Recombinant Human Adenovirus Type Research Articles

Langerin negative dendritic cells promote potent CD8 + T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

Bovine Type III Interferon Significantly Delays and Reduces the Severity of Foot-and-Mouth Disease in Cattle

Interferons (IFNs) are the first line of defense against viral infections. Although type I and II IFNs have proven effective to inhibit foot-and-mouth disease virus (FMDV) replication in swine, a similar approach had only limited efficacy in cattle. Recently, a new family of IFNs, type III IFN or IFN-λ, has been identified in human, mouse, chicken, and swine. We have identified bovine IFN-λ3 (boIFN-λ3), also known as interleukin 28B (IL-28B), and demonstrated that expression of this molecule using a recombinant replication-defective human adenovirus type 5 (Ad5) vector, Ad5-boIFN-λ3, exhibited antiviral activity against FMDV in bovine cell culture. Furthermore, inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and upregulation of IFN-stimulated gene expression in the upper respiratory airways and skin. In the present study, we demonstrated that disease could be delayed for at least 6 days when cattle were inoculated with Ad5-boIFN-λ3 and challenged 24 h later by intradermolingual inoculation with FMDV. Furthermore, the delay in the appearance of disease was significantly prolonged when treated cattle were challenged by aerosolization of FMDV, using a method that resembles the natural route of infection. No clinical signs of FMD, viremia, or viral shedding in nasal swabs was found in the Ad5-boIFN-λ3-treated animals for at least 9 days postchallenge. Our results indicate that boIFN-λ3 plays a critical role in the innate immune response of cattle against FMDV. To this end, this work represents the most successful biotherapeutic strategy so far tested to control FMDV in cattle.

Early detection and visualization of human adenovirus serotype 5-viral vectors carrying foot-and-mouth disease virus or luciferase transgenes in cell lines and bovine tissues

Recombinant replication-defective human adenovirus type 5 (Ad5) vaccines containing capsid-coding regions from foot-and-mouth disease virus (FMDV) have been demonstrated to induce effective immune responses and provide homologous protective immunity against FMDV in cattle. However, basic mechanisms of Ad5-FMDV vaccine function including virus tropism, transgene expression, and antigen presentation, remain incompletely understood. The current study characterized the dynamics of Ad5 viral vector (Ad5-FMDV-A24 and Ad5-luciferase) infection in cell lines and early post-inoculation vector-host interactions in cattle. Adenovirus dissemination was described utilizing novel rPCR, rRT-PCR, luminometry, and immunomicroscopy techniques. In vitro infection of human and bovine cells with both Ad5 vectors resulted in dose-dependent detection of vector DNA, mature mRNA transcripts, and transgene-encoded proteins. Subsequent to intramuscular inoculation of cattle, Ad5 and transgene products were detected at the injection sites of all animals at all time-points examined (6, 24, and 48 hpi). Microscopically, injection sites were characterized by marked infiltrates of interstitium consisting of predominantly large mononuclear cells. Immunomicroscopy indicated these cells infrequently contained adenovirus and/or transgenic proteins and were phenotypically consistent with antigen-presenting cells (macrophages and dendritic cells). Vector DNA and mature mRNA transcripts were first detected at the draining and local lymph nodes as early as 6 hpi and systemically at 24 hpi. These results provide novel insights for understanding Ad5-mediated immunity against FMDV using novel techniques that will contribute to ongoing efforts for the improvement of future Ad-FMDV vaccine platforms.

CD8+ T-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells

AbstractWe have recently reported that CD8+ T-cell memory maintenance after immunization with recombinant human adenovirus type 5 (rHuAd5) is dependent upon persistent transgene expression beyond the peak of the response. In this report, we have further investigated the location and nature of the cell populations responsible for this sustained response. The draining lymph nodes were found to be important for primary expansion but not for memory maintenance, suggesting that antigen presentation through a nonlymphoid source was required. Using bone marrow chimeric mice, we determined that antigen presentation by nonhematopoietic antigen-presenting cells (APCs) was sufficient for maintenance of CD8+ T-cell numbers. However, antigen presentation by this mechanism alone yielded a memory population that displayed alterations in phenotype, cytokine production and protective capacity, indicating that antigen presentation through both hematopoietic and nonhematopoietic APCs ultimately defines the memory CD8+ T-cell response produced by rHuAd5. These results shed new light on the immunobiology of rHuAd5 vectors and provide evidence for a mechanism of CD8+ T-cell expansion and memory maintenance that relies upon both hematopoietic and nonhematopoietic APCs.

An adenovirus type 5 (AdV5) vector encoding an envelope domain III-based tetravalent antigen elicits immune responses against all four dengue viruses in the presence of prior AdV5 immunity

Dengue is a mosquito-borne viral disease caused by four antigenically distinct serotypes of dengue viruses (DENVs). This disease, which is prevalent in over a hundred tropical and sub-tropical countries of the world, represents a significant global public health problem. A tetravalent dengue vaccine capable of protecting against all four DENV serotypes has been elusive so far. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we have utilized a discrete carboxy-terminal region of the major DENV envelope (E) protein, known as domain III (EDIII), which mediates virus entry into target cells and contains multiple serotype-specific neutralizing epitopes, to create a chimeric tetravalent antigen. This antigen derived by in-frame fusion of the EDIII-encoding sequences of the four DENV serotypes was expressed using a replication-defective recombinant human adenovirus type 5 (rAdV5) vaccine vector. This rAdV5 vector induced cell-mediated immune responses and virus-neutralizing antibodies specific to each of the four DENVs in mice. Interestingly, anti-AdV5 antibodies did not suppress the induction of DENV-specific neutralizing antibodies. We observed that anti-AdV5 antibodies in the sera of immunized mice could promote uptake of a rAdV5-derived reporter vector into U937 cells, suggesting that pre-existing immunity to AdV5 may in fact facilitate the uptake of rAdV5 vectored vaccines into antigen presenting cells. This work presents an alternative approach to developing a single component tetravalent vaccine that bypasses the complexities inherent in the currently adopted four-in-one physical mixture approach.

Generation and efficacy evaluation of a recombinant adenovirus expressing the E2 protein of classical swine fever virus

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which causes significant economic losses to the pig industry worldwide. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. This study was aimed to develop a recombinant human adenovirus type 5 expressing the CSFV E2 gene (rAdV-E2) and evaluate its efficacy in rabbits and pigs. The results showed that the rabbits and the pigs immunized with the rAdV-E2 developed high-level CSFV-specific neutralizing antibodies. The rAdV-E2-immunized rabbits were protected from fever induced by infection with C-strain, which is pathogenic to the rabbit, and the rAdV-E2-immunized pigs were protected from lethal challenge with highly virulent Shimen strain. This indicates that the recombinant adenovirus can be an attractive candidate vaccine for preventing CSF.

A recombinant replication-defective human adenovirus type 3: A vaccine candidate

Human adenovirus type 3 (HAdV-3) cause respiratory infections globally. There is no safe and efficient HAdV-3 vaccine thus far. In this report, a replication-defective human adenovirus type 3 was constructed by deletion of the whole E1 gene. A HEp-2 cell-based cell line designated HEp-2/E1 was generated and was shown to complement the function of E1 deleted mutant. A partial genome (9.5–100 mu) of HAdV-3 was cloned into pPolyII vector to be a backbone plasmid. A shuttle vector carrying eGFP gene was also constructed. The backbone and shuttle plasmids were co-transfected into the HEp-2/E1 cell line and 293 cell line separately. Typical cytopathic effect (CPE) appeared in the HEp-2/E1, but not in 293 cells 2 weeks after transfection. Wild-type HAdV-3 is neutralized by the sera from the mice immunized with the recombinant HAdV-3. The results demonstrated that the recombinant adenovirus type 3 may serve as a HAdV-3 vaccine candidate.

Mitochondrially targeted wild-type p53 induces apoptosis in a solid human tumor xenograft model

Classic but also novel roles of p53 are becoming increasingly well characterized. We previously showed that ex vivo retroviral transfer of mitochondrially targeted wild type p53 (mitop53) in the Eμ myc mouse lymphoma model efficiently induces tumor cell killing in vivo. In an effort to further explore the therapeutic potential of mitop53 for its pro-apoptotic effect in solid tumors, we generated replication-deficient recombinant human Adenovirus type 5 vectors. We show here that adenoviral delivery of mitop53 by intratumoral injection into HCT116 human colon carcinoma xenograft tumors in nude mice is surprisingly effective, resulting in tumor cell death of comparable potency to conventional p53. These apoptotic effects in vivo were confirmed by Ad5-mitop53 mediated cell death of HCT116 cells in culture. Together, these data provide encouragement to further explore the potential for novel mitop53 proteins in cancer therapy to execute the shortest known circuitry of p53 death signaling.

Immune response in rhesus macaques after mixed modality immunisations with DNA, recombinant adenovirus and recombinant gp120 from human immunodeficiency virus type 1

The establishment of effective regimens for a vaccine against human immunodeficiency virus type 1 (HIV-1) is urgently needed. In the present study we have produced HIV-1 gp120 from a vaccine-relevant primary R5 isolate in recombinant vaccinia (rVV)-infected Vero cells. We have investigated the effect of boosting with this protein in mixed modality immunisations of rhesus macaques following different immunisation. As reported earlier, animals were primed with codon-optimised HIV-1(BX08)env DNA delivered as plasmid or as replication-deficient recombinant human adenovirus type 5 (rAd5), which both induced specific antibody and cellular immune responses (1). Boosting with rAd5 temporarily had increased the anti-gp120 antibody titres approximately 1 log (rAd5+rAd5) or 3 log (DNA+rAd5) (1). However, secondary rAd5 boosting showed less effect due to the induced vector-specific immunity. To further boost the antibody response, the rgp120(BX08) was injected with Quadri A saponin adjuvant. The protein boosting resulted in a 1-2 log antibody increase and also boosting of the cell-mediated immune response. Neutralising antibodies to the heterologous HIV-1(MN) were detected; however, neutralising antibodies to the primary HIV-1(Bx08) isolate were seen only transiently after rAd5 but not the rgp120 immunisation. It is concluded that the rgp120(Bx08) reagent from rVV-infected Vero cells is functional and immunogenic in macaques, inducing both antibody and cellular immunity. The rgp120(Bx08) is a relevant model antigen that may be used to boost antibody and cellular immunity in mixed modality vaccine regimens against HIV-1 in higher animals.

Low-dose adenovirus vaccine encoding chimeric hepatitis B virus surface antigen-human papillomavirus type 16 E7 proteins induces enhanced E7-specific antibody and cytotoxic T-cell responses.

Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (10(6) infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8(+)-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein.

Bovine herpesvirus 1 glycoprotein D expression in bovine upper respiratory tract mediated by a human adenovirus type 5

Bovine herpesvirus 1 glycoprotein D (gD) gene expression by recombinant replication defective human adenovirus type 5 (HAdV-5) was investigated in calves using indirect immunofluorescence microscopy (IIFM), confocal laser scanning microscopy (CLSM) and RT-PCR. One fold intranasal instillation of HAdV-5-expressing gD in the cattle upper respiratory tract showed a short term expression of at least 5 days, but not 10 days, limited only to epithelial cells localised in the epithelium of the nasal mucosa in one out of six calves. Observed limited gene transfer into well differentiated cattle airway epithelial cells must be taken into consideration in order to enhance transfection efficiency, and consequently the vaccine potential of this vector.

Immunogenicity in Mamu-A*01 rhesus macaques of a CCR5-tropic human immunodeficiency virus type 1 envelope from the primary isolate (Bx08) after synthetic DNA prime and recombinant adenovirus 5 boost.

Envelopes of primary R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates may be particularly relevant for vaccine purposes and should be evaluated for immunogenicity in animals including macaques before carrying out human vaccine trials. In the present study, the immunogenicities of synthetic HIV-1 env DNA vaccines, which had been derived from the early primary isolate Bx08 and contain humanized codons, were evaluated in mice, guinea pigs and rhesus macaques. Neutralization sensitivity of the HIV-1(Bx08) isolate was found to resemble that of other primary isolate prototypes. Immunogenicity of gp120 delivered as codon-optimized DNA vaccine was comparable to that of recombinant gp120 protein plus adjuvant in mice. Similarly, DNA vaccination of guinea pigs with synthetic gp140(Bx08) and gp150(Bx08) DNA induced a strong antibody response independent of the gene construct and DNA immunization route. Mamu-A*01 rhesus macaques were DNA vaccinated with synthetic gp150(Bx08) or gp140(Bx08) DNA and boosted with a replication-deficient recombinant human adenovirus type 5 expressing a synthetic gp120(Bx08) gene. DNA-vaccinated rhesus macaques developed specific CD8+ T lymphocyte responses and anti-rgp120(IIIb) antibody responses. Both the humoral and cellular responses were significantly improved following intramuscular boosting with the recombinant adenovirus. The demonstrated humoral and cellular immunogenicities of these HIV Bx08 Env vaccines in non-human primates encourages their further development as one component in candidate HIV vaccines for humans.

Budding and secretion of HIV Gag–Env virus-like particles from recombinant human adenovirus infected cells

We have characterized the assembly, budding and extra-cellular release of human immunodeficiency virus (HIV) Gag–Env virus-like particles (VLPs) from human embryonic kidney cells (293 cells expressing the E1a protein of adenovirus) infected with recombinant replication-defective human adenovirus type 5. Recombinant human adenovirus vectors expressing the chimeric Gag–Env protein were constructed by inserting the gag– env fusion gene into the E1a region of the human adenovirus type 5. Biochemical and immunological analyses of VLPs recovered from the culture supernatant revealed that these particles contain the HIV-2 Gag protein and segments of Env protein from the HIV-1 gp120. This chimeric Gag–Env protein interacted with HIV-1 positive patient sera and with HIV-2 Gag monoclonal antibody. Immunoelectron microscopy of the 293 cells infected with the recombinant adenoviruses showed that the HIV Gag–Env antigen is present in the VLPs. Thin-section electron microscopy (EM) revealed that the Gag–Env VLPs bud through the cytoplasmic membrane, as well as through membranes of intracellular vacuoles. The thin-section EM showed that the Gag–Env VLPs have a spherical morphology with an electron-dense ring. The size of VLPs range from 110 to 140 nm in diameter, which is slightly larger than that of the Gag particles without Env protein fusion. Mice immunized with recombinant adenoviruses generated antibodies that specifically reacted with Gag–Env chimeric proteins. Our results support the idea that the replication-defective adenovirus could be used to induce immune responses that might be useful in a vaccine against HIV/AIDS.

Inhibition of duck hepatitis B virus replication by intrahepatic expression of an antiviral resistance gene.

Resistance genes coding for inhibitors of hepadnaviral replication, such as ribozymes, antisense RNA, and dominant negative mutants have been shown to be effective in transfected hepatoma cells. In vivo studies, however, are not available to date. Here we expanded the use of the duck hepatitis B virus (DHBV) model for studying antiviral resistance genes in vivo. Animals were experimentally infected by intravenous injection of DHBV-positive serum in ovo. The use of recombinant human adenovirus type 5 and avian adenovirus CELO for gene transfer was evaluated. Adenovirus type 5 transduced more than 95% and CELO less than 1% of embryonic hepatocytes in vivo. Adenovirus type 5 interfered with DHBV replication (viral cross-talk), but this effect was moderate and did not preclude analysis of specific antiviral effects. Thus adenoviral transfer of a dominant negative mutant prior to DHBV infection (intracellular immunization) yielded 100-fold suppression of viral replication compared to the green fluorescent protein marker gene. Neither gene was toxic. These data demonstrate that a prototype anthepadnaviral resistance gene is functional in vivo. Duck embryos represent a useful model for evaluating gene therapeutic strategies in vivo without the need for large scale preparations of gene delivery vehicles.

Novel vaccine delivery systems: solutions to HIV vaccine dilemmas?

The history of successful vaccination against severe viral diseases such as smallpox poliomyelitis or measles led to the initial hope that a vaccine against AIDS would be developed quickly. However an effective vaccine against HIV needs to overcome substantial obstacles that emerged as research progressed. Due to life cycle HIV can effectively hide from the host immune response integrating itself as proviral DNA in the host cell genome. A strategy pursued to deal with this difficulty is to include early viral regulatory proteins such as Tat Rev or Nef as vaccine antigens for induction of immune responses that can recognize and destroy HIV-infected cells as soon as the virus life cycle is activated [12]. The virus preferentially targets and destroys host immune cells such as T-helper lymphocytes macrophages and dendritic cells that are probably essential to maintain an effective antiviral immune response. This would imply that vaccine-elicited immunity unlikely to be able to prevent infection itself must be able to quickly control virus replication to prevent harm to the immune system. The high antigenic variability of HIV can be considered as an extremely effective immune-evasion strategy. Because of the low fidelity of the viral RNA polymerase virus progeny always represents a collection of RNA genomes (quasi-species) with random mutations. In vivo selection of immunodeficiency virus variants that can evade the recognition of neutralizing antibodies is common and strong virus-specific cytotoxic T-cell responses can select for escape variants already during resolution of primary viremia [3]. Most HIV infections are acquired sexually via the genital or rectal mucosae; however at these entry sites it appears difficult to induce strong antiviral immunity by vaccination. Finally HIV infection is a poverty-related disease that is particularly threatening health in societies of the developing world. Therefore vaccine candidates must be safe and feasible in production and administration to be eligible for use where most needed. (excerpt)

Human adenovirus type 5 vectors expressing rabies glycoprotein

The prevalence of wildlife rabies throughout the world and the continued spread of this disease in North America highlights the need for oral vaccines which may be used safely and effectively to vaccinate a number of species that are reservoirs or vectors of rabies. We have previously shown that AdRG1, a replication competent recombinant human adenovirus type 5 (Ad5) expressing a rabies glycoprotein (RG), can induce immunity to rabies in rodent, canine, and skunk model systems. To improve the Ad5 vector system as a potential oral vaccine, we have constructed additional Ad5 recombinant vectors and compared RG expression in cell culture and immunogenicity in animals. Two new replication competent vectors are compared. AdRG1.3, which carries RG with accompanying SV40 polyA addition sequences within an E3 deletion, and AdRG4, which has RG in the E3 deletion but under the control of an exogenous Ad2 major late promoter, both express higher levels of RG in permissive cell culture than did AdRG1 and both elicit high levels of serum anti-rabies antibodies by parenteral or oral routes in animals. AdRG1.3 may be a more effective vaccine vector in species which are non-permissive for the replication of human Ad5.

Heat-shock enhanced reactivation of a UV-damaged reporter gene in human cells involves the transcription coupled DNA repair pathway.

A recombinant nonreplicating human adenovirus type 5, Ad5HCMVsp1lacZ, expressing the lacZ gene under control of the human cytomegalovirus immediate early promoter, was used to assess the effect of heat-shock (HS) on DNA repair of a UV-damaged reporter gene. Host cell reactivation (HCR) of beta-galactosidase (beta-gal) activity for UV-irradiated Ad5HCMVsp1lacZ was used as an indicator of DNA repair in the transcribed strand of an active gene. Repair was examined in heat-shock (HS) pretreated and mock-treated normal fibroblasts, normal lung epithelial cells, xeroderma pigmentosum group A, C, D and G fibroblasts (XP-A, XP-C, XP-D and XP-G), Cockayne's syndrome group A fibroblasts (CS-A), SV40-transformed normal fibroblasts (GM637f) and 5 tumour cell lines (SKOV-3, HeLa, HT29, SCC-25 and U20S). HS enhanced reactivation (HSER) of the reporter gene was detected in normal cells, HT29 tumour cells and XP-C fibroblasts. HSER was reduced or absent in all other XP, CS and tumour cell lines tested. HSER in normal and XP-C cell lines, but not CS-A, XP-A, XP-D or XP-G cells, suggests that HS treatment can enhance the repair of UV-damaged DNA through an enhancement of transcription coupled repair (TCR) or a mechanism which involves the TCR pathway. Since this response was absent in the SV40-transformed fibroblast cell line and 4 of 5 tumour cell lines examined, HSER of beta-gal activity for UV-irradiated Ad5HCMVsp1lacZ also requires some cellular function(s) affected by transformation.

Vaccination of pigs with replication-defective adenovirus vectored vaccines: the example of pseudorabies

The efficacy of a recombinant human adenovirus type 5 expressing gD, one of the immunogenic glycoprotein of pseudorabies virus, was tested in pigs. Due to the deletion of the E1a gene, the recombinant virus is unable to replicate in non transcomplementing cells but is capable of eliciting an immune response against gp50 after inoculation into animals. The virus was formulated in a water/oil/water emulsion, a strategy previously shown to enhance the immune response against the virus-induced gp50. Pigs of 18–25 kg were vaccinated twice and the recombinant virus was not isolated from nasal and rectal swabs taken after each injection of the vaccine. High levels of neutralizing antibodies were induced by the vaccination. Protection against a severe challenge was effective, as measured by growth performance (dG = 1.73), and reduction of the time of excretion of the challenge strain (mean time: 4.4 days for the vaccinated and 7.9 days for the control pigs). These results show that non replicating adenoviruses are able to induce a strong protective immune response against foreign genes in pigs, which may be of general interest for the design of pig vaccines.

Immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees.

Recombinant human adenovirus (Ad) type 4-, 5-, and 7-vectored vaccines expressing either the HIV env or gag-protease genes were tested for immunogenicity in three chimpanzees. The first phase of the vaccination protocol consisted of a primary and two booster immunizations with Ad-HIVs by the oral route of administration, followed by a single booster immunization with Gag and/or Env subunit vaccines. The second phase of the vaccination protocol consisted of intranasal administration of Ad-HIVs previously administered by the oral route. Following the first phase adenovirus was shed into stools for only 1-7 days and modest type-specific anti-adenovirus neutralizing antibody titers were induced. Strong anti-Env binding antibody responses were detected in all three animals following the second oral booster immunization. One chimpanzee responded with a low-titered type-specific neutralizing antibody response to HIV. Cell-mediated immune responses to Env were not detected after the primary vaccination, but were detected following all booster immunizations. Administration of the Gag subunit vaccine boosted both humoral and cell-mediated immune responses to Gag antigens. In contrast, the Env subunit vaccine boosted cellular but not humoral immune responses. In the second phase of the vaccination protocol, both virus shedding and anti-adenovirus responses were enhanced. All three chimpanzees responded to the intranasal administration of Ad7-HIVs with boosted anti-HIV serum responses, including low-titered type-specific neutralizing antibodies, elicited anti-HIV antibodies at secretory sites, and stimulated cell-mediated immune responses to both Gag and Env antigens.

Use of Human Adenovirus-based Vectors for Antigen Expression in Animals

An infectious recombinant human adenovirus type 5 (Ad5) vector, AdG12, which carries the glycoprotein gene of vesicular stomatitis virus (VSV) and expresses that gene in cultured HeLa cells was used to examine the host range of insert expression by human Ad vectors. The VSV glycoprotein was expressed in bovine, canine and murine cells when infected with AdG12 in culture. These cell lines are respectively permissive, non-permissive and semi-permissive for human Ad5 replication. Administration of the AdG12 vector to calves, piglets or dogs by either the subcutaneous or oral route resulted in the production of high titres of neutralizing antibodies to VSV. Mice injected intraperitoneally with the vector produced neutralizing antibodies and were protected against subsequent intravenous challenge with normally lethal doses of VSV. This work demonstrates the utility of human adenoviral vectors for antigen expression in a number of non-human cell lines and for the induction of an immune response to the delivered antigen in a number of species.