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- Effect of age and prior COVID-19 on BBIBP-CorV (Sinopharm) vaccination-induced antibody responses in a region with high seroprevalencePublication . Hasan, Zahra; Masood, Kiran Iqbal; Qaiser, Shama; Khan, Erum; Hussain, Areeba; Ghous, Zara; Khan, Unab; Hassan, Imran; Yameen, Maliha; Memon, Haris Ali; Ali, Shiza; Balouch, Sadaf; Nasir, Muhammad Imran; Qazi, Muhammad Farrukh; Veldhoen, Marc; Simas, J. Pedro; Bhutta, Zulfiqar A.; Mahmood, Syed Faisal; Hussain, Rabia; Ghias, KulsoomIntroduction: Sinopharm (BBIBP-CorV) inactivated virus vaccination for COVID-19 has been administered widely in Pakistan. We investigated the dynamics of BBIBP-CorV -induced antibody responses over a 24 week period in a region with a high seroprevalence. Methods: Study subjects (n = 312) were followed up over a 24-week period between May and August 2021. Sera were tested for IgG antibodies to spike and the receptor binding domain (RBD). Results: Study subjects were 62% female. Twenty-two percent had a prior history of COVID-19. At 4-, 8- 16- and 24- weeks post-vaccination, the rate of IgG antibodies positive to spike was 57%, 87%, 66% and 90% of individuals, compared with to RBD which was 48%, 62%, 68% and 85% of subjects, respectively,. IgG to spike and RBD showed a positive correlation at each interval (rho > 0.6, p < 0.0001). Seropositivity to both spike and RBD was reduced in those aged 50 years and over for up until 16 weeks post-vaccination (p < 0.05). Individuals with prior COVID-19 infection showed greater antibody responses for up to 16 weeks post-vaccination (p < 0.05). SARS-CoV-2 infections were observed with a mean interval of 16 weeks postvaccination. Antibody responses did not wane for up to 6 months post-vaccination. Conclusions: Sinopharm vaccination-induced antibody responses were negatively impacted by age and positively impacted by prior COVID-19 for 16 weeks after vaccination. Importantly, we did not find waning of IgG antibodies to RBD over the study period. Maintenance of antibodies may be the result of continued community exposure and boosting with COVID-19 vaccination.
- A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serumPublication . Abidi, Syed Hani; Imtiaz, Kehkeshan; Kanji, Akbar; Qaiser, Shama; Khan, Erum; Iqbal, Kiran; Veldhoen, Marc; Ghias, Kulsoom; Simas, J. Pedro; Hasan, ZahraBackground: Individuals recovering from COVID-19 are shown to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability and some may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays that allow the determination of virus neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods: The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro‐neutralization assay, 19 serum samples, with positive IgG titers against Spike receptor binding domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of the cytopathic effect, then lysed and RNA RT-PCR of SARS-CoV-2. The Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results: Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, while 4 samples gave neutralization at lower dilution, while one sample did not give any neutralization. The correlation between RBD OD and neutralization potential was found to be statistically correlated. Conclusion: We describe a rapid RT-PCR based SARS-CoV-2 microneutralization assay for detection of neutralizing antibodies. This can effectively be used to test anti-viral activity of serum antibodies for investigation of both disease-driven and vaccine-induced responses.
- Modelling the transmission dynamics of H9N2 avian influenza viruses in a live bird marketPublication . Pinotti, Francesco; Kohnle, Lisa; Lourenço, José; Gupta, Sunetra; Hoque, Md Ahasanul; Mahmud, Rashed; Biswas, Paritosh; Pfeiffer, Dirk; Fournié, GuillaumeH9N2 avian influenza viruses (AIVs) are a major concern for the poultry sec24 tor and human health in countries where this subtype is endemic. By fitting a 25 model simulating H9N2 AIV transmission to data from a field experiment, we 26 characterise the epidemiology of the virus in a live bird market in Bangladesh. 27 Many supplied birds arrive already exposed to H9N2 AIVs, resulting in many 28 broiler chickens entering the market as infected, and many indigenous back29 yard chickens entering with pre-existing immunity. Most susceptible chickens 30 become infected within one day spent at the market, owing to high levels of 31 viral transmission within market and short latent periods, as brief as 5.3 hours. Although H9N2 AIV transmission can be substantially reduced under moderate 33 levels of cleaning and disinfection, effective risk mitigation also requires a range 34 of additional interventions targeting markets and other nodes along the poultry 35 production and distribution network.
- Influenza A virus hemagglutinin remodels membranes into a vRNP clustering platformPublication . Wachsmuth-Melm, Moritz; Peterl, Sarah; Makroczyová, Jana; Vale-Costa, Sílvia; Amorim, Maria João; Chlanda, PetrPandemics caused by influenza A virus are associated with co-infections of zoonotic and humanadapted viruses. To form an infectious entity, the virus must incorporate eight different ribonucleoproteins (vRNPs) into virions budding at the plasma membrane. The process of vRNP clustering is poorly understood and has not been investigated at a molecular resolution. Here we employ in situ cryo-correlative light and electron microscopy at late stages of viral infection to identify hemagglutinin and neuraminidase as drivers of membrane remodelling. We show that hemagglutinin, but not neuraminidase-remodelled compartments serve as vRNP clustering platforms. Our nearestneighbour analysis demonstrates that vRNPs cluster on hemagglutinin-remodelled membranes in a Rab11a-dependent manner. Additionally, we uncover that matrix protein 1 (M1) forms multi-layered cylindrical assemblies in the nucleus which restructure in the cytoplasm. Further, we show that M1 layer assembly precedes its attachment to the plasma membrane and M1 thereby coordinates the closure of the virion budding neck. In summary, we demonstrate that hemagglutinin is involved in intracellular vRNP clustering by inducing membrane remodelling to provide a large interaction surface. Thus hemagglutininmembrane remodelling may play a role during vRNP sorting and reassortment that lead to the adaptation of zoonotic influenza A viruses.
- Bioengineered 3D microvessels reveal novel determinants of Trypanosoma congolense sequestrationPublication . Porqueddu, Teresa; Niz, Mariana De; Casas-Sánchez, Aitor; Introini, Viola; Zorrinho-Almeida, Maria; Sender, Silvia Sanz; Carrasqueira, Diana; Figueiredo, Luisa; Bernabeu, Maria; Pereira, Sara SilvaIn the mammalian host, Trypanosoma congolense cytoadheres to the vascular endothelium in a process known as sequestration. Although sequestration influences clinical outcome, disease severity and organ pathology, its determinants and mediators remain unknown. Challenges such as the variability of animal models, the only-recently developed tools to genetically manipulate the parasite, and the lack of physiologically relevant in vitro models have hindered progress. Here, we engineered brain and cardiac 3D bovine endothelial microvessel models that mimic the bovine brain microvasculature and the bovine aorta, respectively. By perfusing these models with two T. congolense strains, we simulated physiologically relevant conditions and investigated the roles of flow for parasite sequestration and tropism for different endothelial beds. We discovered that sequestration is dependent on cyclic AMP signalling, closely linked to parasite proliferation, but not associated with parasite transmission to the tsetse fly vector. Finally, by comparing the expression profiles of sequestered and non-sequestered parasites collected from a rodent model, we showed gene expression changes in sequestered parasites, including of the surface variant antigens. This work presents a physiologically-relevant platform to study trypanosome interactions with the vasculature and provides a deeper understanding of the molecular and biophysical mechanisms underlying T. congolense sequestration.
- Land and climate suitability for West Nile virus in Atlantic archipelagos guided by historical data from EuropePublication . Geraldes, Martim Afonso; Giovanetti, Marta; Cunha, Mónica Vieira; Lourenço, JoséWest Nile (WNV) is a zoonotic mosquito-borne virus with an expanding geographical range and epidemic activity worldwide. Computational studies have contributed to the understanding of factors driving WNV occurrence, particularly in North America and Europe, providing invaluable insights towards surveillance and preparedness. Archipelagos have largely been overlooked, despite the risks WNV poses to unique local avian species and human populations. In this study, we apply a machine learning-based ecological niche approach, trained on WNV occurrence and (a)biotic factors from Portugal, Spain, and Italy, to estimate ecological suitability for WNV occurrence across several Atlantic archipelagos. The approach gives weight to the temporal dimension, moving beyond conventional spatial suitability estimations, and generating novel insights on seasonality both for Europe and the archipelagos. For Portugal, Spain and Italy, modelling results align with previous findings on spatial hotspots and (a)biotic drivers of WNV occurrence, while further unraveling properties of at-risk human populations within dynamically suitable land areas. For Atlantic archipelagos, results constitute a novel and detailed perspective on local ecological suitability for WNV occurrence, providing a data-driven framework that identifies spatial hotspots, defines seasonal patterns and quantifies the local population at risk. The synthetic data generated in this study supports the development of targeted preparedness, surveillance and mitigation plans tailored to the unique ecological and seasonal dynamics of each region under study.
- AlphaFold models of host-pathogen interactions elucidate the prevalence and structural modes of molecular mimicryPublication . Baptista, Delora; Gomez-Lucas, Lídia; Jänes, Jürgen; Krogan, Nevan J.; Amorim, Maria João; Ivarsson, Ylva; Beltrão, PedroPathogens exploit host cellular machinery through protein-protein interactions (PPIs), often using molecular mimicry to hijack host cellular processes. While there have been thousands of host-pathogen PPIs determined to date, the lack of structural information for these impedes the study of the prevalence of molecular mimicry and convergent evolution of protein interaction interfaces. To address this, we benchmarked AlphaFold2 and 3 for prediction of structures of host-pathogen interactions observing that accurate models can be retrieved when ranking by modelling confidence, despite an overall low performance. We predicted structures for 6,782 pathogen-human PPIs yielding 803 models of higher confidence. Most pathogen proteins interacting with a common human protein are predicted to do so via the same interface, suggesting a high degree of convergent evolution of protein interaction interfaces. When comparing structural models from host-pathogen and host-host interactions, we observe that a majority of pathogen proteins are predicted to target existing human PPI interfaces. We categorized instances of mimicry into different modes, occurring at different frequencies: 1) via the same domain family (least common); 2) via a similar structural motif; and 3) via a similar linear motif (most common). We selected examples of linear motif interactions for binding assay testing, confirming 8 out of 12 predicted interfaces, including 3 viral linear motif interactions. This validates AlphaFolds ability to model some host-pathogen interactions and the mechanisms underlying molecular mimicry. This work showcases the value of large-scale structural modelling to study convergent evolution of host-pathogen interactions and how molecular mimicry may contribute to infection or host defense.
- Climate-driven spatiotemporal dynamics of Aedes infestation and dengue transmission in Porto Alegre, Southern Brazil.Publication . Silva, Adryan Aparecido da; Ferreira, Álvaro Gil Araujo; Lourenco, José; Freitas, Amanda Cupertino deDengue transmission is strongly influenced by climatic conditions that affect mosquito population dynamics and virus circulation. In Southern Brazil, where dengue historically occurred at low levels, recent climatic anomalies may be contributing to the expansion of Aedes vectors and an increase in local dengue incidence. This study investigated the spatiotemporal association between climatic variables, Aedes mosquito infestation and dengue cases in Porto Alegre (Southern Brazil, 2018 to 2025). Entomological, surveillance and climatic data were analyzed using Morans I and LISA for spatial association, Kendall correlation, polynomial regression and LASSO to identify relevant drivers and develop predictive models of mosquito infestation and dengue incidence. A strong spatial association between Aedes aegypti and Aedes albopictus was observed, with persistent local clusters detected across all years. Annual climatic variables were associated with mosquito abundance in several districts. Overall, rainfall frequency had a stronger effect on Aedes aegypti abundance than accumulated rainfall. Temperature and lagged infestation indices showed strong association with both species and dengue incidence, with effects observed up to four weeks prior. Predictive models demonstrated good agreement between observed and predicted values, particularly within low to moderate infestation levels. Lagged variables were consistently retained in both mosquito infestation abundance and dengue incidence models, highlighting the importance of temporal predictors for anticipating vector dynamics and dengue risk. This approach is generally applicable for predicting Aedes infestation and disease incidence and emphasizes the importance of integrating entomological and climatic surveillance data to improve anticipation and detection of dengue risk periods and support more effective public health interventions. Author summaryDengue is a viral disease transmitted by mosquitos that affects millions of people worldwide. Its spread is strongly influenced by climatic conditions that modulate mosquito life cycles. In Southern Brazil, where dengue historically occurred at low levels, recent climate variability and extreme weather events may be creating favorable conditions for the expansion of Aedes mosquitos and local dengue activity. In this study, we analyzed mosquito, dengue, and climatic surveil in Porto Alegre, Brazil, from 2018 to 2025 to understand how climate influences mosquito infestation and dengue incidence. We found that mosquito infestation and dengue cases show clear spatial patterns across the city and are strongly influenced by temperature and rainfall. Interestingly, the number of rainy days had a stronger effect on mosquito abundance than the total amount of rainfall. We also observed that higher mosquito infestation was associated with an increase in dengue cases up to four weeks later. These findings show that combining climatic and mosquito surveillance data can help anticipate periods of higher dengue risk and support more effective mosquito control and public health actions.
- Direct suppression of host gluconeogenesis by Trypanosoma bruceiPublication . Figueiredo, Luísa M.; Amin, Abdulbasit; Colaço, João; Machado, Henrique; Nunes-Cabaço, Helena; Trindade, Sandra; Scherer, Philipp; Pereira, Sara SilvaThe cause of hypoglycaemia in African trypanosomiasis remains unresolved. We found that infected mice show normal carbohydrate digestion, intestinal glucose absorption, and tissue glucose uptake, but a profound reduction of host glucose production via gluconeogenesis. Liver transcriptomics uncovered coordinated repression of gluconeogenic pathways alongside inflammatory activation. Infection of Rag2⁻/⁻ mice showed that adaptive immunity contributes only minimally to suppression of host gluconeogenesis. In contrast, therapeutic parasite clearance completely restored host glycaemia and gluconeogenesis despite persistent immune activation. These results demonstrate that live parasites directly repress gluconeogenesis in vivo. Using a co-culture assay, we showed that T. brucei is sufficient to directly repress gluconeogenic gene expression in primary hepatocytes in vitro. Finally, we found that providing glycerol in drinking water to infected animals stimulated gluconeogenesis, elevated glycaemia, and improved survival without impacting parasitaemia. This study reveals a previously unrecognised pathogenic strategy in which a eukaryotic parasite directly suppresses host gluconeogenesis.
- A 3D tumor-on-a-chip platform to identify drugs that block breast cancer cell intravasationPublication . Perera, Narmada; Coutinho, Diogo; Morais, Carolina; Faria, Maria; Neto, Raquel; Roman, William; Gomes, E. R.; Franco, Cláudio A.; Costa, Luís; Barata, David; Serre, Karine; Dias, Sérgio; Magalhães, AnaMetastasis is the leading cause of death in breast cancer patients, yet there are no drugs specifically designed to block cancer cell intravasation, an early step of the metastatic cascade that originates circulating tumour cells (CTCs). A major challenge in developing anti-intravasation drugs is the scarcity of relevant in vitro platforms suitable for predictable drug discovery. Intravasation is a fundamental step of metastasis and involves the crossing of cancer cells through an endothelial barrier to enter the blood circulation. Here we developed an intravasation-on-a-chip model with controlled extracellular matrix composition, fluid flow and shear stress, which mimics the dynamic tumour-endothelium interface. The systems allows real-time imaging of intravasation and the isolation and quantification of intravasated cancer cells. As a proof-of-concept for drug testing, we show that perfusion with the PI3K/mTOR inhibitor Dactolisib, significantly reduced intravasation without compromising endothelial cell viability. The system also provides the capability to evaluate inhibitor on-target activity via imaging analysis. This intravasation-on-a-chip model offers a powerful, scalable, and imaging-compatible platform for discovering and evaluating anti-intravasation compounds.
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