Biology subjects

Romeo, O.

Publications and source records attributed to Romeo, O..

4 recordsLinked to original sources

bioRxiv2025

Differential importance of MSP4 and MSP5 for infection of red blood cells between human infecting malaria parasites

Plasmodium species malaria parasites require invasion and replication within red blood cells to cause disease. Merozoite surface proteins (MSPs) are proposed to play a role in attachment of merozoites to RBCs and have long been considered as potential vaccine targets, but their functions during invasion are largely unknown. We applied targeted gene editing to investigate MSP4 and 5 function in P. falciparum, which causes most malaria mortality, and P. knowlesi, an in vitro culturable zoonotic species closely related to the widespread P. vivax. CRISPR-Cas9 gene-editing revealed that P. knowlesi MSP4 was not required for parasite growth in vitro. While P. knowlesi MSP5 could be functionally replaced by P. vivax MSP5, it was refractory to gene deletion. We confirmed the opposite for two different P. falciparum laboratory isolates where MSP4 is essential but MSP5 is dispensable. Attempts to select for reliance on the non-essential MSP (e.g. P. knowlesi MSP4 or P. falciparum MSP5) through long-term growth of inducible knock-out parasites, or via chimeric complementation of the essential MSP4 or 5 with the essential MSP from the other species, were unsuccessful. Live cell filming revealed a severe cell-entry defect with conditional knock-down of MSP5 protein expression in P. knowlesi. This study demonstrates differential importance of MSP4 and MSP5 during merozoite RBC invasion across human infecting malaria species, emphasises that vaccine candidates must be considered individually for the two most prominent human malarias and promotes MSP5 as a potential vaccine candidate for P. knowlesi and P. vivax. SignificanceFor a malaria parasite to cause disease, the merozoite form of the lifecycle has to infect and replicate within human red blood cells. Proteins on the surface of the merozoite are considered as promising vaccine candidates, but the functions of these proteins are poorly understood. Here we demonstrate that two structurally similar merozoite surface proteins (MSP), MSP4 and MSP5, have differential importance between one human infecting malaria species compared to a second. The finding that MSP4 is essential for growth in one species, and MSP5 in the other, has implications for understanding invasion biology of malaria parasites and highlights that even structurally similar vaccine targets may need to be chosen specifically for each human infecting malaria species.

microbiology↗

bioRxiv2025

An abundant merozoite surface protein of Plasmodium falciparum modulates susceptibility to inhibitory antibodies

Malaria merozoite surface proteins (MSPs), are thought to have important roles in red blood cell (RBC) invasion and their exposure on the parasite surface makes them attractive vaccine candidates. However, their role in invasion has not been directly demonstrated and their biological functions are unknown. One of the most abundant proteins is PfMSP2, which is likely an ancestral protein that has been maintained in the Plasmodium falciparum lineage and is a focus of vaccine development, whose function remains unknown. Using CRISPR-Cas9 gene-editing, we removed PfMSP2 from two different P. falciparum lines with no impact on parasite replication or phenotype in vitro, demonstrating that it is not essential for RBC invasion. However, loss of PfMSP2 led to increased inhibitory potency of antibodies targeting other merozoite proteins involved in invasion, particularly PfAMA1. In a solid-phase model, increasing concentrations of PfMSP2 protein reduced binding of different antibodies against PfAMA1 in a dose dependent manner. These data suggest that PfMSP2 can modulate the susceptibility of merozoites to protective inhibitory antibodies. The results of this study change our understanding of the potential functions of PfMSP2 and establishes a new concept in malaria where a surface protein can reduce the protective efficacy of antibodies targeting a different antigen. These findings have important implications for understanding malaria immunity and informing vaccine development.

microbiology↗

bioRxiv2022

Human Liver Organoids as a Patient-derived Model for HBV Infection and Cellular Response

Background & AimsCurrent HBV in vitro model systems suffer from many physiological limitations that restrict understanding of complex viral-host interactions and thus prohibit prediction of disease in vivo. We developed and assessed adult stem cell (AdSC) derived liver organoids as a novel model system for characterisation of the HBV lifecycle, the cellular response to infection and demonstrate their utility in assessing antiviral and immunomodulator response. This model system has the potential to be used in predicting individual HBV responses to antivirals and viral reactivation in the setting of immunosuppressive agents. MethodsDuctal stem cells were isolated from healthy tissue acquired from liver resections or biopsy (n=12). Wnt3a & RSPO-1 containing medium was used to stimulate ductal stem cell expansion into organoids which were subsequently differentiated into hepatocyte-like cells. Mature hepatocyte metabolic markers (albumin, CYP3A4) and HBV entry receptor (Na-taurocholate co-transporting polypeptide, NTCP) expression were evaluated throughout differentiation using qRT-PCR and confocal microscopy. We assessed the organoids culture conditions required for HBV infection and HBV life cycle using HepAD38 (genotype D) and plasma derived HBV (genotype B & C). HBV infection was confirmed using immunofluorescence staining (HBcAg), qRT-PCR (RNA, cccDNA, extracellular DNA) and ELISA (HBsAg and HBeAg). We also assessed drug responsiveness using antivirals and an immunosuppressive agent, and cellular responses (interferon-stimulated genes) using interferon- and viral mimic (PolyI:C). ResultsFollowing differentiation, organoids underwent structural remodelling and changes in cellular polarity, accompanied with an increase in albumin, CYP3A4 and NTCP mRNA expression. Optimal HBV infection was achieved in well-differentiated organoids using spinoculation of at least 200 copies/cell of AD38 derived HBV. Infected organoids demonstrate time and donor dependent increase in HBV RNA, cccDNA, extracellular DNA, HBe and HBsAg consistent with viral replication and antigen secretion. Using these markers we assessed drug-responsiveness to the HBV entry inhibitor, Myrcludex B and the JAK inhibitor, Baricitinib. Despite having a very robust interferon stimulated gene response to interferon- and PolyI:C stimulation, HBV infection in liver organoids did not reveal innate immune activation. ConclusionsAdSC derived liver organoids support the full life cycle of HBV with significant donor dependent variation in viral replication and cellular responses. These features can be utilised for development of personalised drug testing platform for antivirals. Lay SummaryHuman liver organoid culture provides a personalised assessment of HBV infection, replication and responsiveness to antiviral therapy. This model system has a robust innate immune response and could be used to assess novel immune-modulating curative therapy.

microbiology↗

bioRxiv2022

Constitutive expression and distinct properties of IFN-epsilon protect the female reproductive tract from Zika virus infection

The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFN{varepsilon}) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs , {beta} and {lambda}. We show the necessity of IFN{varepsilon} for Zika Virus (ZIKV) protection by: increased susceptibility of IFN{varepsilon}-/- mice; their "rescue" by intravaginal recombinant IFN{varepsilon} treatment and blockade of protective endogenous IFN{varepsilon} by neutralising antibody. Complementary studies in human FRT cell lines showed IFN{varepsilon} had potent anti-ZIKV activity, associated with transcriptome responses similar to IFN{lambda} but lacking the proinflammatory gene signature of IFN. IFN{varepsilon} activated STAT1/2 pathways similar to IFN and {lambda} that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFN{varepsilon} exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFN{varepsilon}. However, the IFN{varepsilon} expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFN{beta} or {lambda}. Thus, the constitutive expression of IFN{varepsilon} provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFN{varepsilon} provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy. Author SummaryThe female reproductive tract (FRT) is vulnerable to sexually transmitted infections and therefore a well-tuned immune surveillance system is crucial for maintaining a healthy FRT. However, our understanding of the factors that impact viral infection of the FRT and the host response are not well understood. In this work we investigate the role of a hormonally regulated type I interferon, IFN epsilon (IFN{varepsilon}) in control of Zika virus (ZIKV) infection of the FRT. IFN{varepsilon} is unique compared to other canonical type-I IFNs in that it is constitutively expressed by epithelial cells of the FRT with expression levels controlled by progesterone and not in response to viral infection. We demonstrate that IFN{varepsilon} has anti-ZIKV properties using a combination of IFN{varepsilon} KO mice, blockade of endogenous IFN{varepsilon} by neutralising Abs and rescue of IFN{varepsilon} KO mice by recombinant IFN{varepsilon} administered directly to the FRT. Furthermore, we complemented our in vivo studies using human FRT derived cell lines. Importantly, ZIKV NS proteins did not block IFN{varepsilon} expression despite their ability to antagonise the expression of IFN{beta} or {lambda}. Collectively this work implicates IFN{varepsilon} as a key type-I IFN that provides a distinct homeostatic antiviral environment in the FRT.

immunology↗