The Adenovirus E4 ORF3 Protein Binds and Reorganizes the TRIM Family Member Transcriptional Intermediary Factor 1 Alpha

One of the most interesting functions attributed to the adenovirus early region 4 open reading frame 3 (E4 ORF3) protein is its reorganization of promyelocytic leukemia (PML) protein nuclear bodies. These normally punctate structures are reorganized by E4 ORF3 into tracks that eventually surround viral replication centers. PML rearrangement is an evolutionarily conserved function of E4 ORF3, yet its cause and functional relevance remain mysteries. The E4 ORF3 protein coimmunoprecipitates with the PML protein, yet E4 ORF3 still forms tracks in cells that lack PML. The PML protein is a member of a larger protein family termed tripartite motif (TRIM) proteins. TRIM proteins contain a tripartite domain structure in proximity to their N termini that consists of a RING finger domain, followed by one or two B box domains and a C-terminal coiled-coil domain (collectively termed the RBCC domain). The order and spacing of these domains are evolutionarily conserved and thought to mediate protein-protein interactions and other functions. We implemented a proteomic approach to isolate cellular proteins that bind to E4 ORF3. We identified a novel interaction between E4 ORF3 and another TRIM family member, transcriptional intermediary factor 1 alpha (TIF1α). TIF1α functions by recruiting coactivators and/or corepressors to modulate transcription. The interaction between E4 ORF3 and TIF1α was validated by coimmunoprecipitation and binding of recombinant proteins. Indirect immunofluorescence assays demonstrated that TIF1α is reorganized into track structures that contain PML upon E4 ORF3 expression. The RBCC domain of TIF1α is sufficient for E4 ORF3-induced rearrangement, and TIF1α reorganization is conserved across adenovirus serotypes

Budding Capability of the Influenza Virus Neuraminidase Can Be Modulated by Tetherin▿

We have determined that, in addition to its receptor-destroying activity, the influenza virus neuraminidase is capable of efficiently forming virus-like particles (VLPs) when expressed individually from plasmid DNA. This observation applies to both human subtypes of neuraminidase, N1 and N2. However, it is not found with every strain of influenza virus. Through gain-of-function and loss-of-function analyses, a critical determinant within the neuraminidase ectodomain was identified that contributes to VLP formation but is not sufficient to accomplish release of plasmid-derived VLPs. This sequence lies on the plasma membrane-proximal side of the neuraminidase globular head. Most importantly, we demonstrate that the antiviral restriction factor tetherin plays a role in determining the strain-specific limitations of release competency. If tetherin is counteracted by small interfering RNA knockdown or expression of the HIV anti-tetherin factor vpu, budding and release capability is bestowed upon an otherwise budding-deficient neuraminidase. These data suggest that budding-competent neuraminidase proteins possess an as-yet-unidentified means of counteracting the antiviral restriction factor tetherin and identify a novel way in which the influenza virus neuraminidase can contribute to virus release

DataSheet_1_Exacerbated lung inflammation following secondary RSV exposure is CD4+ T cell-dependent and is not mitigated in infant BALB/c mice born to PreF-vaccinated dams.pdf

Respiratory syncytial virus (RSV) is the leading cause of childhood hospitalizations due to bronchiolitis in children under 5 years of age. Moreover, severe RSV disease requiring hospitalization is associated with the subsequent development of wheezing and asthma. Due to the young age in which viral protection is needed and risk of vaccine enhanced disease following direct infant vaccination, current approaches aim to protect young children through maternal immunization strategies that boost neutralizing maternal antibody (matAb) levels. However, there is a scarcity of studies investigating the influence of maternal immunization on secondary immune responses to RSV in the offspring or whether the subsequent development of wheezing and asthma is mitigated. Toward this goal, our lab developed a murine model of maternal RSV vaccination and repeat RSV exposure to evaluate the changes in immune response and development of exacerbated lung inflammation on secondary RSV exposure in mice born to immunized dams. Despite complete protection following primary RSV exposure, offspring born to pre-fusion F (PreF)-vaccinated dams had exaggerated secondary ILC2 and Th2 responses, characterized by enhanced production of IL-4, IL-5, and IL-13. These enhanced type 2 cellular responses were associated with exaggerated airway eosinophilia and mucus hyperproduction upon re-exposure to RSV. Importantly, depletion of CD4+ T cells led to complete amelioration of the observed type 2 pathology on secondary RSV exposure. These unanticipated results highlight the need for additional studies that look beyond primary protection to better understand how maternal immunization shapes subsequent immune responses to repeat RSV exposure.</p

DataSheet_1_Prior respiratory syncytial virus infection reduces vaccine-mediated Th2-skewed immunity, but retains enhanced RSV F-specific CD8 T cell responses elicited by a Th1-skewing vaccine formulation.pdf

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered due the risk of enhanced respiratory disease (ERD) following natural RSV exposure and the young age (<6 months) at which children would require protection. Risk factors linked to the development of ERD include poorly neutralizing antibody, seronegative status (never been exposed to RSV), and a Th2-type immune response. Stabilization of the more antigenic prefusion F protein (PreF) has reinvigorated hope for a protective RSV vaccine that elicits potent neutralizing antibody. While anecdotal evidence suggests that children and adults previously exposed to RSV (seropositive) are not at risk for developing vaccine associated ERD, differences in host immune responses in seropositive and seronegative individuals that may protect against ERD remain unclear. It is also unclear if vaccine formulations that skew towards Th1- versus Th2-type immune responses increase pathology or provide greater protection in seropositive individuals. Therefore, the goal of this work was to compare the host immune response to a stabilized prefusion RSV antigen formulated alone or with Th1 or Th2 skewing adjuvants in seronegative and seropositive BALB/c mice. We have developed a novel BALB/c mouse model whereby mice are first infected with RSV (seropositive) and then vaccinated during pregnancy to recapitulate maternal immunization strategies. Results of these studies show that prior RSV infection mitigates vaccine-mediated skewing by Th1- and Th2-polarizing adjuvants that was observed in seronegative animals. Moreover, vaccination with PreF plus the Th1-skewing adjuvant, Advax, increased RSV F85-93-specific CD8 T cells in both seronegative and seropositive dams. These data demonstrate the importance of utilizing seropositive animals in preclinical vaccine studies to assess both the safety and efficacy of candidate RSV vaccines.</p

Engineered dityrosine-bonding of the RSV prefusion F protein imparts stability and potency advantages

Viral fusion proteins facilitate cellular infection by fusing viral and cellular membranes, which involves dramatic transitions from their pre- to postfusion conformations. These proteins are among the most protective viral immunogens, but they are metastable which often makes them intractable as subunit vaccine targets. Adapting a natural enzymatic reaction, we harness the structural rigidity that targeted dityrosine crosslinks impart to covalently stabilize fusion proteins in their native conformations. We show that the prefusion conformation of respiratory syncytial virus fusion protein can be stabilized with two engineered dityrosine crosslinks (DT-preF), markedly improving its stability and shelf-life. Furthermore, it has 11X greater potency as compared with the DS-Cav1 stabilized prefusion F protein in immunogenicity studies and overcomes immunosenescence in mice with simply a high-dose formulation on alum

DataSheet_3_Prior respiratory syncytial virus infection reduces vaccine-mediated Th2-skewed immunity, but retains enhanced RSV F-specific CD8 T cell responses elicited by a Th1-skewing vaccine formulation.pdf

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered due the risk of enhanced respiratory disease (ERD) following natural RSV exposure and the young age (<6 months) at which children would require protection. Risk factors linked to the development of ERD include poorly neutralizing antibody, seronegative status (never been exposed to RSV), and a Th2-type immune response. Stabilization of the more antigenic prefusion F protein (PreF) has reinvigorated hope for a protective RSV vaccine that elicits potent neutralizing antibody. While anecdotal evidence suggests that children and adults previously exposed to RSV (seropositive) are not at risk for developing vaccine associated ERD, differences in host immune responses in seropositive and seronegative individuals that may protect against ERD remain unclear. It is also unclear if vaccine formulations that skew towards Th1- versus Th2-type immune responses increase pathology or provide greater protection in seropositive individuals. Therefore, the goal of this work was to compare the host immune response to a stabilized prefusion RSV antigen formulated alone or with Th1 or Th2 skewing adjuvants in seronegative and seropositive BALB/c mice. We have developed a novel BALB/c mouse model whereby mice are first infected with RSV (seropositive) and then vaccinated during pregnancy to recapitulate maternal immunization strategies. Results of these studies show that prior RSV infection mitigates vaccine-mediated skewing by Th1- and Th2-polarizing adjuvants that was observed in seronegative animals. Moreover, vaccination with PreF plus the Th1-skewing adjuvant, Advax, increased RSV F85-93-specific CD8 T cells in both seronegative and seropositive dams. These data demonstrate the importance of utilizing seropositive animals in preclinical vaccine studies to assess both the safety and efficacy of candidate RSV vaccines.</p