Direct administration in the respiratory tract improves efficacy of broadly neutralizing anti-influenza virus monoclonal antibodies.

The emergence of influenza virus strains resistant to approved neuraminidase inhibitors and the time constrains after infection when these drugs can be effective constitute major drawbacks for this class of drugs. This highlights a critical need to discover new therapeutic agents that can be used for the treatment of influenza virus-infected patients. The use of broadly neutralizing anti-influenza monoclonal antibodies (MAbs) has been sought as an alternative immunotherapy against influenza infection. Here, we tested in mice previously characterized broadly neutralizing anti-hemagglutinin (HA) stalk MAbs prophylactically and therapeutically using different routes of administration. The efficacy of treatment against an influenza H1N1 pandemic virus challenge was compared between two systemic routes of administration, intraperitoneal (i.p.) and intravenous (i.v.), and two local routes, intranasal (i.n.) and aerosol (a.e.). The dose of MAb required for prophylactic protection was reduced by 10-fold in animals treated locally (i.n. or a.e.) compared with those treated systemically (i.p. or i.v.). Improved therapeutic protection was observed in animals treated i.n. on day 5 postinfection (60% survival) compared with those treated via the i.p. route (20% survival). An increase in therapeutic efficacy against other influenza virus subtypes (H5N1) was also observed when a local route of administration was used. Our findings demonstrate that local administration significantly decreases the amount of broadly neutralizing monoclonal antibody required for protection against influenza, which highlights the potential use of MAbs as a therapeutic agent for influenza-associated disease.

Assessment of influenza virus hemagglutinin stalk-based immunity in ferrets.

UNLABELLED: Therapeutic monoclonal antibodies that target the conserved stalk domain of the influenza virus hemagglutinin and stalk-based universal influenza virus vaccine strategies are being developed as promising countermeasures for influenza virus infections. The pan-H1-reactive monoclonal antibody 6F12 has been extensively characterized and shows broad efficacy against divergent H1N1 strains in the mouse model. Here we demonstrate its efficacy against a pandemic H1N1 challenge virus in the ferret model of influenza disease. Furthermore, we recently developed a universal influenza virus vaccine strategy based on chimeric hemagglutinin constructs that focuses the immune response on the conserved stalk domain of the hemagglutinin. Here we set out to test this vaccination strategy in the ferret model. Both strategies, pretreatment of animals with a stalk-reactive monoclonal antibody and vaccination with chimeric hemagglutinin-based constructs, were able to significantly reduce viral titers in nasal turbinates, lungs, and olfactory bulbs. In addition, vaccinated animals also showed reduced nasal wash viral titers. In summary, both strategies showed efficacy in reducing viral loads after an influenza virus challenge in the ferret model. IMPORTANCE: Influenza virus hemagglutinin stalk-reactive antibodies tend to be less potent yet are more broadly reactive and can neutralize seasonal and pandemic influenza virus strains. The ferret model was used to assess the potential of hemagglutinin stalk-based immunity to provide protection against influenza virus infection. The novelty and significance of the findings described in this report support the development of vaccines stimulating stalk-specific antibody responses.

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.

Lung ILC2s are activated in BALB/c mice born to immunized mothers despite complete protection against respiratory syncytial virus.

Activated lung ILC2s produce large quantities of IL-5 and IL-13 that contribute to eosinophilic inflammation and mucus production following respiratory syncytial virus infection (RSV). The current understanding of ILC2 activation during RSV infection, is that ILC2s are activated by alarmins, including IL-33, released from airway epithelial cells in response to viral-mediated damage. Thus, high levels of RSV neutralizing maternal antibody generated from maternal immunization would be expected to reduce IL-33 production and mitigate ILC2 activation. Here we report that lung ILC2s from mice born to RSV-immunized dams become activated despite undetectable RSV replication. We also report, for the first time, expression of activating and inhibitory Fcgamma receptors on ILC2s that are differentially expressed in offspring born to immunized versus unimmunized dams. Alternatively, ex vivo IL-33-mediated activation of ILC2s was mitigated following the addition of antibody: antigen immune complexes. Further studies are needed to confirm the role of Fcgamma receptor ligation by immune complexes as an alternative mechanism of ILC2 regulation in RSV-associated eosinophilic lung inflammation.

Biophysical and functional analyses suggest that adenovirus E4-ORF3 protein requires higher-order multimerization to function against promyelocytic leukemia protein nuclear bodies.

The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT·L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity.

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.

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.

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.

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.

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.

Formulation of the prefusion RSV F protein with a Th1/Th2-balanced adjuvant provides complete protection without Th2-skewed immunity in RSV-experienced young mice.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections among infants with most infections occurring in the first year of life. Multiple RSV exposures are required for children to mount adult-like immune responses. Although adult RSV immunity is associated with less severe disease, the protection induced through natural infection is short-lived. Therefore, vaccination of RSV-experienced young children may accelerate immunity and provide long-term protection from RSV reinfection. However, the extent to which different Th-biased vaccine regimens influence pre-existing humoral and cellular immunity in RSV-experienced young children is unknown. To address this question, infant BALB/c mice were RSV-infected and subsequently immunized with the prefusion RSV F (PreF) antigen formulated with either a Th2-skewing (Alum) or Th1/Th2-balanced (Advax-SM) adjuvant. These studies show that both adjuvants boosted neutralizing antibody and protected from RSV reinfection, but Advax-SM adjuvant prevented the Th2-skewed immunity observed in RSV-experienced young mice immunized with PreF/Alum.

Prefusion RSV F Immunization Elicits Th2-Mediated Lung Pathology in Mice When Formulated With a Th2 (but Not a Th1/Th2-Balanced) Adjuvant Despite Complete Viral Protection.

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered by the risk of developing enhanced respiratory disease (ERD) upon natural exposure to the virus. Generation of higher quality neutralizing antibodies with stabilized pre-fusion F protein antigens has been proposed as a strategy to prevent ERD. We sought to test whether there was evidence of ERD in naïve BALB/c mice immunized with an unadjuvanted, stabilized pre-fusion F protein, and challenged with RSV line 19. We further sought to determine the extent to which formulation with a Th2-biased (alum) or a more Th1/Th2-balanced (Advax-SM) adjuvant influenced cellular responses and lung pathology. When exposed to RSV, mice immunized with pre-fusion F protein alone (PreF) exhibited increased airway eosinophilia and mucus accumulation. This was further exacerbated by formulation of PreF with Alum (aluminum hydroxide). Conversely, formulation of PreF with a Th1/Th2-balanced adjuvant, Advax-SM, not only suppressed RSV viral replication, but also inhibited airway eosinophilia and mucus accumulation. This was associated with lower numbers of lung innate lymphocyte cells (ILC2s) and CD4+ T cells producing IL-5+ or IL-13+ and increased IFNγ+ CD4+ and CD8+ T cells, in addition to RSV F-specific CD8+ T cells. These data suggest that in the absence of preimmunity, stabilized PreF antigens may still be associated with aberrant Th2 responses that induce lung pathology in response to RSV infection, and can be prevented by formulation with more Th1/Th2-balanced adjuvants that enhance CD4+ and CD8+ IFNγ+ T cell responses. This may support the use of stabilized PreF antigens with Th1/Th2-balanced adjuvants like, Advax-SM, as safer alternatives to alum in RSV vaccine candidates.

Maternal immunization with adjuvanted RSV prefusion F protein effectively protects offspring from RSV challenge and alters innate and T cell immunity.

Respiratory syncytial virus (RSV) commonly causes severe respiratory tract infections in infants, peaking between 2 and 6 months of age; an age at which direct vaccination is unlikely to be effective. Maternal immunization can deliver high levels of antibodies to newborns, providing immediate protection. Following natural infection, antibodies targeting the prefusion conformation of RSV F protein (PreF) have the greatest neutralizing capacity and thus, may provide infants with a high degree of RSV protection when acquired through maternal vaccination. However, the influence of anti-PreF maternal antibodies on infant immunity following RSV exposure has not been elucidated. To address this knowledge gap, offspring born to dams immunized with a RSV PreF vaccine formulation were challenged with RSV and their immune responses were analyzed over time. These studies demonstrated safety and efficacy for RSV-challenged, maternally-immunized offspring but high and waning maternal antibody levels were associated with differential innate and T cell immunity.

Modulation of an ectodomain motif in the influenza A virus neuraminidase alters tetherin sensitivity and results in virus attenuation in vivo.

We previously demonstrated that ectodomain residue Asp286 in N2 neuraminidase (NA; Asp268 in N1 NA) present in budding-capable NA proteins contributes to productive NA plasma membrane transport partly by mediating escape from tetherin restriction [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480-2491]. Budding-incapable NA proteins contain a G at this position and either co-expression of human immunodeficiency virus type 1 vpu or siRNA-mediated depletion of tetherin rescued budding capabilities in these proteins [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480-2491]. Furthermore, replacement of D286 with G in budding-capable NA proteins caused loss of function, preventing release of NA virus-like particles (VLPs). Here, we show that mutation of this residue specifically modulates the ability of NA to escape tetherin restriction at the plasma membrane and results in virus attenuation in vivo. Based on immunogold electron microscopy and co-immunoprecipitation assays, both NAD286-containing and NAD286G-containing proteins associated with tetherin in the endoplasmic reticulum (ER). However, the NAD286G loss-of-function mutant also associated with the host factor outside the ER and in plasma-membrane-localized VLPs as visualized using immunogold electron microscopy. We conclude that the presence of aspartate at residue 286 liberates NA from tetherin-dependent restriction upon exit from the ER compartment thus preventing restriction at the plasma membrane. Underscoring the importance of these observations, knockdown of tetherin resulted in a 1-1.5 log increase in influenza virus growth. Additionally, the loss-of-function mutation conferred attenuation in a mouse model of influenza infection as evidenced by a 5-fold increase in LD50 and increases in either percent survival or time to death dependent on the administered dose in vivo.

Adenovirus E4-ORF3-dependent relocalization of TIF1α and TIF1γ relies on access to the Coiled-Coil motif.

The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1α), but not homologous TIF1ß. Here, we introduce TIF1γ as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1γ in virus-infected cells in vivo and binds to TIF1γ in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1α, TIF1ß, and TIF1γ, and that the C-terminal half of TIF1ß interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.

Un-"ESCRT"-ed budding.

In their recent publication, Rossman et al. describe how the inherent budding capability of its M2 protein allows influenza A virus to bypass recruitment of the cellular ESCRT machinery enlisted by several other enveloped RNA and DNA viruses, including HIV, Ebola, rabies, herpes simplex type 1 and hepatitis B. Studies from the same laboratory and other laboratories indicate that budding of plasmid-derived virus-like particles can be mediated by the influenza virus hemagglutinin and neuraminidase proteins in the absence of M2. These events are also independent of canonical ESCRT components. Understanding how intrinsic properties of these influenza virus proteins permit ESCRT-independent budding expands our understanding of the budding process itself.

Influenza virus vaccine based on the conserved hemagglutinin stalk domain.

Although highly effective in the general population when well matched to circulating influenza virus strains, current influenza vaccines are limited in their utility due to the narrow breadth of protection they provide. The strain specificity of vaccines presently in use mirrors the exquisite specificity of the neutralizing antibodies that they induce, that is, antibodies which bind to the highly variable globular head domain of hemagglutinin (HA). Herein, we describe the construction of a novel immunogen comprising the conserved influenza HA stalk domain and lacking the globular head. Vaccination of mice with this headless HA construct elicited immune sera with broader reactivity than those obtained from mice immunized with a full-length HA. Furthermore, the headless HA vaccine provided full protection against death and partial protection against disease following lethal viral challenge. Our results suggest that the response induced by headless HA vaccines is sufficiently potent to warrant their further development toward a universal influenza virus vaccine.

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 (TIF1alpha). TIF1alpha functions by recruiting coactivators and/or corepressors to modulate transcription. The interaction between E4 ORF3 and TIF1alpha was validated by coimmunoprecipitation and binding of recombinant proteins. Indirect immunofluorescence assays demonstrated that TIF1alpha is reorganized into track structures that contain PML upon E4 ORF3 expression. The RBCC domain of TIF1alpha is sufficient for E4 ORF3-induced rearrangement, and TIF1alpha reorganization is conserved across adenovirus serotypes.