Final Report Summary - AIDS-COVAC (Generation of a coronavirus-based multigene AIDS vaccine and evaluation in a preclinical SIV model)
Coronaviruses spread via mucosal surfaces and can infect dendritic cells. These features and their exceptional transcription strategy make them extremely promising candidate vaccine vectors to overcome known problems of current HIV vaccine approaches. The National Institutes of Health, Bethesda, United States (US), have supported partner 1 during the years 2004-2006 to pursue this research line in the murine coronavirus system because of its innovation potential and since US researchers had not yet started to work in this direction. The European perspective of this project was thus to further promote this specific research line in Europe in order to pave the way for the generation of coronavirus-based HIV vaccines in humans. The AIDS-COVAC consortium's major aim was to generate a novel coronavirus-based HIV vaccine vector that is optimised for host entry by targeting professional antigen-presenting cells, namely Dendritic cells (DCs). Recombinant coronavirus vectors in the context of a simian model could serve as a paradigm for the development and evaluation of coronavirus-based HIV vaccines.
The consortium consisted of three scientific partners with well-matched, complementary, expertise and resources covering:
(i) the knowledge on coronavirus biology, reverse genetics and vector construction;
(ii) an ample expertise on DC-based vaccination in murine models and human clinical trials;
(iii) state-of-the-art technology to assess vector-DC interactions; and
(iv) animal facilities and comprehensive experience for the evaluation of candidate AIDS vaccines in pre-clinical studies in monkeys.
Background
The Human immunodeficiency virus (HIV) pandemic with approximately 40 million people infected worldwide and more than 4 million deaths per year, represents a major human health problem. The majority of the infections occur in Africa and HIV-induced Acquired immunodeficiency syndrome (AIDS) is the leading cause of death among adults aged 15-49 years in this region. Antiviral drug treatment has increased life expectancy and quality in Western countries, but this expensive medication is usually not accessible for infected individuals in developing countries. There is thus an urgent need for an efficient and affordable vaccine.
The knowledge of HIV biology generated over the last two decades has paved the way for rational vaccine design. Progress in the understanding of basic immunological mechanisms underlying antigen presentation, lymphocyte trafficking and activation, and immunological memory has been instrumental for the identification of parameters that ensure induction of protective antiviral immunity. Taken together, an HIV vaccine should:
- target and activate DCs;
- contain immunodominant CTL and Th cell antigens;
- display antigenic determinants to induce broadly neutralising antibody responses;
- be applicable via mucosal surfaces.
Coronaviral vectors display a number of features that make them particularly promising candidates for a new class of highly immunogenic viral multigene expression vectors. First, replication of these positive-stranded RNA viruses is restricted to the cytoplasm without a DNA intermediary, making insertion of viral sequences into the host cell genome unlikely. Second, there is accumulating knowledge on how to attenuate coronaviruses in order to provide biosafe vectors. Third, coronavirus genomes with a size of 27-31 kb represent the largest autonomously replicating RNAs known to date and thus offer a cloning capacity of more than 6 kb. Fourth, the unique transcription process generates 6-8 subgenomic mRNAs encoding for the canonical four structural proteins and a variable number of accessory proteins which can be replaced to encode multiple heterologous proteins. Fifth, the first target of coronaviruses in their natural hosts is the mucosa, suggesting that coronavirus vectors are applicable via mucosal surfaces. Finally and certainly most intriguing, cell surface receptors of human and murine coronaviruses are expressed on human and murine DCs, respectively.
Aim
The AIDS-COVAC consortium has focussed its objectives on the development of a novel coronavirus-based HIV vaccine vector that is optimised for vector entry into the host by targeting and activation of DCs. In addition, it was planned to provide proof-of-principle for the efficacy of this strategy by testing the prototype vaccine in the simian AIDS model.
General summary of AIDS-COVAC achievements
The AIDS-COVAC collaborative and complementary research was divided into two scientific work packages (WPs).
WP1 (Vector generation and optimisation) involved the determination of the best suitable surface glycoprotein protein for optimal vector entry into simian DCs and the analysis of vector-DC interactions by expression profiling, gene-clustering and advanced data mining. Extensive studies by using three known Human coronaviruses (HCoV), namely HCoV-229E, HCoV-NL63, and HCoV-OC43, revealed that, unexpectedly, none of these viruses could infect simian DCs. Therefore, the surface glycoprotein of the Vesicular-stomatitis-virus (VSV-G) was incorporated into coronavirus-based vectors in order to mediate transduction of simian DCs. In addition, the interaction of coronaviruses with DCs was studied by functional genomics. Expression profiling and gene clustering analyses of murine DC-coronavirus and macrophage-coronavirus interaction was used to define specific molecular signatures. These studies revealed that the molecular signatures of DCs and macrophages following coronavirus infection show remarkable differences and provide the basis for future studies aimed at correlating the immunogenicity of coronavirus-based vaccines with specific transcriptional responses in professional antigen-presenting cells such as DCs and macrophages.
WP2 (Vector evaluation) studied the efficacy of vector-mediated gene transfer into murine and simian DCs in vitro and the efficacy of this vaccination approach in vivo. Detailed evaluation of coronavirus-based vectors in the murine system revealed that they can indeed efficiently mediate expression of antigens and immunostimulatory molecules in DCs. In vivo, murine coronavirus-based vectors can deliver multiple antigens and immunostimulatory cytokines almost exclusively to CD11c+ DCs within secondary lymphoid organs. Moreover, immunisation with only low numbers of particles elicited potent CD8+ T cell responses that provided long-lasting protection against viral challenge. A pronounced immunostimmulatory effect was encountered by coronavirus vectors encoding GM-CSF that mainly fosters DC maturation and survival and thereby facilitates efficient and prolonged antigen presentation. Finally, a VSV-G-expressing coronavirus-based vector was used for immunisation in rhesus macaques, and first results demonstrated that application of both live and heat inactivated vectors was well tolerated without any adverse effects. Enumeration of leukocytes by flow cytometry revealed a significant reduction of blood lymphocyte counts during the first 2 days of immunisation with the live vector, consistent with activation-induced redistribution of lymphocytes to lymphoid organs. In addition, expression of CD183, the receptor for the chemokine IP-10, was reduced on T-cells and B-cells within the first 48 hours after inoculation with live vector but not of heat-inactivated vector, indicative of a vector-induced strong interferon response in vivo. In accordance with these findings the analysis of humoral responses revealed that a single administration of live, but not inactivated, VSV-G vectors elicited VSV-neutralising antibody titers. Currently, further analyses on cellular and humoral immune responses are being performed, and a second immunisation (boost) is scheduled.
The consortium consisted of three scientific partners with well-matched, complementary, expertise and resources covering:
(i) the knowledge on coronavirus biology, reverse genetics and vector construction;
(ii) an ample expertise on DC-based vaccination in murine models and human clinical trials;
(iii) state-of-the-art technology to assess vector-DC interactions; and
(iv) animal facilities and comprehensive experience for the evaluation of candidate AIDS vaccines in pre-clinical studies in monkeys.
Background
The Human immunodeficiency virus (HIV) pandemic with approximately 40 million people infected worldwide and more than 4 million deaths per year, represents a major human health problem. The majority of the infections occur in Africa and HIV-induced Acquired immunodeficiency syndrome (AIDS) is the leading cause of death among adults aged 15-49 years in this region. Antiviral drug treatment has increased life expectancy and quality in Western countries, but this expensive medication is usually not accessible for infected individuals in developing countries. There is thus an urgent need for an efficient and affordable vaccine.
The knowledge of HIV biology generated over the last two decades has paved the way for rational vaccine design. Progress in the understanding of basic immunological mechanisms underlying antigen presentation, lymphocyte trafficking and activation, and immunological memory has been instrumental for the identification of parameters that ensure induction of protective antiviral immunity. Taken together, an HIV vaccine should:
- target and activate DCs;
- contain immunodominant CTL and Th cell antigens;
- display antigenic determinants to induce broadly neutralising antibody responses;
- be applicable via mucosal surfaces.
Coronaviral vectors display a number of features that make them particularly promising candidates for a new class of highly immunogenic viral multigene expression vectors. First, replication of these positive-stranded RNA viruses is restricted to the cytoplasm without a DNA intermediary, making insertion of viral sequences into the host cell genome unlikely. Second, there is accumulating knowledge on how to attenuate coronaviruses in order to provide biosafe vectors. Third, coronavirus genomes with a size of 27-31 kb represent the largest autonomously replicating RNAs known to date and thus offer a cloning capacity of more than 6 kb. Fourth, the unique transcription process generates 6-8 subgenomic mRNAs encoding for the canonical four structural proteins and a variable number of accessory proteins which can be replaced to encode multiple heterologous proteins. Fifth, the first target of coronaviruses in their natural hosts is the mucosa, suggesting that coronavirus vectors are applicable via mucosal surfaces. Finally and certainly most intriguing, cell surface receptors of human and murine coronaviruses are expressed on human and murine DCs, respectively.
Aim
The AIDS-COVAC consortium has focussed its objectives on the development of a novel coronavirus-based HIV vaccine vector that is optimised for vector entry into the host by targeting and activation of DCs. In addition, it was planned to provide proof-of-principle for the efficacy of this strategy by testing the prototype vaccine in the simian AIDS model.
General summary of AIDS-COVAC achievements
The AIDS-COVAC collaborative and complementary research was divided into two scientific work packages (WPs).
WP1 (Vector generation and optimisation) involved the determination of the best suitable surface glycoprotein protein for optimal vector entry into simian DCs and the analysis of vector-DC interactions by expression profiling, gene-clustering and advanced data mining. Extensive studies by using three known Human coronaviruses (HCoV), namely HCoV-229E, HCoV-NL63, and HCoV-OC43, revealed that, unexpectedly, none of these viruses could infect simian DCs. Therefore, the surface glycoprotein of the Vesicular-stomatitis-virus (VSV-G) was incorporated into coronavirus-based vectors in order to mediate transduction of simian DCs. In addition, the interaction of coronaviruses with DCs was studied by functional genomics. Expression profiling and gene clustering analyses of murine DC-coronavirus and macrophage-coronavirus interaction was used to define specific molecular signatures. These studies revealed that the molecular signatures of DCs and macrophages following coronavirus infection show remarkable differences and provide the basis for future studies aimed at correlating the immunogenicity of coronavirus-based vaccines with specific transcriptional responses in professional antigen-presenting cells such as DCs and macrophages.
WP2 (Vector evaluation) studied the efficacy of vector-mediated gene transfer into murine and simian DCs in vitro and the efficacy of this vaccination approach in vivo. Detailed evaluation of coronavirus-based vectors in the murine system revealed that they can indeed efficiently mediate expression of antigens and immunostimulatory molecules in DCs. In vivo, murine coronavirus-based vectors can deliver multiple antigens and immunostimulatory cytokines almost exclusively to CD11c+ DCs within secondary lymphoid organs. Moreover, immunisation with only low numbers of particles elicited potent CD8+ T cell responses that provided long-lasting protection against viral challenge. A pronounced immunostimmulatory effect was encountered by coronavirus vectors encoding GM-CSF that mainly fosters DC maturation and survival and thereby facilitates efficient and prolonged antigen presentation. Finally, a VSV-G-expressing coronavirus-based vector was used for immunisation in rhesus macaques, and first results demonstrated that application of both live and heat inactivated vectors was well tolerated without any adverse effects. Enumeration of leukocytes by flow cytometry revealed a significant reduction of blood lymphocyte counts during the first 2 days of immunisation with the live vector, consistent with activation-induced redistribution of lymphocytes to lymphoid organs. In addition, expression of CD183, the receptor for the chemokine IP-10, was reduced on T-cells and B-cells within the first 48 hours after inoculation with live vector but not of heat-inactivated vector, indicative of a vector-induced strong interferon response in vivo. In accordance with these findings the analysis of humoral responses revealed that a single administration of live, but not inactivated, VSV-G vectors elicited VSV-neutralising antibody titers. Currently, further analyses on cellular and humoral immune responses are being performed, and a second immunisation (boost) is scheduled.