Intranasal Vaccination with Recombinant Adeno-Asso
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病菌学杂志 2006年第6期
Infection and Cancer Programme, German Cancer Research Center, Heidelberg, Germany
ABSTRACT
Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy in many preclinical studies, as well as in clinical trials. However, only a few approaches have used recombinant AAV (rAAV) to deliver vaccine antigens. We generated an rAAV encoding the major capsid protein L1 (L1h) from the human papillomavirus type 16 (HPV16), aiming to develop a prophylactic vaccine against HPV16 infections, which are the major cause of cervical cancer in women worldwide. A single dose of rAAV5 L1h administered intranasally was sufficient to induce high titers of L1-specific serum antibodies, as well as mucosal antibodies in vaginal washes. Seroconversion was maintained for at least 1 year. In addition, a cellular immune response was still detectable 60 weeks after immunization. Furthermore, lyophilized rAAV5 L1h successfully evoked a systemic and mucosal immune response in mice. These data clearly show the efficacy of a single-dose intranasal immunization against HPV16 based on the recombinant rAAV5L1h vector without the need of an adjuvant.
INTRODUCTION
Genital infections with human papillomaviruses (HPV) are among the most common viral sexually transmitted infections in humans. It has been estimated that at least 50% of sexually active adults had a genital HPV infection. More than 120 different genotypes have been described thus far, 15 of which (i.e., HPV type 16 [HPV16] and HPV18) were identified as causative agents of at least 90% of cancers of the cervix and were also linked to more than the half of other anogenital cancers. Cervical cancer is the second most frequent malignant tumor in women worldwide (for a review, see reference 74).
Several vaccination models against HPV have been evaluated aiming to generate neutralizing antibodies. The degree of protection is directly proportional to the amount of neutralizing antibodies detected at the virus entry site and protection lasts as long as neutralizing antibodies persist. Currently, a plethora of immunization agents such as virus-like particles (VLP) (2, 3, 29, 39-41), recombinant fusion proteins (13, 32, 36) and peptides (15, 37, 38), live recombinant bacteria (4, 54), recombinant viruses (43, 49), or naked DNA (42, 61) are being scrutinized for vaccination purposes.
Among the viral based vaccines, recombinant adeno-associated virus (rAAV) emerged as a promising candidate. AAV was mainly used to amend genetic and acquired human diseases such as cystic fibrosis, hemophilia, muscular dystrophy or diabetes mellitus (for a review, see references 22, 30, and 66). In particular, its high clinical safety record in humans, the absence of significant inflammation upon gene delivery, the broad tissue tropism, the ability to infect dividing and quiescent cells, and the long-term expression are attractive properties of this vector system.
Despite reports that AAV induces only weak immune responses against the vector and the expressed transgene in gene therapy approaches (6, 64), there is evidence that rAAV vectors also are efficient in genetic vaccination (63). The induction of both cellular and humoral immune responses against several antigens administered by different routes has been reported (9, 45, 71). Recently, a rAAV2 vector expressing the human immunodeficiency virus type 1 env gene was orally administered and shown to induce systemic and regional immunity (70). A rAAV2 vaccine encoding simian immunodeficiency virus (SIV) elicited protective SIV-specific T cells and antibodies in macaques after a single intramuscular dose (35). An AAV vector-based system was also used for vaccination against HPV infections. A rAAV2 encoding HPV16 E7 fused to a heat shock protein was administered in a therapeutic approach, leading to a specific cellular immune response (44). In a recent study a prophylactic vaccination approach against HPV infections was investigated. The intramuscular application of a rAAV2 vaccine encoding the capsid protein L1 from HPV16, together with a recombinant Adenovirus encoding murine granulocyte-macrophage colony-stimulating factor, led to the induction of neutralizing L1 antibodies (43). All of these studies combined have been performed with AAV2 vectors. It has been estimated that up to 80% of all humans are seropositive for AAV2 and that a significant portion among them carry neutralizing antibodies against AAV2, which may prevent an efficient AAV2-based therapy (7, 16, 20, 47, 62; for reviews, see references 11 and 63). Different AAV serotypes may overcome these limitations due to their ability to evade the immune response established against, i.e., AAV2 (for a review, see reference 22). Thus far, 11 different serotypes isolated from primate sources have been described, which revealed an interesting difference in cellular tropisms and transduction efficiency (19, 21, 50). Antibodies to AAV2 are prevalent in the human population; thus, one has to use a heterologous AAV for gene transfer. AAV5 was chosen due to its reported high ability to infect airway epithelia (1, 68, 72).
Here, we present the results of intranasal vaccinations of female C57BL/6 mice using an AAV2-based vector construct containing the codon-optimized major capsid gene L1 (L1h) from HPV16 under the control of the cytomegalovirus immediate-early promoter (42) pseudotyped into AAV5 capsids (rAAV5 L1h). In a previous study we demonstrated that the use of codon-optimized L1 is the prerequisite for efficient DNA immunization (42). Our results show that rAAV5 L1h induced both long-lasting humoral and cellular immune responses against HPV16 L1 after a single intranasal application. Furthermore, lyophilized rAAV5 L1h successfully induced a systemic and mucosal immune response in mice.
MATERIALS AND METHODS
Animals, cell lines, and cell culture. For all animal experiments 4- to 6-week-old female C57BL/6 (H-2b) mice were used. Mice were purchased from Charles River Wiga (Sulzfeld, Germany) and kept in an isolator at the animal facilities of the DKFZ. Mice were housed in accordance to the institutional guidelines.
HeLa and 293T cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal-calf serum, 100 U of penicillin/ml, and 100 μg of streptomycin/ml at 37°C in 5% CO2.
Vector production and purification. AAV vector production was carried out in 293T cells by calcium phosphate transfection as previously described (23). Briefly, 107 293T seeded in 150-mm dishes (Nunc) were cotransfected with the packaging plasmid (pDP5) and the vector plasmid UF2 green fluorescent protein (Gfp) or UF3 L1h. Cells were incubated for 48 h at 37°C and 5% CO2. Cells were collected, dispersed in TBSM buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2 [pH 8.0]), and disrupted by three freeze-thaw cycles using liquid nitrogen. The tissue debris was removed by centrifugation (2,000 x g, 20 min, 4°C), and the supernatant was digested with Benzonase (50 U/ml; Sigma, Germany, Taufkirchen) for 30 min at 37°C.
Fractionation was performed on CsCl density gradients. Freeze-thaw lysates were adjusted with CsCl to a refraction index of = 1.37 and underlaid with 0.5 ml of a 1.5-g/ml CsCl solution. Samples were centrifuged in a Kontron TST65.13 rotor for 24 h at 300,000 x g at 20°C in 12.5-ml Quickseal tubes. One-milliliter fractions of the gradients were collected from the bottom of the tube. The AAV-containing fractions were detected by dot blot Western analysis. For this purpose, 50 μl of each fraction was added to 50 μl of 2x sodium dodecyl sulfate (SDS) loading buffer (2 mM EDTA, 100 mM Tris-HCl [pH 8.0], 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue), heated for 5 min at 95°C, and transferred onto a nitrocellulose membrane. The membrane was blocked for 1 h in phosphate-buffered saline (PBS) containing 6% skimmed milk powder and then incubated for 1 h at room temperature with antibody B1 (69) (diluted 1:10 in 6% skim milk). The membranes were washed subsequently in PBS for 30 min at room temperature and incubated with the secondary peroxidase-coupled goat anti-mouse antibody (Dianova, Hamburg, Germany; diluted 1:5,000 in PBS). The AAV capsid proteins were detected and visualized by using an enhanced chemiluminescence detection kit from NEB (Frankfurt, Germany). Fractions containing full rAAV particles were pooled, and a second CsCl centrifugation was performed under the same conditions. The rAAV-containing fractions were pooled and dialyzed against PBS overnight at room temperature.
The remaining HPV16 L1 protein in the vector stocks was removed by an immunodepletion step. Hence, 60 mg of total immunoglobulin G (IgG), isolated via protein A-Sepharose from a polyclonal rabbit serum raised against native and denatured HPV16 L1 VLP, was coupled to NHS-activated Sepharose (Amersham, Braunschweig, Germany) according to the manufacturer's protocol. The matrix was equilibrated and incubated with the virus stock for 1 h at room temperature under agitation. The virus-containing flowthrough was collected, the bound L1 protein was eluted with 10 matrix volumes of 3 M KSCN (Sigma, Taufkirchen, Germany), and the matrix was re-equilibrated by washes with 10 volumes of PBS. This depletion step was repeated at least three times until all L1 material was removed. AAV5 Gfp vector stocks were depleted once. Finally, the virus stock was concentrated in a Vivaspin 20 (Vivascience, Gottingen, Germany) concentrator according to the manufactures protocol to a titer of 5 x 1013 genome-containing particles (gp)/ml for AAV5 L1h and 5 x 1012 gp/ml for AAV5 Gfp. Samples were stored frozen at –80°C. Vector stocks were frozen in liquid nitrogen and lyophilized overnight. Lyophilized samples were stored at room temperature and reconstituted with an appropriate volume of distilled water prior to administration.
Vector titration. Vector genome titers were determined by DNA dot blot hybridization as previously reported (24). Alternatively, vector genome titers were determined by taking the average of three quantitative real-time PCR determinations described formerly (67). A cytomegalovirus enhancer DNA fragment of 135 bp was amplified, and the plasmid pTRUF2 was used as a standard in serial 10-fold dilutions in the range of 101 to 107 copies. The primers used were as follows: forward primer, 5'-TGCCCAGTACATGACCTTATGG-3'; reverse primer, 5'-GGAAATCCCCGTGAGTCAAAC-3'; and probe, Fam-MGB probe 5'-AGTCATCGCTATTACCATGG-3'. All materials for the quantitative real-time PCR were purchased form Applied Biosystems. The PCR conditions were 2 min at 50°C (destruction of contaminating PCR product by AmpErase) and 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. PCR samples were analyzed by ABI Prism 7700 sequence detection system and SDS 2.1 software.
Analysis of vector transduction in vitro. In vitro transduction activities were assessed by infecting HeLa cells for 1 h in serum-free medium with each vector (103 gp/cell). Subsequently, the medium was supplemented with an equal volume DMEM-20% fetal calf serum containing adenovirus 5 (multiplicity of infection [MOI] = 10). Infected cells were incubated for 48 h at 37°C and 5% CO2, harvested, washed with PBS, and then lysed in 2x SDS loading buffer by heating to 95°C for 5 min. Aliquots of equal cell equivalents were subjected to Western blotting onto nitrocellulose membranes. Expressed L1 protein was detected as described below with Camvir-1.
Western blot analysis. For immunodetection of AAV capsid proteins or HPV16 L1 protein, equal aliquots of vector stocks were subjected to Western blot analysis. AAV capsid proteins were detected by incubation for 1 h at room temperature with the polyclonal rabbit serum VP51 diluted 1:200 in PBS-1% bovine serum albumin (BSA) (23). For the detection of HPV16 L1 protein, membranes were incubated for 1 h at room temperature with the L1-specific monoclonal antibody Camvir-1 (BD Bioscience, Heidelberg, Germany) (48) diluted 1:4,000 in PBS-1% BSA. Secondary peroxidase-coupled goat anti-rabbit and goat anti-mouse antibodies (Dianova, Hamburg, Germany), respectively, were diluted 1:5,000 in PBS-1% BSA. Membranes were washed, and the antibody reaction was visualized by using an enhanced chemiluminescence detection kit (NEB, Frankfurt, Germany).
Immunization of mice. Baculovirus-derived HPV16 L1 VLP were purified as described previously (52) and diluted with PBS to a final concentration of 5 μg/10 μl. Capsomeres were obtained as described elsewhere (55) and diluted to a concentration of 10 μg/10 μl. For intranasal vaccination, female mice were anesthetized by intraperitoneal injection with 100 μl each of both 0.2% xylazine hydrochloride (Rompun; Bayer) in PBS and 10 μg of ketamine hydrochloride per ml (Ketavet; Parke-Davis) per 10 g of body weight. An inoculum of 10 μl (viral vector, VLP, or capsomeres) was instilled dropwise into one nostril. Blood samples were taken by retro-orbital puncture, and vaginal washes were obtained by gently pipetting 300 μl of PBS up and down with protease inhibitors (Complete, Mini, EDTA-free; Roche, Germany) with a blunt-tipped Pasteur pipette. Samples were centrifuged for 5 min at 10,000 x g to pellet cellular material, and the antibody titer was determined in an enzyme-linked immunosorbent assay (ELISA). All samples were stored at –20°C.
ELISA techniques. For endpoint titration assays of sera and vaginal washes obtained from immunized mice, a VLP-based ELISA was carried out for the detection of HPV16 L1-specific antibodies described previously (12). VLP were produced and purified according to the method of Müller et al. (52). A peroxidase-coupled goat anti-mouse IgG (-chain specific) and an anti-mouse IgA (Southern Biotechnology, Birmingham, MA) were used as secondary antibodies. Specific IgG titers are expressed as the reciprocal of the highest dilution that yielded an optical density at 450 nm four times that of control mice. Extinction at 450 nm was measured after 5 to 20 min in a Titertek automated plate reader.
To verify the absence of assembled HPV16 L1 protein in the vector stocks, an antigen capture ELISA was carried out with antibody 1.3 specific for HPV16 L1 VLP and capsomeres as described before (52). HPV16 L1 VLP were used in a serial twofold dilution beginning with 50 ng of total protein titrated to 0.78 ng of protein as a standard.
AAV-based ELISA. Endpoint titration assays were performed to detect AAV5-specific antibodies. Microtiter plates were coated overnight with 109 empty particles of AAV5/well. Plates were blocked (5% skim milk in PBS for 1 h at 37°C), and mouse sera or vaginal washes were added in serial twofold dilutions starting with a 1:160 to 1:20,480 dilution or with a 1:2 to 1:265 dilution, respectively, for the vaginal washes, and incubated for 1 h at 37°C. Nonspecific binding was determined by using the same dilutions on plates coated with PBS only. Plates were washed and horseradish peroxidase-conjugated goat anti-mouse IgG antibody (-chain specific; Southern Biotechnology, Birmingham, AL) was added at a 1:5,000 dilution in PBS. Plates were incubated for 1 h at 37°C, washed, and stained with TMB (3,3',5,5'-tetramethylbenzidine) substrate solution (Sigma). Plates were measured, and specific IgG titers were expressed as described above.
AAV empty capsid production. At least 10 150-mm dishes (Nunc) were seeded with 5 x 106 293T cells per dish and incubated at 37°C and 5% CO2 overnight. Cells were transfected with 40 μg of the packaging plasmid (pDP5)/dish. Cells were harvested in TBSM buffer at 48 h posttransfection, subjected to repeated freeze-thaw cycles, and digested with 50 U of Benzonase/ml for 30 min at 37°C. Tissue debris was removed by centrifugation at 2,000 x g for 20 min at 4°C. The supernatant was fractionated on a sucrose cushion by underlaying the supernatant with 450 μl of 30% sucrose (in Tris-EDTA) followed by 450 μl of 50% sucrose in Tris-EDTA. The gradients were centrifuged at 273,000 x g for 2.5 h at 4°C by using an SW60 rotor (Beckman, Germany). The pellet was resuspended in TBSM buffer and purified on a second sucrose cushion. The resulting pellet was resuspended in an appropriate volume of TBSM buffer and then analyzed by electron microscopy.
IFN--enzyme-linked immunospot assay (ELISPOT) assay. MultiScreen IP sterile plates (96 well; Millipore, Eschborn, Germany) were presoaked with 70% ethanol for 1 min, and the ethanol was removed by extensive rinsing with PBS. The plates were coated with 200 ng of anti-mouse gamma interferon (IFN-) capture antibody (clone R4-6A2; BD Pharmingen, Heidelberg, Germany) in 100 μl of PBS overnight at 4°C. Unbound antibody was removed by washing twice with sterile Milli-Q water. Plates were blocked for 2 h with 100 μl of medium (RPMI, 10% fetal calf serum antibiotics) at 37°C, and splenocytes were seeded in triplicates in serial dilutions ranging from 200,000 to 25,000 cells per well in 100 μl of medium. For each triplicate, splenocytes in one well were left untreated (background control), cells in the next well were stimulated with 200 ng of pokeweed mitogen (Sigma) in 20 μl of medium (positive control), and cells in the last well were treated with 30 μM L1165-173 peptide in 20 μl of medium (test samples). Plates were incubated for 18 to 20 h at 37°C. Cells were removed by six washes with PBS-0.01% Tween 20. Then, 20 ng of sterile-filtered biotinylated anti-mouse IFN- detection antibody (clone XMG1.2; BD Pharmingen) in 100 μl of PBS-0.5% BSA was added per well, and the plates were kept at 4°C overnight. The plates were washed six times with PBS-0.01% Tween 20, and this was followed by the addition of 100 μl of a 1:1,000 dilution of streptavidin-alkaline phosphatase (BD Pharmingen) in PBS. Plates were incubated for 45 min at room temperature and then washed three times with PBS-0.01% Tween 20, followed by three washing steps with PBS alone. Plates were developed for 2 to 20 min with 100 μl of 5-bromo-4-chloro-3-indolylphosphate (BCIP/Nitro Blue Tetrazolium Liquid Substrate System; Sigma). The reaction was stopped by rinsing the plates with water. Spots were quantified by using an ELISPOT reader (ELISPOT Reader System ELR02; AID GmbH, Strasbourg, Germany). Wells with medium alone and no splenocytes were assayed in parallel as negative controls. Counts of the background control wells were subtracted from the samples.
Tissue distribution analysis of vector DNA. Mice were sacrificed, and the following tissues were excised and immediately frozen in liquid nitrogen: heart, liver, kidney, and lung. Genomic DNA was extracted from approximately 10 to 25 mg of each tissue by using the DNeasy Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and then eluted from the supplied columns in 100 μl of the provided elution buffer. Total DNA concentrations were quantified by spectrophotometry (Ultraspec III; Pharmacia).
PCR with a L1-specific probe was carried out with the forward primer 5'-CCCATCAAGAAGCCCAACAAC-3' and the reverse primer 5'-GGGGCTTGCAGCCGATCAGG-3', yielding a 339-bp PCR product. PCR conditions were 2 min at 95°C, followed by 30 cycles of 30 s at 95°C and 30 s at 62°C and elongation for 1 min at 72°C, with a final elongation for 5 min at 72°C. Samples were analyzed by agarose gel electrophoresis.
Preparation of papillomavirus pseudovirions and neutralization assay. Presence of neutralizing antibodies in sera of immunized animals was determined by using a protocol adapted from Buck et al. (10). Briefly, pseudovirions were prepared by transfecting 7 x 106 293T cells (cultivated in DMEM containing 50 μg of hygromycin/ml) with a plasmid encoding for the humanized HPV16 L1 and L2 genes, together with a plasmid containing the gene for secreted alkaline phosphatase under the control of the hCMV promoter. After 4 days, cells were harvested by using trypsin and washed with PBS. Cells were then adjusted to 5 x 107 per ml and lysed by 0.5% Brij58 in the presence of Benzonase (2,000 U/ml) for 5 min on ice. After the NaCl concentration was increased to 850 mM, the cells were centrifuged and the cleared supernatant containing the pseudovirions was used for infection of 293T cells. For infection, pseudovirion stocks were diluted 1:5,000 in complete medium and preincubated with the sera (1:50 dilution) for 15 min at room temperature. Pseudovirions were then added to the cells, followed by incubation at 37°C for 4 days. Secreted alkaline phosphatase activity in the supernatant was measured by using a commercial assay (Roche, Mannheim, Germany) according to the manufacturer's recommendations.
RESULTS
Characterization of the vector stocks. We sought here to develop an AAV vector for efficient induction of humoral immune responses against HPV16 infection after intranasal immunization. Therefore, the codon-optimized HPV16 L1 gene (L1h) introduced into an AAV2 vector (42) was pseudotyped into AAV5 capsids (Fig. 1a). A vector expressing the Gfp gene was generated as a negative control. Crude vector preparations were prepared as described before (23) and fractionated by two successive CsCl density centrifugations. Western blot analysis revealed that the vector containing fractions were still contaminated with HPV16 L1 protein generated during rAAV5 L1h production in 293T cells (data not shown). Hence, we depleted the vector preparations from residual L1 protein by immunoaffinity purification of matrix-immobilized IgG of a polyclonal rabbit serum raised against native and denatured HPV16 L1 VLP.
Western blot analysis of 5 x 1011 gp of a purified vector preparation using the AAV-specific polyclonal rabbit serum VP51 (23) and Camvir-1 (48) verified the presence of intact viral capsid proteins and demonstrated that it was free of detectable HPV16 L1 protein (Fig. 1b and c, respectively). The vector stock was further analyzed by an antigen capture-ELISA based on the monoclonal antibody 1.3 (52). No traceable amounts of L1 were found in the rAAV5 L1h vector stock. Spiking experiments revealed that the detection limit for this assay was 0.8 ng of VLP present in 5 x 1011 gp (data not shown). Transduction of HeLa cells with the rAAV5 L1h vector and subsequent Western blot analysis with an anti-L1 antibody (Camvir-1) demonstrated that the vector was functional, expressing an L1 protein with the expected size (Fig. 1d).
Intranasally delivered rAAV5 L1h evokes systemic and mucosal immune responses in mice. The intention of the present study was to establish an efficient and easy-to-apply vaccination model. Three groups of C57BL/6 mice (labeled as single, double, and triple; a total of 10 mice per group in two independent experiments) were immunized intranasally with one, two (second at day 7), or three (third at day 42) doses of 5 x 1010 gp. In addition, one group (rAAV5 L1h lyo) of mice was inoculated with three doses (5 x 1010 gp/dose) of the lyophilized rAAV5 L1h vector, and another group (rAAV5 L1h high) received three doses of 5 x 1011 gp/dose. As a negative control group, mice were inoculated three times with PBS alone or with rAAV5 Gfp (5 x 1010 gp/dose), while three immunizations with L1-VLP (5 μg/dose) or L1-capsomeres (10 μg/dose) served as positive controls. At 3 months after the first immunization, sera and vaginal washes were collected and screened for the presence of L1-specific antibodies by using a VLP-based ELISA. The vaccination trial was repeated in two independent experiments. Figure 2a shows the representative results for serum IgG of one experiment, and in Table 1 the results of both experiments are summarized.
Mice treated with VLP or capsomeres alone (9 of 10 or 5 of 5 animals, respectively) had L1-specific serum antibodies (Fig. 2a and Table 1). All sera of mice immunized with rAAV5 L1h also developed L1-specific serum antibodies. Serum titers of mice inoculated with the lyophilized vector were lower but still significantly (P = 0.02) above the background obtained from sera of mice vaccinated with PBS or rAAV5 Gfp. In one experiment the highest L1-specific serum titer was measured in the mice that had received 5 x 1011 gp/dose of rAAV5 L1h (Fig. 2a). In both experiments combined highest L1-specific titers were achieved in the rAAV5 L1h triple groups (5 x 1010 gp/dose) (Table 1). Neutralization assays using pseudovirions with secreted alkaline phosphatase as a reporter demonstrated that all tested antibody-positive sera were also neutralizing (Fig. 2b). In conclusion, a successful protective seroconversion was achieved after intranasal application of rAAV5 L1h vectors.
The immune response at the viral entry site is a crucial factor in establishing protective immunity against HPV. Therefore, vaginal washes were collected and analyzed for the presence of L1-specific mucosal immunoglobulins. Three of five mice inoculated with capsomeres developed L1-specific IgG antibodies in the vaginal washes, whereas within the VLP-treated group only two of ten mice revealed mucosal antibodies (Table 1). Also, the groups treated with rAAV5 L1h responded with the formation of mucosal antibodies in the vaginal washes. However, only the group with the highest vector dose showed a reaction in all animals. Antibody titers in the vaginal washes corresponded to the serum titers of the different groups. We also found L1-specific IgA antibodies but, similar to findings presented by others in humans (59) and mice (3), the levels of these antibodies were lower but consistent with the mucosal IgG titers (Table 1).
In conclusion, the results show that the intranasal delivery of the rAAV5 L1h-based vector efficiently induces L1-specific antibodies both in serum and in vaginal washes. The presence of L1-specific mucosal antibodies in the vaginal washes represents a prerequisite to preventing HPV16 infections at the viral entry site.
rAAV5 L1h induce long-lasting L1-specific humoral immunity. In order to monitor the persistence of the humoral response over time, sera were taken repeatedly for 60 weeks after the first immunization. The single vaccine dose (rAAV5 L1h single) administered initially was sufficient to induce and maintain high serum antibody titers for this prolonged period of time (Fig. 3). The highest antibody titers were found in mice which had received two doses (rAAV5 L1h double). Titers were significantly higher compared to the titers of the group receiving a single dose (P = 0.03). However, the difference in titers compared to the group that received three doses was not significant (P = 0.07). This may reflect the effect of an AAV-specific immune response already established at the time of third immunization, preventing a further boosting against the L1 protein (Fig. 3). In contrast, the anti-L1 titer in mice that had received three doses of VLP had dropped below the detection limit. The sera of the negative control mice treated with PBS or rAAV5 Gfp (not shown) remained negative throughout the experiment. In summary, vaccination with AAV5 L1h induces a long-lasting seroconversion even after a single administration.
L1-specific cellular immune responses are present 60 weeks after vaccination. Cellular immune responses are pivotal for the elimination of virus-infected cells. The HPV16 L1-specific lymphocytes were evaluated at the completion of the vaccination experiment (60 weeks after the first immunization). Splenocytes from mice immunized with rAAV5 L1h (single, double, and triple), as well as from VLP-inoculated mice, were isolated, and the frequency of IFN--producing L1-specific lymphocytes was analyzed ex vivo by ELISPOT (Fig. 4). The mice that had received VLP had no measurable levels of L1-specific lymphocytes. In contrast, all mice treated with the rAAV5 L1h vector, regardless of the number of doses, revealed a notable fraction of IFN--producing L1-specific lymphocytes. The amounts of specific lymphocytes were significantly higher in the rAAV5L1h single (P = 0.049), double (P = 0.03), and triple (0.03) group compared to the VLP-treated mice. This clearly demonstrates the usefulness of an AAV-based vaccine to induce and maintain a long-term, specific cellular immune response against the delivered transgene.
Vaccination with rAAV5 elicits vector-directed humoral antibodies. Immune responses raised against the delivery system itself may inhibit further vaccinations. An AAV-based ELISA assessed the immune responses that were induced against the AAV vectors. After 3 months the sera of all mice immunized with the AAV vectors revealed high titers of AAV-specific antibodies (Fig. 5a). No remarkable differences in antibody titers were measured in mice receiving single, double, or triple doses. The group immunized with a 10-fold-greater vector dose (rAAV5 L1h high) revealed also higher AAV-specific titers (Fig. 5a). AAV-specific antibodies were already detectable after 42 days (data not shown), which may have partially neutralized the third immunization dose in some experiments. This may explain the L1-specific antibody levels found in mice after 60 weeks. The immunogenicity of the AAV vector was also demonstrated by the specific mucosal immune responses present in the groups of mice treated with the rAAV5 vectors (Fig. 5b). Mice that had received PBS, VLP, or capsomeres were negative (Fig. 5b).
The presence of antibodies raised against a specific AAV serotype can influence the success of a repeated administration of the same or other serotype-based vectors (26, 28, 46, 51, 56). Therefore, groups of five mice were intranasally immunized with empty capsids of AAV2, AAV4, or AAV5. After 6 weeks, seroconversion against the respective serotype was confirmed by an AAV-based ELISA (data not shown), and mice were inoculated intranasally with a single dose of rAAV5 L1h (5 x 1010 gp). Six weeks later, sera were screened for L1-specific antibodies by using a VLP-based ELISA (Fig. 5c). Mice seropositive for AAV2 or AAV4 could be immunized successfully by administration of rAAV5 L1h vectors, eliciting an L1-specific humoral immune response, whereas readministration of the same AAV5 serotype failed to induce L1-specific antibodies. In contrast, in mice treated with rAAV5 Gfp 60 weeks previously, readministration of rAAV5 L1h induced successfully L1-specific antibodies both in serum and in mucosa (data not shown). This may be linked to the presumably reduced AAV-specific antibody titers after 60 weeks. In conclusion, administration of an AAV5-based vector in mice that are seropositive for AAV2 or AAV4 is possible; however, a readministration of vector to mice with preexisting antibodies against AAV5 is only possible if the AAV5 titer drops below a protective level.
Recombinant vector DNA is detected in lung tissues. The distribution of viral vectors in the body after application is one major biosafety concern. Hence, several tissues were examined for the presence of L1h vector DNA after intranasal rAAV5 L1h administration. Genomic DNA from liver, heart, kidney, and lung tissue was extracted from five individual mice of each group (see above) sacrificed 3 months after the first intranasal immunization. The presence of vector DNA in these tissues was assessed by PCR with a L1-specific primer resulting in a 339-bp fragment. The detection limit was in the range of 0.5 pg of vector DNA. The samples prepared from liver, heart, and kidney tissue of all mice from each group revealed no vector DNA. AAV vector DNA could only be found in DNA probe from the lung samples of the rAAV5 L1h high group (Fig. 6). This clearly indicates that the intranasal application of an rAAV prevents the systemic spread of vector DNA that occurred after intravenous administration (25) and represents a further safety benefit of mucosal AAV-based vaccination.
DISCUSSION
The vast majority of pathogenic infections occur at and progress from mucosal surfaces by colonizing or penetrating through the mucosa of the gastrointestinal, respiratory, or genital tract. In these cases, the local application of a vaccine is generally more efficient to induce a protective immune response, since systemic vaccination does not usually increase the mucosal antibody levels (59). However, in practice it has often proven to be rather difficult to stimulate strong mucosal immune responses by oral-mucosal administration of antigens; thus, the parenteral route of immunization is still the standard route of vaccination. Indeed, relatively few of the current vaccines that are approved for human use are administered mucosally. However, for reasons of simplicity, the two most attractive routes of vaccination in humans would be the oral and nasal application routes. Intranasal vaccination has emerged as the optimal vaccination strategy in rodents for the induction of antibody responses in genital tissues (3, 34, 54, 65) and was also shown to be successful in humans (5, 14, 17, 31, 33, 34, 41, 60). Mucosal antibodies are crucial in the protection against sexually transmitted infections and form the first line of defense. Among the most common viral sexually transmitted diseases are genital HPV infections (for a review, see reference 8).
Prevention and control of cervical cancer on a global basis is most easily envisaged through vaccine-mediated prevention of HPV infection and/or elimination of persistent infection at sites of high risk for development of cervical cancer. A suitable way to induce protective neutralizing antibodies in vaginal secretions of rodents and humans against applied antigens was shown by intranasal vaccination (3, 5, 14, 31, 33, 34, 54, 60, 65). Additional important benefits of intranasal application are that it is "needle-less," simple, and noninvasive; this is especially important in light of the high prevalence of HPV infections in developing countries. Different approaches for prophylactic and therapeutic immunizations against HPV are currently being developed, mostly based upon VLP. We intended to establish an alternative genetic vaccination model to the systems currently being investigated. Genetic vaccinations are supposed to mediate a long-term expression linked to a long-lasting immunity.
The development of new generations of vaccines and the delivery of peptides and proteins in general has been impeded by a lack of appropriate delivery systems. Here, we demonstrate the efficacy of an rAAV5-based vaccine, encoding the codon-optimized major capsid gene L1h of HPV16, administered intranasally to female C57BL/6 mice. A single dose of rAAV5 L1h induced a strong humoral and cellular immune response against HPV16 L1; both responses were maintained for more than 1 year. In particular, the presence of an ex vivo detectable L1-directed specific cellular immune response after 60 weeks measured by IFN- production on a single-cell level raises the hope for a successful therapeutic application of this vector system. L1-specific mucosal antibodies were also detected in the vaginal washes of mice, suggesting the presence of a protective immune response against HPV16 infections. It is noteworthy that both IgG and IgA were detected in the vaginal washes, but the IgG responses dominated over the IgA response, which was also reported after oral or nasal vaccination of humans with cholera vaccine (59). We have no information about the transport of IgG into the vaginal lumen. Receptor-mediated transcytosis has been reported to occur, but transudation is another likely mechanism (58). In general, the distribution of antibody classes varies on the different mucosal surfaces in humans, i.e., IgA is the major isotype in the gut, whereas IgA and IgG are found in comparable amounts in nasal and vaginal secretions (57).
AAV5-specific immune responses in mice, both in serum and mucosa, were observed 42 days after the first inoculation. Potential reapplication of the vaccine can be prevented if a protective immunity against the carrier system is established (22, 26-28). We assume that the third immunization dose given at day 42 may already be neutralized (in some experiments) by the antibodies developed against AAV5, leading to the lower level of L1-specific serum antibodies detected after 60 weeks. In addition, application of the third dose may boost an AAV5-specific cellular immune response eliminating transduced cells. Thus, preimmunized mice with AAV5 could not be successfully vaccinated by readministration of rAAV5 L1h, but mice immunized with rAAV5 Gfp vectors 60 weeks ago could be readministered successfully. This finding strongly indicates the presence of a stronger AAV5 directed immune response established after 42 days that may have declined with time to a level which permits the successful readministration of rAAV5 L1h (1). In contrast, a preexisting immune response against AAV2, which has a high prevalence in humans (7, 11, 16, 20, 47, 62), as well as against AAV4, had no impeding influence on rAAV5 L1h-based immunizations. The use of different serotype combinations for repeated administration is extensively investigated and was shown to bypass acquired immunity against a certain AAV serotype (for a review, see reference 22). Vaccination of mice with AAV4 L1h was also able to induce both humoral and mucosal immune responses (unpublished observation), but vaccination or readministration with AAV4 L1h seemed to be unlikely for application in humans due to the inefficient transduction of human cells by AAV4-based vectors (23).
The detection of vector DNA only in lung tissue in vaccinated mice and the lack of vector spread in tissues other than the lung are coincident with prior findings in rabbits after airway application of AAV vector. Several other rabbit tissues were screened for recombinant vector DNA; however, it was found mainly in the airway surface epithelium (18). Moreover, the presence of vector DNA in the lung tissue may elucidate the long-lasting L1 antibody titers presumably resulting from persisting transgene expression. Further, the local distribution may also support the biosafety specifications for vaccines, especially with regard to the concerns of AAV vector integration in the host genome (53).
Recently, covaccination of an intramuscularly applied AAV2 L1 together with an adenovirus encoding murine granulocyte-macrophage colony-stimulating factor was reported to elicit a strong and prolonged immune response (43). However, previous observations showed that gene expression of the original HPV16 L1 sequence was hardly detectable after transfections (42). Further, it could be assumed that contaminating L1 protein present in the vector stock was the potential immunogenic agent. Liu et al. did not control their study for this possibility. Our own experience has proven that all AAV L1h vector stocks contained high amounts of L1 protein—presumably as VLP—even after density gradient purification. This made it always difficult to assign the immune response specifically to either the L1 protein or the rAAV.
In addition to the usage of AAV vectors in many applications of gene therapy, AAV was also shown to be successful in oral and nasal vaccinations of mice against human immunodeficiency virus (70, 71), herpes simplex virus (45), and Alzheimer's disease (73). We present here a novel mucosal application of rAAV5 for the vaccination against HPV. The possibility to lyophilize the recombinant vector renders the maintenance of expensive cold-chain storage unnecessary. Further advantages of this vector system include the noninvasive application route of an adjuvant-independent vector eliciting a long-lasting humoral and cellular immune response. Given these properties, recombinant HPV L1-rAAV may be suitable for use in developing countries provided that the manufacturing costs per vaccination dose can be reduced.
ACKNOWLEDGMENTS
We thank Corinna Klein for help with the ELISPOT assay, Petra Galmbacher for the L1-VLP production, Martin Friedel and Werner Nicklas from the DKFZ animal facility, and Ute Koch for critically reading the manuscript. We especially thank Harald zur Hausen for his continuous support.
The project was supported by a grant from the Deutsche Krebshilfe (10-1912-Kl I).
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ABSTRACT
Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy in many preclinical studies, as well as in clinical trials. However, only a few approaches have used recombinant AAV (rAAV) to deliver vaccine antigens. We generated an rAAV encoding the major capsid protein L1 (L1h) from the human papillomavirus type 16 (HPV16), aiming to develop a prophylactic vaccine against HPV16 infections, which are the major cause of cervical cancer in women worldwide. A single dose of rAAV5 L1h administered intranasally was sufficient to induce high titers of L1-specific serum antibodies, as well as mucosal antibodies in vaginal washes. Seroconversion was maintained for at least 1 year. In addition, a cellular immune response was still detectable 60 weeks after immunization. Furthermore, lyophilized rAAV5 L1h successfully evoked a systemic and mucosal immune response in mice. These data clearly show the efficacy of a single-dose intranasal immunization against HPV16 based on the recombinant rAAV5L1h vector without the need of an adjuvant.
INTRODUCTION
Genital infections with human papillomaviruses (HPV) are among the most common viral sexually transmitted infections in humans. It has been estimated that at least 50% of sexually active adults had a genital HPV infection. More than 120 different genotypes have been described thus far, 15 of which (i.e., HPV type 16 [HPV16] and HPV18) were identified as causative agents of at least 90% of cancers of the cervix and were also linked to more than the half of other anogenital cancers. Cervical cancer is the second most frequent malignant tumor in women worldwide (for a review, see reference 74).
Several vaccination models against HPV have been evaluated aiming to generate neutralizing antibodies. The degree of protection is directly proportional to the amount of neutralizing antibodies detected at the virus entry site and protection lasts as long as neutralizing antibodies persist. Currently, a plethora of immunization agents such as virus-like particles (VLP) (2, 3, 29, 39-41), recombinant fusion proteins (13, 32, 36) and peptides (15, 37, 38), live recombinant bacteria (4, 54), recombinant viruses (43, 49), or naked DNA (42, 61) are being scrutinized for vaccination purposes.
Among the viral based vaccines, recombinant adeno-associated virus (rAAV) emerged as a promising candidate. AAV was mainly used to amend genetic and acquired human diseases such as cystic fibrosis, hemophilia, muscular dystrophy or diabetes mellitus (for a review, see references 22, 30, and 66). In particular, its high clinical safety record in humans, the absence of significant inflammation upon gene delivery, the broad tissue tropism, the ability to infect dividing and quiescent cells, and the long-term expression are attractive properties of this vector system.
Despite reports that AAV induces only weak immune responses against the vector and the expressed transgene in gene therapy approaches (6, 64), there is evidence that rAAV vectors also are efficient in genetic vaccination (63). The induction of both cellular and humoral immune responses against several antigens administered by different routes has been reported (9, 45, 71). Recently, a rAAV2 vector expressing the human immunodeficiency virus type 1 env gene was orally administered and shown to induce systemic and regional immunity (70). A rAAV2 vaccine encoding simian immunodeficiency virus (SIV) elicited protective SIV-specific T cells and antibodies in macaques after a single intramuscular dose (35). An AAV vector-based system was also used for vaccination against HPV infections. A rAAV2 encoding HPV16 E7 fused to a heat shock protein was administered in a therapeutic approach, leading to a specific cellular immune response (44). In a recent study a prophylactic vaccination approach against HPV infections was investigated. The intramuscular application of a rAAV2 vaccine encoding the capsid protein L1 from HPV16, together with a recombinant Adenovirus encoding murine granulocyte-macrophage colony-stimulating factor, led to the induction of neutralizing L1 antibodies (43). All of these studies combined have been performed with AAV2 vectors. It has been estimated that up to 80% of all humans are seropositive for AAV2 and that a significant portion among them carry neutralizing antibodies against AAV2, which may prevent an efficient AAV2-based therapy (7, 16, 20, 47, 62; for reviews, see references 11 and 63). Different AAV serotypes may overcome these limitations due to their ability to evade the immune response established against, i.e., AAV2 (for a review, see reference 22). Thus far, 11 different serotypes isolated from primate sources have been described, which revealed an interesting difference in cellular tropisms and transduction efficiency (19, 21, 50). Antibodies to AAV2 are prevalent in the human population; thus, one has to use a heterologous AAV for gene transfer. AAV5 was chosen due to its reported high ability to infect airway epithelia (1, 68, 72).
Here, we present the results of intranasal vaccinations of female C57BL/6 mice using an AAV2-based vector construct containing the codon-optimized major capsid gene L1 (L1h) from HPV16 under the control of the cytomegalovirus immediate-early promoter (42) pseudotyped into AAV5 capsids (rAAV5 L1h). In a previous study we demonstrated that the use of codon-optimized L1 is the prerequisite for efficient DNA immunization (42). Our results show that rAAV5 L1h induced both long-lasting humoral and cellular immune responses against HPV16 L1 after a single intranasal application. Furthermore, lyophilized rAAV5 L1h successfully induced a systemic and mucosal immune response in mice.
MATERIALS AND METHODS
Animals, cell lines, and cell culture. For all animal experiments 4- to 6-week-old female C57BL/6 (H-2b) mice were used. Mice were purchased from Charles River Wiga (Sulzfeld, Germany) and kept in an isolator at the animal facilities of the DKFZ. Mice were housed in accordance to the institutional guidelines.
HeLa and 293T cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal-calf serum, 100 U of penicillin/ml, and 100 μg of streptomycin/ml at 37°C in 5% CO2.
Vector production and purification. AAV vector production was carried out in 293T cells by calcium phosphate transfection as previously described (23). Briefly, 107 293T seeded in 150-mm dishes (Nunc) were cotransfected with the packaging plasmid (pDP5) and the vector plasmid UF2 green fluorescent protein (Gfp) or UF3 L1h. Cells were incubated for 48 h at 37°C and 5% CO2. Cells were collected, dispersed in TBSM buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2 [pH 8.0]), and disrupted by three freeze-thaw cycles using liquid nitrogen. The tissue debris was removed by centrifugation (2,000 x g, 20 min, 4°C), and the supernatant was digested with Benzonase (50 U/ml; Sigma, Germany, Taufkirchen) for 30 min at 37°C.
Fractionation was performed on CsCl density gradients. Freeze-thaw lysates were adjusted with CsCl to a refraction index of = 1.37 and underlaid with 0.5 ml of a 1.5-g/ml CsCl solution. Samples were centrifuged in a Kontron TST65.13 rotor for 24 h at 300,000 x g at 20°C in 12.5-ml Quickseal tubes. One-milliliter fractions of the gradients were collected from the bottom of the tube. The AAV-containing fractions were detected by dot blot Western analysis. For this purpose, 50 μl of each fraction was added to 50 μl of 2x sodium dodecyl sulfate (SDS) loading buffer (2 mM EDTA, 100 mM Tris-HCl [pH 8.0], 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue), heated for 5 min at 95°C, and transferred onto a nitrocellulose membrane. The membrane was blocked for 1 h in phosphate-buffered saline (PBS) containing 6% skimmed milk powder and then incubated for 1 h at room temperature with antibody B1 (69) (diluted 1:10 in 6% skim milk). The membranes were washed subsequently in PBS for 30 min at room temperature and incubated with the secondary peroxidase-coupled goat anti-mouse antibody (Dianova, Hamburg, Germany; diluted 1:5,000 in PBS). The AAV capsid proteins were detected and visualized by using an enhanced chemiluminescence detection kit from NEB (Frankfurt, Germany). Fractions containing full rAAV particles were pooled, and a second CsCl centrifugation was performed under the same conditions. The rAAV-containing fractions were pooled and dialyzed against PBS overnight at room temperature.
The remaining HPV16 L1 protein in the vector stocks was removed by an immunodepletion step. Hence, 60 mg of total immunoglobulin G (IgG), isolated via protein A-Sepharose from a polyclonal rabbit serum raised against native and denatured HPV16 L1 VLP, was coupled to NHS-activated Sepharose (Amersham, Braunschweig, Germany) according to the manufacturer's protocol. The matrix was equilibrated and incubated with the virus stock for 1 h at room temperature under agitation. The virus-containing flowthrough was collected, the bound L1 protein was eluted with 10 matrix volumes of 3 M KSCN (Sigma, Taufkirchen, Germany), and the matrix was re-equilibrated by washes with 10 volumes of PBS. This depletion step was repeated at least three times until all L1 material was removed. AAV5 Gfp vector stocks were depleted once. Finally, the virus stock was concentrated in a Vivaspin 20 (Vivascience, Gottingen, Germany) concentrator according to the manufactures protocol to a titer of 5 x 1013 genome-containing particles (gp)/ml for AAV5 L1h and 5 x 1012 gp/ml for AAV5 Gfp. Samples were stored frozen at –80°C. Vector stocks were frozen in liquid nitrogen and lyophilized overnight. Lyophilized samples were stored at room temperature and reconstituted with an appropriate volume of distilled water prior to administration.
Vector titration. Vector genome titers were determined by DNA dot blot hybridization as previously reported (24). Alternatively, vector genome titers were determined by taking the average of three quantitative real-time PCR determinations described formerly (67). A cytomegalovirus enhancer DNA fragment of 135 bp was amplified, and the plasmid pTRUF2 was used as a standard in serial 10-fold dilutions in the range of 101 to 107 copies. The primers used were as follows: forward primer, 5'-TGCCCAGTACATGACCTTATGG-3'; reverse primer, 5'-GGAAATCCCCGTGAGTCAAAC-3'; and probe, Fam-MGB probe 5'-AGTCATCGCTATTACCATGG-3'. All materials for the quantitative real-time PCR were purchased form Applied Biosystems. The PCR conditions were 2 min at 50°C (destruction of contaminating PCR product by AmpErase) and 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. PCR samples were analyzed by ABI Prism 7700 sequence detection system and SDS 2.1 software.
Analysis of vector transduction in vitro. In vitro transduction activities were assessed by infecting HeLa cells for 1 h in serum-free medium with each vector (103 gp/cell). Subsequently, the medium was supplemented with an equal volume DMEM-20% fetal calf serum containing adenovirus 5 (multiplicity of infection [MOI] = 10). Infected cells were incubated for 48 h at 37°C and 5% CO2, harvested, washed with PBS, and then lysed in 2x SDS loading buffer by heating to 95°C for 5 min. Aliquots of equal cell equivalents were subjected to Western blotting onto nitrocellulose membranes. Expressed L1 protein was detected as described below with Camvir-1.
Western blot analysis. For immunodetection of AAV capsid proteins or HPV16 L1 protein, equal aliquots of vector stocks were subjected to Western blot analysis. AAV capsid proteins were detected by incubation for 1 h at room temperature with the polyclonal rabbit serum VP51 diluted 1:200 in PBS-1% bovine serum albumin (BSA) (23). For the detection of HPV16 L1 protein, membranes were incubated for 1 h at room temperature with the L1-specific monoclonal antibody Camvir-1 (BD Bioscience, Heidelberg, Germany) (48) diluted 1:4,000 in PBS-1% BSA. Secondary peroxidase-coupled goat anti-rabbit and goat anti-mouse antibodies (Dianova, Hamburg, Germany), respectively, were diluted 1:5,000 in PBS-1% BSA. Membranes were washed, and the antibody reaction was visualized by using an enhanced chemiluminescence detection kit (NEB, Frankfurt, Germany).
Immunization of mice. Baculovirus-derived HPV16 L1 VLP were purified as described previously (52) and diluted with PBS to a final concentration of 5 μg/10 μl. Capsomeres were obtained as described elsewhere (55) and diluted to a concentration of 10 μg/10 μl. For intranasal vaccination, female mice were anesthetized by intraperitoneal injection with 100 μl each of both 0.2% xylazine hydrochloride (Rompun; Bayer) in PBS and 10 μg of ketamine hydrochloride per ml (Ketavet; Parke-Davis) per 10 g of body weight. An inoculum of 10 μl (viral vector, VLP, or capsomeres) was instilled dropwise into one nostril. Blood samples were taken by retro-orbital puncture, and vaginal washes were obtained by gently pipetting 300 μl of PBS up and down with protease inhibitors (Complete, Mini, EDTA-free; Roche, Germany) with a blunt-tipped Pasteur pipette. Samples were centrifuged for 5 min at 10,000 x g to pellet cellular material, and the antibody titer was determined in an enzyme-linked immunosorbent assay (ELISA). All samples were stored at –20°C.
ELISA techniques. For endpoint titration assays of sera and vaginal washes obtained from immunized mice, a VLP-based ELISA was carried out for the detection of HPV16 L1-specific antibodies described previously (12). VLP were produced and purified according to the method of Müller et al. (52). A peroxidase-coupled goat anti-mouse IgG (-chain specific) and an anti-mouse IgA (Southern Biotechnology, Birmingham, MA) were used as secondary antibodies. Specific IgG titers are expressed as the reciprocal of the highest dilution that yielded an optical density at 450 nm four times that of control mice. Extinction at 450 nm was measured after 5 to 20 min in a Titertek automated plate reader.
To verify the absence of assembled HPV16 L1 protein in the vector stocks, an antigen capture ELISA was carried out with antibody 1.3 specific for HPV16 L1 VLP and capsomeres as described before (52). HPV16 L1 VLP were used in a serial twofold dilution beginning with 50 ng of total protein titrated to 0.78 ng of protein as a standard.
AAV-based ELISA. Endpoint titration assays were performed to detect AAV5-specific antibodies. Microtiter plates were coated overnight with 109 empty particles of AAV5/well. Plates were blocked (5% skim milk in PBS for 1 h at 37°C), and mouse sera or vaginal washes were added in serial twofold dilutions starting with a 1:160 to 1:20,480 dilution or with a 1:2 to 1:265 dilution, respectively, for the vaginal washes, and incubated for 1 h at 37°C. Nonspecific binding was determined by using the same dilutions on plates coated with PBS only. Plates were washed and horseradish peroxidase-conjugated goat anti-mouse IgG antibody (-chain specific; Southern Biotechnology, Birmingham, AL) was added at a 1:5,000 dilution in PBS. Plates were incubated for 1 h at 37°C, washed, and stained with TMB (3,3',5,5'-tetramethylbenzidine) substrate solution (Sigma). Plates were measured, and specific IgG titers were expressed as described above.
AAV empty capsid production. At least 10 150-mm dishes (Nunc) were seeded with 5 x 106 293T cells per dish and incubated at 37°C and 5% CO2 overnight. Cells were transfected with 40 μg of the packaging plasmid (pDP5)/dish. Cells were harvested in TBSM buffer at 48 h posttransfection, subjected to repeated freeze-thaw cycles, and digested with 50 U of Benzonase/ml for 30 min at 37°C. Tissue debris was removed by centrifugation at 2,000 x g for 20 min at 4°C. The supernatant was fractionated on a sucrose cushion by underlaying the supernatant with 450 μl of 30% sucrose (in Tris-EDTA) followed by 450 μl of 50% sucrose in Tris-EDTA. The gradients were centrifuged at 273,000 x g for 2.5 h at 4°C by using an SW60 rotor (Beckman, Germany). The pellet was resuspended in TBSM buffer and purified on a second sucrose cushion. The resulting pellet was resuspended in an appropriate volume of TBSM buffer and then analyzed by electron microscopy.
IFN--enzyme-linked immunospot assay (ELISPOT) assay. MultiScreen IP sterile plates (96 well; Millipore, Eschborn, Germany) were presoaked with 70% ethanol for 1 min, and the ethanol was removed by extensive rinsing with PBS. The plates were coated with 200 ng of anti-mouse gamma interferon (IFN-) capture antibody (clone R4-6A2; BD Pharmingen, Heidelberg, Germany) in 100 μl of PBS overnight at 4°C. Unbound antibody was removed by washing twice with sterile Milli-Q water. Plates were blocked for 2 h with 100 μl of medium (RPMI, 10% fetal calf serum antibiotics) at 37°C, and splenocytes were seeded in triplicates in serial dilutions ranging from 200,000 to 25,000 cells per well in 100 μl of medium. For each triplicate, splenocytes in one well were left untreated (background control), cells in the next well were stimulated with 200 ng of pokeweed mitogen (Sigma) in 20 μl of medium (positive control), and cells in the last well were treated with 30 μM L1165-173 peptide in 20 μl of medium (test samples). Plates were incubated for 18 to 20 h at 37°C. Cells were removed by six washes with PBS-0.01% Tween 20. Then, 20 ng of sterile-filtered biotinylated anti-mouse IFN- detection antibody (clone XMG1.2; BD Pharmingen) in 100 μl of PBS-0.5% BSA was added per well, and the plates were kept at 4°C overnight. The plates were washed six times with PBS-0.01% Tween 20, and this was followed by the addition of 100 μl of a 1:1,000 dilution of streptavidin-alkaline phosphatase (BD Pharmingen) in PBS. Plates were incubated for 45 min at room temperature and then washed three times with PBS-0.01% Tween 20, followed by three washing steps with PBS alone. Plates were developed for 2 to 20 min with 100 μl of 5-bromo-4-chloro-3-indolylphosphate (BCIP/Nitro Blue Tetrazolium Liquid Substrate System; Sigma). The reaction was stopped by rinsing the plates with water. Spots were quantified by using an ELISPOT reader (ELISPOT Reader System ELR02; AID GmbH, Strasbourg, Germany). Wells with medium alone and no splenocytes were assayed in parallel as negative controls. Counts of the background control wells were subtracted from the samples.
Tissue distribution analysis of vector DNA. Mice were sacrificed, and the following tissues were excised and immediately frozen in liquid nitrogen: heart, liver, kidney, and lung. Genomic DNA was extracted from approximately 10 to 25 mg of each tissue by using the DNeasy Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and then eluted from the supplied columns in 100 μl of the provided elution buffer. Total DNA concentrations were quantified by spectrophotometry (Ultraspec III; Pharmacia).
PCR with a L1-specific probe was carried out with the forward primer 5'-CCCATCAAGAAGCCCAACAAC-3' and the reverse primer 5'-GGGGCTTGCAGCCGATCAGG-3', yielding a 339-bp PCR product. PCR conditions were 2 min at 95°C, followed by 30 cycles of 30 s at 95°C and 30 s at 62°C and elongation for 1 min at 72°C, with a final elongation for 5 min at 72°C. Samples were analyzed by agarose gel electrophoresis.
Preparation of papillomavirus pseudovirions and neutralization assay. Presence of neutralizing antibodies in sera of immunized animals was determined by using a protocol adapted from Buck et al. (10). Briefly, pseudovirions were prepared by transfecting 7 x 106 293T cells (cultivated in DMEM containing 50 μg of hygromycin/ml) with a plasmid encoding for the humanized HPV16 L1 and L2 genes, together with a plasmid containing the gene for secreted alkaline phosphatase under the control of the hCMV promoter. After 4 days, cells were harvested by using trypsin and washed with PBS. Cells were then adjusted to 5 x 107 per ml and lysed by 0.5% Brij58 in the presence of Benzonase (2,000 U/ml) for 5 min on ice. After the NaCl concentration was increased to 850 mM, the cells were centrifuged and the cleared supernatant containing the pseudovirions was used for infection of 293T cells. For infection, pseudovirion stocks were diluted 1:5,000 in complete medium and preincubated with the sera (1:50 dilution) for 15 min at room temperature. Pseudovirions were then added to the cells, followed by incubation at 37°C for 4 days. Secreted alkaline phosphatase activity in the supernatant was measured by using a commercial assay (Roche, Mannheim, Germany) according to the manufacturer's recommendations.
RESULTS
Characterization of the vector stocks. We sought here to develop an AAV vector for efficient induction of humoral immune responses against HPV16 infection after intranasal immunization. Therefore, the codon-optimized HPV16 L1 gene (L1h) introduced into an AAV2 vector (42) was pseudotyped into AAV5 capsids (Fig. 1a). A vector expressing the Gfp gene was generated as a negative control. Crude vector preparations were prepared as described before (23) and fractionated by two successive CsCl density centrifugations. Western blot analysis revealed that the vector containing fractions were still contaminated with HPV16 L1 protein generated during rAAV5 L1h production in 293T cells (data not shown). Hence, we depleted the vector preparations from residual L1 protein by immunoaffinity purification of matrix-immobilized IgG of a polyclonal rabbit serum raised against native and denatured HPV16 L1 VLP.
Western blot analysis of 5 x 1011 gp of a purified vector preparation using the AAV-specific polyclonal rabbit serum VP51 (23) and Camvir-1 (48) verified the presence of intact viral capsid proteins and demonstrated that it was free of detectable HPV16 L1 protein (Fig. 1b and c, respectively). The vector stock was further analyzed by an antigen capture-ELISA based on the monoclonal antibody 1.3 (52). No traceable amounts of L1 were found in the rAAV5 L1h vector stock. Spiking experiments revealed that the detection limit for this assay was 0.8 ng of VLP present in 5 x 1011 gp (data not shown). Transduction of HeLa cells with the rAAV5 L1h vector and subsequent Western blot analysis with an anti-L1 antibody (Camvir-1) demonstrated that the vector was functional, expressing an L1 protein with the expected size (Fig. 1d).
Intranasally delivered rAAV5 L1h evokes systemic and mucosal immune responses in mice. The intention of the present study was to establish an efficient and easy-to-apply vaccination model. Three groups of C57BL/6 mice (labeled as single, double, and triple; a total of 10 mice per group in two independent experiments) were immunized intranasally with one, two (second at day 7), or three (third at day 42) doses of 5 x 1010 gp. In addition, one group (rAAV5 L1h lyo) of mice was inoculated with three doses (5 x 1010 gp/dose) of the lyophilized rAAV5 L1h vector, and another group (rAAV5 L1h high) received three doses of 5 x 1011 gp/dose. As a negative control group, mice were inoculated three times with PBS alone or with rAAV5 Gfp (5 x 1010 gp/dose), while three immunizations with L1-VLP (5 μg/dose) or L1-capsomeres (10 μg/dose) served as positive controls. At 3 months after the first immunization, sera and vaginal washes were collected and screened for the presence of L1-specific antibodies by using a VLP-based ELISA. The vaccination trial was repeated in two independent experiments. Figure 2a shows the representative results for serum IgG of one experiment, and in Table 1 the results of both experiments are summarized.
Mice treated with VLP or capsomeres alone (9 of 10 or 5 of 5 animals, respectively) had L1-specific serum antibodies (Fig. 2a and Table 1). All sera of mice immunized with rAAV5 L1h also developed L1-specific serum antibodies. Serum titers of mice inoculated with the lyophilized vector were lower but still significantly (P = 0.02) above the background obtained from sera of mice vaccinated with PBS or rAAV5 Gfp. In one experiment the highest L1-specific serum titer was measured in the mice that had received 5 x 1011 gp/dose of rAAV5 L1h (Fig. 2a). In both experiments combined highest L1-specific titers were achieved in the rAAV5 L1h triple groups (5 x 1010 gp/dose) (Table 1). Neutralization assays using pseudovirions with secreted alkaline phosphatase as a reporter demonstrated that all tested antibody-positive sera were also neutralizing (Fig. 2b). In conclusion, a successful protective seroconversion was achieved after intranasal application of rAAV5 L1h vectors.
The immune response at the viral entry site is a crucial factor in establishing protective immunity against HPV. Therefore, vaginal washes were collected and analyzed for the presence of L1-specific mucosal immunoglobulins. Three of five mice inoculated with capsomeres developed L1-specific IgG antibodies in the vaginal washes, whereas within the VLP-treated group only two of ten mice revealed mucosal antibodies (Table 1). Also, the groups treated with rAAV5 L1h responded with the formation of mucosal antibodies in the vaginal washes. However, only the group with the highest vector dose showed a reaction in all animals. Antibody titers in the vaginal washes corresponded to the serum titers of the different groups. We also found L1-specific IgA antibodies but, similar to findings presented by others in humans (59) and mice (3), the levels of these antibodies were lower but consistent with the mucosal IgG titers (Table 1).
In conclusion, the results show that the intranasal delivery of the rAAV5 L1h-based vector efficiently induces L1-specific antibodies both in serum and in vaginal washes. The presence of L1-specific mucosal antibodies in the vaginal washes represents a prerequisite to preventing HPV16 infections at the viral entry site.
rAAV5 L1h induce long-lasting L1-specific humoral immunity. In order to monitor the persistence of the humoral response over time, sera were taken repeatedly for 60 weeks after the first immunization. The single vaccine dose (rAAV5 L1h single) administered initially was sufficient to induce and maintain high serum antibody titers for this prolonged period of time (Fig. 3). The highest antibody titers were found in mice which had received two doses (rAAV5 L1h double). Titers were significantly higher compared to the titers of the group receiving a single dose (P = 0.03). However, the difference in titers compared to the group that received three doses was not significant (P = 0.07). This may reflect the effect of an AAV-specific immune response already established at the time of third immunization, preventing a further boosting against the L1 protein (Fig. 3). In contrast, the anti-L1 titer in mice that had received three doses of VLP had dropped below the detection limit. The sera of the negative control mice treated with PBS or rAAV5 Gfp (not shown) remained negative throughout the experiment. In summary, vaccination with AAV5 L1h induces a long-lasting seroconversion even after a single administration.
L1-specific cellular immune responses are present 60 weeks after vaccination. Cellular immune responses are pivotal for the elimination of virus-infected cells. The HPV16 L1-specific lymphocytes were evaluated at the completion of the vaccination experiment (60 weeks after the first immunization). Splenocytes from mice immunized with rAAV5 L1h (single, double, and triple), as well as from VLP-inoculated mice, were isolated, and the frequency of IFN--producing L1-specific lymphocytes was analyzed ex vivo by ELISPOT (Fig. 4). The mice that had received VLP had no measurable levels of L1-specific lymphocytes. In contrast, all mice treated with the rAAV5 L1h vector, regardless of the number of doses, revealed a notable fraction of IFN--producing L1-specific lymphocytes. The amounts of specific lymphocytes were significantly higher in the rAAV5L1h single (P = 0.049), double (P = 0.03), and triple (0.03) group compared to the VLP-treated mice. This clearly demonstrates the usefulness of an AAV-based vaccine to induce and maintain a long-term, specific cellular immune response against the delivered transgene.
Vaccination with rAAV5 elicits vector-directed humoral antibodies. Immune responses raised against the delivery system itself may inhibit further vaccinations. An AAV-based ELISA assessed the immune responses that were induced against the AAV vectors. After 3 months the sera of all mice immunized with the AAV vectors revealed high titers of AAV-specific antibodies (Fig. 5a). No remarkable differences in antibody titers were measured in mice receiving single, double, or triple doses. The group immunized with a 10-fold-greater vector dose (rAAV5 L1h high) revealed also higher AAV-specific titers (Fig. 5a). AAV-specific antibodies were already detectable after 42 days (data not shown), which may have partially neutralized the third immunization dose in some experiments. This may explain the L1-specific antibody levels found in mice after 60 weeks. The immunogenicity of the AAV vector was also demonstrated by the specific mucosal immune responses present in the groups of mice treated with the rAAV5 vectors (Fig. 5b). Mice that had received PBS, VLP, or capsomeres were negative (Fig. 5b).
The presence of antibodies raised against a specific AAV serotype can influence the success of a repeated administration of the same or other serotype-based vectors (26, 28, 46, 51, 56). Therefore, groups of five mice were intranasally immunized with empty capsids of AAV2, AAV4, or AAV5. After 6 weeks, seroconversion against the respective serotype was confirmed by an AAV-based ELISA (data not shown), and mice were inoculated intranasally with a single dose of rAAV5 L1h (5 x 1010 gp). Six weeks later, sera were screened for L1-specific antibodies by using a VLP-based ELISA (Fig. 5c). Mice seropositive for AAV2 or AAV4 could be immunized successfully by administration of rAAV5 L1h vectors, eliciting an L1-specific humoral immune response, whereas readministration of the same AAV5 serotype failed to induce L1-specific antibodies. In contrast, in mice treated with rAAV5 Gfp 60 weeks previously, readministration of rAAV5 L1h induced successfully L1-specific antibodies both in serum and in mucosa (data not shown). This may be linked to the presumably reduced AAV-specific antibody titers after 60 weeks. In conclusion, administration of an AAV5-based vector in mice that are seropositive for AAV2 or AAV4 is possible; however, a readministration of vector to mice with preexisting antibodies against AAV5 is only possible if the AAV5 titer drops below a protective level.
Recombinant vector DNA is detected in lung tissues. The distribution of viral vectors in the body after application is one major biosafety concern. Hence, several tissues were examined for the presence of L1h vector DNA after intranasal rAAV5 L1h administration. Genomic DNA from liver, heart, kidney, and lung tissue was extracted from five individual mice of each group (see above) sacrificed 3 months after the first intranasal immunization. The presence of vector DNA in these tissues was assessed by PCR with a L1-specific primer resulting in a 339-bp fragment. The detection limit was in the range of 0.5 pg of vector DNA. The samples prepared from liver, heart, and kidney tissue of all mice from each group revealed no vector DNA. AAV vector DNA could only be found in DNA probe from the lung samples of the rAAV5 L1h high group (Fig. 6). This clearly indicates that the intranasal application of an rAAV prevents the systemic spread of vector DNA that occurred after intravenous administration (25) and represents a further safety benefit of mucosal AAV-based vaccination.
DISCUSSION
The vast majority of pathogenic infections occur at and progress from mucosal surfaces by colonizing or penetrating through the mucosa of the gastrointestinal, respiratory, or genital tract. In these cases, the local application of a vaccine is generally more efficient to induce a protective immune response, since systemic vaccination does not usually increase the mucosal antibody levels (59). However, in practice it has often proven to be rather difficult to stimulate strong mucosal immune responses by oral-mucosal administration of antigens; thus, the parenteral route of immunization is still the standard route of vaccination. Indeed, relatively few of the current vaccines that are approved for human use are administered mucosally. However, for reasons of simplicity, the two most attractive routes of vaccination in humans would be the oral and nasal application routes. Intranasal vaccination has emerged as the optimal vaccination strategy in rodents for the induction of antibody responses in genital tissues (3, 34, 54, 65) and was also shown to be successful in humans (5, 14, 17, 31, 33, 34, 41, 60). Mucosal antibodies are crucial in the protection against sexually transmitted infections and form the first line of defense. Among the most common viral sexually transmitted diseases are genital HPV infections (for a review, see reference 8).
Prevention and control of cervical cancer on a global basis is most easily envisaged through vaccine-mediated prevention of HPV infection and/or elimination of persistent infection at sites of high risk for development of cervical cancer. A suitable way to induce protective neutralizing antibodies in vaginal secretions of rodents and humans against applied antigens was shown by intranasal vaccination (3, 5, 14, 31, 33, 34, 54, 60, 65). Additional important benefits of intranasal application are that it is "needle-less," simple, and noninvasive; this is especially important in light of the high prevalence of HPV infections in developing countries. Different approaches for prophylactic and therapeutic immunizations against HPV are currently being developed, mostly based upon VLP. We intended to establish an alternative genetic vaccination model to the systems currently being investigated. Genetic vaccinations are supposed to mediate a long-term expression linked to a long-lasting immunity.
The development of new generations of vaccines and the delivery of peptides and proteins in general has been impeded by a lack of appropriate delivery systems. Here, we demonstrate the efficacy of an rAAV5-based vaccine, encoding the codon-optimized major capsid gene L1h of HPV16, administered intranasally to female C57BL/6 mice. A single dose of rAAV5 L1h induced a strong humoral and cellular immune response against HPV16 L1; both responses were maintained for more than 1 year. In particular, the presence of an ex vivo detectable L1-directed specific cellular immune response after 60 weeks measured by IFN- production on a single-cell level raises the hope for a successful therapeutic application of this vector system. L1-specific mucosal antibodies were also detected in the vaginal washes of mice, suggesting the presence of a protective immune response against HPV16 infections. It is noteworthy that both IgG and IgA were detected in the vaginal washes, but the IgG responses dominated over the IgA response, which was also reported after oral or nasal vaccination of humans with cholera vaccine (59). We have no information about the transport of IgG into the vaginal lumen. Receptor-mediated transcytosis has been reported to occur, but transudation is another likely mechanism (58). In general, the distribution of antibody classes varies on the different mucosal surfaces in humans, i.e., IgA is the major isotype in the gut, whereas IgA and IgG are found in comparable amounts in nasal and vaginal secretions (57).
AAV5-specific immune responses in mice, both in serum and mucosa, were observed 42 days after the first inoculation. Potential reapplication of the vaccine can be prevented if a protective immunity against the carrier system is established (22, 26-28). We assume that the third immunization dose given at day 42 may already be neutralized (in some experiments) by the antibodies developed against AAV5, leading to the lower level of L1-specific serum antibodies detected after 60 weeks. In addition, application of the third dose may boost an AAV5-specific cellular immune response eliminating transduced cells. Thus, preimmunized mice with AAV5 could not be successfully vaccinated by readministration of rAAV5 L1h, but mice immunized with rAAV5 Gfp vectors 60 weeks ago could be readministered successfully. This finding strongly indicates the presence of a stronger AAV5 directed immune response established after 42 days that may have declined with time to a level which permits the successful readministration of rAAV5 L1h (1). In contrast, a preexisting immune response against AAV2, which has a high prevalence in humans (7, 11, 16, 20, 47, 62), as well as against AAV4, had no impeding influence on rAAV5 L1h-based immunizations. The use of different serotype combinations for repeated administration is extensively investigated and was shown to bypass acquired immunity against a certain AAV serotype (for a review, see reference 22). Vaccination of mice with AAV4 L1h was also able to induce both humoral and mucosal immune responses (unpublished observation), but vaccination or readministration with AAV4 L1h seemed to be unlikely for application in humans due to the inefficient transduction of human cells by AAV4-based vectors (23).
The detection of vector DNA only in lung tissue in vaccinated mice and the lack of vector spread in tissues other than the lung are coincident with prior findings in rabbits after airway application of AAV vector. Several other rabbit tissues were screened for recombinant vector DNA; however, it was found mainly in the airway surface epithelium (18). Moreover, the presence of vector DNA in the lung tissue may elucidate the long-lasting L1 antibody titers presumably resulting from persisting transgene expression. Further, the local distribution may also support the biosafety specifications for vaccines, especially with regard to the concerns of AAV vector integration in the host genome (53).
Recently, covaccination of an intramuscularly applied AAV2 L1 together with an adenovirus encoding murine granulocyte-macrophage colony-stimulating factor was reported to elicit a strong and prolonged immune response (43). However, previous observations showed that gene expression of the original HPV16 L1 sequence was hardly detectable after transfections (42). Further, it could be assumed that contaminating L1 protein present in the vector stock was the potential immunogenic agent. Liu et al. did not control their study for this possibility. Our own experience has proven that all AAV L1h vector stocks contained high amounts of L1 protein—presumably as VLP—even after density gradient purification. This made it always difficult to assign the immune response specifically to either the L1 protein or the rAAV.
In addition to the usage of AAV vectors in many applications of gene therapy, AAV was also shown to be successful in oral and nasal vaccinations of mice against human immunodeficiency virus (70, 71), herpes simplex virus (45), and Alzheimer's disease (73). We present here a novel mucosal application of rAAV5 for the vaccination against HPV. The possibility to lyophilize the recombinant vector renders the maintenance of expensive cold-chain storage unnecessary. Further advantages of this vector system include the noninvasive application route of an adjuvant-independent vector eliciting a long-lasting humoral and cellular immune response. Given these properties, recombinant HPV L1-rAAV may be suitable for use in developing countries provided that the manufacturing costs per vaccination dose can be reduced.
ACKNOWLEDGMENTS
We thank Corinna Klein for help with the ELISPOT assay, Petra Galmbacher for the L1-VLP production, Martin Friedel and Werner Nicklas from the DKFZ animal facility, and Ute Koch for critically reading the manuscript. We especially thank Harald zur Hausen for his continuous support.
The project was supported by a grant from the Deutsche Krebshilfe (10-1912-Kl I).
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