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. 2013 Jan 8;21(3):680–687. doi: 10.1038/mt.2012.260

Oral Vaccination With Adeno-associated Virus Vectors Expressing the Neu Oncogene Inhibits the Growth of Murine Breast Cancer

Jason C Steel 1,2, Giovanni Di Pasquale 3, Charmaine A Ramlogan 1,2, Vyomesh Patel 4, John A Chiorini 3, John C Morris 1,2,*
PMCID: PMC3589150  PMID: 23295951

Abstract

Recombinant adeno-associated viruses (AAV) have been used for therapeutic gene transfer. These vectors offer a number of advantages including resistance to the effects of pH, a broad cellular tropism, efficient gene transfer, persistence of gene expression, and little toxicity. AAV vectors; however, at high doses can induce humoral and cellular immune responses. While potentially problematic for replacement gene therapy, this effect may be advantageous for antitumor vaccination. We examined the activity of an oral and intramuscular antitumor vaccination using AAV serotypes 5 and 6 expressing a truncated neu oncogene in a neu-positive murine TUBO breast cancer model. Mice receiving a single oral administration of AAV5-neu or AAV6-neu demonstrated improved survival. Oral vaccination significantly improved survivals compared with intramuscular vaccination. Mice vaccinated with AAV6-neu survived longer than those treated with AAV5-neu. Vaccination with AAV5-neu or AAV6-neu induced both humoral and cellular immune responses against the NEU antigen. These responses were more robust in the mice undergoing oral vaccination compared with mice receiving the intramuscular vaccination. Protection from tumor was long lasting with 80% of the animals treated with oral AAV6-neu surviving a re-challenge with TUBO cells at 120 and 320 days post-vaccination. Further evaluation of AAV-based vectors as tumor vaccines is warranted.

Introduction

Adeno-associated virus (AAV) is member of the Parvoviridae. AAV can infect a wide variety of human and nonhuman cells and has not been associated with any known disease. This safety profile made AAV an attractive vector for therapeutic gene transfer studies. Being devoid of all viral genes and consisting only of the exogenous transgene flanked by the viral inverted terminal repeats, recombinant AAV vectors were thought to have limited immunogenicity.1 Indeed, early gene therapy studies showed limited immune responses making AAV a promising vector for replacement gene therapy.2,3,4 Subsequently, it was shown that AAV vectors can induce cell-mediated and humoral immune responses to the transgene depending on dose, route of administration, and serotype.5,6 These responses can limit the expression of the transgene and result in the elimination of the virally transduced cells. While this is a potential drawback for replacement gene therapy, these characteristics could be exploited for the development of vaccines.

AAV is currently being investigated as a potential vaccine platform largely for infectious diseases such as HIV.7,8,9,10 A clinical trial using an AAV-based vaccine targeting HIV has been recently completed.7 In this study, AAV serotype 2 (AAV2) encoding HIV-1 subtype C gag, protease, and part of reverse transcriptase was intramuscularly administered. The vaccine was well tolerated and 38% of patients in the high-dose vaccine cohort exhibited T-cell responses against HIV.7 While this study confirmed AAV to be safe, the limited immune responses observed were disappointing. There is clearly a need to examine alternative AAV serotypes, routes of administration, and other targets for AAV-based vaccines.

In the current study, we examined the activity of AAV serotypes 5 and 6 as a platform for the oral vaccination against the neu oncogene in a model of NEU-expressing breast cancer. This is the first reported study utilizing oral delivery of AAV as a cancer vaccine. AAV5 has been shown to transcytose gut epithelial cells allowing systemic delivery of the vector, while AAV6 has been shown to transduce gut cells allowing a localized delivery.11 AAV has also been shown to be resistant to pH and temperature.12 These characteristics enhance the potential for AAV as an oral vaccine vector. We compared the activity of oral and intramuscular antitumor vaccination of AAV serotypes 5 and 6 and showed that mice receiving a single oral administration of AAV5-neu or AAV6-neu demonstrated improved survival. Oral vaccination resulted in significantly longer survival than intramuscular vaccination. Mice vaccinated with AAV6-neu survived longer than those treated with AAV5-neu. Vaccination induced both humoral and cellular immune responses against the NEU antigen. The responses were more robust in the mice undergoing oral vaccination compared with mice receiving the intramuscular vaccination and also resulted in greater protection from tumors and against tumor re-challenge.

Results

In vitro transduction with AAV5 or AAV6 vectors encoding neu results in surface expression of NEU protein in several cell types

HeLa cells transduced with AAV5-neu or AAV6-neu were stained with monoclonal anti-NEU antibody and examined by flow cytometry for the surface expression of NEU. HeLa cells transduced with either AAV5-neu or AAV6-neu at an multiplicity of infection (MOI) of 100 expressed surface NEU protein in 48 and 67% of cells, respectively (Figure 1a).

Figure 1.

Figure 1

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In vitro and in vivo transduction of AAV5- or AAV6-based vectors. (a) HeLa cells were transduced with AAV5-neu or AAV6-neu at an MOI = 100, and analyzed by flow cytometry for the surface expression of NEU. (b) Caco-2 cells were transduced with AAV2-luc, AAV5-luc or AAV6-luc at an MOI = 1,000 and assayed for luciferase expression. (c) Murine-derived dendritic cells were transduced with AAV5-GFP (left panel) and AAV6-GFP (right panel) at an MOI = 1,000 and assayed for transduction efficiency by flow cytometry. (d) Groups (N = 3) of 6–8 weeks old female BALB/c mice received a single oral administration of 1 × 1011 VG of AAV5-neu or AAV6-neu. One week later, the animals were killed and tissues were examined by qPCR for viral DNA; *P < 0.05. (e,f) Groups (N = 3) of BALB/c mice received a single oral administration of 1 × 1011 VG of AAV6-Luc. Two weeks later, the animals were killed and tissues were examined for luciferase expression using a Xenogen imaging system. AAV, adeno-associated virus; GFP, green fluorescent protein; MOI, multiplicity of infection; qPCR, quantitative PCR; RLU, relative light unit; VG, viral genome.

Next, we examined the levels of gene expression that could be achieved with vectors based on AAV serotypes 2, 5 or 6 in the human Caco-2 colon carcinoma cell line using a luciferase reporter gene. Caco-2 cells grown in transwells have been shown to differentiate and polarize and have been used as a representative of gastrointestinal epithelia. We achieved limited expression using both AAV2-luc and AAV5-luc compared with AAV6-luc in Caco-2 cells (Figure 1b). AAV6 demonstrated greater than a log higher luciferase expression (54,672 relative light units) when compared with AAV2 (3,234 relative light units) or AAV5 (876 relative light units).

The ability of AAV serotypes 5 and 6 to transduced murine dendritic cells (DC) was examined. We showed that both AAV5 and AAV6 were able to successfully transduce bone marrow-derived murine DC (Figure 1c). At an MOI of 103 viral genome (VG), AAV5-green fluorescent protein (GFP) transduced 20.6 ± 3.2% of DC and AAV6-GFP transduced 14.7 ± 5.2% of DC. The transfection efficiencies of AAV5 and AAV6 were not significantly different (P > 0.05).

AAV5 and AAV6 demonstrate differing biodistribution following oral administration

In order to examine biodistribution of AAV5 and AAV6 in a mouse model, we orally administered 1 × 1012 VG/ml of each serotype in 100 µl of phosphate-buffered saline (PBS). After 1 week, we removed the lungs, heart, liver, draining lymph nodes, kidneys, and stomach and examined them by quantitative PCR for the presence of AAV genomes (Figure 1d). Mice treated with AAV5 had detectable levels of viral DNA in each of the organs examined with the greatest concentration found in the stomach and liver. Similarly, mice treated with AAV6 had detectable levels of AAV in each of the tissues examined with the majority found in the stomach and liver. Interestingly, mice treated with AAV6 had significantly more viral DNA in the lymph nodes and significantly less viral DNA in the lungs and kidneys compared with AAV5-treated mice (P < 0.05).

In vivo imaging of mice transduced with AAV6-encoding luciferase 2 weeks after oral delivery that was confirmed the quantitative PCR (Figure 1e,f). Using xenogen imaging, the majority of luciferase expression was localized to the stomach and liver. There was no significant difference in the expression between these tissues. No luciferase expression was detected in the lungs, heart, sternum or kidneys.

Vaccination with AAV-neu–protected mice against challenge with neu-expressing TUBO mammary cancer cells

Mice were followed for the development of tumors and survival after intramuscular (i.m.) or oral (p.o.) treatment with AAV5-neu, AAV6-neu or adenovirus-neu (Ad5.Neu) and challenge with the neu-expressing TUBO breast cancer cell line. Animals treated i.m. with either AAV5-neu or AAV6-neu at 1012 VG demonstrated inhibition of tumor growth and increased overall survival compared with animals treated with PBS, or those treated at lower doses, P < 0.05 (Figure 2a). All animals treated with AAV6-neu at 1012 VG survived for greater than 100 days following a single treatment, while only 50% of animals treated i.m. with AAV5-neu survived greater than 100 days post-tumor implantation. Animals treated i.m. with AAV5-neu or AAV6-neu at 1011 VG showed no survival advantage over PBS controls. These results suggest that oral delivery is superior to i.m. delivery in triggering an effective antitumor vaccine response.

Figure 2.

Figure 2

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Vaccination of BALB/c mice with AAV5-neu and AAV6-neu significantly delayed or prevented the development of mammary tumors resulting in increased survival. Groups (N = 10) of 6–8 weeks old female BALB/c mice received a single vaccination with 1 × 1011 or 1 × 1012 VG of AAV5-neu, AAV6-neu, Ad5-neu or PBS control (100 µl), either (a) intramuscularly (i.m.), or (b) via oral gavage (p.o.). Eight weeks following vaccination, the mice were subcutaneously injected with 1 × 106 TUBO cells. The mice were evaluated twice weekly for tumor growth. AAV, adeno-associated virus; PBS, phosphate-buffered saline; VG, viral genome.

When looking at the mice receiving the oral vaccination, animals treated with AAV5-neu or AAV6-neu demonstrated a significant survival advantage over PBS- or Ad5.Neu-treated animals, P < 0.05 (Figure 2b). All animals treated with AAV6-neu at 1012 VG, and 80% of animals treated with AAV5-neu at 1012 VG survived greater than 100 days, while only 50 and 10% of animals treated with AAV6-neu and AAV5-neu at 1011 VG, respectively survived the tumor challenge. At the 1011 VG dose, animals administered the oral vaccine demonstrated a significantly greater overall survival than mice vaccinated i.m. (P < 0.05). There was no survival advantage to using Ad5.Neu orally. AAV5- or AAV6-neu–vaccinated animals challenged with the TS/A, a neu-negative cell line, did not show a survival advantage over PBS-vaccinated animals (Supplementary Figure S1a).

AAV-neu vaccination induced anti-neu antibodies

We examined the effect of the different vaccines on the levels of serum anti-NEU antibodies. Mice vaccinated with AAV5-neu either p.o. or i.m. demonstrated significant increases in the levels of serum anti-NEU antibodies compared with the PBS, or N202.1E, a control neu− breast cancer cell line (Figure 3a, Supplementary Figure S1b). The induction of antibody was virus dose-dependent with animals receiving 1012 VG developing higher antibody levels than those receiving 1011 VG. This finding was consistent for both p.o. and i.m. delivery. When comparing these two routes of delivery, animals undergoing oral vaccination developed significantly greater circulating anti-NEU antibodies compared with those receiving the vaccine i.m. A similar effect was also seen in the mice vaccinated with AAV6-neu (Figure 3b). When we compared animals treated with AAV6-neu to those treated with AAV5-neu, we found there was a greater than twofold increase in the levels of detectable antibody in AAV6-neu–treated mice compared with AAV5-neu–vaccinated animals.

Figure 3.

Figure 3

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Oral vaccination of BALB/c mice with AAV5-neu and AAV6-neu induced greater antibody levels than those induced by intramuscular vaccination. Groups (N = 5) of 6–8 weeks old female BALB/c were vaccinated p.o. or i.m. with a single dose of 1 × 1011 or 1 × 1012 VG of (a) AAV5-neu, or (b) AAV6-neu. The mice were bled and the quantity of anti-NEU antibodies were assayed by a cell-based flow cytometry assay. Ab, antibody; AAV, adeno-associated virus; i.m., intramuscularly; MFI, mean fluorescence intensity; p.o., orally; VG, viral genome.

Vaccination with AAV-neu induces antitumor cell-mediated immune responses

To examine whether the vaccines induced tumor-specific cell-mediated immune responses, we looked at the ability of splenocytes from vaccinated mice to lyse TUBO tumor cells in an ex-vivo cytolytic T-lymphocyte (CTL) assay, as well as to stimulate interferon-γ (IFN-γ) release in response to a CD8-dominant rat NEU peptide epitope. Splenocytes isolated from mice vaccinated with either AAV5-neu or AAV6-neu at 1012 VG induced the lysis of TUBO cells in this assay (Figure 4a,b). Splenocytes isolated from animals undergoing oral vaccination with AAV5-neu induced greater tumor cell lysis than splenocytes from mice receiving the intramuscular vaccination. A similar effect was seen in the animals vaccinated with AAV6-neu. When comparing the splenocytes from AAV6-neu– and AAV5-neu–treated animals, we found those from AAV6-neu–vaccinated mice induced greater CTL killing compared with AAV5-neu–vaccinated mice. No lysis of TS/A, a neu− cell line was observed with splenocyes from AAV5- or AAV6-neu–vaccinated animals (data not shown).

Figure 4.

Figure 4

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Oral vaccination of BALB/c mice with AAV5-neu and AAV6-neu induced greater cell-mediated immune responses than those induced by i.m. vaccination. Groups (N = 5) of 6–8 weeks old female BALB/c were vaccinated p.o. or i.m. with a single treatment of 1 × 1011 or 1 × 1012 VG of (a,c) AAV5-neu or (b,d) AAV6-neu. Eight weeks later, their spleens were removed and assayed for (a,b) CTL and (c,d) IFN-γ secretion. Columns represent the mean of triplicate treatment in one experiment. AAV, adeno-associated virus; CTL, cytolytic T-lymphocyte; IFN, interferon; i.m., intramuscularly; PBS, phosphate-buffered saline; p.o., orally; VG, viral genome.

When looking at the ability to release IFN-γ in response to an immunodominant NEU peptide, we found the results were similar to those seen in the CTL assay. Splenocytes from animals vaccinated either i.m. or p.o. with 1012 VG of AAV5-neu or AAV6-neu released significant levels of IFN-γ (Figure 4c,d). Furthermore, significantly more IFN-γ was released from splenocytes of animals undergoing oral vaccination compared with those receiving the i.m. vaccine. Similarly, more IFN-γ was released from splenocytes of mice treated with AAV6-neu compared with AAV5-neu–vaccinated mice.

CD8+ and CD4+ cells mediate AAV-neu antitumor effects

To further explore the mechanism of AAV-based antitumor vaccination, we administered anti-CD4, anti-CD8, or anti-NK (anti-asialo-GM1) antibodies to groups of BALB/c mice before and after oral vaccination with AAV5-neu at 1011 VG to deplete these cell lineages. Two weeks after the vaccination, the mice were challenged with 1 × 106 TUBO cells and tumor volumes were measured 21 days later (Figure 5). Mice treated with anti-asialo-GM1 or anti-CD4 after vaccination (late), but before tumor implantation did not demonstrate significantly larger tumors compared with the controls indicating these cells are not required for the antitumor effect. However, mice depleted of CD8+ cells before tumor implantation and CD4+ cells before vaccination (early) showed significantly larger tumors than the control mice indicating that these cells are required for the antitumor response to the vaccine.

Figure 5.

Figure 5

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CD4+ T-cells and CD8+ T-cells are required for the generation of antitumor responses following vaccination with AAV5-neu. CD4+, CD8+, and NK cells were depleted from groups of BALB/c mice vaccinated with AAV5-neu using injections of 200 µg of anti-CD4 (GK1.5), 5 days before vaccination (early) or 5 days before tumor implantation (late) or anti-CD8 (2.43) or 50 µg of anti-NK (anti-asialo GM1) antibodies as described in Materials and Methods. Eight weeks after the vaccination, mice were injected with 1 × 106 TUBO cells. Tumor size was determined 3 weeks following tumor inoculation (10 mice per group). Mean ± SD. *P < 0.05. AAV, adeno-associated virus; NK, natural killer cell.

Vaccination with AAV-neu induces long-term immunity to tumor

To examine whether the protective effect induced by AAV-neu was durable, we re-challenged vaccinated mice that were tumor-free after the initial challenge with TUBO cells at 120 days (Figure 6a) and then again 320 days after vaccination (Figure 6b). All animals vaccinated with AAV6-neu survived re-challenge 120 days post-vaccination and survived tumor-free >300 days. Sixty percent of animals vaccinated with AAV5-neu survived re-challenge to 300 days (Figure 6a). AAV6-neu–vaccinated animals that were tumor-free, were again re-challenged at 320 days with TUBO with 100% surviving a further 60 days and 80% surviving the full 420 days post-vaccination (Figure 6b).

Figure 6.

Figure 6

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Vaccination responses with AAV were durable and long lasting. In order to examine long-term protection and immunological memory, the surviving tumor-free mice were re-challenged with 1 × 106 TUBO cells at (a) 120 days and (b) 320 days following the initial tumor implantation. AAV, adeno-associated virus; VG, viral genome.

Discussion

Development of an effective vaccine for the prevention or treatment of breast cancer would represent a major step forward. An oral vaccine would be a particularly exciting development. To date, the majority of experimental oral vaccines have centered on the use of live, attenuated strains of bacteria such as Salmonella, Listeria monocytogenes, Escherichia coli, and Shigella.13 However, the use of live bacterial vectors may pose safety concerns. AAV has recently been studied as a potential oral vaccine vector.10,14,15 AAV is non-pathogenic and the recombinant vectors retain none of their viral genes making it a safer alternative to live bacterial strains. The lack of AAV's Rep gene also limits the vector's integration potential and the vast majority of AAV vectors are thought to remain episomal.16

We evaluated the use of recombinant AAV serotypes 5 and 6 encoding a non-signaling truncated rat neu oncogene as a platform for antitumor vaccination in a murine mammary tumor model. We showed that our vectors were functional by successfully transducing several cell types with AAV5-neu and AAV6-neu vectors resulting in high levels of the NEU protein on the surface of the cells including gut and DC. We found that AAV6 could transduce gastrointestinal Caco-2 cells with higher activity compared with the AAV2 and AAV5 serotypes likely as a result of the high levels of the epidermal growth factor receptor on their cell surface which was recently identified as a coreceptor for AAV6.17,18 AAV5 utilizes platelet-derived growth factor receptor for virus entry, which is poorly expressed on Caco-2 cells.19 Rather, AAV5 has been shown to transcytose through Caco-2 cells, in vitro, and be released intact on the basolateral surface of the cell, potentially allowing for a broader dissemination of this vector to more permissive cell types.11

To examine whether AAV5-neu and AAV6-neu could be used as an oral vaccine for cancer, we compared oral vaccination with i.m. delivery of both vectors. Both AAV5-neu and AAV6-neu when p.o. delivered at 1011 VG resulted in superior tumor protection compared with i.m. delivery. The gastrointestinal tract and surrounding tissues contain high numbers of antigen-presenting DC. DC are found in Peyer's patches as well as in the lamina propria of the gastrointestinal tract where they sample and process antigens for presentation to the immune system.20 In this study, we showed that both AAV5 and AAV6 successfully transduce DC in vitro. This is in line with previous reports showing that both AAV5 and AAV6 have a natural tropism for DC and can induce potent immune responses to viral antigens as well as the expressed transgene.9,21,22 Oral delivery of AAV may allow for greater access to DC compared with i.m. delivery leading to improved antitumor immune responses. Furthermore, it has been reported that i.m. injections of high concentrations of AAV may actually lead to the induction of immune tolerance.23,24 Indeed, when we examined for humoral- or cell-mediated immune responses, we found that mice p.o. vaccinated with either AAV5-neu or AAV6-neu demonstrated significantly higher levels of circulating anti-NEU antibodies and stronger antitumor CTL responses compared with those receiving the i.m. vaccine (Figures 3 and 4).

When comparing AAV serotypes 5 and 6 for their efficacy as an oral vaccine, AAV6-neu induced greater cell-mediated and humoral immune responses than AAV5-neu. The higher levels of circulating anti-NEU antibodies as well as greater CTL activity correlated with increased survival in the AAV6-neu–treated animals. We posited that AAV5 might be broadly disseminated due to its ability to transcytose through gut epithelium and as such may not be taken up by antigen-presenting cells in the gastrointestinal tract as efficiently as AAV6 that does not transcytose as efficiently. As evidence for this, we showed that animals treated with AAV5 had significantly greater amounts of viral DNA at distant sites such as the lung and kidneys indicating a greater dissemination of the virus. While we have not been able to directly show that AAV6 is taken up by DC or other antigen-presenting cells in the gut, we did see that animals treated with AAV6 had significantly more viral DNA in the draining lymph nodes compared with AAV5-treated animals. This is indirect evidence of superior transduction of DC with AAV6, which can then travel to the mesenteric lymph nodes. Similar results have been reported with Salmonella typhimurium-based oral vaccines.25 It also may explain why AAV6 induces greater immune responses compared with AAV5.

The ability of AAV-based vectors to induce both CTL and antibody responses represents an advance over adenoviral-based vaccines in this model. Adenoviral vectors, expressing the same neu transgene, used subcutaneously,26 or as a DC vaccine,27,28 have not been shown to effectively induce CTL responses in this model. Rather, the antitumor responses appear to be predominantly antibody-mediated. Similarly, we showed that CD4+ T-cell help was important, especially shortly after the vaccine was administered where it functions to prime B-cells for antibody production. In contrast to the adenoviral-based vaccine, AAV-neu vaccination induced tumor cell-specific CD8+ CTL and CD8+ cells were involved in the antitumor response with this vaccine. Furthermore, most adenoviral vectors are acid labile, inhibiting transduction of the gut,29 whereas AAV is resistant to the acid conditions in the gut allowing for successful oral delivery.14

While AAV vectors were initially touted for their ability to induce long-term gene expression without inducing CTL responses,2,3,4 recent studies have shown that route of vector administration, vector dose, and serotype can influence CTL and humoral responses in mice and nonhuman primates.5 This is highlighted in the current study where p.o. delivered AAV6 at high doses gave the strongest immune response, and also the strongest inhibition of tumor. The AAV6-neu oral vaccine-induced antitumor protection was also long lasting. Tumor-free animals re-challenged with tumor 120 and 320 days post-vaccination were largely protected from development of tumors with 80% of mice remaining tumor-free at 420 days after the first inoculation of tumor.

The development of an oral vaccine that is safe, effective, and that affords long-lasting protection is the holy grail of vaccine research. In this study, we have demonstrated that a single oral administration of AAV6-neu could induce significant humoral- and cell-mediated immunity that would prevent the establishment of neu-expressing breast cancer in mice. This protection was durable making AAV6-based antitumor vaccination a promising approach for further evaluation.

Materials and Methods

Cell lines. The NEU-expressing mammary cancer cell lines TUBO, TS/A, and N202.1A derived from a BALB-neuT and FVB-neuN neu-transgenic mouse, respectively, were gifts from Dr Patrizia Nanni (University of Bologna, Bologna, Italy),30,31 and were grown in Dulbecco's modified Eagle's medium (BioSource, Gaithersberg, MD) with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA) and 10 µg/ml gentamicin sulfate (BioSource). Human embryonic kidney (HEK293 and HEK-293T) and human colorectal adenocarcinoma (Caco-2) cells were purchased from the American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 10 µg/ml gentamicin sulfate (BioSource).

Recombinant adenovirus and AAVs. The cDNA encoding the extracellular and transmembrane (ECM-tm) domains of an activated rat neu oncogene was provided by Dr Augusto Amici (University of Camerino, Camerino, Italy).32 Ad.Neu is an E1, E3-deleted recombinant adenovirus expressing the rat neu ECM-tm domains driven by a cytomegalovirus promoter. The vector was generated using the AdMax system (Microbix, Toronto, Ontario, Canada), was plaque-isolated, expanded on HEK-293 cells, purified on two-step and continuous CsCl gradients, or anion-exchange column (Sartorius Stedim, Edgewood, NY), titered as plaque-forming units/ml and stored at −70 °C.

AAV5-neu and AAV6-neu are recombinant AAVs serotypes 5 or 6 expressing the neu ECM-tm domains driven by a cytomegalovirus promoter. AAV2-luc, AAV5-luc, and AAV6-luc or AAV5-GFP and AAV6-GFP are recombinant AAVs serotypes 2, 5, and 6, expressing luciferase or GFP driven by a cytomegalovirus promoter. All vectors were generated using a three-plasmid (adenovirus helper plasmid, AAV helper plasmid, and vector plasmid) cotransfection technique.33 Viruses were harvested from HEK-293T cells 48 hours after transfection and purified by CsCl density gradient centrifugation. Viral titers were determined by quantitative PCR and ranged from 1011–1013 VG/ml.

In vitro AAV transduction. HeLa cells were transduced with AAV5-neu or AAV6-neu at an MOI of 100 VG/cell. Seventy-two hours after transduction, the cells were harvested and incubated with anti-rat NEU antibodies (Oncogene Research, La Jolla, CA) for 1 hour on ice. This was followed by incubation with a secondary fluorescein isothiocyanate-labeled rabbit anti-mouse immunoglobulin, or a fluorescein isothiocyanate-labeled isotype control (BD Pharmingen, San Diego, CA). The cells were analyzed by flow cytometry using a FACSCalibur (BD Biosciences, San Jose, CA) and FlowJo software (Tree Star, Ashland, OR).

Caco-2 cells grown on transwells were incubated with AAV2-luc, AAV5-luc or AAV6-luc at an MOI of 103 VG/cell, and 24 hours later, cells were lysed and assayed for luciferase expression using the Bright-Glo luciferase assay system as per the manufacturer's instructions (Promega, Madison, WI).

Bone marrow-derived DC were isolated and grown as previously described.27,28 The DC were transduced with AAV5-GFP or AAV6-GFP at an MOI of 103 VG/cell. The DC were assayed, 24 hours after transduction, for GFP expression by flow cytometry.

In vivo AAV transduction. All animal studies were approved by the Animal Care and Use Committees of the National Cancer Institute, National Institute of Dental and Craniofacial Research, and University of Cincinnati College of Medicine. Female BALB/c mice were obtained from the Division of Cancer Treatment, National Cancer Institute (Frederick, MD) or from the Jackson Laboratory (Bar Harbor, ME). Groups (N = 3) of 6–8 weeks old female BALB/c mice were p.o. administered with 1011 VG of AAV5 or AAV6 in 100 µl of PBS by gavage. The animals were killed 1 week later and their organs were examined for the presence of AAV DNA by quantitative PCR.

In another experiment, groups (N = 3) of 6–8 weeks old female BALB/c mice were orally administered, 1011 VG of AAV6-luc in 100 µl of PBS. The animals were killed 2 weeks later and their organs examined for luciferase expression using a Xenogen live imaging system (Xenogen, Alameda, CA).

Vaccination of mice with AAV5-neu and AAV6-neu. Groups (N = 10) of 6–8 weeks old female BALB/c mice received a single vaccination with 1 × 1011 or 1 × 1012 VG of AAV5-neu, AAV6-neu, an E1, E3-deleted adenovirus expressing the neu ECM-tm (Ad.Neu), or PBS control (100 µl), either i.m., or via p.o. For either i.m. and p.o. administration, AAV and adenovirus were delivered in 100 µl of PBS. Eight weeks following vaccination, the mice were subcutaneously injected with 1 × 106 TUBO cells. The mice were evaluated twice weekly for tumor growth and were killed based on prospective Animal Care and Use Committee-approved criteria and the survival times for each treatment were determined.

In order to examine long-term protection and immunological memory, surviving tumor-free mice were re-challenged with 1 × 106 TUBO cells at 120 and 320 days post-vaccination.

Detection of serum anti-NEU antibodies. Blood was drawn before vaccination and 1 month after vaccination. Serum was separated and stored at −20 °C until assayed. N202.1A cells (neu+) were used to quantify anti-NEU antibodies as previously described.28,34 Briefly, 2 × 105 N202.1A cells were incubated with test sera diluted 1:10 in 1% fetal bovine serum in PBS at 4 °C for 1 hour. Cells were washed and incubated with fluorescein isothiocyanate-labeled rabbit anti-mouse immunoglobulin antibody (BD Pharmingen) and mean fluorescence intensity was measured by flow cytometry. N202.1E cells (neu−) were used as a control.

Detection of cell-mediated responses. To detect antigen-specific cytolytic responses, splenocytes were isolated 1 month after vaccination with either AAV5-neu, AAV6-neu or PBS. Effector cells were restimulated by co-culturing 3 × 106 splenocytes with mitomycin C-treated TUBO stimulator cells in RPMI 1640 (BioSource) supplemented with 20 units/ml of IL-2 (PeproTech, Rocky Hill, NJ) for 5 days. Effector cells were assayed for their ability to lyse TUBO or T/SA cells at E:T (effector:target) ratios of 100:1, 10:1, and 1:1. Cytotoxicity was quantified by lactate dehydrogenase release (CytoTox96 Non-Radioactive Cytotoxicity Assay; Promega) as per the manufacturer's protocol. The percent cytotoxicity was calculated as follows: 100 × ((experimental release) − (effector spontaneous release) − (target spontaneous release))/((target maximum release) − (target spontaneous release)).

To detect a CD8+ response against the NEU antigen, splenocytes were assayed for IFN-γ secretion after stimulation. Splenocytes from groups of animals (N = 3) vaccinated with AAV5-neu, AAV6-neu or PBS were pooled and plated at 2 × 106 cells per well in 24-well plates in triplicate. Splenocytes were co-cultured for 72 hours with 10 µg/ml of the CD8-dominant rat NEU epitope peptide p66 (TYVPANASL) or the irrelevant OVA257-264 peptide (SIINFEKL) as a control. Supernatants were collected and IFN-γ was measured by ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. All samples were assayed in triplicate.

Depletion of specifc immune cell populations. Groups of BALB/c mice were depleted of specific immune cell subsets using antibodies before and after oral vaccination with AAV5-neu at 1011 VG.28 Briefly, CD4+ or CD8+ cells were depleted with anti-CD4 or anti-CD8 antibodies purified from the supernatants of hybridomas GK1.5 (American Type Culture Collection) and 2.43 (American Type Culture Collection), respectively. Mice were depleted of CD4+ cells 5 days before vaccination (early) and 5 days before tumor implantation (late) by intraperitoneal injection of 200 µg of the anti-CD4 antibody for three consecutive days and continued every 3 days thereafter for the duration of the experiment. CD8+ cells were depleted 5 days before tumor implantation with 200 µg of antibody for three consecutive days and continued every 3 days thereafter for the duration of the experiment. To deplete natural killer cells, anti-asialo GM1 50 µg (Wako, Richmond, VA) was administered at the beginning 5 days before tumor implantation for three consecutive days and then continued every 3 days thereafter. Greater than 95% depletion of specific lymphocyte populations was confirmed by peripheral blood flow cytometry. Mice were inoculated with 1 × 106 TUBO cells, 8 weeks after the vaccination. Tumor size was determined 3 weeks following tumor inoculation (10 mice per group). Tumor volumes were calculated using the following formula: V = (l × w2)/2.35

Statistical analysis. Statistical analysis was performed using JMP Statistical Software version 5.1 (SAS Institute, Cary, NC). Kaplan–Meier nonparametric regression analyses were performed for tumor prevention experiments with significance determined by the log-rank test. The comparison of the effect of vaccination on antibody titers among different groups was analyzed by one-way analysis using Tukey–Kramer honestly significant difference and nonparametric Wilcoxon/Kruskal–Wallis tests. A P value of <0.05 was considered significant.

SUPPLEMENTARY MATERIAL Figure S1. Vaccination of BALB/c mice with AAV5-neu and AAV6-neu failed to prevent the development of neu− TS/A mammary tumors or induce antibodies targeting neu− N202.1E cells.

Acknowledgments

We thank Alfredo Molinolo for technical help in isolating the stomach-associated lymph nodes. This work is supported in part by National Institutes of Health, National Cancer Institute, National Institute of Dental and Craniofacial Research intramural grants to J.A.C., and the Division of Hematology-Oncology, University of Cincinnati. The authors declared no conflict of interest.

Supplementary Material

Figure S1.

Vaccination of BALB/c mice with AAV5-neu and AAV6-neu failed to prevent the development of neu− TS/A mammary tumors or induce antibodies targeting neu− N202.1E cells.

Click here for additional data file. (73.7KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Vaccination of BALB/c mice with AAV5-neu and AAV6-neu failed to prevent the development of neu− TS/A mammary tumors or induce antibodies targeting neu− N202.1E cells.

Click here for additional data file. (73.7KB, pdf)

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