Mediation of antitumor activity by AZD4820 oncolytic vaccinia virus encoding IL-12.

Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase-treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti-PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors.

Development of a potent Zika virus vaccine using self-amplifying messenger RNA.

Zika virus (ZIKV) is the cause of a pandemic associated with microcephaly in newborns and Guillain-Barre syndrome in adults. Currently, there are no available treatments or vaccines for ZIKV, and the development of a safe and effective vaccine is a high priority for many global health organizations. We describe the development of ZIKV vaccine candidates using the self-amplifying messenger RNA (SAM) platform technology delivered by cationic nanoemulsion (CNE) that allows bedside mixing and is particularly useful for rapid responses to pandemic outbreaks. Two immunizations of either of the two lead SAM (CNE) vaccine candidates elicited potent neutralizing antibody responses to ZIKV in mice and nonhuman primates. Both SAM (CNE) vaccines protected these animals from ZIKV challenge, with one candidate providing complete protection against ZIKV infection in nonhuman primates. The data provide a preclinical proof of concept that a SAM (CNE) vaccine candidate can rapidly elicit protective immunity against ZIKV.

A novel mode of poxvirus superinfection exclusion that prevents fusion of the lipid bilayers of viral and cellular membranes.

UNLABELLED: Superinfection exclusion is a widespread phenomenon that prevents secondary infections by closely related viruses. The vaccinia virus A56 and K2 proteins in the cell membrane can prevent superinfection by interacting with the entry-fusion complex of subsequent viruses. Here, we described another form of exclusion that is established earlier in infection and does not require the A56 or K2 protein. Cells infected with one or more infectious virions excluded hundreds of superinfecting vaccinia virus particles. A related orthopoxvirus, but neither a flavivirus nor a rhabdovirus, was also excluded, indicating selectivity. Although superinfecting vaccinia virus bound to cells, infection was inhibited at the membrane fusion step, thereby preventing core entry into the cytoplasm and early gene expression. In contrast, A56/K2 protein-mediated exclusion occurred subsequent to membrane fusion. Induction of resistance to superinfection depended on viral RNA and protein synthesis by the primary virus but did not require DNA replication. Although superinfection resistance correlated with virus-induced changes in the cytoskeleton, studies with mutant vaccinia viruses indicated that the cytoskeletal changes were not necessary for resistance to superinfection. Interferon-inducible transmembrane proteins, which can inhibit membrane fusion in other viral systems, did not prevent vaccinia virus membrane fusion, suggesting that these interferon-inducible proteins are not involved in superinfection exclusion. While the mechanism remains to be determined, the early establishment of superinfection exclusion may provide a "winner-take-all" reward to the first poxvirus particles that successfully initiate infection and prevent the entry and genome reproduction of defective or less fit particles. IMPORTANCE: The replication of a virus usually follows a defined sequence of events: attachment, entry into the cytoplasm or nucleus, gene expression, genome replication, assembly of infectious particles, and spread to other cells. Although multiple virus particles may enter a cell at the same time, mechanisms exist to prevent infection by subsequent viruses. The latter phenomenon, known as superinfection exclusion, can occur by a variety of mechanisms that are not well understood. We showed that superinfection by vaccinia virus was prevented at the membrane fusion step, which closely followed virion attachment. Thus, neither gene expression nor genome replication of the superinfecting virus occurred. Expression of early proteins by the primary virus was necessary and sufficient to induce the superinfection-resistant state. Superinfection exclusion may be beneficial to vaccinia virus by selecting particles that can infect cells rapidly, excluding defective particles and synchronizing the replication cycle.

The membrane fusion step of vaccinia virus entry is cooperatively mediated by multiple viral proteins and host cell components.

For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.

Assembly and biological and immunological properties of Newcastle disease virus-like particles.

Virus-like particles (VLPs) released from avian cells expressing the Newcastle disease virus (NDV) strain AV proteins NP, M, HN (hemagglutinin-neuraminidase), and F were characterized. The VLP-associated HN and F glycoproteins directed the attachment of VLPs to cell surfaces and fusion of VLP membranes with red blood cell membranes, indicating that they were assembled into VLPs in an authentic conformation. These particles were quantitatively prepared and used as an immunogen, without adjuvant, in BALB/c mice. The resulting immune responses, detected by enzyme-linked immunosorbent assay (ELISA), virus neutralization, and intracellular cytokine staining, were comparable to the responses to equivalent amounts of inactivated NDV vaccine virus. HN and F proteins from another strain of NDV, strain B1, could be incorporated into these VLPs. Foreign peptides were incorporated into these VLPs when fused to the NP or HN protein. The ectodomain of a foreign glycoprotein, the Nipah virus G protein, fused to the NDV HN protein cytoplasmic and transmembrane domains was incorporated into ND VLPs. Thus, ND VLPs are a potential NDV vaccine candidate. They may also serve as a platform to construct vaccines for other pathogens.

Lipid membranes in poxvirus replication.

Poxviruses replicate in the cytoplasm, where they acquire multiple lipoprotein membranes. Although a proposal that the initial membrane arises de novo has not been substantiated, there is no accepted explanation for its formation from cellular membranes. A subsequent membrane-wrapping step involving modified trans-Golgi or endosomal cisternae results in a particle with three membranes. These wrapped virions traverse the cytoplasm on microtubules; the outermost membrane is lost during exocytosis, the middle one is lost just prior to cell entry, and the remaining membrane fuses with the cell to allow the virus core to enter the cytoplasm and initiate a new infection.

Appraising the apoptotic mimicry model and the role of phospholipids for poxvirus entry.

Entry of vaccinia virus (VACV) into cells occurs by fusion with the plasma membrane and via a low pH-dependent endosomal pathway, presumably involving unidentified cellular receptors. In addition to approximately 25 viral proteins, the membrane of VACV mature virions contains several phospholipids including phosphatidylserine (PS). A recent model posits that PS flags virions as apoptotic debris to activate a common cellular uptake pathway to gain cell entry, perhaps through an interaction with a PS-specific cell surface receptor. To evaluate the apoptotic mimicry model, we reconstituted the membrane of detergent-extracted virions with several different phospholipids. Although the ability of the L-stereoisomer of PS to reconstitute infectivity was confirmed, the nonbiologically relevant D-stereoisomer of PS, and phosphatidylglycerol, which are not normally present in the virion membrane, functioned as well. Regardless of which phospholipid reconstituted infectivity, virus entry was inhibited by a neutralizing monoclonal antibody to a virion surface protein and by the drugs blebbistatin and bafilomycin A1, suggesting that in each case virus uptake was specific and occurred by a similar mechanism involving macropinocytosis and a low-pH endocytic pathway. Lipid-reconstituted and nonreconstituted, membrane-extracted virions were equally capable of binding to cells. However, the physical association of phospholipids with virus particles during membrane reconstitution correlated directly with rescue of particle infectivity and cell entry capability. Our results support a role for PS in poxvirus entry, but demonstrate that other phospholipids, not known to signal uptake of apoptotic debris, can function similarly.

Incorporation of functional HN-F glycoprotein-containing complexes into newcastle disease virus is dependent on cholesterol and membrane lipid raft integrity.

Newcastle disease virus assembles in plasma membrane domains with properties of membrane lipid rafts, and disruption of these domains by cholesterol extraction with methyl-beta-cyclodextrin resulted in the release of virions with irregular protein composition, abnormal particle density, and reduced infectivity (J. P. Laliberte, L. W. McGinnes, M. E. Peeples, and T. G. Morrison, J. Virol. 80:10652-10662, 2006). In the present study, these results were confirmed using Niemann-Pick syndrome type C cells, which are deficient in normal membrane rafts due to mutations affecting cholesterol transport. Furthermore, cholesterol extraction of infected cells resulted in the release of virions that attached to target cells at normal levels but were defective in virus-cell membrane fusion. The reduced fusion capacity of particles released from cholesterol-extracted cells correlated with significant loss of HN-F glycoprotein-containing complexes detected in the virion envelopes of these particles and with detection of cell-associated HN-F protein-containing complexes in extracts of cholesterol-extracted cells. Extraction of cholesterol from purified virions had no effect on virus-cell attachment, virus-cell fusion, particle infectivity, or the levels of glycoprotein-containing complexes. Taken together, these results suggest that cholesterol and membrane rafts are required for the formation or maintenance of HN-F glycoprotein-containing complexes in cells but not the stability of preformed glycoprotein complexes once assembled into virions.

Integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus particles.

Membrane lipid raft domains are thought to be sites of assembly for many enveloped viruses. The roles of both classical lipid rafts and lipid rafts associated with the membrane cytoskeleton in the assembly of Newcastle disease virus (NDV) were investigated. The lipid raft-associated proteins caveolin-1, flotillin-2, and actin were incorporated into virions, while the non-lipid raft-associated transferrin receptor was excluded. Kinetic analyses of the distribution of viral proteins in lipid rafts, as defined by detergent-resistant membranes (DRMs), in non-lipid raft membranes, and in virions showed an accumulation of HN, F, and NP viral proteins in lipid rafts early after synthesis. Subsequently, these proteins exited the DRMs and were recovered quantitatively in purified virions, while levels of these proteins in detergent-soluble cell fractions remained relatively constant. Cholesterol depletion of infected cells drastically altered the association of viral proteins with DRMs and resulted in an enhanced release of virus particles with reduced infectivity. Decreased infectivity was not due to effects on subsequent virus entry, since the extraction of cholesterol from intact virus did not significantly reduce infectivity. Particles released from cholesterol-depleted cells had very heterogeneous densities and altered ratios of NP and glycoproteins, demonstrating structural abnormalities which potentially contributed to their lowered infectivity. Taken together, these results indicate that lipid rafts, including cytoskeleton-associated lipid rafts, are sites of NDV assembly and that these domains are important for ordered assembly and release of infectious Newcastle disease virus particles.