Molecular Basis of Antigenic Drift in Serotype O Foot-and-Mouth Disease Viruses (2013–2018) from Southeast Asia

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals with serious economic consequences. FMD is endemic in Southeast Asia (SEA) and East Asia (EA) with the circulation of multiple serotypes, posing a threat to Australia and other FMD-free countries. Although vaccination is one of the most important control measures to prevent FMD outbreaks, the available vaccines may not be able to provide enough cross-protection against the FMD viruses (FMDVs) circulating in these countries due to the incursion of new lineages and sub-lineages as experienced in South Korea during 2010, a FMD-free country, when a new lineage of serotype O FMDV (Mya-98) spread to the country, resulting in devastating economic consequences. In this study, a total of 62 serotype O (2013–2018) viruses selected from SEA and EA countries were antigenically characterized by virus neutralization tests using three existing (O/HKN/6/83, O/IND/R2/75 and O/PanAsia-2) and one putative (O/MYA/2009) vaccine strains and full capsid sequencing. The Capsid sequence analysis revealed three topotypes, Cathay, SEA and Middle East-South Asia (ME-SA) of FMDVs circulating in the region. The vaccines used in this study showed a good match with the SEA and ME-SA viruses. However, none of the recently circulating Cathay topotype viruses were protected by any of the vaccine strains, including the existing Cathay topotype vaccine (O/HKN/6/83), indicating an antigenic drift and, also the urgency to monitor this topotype in the region and develop a new vaccine strain if necessary, although currently the presence of this topotype is mainly restricted to China, Hong Kong, Taiwan and Vietnam. Further, the capsid sequences of these viruses were analyzed that identified several capsid amino acid substitutions involving neutralizing antigenic sites 1, 2 and 5, which either individually or together could underpin the observed antigenic drift.

Coevolution of Adeno-associated Virus Capsid Antigenicity and Tropism through a Structure-Guided Approach

ABSTRACT Adeno-associated viruses (AAV) are composed of nonenveloped, icosahedral protein shells that can be adapted to package and deliver recombinant therapeutic DNA. Approaches to engineer recombinant capsids for gene therapy applications have focused on rational design or library-based approaches that can address one or two desirable attributes; however, there is an unmet need to comprehensively improve AAV vector properties. Such cannot be achieved by utilizing sequence data alone but requires harnessing the three-dimensional (3D) structural properties of AAV capsids. Here, we solve the structures of a natural AAV isolate complexed with antibodies using cryo-electron microscopy and harness this structural information to engineer AAV capsid libraries through saturation mutagenesis of different antigenic footprints. Each surface loop was evolved by infectious cycling in the presence of a helper adenovirus to yield a new AAV variant that then serves as a template for evolving the next surface loop. This stepwise process yielded a humanized AAV8 capsid (AAVhum.8) displaying nonnatural surface loops that simultaneously display tropism for human hepatocytes, increased gene transfer efficiency, and neutralizing antibody evasion. Specifically, AAVhum.8 can better evade neutralizing antisera from multiple species than AAV8. Further, AAVhum.8 displays robust transduction in a human liver xenograft mouse model with expanded tropism for both murine and human hepatocytes. This work supports the hypothesis that critical properties, such as AAV capsid antibody evasion and tropism, can be coevolved by combining rational design and library-based evolution for clinical gene therapy. IMPORTANCE Clinical gene therapy with recombinant AAV vectors has largely relied on natural capsid isolates. There is an unmet need to comprehensively improve AAV tissue tropism, transduction efficiency, and antibody evasion. Such cannot be achieved by utilizing capsid sequence data alone but requires harnessing the 3D structural properties of AAV capsids. Here, we combine rational design and library-based evolution to coevolve multiple, desirable properties onto AAV by harnessing 3D structural information.

Human Norovirus Epitope D Plasticity Allows Escape from Antibody Immunity without Loss of Capacity for Binding Cellular Ligands

ABSTRACT Emergent strains of human norovirus seed pandemic waves of disease. These new strains have altered ligand binding and antigenicity characteristics. Study of viral variants isolated from immunosuppressed patients with long-term norovirus infection indicates that initial virus in vivo evolution occurs at the same antigenic sites as in pandemic strains. Here, cellular ligand binding and antigenicity of two cocirculating strains isolated from a patient with long-term norovirus infection were characterized. The isolated GII.4 viruses differed from previous strains and from each other at known blockade antibody epitopes. One strain had a unique sequence in epitope D, including loss of an insertion at residue 394, corresponding to a decreased relative affinity for carbohydrate ligands. Replacement of 394 with alanine or restoration of the contemporary strain epitope D consensus sequence STT improved ligand binding relative affinity. However, monoclonal antibody blockade of binding potency was only gained for the consensus sequence, not by the alanine insertion. In-depth study of unique changes in epitope D indicated that ligand binding, but not antibody blockade of ligand binding, is maintained despite sequence diversity, allowing escape from blockade antibodies without loss of capacity for binding cellular ligands. IMPORTANCE Human norovirus causes ∼20% of all acute gastroenteritis and ∼200,000 deaths per year, primarily in young children. Most epidemic and all pandemic waves of disease over the past 30 years have been caused by type GII.4 human norovirus strains. The capsid sequence of GII.4 strains is changing over time, resulting in viruses with altered ligand and antibody binding characteristics. The carbohydrate binding pocket of these strains does not vary over time. Here, utilizing unique viral sequences, we study how residues in GII.4 epitope D balance the dual roles of variable antibody binding site and cellular ligand binding stabilization domain, demonstrating that amino acid changes in epitope D can result in loss of antibody binding without ablating ligand binding. This flexibility in epitope D likely contributes to GII.4 strain persistence by both allowing escape from antibody-mediated herd immunity and maintenance of cellular ligand binding and infectivity.

Crystal structure of the 3C protease from Southern African Territories type 2 foot-and-mouth disease virus

The replication of foot-and-mouth disease virus (FMDV) is dependent on the virus-encoded 3C protease (3Cpro). As in other picornaviruses, 3Cproperforms most of the proteolytic processing of the polyprotein expressed from the large open reading frame in the RNA genome of the virus. Previous work revealed that the 3Cprofrom serotype A—one of the seven serotypes of FMDV—adopts a trypsin-like fold. On the basis of capsid sequence comparisons the FMDV serotypes are grouped into two phylogenetic clusters, with O, A, C, and Asia 1 in one, and the three Southern African Territories serotypes, (SAT-1, SAT-2 and SAT-3) in another, a grouping pattern that is broadly, but not rigidly, reflected in 3Cproamino acid sequences. We report here the cloning, expression and purification of 3C proteases from four SAT serotype viruses (SAT2/GHA/8/91, SAT1/NIG/5/81, SAT1/UGA/1/97, and SAT2/ZIM/7/83) and the crystal structure at 3.2 Å resolution of 3Cprofrom SAT2/GHA/8/91.

The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver

Although adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-α, monocyte chemotactic protein-1, IFN-γ inducible protein-10, and IFN-α/β expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.

Self-Complementary AAV Vectors Cause a Substantially Heightened TLR9-Dependent Innate Immune Response In the Liver

Abstract 252 Successful gene therapy for hemophilia requires therapeutic expression in the absence of destructive immune responses. Clinical trials have revealed that adaptive immune responses eliminated therapeutic levels of factor IX (F.IX) following high-dose hepatic in vivo gene transfer using single stranded (ss) AAV serotype 2 vector. More efficient AAV vectors have been developed using AAV2 with Y-F capsid mutations, alternate serotypes such as AAV8, and self-complimentary (sc) rAAV vectors. Innate immune responses to AAV in the liver are thought to be weak and highly transient. Our study sought to investigate whether variations in the capsid or genome will alter the immune profile. AAV vectors were delivered into the portal circulation of C3H/OuJ mice at 1011 vector genomes per mouse. We tested ssAAV-2, ssAAV2-triple Y-F mutant, ssAAV-8 vectors as well as scAAV-2 and scAAV-8 vectors, all expressing the hF.IX transgene. Quantitative RT-PCR array showed a mild transient up-regulation of MyD88, TLR-9, TNF-α, MCP-1, IP-10 and IFN-α/β within 2 hrs, which subsided by 6 hrs following delivery of ssAAV vectors, regardless of the capsid sequence/efficiency of hepatocyte transduction. In contrast, scAAV-2 and scAAV-8 vectors induced 4- to 8-fold increases in TLR-2, TLR-9, MyD88, TNF-α, MCP-1, IP-10, and IFN-α/β expression when compared to the ssAAV vectors. In addition, scAAV vectors induced expression of pro-inflammatory cytokines and chemokines not detected for ssAAV: IL-6, IL-12α, MIP-1, and RANTES. Paralleling this data was a 4-fold increase in local protein levels of IL-6, TNF-α, and MCP-1, and a systemic increase in IL-6. None of the vectors activated TLR-4, indicating that these effects cannot be attributed to LPS contamination. Several other innate response genes showed no differences in expression compared to PBS injected mice, which included IL-1α, IL-1β, KC, TLR-1, and TLR-3 to -8. The heightened innate response to scAAV vectors was further characterized by a 5-fold increase in macrophage and neutrophil infiltrates in liver sections, illustrating the potential for hepatotoxicity. Innate responses were absent in TLR-9 deficient C57BL/6 mice and in wild-type mice that had received an oligonucleotide ligand inhibitory to TLR-9. Compared to wild-type mice, hFIX levels were increased 2-fold in TLR-9 deficient mice following scAAV vector delivery and antibody formation against capsid was delayed. Additionally, in vitro studies with a TLR-9 reporter cell line showed significantly increased TLR-9 signaling for scAAV2 compared to ssAAV vectors, independent of the transgene/expression cassette. Neither vector activated the inflammasome. Further in vivo studies showed that the innate response to scAAV was, with the exception of TNF-a expression, Kupffer cell dependent, likely owing to the importance of these cells to sequester viral particles in the liver. Our results have several implications. Gene therapy applications based on administration of high-dose scAAV may be limited by immunotoxicities, which, however, can be prevented by inhibition of TLR-9 signaling. Of general importance, changing the genome configuration of a virus from single- to double-stranded DNA increased sensing by the endosomal receptor TLR-9, thereby enhancing innate immunity. Disclosures: Herzog: Genzyme Corp: Patents & Royalties.

Evolution and Phylogeography of the Nonpathogenic Calicivirus RCV-A1 in Wild Rabbits in Australia

ABSTRACT Despite its potential importance for the biological control of European rabbits, relatively little is known about the evolution and molecular epidemiology of rabbit calicivirus Australia 1 (RCV-A1). To address this issue we undertook an extensive evolutionary analysis of 36 RCV-A1 samples collected from wild rabbit populations in southeast Australia between 2007 and 2009. Based on phylogenetic analysis of the entire capsid sequence, six clades of RCV-A1 were defined, each exhibiting strong population subdivision. Strikingly, our estimates of the time to the most recent common ancestor of RCV-A1 coincide with the introduction of rabbits to Australia in the mid-19th century. Subsequent divergence events visible in the RCV-A1 phylogenies likely reflect key moments in the history of the European rabbit in Australia, most notably the bottlenecks in rabbit populations induced by the two viral biocontrol agents used on the Australian continent, myxoma virus and rabbit hemorrhagic disease virus (RHDV). RCV-A1 strains therefore exhibit strong phylogeographic separation and may constitute a useful tool to study recent host population dynamics and migration patterns, which in turn could be used to monitor rabbit control in Australia.

Strain-Specific Differences in the Impact of Human TRIM5α, Different TRIM5α Alleles, and the Inhibition of Capsid-Cyclophilin A Interactions on the Infectivity of HIV-1

ABSTRACT HIV-1 infectivity is strongly restricted by TRIM5α from certain primate species but has been described as being only marginally susceptible to human TRIM5α. In this study, we evaluated the effects of the modulation of human TRIM5α activity (pretreatment of target cells with alpha interferon, expression of a pre-miRNA targeting TRIM5α, and/or overexpression of TRIM5γ), the inhibition of cyclophilin A (CypA)-CA interactions, and the expression of different allelic variants of human TRIM5α on the infectivity of a series of recombinant viruses carrying different patient-derived Gag-protease sequences. We show that HIV-1 displays virus-specific differences in its sensitivity to human TRIM5α and in its sensitivity to different TRIM5α alleles. The effect of inhibiting CypA-CA interactions is also strain specific, and blocking these interactions can either inhibit or improve viral infectivity, depending on the isolate studied. The inhibition of CypA-CA interactions also modulates viral sensitivity to human TRIM5α. In the absence of CypA-CA interactions, most viruses displayed increased sensitivity to the inhibitory effects of TRIM5α on viral replication, but one isolate showed a paradoxical decrease in sensitivity to TRIM5α. Taken together, these findings support a model in which three interlinked factors—capsid sequence, CypA levels, and TRIM5α—interact to determine capsid stability and therefore viral infectivity.