Optimizing HIV-1-specific CD8+ T-cell induction by recombinant BCG in prime-boost regimens with heterologous viral vectors.

The desire to induce HIV-1-specific responses soon after birth to prevent breast milk transmission of HIV-1 led us to propose a vaccine regimen which primes HIV-1-specific T cells using a recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) vaccine. Because attenuated live bacterial vaccines are typically not sufficiently immunogenic as stand-alone vaccines, rBCG-primed T cells will likely require boost immunization(s). Here, we compared modified Danish (AERAS-401) and Pasteur lysine auxotroph (222) strains of BCG expressing the immunogen HIVA for their potency to prime HIV-1-specific responses in adult BALB/c mice and examined four heterologous boosting HIVA vaccines for their immunogenic synergy. We found that both BCG.HIVA(401) and BCG.HIVA(222) primed HIV-1-specific CD8(+) T-cell-mediated responses. The strongest boosts were delivered by human adenovirus-vectored HAdV5.HIVA and sheep atadenovirus-vectored OAdV7.HIVA vaccines, followed by poxvirus MVA.HIVA; the weakest was plasmid pTH.HIVA DNA. The prime-boost regimens induced T cells capable of efficient in vivo killing of sensitized target cells. We also observed that the BCG.HIVA(401) and BCG.HIVA(222) vaccines have broadly similar immunologic properties, but display a number of differences mainly detected through distinct profiles of soluble intercellular signaling molecules produced by immune splenocytes in response to both HIV-1- and BCG-specific stimuli. These results encourage further development of the rBCG prime-boost regimen.

Novel recombinant Mycobacterium bovis BCG, ovine atadenovirus, and modified vaccinia virus Ankara vaccines combine to induce robust human immunodeficiency virus-specific CD4 and CD8 T-cell responses in rhesus macaques.

Mycobacterium bovis bacillus Calmette-Guérin (BCG), which elicits a degree of protective immunity against tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo T-cell assays, BCG.HIVA(401) as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA(401) was used as a priming component in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell responses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited into the memory pool. These desirable features are thought to be associated with good control of HIV-1 infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA(401) and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration.

Cryoelectron microscopy map of Atadenovirus reveals cross-genus structural differences from human adenovirus.

A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-A resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent "knobs" on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.

Production and release testing of ovine atadenovirus vectors.

Gene-directed enzyme prodrug therapy (GDEPT) is an emerging approach for the treatment of cancers. A variety of viral vectors have been used to deliver genes that encode the relevant enzymes, and some have been tested in clinical trials. To ensure the potency and efficacy of such vectors and to obtain regulatory approval to administer them to humans, it is necessary to develop a suite of assays that provide quality assurance. New GDEPT vectors based on ovine atadenovirus and Escherichia coli purine nucleoside phosphorylase (PNP) have been developed for first time use in humans in a phase I trial for the treatment of prostate cancer. Here we describe methods for their production together with several quality-control assays. In particular, a functional cell killing assay was devised to measure the potency of PNP-GDEPT vectors, the principles of which could easily be adapted to other systems.

PKC alpha is activated but not required during glucose-induced insulin secretion from rat pancreatic islets.

The role of protein kinase C (PKC) in glucose-stimulated insulin secretion (GSIS) is controversial. Using recombinant adenoviruses for overexpression of PKC alpha and PKC delta, in both wild-type (WT) and kinase-dead (KD) forms, we here demonstrate that activation of these two PKCs is neither necessary nor sufficient for GSIS from batch-incubated, rat pancreatic islets. In contrast, responses to the pharmacologic activator 12-O-tetradecanoylphorbol-13-acetate (TPA) were reciprocally modulated by overexpression of the PKC alpha WT or PKC alpha KD but not the corresponding PKC delta adenoviruses. The kinetics of the secretory response to glucose (monitored by perifusion) were not altered in either cultured islets overexpressing PKC alpha KD or freshly isolated islets stimulated in the presence of the conventional PKC (cPKC) inhibitor Go6976. However, the latter did inhibit the secretory response to TPA. Using phosphorylation state-specific antisera for consensus PKC phosphorylation sites, we also showed that (compared with TPA) glucose causes only a modest and transient functional activation of PKC (maximal at 2-5 min). However, glucose did promote a prolonged (15 min) phosphorylation of PKC substrates in the presence of the phosphatase inhibitor okadaic acid. Overall, the results demonstrate that glucose does stimulate PKC alpha in pancreatic islets but that this makes little overall contribution to GSIS.

Calcitonin-specific transcription and splicing targets gene-directed enzyme prodrug therapy to medullary thyroid carcinoma cells.

Recurrent and metastatic medullary thyroid carcinoma (MTC) remains difficult to treat due to its limited responsiveness to chemotherapy, radiotherapy, and imaging. To investigate an alternative therapeutic approach, we examined the feasibility of targeting gene-directed enzyme/prodrug therapy delivered by adenoviral vectors to MTC. We previously described a modified human calcitonin (CT)/CT gene-related peptide promoter that produced increased expression while maintaining specificity for MTC cells. In this study, we introduced an additional level of specificity by using cell-specific splicing and examined whether the selectivity of the gene-directed enzyme/prodrug therapy for MTC was enhanced when both the promoter and splicing features were combined in a single transcription unit. Two replication-defective adenoviruses were constructed that expressed the Escherichia coli purine nucleoside phosphorylase (PNP) gene under the transcriptional control of a modified T2 promoter (Ad.T2-PNP) or the T2 promoter in combination with a CT minigene cassette in which the PNP gene was imbedded within the CT gene exon 4 (Ad.T2-CT/PNP). The specificity of PNP expression by Ad.T2-PNP, Ad.T2-CT/PNP, and control viruses in the MTC cell line, TT, and in a panel of non-MTC cell lines was evaluated. The highest level of PNP gene expression and the most effective cell killing in the presence of prodrug occurred in TT cells infected with Ad.T2-PNP, followed by Ad.T2-CT/PNP. Infection of most non-MTC cell lines, even with high multiplicities of Ad.T2-PNP, produced only low-level PNP expression that resulted in minimal cell killing in the presence of prodrug. High-level expression of PNP and effective cell killing was observed with both adenoviral gene constructs. The highest level of cell specificity was achieved with the combined use of promoter and splicing regulation in the Ad.T2-CT/PNP virus.

Transcription-targeted gene therapy for androgen-independent prostate cancer.

The Escherichia coli enzyme (purine nucleoside phosphorylase, PNP) gene is delivered directly into PC3 tumors by one injection of replication-deficient human type-5 adenovirus (Ad5). Expressed PNP converts the systemically administered prodrug, 6MPDR, to a toxic purine, 6MP, causing cell death. We sought to increase the specificity of recombinant Ad vectors by controlling PNP expression with the promoter region from the androgen-dependent, prostate-specific rat probasin (Pb) gene. To increase its activity, the promoter was combined with the SV40 enhancer (SVPb). Cell lines were transfected with plasmids containing both a reporter gene, under SVPb control, and a reference gene cassette to allow normalization of expression levels. Plasmids expressed approximately 20-fold more reporter in prostate cancer than in other cells, but surprisingly, the SVPb element was both androgen-independent and retained substantial prostate specificity. Killing by Ad5-SVPb-PNP vector of cell lines cultured with 6MPDR for 6 days was 5- to 10-fold greater in prostate cancer than in liver or lung cells. In vivo, a single intratumoral injection of Ad5-SVPb-PNP (4 x 10(8) pfu), followed by 6MPDR administration twice daily for 6 days, significantly suppressed the growth of human prostate tumors in nude mice and increased their survival compared to control animals. Thus, the androgen-independent, prostate-targeting Ad5 vector reduces human prostate cancer growth significantly in vitro and in vivo. This first example of an androgen-independent vector points the way toward treatment of emerging androgen-independent prostate cancer in conjunction with hormone ablation therapy at a time when the tumor burden is low.

Advances in the development of non-human viral DNA-vectors for gene delivery.

Within the last two decades, various vectors based on human viruses have been developed as gene transfer vehicles for gene therapy applications and vaccination. However, one yet unresolved problem connected to the use of viral vectors in humans is the pre-existing immunity to most of these vectors in the vast majority of the population which can result in impaired gene transfer efficiency and increased secondary toxicity. One approach to solve this problem is the development of recombinant viruses of non-human origin as vectors for gene transfer. The major rationale for using such vectors is the avoidance of vector neutralization by pre-existing antibodies directed against the virus on which the vector is based. Use of vectors based on non-human viruses may therefore allow the use of lower initial vector doses to achieve efficient gene transfer. Side-effects caused by interactions between vectors derived from human viruses with a primed immune system or with blood components could also be reduced. Furthermore, these vectors might show new cell type tropisms and could therefore infect tissue and organs that are not accessible to current viral vectors. This review outlines some of the problems inherent in the human origin of current viral vectors and describes features and progress with non-human adenovirus and baculovirus-derived vectors that may provide alternatives.

Potent antietumor immunity in mice induced by vaccination with an ovine atadenovirus vector.

Identification of adenoviral isolates of nonhuman origin has fostered development of vectors with potential to overcome preexisting immunity in the human population that may affect clinical applications. Ovine adenoviral isolate, OAdV287 (OAdV7), the prototype of the genus Atadenovirus, has been previously characterized as a gene delivery vector although the receptor(s) used for infection remain to be identified. Here, we report the first use of recombinant OAdV7 as a vaccine for inducing an antitumor immune response in a mouse model. Treatment of murine BMDC with OAdV7 vectors expressing ovalbumin (OVA) resulted in upregulation of costimulatory markers and production of IL-12. Splenocytes isolated from immunized mice responded to antigen restimulation in vitro by proliferation and production of IFNγ. In vivo cytotoxicity assays revealed efficient killing of antigenic peptide-pulsed target cells 1 week after immunization, with an average killing efficiency of 75%. In mice inoculated with B16-OVA tumor cells immunization with OAdV7-OVA retarded and essentially prevented tumor growth in prophylactic and therapeutic tumor trials, respectively. Generation of a robust memory response was confirmed on tumor rechallenge in the prophylactic model. Therefore, OAdV7 is a novel vector with potential for further development of tumor vaccines.

Induction of both cellular and humoral immunity following a rational prime-boost immunization regimen that incorporates recombinant ovine atadenovirus and fowlpox virus.

Recombinant fowlpox viruses (rFPV) and ovine atadenoviruses (rOAdV) are being developed as safe, nonpathogenic, prophylactic and therapeutic vaccine vectors. There is scope, however, to improve the limited immune responses elicited by each of these vaccine vectors. Using previously determined and optimized routes of administration and viral doses, we characterized the primary adaptive immune responses elicited by recombinant variants of each virus. We demonstrate the contrasting nature of the response elicited by each recombinant virus. Whereas rFPV generates predominately cell-mediated immunity to our nominal target antigen, ovalbumin (OVA), rOAdV drives strong humoral responses. By defining the time taken to achieve maximal cytotoxic T cell responses and by studying the different patterns and kinetics of major histocompatibility complex class I-restricted OVA antigen expression postimmunization, we proposed a heterologous prime-boost regimen of immunization with rOAdV followed by rFPV. The subsequent experimental results showed that this approach produced robust cell-mediated and humoral immune responses against OVA that, importantly, were accompanied by weak anti-viral vector antibody responses. These results, therefore, represent a novel and potentially clinically applicable way to achieve broadly based and effective immunity to the antigens encoded by vectored vaccines.

Gene-directed enzyme prodrug therapy for cancer: a glimpse into the future?

Gene-directed enzyme prodrug therapy offers a new approach to treating some cancers. Clinical trials have been completed for brain and prostate cancers and the first product for post-surgical treatment of some brain tumors is awaiting marketing approval. Recent innovations provide a glimpse into the possible future evolution of a new gene medicine.

Recent progress in gene-directed enzyme prodrug therapy: an emerging cancer treatment.

The principle of gene-directed enzyme prodrug therapy (GDEPT) has existed for many years but, while simple in concept, the effective practical application of this therapy has proven to be challenging. Improvements in the efficacy of GDEPT have been achieved principally through the choice and development of more effective vectors, by optimizing and controlling gene expression and by increasing the activity of the delivered enzyme through mutation. While innovation continues in this field, the pioneering GDEPT systems designed to treat glioma and prostate cancer have completed or are now entering late-stage clinical trials, respectively. As the pace of innovation in GDEPT technology far exceeds its clinical application, these initial products are anticipated to be replaced by next-generation biologicals. This review highlights recent progress in the strategies and development of GDEPT and summarizes the status of current clinical trials. With the first GDEPT product for treatment of resected gliomas poised to gain marketing approval, a new era in cancer gene medicine is emerging.

Ovine atadenovirus, a novel and highly immunogenic vector in prime-boost studies of a candidate HIV-1 vaccine.

Ovine adenovirus type 7 (OAdV) is the prototype member of the genus Atadenovirus. No immunity to the virus has so far been detected in human sera. We describe the construction and evaluation of a candidate HIV-1 vaccine based on OAdV and its utilisation alone and in combination with plasmid-, human adenovirus type 5 (HAdV5; a Mastadenovirus)-, and modified vaccinia Ankara (MVA)-vectored vaccines. All vectors expressed HIVA, an immunogen consisting of HIV-1 clade A consensus Gag-derived protein coupled to a T cell polyepitope. OAdV.HIVA was genetically stable, grew well and expressed high levels of protein from the Rous sarcoma virus promoter. OAdV.HIVA was highly immunogenic in mice and efficiently primed and boosted HIV-1-specific T cell responses together with heterologous HIVA-expressing vectors. There were significant differences between OAdV and HAdV5 vectors in priming of naïve CD8(+) T cell responses to HIVA and in the persistence of MHC class I-restricted epitope presentation in the local draining lymph nodes. OAdV.HIVA primed T cells more rapidly but was less persistent than AdV5.HIVA and thus induced a qualitatively distinct T cell response. Nevertheless, both vectors primed a response in mice that reduced viral titres in a surrogate challenge model by three to four orders of magnitude. Thus, OAdV is a novel, underexplored vaccine vector with potential for further development for HIV-1 and other vaccines. The data are discussed in the context of the latest HIV-1 vaccine developments.

Increased complexity of wild-type adeno-associated virus-chromosomal junctions as determined by analysis of unselected cellular genomes.

Adeno-associated virus (AAV) undergoes preferential Rep-mediated integration into the AAVS1 region of human chromosome 19 during latent infection, at least in highly-selected cell cultures. However, integration at the level of the whole eukaryotic genome in unselected cells has not yet been monitored for AAV as it has been for retro- and lentiviruses. Here we have used ligation-mediated PCR (LMPCR) to monitor the formation of AAV-chromosome junctions within unselected genomic DNA after infection. Our analyses show that, in the absence of selection, the complexity of junction formation is much greater than for selected cells. Sequencing of more than 50 authentic LMPCR clones showed that AAV formed junctions with many different chromosomal sites via DNA micro-homologies that frequently involved GGTC motifs located within the AAV p5 element. One site at position 280 was preferred. Even greater complexity was found when unselected junctions identified by LMPCR were analysed by direct PCR amplification and cloning of genomic DNA. No clones containing AAV-AAVS1 chromosome 19 junctions were identified among the LMPCR clones, although they were readily obtained using chromosomal PCR primers, suggesting that junctions with AAVS1 constituted only a small portion of the total. Thus, we have identified an additional means by which AAV sequences may join to human chromosomes, although the detailed molecular mechanisms remain to be elucidated. These data may have implications for the design of new-generation AAV vectors.

An adenovirus serotype 5 vector with fibers derived from ovine atadenovirus demonstrates CAR-independent tropism and unique biodistribution in mice.

Many clinically important tissues are refractory to adenovirus (Ad) infection due to negligible levels of the primary Ad5 receptor the coxsackie and adenovirus receptor CAR. Thus, development of novel CAR-independent Ad vectors should lead to therapeutic gain. Ovine atadenovirus type 7, the prototype member of genus Atadenovirus, efficiently transduces CAR-deficient human cells in vitro, and systemic administration of OAdV is not associated with liver sequestration in mice. The penton base of OAdV7 does not contain an RGD motif, implicating the long-shafted fiber molecule as a major structural dictate of OAdV tropism. We hypothesized that replacement of the Ad5 fiber with the OAdV7 fiber would result in an Ad5 vector with CAR-independent tropism in vitro and liver "detargeting" in vivo. An Ad5 vector displaying the OAdV7 fiber was constructed (Ad5Luc1-OvF) and displayed CAR-independent, enhanced transduction of CAR-deficient human cells. When administered systemically to C57BL/6 mice, Ad5Luc1-OvF reporter gene expression was reduced by 80% in the liver compared to Ad5 and exhibited 50-fold higher gene expression in the kidney than the control vector. To our knowledge, this is the first report of a fiber-pseudotyped Ad vector that simultaneously displays decreased liver uptake and a distinct organ tropism in vivo. This vector may have future utility in murine models of renal disease.

LH3, a "homologue" of the mastadenoviral E1B 55-kDa protein is a structural protein of atadenoviruses.

Ovine adenovirus serotype 7 (OAdV), the prototype atadenovirus, has gene homologues for most mastadenovirus structural proteins but lacks proteins V and IX. Instead, OAdV has structural proteins of 32 and 42 kDa although the gene encoding the latter had not previously been identified. The presently reported studies of OAdV virions have now identified a minor structural polypeptide of approximately 40 kDa as the product of the L1 52/55-kDa gene and, more surprisingly, shown that the 42-kDa protein is encoded by LH3. This gene product was previously thought to be a homologue of mastadenovirus E1B 55 kDa, which is a multi-functional, non-structural protein that cooperates with E1A in cell transformation. The lack of transforming activity previously demonstrated for OAdV combined with a structural role for the LH3 product indicates that the protein has a different function in atadenoviruses. We discuss the abundance and likely core location of LH3 in the virion and the possible derivation of the E1B 55-kDa gene from the LH3 gene.

Ovine atadenovirus: a review of its biology, biosafety profile and application as a gene delivery vector.

The ovine adenovirus isolate OAdV287 is the prototype of the newly recognized genus of atadenoviruses. Although not as well studied as human mastadenoviruses, a substantial amount of work has now been carried out with this virus and an understanding of its interesting and unique properties is beginning to emerge. In this article the biology and biosafety profile of the virus is reviewed. This knowledge underpins the exploitation of the virus as a gene delivery vector. Its potential as a vaccine vector and its application to the treatment of prostate cancer is summarized and discussed.

Identification of a unique family of F-box proteins in atadenoviruses.

Ovine adenovirus isolate 287 (OAdV-7) is the prototype of the atadenoviruses, a genus whose strategy for infection and replication is still being elucidated. A transcription unit at the right end of the genome contains four related genes (ORFs RH1, 2, 4, and 6), at least three of which are nonessential for replication in vitro. Related genes are also present in the genomes of bovine and avian atadenoviruses. To investigate how these apparently redundant genes are decoded, a more detailed transcription map of the right end of the OAdV-7 genome has been deduced. Eight transcripts that were derived from a promoter in the terminal repeat sequence were identified. Five were potentially bicistronic. The transcripts could encode all the potential proteins of the region subject to efficient reinitiation of translation. However, the most interesting and surprising finding in this work was that the related RH proteins carry an F-box motif. This was first identified in OAdV-7 RH1 and subsequently found in RH2, 4, and 6 proteins and the related reading frames from the bovine and avian atadenoviruses. Although very rare among viral proteins, several hundred cellular proteins contain F-box motifs. F-box proteins facilitate the degradation of a variety of important regulatory proteins via SCF ubiquitin ligase complexes. Thus, it appears that atadenoviruses have adopted a strategy to regulate a key cellular pathway(s) that distinguishes them from the other adenovirus genera and from most viruses in general.

Complementation of a defective human adenovirus by an otherwise incompatible ovine adenovirus recombinant carrying a functional E1A gene.

All known human adenoviruses are classified as mastadenoviruses, while the ovine adenovirus (OAdV) serotype 7 is the prototype of the atadenoviruses, a proposed new genus. OAdV replicates abortively in human cell types and has potential as a gene transfer vector. However, the function of OAdV nonstructural genes is poorly understood and it is unclear whether OAdV replication might be complemented by a replicating human AdV in coinfected cells. To investigate possible interactions three human cell lines were singly infected with OAdV or human AdV5 or doubly infected. The development of a cytopathic effect and genome replication was monitored over three passages in each cell type. No significant OAdV replication occurred in any of the cell types examined either in the presence or in the absence of replicating AdV5. No aberrant AdV5 genome products were detected in coinfected cells. In contrast, in coinfected cells an OAdV recombinant that expressed the AdV5 E1A gene was able to promote the replication of an AdV5 E1A-deficient mutant, demonstrating trans-complementation between appropriate viruses. These findings have implications for the biosafety of OAdV vectors and their possible utility for enhancing gene delivery.