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Several lines of evidence suggest that nitrogen in most tropical forests is relatively more available than N in most temperate forests, and even that it may function as an excess nutrient in many tropical forests. If this is correct, tropical forests should have more open N cycles than temperate forests, with both inputs and outputs of N large relative to N cycling within systems. Consequent differences in both the magnitude and the pathways of N loss imply that tropical forests should in general he more 15N enriched than are most temperate forests. In order to test this hypothesis, we compared the nitrogen stable isotopic composition of tree leaves and soils from a variety of tropical and temperate forests. Foliar δ15N values from tropical forests averaged 6.5‰ higher than from temperate forests. Within the tropics, ecosystems with relatively low N availability (montane forests, forests on sandy soils) were significantly more depleted in 15N than other tropical forests. The average δ15N values for tropical forest soils, either for surface or for depth samples, were almost 8‰ higher than temperate forest soils. These results provide another line of evidence that N is relatively abundant in many tropical forest ecosystems.

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection., (© 2024. The Author(s).)

Ebolavirus disease (EVD) is caused by multiple species of Ebolavirus . Monoclonal antibodies (mAbs) against the virus glycoprotein (GP) are the only class of therapeutic approved for treatment of EVD caused by Zaire ebolavirus (EBOV). Therefore, mAbs targeting multiple Ebolavirus species may represent the next generation of EVD therapeutics. Broadly reactive anti-GP mAbs were produced; among these, mAbs 11886 and 11883 were broadly neutralizing in vitro . A 3.0 Å cryo-electron microscopy structure of EBOV GP bound to both mAbs shows that 11886 binds a novel epitope bridging the glycan cap (GC), 3 10 pocket and GP2 N-terminus, whereas 11883 binds the receptor binding region (RBR) and GC. In vitro , 11886 synergized with a range of mAbs with epitope specificities spanning the RBR/GC, including 11883. Notably, 11886 increased the breadth of neutralization by partner mAbs against different Ebolavirus species. These data provide a strategic route to design improved mAb-based next-generation EVD therapeutics., Competing Interests: Declaration of Interests DJL, VO’D are employees of UCB Pharma. DJL holds stock and/or stock options in UCB Pharma. FRD, SJD, DJL, VO’D, ART, PR and LS are inventors on patent applications relating to anti-Ebolavirus GP antibodies.

Antibody-mediated immunity plays a key role in protection against SARS-CoV-2. We characterized B-cell-derived anti-SARS-CoV-2 RBD antibody repertoires from vaccinated and infected individuals and elucidate the mechanism of action of broadly neutralizing antibodies and dissect antibodies at the epitope level. The breadth and clonality of anti-RBD B cell response varies among individuals. The majority of neutralizing antibody clones lose or exhibit reduced activities against Beta, Delta, and Omicron variants. Nevertheless, a portion of anti-RBD antibody clones that develops after a primary series or booster dose of COVID-19 vaccination exhibit broad neutralization against emerging Omicron BA.2, BA.4, BA.5, BQ.1.1, XBB.1.5 and XBB.1.16 variants. These broadly neutralizing antibodies share genetic features including a conserved usage of the IGHV3-53 and 3-9 genes and recognize three clustered epitopes of the RBD, including epitopes that partially overlap the classically defined set identified early in the pandemic. The Fab-RBD crystal and Fab-Spike complex structures corroborate the epitope grouping of antibodies and reveal the detailed binding mode of broadly neutralizing antibodies. Structure-guided mutagenesis improves binding and neutralization potency of antibody with Omicron variants via a single amino-substitution. Together, these results provide an immunological basis for partial protection against severe COVID-19 by the ancestral strain-based vaccine and indicate guidance for next generation monoclonal antibody development and vaccine design., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8 + T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4 + and CD8 + T cells enhance CD8 + T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8 + T-cell infiltration. We further show that SEMA3A affects CD8 + T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8 + T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours., (© 2024. The Author(s).)

Background: Epimers of ginsenoside Rg3 (Rg3) have a low bioavailability and are prone to deglycosylation, which produces epimers of ginsenoside Rh2 (S-Rh2 and R-Rh2) and protopanaxadiol (S-PPD and R-PPD). The aim of this study was to compare the efficacy and potency of these molecules as anti-cancer agents., Methods: Crystal violet staining was used to study the anti-proliferatory action of the molecules on a human epithelial breast cancer cell line, MDA-MB-231, and human umbilical vein endothelial cells (HUVEC) and compare their potency. Cell death and cell cycle were studied using flow cytometry and mode of cell death was studied using live cell imaging. Anti-angiogenic effects of the drug were studied using loop formation assay. Molecular docking showed the interaction of these molecules with vascular endothelial growth factor receptor-2 (VEGFR2) and aquaporin (AQP) water channels. VEGF bioassay was used to study the interaction of Rh2 with VEGFR2, in vitro ., Results: HUVEC was the more sensitive cell line to the anti-proliferative effects of S-Rh2, S-PPD and R-PPD. The molecules induced necroptosis/necrosis in MDA-MB-231 and apoptosis in HUVEC. S-Rh2 was the most potent inhibitor of loop formation. In silico molecular docking predicted a good binding score between Rh2 or PPD and the ATP-binding pocket of VEGFR2. VEGF bioassay showed that Rh2 was an allosteric modulator of VEGFR2. In addition, SRh2 and PPD had good binding scores with AQP1 and AQP5, both of which play roles in cell migration and proliferation., Conclusion: The combination of these molecules might be responsible for the anti-cancer effects observed by Rg3., Competing Interests: The authors declare no conflict of interest., (© 2024 The Korean Society of Ginseng. Publishing services by Elsevier B.V.)

Influenza virus-specific tissue-resident memory (Trm) CD8 + T cells located along the respiratory tract provide cross-strain protection against a breadth of influenza viruses. We show that immunization with a single-cycle influenza virus vaccine candidate (S-FLU) results in the deposition of influenza virus nucleoprotein (NP)-specific CD8 + Trm along the respiratory tract that were more cross-reactive against viral variants and less likely to drive the development of cytotoxic T lymphocyte (CTL) escape mutants, as compared to the lung memory NP-specific CD8 + T cell pool established following influenza infection. This immune profile was linked to the limited inflammatory response evoked by S-FLU vaccination, which increased TCR repertoire diversity within the memory CD8 + T cell compartment. Cumulatively, this work shows that S-FLU vaccination evokes a clonally diverse, cross-reactive memory CD8 + T cell pool, which protects against severe disease without driving the virus to rapidly evolve and escape, and thus represents an attractive vaccine for use against rapidly mutating influenza viruses.

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14, and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications. IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

Antibody-mediated immunity plays a crucial role in protection against SARS-CoV-2 infection. We isolated a panel of neutralizing anti-receptor-binding domain (RBD) antibodies elicited upon natural infection and vaccination and showed that they recognize an immunogenic patch on the internal surface of the core RBD, which faces inwards and is hidden in the "down" state. These antibodies broadly neutralize wild type (Wuhan-Hu-1) SARS-CoV-2, Beta and Delta variants and some are effective against other sarbecoviruses. We observed a continuum of partially overlapping antibody epitopes from lower to upper part of the inner face of the RBD and some antibodies extend towards the receptor-binding motif. The majority of antibodies are substantially compromised by three mutational hotspots (S371L/F, S373P and S375F) in the lower part of the Omicron BA.1, BA.2 and BA.4/5 RBD. By contrast, antibody IY-2A induces a partial unfolding of this variable region and interacts with a conserved conformational epitope to tolerate all antigenic variations and neutralize diverse sarbecoviruses as well. This finding establishes that antibody recognition is not limited to the normal surface structures on the RBD. In conclusion, the delineation of functionally and structurally conserved RBD epitopes highlights potential vaccine and therapeutic candidates for COVID-19., (© 2023. The Author(s).)

To combat future severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and spillovers of SARS-like betacoronaviruses (sarbecoviruses) threatening global health, we designed mosaic nanoparticles that present randomly arranged sarbecovirus spike receptor-binding domains (RBDs) to elicit antibodies against epitopes that are conserved and relatively occluded rather than variable, immunodominant, and exposed. We compared immune responses elicited by mosaic-8 (SARS-CoV-2 and seven animal sarbecoviruses) and homotypic (only SARS-CoV-2) RBD nanoparticles in mice and macaques and observed stronger responses elicited by mosaic-8 to mismatched (not on nanoparticles) strains, including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization showed equivalent neutralization of SARS-CoV-2 variants, including Omicrons, and protected from SARS-CoV-2 and SARS-CoV challenges, whereas homotypic SARS-CoV-2 immunization protected only from SARS-CoV-2 challenge. Epitope mapping demonstrated increased targeting of conserved epitopes after mosaic-8 immunization. Together, these results suggest that mosaic-8 RBD nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers.

Aim: Reviewing outcomes of regorafenib use in metastatic colorectal cancer using real-world data from the South Australian Metastatic Colorectal Cancer Registry., Methods: A retrospective review of the characteristics and outcomes of patients who received regorafenib in the Registry up to December 2018. The registry started in February 2006., Results: Fifty-three patients received regorafenib therapy since approved by the therapeutic goods administration in November 2013. The median age was 66 (range 34-82). 66% were male, 66% had stage IV disease at diagnosis, 53% had liver only involvement, whereas 13% had liver and lung disease and 6% had lung only involvement. 75% had left-sided primary. KRAS was available in 35/53 patients with 49% of them being WT. BRAF status was known in 8/53 with 25% of them having a mutated variant. MSI testing was known in 14 patients in whom 21% of them had MSI-High tumors. Prior lines of treatment received: one line 4%, two 9%, three 23%, four 26%, >four 37%. Prior biological use: bevacizumab 72%, anti-EGFR 100% (for RAS WT). Median survival from diagnosis was 3.3 years (95% CI, 2.8-3.8 years). Median survival from the start of regorafenib was 7.1 months (95% CI, 4.8-9.4 months) and the 12-month survival rate was 28%., Conclusion: The survival outcome for those patients from our population-based registry who access regorafenib is in keeping with reports from large, randomized trials. Thus, clinicians can quote local real world data when discussing efficacy and access to regorafenib therapy for mCRC patients., (© 2021 John Wiley & Sons Australia, Ltd.)

On 24 th November 2021, the sequence of a new SARS-CoV-2 viral isolate Omicron-B.1.1.529 was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titers of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic Alpha, Beta, Gamma, or Delta are substantially reduced, or the sera failed to neutralize. Titers against Omicron are boosted by third vaccine doses and are high in both vaccinated individuals and those infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of the large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses and uses mutations that confer tight binding to ACE2 to unleash evolution driven by immune escape. This leads to a large number of mutations in the ACE2 binding site and rebalances receptor affinity to that of earlier pandemic viruses., Competing Interests: Declaration of interests G.R.S. sits on the GSK Vaccines Scientific Advisory Board and is a founder member of RQ Biotechnology. J.Z. and G.S. declare the Israel patent application no. 23/09/2020—277,546 and United States patent application no. 16/12/2020—63/125,984, entitled methods and compositions for treating coronaviral infections. Oxford University holds intellectual property related to the Oxford-Astra Zeneca vaccine. A.J.P. is Chair of UK Dept. Health and Social Care’s (DHSC) Joint Committee on Vaccination & Immunisation (JCVI) but does not participate in the JCVI COVID-19 committee, and is a member of the WHO’s SAGE. The views expressed in this article do not necessarily represent the views of DHSC, JCVI, or WHO. The University of Oxford has entered into a partnership with AstraZeneca on coronavirus vaccine development. The University of Oxford has protected intellectual property disclosed in this publication. S.C.G. is co-founder of Vaccitech (collaborators in the early development of this vaccine candidate) and is named as an inventor on a patent covering use of ChAdOx1-vectored vaccines and a patent application covering this SARS-CoV-2 vaccine (PCT/GB2012/000467). T.L. is named as an inventor on a patent application covering this SARS-CoV-2 vaccine and was a consultant to Vaccitech for an unrelated project during the conduct of the study. S.J.D. is a Scientific Advisor to the Scottish Parliament on COVID-19., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) incidence in young adults is rising. Identifying genetic risk factors is fundamental for the clinical management of patients and their families. This study aimed to identify clinically significant germline variants among young adults with CRC. Whole-exome sequencing data of blood-derived DNA from 133 unrelated young CRC patients (<55 years of age) underwent a comprehensive analysis of 133 cancer-predisposition/implicated genes. All patient tumors were evaluated for mismatch repair deficiency (dMMR). Among 133 patients (aged 16-54 years), 15% (20/133) had clinically actionable pathogenic or likely pathogenic (P/LP) variants in at least 1 well established cancer-predisposing gene: dMMR genes (6), MUTYH [bi-allelic (2), mono-allelic (3)], RNF43 (1), BMPR1A (1), BRCA2 (4), ATM (1), RAD51C (1), and BRIP1 (1). Five patients (4%) had variants in genes implicated in cancer but where the significance of germline variants in CRC risk is uncertain: GATA2 (1), ERCC2 (mono-allelic) (1), ERCC4 (mono-allelic) (1), CFTR (2). Fourteen (11%) had dMMR tumors. Eighteen (14%) reported a first-degree relative with CRC, but only three of these carried P/LP variants. Three patients with variants in polyposis-associated genes showed no polyposis (one each in MUTYH [bi-allelic], RNF43, and BMPR1A). Approximately one in five young adults in our series carried at least one P/LP variant in a cancer-predisposing/implicated gene; 80% of these variants are currently considered clinically actionable in a familial cancer setting. Family history and phenotype have limitations for genetic risk prediction; therefore multigene panel testing and genetic counseling are warranted for all young adults with CRC regardless of those two factors., (© 2021 Wiley Periodicals LLC.)

Background: Effective targeting of RAS mutations has proven elusive until recently. Novel agents directly targeting KRAS G12C have shown promise in early-phase clinical trials that included patients with metastatic colorectal cancer. Prior reports have suggested that G12C mutation may be predictive of poor outcome., Objective: Assessment of the specific characteristics and prognostic implications of individual RAS mutation subtypes in patients with metastatic colorectal cancer., Patients and Methods: Retrospective review of individual RAS mutation types from the South Australian Metastatic Colorectal Registry between 2006 and 2020., Results: Of the 5165 patients entered onto the registry, 2305 (45%) had RAS mutation results available. 772 (33%) had a RAS mutation. The nature of the RAS mutation was available in 668 (87% of those with RAS mutation). Rare mutations (outside codons 12 and 13) made up 12.6% of the total. There were numerical differences in survival between the specific RAS mutation subgroups, with the longest median overall survival (30 months) observed in those with G12S mutations. However, there was no statistical difference in survival when comparing the various RAS mutations, including the comparison of G12C to G12S (p = 0.38). Patients with cancer harbouring rare RAS mutations had a median survival of 30 months., Conclusions: Whilst the G12S mutation was associated with the longest survival numerically, the observed survival for patients with the most common RAS mutations (G12C, G12V, G12A, G12D and G13D) did not significantly differ., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Administration of potent anti-receptor-binding domain (RBD) monoclonal antibodies has been shown to curtail viral shedding and reduce hospitalization in patients with SARS-CoV-2 infection. However, the structure-function analysis of potent human anti-RBD monoclonal antibodies and its links to the formulation of antibody cocktails remains largely elusive. Methods: Previously, we isolated a panel of neutralizing anti-RBD monoclonal antibodies from convalescent patients and showed their neutralization efficacy in vitro . Here, we elucidate the mechanism of action of antibodies and dissect antibodies at the epitope level, which leads to a formation of a potent antibody cocktail. Results: We found that representative antibodies which target non-overlapping epitopes are effective against wild type virus and recently emerging variants of concern, whilst being encoded by antibody genes with few somatic mutations. Neutralization is associated with the inhibition of binding of viral RBD to ACE2 and possibly of the subsequent fusion process. Structural analysis of representative antibodies, by cryo-electron microscopy and crystallography, reveals that they have some unique aspects that are of potential value while sharing some features in common with previously reported neutralizing monoclonal antibodies. For instance, one has a common VH 3-53 public variable region yet is unusually resilient to mutation at residue 501 of the RBD. We evaluate the in vivo efficacy of an antibody cocktail consisting of two potent non-competing anti-RBD antibodies in a Syrian hamster model. We demonstrate that the cocktail prevents weight loss, reduces lung viral load and attenuates pulmonary inflammation in hamsters in both prophylactic and therapeutic settings. Although neutralization of one of these antibodies is abrogated by the mutations of variant B.1.351, it is also possible to produce a bi-valent cocktail of antibodies both of which are resilient to variants B.1.1.7, B.1.351 and B.1.617.2. Conclusions: These findings support the up-to-date and rational design of an anti-RBD antibody cocktail as a therapeutic candidate against COVID-19., Competing Interests: Competing Interests: This work has been described in pending patent applications (Taiwan, File No. 110116981; PCT, File No. PCT/CN2021/093083)., (© The author(s).)

The role of RNF43 as a cause of an inherited predisposition to colorectal cancer (CRC) is yet to be fully explored. This report presents our findings of two individuals with CRC from a single family carrying a likely-pathogenic inherited germline variant in RNF43. The proband (III:1) and the proband's mother (II:2) were diagnosed with mismatch repair proficient CRCs at the age of 50 years and 65 years, respectively. Both patients had BRAF V600E mutated colon tumours, indicating that the CRCs arose in sessile serrated lesions. The germline variant RNF43:c.375+1G>A was identified in both patients. RNA studies showed that this variant resulted in an aberrantly spliced transcript, which was predicted to encode RNF43:p.Ala126Ilefs*50 resulting in premature termination of protein synthesis and was classified as a likely-pathogenic variant. Our report adds further evidence to the hereditary role of RNF43 as a tumour suppressor gene in colorectal tumorigenesis and supports the inclusion of RNF43 as a gene of interest in the investigation of CRC predispositions outside the setting of serrated polyposis., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies (mAbs). In this study we demonstrated that prophylactic intravenous administration of 15 mg/kg of porcine mAb pb18, against the K160-163 site of the hemagglutinin, significantly reduced lung pathology and nasal virus shedding and eliminated virus from the lung of pigs following H1N1pdm09 challenge. When given at 1 mg/kg, pb18 significantly reduced lung pathology and lung and BAL virus loads, but not nasal shedding. Similarly, when pb18 was given in combination with pb27, which recognized the K130 site, at 1 mg/kg each, lung virus load and pathology were reduced, although without an apparent additive or synergistic effect. No evidence for mAb driven virus evolution was detected. These data indicate that intravenous administration of high doses was required to reduce nasal virus shedding, although this was inconsistent and seldom complete. In contrast, the effect on lung pathology and lung virus load is consistent and is also seen at a one log lower dose, strongly indicating that a lower dose might be sufficient to reduce severity of disease, but for prevention of transmission other measures would be needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Paudyal, McNee, Rijal, Carr, Nunez, McCauley, Daniels, Townsend, Hammond and Tchilian.)

Nucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1 mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1 mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1's Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ebolaviruses continue to pose a significant outbreak threat, and while Ebola virus (EBOV)-specific vaccines and antivirals have been licensed, efforts to develop candidates offering broad species cross-protection are continuing. The use of pseudotyped virus in place of live virus is recognised as an alternative, safer, high-throughput platform to evaluate anti-ebolavirus antibodies towards their development, yet it requires optimisation. Here, we have shown that the target cell line impacts neutralisation assay results and cannot be selected purely based on permissiveness. In expanding the platform to incorporate each of the ebolavirus species envelope glycoprotein, allowing a comprehensive assessment of cross-neutralisation, we found that the recently discovered Bombali virus has a point mutation in the receptor-binding domain which prevents entry into a hamster cell line and, importantly, shows that this virus can be cross-neutralised by EBOV antibodies and convalescent plasma.

The extent to which immune responses to natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immunization with vaccines protect against variants of concern (VOC) is of increasing importance. Accordingly, here we analyse antibodies and T cells of a recently vaccinated, UK cohort, alongside those recovering from natural infection in early 2020. We show that neutralization of the VOC compared to a reference isolate of the original circulating lineage, B, is reduced: more profoundly against B.1.351 than for B.1.1.7, and in responses to infection or a single dose of vaccine than to a second dose of vaccine. Importantly, high magnitude T cell responses are generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. Vaccination is required to generate high potency immune responses to protect against these and other emergent variants., (© 2021. The Author(s).)

Cyclic GMP-AMP (cGAMP) is an immunostimulatory molecule produced by cGAS that activates STING. cGAMP is an adjuvant when administered alongside antigens. cGAMP is also incorporated into enveloped virus particles during budding. Here, we investigate whether inclusion of cGAMP within viral vaccine vectors enhances their immunogenicity. We immunise mice with virus-like particles (VLPs) containing HIV-1 Gag and the vesicular stomatitis virus envelope glycoprotein G (VSV-G). cGAMP loading of VLPs augments CD4 and CD8 T-cell responses. It also increases VLP- and VSV-G-specific antibody titres in a STING-dependent manner and enhances virus neutralisation, accompanied by increased numbers of T follicular helper cells. Vaccination with cGAMP-loaded VLPs containing haemagglutinin induces high titres of influenza A virus neutralising antibodies and confers protection upon virus challenge. This requires cGAMP inclusion within VLPs and is achieved at markedly reduced cGAMP doses. Similarly, cGAMP loading of VLPs containing the SARS-CoV-2 Spike protein enhances Spike-specific antibody titres. cGAMP-loaded VLPs are thus an attractive platform for vaccination., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Key problems of chemotherapies, as the mainstay of treatment for triple-negative breast cancer (TNBC), are toxicity and development of tumour resistance. Using response surface methodology, we previously optimised the combination of epimers of ginsenoside Rg3 (Rg3) for anti-angiogenic action. Here, we show that the optimised combination of 50 µM SRg3 and 25 µM RRg3 (C3), derived from an RSM model of migration of TNBC cell line MDA-MB-231, inhibited migration of MDA-MB-231 and HCC1143, in 2D and 3D migration assays ( p < 0.0001). C3 inhibited mammosphere formation efficiency in both cell lines and decreased the CD44 + stem cell marker in the mammospheres. Molecular docking predicted that Rg3 epimers had a better binding score with IGF-1R than with EGFR, HER-2 or PDGFR, and predicted an mTOR inhibitory function of Rg3. C3 affected the signalling of AKT in MDA-MB-231 and HCC1143 mammospheres. In a mouse model of metastatic TNBC, an equivalent dose of C3 (23 mg/kg SRg3 + 11 mg/kg RRg3) or an escalated dose of 46 mg/kg SRg3 + 23 mg/kg RRg3 was administered to NSG mice bearing MDA-MB-231-Luc cells. Calliper and IVIS spectrum measurement of the primary and secondary tumour showed that the treatment shrunk the primary tumour and decreased the load of metastasis in mice. In conclusion, this combination of Rg3 epimers showed promising results as a potential treatment option for TNBC patients.

Background: The Oncotype DX 21-gene Recurrence Score is predictive of adjuvant chemotherapy benefit for women with early-stage, estrogen receptor (ER)-positive, HER2-negative breast cancer. In premenopausal women, fluctuations in estrogen and progesterone during the menstrual cycle impact gene expression in hormone-responsive cancers. However, the extent to which menstrual cycling affects the Oncotype DX 21-gene signature remains unclear. Here, we investigate the impact of ovarian cycle stage on the 21-gene signature using a naturally cycling mouse model of breast cancer., Methods: ER-positive mammary tumours were dissected from naturally cycling Mmtv-Pymt mice at either the estrus or diestrus phase of the ovarian cycle. The Oncotype DX 21-gene signature was assessed through quantitative real time-PCR, and a 21-gene experimental recurrence score analogous to the Oncotype DX Recurrence Score was calculated., Results: Tumours collected at diestrus exhibited significant differences in expression of 6 Oncotype DX signature genes (Ki67, Ccnb1, Esr1, Erbb2, Grb7, Bag1; p ≤ 0.05) and a significant increase in 21-gene recurrence score (21.8 ± 2.4; mean ± SEM) compared to tumours dissected at estrus (15.5 ± 1.9; p = 0.03). Clustering analysis revealed a subgroup of tumours collected at diestrus characterised by increased expression of proliferation- (p < 0.001) and invasion-group (p = 0.01) genes, and increased 21-gene recurrence score (p = 0.01). No correlation between ER, PR, HER2, and KI67 protein abundance measured by Western blot and abundance of mRNA for the corresponding gene was observed, suggesting that gene expression is more susceptible to hormone-induced fluctuation compared to protein expression., Conclusions: Ovarian cycle stage at the time of tissue collection critically affects the 21-gene signature in Mmtv-Pymt murine mammary tumours. Further studies are required to determine whether Oncotype DX Recurrence Scores in women are similarly affected by menstrual cycle stage.

Purpose: Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells., Methods: Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro., Results: In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines., Conclusions: Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

We previously showed how triterpene saponin bacopaside (bac) II, purified from the medicinal herb Bacopa monnieri , induced cell death in colorectal cancer cell lines and reduced endothelial cell migration and tube formation, and further demonstrated a synergistic effect of a combination of bac I and bac II on the inhibition of breast cancer cell line growth. Here, we assessed the effects of bac I and II on the colorectal cancer HT-29 cell line, and mouse (2H-11) and human umbilical vein endothelial cell (HUVEC) lines, measuring outcomes including cell viability, proliferation, migration, tube formation, apoptosis, cytosolic Ca 2+ levels and plasma membrane integrity. Combined bac I and II, each applied at concentrations below IC 50 values, caused a synergistic reduction of the viability and proliferation of HT-29 and endothelial cells, and impaired the migration of HT-29 and tube formation of endothelial cells. A significant enhancement of apoptosis was induced only in HUVEC, although an increase in cytosolic Ca 2+ was detected in all three cell lines. Plasma membrane integrity was compromised in 2H-11 and HUVEC, as determined by an increase in propidium iodide staining, which was preceded by Ca 2+ flux. These in vitro findings support further research into the mechanisms of action of the combined compounds for potential clinical use.

Tumour angiogenesis plays a key role in tumour growth and progression. The application of current anti-angiogenic drugs is accompanied by adverse effects and drug resistance. Therefore, finding safer effective treatments is needed. Ginsenoside Rg3 (Rg3) has two epimers, 20(S)-Rg3 (SRg3) and 20(R)-Rg3 (RRg3), with stereoselective activities. Using response surface methodology, we optimised a combination of these two epimers for the loop formation of human umbilical vein endothelial cell (HUVEC). The optimised combination (C3) was tested on HUVEC and two murine endothelial cell lines. C3 significantly inhibited the loop formation, migration, and proliferation of these cells, inducing apoptosis in HUVEC and cell cycle arrest in all of the cell lines tested. Using molecular docking and vascular endothelial growth factor (VEGF) bioassay, we showed that Rg3 has an allosteric modulatory effect on vascular endothelial growth factor receptor 2 (VEGFR2). C3 also decreased the VEGF expression in hypoxic conditions, decreased the expression of aquaporin 1 and affected AKT signaling. The proteins that were mostly affected after C3 treatment were those related to mammalian target of rapamycin (mTOR). Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) was one of the important targets of C3, which was affected in both hypoxic and normoxic conditions. In conclusion, these results show the potential of C3 as a novel anti-angiogenic drug.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC 50  < 0.1 μg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models., Competing Interests: Declaration of interests M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation and is on the Scientific Advisory Boards of Moderna and Immunome. The M.S.D. laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. G.R.S. sits on the GSK Vaccines Scientific Advisory Board. A.J.P. is Chair of UK Department Health and Social Care’s (DHSC) Joint Committee on Vaccination & Immunisation (JCVI), but does not chair or participate in the JCVI COVID19 committee, and is a member of the WHO’s SAGE. The views expressed in this article do not necessarily represent the views of DHSC, JCVI, NIHR, or WHO. The University of Oxford has entered into a partnership with AstraZeneca on coronavirus vaccine development. All other authors declare no competing financial interests. The University of Oxford has protected intellectual property disclosed in this publication., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations., Competing Interests: The authors have declared that no competing interests exist.

Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 3.5% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-receptor-binding domain (RBD), three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two recovered patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. Finally, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: KYAH files a patent application on anti-spike and anti-nucleocapsid antibodies described herein.

Background: Colorectal cancer (CRC) is rising in incidence in young adults, and this observation is currently unexplained. We investigated whether having a personal history of type 2 diabetes mellitus (T2D) was a potential risk factor for young-onset colorectal cancer (YOCRC)., Methods: The South Australian Young Onset (SAYO) CRC study is a series of young adults with CRC below age 55. Ninety unrelated YOCRC cases were recruited to the study. Personal history and detailed family history of T2D were obtained at face-to-face interview and confirmed from medical records. Whole exome sequencing was conducted on germline DNA from each CRC case. Controls for personal history studies of T2D were 240 patients with proven clear colonoscopies and no known CRC predispositions., Results: The median age of YOCRC cases was 44 years (18-54) and of controls was 45 years (18-54), and 53% of both cases and controls were females (P = 0.99). Left-sided (distal) CRC was seen in 67/89 (75%) of cases. A personal history of T2D was confirmed in 17/90 (19%) YOCRC patients compared with controls (12/240, 5%; P < 0.001; odds ratio = 4.4; 95% confidence interval, 2.0-9.7). YOCRC patients frequently reported at least one first-degree relative with T2D (32/85, 38%). Ten of 87 (12%) of YOCRC cases had CRC-related pathogenic germline variants, however, no pathogenic variants in familial diabetes-associated genes were seen., Conclusions: Though the mechanism remains unclear, our observations suggest that there is enrichment for personal history of T2D in YOCRC patients., Impact: A diagnosis of T2D could therefore potentially identify a subset of young adults at increased risk for CRC and in whom early screening might be appropriate., (© 2020 John Wiley & Sons Australia, Ltd.)

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

Matching of symmetry at interfaces is a fundamental obstacle in molecular assembly. Virus-like particles (VLPs) are important vaccine platforms against pathogenic threats, including Covid-19. However, symmetry mismatch can prohibit vaccine nanoassembly. We established an approach for coupling VLPs to diverse antigen symmetries. SpyCatcher003 enabled efficient VLP conjugation and extreme thermal resilience. Many people had pre-existing antibodies to SpyTag:SpyCatcher but less to the 003 variants. We coupled the computer-designed VLP not only to monomers (SARS-CoV-2) but also to cyclic dimers (Newcastle disease, Lyme disease), trimers (influenza hemagglutinins), and tetramers (influenza neuraminidases). Even an antigen with dihedral symmetry could be displayed. For the global challenge of influenza, SpyTag-mediated display of trimer and tetramer antigens strongly induced neutralizing antibodies. SpyCatcher003 conjugation enables nanodisplay of diverse symmetries towards generation of potent vaccines., Competing Interests: M.H. is an inventor on a patent regarding spontaneous amide bond formation (EP2534484) and a patent application regarding SpyTag003:SpyCatcher003 (UK Intellectual Property Office 1706430.4). M.H. and S.J.D. are SpyBiotech co‐founders, shareholders and consultants. A.R.T. is an inventor on a patent relating to S‐FLU (EP2758525A2)., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Background: Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (cetuximab or panitumumab) are today increasingly used in the first- or second-line setting for RAS wild-type metastatic colorectal cancer (CRC) patients. Following progression beyond third- or fourth-line therapy, some patients are unsuitable for further chemotherapy because of poor performance status or patient choice. However, a significant number of patients are still candidates for further therapy despite limited standard options being available. The role of rechallenge with anti-EGFR therapy, particularly in patients who had previously responded, is often considered, but there is limited evidence in the literature to support such a strategy., Objective: This retrospective study aims to review the outcome of metastatic CRC patients who had anti-EGFR rechallenge., Patients and Methods: Patients who had been rechallenged with anti-EGFR therapy were identified from the South Australian metastatic CRC database. Patient characteristics were recorded and tumor response was retrospectively assessed using Response Evaluation Criteria in Solid Tumors (RECIST). Kaplan-Meier analysis was used to assess progression free survival (PFS) for each rechallenge and overall survival (OS)., Results: Twenty-two patients were eligible for inclusion in this analysis. Disease control rate (stable disease and partial response) was 45.4% (ten patients) for patients who received rechallenge anti-EGFR. Seven patients received a second rechallenge and disease control rate was 28.6% (two patients). The median interval time between initial anti-EGFR therapy and rechallenge was 13.5 months. The median PFS after rechallenge 1 was 4.1 months and after rechallenge 2 was 3.5 months. The median OS was 7.7 months from date of rechallenge., Conclusions: Anti-EGFR rechallenge provides clinical benefit in patients with RAS wild-type metastatic CRC.

When recombinant simian immunodeficiency virus (SIV) is pseudotyped with the F and HN glycoproteins from murine respiratory Sendai virus (rSIV.F/HN), it provides efficient lung cell targeting and lifelong transgene expression in the murine airways. We have shown that a single dose of rSIV.F/HN can direct stable expression of neutralising antibody against influenza in the murine airways and systemic circulation, and protects mice against two different influenza strains in lethal challenge experiments. These data suggest that rSIV.F/HN could be used as a vector for passive immunisation against influenza and other respiratory pathogens., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Ginsenoside Rg3 (Rg3) is a member of the ginsenoside family of chemicals extracted from Panax ginseng . Like other ginsenosides, Rg3 has two epimers: 20(S)-ginsenoside Rg3 (SRg3) and 20(R)-ginsenoside Rg3 (RRg3). Rg3 is an intriguing molecule due to its anti-cancer properties. One facet of the anti-cancer properties of Rg3 is the anti-angiogenic action. This review describes the controversies on the effects and effective dose range of Rg3, summarizes the evidence on the efficacy of Rg3 on angiogenesis, and raises the possibility that Rg3 is a prodrug.

Here, we investigate a monoclonal antibody, Z2B3, isolated from an H7N9-infected patient, that exhibited cross-reactivity to both N9 (group 2) and a broad range of seasonal and avian N1 (group 1) proteins but lost activity to the N1 with the substitution K432E. This substitution exists in 99.25% of seasonal influenza strains after 2013. The NA-Z2B3 complex structures indicated that Z2B3 binds within the conserved active site of the neuraminidase (NA) protein. A salt bridge between D102 in Z2B3 and K432 in NA plays an important role in binding. Structure-based modification of Z2B3 with D102R in heavy chain reversed the salt bridge and restored the binding and inhibition of N1 with E432. Furthermore, Z2B3-D102R can protect mice from A/Serbia/NS-601/2014 H1N1 virus (NA contains E432) infection while the wild-type Z2B3 antibody shows no protection. This study demonstrates that a broadly reactive and protective antibody to NA can be in principle edited to restore binding and inhibition to recently drifted N1 NA and regain protection against the variant influenza strain. IMPORTANCE The immune system produces antibodies to protect the human body from harmful invaders. The monoclonal antibody (MAb) is one kind of effective antivirals. In this study, we isolated an antibody (Z2B3) from an H7N9 influenza virus-infected child. It shows cross-reactivity to both group 1 (N1) and group 2 (N9) neuraminidases (NAs) but is sensitive to N1 NA with a K432E substitution. Structural analysis of the NA-antibody fragment antigen-binding (Fab) complex provides a clue for antibody modification, and the modified antibody restored binding and inhibition to recently drifted N1 NA and regained protection against the variant influenza strain. This finding suggests that antibodies to NA may be a useful therapy and can be in principle edited to defeat drifted influenza virus., (Copyright © 2020 Jiang et al.)