Biology subjects

da Costa, K.

Publications and source records attributed to da Costa, K..

6 recordsLinked to original sources

bioRxiv2026

Pseudotyped virus-based platform and structural analysis reveal potential cross-reactivity sites between influenza C and D viruses

Influenza C (ICV) and influenza D (IDV) viruses belong to the Orthomyxoviridae family and are classified in the genera Gammainfluenzavirus and Deltainfluenzavirus, respectively. Although the main reservoir of ICV is humans, IDV is mainly found in cattle. To date, the zoonotic potential of IDV has not been fully elucidated. ICV and IDV share about 50% homology at the genetic level, and both express hemagglutinin esterase fusion (HEF) glycoproteins on the surface for the dual purpose of binding the receptor and releasing new virions. Using pseudotyped viruses (PVs) in a pseudotyped virus-based microneutralisation assay (pMN), some bovine serum samples showed strong neutralisation of both ICV and IDV. In silico analyses were performed to explore the molecular basis of this phenomenon. HEF structures were recovered from the Protein Data Bank, epitopes were predicted using BepiPred, and sialic acid receptor docking was evaluated with HDOCK. Five potential epitopes were selected, and mutual substitutions of amino acid residues were introduced to generate mutant ICV and IDV HEFs and corresponding PVs. Although only mutant IDV PVs were successfully produced, a reference ICV antiserum showed high neutralising activity against one construct, indicating the exposure of an ICV-like antigenic site within the IDV framework. Herein we provide evidence consistent with the existence of antigenic sites shared between ICV and IDV, which could be exploited for cross-protective vaccine design, through integrated computational and experimental investigations.

immunology↗

bioRxiv2025

Cross-Neutralizing Monoclonal Antibodies with Broad Activity Against Human and Bat-Derived SARS-Related Coronaviruses

Effective therapeutics for severe cases of SARS-CoV-2 are still needed. As new variants of concern emerged an increase in hospitalizations was observed, especially in non-vaccinated individuals, immunocompromised individuals and the elderly, whereby treatment options became challenging. Several monoclonal antibodies (mAb) are being evaluated for approval due to their ability to neutralize the virus. Here, we report monoclonal antibodies targeting the Spike (S) with a strong neutralization profile against lentiviral and VSV pseudotypes displaying the Spikes of SARS-CoV-1, SARS-CoV-2 and variants of concern, in addition to two bat coronaviruses. We found several mAbs that were able to bind and neutralize a broad set of variants, with one mAb able to neutralize SARS-CoV-1, SARS-CoV-2, RaTG13 and WIV16. Their binding affinities were also characterized, and several mAbs were in the picomolar range against different variants. These results indicate that their use as a cocktail of monoclonal antibodies to treat patients infected with variants associated with severe disease and immune escape can be explored as an option. Furthermore, the cross reactivity observed by one mAb may reveal further insight into vulnerable portions of the Spike protein, which may become valuable future targets.

microbiology↗

bioRxiv2025

Expanded adaptive NKG2C+ NK cells exhibit potent ADCC and functional responses against HBV-infected hepatoma cell lines

BackgroundHepatitis B virus (HBV) infection remains a significant global health challenge, leading to chronic liver disease and hepatocellular carcinoma (HCC). Natural killer (NK) cells play an important role in the clearance of HBV-infected cells, but their efficacy is often compromised during chronic infection. Adaptive NK cells, characterised by NKG2C expression and enhanced functional responses, represent a promising therapeutic avenue for enhancing anti-HBV immunity and responses to HBV-driven cancers. MethodsWe applied an established protocol, involving K562-HLA-E expressing feeder cells and cytokines (IL-2), for the expansion of adaptive NK cells from cryopreserved T- and B cell depleted peripheral blood mononuclear cells (PBMCs) derived from donors with chronic HBV infection alone or with Human Immunodeficiency Virus (HIV) co-infection. We evaluated the adaptive profile of expanded NK cells, their antibody-dependent cellular cytotoxicity (ADCC) capacity and functional responses against hepatoma cell lines in the presence or absence of HBV infection. ResultsExpanded NK cells achieved >97% purity, with the NKG2C positive population exhibiting a mean 100-fold expansion. These cells demonstrated a predominantly adaptive phenotype with high surface expression of NKG2C and cytotoxic potential (Granzyme B). They maintained high levels of CD16 surface expression and upregulated CD2, essential for ADCC. Functionally, expanded adaptive NK cells showed enhanced ADCC capacity and functional responses to K562 targets, naive, HBV integrant-expressing, and de novo infected hepatoma cell lines. TGF-{beta} preconditioning induced tissue-resident features (CD103, CD49a) in expanded adaptive NK cells, while preserving their adaptive phenotype and functionality, enhancing their potential for liver targeted immunotherapy. Further, expanded adaptive NK cells demonstrated minimal reactivity against autologous activated T cells, suggesting limited off-target effects. ConclusionsOur study demonstrates the first successful expansion of adaptive NK cells with robust functional responses from donors with chronic viral infection. This approach creates opportunities for NK cell-based therapies alone or in combination with monoclonal antibodies contributing to HBV functional cure strategies and the treatment of HBV-driven cancers.

immunology↗

bioRxiv2025

Development and optimisation of Influenza C and Influenza D pseudotyped viruses

To facilitate the study of influenza C (ICV) and influenza D (IDV) viruses, we generated lentiviral pseudotyped viruses (PVs) expressing the hemagglutinin-esterase fusion (HEF) glycoprotein from ICV (C/Minnesota/33/2015) and IDV (D/Swine/Italy/199724-3/2015, D/Bovine/France/5920/2014, and D/Bovine/Ibaraki/7768/2016). The production of these PVs was optimised using different amount of human airway trypsin-like (HAT) protease to enhance HEF maturation, and the transduction efficiency was evaluated in multiple cell lines. Using these PVs, we established a pseudovirus-based microneutralisation (pMN) assay to measure neutralising antibody responses and adapted an esterase activity assay to evaluate PV. Specific antisera neutralised PVs but failed to inhibit esterase activity. These findings confirm that ICV and IDV PVs provide a scalable, sensitive, and safe tool for antiviral screening, and sero-epidemiological research.

immunology↗

bioRxiv2025

Investigating Factors Driving Shifts in Subtype Dominance within H5Nx Clade 2.3.4.4b High-Pathogenicity Avian Influenza viruses

H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs) have decimated wild bird and poultry populations globally since the autumn of 2020. In the United Kingdom (UK) and in continental Europe, the H5N8 subtype predominated during the first epizootic wave of 2020/21, with few detections of H5N1. However, during the second (2021/22) and third (2022/23) epizootic waves, H5N1 was the dominant subtype. The rapid shift in dominance from H5N8 to H5N1 was likely driven by a combination of virological, immunological, and/or host-related factors. In this study, we compared viral fitness and immunological responses in ducks, a key reservoir species, using dominant genotypes of H5N1 (genotype AB) and H5N8 (genotype A) from the second wave. While viral shedding dynamics were similar for both viruses, H5N8 was more pathogenic. Antigenic analysis of post-infection duck sera revealed that the haemagglutinin (HA) protein was antigenically similar across clade 2.3.4.4b H5 HPAIVs, but neuraminidase (NA) proteins displayed different patterns of cross-reactivity. We also modelled a scenario where ducks were pre-exposed to H5N1 (genotype C) or H5N8 (genotype A) from the first wave and subsequently challenged with either homologous or heterologous subtypes from the second wave (genotype AB or A). Despite the absence of seroconversion, pre-exposure to different subtypes resulted in varying clinical outcomes following challenge. These findings indicate that both viral and immunological factors likely played significant roles in the emergence and spread of H5Nx HPAIVs in wild bird populations.

microbiology↗

bioRxiv2021

Establishment of pan-Influenza A (H1-H18) and pan-Influenza B (pre-split, Vic/Yam) Pseudotype Libraries for efficient vaccine antigen selection

We have developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18, and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza serological and neutralization assays. We demonstrate its utility in detecting serum response to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further pre-clinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objectives that contribute to the strengthening of global influenza surveillance, expansion of seasonal influenza prevention and control policies, and strengthening pandemic preparedness and response.

immunology↗