Federated learning enables big data for rare cancer boundary detection.

Although machine learning (ML) has shown promise across disciplines, out-of-sample generalizability is concerning. This is currently addressed by sharing multi-site data, but such centralization is challenging/infeasible to scale due to various limitations. Federated ML (FL) provides an alternative paradigm for accurate and generalizable ML, by only sharing numerical model updates. Here we present the largest FL study to-date, involving data from 71 sites across 6 continents, to generate an automatic tumor boundary detector for the rare disease of glioblastoma, reporting the largest such dataset in the literature (n = 6, 314). We demonstrate a 33% delineation improvement for the surgically targetable tumor, and 23% for the complete tumor extent, over a publicly trained model. We anticipate our study to: 1) enable more healthcare studies informed by large diverse data, ensuring meaningful results for rare diseases and underrepresented populations, 2) facilitate further analyses for glioblastoma by releasing our consensus model, and 3) demonstrate the FL effectiveness at such scale and task-complexity as a paradigm shift for multi-site collaborations, alleviating the need for data-sharing

Author Correction: Federated learning enables big data for rare cancer boundary detection.

10.1038/s41467-023-36188-7NATURE COMMUNICATIONS14

Federated Learning Enables Big Data for Rare Cancer Boundary Detection

Although machine learning (ML) has shown promise across disciplines, out-of-sample generalizability is concerning. This is currently addressed by sharing multi-site data, but such centralization is challenging/infeasible to scale due to various limitations. Federated ML (FL) provides an alternative paradigm for accurate and generalizable ML, by only sharing numerical model updates. Here we present the largest FL study to-date, involving data from 71 sites across 6 continents, to generate an automatic tumor boundary detector for the rare disease of glioblastoma, reporting the largest such dataset in the literature (n = 6, 314). We demonstrate a 33% delineation improvement for the surgically targetable tumor, and 23% for the complete tumor extent, over a publicly trained model. We anticipate our study to: 1) enable more healthcare studies informed by large diverse data, ensuring meaningful results for rare diseases and underrepresented populations, 2) facilitate further analyses for glioblastoma by releasing our consensus model, and 3) demonstrate the FL effectiveness at such scale and task-complexity as a paradigm shift for multi-site collaborations, alleviating the need for data-sharing

Capsid Protein C of Tick-Borne Encephalitis Virus Tolerates Large Internal Deletions and Is a Favorable Target for Attenuation of Virulence

Deletions ranging in size from 4 to 21 amino acid residues were introduced into the capsid protein of the flavivirus tick-borne encephalitis (TBE) virus. These deletions incrementally affected a hydrophobic domain which is present at the center of all flavivirus capsid protein sequences and part of which may form an amphipathic alpha-helix. In the context of the full-length TBE genome, the deletions did not measurably affect protein expression and up to a deletion length of 16 amino acid residues, corresponding to almost 17% of mature protein C, viable virus was recovered. This virus was strongly attenuated but highly immunogenic in adult mice, revealing capsid protein C as a new and attractive target for the directed attenuation of flaviviruses. Apparently, the larger deletions interfered with the correct assembly of infectious virus particles, and this disturbance of virion assembly is likely to be the molecular basis of attenuation. However, all of the mutants carrying large deletions produced substantial amounts of subviral particles, which as judged from density gradient analyses were identical to recombinant subviral particles as obtained by the expression of the surface proteins prM and E alone. The structural and functional flexibility of protein C revealed in this study and its predicted largely alpha-helical conformation are reminiscent of capsid proteins of other enveloped viruses, such as alphaviruses (N-terminal domain of the capsid protein), retroviruses, and hepadnaviruses and suggest that all of these may belong to a common structural class, which is fundamentally distinct from the classical β-barrel structures of many icosahedral viral capsids. The possibility of attenuating flaviviruses by disturbing virus assembly and favoring the production of noninfectious but highly immunogenic subviral particles opens up a promising new avenue for the development of live flavivirus vaccines

Functional Analysis of the Tick-Borne Encephalitis Virus Cyclization Elements Indicates Major Differences between Mosquito-Borne and Tick-Borne Flaviviruses

The linear, positive-stranded RNA genome of flaviviruses is thought to adopt a circularized conformation via interactions of short complementary sequence elements located within its terminal regions. This process of RNA cyclization is a crucial precondition for RNA replication. In the case of mosquito-borne flaviviruses, highly conserved cyclization sequences (CS) have been identified, and their functionality has been experimentally confirmed. Here, we provide an experimental identification of CS elements of tick-borne encephalitis virus (TBEV). These elements, termed 5′-CS-A and 3′-CS-A, are conserved among various tick-borne flaviviruses, but they are unrelated to the mosquito-borne CS elements and are located at different genomic positions. The 5′-CS-A element is situated upstream rather than downstream of the AUG start codon and, in contrast to mosquito-borne flaviviruses, it was found that the entire protein C coding region is not essential for TBEV replication. The complementary 3′-CS-A element is located within the bottom stem rather than upstream of the characteristic 3′-terminal stem-loop structure, implying that this part of the proposed structure cannot be formed when the genome is in its circularized conformation. Finally, we demonstrate that the CS-A elements can also mediate their function when the 5′-CS-A element is moved from its natural position to one corresponding to the mosquito-borne CS. The recognition of essential RNA elements and their differences between mosquito-borne and tick-borne flaviviruses has practical implications for the design of replicons in vaccine and vector development

Construction and Mutagenesis of an Artificial Bicistronic Tick-Borne Encephalitis Virus Genome Reveals an Essential Function of the Second Transmembrane Region of Protein E in Flavivirus Assembly

Flaviviruses have a monopartite positive-stranded RNA genome, which serves as the sole mRNA for protein translation. Cap-dependent translation produces a polyprotein precursor that is co- and posttranslationally processed by proteases to yield the final protein products. In this study, using tick-borne encephalitis virus (TBEV), we constructed an artificial bicistronic flavivirus genome (TBEV-bc) in which the capsid protein and the nonstructural proteins were still encoded in the cap cistron but the coding region for the surface proteins prM and E was moved to a separate translation unit under the control of an internal ribosome entry site element inserted into the 3′ noncoding region. Mutant TBEV-bc was shown to produce particles that packaged the bicistronic RNA genome and were infectious for BHK-21 cells and mice. Compared to wild-type controls, however, TBEV-bc was less efficient in both RNA replication and infectious particle formation. We took advantage of the separate expression of the E protein in this system to investigate the role in viral assembly of the second transmembrane region of protein E (E-TM2), a second copy of which was retained in the cap cistron to fulfill its other role as an internal signal sequence in the polyprotein. Deletion analysis and replacement of the entire TBEV E-TM2 region with its counterpart from another flavivirus revealed that this element, apart from its role as a signal sequence, is important for virion formation

Humoral and Cellular Immune Response to RNA Immunization with Flavivirus Replicons Derived from Tick-Borne Encephalitis Virus

A new vaccination principle against flaviviruses, based on a tick-borne encephalitis virus (TBEV) self-replicating noninfectious RNA vaccine that produces subviral particles, has recently been introduced (R. M. Kofler, J. H. Aberle, S. W. Aberle, S. L. Allison, F. X. Heinz, and C. W. Mandl, Proc. Natl. Acad. Sci. USA 7:1951-1956, 2004). In this study, we evaluated the potential of the self-replicating RNA vaccine in mice in comparison to those of live, attenuated vaccines and a formalin-inactivated whole-virus vaccine (ImmunInject). For this purpose, mice were immunized using gene gun-mediated application of the RNA vaccine and tested for CD8(+) T-cell responses, long-term duration, neutralizing capacity, and isotype profile of specific antibodies and protection against lethal virus challenge. We demonstrate that the self-replicating RNA vaccine induced a broad-based, humoral and cellular (Th1 and CD8(+) T-cell response) immune response comparable to that induced by live vaccines and that it protected mice from challenge. Even a single immunization with 1 μg of the replicon induced a long-lasting antibody response, characterized by high neutralizing antibody titers, which were sustained for at least 1 year. Nevertheless, it was possible to boost this response further by a second injection with the RNA vaccine, even in the presence of a concomitant CD8(+) T-cell response. In this way it was possible to induce a balanced humoral and cellular immune response, similar to infection-induced immunity but without the safety hazards of infectious agents. The results also demonstrate the value of TBEV replicon RNA for inducing protective long-lasting antiviral responses

Isolation of Capsid Protein Dimers from the Tick-Borne Encephalitis Flavivirus and In Vitro Assembly of Capsid-Like Particles

Flaviviruses have a spherical capsid that is composed of multiple copies of a single capsid protein and, in contrast to the viral envelope, apparently does not have an icosahedral structure. So far, attempts to isolate distinct particulate capsids and soluble forms of the capsid protein from purified virions as well as to assemble capsid-like particles in vitro have been largely unsuccessful. Here we describe the isolation of nucleocapsids from tick-borne encephalitis (TBE) virus and their disintegration into a capsid protein dimer by high-salt treatment. Purified capsid protein dimers could be assembled in vitro into capsid-like particles when combined with in vitro transcribed viral RNA. Particulate structures could also be obtained when single-stranded DNA oligonucleotides were used. These data suggest that the dimeric capsid protein functions as a basic building block in the assembly process of flaviviruses

Spontaneous Mutations Restore the Viability of Tick-Borne Encephalitis Virus Mutants with Large Deletions in Protein C

The capsid protein, C, of tick-borne encephalitis virus has recently been found to tolerate deletions up to a length of 16 amino acid residues that partially removed the central hydrophobic domain, a sequence element conserved among flaviviruses which may be crucial for virion assembly. In this study, mutants with deletion lengths of 19, 21, 27, or 30 residues, removing more or all of this hydrophobic domain, were found to yield viable virus progeny, but this was without exception accompanied by the emergence of additional mutations within protein C. These point mutations or sequence duplications were located downstream of the engineered deletion and generally increased the hydrophobicity, suggesting that they may compensate for the loss of the central hydrophobic domain. Two of the second-site mutations, together with the corresponding deletion, were introduced into a wild-type genetic backbone, and the analysis of these “double mutants” provided direct evidence that the viability of the deletion mutant indeed depended on the presence of the second-site mutation. Our results corroborate the notion that hydrophobic interactions of protein C are essential for the assembly of infectious flavivirus particles but rule out the possibility that individual residues of the central hydrophobic domain are absolutely required for infectivity. Furthermore, the double mutants were found to be highly attenuated and capable of inducing a protective immune response in mice at even lower inoculation doses than the previously characterized 16-amino-acid-residue deletion mutant, suggesting that the combination of large deletions and second-site mutations may be a superior way to generate safe, attenuated flavivirus vaccine strains