A Chimpanzee-Origin Adenovirus Vector Expressing the Rabies Virus Glycoprotein as an Oral Vaccine against Inhalation Infection with Rabies Virus

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doi:10.1016/j.ymthe.2006.03.027

A Chimpanzee-Origin Adenovirus Vector Expressing the Rabies Virus Glycoprotein as an Oral Vaccine against Inhalation Infection with Rabies Virus Dongming Zhou, Ann Cun, Yan Li, Zhiquan Xiang, and Hildegund C. J. Ertl* The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA *To whom correspondence and reprint requests should be addressed. Fax: +1 215 898 3868. E-mail: [email protected].

Available online 22 June 2006

Rabies has the highest fatality rate of all human viral infections and the virus could potentially be disseminated through aerosols. Currently licensed vaccines to rabies virus are highly effective but it is unknown if they would provide reliable protection to rabies virus transmitted through inhalation, which allows rapid access to the central nervous system upon entering olfactory nerve endings. Here we describe preclinical data with a novel vaccine to rabies virus based on a recombinant replicationdefective chimpanzee-origin adenovirus vector expressing the glycoprotein of the Evelyn Rokitniki Abelseth strain of rabies virus. This vaccine, termed AdC68rab.gp, induces sustained central and mucosal antibody responses to rabies virus after oral application and provides complete protection against rabies virus acquired through inhalation even if given at a moderate dose. Key Words: rabies, adenovirus vector, vaccine, mouse model, bioterror

INTRODUCTION Rabies is an ancient disease recorded as early as in Babylonian scriptures. The name rabies is derived from the Latin word for rage, reflecting the symptoms commonly seen in infected dogs, which become aggressive and bite. Bites transmit the rabies virus, which replicates in the central nervous system and spreads to the salivary glands. Rabies virus has the highest fatality rate of all known human viral pathogens. Humans that develop rabies almost inevitably die, with few known exceptions. Most of the few survivors that have been reported were found to have extensive brain damage following the infection [1]. Exposure typically occurs through a bite from an infected animal or through mucosal contact with infectious fluids. The latter route of infection could be utilized for a bioterror attack. One possible scenario could involve spreading airborne rabies virus, which rapidly gains access to the central nervous system by entering the nerve endings of the olfactory bulb upon inhalation [2]. Other bio-attack scenarios could involve the release of infected companion animals in densely populated urban areas. Rabies virus has therefore been classified as a bCQ list bioterror agent. Rabies virus is a simple negative-stranded RNA virus that encodes five structural antigens. Of those, the rabies virus glycoprotein is the sole target for virus neutralizing 662

antibodies (VNAs), which provide full protection against virus challenge [3–5]. Due to efforts spearheaded by the World Health Organization (WHO), standardized assays to measure VNA titers are available [6]. Titers of 0.5 international units (IU) or above as determined by an infectious foci reduction assay against a WHO reference serum are protective in mammalian species tested to date. Efficacious rabies vaccines are commercially available. In developed countries, the vaccines are based on fixed strains of rabies virus that are grown in tissue culture [7]. These vaccines contain inactivated virus and adjuvant. They must be given three times in a prophylactic treatment to achieve protective immunity. Upon exposure to rabies virus, the vaccines must be given four or five times, and in cases of severe exposure they must be combined with an immunoglobulin to rabies virus (RIG). RIG, which should be of human origin, is in limited supply. The current postexposure treatment, although highly efficacious and in general well tolerated, remains very costly and requires the patient to seek medical treatment four or five times. Some of the less developed countries still use the so-called Semple vaccine derived from rabies virus propagated in the brains of infected sheep or goat and then inactivated by phenol treatment [8]. The Semple vaccine requires 14–21 daily injections given subcutaneously into the abdominal wall. The Semple vaccine is cheaper than the vaccines grown in tissue

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culture, but it has a potential for serious side effects in the form of vaccine-induced autoimmune encephalomyelitis, which afflicts ~1:200 vaccinees [9]. Although current rabies vaccines protect efficiently against exposure through the bite of a rabid animal, the effectiveness of their protection against weaponized rabies virus spread by aerosols remains unknown. Current vaccines are applied systemically in both less developed countries and developed countries. During an emergency situation such as terrorist release of an infectious agent, a more convenient route of application, such as oral application, may greatly facilitate rapid preventative vaccine distribution to a large number of humans at risk. Furthermore, oral vaccination may promote induction of mucosal antibody responses, which may provide superior protection to rabies virus acquired by inhalation. Here we describe preclinical results obtained with oral delivery of a vaccine to rabies virus based on a replication-defective chimpanzee-origin adenovirus vector expressing the rabies virus glycoprotein, called AdC68rab.gp, which was used in comparison to RabAvert, a commercial vaccine produced by Chiron Behring GmbH & Co (Germany), which is given in a three-dose regimen for preventative immunizations. Our results show that upon oral application a single dose of AdC68rab.gp induces a sustained mucosal antibody response that provides complete protection against intranasal infection with rabies virus.

RESULTS Basic Characteristics of the Vaccine Constructs RabAvert, the commercial vaccine used as a positive control in our studies, is made from the fixed Flury low egg passage (LEP) strain of rabies virus that is grown on primary cultures of chicken fibroblasts. The virus is inactivated with h-propionolactone and then purified by sucrose gradient centrifugation. Antigenically LEP is closely related to the Evelyn Rokitniki Abelseth (ERA) strain of rabies virus from which the glycoprotein inserted into the AdC68 vector originated. ERA and LEP are furthermore antigenically closely related to the challenge virus standard (CVS) strains of virus used for infection of mice [10]. E1-deleted adenoviral recombinants of common human serotypes such as 5 have yielded highly promising results as vaccine carriers for a variety of viral antigens, including the rabies virus glycoprotein, in adult and neonatal rodents, primates, and canines [11–17]. Preexisting immunity to the same adenovirus serotypes found in a significant percentage of humans was shown to interfere with the efficacy of such vaccines in this target species. To circumvent this potential problem, we used adenoviral vaccine carriers based on AdC68 virus that had been isolated from a different but closely related species, i.e., chimpanzees [18]. The AdC68 virus has ~90%

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sequence homology with the known sequences of adenovirus of the human serotype 4. Humans residing in the United States do not carry neutralizing antibodies to AdC68 virus, whereas N80% of the tested chimpanzee sera have such antibodies, demonstrating that this virus is indeed a chimpanzee virus [19]. Unlike vaccines derived from human serotypes of adenovirus, the immunogenicity of the E1-deleted AdC68 vaccine carrier is not strongly impaired by preexisting immunity to common human serotypes of adenovirus, at least in mice [20]. Biodistribution and Persistence of ADC68rab.gp Vector Upon Mucosal Application Replication-defective adenovirus recombinants have been tested extensively upon systemic application and upon administration to the airways, but their biodistribution over time upon oral application has not yet been tested in depth. We addressed this by immunizing groups of ICR mice (five adult female mice per time point) with 108 plaque-forming units (pfu) of AdC68rab.gp vector given orally or 2  107 pfu of AdC68rab.gp given intranasally. We chose a higher dose for oral administration of vector to compensate for technical inaccuracies using this route of immunization. Control mice received phosphatebuffered saline (PBS) instead. We quantified the copy number of rabies virus glycoprotein (rab.gp) in a number of tissues by a semiquantitative nested PCR at different times after vector application. We standardized samples to glyceraldehyde-3-phosphate dehydrogenase (GAPD) copy numbers determined by a real-time PCR. The nested semiquantitative PCR, which has been described in detail previously [21] and which was used to detect the sequences of the transgene of the AdC68rab.gp vector, is exquisitely sensitive and detects 2–10 copies per sample. On day 4 after oral vaccination, the rab.gp sequence could be detected in all tissues but for brain, but levels were markedly higher in lung, nasal-associated lymphoid tissue (NALT), trachea, tongue, esophagus, and soft tissues of the mouth cavity. By day 10, copy numbers of rab.gp had decreased significantly in most tissues; substantial copy numbers could still be detected in NALT, tongue, trachea, and lung, and low numbers were present in the mouth cavity, esophagus, and PeyerTs patches. In other tissues copy numbers had declined to levels below detectability. By day 28, rab.gp sequences remained detectable at levels comparable to those of day 10 in NALT and lung and low levels could be detected in trachea. By day 90 the level of rab.gp sequences had declined further in lung and NALT and had remained at the limit of detectability in trachea (Fig. 1). Overall these results indicate that initially after oral application AdC68 is widely distributed although most of the uptake occurs locally by resident cells in the mouth cavity, NALT, and lungs. From most tissues vectors or vector-carrying cells are rapidly removed. Nevertheless, low levels of vectors persist in cells of local lymphoid tissues (i.e., NALT) and in lungs.

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FIG. 1. Relative amounts of AdC68rab.gp sequences in different tissues. Groups of five ICR mice were inoculated with 108 or 2  107 pfu of AdC68rab.gp vector given orally or intranasally, respectively. Fourteen kinds of tissues were harvested 4, 10, 28, and 90 (orally only) days later, and genomic DNA was isolated from tissues of individual mice. DNA was amplified by a regular PCR using rabies virus glycoprotein gene-specific primers. After the PCR samples were checked by agarose gel electrophoresis, none of the samples showed an amplicon of the expected size, demonstrating that the first PCR had not surpassed the phase of exponential amplification. 0.1 Al of the amplicon was used as a template for a nested PCR. Genomic DNA was also amplified using primers for GAPD in a single real-time PCR and data were adjusted to indicate relative number of AdC68rab.gp DNA copies per 1  103 molecules of GAPD. The different symbols show results for individual mice.

We conducted similar studies with mice that received AdC68rab.gp vector intranasally. On day 4 after vector application high levels of rab.gp sequences could be detected in NALT, trachea, and esophagus, while low levels were found in the ovaries. By day 10 rab.gp sequences remained detectable in NALT and at low levels in the trachea and the esophagus. By day 28 most of the vector had been cleared and only very low levels were detectable in some of the mice in trachea (Fig. 1). The more rapid loss upon intranasal compared to oral administration may in part reflect that a higher dose of vector had been used for oral application. We tested groups of naive mice in parallel; rab.gp sequences could not be amplified from these mice at any of the time points (data not shown). Induction of Rabies Virus-Specific Antibodies by the ADC68rab.gp Vector We tested for induction of antibody responses to rabies virus in mice immunized with the AdC68rab.gp vector given mucosally by a number of assays. We used enzyme-

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linked immunoadsorbent assay (ELISA) on plates coated with whole rabies virus to determine the kinetics of the response in sera, saliva, and vaginal lavage upon oral application of vector (Fig. 2). We determined the biological quality of the response by measuring sera for rabies VNAs (Fig. 3) and assessed the biodistribution of B cells secreting different types of antibodies to rabies virus by ELISpot assays (Fig. 4). To test the longevity of the antibody response upon oral immunization, we immunized groups of adult ICR mice orally with 106 or 108 pfu of AdC68rab.gp vector; control mice received PBS instead. We collected sera, saliva, and vaginal wash of individual mice in 2- to 9week intervals for 6 months after vaccination and analyzed them at serial dilutions by ELISA. Figs. 2A and 2B show data for the week 2, 5, 9, 16, and 25 time points for individual sera diluted to 1:200, saliva diluted to 1:20, and vaginal lavage diluted to 1:8. It should be pointed out that saliva was harvested upon drug-induced hypersalivation, while vaginal lavage was harvested by rinsing the vaginal cavity of each mouse with 150 Al of PBS. The

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actual dilution of saliva and vaginal lavage was thus higher. Most mice immunized orally with either of the two doses of AdC68rab.gp vector developed high serum antibody levels and in each group 90% of animals had positive serum responses at least at one of the time points assayed. Vaccination with the high dose of AdC68rab.gp vector also resulted in detectable antibodies to rabies virus in vaginal secretions and in saliva in 90% of the animals. The one nonresponder to the high dose of vaccine failed to develop antibodies in sera or at mucosal sites. In the low-dose vaccine group, only 70% of animals developed antibodies that could be detected in vaginal lavage, while all of the saliva samples scored positive for at least one time point. The response came up rapidly in sera, which scored positive after 2 weeks, the earliest time point tested, while mucosal fluids became positive later. Peak responses were detected after 5 weeks and the magnitude of antibodies correlated with the vaccine dose. Antibody levels remained remarkably stable over time in sera but decreased in the mucosal fluids of several animals. There was no strict correlation between levels of antibodies in sera and those measured from saliva or vaginal lavage. We measured VNA titers from sera of different cohorts of mice immunized 5 weeks earlier with 5  106 or 5  107 pfu of AdC68rab.gp given orally, intranasally, or intramuscularly (im) (Fig. 3A). Titers induced by either route were comparable and even at the lower vector dose AdC68rab.gp vector given orally achieved protective VNA titers above 0.5 IU in four of five animals. Deviations in levels of titers assessed from sera of individual mice became markedly more pronounced at the lower vector dose in both the intranasally and the orally immunized groups, which may reflect in part the use of outbred animals and in part inaccurate dosing using mucosal routes in mice. We used a commercially available rabies virus vaccine called RabAvert (Chiron) in comparison. We vaccinated mice according to the vaccine userTs manual three times im at 2-week intervals with 1/20 or 1/50 of the dose used in humans. One intramuscular or intranasal application of 5  107 pfu of the AdC68rab.gp vector induced titers that were comparable to those achieved with the commercial vaccine upon three applications of doses that were exceedingly high based on the weight of the immunized animals. Antibody titers induced by oral immunization were more variable; nevertheless, two of five animals developed titers comparable to those elicited by the commercial vaccine given three times im. To compare mucosal antibody responses induced by the vaccines given through different routes, we tested saliva from mice that had been immunized 6 weeks earlier with AdC68rab.gp vector given at 107 pfu orally or intranasally and from mice that 6 weeks previously had completed the three-dose RabAvert regimen for antibodies to rabies virus by an ELISA. It should

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be pointed out that VNAs could not be detected reliably in lavage fluids, which most likely reflects that this assay lacks the sensitivity of the ELISA. As shown in Fig. 3B, mice immunized with AdC68rab.gp vector given orally or intranasally had comparable levels of antibodies in their saliva, while such antibodies could not be detected in mice immunized with RabAvert. Using an isotype typing assay, we showed that both oral and intranasal immunization with AdC68rab.gp induced IgA and IgG antibodies to rabies virus in saliva, while RabAvert induced mainly IgG2A and some IgG2B. It should be pointed out that the assay used for isotyping is again more sensitive than the titration assay used for Fig. 3B. We determined the distribution of rabies virus-specific IgA- and IgG-secreting B cells by an ELISpot assay with lymphocytes isolated 2 and 4 weeks after immunization from NALT, the interstitium of lungs, spleens, mesenteric lymph nodes, PeyerTs patches (Fig. 4A), and interstitial lymphocytes of the intestine (not shown). Some rabies virus-specific Ig-producing cells could be detected 2 weeks after immunization, nevertheless numbers increased markedly by 4 weeks. Oral immunization induced very high numbers of IgA-producing B cells in NALT, which also contained IgG2b-, IgG2a-, and IgG1secreting cells. Percentages of B cells secreting rabies virus-specific antibodies over all Ig-secreting B cells ranged between 14 and 20% in NALT (Fig. 4B) but remained below 5% in all other tissues. Low numbers of IgA- and IgG-secreting cells could be detected in cells isolated from the lungs and spleens. Oral immunization did not result in antibody-producing cells in gut-associated lymphoid tissues. Frequencies of rabies virus Igsecreting cells were comparatively modest in the analyzed tissues upon intramuscular immunization, which may in part reflect homing of plasma cells to bone marrow. Upon im immunization, IgG-secreting cells could be detected in the spleen but neither in gutassociated lymphoid tissues nor in lungs. Induction of Protective Immunity Vaccine-induced protection to rabies virus transmitted by a bite correlates with serum VNA titers, and titers above 0.5 IU are known to prevent disease following a peripheral challenge with an otherwise lethal dose of rabies virus. Rabies virus can infect through mucosal surfaces and it is currently unknown if the titers of serum neutralizing antibodies that are known to protect against peripheral challenge will also provide protection against mucosal challenge. We addressed this by immunizing mice once with AdC68rab.gp vector given either orally or intramuscularly. We chose moderate doses of vaccine that were below those needed to achieve complete protection of all of the animals to allow for an assessment of whether serum VNA titers to rabies virus correlate with protection against rabies virus acquired by inhalation. We tested antibody titers 3–4

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weeks after vaccination from sera and saliva of individual mice. Serum VNA titers are shown in Fig. 5. Levels of antibodies in saliva tested for by ELISA on plates coated with rabies virus or anti-mouse Ig, the latter to compensate for sampling differences, were too low to yield reliable data and are not shown. We challenged the mice 6 weeks after immunization intranasally with CVS-N2C rabies virus. Cohorts of naRve animals and of mice immunized im with a commercial vaccine to rabies

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virus (3 in 2-week intervals used at 1/20 and 1/50 of the human dose) were challenged in parallel. All of the mice immunized with the commercial vaccine survived, while all of the unvaccinated animals died following challenge. AdC68rab.gp given intramuscularly at 5  105 pfu/mouse protected 80% of animals, while a reduced dose of 5  104 pfu protected 40% of mice. Upon oral immunization with 5  105 pfu all of the animals survived the challenge, while ~80% were

FIG. 2. Antibody responses to AdC68rab.gp. Groups of 10 or 9 mice were immunized orally with (A) 108 or (B) 106 pfu of AdC68rab.gp vector, respectively. Mice were bled at the indicated intervals after vaccination. On the same dates vaginal lavage and saliva were collected. Antibodies to rabies virus were assessed from sera, vaginal lavage, and saliva from individual mice by an ELISA. Pooled sera, vaginal lavage fluids, and saliva from a cohort of naRve mice were used as control (X). Throughout the graphs, the symbols are kept identical for each individual mouse. In addition the y axis is the same for the different samples. Data show mean results of duplicate wells. Standard deviations were generally below 10% of the mean and are not shown. Samples had to meet the following criteria to be scored as positive: sera, the mean adsorbance had to be N0.1 and/or higher than the mean of the control serum + 3 SD of the mean of the experimental sample; saliva and vaginal lavage, the mean adsorbance had to be above 0.05 and/or higher than the mean of the control samples + 3 SD of the mean of the experimental sample.

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FIG. 2 (continued).

protected upon vaccination with the lower dose of 5  104 pfu. Serum VNA titers measured from individual mice did not fully correlate with protection against the intranasal challenge. Mice that had titers N5 IU were protected. Nevertheless, several of the mice that had undetectable VNA titers in sera survived (Fig. 5A), while some animals that had titers above 0.5 IU succumbed (Fig. 5B) to the infection.

DISCUSSION Our goal is to develop an improved vaccine to rabies virus that rapidly, after a single oral application, induces titers of rabies virus neutralizing antibodies that protect against the virus given through the bite of infected animals or

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through aerosol upon deliberate dissemination of the virus during a bioterror attack. In the United States, due to mandatory pet vaccinations and public awareness of potential transmission through wild carnivores, rabies in humans is rare. The few cases that are reported each year are caused by exposures to rabid bats [22,23]. These exposures, which may result in minor lesions, can be overlooked. Other cases are imported or result from organ transplants [24]. In Asia and Africa, rabies remains a major public health problem. Asia reports 35,000 deaths due to rabies annually and uses more than 10,000,000 doses of rabies vaccines for postexposure treatment. Approximately 48% of the rabies vaccine doses used in Asia are still based on brain-derived virus. Current predictions estimate 24,000

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FIG. 3. Antibody responses to rabies virus vaccines. (A) Groups of five mice were immunized orally, intranasally, or intramuscularly with the indicated amount of AdC68rab.gp vector. Additional mice were vaccinated three times with two different doses of the commercially available rabies virus vaccine called RabAvert, which is licensed for use in humans. The graph shows virus neutralizing antibody titers determined 5 weeks later from sera of individual mice (squares). The geometric mean of the titers is shown as X. Titers were tested in comparison to a WHO standard serum and adjusted to international units (IU). (B) To compare the vaccines for induction of mucosal antibody responses, mice were immunized with 5  107 pfu of AdC68rab.gp given intranasally or orally. Other mice were vaccinated with the three-dose regimen of RabAvert. Saliva was harvested 6 weeks later from immunized and naRve control mice and tested for antibodies to rabies virus by ELISA. The curves show the results for pooled samples tested in duplicate F standard deviations. (C) Saliva from the same groups of mice described in (B) was tested for the isotypes of rabies virus-specific antibodies. The bar graph shows mean results of pooled samples F standard deviation.

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deaths due to rabies in Africa, where rabies surveillance is virtually absent in many areas and where death rates thus remain underreported [25]. WHO estimates 3.3 billion humans at risk for exposure to rabies virus with a currently predicted annual fatality rate of 55,000. Without postexposure vaccine treatment this rate would raise to 303,000. On the DALY score (disability adjusted life years) rabies with current treatment ranks higher than trypanosomiasis or Dengue fever [25]. In Africa and Asia, rabies virus is most commonly transmitted though the bite of a rabid dog and in ~50% of cases infects and kills children below the age of 15. Correlates of protection are well defined for rabies virus. VNAs directed against the viral glycoprotein at titers of or above 0.5 IU provide full protection to viral challenge [3,4]. In the past, some of our efforts focused on E1-deleted adenoviral recombinants of the human serotype 5 (AdHu5) [11–13]. E1-deleted AdHu5 recombinants induce, even if given at moderate doses, superb B and CD8+ T cell responses in experimental animals. The immune responses to the transgene product far surpass those achieved with other types of subunit vaccines such as vaccinia virus recombinants or DNA vaccines [12,14]. In a mouse model for rabies, full and long-lasting protection against a severe challenge with rabies virus could be induced with a single moderate dose of an AdHu5 vaccine expressing the rabies virus glycoprotein (AdHu5rab.gp), while in contrast the currently used vaccines require three doses. Comparable results were obtained with an E1-deleted AdC68 vector expressing the rabies virus glycoprotein. Our goal was to test the performance of the AdC68rab.gp vector as an oral vaccine delivery vehicle. We initially conducted studies to determine vector biodistribution over time in groups of mice that had been immunized through the oral or intranasal route. Shortly after oral or intranasal application, AdC68rab.gp sequences could be detected, mainly locally. Oral application resulted in distribution of vector into the respiratory tract, presumably due to inadvertent inhalation of some of the vaccine. In addition, orally applied vector could be detected transiently in distant tissues including gonads, which we assume to reflect trafficking of transduced lymphoid cells rather than dissemination of the replication-defective vector. Neither oral nor intranasal vaccination resulted in transfer of vector into the central nervous system [26]. Only small amounts of vector could be detected in the intestinal tract after oral vaccination, suggesting that AdC68 vectors are destroyed during passage through the stomach. In most tissues AdC68rab.gp vector-transduced cells are rapidly eliminated, presumably by the activity of transgene or vector antigen-specific CD8+ T cells [27]. Low-level persistence was observed in NALT and lungs for at least 90 days in orally vaccinated animals. Adenoviruses acquired by natural infections are known to persist in T cells for

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FIG. 4. B cell responses to AdC68rab.gp vaccination. Mice were vaccinated orally or intramuscularly with 108 pfu of AdC68rab.gp vector. Tissues were harvested 2 and 4 weeks later, and lymphocytes were isolated and tested for rabies virus-specific B cells in an ELISpot assay. (A) Mean frequencies of rabies virus-specific B cells secreting antibodies of different isotypes F standard deviations per 106 mononuclear cells. (B) In addition lymphocytes from the different tissues were tested for secretion of IgA, IgG1, IgG2a, and IgG2b on plates coated with antibodies to the different antibody isotypes. The ratios of rabies virus-specific B cells to B cells secreting Ig in NALT are shown. In all other tissues this ratio was b5% and data are not shown.

decades [28,29] and AdC68 vectors may in part share this trait. The exact cell type that harbors AdC68 sequences and the mechanism that allows for their escape from immunosurveillance remains to be elucidated. Oral immunization was investigated in more depth than intranasal immunization mainly for logistic reasons. Although an intranasal vaccine in now on the market for influenza virus [30] we would expect that an oral vaccine could be applied with more ease under field conditions or in emergency situations. Here we show that oral vaccination results in sustained antibody titers in sera and at mucosal surfaces and we speculate that the longevity of the antibody response may in part relate to the persistence of AdC68-transduced cells. Such cells may continue to transcribe the transgene and thus provide continued stimuli to the immune system. Adenoviral vectors, including those of the human serotype 5, have been shown repeatedly to induce exceptionally sustained

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CD8+ T cell responses that fail to contract after the initial effector phase [31], supporting the hypothesis of continued production of antigen. The distribution of rabies virus Ig-secreting B cells differs markedly depending on the route of immunization. Oral, unlike systemic, immunization results in a robust local B cell response in NALT and lungs and most of the B cells detected there secrete IgA. Accordingly, oral immunization with very modest doses of AdC68 provides superior protection to intranasal challenge compared to intramuscular immunization. There is no strict correlation between rabies virus-specific VNA titers in serum and protection against rabies virus acquired by inhalation. We assume that local antibody titers within nasal secretion may be more predictive for the outcome of an intranasal challenge with rabies virus. This could not be confirmed in our preclinical mouse model, which does not allow access to nasal secretions. Nevertheless, this hypothesis is sup-

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FIG. 5. AdC68rab.gp vaccine induces protection against intranasal challenge with rabies virus. Groups of ICR mice were vaccinated with 5  105 or 5  104 pfu of AdC68rab.gp vector given im or orally. Control mice were left unvaccinated. The group sizes were as follows: im either dose, 10 mice; oral 5  105 pfu, 15 mice; oral 5  104 pfu, 33 mice; unvaccinated, 20 mice. (A) All mice of each group were bled 4–5 weeks later and VNA titers were determined. VNA titers in IU in individual mice are shown. (B) Mice were challenged with a lethal dose of rabies virus given intranasally 6 weeks after vaccination and disease-free survival was recorded. Mice were euthanized once they developed hind-leg paralyses indicative of an end stage infection with rabies virus. The vaccine protocols are shown in the legend next to the graph.

ported by the high frequencies of rabies virus-specific B cell response that could be detected in NALT. In summary our results show that oral application of the AdC68rab.gp vector induces sustained central and mucosal antibody responses and most importantly complete protection against rabies virus given intranasally. It is uncertain if aerosolized rabies virus will ever be used in a bioterror attack. Notwithstanding, the use of an orally applied adenoviral vaccine vector has broader applicability to other viruses that spread through aerosols and for which neutralizing antibodies have been identified as correlates of protection. One such example is influenza virus, including the avian H5N1 strain that is feared to trigger a global epidemic with high fatality rates once antigenic shift of the virus currently circulating in birds allows for its sustained transmission from humans to humans [32].

MATERIALS

AND

METHODS

Mice. Female ICR mice at 6–8 weeks of age were purchased from Charles River Laboratories (Boston, MA, USA) and kept in the Animal Facility of

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the Wistar Institute (Philadelphia, PA, USA). Animals were treated according to institutional guidelines. Cell lines. HEK 293 cells and BHK-21 cells were grown in DulbeccoTs modified EaglesT medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Rabies virus. Rabies virus of the ERA and CVS-11 strains were propagated on BHK-21 cells. ERA was purified by sucrose gradient centrifugation and inactivated by treatment with h-propionolactone (BPL). The protein concentration of inactivated rabies virus (ERA-BPL) was measured and adjusted to 0.1 mg/ml. The CVS-N2C strain of rabies virus [33], kindly provided by Dr. Dietzschold (Thomas Jefferson University, Philadelphia, PA, USA), was propagated in brains of newborn mice, and the lethal dose (LD) was determined by titration in adult ICR mice. Generation and propagation of adenoviral recombinants. The E1-deleted recombinant adenovirus based on chimpanzee serotype 68, termed AdC68rab.gp, expressing the glycoprotein of rabies virus strain ERA, has been described previously [34]. The adenovirus vector was propagated on HEK 293 cells and purified by CsCl gradient centrifugation, the number of virus particles was determined by spectrophotometry, and plaque-forming units were determined by titration on HEK 293 cells [35]. For some experiments unpurified vector was used. Immunization of mice. In the biodistribution study, adult female ICR mice received 108 or 2  107 pfu of AdC68rab.gp vector in saline, given once orally or intranasally. On days 4, 10, 28, and 90 after vector

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application, mice were sacrificed and perfused with cold PBS to remove blood from organs. Fourteen different types of tissues (ovaries, PeyerTs patches, small intestine, large intestine, stomach, spleen, liver, lung, trachea, esophagus, soft tissues of mouth cavity, tongue, nasal lymphoid, and brain) were harvested from individual mice and stored at 808C until use. To determine vaccine immunogenicity and efficacy, female ICR mice were vaccinated orally, intranasally, or im with various doses of AdC68rab.gp vector in saline. Rabies vaccine RabAvert (Chiron, Emeryville, CA, USA) was used as a positive control for which the dosage was adjusted for mice. Oral immunizations were performed with a 1-cc syringe to which a plastic feeding tube had been attached. The feeding tube was positioned into the esophagus and fluid (100 Al) was applied slowly. The procedure was repeated ~1 min later and an additional 100 Al was applied. For intranasal application, vector was diluted in 20 Al of saline and applied into the nostrils with the help of an Eppendorf pipette and a blunted tip. For intramuscular immunization animals were injected with vaccine diluted in 100 Al of saline into the left upper leg muscles. Collection of samples. Blood was collected by retro-orbital puncture. Sera were collected and heat inactivated prior to testing in a neutralization assay. To collect saliva, mice were injected intraperitoneally with 0.1 mg of pilocarpine hydrochloride (P6503; Sigma, St. Louis, MO, USA) in 100 Al of sterile saline. After ~5 min when mice started salivating, saliva was collected from the oral cavity using plastic Pasteur pipettes. Vaginal lavage was harvested by rinsing the vaginal cavity two times with 75 Al of sterile saline applied and collected with an Eppendorf pipette and a blunted tip. Saliva and vaginal lavage samples were centrifuged for 3 min at 10,000 rpm in an Eppendorf centrifuge to remove cells and debris prior to their testing for antibodies. Tissues were harvested from exsanguinated mice. PeyerTs patches were harvested after the intestines had been recovered. Nasal-associated lymphoid tissues were harvested after the bony parts of the nose had been removed. Cells were recovered from lymph nodes, including PeyerTs patches and spleens, upon grinding tissues through a fine wire mesh with the help of forceps. Lymphocytes were isolated from the lungs upon digestion with collagenase (20 mg/ml in serum-free medium, 3 ml per lung, treated for 1 h at 378C). Cells were then purified by Percoll gradient centrifugation. Average recoveries of mononuclear cells from the various tissues per mouse were ~108 cells/spleen, 0.5–2  106 cells/lung, 106 cells/ NALT, 2  106 cells/set of PeyerTs patches. Challenge of mice. Six weeks after vaccination, mice were challenged with rabies virus CVS-N2C given intranasally at 10 LD50. After challenge, mice were checked daily and recorded individually for at least 3 weeks. They were euthanized once they developed complete hind-leg paralysis indicative of terminal rabies encephalitis. Quantification of rab.gp in tissues. Genomic DNA of tissues harvested from vaccinated mice was extracted using DNeasy Tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturerTs instructions. The glycoprotein gene of rabies virus ERA strain in each tissue was amplified, first by regular PCR (5V and 3V primers were 5VCCTGGAGCCCGATTGACATAC-3V and 5V-ACAAGGTGCTCAATTTCGTC3V, respectively). The PCR consisted of 30 cycles of 948C for 50 s, 558C for 50 s, and 728C for 1 min. The amplicon (0.1 Al) from the first PCR product was then used as template for a second real-time PCR to semiquantify the rab.gp gene in different tissues. Nested primers were used to amplify a 266-bp fragment in rab.gp (5V and 3V primers were 5VAAAGCATTTCCGCCCAACAC-3V and 5V-GGTTAGTGGAGCAGTAGGTAA3V, respectively). The second PCR was run for 40 cycles at 958C for 5 s, 638C for 10 s, 728C for 15 s, and 858C for 4 s. The copy numbers of rab.gp in each tissue were normalized in comparison to GAPD sequences quantified by a real-time PCR as described [10]. ELISA. Sera, saliva, and vaginal lavage were tested on plates coated with inactivated rabies virus. Briefly, round-bottom ELISA plate wells were coated overnight with 0.2 Ag of ERA-BPL virus diluted in 100 Al of coating buffer (15 mM Na2CO3, 35 mM NaHCO3, and 3 mM NaN3, pH 9.6). The next day plates were blocked with 150 Al PBS containing 3% BSA for 24 h. Plates were washed with PBS twice, dried, and kept at 208C. Sera, saliva,

MOLECULAR THERAPY Vol. 14, No. 5, November 2006 Copyright C The American Society of Gene Therapy

and vaginal lavage samples were serially diluted in PBS supplemented with 3% BSA. Dilutions of samples were incubated in duplicates at 100 Al/ well on the ERA-BPL-coated plates for 1 h at room temperature. Plates were washed, and a 1:200 dilution of alkaline phosphatase-conjugated goat anti-mouse Ig (Cappel, Irvine, CA, USA) was added to each well for 1 h at room temperature. After being washed, plates were incubated for 20 min with substrate (10 mg d-nitrophenyl phosphate disodium dissolved in 10 ml of 1 mM MgCl2, 3 mM NaN3, and 0.9 M diethanolamine, pH 9.8) and then read in an automated ELISA reader at 405 nm. ELISpot assay. ELISpot assays were used to quantify plasma cells secreting rabies virus-specific antibodies and to determine the isotypes of these antibodies. Adult ICR mice were immunized orally with 108 pfu AdC68rab.gp vector. Two and 4 weeks after vaccination, mice were euthanized, and cells from NALT, lung interstitium, spleen, mesenteric lymph node, and PeyerTs patches were harvested as described previously [36] and tested for antibody-secreting B cells in an ELISpot assay. Briefly, Multiscreen 96-well filtration plates (Millipore, Bedford, MA, USA) were coated with 2 Ag/ml ERA-BPL or 10 Ag/ml goat-derived capture antibodies to total mouse IgTs (Calbiochem Hybridoma Subisotyping Kit; Calbiochem, San Diego, CA, USA) overnight. They were then washed with DMEM supplemented with 10% FBS. The same medium was added and plates were incubated at 378C for 4 h. The medium was removed and different concentrations (106 to 104 cells in 100 Al per well) of lymphocytes in DMEM with 10% FBS were added in duplicate. Plates were incubated in a CO2 incubator overnight and washed four times with PBS containing 0.05% Tween 20. Rabbit anti-mouse IgA, IgG1, IgG2a, and IgG2b sera were added. After 2 h of incubation at room temperature, plates were washed with PBS, and HRP-conjugated goat anti-rabbit antibody was added into each well. After 1 h of incubation at room temperature, followed by washing with PBS, color was developed with stable DAB solution (Invitrogen, Carlsbad, CA, USA). The Ig-secreting cells, visualized as dark red spots, were counted and analyzed in an automated ELISpot counter (CTL Analyzers, Cleveland, OH, USA) and the frequency was determined in relation to the total number of nucleated cells added to the well. Virus neutralization assay. Sera of mice were tested on BHK-21 cells for neutralization of CVS-11 virus, which is antigenically closely related to the ERA virus, as described previously [4]. Assays were standardized by inclusion of a reference serum from the World Health Organization. Data are expressed as international units. Statistical analysis. Experiments were conducted at least twice using at least five mice per group. Sera, saliva, and vaginal lavage were tested by ELISA or neutralization assay in duplicates. Results show the means F SD. Differences between two groups were analyzed by StudentTs t test. Data with P b 0.05 were viewed as showing a statistically significant difference.

ACKNOWLEDGMENTS We thank Nia Tatsis (Wistar Institute) and Marcio Lasaro (Wistar Institute) for helpful discussion, Bernhard Dietzschold (Jefferson University, Philadelphia, PA, USA) for providing stocks of the challenge virus, and Colin Barth for assembly of the manuscript. This work was supported by grants from NIAID/NIH. RECEIVED FOR PUBLICATION JANUARY 14, 2006; REVISED MARCH 17, 2006; ACCEPTED MARCH 27, 2006.

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