IgG antibody titers (P < 0.001)than both i.n. immunization does (20 and 100 µg) (Fig.1A) but failed to induce detectable IgA titers (Fig. 1B). IgAantibody titers (1:250, on average), instead, were detectedin sera of mice immunized by the i.n. route, and no statisticallysignificant difference was observed between the two HIV-VLP_Asdoses (20 and 100 µg) (P > 0.5) (Fig. 1B).

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FIG. 1. Levels of specific humoral response induced in mice immunized with HIV-VLP_As. Geometric mean titers of specific serum antibodies induced in each BALB/c mouse group by administration of different doses of HIV-VLP_As via parenteral and mucosal routes in two independent experiments. At 3, 7, and 9 weeks following the primary immunization, each group was administered the same amount of antigen, and a week later, blood samples were drawn from the retro-orbital vein. Serum samples were analyzed for specific IgG (A) and IgA (B) antibodies by ELISA, performed in triplicate on 96-well plates coated with 2.5 µg of HIV-VLP_As. In each panel, results are expressed as the individual average reciprocal last dilution with an OD at 492 nm threefold that of the preimmune sera. Data from the two independent experiments, with three mice per group per experiment, are indicated by open and full symbols. Bars represent the cumulative mean of each group ± the standard deviation. P values are indicated for intergroup comparisons.

ELISAs performed on microwell plates coated with HIV-1 peptidesconfirmed the same antibody titers for Gag-specific epitopesbut showed 1- to 2-dilution-lower titers for Env-specific epitopes.Reactions were considered positive when the absorbance at 492nm exceeded the average absorbance of equal dilutions of seracollected from control animals by a factor of 3 (data not shown).Within each group, the standard deviations of the antibody responseswere always <10% of the mean value. In addition, a standarddeviation <10% was observed between the same groups in thetwo independent experiments performed.

Induction of mucosal antibody responses by HIV-VLP_As injected by different routes. In order to evaluate the antibody response at the mucosal level,vaginal washes and fecal pellets were collected from each animala week after each HIV-VLP_A vaccination dose and evaluated inan ELISA system. Given the low antibody titers at mucosal sitesreported by several authors (54, 48, 60), ELISAs were performedat a single dilution, 1:5, and results were expressed as ODvalues (54). Reactions were considered positive when the absorbanceat 492 nm exceeded the average absorbance of samples from controlanimals by a factor of 2. The results showed that, at the intestinallevel, the strongest IgG response was induced by the i.p. immunizationroute (P < 0.001), whereas the IgA response was equally inducedby the i.p and i.n. routes (P > 0.05) (Fig. 2A). In contrast,at the vaginal level, the 20-µg dose administered by thei.n. route induced the strongest IgG and IgA responses, comparedto both the i.p. route and the 100-µg i.n. dose (P <0.001) (Fig. 2B). Consistent with the systemic results, oraladministration of HIV-VLP_As did not result in a detectable antibodyresponse at either the intestinal or the vaginal level.

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FIG. 2. Levels of specific antibodies in the feces and vaginal washes of mice immunized with HIV-VLP_As. At 3, 7, and 9 weeks following the primary immunization, each group of mice was administered the same amount of antigen, and a week later, fecal samples and vaginal washes were obtained for each animal. Levels of specific IgG and IgA antibodies in the feces (A and B) and in the vaginal washes (C and D) were evaluated by ELISA, performed in triplicate on 96-well plates coated with 2.5 µg of HIV-VLP_As. In each panel, results are represented as the average OD at 492 nm observed in individual animals at a 1:5 dilution of mucosal samples. Data from the two independent experiments, with three mice per group per experiment, are indicated by open and full symbols. Reactions were considered positive when the absorbance exceeded the average absorbance of samples from control animals by a factor of 2. Bars represent the cumulative mean of each group ± the standard deviation. P values are indicated for intergroup comparisons.

Also for the mucosal antibody response, the standard deviationswithin each group were always <10% of the mean value, anda standard deviation <10% was observed between the same groupsin the two independent experiments performed.

Neutralization activity against autologous and heterologous primary field isolates. The neutralization activities of antibodies induced by HIV-VLP_Asin mice immunized via different routes were verified ex vivoagainst primary field isolates characterized in our laboratory:the autologous Ugandan clade A isolate, whose gp120 was expressedon the HIV-VLP_As (10), and a heterologous clade B strain identifiedfrom an Italian intravenous drug user. The results show thatimmune sera derived from mice immunized via the i.p. or i.n.route are able to inhibit the replication of both autologousand heterologous isolates in CCR5-expressing U87 cells, up toa final dilution of 1:40 (i.p.) or 1:20 (i.n.). In particular,such serum dilutions show an average neutralization capacity>75% against the autologous isolate for the entire periodof observation. In contrast, the next dilutions (1:80 i.p. and1:40 i.n.) show a transient 55% neutralization on day 4 p.i.which is no longer detectable on day 7 p.i. (Fig. 3A and B).In parallel, the neutralizing serum dilutions are able to inhibitthe replication kinetics of the heterologous clade B primaryisolate, showing an average 64% neutralization on days 4 and7 p.i. (Fig. 3C and D). Furthermore, moderate neutralizing activity(approximately 57%) against both autologous and heterologousprimary isolates was detected, at a 1:5 dilution, in both vaginaland intestinal fluids from mice immunized by the i.n. route(Fig. 4A to D) and only in vaginal fluids from mice immunizedby the i.p. route (Fig. 4B and D). In all the neutralizationassays, the mean standard deviation of the samples' triplicatecultures, in both control and immunized groups, was always lowerthan 5% of the mean value. The mean standard deviation of thecombined results from the corresponding groups of the two independentexperiments was always lower than 10% of the mean value.

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FIG. 3. Autologous and heterologous neutralization efficiencies of sera from mice immunized with HIV-VLP_As. The HIV-1 neutralization assay was performed on CCR5-expressing U87 cells against autologous and heterologous primary isolates, clade A Ugandan and clade B Italian HIV-1 isolates, respectively. Ten TCID₅₀ of each isolate were pretreated with serial dilutions of the immune sera, collected at the end of the complete immunization schedule, from the mouse groups immunized with 20 µg of HIV-VLP_As via the i.p. or the i.n. route. The neutralization activities of serum antibodies, at different dilutions, induced via the i.p. (A and C) or the i.n. (B and D) route are represented as percentages of the virus replication in control samples (vertical bars). Fifty percent neutralization has been considered the lowest limit for scoring the test positive. In all the panels the kinetics of virus growth during the 7-day neutralization assay is shown (lines). Each serum dilution was evaluated in triplicate, and the reported results include the cumulative data from two independent experiments with three mice per group per experiment.

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FIG. 4. Autologous and heterologous neutralization efficiencies of mucosal antibodies from mice immunized with HIV-VLP_As. The HIV-1 neutralization assay was performed on CCR5-expressing U87 cells against autologous and heterologous primary isolates, clade A Ugandan and clade B Italian HIV-1 isolates, respectively. Ten TCID₅₀ of each isolate were pretreated with a 1:5 dilution of fecal (A and C) or vaginal (B and D) extracts, collected from the mouse groups immunized with 20 µg of HIV-VLP_As via the i.p. or the i.n. route. Neutralization activity is represented as a percentage of the virus replication in control samples (vertical bars). Fifty percent neutralization has been considered the lowest limit for scoring the test positive. In all the panels the kinetics of virus growth during the 7-day neutralization assay is shown (lines). Each dilution was evaluated in triplicate, and the reported results include the cumulative data from two independent experiments with three mice per group per experiment.

Both primary field isolates, treated with preimmune sera oruntreated, showed similar effective kinetics of replicationin the 7-day-p.i. experiment, characterized by a progressiveincrease in virus production (by measurement of p24 or reversetranscriptase activity) and a final p24 concentration $≥$ 130 pg/ml.

CTL response to HIV-1 epitopes. To assess the induction of Env- and Gag-specific CTLs, spleenswere removed from immunized as well as control mice. Purifiedsplenocytes (effector cells) were restimulated ex vivo for 2weeks with ${gamma}$ -irradiated autologous splenocytes infected witheither Env- or Gag-vaccinia virus recombinants. The vacciniavirus recombinants express the env sequence derived from a Ugandanclade A HIV-1 isolate (44) and the gag sequence from the cladeB HIV-1_BH10 isolate (24). The cytotoxic activities of effectorswere then evaluated in a ⁵¹Cr release assay against syngeneic(H-2^d) P815 cells (targets) infected with the same Env- or Gag-vacciniavirus recombinants by using an E:T ratio ranging from 25:1 to3.125:1. As previously reported (13), a 30 to 35% CTL responsewas detected at the E:T ratio of 25:1 in each paired experimentperformed with splenocytes from animals immunized via the i.p.route. Significantly lower CTL activity (16 to 20%) (P <0.001) was detected at the same E:T ratio when the experimentwas performed with splenocytes from animals immunized via thei.n. route. No CTL activity was induced by oral administrationof HIV-VLP_As (Fig. 5A and B). The HIV-1 Env and Gag epitopeswere used independently for both the ex vivo restimulation andthe lysis assay. In contrast, <5% lysis was observed whenthe effectors were restimulated ex vivo with Env and mixed withGag-expressing targets (and vice versa), indicating that thereaction was specifically driven ex vivo by antigen-restimulatedCTL clones (data not shown). The correlation between the percentageof observed lysis and the E:T ratio was approximately linearwithin the range analyzed. As expected, no cytotoxic activitywas detected against parental P815 cells. Each end point foreach animal was evaluated in triplicate, and the standard deviationwas always lower than 5% of the mean value. The standard deviationof the mean was always lower than 8% in each experimental groupof three animals and 10% between the two corresponding groupsof the two independent experiments.

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FIG. 5. Cytotoxic T-cell activation (CTL analysis) in mice immunized with HIV-VLP_As. CTL response was evaluated in splenocytes from mice immunized with HIV-VLP_As. Splenocyte cultures were restimulated in vitro with ${gamma}$ -irradiated splenocytes infected with either Env (A)- or Gag (B)-expressing vaccinia virus recombinants. Effector cells were then assayed for lysis of P815 target cells presenting either HIV-1 gp160_A or Gag_B epitopes, at an E:T ratio ranging from 25:1 to 3.125:1. Parental P815 cells were used as a negative control in both experiments. Specific cell lysis was measured as ⁵¹Cr release and expressed as the percentage of total lysis from acid-disrupted target cells. Data are means of triplicate cultures from all animals included in the two independent experiments with three mice per group per experiment.

DISCUSSION

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Discussion

It has been reported previously that a candidate HIV-1 preventive/therapeuticvaccine recently developed in our laboratory, based on VLPsexpressing a gp120 glycoprotein derived from an HIV-1 cladeA isolate (HIV-VLP_As) (10, 12), shows strong in vivo immunogenicityin BALB/c mice and induces both a CTL response and cross-cladeneutralizing antibodies, in the absence of adjuvants (13).

In the present study, it is shown that the recombinant HIV-VLP_As,administered by the i.p. or i.n. route in the absence of adjuvants,stimulate IgA and/or IgG antibody responses in sera as wellas at vaginal and intestinal mucosal sites. Moreover, as alreadyreported for the i.p. route, the i.n. immunization route alsoprovides a way to induce CTL responses, although to a much lowerextent.

An immunization protocol based on a 4-dose regimen was performedin parallel on four animal groups with two different doses ofHIV-VLP_As (20 and 100 µg, respectively) administered bythe i.n. or oral route. A group was immunized under standardconditions (20 µg by the i.p. route) (13), and the controlgroup was i.p. injected with PBS alone. The booster doses elicitedincreasing serum levels of specific IgG antibodies in animalsimmunized by the i.p. or i.n. route, while IgA antibodies wereinduced only by the i.n. route of administration. The i.n. 20-µgdose appeared to be optimal, given that a fivefold-higher dose(100 µg) does not correlate with a higher antibody titer,probably due to a tolerance effect. Local production of specificantibodies was demonstrated at the intestinal and vaginal levels,showing that the i.p. administration route is able to stimulatean IgG response preferentially, whereas the i.n. administrationroute induces both IgG and IgA responses. In the present study,oral administration of HIV-VLP_As failed to stimulate systemicas well as mucosal immune responses. In this regard, althoughmice were gavaged with bicarbonate buffer immediately priorto each oral inoculation with VLPs, this treatment could havebeen insufficient to neutralize stomach acidity, resulting insignificant antigen degradation.

Immune sera were tested for ex vivo neutralization activityagainst primary field isolates, the autologous Ugandan clade-Aisolate and a heterologous Italian clade B isolate, using theCR-expressing U87 human glioma cell line as a target (14). Serafrom mice immunized with 20 µg of HIV-VLP_As by the i.p.route showed neutralization activity against both the autologousand heterologous field isolates at a final dilution of 1:40.Equivalent neutralization activity against both HIV-1 fieldisolates was obtained also with a 1:20 dilution of sera fromanimals immunized by the i.n. administration route. Furthermore,moderate neutralizing activity (approximately 57%) against bothautologous and heterologous primary isolates was detected invaginal and intestinal fluids from mice immunized by the i.n.route and only in intestinal samples from mice immunized bythe i.p. route. The breadth of this cross-clade neutralizationactivity is currently being evaluated using an expanded panelof primary HIV-1 isolates of different subtypes. The neutralizationefficacy against HIV-1 primary isolates observed in this studyis extremely significant, considering the poor neutralizationactivity against primary isolates in comparison to T-cell-adaptedstrains consistently reported in the literature (43, 58), andstrongly suggests the potential applications of the VPL as avaccine approach in countries characterized by multiclade HIV-1epidemics.

Furthermore, the immunization of mice with HIV-VLP_As by thei.p. and, to a lesser extent, the i.n. administration routewas able to induce in vivo CD8⁺ CTLs with specific cytolyticactivity against target cells expressing either Env or Gag moleculesderived from HIV-1 isolates of clade A or B, respectively.

All the results were obtained in two independent immunizationexperiments, where the standard deviation was between 5 and10% of the mean value, and the standard deviation of the meanwas <5% of the mean value, indicating significant consistencyof the independent observations.

The data confirm that these HIV-VLP_As are able to induce a stronghumoral as well as a cellular immune response, likely due toefficient presentation in a major histocompatibility complexclass II context with a resulting stimulation of T-helper-cellresponses, as previously reported (13). In particular, the inductionof immune responses in mice immunized by the i.n. administrationroute, without adjuvants, shows that nonreplicating VLPs areimmunologically more effective than monomeric proteins or linearpeptides, probably due to their particulate structure (6, 30,54). Most proteins administered alone by mucosal routes, infact, induce poor if measurable immune responses, and only afew natural antigens, including bacterial toxins such as choleratoxin or E. coli labile toxin, consistently stimulate strongmucosal responses (22). Furthermore, the lower serum antibodytiters and CTL activity detected in animals immunized by thei.n. route compared to those in animals immunized by the i.p.route, as well as the lack of HIV-VLP_A immunogenicity followingoral administration, suggest the need to optimize the immunizationprotocols. This goal will be pursued by exploring prime/booststrategies (i.e., DNA/HIV-VLP) as well as introducing nontoxicadjuvants in order to enhance the immunogenicity of mucosalimmunizations and reduce the dose administered, which shouldalso result in lower potential toxicity.

The results obtained with our HIV-VLP_As, in particular the inductionof antibody responses with ex vivo cross-clade neutralizationactivity, suggest a possible broad in vivo protection whichwould make this strategy a good candidate for a mono- as wellas a multicomponent worldwide vaccine approach not limited tosub-Saharan countries with high incidences of HIV-1 clade Aisolates.

ACKNOWLEDGMENTS

Recombinant vaccinia virus, expressing gp160 of HIV-1 cladeA (EVA 289.1-4) and Pr55^gag of HIV-1 clade B (ARP 253), thegp120 V3 peptide (EVA7017.1), and overlapping peptides spanningthe entire p24^gag protein (ARP788.1-22) were obtained throughthe NIBSC Centralised Facility for AIDS Reagents, supportedby EU Program EVA (contract BMH4 97/2515) and the UK MedicalResearch Council. The original donors of the reagents were M.Esteban (EVA 289.1-4) and D. Nixon (ARP 253). We thank JohnMcKnight for revising the English style of the manuscript.

This study was supported by grants from the Ministero Italianodella Sanità (Ricerca Corrente and Progetto FinalizzatoAIDS 2000) and the ICSC-World Lab, Lausanne, Switzerland (projectMCD-2/7).

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Viral Oncology and AIDS Reference Center, Istituto Nazionale Tumori "Fond. G. Pascale," Via Mariano Semmola, 1, 80131 Naples, Italy. Phone: 39-081-5903.830. Fax: 39-081-5451276. E-mail: irccsvir{at}unina.it.

REFERENCES

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