Human immune responses to traditional and novel rabies vaccines.

Traditional rabies vaccines given preventatively or after exposure to the virus induce cluster of differentiation 4+ (CD4+) T cell responses that promote the induction of long-lived memory B cells and neutralising antibody-secreting plasma cells. The high cost of rabies vaccines, combined with the complexity of immunisation protocols, is partially to blame for their under-use in exposed individuals and prevents the vaccines' widespread use in preventative childhood immunisation programmes in areas where rabies remains common. Novel vaccines or vaccine adjuvants that reduce the cost of rabies vaccination and afford protective immunity, as well as sustained immunological memory, after a single dose are being developed and may very well reduce the human death toll of rabies.

Les vaccins antirabiques classiques administrés préventivement ou suite à une exposition virale déclenchent une réponse cellulaire des lymphocytes T CD4+ induisant l'activation des lymphocytes B mémoire à longue durée de vie et des plasmocytes sécréteurs d'anticorps neutralisants. Le coût élevé des vaccins contre la rage et la complexité des protocoles de vaccination se traduisent par une sous-utilisation chez les individus exposés et font obstacle à l'emploi généralisé de ces vaccins dans les programmes d'immunisation préventive des enfants dans les régions où la rage est endémique. Des vaccins ou adjuvants innovants sont en cours de développement, qui pourraient réduire les coûts de la vaccination antirabique tout en conférant une immunité protectrice et en renforçant la mémoire immunitaire après l'administration d'une dose unique, ce qui contribuerait à réduire considérablement le tribut en vies humaines payé à la rage.

Las vacunas antirrábicas tradicionales, administradas con fines preventivos o tras la exposición al virus, inducen, en linfocitos T que expresan el cúmulo de diferenciación 4 (linfocitos CD4+), una respuesta que promueve la inducción de células B de memoria de larga vida y células plasmáticas secretoras de anticuerpos neutralizantes. El hecho de que las vacunas antirrábicas se utilicen menos de lo debido en personas expuestas se explica en parte por su elevado costo, que, sumado a la complejidad de los protocolos de inmunización, obstaculiza su empleo generalizado para programas preventivos de inmunización infantil en zonas donde la rabia sigue siendo frecuente. Ahora se están obteniendo adyuvantes o vacunas de nuevo cuño que reducen el costo de la vacunación antirrábica, ofrecen inmunidad protectora y confieren una memoria inmunológica duradera tras una sola dosis, lo que muy bien podría aligerar el duro tributo que se cobra la rabia en vidas humanas.

Adenovirus-mediated artificial MicroRNAs targeting matrix or nucleoprotein genes protect mice against lethal influenza virus challenge.

Influenza virus (IV) infection is a major public health problem, causing millions of cases of severe illness and as many as 500 000 deaths each year worldwide. Given the limitations of current prevention or treatment of acute influenza, novel therapies are needed. RNA interference (RNAi) through microRNAs (miRNA) is an emerging technology that can suppress virus replication in vitro and in vivo. Here, we describe a novel strategy for the treatment of infuenza based on RNAi delivered by a replication-defective adenovirus (Ad) vector, derived from chimpanzee serotype 68 (AdC68). Our results showed that artificial miRNAs (amiRNAs) specifically targeting conserved regions of the IV genome could effectively inhibit virus replication in human embryonic kidney 293 cells. Moreover, our results demonstrated that prophylactic treatment with AdC68 expressing amiRNAs directed against M1, M2 or nucleoprotein genes of IV completely protected mice from homologous A/PR8 virus challenge and partially protected the mice from heterologous influenza A virus strains such as H9N2 and H5N1. Collectively, our data demonstrate that amiRNAs targeting the conserved regions of influenza A virus delivered by Ad vectors should be pursued as a novel strategy for prophylaxis of IV infection in humans and animals.

Adenovirus-based vaccines: comparison of vectors from three species of adenoviridae.

In order to better understand the broad applicability of adenovirus (Ad) as a vector for human vaccine studies, we compared four adenovirus (Ad) vectors from families C (Ad human serotype 5 [HAdV-5; here referred to as AdHu5]), D (HAdV-26; here referred to as AdHu26), and E (simian serotypes SAdV-23 and SAdV-24; here referred to as chimpanzee serotypes 6 and 7 [AdC6 and AdC7, respectively]) of the Adenoviridae. Seroprevalence rates and titers of neutralizing antibodies to the two human-origin Ads were found to be higher than those reported previously, especially in countries of sub-Saharan Africa. Conversely, prevalence rates and titers to AdC6 and AdC7 were markedly lower. Healthy human adults from the United States had readily detectable circulating T cells recognizing Ad viruses, the levels of which in some individuals were unexpectedly high in response to AdHu26. The magnitude of T-cell responses to AdHu5 correlated with those to AdHu26, suggesting T-cell recognition of conserved epitopes. In mice, all of the different Ad vectors induced CD8(+) T-cell responses that were comparable in their magnitudes and cytokine production profiles. Prime-boost regimens comparing different combinations of Ad vectors failed to indicate that the sequential use of Ad vectors from distinct families resulted in higher immune responses than the use of serologically distinct Ad vectors from the same family. Moreover, the transgene product-specific antibody responses induced by the AdHu26 and AdC vectors were markedly lower than those induced by the AdHu5 vector. AdHu26 vectors and, to a lesser extent, AdC vectors induced more potent Ad-neutralizing antibody responses. These results suggest that the potential of AdHu26 as a vaccine vector may suffer from limitations similar to those found for vectors based on other prevalent human Ads.

Chimpanzee-origin adenovirus vectors as vaccine carriers.

Vaccines based on replication-defective adenoviral vectors are being developed for infectious agents and tumor-associated antigens. Early work focused on vaccines derived from a common human serotype of adenovirus, that is, adenovirus of the serotype 5 (AdHu5). Neutralizing antibodies against AdHu5 virus, present in a large percentage of the human population, dampen the efficacy of vaccines based on this carrier. To circumvent this problem, we generated vectors derived from chimpanzee adenoviruses. Here we describe some basic parameters of vectors derived from chimpanzee adenoviruses C68 and C7, including growth characteristics, yields of infectious particles, effects of additional deletions in E3 and E4 and lengths of the inserted foreign sequence as they relate to the suitability for their eventual development as vaccine carriers for clinical use.

DNA immunization using a non-viral promoter.

Most DNA vaccines rely on strong viral promoters to optimize levels of transgene expression. Some studies have demonstrated that the potency of viral promoters does not necessarily correlate with DNA vaccine efficacy in vivo. This has partly been attributed to downregulation of these promoters by cytokines such as interferon gamma induced by the CpG motives of these vaccines. In an attempt to avoid downregulation of viral promoters by IFN-gamma, we tested vaccine vectors driven by the MHC class II promoter. To enhance the activity of this promoter, another plasmid expressing the human MHC class II transactivator driven by a viral promoter, the native IFN-gamma inducible CIITA type IV promoter (PIV) or a synthetic promoter containing IFN-gamma inducible elements was co-inoculated. Our data show that a non-viral promoter such as the MHC class II promoter tested in this study can indeed be used in DNA vaccines.

Adenoviral gene delivery for HIV-1 vaccination.

The AIDS epidemic continues to spread throughout nations of Africa and Asia and is by now threatening to undermine the already frail infrastructure of developing countries in Sub-Saharan Africa that are hit the hardest. The only option to stem this epidemic is through inexpensive and efficacious vaccines that prevent or at least blunt HIV-1 infections. Despite decades of pre-clinical and clinical research such vaccines remain elusive. Most anti-viral vaccines act by inducing protective levels of virus-neutralizing antibodies. The envelope protein of HIV-1, the sole target of neutralizing antibodies, is constantly changing due to mutations, B cell epitopes are masked by heavy glycosylation and the protein's structural unfolding upon binding to its CD4 receptor and chemokine co-receptors. Efforts to induce broadly cross-reactive virus-neutralizing antibodies able to induce sterilizing or near sterilizing immunity to HIV-1 have thus failed. Studies have indicated that cell-mediated immune responses and in particular CD8+ T cell responses to internal viral proteins may control HIV-1 infections without necessarily preventing them. Adenoviral vectors expressing antigens of HIV-1 are eminently suited to stimulate potent CD8+ T cell responses against transgene products, such as antigens of HIV-1. They performed well in pre-clinical studies in rodents and nonhuman primates and are currently in human clinical trials. This review summarizes the published literature on adenoviral vectors as vaccine carriers for HIV-1 and discusses advantages and disadvantages of this vaccine modality.

Human immunodeficiency virus type 1-specific immune responses in primates upon sequential immunization with adenoviral vaccine carriers of human and simian serotypes.

Two triple immunization vaccine regimens with adenoviral vectors with E1 deleted expressing Gag of human immunodeficiency virus type 1 were tested for induction of T- and B-cell-mediated-immune responses in mice and in nonhuman primates. The vaccine carriers were derived from distinct serotypes of human and simian adenoviruses that fail to elicit cross-neutralizing antibodies expected to dampen the effect of booster immunizations. Both triple immunization regimens induced unprecedented frequencies of gamma interferon-producing CD8(+) T cells to Gag in mice and monkeys that remained remarkably stable over time. In addition, monkeys developed Gag-specific interleukin-2-secreting T cells, presumably belonging to the CD4(+) T-cell subset, and antibodies to both Gag and the adenoviral vaccine carriers.

CpG methylation of a plasmid vector results in extended transgene product expression by circumventing induction of immune responses.

Gene therapy has the potential to cure inherited diseases if the delivered genes achieve long-term expression at therapeutic levels in the targeted tissues. Expression is commonly short-lived due to induction of cell-mediated immune responses to the gene therapy vehicle and/or the transgene product, which can be perceived as "foreign" by the host's immune system. Plasmid expression vectors have been used to deliver genes. Bacterial DNA carries immunostimulatory sequences in the form of unmethylated CpG motifs, which induce an inflammatory reaction that in turn promotes activation of transgene product-specific B and T cells. Elimination or methylation of immunostimulatory CpG sequences in plasmid expression vectors prevents the stimulation of transgene product-specific immune responses without necessarily reducing transgene expression. In this study, we tested if a CpG-methylated plasmid expression vector expressing the highly immunogenic glycoprotein of rabies virus can achieve prolonged transgene product expression by circumventing immune recognition. Our data show that mice inoculated with a CpG-methylated plasmid expression vector show delayed clearance of transfected cells and fail to mount a strong immune response to the transgene product. Gene transfer with a CpG-methylated plasmid results in a state of immunological low responsiveness to the transgene product, which may facilitate readministration of the transgene. Nevertheless, mice remain responsive to the transgene product delivered by a viral vector.

Induction of CD8+ T cells to an HIV-1 antigen upon oral immunization of mice with a simian E1-deleted adenoviral vector.

An E1-deleted adenoviral recombinant derived from the chimpanzee serotype 6 expressing a codon-optimized truncated form of gag of human immunodeficiency virus type 1 (HIV-1) was tested for induction of a transgene product-specific CD8+ T cell response upon oral immunization of mice. The vector was shown to induce gag-specific CD8+ T cells detectable at moderate frequencies of approximately 0.5-1.0% in the spleens and to provide partial protection in a surrogate challenge model based on intraperitoneal (i.p.) infection of mice with a vaccinia virus recombinant expressing gag (VVgag) of HIV-1. Frequencies of gag-specific CD8+ T cells could be augmented by using a different, i.e., heterologous, vaccine carrier based on a distinct recombinant virus or an alternative adenoviral serotype expressing the same form of gag for oral or systemic-booster immunization.

Chemokines and TRANCE as genetic adjuvants for a DNA vaccine to rabies virus.

An adaptive immune response is initiated by mature dendritic cells presenting processed antigen to nai;ve T cells. Assuming that the magnitude of the immune response is influenced by the number and type of antigen-presenting dendritic cells and by the duration of antigen presentation, we tested if chemokines that bind to receptors expressed on immature dendritic cells or TRANCE, a survival factor for mature dendritic cells, can serve as adjuvants. None of the immunomodulaters given as genetic adjuvants with a DNA vaccine encoding the full-length rabies virus glycoprotein augmented the transgene product-specific response. However, RANTES, MCP-1, MIP 1-beta, and TRANCE given together with a DNA vaccine expressing a truncated and thus secreted version of the rabies virus glycoprotein enhanced the response suggesting that the tested genetic adjuvants promoted preferentially presentation of reprocessed antigen originating from transduced tissue cells.

Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier.

Adenovirus vectors with E1 deleted of the human serotype 5 (AdHu5) and the chimpanzee serotype 68 (AdC68) expressing the glycoprotein of the Evelyn Rokiniki Abelseth strain of rabies virus were tested upon oral application for induction of systemic and mucosal transgene product-specific antibody responses in mice. Both vectors induced systemic and mucosal antibodies to rabies virus, including virus-neutralizing antibodies and protection against a severe intracerebral challenge with a mouse-adapted strain of rabies virus. Pre-existing immunity of AdHu5 virus, which dampens induction of transgene product-specific immunity elicited by AdHu5 vectors given systemically did not impair the response induced by oral vaccination. Oral priming-boosting regimens with either heterologous or homologous adenoviral vectors used sequentially increased both mucosal and systemic antibody titers to rabies virus [corrected]

T helper cell-independent antibody responses to the transgene product of an e1-deleted adenoviral vaccine require NK1.1 T cells.

Mice lacking CD4(+) T cells due to a knock-out mutation respond to vaccination with a replication-defective adenoviral recombinant expressing the glycoprotein of rabies virus with a long-lasting virus-neutralizing antibody response. The vaccine-induced B cells secrete antibodies that are mainly of IgG isotypes. The response can be enhanced upon booster immunization, indicating the induction of B cell memory in the absence of CD4(+) T cells. The antibody response is independent of CD8(+) T cells but requires the presence of CD3(+) cells carrying the NK1.1 markers.

An experimental vaccine expressing wild-type p53 induces protective immunity against glioblastoma cells with high levels of endogenous p53.

Inoculation of mice with a recombinant vaccinia virus expressing the full-length mouse wild-type p53 protein (Vp53-wt) was shown to induce partial protection against peripheral challenge with a mouse glioblastoma cell line, termed GL261, expressing high levels of nuclear, endogenous wild-type p53. In vivo experiments with knockout (KO) mice and mice treated with depleting doses of antibodies specific to lymphocyte subsets revealed that vaccine efficacy depended on CD4+ and CD8+ T cells as well as on natural killer (NK) cells. Vp53-wt virus-vaccinated mice that failed to develop tumours upon challenge with a minimal tumourigenic dose of GL261 cells remained completely resistant to further challenge with increased doses of GL261 cells. The efficacy of the Vp53-wt vaccine was improved by adding recombinant mouse interleukin-12 (rIL-12) as an adjuvant at the time of tumour challenge. The induction of T cells to p53 in Vp53-wt virus-immune mice was also demonstrated at the tumour site by immunochemistry and was further confirmed by a delayed-type hypersensitivity response to the p53 protein, although in vitro experiments using splenocytes from vaccinated mice failed to demonstrate CD4+ or CD8+ T-cell activity to p53.