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.

Novel chimpanzee adenovirus-vectored respiratory mucosal tuberculosis vaccine: overcoming local anti-human adenovirus immunity for potent TB protection.

Pulmonary tuberculosis (TB) remains to be a major global health problem despite many decades of parenteral use of Bacillus Calmette-Guérin (BCG) vaccine. Developing safe and effective respiratory mucosal TB vaccines represents a unique challenge. Over the past decade or so, the human serotype 5 adenovirus (AdHu5)-based TB vaccine has emerged as one of the most promising candidates based on a plethora of preclinical and early clinical studies. However, anti-AdHu5 immunity widely present in the lung of humans poses a serious gap and limitation to its real-world applications. In this study we have developed a novel chimpanzee adenovirus 68 (AdCh68)-vectored TB vaccine amenable to the respiratory route of vaccination. We have evaluated AdCh68-based TB vaccine for its safety, T-cell immunogenicity, and protective efficacy in relevant animal models of human pulmonary TB with or without parenteral BCG priming. We have also compared AdCh68-based TB vaccine with its AdHu5 counterpart in both naive animals and those with preexisting anti-AdHu5 immunity in the lung. We provide compelling evidence that AdCh68-based TB vaccine is not only safe when delivered to the respiratory tract but, importantly, is also superior to its AdHu5 counterpart in induction of T-cell responses and immune protection, and limiting lung immunopathology in the presence of preexisting anti-AdHu5 immunity in the lung. Our findings thus suggest AdCh68-based TB vaccine to be an ideal candidate for respiratory mucosal immunization, endorsing its further clinical development in humans.

Adenovirus-based vaccines against rhesus lymphocryptovirus EBNA-1 induce expansion of specific CD8+ and CD4+ T cells in persistently infected rhesus macaques.

UNLABELLED: The impact of Epstein-Barr virus (EBV) on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA-1) is an important target for vaccination because it is the only protein expressed in all EBV-associated malignancies. We have designed and tested two therapeutic EBV vaccines that target the rhesus (rh) lymphocryptovirus (LCV) EBNA-1 to determine if ongoing T cell responses during persistent rhLCV infection in rhesus macaques can be expanded upon vaccination. Vaccines were based on two serotypes of E1-deleted simian adenovirus and were administered in a prime-boost regimen. To further modulate the response, rhEBNA-1 was fused to herpes simplex virus glycoprotein D (HSV-gD), which acts to block an inhibitory signaling pathway during T cell activation. We found that vaccines expressing rhEBNA-1 with or without functional HSV-gD led to expansion of rhEBNA-1-specific CD8(+) and CD4(+) T cells in 33% and 83% of the vaccinated animals, respectively. Additional animals developed significant changes within T cell subsets without changes in total numbers. Vaccination did not increase T cell responses to rhBZLF-1, an immediate early lytic phase antigen of rhLCV, thus indicating that increases of rhEBNA-1-specific responses were a direct result of vaccination. Vaccine-induced rhEBNA-1-specific T cells were highly functional and produced various combinations of cytokines as well as the cytolytic molecule granzyme B. These results serve as an important proof of principle that functional EBNA-1-specific T cells can be expanded by vaccination. IMPORTANCE: EBV is a common human pathogen that establishes a persistent infection through latency in B cells, where it occasionally reactivates. EBV infection is typically benign and is well controlled by the host adaptive immune system; however, it is considered carcinogenic due to its strong association with lymphoid and epithelial cell malignancies. Latent EBNA-1 is a promising target for a therapeutic vaccine, as it is the only antigen expressed in all EBV-associated malignancies. The goal was to determine if rhEBNA-1-specific T cells could be expanded upon vaccination of infected animals. Results were obtained with vaccines that target EBNA-1 of rhLCV, a virus closely related to EBV. We found that vaccination led to expansion of rhEBNA-1 immune cells that exhibited functions fit for controlling viral infection. This confirms that rhEBNA-1 is a suitable target for therapeutic vaccines. Future work should aim to generate more-robust T cell responses through modified vaccines.

Protection of non-human primates against rabies with an adenovirus recombinant vaccine.

Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials.

Influenza virus specific CD8⁺ T cells exacerbate infection following high dose influenza challenge of aged mice.

Influenza viruses cause severe illnesses and death, mainly in the aged population. Protection afforded by licensed vaccines through subtype-specific neutralizing antibodies is incomplete, especially when the vaccine antigens fail to closely match those of the circulating viral strains. Efforts are underway to generate a so-called universal influenza vaccine expressing conserved viral sequences that induce broad protection to multiple strains of influenza virus through the induction of CD8⁺ T cells. Here we assess the effect of a potent antiviral CD8⁺ T cell response on influenza virus infection of young and aged mice. Our results show that CD8⁺ T cell-inducing vaccines can provide some protection to young mice, but they exacerbate influenza virus-associated disease in aged mice, causing extensive lung pathology and death.

CD4+ and CD8+ T-cell responses to latent antigen EBNA-1 and lytic antigen BZLF-1 during persistent lymphocryptovirus infection of rhesus macaques.

Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4(+) and/or CD8(+) T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4(+) T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8(+) T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines.

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.

Intramuscular rather than oral administration of replication-defective adenoviral vaccine vector induces specific CD8+ T cell responses in the gut.

Gut-associated lymphoid tissue (GALT) is the primary replication site for HIV-1, resulting in a pronounced CD4(+) T cell loss in this tissue during primary infection. A mucosal vaccine that generates HIV-specific CD8(+) T cells in the gut could prevent the establishment of founder populations and broadcasting of virus. Here, we immunized mice orally and systemically with a chimpanzee derived adenoviral vector expressing HIV gag (AdC68gag) and measured frequencies of gag-specific interferon-gamma (IFN-gamma) producing CD8(+) T cells in the GALT. A single oral administration was inefficient at eliciting responses in the mesenteric lymph nodes and Peyer's Patches, while a single intramuscular administration elicited strong systemic and detectable mucosal responses. The gag-specific CD8(+) T cell responses were present in both acute and memory phases following intramuscular administration.

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.

Replication-defective vector based on a chimpanzee adenovirus.

An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.