Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Laboratory-Scale Emulsification Process of a Recombinant Adenovirus Vaccine with a Water-In-Oil-In-Water Adjuvant

Adjuvants play an important role to enhance the efficacy of vaccines and are often required to direct immune responses toward specific long-term ...

INRA ENVA ANSES

ANSES, Ecole Nationale Vétérinaire d'Alfort

Therapeutic vaccination aims at a strong stimulation of antigen-specific CD8(+) T-cells, so that they differentiate into effectors active in vivo against antigenic targets. Two adenovirus vectors (Ad) encoding two HLA-A*0201-restricted HIV epitope sequences (pol 476 and pol 589) were constructed. The Ad differ by the presence or absence of a ubiquitin monomer sequence (AdUb(+) and AdUb(-)). The effect of transgene product ubiquitination was analyzed on (1) in vivo, the immunization of Ad vaccinated HLA-A*0201 humanized HHD mice and (2) in vitro, the presentation of the transgene encoded peptides by transduced human dendritic cells (DC). In vivo, we found that immunization of humanized HHD mice with AdUb(+) elicited a transgene product-specific interferon (INF)-gamma CD8(+) T-cell response detectable by enzyme-linked immunospot (ELISPOT), whereas the AdUb(-) construction did not. Antigen-specific cytotoxic T lymphocytes (CTL) were also generated in HHD mice immunized with AdUb(+) and not with AdUb(-). In vitro, using human AdUb(+)-transduced DC, a sizeable expansion of pol 476 and pol 589 tetramer positive CD8(+) T cells as well as CD8(+) CTL were obtained in healthy donors. Compared to AdUb(-)-transduced DC, AdUb(+)-transduced DC triggered a higher number of pol 476-specific IFN-gamma-secreting CD8(+) T cells. In agreement, AdUb(+) transduced DC, used as target in a (51)Cr-release assay, were more efficiently lysed by peptide-specific CTL than AdUb(-)-transduced DC. In conclusion, the addition of an ubiquitin sequence to the adenoviral transgene, used as an antigen source, resulted in both in vivo enhanced CD8(+) T-cell immunogenicity in HHD mice and in vitro increased HLA class I-restricted presentation of encoded peptides by human DC.

Adenoviral transgene ubiquitination enhances mouse immunization and class I presentation by human dendritic cells.

Formation of a fully functional artery proceeds through a multistep process. Here we show that Notch3 is required to generate functional arteries in mice by regulating arterial differentiation and maturation of vascular smooth muscle cells (vSMC). In adult Notch3-/- mice distal arteries exhibit structural defects and arterial myogenic responses are defective. The postnatal maturation stage of vSMC is deficient in Notch3-/- mice. We further show that Notch3 is required for arterial specification of vSMC but not of endothelial cells. Our data reveal Notch3 to be the first cell-autonomous regulator of arterial differentiation and maturation of vSMC.

Notch3 is required for arterial identity and maturation of vascular smooth muscle cells.

The preadipocyte cell lines 3T3-L1 and 3T3-F442A are widely used to study the cellular mechanisms of preadipocyte differentiation and mature adipocyte functions. However, transfection with naked DNA is inefficient in these cell lines. Adenoviral gene transfer is a powerful technique to induce high levels of transgene expression. After failing to obtain 3T3-F442A stable transfectants, we studied different techniques designed to enhance the efficiency of adenoviral transduction in fat cells. First, we compared the effects of two agents known to significantly enhance adenoviral transgene transduction, namely the cationic lipid lipofectamine and the cationic polymer polylysine. We show here that lipofectamine-assisted adenoviral transduction was more efficient in 3T3-F442A than in 3T3-L1 preadipocytes at all tested multiplicity of infection. Lipofectamine, and more efficiently polylysine, yielded high and sustained levels of adenoviral transgene expression in 3T3-F442A preadipocytes. Adenoviral transgene expression was maintained throughout the differentiation process. Furthermore, the two agents also efficiently enhanced adenoviral transduction in mature 3T3-F442A adipocytes. Interestingly, neither protocol affected the differentiation process, morphological features or protein expression of mature adipocytes. These approaches could be of interest to study fat cell differentiation and the functions of mature adipocytes.

Efficient adenoviral transduction of 3T3-F442A preadipocytes without affecting adipocyte differentiation.

The recent advances in molecular genetics, pathogenesis and immunology have provided an optimal framework for developing novel approaches in the rational design of vaccines effective against viral epizootic diseases. This paper reviews most of the viral-vector based antigen delivery systems (ADSs) recently developed for vaccine testing in veterinary species, including attenuated virus and DNA and RNA viral vectors. Besides their usefulness in vaccinology, these ADSs constitute invaluable tools to researchers for understanding the nature of protective responses in different species, opening the possibility of modulating or potentiating relevant immune mechanisms involved in protection.

Antigen delivery systems for veterinary vaccine development. Viral-vector based delivery systems.

The evaluation of vaccine strategies in animal models is essential for the development of a vaccine against HIV. In efficacy trials conducted in non-human primate models of AIDS, vaccines based on adenoviruses compared favourably with other vaccine vectors. To determine whether this strategy could be transposed to another animal model, and by extension, to humans, we have evaluated the efficacy of adenoviral vectors in a natural model of AIDS, infection of the cat by the feline immunodeficiency virus (FIV). Recombinant canine adenoviruses expressing the envelope glycoproteins or the Gag protein of a primary strain of FIV were constructed. Three groups of six cats were immunised twice with vectors expressing FIV antigens or with a vector expressing an irrelevant antigen, green fluorescent protein, by intramuscular and subcutaneous routes. Humoral responses were elicited against the transgene product in 6/6, 3/6 and 0/6 cats after immunisation against green fluorescent protein, Gag or the envelope glycoproteins, respectively. Six weeks after the second administration, cats were challenged by the intraperitoneal route with the homologous strain, and viral burden in plasma was followed by quantitative RT-PCR. Immunisation with FIV antigens did not afford protection. Rather, viral RNA was detected at earlier time points in cats immunised against Gag than in cats immunised with a vector expressing an irrelevant antigen. Such immune-mediated enhancement did not appear to have a long-range impact on viral set point or inversion of the CD4(+)/CD8(+) ratio. Thus, in the feline AIDS model pre-existing immunity against a viral antigen exacerbated acute phase infection.

Gag-specific immune enhancement of lentiviral infection after vaccination with an adenoviral vector in an animal model of AIDS.

Vectors derived from canine adenovirus type 2 (CAV-2) are attractive candidates for gene therapy and live recombinant vaccines. CAV-2 vectors described thus far have been generated by modifying the virus genome, most notably early regions 1 and 3 or the fiber gene. Modification of these genes was underpinned by previous descriptions of their mRNA and protein-coding sequences. Similarly, the construction of new CAV-2 vectors bearing changes in other genomic regions, in particular many of those expressed late in the viral cycle, will require prior characterization of the corresponding transcriptional units. In this study, we provide a detailed description of the late transcriptional organization of the CAV-2 genome. We examined the major late transcription unit (MLTU) and determined its six families of mRNAs controlled by the putative major late promoter (MLP). All mRNAs expressed from the MLTU had a common non-coding tripartite leader (224 nt) at their 5' end. In transient transfection assays, the predicted MLP sequence was able to direct luciferase gene expression and the TPL sequence yielded a higher amount of transgene product. Identification of viral transcriptional products following in vitro infection confirmed most of the predicted protein-coding regions that were deduced from computer analysis of the CAV-2 genome. These findings contribute to a better understanding of gene expression in CAV-2 and lay the foundation required for genetic modifications aimed at vector optimization.

Functional organization of the major late transcriptional unit of canine adenovirus type 2.

Safe and efficient vaccination is important for rabies prevention in domestic animals. Replicative vectors expressing the rabies virus glycoprotein, derived from canine adenovirus have been reported to be promising vaccines in various animal models. In this paper we compare the potential of a replicative and a non-replicative vector, both based on canine adenovirus type 2 and expressing the rabies glycoprotein. Upon inoculation in sheep, immune responses against the rabies virus protein elicited by recombinant vectors were monitored. All immunised sheep produced a rapid and potent neutralizing antibody response against rabies virus after a single inoculation of either replicative or non-replicative recombinant canine adenovirus type 2. In addition, the non-replicative vector expressing the rabies glycoprotein stimulated antigen-specific CD4(+) and CD8(+) lymphocyte proliferation as well as IFN-γ production. These results suggest that vectors derived from canine adenovirus 2 could be considered for the development of promising vaccines in the ruminant species.

Canine adenoviruses elicit both humoral and cell-mediated immune responses against rabies following immunisation of sheep.

Different subsets of dendritic cells (DC) elicit qualitatively different immune responses. In mice, two lymphoid tissue-resident subsets, CD8α(+) and CD8α(-), have been implicated in the induction of T helper 1 (Th1) or Th2 responses, respectively. Moreover, CD8α(+) DC appear to play a major role in priming CD8(+) T lymphocyte responses to viral antigens in the course of diverse viral infections. These considerations have been less extensively explored for vaccine vectors derived from viruses. Despite inefficient ex vivo transduction of DC, vectored vaccines derived from human adenoviruses of serotype 5 (Ad5) elicit robust immune responses, predominantly of the Th1 orientation, in humans and mice. At present it is unknown whether Ad5 interacts with DC subsets in a differential manner, thereby influencing the quality of the elicited IR. To address this issue, successive steps (attachment, transgene expression, MHC class I antigen presentation and activation of antigen-specific T lymphocytes) involved in induction of immune responses by Ad5-based vectors have been examined in CD8α(+) and CD8α(-) murine DC subsets. Although in both ex vivo and in vivo experiments CD8α(+) and CD8α(-) DC subsets captured an Ad5-based vector to a similar extent, transgene expression and subsequent MHC class I display of a transgene-encoded antigen were more efficient in CD8α(+) DC. Moreover, following in vivo and ex vivo transduction with an Ad5-based vaccine, antigen-specific CD8(+) T lymphocytes were more efficiently activated by CD8α(+) DC than by CD8α(-) DC. Thus, superior antigen expression and MHC class I display in CD8α(+) DC may contribute to preferred priming of antigen-specific CD8(+) lymphocytes by Ad5-transduced CD8α(+) DC.

Antigen encoded by vaccine vectors derived from human adenovirus serotype 5 is preferentially presented to CD8+ T lymphocytes by the CD8α+ dendritic cell subset.

Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b(+) -type and CD103(+) -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b(+) -type DCs was far higher and broader than in the CD103(+) -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b(+) -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b(+) DC type is more responsive to CAV2 than the CD103(+) DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness.

Bernard Klonjkowski

UMR Viroligie

INRA ENVA ANSES

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Laboratory-Scale Emulsification Process of a Recombinant Adenovirus Vaccine with a Water-In-Oil-In-Water Adjuvant

Bernard Klonjkowski

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INRA ENVA ANSES

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Laboratory-Scale Emulsification Process of a Recombinant Adenovirus Vaccine with a Water-In-Oil-In-Water Adjuvant

UMR Viroligie