Case reports and series suggested an association between SARS-CoV-2 and Guillain-Barré syndrome (GBS). However, the GBS epidemic which was predicted from early risk estimates did not materialize in overall case numbers, and no plausible mechanism for any link has been established. An increased risk of GBS following adenoviral vector-based COVID-19 vaccination has been more consistently demonstrated, but a pathophysiological explanation for this association has also not yet emerged. Here, we sought to identify whether patients with GBS following COVID-19 infection or vaccination had any distinct clinical or serological features differentiating them from one another or non-pandemic GBS, and to explore the potential mechanisms of any associations. Between March 2020 and October 2021, sera from patients with GBS (n = 64) and controls (n = 70) were collected. Clinical features were retrieved from medical records. GBS cases were evaluated for diagnostic certainty by Brighton criteria and classified as non-COVID-19 associated (GBS-NC, n = 20), GBS after COVID-19 infection (GBS-AC, n = 10), or GBS after COVID-19 vaccination (GBS-AV, n = 34). The humoral responses to SARS-CoV-2 proteins and putative peripheral nerve antigens, and the cytokine profile of each group were established and compared. Antibodies cloned from the acute-phase plasmablasts of an individual with GBS-AC were also assessed for reactivity against SARS-CoV-2 and peripheral nerve antigens. Sera from GBS patients and from individuals who received COVID-19 vaccinations (n = 36: 16 ChAdOx1, 10 Ad26.COV2.S/Janssen and 10 tozinameran/Pfizer-BioNTech) without developing GBS were tested for IgG reactivity against SARS-CoV-2 and adenoviral proteins. There were no clinical differences between the GBS groups. Patients with GBS-AC had a greater IgG reactivity to the S1 component of the SARS-CoV-2 spike protein compared to non-GBS COVID-19 controls. A minority of antibodies from cloned plasmablasts targeted SARS-CoV-2 proteins but there was no reactivity or cross reactivity with peripheral nerve antigens or tissue. There were no other serological or immunological differences between the GBS groups. However, when compared to uncomplicated vaccine recipients, GBS patients in toto, and each group individually, demonstrated significantly greater antibody reaction to a range of human adenoviral proteins. Compared to controls exposed to the same immunological stimulus, antibody reactivities to viral antigens are enhanced in patients with GBS. However, we found no mechanistic link between S1 and peripheral nerve reactivity or pathology. Serological responses to adenoviral proteins may be directly involved in the pathogenesis of Guillain-Barré syndrome, potentially contributing to cases with currently unexplained aetiology.

Abstract Diverse human viruses deploy a common immune-evasion strategy: viral 2’-O-methylation coupled with STAT2 antagonism. Yet the importance of this coupling and whether targeting the coupled functions offers benefits in vaccine development, remains unknown. Using deep mutational scanning of the dengue NS5 protein, we discovered that the two immune-evasion functions work synergistically to cripple interferon signaling. Disrupting this synergy exposes a reciprocal regulatory relationship between the host proteins IFIT1 and STAT2 that controls both viral restriction and immune cell activation. Dengue viruses with mutations that disrupt the synergy are potently attenuated yet trigger antigen-presenting cell activation that exceeds a licensed dengue vaccine benchmark. Our results establish a generalizable principle: targeting synergies between viral immune evasion mechanisms can simultaneously achieve maximal viral attenuation and innate immune priming. This work provides a rational design framework for developing high-performance vaccines against many human viruses that exploit the IFIT1-STAT2 axis. Teaser Breaking synergistic viral immune evasion unlocks potent innate immunity for next-generation live-attenuated vaccines.

Abstract Dengue virus (DENV) is a major global health threat, with secondary heterotypic infections potentially inducing detrimental memory immune responses. Antigen-specific CD8 + T cells contribute to both protection and pathogenicity, yet how their phenotypic heterogeneity relates to disease severity remains unclear. Here, we performed plate-based single-cell RNA sequencing of circulating DENV-specific CD8 + T cells identified by HLA tetramers loaded with DENV NS3-derived epitopes. Using tetramer binding to peptides corresponding to the currently and serologically inferred dominant previously infecting serotypes, we identify distinct CD8 + T cell subsets associated with disease severity. Asymptomatic dengue is enriched for lower tetramer binding cells with moderate cytotoxic programs, whereas dengue hemorrhagic fever is associated with high tetramer binding CX3CR1 + CD8 + T cells exhibiting enhanced expression of genes related to T cell receptor signaling and cytotoxicity. T cell receptor repertoires are similar among symptomatic cases but displayed temporal dynamics. Overall, DENV NS3-specific CD8 + T cells across disease severity and time are associated with distinct transcriptomic states and T cell receptor features.

The majority of SARS-CoV-2 genomes obtained during the pandemic were derived by amplifying overlapping windows of the genome (‘tiled amplicons’), reconstructing their sequences and fitting them together. This leads to systematic errors in genomes unless the software is both aware of the amplicon scheme and of the error modes of amplicon sequencing. Additionally, over time, amplicon schemes need to be updated as new mutations in the virus interfere with the primer binding sites at the end of amplicons. Thus, waves of variants swept the world during the pandemic and were followed by waves of systematic errors in the genomes, which had significant impacts on the inferred phylogenetic tree. Here we reconstruct the genomes from all public data as of June 2024 using an assembly tool called Viridian ( https://github.com/iqbal-lab-org/viridian ), developed to rigorously process amplicon sequence data. With these high-quality consensus sequences we provide a global phylogenetic tree of 4,471,579 samples, viewable at https://viridian.taxonium.org . We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny.

IntroductionThe four serotypes of dengue virus (DENV1-4) are the leading cause of arboviral diseases in humans. Currently, developing a safe and effective DENV vaccine remains a challenge. Previously we reported potently neutralizing human monoclonal antibodies (mAbs) preferentially recognize mature DENV particles, on which there is limited access to the fusion loop (FL) epitope and no premembrane (prM) protein. As FL and prM antibodies are weakly- or non-neutralizing and contribute to antibody-dependent enhancement, mature DENV particles represent an ideal DENV vaccine candidate. Several inactivated flavivirus vaccines, generated using formalin, ultraviolet or other inactivation methods, have progressed through preclinical and clinical studies. Little is known about how different inactivation methods affect viral epitopes and the quality of antibodies induced.MethodsWe investigated epitopes on Tween 20-inactivated mature DENV1 particles by testing a panel of well-characterized human mAbs and membrane integrity by sucrose-gradient ultracentrifugation and protein K digestion. We examined the mechanisms of Tween 20 inactivation by measuring RNA copy numbers, virus binding to cells and acid exposure, and antibody responses induced by Tween 20-inactivated mature DENV1 particles in mice.ResultsTween 20 inactivation better preserved the epitopes recognized by potently neutralizing mAbs compared with other methods. Sucrose-gradient ultracentrifugation and protein K digestion revealed no disruption of membrane integrity by Tween 20. Mechanistically, Tween 20 treatment caused reduced virus binding to cells and RNA degradation, which was reverted by pre-treatment with RNAseOUT, suggesting the involvement of extracellular RNase, and prevented the envelope protein conformational changes induced by acid exposure. Moreover, Tween 20-inactivated mature DENV1 particles induced a neutralizing antibody response to all four DENV serotypes characterized by competition with several potently neutralizing mAbs and limited recognition of FL.ConclusionOur results suggest that Tween 20-inactivated mature particles are a promising strategy for DENV vaccine development.

Adenoviral (Ad) vectors and mRNA vaccines exhibit distinct patterns of immune responses and reactogenicity, but underpinning mechanisms remain unclear. We longitudinally compared homologous ChAdOx1 nCoV-19 and BNT162b2 vaccination, focusing on cytokine-responsive innate-like lymphocytes-mucosal-associated invariant T (MAIT) cells and Vδ2+ γδ T cells-which sense and tune innate-adaptive cross-talk. Ad priming elicited robust type I interferon (IFN)-mediated innate-like T cell activation, augmenting T cell responses (innate-to-adaptive signaling), which was dampened at boost by antivector immunity. Conversely, mRNA boosting enhanced innate-like responses, driven by prime-induced spike-specific memory T cell-derived IFN-γ (adaptive-to-innate signaling). Extending the dosing interval dampened inflammation at boost because of waning T cell memory. In a separate vaccine trial, preboost spike-specific T cells predicted severe mRNA reactogenicity regardless of the priming platform or interval. Overall, bidirectional innate-like and adaptive cross-talk, and IFN-γ-licensed innate-like T cells, orchestrate interval-dependent early vaccine responses, suggesting modifiable targets for safer, more effective regimens.

AbstractKey functions of antibodies, such as viral neutralisation, depend on high-affinity binding. However, viral neutralisation poorly correlates with antigen affinity for reasons that have been unclear. Here, we use a new mechanistic model of bivalent binding to study >45 patient-isolated IgG1 antibodies interacting with SARS-CoV-2 RBD surfaces. The model provides the standard monovalent affinity/kinetics and new bivalent parameters, including the molecular reach: the maximum antigen separation enabling bivalent binding. We find large variations in these parameters across antibodies, including reach variations (22–46 nm) that exceed the physical antibody size (~15 nm). By using antigens of different physical sizes, we show that these large molecular reaches are the result of both the antibody and antigen sizes. Although viral neutralisation correlates poorly with affinity, a striking correlation is observed with molecular reach. Indeed, the molecular reach explains differences in neutralisation for antibodies binding with the same affinity to the same RBD-epitope. Thus, antibodies within an isotype class binding the same antigen can display differences in molecular reach, substantially modulating their binding and functional properties.

A strong and effective COVID-19 and future pandemic responses rely on global efforts to carry out surveillance of infections and emerging SARS-CoV-2 variants and to act accordingly in real time. Many countries in Southeast Asia lack capacity to determine the potential threat of new variants, or other emerging infections. Funded by Wellcome, the Southeast Asia initiative to combat SARS-CoV-2 variants (SEACOVARIANTS) consortium aims to develop and apply a multidisciplinary research platform in Southeast Asia (SEA) for rapid assessment of the biological significance of SARS-CoV-2 variants, thereby informing coordinated local, regional and global responses to the COVID-19 pandemic. Our proposal is delivered by the Vietnam and Thailand Wellcome Africa Asia Programmes, bringing together a multidisciplinary team in Indonesia, Thailand and Vietnam with partners in Singapore, the UK and the USA. Herein we outline five work packages to deliver strengthened regional scientific capacity that can be rapidly deployed for future outbreak responses.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires ongoing monitoring to judge the ability of newly arising variants to escape the immune response. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal serum samples. We compared 18 datasets generated using human, hamster, and mouse serum and six different neutralization assays. Datasets using animal model serum samples showed higher titer magnitudes than datasets using human serum samples in this comparison. Fold change in neutralization of variants compared to ancestral SARS-CoV-2, immunodominance patterns, and antigenic maps were similar among serum samples and assays. Most assays yielded consistent results, except for differences in fold change in cytopathic effect assays. Hamster serum samples were a consistent surrogate for human first-infection serum samples. These results inform the transition of surveillance of SARS-CoV-2 antigenic variation from dependence on human first-infection serum samples to the utilization of serum samples from animal models.

OBJECTIVES:We studied the immunogenicity after primary and booster vaccinations of the Abdala COVID-19 vaccine, a receptor-binding domain protein subunit vaccine, in Vietnamese people by determining the level of neutralization and cross-neutralization activities against the ancestral SARS-CoV-2 and its variants and SARS-CoV-1. METHODS:We performed a prospective observational study, enrolling adults aged 19-59 years in Dong Thap province, southern Vietnam, and collected blood samples from baseline until 4 weeks after the booster dose. We measured anti-nucleocapsid, anti-spike, and neutralizing antibodies against SARS-CoV-2 and assessed the cross-neutralization against 14 SARS-CoV-2 variants and SARS-CoV-1. Complementary antibody data came from Vietnamese health care workers fully vaccinated with ChAdOx1-S. RESULTS:After primary vaccination, anti-spike antibody and neutralizing antibodies were detectable in 98.4% and 87% of 251 study participants, respectively, with neutralizing antibody titers similar to that induced by ChAdOx1-S vaccine. Antibody responses after a homologous (Abdala COVID-19) or heterologous (messenger RNA BNT162b2) booster could neutralize 14 SARS-CoV-2 variants (including Omicron) and SARS-CoV-1. CONCLUSIONS:Abdala COVID-19 vaccine is immunogenic in Vietnamese people. Enhanced antibody response after a booster dose could cross-neutralize 14 SARS-CoV-2 variants and SARS-CoV-1. Our results have added to the growing body of knowledge about the contribution of protein subunit vaccine platforms to pandemic control.

A strong and effective COVID-19 and future pandemic responses rely on global efforts to carry out surveillance of infections and emerging SARS-CoV-2 variants and to act accordingly in real time. Many countries in Southeast Asia lack capacity to determine the potential threat of new variants, or other emerging infections. Funded by Wellcome, the Southeast Asia initiative to combat SARS-CoV-2 variants (SEACOVARIANTS) consortium aims to develop and apply a multidisciplinary research platform in Southeast Asia (SEA) for rapid assessment of the biological significance of SARS-CoV-2 variants, thereby informing coordinated local, regional and global responses to the COVID-19 pandemic. Our proposal is delivered by the Vietnam and Thailand Wellcome Africa Asia Programmes, bringing together a multidisciplinary team in Indonesia, Thailand and Vietnam with partners in Singapore, the UK and the USA. Herein we outline five work packages to deliver strengthened regional scientific capacity that can be rapidly deployed for future outbreak responses.

Viral entry is mediated by oligomeric proteins on the virus and cell surfaces. The association is therefore open to multivalent interactions between these proteins, yet such recognition is typically rationalised as affinity between monomeric equivalents. As a result, assessment of the thermodynamic mechanisms that control viral entry has been limited. Here, we use mass photometry to overcome the analytical challenges consequent to multivalency. Examining the interaction between the spike protein of SARS-CoV - 2 and the ACE2 receptor, we find that ACE2 induces oligomerisation of spike in a variant-dependent fashion. We also demonstrate that patient-derived antibodies use induced-oligomerisation as a primary inhibition mechanism or to enhance the effects of receptor-site blocking. Our results reveal that naive affinity measurements are poor predictors of potency, and introduce a novel antibody-based inhibition mechanism for oligomeric targets. One-Sentence Summary Multivalent interactions between viral proteins, cell-surface receptors, and anti-viral antibodies regulate infection and inhibition.

Under pressure from neutralising antibodies induced by vaccination or infection the SARS-CoV-2 spike gene has become a hotspot for evolutionary change, leading to the failure of all mAbs developed for clinical use. Most potent antibodies bind to the receptor binding domain which has become heavily mutated. Here we study responses to a conserved epitope in sub-domain-1 (SD1) of spike which have become more prominent because of mutational escape from antibodies directed to the receptor binding domain. Some SD1 reactive mAbs show potent and broad neutralization of SARS-CoV-2 variants. We structurally map the dominant SD1 epitope and provide a mechanism of action by blocking interaction with ACE2. Mutations in SD1 have not been sustained to date, but one, E554K, leads to escape from mAbs. This mutation has now emerged in several sublineages including BA.2.86, reflecting selection pressure on the virus exerted by the increasing prominence of the anti-SD1 response.

Natural killer cells (NK cells) are the front line of immune cells to combat pathogens and able to influence the subsequent adaptive immune responses. One of the factors contributing to pathogenesis in dengue hemorrhagic fever (DHF) disease is aberrant immune activation during early phase of infection. This study explored the profile of NK cells in dengue infected pediatric patients with different degrees of disease severity. DHF patients contained higher frequency of activated NK cells but lower ratio of CD56dim:CD56bright NK subsets. Activated NK cells exhibited alterations in several NK receptors. Interestingly, the frequencies of NKp30 expressing activated NK cells were more pronounced in dengue fever (DF) than in DHF pediatric patients. In vitro functional analysis indicated that degranulation of NK cells in responding to dengue infected dendritic cells (DCs) required cell-cell contact and type I IFNs. Meanwhile, Interferon gamma (IFN-γ) production initially required cell-cell contact and type I IFNs followed by Interleukin-12 (IL-12), Interleukin-15 (IL-15) and Interleukin-18 (IL-18) resulting in the amplification of IFN-γ producing NK cells over time. This study highlighted the complexity and the factors influencing NK cells responses to dengue virus. Degree of activation, phenotypes of activated cells and the crosstalk between NK cells and other immune cells, could modulate the outcome of NK cells function in the dengue disease.

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 4,471,579 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of June 2024, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

BACKGROUND:Little is known about culture-negative subclinical pulmonary tuberculosis (TB), and its diagnosis remains challenging. Therefore, this study aimed to identify the characteristics and the extent of disease associated with culture-negative subclinical pulmonary TB.METHODS:This retrospective cohort study was conducted on immunocompetent individuals with subclinical pulmonary TB at a university hospital in Thailand from January 2014 to December 2019. Subclinical pulmonary TB was diagnosed based on the presence of radiographic abnormalities consistent with TB in the absence of TB symptoms. All subjects demonstrated significant improvement or resolution of radiographic abnormalities following the completion of treatment. At least two negative sputum cultures were needed to fulfill the definition of culture-negative pulmonary TB. Data were analyzed using univariate and multiple logistic regression analyses to determine the characteristics of those with culture-negative subclinical pulmonary TB compared to culture-positive ones.RESULTS:Out of the 106 individuals identified with subclinical pulmonary TB, 84 met the criteria for inclusion in the analysis. The study found lower radiographic extent and increasing age were key attributes of culture-negative subclinical pulmonary TB. The odds ratios (95% confidence interval) were 7.18 (1.76 to 29.35) and 1.07 (1.01 to 1.13), respectively. They tend to have lower rates of bilateral involvement in both chest x-ray (8.5% vs. 32.0%, p=0.006) and computed tomography (15.4% vs. 42.9%, p=0.035). However, no other specific radiographic findings were identified.CONCLUSIONS:People with culture-negative subclinical pulmonary TB were likely to have less radiographic -severity, reflecting early disease. Nevertheless, no radiographic patterns, except for unilaterality, were related to culture-negative subclinical pulmonary TB.

Cellular processes are controlled by the thermodynamics of the underlying biomolecular interactions. Frequently, structural investigations use one monomeric binding partner, while ensemble measurements of binding affinities generally yield one affinity representative of a 1:1 interaction, despite the majority of the proteome consisting of oligomeric proteins. For example, viral entry and inhibition in SARS-CoV-2 involve a trimeric spike surface protein, a dimeric angiotensin-converting enzyme 2 (ACE2) cell-surface receptor and dimeric antibodies. Here, we reveal that cooperativity correlates with infectivity and inhibition as opposed to 1:1 binding strength. We show that ACE2 oligomerizes spike more strongly for more infectious variants, while exhibiting weaker 1:1 affinity. Furthermore, we find that antibodies use induced oligomerization both as a primary inhibition mechanism and to enhance the effects of receptor-site blocking. Our results suggest that naive affinity measurements are poor predictors of potency, and introduce an antibody-based inhibition mechanism for oligomeric targets. More generally, they point toward a much broader role of induced oligomerization in controlling biomolecular interactions.

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross -reac-tivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.