A comprehensive analysis of interferon-beta's role in shaping the immunogenicity and pathogenicity of rabies virus: Development of a novel vaccine vector
The central goal of this thesis is to advance the development of rabies virus (RABV) as a vaccine vector. In order to generate an efficacious and safe vaccine, we aim to improve the immunogenicity of the RABV vector and reduce its pathogenicity. The innate immune response has a critical role in activating the adaptive arm of immunity, and thus must be well understood in order to manipulate the host response to the RABV vector. The overall hypothesis in this research project is that the addition of a molecular adjuvant capable of altering the host's innate immune response can increase the adaptive immune response to recombinant RABV expressing foreign genes.
We began our study by determining the efficacy of our current RABV-based vaccines in rhesus macaques. For this approach, highly attenuated RABV vectors expressing either SIVmac239-GagPol or SIVmac239-Env was used to immunize Mamu-A*01 negative rhesus macaques prior to challenge with the pathogenic SIVmac251 virus. Sixteen weeks after challenge, we saw that immunized macaques had 1.3-1.6-log fold decrease in viral set point compared to control animals. Analysis of the immune response revealed that compared to control animals vaccinated macaques had a more rapid induction of SIVmac251 neutralizing antibodies and CD8+ T cell responses to various SIV epitopes. These findings indicate promise for the use of RABV based vectors against elusive pathogens such as HIV.
We next sought to improve the immunogenicity of the RABV vector, which requires a detailed understanding of viral-host interaction and activation of the host's immune system. Here we focused on the host's recognition of virus by innate immune cells early after a RABV infection. We hypothesize that viral recognition by innate immune receptors on dendritic cells will occur in the cytoplasm and affect viral pathogenicity by directing the host's antiviral response. To determine the receptor responsible for innate cell activation, we utilized bone marrow derived dendritic cells (BMDC) as a model. BMDCs were infected with RABV and expression of co-stimulatory molecules and production of type I interferon (IFN) was quantified. Our results indicate that BMDC activation and type I IFN production following a RABV infection occurs independent of toll-like receptor (TLR)-3 and myeloid differentiation factor 88 (MyD88) signaling. However, the adaptor protein interferon beta promoter stimulator-1 (IPS-1) was required for both BMDC activation and IFN production. To further identify the receptor recognizing RABV after infection we analyzed BMDC from mice deficient in melanoma differentiation-associated gene-5 (Mda-5) or retinoic acid-inducible gene I (RIG-I), as both of these receptors signal via IPS-1. In the absence of either receptor there is a significant decrease in BMDC activation induced at 12h. Alternatively, only RIG-I -/- derived BMDCs exhibited a delay in type I IFN production at early timepoints. However, by 48h post infection both the RIG-I-/- and Mda-5-/- cells make type I IFN and express co-stimulatory molecules to levels equivalent to wildtype BMDCs. These data indicate that following infection any perturbation to IPS-1 activation is deleterious and that in wildtype cells both RIG-I and Mda-5 are able to recognize RABV and activate the innate immune response. Furthermore, having identified the host receptors that are activated following a RABV infection, we can target the unused receptors with recombinant RABV in order to increase the innate immune response and potentially enhance RABV immunogenicity.
Once we had identified the host receptors responsible for activating the innate immune response following a RABV infection, we sought to increase the adaptive immune response to our vaccine vector by artificially increasing the type I IFN production. IFN-β is known to activate dendritic cells and increase the functionality of effector CD8+ T cells. Thus, we decided to explore the use of IFN-β as an adjuvant for the RABV vector. Our results indicate that viral replication of a recombinant RABV expressing HIV-1 Gag and IFN-β (IFN(+)) is reduced approximately 100-fold when compared to a control RABV not expressing functional IFN-&β Despite the decrease in vector replication, mice immunized with IFN(+) had a significantly greater number of activated CD8+ T cells during the primary immune response. However, when IFN(+) immunized mice were challenged with vaccinia expressing Gag there was no significant difference in the number or functionality of Gag-specific CD8+ T cells compared to control mice. The increased CD8+ T cell activation in the presence of IFN-β, even with greatly reduced viral replication, indicates the beneficial effect of IFN-β for the host. Taken together, the findings presented in this thesis highlight the safety of RABV vaccines and support the use of RABV as a vaccine vector in both prophylactic and therapeutic immunization strategies.